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This is to certify that the

dissertation entitled

Regulation of Carbohydrate Metabolism in
Asparagus Spears (Asparagus officinalis L.)

presented by
Silvanda de Melo Silva

has been accepted towards fulfillment
ofthe requirements for

Ph. D. degree in HortiCUlture

 

 

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REGULATION OF CARBOHYDRATE METABOLISM IN ASPARAGUS
SPEARS (Asparagus officinalis L.)
By

Silvanda de Melo Silva

A DISSERTATION
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1998

ABSTRACT

REGULATION OF CARBOHYDRATE METABOLISM IN ASPARAGUS
SPEARS (Asparagus officinalis L.)

By

SILVANDA DE MELO SILVA

Sucrose depletion in harvested asparagus (Asparagus oflicinalis L.) spear tips is
rapid and may trigger senescence. This research focuses on investigating how
carbohydrate metabolism in asparagus spear tips, stored at 1 °C, is regulated. The
research was divided in two parts. The first portion was an investigation into the effects
of low 02 and high CO2 on respiratory and fermentative metabolism, sucrose-metabolizing
and glycolytic enzymes, and phosphate and adenylate levels in spear tips. The second
portion was an evaluation of the effects of exogenous cytokinin on sucrose metabolism in
harvested asparagus spears.

For this portion of the research, spears were stored with a range of O2 partial
pressures combined with CO2 treatments of O, 5, 10, and 20 kPa using a system
combining modified atmosphere packages in flow-through containers for a period of 6
days. The respiratory quotient increased and ethanol, acetaldehyde, and lactate
accumulated below 2 kPa. During 6 days of storage, sucrose declined markedly, relative
to harvest. Sucrose content was highest for spears exposed to 5 kPa C02. Sucrose
synthase activity was higher at harvest; however, bound acid invertase activity was the
highest after the storage period, indicating that sucrose may be degraded postharvest
mostly in the apoplast. S kPa CO2 reduced sucrose synthase, invertase and hexokinase

activities, which may account for the reduction in sucrose utilization. Alcohol

dehydrogenase (ADH) and pyruvate decarboxylase activities increased for 02 levels below
2 kPa. Lactate dehydrogenase activity was lower than ADH activity; however, it also
increased below 2 kPa 02. Visual quality (VQ) was reduced by C02, but increased as 02
increased. VQ was best for spears stored in near-ambient O2 and 5 kPa C02.

Low 02 enhanced the interconversion of phosphoenolpyruvate (PEP) to pyruvate
(PYR) and F6P to fructose-1,6-bisphosphate (F1,6P2), indicating a promotion of
glycolysis with a resulting loss of carbon in the sugar pool. Low 02 caused an increase in
pyruvate kinase (PK) activity. However, high CO2 besides having an effect on PK,
appeared to affect conversion of F6P to Fl,6P2 at 02 levels below 2 kPa.

PPi and ATP decreased below 2 kPa 02, with a concomitant increase in Pi, ADP,
and AMP. Low 02 also reduced adenylate energy charge (AEC) relative to high 02.
Higher C02 reduced AEC for 02 levels greater than 2 kPa. Decreases in PPi, ATP, and
ABC, and increases in Pi, ADP, and AMP in response to O2 deficiency suggest impairment
of oxidative phosphorylation and unbalanced cell metabolism, which may limit asparagus
spear survival. Below 2 kPa 02 the cells were under stress leading to fermentation.
Below 1 kPa 02 the spear tip tissues were experiencing severely limiting energy
availability.

6-BAP reduced respiration rate of spears with intact tips, slowed changes in
fluorescence, and slowed chlorophyll degradation during 31d of storage at 1°C. 6-BAP
also slowed the decline in sucrose content in spears with tips. The visual quality was

highest for spears with tips that were treated with 6-BAP.

To my parents Jose’ Jorge and Eliza

(In memoriam )

To my husband, Djail and my son, Lucas,

for their love and support

iv

ACKNOWLEDGMENTS

I would like to express my gratitude to my major professors, Dr. Robert Hemer
and Dr. Randy Beaudry. Dr. Hemer provided me with guidance, enthusiastic support,
encouragement, and friendship throughout my graduate program. Dr. Beaudry supported
me with friendship and enlightening guidance. His sharp vision in pursuing new research
avenues provided a foundation for my scientific development and is genuinely appreciated.

I am grateful to the members of my guidance committee: Dr. Wayne Loescher,
whose vision and advices greatly improved this work; Dr. Ken Pot? and Dr. Mark
Uebersax, for their constant encouragement and for bringing insightful suggestions into
the proposed research. In addition, I would like to thank Dr. D.R. Dilley. His scientific
rigor will remain as an example for my career.

A special thanks goes to Dr. John Everard for his valuable technical expertise and
for his constructive comments and suggestions. This work benefited greatly from his
knowledge and willingness to help. I also wish to thank the Postharvest group, especially
Dr. Dina Kadyrzhanova and Dr. Nazir Mir, for for their constant help.

I also wish to thank Michigan State University for providing me the unique
opportunity to participate in the doctoral program in Postharvest Physiology offered by
the Department of Horticulture. The financial support of the Ministry of Education of
Brazil, through the CAPES (Fundacao Coordenacao de Aperfeicoamento de Pessoal de
Nivel Superior) fellowship program and Universidade Federal da Paraiba (UFPB) made

possible the completion of my degree.

I thank my graduate fellows in the Department of Horticulture, especially Stacie
Wee, Jennifer Dwyer, and Rijida Leepipattanawit for their valuable friendship, constant
support and encouragement.

I would like to thank to the Main Office of the Department of Horticulture,
specially to Ms. Joyce Jackson, Sherry Mulvaney and Lorri Busick for their assistance.

I want to thank my fiiends and colleagues at UFPB, especially Rita de Cassia
Queiroga, Roberto Gennano Costa, An'naldo Frazao, and Genival Azeredo for their
encouragement and support. I wish to thank the many friends of the Brazilian community
at Michigan State University. Special thanks goes to J osé Lima and family, for their
fiiendship and precious help in getting started at MSU; Dorotéia Paglis and family, for
their fiiendship and for being close by.

Finally, I wish to thank my sister (Cirlene) and brothers (Cicero, Cidene,

Bartolomeu, Civaldo, e Civanildo) for always being there and believing in me.

vi

TABLE OF CONTENTS

LIST OF FIGURES ................................................... ix
INTRODUCTION .................................................... 1
CHAPTER 1. LITERATURE REVIEW .................................... 4
Asparagus Botanical Background .................................... 4
Biology of Growth and Carbon Allocation ............................. 5
Carbon Metabolism in Harvested Asparagus ........................... 7
Sucrose Metabolism .............................................. 9
Respiratory metabolism .......................................... 17
Glycolysis ............................................... l9

Fennentative metabolism ................................... 29

Phosphate and adenylate metabolism .......................... 33

Cytokinin Metabolism in Asparagus ................................. 37
Literature Cited ................................................ 39

CHAPTER 2. RESPIRATORY AND FERMENTATIVE METABOLISMS IN
ASPARAGUS TIPS UNDER LOW OZ/HIGH CO2 ATMOSPHERES

INTRODUCTION ................................................... 51
MATERIAL AND METHODS .......................................... 53
RESULTS .......................................................... 60
DISCUSSION ....................................................... 63
LITERATURE CITED ................................................ 70

CHAPTER 3. REGULATION OF THE STEADY STATE 02 IN ASPARAGUS SPEAR
TIPS: GLYCOLYTIC METABOLISM AND SUCROSE-
METABOLIZING ENZYMES UNDER LOW OZ/HIGH CO2

ATMOSPI-IERES
INTRODUCTION ................................................... 86
MATERIAL AND METHODS .......................................... 88
RESULTS .......................................................... 95
DISCUSSION ....................................................... 99
LITERATURE CITED ............................................... 109

vii

CHAPTER 4. PHOSPHATE AND NUCLEOTIDE METABOLISM UNDER LOW
OZ/HIGH CO2 PARTIAL PRESSURES IN THE TIPS OF HARVESTED
ASPARAGUS SPEARS STORED AT 1 °C

INTRODUCTION .................................................. 123
MATERIAL AND METHODS ......................................... 126
RESULTS ......................................................... 130
DISCUSSION ...................................................... 133
LITERATURE CITED ............................................... 142

CHAPTER 5. EFFECTS OF 6-BENZYLAMINOPURINE ON SUGAR PROFILE AND
SENESCENCE OF ASPARAGUS (Asparagus oflicinalis L.) SPEARS
STORED AT 1 ° C

INTRODUCTION .................................................. 1 5 5
MATERIAL AND METHODS ......................................... 157
RESULTS AND DISCUSSION ........................................ 162
LITERATURE CITED ............................................... 169

viii

LIST OF FIGURES

Figure 1.1. Proposed cellular pathways of sugar transport and metabolism in a sink cell of
asparagus supplied with sucrose via symplastic and apoplastic routes. Enzymes are:
bound sucrose synthase (SUSY B) and soluble sucrose synthase (SUSY F), invertases
(VI, cytosolic neutral; VI, vacuolar acid; CWI, cell wall bound acid), and hexokinase
(HK). Substrates are sucrose (S), glucose (G), fi'uctose (F). Sugar transporters and
cellulose synthase are represented by black dots. Adapted from Krausgrill et al. (1996) 12

Figure 1.2. Glycolysis in plant tissues showing the relationship with oxidative pentose
phosphate (OPP) pathway and TCA cycle. The numbers represent enzymes as follows: 1,
hexokinase; 2 fructokinase; 3, phosphoglucoisomerase; 4, ATPzPFK; 4a, PPizPFK; 5,
aldolose; 6, triosephosphate isomerase; 7, glyceraldehyde-3 -phosphate dehydrogenase; 8,
phosphoglycerate kinase; 9, phosphoglycerate mutase; 10, enolase; ll, pyruvate kinase;
12, pyruvate decarboxylase; 13, alcohol dehydrogenase; l4, lactate dehydrogenase; 15,
intracellular invertase; 15a, extracellular invertase; 16, sucrose synthase; 17, UDP-glucose
pyrophosphorylase; l8, phosphoglucomutase; 19, glucose-6-phosphate dehydrogenase.
Adapted from Ashihara et al. (1988) ...................................... 20

Figure 1.3. Responses to the cellular energy charge of regulatory enzymes in metabolic
pathways in which ATP is produced (curve 1) and in which it is utilized (curve 2). From
Plaxton (1990) ....................................................... 36

Figure 2.1. Proposed pathway of fennentative metabolism is asparagus spears regulated
by low Ozlhigh CO2 stress. ADH (alcohol dehydrogenase); ETC (electron transport
chain); G-6-P (glucose-6-phosphate); LDH (lactic dehydrogenase); PDC (pyruvate
decarboxylase); TCA (tricarboxylic acid cycle); ---+---> , induction and /or activation;
--_--->, reduction and/or inhibition. Adapted from Ke et al. (1995) .............. 75

Figure 2.2. Schematic drawing of experimental apparatus allowing adjustment of a
package gas composition to target levels by controlling composition of the atmosphere in
the exterior chamber .................................................. 76

Figure 2.3. The effects of storage 02 and CO2 on 02 uptake, CO2 production, and
respiratory quotient (RQ) in harvested asparagus spears held at 1 °C for 6 days ...... 77

Figure 2.4. The effects of storage 02 and CO2 on non-oxidative CO2 in harvested
asparagus spears held at 1 °C for 6 days .................................... 78

Figure 2.5. The effects of storage 02 and CO2 on the contents of fi'uctose, glucose, and

sucrose (fresh mass basis), relative to harvest in the most acropetal 45 mm of the tip of
asparagus spears held at 1 °C for 6 days .................................... 79

ix

Figure 2.6. The effects of storage 02 and CO2 on package headspace acetaldehyde and
ethanol for asparagus spears held at 1 °C for 6 days ........................... 80

Figure 2.7. The effects of storage 02 and CO2 on the content of lactate (fresh mass basis),
relative to harvest in the most acropetal 45 mm of the tip of asparagus spears held at 1 °C
for 6 days .......................................................... 81

Figure 2.8. The effects of storage 02 and CO2 on the activity of lactic dehydrogenase
(fresh mass basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus
spears held at 1 °C for 6 days ............................................ 82

Figure 2.9. The effects of storage 02 and CO2 on the activity of alcohol dehydrogenase
(fresh mass basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus
spears held at 1 °C for 6 days ............................................ 83

Figure 2.10. The effects of storage 02 and CO2 on the activity of pyruvate decarboxylase
(fresh mass basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus
spears held at 1 °C for 6 days ............................................ 84

Figure 2.11. The effects of storage 02 and CO2 on visual appearance in asparagus spears
held at 1 °C for 6 days. Value 6 represents the visual appearance threshold. ......... 85

Figure 3.1. The effects of storage 02 and CO2 on the activity of acid, bound acid, neutral
invertases, and sucrose synthase, (fresh mass basis) relative to harvest in the most
acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ............ 113

Figure 3.2. The effects of storage 02 and CO2 on the contents of glucose-6P, fructose-6-
P, and fi'uctose-l,6-P2 (fresh mass basis), relative to harvest in the most acropetal 45 mm
of the tip of asparagus spear held at 1 °C for 6 days .......................... 114

Figure 3.3. The effects of storage 02 and CO2 on the ratios of glucose-6-P to fructose-6-
P and fructose-1,6-P2 to fructose-6-P (fresh mass basis), relative to harvest in the most
acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ............ 115

Figure 3.4. The effects of storage 02 and CO2 on the contents of glyceraldehyde-3-P and
dehydroxyacetone—P (fiesh mass basis), relative to harvest in the most acropetal 45 mm of
the tip of asparagus spear held at 1 °C for 6 days ............................ 116

Figure 3.5. The effects of storage 02 and CO2 on the contents of 1,3 phosphoglyceratic,
3-phosphogliceric, and 2-phosphoglyceric acids (fi'esh mass basis), relative to harvest in
the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ..... 117

Figure 3.6. The effects of storage 02 and CO2 on the contents of phosphoenolpyruvate
(PEP), pyruvate (PYR) (fresh mass basis), and the PYR/PEP ratio, relative to harvest in
the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ..... 118

Figure 3.7. Crossover plot comparing lower 02 levels with the highest 02 level (16 kPa)
for CO2 levels of 20 (A), 10 (B), 5 (C), and 0 (D) kPa, in the most acropetal 45 mm of the
tip of asparagus spear held at 1 °C 6 for days ............................... 119

Figure 3.8. Crossover plot comparing higher CO2 levels (20 (A), 10 (B), and S (C) kPa)
with the lowest CO2 level (0 kPa), in the most acropetal 45 mm of the tip of asparagus
spear held at 1 °C 6 for days ........................................... 120

Figure 3.9. The effects of storage 02 and CO2 on the activities of hexokinase,
fi'uctokinase, and glucose-6-P dehydrogenase (fresh mass basis), relative to harvest in the
most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ........ 121

Figure 3.10. The effects of storage 02 and CO2 on the activities of
ATPzphosphofructokinase, PPizphosphofiuctokinase, and pyruvate kinase (fresh mass
basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus spear held at
1 °C for 6 days ..................................................... 122

Figure 4.1. The effect of storage 02 and CO2 on the content of PPi (fresh mass basis),
relative to harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C
for 6 for days ....................................................... 146

Figure 4.2. Pi status measured by 31P-NMR, relative to harvest in 45 mm long asparagus
spear tips under 1 kPa steady state 02 and 20 kPa CO2 held at 1 °C, Peak assignments are
as follows: I, glucose-6-phosphate (G6P); II, cytoplasmic inorganic phosphate (Pi); III,
vacuolar phosphate; IV, y-P of ATP and B-P of ADP; V, a-P of ATP and of ADP; VI, [3-
P of ATP. Insert shows an amplified scale spectrum to improve peak discrimination 147

Figure 4.3. The effect of storage 02 and CO2 on the content of Pi (fresh mass basis),
relative to harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C
for 6 days. Insert shows the effect of O2 partial pressures on the PPi/Pi ratio ....... 148

Figure 4.4. The effect of storage 02 and CO2 on the content of ATP (fresh mass basis),
relative to harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C
for 6 days. Inserts show: A) the effect of O2 partial pressures on the PPi/ATP ratio and B)
the effect of 02 uptake on the content of ATP .............................. 149

xi

Figure 4.5. The effect of storage 02 and CO2 on the content of ADP (fresh mass basis),

relative to harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C
for 6 days ......................................................... 150

Figure 4.6. The effect of storage 02 and CO2 on the content of AMP (fresh mass basis),

relative to harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C
for 6 days ......................................................... 151

Figure 4.7. The effect of storage 02 and CO2 on the AEC, relative to harvest in the most
acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ............ 152

Figure 4.8. The effect of storage 02 and CO2 on the ATP/ADP ratio, relative to harvest in
the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ..... 153

Figure 4.9. The effect of ABC on the ATP/ADP ratio, relative to harvest in the most
acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days ............ 154

Figure 5.1. Changes in CO2 production in asparagus spears with and without 6-BAP, with
and without 45 mm long tip segments during storage at 1 °C. Bar = 3: SE(n=3) . . . . 173

Figure 5.2. Changes in fructose content (fresh mass basis) in asparagus spears with tips,
with and without 6-BAP, during storage at 1 °C. Bar = :1: SE (n=3) .............. 174

Figure 5.3. Changes in fructose content (fresh mass basis) in asparagus spears without 45
mm long tip segments, with and without 6-BAP, during storage at 1 °C. Bar = :1: SE
(n=3) ............................................................. 175

Figure 5.4. Changes in glucose content (fresh mass basis) in asparagus spears with tips,
with and without 6-BAP, during storage at 1 °C. Bar = d: SE (n=3) .............. 176

Figure 5.5. Changes in glucose content in asparagus spears without 45 mm long tip
segments, with and without 6-BAP, during storage at 1 °C. Bar = d: SE (n=3) ..... 177

Figure 5.6. Changes in sucrose content in asparagus spears with tips, with and without 6-
BAP, during storage at 1 °C. Bar = i SE (n=3) ............................. 178

Figure 5.7. Changes in sucrose content in asparagus spears without 45 mm long tip
segments, with and without 6-BAP, during storage at 0 °C. Bar = :E SE (n=3) ..... 179

Figure 5.8. Changes in total chlorophyll content (fresh mass basis) in asparagus spears
with tips, with and without 6-BAP, during storage at 1 oC. Bar = d: SE (n=3) ...... 180

xii

Figure 5.9. Changes in total chlorophyll content (fresh mass basis) in asparagus spears
without 45 mm long tip segments, with and without 6-BAP, during storage at 1 °C. Bar =
a; SE (n=3) ........................................................ 181

Figure 5.10. Changes in the basal chlorophyll fluorescence (F0) in asparagus spears with
and without 6-BAP, with and without 45 mm long tip segments, during storage at 1 °C.
Bar = t SE (n=3) .................................................... 182

Figure 5.11. Changes in the maximum chlorophyll fluorescence (Fm) in asparagus spears
with and without 6-BAP, with and without 45 mm long tip segments, during storage at 1
°C. Bar = d: SE (n=3) ................................................ 183

Figure 5.12. Changes in the quantum yield maximum (Fv/F m) in asparagus spears with
and without 6-BAP, with and without 45 mm long tip segments, during storage at 1 °C.
Bar = 3: SE (n=3) .................................................... 184

Figure 5.13. Changes in length relative to the initial storage time in asparagus spears with
and without 45 mm long tip segments, with and without 6-BAP, during storage at 1 °C.
Bar-=21: SE (n=10) ................................................... 185

Figure 5.14. Changes in visual quality in asparagus spears with and without 6-BAP, with
and without 45 mm long tip segments, during storage at 1 °C. Value 6 represents the
visual quality threshold. Bar = :t SE (n=3) ................................. 186

xiii

INTRODUCTION

Harvesting and handling of horticultural crops can impose a series of stresses,
including wounding, dehydration and nutrient deprivation. Harvest stresses are
particularly severe on organs that are actively growing at harvest. Harvested actively
growing tissues tend to senesce rapidly due to an inability to maintain metabolic
homeostasis (Davis and King, 1993).

Harvested asparagus spears deteriorate rapidly, having a shelf-life of only 3-5 days
at room temperature (Lipton, 1990). The spear tip is composed of mostly men'stematic
tissue and is usually the first portion of the harvested spear to deteriorate (Davis and King,
1993). This postharvest deterioration is accompanied by numerous physiological and
biochemical changes (Lipton, 1990). Following harvest, the level of soluble
carbohydrates, especially sucrose, decline rapidly, the respiration rate declines markedly,
protein is lost, free amino acids increase (specially asparagine) and ammonia can
accumulate (Hurst et al., 1993; King et al., 1990). Indeed, sucrose content declines
approximately 60% within 6 h after harvest at 20 °C (Irving and Hurst, 1993), and
changes in gene expression also occur very early, including de novo induction of specific
genes (King and Davis, 1992).

Postharvest research is largely directed at maintaining quality and prolonging the
storage life of harvested whole and minimally processed crops. In many cases the
fimdamental physiological and biochemical processes that result in changes in quality are

poorly understood. Many of these processes are inter-related or interdependent.

Improving our understanding of the fundamental processes or steps that exert control over
factors that limit quality will likely provide opportunities for improving storability.

Glycolysis is an ubiquitous pathway that operates under both aerobic and
anaerobic conditions (Kennedy et al., 1992) and its central importance in the generation of
energy and the regulation of carbon flux has obvious implications on storability. Reduced
02 levels and/or elevated CO2 can alter glycolysis via effects on respiration rate (Kader,
1986). Lowering O2 partial pressures too far, however, may induce the fermentative
pathway. If 02 levels are sufficiently low, fermentation can replace the Krebs cycle as the
main source of metabolic energy. The relatively energy inefficient fermentative
metabolism may alter cellular energetics. Other undesirable physiological changes, such
as off-flavors may also occur (Ke et al., 1993). Understanding the physiology of sucrose
metabolism, the role of glycolytic enzymes, and changes in energy charge should elucidate
how harvested asparagus spears coordinate carbohydrate partitioning, metabolic capacity,
and carbohydrate utilization processes.

The main focus of this research is to investigate how carbohydrate metabolism is
regulated during storage in harvested asparagus spears. To accomplish this, the research
project was divided into four main topics: 1) determining how utilization of sucrose and
carbon flux govern fermentative metabolism; 2) evaluating the implications of reduced
levels of O2 and elevated CO2 on carbon flow and the glycolytic pathway; 3) evaluating
the impact of reduced 02 and elevated CO2 on phOSphate and adenylate energy supply;
and 4) evaluating the effects of exogenous cytokinin on sucrose metabolism in harvested

asparagus spears.

LITERATURE CITED

Davis, K. M. and GA. King. 1993. Isolation and characterization of a cDNA clone for a

harvest-induced asparagine synthetase from Asparagus officinalis L. Plant
Physiol. 102:1337-1340.

Hurst, P.L., L.M Hyndman, and PI Hannah. 1993. Sucrose synthase, invertases, and
sugars in growing asparagus spears. NZ. J. Crop Hort. Sci. 21:331-336.

Irving, DE. and PL. Hurst. 1993. Respiration, soluble carbohydrates and enzymes of
carbohydrate metabolism in tips of harvested asparagus spears. Plant Sci. 94:89-
97.

Kader, AA. 1986. Biochemical and physiological basis for effects of controlled and
modified atmospheres on fruits and vegetables. Food Tech. 40299-100 & 102-104.

Ke, D., M. Mateos, and AA. Kader. 1993. Regulation of fermentative metabolism in
fruits and vegetables by controlled atmospheres. Proc. 6th Int. Cont. Atm. Res.
Conf. 1:15-17.

Kennedy, R.A., M.E. Rumpho, and TC. Fox. 1992. Anaerobic metabolism in plants.
Plant Physiol. 100: 1-6.

King, G.A., D.C. Woollard, D.E. Irving, and W.M. Borst. 1990. Physiological changes
in asparagus spears tips after harvest. Physiol. Plant. 80:393-400.

King, GA. and KM. Davis. 1992. Identification, cDNA cloning, and analysis of mRNAs

having altered expression in tips of harvested asparagus spears. Plant Physiol.
100:1661-1669.

Lipton, W.J. 1990. Postharvest biology of fresh asparagus. Hort. Rev. 12269-155.

CHAPTER 1

LITERATURE REVIEW

Numerous physiological and biochemical processes are involved in metabolism of
harvested asparagus spears. Many of those processes directly impact spear quality and
storability. The acquisition and storage of energy and the utilization of this stored energy
are central processes in the control of the plant metabolism. In intact plants, there is an
interdependence between organs that supply and utilize energy. The harvest disrupts this
interdependence and may, therefore, influence postharvest behavior. For example,
acquisition of energy through photosynthesis and utilization via respiration are opposing
forces in the intact plant, and photosynthesis does not occur in all products after harvest.
To understand these distinct and interdependent processes and their roles in supporting life
of harvested asparagus spear, carbon allocation, sucrose metabolism, and respiration need
to be assessed simultaneously and described in detail.

Asparagus Botanical Background

Asparagus (Asparagus oflicinalis L.) is grown for its succulent fleshy shoots
(spears), which appear after a winter resting period. Asparagus is an European-Siberian
continental plant related to the East Mediterranean vegetation. It is a monocotyledonous
(Robb, 1984), member of the Liliaceae (lily) family that has been cultivated in Europe and
Asia for more than 2000 years (Zandstra et al., 1992). The common name and the Latin
name are identical. ‘Officinalis’ means ‘medicinal’, a property for which asparagus was

first noted. (Nonnecke, 1989). Cultivated asparagus is a diploid (n=10) species (Robb,

ll

1984). Asparagus is dioecious (male and female flowers occur in separate plants) with a
sex ratio of 1:1 (Reuther, 1985). However, male plants have flowers that vary from
staminate (no functional carpels) to hermaphroditic (Robb, 1984). New all-male hybrids
plants produce higher spear yield (Zandstra et al., 1992).

Biology of Growth and Carbon Allocation

Asparagus is an herbaceous perennial, and the aboveground foliage is known as
the fern, the underground rhizomatous stem is termed the crown (Nonnecke, 1989). The
spears develop into fem-like shoots, each of which developed from a separate bud on the
underground rhizomatous stem. Each shoot consists of a central stem which subtends
many fine, needle-like branches or cladophylls. The leaves in the cladophylls have been
reduced to very small scales (Pressman et al., 1989).

As in other monocotylenoneus species, the crown of the asparagus plant is the
critical growth center (Robb, 1984), in which fructo-oligosaccharides and fructo-
polysaccharides (fi'uctans) are the storage carbohydrates (Shiomi, 1992). The roots of the
plant consist of underground stems (rhizomes), fleshy roots, and fibrous roots. The fleshy
roots serve as storage organs and the fibrous roots as absorption organs. The fibrous
roots die after each year’s growth (Nonnecke, 1989). The fleshy roots remain to provide
nourishment for the next generation of spears. During the fall and winter, low
temperatures induce dormancy in the asparagus plant, accompanied by die-back of the
mature fern. With the coming of the spring and the end of dormancy period, the fi'uctan
content in the crown decreases with the end of dormancy period concomitant with spear

(young shoot) production and harvest. Growth of new spears is presumed to be at the

expense of previously stored carbohydrates, which are replenished later in the season
following canopy development and maturation (Pressman et al., 1989). Although fi'uctans
are mostly fructose polymers, feeding l‘COZ to tissue accumulating fructans leads to the
initial appearance of radioactivity in sucrose, followed by the progressive appearance of
label in fructans (Pollock et al., 1996). The fructans are synthesized from sucrose via a
trisaccharide intermediate, by the combined action of two fructosyl tranferases: 1.
sucrosezsucrose fructosyl transferase (SST, EC 2,4199) and 2. fructanzfi'uctan fructosyl
tranferase (FFT, BC 2.4. 1.100). In asparagus crowns, fructans account for 85% of the
total carbohydrates, with sucrose and smaller oligofructans representing the remaining
15%. Of fi'uctans, approximately 91% are polymers containing five or more fi'uctose units
(Cairns, 1992). Of the remaining 9%, the degree of polymerization generally ranges
from 12 to 21 fructose units (Shiomi, 1993). These longer chain fructans consist mainly
of iso-kestose and neo-kestose (Shiomi, 1992). The presence of sucrose and small
oligofiuctans in the crown suggests that the crown, in addition to being a storage organ,
may serve as a passage for the assimilate stream between the above and underground part
of the plant (Martin, 1990). In spears, fructose, glucose, and sucrose are the predominant
carbohydrates (Irving and Hurst, 1993). Fructans have been detected in the spear in some
studies (Martin, 1990), but not in others (Pressman et al., 1989; Silva et al., 1998). Upon
maturation of the shoot, fructan content in the roots increases again.

