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This is to certify that the
thesis entitled

Factors affecting color and anthocyanin content of “Michigan
Purple' potato tubers during tuber development and storage

presented by
Elzette van Rooyen

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MS. degree in Horticulture

 

 

 

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FACTORS AFFECTING COLOR AND ANTHOCYANIN CONTENT OF
‘MICHIGAN PURPLE’ POTATO TUBERS DURING TUBER DEVELOPMENT
AND STORAGE

By

Elzette van Rooyen

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

2004

ABSTRACT

FACTORS AFFECTING COLOR AND ANTHOCYANIN CONTENT OF
‘MICHIGAN PURPLE’ POTATO TUBERS DURING TUBER DEVELOPMENT
AND STORAGE

By

Elzette van Rooyen

Achieving vivid purple color at harvest and maintaining color during storage are
important quality factors for purple colored potatoes if growers wish to receive
premium prices. To ensure the marketability of the ‘Michigan Purple’ potato
cultivar, we investigated color stability and anthocyanin concentration of
‘Michigan Purple’ potato tubers during storage and compared it with other
colored potato cultivars. In addition, we investigated color and anthocyanin
concentrations during tuber development and tentatively identified and quantified
the anthocyanins responsible for the purple color in this cultivar. We also
investigated pre-and postharvest tools that could possibly enhance the tuber
skin-color at harvest and in storage. Preharvest investigations include the
evaluation of soil type, plant spacing, nitrogen fertilizer type and application rate,
and 2,4-dichlorophenoxyacetic acid (2,4-D) application rate. Postharvest
investigations include the evaluation of storage temperature and ethylene

application.

Dedicated to my dear parents,

Wilhelm and Ellena van Rooyen.

iii

ACKNOWLEDGEMENTS

I want to thank my parents, Ellena and Wilhelm van Rooyen, my brother
Michael and my sister Francoinette for believing in me and constantly giving me
the support that I needed to make it through this challenging time in my life. It
wasn’t easy, but I made it, thanks to you! I also want to thank my friends in South
Africa for their love and constant support.

I would like to express my appreciation to Dr. Deirdre Holcroft for inviting
me to study with her here at Michigan State University. Thank you for giving me
the opportunity to spread my wings. I know you had to leave, but I’m truly sorry
that we couldn’t keep working together. Fortunately, this gave me the opportunity
to work with a very interesting person, Dr. Randy Beaudry. Randy, thank you for
your inputs and support and patiently helping me achieve my goal. I enjoyed
working with you and learning more about postharvest in your lab.

I would also like to thank the other members of my committee, Dr.
Sieglinde Snapp and Dr. Johan Brink for all their help and inputs. Special thanks
to Karen and Johan Brink for being so understanding and supportive. I’m glad
you decided to live in East Lansing.

There are many special people that I met working at MSU: Kasey and
Nick Wierzba, we went through a lot together, good times and bad times,
Mauricio Cafloles and Marlene Ayala, you were my second committee, thanks for

all the help and support, I don’t think I would have been able to do it without the

iv

two of you, Leah Cleanrvater, another great person I met here, thanks for the
good times, Alejandra and Robert, Jen Dwyer and John Treloar.

Lastly I would like to thank a very special person in my life, Conrad
Schutte. Thank you for following me to America and being here everyday for the

last year to help me. Thanks for all your patience and love.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................................. viii
LIST OF FIGURES ................................................................................................ xi
INTRODUCTION .................................................................................................... 1
References .................................................................................................. 4
LITERATURE REVIEW
Introduction .................................................................................................. 6
Potato history and importance ..................................................................... 6
Tuber quality ................................................................................................ 8
Preharvest enhancement of anthocyanins ................................................. 9
Postharvest enhancement of anthocyanins ............................................... 11
Biology and biosynthesis of anthocyanins ................................................. 12
Induction of anthocyanin biosynthesis ............................................. 14
The role of glutathione—s-transferases in the accumulation
of anthocyanins ............................................................................... 17
Primary structures of anthocyanins ................................................. 18
Color changes through structure change ......................................... 19
Stability ............................................................................................ 20
Analytical techniques for anthocyanin identification and/or quantification
......................................................................................................... 22
High performance liquid chromatography (HPLC), ......................... 22
Spectrophotometry, ........................................................................ 23
Nuclear magnetic resonance spectroscopy (NMR) ......................... 23
Anthocyanin functions in plants .................................................................. 24
Anthocyanins in underground organs ......................................................... 25
Potato .............................................................................................. 25
Onion ............................................................................................... 27
Red Radish ...................................................................................... 28
Sweet Potato and yams ................................................................... 28
Garlic ............................................................................................... 29
Research Hypotheses and Objectives ....................................................... 30
References ................................................................................................. 32

vi

CHAPTER 1
CHANGE IN TUBER SKIN-COLOR OF ‘MICHIGAN PURPLE” AND OTHER
COLORED POTATO CULTIVARS DURING DEVELOPMENT AND STORAGE

Introduction ................................................................................................. 49

Materials and Methods ............................................................................... 50

Results and Discussion .............................................................................. 54

Conclusions ................................................................................................ 59

References ................................................................................................. 60
CHAPTER 2

PREHARVEST TECHNIQUES TO IMPROVE PIGMENT CONCENTRATION
AND COLOR OF ‘MICHIGAN PURPLE’ POTATOES AT HARVEST AND IN

STORAGE
Introduction ................................................................................................. 71
Materials and Methods ............................................................................... 74
Results and Discussion .............................................................................. 79
Conclusions ................................................................................................ 83
References ................................................................................................. 85
CHAPTER 3

Postharvest techniques to improve pigment concentration and color of ‘Michigan
Purple’ potatoes at harvest and in storage

Introduction ................................................................................................. 97
Materials and Methods ............................................................................... 99
Results and Discussion ............................................................................. 100
Conclusions ............................................................................................... 102
References ................................................................................................ 104
CHAPTER 4
Summary .............................................................................................................. 111
APPENDIX A ........................................................................................................ 1 13
APPENDIX B ........................................................................................................ 129
APPENDIX C ....................................................................................................... 134

vii

LIST OF TABLES

CHAPTER 1

Table 1. A comparison between the rates of change in hue angle values of over 6
months of storage in ‘Michigan Purple’, ‘Dakota Rose’, ‘California Red‘,
and ‘Norland’ potatoes stored at 10 °C. Standard errors are given in
parentheses. ............................................................................................. 62

Table 2. A comparison between the rates of change in hue values in the first 3
months, the last 3 months and the overall 6 months of storage at 4 °C in
‘Michigan Purple’, ‘Dakota Rose’, and ‘Chieftain’ potatoes. Standard errors
are given in parentheses. . ........................................................................ 63

CHAPTER 2

Table 1. Four nitrogen schemes with different fertilizer or amendment
concentrations (kg.ha'1) applied at given times to ‘Michigan Purple’ potato
plots. Amount of actual N applied (kg.ha'1) given in parentheses. ............. 88

Table 2. The effect of soil type and plant spacing on rate of hue angle change in
‘Michigan Purple’ potato tubers grown in organic and mineral soils at 0.3,
0.2, and 0.33 m respectively during first two months and last two months
of 4 months storage at 10 °C. Standard errors are given in parentheses.
................................................................................................................... 88

Table 3. A comparison between the rates of change in hue angle in ‘Michigan
Purple’ potatoes treated with four different rates of nitrogen: 200 kg.ha",
300 kg.ha'1, 200 kg.ha'1 poultry manure, and 200 kg.ha' slow releasing
nitrogen. Standard errors are given in parentheses. ................................ 89

Table 4. Effect of foliar applications of 2,4-D concentrations on the anthocyanin
concentration, hue, lightness, and chroma of the skin of ‘Michigan Purple’

viii

potatoes. Tubers were harvested 12 days after the second 2,4-D
application. Anthocyanin analyses were done by using a
spectrophotometer. Standard errors are given in parentheses. ................. 89

CHAPTER 3

Table 1. Anthbcyanin (pg/g) concentrations in the skin of ‘Michigan Purple’ potato
tubers after 60 days of storage at different combinations of 4 °C. 20 °C
and 30 °C. Analyses were done by using a spectrophotometer. Standard
errors are given in parentheses. .............................................................. 107

Table 2. Differences in hue angle, lightness, and chroma between ‘Michigan
purple‘ potatoes stored under different conditions. Standard errors are
given in parentheses. .............................................................................. 108

APPENDIX A

Table 1. Impact of storage duration at 10 °C on total anthocyanins (pg/g), of
‘Michigan Purple’, “Dakota Rose’, ‘CaIifomia Red’, and ‘Norland’ potatoes.
HPLC absorbance values of total anthocyanins are given. HPLC
absorbance values (au) at 530 nm also are given for comparison between
cultivars.
.................................................................................................................. 114

Table 2. Impact of storage duration on anthocyanin and anthocyanidin content of
‘Michigan Purple’, ‘California Red’, ‘Norland’, and ‘Dakota Rose’ potato
tubers stored at 10 °C. ............................................................................ 116

Table 3. Impact of storage duration on the anthocyanin and anthocyanidin
content of ‘Michigan Purple’, ‘California Red’, ‘Norland’, and ‘Dakota Rose’
potato tubers stored at 10 °C. .................................................................. 117

Table 4. The effect of storage duration on the pigment (anthocyanin and
anthocyanidin) concentration (ug/g), hue, lightness, and chroma of the
skin of potato cultivars ‘Chieftain’, ‘Dakota Rose’ and “Michigan Purple'
stored at 4 °C in 2003. ............................................................................. 118

ix

APPENDIX B

Table 1.The effect of soil type and plant spacing on anthocyanin and
anthocyanidin (pg/g) concentrations in the skin of ‘Michigan Purple’
potatoes at harvest and after 2 and 4 months of storage at 10 °C. ........... 130

Table 2. The effect of soil type and plant spacing on anthocyanin and
anthocyanidin (pg/g) concentrations in the skin of ‘Michigan Purple’
potatoes at harvest and after 2 and 4 months of storage at 10 °C. .......... 131

Table 3. The effect of different soil types on the total pigments (anthocyanin and
anthocyanidin) concentration, total absorbance at 530 nm, hue (h°),
lightness, and chroma of the skin of ‘Michigan Purple’ tubers stored at 10
°C. ........................................................................................................... 132

Table 4. The anthocyanins concentration and color of the skin of ‘Michigan
Purple’ tubers as a function of nitrogen source and rate, and storage
duration at 4 °C. Treatments were 1 = 200 kg.ha‘1 N, 2 = 300 kg.ha‘1 N, 3
= 200 kg.ha'1 poultry manure and 4 = 200 kg.ha'1 slow releasing N. ........ 133

LIST OF FIGURES

LITERATURE REVIEW

Figure 1. The different types of flavonoids and their structures. .......................... 42

Figure 2. The basic structure of all flavonoids. The A-ring is formed from 3
acetate units (via the malonic acid pathway) and the B-ring with the 3-
carbon bridge is constructed from phenylalanine via the shikimic acid
pathway. ................................................................................................... 43

Figure 3. The phenylpropanoid pathway leading to the synthesis of anthocyanins.
The enzymes involved in the reactions are: PAL: phenylalanine
ammonialyase; C4H: cinnamate-4-hydroxylase; 4CL: 4-coumaryI-CoA
Iigase; CHS: chalcone synthase; CHI: chalcone-flavanone isomerase;

F3H: flavanone 3-hydroxylase; F3‘H: flavonoid 3‘-hydroxylase; F3‘5'H:
flavonoid 3‘,5'-hydroxylase; DFR: dihydroflavonol reductase; ANS:
anthocyanidin synthase; GT: UDPG-flavonoid-3-glucosyltransferase; RT:
anthocyanidin-3glucoside rhamnosyltransferase; FS: flavone synthase

and FLS: flavonol synthase. ..................................................................... 44

Figure 4. The structures of the six most common anthocyanidins, differing in
hydroxylation and methylation positions. .................................................. 46

Figure 5. The structural transformation of anthocyanins in aqueous solutions
(adapted from Brouillard, 1983). ............................................................... 47

CHAPTER 1

Figure 1. The effect of storage at 10 °C on the pigment (anthocyanin and
anthocyanidin) concentrations of the cultivars ‘Michigan Purple’ (MIP),
‘Dakota Rose’ (DR) “California Red” (CR), and ‘Norland’ (N). Vertical bars
refer to standard error of the means (n=20). Anthocyanin analyses were
done by using a spectrophotometer. ........................................................ 64

xi

Figure 2. The effect of storage at 10 °C on (a) the hue angle values, (b) the
lightness and (c) chroma of the skin of ‘Michigan Purple’ (MIP), ‘Dakota
Rose’ (DR), ‘California Red’ (CR), and ‘Norland’ (N) potatoes. Vertical
bars refer to standard error of the means (n=20). ..................................... 65

Figure 3. The effect of storage at 4 °C on the pigment (anthocyanin and
anthocyanidin) concentrations in the skin of ‘Michigan Purple’ (MIP),
‘Dakota Rose’ (DR), and ‘Chieftain’ (CHT) tubers. Vertical bars refer to
standard error of the means (n=30). Anthocyanin analyses were done by
using a spectrophotometer. ...................................................................... 67

Figure 4. The effect of storage at 4 °C on the (a) hue angle values, (b) lightness,
and (c) chroma of the skin of ‘Michigan Purple’ (MIP), ‘Dakota Rose” (DR),
and ‘Chieftain’(CHT) potatoes. Vertical bars refer to standard error of the
means (n=30). .......................................................................................... 68

Figure 5. The effect of stage of development of the pigment content (pg/g) in the
skin of ‘Michigan Purple’ tubers. Vertical bars refer to standard errors of
the means (P < 0.05, LSD). Anthocyanin analyses were done by using a
spectrophotometer. .................................................................................. 69

CHAPTER 2

Figure 1. The effect of storage at 10 °C on the anthocyanin and anthocyanidin
(pg/g) concentration in the skins of ‘Michigan Purple’ potato tubers grown
in organic soil, 0.3 m apart, and in mineral soil, 0.2 and 0.33 m apart.
Vertical bars refer to standard errors of the means (n=40). Anthocyanin
analyses were done by using HPLC. ........................................................ 90

Figure 2. The effect of storage at 10 °C on the (a) hue angle, (b) lightness, and
(c) chroma of the skins of ‘Michigan Purple’ tubers grown in organic soil,
0.3 m apart, and in mineral soil, 0.2 and 0.33 m apart. Vertical bars refer
to standard errors of the means (n=40). ................................................... 91

Figure 3. The effect of different nitrogen treatments: 200 kg.ha'1, 300 kg.ha", 200
kg.ha'1 poultry manure, and 200 kg.ha'1 slow-releasing nitrogen, on the
anthocyanin and anthocyanidin (pg.g'1) content in the skins of ‘Michigan
Purple’ potato tubers at harvest and in storage. Vertical bars refer to

xii

standard error of the means (n=20). Anthocyanin analyses were done by
using a spectrophotometer. ...................................................................... 93

Figure 4. The effect of different nitrogen treatments: 200 kg.ha'1, 300 kg.ha", 200
kg.ha'1 poultry manure, and 200 kg.ha'1 slow-releasing nitrogen, on the (a)
hue angle, (b) lightness and (c) chroma of the skins of ‘Michigan Purple’
tubers at harvest and in storage. Vertical bars refer to standard error of the
means (n=20). ........................................................................................... 94

CHAPTER 3

Figure 1. The effect of exposure of 100 ppm ethylene for 7 days on the hue angle
of ‘Michigan Purple’ potatoes stored at 4 °C after receiving treatment.
Vertical bars refer to the standard errors (n=25). ..................................... 110

APPENDIX A

Figure 1. The HPLC chromatogram of ‘Michigan Purple’ potato (a) and the
spectra of (b) peak 1, (c) peak 2, (d) peak 3 and (e) the standard,
petunidin-chloride (0.25 ug/pl), used to quantify the pigment content in
‘Michigan Purple’ potatoes. ..................................................................... 119

Figure 2. The HPLC chromatogram (a) of ‘California Red’ potato with the spectra
of (b) peak 1, (c) peak 2, and (d) peak 3. ................................................ 122

Figure 3. The HPLC chromatogram (a) of ‘Norland’ potato with the spectra of (b)
peak 1 and (c) peak 2. ............................................................................. 125

Figure 4. The HPLC chromatogram of “Dakota Rose’ potato (a) and the spectra
of (b) peak 1, (c) peak 2, (d) peak 3 and (e) the standard, pelargonidin 3,5-
diglucoside (1.5 ug/ul).used to quantify the pigment content in ‘Norland’,
“California Red’, and ‘Dakota Rose’ potatoes. ......................................... 126

APPENDIX C

xiii

Figure 1. The effect of 60 days storage at different temperatures on the hue
angle of ‘Michigan Purple’ potato tuber skins. Vertical bars refer to
standard errors of the means (n=30). ...................................................... 135

xiv

INTRODUCTION

Pigmented potatoes and other colored underground organs are currently
receiving more market interest for their aesthetic and health properties,
especially in the food and restaurant industry. Colored potatoes currently account
for 10% of the $ 3.06 billion production in the United States (USDA, 2003). The
potato (Solanum tuberosum L) is an important crop worldwide, ranking fourth in
importance after rice, wheat, and maize.

Color intensity at harvest and during storage is an essential quality factor
for colored potatoes if growers want to receive premium prices. In 2001, a purple
variety, ‘Michigan Purple’ was released by the Michigan State University
breeding program (Douches et al., 2001 ). This is an attractive round potato with
white flesh and purple skin color, rather than red, which makes it unusual in the
tablestock market. The color has been reported to degrade in storage. Thus, to
ensure adequate color, it is important to develop pre- and postharvest strategies
to maintain or increase the stability and the concentration of anthocyanin
pigments.

