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dissertation entitled

MOLECULAR ASPECTS OF FRUIT ABSCISSION IN MALUS
DOMESTICA AND FLORAL ORGAN ABSCISSION IN
ARABIDOPSIS THALIANA

presented by

Lingxia Sun

has been accepted towards fulfillment
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PhD degree in Horticulture

 

 

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MOLECULAR ASPECTS OF FRUIT ABSCISSION TN MAL US DOMESTIC/1 AND FLORAL
ORGAN ABSCISSION IN ARABIDOPSIS T HALIANA

By

Lingxia Sun

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Horticulture

2009

ABSTRACT

MOLECULAR ASPECTS OF FRUIT ABSCISSION IN MALUS AND FLORAL
ORGAN ABSCISSION IN ARABIDOPSIS THALIANA

By
Lingxia Sun

Organ abscission is a developmentally and environmentally regulated cell
separation process initiated in specialized tissues, the abscission zones (AZs).
Understanding the basis of organ abscission is of fimdamental interest to elucidate
underlying mechanism of organ abscission and is of applied interest as abscission
regulation is critical to achieve optimized market value. A model developed from
physiological studies has established that the balance between auxin and ethylene within
the A23 regulates leaf abscission. Genetic analyses in model plant species have
identified both ethylene-dependent and ethylene-independent pathways to regulate floral
organ abscission. In this dissertation, I sought to: investigate natural variation in fruit
abscission-related traits in Malus species, identify gene expression changes within the
pedicel abscission zone during apple fi'uit abscission, analyze promoter activity of
members of the PECTATE LYASE-LIKE gene family in cell separation and wall
loosening in Arabidopsis. In the first study, I evaluated 144 Malus accessions
representing wild species, domestic cultivars, and hybrids for abscission-related traits. I
found that seasonal timing of fruit abscission in wild species and hybrids showed a broad
distribution similar to that seen for domestic cultivars, and that internal ethylene
concentration at the time of abscission varied by over three orders of magnitude. Wild
species, domestic cultivars, and hybrids all included representatives that showed

abscission of fruit prior to substantial production of ethylene, as well as accessions that

retained fiuit for a significant period of time following ethylene production. For all
accessions that retained fruit, fruit removal resulted in abscission of the pedicel, and
exogenous ethylene promoted abscission, suggesting that the abscission zone was
functional. Our results suggest important roles for mechanisms independent of fruit
ethylene production in abscission. In the second study, we identified transcriptional
changes accompanying the transition from competent-quiescent to activated A23 in the
apple fi'uit pedicel. The abscission-associated genes identified in this work contribute to
our understanding of fruit abscission, while suggesting a common molecular mechanism
of fruit abscission induced under various conditions. In the third study, we documented
the spatial and temporal promoter activity of 23 of the 26 Arabidopsis PLL genes
throughout development. Numerous gene family members showed activity in localized
domains programmed for abscission, such as the abscission zones (AZs) of the sepal,
petal, and stamen, and seed, as well as the fi'uit dehiscence zone. Several other members
showed activity in cell types expected to facilitate separation, including the endosperm
layers during seed germination, and root endodermal and cortical layers during lateral
root emergence. Other PLL promoters were active in domains not obviously
programmed for separation, including the apparent vestigial AZs of the branch and
pedicel. These results suggest potential for unique and overlapping activity of PLL genes,

and provide guidance for analysis of individual gene function through reverse genetics.

DEDICATION

T 0 my family, for their support and love, and to my husband, Y iyong, for his
understanding, encouragement, and endless love

iv

ACKNOWLEDGEMENTS

I would like to thank my committee members; Drs Steve van Nocker, Randy
Beaudry, Martin J Bukovac, Jianping Hu, and Rebecca Grumet, for their guidance
throughout my graduate studies. I would like to give special thanks to my advisor, Dr.
Steve van Nocker, for providing me the support and encouragement I needed during my

study in his lab.

I would like to thank Philip Forsline and Bill Srmack from USDA-ARS Plant
Genetic Resources Unit for their help during the time I stay there for my research. I also
would like to thank Dr. Chris Watkins from Cornell University for allowing me using his

instrument.