Early products of the photosynthetic carbon reduction (PCR) cycle in the
chloroplasts are phosphoglycerate and dihydroxyacetone phosphate. These sugar

phosphates are biochemically and spatially partitioned between two major biosynthetic

pathways, one leading to the synthesis and then retention of carbohydrate reserves, such as
starch in plastids, and another to sucrose synthesis in the cytoplasm (Gifford et al., 1984).
Asparagus has a C3 phothosynthetic pathway and the cladophylls are asparagus’s main
site of carbon assimilation. Carbon assimilation is very dependent on the amount of solar
energy available, and the optimum temperature for photosynthesis is about 18 °C (Sawada
et al., 1962). The yield of asparagus is not directly the result of current photosynthesis but
is instead a function of carbohydrate reserves, produced by the previous year’s ferns
(Downton and Torokfalvy, 1975). Thus, yield is largely determined the year prior to
harvest.
Carbon Metabolism in Harvested Asparagus

Sucrose is the primary transport form of reduced carbon in many higher plants
(Huber and Akazawa, 1986). Thus, it is necessary to understand the pathway of sucrose
metabolism as a means to understand processes regulating energy production and carbon
flux at the biochemical level. Asparagus spears utilize imported sucrose to provide energy
and carbon skeletons for growth. In the spear, the tip is the most metabolically active
zone and is the strongest carbohydrate sink, which demand more translocated assimilates.
The base of the spear, however accumulates carbohydrates and is a storage sink (Hurst et
al., 1993). In asparagus spears, high levels of sucrose at harvest (Irving and Hurst, 1993;
Silva et al., 1998) together with the absence of fructans in the spears (Pressman et a1.
1989, Silva et al., 1998) suggest that sucrose is the major carbohydrate being translocated
fi'om roots to spears and, during carbon assimilation, from the spears to the roots. In

harvested asparagus, sucrose movement has been suggested to be from the base to the

upper parts of spears (Saltveit and Kasmire, 1985). Harvesting asparagus immediately
removes the source of respiratory substrates and, due to its very high metabolic rate
(Irving and Hurst, 1993), leads to rapid loss in carbohydrate. After 7 d storage at 1 °C,
the respiratory demands of spear tips had outstripped the rate of use of soluble
carbohydrate available in that tissue, suggesting that substrates are transported from the
base to the tip during postharvest storage or that material other than sugars may be used
to sustain metabolism (Silva et al., 1998). Such rapid depletion in sugars apparently leads
to carbohydrate starvation typically seen in senescing or sugar-starved plant tissues
(Brouquisse et al., 1991). Other studies on sycamore cells and excised maize root tips,
have provided evidence that cell contents are degraded to sustain metabolism during
carbohydrate starvation. In those tissues, loss of carbohydrates, proteins and lipids was
accompanied by an increase in free amino acids, inorganic phosphate, and
phosphorylcholine, and reduction in activity of glycolytic enzymes (Dome, 1987; Genix et
al., 1991). The decline in sucrose content in asparagus is accompanied by a drop in
respiration rate; these are two of the earliest physiological events detected after harvest
(Irving and Hurst, 1993; Silva et al., 1998). Sucrose depletion is suggested to trigger
senescence (King et al., 1993). Thus, the sucrose-metabolizing enzymes may control
sucrose decline in harvested spears. Understanding the role and regulation of enzymes in
sucrose catabolism could provide a means to control loss of sucrose and, therefore, extend
the shelflife of asparagus.

Sugar-regulated genes provide a means for the plant to integrate the cellular

response to transport sugars and to coordinate changes in resource utilization and

allocation among parts. To this end, genes upregulated by increased carbohydrate
availability in plants include many that would enhance sucrose utilization through storage,
respiration, and biosynthetic processes (Koch, 1996). Also, a major contribution to
regulation of carbon partitioning is the expression of genes of the sucrose-metabolizing
enzymes (Geiger et al., 1996). In addition, glucose and fructose from sucrose breakdown
are thought to repress the transcription of photosynthetic genes in plants (Sheen, 1990),
probably acting via a hexose sensor (Jang and Sheen, 1997; Jang et al., 1997).

In asparagus, sugar depletion may also regulate the expression of genes involved in
specific carbon and nitrogen remobilization processes (King et al., 1995; King and Davis,
1995). In response to carbohydrate stress, asparagine synthetase (EC 6.3.5.4) mRNA
levels begin to increase in asparagus tip tissues within 2 h following harvest (King and
Davis, 1995). The increase in asparagine synthetase message precedes the accumulation
of asparagine, which occurs approximately 6 h after harvest King and Davis, 1995).
Asparagine accumulation is considered to be a postharvest senescence symptom (Eason et
al., 1996). During senescence, visual appearance of the spear changes; symptoms include
feathering and browning of bracts, tissue flaccidity and cellular breakdown (King et al.,
1990)

Sucrose Metabolism

Photoassimilate partitioning to sinks is a highly regulated process (Stitt and
Sonnewald, 1995) that includes sugar transporters (Kruger, 1990) and sucrose-
metabolizing enzymes (Sturm, 1996) (Fig. 1). Sucrose and starch are two of the most

important end-products of photosynthesis (Quick and Schaffer, 1996). Sucrose, however,

is the major form in which carbon is translocated (Stitt and Sonnewald, 1995) in addition
to being an important storage carbohydrate in plants (Ehness and Roitsch, 1997). Sucrose
transport in the phloem is suggested to be driven by a turgor pressure gradient generated
by a sucrose concentration gradient, with high sucrose concentrations at the site of phloem
loading and lower concentrations at the site of phloem unloading (Ho and Baker, 1982).
Transport of sucrose from the apoplast into the phloem is catalyzed by a H‘lsucrose co-
transporter of the companion cell plasma membrane and driven by ATP hydrolysis at the
cytoplasmic side of the transport protein (Daie, 1989).

Once translocated, sucrose enters in the sink cells by either crossing the plasma
membrane apoplastically via sucrose carrier or by diffusion, and/or sucrose enters
symplastically through plasmadesmata (Krausgrill et al., 1996; Sung et al., 1989). If
sucrose is the primary form in which carbohydrates are stored, high sugar levels may be
favored in the cell because sucrose contributes half of the osmolarity of the equivalent of
glucose and fructose (Steingrdver, 1983) and may be less metabolically accessible to
respiratory loss than hexose sugars (Sung et al., 1989). As proposed by Ho (1988), the
flow of sucrose into a tissue (sink strength) is regulated by the rate of sucrose degradation.
In carrots, sink strength is generated by a common action of sucrose synthase and
vacuolar invertase and, especially in the case of apoplastic unloading, by the active
transport of sucrose across the membrane (Sturrn, 1996). It is through sucrose
degradation that glycolysis is initiated in most plants (Kruger, 1990).

Two pathways for the degradation of sucrose exist and provide the basis for a

flexible system that can markedly affect the partitioning and metabolism of assimilated

10

carbon in sink tissues (Huber and Akazawa, 1986) (Fig. 1.1). Sucrose can be degraded
and metabolized in higher plants by sucrose synthase (SUSY, EC 2.4.1.13) or invertases
(B-fructosidase, EC 3.2.1.26). Specific sucrose-metabolizing enzymes are associated with
specific sink functions. For example, invertases predominate in storage sinks while SUSY
predominates where cell wall expansion is active (Sung et al.,1989).

Sucrose metabolism is responsive to developmental patterns in cell division and
differentiation. During rapid cell wall biosynthesis, the incoming sucrose is preferentially
channeled to cellulose (Delmer and Amor, 1995). In storage tissues, sucrose is largely
diverted to carbohydrate reserve and/or soluble sugars (Krausgrill et al., 1996). In these
tissues sucrose is transported into the vacuole (Kruger, 1990). In storage tissue sucrose
hydrolysis to fructose and glucose is by vacuolar invertase (Krausgrill et al., 1996); the
hexoses produced are part released in the cytoplasm and part retained in the vacuole.

Invertase hydrolysis of sucrose to hexose sugars plays a firndamental role in the
energy requirements for plant growth and maintenance (Klann et al., 1996). The reaction
is highly exothermic and irreversible (Avigard, 1982; Kruger, 1990). The functions of
invertases in sink tissues are likely complex as reflected by the existence of difi‘erentially
expressed isoforms (Sturm et al., 1995), some of which are inactivated by inhibitory
proteins at certain stages of plant development (Greiner et al., 1998; Krausgrill et al.,
1996). Isoforms may differ in their cellular location (vacuole, cytoplasm, apoplast), their
spacial expression during development (Sturm et al., 1995), and their expression in the
presence of different glucose concentrations as in ‘feast’ and ‘famine’ isoforms of vacuolar

invertase in maize (Koch et al., 1996). In addition, specific isozyme genes are also

11

 

 

 

pl »$——-Di—CWI_| sink

cefl

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(Ere <—
. SUSY F
F r——
cellulose HK * F G K?
L____, L ire-spinner; V respiration
T __ / L__.> 5 ——> C1

 

 

 

 

 

 

V
s i
i i

sieve L vacuole
tube L j

i Fructans
S 4—

Roots

 

 

 

 

 

 

 

 

 

 

Figure 1.1. Proposed cellular pathways of sugar transport and metabolism in a

sink cell of asparagus supplied with sucrose via symplastic and apoplastic routes.

Enzymes are: bound sucrose synthase (SUSY B) and soluble sucrose synthase
(SUSY F), invertases (VI, cytosolic neutral; VI, vacuolar acid; CW1, cell wall
bound acid), and hexokinase (HK). Substrates are sucrose (S), glucose (G), and
fructose (F). Sugar transporters and cellulose synthase are represented by black
dots. Adapted from Krausgrill et al. (1996).

12

expressed when carbohydrate supplies are limited. For example, Chenopodium rubrum
invertase isozyme genes have at least three members (Ehness and Roitsch, 1997).

There are two types of acid invertases in plants: vacuolar soluble (VI) and cell
wall-bound (CWI) invertases, which have optimum activity about pH 5.0, and are present
in the vacuole and in cell wall, respectively. Cytosolic (neutral) invertase (CI), also termed
alkaline invertase, has optimum activity at pH 7 0-75 and is localized in the cytoplasm
(Klann et al., 1996). High activity of CWI, which is ionically bound to the cell wall, is
usually associated with rapidly growing tissues, where these enzymes are said to play a
major role in maintaining the source-to-sink gradient in sucrose concentration by rapidly
hydrolyzing incoming sucrose (Krausgrill et al., 1996). CWI has been proposed also to be
important for phloem unloading and sink strength (Ho, 1988). CWI has been cloned and
it has been shown that the mRN A levels increases in response to sugars (Karrer and
Rodriguez, 1992), giving further evidence that invertases also belong to the group of
enzymes that are under metabolic control by carbohydrates. CWI expression is often high
in young growing tissues (Sturm et al., 1995). However, it seems that high CWI activity
is not necessarily linked to apoplastic sucrose unloading from the phloem. Rather it seems
that even in symplastically supplied sinks, a considerable amount of sucrose may diffuse
from the sink cells and be hydrolyzed in the apoplast (Krausgrill et al., 1996). VI has a
higher activity in the rapidly elongating region of the asparagus spear than in the more
apical or basal tissues, which is 6 cm below the spear tip (Hurst et al., 1993).

SUSY catalyzes an equilibrium reaction that degrades sucrose to UDP-glucose

and fructose and so conserves energy equivalent to one ATP in the glycolytic bond

13

relative to invertase (Geiger et al., 1996). UDP-glucose can be converted to respiratory
intermediates by being converted to glucose-l-P or glucose and used throughout
respiration (Kruger, 1990), the UDP-glucose generated from sucrose by SUSY is a
intermediate in the biosynthesis of cell wall polysacharides, amino acids, lipids, etc (Sturm,
1996). SUSY activity is three times higher than neutral invertase in asparagus spear tips
at harvest (Irving and Hurst, 1993). High SUSY activity has also been reported to be
present in other rapidly growing tissues (Ap Rees, 1984; Avigard, 1982; Geigenberg and
Stitt, 1993), and it has been suggested that this enzyme activity can be used as a measure
for sink strength (Sung et al., 1989). SUSY is localized in the cytosol, or attached to the
inner side of plasma membrane (Delmer and Amor, 1995).

Sucrose synthase, in contrast to invertase, catalyzes a freely reversible reaction,
with a theoretical equilibrium constant (Keq) in the direction of sucrose degradation
([UDP-glucose] [fructose])/([sucrose]) of 0. 15-0.56 (Morell and Copeland, 1985).
Enzymes that catalyze irreversible reactions are generally considered good candidates for
regulation. Conversely, enzymes that catalyze near-equilibrium reaction are considered to
be in excess and, hence, poor candidates for regulation (Stitt, 1990). However, a readily
reversible reaction can be rendered effectively irreversible in vivo by removing one of the
products. The reaction catalyzed by SUSY may be displaced from equilibrium in vivo,
due to the rapid removal of fructose or UDP-glucose. For instance, in intact growing
potato tubers, the SUSY reaction may be displaced from equilibrium in favor of sucrose
degradation (Geigenberger and Stitt, 1993). The flux through SUSY responds promptly

to the supply of sucrose and the demand of sucrose in the cell.

14

Tissues that have high metabolic activity such as asparagus spear tips have a high
demand for sucrose in order to maintain the carbon supply for respiratory and biosynthetic
pathways. In sycamore cells, SUSY causes the rate of sucrose degradation to respond
sensitively and automatically to those demands (Huber and Akazawa, 1986), but, at least
in potato tubers, SUSY does not regulate the rate of sucrose degradation (Geigenberger
and Stitt, 1993). In some cases, SUSY may operate in parallel with irreversible reactions
catalyzed by sucrose phosphate synthase or invertase (Xu et al., 1989).

In the case of sucrose being metabolized by invertase, it is essential that this
reaction is regulated. High invertase activity could inhibit sucrose movement into a sink,
because one molecule of sucrose is replaced by glucose and fructose, two osmotically
active products (Geigenberger and Stitt, 1993). If these hexoses accumulate to significant
levels, they will alter the regulation of turgor and water movement within the symplast and
between symplast and apoplast (Heineke et al., 1992). Hence, sucrose metabolization via
invertase will presumably require sophisticated regulatory mechanisms to coordinate
invertase and hexokinase (EC 2.7.1.l)/fructokinase (EC 2.7.1.4) activity with cell growth
and turgor (Geigenberger and Stitt, 1993; Kruger, 1990). The two latter enzymes are also
suggested as having a glycolytic regulatory role in viva (Jang et al., 1997). SUSY plays a
critical role in anoxic tolerance in maize seedling roots, since it is the main enzyme for
metabolizing sucrose under 02 deprivation (Ricard et al., 1998). However, it seems that
different genes and signal mechanisms for SUSY are expressed under hypoxia and anoxia,
in a manner that parallels the induction of “feast and famine” SUSY genes by hypoxia and

anoxia, respectively (Zeng et al., 1998).

15

There are analogies between SUSY and another unusual enzyme in plant
metabolism, pyrophosphate:fmctose-6-phosphate phosphotransferase (PPi:PFK, EC
2.7.1.90). Both of these enzymes are PPi-dependent, and catalyze a readily reversible
reaction which, in effect, short-circuits an irreversible reaction. Furthermore, both
enzymes are highly regulated. SUSY is inhibited by free glucose and fructose, and also
shows large changes in activity during development and in response to sugars (F arrar,
1991), presumably due to regulation at the level of gene expression (Maas et al., 1990)
and by post-transcriptional mechanisms (Doehlert and Chourey, 1991). PPi:PFK is
regulated by substrates and products, and fructose-2,6-bisphosphate, and also undergoes
developmentally regulated changes, being higher in metabolically active tissues (Stitt,
1990)

Reduced levels of 02 and/or elevated levels of CO2 are known to affect respiratory
and enzyme activities in plant tissues (Kato-Noguchi and Watada, 1996a, b; Kerbel et al.,
1988). Under anoxia, sucrose metabolism seems to take place mainly through SUSY in
rice seedlings (Guglielminetti et al., 1995; Ricard et al., 1994), and maize, wheat and
barley seeds (Guglielminetti et al., 1997). The degradation of sucrose may be reduced if
asparagus spears are stored under limiting O2 and CO2 levels as a result of decreasing the
activity of the enzymes involved in sucrose catabolism. Investigating the effect of Oz and
CO2 on the metabolism of sugars in asparagus spear tips may be a way to identify by

which means the carbon flux is regulated.

16

Respiratory Metabolism

Respiration is the anabolic process that involves the oxidative breakdown of
carbohydrates, organic acids, lipids, and, on occasion, proteins to CO2 and H20. This
process results in concomitant generation of energy in the form of reducing power and
ATP, and the formation of carbon skeletons to be used by the cell for biosynthetic
reactions, maintenance of cellular organization and membrane integrity, preservation of
ion gradients and protein turnover. This oxidative process can be subdivided in three
stages: 1) glycolysis, 2) the tricarboxylic acid (TCA) cycle, and (3) the electron transport
chain (Taiz and Zeiger, 1991). From a postharvest point of view, rate of respiration is
important not only because of those processes, but also because it also gives an indication
of the overall rate of metabolism of the plant or plant part (Kays, 1991).

Controlled atmosphere (CA), modified-atmosphere (MA), and modified
atmosphere packaging (MAP) systems refer to techniques for creating gas mixtures
differing from that of air and have been used as tools to minimize deterioration, improving
quality in fruits and vegetables (Kader, 1986; Lakakul, 1994). Depending on the degree
of atmosphere modification, there can be positive or detrimental effects to the
commodities (Cameron et al., 1994). High levels of CO2 can either slow or elevate
respiratory rates depending on the commodity and concentration of CO2 employed
(Beaudry, 1993; Kerbel et al., 1988). An elevation of the respiratory quotient (RQ) can
also take place due to COz-enhanced synthesis of ethanol and acetaldehyde (Thomas,
1925). The effect of CO2 on the synthesis of fermentation products is further modulated

by the presence of O2 (Shirazi et al., 1991; Thomas, 1925). A variety of techniques have

17

been used to investigate the effects of reduced 02 and elevated CO2 levels on respiration
(Baxter and Waters, 1991; Beaudry, 1993; Corrigan and Carpenter, 1993; King et al.,
1986; Lakakul; 1994). The use of Oz/CO2 barrier films may have potential for MAP of a
perishable horticultural crop like asparagus. However, a film that matches the respiration
rate of asparagus should be carefully tested to control possible anaerobic respiration
during storage. Systems using a flow through design in which small differences between
inlet and outlet gas concentrations are determined, permit analysis of O2 and CO2 on
respiration, however the accuracy can be poor. On the other hand, modified-atmosphere
packaging (MAP) systems as respirometers, like flow-through systems, achieve steady
state conditions and allow the evaluation of the effects of a broad range of O2 and C02.
The fluxes of O2 and CO2 in a package system are calculated from the respiration-driven
gradients of O2 and CO2 across the film (Beaudry et al., 1993). This technique allows
establishing accurate ranges of O2 and CO2 in a packaging system, and also allows
accurate respiratory measurements despite high backgrounds levels of O2 and C02. In
asparagus, however, much of CA and MAP research has been directed toward the
determination of the effects and optimum conditions of lower 02/ higher CO2 for
postharvest storage (Baxter and Waters, 1991; Corrigan and Carpenter, 1993; King et al.,
1986; King et al., 1993; Lipton, 1965). Importantly, gas concentrations achieved in MAP
under commercial conditions may differ significantly from optimal conditions (Cameron et
al., 1994). The postharvest basis of using low 02 and/or high CO2 for storage of
commodities has been assessed at biochemical (Kato-Nogushi and Watada, 1996a, b; Ke

et al., 1995; Kerbel et al., 1988; Lange and Kader, 1997a, b, c) and physiological levels

18

(Beaudry, 1993; Cameron et al., 1994; Gran and Beaudry, 1993). However, biochemical
studies have been restricted to 2 or 3 levels of 02 and/or C02, which limits a broader
evaluation of the processes involved. Furthermore, little is known regarding atmosphere
effects on asparagus carbohydrate metabolism (Hurst et al., 1997).

Glycolysis. Glycolysis is carried out by a group of soluble enzymes located in the
cytosol (Fig. 1.2). Glycolysis functions to fulfill two fundamental roles. It oxidizes
hexoses to generate ATP, reductant, and pyruvate, and it produces carbon skeletons for
synthetic reactions. Chemically, hexoses (derived primarily from starch, sucrose, fructans,
and other sugars) undergo a limited amount of oxidation to produce two molecules of
pyruvate, two molecules of ATP, and two molecules of the reduced pyridine nucleotide,
NADH (Taiz annd Zieger, 1991). Thus, classical glycolysis is the cytosolic linear
sequence of 10 enzymatic reactions that catalyze the net reaction:

Glucose + 2ADP + 2 Pi + 2 NAD’ ------ > 2 pyruvate + 2 ATP + 2 NADH [l].

Glycolysis is recognized as having a central role in carbon metabolism and is
present, at least in part, in all organisms. Glycolysis is directly involved in many
biochemical adaptations of plant and nonplant species to environmental stresses such as
osmotic stress, drought, cold/freezing, hypoxia and anoxia (Plaxton, 1996). Glycolysis
also can function in reverse to generate hexoses from various low-molecular—weight
compounds in energy-dependent gluconeogenesis. Both processes are tightly regulated in
vivo (Plaxton et al., 1996).

Glycolysis and gluconeogenesis, in conjunction with the pentose phosphate

pathway, are at the central point of the allocation of carbon to the various biosynthetic

l9

 

 

 

 

 

( 153)
Sugrose g Gluicose . Frugctose (Apoplast)
i 3 i
Sucrose—(LL Glucose . Fructose { C“ 0
na\\ an (m) i”) (W)
Fructose UDPGQGI PEG P N ADP-.9 NADPH ‘1". Gluconate-6-P
(2) (3) (2) (OPP)
-F P 4
Ma) 1M)
1.68P
(5)

DHAPgfi 3P

1.3PGA
(a)

3313::

ZPGA
$00)

PEP

(72) law {74}
AcetaldehydeE'Pyruvate——'Lactate
@032
Ethanol

(TCA Cycle)

Figure 1.2. Glycolysis in plant tissues showing the relationship with oxidative
pentose phosphate (OPP) pathway and TCA cycle. The numbers represent
enzymes as follows: 1, hexokinase; 2 fructokinase; 3, phosphoglucoisomerase; 4,
ATPzPFK; 4a, PPi:PFK; 5, aldolose; 6, triosephosphate isomerase; 7,
glyceraldehyde-3-phosphate dehydrogenase; 8, phosphoglycerate kinase; 9,
phosphoglycerate mutase; 10, enolase; l 1, pyruvate kinase; 12, pyruvate
decarboxylase; 13, alcohol dehydrogenase; 14, lactate dehydrogenase; 15,
intracellular invertase; 15a, extracellular invertase; 16, sucrose synthase; 17, UDP-
glucose pyrophosphorylase; 18, phosphoglucomutase; 19, glucose-6-phosphate
dehydrogenase (Adapted from Ashihara et al., 1988).

20

pathways in plants. The metabolites comprising these pathways can be divided into a
number of pools of phosphorylated intermediates and hexoses (Taiz and Zieger, 1991).
The entry and exit of metabolites from pools are highly regulated, and this regulation is
complicated by the existence of separate pools within the cytosol and plastids (Blakeley
and Dennis, 1993). However, a fundamental characteristic of cytosolic glycolysis is the
existence of two previously mentioned reactions that can utilize PPi rather than nucleoside
triphosphates (NTPs) as a phosphoryl donor (Plaxton, 1996).

The ability to regulate the rates of metabolic processes in response to changes in
the internal and or external environment is a firndamental feature that is inherent to plant
material and allows the maintenance of an efficient functional state in all organisms
(Plaxton, 1996). The flux of metabolites through any given pathway must be closely
coordinated with the needs of the cell, tissue, or organism for the products of the
pathway. A common way to accomplish such regulation is through altering the activity of
at least one rate-limiting enzyme of the pathway (Blakeley and Dennis, 1993). The
identification and characterization of key enzyme that function as rate limiting enzymes or
pacemakers has been a major focus of research in glycolysis. The rate-limiting steps of a
pathway are essentially irreversible (i.e., have high negative fi'ee energy) in viva, have a
low activity overall, and frequently occur at the first committed step of a pathway, directly
before major branch points (Plaxton, 1990). The control analysis theory developed by
Kacser and Burns (1973) attempts to provide a quantitative mechanism for probing intact
biological systems and interpreting results without taking into account which enzymes

were preconceived as ‘rate-limiting steps’ or ‘pacemakers’ in the pathway. These authors

21

have established the concept of the flux afcantral coefficient (C’E) whose value specifies
the change in metabolic flux (A!) to small changes in the activity of any enzyme (AE) in
the metabolic system as follows:

C’ E = (A!) (A5)" [2].

Those enzymes with a control coefficient approaching 1 have a major control
firnction in the pathway, while those with a coefficient approaching zero have little
control. In this way, this theory assigns a flux control coefficient for each enzyme. In
fact, according to Blakeley and Dennis (1993), it has become apparent that all enzymes of
a pathway may exert some control on the flux. However, if one of the components of a
pathway has a large C’ , flux through the pathway is particularly sensitive to small
pertubations in the activity of that particular enzyme (Anderson, 1974).

‘Coarse’ and ‘fine’ metabolic controls are the two basic mechanisms potentially
used by the cell to vary the reaction velocity of a particular enzyme. Coarse metabolic
control is a long term (hours or days), energetically expensive response that is achieved
through changes in the total cellular population of enzyme molecules. Coarse control may
be important in long term environmental (adaptative) changes, such as imposition of lower
Ozlhigher CO2 atmosphere. The reaction rate of a given enzyme is dependent on the rates
of its biosynthesis versus degradation. Thus, any changes in the rate of transcription,
translation, mRN A processing or degradation, or proteolysis contribute to coarse
metabolic control (Plaxton, 1990).

Fine metabolic control often involves fast (seconds to minutes), energetically

inexpensive regulatory devices which modulate the activity of the pre-existing enzyme

22

molecules (Cohen, 1983). Fine control are seen as “metabolic transducers” that sense the
momentary metabolic requirements of the cell, and modulate the rate of metabolic flux
through the pathways (Plaxton, 1990). There are several ways by which fine metabolic
control can be exerted in plants: i) alteration in substrate or cosubstrate concentration,
which modifies the of pathway flux, most significantly for enzymes that show sigmoidal
substrate kinetics; ii) variation of pH, above or below the enzyme’s maximal activity; iii)
allosteric regulation, by which specific inhibitor or activator metabolites can reversibly
bind in allosteric sites often present in multisubunit regulatory enzymes (adenine
nucleotides are examples of allosteric regulators for ATP—PFK in plants); iv) covalent
modification, which firnctions cooperatively with allosteric regulation and results in
formation of stable covalent bonds, often by the action of two enzymes as in the case of
dithiol-disulfide interconversion and phosphorylation-dephosphorylation (Loewe et al.,
1996), which are reversible covalent modifications; v) subunit association-dissociation; vi)
reversible associations of metabolically sequential enzymes. Essentially all rate-limiting
enzymes are at least partially controlled by allosteric effectors (Plaxton, 1996).

Enzymatic analysis is a valuable tool for determining kinetics of the reactions and
the influence of regulators. Ofien such work is necessary to demonstrate the existence of
a particular enzyme or a pathway (Carnal and Black, 1979) and indicate the involvement
in glycolysis. However, in many studies, the activity of glycolytic enzymes extracted
directly from a tissue is used as a means to detect changes in enzyme activity during
development or following a particular treatment. Such studies are subject to reasonable

criticism when the attempt is made to extrapolate in vitra activity measurements to in viva

23

situations (Kruger, 1995).

Quite often enzymatic analysis is supported by analysis of intermediate, substrate,
and end-product contents or their production rates. Modification in the pattern of
intermediate levels can be readily interpreted by comparing changes in the relative
concentrations of successive intermediates along a biochemical pathway between two
treatments. A convenient means to do this is via the crossover plot (Adams and Rowan,
1970; Chance et al., 1958; Ghosh and Chance, 1964), applying the following equation:

Crossover value = ([C2] -[C1])‘(([C2] + [Cm/2T1 [3],
where Cl and C2 are initial and final concentrations of each intermediate at the start and
end of a treatment. Changes in intermediate concentrations that cause the line connecting
crossover values from two successive intermediate pools in a biochemical pathway to
‘cross over’ the zero line is interpreted to indicate a change in regulation and thereby
indicating a regulatory enzymatic step. An increase in values from negative to positive
means that the step was stimulated. Conversely, a decrease in values from positive to
negative means that the step was inhibited. Use of the crossover theorem has proven to
yield fairly consistent results in a variety of plant materials in that two glycolytic
interconversions from fi’uctose-6-phosphate to fructose- l ,6-bisphosphate and fiom
phosphoenolpyruvate to pyruvate are regularly recognized as major control points in the
glycolytic sequence (Adams and Rowan, 1970; Kato-Nogushi and Watada, 1996; Kerbel
et al., 1988). There are several regulatory interconversions in the glycolytic and pentose
phosphate pathways:

Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) is the key enzyme

24

controlling carbon flux into the oxidative pentose phosphate (OPP) pathway. Glucose-6-
phosphate can flow either into glycolysis or through OPP (Taiz and Zieger, 1991).
G6PDH catalyzes the oxidation of glucose-6-P to b-glucono—1,5-lactone-6-phosphate
with the concomitant reduction of NADP’ (Stitt, 1990). In higher plants, elevated activity
of G6PDH is associated with the production of NADPH for reductive biosyntheses, which
may be regulated independently of glycolysis (Ap Rees, 1984).

Hexose kinase (HK) catalyzes the production of hexose monophosphates from
glucose and fructose utilizing a nucleoside tri-phosphate (NTP) as a phosphoryl donor
(Gardner et al., 1992). HK is thought to be the sensor in the signaling pathway in which
glucose and other hexoses act to repress photosynthetic genes (Jang et al., 1997; Sheen,
1990). The phosporylation of hexoses by HK appears to be critical for signaling because
only the hexose and glucose analogs that can be phosphorylated by I-IK are effective in
signal transduction (J ang et al., 1997). There are a variety of hexose kinases in plant
tissues (Turner and Turner, 1980). These enzymes may be subclassified on the basis of
their substrate specificity. An enzyme that can effectively utilize several hexoses is termed
hexokinase (HK, EC 2.7.1.1), while that which preferentially metabolizes glucose is
termed glucokinase (GK, EC 2.7.1.2), and that which phosphorylates fructose is
designated fructokinase (FK, EC 2,7.1.4) (Kruger, 1990). For example, in pea seeds,
fructokinase fraction IV, was specific for fructose as a substrate, having little activity with
glucose (Turner et al., 1977). In this case, this enzyme is probably responsible for the
metabolism of fructose derived from the cleavage of sucrose by sucrose synthase (Kruger,

1990). In tomato fiuits undergoing starch synthesis, FK is inhibited by fiuctose (Schaffer

25

and Petreikov, 1997). GK and FK phosphorylating activities is inhibited by anoxia in
maize roots (Bouny and Saglio, 1996).