The anthocyanin pigments responsible for the flesh and skin color of
various novelty potatoes have recently been identified (Fossen and Andersen,
2000; Lewis et al., 1998 a, b; Naito et al., 1998; Rodriguez-Saona et al., 1998).
The anthocyanins in ‘Michigan Purple’ have not been identified or quantified

despite the importance of color for successful marketing of this variety.

Currently there is no data available on the extent of color loss in the skin
during storage, or on pre- or postharvest factors to maintain or enhance
anthocyanins.

This project aims to investigate factors affecting color development and
stability during production and storage which may be important for the
marketability of this new purple-skinned variety. Specifically, the results from this
research will provide the information on changes in color of ‘Michigan Purple’
during storage that will allow growers and storage operators to predict the
storage life of the tubers, relative to visual quality. In addition, the research
results provides insights into preharvest recommendations for improving tuber
skin-color such as soil type and planting space, the use of growth regulator 2,4-
D, and the application of nitrogen fertilizer and manure. Postharvest
recommendations will include optimum storage temperature and the possible
application of ethylene to improve and/or maintain color during production and

storage.

References

Douches, D. S., K. J. Felcher, J. Coombs, C. Long, R. Hammerschmidt and W.
W. Kirk. 2001. Michigan Purple: A purple-skinned tablestock variety.
Amer. J. Potato Res. 78:421-424.

Fossen, T. and O. M. Andersen. 2000. Anthocyanins from tubers and shoots of
the purple potato, Solanum tuberosum. J. Hort. Sci. Biotechnol. 75:360-
363.

Lewis, C. E., J. R. L. Walker, J. E. Lancaster and A. J. Conner. 1998a. Light
regulation of anthocyanin, flavonoid, and phenolic acid biosynthesis in
potato minitubers in vitro. Aust. J. Plant Physiol. 25:915-922.

Lewis C. E., J. R. L. Walker, J. E. Lancaster and K. H. Sutton. 1998b.
Determination of anthocyanins, flavonoids, and phenolic acids in potatoes.
l: Colored cultivars of Solanum tubersosum L. J. Sci. Food Agr. 77:45-57.

Naito, K., U. Yoshiki, M. Mori, T. Sumida, T. Okada., N. Takamatsu, O. Yutaka,
K. Hayashi, N. Saito and T. Honda. 1998. Acylated pelargonidin
glycosides from a red potato. Phytochemistry 472109-112.

Rodriguez-Saona, L. E., M. M. Giusti and R. E. Wrolstad. 1998. Anthocyanins in
vegetative tissues: A unified function in photoprotection. New Phytol.
155:349-361.

United States Department of Agriculture. 2003. Potatoes 2002. National
Agricultural Statistics Service. http:/www.usda/nass 12/10/2003

LITERATURE REVIEW

Introduction

Color is an important aesthetic attribute of most fresh and processed fruit
and vegetables. In addition, colored commodities are becoming increasingly
popular since the discovery of the health benefits of the pigments that are
responsible for the color. Hence, interest in pigmented potatoes and products
from pigmented potatoes is growing. These potatoes represent a niche market
with potential for the gourmet industry based on color. Fortunately, the presence
of color (i.e. anthocyanin pigments) in the tuber skin and/or the tuber flesh makes
them of potential benefit to human health. Available evidence suggests that
anthocyanins could exhibit multiple biological effects such as anti-inflamatory
action, antioxidant-antiradical activity, inhibition of blood platelet aggregation and
antimicrobial activity, treatment of diabetic retinopathy, and prevention of
cholesterol-induced atherosclerosis (Clifford, 2000; Espin et al., 2000; Mazza and

Miniati, 1993; Wang et al., 1997).

Potato History and Importance

Potatoes are the fourth most important crop in the world after rice, wheat,
and maize (Salazar and Busch, 2001). It is believed that the potato originated in
South America, specifically in the Andean highlands of Bolivia, Ecuador and Peru
and was first domesticated more than 6000 years ago in the area around Lake
Titicaca (Burton, 1948). In 1532, the Spanish took the potato to Europe where it

adapted very well to the climate and soon became an important crop there. It is

believed that the potato arrived in North America about 50 years later. Native
Andean potatoes are still cultivated today and display an extraordinary diversity
of taste and texture and come in a fascinating array of shapes and colors.

In the United States today, about 1.3 million acres are planted with
potatoes and the industry itself has an estimated value of $3 billion (USDA,
2003). Approximately 37% are utilized as frozen products, 12% as dehydrated
products, 12% as chips and shoestrings, 31% as table stock, 6% as seed and
2% for other uses such as starch, flour and canned products (Salazar and Busch,
2001)

In the United States, potatoes are classified by the USDA into three
categories: round whites, russets and red-skinned. Potatoes in each category
share similar characteristics for example the red-skinned category generally have
a low dry matter content and are targeted to the fresh market. Red potatoes
comprise approximately 10% of the total potato acreage in the United States
today (Andersen et al., 2002). Currently, the most popular red-skinned varieties
are ‘Red Pontiac’ and ‘Norland’ (Salazar and Busch, 2001).

In contrast to the past, there is an increase in the amount of colored
potatoes available on the market today. Varieties with purple, blue, and red skin
and flesh have been released with names such as ‘French Fingerling’, ‘AII Blue’,
‘Chieftain,’ ‘Norland,’ ‘Dark Red Nonand’, and ‘Red Pontiac’. These colored
potatoes are referred to as ‘specialty’ potatoes and are intended to appeal to

restaurateurs, caterers, and home chefs.

Tuber Quality

Visual appeal, consumer preference, and ability to meet market
specifications are criteria of tuber quality. Tuber quality assessment differs
depending on the intended use of the tubers; potatoes targeted for the fresh
market or for the French fry or chip industries are evaluated differently. In 1991,
The United States Department of Agriculture released ‘The United States
Standards for Grades of Potatoes’ in which details regarding potato grades are
reported (USDA, 1991 ). Here follows a summary of some of the factors that are
taken into account to determine quality.

Fresh market: Important tuber quality factors are uniformity in size and
shape, unmarked skin, flavor, flesh firmness, shallow eyes, and absence of
diseases or defects such as internal brown spot (internal rust spot), internal heat
necrosis, glycoalkoloids (greening), and mechanical damage.

‘French fry industry. The most important quality factors are flavor, absence
of defects, concentration of reducing sugar (sugars that contain aldehyde groups
that can be oxidized to carboxylic acids), and dry matter. Dry matter is measured
as specific gravity, which increases as the water content of the potato decreases.
The French frying process dehydrates the tuber flesh, thus tubers with low
specific gravity (high water content) will result in major water loss and lower fry
yield. Specific gravity also affects oil absorption. Tubers with low specific gravity
absorb more oil than those with high specific gravity, resulting in greasy fries.
The reducing sugar concentration influences the color of the fries. Tubers with a

high reducing sugar concentration become dark French fries since the sugars

react with amino acids in the tuber in a non-enzymatic Maillard-type browning
reaction.

Chip industry. The best chip potatoes have good flavor, absence of
bitterness and off-flavors, low reducing sugars concentration, and high specific
gravity. The color of chips depends on the concentration of the reducing sugars
in the potato for the same reasons mentioned for French fries; high reducing
sugar concentration will result in dark chips. High specific gravity is extremely
important in the chipping industry since chip crispness and lack of oiliness greatly
depends on it.

Color is a factor that has increased in importance since colored varieties
started entering the mainstream market. Although potatoes of different color and
shape, apart from some red-skinned cultivars, are not yet readily available in big
retail stores, these potatoes can be found in smaller specialty stores. Since the
primary selling point of pigmented cultivars is color, good color is a necessity for
cultivars such as ‘Red Pontiac’, ‘Norland’ and ‘All Blue’. Good color development
starts in the field, thus growers can make use of certain growing practices to

enhance the anthocyanin synthesis in the field.

Preharvest Enhancement of Anthocyanins

In order to sustain quality after harvest, growers have to make correct
preharvest management decisions. Nitrogen (N) management is important for
obtaining appropriate plant productivity and color. Nitrogen is a critical nutrient for

potato plants since it has a great effect on yield and tuber quality. Inadequate

amounts of N will reduce yields, whereas an excess can reduce tuber quality and
tuber yield.

Nitrogen fertilizer sources include ammonium sources such as urea,
ammonium sulfate, diammonium phosphate, monoammonium phosphate, and
ammonium polyphosphate. These sources are used at planting time since the
nitrogen is in the ammonium form (NH4’) which is less likely to leach into the soil,
as is the case with ammonium nitrate or calcium nitrate (NOa') (MSU extension,
2004). Organic matter such as manure can also be used as a source of nitrogen.

Callus and cell suspension culture studies on grapes have shown that the
total amounts of nitrogen, as well as the ratio of N03- to NH4+ strongly affect
anthocyanin production and cell growth (Kim and Kim, 2002). To our knowledge,
the impact of nitrogen form and application on the development of the
anthocyanins in the tuber skin during tuber development and in storage has
never been documented.

The second potential preharvest color enhancement strategy is the use of
2, 4-dichlorophenoxyacetic acid (2,4-D). Foliar applications of hormones such as
this synthetic auxin have been used commercially to enhance the color of the red
potatoes. 2,4-D stimulates the synthesis of ethylene, which is thought to promote
the production of anthocyanins in the periderrn (Nelson and Bristol, 1975)

The use of 2,4-D as a potential color enhancer became of interest in the
19403. One of the first reports of auxin as a color enhancer was from that of Fults
and Schaal (1948). They observed an improvement in the skin color of red-

skinned ‘Bliss Triumph’ aka ‘Red Bliss’ in plants fertilized with 20 lbs per acre of

10

the sodium and ammonium salts and the butyl ester of 2,4-D. Payne et al. (1950)
later proved that 2,4-D also improves the color of ‘Red McClure’ and that the
improved skin color did remain stable in storage. The efficiency of 2,4-D as a
color enhancer, depends on the application rate and the stage of development of

the plant at the time of application (Nylund, 1955).

Postharvest Enhancement of Anthocyanins

Maintaining storage conditions after harvest affect the appearance of the
potato including color and anthocyanin concentration. Appropriate temperature
and ventilation are extremely important for maintaining tuber quality. Tubers
intended for table use are usually stored at 4°C and at high relative humidity
(RH). Those intended for processing are stored at higher temperatures such as
10°C and high RH in order to prevent starch converting to sugar, which as
previously noted, can cause potato tissue to darken when fried or dehydrated
(Sowokinos, 2001).

In addition, storage temperature is also important to maintain good tuber
color. Lewis et al. (1999) found that anthocyanins increased in the skin and/or the
flesh of ‘Arran Victory’ (purple skin/white flesh), ‘Desiree’ (pink skin/ white flesh)
and ‘Urenika’ (purple skin/ purple flesh) tubers over long term storage at 4°C,
while tubers stored at 10, 18, and 26 °C did not have an increase in
anthocyanins. They argued that reducing sugars act as precursors for
anthocyanins and low temperatures stimulate the synthesis of sugars and thus

increase the formation of anthocyanins. Sugars can act as precursors to

11

anthocyanins by either providing the carbon skeletons of the anthocyanins
molecule, or through the glycosylation of the anthocyanins. Conversely,
Andersen et al. (2002) found that color and anthocyanin concentration decreased
in red-skinned ‘Norland’ tubers after one month of storage at 4 °C. It is possible
that this discrepancy in results is due to differences in response of different
varieties to temperature.

In apples it has been found that ethylene can increase the rate of
anthocyanin synthesis (Faragher and Brohier, 1984). Ethephon, an ethylene
generating compound, is commonly applied preharvest as a foliar application to
apple trees shortly before harvest to advance the marketing season. It results in
an increase in the concentration of anthocyanins, presumably through the
increase of the phytohormones chalcone isomerase (CHI) and phenylalanine-
ammonialyase (PAL) (Li et al., 2002). To our knowledge, applying ethylene
postharvest to increase the periderm color of colored potatoes has not been

tested.

Biology and Biosynthesis of Anthocyanins

The term ‘anthocyanin’ was first used by Marquart (1835) to describe blue,
violet, and red pigments derived from plants. Anthocyanins are part of a diverse
class of secondary metabolites known as phenolic compounds. Phenolic
compounds, synthesized primarily from the shikimic acid pathway, presumably in
the chloroplast (Taiz and Zeiger, 1998), accumulate in the vacuole of the plant

cell. Phenolic compounds can be divided into two classes based on their

12

structure: non-flavonoids and flavonoids. Non-flavonoids include phenolic acids
and coumarins; flavonoids include anthocyanins, flavonols, flavan-3-ols,
flavones, flavanones, flavanonols, condensed tannins, flavan-3-4-diols,
chalcones, and isoflavanoids (Figure 1). Anthocyanins, therefore, are
synthesized in the cytoplasm but are ultimately localized in the vacuoles
(Hrazdina, 1992; Matem et al., 1986; Wagner and Hrazdina, 1984).

Anthocyanins are the most widespread group of pigmented flavonoids and
are responsible for most of the red, pink, purple, and blue colors observed in
various plant organs such as the flowers, fruit, roots, stems, leaves, and bracts
(Taiz and Zeiger, 1998).

Flavonoids have a basic carbon skeleton containing 15 carbons (C6-C3-
C6) with 2 aromatic rings connected by a 3-C bridge (Taiz and Zeiger, 1998)
(Figure 2). The carbon skeleton of all flavonoids is derived from acetyl CoA and
phenylalanine. The A-ring is formed from three acetate units and phenylalanine
gives rise to the B-ring and the 3-C chain of the heterocyclic C-ring (Macheix et
al., 1990). The flavonoid biosynthesis pathway starts with phenylalanine which is
metabolized to cinnamate by the enzyme phenylalanine ammonia lyase (PAL).
Cinnamate in turn forms p-coumarate through the enzyme cinnamate 4-
hydroxylase (C4H). p-coumarate eventually forms p-coumaroyl coenzyme A
(CoA) through the action of enzyme 4-coumarate: CoA Iigase (4CL) (Lewis et al.,
1998a) (Figure 3).

The next step is the formation of the yellow C-15 skeleton naringenin

chalcone (4,2’,4’,6’ -tetrahydroxychalcone) through the condensation of one

13

molecule of 4-coumaroyl CoA with three molecules of malonyl CoA. This step is
catalyzed by chalcone synthase (CHS) (Koes et al., 1994). The malonyl CoA
needed for the reaction is derived through the carboxylation of acetyl CoA.
Naringenin chalcone or the isomeric flavanone is the central intermediate in the
synthesis of all flavonoids (Macheix et al., 1990). Naringenin chalcone is then
isomen'zed by chalcone isomerase (CSI) to the colorless naringenin (5, 7, 4’-
trihydroxyflavanone). Naringenin is converted to dihydrokaempferol (DHK) by
flavanone 3-hydroxylase (F3H) or alternatively to the flavones by flavone
synthase (FS).

From here, DHK can be hydroxylated by flavonoid 3’hydroxylase (F3’H) to
produce dihydroquercetin (DHQ) or by flavonoid 3’, 5’hydroxylase (F3’5’H) to
form dihydromyricetin (DHM) (Holton and Cornish, 1995). Flavonol synthase
(FLS) can catalizes the formation of flavonols kaempherol, quercetin and
myricitin from DHK, DHQ and DHM, respectively. In order to form anthocyanins,
the enzyme dihydroflavonol reductase (DFR) reduces the dihydroflavonols (DHK,
DHQ and DHM) to flavan-3,4-diols (leucoanthocyanidins) (Seigler, 1998).
Anthocyanidin synthase (ANS) catalyzes oxidation and dehydration of these
compounds to produce anthocyanidins. Anthocyanins are formed by adding a
sugar to the aglycone by UDP-glucose-flavonoid-3-glycosyltransferase (3GT).
The enzyme, anthocyanidin-3-glucoside rhamnosyltransferase (RT), catalyzes

the addition of rhamnosyl to the glucose of the anthocyanin molecule.

Induction of anthocyanin biosynthesis

14

Photoinduction by wavelengths in the UV, visible and far-red regions is
probably the most important environmental factor to play a role in anthocyanin
biosynthesis in above-ground plant parts. It has been found that in most systems
little or no anthocyanin is formed in darkness (Camm et al., 1993; Dong et al.,
1998; Grisebach, 1982; Kakegawa et al., 1987) and that light enhances the rate
of synthesis of anthocyanins and flavonoids (Lewis et al., 1998).

The enzymes in the phenylpropanoid pathway (phenylalanine ammonia-
lyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-coumarate: CoA Iigase
(4CL) reach maximal activity after 15 hours of irradiation (Hahlbrock et al. 1976).
Anthocyanin accumulation is directly associated with phenylalanine ammonia-
lyase (PAL) activity in apples (Mazza and Miniati, 1993). Furthermore, PAL
activity has only been observed in the red part of apple skin.

Other triggers for anthocyanin biosynthesis are cold stress (Shichijo et al.,
1993; Christie et al., 1994; Graham, 1998), osmotic stress induced by sucrose
(Corrnier et al., 1989; Decendit and Merillon, 1996), glucose (T holakalabavi et al.,
1997) and mannitol (T holakalabavi et al., 1994), deficiency in nitrogen (Bongue-
Bartelsman and Philips, 1995; Do and Corrnier, 1991), deficiency in phosphorus
(Dedaldechamp et al., 1995; Rajendran et al., 1992; Trull et al., 1997), low pH
(Suzuki, 1995), exposure to methyl jasmonate (Franceschi and Grimes, 1991),
wounding (Ferreres et al., 1997), pathogen infection (Dixon et al., 1994), and
exogenous growth regulators such as auxin and cytokinins (Rajendran et al.,

1992; Sakamoto et al., 1994).

15

The biosynthesis of color in underground organs is not well understood.
Lewis et al. (1998a) investigated the factors that regulate the biosynthesis of
anthocyanins and flavonoids in colored potato tubers. They hypothesize that the
exposure of the aerial parts of the plants to light induces a ’trigger' molecule
which is then transported to the tubers to initiate anthocyanin biosynthesis.