I would like to express my gratitude to my fellow labmates, past and present, Hua
Zhang, Philip Ludwig, Jullisa Ek-Ramos, YingYan, Sookyung Oh, Sunchung Park, for
their technical and moral support. Also, I would like to thank Kaori Ando fi'om Rebecca
Grumet’s lab, Ann Armenia and Veronica Vallejo from Ning Jiang’s lab for their

friendship and moral support.

I also would like to thank College of Agricultural Natural Resources for the
Dissertation Completion Fellowship, and Office for International Students & Scholar for

tuition fee assistance.

TABLE OF CONTENTS

LIST OF FIGURES .................................................................................... x
CHPATER 1. Literature Review .................................................................... 1
l. Introductronl

2. Processes of organ abscission ........................................................... 3

2.1 Differentiation of abscission zone ........................................... 3

2.2 Activation of abscission zone ................................................ 6

2.3 Separation of abscission zone ............................................... 20

3. Hormonal regulation of organ abscission in Agricultural Practices .............. 22

3.1 Fruit thinning. .................................................................................... 23

3.2 Preharvest fruit drop ............................................................................ 25

4. References .......................................................................................................... 27
CHAPTER 2. Natural variation in hit abscission-related traits in apple ......................... 37
Abstract .................................................................................................................. 37
Introduction ............................................................................................................ 38
Material and Methods ............................................................................................ 43

Plant material ............................................................................................. 43

Analysis of fruit abscission ........................................................................ 43

Measurement of internal ethylene concentration, firmness, and starch ..... 44

MdA CSI genotyping .................................................................................. 44

Evaluation of pedicel abscission and ethephon-promoted abscission ...... 45

Results ................................................................................................................... 46
Seasonal timing of fruit abscission ........................................................... 46

Variation in fruit ethylene concentration at abscission ............................. 49

Abscission in response to fruit removal and exogenous ethylene ............. 54

Discussion .............................................................................................................. 59
References .............................................................................................................. 64

CHAPTER 3. Molecular analysis of abscission zone development in apple fruit

pedicels .............................................................................................................................. 68
Abstract .................................................................................................................. 68
Introduction ............................................................................................................ 69
Material and Methods ............................................................................................ 71

Plant material and treatments ..................................................................... 71
Preparation of cDNA arrays, probe preparation, and hybridization .......... 72
Experimental designs and data analysis ..................................................... 73
Reverse transcription (RT)-PCR analysis ................................................. 74
Results and Discussion .......................................................................................... 75
Abscission kinetics of apple fruit pedicel .................................................. 75
Analysis of gene expression changes in abscission of fruit pedicels ......... 75
Chemical fruit thinning .............................................................................. 80

Preharvest fruit drop .................................................................................. 83
Implications of differentially expressed genes in abscission ..................... 85
References .............................................................................................................. 97

CHAPTER 4. Analysis of promoter activity of members of the PECTA TE LYASE-LIKE

(PLL) gene family in cell separation and wall loosening in Arabidopsis ........................ 102
Abstract ............................................................................................................... 102
Introduction ......................................................................................................... 103
Material and Methods ......................................................................................... 105

Plant growth conditions .......................................................................... 105
Phylogenetic analysis and protein domain identification ....................... 106
Transgenic plant construction. ................................................................ 106
Histochemical GUS assay ....................................................................... 108
Expression analysis of AtGenExpress data ............................................. 108
Results ................................................................................................................ 109
Phylogenetic analysis .............................................................................. 109
Estimation of PLL gene expression through analysis of public
transcriptome data .................................................................................... 109
Analysis of PLL promoter activity .......................................................... 111
PLL:G US expression associated with cell separation ............................. 113
PLL-GUS expression in the cell wall loosening ..................................... 117
Discussion ............................................................................................... 125
References ............................................................................................................ 131
CONCLUSION ................................................................................................................ 137

vii

LIST OF TABLES
Table 2.1 Number of wild species, domestic cultivars, and hybrids used in this study...47