Three enzymes operate at the reversible interconversion of F~6-P to F-l,6-P2. This
step in carbon metabolism exerts control over glycolytic flux and is considered to be
rating-limiting for glycolysis for some tissues/conditions. Pyrophosphate-dependent 6-
phosphofructose l-phosphotransferase (PPi-PFK, EC 2.7.1.90) catalyzes the
interconversion of PPi and F-6-P to Pi and F l ,6P2 in plants (Smith et al., 1984). PPi:PFK
was discovered in 1974 in amoeba (Reeves et al., 1974) and in 1975 in bacteria (O’Brian
et al., 1975). PPi:PFK was later found to be present in plant tissue and exhibited similar
regulatory properties (Carnal and Black, 1979). This reaction is firlly reversible (Nielsen,
1995), however, PPi:PFK participates in glycolysis through the use of PPi both as a
phosphate donor and as an energy source (Smyth et al., 1984). PPi:PFK can be
interconverted between an active 260 kDa tetramer, and a less active 130 kDa dimer
(Black et al., 1987). The state of aggregation of this enzyme is dependent on the
concentrations of its allosteric activator, fructose 2,6-bisphosphate (F 2,6P2), and the
product of the forward reaction, Pi (Plaxton, 1996). The activity of tetrameric form of the
enzyme is enhanced by the presence of F2,6P2 (Xu et al., 1984). Alternatively, PPi:PFK
was demonstrated to be activated in barley leaves by F1,6P2 in presence of aldolase
(Nielsen, 1995). The dimeric form of PPi:PFK, however, as well as its activity in the
reverse, or gluconeogenic direction, is promoted by Pi (Dennis and Greyson, 1987).
PPi:PFK activity is inhibited by increased levels of Pi in the cytosol (Plaxton, 1996).

ATP:PFK (ATP: D-fructose-6-phosphate l-phosphotransferase, EC 2.7.1.11)

26

catalyzes the interconvertion of ATP and F -6-P to ADP and F1,6P2 in higher plants and is
essentially irreversible under physiological conditions (Plaxton, 1996). ATP:PFK operates
in the glycolytic direction, in a non-equilibrium reaction. Fructose-1,6-bisphosphatase
(FBPase, EC 3.1.3.11) functions gluconeogenically to catalyze the interconversion of
F1,6P2 to F-6-P. In contrast to PPi:PFK, FBPase is inhibited by F2,6P2 (Plaxton, 1996).
ATP:PFK and FBPase were called “maintenance enzymes” for the regulation of sugar
metabolism (Black et al., 1987) due to their roles of remaining fairly stable in a given
tissue throughout development for a variety of species.

ATP:PFK is under complex allosteric regulation. ATP is not only the substrate for
ATP:PFK, but also inhibits its activity when present in high levels by binding to an
allosteric site and lowering the affinity of the enzyme for its substrate F-6-P (Plaxton,
1996). High citrate levels increase the inhibitory effect of ATP, firrther reducing the
carbon flux through glycolysis. In contrast, ADP and AMP, which rise in concentration
when the consumption of ATP outspaces its production, act allosterically to relieve this
inhibition by ATP (Dennis and Greyson, 1987). Phosphoenolpyruvate (PEP) inhibits the
activity of ATP:PFK; this inhibition is relieved by Pi (Plaxton, 1996). In addition,
ATP:PFK activity may be inhibited by low pH. Low 02 or high CO2 reduce cytoplasmic
and vacuolar pH and ATP levels in lettuce (Siriphanich and Kader, 1986), which, in turn,
affected the activity of ATP:PFK and PPi: PFK (Hess et al., 1993).

Pyruvate kinase (PK, ATPzpyruvate 2-O-phosphotransferase, EC2.7. 1.40), is a
regulatory enzyme of glycolysis that catalyzes the irreversible synthesis of pyruvate (PYR)

and ATP fi'om PEP and ADP (Knowles et al., 1998). This step is now recognized as the

27

primary control site of glycolytic flux to pyruvate in plants (Beaudry et al., 1989 and 1987;
Plaxton, 1996). This enzyme is localized in the cytoplasm (PK,) and in the plastids (PKp),
where they differ by immunological and kinetic properties (Blakeley and Dennis, 1993).
PK, is suggested to play a key role in C3 plants regulation of glycolytic flux, thereby
affecting mitochondrial respiration and carbon skeleton generation for anabolic reactions
(Knowles et al., 1998; Plaxton, 1996). Studies on PK, and PK, at the mRNA and protein
levels in developing tobacco seeds demonstrated that the expression of these isozymes
may be controlled by independent transcriptional and posttranscriptional mechanisms, and
PK, has a much greater rate of turnover than PK, (Blakeley and Dennis, 1993). However,
PK, activity also may be modulated by fine control mechanisms (Plaxton, 1996). For
example, high levels of ATP inhibit PK allosterically in higher plants, by decreasing the
affinity of the enzyme for its substrate PEP (Plaxton, 1996; Turner and Turner, 1980). PK
is also inhibited by acetyl-CoA and by long-chain fatty acids (Plaxton, 1996).

High levels of CO2 can either slow or elevate respiratory rates depending on the
commodities and the concentration of CO2 employed (Beaudry, 1993; Cameron et al.,
1994; Gran and Beaudry, 1993; Kerbel et al., 1988). Lower 02 levels also reduce the
respiration rate of fresh fruits and vegetables. Storage of fruits and vegetables in less than
15 kPa CO2 can cause changes in the activity of respiratory metabolism and may have an
uncoupling effect on oxidative phosphorylation (Lange and Kader, 1997a). Air plus 10
kPa CO2 inhibits glycolysis in intact pear fruit, probably due to CO2 effects on the
glycolytic enzymes, especially for ATP:PFK and PPi:PFK (Kerbel et al., 1988).

According to Baxter and Waters (1991), asparagus stored under controlled atmosphere

28

(CA) of 5 kPa O2 and 10 kPa CO2 retained more sugars than spears stored in air at room
temperature. The greater retention of sugars for CA-stored spears compared to air-stored
spears implicates a general impairment of glycolytic reactions by C02.

In asparagus spears tips, effects of O2 and CO2 on the pool of intermediate and the
activity of glycolytic enzymes has not been studied. This effect is expected to be more
pronounced for PFK and PK. The effects of low Oz/high CO2 levels on intermediate pools
and enzymes of glycolysis in asparagus is not known. The response is addressed in
Chapter 4.

Fermentative metabolism. Low 02 dramatically affects plant metabolism by
impairing the oxidative phosphorylation pathway. The enzymes and cofactors for this
pathway are located in the mitochondrial membrane. The oxidative phosphorylation
pathway is normally responsible for the regeneration of NADH and NADPH in the form
of reducing power, which is critical for glycolysis, with a concomitant production of ATP.
Limited availability of 02 as terminal electron acceptor for oxidative phosphorylation stops
ATP production via this pathway and depletes the resources of NAD’ and NADP"
(Dolferus et al., 1997). Earlier views of the response to limiting levels of 02 at the cell
level were formalized in the concept of the Pasteur effect. As ATP generation by
oxidative phosphorylation begins to fall off due to insufficient 02, the energetic deficit is
made up by activation of anaerobic ATP supply pathways. In some cases, this kind of O2
sensing would mean activation of anaerobic glycolysis (since this is the only known
mechanism for ATP generation without 02 in the cell). Contrary to this point of view, as

a means for sensing hypoxia, it is argued that hypoxia tolerant tissues use anaerobic

29

metabolism not to make up energy deficits but to sustain a reduced energy turnover state
instead (Hochachka et al., 1996).

In passive MAP of fresh produce, there is a risk that oxygen levels could fall below
critical levels and cause product fermentation or even favor the growth of harmful human
pathogens (Cameron et al., 1994). There is considerable risk associated with MAP
systems when attempts are made to reduce the 02 concentration inside the package to low
levels at low temperatures because anaerobic conditions can develop if temperature
increases during subsequent storage or handling (Cameron et al., 1994). In asparagus
stored at elevated temperatures, the rate of loss of carbohydrates, proteins, and amino
acids are also more accentuated than at lower temperatures (Saltveit and Kasmire, 1985).

Anaerobiosis increases the rate of quality loss of asparagus during storage (Torres-
Penaranda and Saltveit, 1994). Anaerobic respiration develops below a critical 02 level of
2.3 kPa at 20 °C as shown by an increase in CO2 production relative to aerobic respiration
(Platenius, 1943). Asparagus spears exposed to anaerobiosis for 6 h at 20°C and for 4h at
2.5 °C resulted in a stimulation followed by a decline in CO2 production. The
physiological cause for that was not explained (Torres-Penaranda and Saltveit, 1994),
however, it is possible that accumulated products of anaerobic respiration may be
responsible for such response. Very low levels of 02 may cause the accumulation of
ethanol and acetaldehyde in hits and vegetables (Ke and Kader, 1990). In asparagus held
at 20 °C, an alcoholic aroma started to appear for 02 levels below 2 kPa (Platenius, 1943).

The effect of 02 on the synthesis of fermentation products is further modulated by the

presence of CO2 (Shirazi et al., 1991; Thomas, 1925). In addition, the combination of low

30

O2 and high CO2 had a synergistic effect on inhibiting ATP biosynthesis (Hess et al.,
1993). Lipton (1965) found that an atmosphere of l kPa 02 for 7 days at 6 °C reduced
soft rot, which was eliminated by 0 kPa 02. However, 0 kPa O2 resulted in low-O2 injury
to the spear. In avocado, anaerobic atmospheres induced the accumulation of ethanol and
acetaldehyde to levels that caused off-flavor and an enhancement of some physiological
disorders (Ke and Kader, 1990). Saltveit and Ballinger (1983) found that ethanol
concentration in asparagus spears increased linearly for up to 4 h under N2 or CO2
atmosphere and exponentially with temperatures from O to 30 °C.

Severe stress levels of 02 substantially reduce NADH flux to the cytochrome
electron transport system and oxidative phosphorylation is greatly reduced. Pyruvate
dehydrogenase activity is reduced and pyruvate flux to the TCA cycle decreases.
Decreased carbon flux through the TCA cycle directs PYR to the fermentation pathway
and cause the accumulation of acetaldehyde and ethanol (Ke et al., 1993). Some plant
species delay or avoid accumulation of ethanol by diverting glycolytic intermediates to
alternate end products such as lactate and malate as a result of limited O2 supply (Kennedy
et al., 1992). According to Kennedy et al., (1992), lactate dehydrogenase (LDH, L-(+)-
lactatezNAD+ oxidoreductase, EC1.1. 1.27), pyruvate decarboxylase (PDC, 2-oxoacid
carboxylase, EC 4.1.1.17), and alcohol dehydrogenase (ADH, alcohol:NAD
oxidoreductase, EC 1.1.1.1) are among the primary enzymes involved in anaerobic
metabolism in plants.

LDH and PDC are generally present in very low amount in aerobic tissues and

often suggested to be limiting (Germain et al., 1997). LDH catalyzes the formation of

31

lactate from pyruvate, is present as multiple isoenzymes, and is anaerobically induced in
several species. Lactate fermentation generally precedes ethanol production as 02 levels
become increasingly hypoxia (Davis, 1980). The relative rate of synthesis of lactate versus
ethanol, however, depends upon the cytoplasmic pH (Davies, 1980). Low O2 and/or high
CO2 stresses may change intracellular pH (Hess et al., 1993). At the onset of
anaerobiosis, PYR is initially converted to lactate, resulting in a drop in cytoplasmic pH.
The drop in pH reduces LDH activity, but activates PDC; subsequently, ethanol is
predominantly synthesized by ADH (Davies, 1980; Germain et al., 1997). Based on this
scenario, Davis, (1980) suggested that LDH and PDC act as a metabolic pH-stat for
regulating fermentative metabolism.

PDC catalyzes the decarboxylation of pyruvate, yielding CO2 and acetaldehyde, the
precursor of ethanol. In maize, PDC activity may increase 5- to 9-fold during anoxia
whereas PDC mRNA may be induced about ZO-fold (Kelley, 1989). ADH governs
ethanol formation. In response to anaerobiosis, ADH activity increases in most plants.
ADH activity may represent a flood tolerance strategy in some species. For instance,
ADH activity seems to be essential for survival during short periods of flooding in maize
(Drew, 1990), and may also be energetically advantageous for survival under 02 and/or
CO2 stresses.

Asparagus responses to very low 02/ high CO2 concentrations by inducing the
fermentative pathway. This may lead to a decrease in intracellular pH and ATP levels and
the accumulation of ethanol. Lactate accumulation is an alternative pathway that can

occur in asparagus. The regulation of ethanol and/or lactate fermentation in asparagus in

32

response to low Ozlhigh CO2 stresses is not known. This response is addressed in Chapter
3.

Phosphate and adenylate metabolism. Plant tissues use molecular oxygen for a
number of biosynthetic or degradative processes (Kennedy et al., 1992), the most
important one being mitochondrial respiration (Ricard et al., 1994). Phosphate
metabolism is a vital component in energy transfer during the reduction of Oz. Phosphate
is also used in metabolic regulation and is an important constituent of macromolecules
such as phospholipids and nucleic acids (Duff et al., 1989). The growth and development
of plants is particularly dependent upon the availability of Pi, and the effects of
environmental Pi deficiency on the rates of photosynthesis and photoassimilate partitioning
in plants has been studied (Duff et al., 1989; Goldschmidt and Huber, 1982; Preiss and
Sivak, 1996). Starch synthesis in leaf cells is mainly controlled by the ADPG—
pyrophosphorylase (EC 2.7.7.27), which catalyzes the interconversion of glucose-6-
phosphate to ADP-glucose. Its activity is largely regulated by the intracellular Pi
concentration and triose phosphate availability (Preiss and Sivak, 1996).

Phosphate starvation is suggested to induce bypass of adenylate- and phosphate-
dependent glycolytic enzymes when intracellular pools of ATP, ADP, and Pi are depleted
(Duff, et al., 1989). Pi content also seems to be altered by sucrose levels. For instance, in
sycamore cells, when sucrose levels dropped to critical levels in the cell, the levels of
intracellular Pi increased (Re’beille' et al., 1985). Pi content is also affected by 02 and CO2
partial pressures. Its content increases in avocado tissues in response to elevated CO2

levels (Lange and Kader, 1997b) and in minimally processed carrots treated with 0.5 and

33

2.0 kPa 02, at 5 and 15 °C, when compared to air (Kato-Nogushi and Watada, 1996b).

Perphosphate (PPi) is thought to be controlled in the cytoplasm by PPi:PFK,
which produces PPi during gluconeogenic carbon flux. PPi concentration may enhance or
regulate sucrose breakdown via sucrose synthase. PPi:PFK is also able to maintain PPi
levels in the cytoplasm according to its supply and demand (Dancer and Ap Rees, 1989).
PPi, therefore, serves as energy source for glycolysis (Carnal and Black, 1979). PPi is
also a by-product of a host of reactions involved in macromolecule biosynthesis. One
dogma of cellular bioenergetics is that the anhydride bond of PPi is never utilized and that
PPi produced in biosynthesis is always removed by the hydrolytic action of an inorganic
alkaline pyrophosphatase, thereby providing a thermodynamic “pull” for biosynthetic
processes (Plaxton, 1996). PPi-dependent processes may be a crucial mechanism for
metabolic adaptation of plants to environmental stresses that reduce NTP availability via
increased activity of sucrose synthase (Guglielminetti et al., 1995; Huber and Akazawa,
1986), PPi;PFK (Duff et al., 1989), and the tonOplast Hi-PPiase (Plaxton, 1996).

Energy transduction and energy storage, involving the adenine nucleotides (ATP,
ADP, AMP), are fundamental features of metabolism. Adenine nucleotides form an
extensive systems of allosteric regulators that operate on a large number of reactions to
adjust the rate of ATP synthesis to its utilization (Atkinson, 1977; Pradet and Raymond,
1983). Adenine nucleotides are maintained in equilibrium by the enzyme adenylate kinase
(EC 2.7.4.3) which catalyzes the reaction,

ATP +AMP <----> 2 ADP [4].

Therefore, a decrease in the concentration of ATP will occur following an increase in that

34

of AMP, and vice versa. The concept of ‘adenylate energy charge’ (AEC), which
represents the relative saturation of the adenylate pool in phosphoanhydride bands, was
introduced by Atkinson (1977), which is expressed as:

AEC = ([ATP] + 0.5 ' [ADP]) ([ATP] +[ADP] + [AMPD‘l [5].

ABC can theoretically vary between 0 and 1. Usually, the ABC of actively
metabolizing plant tissues varies from 0.8 to 0.85 (Pradet and Raymond, 1983).
Regulatory enzymes that occur in pathways in which ATP is utilized respond to changes in
energy charge in the general way shown by curve 2 of Fig. 1.3. Conversely, regulatory
enzymes that participate in pathways in which ATP is regenerated respond to the pattern
shown in curve 1 (Plaxton, 1990). According to Fig. 1.3, in a healthy cell, any tendency
for ABC to fall would be prevented through ATP-regenerating pathways (curve 1) and by
the decrease in flux through ATP-utilizing pathways (curve 2). Plant tissue under anoxia
generally results in a large decrease in ABC (Saglio et al., 1980). Under hypoxia, tissue
metabolic activity is limited by the availability of oxygen, and it appears that the level of
ABC is clearly correlated with metabolic activity (Pradet and Raymond, 1983).

The ATP/ADP ratio is a fundamental regulatory and regulated value, which is
sensed by the enzymes and is related to the thermodynamic state of the adenine nucleotide
pools (Pradet and Raymond, 1983). For instance, low ATP/ADP ratios of anoxic tissues
resulted in strong inhibition of HK (Renz and Stitt, 1993), and, thus, glycolytic flux. The
levels of ADP in the cell are substrates for oxidative phosphorylation in the mitochondria,
hence, the absolute cytoplasmic concentration of ADP available for uptake into that

organelle controls the rate of respiration (Rébeille’ et al., 1985). In anoxic maize roots,

35

 

 

    

 

 

 

AVG.E.C.oI
HOST CELLS
).
L: I I
0
o 1.ATP producing/ : I
d reactions , /
> I
z i/
9
r- I
2 / :
a“: 2 ATP t‘l' ' / '
. u r rzrng ,
reactions\ / / .
/ I
av ’ I
L _. —- -" ’ l r I
O 0.5 '°-‘° 1-0
on" Energy Charge (E.C.) only
AMP ATP

Figure 1.3. Responses to the cellular energy charge of regulatory enzymes in
metabolic pathways in which ATP is produced (curve 1) and in which it is utilized
(curve 2). From Plaxton, (1990).

36

10 h of anoxia resulted in increased ADP with a concomitant decrease in ATP content
(Bouny and Saglio, 1996).

In asparagus spear tips, the pool of phosphate and adenylates, and ABC may be
affected when spears are stored under low 02/ high C02. The effects of lower 02 /high
CO2 on phosphates, adenylates and ABC is unknown for asparagus and is addressed in
Chapter 5.

Cytokinin and Carbon Allocation

Senescence in mature plant parts can be induced or enhanced by the influence of
growing organs (e. g. asparagus tips), and this relationship affects the transport process.
In maize leaves, the start of senescence was delayed by interrupting the transport of
metabolites to sink tissues (Muller and Leopold, 1966b). In asparagus, rapid depletion of
sucrose in the tip (King et al., 1990) with concomitant acropetal decline of soluble sugar
(Silva et al., 1998), suggests that the tip may have a regulatory influence on the after
harvest physiology of the remaining spear in acting as a sink that “scavenges” for
metabolites translocated from lower parts of the spear (Saltveit and Kasmire, 1985).

It has been hypothesized that allocation of carbon is mediated by cytokinin and
sucrose (Van der Werf and Nagel, 1996). Exogenous application of cytokinins to plant
tissues results in various responses including a delay in senescence, increased chlorophyll
production, stimulation of chloroplast development, a decline in chlorophyll degradation,
the promotion of protein and nucleic acid synthesis, and the mobilization of nutrients into
the cytokinin-treated area (Taiz and Zeiger, 1991). Cytokinins are also involved in the

maintenance of photosynthetic activity (Wingler et al., 1998) through effects on

37

chlorophyll degradation and inhibition of the loss of photosynthetic proteins (Badenoch-
Jones et al., 1996). Exogenously applied cytokinin promotes production of, and increased
activity of, ribulose bisphosphate carboxylase (Clarke et al., 1994). Cytokinin was also
effective in delaying chlorophyll degradation and decreasing rate of respiration in
harvested broccoli (Clarke et al., 1994; Rushing, 1990). Senescence has been delayed in
transgenic plants by expression of a bacterial gene encoding isopentenyl transferase, the
enzyme catalyzing the first step of cytokinin synthesis (Gan and Amasino, 1995).
However, it has been suggested that cytokinin, in addition to delaying senescence, could
block some responses to changing sugar levels (Jang et al., 1997). Cytokinin was effective
in stimulating a mass-flow in the phloem, attracting substances from untreated tissue and
directing them toward the site of application (Muller and Leopold, 1966a and b). The
interaction of a Supplier region and a mobilizer region involving a redistribution of material
was termed ‘cytokinin-induced directed transport’.

Thus, the evaluation of the physiological changes and mechanisms that may
regulate carbon flux and the rate of utilization of sugars throughout the spears may
contribute much to understanding the deteriorative processes that accompany postharvest
of asparagus. It is possible that cytokinin application in spear tips would enhance carbon
flux in its direction. This would minimize the rate of sucrose degradation in the tip and
improve the quality of asparagus spears stored at 1°C. This response is addressed in

Chapter 6.

38

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50

CHAPTER 2
RESPIRATORY AND FERMENTATIVE METABOLISMS IN ASPARAGUS
SPEAR TIPS UNDER LOW OIIHIGH COz ATMOSPHERES
INTRODUCTION

Asparagus spears senesce rapidly after harvest. The process of senescence is
associated with many physiological and biochemical changes. Among these changes,
sugar depletion is notable. The sugar content of asparagus spears declines rapidly
following harvest. The decline in sugar is most marked in the spear tip, a meristematic
tissue that is recognized to be a very active sink (Hurst et al., 1993b). The predominant
sugars in asparagus at harvest, sucrose (Irving and Hurst, 1993 ), is translocated from the
crown during the period of rapid spear growth (Robb, 1984). Sucrose is utilized to
provide energy for global metabolic processes and to a significant extent, is the source of
carbon skeletons for cell wall formation. The rapid use of imported sucrose sustains a
sucrose concentration gradient between the sink cell and the phloem tissue (Ho, 1988).

It has been suggested that sucrose rapid decline in the spear tip triggers or signals
deterioration in the whole spear (King et al., 1993). Thus, knowledge of the processes
that govern the sugar content may be important in maintaining the quality of asparagus
postharvest. Importantly, understanding the pattern of sucrose decline in the tip could
help elucidate a means to control its loss, and, subsequently, control spear deterioration.
Ultimately, it may be possible to extend the shelflife of asparagus spear.

Exposing harvested horticultural crops to low levels of 02 with or without high

levels of CO2 has been shown to affect many causes of deterioration and influence crops

51

quality and increase storage life (Kader, 1986). Elevated CO2 atmospheres seem to have a
particular effect on sugar content in asparagus (Baxter and Waters, 1991). Treating
asparagus with elevated CO2 for a limited period also has potential as a quarantine
treatment for insect control (Corrigan and Carpenter, 1993). Different commodities
exhibit widely different thresholds to O2 and CO2 that are both temperature- and
concentration-dependent (Saltveit, 1993). However, some undesirable physiological
changes may also occur in plant tissues under stress levels of low 02 and high C02. The
potential benefit of using low Ozlhigh CO2 atmospheres in storage depends on several
variables. High levels of CO2 can either reduce or elevate respiration rates depending on
the commodity and the concentration of CO2 utilized (Beaudry, 1993; Kerbel et al., 1988).
Elevation of the respiratory quotient (RQ) can also occur in response to elevated CO2
levels (Thomas, 1925). The increase in RQ is accompanied by enhanced synthesis of
ethanol and acetaldehyde (Thomas, 1925). The effects of CO2 on the synthesis of
fermentation products is further modulated by the presence of O2 (Beaudry, 1993; Shirazi
et al., 1991; Thomas, 1925).

Higher plants have an absolute requirement for oxygen as a terminal electron
acceptor during respiration (Kennedy et al., 1992). Plant responses to low 02 and high
CO2 levels include induction of fermentative metabolism and a decrease in ATP content
(Ke et al., 1994; Saglio et al., 1980) (Fig. 2.1). One pathway of fermentative metabolism
results in accumulation of acetaldehyde, catalyzed by the enzyme pyruvate decarboxylase
(PDC, EC 4.1.1.1) and ethanol, catalyzed by the enzyme alcohol dehydrogenase (ADH,

EC 1.1.1.1). Some plant species delay or avoid accumulation of ethanol by diverting

52

pyruvate to lactate via a reaction catalyzed by the enzyme lactate dehydrogenase (LDH,
EC 1.1.1.27) (Guglielminetti et al., 1997). It is believed that the primary function of
fermentative metabolism is to use NADH and pyruvate when electron transport and
oxidative phosphorylation are inhibited so that NAD is recycled and glycolysis can
proceed. This allows for continued carbon flux and the production of some ATP, which
will permit the plant tissue to survive temporarily under 02-deficient conditions.
Modification of storage atmosphere has been long used as a means to develop a
better understanding of the biochemical and physiological processes in crops (Kays, 1991).
Although intensive research has been done on anaerobic metabolism in maize, rice, pears,
avocado (Davis, 1980; Ke et al., 1995; Kennedy et al., 1992), the mechanism for
regulation of low 02 and high CO2 on ethanolic fermentation in asparagus has not been
studied (Baxter and Waters, 1991; Hurst et al., 1997; King et al., 1986; Lipton, 1965). In
this research, the effects of C02 (0, 5, 10, and 20 kPa) combined 02 partial pressures
ranging from 0.1 to 16 kPa (1% 02 = 1.013 kPa 02 at 1 atm) were evaluated. The
purpose of this work was to investigate the influence of CO2 and O2 partial pressures on
sugar utilization, respiratory activity, fermentative metabolism, and quality in asparagus

spears stored at 1 °C.

MATERIAL AND METHODS
Plant Material. Spears (Asparagus ajficinalis L. cv. Jersey Giant) were harvested
in May from the Michigan State University Horticultural Research Center between 6:00

and 9200 am. Spears were harvested at a length of 240 mm, trimmed to 180mm, graded,

S3

placed on ice and held at 0 °C. Only straight undamaged spears with closed bracts and no
obvious disease symptoms were used. A set of spears were used for determination of
sugar and lactate contents and enzyme activities at harvest. These spears were trimmed
and flown in liquid N2 in the field. These and all subsequent samples were stored at -80
°C until analysis. Three entirely separate experiments were conducted in three consecutive
asparagus seasons (1995, 1996, 1997) and the obtained data combined. Asparagus spears
were held at 1 °C throughout this study.

Steady State 02 and C 02 and Respiration Rates. The levels of O2 generated
ranged from approximately 0.16 to 16 kPa for each of four different treatment levels of
C02 (0, 5, 10, and 20 kPa). Spears were enclosed within a package composed of low
density polyethylene (LDPE) film and having a surface area of 462 cm2. The packages
for the 5, 10, 20 kPa CO2 treatments were enclosed in 1.95-L glass jars using the method
of Beaudry (1993). Each package was equipped with a gas sampling septum (Boylan-
Pett, 1986). For packages in jars, the septum was attached to the base of a 1.3 cm internal
diameter (ID) x 2.7 cm long glass tube using silicone sealant. The glass tube was
inserted through the jar lid and attached to the surface of the enclosed package (Fig. 2.2).
Chamber lids were also fitted with inlet and outlet ports for purging with gas mixtures.
Chambers were continually purged with a mixture of air, C02, and N2 gases at 30 mL'rnin'l
to generate target atmospheres within the enclosed packages. Packages were designed to
achieve target 02 levels based on measured permeabilities of the LDPE films to O2 and
C02, and by altering film thickness, and the total spear mass within the packages. Package

gas composition was determined daily by withdrawing a IOO-uL gas sample from the

54

package with a 500-uL gas-tight syringe (Hamilton Co. Reno, Nevada) and analyzing for
O2 and CO2 using 02 (Servomex series 1100 with a paramagnetic detection cell) and CO2
(ADC 225-MK3 analytical infrared gas analyzer) analyzers connected in series with N2 as
a carrier gas (flow rate=100 mL'min“). Gas concentrations were converted to partial
pressures. The gas composition of individual packages was monitored until steady state
conditions were reached (approximately six days). 02 and CO2 gradients across the film
were used to calculate steady state diffusion for both gases through the film using
previously obtained permeability data (Lakakul, 1994). Flux data were used to calculate
the rates of 02 uptake and CO2 production (Cameron et al., 1994).

Measured O2 and CO2 gradients across the film were used to calculate steady state
diffusion rates for both gases through the film. Respiration rates were calculated from the
gas diffusion rates by assuming diffusion rates though the package were equal to the O2
and CO2 fluxes in and out of the fruit. The data were combined to ascertain the rates of

respiration using the following formulae:

(P02 A/x)([02]chmbr ' [02]pkg)
RR02 = [1]
W

(Pcoz A/x)([C02]pkg ' [cozichmbr)
RR C02 = [2]
W

 

where RRO2 and R CO, are the rates of 02 uptake and CO2 production (mmol'kg"°hr"),

respectively; P O, and P CO, are measured 02 and CO2 permeability coefficients

55

(mmol'cm'cm‘zhr'l'kPa‘l), respectively, for LDPE at the storage temperature; A is film area
(cmz); x is thickness (cm); [Oz],hmbr and [02]pkg are chamber and package partial pressures
of 02 (kPa), respectively; [C02]pkg and [C02],,,,,,,,, are package and chamber CO2 partial
pressure (kPa), respectively; and W is asparagus spear weight (kg). The RQ was
calculated as R CO, divided by RR02 . Non-oxidative C02 (C02 not used in oxidative
reactions) was calculated by subtracting RR CO, from RRO2 (mmol'kg'l'hr"). Steady state
levels were achieved on day 6. Tissue samples (apical 45 mm section) were taken on day
6 and stored at -80 °C for soluble sugar, lactate and fermentative enzyme determinations.
Spears were combined into three replication jars for each parameter evaluated.

Ethanol and acetaldehyde determinations. Headspace ethanol and acetaldehyde
were measured by a gas chromatograph equipped with a flame ionization detector (Series
400, Hach Carle Co., Series 400, Loveland, CO). A sample of 1 mL was withdrawn
from each package using a gas-tight syringe (Hamilton Co., Reno, Nevada). The
chromatograph column (4 m long x 3 mm I.D.), was packed with 10% DEGS-PS,

80/ 100 mesh Supelcoport (Supelco, Inc., Bellefonte, PA) and was maintained at 110
°C. The carrier gas was He and the flow rate was 40 mL'min". The flow rate of H2
and air were 25 and 200 mL'min", respectively. Standard curves were prepared for
ethanol and acetaldehyde with analytical grade products (Sigma Chemical Co., St.
Louis, MO) and using the method of Buttery et al. (1969).