Although one could speculate that anthocyanins are dependent on
carbohydrates from photosynthesis, plants grown in the dark, supplied with a
carbohydrate source (sucrose), produced tubers, but the tubers did not
accumulate anthocyanins. Further evidence to support a ‘trigger’ molecule
includes the polar nature of anthocyanin accumulation in tubers of plants
exposed to light, seeming to appear at the stem end first and then gradually
accumulating across the tuber over time. Further work is required to identify the
nature of this ‘trigger’ molecule.

After growing potato plants in the dark, it was found that some cultivars
that usually produce highly pigmented tubers still produced limited color in the
tubers, however, less colored cultivars failed to produce any color in the dark.
Both types produced significantly higher amounts of phenolics including
flavonoids and anthocyanins after exposure to white light.

The authors suggest in the dark the precursor(s) of these compounds are
depleted in an earlier part of the pathway such as phenolic acid production
and/or that phenolics, flavonoids, and anthocyanins are all controlled by different
control mechanisms. That the various components of the phenolic pathway are

governed by different control mechanisms is supported by the fact that in the

16

tubers of ‘Desiree’, a less intensely colored variety, the anthocyanin pathway is
virtually inactive while the phenolic and the flavonoid pathways show some
activity, judging by end products.

Lewis et al. (1998a) suggest that light causes the preferable diversion of
the flavonoid intermediates (naringenin-chalcone and naringenin) to synthesize
anthocyanin whereas in the absence of light, only the biosynthetic pathway of the
flavonoid precursors and the branch pathway to catechin (a flavan-3-ol) appear
to be active. After exposure of minitubers to light, there appears to be an
increase in PAL and other enzymes downstream from PAL. This correlates with
the rate of increase in anthocyanin, fiavonoid and phenolic biosynthesis. This
derepression of the enzymes seems to require the exposure of the leaves to

light, and not the tubers.

The role of glutathione-s-transferases in the accumulation of anthocyanins
Glutathione-S-transferases (GSTs) are enzymes responsible for the
conjugation of the tri-peptide glutathione (GSH) to hydrophobic, electrophilic, and
usually cytotoxic substrates (Marrs, 1996). They can be found in both animal and

plant organisms. Plant GSTs were first identified for their ability to detoxify
herbicides. It was found that the genes of a specific subclass of GSTs termed
type III GSTs, are induced by auxin and ethylene treatments and numerous
stresses including oxidative stress, pathogen attack, and heavy-metal toxicity.
The GST enzymes also play a role in the normal metabolism of secondary

metabolites such as anthocyanins. According to lrzyk et al. (1995), anthocyanin

17

pigments require GSH conjugation for transport from the cytoplasm into the
vacuole. If the anthocyanin pigment remains too long in the cytoplasm, it
becomes toxic to the cell and the further production of the anthocyanin is
jeopardized.

Thus, if 2,4-D or ethylene are applied to the plants, it will cause the
upregulation of GST genes and the synthesis of GST enzymes. These enzymes
will conjugate glutathione to the anthocyanin precursors in the cytoplasm and will

eventually allow for it to be transported into the vacuole.

Primary structure of anthocyanins
Anthocyanidins

Anthocyanidins are the structural pigment part (i.e. chromophore) of an
anthocyanin molecule. It is also called the aglycone as opposed to anthocyanins
which are called the glycone. Macheix et al., (1990) define anthocyanindins as
the hydroxylated and methoxylated derivatives of phenyl-2-benzopyrilium. Today
there are 15 known anthocyanidins, although only six (pelargonidin (Pg), cyanidin
(Cy), delphinidin (Dp), peonidin (Pn), petunidin (Pt), and malvidin (Mv)) are
widespread and contribute to pigmentation of plants (Mazza and Miniati, 1993).
These common six anthocyanidins differ in the number of 3’ and 5’ hydroxy and
methoxy groups on the B ring (Macheix et al., 1990) (Figure 4).

Three anthocyanidins, Pg, Cy and Dp, provide the whole range of flower
color from pink, orange and scarlet to mauve, violet and blue, either singly or as

mixtures (Goodwin, 1965). Cyanidin is the most commonly found anthocyanidin

18

in fruits (90%). It is followed in decreasing order by Dp (35%), Pn (30%), Pg

(20%) and Pt and Mv (15%).

Anthocyanins

A relatively unstable anthocyanidin becomes a more stable anthocyanin
when a sugar is added to the aglycone. Anthocyanins can be modified by
glycosylation, methylation, and acylation (Koes et al., 1994). An anthocyanidin
may be glycosylated and acylated by different sugars and acids, therefore there
are 15-20 times more anthocyanins than anthocyanidins. The most common
sugars to bind to anthocyanidins are glucose, galactose, rhamnose, and
arabinose. Glycosylation at position 3 on the C-ring results in monoglycosides.
The most common substitutes for monoglycosides are glucose, arabinose and
galactose (Mazza and Miniati, 1993). For further variation these sugars can also
bind in combinations in the form of di- and trisaccharides, or the sugar residues
can be acylated by c-coumaric, caffeic, ferulic, sinapic, p-hydroxybenzoic,

malonic, oxalic, malic, succinic or acetic acids (Macheix et al., 1990).

Color change through structure change
Hydroxylation, methylation, and glycosylation

Increasing the number of hydroxyl groups on the B-ring causes the visible
absorption maximum of the anthocyanidin to shift to longer wavelengths, causing
a color change from orange to blue (bathochromic shift) and the destabilization of
the molecule (Hrazdina et al., 1970). Methylation of the free hydroxyl groups on

the B-ring (Mazza and Miniati, 1993) and the addition of a sugar to the

19

anthocyanidin molecule (Goodwin, 1965) both cause a perceived reddening in
color, also referred to as a hypsochromic shift. In addition, methylation of the B-

ring will stabilize the molecule (Figure 4).

Stability
Stability in vivo

pH and copigmentation are the primary factors affecting stability and color
of the anthocyanin pigment. Shifts in pH can alter pigment structure (Mazza and
Miniati, 1993). Four basic pH.dependant forms exist (Figure 5). At pH 52, the
anthocyanins exist only in the orange to red flavylium cation (AH’). Raising the
pH to pH 4-5 causes a fast acid-base reaction. The hydroxyl groups at position 4’
and 7’deprotonate to form the blue quinonoidal base (Macheix et al., 1990). The
first deprotonation reaction occurs at about pH 4 (A) and the second at pH 7 (A').

While this reaction is occurring, a slower pseudo-acid-base reaction also
takes place. The water molecules act as nucleophiles, attacking the flavylium
cation at positions 2 and/or 4 and forming the colorless hemiacetal also known as
pseudobase or carbinol (B). This reaction accounts for the color loss of the
pigment. The red flavylium can be restored with acidification, but the colorless
carbinol can open the C-ring to form a yellow or colorless unstable chalcone (C)
which can undergo irreversible degradation (Brouillard et al., 1997).

Copigmentation is a color stabilization mechanism that prevents or
reduces nucleophilic addition of water to the flavium cation. It occurs from pH 1 to

neutrality. A copigment has no color of its own, but when added to a solution

20

containing anthocyanins it will stabilize, enhance, and modify color (Mazza and
Miniati, 1993). Addition of copigments can cause a shift in the max towards a
bluer color (bathochromic shift) and increase color intensity (hyperchromic shift).

There are two kinds of copigmentation: intramolecular and intermolecular.
lntramolecular copigmentation usually refers to the covalent binding of aliphatic
and/or aromatic organic acids to the sugars on the anthocyanin molecule, whilst
intermolecular copigmentation occurs when other compounds binds non-
covalently to the anthocyanin molecule. Compounds that act as copigments
include polyphenols, flavonoids, nucleic acids, and amino acids. The type and
the concentration of the anthocyanin and the co-pigment, as well as the pH and
the temperature of the medium can influence co-pigmentation (Mazza and

Miniati, 1993).

Stability during processing
Enzymatic degradation

According to Macheix et al. (1990) there are several enzyme systems
involved in anthocyanin degradation: IS-glycosidase (also called anthocyanases),
peroxidases and polyphenol oxidases (PPO). B—glycosidase hydrolyzes the
anthocyanin to the anthocyanidin (aglycone) and glycoside, followed by the
decomposition of the anthocyanidin (Huang, 1955). Peroxidase is found to

enhance the degradation of the aglycone.

High temperatures

21

High temperatures are often used to prevent PPO activity in fruit and thus
protecting the anthocyanins from degradation, e.g. blanching fruit prior to
processing or preservation. Conversely, thermal degradation of anthocyanins has
been studied for strawberry (Markakis et al., 1957), black raspberry (Daravingas
and Cain, 1968), raspberry (Tanchev, 1972), Concord grape (Calvi and Francis,
1978), and sour cherry (Cemeroglu et al, 1994). Raising temperatures can
accelerate color loss in fmit juices. However, a short time/high temperature
process is recommended for retention of pigments in red fruit juices since
anthocyanin loss seems negligible for treatments exposed to 100 °C for less than

12 min (Macheix et al., 1990).

Light

Light is needed for the synthesis of anthocyanins, however light can also
accelerate the degradation of anthocyanins. For this reason, fruit juices are
generally stored in the dark. Acylated forms of anthocyanins are the most light
stable. Copigmentation with polyhydroxylated flavonoids, isoflavonoids, and

aurone sulfonates can make anthocyanins light-stable (Macheix et al., 1990).

Analytical Techniques for Anthocyanin Identification and/or Quantification

There are several techniques for isolating and identifying anthocyanins. Here
follows three techniques, see review on analysis techniques by Kong et al.
(2003) for more.

High performance liquid chromatography-HPLC

22

Since the 1980’s the use of HPLC in identifying anthocyanins has become
very popular. The concept of HPLC is based on the separation of a mixture of
components by passage through a chromatographic column. The mobile phase,
which contains the mixture of components, passes through the stationary phase
which is a column filled with solid particles. The components of the solution
migrate according to the non-covalent interactions of the compound with the
column. The chemical interactions of the mobile phase and sample with the
column determine the rate of migration and separation of components contained
in the sample (Fallon et al., 1987).

Spectrophotometry

Spectrophtometry is used to measure the extent to which solutes absorb
light at specified wavelengths in the region of 200—800 nanometers. This
technique has important applications: the amount of light absorbed at a specified
wavelength can produce information on the concentration of a solute; a reaction
rate can be determined by plotting absorbed light as a function of time; a plot of
absorbed light at different wavelengths (absorption spectrum) can give
information on the chromophore (the part of the molecule that absorb light), and
can help in the identification of the compound.

NMR

Nuclear magnetic resonance (NMR) is a phenomenon that occurs when
the nuclei of certain atoms with gyromagnetic properties such as hydrogen are
immersed in a static magnetic field and, when exposed to a second oscillating

magnetic field, will tend to align itself with that field by spinning at a rate that is

23

proportional to the strength of the applied field. If the sample is radiated at the
precessional frequency, absorption of energy to the excited state takes place.
When the sample returns to the normal state, energy will be released and this will
be measured. The energy is directly related to the number and kind of nuclei in
the molecule. A NMR spectrum is constituted from a plot of frequency, relative to
a standard, versus intensity (Mussinan, 1993). This technique is used for
structure determination and peak identification of anthocyanin molecules (Mazza

and Miniati, 1993).

Anthocyanin Functions in Plants

From an ecological point of view, the ability of anthocyanins to add color to
plants is probably their most significant function. Color in plants contributes to
pollination, fertilization, and seed dispersal by animals. Anthocyanins can also
act as a light screen against damaging UV radiation (reviewed by Steyn et al.,
2001) and photoinhibition in leaves, a role sometimes ascribed to kaempferol
(Buchanan et al., 2000). Anthocyanins are also associated with resistance to
pathogens in Brassica species, sunflowers, pea seedlings and maize (Mazza and
Miniati, 1993; Buchanan et al, 2000). The functions of anthocyanins in
underground organs is less clear, however, betalains, the pigments in red beets,
have been associated with the indication of nutrient stress and pathogen
resistance (Mabry, 1980; Piattelli, 1981; Stafford, 1994), and improved viral

defense (Sosnova, 1970).

24

Anthocyanins in Underground Organs

Anthocyanin accumulation in underground tissues can result in the
formation of red, purple and blue organs for potatoes, onions, red radishes,
sweet potatoes, yarns, and garlic. Colored underground organs are now
receiving more market interest for their aesthetic and health properties,
especially in the gourmet industry. Consequently a considerable amount of
research has gone into identifying the anthocyanins in these commodities, as

well as trying to improve the color and stability of these compounds.

Potato

In potatoes, the anthocyanins are found in the periderm and/or the
peripheral cortex of the tuber, depending on the cultivar (Burton, 1989). The
periderm consists of the phellem, phellogen and the phelloderrn (Lulai and
Freeman, 2001) and the peripheral cortex is the starchy center. According to
Howard et al., (1970) tetraploid Solanum tuberosum subsp. tuberosum have
single anthocyanin pigments rather than mixtures as found in Solanum
tuberosum subsp. andigena and cultivated diploid cultivars. Anthocyanins tend to
degrade during storage; however Lewis et al. (1999) found anthocyanin
concentration to increase during storage at 4 °C. Anthocyanins have been found
to decrease during tuber growth (Hung et al. 1997).

The first chemical identification of anthocyanins in a potato was done by
Chmielewska in 1935 on ‘Negresse’ (also known as ‘Congo’), which is a cultivar

with purple-black skin and flesh. The anthocyanin identified were negretein, a

25

malvidin derivative: Mv-3-(p-coumaroyl-rutinoside), and a 3-monoglucoside of the
then unknown petunidin. The petunidin-3-monoglucoside molecule was later
identified as petanin. Andersen et al. (1991) identified the complete structure of
petanin as petunidin 3-O-[6-O-(3-O-E-p-coumaroyl-a-L-rhamnopyranosyl)-,8-D-
glucopyranosidej-5-O-B-D-glucopyranoside. In addition, Fossen et al. (2000)
identified the novel 3-O-[6-(4-ferulyl-O-a -rhamnopyranosyl)B-glucopyranoside]-
5-O-B-glucopyranosides of petunidin and malvidin in this cultivar.

The isolation and characterization of ten anthocyanins from diploid and
tetraploid potato species led to the discovery of p-coumaroyl-3-rutinoside-5-
glucoside derivatives of all six common anthocyanidins, plus the 3-rutinoside
derivatives of Pg, Cy, Dp, and Pt in potato (Harbome, 1960). Harbome (1960)
identified the anthocyanins in red Solanum phureja as the rhamnosylglucosides
of cyanidin and pelargonidin, and in the red Solanum tuberosum, the
rhamnosylglucosides of peonidin and pelargonidin. According to Fossen et al.
(2000) and lshii et al. (1996) the major anthocyanin in red-fleshed tubers is
pelargonidin 3-O-[6-O-(3-O-E-p-coumaroyl-a-L-rhamnopyranosyl)—,8-D-
glucopyranoside]-5—O-B-D-glucopyranoside) also known as pelanin. Naito et al.
(1998) isolated pelanin and pelargonidin 3-O-[6-O-(4-O-E-feruloyI-a-L-
rhamnopyranosyl)-B-D-glucopyranoside]-5-O-fi-D-glucopyranoside from red
tubers of an anthocyanin-rich tetraploid.

Lewis et al. (1998b) identified the anthocyanins in the dark purple cultivar
‘Urenika’ and the medium purple cultivar ‘Arran Victory’ as petunidin-3-(p-

coumaroyl-rutinoside)-5-glucoside and malvidin-3-(p-coumaroyl-rutinoside)—5-

26

glucoside. The petunidin derivative was found in similar concentrations in both
cultivars and the malvidin derivative at a higher concentration in the darker
‘Urenika’.

Hung et al. (1997) determined that more than 90% of the anthocyanidins
in the periderm of ‘Norland’ are pelargonidin and peonidin. Lewis et al. (1998b)
identified the anthocyanins in the red cultivars ‘Desiree’ and ‘Redflesh’ as
pelargonidin 3-(p-coumaroyl-rutinoside)-5-glucoside and peonidin 3-(p-
coumaroyl-rutinoside)-5-glucoside. Sachse (1973) also identified pelargonidin 3-
(p-coumaroyI-rutinoside)-5-glucoside and peonidin feruloyI-3-rutinoside-5-
glucoside in varieties ‘Urgenta’ and ‘Desiree’. Lewis et al. (1998c) analyzed eight
wild Solanum species and found lower anthocyanin levels than in colored
Solanum tuberosum as tubers of these wild species were mostly white, with

some showing slight purple color around the eyes.

Onions

The onion plant (Allium cepa L.) produces white, yellow or purple bulbs,
depending on its genetic makeup. All the anthocyanidins except malvidin have
been found in onions. Anthocyanins can be found in crystalline form in the
epidermal cells (Mazza and Miniati, 1993). Early work done by Robinson and
Robinson (1932) identified a cyanidin 3-pentoseglycoside. Fouassin (1956)
identified three cyanidin derivatives, including mono- and diglucosidic derivatives.

Fossen et al. (1996) analysed three red onions and other Allium spp. and

found them to contain several or all of the following anthocyanins: 3-(6’-malonyl-

27

3’glucosylglucoside), 3-(3’, 6’-dimalonylglucoside), 3-(6’-malonylglucoside), 3-(3’-
malonylglucoside), 3-(3’-glucosylglucoside) and 3—glucoside of cyanidin. They
also found trace amounts of two pelargonidin derivatives and the 3, 5-
diglucosides of cyanidin, and peonidin were reported for the first time in red
onion. Gennaro et al. (2002) reported that cyanidin derivatives constitute more
than 50% of total anthocyanins in ‘Tropea’ red onions (Allium cepa L.) and in

addition detected delphinidin and petunidin derivatives for the first time in onions.

Red Radish.