Table 2.2 Selected accessions showing extremes of IEC in abscising and non-abscising

fruit ......................................................................................................................... 52
Table 2.3 Accessions showing extremes of pedicel abscission ........................................ 55
Table 2.4 Intemal ethylene concentration and abscission in response to ethephon .......... 58
Table 3.1 Differentially expressed genes in the pedicel AZ in fi'uit abscission ............... 91
Table 3.2 Primers used in RT-PCR analysis ..................................................................... 95
Table 3.3 Amino acid sequences of extensions in apple, almond, and Arabidopsis ........ 96
Table 4.1 Primers used for amplification of PLL promoters .......................................... 107
Table 4.2 Summary result of Figure 4.3 to 4.8 .............................................................. 124

viii

LIST OF FIGURES
Figure 1.1 Model of floral organ abscission and genes acting in each step ........................ 2

Figure 2.1 Frequency distribution of accessions evaluated for harvest date relative to
species and weight ............................................................................................................. 48

Figure 2.2 Frequency distribution of accessions evaluated for internal ethylene
concentration (IEC) at peak abscission date (abscising accessions) or final harvest date

(non abscising accessions) ................................................................................................. 50

Figure 2.3 Relationship between harvest date, IEC at abscission, and ACSI allelotype for

studied accessions .............................................................................................................. 53
Figure 3.1 Effect of fruit removal on fruit abscission in apple ......................................... 78
Figure 3.2 Expression profiles of 14 redundant ESTs assembled in the same contig ...... 79

Figure 3.3 Functional categorization of 118 differential expressed genes based on GO
Biological Process .............................................................................................................. 80

Figure 3.4 Classification of 118 AZ differentially expressed genes into five clusters with
similar temporal responses in pedicel abscission ............................................................... 82

Figure 3.5 Expression profiles of selected differentially expressed genes within the A23
and pedicles in pedicel abscission induced by fruit removal ............................................. 83

Figure 3.6 Effects of 6-BA application on fruit thinning (A) and expression profiles of
selected differentially expressed genes within the AZs in fruit abscission induced by 6-
BA(B) ................................................................................................................................. 85

Figure 3.7 Effects of ethephon application on fruit abscission in preharvest season (A),
ethylene production in fruit (B), and expression profiles of selected differential expressed

genes within the MS (C) ................................................................................................... 86
Figure 4.1 Phylogenetic tree of Arabidopsis PLL amino acid sequences ....................... 110
Figure 4.2. Hierarchical clustering of RNA accumulation for 25 PLLs .......................... 1 12

Figure 4.3 Spatial and temporal PLL-GUS expression in the abscission zones of sepal,
petal, and stamen .............................................................................................................. 114

Figure 4.4 PLL:GUS expression in the dehiscence zone of siliques (A) and abscission
zone of mature seeds (B) .................................................................................................. 116

Figure 4.5 PLL:GUS expression in the tissues involved in cell separation processes....l l8

ix

Figure 4.6 PLL: GUS expression in the seedlings .......................................................... 120

Figure 4.7 PLL-GUS expression in the hydathodes (A) and stipules within shoot apex

(B) .................................................................................................................................... 122
Figure 4.8 PLL-GUS expression in the different parts of flower at stage 15 (A) and stage
(11-14) (B) ....................................................................................................................... 123

CHAPTER 1

Literature Review
1. Introduction

Many types of plant organs, such as leaves, flowers, fruits, and seeds, undergo
separation from the main plant body during plant development. Plants have evolved
successful strategies of growth, development, and survival by utilizing organ shedding.
Shedding of ripened fruit and dispersal of seeds facilitate reproduction, and dropping of
senescent leaves and abscising flowers after fertilization allows for nutrient recycling. In
addition, dropping of infected organs protects plants from pathogen invasion, and
shedding of old branches enables remodeling of plant structure. Furthermore, shedding
of excess flower buds and fi'uits ensures optimal growth of remaining organs.