Sugar Determinations. Sugars were extracted from approximately 3.0 g of frozen
tip tissues by adding 3.5 mL of 80% ethanol and grinding with a tissue homogenizer (Ultra

Turrex Model, Tekmar Co., Cincinnati, OH), and held for 15 min at room temperature.

56

The mixture was centrifuged (Model RG-SC, Sorval Instruments, Dupont Co., Newton,
CT) at 2,000 g for 5 min and the supernatant decanted into a capped 50 mL centrifuge
tube. The pellet was resuspended and extracted two additional times. The extract was
then washed out with 5 mL of water. To remove traces of chlorophyll, 5 mL of
chloroform was added to the combined 15.5 mL of ethanol/water mixture. The tubes
were capped, shaken vigorously and then centrifuged at 1,000 g for 3 min. The upper,
clear aqueous phase containing the soluble sugars were transferred to a 15 mL test tube
and evaporated to dryness using a speedvac equipped with a refrigerated condensation
trap (RT4104) maintained at -103 °C (SC200 Savant Speedvac, Savant Instruments, Inc.,
Fanningdale, NY). Fructose, glucose, and sucrose were separated and quantified by a gas
chromatograph (Hewlett Packard Model HP5890 Series II, Hewlett Packard Co., Palo
Alto, CA) fitted with an FID and a capillary colunm (30 m long x 250 um i.d.) having a
0.25 pm film thickness DB 1702 capillary column.

Lactate extraction. Approximately 5 g of frozen tissue per replicate was
transferred to liquid N2 in a precooled mortar and ground with a pestle to a fine powder.
Ten mL of ice-cooled 0.8 N (v/v) HCIO, were added to the powder, which was extracted
for 30 min at 0 °C with occasional shaking, followed by centrifugation at 30,000 g for 30
min at 0 °C. Supernatant pH was adjusted to 7.0 to 7.2 with 5 N KZCO3, allowed to sit on
ice for 10 min, and clarified by centrifugation at 30,000 g for 10 min. The resulting
supernatant was used for lactate analysis. To estimate losses of this metabolite during
extraction, measured amounts of pure lactate roughly equal to the amounts present in the

tissue, were added to the extracting medium. Recovery was 91%. The recovery assay

57

was replicated three times.

Lactate assay. Lactate was assayed spectrophotometrically (Hitachi, Model
U3000, Hitachi Inc., Tokyo, Japan) at 25 °C by measuring changes in the absorbance of
pyridine nucleotides at 340 nm due to oxidation/reduction following adaptations from
procedures by Noll (1984) and Scott et al., (1995), using a double-beam
spectrophotometer (Hitachi, Model U3000, Hitachi Inc., Tokio, Japan), equipped with
enzyme module. The determination of lactate was assayed in a final volume of 1 mL
containing 40 mM glycylglycine/L-glutamate buffer (pH 9.0), 0.5 mM NAD, 2 units L-
alanine: 2-oxyglutarate aminotransferase, and 2 units lactic dehydrogenase (LDH).

Extraction of Pyruvate Decarboxylase (PDC), A [coho] Dehydragenase (ADH) and
LDH. Approximately 3 g of frozen tip tissue was ground as described previously and
mixed with a 10 mL ice-cold 100 mM 2-(N-morpholina) ethane-sulfonic acid (MES)
buffer (pH 6.5), containing 2 mM dithiothreitol, 50 mg/g tissue of insoluble (40,000 mw)
PVPP, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 10 % (v/v) glycerol. The mixture
was centrifuged at 30,000 g for 10 min. The supernatant was used as enzyme extract for
the immediately measurement of PDC, ADH, and LDH activities (Ke et al., 1994).

Enzyme Assays. Assays for enzyme activities were run immediately after
extraction in a final volume of 1 mL based in modifications from Ke et al. (1994). The
assay cocktails were as follows: Alcohol dehydrogenase: 80 mM MES (pH 6.5), 0.2 mM
NADH, 50 11L of the enzyme extract, and 10 mM of acetaldehyde (Ashihara et al., 1989).
Lactate dehydrogenase: 74 mM MES (pH 6.5), 0.2 mM NADH, 4 mM pyruvate, 3 mM of

4-methylpyrazol, 3 mM KCN, and 100 11L of extract (Ke et al., 1994). Pyruvate

58

decarboxylase: 60 mM MES (pH 6.5), 0.5 mM thiamine pyrophosphate, 5 mM MgClz, 0.5
mM NADH, 15 units ADH, 50 11L of enzyme extract, and 10 mM pyruvate (start
reaction) (Nanos et al., 1992). The measurement of the oxidation of NADH for ADH and
LDH was followed at 30 °C by recording the decrease in absorbance at 340 nm as
previously described.

Sensory quality. Sensory quality was rated for the whole spears upon opening
each LDPE bags and exposing the spear to room temperature for 12 hr after the steady
state 02 was reached (following 6 days of storage). Each treatment replication was rated
independently by the subjective evaluation of four observers using the following scale
adapted from Hurst et al. (1993a):

9= fresh like;

7: slight browning of tips and scales, slight flaccidity and/or very slight wrinkle on
stems, absence of strange flavors;

5= moderate browning of tips and/or stem bracts browning, more pronounced
wrinkle, loss of pink blush at the base, slight ethanol odor;

3= extensive browning and pronounced flaccidity of tips and stems bracts
(feathering), wrinkle and wilting of stems, moderate fermentative odor;

1= extensive browning and flaccidity of tip and stems bracts, complete loss of pink
blush at the base, extensive wrinkle and wilting stems and base, brown spot in stems when
exposed to air, strong fermentative odor.

Asparagus rating of 6 was considered unacceptable for sale although still edible.

59

RESULTS

Respiration rate. The rate of 02 uptake declined with decreasing Oz partial
pressures with the rate of decline being most rapid below 2 kPa 02 (Fig. 2.3). 02
uptake was apparently unaffected by C02 partial pressures. CO2 production behaved
similarly to 02 uptake with respect to O2 partial pressures. However, CO2 production
was enhanced by the 20 kPa CO2 treatment. Above 2 kPa 02, the RQ was
approximately 1 for 0, 5, and 10 kPa CO2 treatment. The RQ averaged 1.2 for the 20
kPa CO2 treatment in this 02 range. Below 2 kPa 02, the RQ increased markedly as
CO2 levels increased. Non-oxidative CO2 production tended to increase as CO2 levels
increased. Non-oxidative CO2 production also tended to increase as 02 levels declined,
with the most rapid increase occurring below 2 kPa 02 (Fig. 2.4), following the same
pattern of RQ. Interestingly, it appeared that the profile of the non-oxidative CO2
production peaked at approximately 1 kPa 02 for all four CO2 treatments, declining
sharply as the O2 partial pressure dropped below 1 kPa.

Sugar Metabolism. There was a drop in glucose, fructose and sucrose levels in
spear tips following harvest in the time required to achieve steady state 02 conditions.
Sucrose was the prevalent sugar at harvest and the one that declined most markedly after
6 d of storage at 1 °C (Fig. 2.5). For spear stored at near-ambient conditions (> 16 kPa
02, O kPa C02) the reduction in sucrose, glucose and fructose was 85, 75 and 45% of at-
harvest contents, respectively. The decline in sucrose was greatest when asparagus spear
tips were exposed to 20 kPa CO2 although the reduction in sucrose for the 0 kPa CO2

treatment was nearly as extensive (Fig. 2.5). The greatest retention of sucrose occurred

60

for spears exposed to 5 and 10 kPa C02. At 02 levels greater than 2 kPa, fructose and
glucose tended to be similar across the 02 range and largely unaffected by C02. Below
approximately 2 kPa Oz, fructose tended to accumulate. For the 20 kPa CO2 treatment,
low 02 further decreased sucrose content.

Fermentative Metabolism. Acetaldehyde and ethanol accumulation in the
package headspace were well correlated and were also clearly dependent on both 02
and CO2 levels (Fig 2.6). Acetaldehyde and ethanol levels increased as 02 declined,
increasing most rapidly below 1 kPa. CO2 tended to enhance the accumulation of both
fermentation products at 02 levels below 1 kPa. Headspace acetaldehyde levels were
approximately 1/3 those of ethanol (Fig. 2.6). The fermentation threshold, when
assessed as the O2 partial pressure which causes a 20% increase in the RQ relative to
the aerobic RQ, was approximately 1 kPa 02 for all CO2 treatments. Based on the RQ
and non-oxidative C02, however, the lower 02 limit for asparagus could be estimated
to be approximately 1, 1.5, 2, and 3 kPa 02 for 0, 5, 10 and 20 kPa CO2 treatments,
respectively.

The lactate content increased from around 50 nmol'g" on a fresh mass basis at
harvest to 200 nmol‘g‘l fresh mass at O2 partial pressures below 1 kPa (Fig. 2.7). CO2
caused lactate to accumulate at O2 partial pressures greater than 4 kPa. The LDH
activity declined with storage for spears stored in near-ambient atmosphere (Fig. 2.8).
There was an increase in activity as the CO2 partial pressure increased. LDH activity
declined as 02 declined to 2 kPa for the C02-treated spears. As 02 declined below 2
kPa, LDH activity increased to a maximum at 1 kPa O2 and declined again as the O2

61

partial pressure dropped below 0.5 kPa 02.

ADH activity declined with storage for spears in near-ambient atmosphere (Fig.
2.9). As the CO2 partial pressures increased, ADH activity increased. Activity
declined as the O2 partial pressure decline to 4 kPa for the 10 and 20 kPa treatments.
ADH activities were similar for all CO2 treatments between 4 and 1 kPa O2 and
increased as the O2 partial pressure declined below 1 kPa.

PDC activity exhibited much the same pattern as LDH, increasing as CO2
increased for 02 levels greater than 4 kPa and having a peak in the activity between 0.5
and 2 kPa 02 (Fig. 2.10). PDH activity enhancement around that 02 range, however,
was prevalent for 20 kPa CO2 treatment. ADH activity was approximately 3-fold
higher than PDC and 10-fold higher than LDH (Figs.2.8, 2.9, and 2.10). In addition,
in contrast to PDC and LDH, ADH activity increased markedly below 1 kPa 02
without showing subsequent decrease, notedly for 20 kPaCOz.

Sensory quality. From the results of this research at 1 °C, when spears reached
the steady state at 6 (1 storage, the sensorial quality was best as 02 levels increased and
CO2 levels decreased (Fig. 2.11). The lowest level of sensorial quality was detected at
20 kPa C02, which showed marked alteration of visual apparency and strong off-
flavor, probably due to accumulation of fermentative products. The best quality
acceptance was found for 5 kPa CO2 treatment, which crossed the sensorial threshold
(grade 6) at approximately 5 kPa 02. At 0 kPa C02, the visual quality was acceptable

above 6 kPa 02,

62

DISCUSSION

The imposition of O2 limitation, which results in inhibition of oxidative
phosphorylation causes a rapid decline in energy metabolism is asparagus spears, and
may result in death of the tissues if anoxia exists for a prolonged period of time. This
may be occurred in asparagus spear tips for 02 below 1 kPa, when 02 uptake and CO2
production markedly decreased. This energy drop may be correlated with a decline in
respiratory substrates.

Genes upregulated by carbohydrate availability in plants include many that would
enhance sucrose utilization throughout storage, respiration, and biosynthetic processes.
Sugar-regulated genes carries information on carbohydrate status for coordinating changes
in resource utilization and allocation (Koch, 1996), which may be reflected in metabolite
pools and enzyme activities. In tissues, sucrose serves as the source of organic carbon
for building structural elements, is used as a metabolic fuel, or it may accumulate
(Avigad, 1982). In asparagus, sucrose is thought to be translocated from the roots of
growing spears (Hurst et al., 1993b) and from lower segments (Salveit and Kasmire,
1985) throughout the spear toward the tip, where it is subsequently metabolized to
glucose and fructose. In asparagus spear tips, the relatively lower rate of
metabolization of fructose compared to sucrose and glucose may be due to limited
availability of UTP, an essential cofactor for fructose metabolism (Irving and Hurst,
1993). Furthermore, in plants, cleavage of the glycosidic bond in sucrose occurs by
either sucrose synthase (EC 2.4,1,13) or invertase (EC 3.2.1.26). Sucrose synthase
requires UDP as a co—substrate and produce fructose and UDP-glucose, which may go

63

to cell wall biosynthesis (Copeland, 1990) or may be further metabolized in several
ways. In addition to its function as a glucosyl donor in glucosylation reactions, UDP-
glucose may also be oxidized to UDP-Glucuronic acid for the synthesis of several
structural polysaccharides and several amino acids, or may be converted by UDP-
glucose pyrophosphorylase (EC 2.7.7.9) to glucose-l-P and incorporated into the
glycolytic or pentose phosphate pathways (Stewart and Copeland, 1998). In contrast,
invertase simply splits sucrose into glucose and fructose (Capeland, 1990). The higher
sucrose content found in asparagus tips at harvest may favor higher glucose and
fructose contents because sucrose may be less metabolically accessible to respiratory
loss than hexose sugars (Salerno and Pontis, 1978). Here, the drop in sucrose
concentration seemed to parallel a decline in respiration rate observed in asparagus spear
sections (Silva et al., 1998), and in spear tips (Irving and Hurst, 1993). The more marked
decline in the glucose content relative to fructose indicates that it may be preferentially
utilized in glycolysis or lost to cell wall biosynthesis as sucrose is degraded by sucrose
synthase to fructose and UDP-glucose. These results indicate that sucrose supplies not
only demands for synthesis of structural polysaccharides and other synthetic processes
but also the demand of the respiratory pathways. Maintenance of fructose levels may
be explained by the recycling of fructose derived from sucrose.

Although all sugars declined when compared to at harvest, hexoses were still
higher when compared to sucrose. Irving and Hurst (1993) found a similar ratio of
sugars in growing asparagus spears. These improved retention of sucrose by 5 kPa C02
relative to O kPa CO2 may result from a decline in the activity of sucrose-metabolizing

64

enzymes. No decline in respiration was evident for 5 kPa CO2 treated spears, however.
It is possible that the decline in respiration that occur soon after harvest was accelerated
by 5 kPa CO2 on that this CO2 levels caused a shift in the respiratory substrate to
something other than sucrose.

Increases in acetaldehyde and ethanol at less than 1 kPa 02 are consistent with
the observed increase in the RQ. The effect of CO2 on enhanced ethanol and
acetaldehyde accumulation is similar to the effect reported by Thomas (1925) as C02-
zymasis, in which CO2 increased the rate of fermentation. Although some ethanol may
accumulate can occur without decreasing sensorial quality, accumulation of
acetaldehyde and ethanol for prolonged periods may be detrimental.

In response to CO2 exposure, an initial drop in pH may be attributed to lactate
accumulation (Kennedy etal., 1992). The pH—stat hypothesis (Davis et al., 1974;
Roberts et al., 1984) proposes that, in response to low 02, lactate is initially produced
from pyruvate leading to its accumulation and a corresponding fall in pH. The fall in
pH activates PDC and may initiate competition between LDH and PDC for pyruvate
(Davis et al., 1974). The drop in pH and shift in enzyme activity may trigger ethanolic
fermentation (Kennedy et al., 1992). Thus, ethanol biosynthesis could be assigned as a
mechanism to avoid acidosis, rather than to avoid anaerobiosis by itself (Kimmerer and
MacDonald, 1987). Since higher plants absolutely require oxygen to sustain
metabolism, most plant tissues will tolerate anoxia only for a short period of time
before irreversible damage occurs (Kennedy et al., 1992). In avocado, higher C02
levels resulted in reduction in pH (Hess et al., 1993; Ke et al., 1995). Furthermore, an

65

additive effect on pH reduction was noted when high 02 levels were combined with
lower 02 (Hess et al., 1993). At some degree of hypoxia, three factors may slow the
decline in pH: transport of lactate into the vacuole, enhanced activity of PDC and
ADH, or reduced activity of LDH at lower pH values (Kennedy et al. , 1992). In intact
tissues, the production of lactate may precede the production of ethanol. In some cases
(Davis et al., 1974), as reported here, ethanol and lactate accumulated together (Figs.
2.6 and 2.7). In asparagus spears ethanol was the main product of anaerobic
metabolism; however and a smaller amounts of lactate were also detected at harvest and
at near—ambient conditions.

ADH gene expression and enzyme activity are upregulated during hypoxia and
anoxia (Andrews et al., 1993); however, the ADH genes adh-Z has been reported to
increase during ripening (Chen and Chase, 1993). In tomatoes, adh-I is apparently
responsible for most ADH activity under hypoxia, whereas adh-Z is not induced
(Tanksley, 1979). The results of the current work show that, besides being actives
below and above 2 kPa 02, ADH and LDH activities were also present in asparagus
spear tip tissues at harvest. However, 20 kPa CO2 caused an increase in ADH activity
in asparagus tips at 16 kPa Oz. Ethanol production and accumulation was correlated
with ADH activity only when 02 levels were below the RQ breakpoint (approximately
1 kPa 02), however. At higher 02 partial pressures, ADH activity was not correlated
with ethanol present in the tissues. This suggests strict control over carbon flux
through ADH and that more than one gene is expressed at different ranges of O2 partial
pressure. Such hypoxic responses, with emphasis primarily on ethanol production by

66

ADH, have been extensively studied in maize (Andrews et al. , 1993; Robert et al. ,
1984; Saglio et al., 1980), and fruits (Ke etal., 1995; Nanos et al., 1992). However,
hypoxia or anoxia in asparagus has received little attention. ADH activity seems to be
essential for hypoxic survival, since adh null mutants do not survive 24 h of anoxia
(Lemke-Keyes and Sachs, 1989).

In asparagus spear tips, the drop in LDH activity around 4 kPa 02 for all C02
treatments suggests that decrease in the amount of LDH may occurred. This decline in
LDH activity may also suggest a switch in gene expression around 4 kPa 02. The
increase in LDH activity around 2 kPa 02, in contrast to thr Rivoal and Hanson (1994)
report, suggests that LDH in asparagus tips LDH also has a role in a long-term hypoxic
response. The work here identified two features of asparagus LDH: a) extractable
LDH activity changed in response to a wide range 02 partial pressures; b) ethanol
present in the tissues of asparagus spears tips does not block LDH activity, implying
the presence of a positive regulatory system, as also reported by Christopher and Good
(1996). C02 enhanced LDH activity at O2 ranging from 5 to 16 kPa 02, however, 02
rather than CO2 appeared affect most LDH activity below 2 kPa. The LDH activity
behavior followed a similar pattern to that of lactate in response to O2 partial pressures
that ranged from near-ambient to near anoxic. The similarity in patterns suggests little
allosteric regulation and that changes in carbon flux to lactate are largely determined by
enzyme amount. The increase in LDH activity below 2 kPa 02 suggests that, in
asparagus spear tips, there was changes in gene expression under those 02-deficient
conditions.

67

The profile for PDC activity below 2 kPa 02 does not close correlate with
headspace acetaldehyde, since acetaldehyde increased below 1 kPa 02 without showing
subsequent decrease.

From the results of the research here and references cited, a model of action of
low 02 /high CO2 stresses on fermentative metabolism in asparagus is presented (Fig.
2.1). Low Ozlhigh CO2 partial pressures cause a substantial reduction in NADH flux
through the electron transport chain (ETC). As 02 becomes limiting or CO, becomes
toxic, concentrations of NAD and ATP decrease while NADH levels increase. As
cytoplasmic pH decreases, LDH activity is enhanced and a new ADH isozyme is
induced. PDC activity is also enhanced by an increase in pyruvate concentration,
which directs pyruvate to the production of acetaldehyde. Although a decrease in pH
would tend to inhibit ADH, the increase in acetaldehyde and NADH concentrations and
decrease in NAD drive the ADH reaction, resulting in ethanol accumulation, the major
fermentation product in asparagus spears (Fig. 2.6). The increase in pyruvate and
NADH concentrations, and the decreases in NAD and ATP in conjunction with a
limited range of pH drop, activates LDH and direct pyruvate into lactate.

Fermentative metabolism can be regulated by two mechanisms: molecular
control of the levels of PDC, ADH, and LDH, and metabolic control of the actual
functions of these enzymes in plant tissue under low and/or high CO2 stresses (Chang et
al., 1983; Ke et al., 1993). According to Hochachka, et al. (1996), Oz-sensing and
signal transduction control systems may be basic to the hypoxic up-regulation of genes
for glycolytic enzymes, and for hypoxic suppression of Krebs cycle enzymes. In

68

avocado, a decrease in cytoplasmic pH substantially activated PDC but not LDH, and
ADH had more affinity for NADH than that for LDH (Ke et al.,1995). In here pH
was not measured; however, the results suggest that acetaldehyde produced from PDC
reaction was easily converted to ethanol due to the 3—fold higher activity of ADH and
predominant accumulation of ethanol over other fermentation products. Therefore, in
asparagus spears, as the level of 02 was reduced and CO2 increased, the ethanol
pathway was favored relative to the lactate pathway. Similar results have been
obtained for other plant tissues such as lettuce (Siriphanich and Kader, 1986), avocado
(Ke et al., 1995), and pears (Nanos et al., 1992). In addition, the results here indicate
that CO2 may have influence on the activity and amount of LDH, PDC, and ADH, as
indicated by measured extractable enzyme activities as well as by the content of lactate,
acetaldehyde, and ethanol.

Exposure of fruits to very low 02 and high CO2 concentrations at certain levels
may have some effect in maintaining the quality of commodities (Saltveit, 1993). The
shelflife of asparagus spears stored in air for 105 hr (approximately 4.5 d) at 0 °C was
about 5 (I when transferred to 20 °C (Brash et al., 1995). Recommendations for
controlled atmosphere/ modified atmosphere indicate that the optimum range for
asparagus storage is to hold in 21 kPa O2 and 10 to 14 kPa CO2 at the range of 1 to 5
°C (Saltveit, 1993). The results here clearly show that combination of near-ambient
02 level with 5 kPa CO2 slowed down metabolism, as demonstrated by reductions in

sucrose depletion, and also provided a better visual quality.

69

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71

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74

Lactate

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+ NAD
first: :: mi“ NADH
I I - p A A - -
yruvate a a G 5 p
, 1
. 1 / 0 fl
' i TCA ATP ADP NADH NAD
l
I
I
1 ' Cycle Acetaldehyde g» Ethanol
1 : 0 [PDC All—311i}
: 1 NADH NAD i 1+ I i
I I U +| I _[ 1+
l I l l | l
1 ' ETC ' ' 1 '
l l | l | l
1 1 1 1
; ----1 ...... ,__,______,,,, ..... .1 .
I 7 I |
—. ----- ATPI ' i '
1 + : I '
I
',_ _____ J _____ J____ rowoy _____,
HIGHco2

 

 

 

Figure 2.1. Proposed pathway of fermentative metabolism is asparagus spears
regulated by low Ozlhigh CO2 stress. ADH (alcohol dehydrogenase); ETC
(electron transport chain); G-6-P (glucose-6-phosphate); LDH (lactic
dehydrogenase); PDC (pyruvate decarboxylase); TCA (tricarboxylic acid cycle);
---+---> , induction and /or activation; ---_--->, reduction and/or inhibition.
Adapted from Ke et al. (1995).

75

 

 

 

 

 

 

Figure 2.2. Schematic drawing of experimental apparatus allowing adjustment of a
package gas composition to target levels by controlling composition of the
atmosphere in the exterior chamber.

76

Respiration Rate (nmol'kg'l‘hr'l)

 

 

 

 

 

 

 

 

 

 

 

3.0 7
. O Uptake
2.5 -1' 0 C62 Production 20 kPa C02 '- 6
2.0 N ' ' R0 7 5
15 — g o 3 00 99 _ 4 g
. O l—
1.0— 632.. 3 .ae .- 8 _§
0.5 0‘' ' V W W W V — 1
01) T 1 1 1 *T 1 1 0
2.0 6
- — 4
10 I 9 a 9 9,9 ‘9 - 3 o
05 _ 2
' V v V & wv V - 1
0-0 1 1 1 1 1 1 1 1 0
2.0 v 5
15 J SkPa co2 _ 4
i " o m - 3
10 — ' 00 0° 99 G9 0
9 0 o O - 2 m
0.5 V v V w '7 v W W V W I" 1
0-0 T 1 1 1 r 1 1 1 O
2.0 5
Old? (SCI L
1.5 —' a 2 4
1.0 _ CD CD 090090 6&0 % (Q — 3 o
o — 2 :4
0-5 ‘ ‘v' wvvvv my ‘7' V P 1
0-0 1 1 1 1 l 1 1 1 0

 

 

0 2 4 6 8 10 12 14 16
Steady State 02 Partial Pressure (kPa)

18

Figure 2.3. The effects of storage 02 and CO2 on 02 uptake, CO2 production, and
respiratory quotient (RQ) in harvested asparagus spears held at 1 °C for 6 days.

77

Non-oxidative C02 (mmol'kg'l'hr'l)

 

1.25

Non- Oxidative C02

 

20 kPa C02
10 kPa C02
5 kPa C02
0 kPa C02

040.

 

 

 

 

 

 

 

Steady State 02 Partial Pressure (kPa)

Figure 2.4. The effects of storage 02 and CO2 on non-oxidative CO2 in harvested
asparagus spears held at 1 °C for 6 days.

78

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

12— Fructose

101 7/

8 - / 5 ‘00 @qg'v 5% v y

6‘ O .?<> .0

4—1

2.—
A 0 % 1 1 T 1 n 1 1 1 1
"1‘ 12
2': 10 - 7 Glucose <3 2:222:
V 8— a
._ A O ::.::.:::
4" é 0a- a
C2 12

mi // Sucrose

2: Z

4% Z c: v @vg‘b 5%} v g

2— ,0 6

O Q .I I” I<> I. I I I I

O 2 4 6 8 10 12 l4 16 18
Harvest

Steady State 02 Partial Pressure (kPa)

Figure 2.5. The effects of storage 02 and CO2 on the contents of fiuctose,

glucose, and sucrose (fresh mass basis), relative to harvest in the most acropetal 45
mm of the tip of asparagus spears held at 1 °C for 6 days.

79

 

 

 

Acetaldehyde (ppm)

350

 

80 _ O Acethaldehyde

OkPa C02
0 Ethanol

 

 

 

 

1- 300
- 250

 

100

80 SkPa C02
60

4o

20

0 1 Ore 1 Ha—

 

*- 150

 

100 *
60
40 O
20 O

(D
1 1 TT 1 1 1

350
- 300
- 250
- 200
- 150
- 100
- 50

 

350
F 300
+- 250
- 200
— 150
e 100
r- 50

 

100
80 20 kPa C02
60
40 Q
20 .
0 I {.6 1 149 1 9L

0

 

 

O 2 4 6 8 10 12 14 16 1
Steady State 02 Partial Pressure (kPa)

8

Ethanol (ppm)

Figure 2.6. The effects of storage 02 and CO2 on package headspace acetaldehyde

and ethanol for asparagus spears held at 1 °C for 6 days.

80

Lactate (nmol‘g'l)

220

 

 

 

 

 

 

 

 

 

+ 20 kPa C02

200 — —O— 10 kPa C02
130 “ + SkPa coz
160 — + OkPa C02
140 —
120 - "
100 -

80 — 3 c
60 -i

40 —

20 I Lactate

0 I I 1 1 1 1 1 1 1

 

Harvest 0 2 4 6 8 10 12 14 16 18
Steady State 02 Partial Pressure (kPa)

Figure 2.7. The effects of storage 02 and CO2 on the content of lactate (fresh
mass basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus
spears held at 1 °C for 6 days.

81

0.6

 

 

Lactate Dehydrogenase
o 5 + 20 kPa C02
' —o— 10 kPa €02
+ 5 kPa C02
0.4 '— 9 + 0 kPa C02

 

 

 

 

Enzyme Activity (umol'min'l'g'l)

 

 

 

 

 

0.3 - i"
43
1/ - - a
0.2 — '5"
0.1 -
0-0 I ' l I I I l l l 7 1

Harvest 0 2 4 6 8 10 12 14 16 18
Steady State 02 Partial Pressure (kPa)

Figure 2.8. The effects of storage 02 and CO2 on the activity of lactate
dehydrogenase (fresh mass basis), relative to harvest in the most acropetal 45 mm
of the tip of asparagus spears held at 1 °C for 6 days.

82

Enzyme Activity (umol'min'l'g'l)

 

 

 

 

 

9 _ Alcohol Dehydrogenase
s o -------o 20 kPa coz
9 —o—- 10 kPa 002
7 - _._. SkPa coz
6 '3 + 0kPa C02
5- i
. e
4 - ...§
...... ........... ,a-‘O
3 — /@‘“—OO”
-4—_ *4
2 _‘ _fi\.\ \‘
l _
0 I l l I l l l T l I

 

 

 

 

Harvest 0 2 4 6 8 10 12 14 16 18
Steady State 02 Partial Pressure (kPa)

Figure 2.9. The effects of storage 02 and CO2 on the activity of alcohol
dehydrogenase (fresh mass basis), relative to harvest in the most acropetal 45 mm
of the tip of asparagus spears held at 1 °C for 6 days.

83

 

3.0
Pyruvate Decarboxylase

 

 

 

 

g:
u:
I
a

F?“ 25 _ + 20 kPa C02
90 —O— 10 kPa C02
v-I ,0
1: .12 _ + SkPa C02
.2 2-0 “ 1. + 0kPa C02
'5 ~ ‘
E I
3 l
5 15 4
E
H
it) 10 — "' '
Q . u (. ewe, (.9
E .
>3
N
G
II-I

 

 

 

 

 

0.0 //4

I I l I I I I I I I

Harvest O 2 4 6 8 10 12 14 16 18

Steady State 02 Partial Pressure (kPa)

Figure 2.10. The effects of storage 02 and CO2 on the activity of pyruvate
decarboxylase (fresh mass basis), relative to harvest in the most acropetal 45 mm
of the tip of asparagus spears held at 1 °C for 6 days.

84

Visual Appearance (1 - 9)

 

Visual Appearance 0
— 0 GO A o A.
O O
2 G O D O A 0 O ‘0 A
o o
469 ‘ ..... O .......... Ammuo Q ......................... Q ...............................