Radishes occur in a variety of color such as white, purple, yellow, and
black. However, it is the red radishes that receive the most attention as a natural
alternative to artificial colorants. Giusti et al. (1998) identified the major pigments
in 27 red radish cultivars to be pelargonidin 3-sophoroside-5-glucoside, mono— or
di-acylated with cinnamic and malonic acids. Harbome et al. (1958) found
pelargonidin 3-sophoroside—5-glucoside in red radish to be acylated with p-
coumaric and ferrulic acid, referred to as Raphanusin A and B respectively. In
purple radishes, the anthocyanin composition is the same as in red radish;

however the aglycone moiety pelargonidin is replaced by cyanidin.

Sweet potato and yams
Sweetpotatoes come in an array of colors. The anthocyanins of the purple
sweetpotato (lpomoea batatas L.) cultivar ‘Ayamurasaki’ were identified as 3-(6,

6’-caffeylferulylsophoroside)-5-glucoside of cyanidin and peonidin (Yoshimoto et

28

al., 2001). In addition, Terahara et al. (2000) identified another anthocyanin in
this cultivar as cyanidin 3-O-(2-O-(6-O-(E)—p-coumaroyI-B-D-glucopyranosyl)-B-D-
glucopyranoside)—5—O-B—D-glucopyranoside.

Odake et al. (1992) identified the chemical structures of two major
anthocyanins in ‘Yamagawamurasaki’ as 3-caffeylferulysophoroside-5-glucoside
of cyanidin and peonidin. Goda et al. (1997) isolated two new acylated
anthocyanins from the same purple cultivar and through spectroscopic analysis
identified these compounds as 3-O-(6-O-trans-caffeyI-2-O- B—glucopyranosyl- ,8-
glucopyranoside)-5-O- B—glucoside of cyanidin and peonidin.

The popular edible yam (Dioscorea alata L.) usually has white tubers,
however, some varieties contain purple pigment. According to Shoyama et al.
(1990) the following anthocyanins have been isolated in D. alata: cyanidin 3-O-
glucoside, cyanidin 3-O-diglucoside, cyanidin 3-O—rhamnoside, cyanidin 3-O-
gentiobioside and cyanidin 3-O—glucoside acylated with ferulic acid. In addition,

Shoyama et al. (1990) isolated cyanidin and peonidin 3-O-gentiobioside.

Garlic

Pigmentation of garlic (Allium sativum L.) is due to the presence of
carotenoids, chlorophylls and anthocyanins, the latter being localized in the inner
scales of leaves of the bulb (Mazza and Miniati, 1993).The three major
anthocyanins are cyanidin 3-glucoslde, cyanidin 3-glucoside monoacylated, and
cyanidin 3-glucoside diacylated with an aliphatic acid. Fossen and Andersen

(1996) found that acidified methanolic extract of inner scale leaves of garlic

29

contain mostly anthocyanins with aliphatic acylation. These are the rare 3’, 6’-
dimalonylglucoside (13%) and 3’-malonylglucoside (3%) of cyanidin, in addition

to cyanidin 3-(6’-malonylglucoside) (71%) and cyanidin 3-glucoside (12%).

Research Hypotheses and Objectives

Demand for plant products with greater health and aesthetic appeal
suggests the development of pre- and postharvest intervention strategies to
improve the development and stability of anthocyanin pigments in novelty
potatoes, such as ‘Michigan Purple’. With this in mind we wanted to investigate
the development of anthocyanins during tuber development, as well as in storage
and compare this with other colored potatoes. We hypothesized that the
preharvest factors that may impact stress responses such as soil type and
planting space, rate of nitrogen application, and application of the synthetic auxin
2,4-D, will affect the synthesis and stability of anthocyanins. In addition, we
hypothesized that storage temperature regime will impact anthocyanin stability,
and that postharvest ethylene application through initiation of stress responses,
will result in increased biosynthesis of anthocyanins in the skins of ‘Michigan

Purple’ potatoes.

Objectives

30

. Quantity changes in color of ‘Michigan Purple’ potatoes during storage
and compare these to changes in other colored potatoes: ‘Dakota Rose’,
‘Chieftain’, ‘Norland’ and ‘California Red’.

. Quantify the changes in purple color during tuber development in
‘Michigan Purple’ potatoes

. Investigate and compare color of ‘Michigan Purple tubers grown in muck
and mineral soils, and investigate the effect of different planting spaces in
mineral soils.

. Investigate and compare color of ‘Michigan Purple‘ tubers grown under
different nitrogen fertilization rates.

. Investigate and compare tuber color of “Michigan Purple’ plants sprayed
with 3 different 2, 4-D rates.

. Investigate the effect of postharvest temperature manipulation on the
synthesis of anthocyanins in tubers of ‘Michigan Purple‘ and red-skin
cultivars.

. Investigate the change in color of ‘Michigan Purple’ tubers exposed to

postharvest ethylene.

31

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41

 

OH
Flavan-3-ol Flavanonol

”° 0 ”
OH

Condensed tannins

Figure 1. The different types of flavonoids and their structures.

42

 

Figure 2. The basic structure of all flavonoids. The A-ring is formed from 3
acetate units (via the malonic acid pathway) and the B-ring with the 3-carbon
bridge is constructed from phenylalanine via the shikimic acid pathway.

43

Figure 3. The phenylpropanoid pathway leading to the synthesis of anthocyanins.
The enzymes involved in the reactions are: PAL: phenylalanine ammonialyase;
C4H: cinnamate-4-hydroxylase; 4CL: 4-coumaryI-CoA Iigase; CHS: chalcone
synthase; CHI: chalcone-flavanone isomerase; F3H: flavanone 3-hydroxylase;
F3'H: flavonoid 3'-hydroxylase; F3'5'H: flavonoid 3‘,5'-hydroxylase; DFR:
dihydroflavonol reductase; ANS: anthocyanidin synthase; GT: UDPG-flavonoid-3-
glucosyltransferase; RT: anthocyanidin-3-glucoside rhamnosyltransferase; FS:
flavone synthase and FLS: flavonol synthase.

 

44

 

 

Phenylalanine

' PAL
Cinnamate
I C4H
p'cm‘ma'a‘" 3Malonyl CoA
I 4CL
/p-CoumaroyICoA \<‘CI:3 OH

Phenolic acids
(Chlorogenic acid, etc)

  

 

Flavones
Naringenin/ SKaempherol
Quercetin :O\(;:(c;.q:: W.” ‘HS‘
OH. O . OH O OH O
DFR DFR 1 0” DFRl
,. .0 O
Leucocyanldln O Leucopelargonidin Leucodelphinidin
/ OH
ANS OH OH ANS ANS
Cyani din Delphlnldln

 

O 3GT
3GT +
HO O I O\

/ O-glucose I

 

 

II

Cyanidin-O-glucoside OH OH 1 Pelargonidin-3-O-glucoside Delphinidin-O—glucoside
RT

     

HO RT
O-glucose-rhamnose

Cyanidin-O-rutinoside OH OH Pelargonidin-3-O-rutinoside Delphinidin-O-rutinoside

45

 

Figure 4. The structures of the six most common anthocyanidins, differing in
hydroxylation and methylation positions.

46

 

 

 

 

 

 

 

 

 

 

blue quinonoidal base
pH4-5

 

 

 

 

 

red flavylium cation
pH1-2

 

 

 

colorless carbinol
pseudobase

pH3-6

 

 

 

 

 

colorless to
yellow chalcone
pseudobase

pH3-6
OH CE unstable

 

 

Figure 5. The structural transformation of anthocyanins in aqueous solutions
(adapted from Brouillard, 1983).

47

 

 

 

 

CHAPTER I

CHANGE IN TUBER SKIN-COLOR OF ‘MICHIGAN PURPLE’ AND OTHER

COLORED POTATO CULTIVARS DURING DEVELOPMENT AND STORAGE

48

INTRODUCTION

Colored potatoes currently comprise 10 % of the total acreage in the
United States (USDA, 2003). The skin color of colored potatoes is due to the
presence of anthocyanins in the periderm (Burton, 1989). If the color of red
potatoes is pink at harvest or fades during storage, it can result in potatoes with
lower value. Anthocyanin concentrations can change during growth and
development due to environmental and growth conditions such as temperature,
disease, and nutrition levels. However, when harvested, factors such as handling
conditions during harvest, storage conditions and duration may also alter
anthocyanin concentrations.

Anthocyanins can be degraded in plant tissues due to the following
enzymes: glycosidases, polyphenoloxidase (PPO), and peroxidase (Francis,
1989; Macheix et al., 1990). Glycosidase hydrolyzes the anthocyanin to the
anthocyanidin (aglycone) and glycoside after which the anthocyanidin is more
prone to further degradation (Huang, 1955). Peroxidase is found to enhance the
degradation of the aglycone. As senescence continues during storage, cells will
become more vulnerable to degradation of anthocyanins as a result of the
interaction of these enzymes with anthocyanins (Mayer and Harel, 1979; Walker
and Ferrar, 1988).

Lewis et al. (1999) found anthocyanins to increase in ‘Arran Victory’

(purple skinl white flesh), ‘Desirée’ (pink skinl white flesh), ‘Red Flesh’ (red skinl

49

pink flesh), and ‘Urenika‘ (purple skinl purple flesh) varieties stored at 4 °C.
Conversely, Andersen et al., 2002 found that anthocyanins decreased in
‘Nonand‘ (red skinl white flesh) after 1 month in storage at 4 °C. This discrepancy
in results is probably due to the different responses of varieties to cold-storage.
The response of ‘Michigan Purple’ pigment content to low temperature storage is
not known.

Hung et al. (1997) found anthocyanin concentrations and chroma of
‘Norland’ potatoes to decrease with increasing tuber size. Hung et al. (1997) did
not find a change in hue with increase in tuber size. Lewis et al. (1999) found a
slight increase in anthocyanin concentrations with initial tuber weight increase in
‘Desiree tubers’, after which anthocyanin content stabilized.

In this paper, we investigate the change in anthocyanins in the skins of
‘Michigan Purple’ during development of the tubers as well as during storage at
4°C and 10 °C for 6 months. We compare the behavior of purple ‘Michigan
Purple’ tubers in storage with red cultivars ‘Dakota Rose’, ‘California Red’,
‘Chieftain’, and ‘Norland’. We hypothesize that storage of potatoes at low
temperatures would result in an increase in anthocyanin concentration in the

tuber skins.

MATERIALS AND METHODS

50

Site description and experimental design. In 2002 ‘Michigan Purple‘,
‘Dakota Rose’, ‘CaIifomia Red‘ and ‘Norland’ were grown in black organic soil at
Krummrey & Sons, Inc., Stockbridge, Michigan. The area is classified as an OsB-
Oshtemo sandy loam with a 0-6 percent slope (USDA, 1979). In 2003, ‘Michigan
Purple’, ‘Dakota Rose’, and ‘Chieftain’ tubers were grown in Montcalm/McBride
sandy loam soil at Montcalm Research Farm, Entrican, Michigan. In addition, we
implemented a developmental study in 2003 with the goal of determining whether
intensity of color is affected by the stage of tuber development.

Storage conditions. In 2002, 40 tubers of each cultivar were washed, air
dried, and placed into trays. A circle, 1.5 cm in diameter, was drawn on each
potato to indicate the area where color was to be measured. The tubers were
stored at 10 °C and 80-90% relative humidity (RH) for 6 months. In 2003, 20
tubers of each cultivar were numbered, marked with a circle, and randomly
placed into 3 trays to be stored at 4 °C and 85-90% RH for 6 months. In addition,
extra tubers were stored for extraction purposes at 0, 2, 4 and 6 months: three
replicates of 5-10 tubers of each cultivar were placed into brown paper bags
immediately after harvest and stored under same conditions as trays in both
years.

Color measurement. In 2002, color was measured every 2 weeks in
circled areas with a colorimeter (Colorimeter CR 300, Minolta, Japan). Chroma,
hue angle, and lightness values were recorded. Chroma refers to the intensity of
the color and hue angle (h° = arcangle[b/a]) refers to the angle formed byline

from the origin to the intercept of the a (x-axis) and b (y-axis) co-ordinates, where

51

0° = red-purple, 90° = yellow, 180° = bluish—green, and 270° = blue (McGuire,
1992)

Anthocyanin extraction: In 2002, an extraction protocol was followed for
quantification and identification of anthocyanins with high-performance liquid
chromatography (HPLC). Tubers were taken from storage at the appropriate
times and the periderm removed by scraping with a scalpel. The skin was
' immediately frozen in liquid nitrogen and stored at -80 °C until analyzed. Two
grams of potato periderm were ground to a powder in a mortar and pestle using
liquid nitrogen. Samples were extracted in 20 mL of 2.5% (v/v) formic acid in
methanol and placed in a shaker for 60 min at 4 °C. Samples were centrifuged at
3000 rpm for 15 min at 4 0° and the supernatant decanted. The residue was
extracted three more times with 10 mL extraction solvent, centrifuging for 10 min
at 3000 rpm, and decanting the supernatant.

The combined extracts were filtered with a 0.8 pm filter into a clean test
tube. Samples were evaporated to dryness at 35 °C and redissolved in 2 mL
2.5% (v/v) formic acid in water. Filter cartridges (Sep Pak® Plus C18, Waters,
Ireland) were prepared by dripping 5 mL 100% methanol through and rinsing with
5 mL 2.5% (v/v) formic acid in water. The sample was slowly loaded onto the
filter cartridge and rinsed twice with 5 mL 2.5% (v/v) formic acid in water. The
sample was eluted with 1.8 ml 2.5% (v/v) formic acid in methanol and filtered
through a 0.2 filter into an auto-sampling vial (Alltech associates, lnc., IL).

Anthocyanins were analyzed using analytical reversed-phase HPLC with a

separations module (Waters 2690) with a photodiode array detector (Waters

52

2994) and a 4.6 x 150 mm column (XTerra® RP-18 column), maintained at 30
°C. Sample volume was 15-20 uL. Elution solvents used were: A, 2.5% formic
acid in methanol and B, 2.5 % formic acid in water. A flow rate of 1 mL.min'1 was
maintained. Samples were eluted using the following program: 70% A to 35% A
and 30% B to 65 % B from 0 min to 17 min; 0% A and 100% B at 19 min and
70% A and 30 % B at 21 min. The eluted compounds were monitored at 530 nm
for anthocyanins.

Four standards were prepared and run on the HPLC for our identifications
properties: petunidin chloride (Apin chemicals, UK), pelargonidin 3,5-diglucoside
(Apin chemicals, UK), cyanidin 3-glucoside, and cyanidin 3-rutinoside (Sigma, St.
Louis, US).

For additional identification purposes, HPLC analysis was done with a 4.6
x 150 mm Capsell RP-18 column. The elution solvents were on a v/v basis: A,
0.1 % trifluroacetic acid in water and B, 50.4 % water, 48.5 % acetonitrile, 1 %
acetic acid and 0.1 % trifluroacetic acid. The samples were eluted using the
following program: 80% A to 40 % A and 20 % B to 60 % B from 0 min to 26 min;
80% A and 20% B at 30 min, held until 45 min.

Based on their retention times, spectra, and a library comparison, peaks 3
and 4 were identified as the anthocyanidins delphinidin and cyanindin,
respectively. Peak 1 was identified as an anthocyanin. Peak 1 had the same
retention time as pelargonidin 3, 5-diglucoside.

In 2003, extractions were based on the procedure of Siegelman and

Hendricks (1958). Skin samples were frozen and ground as described above. 0.5

53

g of ground sample was added to 15 mL of 1% (v/v) HCI in methanol solution and
left for 18 hours at 5 °C in the dark. Samples were centrifuged for 10 min at 3000
rpm. Absorbance of the supernatant was measured at 530 nm on a
spectrophotometer (U-3000, Hitachi, Japan) and compared to the standards
pelargonidin 3, 5-diglucoside and petunidin chloride (Apin chemicals, UK).

Development experiment. In 2003, starting eight weeks after planting,
three ‘Michigan Purple’ plants were randomly selected from the plots at
Montcalm Research Farm, Entrican, Michigan on July 25, August 7 and 28. All
tubers of every plant were harvested and divided into four classes according to
their weight: 55 g; 5-24.9g; 25-49.9 9; >50 9 (Hung et al., 1997). Color
measurements and anthocyanin extractions were done as described above.

Statistical analysis. Data was analyzed using a Statistical Analysis System
(SAS Institute, Inc., Version 8.0, 2000) to conduct least significant differences
between treatments using PROC MIXED and PROC GLM. Slope analyses were
done using PROC MIXED.

RESULTS AND DISCUSSION

Effect of storage at 10 °C. There was considerable variation in the total pigment
concentration (anthocyanin and anthocyanidins) (5 peaks combined) of the skins
of the four cultivars during storage and no consistent change in concentration

could be measured over the 6 months storage period (Figure 1). (See Appendix

A Tables 2 and 3 for complete analysis of HPLC peaks and Figures 1-4 for HPLC
chromatogram with spectra of the individual peaks of each cultivar).

‘California Red’ had the highest concentration of total anthocyanins at 0
and 6 months, differing significantly from the other cultivars. After two months of
storage, ‘Michigan Purple’ had a statistically lower concentration, but there was
no statistical difference between the other three cultivars. However, after four
months both ‘California Red‘ and ‘Dakota Rose’ had significantly higher pigment
concentrations than the other cultivars and ‘Michigan Purple’ was significantly
lower than ‘Norland’. After six months in storage, ‘Michigan Purple’ was
significantly lower in pigment concentration than ‘California Red’ and ‘Dakota
Rose”, but not ‘Norland’. These data suggest that storage duration, when the
storage temperature was 10 °C did not affect the anthocyanin content of
potatoes, even after six months of storage.

Since the anthocyanin concentration of ‘Michigan Purple‘ was quantified
using petunidin chloride (an anthocyanidin) and the other three potato cultivars
were quanitified using pelargonidin 3,5-diglucoside (an anthocyanin), direct
comparisons on their concentration are difficult. Different anthocyanin standards
can result in very different readings (Holcroft and Kader, 1999). Consequently, a
comparison of peak areas (AU) at 530 nm is included to give a better indication
of the anthocyanin intensity of ‘Michigan Purple‘ (Appendix A Table 1). There
was no change in AU values in any of the cultivars between months 0 and 6.