Abscission is a cell separation process that occurs at a pre-detennined position,
called the abscission zone (AZ). This is generally arranged transversely to the axis of the
distal organs (Addicott, 1982) (Figure 1.1). Abscission is a highly co-ordinated and
active process including changes in cell structure, metabolism and gene expression within
the AZ in response to either developmental signals or various environmental conditions.
Early work mainly focused on morphological and biochemical events occurring in
abscission and manipulation of abscission through the use of the plant hormones,
ethylene and auxin. Recent technological advances in plant genomics allows for
identification of genes regulating abscission of floral organs, mostly in Arabidopsis.
These studies have led to a general model of the organ abscission process across plant
species (Figure 1.1) (Sexton and Roberts, 1982; Osborne, 1989; Patterson, 2001; Robert

et al., 2002). In this model, cells at the base of abscising organs undergo patterning and

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Figure 1.1 Model of floral organ abscission and genes acting in each step in
Arabidopsis. Step 1: Initially, cells at the base of floral organs undergo patterning and
differentiation to form morphological distinct abscission zone (AZ) (shown in yellow).
BOP genes regulate AZ formation indirectly through influencing proximal identity of
floral organs. Step 2: Next, multiple components and pathways are involved in AZ
activation. E TR] and EIN2 in the ethylene signaling pathway promote abscission. ARF2,
along with ARFI, ARF 7 and ARFI9, also stimulate abscission, and ARF2 promotes
abscission in part by stimulating ethylene biosynthesis. AOS also promotes abscission
presumably through interacting with the ethylene signaling pathway. An ethylene-
independent pathway from ligand IDA/IDLI to receptors HAE/HSLI to downstream
effectors has been shown to be crucial for AZ activation. ARP4/ARP7 also promotes
abscission in an ethylene-independent way presumably through chromatin modification.
DABs specifically influence the timing of floral organ abscission through an unknown
mechanism. AGL15 acts as an inhibitor of floral organ abscission probably through
maintaining cell in an embryonic state. Step 3: Following AZ activation, many cell wall
modifying enzymes are associated with the cell separation process. ADPG and QRT 2
promote abscission through loosening the middle larnella between adjacent cells. Step 4:
Following cell separation, protective layers form at the site of separation to prevent plant
from pathogen attack. Many defense related genes are associated with this step.

differentiation to form morphologically distinct layers of small, cytoplasmically dense
AZ cells. The AZ is then activated by developmental, hormonal, or environmental cues.
This is followed by the activation of cell wall modifying enzymes within the AZ
triggering the cell separation process. Lastly, protective layers are formed at the site of
organ detachment to protect plants from pathogen attack. In the first part of this literature
review, I will address molecular events occurring in each step of organ abscission and
hormonal factors regulating abscission. In the second part, I will briefly review chemical

fi'uit thinning and preharvest fruit drop in apple.

2. Processes of Organ Abscission
2.1 Differentiation of the Abscission Zone

During plant development, the AZ is precisely differentiated at the base of many
types of organs to be shed, and currently the A23 of leaf, flower, and fruit are well-
studied at the morphological level. The AZs in these organs are morphologically distinct
from neighboring tissues and characterized by layers of small, densely cytoplasmic cells
prior to the onset of abscission (Addicott, 1982, Sexton and Roberts, 1982; Osborne,
1989). The size of the AZ varies significantly among organs and species, for example, 1-
2 rows in the AZs of sepals, petals, and stamens in Arabidopsis, and 20-30 rows in the
fruit pedicel A23 in oil palm (Patterson, 2001; Henderson et al., 2001). The timing of AZ
cell differentiation also exhibits a wide range among different organs in the various plant
species. For example, differentiation of bean leaf AZ cells may occur in the primordial
leaf, while AZ cells of fi'uit pedicel in pome fruits are differentiated after flower bud

opening (Osborne, 1989).