~00 O AOAOOOO

 

 

 

 

 

 

 

4 ~00!) O O O . 0 kPa C02
0 5 kPa C02
3 7p ©A 0 A 10 kPa C02
0 20 kPa C02
2 ~00 <> ----------- Quality Threshold
1 *KKX’ I I I I I T I I

O 2 4 6 8 10 12 14 16 18
Steady State 02 Partial Pressure (kPa)

Figure 2.11. The effects of storage 02 and CO2 on visual appearance in asparagus
spears held at 1 °C for 6 days. Value 6 represents the visual appearance threshold.

85

CHAPTER 3
REGULATION OF THE STEADY STATE 02 IN ASPARAGUS SPEARS TIPS:
RESPONSES OF GLYCOLYTIC METABOLISM AND SUCROSE-
METABOLIZING ENZYMES UNDER LOW OIIHIGH CO, ATMOSPHERES
INTRODUCTION

Asparagus (Asparagus officinalis L.) spears are rapidly developing shoots when
harvested, with meristematic tips that possesses intense metabolic activity. Harvesting
separates the carbohydrate source from the developing shoots with the results that spear
tips are nearly depleted of sucrose soon after harvest. This sucrose loss is suspected to
limit shelflife and is particularly accentuated in the spear tips (Irving and Hurst, 1993).

Sugars, primarily sucrose, are capable of acting as regulatory signals that affect
gene expression, and thus, metabolism in all organisms (Koch et al., 1996). However, as
the sugar levels do not always correlate with sugar-regulated gene expression, it is
suggested that metabolic flux, rather than accumulated sugar levels, is the important in
triggering sugar-responsive gene expression (Jang and Sheen, 1997). Degradation of
sucrose within plant cell involves sucrose synthase (SS, EC 2.4.1.13) and/or invertases
(IN, EC 3.2.1.26). These enzymes are present in all plant tissues and found at high levels
particularly in sink tissues (Avigad, 1982). The invertase reaction produces glucose and
fructose, which must be phosphorylated before subsequent glycolytic metabolism. In
higher plants, phosphorylation of glucose involves hexokinase (HK, EC 2.7.1.1), in a
nonspecific reaction, or glucokinase (Turner et al., 1977a). Phosphorylation of fructose is

apparently catalyzed by a specific fructokinase (FK, EC 2.7.1.4) (Turner et al., 1977b).

86

Carbohydrates are the principal energy source for most living organisms, and the
main pathway of carbohydrate degradation is through glycolysis (Plaxton, 1996) (Fig.
1.2., p.20). The glycolytic pathway serves in three fundamental roles: to oxidize hexoses
to generate carbon skeletons for building new molecules, to produce energy for cellular
maintenance and growth, and to generate hexoses in gluconeogenesis (Plaxton, 1996).
Glycolysis shares some steps with oxidative pentose phosphate (OPP) pathway. Glucose-
6-phosphate from glycolysis can be diverted to OPP to produce gluconate-6-phosphate
and NADPH, step catalyzed by glucose-6-phosphate dehydrogenase (G6PDH, EC
1.1.1.49). The primary role of OPP is the production of NADPH to be used in
biosynthetic reactions (Ap Rees, 1980).

The first rate-limiting step in glycolysis is the interconversion of fructose-6-P
(F 6P) and fructose-1,6-P2 (F1,6P2), a reaction catalized by these enzymes.
ATPzphosphofructokinase (ATP:PFK, EC 2.7.1.11) operates in the glycolytic direction
(Plaxton, 1996), PFK, PPizphosphofi’uctokinase (PPi:PFP, EC 2.7.1.90) catalyzes the
reversible conversion of F6P to F 1,6P2 (Smith et al., 1984). Another rate- limiting step in
glycolysis is the conversion of phosphoenolpyruvate (PEP) to pyruvate (PYR) by pyruvate
kinase (PK, EC 2.7.1.40) (Plaxton, 1996). Glycolytic flux may be regulated at either or
both of these two steps.

Exposure of harvested horticultural crops to low OZ/high CO2 environments can
reduce deterioration and influence quality and storage life by reducing metabolic activity
(Kader, 1986). However, much has yet to be understood regarding on the biochemical

effects of the application of combined levels of these two gases. A reduced metabolic rate

87

may lead to reduced sugar depletion in asparagus during storage. The evaluation of the
changes in the metabolite pools and activities of sucrose-metabolizing and glycolytic
enzymes in crops exposed to low OZ/high CO2 levels may provide information of the
control of glycolytic carbon flux. Thus, it must help to elucidate the processes through
which sugars are utilized and the mode of action of low Oz/high CO2 in a commodity.

Our objective was to investigate how glycolytic intermediate pools and activity of
the sucrose-metabolizing and glycolytic enzymes in these tissues were influenced by low
Ozlhigh CO2 partial pressures. These data were to be used to evaluate the mechanisms
governing carbon flux though glycolysis and to identify reactions which may exert
significant control over sugar content in asparagus spear tips stored under the above
conditions at l 0C.

MATERIAL AND METHODS

Plant material. Asparagus spears (Asparagus officinalis L. cv. Jersey Giant) were
harvested early in May from the Michigan State University Horticultural Research Center
between 6:00 and 9:00 am. and stored at 1 °C. Samples were taken for analyses at
harvest and after storage. Spears selected to represent harvest conditions were frozen in
liquid N2 in the field. Spears were harvested when they reached a minimum length of 240
mm and were trimmed to 180 mm. Only straight undamaged spears with closed bracts
and no obvious symptoms of disease were used. Spears to be placed on storage were
chilled with ice while still in the field and transported to the laboratory within the hour.
After storage, spears were frozen in liquid N. After freezing, all spears were stored at -80

°C until analysis. Three replicate experiments were conducted in consecutive asparagus

88

seasons ( 1995, 1996, 1997). Samples comprised the most apical 45 mm of the spear.

Atmosphere generation. 02 partial pressures ranging from a low of a 0.18 kPa to
a high of 16 kPa were generated in packages for four CO2 treatment levels (0, 5, 10, and
20 kPa). 02 levels were obtained by sealing spears in packages composed of low density
polyethylene (LDPE) of varying film thickness and placing the packages in air (0 kPa C02)
or enclosing the packages in glass chambers with 20.7 kPa O2 and 5, 10, and 20 kPa CO2
(Beaudry, 1993). Chamber lids were fitted with inlet and outlet ports for purging with gas
mixtures; a glass tube (1.3 cm id. x 2.7 cm long) was inserted through the lid and glued to
a piece of electrical tape on the surface of the enclosed package with silicone sealant
(Beaudry, 1993). The silicone sealant formed a septum through which gas samples could
be obtained from within the package. Chambers were continually purged with gas mixture
of 02, C02, and N2 gases at 30 mL'min" to generate the target atmospheres within the
enclosed packages. Actual CO2 levels in the packages were slightly higher than reported
values, but for clarity of presentation, are reported as treatment values. The gas
composition of individual packages was monitored until steady state conditions were
reached. Steady state conditions were achieved on day six of the study. 02 and CO2
gradients across the film were used to calculate the flux for both gases through the film
using previously obtained permeability data (Lakakul, 1994). Flux data were used to
calculate the rates of 02 uptake and CO2 production (Cameron et al., 1994).

Glycolytic intermediate extraction. Approximately 5 g of frozen tissue per sample
were transferred to liquid N2 in a precooled mortar and ground with a pestle to a fine

powder. Ten mL of ice-cold 0.8 N (v/v) HCIO4 were added to the powder, which was

89

extracted for 30 min at 0 °C with occasional shaking, followed by centrifugation at 30,000
g for 30 min at 0 °C. Supernatant pH was adjusted to pH 7.0 to 7.2 with 5 N K2C03,
allowed to sit on ice for 10 min, and clarified by centrifugation at 30,000 g for 10 min.

The resulting supernatant was used for analysis of intermediates. To estimate losses of
metabolites during extraction, measured amounts of pure glycolytic intermediates roughly
equal to the amounts present in the tissue, were added to the extracting medium.
Recoveries were 96%, 95%, 90%, 89%, 87%, 89%, 90%, 96%, 92% for G6P, F6P,
F1,6P, DHAP, 3PGA, 1,3PGA, 2PGA, PEP, and PYR, respectively. The recovery assays
were replicated in six times.

Glycolytic intermediates assay. The glycolytic intermediates were assayed
spectrophotometrically (Hitachi, Model U3000, Hitachi Inc., Tokio, Japan) at 25 °C using
a temperature controlled circulator (Polystat, Cole-Palmer Instrument Co., Chicago, IL).
The oxidation/reduction of pyridine nucleotides was assayed by measuring changes in
absorbance of the assay cocktails at 340nm according to procedures adpted from Kato-
Noguchi and Watada (1986), Kerbel et al. (1988), and Bergmeyer (1983). Measurements
were taken for 15 to 20 min in lmL of triethanolamine buffer (TEA, pH 7.4) and other
additions specified for each intermediate. Depending on the intermediate, 50 to 100 uL
enzyme extract was used. Assay cocktails for the intermediate were as follows: Glucose-
6-Phosphate (G6P): 250 mM TEA, 0.25 mM of NADP, 4 mM MgC12, 0.5 units glucose-
6-P dehydrogenase; Fructose-6-phosphate(F6P): the above mixture for G6P plus 1 unit of
phosphoglucose isomerase; Glyceraldehyde-3-Phosphate (GAP): 250 mM TEA, 20 mM

EDTA, 0.13 mM NADH, 0.5 units glycerol-3-phosphate dehydrogenase (GDH);

90

Dihydroxyacetone phosphate (DHAP): the previous mixture for GAP plus 1.5 units of
triosephosphate isomerase (TIM); Fructose-1,6-bisphosphate (F16P2): the previous
mixture for DHAP plus 1 unit of aldolase; Gcherate-I,3-bisphosphate (1,3PGA): 200
mM TEA, lmM EDTA, 0.13 mM NADH, and 10 units glyceraldehyde-3-phosphate
dehydrogenase (GAPDH); Glycerate-3-phosphate (3 PGA): 200 mM TEA, 2 mM EDTA,
0.13mM NADH, 8 mM Na2 $04, 8 mM ATP, 4 units of GAPDH, 1 unit GDH, 25 units of
TIM, 16 units phosphoglycerate kinase; Pyruvate (PYR): 200 mM TEA, lmM EDTA,
0.13 mM NADH, 12 mM Mg S0,, 45 mM KCl, 2 mM ADP, and 10 units lactate
dehydrogenase; Phosphoenolpyruvate (PEP): the previous mixture for PYR plus 5 units
of pyruvate kinase; 2-Phosphog1yceric acid (2PGA): the previous mixture for PEP plus 4
units of enolase.

Sucrose synthase and invertase extraction solutions. The solutions used for
sucrose synthase (SS, EC 2.4.1.13) and invertase (EC 3.2.1.26) extractions were
modifications from Irving and Hurst (1993). Solution A contained 40 mM Hepes-NaOH
(pH 7.6), 2 mM NazEDTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluoride
(PMSF), and 10% of glycerol. Solution B was buffer A minus PMSF and Solution C was
Solution B containing 1 M NaCl.

Sucrose synthase and soluble invertase extractions. Approximately 5 g of frozen
tissue, to which 50 mg/g tissue of insoluble (40,000 mw) PVPP was added, was ground as
described previously and mixed with 10 mL of ice-cold Solution A. The mixture was
centrifuged at 30,000 g for 30 mim. The supernatant was collected and 1.3-mL samples

were passed through a column of of Sephadex G-25 (Pharmacia Uppsala, Sweden)

91

medium-sized beads (9 mL bed volume), previously swollen overnight in 40 mM Hepes-
NaOH (pH 7.6) and equilibrated in Solution B (Helmerhorst and Stokes, 1980; Neal and
Florini, 1973) for removing salt and sugars. The eluate was assayed for sucrose synthase
and acid and neutral soluble invertase activities. Substrates and coupling enzymes were
purchased from a commercial source (Sigma Chemical Co., St. Louis, MO).

Bound acid invertase extraction. Two-hundred milligrams (fresh mass basis) of
the cell debris collected from the bottom of the centrifuge tube described previously were
transferred to 2 mL microcentrifuge tubes and resuspended (30 sec vortex) in 1 mL of
Solution A. After microcentrifiiging at 3,000 g for 30 sec, the pellet was washed three
times with 1 mL of Solution B. The pellet was resuspended (l min vortex) in lmL of
Solution C and kept overnight at 4 °C to release bound protein. The supernatant was
assayed for acid invertase activity.

Sucrose synthase and invertase assays. Assays for enzyme activities were assayed
in a final volume of 1 mL based on procedures adapted from Huber and Akazawa (1986).
Blanks were prepared for each assay. The change in absorbance was monitored
spectrophotometrically (Hitachi, Model U3000, Hitachi Inc., Tokio, Japan, double-bean)
at 30 °C by following the oxidation/reduction of pyridine nucleotides at 340nm.
Continuously monitored reactions were linear for at least 5 min under the assay
conditions. Activities of all enzymes were expressed as umol'min"'g" of tissue on fresh
mass basis.

The sucrose synthase cocktail included: 50 mM Hepes-KOH (pH 7.4), 4 mM

MgC12, 1 mM EDTA, 25 mM sucrose, 0.13 mM of NAD, 3 mM UDP (omitted from the

92

blank), lmM ATP, 15 mM KCl, 25 units phosphoglucose isomerase, 10 units glucose-6—
phosphate dehydrogenase, and 5 units hexokinase. The reaction was started by addition of
75 uL extract.

Acid invertase: 50 mM sodium acetate (pH 5.0), 2 mM MgCl2, O. 13 mM NAD, 20
units phosphoglucose isomerase, 5 units glucose-6-P dehydrogenase, 5 units hexokinase, 1
mM Mg.ATP, 100 uL of extract, and 33 mM sucrose (started the reaction). The reaction
mixture was incubated for 30 min at 30 °C. The reaction, which was linear with respect to
time and volume of extract, was stopped by boiling for 5 min. Blanks contained all the
above and boiled enzyme.

Neutral invertase (NI, EC 3.2.1.26): The reaction was the same as for acid
invertase except that the buffer was 50 mM Hepes-NaOH (pH 7.0).

Glycolytic enzymes extraction. Approximately 5 g of frozen tissue per replicate
was ground as described above and mixed with a 10 mL ice-cold solution containing 60
mM Tris-Hcl (pH 7.6), 5 mM NazEDTA, 10 mM MgC12, 5 mM of dithiothreitol, 0.1 mM
phenylmethylsulfonylfluon'de (PMSF), 10 % (v/v) glycerol, and 50 mg/g tissue of
insoluble (40,000 mw) PVPP. The mixture was centrifiiged at 30,000x g and part of the
supernatant used immediately for the measurement of the activities of glycolytic enzymes.

Glycolytic enzyme assays. The change in absorbance was monitored
spectrophotometrically 30 °C by following the oxidation/reduction of pyridine nucleotides
at 340nm. The reaction mixture (lmL) for all assays contained 60 mM Hepes-NaOH (pH
7.4), 2 mM MgC12, 1 mm EDTA, 15 mM KCl, and other additions specified for each

enzyme. Depending on the enzyme, 50 to 75 uL of extract per reaction was used. The

93

enzyme assay cocktails are as follows: hexokinase (HK, EC 2.7.1.1): 0.13 mM NAD, 4
mM ATP, 15 units glucose-6-phosphate dehydrogenase, and 15 mM glucose (omitted
from the blank, otherwise used to start the reaction). F ructokinase (F K, EC2.7. 1 .4): 0.13
mM NAD, 7 mM UTP, 10 units glucose-6-phosphate dehydrogenase, 15 units
phosphoglucose isomerase, and 15 mM fructose (omitted from the blank, otherwise used
to start the reaction). ATP-dependent phosphofi'uctokinase: 2 mM MgC12, 0.2 mM
NADH, lmM ATP, 5 units aldolase, 6 units glycerol-3-phosphate dehydrogenase, 20 units
triosephosphate isomerase, and 3 mM fructose-6-phosphate (omitted from the blank,
otherwise used to start the reaction). PPi-dependent phosphofructokinase: 2 mM MgC12,
0.2 mM NADH, 1 mM inorganic pyrophosphate (PPi used to start the reaction), 5 units
aldolase, 6 units glycerol-3 -phosphate dehydrogenase, 20 units triosephosphate isomerase,
3 mM fructose-6-phosphate, and 20 uM fructose-2,6-bisphosphate (omitted from the
blank). Pyruvate kinase: 0.2 NADH, 1.5 mM ADP (omitted from the blank), 15 mM KC],
10 units lactate dehydrogenase, and 1,5 mM phosphoenolpyruvate (to start the reaction).
Glucose-6-phosphate dehydrogenase: 0.25 mM NADP and 2 mM glucose-6-phosphate
(omitted from the blank). The reaction was started by addition of extract.

The recovery of enzyme activity through the quantification process was above
91% and below 99%, as determined by 3 to 5 repeated assays with pure enzymes added to
the extraction medium.

Crossover plot. A crossover plot (Chance et al., 1958; Kerbel et al 1988) was
used to evaluate stimulation or inhibition of the irreversible reactions regulating the rate of

respiration. Crossover usually ranges from -1 to +1. Data on the 0 line indicates that

94

there is no difference between treatments. In this research, the broad range of O2 partial
pressures combined with four CO2 levels allowed, by a crossover evaluation of the
intermediate pools, separations of the effects of O2 and CO2 to detect the control points of
glycolysis in asparagus spears tips. Two crossover plot were constructed: 1. comparing
the highest 02 level (16 kPa 02), with 02 levels below that for each of the four CO2

treatments (Fig. 3.7); and 2. comparing 0 kPa CO2 with higher CO2 treatments (Fig. 3.8).

RESULTS

Sucrose-metabolizing enzymes. SS and BAI activities were higher than those of
AI and NI at harvest (Fig. 3.1). From 2 to 16 kPa 02, at 20 kPa C02, however, BAI
activity was maintained higher than SS. After 6 d at 1 °C, the spears stored at near
ambient atmosphere (16 kPa Oz, 0 kPa C02) experienced an increase in the invertase, but
no change in SS activity. However the activities AI, BAI, NT, and SS were markedly
reduced at the lowest 02 levels, for all CO2 treatments (Fig. 3.1). In addition, the activities
of these sucrose-metabolizing enzymes were altered by C02 treatments. AI, BAI, and SS
activities were enhanced by 20 kPa CO2 relative to 0 kPa Oz. The activities of these
enzymes was reduced by 5 kPa C02, which is consistent with higher sucrose content
found under similar conditions (Silva et al., 1998a). Interestingly, N1 was not affected in
the same manner by C02; its activity was reduced by 5 and 20 kPa CO2 and enhanced by
10 kPa C02.

Intermediates. Of the hexose-phosphates, G6P was present in the highest level at

harvest and accumulated to the greatest extent (z 3-fold) for spears stored at near ambient

95

atmosphere (0 kPa C02, 16 kPa 02) (Fig. 3.2). 02 partial pressure below 1 kPa caused a
decline in the contents of G6P, F6P, and F16P2, for all CO2 treatments. G6P and F6P
contents decreased as CO2 levels increased.

F16P2 was not affected by storage at 1 °C. 02 partial pressures below 1 kPa
caused a reduction in F16P2 for all CO2 treatments. F16P2 content was enhanced by 10
and 5 kPa CO2 but reduced by 20 kPa C02. The ratio G6P/F 6P was approximately 4 at
harvest, which is near equilibrium (Fig. 3.3). This ratio declined with storage and with
increasing CO2 partial pressures. At 02 levels below 1 kPa, however, G6P/F 6P ratio
tended to increase as CO2 increased.

The Fl6P2/F 6P ratio declined relative to harvest (Fig. 3.3). This ratio increased
slightly as CO2 partial pressures increased. At 02 levels below 1 kPa, the F16P2/F 6P ratio
increased, declining again below 0.5 kPa Oz.

The GAP and DHAP contents after storage were similar to those at harvest (Fig.
3.4). The content of these triose-P intermediates declined as the O2 partial pressure
dropped below 4 kPa. The amount of GAP and DHAP was not affected by C02.

The contents of 1,3-PGA, 3-PGA, and 2-PGA tended to increase with storage at
near ambient O2 and CO2 levels. Their content tended to decline as CO2 partial pressure
increased. At 02 partial pressures greater than 1 kPa, the content of these intermediates
was largely unaffected by 02, with the possible exception of 3-PGA for spears treated with
20 kPa C02. Below 1 kPa 02, the contents of 1,3-PGA, 3-PGA, and 2-PGA were
dramatically (Fig. 3.5).

The contents of PYR was approximately 1/3 that of PEP at harvest (Fig. 3.6).

96

Following storage, this ratio declined to 1 for spears stored at near ambient atmospheres.
The content of both compounds declined approximately 50% as CO2 increased to 20 kPa.
The effect of 02 on both intermediates was complex. PEP tended to be enhanced by 02
levels between 0.5 and 2 kPa. The PYR/PEP tended to decline between 2 and 4 kPa 02
relative to higher 02 levels; this ratio tended to increase between 1 and 2 kPa 02.

A crossover plot shows that, for all of CO2 treatments, the interconversion of PEP
and PYR was the only step in glycolysis to result in a crossover event (Fig. 3.6). A
crossover took place only at 02 levels below 1.5 kPa. As the 02 level declined, the levels
of intermediates declined and the effect tended to be greater for those intermediates
‘earlier’ in the glycolytic pathway. When comparing higher CO2 (20, 10, and 5 kPa) to the
lowest (0 kPa) CO2 level no clear pattern was observed (Fig. 3.8 AB, and C). The most
obvious crossover event was detected at the conversion of F6P and Fl,6P2, at higher 02
levels for 5 and 10 kPa C02. Fl ,6P2 tended to accumulate relative to F6P under these
atmospheric conditions.

Glycolytic enzymes. HK activity increased 4-fold with storage under near-ambient
conditions (Fig. 3.9). Relative to harvest, the activity was markedly higher for 0 kPa CO2
followed in order by 10, 5, and 20 kPa C02. Activity declined as 02 below 4 kPa.

Compared to HK, FK activity was about 10 times higher at harvest (Fig. 3.9).
However, the activity of FK declined with storage. The effects of CO2 and 02 were nearly
identical for PK and HK, with activity of FK declining in the same order in response to
CO2 and declining as 02 dropped below 4 kPa.

G6PDH activity increased slightly relative to harvest for spears stored under near

97

ambient conditions (Fig. 3.9). Activity declined as CO2 increased in the 02 range of 4 to
16 kPa. The effect of 02 was complex. As 02 declined below 8 kPa to 2 kPa Oz, G6PDH
activity declined. Below 2 kPa 02, there was an increase in activity, which peaked from
0.5 to 1 kPa O2 and declined as 02 declined further.

ATP:PFK activity declined by approximately 1/3 compared to harvest for spears
stored under near-ambient atmospheric conditions (Fig. 3.10). ATP:PFK activity declined
as CO2 partial pressures increased. ATP:PFK activity also declined as 02 partial pressure
decreased. The 02-dependet decline was greatest below 2 kPa 02 for all CO2 treatments
except the 20 kPa treatment, which exhibited marked inhibition below 6 kPa 02.

At harvest, the activity of PPi-PFK was approximately 4-fold higher than ATP-
PFK (Fig. 3.10). Storage did not appear to affect activity for spears held in near-ambient
conditions. The higher activity for PPi:PFK over ATP:PFK was maintained accros the 02
range in spear tips for all CO2 treatments. After storage, the highest PPi:PFK was for the
10 kPa C02, declining, in order, for O, 5, and 20 kPa C02. The pattern relative to 02
levels was nearly identical to that for G6PDH activity.

PK activity did not change in response to storage when spears were held in near-
ambient atmospheres. Activity declined as CO2 partial pressures increased (Fig. 3.10).
There was a marked inhibition by even 5 kPa CO2 for spears held in 4 to 14 kPa 02. For
each of the 3 CO2 treatments, PK activity declined as 02 declined in the range of 16 to 2
kPa 02. At 20 kPa CO2 PK activity was reduced by 1/3. For 0 kPa CO2 treatment, PK
activity declined only below 6 kPa Oz. PK activity increased at 02 levels below 2 kPa,

similarly to G6PDH and PPi:PFK. However, for PK this increase seemed to be most

98

pronounced for the 20 kPa CO2 treatment and the increase relative to activity at 2 kPa 02
was higher when compared to the other two enzymes.
DISCUSSION

Sugar-metabolizing enzymes. In asparagus spears tips, as CO2 levels increased
and 02 levels decreased the content of sucrose decreased (Silva et al., 1998a). This sugar
was most markedly affected during storage. Sucrose is metabolized by sucrose synthase,
acid and/or neutral invertases. The genes for these enzymes are modulated by sugars
(Koch et al., 1996), and their isozyme forms have contrasting carbohydrate responses
(Geiger et al., 1996). In asparagus spear tips, the activity of these enzymes seemed to be
affected differently by the availability of sugars and by 02 and CO2 levels. In maize,
expression of invertase and sucrose synthase genes were not only modulated by the
availability of carbohydrates, but also by different genes. For example, expression of the
[WI invertase and Sh! sucrose synthase were starvation-tolerant and could be repressed
by sugar. In contrast, Irv2 ans Susl expression was enhanced by increasing sugar
availability and markedly sensitive to carbohydrate depletion (Geiger et al., 1996).

The results indicate that in asparagus spear tips, hydrolysis of sucrose to feed
glycolytic carbon flux probably occurs mostly in the cytosol (via SS and N1), and in the
vacuole and apoplasts (via BAI). In maize seedlings, sucrose synthase is the major
sucrose-metabolizing enzyme, which seems to be induced at transcriptional level
(Guglielminetti, et al., 1997). In maize leaf, phosphorylation of serine-lS of SS in vivo
selectively activates the cleavage reaction by increasing the apparent affinity of the enzyme

for sucrose and UDP, suggesting that phoshorylation may have regulatory significance

99

(Huber et al., 1996). The inhibition in activity of NI by higher CO2 levels, in contrasting
with enhanced activity detected for SS and other invertases, suggests an inhibitory CO2
effect or a reduction of the amount of the enzyme. In vivo, changes in the environment
can change the pH in the cytoplasm. It is known that pH regulates the activity of key
enzymes and metabolic steps. Anaerobiosis induces cytoplasmic pH decrease (Roberts et
al., 1984). In addition, CO2 at concentrations around 1% and above is known to acidify
the cytoplasm (Martin et al., 1993).

In sycamore cells, SS synthase has much more affinity for sucrose as compared
with NI. Consequently, the SS pathway may be more important when sucrose availability
is limiting, as probably in case of asparagus. Sucrose synthase pathway is also more
energetically efficient, since it requires the input of three ATP to metabolize one molecule
of sucrose to triose—P, compared to four in the invertase pathway (Huber and Akazawa,
1986). Nevertheless, BAI activity was the highest for 0 kPa OZ, and was enhanced
relative to harvest, SS was the enzyme with the highest activity at harvest, which is
consistent with the metabolic activity in growing asparagus. The data from this
experiment strongly suggest that BAI, rather than other invertases, plays the major role in
metabolizing the sucrose delivered to the spear tips under storage conditions. The
increased BAI activity with storage indicates that a significant part of the sucrose
metabolism in asparagus occurs via the apoplast. In tomato fruit, the hydrolysis of sucrose
by acid invertase is proposed to influence the rate and extent of sugar storage in fruit by
creating a sucrose concentration gradient between the site of phloem unloading and

storage cells (Walker et al., 1978). Since sucrose is the one of the major form of

100

translocated carbon in plants (Ap Rees, 1980), and the compound that initiates sucrolysis
and glycolysis in plant cells (Avigard, 1980; Sung et al., .1989), one biochemical
determinant of sink strength is the ability of the sink to metabolize sucrose.

Asparagus spear tips are known to be a very active metabolic sink (Irving and
Hurst, 1993). When the spear is harvested, sucrose translocation from the crown is
suppressed, having the spear to relay on its stored carbohydrate supply to support the tip’s
high metabolic and biosynthetic demands. According to Sung et al. (1989), tissues where
biosynthesis is predominant, the SS activity tends to be greater than invertase activity. In
rapidly growing tissues in which the sucrose content was low or rapidly declining, such as
in carrots roots, AI activity was higher when compared to N1 (Ricardo and Ap Rees,
1970). In case of asparagus tips, the higher activity at harvest detected for BAI and SS
relative to AI and NT may be explained as a means to support spear’s high metabolic rate
and biosynthetic activity.

Intermediates. The sharply reduced levels of G6P, F6P, and F16P2 below 1 kPa
02 observed in asparagus may indicate an enhancement of glycolytic carbon flux, as also
suggested by the increased G6P/F6P and decreased F16P2/F6P ratios relative to harvest at
that 02 range. Typically the ratio of G6P/F6P is 4:1 in the healthy tissue and is near
equilibrium for phosphoglucose isomerase (PGI, EC 5.3.1.9), the enzyme catalyzing this
reaction (Turner and Turner, 1980). The reduction in G6P/F6P ratio relative to harvest in
asparagus spears tips may, therefore, indicate that the enzymatic status of P61 was
altered. In addition, 02 levels below 1 kPa caused accumulation of fermentation products

ethanol and lactate (Silva et al., 1998a). Anoxia stimulates glycolysis, at least in part, via

10]

the reaction that converts F6P to Fl,6P2 (Ap Rees et al., 1985). The increase in the
F2,6P2/F 6P ratio at O2 partial pressures below 1 kPa supports a role for regulation of
fermentative carbon flux at this step.

The unchanged amounts of GAP relative to DHAP throughout the all 02 range and
CO2 treatments may indicate that the equilibrium for triose phosphate isomerase (TPI, EC
5.3.1.1) was not altered by 02 and/or CO2 treatments.