‘Michigan Purple’ samples had significantly higher AU values than ‘Norland’ at

55

month 0, but were not different from ‘California Red’ or ‘Dakota Rose’. “California
Red’ had the highest AU value after 4 months of storage.

Hue angle (h°) increased over the six month storage period at 10 °C in all
cultivars except ‘Norland’ (Figure 2; Appendix A Table 1), indicating a reddening
during storage. As expected, ‘Michigan Purple’ had a consistently lower h° value
than the red cultivars which reflected its purple coloration. The slope of the h°
curve over months in storage was used as a means to describe the rate of
change in skin color of the stored potato (Table 1). The slope of ‘Michigan Purple’
was steeper (0.81 hue. week") than the red cultivars (0.41, 0.41 and 0.18 hue.
week"), indicating a faster rate of change. However, since the h° of ‘Michigan
Purple’ was initially lower, the h° after storage was still significantly lower (more
purple) than the red cultivars. The slope of the red cultivars did not differ from
one another, indicating that there was no difference in rate of color change.

Lightness increased and remained significantly higher after two months in
storage in the cultivars ‘Michigan Purple‘ and ‘Dakota Rose‘ (Figure 2; Appendix
A Table 1). No significant changes were recorded for ‘California Red’ and
‘Norland’. Significant differences in lightness were recorded at harvest (month 0)
between ‘Michigan Purple’, “California Red’ and ‘Norland’ and after two months
between ‘Michigan Purple’ and all three red cultivars; however, after four and six
months in storage, no significant differences were recorded. These changes in
lightness, though significant, were still very small and probably did not contribute

to major color changes.

56

Chroma did not change significantly after six months in storage in any of
the cultivars. ‘Michigan Purple’ had a lower chroma value than the red cultivars at
all times (Figure 2; Appendix A Table 1). The chroma of the red cultivars did not
differ between 0 and 6 months although ‘Dakota Rose' differed from ‘Norland’ at
months four and six. These data indicate that storage at 10 °C did not influence
chroma or anthocyanin concentration, but with time a change in perceived color
of red and purple potatoes was observed.

Effect of storage at 4 °C. Of the four cultivars tested, ‘Dakota Rose’ was
the only cultivar that showed significant increases in pigment (anthocyanidin and
anthocyanin) concentration after two, four, and six months (Figure 3; Appendix A
Table 4). A similar response of potatoes to cold storage has been reported by
Lewis et al. (1999) who suggested that this increase in anthocyanin
concentrations at 4 °C is due to the conversion of starch to sugars in the tubers.
Since sugars are the precursors of anthocyanins, this leads to increased
anthocyanin synthesis. ‘Michigan Purple’ had the lowest anthocyanin
concentration at all times and there was no increase in storage at 4 °C. This data
agrees with Andersen et al. (2002) who found that anthocyanin concentration in
‘Norland’ decreased in storage at 4 °C. This discrepancy in results could be due
to the response of different cultivars to temperature.

Hue angle values of all the cultivars were significantly higher after 2
months in storage at 4 °C and remained so for the duration of the storage time
(Figure 4, Appendix Table 4). ‘Chieftain’ had the highest hue angle throughout

the storage duration, while the hue angle values of ‘Michigan Purple’ and ‘Dakota

57

 

 

Rose’ were significantly lower than ‘Chieftain‘. The hue angle of ‘Michigan Purple’
increased during storage and reached the same value as ‘Dakota Rose’ after 6
months. Interestingly, the skin color, as judged by the human eye, did not
resemble each other suggesting the colorimeter was responding to pigments not
readily seen by the human eye, perhaps located beneath the surface layer. The
rate change in hue was greatest in ‘Michigan Purple‘ during 6 months of storage
(Table 2). It appears that the color of the potato cultivars stabilized after 3 months
of storage as indicated by the lower slope values (Table 2).

The hue angle of ‘Michigan Purple’ increased from 109° to 26.44°after six
months at 4 °C. and from 359.97° to 6.45 °after 6 months at 10 °C. These data
suggest that storage at higher temperature of 10 °C is better for maintaining a
more purple color.

All three cultivars became significantly darker after 2 months of storage
but of these, ‘Chieftain’ was significantly lighter than ‘Michigan Purple’ and
‘Dakota Rose’ after 2 months of storage. All cultivars had significantly higher
chroma values after 2 months in storage indicating a more intense color.

Development experiment. Tubers in stage 3 (5 g to 25 9) had the highest
concentration of pigments (Figure 5). All stages differed significantly from each
other. Anthocyanin and anthocyanidin content underwent a decreasing trend as
tuber weight increased similar to observations by Hung et al. (1997). There was a
negative correlation between pigment concentration and h°, lightness and

chroma measurements.

58

CONCLUSIONS

From this study we now know that ‘Michigan Purple’ potatoes have both
anthocyanin and anthocyanidin pigments. We concluded that storage at low
temperatures did not affect anthocyanin concentrations in ‘Michigan Purple‘
potato tubers up to six months in storage at 4 or 10 °C. However, color (hue)
change was cultivar-dependant and was faster at 4 °C than at 10 °C, suggesting
that reducing storage temperature, while not benefiting anthocyanin content,
does improve color stability.

In addition, we found anthocyanin concentrations to be different in tubers
from different developmental stages, with the smaller tubers having the highest
concentrations. Hence, ‘Michigan Purple’ might be best marketed as new
potatoes since they have the highest anthocyanin concentrations when tubers

are small, and thus the most health benefits.

59

REFERENCES

Andersen, A. W., C. S. Tong and D. E Krueger. 2002. Comparison of periderm
color and anthocyanins of four red potato varieties. Amer. J. Potato Res.
79:249-253.

Burton, W. G. 1989. The potato. 3rd ed. Longman, Herlow, UK.

Francis, F. J. 1989. Food colorants: anthocyanins. Crit. Rev. Food Sci. Nutr.
28:273-314.

Holcroft, D.M. and A. A .Kader. 1999. Carbon dioxide-induced changes in color
and anthocyanin synthesis of stored strawberry fruit. HortScience
34:1244-1248.

Hung, Q, J. R. Murray, S. M. Ohmann and CBS. Tong. 1997. Anthocyanin
accumulation during potato tuber development. J. Amer. Soc. Hort. Sci.
122:20-23.

Huang, H. T. 1955. Decolorization of anthocyanins by fungal enzymes. J.
Agric. Food Chem. 32141-146.

Lewis C. E., J. R. L. Walker and J. E. Lancaster. 1999. Changes in anthocyanin,
flavonoid and phenolic concentrations during development and storage of
coloured potato (Solanum tubersosum) tubers. J. Sci. Food Agri. 79:311-
316.

Macheix, J. J., A. Fleuriet and J. Billot. Fruit Phenolics. 1990. CRC Press, Boca
Raton. Fla.

Mayer, A. M. and E. Harel. 1979. Polyphenol oxidases in plants. Phytochemistry
8:193-215.

60

McGuire, R. G. 1992. Reporting of objective color measurements. HortScience
27:1254-1255.

Siegelman, H. W. and S. B. Hendricks. 1958. Photocontrol of anthocyanin
synthesis in apple skin. Plant Physiol. 33:185-190.

United States Department of Agriculture. 1979. Soil Survey of lngham County,
Michigan. US. Dept. Agr., Washington, DC.

United States Department of Agriculture. 2003. Potatoes 2002. National
Agricultural Statistics Service. http:/www.usda/nass. 12/10/2003

Walker J. R. L. and P. H. Ferrar. 1988. Diphenol oxidases, enzyme-catalysed
browning and plant disease resistance. Biotechnol. Genet. Eng. Rev.
15:457-498.

61

Table 1. A comparison between the rates of change in hue angle values of over 6
months of storage in ‘Michigan Purple’, ‘Dakota Rose’, ‘California Red’, and
‘Norland‘ potatoes stored at 10 °C. Standard errors are given in parentheses.

 

 

Cultivar Slope
hue.week'1
Michigan Purple 0.81 (0.19) a
Dakota Rose 0.41 (0.10) b
California Red 0.41 (0.10) b
Norland 0.18 (0.08) b

 

Means separation within column (P< 0.05, LSD, n=30).

62

Table 2. A comparison between the rates of change in hue angle values in the
first 3 months, the last 3 months and the overall 6 months of storage at 4 °C in
‘Michigan Purple’, ‘Dakota Rose’, and ‘Chieftain’ potatoes. Standard errors are
given in parentheses

 

 

Cultivar Date Slope

hue.week’1
Michigan Purple First 3 months 1.92 (0.02) a
Chieftain 0.70 (0.02) b
Dakota Rose 0.76 (0.02) b
Michigan Purple Last 3 months 0.72 (0.06) a
Chieftain 0.37 (0.05) b
Dakota Rose 0.22 (0.03) 0
Michigan Purple Overall 6 months 1.33 (0.08) a
Chieftain 0.57 (0.03) b
Dakota Rose 0.46 (0.03) b

 

Means separation within column (P< 0.05, LSD, n=30).

63

 

 

 

 

 

m 2000

C
6 1800 - +M'P
'E +CR

g 1600 — +~ .
8 1400 +DR
TE 1200 /
.‘c", E

c i U) 1000 ‘ "I

m 3

m 800 -

.E

g 600 ~

>

E) 400 -

‘E’ 200 ~ I'\r\§/«I

< o . .

0 2 4

Months in storage

Figure 1. The effect of storage at 10 °C on the pigment (anthocyanin and
anthocyanidin) concentrations of the cultivars ‘Michigan Purple’ (MIP), ‘Dakota
Rose’ (DR) ‘California Red’ (CR), and ‘Norland’ (N). Vertical bars refer to

standard error of the means (n=20). Anthocyanin analyses were done by using a
spectrophotometer.

64

 

 

Figure 2. The effect of storage at 10 °C on (a) the hue angle values, (b) the
lightness and (c) chroma of the skin of ‘Michigan Purple’ (MIP), ‘Dakota Rose’
(DR), ‘California Red’ (CR), and ‘Norland’ (N) potatoes. Vertical bars refer to

standard error of the means (n=20).

65

Figure 2

Hue angle (°)

Ughm

30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I
25 .
20 -
+MIP
15 - +CR
+CR
1o - A +N
5 1
o T l T
g 2 4 6
355
Months in storage
45
44 4
43 ~
,,
42 -
41 -
40‘ :I
39 1
38 1 +MIP
37 - -°-DR
+CR
36 . +N
35 I T .
0 2 4 6
Months in storage
30
25‘ W
gal
C
15 -
{\1 M
1* x
10 ~
+MIP
5 1 +DR
+CR
+N
0 . . 4
0 2 4 6

Months in storage

66

 

 

 

 

 

20000

 

+MIP
+DAK
1 6000 - +CHT

1 8000 1

1 4000 1
1 2000 1

1 0000 1

and anthocyanidins

lug/9)

8000 1
6000 1

4000 1

Anthocyanins

2000 1 Tk

 

0 i

 

 

Months in storage

Figure 3. The effect of storage at 4 °C on the pigment (anthocyanin and
anthocyanidin) concentrations in the skin of ‘Michigan Purple’ (MIP), ‘Dakota

Rose’ (DR), and ‘Chieftain’ (CHT) tubers. Vertical bars refer to standard error of

the means (n=30). Anthocyanin analyses were done by using a
spectrophotometer.

67

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

so
40 4 A
I
5‘ 30 4
v
9
2‘ 20
<0
9 +MIP
I 10 - +DR
+CHT
° 1
‘ o 2 4 6
-10 1'
Months in storage
90
80 ., B +MIP
+CR
+CHT
E 70 1
o: 60 :
III
50 1 A -
40 I
30 1 .
o 2 4 6
Months in storage
25 1
C
8 20 1
E 15 -
| x -
1o - fl
+MIP
5 - +DR
+CHT
0 I I r
o 2 4 6

Months in storage

Figure 4. The effect of storage at 4 °C on the (a) hue angle values, (b) lightness,
and (c) chroma of the skin of ‘Michigan Purple’ (MIP), ‘Dakota Rose’ (DR), and
‘Chieftain’(CHT) potatoes. Vertical bars refer to standard error of the means
(n=30)

68

Anthocyanin and Anthocyanidin (pg/g)

 

 

<49 5.24.9 25-49.5 >50

Stage of development (9)

Figure 5. The effect of stage of development of the pigment content (pg/g) in the
skin of “Michigan Purple’ tubers. Vertical bars refer to standard errors of the
means (P < 0.05, LSD). Anthocyanin analyses were done by using
spectrophotometer.

69

CHAPTER II

PREHARVEST TECHNIQUES TO IMPROVE PIGMENT CONCENTRATION AND
COLOR OF ‘MICHIGAN PURPLE’ POTATOES AT HARVEST AND IN STORAGE

70

INTRODUCTION

Intense tuber skin-color is an important quality factor for growers who wish
to obtain high prices for their niche market potatoes (Solanum tuberosum L.).
Three preharvest tools that could improve the outcome of tuber skin-color at
harvest are soil type and plant spacing, the application of different sources of
nitrogen (inorganic and organic), and the application of different rates of 2,4-
dichlorophenoxyacetic acid (2,4-D).

We investigated the difference in skin color of ‘Michigan Purple’ potatoes
grown in mineral and organic soils at harvest and in storage. Mineral and organic
soils differ markedly in their physical and nutritional make up. Mineral soils form
from rock, sediment and a mixture of hundreds of minerals, and are made up
from organic matter, mineral matter, water, and air. Organic soils, also known as
muck or peat soils, have much more organic carbon (usually > 3-10%), are made
from peat and plant remains, and usually have a darker color than mineral soils
(Miller and Donahue, 1995; Pierzynski et al., 1994)

In addition, we investigated whether different planting spaces could have
an effect on skin-color. Deficiencies in nutrients such as phosphorus have been
found to increase anthocyanin biosynthesis in grapes (Dedaldechamp et al.
1995), carrots (Rajendran et al. 1992), and Arabidopsis thaliana (Trull et al.,
1997), and deficiencies in nitrogen have been found to increase anthocyanin
biosynthesis in tomato (Bongue-Bartelsman and Philips, 1995), and grape (Do

and Corrnier, 1991). Therefore, we hypothesize that plants grown closer together

71

would compete for nutrients and sunlight and hence be under more nutrient
stress, resulting in increased anthocyanin concentrations in tubers. To our
knowledge, the effect of soil type and plant spacing on potato tuber skin-color
has not been investigated.

Nitrogen (N) is a critical nutrient for potato plants since it has a great effect
on yield and tuber quality. Inadequate amounts of N will reduce yields, whereas
an excess can reduce tuber quality and tuber yield. A nitrogen deficiency has
also been found to increase anthocyanin synthesis in other commodities such as
grape (Do and Cormier. 1991); however, it is not clear whether excessive
amounts of nitrogen could suppress anthocyanin formation and thus contribute to
less intensely colored tubers. We investigated the application of different rates
and sources of nitrogen within the range of commercially acceptable fertilization
levels on the tuber skin-color of ‘Michigan Purple’. We expected to see the best
color in the treatments with the least amount of nitrogen since nitrogen
deficiencies result in an accumulation of carbohydrates. Since these
carbohydrates cannot be used in the synthesis of amino acids or other nitrogen
compounds, they may be used in anthocyanin production (T aiz and Zeiger,
1998). We hypothesized that tubers receiving higher amounts of nitrogen will
have lower anthocyanin concentrations than the tubers receiving lower nitrogen
concentrations.

Anthocyanin synthesis is an energy expensive process and significant
production only occurs during specific developmental stages of plant organs or in

response to pre- or postharvest environmental stresses. Many stress responses

72

act through ethylene, which itself stimulates anthocyanin synthesis (Faragher
and Brohier, 1984; Faragher and Chalmers, 1977; Li et al., 2002). In addition to
ethylene, auxin has been shown to induce the biosynthesis of anthocyanins in
cell culture (Rajendran et al., 1992; Sakamoto et al., 1994). Auxin causes the
stimulation of ethylene biosynthesis through enhancing the conversion of
S-Adenosyl-methionine (Adomet) to 1- Aminocyclopropane-1-carboxylic acid
(ACC).

Ethylene stimulates anthocyanin biosynthesis by increasing the activity of
phenylalanine ammonia-lyase (PAL) (Faragher and Brohier, 1984), chalcone
isomerase (CHI) (Li et al., 2002), and UDP glucose-flavonoid 3-O-
glucosyltransferase (UFGT) (Kereamy et al., 2002). The ethylene-generating
compound, ethephon, has been used on many solanaceous fruit, including
tomato (Lycopersicon esculentum Mill. (Burg and Burg, 1965; Edwards et al.,
1984) and pepper (Capsicum annuum L.) (Armitage, 1989; Cantliffe and
Goodwin, 1975; Knavel and Kemp, 1973 and Worku et al., 1975) to advance
ripening and promote anthocyanin accumulation.

We investigated the efficacy of preharvest 2,4-D applications on the purple
variety ‘Michigan Purple’ as a means to increase tuber skin-color. The synthetic
auxin, 2,4 -D has been proven to enhance the skin-color of red potatoes when
applied at pre-bloom stage at 0.4 kg.ha'1 in sodium salt form (76% acid
equivalent) (Fults and Schaal, 1948; Payne et al., 1950). 2,4-D is labeled for
commercial use as a means to increase tuber color of red potato. The efficacy of

2,4-D on anthocyanin accumulation in purple-skinned cultivars has not been

73

demonstrated. We hypothesize that preharvest 2,4-D applications will result in
tubers with higher anthocyanin concentrations.

This paper will therefore focus on three cultural tools to increase
anthocyanin accumulation in ‘Michigan Purple’ potatoes: planting site selection
(organic vs. mineral), soil nutrition (nitrogen fertilization) and growth regulator

(2,4-D) application.

MATERIALS AND METHODS

Experiment 1. The effect of organic and mineral soils on tuber skin-color.