Recent genetic approaches in model plant species, Arabidopsis and tomato, have
identified several genes regulating AZ differentiation. JOINTLESS (J) and J-Z are best
known for their roles in promoting pedicel AZ formation in tomato (Mao et al., 2000;
Yang, et al., 2005). Tomato sheds flowers and fruit at a swollen ‘joint’ on the pedicel, a
site called the pedicel AZ. The j mutant fails to form the pedicel AZ, and this leads to a
complete blockage of abscission of flower and fruit (Butler, 193 6). JOINT LESS encodes
a MADS-domain transcriptional factor and functions in directing differentiation of the
pedicel AZ (Mao et al., 2000). Besides the lack of pedicel AZ formation, the
inflorescence meristems in the j mutants converts to the vegetative growth after forming
only a few flowers and remain determinate (Rick and Sawant, 1955). J is preferentially
expressed in the main shoot and floral meristems and weakly expressed in the axillary
and sympodial meristems (Szymkowiak et al., 2006). However, there was no detectable
expression of J in any stage of the developing flower pedicels (Szymkowiak and Irish,
2006). These results implied that lack of AZ formation in the j pedicels presumably
results from an indirect effect of the suppression of sympodial meristem identity in the
inflorescence (Szymkowiak and Irish, 2006). The j-Z mutant also lacks the formation of
the pedicel AZ (Rick, 1956). In addition, the j-2 mutant also has pleiotropic defects in
shoot architectures including the production of many flowers and the conversion of sepals
to leafy structures (Reynard, 1961). Currently, the J-2 gene has not been identified and is
only known being localized at centromeric region of tomato chromosome 12 (Budirnan et
al., 2004).

Arabidopsis shed petals, stamens, and sepals afler pollination at the AZ located at

the base of these organs (Bleecker and Patterson, 1997). BLADE-ON-PE T IOLE I (BOP!)

and BOP2 are redundant transcriptional regulators of leaf and floral patterning in
Arabidopsis and required for the formation of the A23 (Ha et al., 2004; Hepworth et al.,
2005; Norberg et al., 2005; Ha et al., 2007; Mckim et al., 2008). The bopI bop2 double
mutants fail to abscise their floral organs (Hepworth et al., 2005; Norberg et al., 2005).
Histological analysis revealed that the A23 of floral organs and cauline leaves are not
properly differentiated in the bop] bop2 double mutants, suggesting that BOP] and
BOP2 are essential for differentiation of the A23 in the floral organs and leaves (McKim
et al., 2008). In addition, the bop] bop2 double mutants are abnormal in organ patterning
including formation of two sepal/petal mosaic organs and leafy projections from petioles
and floral bracts (Hepworth et al., 2005; Norberg et al., 2005; Ha et al., 2007).
Furthermore, nectary development is deficient in the bop] bop2 double mutants (Mckim
et al., 2008). Consistent with its role in the AZ development, BOP] is expressed at the
base of sepals, petals, and stamens (McKim et al., 2008). These results suggest that BOP
1 and 2 are not specifically committed to AZ differentiation, but appearing have roles in
suppressing cell differentiation in the multiple developmental events.

Arabidopsis produces dry dehiscent fi'uit that is composed of the replum with its
attached seeds, the carpel valves, and the valve margins. Seed dispersal requires two
sequential cell separation events: fruit dehiscence and seed detachment. Fruit dehiscence
takes place along the valve margins at the dehiscence zone (DZ); this process is
controlled by a group of transcriptional factors (Liljegren et al., 2004). Mature seeds are
released from the funiculus, a stalk-like structure connecting seeds to the replum, at a site
referred to as the seed AZ (Pinyopich et al., 2003). The seedstick (stk) mutants do not

properly differentiate the seed A25 and fail to release seeds from mature fi'uit (Pinyopich

et al., 2003). In addition, the stk mutants exhibit shorter fruit and enlarged fimiculus
associated with increased cell expansion and cell division (Pinyopich et al., 2003). These
results suggest that STK, a MADS domain transcriptional factor, may participate in
differentiation of the seed AZ through directing development of the funiculi (Pinyopich et

al., 2003).

2.2 Activation of the Abscission Zone

Early studies have shown that the plant hormones ethylene and auxin are the main
regulators of organ abscission. Those studies mainly addressed the physiological actions
of hormonal regulation and applications in agricultural practice (Addicott, 1982).
Forward and reverse genetics approaches have identified many mutants with defects in
abscission activation in model plant species (Figure 1.1). Many components in ethylene
biosynthesis and perception are known to be involved in organ abscission. A few
components in auxin and jasmonate signaling in Arabidopsis presumably regulate organ
abscission through an ethylene-dependent pathway. A recently identified ethylene-
independent pathway has been show to be crucial for abscission. A few additional genes
regulate abscission in an ethylene-independent manner through unknown mechanisms.