The higher 3-PGA content at the hypoxic 02 range may suggest an inhibition of
the cytosolic FBPase (Stitt, 1990), which, combined with the fact that the highest activity
of PPi:PFK in the tip is relatively higher than ATP:PFK, suggests carbon flux at that point
was directed toward glycolysis. Barker et al. (1966) discussing the Pasteur effect,
generated by anoxia in pea seedlings, attributed a decrease in the levels of 3-PGA and PEP
to higher rates of glycolysis. They suggested that those changes reflected an acceleration
of the glycolytic flux resulting in formation of fermentation products as a short term
alternative for the cell resupply/keep its energy. As a result of anoxia, the contents of,
G6P, F6P, and pyruvate increase (Scott et al., 1995). In this experiment, an increase in
PEP at range of 2 to 4 kPa 02 was detected, which was followed by a marked decrease at
02 below 1 kPa (Fig. 3.6) for all CO2 levels. The pattern for PYR was converse to that
for PEP, showing an increase in PYR contents when contents of PEP were lower (below 1
kPa 02). This data is also reflected in the crossover plots and suggests that glycolysis
was regulated at that step for asparagus spear tips experiencing limiting levels of 02.

Glycolytic enzymes. HK are enzymes recognized to be involved in short-term

acute regulation of glycolysis (Meyrick, 1990), possibly acting as the sugar sensor in

102

higher plants (Jang et al., 1997), by transducing the signal that connects glycolysis with
photosynthesis (Jang and Sheen, 1997). HK are also the enzymes that metabolize most of
the glucose and fructose produced by the hydrolysis of sucrose, which is sent forward
throughout glycolysis. The level of 20 kPa CO2 caused a reduction in HK activity. The
reduced activity of HK is consistent with the lower contents of G6P and F 6P, and by the
reduced sucrose content found in the same CO2 level. Bouny and Saglio (1996) found
that under anaerobiosis, phosphorylation of hexoses by HK was the major limiting step in
glycolysis when the pH of the cytoplasm dropped to around 6.5. The decreased

glycolytic flux was attributed not to an increase in proteolytic activity but to the reduction
in the levels of ATP and pH. Low pH acts in the cell as a fine metabolic control, which
modulates the activity of enzymes, and therefore affects the flux of metabolites through
the pathway (Plaxton, 1996). As the pH was not measured in this experiment, and
considering the saturated conditions in which the enzyme assays were conducted, these
results also can be attributed to a change in the amount of this enzyme. Furthermore,
similarly to Bouny and Saglio (1996), in asparagus spear tips ATP declined at O2 partial
pressure below 1 kPa (Silva et al., 1998b), which supports the reduced hexokinase activity
observed at similar 02 levels.

FK phosphorylates fructose specifically (Turner et al., 1977b) as the initial step of
glycolysis, following the split of sucrose into glucose and fructose. In immature tomato
fi'uit (Schaffer and Petreteikov, 1997), FK is reported to be the one of the key enzymes for
sucrose metabolism, in an apparently coordinated manner with SS. In asparagus tips,

much of thee incoming carbon is metabolized to cell walls. The UDP-glucose formed by

103

SS is rapidly consumed for cell wall formation (Huber and Akazawa, 1986). The higher
FK activity found at harvest, may indicate that was a need to metabolize fiuctose at that
time, and sucrose was metabolized mostly via SS. However, conversely to HK, FK
activity was reduced more than half at the steady state 02.

G6PDH is the enzyme that catalyzes the first reaction from the pentose phosphate
pathway, which primary fiinction is thought to be supplying NADPH to the cytoplasm for
use in biosynthetic processes (Turner and Turner, 1975). In this way, elevated activity of
this enzyme is associated with tissues that possess high biosynthetic activity (Plaxton,
1996). Changing the levels of CO2 and 02 may result in changes in the amount of
enzymes and/or expression of different genes for the regulation of biosynthetic processes.
The data from this experiment shows that most of the changes in metabolite pools and
enzyme activities was detected below 2 kPa 02. This may indicate that changes in the
expression of different genes for de novo synthesis of several isozymes may be occurring
around this 02 level. This implies a large requirement for reducing power to supply
demanding biosynthetic processes for growth and protein synthesis, which may explain the
high G6PDH activity in asparagus tips and the sudden increase in activity below 2 kPa 02.

Low 02 partial pressure (below 2 kPa 02) caused a reduction in the content of
both F6P and Fl,6P2, relative to the harvest, which appeared to be independent of CO2
(Fig. 3.2). Low 02 caused elevated levels of Fl,6P2 relative to F6P, which is interpreted
to indicate a promotion of glycolysis by one or more of the two enzymes catalyzing this
interconversion: ATP-PFK and PPi-PFK. However, in asparagus spears tips, only

PPi:PFK underwent an increase in activity below 2 kPa 02. High CO2 concentrations

104

affected levels of intermediate that have regulatory roles. Plant ATP-PFK, for instance,
can be inhibited by ATP and PEP (Turner and Turner, 1980). In pea seed, for example,
ATP:PFK is inhibited by PEP (Kelly and Turner, 1970). This inhibition is relieved by F6P,
MgCl2 , and Pi. However, ATP:PFK is not regulated by the simple adenylate energy
charge, but, although it requires ATP as substrate, it is inhibited by high concentrations of
ATP (Dennis and Greyson, 1987). The principal regulation of plant ATP:PFK appears to
be by the PEP/Pi ratio. However, metabolites closely related to PEP, 3-phosphoglycerate
and 2-phosphoglycerate also inhibit the enzyme (Dennis and Greyson, 1987). In carrots,
ATP:PFK enhances the flux of metabolites through the glycolytic pathway, which is
associated with an increase in respiration (Adams and Rowan, 1970). Alternatively,
regulation of glycolysis by ATP-PFK, which functions strictly in the glycolytic direction,
depends to a great extent on the pH (Turner and Turner, 1980). High CO2 results in a
slight reduction in pH (Hess et al., 1993) and decline of the activity of both ATP- and
PPi-PFK (Kerbel et al., 1988), as also demonstrated in this experiment by the crossover
plot (Figs. 3.7 and 3.8). In this work, pH was not measured, however, a sudden increase
in PEP at 2 to 4 kPa 02 (Fig. 3.6) coincided with a concomitant reduction in the ATP:PFK
activity. From 2 to 16 kPa Oz, ATP:PFK activity was higher as CO2 levels decreased,
suggesting that CO2 caused an inhibition on its activity. In asparagus tips, ATP was found
to decrease as 02 decreased (Silva et al., 1998b), that is consistent with similar decrease in
ATP:PFK activity. It can be that, as 02 decreased, the decline in ATP resulted in the
displacement of the ATP:PFK reaction from equilibrium and accumulation of F2,6P2

relative to F6P, slowing glycolytic flux in asparagus spear tips.

105

PPi:PFK is a near-equilibrium reaction and could in principle, catalyze a net flux of
carbon in the direction of glycolysis or gluconeogenesis, and is involved in the regulation
of cytosolic PPi (Stitt, 1990). However, it may catalyzes a reaction that is directed toward
glycolysis primarily in tissues dominated by biosynthesis and the use of respiratory
intermediates for the synthesis of cellular material (Ap Rees et al., 1985) as occur is
asparagus spear tips. ATP:PFK is involved in glycolysis when metabolites for
mitochondrial energy metabolism are required, while PPi:PFK activity exceeds ATP:PFK
activity in tissues engaged in biosynthetic activity (Dennis and Greyson, 1987). The high
metabolic rate reported for asparagus explains the higher PPi:PFK activity relative to
ATP:PFK found in spear tips at harvest. The enhancement of PPi:PFK activity below 2
kPa 02 was probably due to an enhancement of the glycolytic flux, and a possible increase
in protein synthesis. Below 2 kPa Oz, PEP content decreased while an increase in PYR
content relative to harvest was detected for all CO2 treatments (Fig. 3.6). This decrease
in PEP might, therefore, be also resulted in increased glycolytic carbon flux through a
stimulation of the F6P/F1,6P2 interconversion step via regulation of ATP-PFK, which is
strongly inhibited by micromolar quantities of PEP (Turner and Turner, 1980).
Furthermore, assuming the rate of photosynthesis and sucrose synthesis were minimal
under the conditions this experiment was performed, the increase in F6P (Fig. 3.2) and PPi
and lower Pi, in range of O2 partial pressure greater than 2 kPa (Silva et al., 1998b) would
favor reaction of PPi:PFK toward glycolysis (Stitt, 1990; Theodorou and Plaxton, 1996).
In Brassica nigra, a large induction of PPi:PFK activity appear to be based on de novo

synthesis of the a subunit of the enzyme, leading to a significant enhancement in activation

106

by F2,6P2 (Theodorou, et al., 1992). F2,6P2 levels were not measured in this work,
however, the increase in PPi:PFK activity as the level of 02 decreased below 2 kPa
supports the contention that glycolysis was promoted as 02 became limiting.

The crossover plot comparing 0 kPa CO2 with higher CO2 treatments (Fig. 3.8 A,
B, and C) indicate that the conversion of F6P to Fl,6P2 was the step more affected by
C02, mainly for 02 levels above 1 kPa. However, a slight enhancement by C02 on the
conversion of PEP to PYR, and thus on PK, was also detected, mainly for 02 below 9
kPa. Furthermore, Fig. 3.8 also suggests that the conversion of F1 ,6P2 to triose-P by
aldolase (EC 4.1.2.13) also was affected by 5 kPa CO2 at the lowest 02 level achieved.

The increase in PK activity below 2 kPa 02 was coincident with a similar increase
in PYR content in the same 02 range (Fig. 3.6). An increase in PYR levels with a
simultaneous decrease in PEP (i.e. an increase in the PYR/PEP) also indicates a promotion
of the conversion of PEP to PYR, which is consistent with the increase in PK activity
(Fig. 3.10).

The crossover plot comparing the highest O2 (16 kPa) with lower 02 levels for
each CO2 treatment (Fig. 3.9 A, B, C, and D) indicates that the conversion of PEP to PYR
(PK) was the step most affected by low 02. PK appears to be much affected as CO2 level
decreases, suggesting that high CO2 may cause an unbalance in the system.

In summary, 02 levels below 1 kPa caused a reduction in all glycolytic
intermediates tested, a islight increase in the ratio G6P/F 6P, and an increase in the ratios of
F1,6P2/F6P and PYR/PEP, whereas storage at 02 levels above 1 kPa led to a decrease in

the ratio of F1,6P2/F 6P. 20 kPa CO2 caused a reduction in sucrose content, a reduction

107

in the G6P/F 6P ratio, and, when combined to low 02, an increase in the ratio PYR/PEP.
At 02 partial pressures below 2 kPa, the decline in PEP and increase in PYR indicates a
high level of control exerted at this conversion. The decline in sucrose was most affected
by 20 kPa C02, which is supported by the higher activities of sucrose synthase and acid
invertases. In contrast, 5 kPa C02, resulted in the least amount of sucrose loss (Silva et
al., 1998a) and had resulted in the lowest SS and invertase activities, suggesting control
over sucrose loss by these enzymes. The disappearance of this respiratory substrate at
lower 02 levels was paralleled by an enhancement of the conversion of PEP to PYR. This
suggests elevated CO2 may enhance glycolysis with a resulting loss in carbon from the
sugar pool.

Collectively, the data suggest carbon flux in asparagus spears stored under low 02
/high CO2 are markedly affected by the levels of CO2 present in the environment. CO2
exert a strong effect on the activities of ATP:PFK, PPi:PFK and PK, enzymes known by
its regulatory role in glycolysis (Plaxton, 1996). Although the effects of low 02 /high CO2
seems to be interactive, the results also suggest clearly separated effects of each gas.
Low 02 seems to have a more pronounced action on PK, end of the glycolytic pathway,
causing acceleration of glycolysis, promoting the shifl from oxidative respiration to
fermentation resulting in the activation of PDC and accumulation of fermentative
products (Silva et al., 1998a). On the other hand, high C02, besides having an effect on
PK, also appeared to have a larger effect on the conversion of F6P to Fl,6P2, via effects
on ATP:PFK and PPi:PFK activities, which strongly suggest an induced promotion of

glycolysis.

108

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112

 

2.0
1.6 4
1.2 -
0.8 "I
0.4 A

0-0 l I I I I I I I I I
3.0

2.5 4 Bound Acid Invertase
2.0 m
1.5 —

1.0 — -r-
0.5 - /

Acid Invertase

 

 

 

 

 

 

 

 

 

 

 

 

 

1'0 ... 20 kPa (:02
()3 — .0. 10 kPa co2 Neutral Invertase

+ 5 kPa CO2
0.6 m

0.4 -
0.2 -

0.0
2.0

 

\
\

 

 

 

 

 

Enzyme Activity (umol'min'l'g'l)

1.5 -

 

1.0 m

0.5 -

 

8\\\\\\*

 

 

 

0.0

 

 

 

0 2 4 6 81012141618
Steady State 02 Partial Pressure (kPa)

Figure 3.1. The effects of storage 02 and CO2 on the activity of acid, bound acid,
neutral invertases, and sucrose synthase (fresh mass basis), relative to harvest in
the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days.

Harvest

113

 

350
300 ._ + 20 kPa CO2 Glucose-6-Phosphate

_ ._o_ 10 kPa C02
250 + 5 kPa C02
200 -

L.— 0 kPa C02
150 -

.
"hf-'0
100 .— , 58%”
50 - 9’“

w
J

    
    
 

4 v ’
0" .0

  

 

 

... 140
120 4

-1

50

§
1

80-
60‘
40*
20‘

 

 

 

\V,

 

 

60
50 -
40 -
30 4
20 -
10 n

Hexose Phosphate (nmol'

 

 

 

 

1R\\\H

 

 

 

 

0 2 4 6 81012141618
Steady State 02 Partial Pressure (kPa)

Harvest

Figure 3.2. The effects of storage 02 and CO2 on the contents of glucose-6P,
fructose-6-P, and fructose-1,6-P2 (fresh mass basis), relative to harvest in the most
acropetal 45 mm of the tip of asparagus spears held at 1 °C for 6 days.

114

Ratio

 

G6P/F 6P

 

 

 

‘\ \\\\\\\

 

 

 

 

 

 

 

 

 

 

 

 

1 _
O I I I I I I I I I
3
... 20 kPa €02 F1,6P2/F6P
2 + 5 kP a C02
1 _
0 // I I l I I I I I I

 

 

 

 

 

 

024681012141618

Harvest ,
Steady State 02 Partial Pressure (kPa)

Figure 3.3. The effects of storage 02 and CO2 on the ratios of glucose-6-P to
fi'uctose-6-P, and fi'uctose-l,6-P2 to fi'uctose-6-P (fresh mass basis), relative to
harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for
6 days.

115

 

120

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100— I
f
801 /
v-f‘ 60- ¢
g 40— é
5 20—
£3 0 '/ I I I I I 1 I I I
..r:
5,1. 120
gm }
g 80— é
E 604 ¢
40— /
20 _ é Dehydroxyacetone-P
O ' I I I I l I I I I
0 2 4 6 81012141618
Harvest

Steady State Oz Partial Pressure (kPa)

Figure 3.4. The effects of storage 02 and CO2 on the contents of glyceraldehyde-
3-P and dehydroxyacetone-P (fresh mass basis), relative to harvest in the most
acropetal 45 mm of the tip of asparagus spear held at 1 0C for 6 days.

116

Triose Phosphate (nmol'g'l)

 

1,3- Phosphoglyceric Acid

 

 

 

 

-8\\\\—I

 

 

 

 

 

 

 

N
O O
1

 

 

 

 

 

 

 

 

 

100
80—
60—
T
40—
20— /
O '/ I I I I I I I I I
Harvest 0 2 4 6 8 10 12 14 16 18

Steady State 02 Partial Pressure (kPa)

Figure 3.5. The effects of storage 02 and CO2 on the contents of 1,3
phosphoglyceratic, 3-phosphogliceric, and 2-phosphoglyceric acids (fresh mass
basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus spear
held at 1 °C for 6 days.

117

Ratio

250
200 —
150 -
‘T‘A 100 —

N
O
O

160 -
120 —

Intermediate (nmol'g

 

Phosphoenolpyruvate

50-

O

 

 

00
O
1

 

.p.
O
I

 

—4
—<
_
_

 

 

 

.0. 10 kPa co2
+ 5 kPa C02

+ o kPa co2

 
 
   
  

 

 

 

 

 

 

0 17%

I I I I I I I I I I
Harvest 0 2 4 6 8 10 12 14 16 18

Steady State 02 Partial Pressure (kPa)

 

Figure 3.6. The effects of storage 02 and CO2 on the contents of
phosphoenolpyruvate (PEP), pyruvate (PYR) (fresh mass basis), and the PYR/PEP
ratio, relative to harvest in the most acropetal 45 mm of the tip of asparagus spear
held at 1 °C for 6 days.

118

 

 

 

 

 

 

 

 

[Lower kPa 02 - 16 kPa Oz] / [Lower kPa 02+ 16 kPa Oz]l2

 

 

 

 

'09 I I I I I r I I I I
a, a. N A. :- < < < e. a:
w a:
8 n- E 5 E 2 82 52 n- E
“‘ G «31 «A «4
Intermediate

.... 0.2mm kPa o2 .....- 0.4to0.6 kPa02 A l.0tol.5kPa02
..o.. 4.0to6.0kPa02 .43.. 8.0to8.5kPaOz ~-A--- 11-4t012-0kl’302

— Crossover Line

Figure 3.7. Crossover plot comparing lower 02 levels with the highest 02 level
(16 kPa) for CO2 levels of 20 (A), 10 (B), 5 (C), and 0 (D) kPa, in the most
acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days.

119

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

$1.
O 0.3
a _ 20 kPa C02 vs0kPa C02
32 0.2 ‘
c 0.0 — .-
...
0.0 A
3
: "0'1“ ."“'lj.1.;2: ‘ ' ‘ .. ,.
5.“ -0-2 + 211.2%” - A
E '03 T. I I I j I I I I I 2
0.3 ON
7 - 0.2 5:
7 - 0.0 a
c
0.0 +
‘ — -0.1 a
_ — -0.2 ;
N I I I I I I I I I I '03 2
>62 03 :1
' SkPaCO szkPaCO
8 0.2 - 2 2 ‘
g 0.0 ‘ “58;“ . ..... .. ........... .
0.0 m!" ":55. - . ,
i a???” ... wt
In '0.1 "7 ."--"..
“" I
g -0.2 — c
i ‘0-3 I I I I I I I I I I
‘1" ‘3- N “-1 9- < < <2 6..
e D-
u a :2 6 E e e e a E
“a G «a, «a «a
Intermediate

...... 0.2t00.3kPa02 . 0.4t00.6kPa02 ------A 1.0tol-5kPa02
o 4.0to6.0kPa02 ...D... 6.5t08.0kPa02 ---A--- 10t013kP302

-— Crossover Line

Figure 3.8. Crossover plot comparing higher CO2 levels (20 (A), 10 (B), and 5
(C) kPa) with the lowest CO2 level (0 kPa), in the most acropetal 45 mm of the tip
of asparagus spear held at 1 °C for 6 days.

120

Enzyme Activity (pmol'mm 1'g"1)

 

(13

(12 -

0.1 -

n

I\ ‘I

. (r. (K.

Hexokinase

49

 

01)

 

,1" ‘-
r l

l l l

 

(15
(14 —
(l3 —
(12 —
(11 *

 

x\\\\\\\-* S

 

 

 

 

 

 

Fructokinase

 

01)

 

 

215
210 -
1&5 -
210 -
L5 —
1L0 -
(15 -

 

 

 

 

 

 

01)

"k\\\*

Harvest

 

O 2 4
Steady State 02 Partial Pressure (kPa)

6 8

10 12 14 16

Figure 3.9. The effects of storage 02 and CO2 on the activities of hexokinase,

fructokinase, and glucose-6-P dehydrogenase (fresh mass basis), relative to harvest
in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days.

121

 

Enzyme Actmty (umol'min'l‘g'l)

1.5

 

ATP:Phosphofructokinase
+ 20 kPa C02

1.0 4 —o— 10 kPa C02

 

  

 

 

 

 

 

 

T + SkPa C02
05 _ y + 0kPa C02
0.0 4
4

 

.\

((0 g Q

 

 

 

 

\ \\\\—+

 

 

 

 

 

 

-a\\\ V

 

 

 

O 2 4 6 81012141618
Steady State ()2 Partial Pressure (kPa)

Harvest

Figure 3.10. The effects of storage 02 and CO2 on the activities of
ATPzphosphofructokinase, PPi:phosphofi'uctokinase, and pyruvate kinase (fresh
mass basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus
spears held at 1 °C for 6 days.

122

CHAPTER 4

PHOSPHATE AND NUCLEOTIDE METABOLISM UNDER LOW O2 IHIGH CO,

PARTIAL PRESSURES IN THE TIPS OF HARVESTED ASPARAGUS SPEARS

STORED AT 1 °C
INTRODUCTION
Asparagus spears are characterized by a very high postharvest metabolic rate (King
et al., 1990; Silva et al., 1998a; Silva et. al., 1997). The rate of metabolism is particularly
high in the developing meristem (spear tip). Harvesting the spear removes the source of
carbohydrate, leading to carbohydrate starvation. Sugar starvation triggers several
physiological, biochemical, and molecular events in plants, among them: 1. the depletion
of intracellular carbohydrate content (Joumet et al., 1986; Saglio et al., 1980); 2. an
increase in inorganic phosphate (Pi) (Joumet et al., 1986); a concomitant decline in
nucleotide tri-phosphate (Saglio et al., 1980); 3. senescence and deterioration, leading to
loss of cell ultrastructure (King et al., 1990; Silva et al., 1998a).
Plant cells, whether photosynthesizing or not, produce enormous quantities of

ATP from ADP, indicating that, in vivo, the utilization of ATP is extremely well adjusted
to its regeneration (Pradet and Raymond, 1983; Saglio et al., 1980). In aerobic
conditions, ATP is generated by the oxidative processes of glycolysis, oxidative
phosphorylation, and oxidative pentose phosphate pathways (Tetlow et al., 1998). In
response to low 02, most plants experience a reduction in oxidative phosphorylation,
which typically results in a marked inhibition of respiratory (i.e. Oz-linked) ATP synthesis

(Raymond et al., 1985). Under conditions of limiting 02, plants shift to a lower overall

123

rate of ATP synthesis via fermentation with a concomitant recycling of NAD (Rébeille’ et
al., 1985). As adenylate metabolism changes in response to low 02, a shift in all the major
phosphate and nucleotide pools occurs.

Low 02 and/or high CO2 stresses induce a number of metabolic and energetic
modifications, linked to modifications of gene expression in an as yet unknown manner
(Ricard et al., 1994). However, determining the relative importance of biochemical shifts
in enzyme activities and metabolite pools in plant performance under stresses is
complicated by the fact that stresses disturb whole networks of biochemical processes
(Rébeille’ et al., 1985). On the other hand, by reducing respiratory rate through low 02
and altering metabolism by elevated CO2 partial pressures, it may be possible to bring a
commodity to a new steady metabolic activity at any level between the highest reached
under aerobic conditions and the lowest level reached under hypoxia. This may allow to
reach a lower metabolic rate, which may result in delaying senescence. However, research
is needed to evaluate how phosphate and adenylate status interact and how those factors
may be affected by respiration and carbohydrate metabolism, in terms of metabolic energy,
under reduced levels of Oz/elevated CO2 partial pressures in harvested asparagus spears.

PPi, Pi, ATP, and ADP are utilized and/or generated in a number of different steps
in the glycolytic pathway (Duff et al., 1989). These metabolites are critical in satisfying
the energy requirement of a tissue (Pradet and Raymond, 1983), and regulating the
mobilization of stored carbohydrates (Re’beille’ et al., 1985).

Pyrophosphate (PPi) is thought to serve as an energy source for glycolysis (Black

et al., 1987). PPi is controlled in the cytoplasm by PPi:PFK, which produces PPi during

124

gluconeogenic carbon flux (Dancer and Ap Rees, 1989). Advantageous features have
been found for some plants which, under energy stress due to anoxia, conserve ATP by
switching to a PPi-based metabolism (Ricard et al., 1991). In agreement with that, in
asparagus tips the extractable activity of PPi-dependent enzymes such as sucrose synthase
and PPi-PFK (Silva et al., 1998b) was increased by OZ-deficiency. In addition,
carbohydrate metabolism has been suggested to be collectively regulated by key
metabolites such as F-2,6-P2, sugar-P, adenylates, and Pi/PPi ratio (Koch, 1996).

Inorganic phosphate (Pi) not only plays a vital role in energy transfer and in
metabolic regulation (Duff et al., 1989). For instance, under Pi starvation, in order to save
energy, plants ‘bypass’ some ATP and ADP glycolytic steps in Brassica nigra (Dufl‘ et al.,
1989) and maize seedlings (Ricard et al., 1991). Pi is also an important constituent of
sugar-P and macromolecules in plants.

The adenine nucleotide content of the tissue and the adenylate energy charge
(AEC) have been used as indicators of cellular metabolic status (Atkinson, 1968; Xia et
al., 1995). ABC represents the relative saturation of the adenylate pools in
phosphoanhydride bonds (Pradet and Raymond, 1993) and it has been proposed to reflect
the availability of ATP relative to ADP and AMP for ATP-utilizing processes and, via feed
back mechanisms, exerts control over respiration (Brouquisse et al., 1991).

This research work details the effects of 12 02 combined with 4 CO2 partial
pressures, ranging from hypoxic to active fermentation levels, on high and low energy
phosphate pools in asparagus spears tips. Our objective was to understand how these

processes are regulated postharvest during spears storage at 1 °C.

125

MATERIAL AND METHODS

Plant material. Asparagus spears (Asparagus officinalis L. cv. Jersey Giant) were
harvested early in May from the Michigan State University Horticultural Research Center
between 6:00 and 9:00 am. and stored at 1 °C. Samples were taken for analyses at
harvest and afier storage. Spears selected to represent harvest conditions were frozen in
liquid N2 in the field. Spears were harvested when they reached a minimum length of 240
mm and were trimmed to 180 mm. Only straight undamaged spears with closed bracts
and no obvious symptoms of disease were used. Spears to be placed on storage were
chilled with ice while still in the field and transported to the laboratory within the hour.
After storage, spears were frozen in liquid N2. After freezing, all spears were stored at ~80
°C until analysis. There were three replications. Three replicate experiments were
conducted in consecutive asparagus seasons (1995, 1996, 1997). Tissue samples
comprised the most apical 45 mm of the spear.

Atmosphere generation. 02 partial pressures ranging from a low of a. 16 kPa to a
high of 16 kPa were generated in packages for four CO2 treatment levels (0, S, 10, and 20
kPa). 02 levels were obtained by sealing spears in packages composed of low density
polyethylene (LDPE) of varying film thickness and placing the packages in air (0 kPa C0,)
or enclosing the packages in glass chambers with 20.7 kPa O2 and 5, 10, and 20 kPa C02
(Beaudry, 1993). Chamber lids were fitted with inlet and outlet ports for purging with gas
mixtures; a glass tube (1.3 cm id. x 2.7 cm long) was inserted through the lid and glued to
a piece of electrical tape on the surface of the enclosed package with silicone sealant

(Beaudry, 1993). The silicone sealant formed a septum through which gas samples could

126

be obtained from within the package. Chambers were continually purged with gas mixture
of 02, C02, and N2 gases at 30 mI.'min'l to generate the target atmospheres within the
enclosed packages. Actual CO2 levels in the packages were slightly higher than reported
values, but for clarity of presentation, are reported as treatment values. The gas
composition of individual packages was monitored until steady state conditions were
reached. Steady state conditions were achieved on day six of the study. 02 and CO2
gradients across the film were used to calculate the flux for both gases through the film
using previously obtained permeability data (Lakakul, 1994). Flux data were used to
calculate the rates of 02 uptake and CO2 production (Cameron et al., 1994).

“P-NMR Analysis. To evaluate the inorganic phosphate (Pi) status in the sample,
three spear tips (approximately 6 g total tissue mass) from the 1.0 kPa 02 x 20 kPa CO2
treatment were fitted into a 10 mm wide x 180 mm long NMR tube immediately after
cutting. The tube was capped and capillary tubing was used to circulate air. NMR
scanning was done at 1 °C, using a NMR spectrophotometer (General Electric Omega
500). Fifteen hundred transients were acquired using a 60 ° pulse, a 1.8 recycle time,
9806 complex time domain points, and a 15.4 Khz spectral width (Weich et al., 1989).
Line broadening of 30 Hz was applied before Fourier transformation. Total acquisition
time was lb and 22 min. Chemical shift in all spectra are reported relative to 85% H3PO,
(as an external standard) and 0.5 M methylenediphosphonic acid (as an internal standard).

PPi and Pi and Adenylate Extractions. Approximately 3 g of frozen tissue per
sample were transferred to liquid N2 in a precooled mortar, in which 5 mL of ice-cooled

0.8 N (v/v) HClO4 were added to the frozen tissue, and ground with a pestle to a fine

127

powder without melting. Five mL of ice-cooled 0.8 N HClO4 (total 10 mL) were then
added to the powder, which was extracted for 30 min at 0 °C with occasional shaking,
followed by centrifugation at 30,000g for 30 min at 0 °C. Supernatant pH was adjusted to
pH 7.0 to 7.2 with 5 N K2C03, allowed to sit on ice for 10 min, and clarified by
centrifugation at 30,000g for 10 min (Scott et al., 1995). Immediately following
extraction, the resulting supernatant was used for analysis of PPi (Edwards et al., 1984),
Pi (Cornell et al., 1979;), NTP (Mohanty et al., 1993; Trautchold, et al., 1985 ), ADP,
and AMP (Jaworek and Welsch, 1985; Scott et al., 1995). To estimate metabolite losses
during extraction, measured amounts of adenylates roughly equal to the amounts present
in the tissue, were added to the extracting medium. Resultant recoveries were 93%, 95%,
98%, 89%, and 91%, for PPi, Pi, ATP, ADP, and AMP, respectively. The recovery
assays were run in five replicates.

PPi and Pi and Adenylate Assays. PPi, Pi, ATP, ADP, and AMP were assayed at
25 °C using a spectrophotometer (Hitachi, Model U3000, Hitachi Inc., Tokio, Japan) with
an attached refrigerator circulator (Polystat, Cole-Palmer Instrument Co., Chicago, IL), by
measuring the absorbance of pyridine nucleotides at 340 nm following adaptations from
procedures described by Kato-Noguchi and Watada (1996), Scott et al., (1995), and
Trautchold, et al., (1985). Measurements were taken for 15 to 20 min in a final volume of
lmL of buffer mixed with other additions specified for each metabolite. Depending on the
metabolite, 50 to 100 uL crude extract was used. Each reaction was allowed to go to
completion. Blank cuvettes for each reaction contained all the reagents listed for each

metabolite except that the extract was replaced with water.