Site description. In 2002 ‘Michigan Purple’ tubers were grown in black
organic soil at Krummrey & Sons, Inc., Stockbridge, Michigan, and in mineral
soils in Rogers City, Michigan. Tubers grown in organic soil were planted 0.3 m
apart, those in mineral soil were planted either 0.2 or 0.33 m apart.

Storage conditions. After harvest, 40 tubers of each treatment were
washed, air dried, and placed into carton trays. A circle, 1.5 cm in diameter, was
drawn on each potato with indelible marker to indicate the position at which color
measurements would be made. Trays were stored at 10 °C and 80-90% RH. In
addition, extra tubers were stored for extraction purposes at 0, 2, 4 and 6
months: three replicates of 5-10 tubers of each cultivar were placed into brown
paper bags immediately after harvest and stored under same conditions as trays.

Color measurements. Color was measured biweekly in circled areas with

a colorimeter (Colorimeter, Minolta CR 300, Japan). Chroma, hue angle, and

74

lightness values were recorded. Chroma refers to the intensity of the color (0
being grayish, 30 dull, and 60 vivid) and hue angle (h° = arcangle[b/aj) refers to
the angle formed by line from the origin to the intercept of the a (x-axis) and b (y-
axis) co-ordinates, where 0° = red-purple, 90° = yellow, 180° = bluish-green, and
270° = blue (McGuire, 1992). Lightness is presented on a scale of 1-100, 1 being
black and 100 being white.

Anthocyanin extraction. Approximately five grams of tuber periderm was
removed from 1 to 10 tubers by scraping with a scalpel. The skin was
immediately frozen in liquid nitrogen and stored at -80 °C until analyzed. Two
grams of potato periderm were ground to a powder in a mortar and pestle using
liquid nitrogen. Samples were extracted in 20 mL of 2.5% (v/v) formic acid in
methanol and placed in a shaker for 60 min at 4 °C. Samples were centrifuged at
3000 rpm for 15 min at 4 0° and the supernatant decanted. Three more
extractions were made by adding 10 mL of the extraction solvent to sample,
centrifuging at 3000 rpm for 10 min and decanting the supernatant.

The combined extracts were filtered with a 50 mm filter into a clean test
tube. Samples were evaporated to dryness at 35 °C and re-dissolved in 2 mL
2.5% (v/v) formic acid in water. Filter cartridges (Sep Pak® Plus C18, Waters,
Ireland) were prepared by dripping 5 mL 100% methanol through the empty
cartridge and rinsing with 5 mL 2.5% (v/v) formic acid in water. The sample was
slowly loaded onto the filter cartridge and rinsed twice with 5 mL 2.5% (v/v)

formic acid in water. The sample was eluted with 1.8 ml 2.5% (v/v) formic acid in

75

methanol and filtered through a 0.2 pm filter into an auto-sampling vial (Alltech
associates, Inc., IL).

Anthocyanin analysis was achieved using analytical reversed-phase
HPLC with a separations module (Waters 2690), a photodiode array detector
(Waters 2994), and a 4.6 x 150 mm column (XTerra® RP-18 column, Waters),
maintained at 30 °C. Sample volume was 15-20 (L. Elution solvents used were:
A, 2.5% formic acid in methanol and B, 2.5 % formic acid in water. A flow rate of
1 mL. min‘1 was maintained. Samples were eluted using the following program:
70% A to 35% A and 30% B to 65 % B from 0 min to 17 min; 0% A and 100% B
at 19 min and 70% A and 30 % B at 21 min. The eluted compounds were
monitored at 530 nm for anthocyanins.

Four standards were prepared and run on the HPLC for our identification
properties: petunidin chloride (Apin chemicals, UK), pelargonidin -3,5 -
diglucoside (Apin chemicals, UK), cyanidin-3-glucoside, and cyanidin-3-
rutinoside (Sigma, St. Louis, US).

For additional identification purposes, HPLC analysis was done with a 4.6
x 150 mm Capsell RP-18 column. The elution solvents were on a v/v basis: A,
0.1 % trifluroacetic acid in water and B, 50.4 % water, 48.5 % acetonitrile, 1 %
acetic acid and 0.1 % trifluroacetic acid. The samples were eluted using the
following program: 80% A to 40 % A and 20 % B to 60 % B from 0 min to 26 min;
80% A and 20% B at 30 min, held until 45 min.

Based on their retention times, spectra, and a library comparison, peaks 3

and 4 were identified as the anthocyanidins delphinidin and cyanindin,

76

respectively. Peak 1 was identified as an anthocyanin. Peak 1 had the same

retention time as pelargonidin -3, 5—diglucoside.

Experiment 2. The effect of nitrogen fertilizer type and rate on tuber skin-color.

Site description and experimental design. In 2003, ‘Michigan Purple'
tubers were grown in a commercial ‘Chieftain’ plot on Montcalm/McBride sandy
loam soil at Montcalm Research Farm, Entrican, Michigan. Nitrogen rates used
were respectively: 200 kg.ha'1 nitrogen (in the form of urea applications), 200
kg.ha"slow releasing nitrogen (in the form of MeisterT102 and an urea
application), 300 kg.ha'1 nitrogen (in the form of urea applications) and
200 kg.ha'1 poultry manure (in the form of 5600 kg.ha'1 poultry manure and urea
applications (I' able 1). Nitrogen application timing was as indicated in Fig.1, in 4
splits. Potatoes were harvested after 5 months in the ground. After harvest tubers
were placed in storage in order to study color changes.

Storage conditions. After harvest, twenty tubers of each treatment were
washed, air dried, and randomly placed into four carton trays. A circle, 1.5 cm in
diameter, was drawn on each potato. Trays were stored at 4 °C and 85-90% RH.
Additional tubers were used for anthocyanin extractions after 0, 2, 4, and 6
months. Three replicates of 5-10 tubers of each treatment were placed into
brown paper bags immediately after harvest and stored under the same
conditions as trays.

Color measurement. Color was measured weekly as described above.

77

Anthocyanin extractions. Extractions were based on the procedure of
Siegelman and Hendricks (1958). Tubers were taken from storage at the
appropriate times and the periderm was removed by scraping with a scalpel. Skin
samples were frozen and ground as described above. Peridenn samples (0.5 g)
were added to 15 mL of 1% (v/v) HCI in methanol solution and left for 18 hours at
5 °C in the dark. Samples were centrifuged for 10 min at 3000 rpm. Absorbance
of the supernatant was measured at 510, 530 and 540 nm on a

spectrophotometer (U-3000, Hitachi, Japan).

Experiment 3. The effect of 2, 4-Dichlorophenoxyacetic acid on tuber skin-color.
Site description and experimental design. The potato variety ‘Michigan
Purple’ was grown in 11.5 cm3 plastic pots in the greenhouse at Michigan State
University. Pots were filled with mixed media, prepared from 60 % sandy soil
from Sandhill research trail field in East Lansing, Michigan and 40 % perlite.
Greenhouse conditions were 16 h daylight period and approximately 28 °C.
Plants were fertilized at planting, 4 weeks after planting, and 8 weeks after
planting with 1.45 g per pot nitrogen, 0.62 g per pot phosphate, and 2.5 g per pot
potassium. Plants were sprayed with 2,4-dichlorophenoxyacetic acid (44% a.i.
2,4-D, Agrisolutions, Agriliance, LLC) using a bench-type spraying machine. A
randomized complete block design was used with four treatments and 5
replications. 2,4-D active ingredient treatment concentrations were: 39.25 g.ha",
78.50 g.ha'1 (recommended dosage), and 157.0 g.ha". The first application was

made when plants were 7-10 inches high (pre-bud stage), and the second

78

application was made 14 days later. Tubers were harvested 9 weeks after
planting.

Color measurements. Color was measured on harvested potatoes as
described above.

Anthocyanin extraction. As described above for nitrogen experiment.

Statistical analysis. Data from all experiments were analyzed using a
Statistical Analysis System (SAS Institute, Inc., Version 8.0, 2000) to conduct
least significant differences between treatments using PROC MIXED and PROC

GLM. Slope analyses were done using PROC MIXED.

RESULTS AND DISCUSSION

Effect of soil type on tuber skin color. Organic soil grown tubers had higher
total pigments than those grown in mineral soils (Figure 1). Tubers grown 0.2 m
and 0.33 m apart in mineral soils did not differ in anthocyanin concentration. The
anthocyanin content in tubers grown in organic soils decreased after 2 months in
storage, however, the anthocyanin content of those grown in mineral soils did not
change. A complete comparison of HPLC peaks between treatments can be
seen in Appendix B Tables 1 and 2.

Hue angle (h°) was lowest in tubers grown in organic soils spaced at
0.3 m compared to those grown at either spacing in mineral soils throughout the
storage period. Hue angle of these tubers increased during the first three months

in storage (Figure 2; Appendix B Table 3). Organic soil grown tubers were also

79

darkest and more intensely colored. Lightness and chroma data shows that these
tubers became significantly lighter and color less intense after one month in
storage.

Tubers grown in mineral soils became slightly darker and the color more
intense after four months of storage. Tubers grown 0.2 m apart in mineral soils
had higher h° values than those planted 0.33 m apart. Although lightness values
were usually not different, the chroma values of the 0.2 m planted tubers were
higher than the 0.33 m planted tubers after one month in storage, meaning that
these tubers were more intensely colored. These data indicate that plants grown
closer together produce purple tubers with a more reddish hue, but a more
intense color. One could hypothesize that the closer spacing resulted in nutrient
stress e.g. phosphorus and nitrogen which lead to increased anthocyanin
synthesis since anthocyanins are synthesized in response to plant stress
(Chalker-Scott, 1999, Leonchenko, 1988, Parker 1962,). However, the
differences of the colorimeter readings, though significant, are very small, and
since the total anthocyanin data indicate no significant difference between
different planting spaces, we can conclude from this study that planting space is
not an effective way of enhancing tuber color in mineral soils.

In order to establish whether tubers grown in organic soils change color at
a different rate than tubers from mineral soils, slope comparisons of the change
in h° over time were made (Table 2). Slopes of organic grown treatments from
both the first 2 months and the last 2 months were significantly higher than

mineral soil grown treatments. However, slopes, thus rate of change in hue, did

80

not differ between 0.2 m and 0.33 m grown treatments, indicating that different

planting spaces do not have an effect on the rate of color change in storage.

Efi‘ect of different nitrogen application rates. Tubers receiving 200 kg.ha'1
N achieved the highest anthocyanin concentration after six months in storage at
4 °C (Figure 3, Appendix B Table 4). The other three treatments reached
maximum anthocyanin concentrations after four months of storage, but
decreased thereafter as can be seen at six months. There was no difference in
anthocyanin concentration between treatments at harvest. After two and four
months tubers supplied with 200 kg.ha'1 slow-releasing N had the highest
anthocyanin concentration. At six months in storage, the 200 kg.ha'1 treatment
had a significantly higher anthocyanin concentration than the other treatments.

Monthly h° values indicate that all treatments affected color significantly as
early as two months in storage (Figure 4, Appendix B Table 4). Initially and after
six months, h° of tubers grown with 200 kg.ha'1 N were higher than those grown
with 200 kg.ha'1 poultry manure and 200 kg.ha'1 slow-releasing N, however,
there was no significant difference in lightness and chroma (intensity) between
treatments after six months in storage.

In order to investigate rate of color change between treatments, a slope
comparison of change in h° was made (Table 3). No significant changes were
noted in the first 3 months of storage or the last 3 months, implying that rate of

color change in storage at 4 ° was not different between treatments.

81

Effect of different 2, 4-D application rates. All plants showed signs of 2,4-D
damage, including the control plants due to drift of 2,4-D. Chlorosis of the leaves,
flower abscission, and foliar damage (e.g. leaf curling), were observed.

Plants that received the recommended rate of 78.50 g.ha'1 a.i. of 2, 4-D
had the highest anthocyanin concentration at harvest (Table 4). Although 78.50
g.ha‘1 2, 4-D differed from the control, it was not significantly different from the
other treatments. The 157.0 g.ha'1 a.i. treatment was not significantly different
from the control or any of the treatments (Table 4).

These results agree with the findings of Fults and Schaal (1948), and
Payne et al. (1950) for red-skinned varieties. This means that anthocyanins can
be increased through the preharvest addition of a synthetic auxin in purple
potatoes as well as red potatoes. The performance of these treated potatoes in
storage was not tested, however, Payne et al. (1950) found that 2,4-D treated
“Red McClure’ tubers retained their color in 6 months of storage at 5 °C. It is
known that auxin can induce ethylene production since auxin enhances the
conversion of S- adenosyl methionine (Adomet) to 1-aminocyclopropane-1-
carboxylic acid (ACC) which in turn leads to the production of enthylene.
Ethylene has been found to enhance the biosynthesis of PAL and CSI, which are
important enzymes in the anthocyanin biosynthesis pathway (Faragher and
Chalmers, 1977; Faragher and Brohier, 1984; Li et al., 2002).

There was no difference in h° between treatments, but the 39.25 and

78.50 g.ha'1 treatments were darker than the control (Table 4). Chroma data

82

indicates that the control had a more intense color than any of the treatments

(Table 4).

CONCLUSIONS

Our study demonstrates that preharvest cultural tools can be used to
successfully modify the anthocyanin concentration and color of potato tubers. We
recommend growing ‘Michigan Purple’ potatoes in organic soils rather than
mineral soils, to enhance color and anthocyanin concentrations (approximately
500 pg.g’1 compared to 100 ug.g'1 in mineral soils). Plant spacing did not result in
differences in anthocyanin concentration, color (hue), or rates of color change in
storage. The effectiveness of different planting spaces in organic soils on tuber
color has not been determined.

Nitrogen fertilizer type and rate did not affect anthocyanin concentration at
harvest. From a nutritional point of view this implies that type of fertilizer will not
affect the health benefits of these colored potatoes. In addition, this gives
growers the freedom to decide on the type of fertilizer to use since there were no
differences between them. Different nitrogen types and rates did affect the color
(hue) of the tubers, but did not affect the rate of color change in skins of stored
potatoes.

Application of 2,4-D altered tuber anthocyanin concentrations in the skins
of ‘Michigan Purple’ tubers in a concentration-dependant manner. The

recommended 2,4-D rate of 78.50 g.ha'1 provided a concentration 2-fold higher

83

than the control. However, 2,4-D resulted in damaged plants. Based on these
greenhouse trials, we recommend using the lowest rate of 38.25 g.ha‘1 since this
rate resulted in anthocyanin concentrations not significantly different than the
recommended rate, and it could result in a decreased level of damage to the

plant.

84

REFERENCES

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ethephon. HortScience 24:962-964.

Bongue-Bartelsman, M. and D. A. Philips. 1995. Nitrogen stress regulates
expression of enzymes in the flavonoid biosynthetic pathway of tomato.
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Burg, S. P. and E. A. Burg. 1965. Ethylene action and ripening of fruits. Science
148:1190-1196.

Cantliffe, D. J. and P. Goodwin. 1975. Red color enhancement of pepper fruits by
multiple applications of ethephon. J. Amer. Soc. Hort. Sci. 100:157-161.

Chalker-Scott, L. 1999. Environmental significance of anthocyanins in plant
stress responses. Photochem. Photobiol. 70:1-9.

Do, C. B. and F. Cormier. 1991. Effects of low nitrate and high sugar
concentrations on anthocyanin content and composition of grape (Vitis
vinifera L.) cell suspension. Plant Cell Rep. 92500-504.

Edwards, J. l., W. R. Henderson and M. E. Saltveit. 1984. Yield and quality of
processing field grown tomatoes as influenced by inhibitors of ethylene
biosynthesis. HortScience 19:579-580.

Faragher, J. D. and D. J. Chalmers. 1977. Regulation of anthocyanin synthesis in
apple skin. Ill. Involvement of phenylalanine ammonia-lyase. Aust. J. Plant
Physiol. 4:133-141.

Faragher, J. D. and R. L. Brohier. 1984. Anthocyanin accumulation in apple skin
during ripening: Regulation by ethylene and phenylalanine ammonia-
Iyase. Sci. Hort. 22:89-96.

85

Fults, J. L. and L. A. Schaal. 1948. Red skin color of ‘Bliss Triumph’ potatoes
increased by the use of synthetic plant hormones. Science 108:411-415.

Kereamy A. E., C. Chervin, J. Souquet, M. Moutounet, M. Monje, F. Nepveu, H.
Mondies, C. M. Ford, R. van Heeswijck and J. Roustan. Ethanol triggers
grape gene expression leading to anthocyanin accumulation during berry
ripening. Plant Sci. 63:449-454.

Knavel, D. E. and T. R. Kemp. 1973. Ethephon and CPTA on color development
in bell pepper fruits. HortScience 82403-404.

Leonchenko, V. G. 1988. Relationship between anthocyanin accumulation and
freezing resistance in apple shoots. Fruit Grow. Viticult. 2226—27.

Li, 2., H. Gemma and S. Iwahori. 2002. Stimulation of ‘Fuji’ apple skin color by
ethepon and phosphorus-calcium mixed compounds in relation to
flavonoid synthesis. Sci. Hort. 94:193-199.

McGuire, R. G. 1992. Reporting of objective color measurements. HortScience
27:1254-1255.

Miller, R. W. and R. L. Donahue. 1995. Soil: Its composition, weathering, and
importance. p. 1-45. In J. U. Miller (ed.). Soils in our environment. 7th ed.
Prentice-Hall, N.J.

Parker, J. 1962. Relationship among cold hardiness, water soluble protein,
anthocyanins and free sugars in Hedera helix L. Plant Physiol. 37:809—
813.

Payne, M. G., J. L. Fults, R. Hay, N. Landblom and L. A. Schaal. 1950. The effect
of storage on color and sprouting of red ‘McClure’ potatoes after 2,4-D
treatment. Amer. Potato J. 292455-464.

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Pierzynski, G. M., J. T. Sims and G. F. Vance. 1994. Introduction to
environmental quality.p.1-16. In: (eds.).Soils and environmental quality.
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Rajendran, L., G. A. Ravishankar, L. V. Venkataraman and K. R. Prathiba. 1992.
Anthocyanin production in callus cultures of Daucus carota as influenced
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Sakomoto, K., I. Kumiko, K. Sawamura, H. Kyoko, A. Yoshihisa, Y. Takafumi and
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Siegelman, H. W. and S. B. Hendricks. 1958. Photocontrol of anthocyanin
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245.

87

Table 1. Four nitrogen schemes with different fertilizer or amendment
concentrations (kg.ha") applied at iven times to ‘Michigan Purple’ potato plots.
Amount of actual N applied (kg.ha' ) given in parentheses.

 

Nitrggen Treatment

 

Fertilizer or Amendment 200 kg 200 kg 200 kg 300kg
Conventional Manure Slow-release Conventional

 

 

Meister T102 400 (160)

(40%N, pre-plant)

Poultry manure compost 5600 (56)

Urea (46-0-0) (N credit)
- at planting-13 May 100 (46) 100 (46) 100 (46) 100 (46)
-at hilling- 28 May 135 (62) 70 (32) 185 (85)
-23 June 135 (62) 70 (32) 185 (85)
-18 July 70 (32) 70 (32) 185 (85)

Total N 200 200 200 300

 

(adapted from Snapp et. al., 2003)

Table 2. Effect of soil type and plant spacing on rate of hue angle change in
‘Michigan Purple’ potato tubers grown in organic and mineral soils at 0.3, 0.2:
and 0.33 m respectively during first two months and last two months of 4 months
storage at 10 °C. Standard errors are given in parentheses.

 

Time Treatment

 

Organic (0.3 m) Mineral (0.2 m) Mineral (0.33 m)

 

First2 months 1.54 (0.08) a 0.61 (0.07) b 0.72 (0.11) b

Last2 months 0.47 (0.12) a -0.08 (0.06) b -0.17 (0.04) b

 

Mean separation within rows (P < 0.05, LSD, n=40).

88

Table 3. A comparison between the rates of change in hue angle in ‘Michigan
Purple’ potatoes treated with four different rates of nitrogen: 200 kg.ha", 300

kg.ha", 200 kg.ha'1 poultry manure, and 200 kg.ha‘1 slow releasing nitrogen.

Standard errors are given in parentheses.

 

 

Cultivar Slope in first Slope in last
3 months 3 months
(hue.week") (hue.week")
200 kg.ha" 2.65 (0.26) a 2.52 (0.09) a
300 kg.ha" 2.87 (0.19) a 2.73 (0.11) a
200 kg.ha'1 poultry manure 2.52 (0.20) a 2.42 (0.13) a
200 kg.ha'1 slow releasing 2.86 (0.22) a 2.67 (0.08) a

 

Mean separation within a column (P < 0.05, LSD, n=20).

Table 4. Effect of foliar applications of 2,4-D concentrations on the anthocyanin
concentration, hue, lightness, and chroma of the skin of ‘Michigan Purple’
potatoes. Tubers were harvested 12 days after the second 2,4-D application.
Anthocyanin analyses were done by using a spectrophotometer. Standard errors
are given in parentheses.

 

 

Treatment Anthocyanins Hue angle (°) Lightness Chroma
190a" m tug/g)
control 2350 b 346.3 a 38.7 a 22.5 a
(46) (2.9) (1.1) (1.2)
39.25 3920 a 354.9 a 35.7 b 14.1 b
(345) (4.2) (0.7) (1.6)
79.5 4360 a 349.8 a 34.8 b 12.7 b
(290) (3.7) (0.6) (1.3)
157 3210 ab 348.1 a 36.7 ab 15.6 b
(182) (3.0) (0.6) (1.5)

 

Mean separation within columns (P < 0.05, LSD).

89

 

600
+ muck 0.3 m
+ mineral 0.2 m

 

 

 

 

 

 

A 500 -
Q +mineral 0.33 m
D)
:
v 400 .
a:
.E
c
(u 300 -
>
o
O
E. 200 -
E
I
100 - 3
0 I l
0 2 4

Months in storage

Figure 1. The effect of storage at 10 °C on the anthocyanin and anthocyanidin

(pg/g) concentration in the skins of ‘Michigan Purple’ potato tubers grown in
organic soil, 0.3 m apart, and in mineral soil, 0.2 and 0.33 m apart. Vertical bars
refer to standard errors of the means (n=40). Anthocyanin analyses were done

by using HPLC.

90

Figure 2. The effect of storage at 10 °C on the (a) hue angle, (b) lightness, and
(c) chroma of the skins of ‘Michigan Purple’ tubers grown in organic soil, 0.3 m
apart, and in mineral soil, 0.2 and 0.33 m apart. Vertical bars refer to standard

errors of the means (n=40).

91

 

 

 

 

 

 

 

 

 

 

 

   
 
 

  

 

 

70 1o
6
.s 65 1 1: 5
E
45 2
A g 60 1 1» 0
3—1 B
9 73
3) ft) 55 1 .4 355
g + mineral 0.2 m
3 50 1 +mineral 0.33m 11 350
I + muck 0.3 m
45 I l 1 T 345
0 1 2 3 4
Months in storage
55
50 1 ’\ I '
”’ B
3 45 4
C
q—l
‘5) W
3 40 '1
35 1 +muck 0.3 m
+ mineral 0.2 m
+mineral0.33m
30 r . . 4
0 1 2 3 4
Months in storage
15
14 1
E
13 1
9
.C
O 12 _ + muck 0.3 m
+ mineral 0.2 m
+ mineral 0.33 m
11 ‘1 C
10 l r T r
0 1 2 3 4
Months in storage
Figure 2

92

Hue angle (°) of muck soil

tubers

3500

 

3000 1

 

2500 1

 

2000 1

 

200 kglha N
-I- 300 kglha N

1500 1
+ 200 kglha poultry manure

Anthocyanins and anthocyanidins(uglg)

+200 kglha slow-releasing N

 

 

 

1000 T . .
0 2 4 6

Months in storage
Figure 3. The effect of different nitrogen treatments: 200 kg.ha", 300 kg.ha", 200
kg.ha‘1 poultry manure, and 200 kg.ha'1 slow-releasing nitrogen, on the
anthocyanin and anthocyanidin (09.9") content in the skins of ‘Michigan Purple‘
potato tubers at harvest and in storage. Vertical bars refer to standard error of
the means (n=20). Anthocyanin analyses were done by using a
spectrophotometer.

93

Figure 4. The effect of different nitrogen treatments: 200 kg.ha", 300 kg.ha", 200
kg.ha'1 poultry manure, and 200 kg.ha'1 slow-releasing nitrogen, on the (a) hue
angle, (b) lightness and (c) chroma of the skins of ‘Michigan Purple’ tubers at
harvest and in storage. Vertical bars refer to standard error of the means (n=20).

94

 

     

 

 

 

 

 

 

 

 

 

  
   

 

 

351 A
30 ..
g" 25 1
v
2 20 1
D)
g 15 1
a: +200kgma~
3 1° ‘ +300 kglheN
I +200 kglha poultry manure
5 1 +200kgflmslow-relm1ng‘ N
o .
355 . V 1 2 3 4 5 6
350
Months 1n storage
48
46 a
44 4
g 42 1
.C
a 101
+200kglha N
38 ‘ +300kg/haN
+200 kglha poultry manure
36 1 +200 kglha slow-releasing N
u I T T T l
1 2 3 4 5 6
Months in storage
15.0
14.5 1 +200| 1 N
14 0 1 +300me
° +200kg/hapoultry manure
13.5 c +200kg/haslow-releasing N
g .
6 12.5 1
12.0 1
11.5 1
11.0 1 .. i
10.5 1
10-0 T I T l T I
0 1 2 3 4 5 6
Months in storage
Figure 4

95

 

 

 

CHAPTER III

THE EFFECT OF POSTHARVEST STORAGE TEMPERATURE REGIMES AND
ETHYLENE EXPOSURE ON THE COLOR AND ANTHOCYANIN CONTENT OF
‘MICHIGAN PURPLE’ POTATO TUBERS

96

INTRODUCTION

Appropriate color and visual appearance of stored potatoes is critical to
consumer purchasing decisions (Maskan, 2001).Temperature is one of the most
important storage factors in postharvest quality control of potatoes. Ineffective
temperature management can lead to postharvest decay and sugar accumulation
as a consequence of excessively low storage temperatures (Kazami et al., 2000;
Spychalla and Desborough, 1990). The colored cultivar “Chieftain” has been
reported to become softer and darker in storage at 4, 8, 12, 16 and 20 °C, but
storage temperatures of 16 and 20 °C resulted in more rapid changes in quality
parameters (Nourian et al., 2003). Lewis et al. (1999) reported that anthocyanin
concentrations increased in the skins of colored potatoes stored at 4 °C, but not
at 10, 18, and 26 °C. Conversely, Andersen et al. (2002) reported a decrease in
the anthocyanin concentrations of potatoes stored at 4 °C. It is likely that this
discrepancy is due to differences in response of different varieties to
temperature.

Lewis et al. (1999) argued that cold temperature induction of sweetening
i.e. the conversion of starch to sugars causes the increase in anthocyanins, since
sugar is an anthocyanin precursor. Hara et al. (2003) found that in the hypocotyls
of radish (Raphanus sativus L.), sugars act as signal molecules that activate the
CH8 (chalcone synthase) and the ANS (anthocyanin synthase) genes to
accumulate anthocyanins. Similar results were also found for CHS-A gene from
petunia in transgenic Arabidopsis (T sukaya et al., 1991). On the other hand, it

has also been found that anthocyanin concentrations can increase when plant

97

tissue is exposed to temperatures high enough to activate heat shock proteins
(T akeda et al., 2003).

Anthocyanin synthesis is an energy expensive process that is usually
influenced by pre- or postharvest environmental stresses. Many of these stress
responses act through ethylene (Faragher and Chalmers, 1977; Faragher and
Brohier, 1984; Li et al., 2002). Ethylene stimulates anthocyanin biosynthesis by
increasing the activity of phenylalanine ammonia-lyase (PAL) (Faragher and
Brohier, 1984), chalcone isomerase (CHI) (Li et al., 2002), and UDP glucose-
flavonoid 3-O-glucosyltransferase (UFGT) (Kereamy et al., 2002). The ethylene-
generating compound, ethephon, has been used on many Solanaceous fruit to
enhance color, including tomato (Lycopersicon esculentum Mill). (Burg and Burg,
1965; Edwards et al., 1984) and pepper (Capsicum annuum L.) (Cantliffe and
Goodwin, 1975; Armitage, 1989). It is also used on apples (Ma/us x domestica)
to enhance color (Awad and de Jager, 2002; Li et al., 2002).

In this paper we investigate the effects of postharvest storage temperature
manipulation on the skin color of ‘Michigan Purple’ potatoes. We hypothesize that
alternating low (4 °C) and high (20 °C) temperatures will result in higher
anthocyanin concentrations than continuous low temperature storage. Our
rationale is based on the idea that enzymes active in anthocyanin biosynthesis
will be synthesized at lower temperatures, and activated at the higher
temperatures. In addition, we investigate the effect of postharvest ethylene

application on the tuber skin-color of ‘Michigan Purple’ potatoes. We hypothesize

98

that postharvest ethylene will enhance the activity of enzymes such as PAL, and

therefore stimulate anthocyanin biosynthesis in the tuber skins.

METHODS AND MATERIALS

Temperature experiment:

Site description and experimental design. ‘Michigan Purple’ potato tubers
were obtained from Montcalm Research Farm, Entrican, Michigan. After harvest,
tubers were washed, air dried and divided into replicates. Following initial color
measurement, tubers were placed into trays in coldrooms. Three replicates of ten
tubers were used for each of the treatments. Treatments were: (1) 60 days at 4
°C, (2) 60 days at 20 °C, (3) 15 days at 4 °C followed by 45 days at 20 °C, (4) 30
days at 4 °C followed by 30 days at 20 °C, (5) 45 days at 4 °C followed by 15
days at 20 °C, and (6) 7 days at 30 °C followed by 53 days at 20 °C. Relative
humidity was kept at 80-90%.

Color measurements: A circle, 1.5 cm in diameter, was drawn on each
potato before placed into storage. Color was measured weekly on the circled
area with a colorimeter (Colorimeter CR 300, Minolta, Japan) in order to trace
color change in the particular area. Chroma, hue angle, and lightness values
were recorded. Colorimeter was calibrated using a standard white plate
(lightness 97.99, chroma 2.03, hue angle 101.0).

Anthocyanin extractions. Extractions were based on the procedure of
Siegelman and Hendricks (1958). Tubers were taken from storage at the

appropriate times and the periderm removed by scraping with a scalpel. The skin

99

was immediately frozen in liquid nitrogen and stored at -80 °C until analyzed. The
periderm was ground to a powder with a mortar and pestle. Periderm samples
(0.5 g) were added to 15 mL of 1% HCI in methanol solution and left for 18 hours
at 5 °C in the dark. Samples were centrifuged for 10 min at 12000 x g.
Absorbance of the supernatant was measured at 510, 530 and 540 nm on a
spectrophotometer (U-3000, Hitachi, Japan).

Ethylene experiment: Freshly harvested potatoes were divided into eight
replicates of five potatoes. A circle, 1.5 cm in diameter, was drawn on each
potato and the potato was numbered. Color was measured as described above

and tubers were placed into glass jars. Four jars were exposed to 100 (uL.L'1)

ethylene for seven days at room temperature while the control was exposed to
air at room temperature. After seven days, tubers were removed from the glass
jars and color was measured every week for the next five weeks.

Statistical analysis. Data was analyzed using commercial Statistical
I Analysis System software (SAS Institute, Inc., Version 8.0, 2000) to calculate
least significant differences between treatments using PROC MIXED and PROC

GLM. Slope analyses were done using PROC MIXED.
RESULTS AND DISCUSSION
Temperature experiment. After 60 days of storage, the amount of

anthocyanin in the skin of the ‘Michigan Purple’ tubers was the highest in the

tubers that were stored at 4 °C for 60 days (Table 1). While exposure to elevated

100

temperatures during the storage period did not always result in significantly lower
anthocyanin levels, treatments 2, 5, and 6 were significantly lower than treatment
1 after 60 days.

Hue angle was not affected by treatments at harvest, and even though
there were differences between treatments after week one and six, after eight
weeks there was still no difference in h° (Table 2; Appendix C Figure 1). Hue
angle increased significantly within every treatment in storage. The color of
tubers stored at 4 °C for 60 days changed significantly after four weeks in
storage and remained at this higher h° until taken out of storage after 8 weeks.
Tubers stored at 20 °C for 60 days changed after one week from 090° to 13.69°,
and remained at this higher hue angle from weeks 4 to 8. Tubers receiving the
heat shock treatment at 30 °C increased significantly in hue angle from 1.75° to
21 .79° after one week and also remained higher than the initial for the 60 days in
storage.

All treatments resulted in darker tubers (decreased lightness) after 8
weeks in storage (Table 2). Although treatments did not differ in lightness at
harvest, tubers receiving treatments 1 and 5 were darker than tubers receiving
treatments 3 and 4. Treatments did not differ in skin-color intensity at harvest, but
after 8 weeks in storage treatment 1 resulted in significantly more intense tubers.
All treatments resulted in more intensely colored tubers after two weeks and
remained more intense until removal from storage. The hue angle of the tubers
did not show a specific shift at this time. Tubers with higher anthocyanin content

were the most intensely colored at the end of the experiment.

101

Ethylene experiment. Initially, when tubers were placed into jars, the
control and the ethylene treatment had significantly different hue values;
however, after two weeks there was no difference between treatments (Figure 1).
Even after 5 weeks of close observation, there was still no difference in color.
Chroma and lightness values did not change over the storage period (data not
shown). We found ‘Michigan Purple’ tubers to be insensitive to postharvest

ethylene application.

CONCLUSIONS

We found that the highest pigment concentration was maintained under
continuous cold temperature of 4 °C, possibly as a result of the conversion of
starch to sugars, which then served as precursors to anthocyanins. Continuous
storage at 20 °C yielded significantly lower pigment concentrations. It seems that
timing of removal of tubers from storage was also important since removal from
4° C after 45 days yielded significantly lower pigment concentration that the
control at 4°C. We suggest that the last 15 days at 20 °C could have resulted in
the deterioration of the pigment, or the initial pigment concentration of the
treatment could have been lower. The heat-shock treatment (7 days at 30 °C) did
not result in higher pigment concentrations. The 30 °C might have been too low
for the activation of the heat-shock proteins. Since the trend in the change of hue

angle values could not be explained by the anthocyanin concentrations after

102

storage, we conclude that hue angle shifts are not entirely associated with
changes in anthocyanin concentrations.

Further studies should be done to determine the pigment concentration at
the point of removal from the initial lower temperature before placement at the
higher temperature since it will be an accurate way of determining when the
changes in pigment concentrations take place.

Postharvest ethylene treatment did not increase or change the color of the
tubers. Further study could include preharvest ethephon applications to
determine if tubers perceive preharvest ethylene and whether pigment

accumulation is enhanced and if so, at which stage they are most responsive.

103

REFERENCES

Andersen, A. W., C. S. Tong and D. E. Krueger. 2002. Comparison of periderm
color and anthocyanins of four red potato varieties. Amer. J. Potato Res.
79:249-253.

Armitage, A. M. 1989. Promotion of fruit ripening of ornamental peppers by
ethephon. HortScience 24:962-964.

Awad, M. A. and A. de Jager. 2002. Formation of flavonoids, especially
anthocyanin and chlorogenic acid in ‘Jonagold’ apple skin: influences of
growth regulators and fruit maturity. Sci. Hort. 93:257-266.

Burg, S. P. and E. A. Burg. 1965. Ethylene action and ripening of fruits. Science
148:1190-1196.