128

Assay cocktails were as follows: PPi: 60 mM Tris-acetate (pH 8.0), 2 mM of
magnesium acetate, 6 mM fiuctose-6-phosphate, 10 uM of fructose-2,6-bisphosphate,
0.13 mM NADH, 1.5 units aldolase (EC 4.1.2.13), 2.5 units g1ycerol-3-phosphate
dehydrogenase (EC 5.3.1.1), 7 units triosephosphate isomerase (EC 5.3.1.1). The reaction
was started by the addition of 0.3 unit PPi:PFK.

Pi: 100 mM TEA (pH 7.6), 1.5 mm EDTA, 2 mM MgC12, 1.5 mM NAD, 15 mM
glucose, 1 mM ADP, 0.5 mM fiuctose-l,6-bisphosphate, 3 units aldolase, 8 units
glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), 2 units hexokinase (EC
2.7.1.1), plus 10 units phosphoglycerate kinase (EC 2.7.2.3). The reaction was started by
adding the extract.

Adenosine 5 '-triphosphate (ATP): 150 mM TEA (pH 7.6), 2.5 mM MgC12, 1.5
mM NADP, 2 mM glucose, 4 units glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 2
units hexokinase. The reaction was started by adding the extract.

Adenosine 5 '-diphosphate and adenosine 5 '-monophosphate (ADP and AMP,
Jaworek and Welsch, 1985): 100 mM TEA (pH 7.6), 1 mM phosphoenolpyruvate, 30 mM
MgC12, 0.12 mM KCl, 0.3 mM NADH, 20 units lactate dehydrogenase (EC 2.7.4.3), 18
units pyruvate kinase (EC 2.7.1.40) for ADP measurement; the above mixture for ATP
plus 16 units myokinase (EC 2.7.4.3) for AMP measurement.

Adenylate Energy Charge (AEC). The ABC was expressed according to Atkinson
(1968) as:

AEC = ([ATP] + 0.5 * [ADP]) - ([ATP] + [ADP] + [AMP])" [1].

129

RESULTS

PPi. In asparagus tips at the steady state, PPi content at 0 kPa CO2 treatment and
16 kPa 02 decreased relative to harvest. The PPi content for the 5 kPa CO2 treatment
tended to be higher than 0 and other CO2 treatments at O2 partial pressures greater than 6
kPa. Within the same 02 range, CO2 partial pressures of 10 and 20 kPa markedly reduced
PPi content. Between 2 and 16 kPa Oz, PPi decreased as CO2 increased and 02 decreased
(Fig. 4.1). Relative to harvest, however, the 20 kPa CO2 treatment resulted in a decline in
PPi of approximately 50 %. 02 levels below 2 kPa, resulted in a large decrease in PPi
(more than 90 %) in a manner that appeared to be independent of CO2 treatment.

Pi. The 31P-NMR inorganic phosphate profile after 6 days at 20 kPa CO2 and 1
kPa 02 indicated of the amounts of Pi present in the vacuole was very high relative to that
present in the cytoplasm (Fig. 4.2). The Pi content in the vacuole was significantly higher
than the nucleotide phosphate pools present in the cytoplasm.

The Pi content at 0 kPa CO2 and 16 kPa 02 was slightly lower than at harvest (Fig.
4.3). However, although Pi at 0 and 5 kPa CO2 were maintained close to harvest content
at 16 kPa 02, Pi increased for 10 and 20 kPa C02. Pi tended to increase as 02 partial
pressure declined. 02 levels below 2 kPa caused a substantial increase in Pi content.
Below 2 kPa Oz, Pi enhancement appeared to be dependent on increased CO2 levels, since
Pi content was enhanced about seven times for 20 kPa CO2 treatment. The PPi/Pi ratio
declined with increasing CO2 and decreasing 02 (Fig. 4.3, insert).

Adenylate. Relative to harvest, ATP content decreased approximately 20% for 0

kPa CO2 and 16 kPa 02 (Fig. 4.4). Although ATP values at 0 and 5 kPa CO2 were close

130

at 16 kPa 02, for 02 levels below 16 kPa O2 and above 6 kPa 02, ATP tended to be higher
for 5 kPa C02. At 16 kPa 02, CO2 partial pressures above 5 kPa CO2 caused ATP to
decrease. From 2 to 16 kPa 02 ATP content was the lowest for 20 kPa CO2 treatment.
Between 6 and 16 kPa Oz, decreasing 02 caused a decline in ATP only for the 20 kPa CO2
treatment. However, ATP appeared to be affected by 20 kPa CO2 treatment at
approximately 14 kPa 02 (Fig. 4.4), when a marked drop in ATP occurred. Below 6 kPa
02, however, ATP content decreased markedly for all CO2 treatments. At the lowest 02
levels achieved, ATP was reduced approximately 90% relative to the highest 02 level for
each CO2 treatment. Relative to ADP and AMP, ATP was the dominant adenine
nucleotide at harvest (compare Figs. 4.4 with 4.5 and 4.6). The PPi/ATP ratio was
maintained around 0.4 between 2 and 16 kPa 02. However, PPi/ATP ratio started to
increase as 02 decreased below 2 kPa Oz, peaking near 1 kPa O2 and declining again at
lower 02 levels. In response to reduced 02 partial pressure, respiration decreased (Silva et
al., 1998a). ATP declined as respiration declined in response to limiting O2 partial
pressures. For a given rate of respiration, ATP content was reduced by C02 partial
pressures of 10 and 20 kPa.

At 0 kPa CO2 and at the steady state 16 kPa 02, both ADP and AMP contents
were close to those found at harvest (Figs. 4.5 and 4.6). At 16 kPa Oz, ADP and AMP
contents increased, as CO2 increased, in pattern similar to Pi, with the lowest ADP and
AMP contents for the 0 kPa CO2 treatment. The effect of decreasing O2 partial pressure
was accentuated for 02 levels below 2 kPa. Between 6 and 16 kPa 02, 02 had no

apparent effect on ADP content, however, ADP tended to increase with decreasing 02 for

131

5, 10, and 20 kPa C02. AMP tended to increase with decreasing 02 for all CO2
treatments. This indicates that CO2 had a marked influence on ADP and AMP
accumulation when the steady state 02 was reached.

At 0 kPa CO2 and 16 kPa 02, ABC was maintained close to that at harvest (Fig. 4.
7). At 16 kPa 02 ABC declined with increasing CO2 partial pressure. In the range of 2 to
16 kPa 02, ABC was dependent both on CO2 and 02 levels, decreasing as CO2 increased
and as 02 decreased (Fig. 4.7). Below 2 kPa 02, ABC declined markedly with decreasing
O2 partial pressure, dropping as low as 0.32. which may reflect some physical minimal
equilibrium between ADP/AMP at that 02 range.

The ATP/ADP ratio for the 0 kPa CO2 treatment and at the steady state 16 kPa 02
was similar to that at harvest (Fig. 4.8). The ATP/ADP ratio decreased as CO2 increased
and 02 decreased. For the 20 kPa CO2 treatment, at 16 kPa 02, there was a reduction of
approximately 80 % in the ATP/ADP ratio. At the lowest 02 partial pressures, the
ATP/ADP ratio decreased more than 95%, relative to the highest 02 partial pressures for
each C02 treatment.

A plot of ATP/ADP ratio against AEC showed that, for the highest AEC measured
(approximately 0.86), ATP/ADP was similar to harvest for 0 kPa CO2 treatment (Fig.
4.9). ATP/ADP ratio was decreased as AEC values decreased and as CO2 increased.

The difference between CO2 treatments was no longer obvious as AEC declined below
0.7. Interestingly, 0 kPa CO2 maintained, at higher AEC, the highest ATP/ADP,

suggesting greater Pi flux per ATP, which indicates much higher ATP regeneration.

132

DISCUSSION

PPi. PPi is present in the cell as a byproduct of the nucleotide-dependent reactions
and is primarily associated with biosynthesis of nucleic acids and proteins (Taiz, 1986).
PPi may be produced or removed by PPi:PFK, which can cycle with either ATP:PFK or
Fru1,6Pase catalyzed reactions. PPi is removed in glycolysis for the production of F1,6P2
and is produced in gluconeogenesis, by Fru1,6Pase, for sucrose production (Dancer et al.,
1990). However, under Oz-deprivation, synthetic processes, such as cell wall and protein
biosynthesis are greatly reduced (Xia et al., 1995). One would expect, therefore, a
concomitant reduction in the rate of PPi biosynthesis relative to its utilization, such that
the PPi pools declines. The data in the present study are consistent with this possibility.
Further, the decrease in PPi with increasing CO2 suggests that elevated CO2 can have a
similar effect to low 02 on synthetic processes.

PPi is known as a potential energy donor in the cytosol of plant cells (Dancer et
al., 1990). The marked increase in the PPi/ATP ratio at decreasing O2 partial pressures
below 2 kPa 02, suggests the predominance of PPi- over ATP-utilizing reactions. In
plants subject to anoxia, the PPi-dependent proton pump activity in tonoplast vesicles is
greatly increased and accompanies an increasing capacity for proton transport, hydrolyzing
PPi in the cytoplasm and pumping H+ inside the vacuole (Carystinos et al., 1995). A drop
in pH might therefore accompany the suggested increased PPi utilization relative to ATP
below 2 kPa 02. Carystinos et al. (1995) reported that, as an additional adaptation to
energy stress, glycolytic enzymes reactions consuming ATP can be at least partially

replaced by reactions utilizing PPi as an energy source. This idea is also supported here by

133

the increase in PPi/ATP ratio below 2 kPa Oz, and parallel increases in PPi:PFK and a
decrease in ATP:PFK activities (Silva et al., 1998b) within this 02 range. PPi is
recognized as a source of energy in plant cells in which PPi: PFK is acting as a glycolytic
enzyme (Dancer and Ap Rees, 1989; Dancer et al., 1990), which would be expected to
result in a decrease in PPi content, as reported in here below 2 kPa 02 (Fig 4.2). Saglio et
al. (1980) reported that PPi:PFK seemed not to be important in driving reactions under
anoxic conditions in acclimated maize root tips. However, in nonacclimated maize root
tips, the activity of PPi:PFK was much higher when compared to hypoxic acclimated ones.
In contrast with these authors, in asparagus spear tips that were at steady-state with
respect to respiration, PPi:PFK activity increased significantly below 2 kPa 02 (Silva et al.,
1998b), without any concomitant increases in PPi, suggesting that PPi:PFK may play a
similar role to alcohol dehydrogenase, which does not limit the capacity of energy
production under anoxia (Xia et al., 1995).

Pi. In asparagus tips at 20 kPa CO2 and l kPa 02, Pi was found mostly in the
vacuole, probably due to the limited interchange of Pi between cytoplasm and plastids
under those conditions. In vivo, in the light plastidic Pi is known to counter-exchange
with imported hexose phosphate (Tetlow et al., 1998). However, in this study, harvested
asparagus spears were stored in dark at 1 °C, which meant that no significant counter-
exchange of Pi occurred.

Pi is a potent feedback inhibitor of PPi:PFK in the glycolytic direction (Dancer et
al., 1990). 02 levels below 1 kPa reduced PPi:PFK activity in asparagus spear tips (Silva

et al., 1998b) when the highest content of inorganic phosphate was measured for all C02

134

treatments (Fig. 4.4). This Pi increase below 2 kPa O2 probably is due associated declines
in phosphorylated organic acids and sugar-phosphates (i.e., glycolytic intermediates)
(Silva et al., 1998b), and tri phosphate nucleotides (Saglio et al., 1980), protein and
phospholipids (Brouquisse et al., 1991), and nucleic acids (Duff et al., 1989), and loss of
selective permeability of membranes as suggested by Brouquisse et al., (1991), as well as
adenylate di- and tri-phosphate hydrolysis. In sycamore cells, under sucrose starvation,
the cytoplasmic-phosphorylated compounds decrease whereas intracellular Pi increases
symmetrically (Rébeillé et al., 1985). In asparagus spear tips, 02 levels below 2 kPa
caused a decline in sucrose content (Silva et al., 1998a). Pi increases in the vacuole were
also correlated to limited sucrose availability and a decline in triphosphate nucleotides
(Roby et al., 1987). The similar responses in asparagus tips below 2 kPa 02 may be
associated either with the observed decline in sucrose or, more likely, a lack in ability to
synthesize ATP. Furthermore, at 16 kPa Oz, Pi content increasing as C02 increased
suggests that, at 02 levels close to ambient, Pi increase may be a C02-dependent process.

Adenylate. Hypoxic treatment cause a decrease in ATP levels in maize root tips
with a concomitant rise in ADP and AMP (Xia and Saglio, 1990), in a manner similar to
that reported here as 02 partial pressures decreased (Figs. 4.4, 4.5, and 4.6). The
responses of PPi and Pi to low 02 were consistent with the drop in ATP and increase in
ADP and AMP below 2 kPa Oz. The increase in the respiratory quotient at these 02
tensions (Silva et al., 1998a) suggests that ATP biosynthesis was compromised. Our data
are consistent 'with this possibility. In asparagus tips, one possible explanation for the

reduced ATP content at higher CO2 treatments, even for higher 02 levels, may be due to

135

an uncoupling effect on oxidative phosphorylation caused by higher CO2 (Fanestil et al.,
1963). Therefore, it seems that 20 kPa C02, even when combined with 02 close to
ambient (16 kPa), could be limiting for the energy supply needed for survival of asparagus
spear. The decline in ATP below 6 kPa 02 for all CO2 treatments, showing a marked
decline below 2 kPa 02, suggests a critical threshold (approximately 0.8 kPa O2 ) where
the ATP seemed not to be regenerated sufficiently to sustain metabolism. This threshold
appeared to coincide with similar 02 level in which increased respiratory quotient, and
lactate, acetaldehyde, and ethanol contents started to be detected in asparagus tissues
(Silva et al., 1998a), and also with a marked decline in glycolytic enzyme activities (Silva
et al., 1998b). These results imply a decline in the glycolytic flux below that 0.8 kPa 02.
This critical threshold for glycolytic flux under those Oz-deficient conditions may reflect
the minimum rate of ATP supply necessary to sustain cell function. This decline in
glycolytic flux is also supported by the low AEC values found below 1 kPa 02, which
reached 0.32 and did not decline lower. This also supports the idea that there is a critical
02 level where the tissues start to suffer irreversible damage through degradation of vital
cell components such as enzymes and nucleic acids, and accompanying a parallel increase
in the activity of acid phosphatases (Roberts et al., 1984), which may result in an increased
total phosphate content.

On the other hand, the observed rise in PPi:PFK, and PK activities around 2 kPa
02, independently of CO2 treatments, (Silva et al., 1998b) suggest an attempt by the tissue
to promote glycolytic flux. The changes in these enzyme activities may indicate that a

transition from hypoxic to Oz-deficient metabolism occurs below 2 kPa 02 for asparagus,

136

leading to accumulation of fermentation products and deficiency in ATP resupply. As
reviewed by Hochachka et al. (1996), as ATP generation by oxidative phosphorylation
begins to fall off under 02 deprivation, the energy deficit is made up by activation of
anaerobic ATP supply pathways, which, as suggested by Saglio et al. (1988), upon
acclimation, may result in a sustained higher glycolytic rate. Maintenance of the glycolytic
flux in these acclimated tissues is argued to be due to a combination of a rise in kinase
activities and decreased inhibition of regulatory enzymes resulting from a higher
cytoplasmic pH and ATP content (Bouny and Saglio, 1996). Furthermore, acclimation of
tissues to Oz-stress conditions results in modification of ATP-utilizing enzymes, which is
measured as an increase their ATP usage through changes in combination of the amounts,
catalytic turnover of these enzymes, and their affinity for ATP (Xia et al., 1995). In this
experiment, it is taken into account that, after 6 (1 storage when the steady state 02/ CO2
was reached, the asparagus spears were acclimated into a steady state condition for each
02/ CO2 combination. Threfore, it may be that in asparagus spears tips, there was an
increase in glycolytic flux around 1 kPa 02, which was immediately followed by a decrease
when 02 levels dropped to a critical level (below 0.8 kPa).

Adenylate Energy Charge (AEC). Under energy-limiting conditions, such as 02-
deficiency, ABC is recognized as an indicator of the rate of ATP regeneration (Saglio et
al., 1980). In the present study, limitation of the respiratory metabolism by low 02
induced a drop in ABC. In maize root tips, ABC has been taken as an indicator of
metabolic activity in cells where the rate of oxidative phosphorylation is Oz-limited (Saglio

et al., 1988). ABC varies from 0 to 1 and ranges from 0.8 to 0.9 in health tissues. At

137

AEC values below 0.5, the relative activity of many ATP-regenerating enzymes is
decreased, while the activity of ATP-utilizing enzymes is increased (Pradet and Raymond,
1983). ABC values around 0.9, such as those found under here (at harvest and for 0 and 5
kPa CO2 treatments with 16 kPa Oz), characterize tissues where respiration is not limited
by 02 (Raymond et al. , 1985). However, small AEC variations at high values (0.75-
0.85), as those measured in the present study for 10 and 20 kPa CO2 at 16 kPa 02, may
correspond to significant variations in the ATP/ADP and ATP/AMP ratios, and may
therefore be an indication of regulatory processes that differ between 02 and CO2 for
enzymatic ATP-utilizing processes (Pradet and Raymond, 1983).

A high AEC value and ATP/ADP ratio indicate that ATP—regenerating pathways
(glycolysis and oxidative phosphorylation) do not limit the energy supply to the metabolic
processes evolved upon imposition of hypoxic stress. However, below a critical 02 level
of approximately 1 kPa glycolysis is diverted to fermentation in asparagus (Silva et al.,
1998a) probably due to an inhibition of oxidative phosphorylation.

Initially, in studies on the effect AEC on enzyme activity, it was argued that the
balance between ATP, ADP, and AMP was the key factor for cellular homeostasis. ABC
was proposed as a means of comparing the physiological role of regulation of enzyme
activities by adenine nucleotide ratios. The ABC expression assumes that the terminal
phosphate groups of ADP and ATP are metabolically equivalent (Atkinson, 1968), which
is true only if the adenylate kinase maintains a near-equilibrium state between adenylates
(Pradet and Raymond, 1983). Nevertheless, in maize root tips acclimated to low-02,

survival is not critically determined by nucleotide levels (Xia et al., 1995).

138

Modification of ATP-utilizing enzymes and glycolytic metabolism are two general
means through which the relationships between nucleotide fluxes and nucleotide
concentrations, or their ratios, could be altered (Xia et al., 1995). In asparagus tips, it
seemed that all sub-ambient lower 02 partial pressures affected AEC. However, the
dramatic decrease in ABC as 02 decreased below 2 kPa (Fig. 4.7), was accompanied by a
similar trend for ATP:PFK and sugar-metabolizing enzyme activities (Silva et al., 1998b).
These apparently coordinated alterations in enzymes and adenylate pool sizes may indicate
that the balance between pathways of ATP regeneration and consumption has been
altered. Furthermore, low 02 may lead to cytoplasmic acidosis, which directly increases
ATP hydrolysis, perhaps through activation of acid phosphatases (Roberts et al., 1984),
resulting in reduced in vivo ATP:PFK activity (Raymond et al., 1985). Plasma membrane
and tonoplast H*-ATPases play an important role in intracellular pH regulation in plants
(Xia and Roberts, 1994). Under acidosis, H*-ATPases consume much more ATP to
extrude protons that leak into the cell (Gout et al., 1992). Therefore, even in asparagus
spear tips acclimated to O2 partial pressures, below 2 kPa 02, the activity of plasma
membrane and tonoplast H*-ATPases would be affected by extreme reduction in
intracellular ATP levels, which may cause significant drop in cytoplasmic pH.
Cytoplasmic pH may be affected by the effect of fermentation to lactate and H+ transport.
In plant tissues exposed to high C02, pH may also be reduced by carbonic acid
dissociations to bicarbonate and hydrogen ion (Roberts et al., 1984). In maize seedling
root tips, anoxia led to ATP breakdown without a concomitant increase in ATP synthesis

via fermentation (Xia and Roberts, 1994). This agrees with the hypoxic and OZ-deficient

139

ATP profiles reported here for asparagus spear tips, where reduced 02 availability
progressively reduced ATP content (Fig. 4.4), AEC values (Fig. 4.7), and ATP/ADP
ratios (Figs. 4.8 and 4.9), with paralleled increases in Pi (Fig. 4.3), ADP (Fig. 4.5) and
AMP (Fig. 4.6). Hence, as also suggested for nonacclimated maize root tips (Xia et al.,
1995), in asparagus tips below 2 kPa Oz, declining ATP may cause the plant material to
respond by reducing hexokinase activity (Bouny and Saglio, 1996); Silva et al, 1998b),
leading to reductions in glycolytic flux below 1 kPa 02. In maize root tips acclimated with
3 kPa 02 the ATP/ADP ratio dropped 30 to 50 % relative to air treatment (Xia and
Roberts, 1994). A similar effect of 02 was detected in asparagus. The decline in ABC
and the ATP/ADP ratio may also indicate that other substrates, such as lipids, proteins and
nucleic acids, were beginning to be utilized. The decrease in ABC here is consistent with
data of Saglio et a1. (1980) in hypoxic- treated maize root tips. Similar results were also
reported by Brouquisse et al. (1991) in maize root tips. The latter authors suggested that,
as the sugar supply is depleted and substrates other than sugars are utilized, enzymes from
carbohydrate metabolism begin to be useless and become substrates for protein
degradation.

In summary, the results here demonstrates that, in asparagus spear tips, as 02
decreases below the critical level of 2 kPa and CO2 partial pressure increases, Pi, ADP,
and AMP contents increase, while PPi, ATP contents and AEC, ATP/ADP ratios decrease
significantly. These alterations were all intensified at O2 partial pressures below 2 kPa,
which makes the transition to active fermentative metabolism. The data suggest 2 kPa is a

critical 02 threshold below which a marked modification in gene expression may occur.

140

These data, and the reduction of ATP:PFK activity (Silva et al., 1998b) and low AEC
found below 1 kPa 02, suggest an intracellular pH reduction. In addition, lower ATP
content, AEC values, and ATP/ADP ratios at 20 kPa C02, at O2 ranging from 2 to 16
kPa, indicate that high CO2 may cause impairment of oxidative phosphorylation and limit
cell survival, even at 16 kPa 02. On the other hand, the marked drop in PPi and ATP
contents associated with reductions in AEC, and ATP/ADP ratio, for all CO2 treatments,
at O2 partial pressures below 2 kPa, indicates the critical role of O2 in asparagus storage.
This also suggests unbalanced cell metabolism, and a low protein turnover rate, which
likely lead to cell death. This suggestion is fiirther supported by the appearance of water
soaked areas in asparagus spear tips under these low 02 tensions (Silva et al., 1998a).
Collectively, this data indicate different roles for CO2 and 02 along the range of steady
state 02 partial pressures studied. CO2 effect seemed to predominate mostly fiom 2 to 16
kPa 02, while 02 effect seemed to predominate over that of CO2 effect below 2 kPa 02.
Finally, the roles that transcriptional and translational events play in determining
the changes in pools of metabolites, metabolic energy, and enzyme activities, have yet to
be established. Some of these modifications, such as reduction in ATP/ADP ratio with
concomitant release of phosphate, may indicate the presence of an O2 “sensor”
(Hochachka et al., 1996) or may play a role in signal transduction. Repression of
metabolic activity, protein synthesis and degradation are adaptative responses to hypoxia.
Nevertheless, the limited number of enzymes directly involved (Ricard et al., 1991) makes
anaerobic induction an attractive system in which to study the molecular mechanism of

cellular adaptation to limiting energy availability.

141

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Tetlow, I.J., K.J. Blissett, M.J. Emes. 1998. Metabolite pools during starch synthesis and

carbohydrate oxidation in amyloplasts isolated from wheat endosperm. Planta
204: 100-108.

Trautschold, I., W. Lamprecht, and G. Schweitzer. 1985. ATP determination: UV-
method with hexokinase and glucose-6-phosphate dehydrogenase. In: H. U.
Bergmeyer, J. Bergmeyer, and M. Grassl (eds), Methods of Enzymatic Analysis.
3"1 ed, Vol. VII, Verlag Chemie Publishers, Deerfield Beach, pp. 346-364.

Xia, J-H and J. KM. Roberts. 1994. Improved cytoplasmic ph regulation, increased
lactate efflux, and reduced cytoplasmic lactate levels are biochemical traits
expressed in root tips of whole maize seedlings acclimated to a low-oxygen
environment. Plant Physiol. 1052651-657.

Xia, J-H. and P. Saglio. 1990. H+ efilux and hexose transport under imposed energy
status in maize roots tips. Plant Physiol. 93: 453-459.

Xia, J-H, P. Saglio, and J. K.M. Roberts. 1995. Nucleotide levels do not critically
determine survival of maize root tips acclimated to a low-oxygen environment.
Plant Physiol. 108: 589-595.

Weich, R.G., P. Lundberg, H.J.Vogel, and P Jensen. 1989. Phosphorus-31 NMR studies

of cell wall-associated calcium-phosphates in Ulva lactuca. Plant Physiol. 90:230-
236.

145

PPi (nmol'g'l)

 

 

 

 

 

 

 

 

%
100— %
%
% +3333?
50- / : swzcoi
Z ... OkPaC02

 

024681012141618
Steady State 02 Partial Pressure (kPa)

Harvest

Figure 4.1. The effect of storage 02 and CO2 on the content of PPi (fresh mass
basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus
spear held at 1 °C for 6 days.

146

Asparagu- PJI m I

 

i
. ... l
l a. 1 °-
l :a l 3 g
x l is :2
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15 10 5 0 -5 -10 -15 -20 -25

PP“I

Chemical Shift (ppm)

Figure 4.2. Pi status measured by 31P-NMR, relative to harvest in 45 mm long
asparagus spear tips under 1 kPa steady state 02 and 20 kPa CO2 held at 1 °C.
Peak assignments are as follows: I, glucose-6-phosphate (G6P); II, cytoplasmic
inorganic phosphate (Pi); III, vacuolar phosphate; IV, y-P of ATP and B-P of
ADP; V, a-P of ATP and of ADP; VI, B-P of ATP. Insert shows an amplified
scale spectrum to improve peak discrimination.

147

 

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8

O
l

 

 

 

      
  

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kit
O
O
l

l
024681012141618

Steady State 02 Partial Pressure (kPa)

+ 20 kPa C02
—0— 10 kPa C02
+ 5 kPa C02
+ 0 kPa C02

....

fill

8
1

Inorganic Phosphate (nmol'g'l)
v—fi N
8 8
O O
l T

“a”
l

 

 

 

 

0 ’I I I I l I I I I I
0 2 4 6 8 10 12 14 16 18

Steady State Oz Partial Pressure (kPa)

Harvest

Figure 4.3. The effect of storage 02 and CO2 on the content of Pi (fresh mass
basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus spear
held at 1 °C for 6 days. Insert shows the effect of O2 partial pressures on the
PPi/Pi ratio.

148

 

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\ a . o .1
02 Uptake (mmol kg h )
0 I I I I I I I I I I

0 2 4 6 8 10 12 14 16 18
Steady State Oz Partial Pressure (kPa)

 

Harvest

Figure 4.4. The effect of storage 02 and CO2 on the content of ATP (fresh mass
basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus spear
held at 1 °C for 6 days. Inserts show: A) the effect of O2 partial pressures on the
PPi/ATP ratio and B) the effect of 02 uptake on the content of ATP.

149

ADP (nmol'g'l)

 

 

 

 

 

400
ADP + 20 kPa C02

350 I" .' —-o— 10 kPa co2

O

" —-— SkPa C02
300 _ «3:0 + (JkPaCO2

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100 -
50 -
0 7 I l l f l l T I I I
Harvest O 2 4 6 8 10 12 14 16 18

Steady State 02 Partial Pressure (kPa)

Figure 4.5. The effect of storage 02 and CO2 on the content of ADP (fresh mass
basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus spear
held at 1 °C for 6 days.

150

 

300
+ 20 kPa CO2

AMP —o— 10 kPa co2
250 - _.._ 5 kPa co,
+ 0 kPa CO2

§

AMP (nmol'g'l)
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)‘(~ “ n ‘
' ‘ k 0
50 '-
0 ' l l l l l l l l I

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Harvest

Figure 4.6. The effect of storage 02 and CO2 on the content of AMP (fresh mass

basis), relative to harvest in the most acropetal 45 mm of the tip of asparagus spear
held at 1 °C for 6 days.

151

Adenylate Energy Charge (AEC)

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Figure 4.7. The effect of storage 02 and CO2 on the AEC, relative to harvest in the

 

 

/

,2

 

 

AEC

 

+ 20 kPa C02
—0— 10 kPa CO2
+ 5 kPa CO2
+ 0 kPa CO2

 

 

Harvest

I I
0 2

l
4

f

6

I I I I
8101214

l

16
Steady State 02 Partial Pressure (kPa)

most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days.

152

 

ATP/ADP

 

+ 20 kPa C02 ATP, ADP
—O— 10 kPa CO2

+ 5 kPa CO2
+ 0 kPa CO2

 

 

 

 

 

 

I 1U I I
0 2 4 6 81012141618

Steady State Oz Partial Pressure (kPa)

Harvest

Figure 4.8. The effect of storage 02 and CO2 on the ATP/ADP ratio, relative to
harvest in the most acropetal 45 mm of the tip of asparagus spear held at 1 °C for
6 days.

153

 

 

 

 

 

10
+ 20 kPa CO2 ATP/ADP .
3— —C>--10kPaCO2 ’
-—A— 5 kPa CO2 ‘
9‘6 ...“ Okraco2 ,. f
G T .'
3 Q
g
< 4 —
2 _ j
0 . I I I I I I

 

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Har est
v Adenylate Energy Charge (AEC)

Figure 4.9. The effect of ABC on the ATP/ADP ratio, relative to harvest in the
most acropetal 45 mm of the tip of asparagus spear held at 1 °C for 6 days.