Cantliffe, D. J. and P. Goodwin. 1975. Red color enhancement of pepper fruits by
multiple applications of ethephon. J. Amer. Soc. Hort. Sci. 100:157-161.

Edwards, J. I., W. R. Henderson and M. E. Saltveit. 1984. Yield and quality of
processing field grown tomatoes as influenced by inhibitors of ethylene
biosynthesis. HortScience 191579-580.

Faragher, J. D. and D. J. Chalmers. 1977. Regulation of anthocyanin synthesis in
apple skin Ill. Involvement of phenylalanine ammonia-lyase. Aust. J. Plant
Physiol. 4:133-141.

Faragher, J. D. and R. L. Brohier. 1984. Anthocyanin accumulation in apple skin
during ripening: Regulation by ethylene and phenylalanine ammonia-
Iyase. Sci. Hort. 22:89-96.

Hara, M., K. Oki, K. Hoshino, and T. Kuboi. 2003. Enhancement of anthocyanin
biosynthesis by sugar in radish (Raphanus sativus) hypocotyl. Plant Sci.
164:259-265.

104

Kazami, D., T. Tsuchiya, Y. Kobayashi and N. Ogura. 2000. Effect of storage
temperature on quality of potato tubers. J. Jpn. Soc. Food Sci. Technol.
47:851-856.

Kereamy, A. E., C. Chervin, J. Souquet, M. Moutounet, M. Monje, F. Nepveu, H.
Mondies, C. M. Ford, R. van Heeswijck and J. Roustan. Ethanol triggers
grape gene expression leading to anthocyanin accumulation during berry
ripening. Plant Sci. 63:449-454.

Lewis, C. E., J. R. L. Walker and J. E. Lancaster. 1999. Changes in anthocyanin,
flavonoid and phenolic concentrations during development and storage of
coloured potato (Solanum tuberosum) tubers. J. Sci. Food Agri. 79:311-
316.

Li, 2., H. Gemma and S. Iwahori. 2002. Stimulation of ‘Fuji’ apple skin color by
ethepon and phosphorus-calcium mixed compounds in relation to
flavonoid synthesis. Sci. Hort. 94:193-199.

Maskan, M. 2001. Kinetics of color change of kiwifruits during hot air and
microwave drying. J. Food Eng. 48:169-175.

Nourian, F ., H. S. Ramaswamy and A. C. Kushalappa. 2003. Kinetics of quality
change associated with potatoes stored at different temperatures.
Lebensm. Wiss U. Technol. 36:49-65.

Siegelman, H. W. and S. B. Hendricks. 1958. Photocontrol of anthocyanin
synthesis in apple skin. Plant Physiol. 33:185-190.

Spychalla, N. P. and S. L. Desborough. 1990. Fatty acids, membrane
permeability, and sugars of stored potato tubers. Plant Physiol. 9421207-
1213.

Takeda, T., M. Inomata, H. Matsuoka, M. Hikuma and S. Furusaki. 2003.
Release of anthocyanin from strawberry cultured cells with heating
treatment. Biochem. Eng. J. 15:205-210.

105

Tsukaya, H., Ohshima, T., Naito, S., Chino and Y. Komeda. 1991. Sugar
dependant expression of the CHS-A gene for chalcone synthase from
petunia in transgenic Arabidopsis. Plant Physiol. 97:1414-1421.

106

Table 1. Anthocyanin (pg/g FW) concentrations in the skin of “Michigan Purple’
potato tubers after 60 days of storage at different combinations of 4 °C, 20 °C
and 30 °C. Analyses were done by using spectrophotometer. Standard errors are
given in parentheses.

 

Treatment Anthocyanin (pg/g)

 

Number Days at 4 °C Days at 30 °C Days at 20 °C

 

1 6o 0 o 2310 (262) a
2 45 0 15 1402 (77) b
3 30 0 30 1760 (146) ab
4 15 o 45 1730 (77) ab
5 0 0 60 1360 (50) b
6 0 7 53 1420 (50) b

 

Mean separation within columns (P < 0.05, LSD).

107

 

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Hue angle

+ control
+ ethylene

 

 

7 14 21 23 28 35

 

 

355

 

Days after ethylene treatment

Figure 1. The effect of exposure of 100 ppm ethylene for 7 days on the hue angle
of ‘Michigan Purple’ potatoes stored at 4 °C after receiving treatment. Vertical
bars refer to the standard errors (n=25).

110

CHAPTER IV

SUMMARY

111

In conclusion, we found the most worthwhile avenue to pursue in order to
achieve intense purple tuber skin-color in ‘Michigan Purple’ potatoes, to be the
use of preharvest cultural tools. Specifically, we found planting in organic soils
rather than mineral soils, and the application of 2,4-D to most dramatically
intensify the tuber skin-color. Plants grown in the greenhouse showed significant
2,4-D damage, therefore, before we recommend any rates to the growers, we
recommend testing 2,4-D under field conditions, since results could vary between
locations.

Postharvest factors that could play a role in maintaining or even increasing
anthocyanin concentrations and color (hue), are storage at low temperatures and
minimizing exposure to higher storage temperatures.

Since auxin and ethylene have the potential to work in similar ways in
stimulating anthocyanin biosynthesis through stimulating the enzymes in
anthocyanin biosynthesis, we suggest testing preharvest foliar and soil
applications of ethephon. If ethephon application is effective, the use of this

product would be preferable to 2,4-D, since 2,4-D damage would be avoided.

112

APPENDIX A

113

Table 1. Impact of storage duration at 10 °C on total anthocyanins (pg/g FW), of
‘Michigan Purple’, ‘Dakota Rose’, ‘California Red’, and ‘Norland’ potatoes. HPLC
absorbance values of total anthocyanins are given. HPLC absorbance values
(au) at 530 nm also are given for comparison between cultivars

114

Table 1.

 

Cultivar

Michigan
Purple
Dakota
Rose
California
Red
Norland

Michigan
Purple
Dakota
Rose
California
Red
Norland

Michigan
Purple
Dakota
Rose
California
Red
Norland

Michigan
Purple
Dakota
Rose
California
Red
Norland

Michigan
Purple
Dakota
Rose
California
Red
Norland

Months in storage at 10 °C

 

 

0 2 4 6
Total Anthocyanins
294.4 B a 198.7 B ab 142.3 C b 209.9 C ab
686.6 B b 1032. A ab 1279.2 A a 963.7 B ab
1147.7 A ab :15000. A b 1471.0 A ab 1584.7 A a
479.8 B a 331.9 A a 761.4 B a 599.1 BC a
Absorbance at 530 nm (AU x 1000)
141 73 A a 1 0645 A a 7560 A a 8652 B a
7352 AB a 1 1 91 1 A a 1 3916 A a 7392 B a
13003 AB a 10870 A a 14900 A a 18350 A a
5870 B a 9662 A a 1 1331 A a 7700 B a
Hue
359.97 B c 7.69 C ab 9.07 B a 6.45 B b
19.28 A b 20.30 B ab 22.19 AB a 21.85 A a
19.56 A b 23.39 AB a 24.14 AB a 22.58 A a
23.74 A a 25.6 A a 25.7 A a 24.76 A a
Lightness
39.00 B b 40.39 B a 40.62 A a 40.69 A a
40.01 B b 41 .24 AB a 40.41 A ab 40.63 A ab
40.97 AB a 41.21 AB a 41.36 A a 40.62 A a
41.77 A a 42.13 A a 41.77 A a 41.89 A a
Chroma
13.46 B a 12.24 C a 12.38 C a 13.34 B a
23.72 A a 24.82 A a 24.19 A a 24.43 A a
24.62 A a 22.88 AB b 22.48 AB b 23.46 A ab
23.16 A a 22.13 B a 22.01 B a 22.57 A a

 

Small letters refer to means separation within a row; capital letters refer to mean
separation within column (P < 0.05, LSD, n=20).

115

Table 2. Impact of storage duration on anthocyanidin content of ‘Michigan
Purple’, ‘California Red’, ‘Norland’, and ‘Dakota Rose' potato tubers stored at 10

°C.

 

Treatment HPLC
peak

Ml Purple

Cal. Red

Norland

Dak. Rose

WAGON-8 (J'l-thA

Ul-h‘flN—i»

Months in storage

 

0
15.7

6.8
39.7
275.9
8.3

99.4
19.9
NA
924.0
104.4

32.5
13.1
23.3
404.0
15.7

34.0
22.4

13.9
512.7
197.5

mmmm 0)

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2
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5.5
15.4
184.3
7.1

106.0
19.3
12.5

788.3
82.4

52.2
23.4
12.1
657.7
94.3

43.2
33.4
17.4
871.1
78.8

mmmmm mama-o- mac-mm

mm

ab

b

4
10.8

4.4
7.8
134.6
5.2

52.3
48.3
19.1

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114.8

42.6
22.6
11.8
651.3
44.6

73.0
38.2
33.8

1015.7

118.5

(DUCT!!! 0)

b
a

a

a
a
a
a

ab

a
a
a
a

ab

6
23.9

NA
12.6
207.9
5.3

164.1
30.5
13.7

1241.7

139.3

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NA

496.1

67.3

81.4
31.2
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787.8
58.8

m

m
mmmcm

mm

a
a
a

ab

b

 

Letters refer to means separation within a column (P < 0.05, n=30). NA=not

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117

Table 4. The effect of storage duration on the pigment (anthocyanin and
anthocyanidin) concentration (pg/g). hue, lightness, and chroma of the skin of
potato cultivars ‘Chieftain’, “Dakota Rose’ and ‘Michigan Purple’ stored at 4 °C in
2003.

 

Months in storage

 

 

Cultivar 0 2 4 6
Total Anthocyanins
Michigan 2184.5 C a 2146.9 B a 2426.5 C a 1612.1 C a
Purple
Chieftain 5526.5 B a 5076.9 B a 5758.4 B a 5499.0 B a
Dakota Rose 9747.2 A d 12005.2 A c 14008.3 A b 16674.1 A a
Hue
Michigan 1.09 C d 13.5 C c 20.56 b 26.44 B
Purple
Chieftain 34.08 A d 39.25 A a 41.96 A b 44.98 A
Dakota Rose 18.49 B c 24.63 B b 26.57 B ab 28.10 B
Lightness
Michigan 77.70 A a 43.62 B b 44.07 B b 44.01 B
Purple
Chieftain 77.62 A a 47.92 A b 48.51 A b 48.48 A
Dakota Rose 76.47 A a 43.32 B c 44.02 B b 44.34 B
Chroma
Michigan 4.46 B c 11.38 C a 10.76 B b 10.60 B
Purple
Chieftain 6.79 A d 21.32 B a 20.24 A b 19.25 A
Dakota Rose 7.44 A d 22.78 A a 21.00 A b 20.09 A

 

Small letters refer to means separation within a row; capital letters refer to mean

seperation within column (P < 0.05, LSD, n=30).

118

 

Figure 1. The HPLC chromatogram of ‘Michigan Purple’ potato (a) and the
spectra of (b) peak 1, (c) peak 2, (d) peak 3 and (e) the standard, petunidin-
chloride (0.25 pg/pl), used to quantify the pigment content in ‘Michigan Purple’
potatoes.

119

 

 

 

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122

 

 

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125

 

 

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126

 

 

 

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1.4% E

1.201
1

1.00-*

3 0-30”.
< .
0.601
0.40:
0.20:

0.002

 

 

 

40000420001440].00460004800050).00520.005400056000’580.00T ‘
nm

Figure 4

128

APPENDIX B

129

63556 docu<2 dvuc .09 .mod v n: 5:28 555, cos—8300 cam—2

 

 

 

 

a de m dd 0 ON m d. E and .2052
3 odd 0 dd? 0 dd 0 0.3 Nd .9052

m mddN m w. 5 <2 0 ad? ad V.03.). 4 5:22
a odd 9 vdp <2 0 mNF mmd .3052
a m. For n 4.9 a NR 0 m.N_. Nd .9055.

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n d. 5 <2 2 dN n md and .9055.
9 odd n we 9 m5 9 vd Nd .9055.

m mNdm m Yum m 4.9 m Pdw dd 0.03.2 o 5:52
4 m N F 9&8 ._6w

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3.33 55298955 new 5cm>oo5:m co @50QO 25.5 new cab :8 ho Soto 05.... min...

130

Table 2. The effect of soil type and plant spacing on anthocyanin and
anthocyanidin (pg/g) concentrations in the skin of ‘Michigan Purple’ potatoes at
harvest and after 2 and 4 months of storage at 10 °C.

 

 

 

Treatment HPLC Months in storage
peak
Soil Planting 0 2 4
space (m)
Muck 0.3 1 13.1 a 8.8 a 10.26 a

2 12.4 a 10.8 a NA
3 57.42 a 46.8 ab 35.3 b
4 362.3 a 310.4 ab 332.6 b

Mineral 0.2 1 5.4 b 12.3 a 16.2 a
2 1.5 a 2.2 a 3.3 a
3 4.8 a 10.4 a 12.0 a
4

60 a 101.5 a 100.6 3

Mineral 0.33 1 5.5 b 13.9 a 13.2 ab
2 3.0 a NA 2.3 a
3 NA 10.4 a 10.5 a

4 61.6 a 85.6 a 91.4 a

 

Mean separation within column (P < 0.05, LSD, n=40, NA=not available).

131

Table 3. The effect of different soil types on the total pigments (anthocyanin and
anthocyanidin) concentration, total absorbance at 530 nm, hue (h°), lightness,
and chroma of the skin of ‘Michigan Purple’ tubers stored at 10 °C.

 

Months in storage

 

Treatments 0 1 2 3 4

 

Soil Planting
space
(m)
Anthocyanins and anthocyanidins (pg/g)

Muck 0.3 460.1 A a NA 397.8 A b NA 368.1 A b
Mineral 0.2 66.7 B a NA 125.7 B a NA 118.8 B a
Mineral 0.33 70.2 B a NA 105.0 B a NA 103.0 B a
Absorbance at 530 nm (AU x 1000)
Muck 0.3 35990 Aab NA 44270 A b NA 25380 A b
Mineral 0.2 3646 B a NA 6616 B a NA 8566 B a
Mineral 0.33 3727 B a NA 5697 B a NA 5432 B a
Hue
Muck 0.3 349.3 C b 358.90 b 1.3 C b 5.3 C a 6.0 Ca
Mineral 0.2 61.4 A b 67.6 A a 67.6 A a 66.8 A a 67.2 Aa

Mineral 0.33 52.8 B b 61.0 Ba 60.9 Ba 61.5 Ba 60.5 Ba

Lightness
Muck 0.3 39.0 B d 40.6 Bc 41.6 Bab 41.5 C b 42.08 Ba
Mineral 0.2 50.1 A a 49.25 Ab 49.3 A b 48.9 A b 49.07 Ab

Mineral 0.33 50.2 A a 48.90 Ab 48.8 A b 47.8 B c 48.40 Ab

Chroma
Muck 0.3 13.8 A a 11.3 C b 10.7 C c 10.5 Bcd 10.4 Cd
Mineral 0.2 13.5 AB c 14.2 A b 14.1 A b 14.3 A a 14.5 Aa

Mineral 0.33 12.8 B c 13.4 Bb 13.4 B b 13.8Aa 13.7 Ba

 

Small letters refer to mean separation within a row; capital letters refer to mean
separation within a column (P< 0.05, LSD, n=40).

132

 

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no < 8.9 u < N29 0 < 8.9 u < 9.9 o < 5.: 0 m< 00.9 a m 94.9 N
o: < 05.9 o < 8.9 o < 04.9 o < 8.9 a < 40.: 0 m 00.9 n m mm. 9 P
050.50
0 < 00.3 0 m< 2...} 0 < no.3. 0 m< 00.3. n m< Nva o < dem : m< Nd... v
:0 < 5.04 0 m 5.0.0 0 < 8.3. 0 m 3.94 o m mm. 3 u < Edd n m 05% m
:0 < 8.04 0 < 04.3 0 < 8.9. 0 < NE? 0 < Nudv u < mmdm 0: < 4.3. N
:0 < 50.3 0 m< N044 0 < no.9. 0 m< 00.3. o m< moNv 0 < Edd : m< mode 9
00053.5
0 m mv.vN 0 m< 0 EN 0 m vaN 0 m< dN. 9N : m< 00.9 o m 5.0 o m <0.va v
0 m omdN 0 m oddN 0 m mmNN : m 00.9 o m 94.9 n m Sam 0 m 3.30 m
0 m< SN 0 m< 94.3 0 m< SEN 0 m< dddN : m< dmdN o m 00.4 .0 m< 0.500 N
n
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0:1
0 m< dva i 0 < SN 1. 0 < dBN i n < dd N v
o o dmdN i 0 < dva i d o 89 i n < 9. N m
0 m. doVN i 0 < ddVN i 0 m< dch i n < dddN N
0 < deN i n < 9¢N i n m EN .1 n < 89 P
«90.5 05205005254
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30.20 5 05:05.

 

133

.2 m5000.0: .550 F.059.
ddN n 4 0:0 05:08 55.28 F.059. SN 0 m .2 P.059. don u N .2 F.059. SN 0. .. 005 05:02:00.... .0.. .10 5.050 000.20 0:0
.050: 0:0 02:00 :095: 2o 8.50:2 0 00 00:3 .0_B:n. c0955.? he :50 05 5o :28 0:0 5205:0050 052050050 0:... .v 0.005

APPENDIX C

134

 

35

 

+4°C2 60d
+4'Cz45d - 20'Cz15d
30 - +4°Cz 30d - 20 °C230d
-<>-4°C:15d - 20’Cz45d
+20’C: 60d

25 4 -G-30°C:7d-20°C:53d ’
”'r-
20 d . /

151

Hue angle (°)

 

7 ' 28 42 60

 

 

 

Days in storage

Figure 1. The effect of 60 days storage at different temperatures on the hue
angle of ‘Michigan Purple’ potato tuber skins. Vertical bars refer to standard
errors of the means (n=30).

135

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