154

CHAPTER 5

EFFECTS OF 6-BENZYLAMINOPURINE ON SUGAR PROFILE AND

SENESCENCE OF ASPARAGUS (Asparagus officinalis L.) SPEARS STORED AT
1 °C
INTRODUCTION
Asparagus spears senesce rapidly postharvest (Lipton, 1990). Postharvest
physiological changes include a decline in respiration rate, a drop in soluble carbohydrates,
mainly sucrose, and losses in chlorophyll, protein and amino acids. Although senescence
is a genetically controlled and programed process (Thomas and Stoddart, 1980), it is
subject to modifications through external influences (Crafis-Brandner, 1984). Exogenous
application of cytokinins to plant tissues results in a variety of responses which may
include a delay in senescence, increased chlorophyll production, a stimulation of
chloroplast development, maintenance of chloroplast activity, a reduced chlorophyll
degradation, the promotion of protein and nucleic acid synthesis, and the mobilization of
nutrients into the cytokinin-treated area (Clarke et al., 1994; Muller and Leopold, 1966;
Wingler et al., 1998). The extension of the shelflife for cytokinin-treated plant tissues has
been attributed to lower respiration rates compared to control (Rushing, 1990); however,
changes in gene expression may also explain those responses (Chen and Leisner, 1985;
Van der Werf and Nagel, 1996).
The senescence-delaying effect of exogenous cytokinin has been derived from

classical experiments which demonstrate the correlative evidence linking endogenous
levels of cytokinins to the progress of leaf senescence in corn leaves (Muller and Leopold,

1966). However the most convincing evidence for role of endogenous cytokinins in

155

senescence comes from transformation experiments in which a significant delay in leaf
senescence was observed in transformed plants containing an active bacterial cytokinin
synthase gene and elevated endogenous cytokinin levels (Clarke et al., 1994). Van der
Werf and Nagel (1996) hypothesized that allocation of carbon is mediated by sucrose and
cytokinins. It has also been suggested by Farrar (1996) that sucrose not only plays a role
in allocation of assimilates between sources and sinks, but also functions as an indicator of
the assimilate status of both source and sink. Sugar concentration may also exert an
influence in the expression of some genes to permit matching the availability of sugars
from the source and the demand of carbon of the sink (Van der Werf and Nagel, 1996).
Since the tip of spear fitnctions as the sink for harvested asparagus (King et al., 1990), and
sugars from the lower portions of the spear supply the carbohydrate demand of metabolic
processes of the tip (Saltveit and Kasmire, 1985), the utilization of carbon by this crop
may be responsive to status of cytokinin and sucrose in the spear tip.

A reduced rate of cell division in leaves leads to a reduced unloading of sucrose
from the phloem into the expanding cells. Consequently, as sucrose concentration in the
phloem nearby the expanding cells increases, it leads to an increase of the turgor pressure
in the cell division zone, at the phloem adjacent cells, resulting in expansion growth.
Supporting this idea, Downes et al., (1969), described the effects of temperature in the
rate of growth of asparagus spears as related to the gain in weight and anatomical
changes. Their results also suggested that the tip may exercise some influence in the rate
of elongation of the stem.

The objective of this research was to evaluate the influence of tip presence and

156

exogenous 6-BAP application on carbohydrate metabolism and visual quality on stored
asparagus spears. CO2 production, chlorophyll fluorescence, chlorophyll content, soluble

sugars, and visual quality were measured during 31 days of storage at 1 °C.

MATERIAL AND METHODS

Plant material and treatments. Asparagus (Asparagus oflicinalis L. cv. Jersey
Giant) spears were harvested in May 1996 no later than 9:00 am. from three year old
plants at the Michigan State University Horticultural Teaching Research Center. Spears
were surface-sterilized in the field by dipping for 1 min in 150 ppm NaOCl followed by a
rinse with tap water. Sterilized spears were placed in contact with absorbent paper to
remove excess moisture. Two treatments were imposed: tip removal and exogenously
applied cytokinin. In addition to control spears, there were three treatment combinations
(6BAP, tipped, 6BAP + tipped). Initially, the length of all spears was adjusted to 180 mm.
Tipped spears had a 45 mm length of the apex was removed using a sharp, stainless steel
knife. Cytokinin was applied in the field immediately following sterilization, using 4.42 x
104 M (100 ppm) of 6-BAP (Sigma Chemical Co., St. Louis, MO, USA) predissolved in
minimal dimethylsulphoxide prior to dilution in water with 0.02% Tween 20 (Nutritional
Biochemical Corporation, Cleveland. OH, USA). 6-BAP was applied by placing cheese
cloth soaked in 6-BAP in contact with the spear for 30 seconds. Excess solution was
removed by absorbent paper. Treated and untreated spears were placed in an ice-cold
chest and transported to the laboratory. Spears were held in the dark at 1 °C. For each

assay date spears were placed in liquid nitrogen at harvest and stored at -80 °C for later

157

measurement.

Spears of each treatment combination were divided into two lots. One lot was
used to monitor the respiratory rate. The second lot was used as a source of spears for
other assays including sugar, chlorophyll fluorescence, chlorophyll content, length, and
visual quality. These assays were performed for spears stored 0, 3, 7, 12, 17, 26, and 31
days.

Respiration rate measurement. Three replicates consisting of ten spears were
enclosed in 1.980 L mason jars ventilated with air at a flow rate of 30 mL'min". The CO2
concentration of the exit gas stream was determined on a 100 uL gas sample taken with a
0.5 mL insulin-type syringe. CO2 analysis was done by an infrared gas analyzer (Model
ADC 225-MK3, The Analytical Development Co. Ltd, Crowborough, Sussex, England)
with N2 as the carrier gas at a flow rate of approximately 100 mL'min (Beaudry, 1993).
Respiration rates were measured approximately 30 min after harvest (time 0), and on a
daily basis for 31 days. Extra jars, similar to those described above, were prepared for
evaluation of the other parameters following described, and at similar time course.

Chlorophyll fluorescence determinations. Chlorophyll fluorescence was measured
at harvest and at regular time intervals from base to tip, using a pulse-modulated
fluorometer (Model OS-500, Opti-Sciences, USA). Measurements were taken from the
center of 45 mm long four segment spears. Three readings in equidistant locations of each
section were taken and averaged. Six spears were used for make up the average for each
replication. Spears were acclimated in the dark for 1 hour room temperature and

measurements were made in a darkened laboratory. Fluorescence was measured using a

158

photodiode in the wavelength range of 710-760 nm. For each measurement run, minimal
fluorescence (F 0) was monitored for 0.2 sec at a rate of 100 readings per second (20
sample points). The excitation (modulated) light (660 nm, filter for > 700nm light)
intensity for the F0 measurement was approximately 0.15 umol'm‘z's’l (OS-500 setting =
60) which was sufficient to get an accurate measurement of F0 (Van Kooten and Snel,
1990). The addition of approximately 3 mW (OS-500 setting = 50) of continuous far red
light (735 nm) did not influence fluorescence, therefore it was assumed that the redox
components of the electron transport chain were fully oxidized. After F0 was determined,
the sampling rate was then increased to 1000's“1 and a saturation light (660 nm) pulse was
supplied via the light guide by a halogen lamp. The light intensity at the peel during the
pulse was estimated to be 2,400 pmol'm'z's" photosynthetically active radiation (PAR)
based on an OS-500 setting of 60; a setting of 255 yields approximately 10,000 umol'm'z's’
lPAR. The pulse duration was 0.8 s and a cut-off filter, blocking light above 700 nm, was
used to prevent saturation of the photodiode. During the pulse, the maximum level of
modulated chlorophyll was designated as Fm. The efficiency of photosystem II (Fv/F m)
was calculated as (Fm-Fo)/Fm (Van Kooten and Snel, 1990).

Chlorophyll determination. Chlorophyll content was determined at the same time
intervals and in the same four 45 mm long spear sections used for chlorophyll
fluorescence. For this determination, approximately 500 mg of the fresh epidermis tissues
were removed and extracted in 500 mL of N,N-dimethylformamide (Sigma Chemical Co.,
St. Louis, MO, USA) for 24 h at 4 °C according to Moran and Porath (1980).

Chlorophyll measurement were made using a spectrophotometer (Model U-3110, Hitachi

159

Ltd, Tokyo, Japan) at 603, 647, and 664 nm. The estimation for chlorophyll a, b, and
total were calculated from formulae described by Moran (1982).

Soluble sugars determination. Soluble sugars were extracted from four 45 mm
long segments which were previously stored at - 80 °C. The segments were the tip, and
proceeding basipetally, 2-4 were successive 45 mm length with segment 4 being the spear
base. Sugars were extracted from grounded spear segments in 3.5 mL of 80% ethanol for
15 minutes at room temperature. The mixture was centrifuged (Model RG-SC, Sorval
Instruments, Dupont Co., Newtown, CT, USA) at 2,000 g for 5 minutes and the
supernatant decanted into a capped 50 mL centrifuge tube. This procedure was repeated
two times and the extract obtained was washed out with 5 mL of water. To remove
chlorophyll, 5 mL of chloroform was added to the combined 15.5 mL ethanol /water
mixture. The tubes were capped, shaken vigorously and then centrifuged at 1,000 g for 3
minutes. The upper, clear aqueous phase containing the soluble sugars was transferred to
a test tube and evaporated to dryness using a speedvac equipped with a refiigerated
condensation trap (at -103 °C) (SC200 Speedvac, Savant Instruments Inc., Farmingdale,
NY).

The driedsoluble carbohydrates were solubilized overnight with agitation in 1 mL
of dry pyridine containing 30 mgmL" hydroxylamine hydrochloride, heated at 70 to 80 °C
for l h, and derivatized. Carbohydrates were assayed using a gas chromatograph (HP
Model 5890 series 11, Hewlett-Packard Co. Palo Alto, CA) fitted with a FID. The column
(DB-1701 capillary column, J &W Scientific, Folsom, CA) had a 0.25-um thick coating,

was 30m in length x 250 um id.

160

Sensory quality. A continuos visual rating scale was constructed to evaluate the
visual quality. Whole spears were rated 12 h after being removed from the jars at room
temperature. Each treatment replication was rated independently by the subjective
evaluation of four observers using the following scale adapted from Silva et al. (1998):

9= fresh like;

7= slight chlorophyll loss in tips and scales, slight flaccidity and/or very slight
wrinkle on stems, absence of strange flavors, no feathering due to irregular growth;

5: moderate chlorophyll loss and browning in tips and/or stem bracts browning,
more pronounced wrinkle, loss of pink blush at the base, slight ethanol odor, slight
feathering due to irregular growth;

3= extensive chlorophyll loss, browning, and pronounced flaccidity of tips and
stems bracts (feathering), wrinkle and wilting of stems, moderate fermentative odor
moderate feathering due to irregular growth;

l= extensive loss of chlorophyll, browning and flaccidity of tip and stems bracts,
complete loss of pink blush at the base, extensive wrinkle and wilting stems and base,
brown spot in stems when exposed to air, strong fermentative odor, extensive feathering
due to irregular growth.

A rating of 6 (threshold quality) was considered unacceptable for sale although still

edible.

161

RESULTS AND DISCUSSION

Carbon dioxide production. Relative to spears with tip, at 0 (1, CO2 production
was 35% higher in spears without tips. CO2 did not differ for spears with and without tips
after 3 d. 6-BAP applications, however, resulted in lower CO2 production in spears, with
and without tips (Fig. 5.1). Wounding of tissues caused by cutting and injuries usually
results in increased CO2 production (Salveit and Kasmire, 1985), an effect observed here
for spears without tips. However, even though CO2 production in spears without tips was
markedly higher than those with tips at the beginning of the storage, the respiratory rates
were lower for cytokinin-treated spears, indicating that cytokinin application reduced
respiration. CO2 production is understood as a physiological indicator of the metabolic
activity of a crop (Kays, 1991) and its decline is usually associated with a general decline
of the metabolic activity of the whole system. Thus, the results presented here suggest
that exogenous cytokinin application has the potential to reduce the metabolic rate of
asparagus spears postharvest. On the other hand, CO2 production did not differ among
treatments after 10 days and throughout the remainder storage period at 1 °C. 6-BAP has
a similar effect on CO2 production in broccoli stored at 16 °C (Rushing, 1990).

Soluble sugars. In spears with intact tips, fructose content in sections 2, 3, and 4
remained lower in cytokinin-treated spears (Fig. 5.2). In the tips, however, fi'uctose
content was higher for cytokinin-treated spears for the first 5d of storage and did not
differ therafter. In spears without tips, fructose did not differ in section 2 for cytokinin-
treated and untreated spears. However, in cytokinin-treated tipped spears, fructose

content was slightly higher in section 3 during the later portion of the storage and in

162

section 4, markedly higher for the first 12 d of storage (Fig. 5.3). For spears with and
without tips, 6BAP did not affect the glucose profile, except in section 4 of intact spears
which experienced a reduction in glucose in response to 6-BAP treatment (Figs. 5.4 and
5.5). Sucrose content was markedly affected by cytokinin treatment, but only in the
location to which it was applied. Its content was higher in the tips of cytokinin-treated
spears (Fig. 5.6). This response differs from that of Irving and Joyce (1995) in broccoli
stored at 23 °C. These results suggest that, in asparagus spears, cytokinin applications
may either cause mobilization of sucrose from lower sections to the tip of spears or reduce
the activities of sucrose-metabolizing enzymes, minimizing degradation of this sugar. It
has been suggested that cytokinin, in addition to delaying senescence, could block some of
the responses to sugar levels (Jang et al., 1997). In addition, carbon allocation to shoots
and roots may be mediated by cytokinin and sucrose (Van der Werf and Nagel, 1996). If
cytokinin causes a change in sucrose allocation, it would be possible, by evaluating the
segmental spear sugar profile, to observe a changed import of sugars into the cytokinin-
responding sink tips. However, since the hexose content in the tip (Figs. 5.3 and 5.5) did
not change much upon cytokinin treatment, it is difficult to determine if the delay in the
sucrose decline in the tip was caused by its import from lower sections or by
gluconeogenesis from other substrates. When submitted to carbohydrate starvation, plant
cells may adapt, and substitute protein and lipid metabolism through autophagic processes
(James et al., 1993; Moriyasu and Ohsumi, 1996; Saglio and Pradet, 1980 ). On the other
hand, in spears without tips, section 2 of the cytokinin-treatments tended to accumulate

more sucrose than untreated in the first 20 d of storage (Fig. 5.6); however, the behavior

163

of the other sections was not much different from non-treated spears. Sucrose utilization
following storage appeared to be reduced in spears without tips, indicating that the tip
may have a role in the postharvest senescence of spears.

Sucrose decline in asparagus tips postharvest has been suggested to trigger
senescence for the whole spear (King et al., 1990). The ability of sink organs to
competitively attract assimilates is termed sink strength (Ho, 1988). Sink strength is
determined by the metabolic activity in the sink tissue, and by a number of enzyme
activities playing a key role in regulation in sink activity (Nielsen and Ulvskov, 1992) and
sink-strength regulation (Wingler et al., 1998). The asparagus spear tip, being comprised
of highly meristematic tissue (Robb, 1984), acts as a strong sink, and is characterized as
having highly active metabolism (King et al., 1990). The change in the pattern of sucrose
utilization in cytokinin-treated spears suggests that in some way cytokinin caused a
blockage in the sensor that triggers sucrose breakdown and utilization. In transgenic
tobacco, for example, it was reported that sugars, cytokinin, and light interact during
senescence by influencing the decline in proteins involved in photosynthetic metabolism
(Wingler et al, 1998). The results in harvested asparagus show that the decline in sucrose
content was, however, closely associated with a decline in respiration rate and with a
decline in chlorophyll content and changes in chlorophyll fluorescence, indicating that a
series of processes may be affected by the sucrose status and sugar utilization in asparagus
spears.

Changes in chlorophyll content. Cytokinins are known to affect photosynthesis

and to stimulate chloroplast biogenesis and chlorophyll biosynthesis, promoting the

164

synthesis of RuBPCase, the differentiation of chloroplasts, and the expression of genes
encoding the small subunit of RuBPCase and the light harvesting chlorophyll a/b-binding
proteins (Genkov et al., 1997; Nielsen and Ulvskov, 1992; Wingler et al., 1998), resulting
in slower rates of chlorophyll degradation. Our results also confirm that applied 6-BAP
delayed the degreening of asparagus spears stored at 1 °C (Figs. 5.8 and 5.9). 6-BAP
application also promoted chlorophyll retention in de-rooted oat seedlings (Badenoch-
Jones, et al., 1996) and in meristematic floral tissues of broccoli (Irving and Joyce, 1995;
Clarke et al., 1994). Chlorophyll a content was the major contributor to total chlorophyll
content, and was four-fold higher than chlorophyll b (data not shown). When treated with
6-BAP, the spear tips appeared to accumulate more chlorophyll a than other sections.
Therefore, application of 6-BAP in asparagus spears tips not only delayed chlorophyll
degradation but also changed the pattern of chlorophyll distribution and metabolization by
the spear.

Changes in chlorophyl fluorescence. Chlorophyll fluorescence has been used as
an indicator of postharvest chilling injury in banana and mango (Srnillie et al., 1994),
tomato (Jung and Steffen, 1997), and green bell peppers (Lurie et al., 1994), and of scald
in apples (DeEll et a1, 1996). In spears with tips, Po, the basal fluorescence of dark-
adapted spears, started to increase after 15 (1 storage in untreated spear tips compared to
more than 25 days in cytokinin-treated spears. F0 was not affected by 6-BAP for spears
without tips (Fig. 5.10). The removal of the tip did seem to affect F0 values for sections
2, 3, and 4 for cytokinin-treated and -untreated spears. In cytokinin-treated spears, Fm,

the maximum fluorescence of dark-adapted spears, did not decrease after 15 (1 storage, but

165

started to decreased after the same period in untreated spears. However, the decline was
much more pronounced is spears without tips (Fig. 5.11). Reduction in the ratio Fv/F m
is manifested as a reduction in the quantum yield of PSII in a decrease in variable
chlorophyll a fluorescence (Demmig and Bjorkman, 1987). In spears with tips, Fv/F m, an
indicator of the photosynthetic capacity of PSII, was significantly higher in cytokinin-
treated than in untreated spears within 10 days of storage. This ratio was also higher for
the section 2 of 6-BAP-treated spears which had the tips removed (Fig. 5.12). Reductions
in Fm and Fv/Fm indicated severe damage in PSII (Franklin et al., 1992). The observed
reduction in Fv/F m and the increase in F 0 during storage for untreated spears, indicated
that there was damage to the PSII centers that was not readily reversible (Jung and
Steffen, 1997). It is unlikely that cellular repair mechanisms could match the rate of
damage to the P811 and hence photosynthetic capacity was impaired (Layne and Flore,
1993). In addition, the decreased efficiency of PSII photochemistry during storage in
untreated spears may reflect the inhibition of PSII function and an increase in thermal
radiationless energy dissipation (Jung and Steffen, 1997), which appeared to be lessened in
cytokinin-treated spears.

Spear growth. Cytokinin promotes the growth of excised cotyledons of various
plants, particularly in darkness (Ulvskov et al., 1992; Chen and Leisner, 1985). The most
common responses of the cotyledons to hormonal stimulation is cell expansion (Huff and
Ross, 1975). The results here indicate that cellular expansion also occurred in asparagus
spears stored at 1 °C, with cytokinin stimulating growth of approximately 8% (Fig.5. 13).

It has been suggested that cytokinins may affect growth by increasing tissue ability to take

166

up, retain or metabolize available assimilates, which would result in changes in sink
strength. This additional growth could be result from an increased biosynthesis of cell
components required for the division and expansion of the cell (Nielsen and Ulvskov,
1992) and promotion of anatomical changes (Downes et al., 1964). Alternatively,
increased solute import could be the basis of decreasing water potential. This would lead
to increased turgor pressure, which potentially could be the driving force for cell
enlargement (Ulvskov et al., 1992). Although the application of cytokinin resulted in
irregular growth of the spears, this did not much influence their qualitative acceptance, as
noted by its visual quality (Fig. 5.14). In addition, the data suggest that the tip has a
marked influence in promoting cell enlargement, since its removal inhibited growth.

Changes in sensorial quality. Application of 6-BAP to the treated surface of the
spear delayed senescence as ascertained by the maintenance of the visual appearance. 6-
BAP-treated spears with tips dropped below the acceptable visual quality threshold 10
days earlier than untreated spears (Fig 5.14). Exogenous cytokinin also delayed
senescence, as measured by appearance, in de-rooted oat and wheat seedlings, and was
suggested to have a role in regulating senescence in intact seedlings (Badenoch-Jones et
al., 1996). However, they reported that such a cytokinin effect is highly species-specific.
The results here indicate that the tip is the segment most affected by senescence, probably
due to its being the most metabolically active tissue. In addition, spears without tips had
much lower acceptance, suggesting that good visual quality for asparagus spears requires
the presence of the tip.

Collectively, cytokinin application to the tips minimized sucrose depletion and

167

other measures of senescence in asparagus. The results showed that the direct application
of 6-BAP delayed chlorophyll loss, decreased respiration rate, delayed changes in
chlorophyll fluorescence, improved the general appearance, and minimized the loss of
sucrose in spears with tips. The carbohydrate data indicates that cytokinins affect carbon
transport, uptake and metabolism. Further research, however, is required to determine
how cytokinin is able to modify the pattern of sucrose utilization by asparagus spears

during storage at 1 °C.

168

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Ho, LC. 1988. Metabolism and compartimentation of imported sugar in sink organs in
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172

 

 

 

 

 

 

  
   
   

 

 

4
With Tips

'7" 3 n
III
e:
um __‘
.3. 2
E
a 1 r
v
e a '0
3 0 I I I I I I I
:t
'8
'5. 4
a Without Tips
:2
E 3
“U
5 2 —~
.8 + Without Cytokinin
a:
Q 1 _ --0- With Cytokinin

0 I I I I I I I

 

0 5 10 15 20 25 30 35
Time (Days)

Figure 5.1. Changes in carbon dioxide production in asparagus spears with and
without 6-BAP, with and without 45 mm long tip segments during storage at 1 °C.
Bar = :t SE(n=3).

173

Fructose (mg’g'l)

 

 

 

+ With Cytokinin
—0— Without Cytokinin

    

Tip

 

 

 

 

 

l l l I l l l

 

 

Section 2

 

 

 

 

 

 

 

 

Section 4

 

 

I I I I I I T
10 1 5 2O 25 30

Time (Days)

35

Figure 5.2. Changes in fiuctose content (fresh mass basis) in asparagus spears
with tips, with and without 6-BAP, during storage at 1 °C. Bar = :1: SE (n=3).

174

 

 

16 - + With Cytokinin
—0— Without Cytokinin

 

 

 

 

 

 

 

 

Fructose (mg'g'l)

 

 

 

 

 

Section 4

 

 

 

0 I I F I I I I
O 5 10 15 20 25 30 35

Time (Days)

Figure 5.3. Changes in fi'uctose content (fresh mass basis) in asparagus spears
without 45 mm long tip segments, with and without 6-BAP, during storage at 1
°C. Bar = :1: SE (n=3).

175

 

 

10 _ + With Cytokinin Tip
—0— Without Cytokinin

 

 

 

 

 

 

 

15 — Section 2

 

 

 

O l l l l l l l

 

Section 3

Glucose (mg‘g'l)
N
o

 

 

 

 

 

 

 

 

0 5 10 15 20 25 30 35
Time (Days)

Figure 5.4. Changes in glucose content (fresh mass basis) in asparagus spears with
tips, with and without 6-BAP, during storage at 1 °C. Bar = :t SE (n=3).

176

 

 

16 ~ + With Cytokinin
—0— Without Cytokinin

 

 

 

 

 

 

 

O l l l l l l l

 

Glucose (mg'g'l)

Section 3

 

 

 

 

Section 4

0 I I I I I I I
0 5 10 15 2O 25 3O 35

 

 

 

Time (Days)

Figure 5.5. Changes in glucose content (fresh mass basis) in asparagus spears
without 45 mm long tip segment, with and without 6-BAP, during storage at 1 °C.
Bar = i SE (n=3).

177

Sucrose (mg‘g'l)

 

 

8 _ + With Cytokinin
—0— Without Cytokinin

 

 

 

 

 

Tip

 

0 I I I I I I #3—4

 

 

 

Section 2

 

 

 

 

 

 

 

 

Section 4

 

 

 

0 5 10 1 5 20 25 30 35
Time (Days)

Figure 5.6. Changes in sucrose content (fresh mass basis) in asparagus spears with
tips, with and without 6-BAP, during storage at 1 °C. Bar = :t SE (n=3).

178

Sucrose (mg‘g'l)

 

 

l

+ With Cytokinin
—<>— Without Cytokinin

l

 

 

l

 

l

l

     
  

 

1

Section 2

l

 

 

 

 

l l

l

l

l J

 
 
     

Section 3

 

 

 

L

1

L11

1

Section 4

l

 

 

 

OI-‘NUJ-RUIGQOO Ot-‘Nw-§UIO\\IOO OF-‘NUJ-fi-UIGQOO
l

 

I I I I I I I
0 5 10 15 20 25 3O 35

Time (Days)

Figure 5.7. Changes in sucrose content (fresh mass basis) in asparagus spears
without 45 mm long tip segments, with and without 6-BAP, during storage at 1
°C. Bar = i SE (n=3).

179

 

 

 

 

 

 

 

 

 

 

100
p 90 _ With Cytokinin] With Tips + “p
'3 80 -
i 70 -
a 60 -
g 50 —
E 40 ""
U 30 '-
3 20 —
O
H 10 r
O l l l l l l l
100
.5“ 90 4 Without Cytokinin/ With Tips
'50
g 80 —
a 70 :—
I: 60 _
8‘ 50 - ~\
'5 40 - °
2 .
o 30 - 'x ‘ g —— ~
5 20 — " \"'\
a 10 — \°
0 l l l l l l l

 

0 5 10 15 20 25 30 35
Time (Days)

Figure 5.8. Changes in total chlorophyll content (fresh mass basis) in asparagus
spears with tips, with and without 6—BAP, during storage at 1 °C. Bar = d: SE
(n=3).

180

 

80

7O _ With Cytokinin/ Without Tips + Section 2
-v- Section 3

60-
50—
40H
304
20-
10—

 

Total Chlorophyll ([lg'g'l)

 

 

 

 

80
7O 4 Without Cytokinin/ Without Tips
60 —
50 -
4O —

20 — ‘

10‘ o

 

Total Chlorophyll (pg'g'l)

 

 

Time (Days)

Figure 5.9. Changes in total chlorophyll content (fi'esh mass basis) in asparagus
spears without 45 mm long tip segments, with and without 6-BAP, during storage
at 1 °C. Bar = :1: SE (n=3).

181

F0

F0

F0

F0

 

500
400 -
300 —
200 r
100 -

With Cytokinin] With Tips

 

+ Section 2
—0— Section 3
+ Section 4

 

l l l l l l l

 

500
400 —
300 —
200 -
100 —

 

 

 

 

500
400 -
300 -
200 4
100 -

 

With Cytokinin/ Without Tips

 

l l l l l l l

 

500
400 -
300 4
200 r
100 -‘

 

Without Cytokinin! Without Tips

  

 

Figure 5.10. Changes in the basal chlorophyll fluorescence (F o) in asparagus
spears with and without 6-BAP, with and without 45 mm long tip segments,

0 5 10 15 20 25 30
Time (Days)

during storage at 1 °C. Bar = 3: SE (n=3).

182

 

Ihn

Fni

Ihn

Ihn

 

 

+ Tip

+ Section 2
—0— Section 3
+ Section 4

 
 
  

 

l l l l I l I

 

 

1400
1200 -
1000 —
800 —
600 -
400 -

 

  

Without Cytokinin] With Tips

 

200

 

1400
1200 -
1000 r
800 u
600 -
400 -

 

  

With Cytokinin/ Without Tips

 

200

l l l l l l I

 

1400
1200 -
1000 r
800 -
600 -
400 -

 

 

Without Cytokinin] Without Tips

 

200

I I I I I I I
0 5 10 15 20 25 30

Time (Days)

 

35

Figure 5.11. Changes in the maximum chlorophyll fluorescence (Fm) in asparagus
spears with and without 6-BAP, with and without 45 mm long tip segments,
during storage at 1 °C. Bar = at SE (n=3).

183

Fv/Fm

Fv/Fm

Fv/F m

Fv/F m

 

1.0
0.8 1
0.6 r
0.4 -
0.2 —

With Cytokinin! With Tips

  

+ Tip

—v— Section 2
—0— Section 3
+ Section 4

 

0.0
1.0

 

0.8 -—
0.6 —
0.4 —
0.2 —

 

 

 

0.0

l l l l

 

1.0
0.8 —
0.6 —
0.4 -
0.2 -

 

With Cytokinin! Without Tips

 

 

0.0
1.0

 

0.8 -
0.6 -
0.4 -
0.2 —

 

0.0

 

 

 

0 5 10 15 20 25 30 35
Time (Days)

Figure 5.12. Changes in the quantum yield maximum (Fv/F m) in asparagus spears
with and without 6-BAP, with and without 45 mm long tip segments, during
storage at 1 °C. Bar = :t SE (n=3).

184

Length Gain (%)

 

 

+ WithoutTips/ Without 6-BAP
7 — -0— Without Tips] With 6-BAP
+ With Tips/ Without 6-BAP
—I:I— With Tips/ With 6-BAP

 

 

 

It

 

 

 

/.
1 " I V ” Length
1 x
0 " I I I I I I
0 5 10 15 20 25 3O 35
Time (Days)

Figure 5.13. Changes in length relative to the initial storage time in asparagus
spears with and without 45 mm long tip segments, with and without 6-BAP,
during storage at 1 °C. Bar = d: SE (n=10).

185

 

 

 

3 _ —0— With Cytokinin
- ~O - Without Cytokinin

Visual Quality (1 to 9)

 

 

 

 

 

 

 

 

 

 

2 T — Visual Qua]. Threshold With Tips
1 I I I I I I I
5?
3
C
5‘
:5
:I
O‘
'3
:r
.2 ‘
> 2 ‘ Without Tips
1 I I I I I I I
O 5 10 15 2O 25 30 35

Time (Days)

Figure 5.14. Changes in visual quality in asparagus spears with and without 6-
BAP, with and without 45 mm long tip segments, during storage at 1 °C. Value 6
represents the visual quality threshold. Bar = t SE (n=3).

186

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