In this section, I will address the major findings of studies of activation of abscission.

2.2.1 The balance model between ethylene and auxin in abscission
Various developmental and environmental factors affecting organ abscission have
been extensively investigated in many plant species (Addicott, 1982; Taylor and

Whitelaw 2001). Aging, fruit ripening, and pollination are three important

developmental factors promoting abscission (Addicott, 1982). In addition, reduced
photoperiod, nutrition deficiency, and water stress from drought, salt, cold, and heat
conditions also promote organ abscission as a result of the decline in the growth and
vigor of the plant (Addicott, 1982). Furthermore, wounding stress from insect and
pathogen attack leads to organ abscission as a function to prevent spread of disease
(Addicott, 1982). All these developmental and environmental signals influencing organ
abscission have been linked to affect the levels of the plant hormones, ethylene and
auxin.

Ethylene is an unsaturated hydrocarbon that binds to copper through a covalent
bond (Abeles, 1992). Ethylene has profound effects on many plant developmental
processes including stimulation of seed germination, inhibition of triple response in dark-
grown seedlings, stimulation of cell expansion, induction of leaf epinasty, organ
senescence and abscission, and flowering in some plant species (Abeles et al., 1992).
Synthetic ethylene-releasing compounds and inhibitors of ethylene biosynthesis and
perception have been used commercially in agricultural practices to manipulate
abscission. It has been shown that ethylene production in fertilized flowers, senescent
leaves and ripening fruit is positively correlated with abscission in many plant species
(Brown, 1997). Fruit abscission in some species occurs without dramatic increase of
ethylene production, suggesting that ethylene sensitivity within the A25 controls
abscission. However, young leaves typically produce more ethylene than older leaves,
but abscise less (Morgan et al., 1992). It has been shown that auxin levels in the distal
organs affect ethylene sensitivity within the AZ (Brown, 1997).

Natural auxins in plants are indole-3-acetic acid (IAA), 4-chloroindole-3-acetic

acid (4-C1-IAA), and indole-3-butyric a cid (IBA); IAA, however, is the most abundant
and physiologically relevant (Taiz and Zeiger, 2003). Auxins affect many developmental
events including promoting formation of lateral roots, regulating floral bud development,
promoting fruit development, and inducing vascular differentiation (Taiz and Zeiger,
2003). Synthetic auxins, such as 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 1-
naphthaleneacetic acid (NAA), have been used to thin fruit and prevent pre-harvest fruit
drop (Greene, 2003). Early leaf-explants studies have shown that the removal of leaf
blades promoted petiole abscission, whereas application of auxin to the cut surface
inhibited abscission (Addicott, 1970; Addicott, 1982). Auxin-transport inhibitors
accelerated leaf abscission in the absence of exogenous ethylene (Morgan and Durham,
1972). IAA content within the A23 of cotton boll was positively correlated with boll
retention in field-grown cotton (Guinn and Brummett, 1987). All of these results support
that auxin prevents organ abscission. Many studies also revealed that ethylene inhibited
basipetal auxin transport, and auxin itself can stimulate ethylene production (Abeles &
Rubinstein, 1964; Beyer and Morgan, 1971). Thus the interaction between ethylene and
auxin appears to regulate activation of organ abscission.

This ethylene-auxin interaction with regard to leaf abscission is outlined in a
model proposed in early studies (Rubinstein and Leopold, 1963; Abeles and Rubinstein,
1964; Sexton and Roberts, 1982). This model revealed that there is no single key
abscission regulator, and that abscission induction is dependent on the complex interplay
between ethylene and auxin signaling. It is generally hypothesized that auxin flow across

the AZ controls the sensitivity to ethylene. According to the model, if basipetal auxin

flux to the AZ is maintained, abscission is inhibited. Loss of auxin flow promotes
abscission by increasing the AZ sensitivity to ethylene. Any factor affecting the supply
of auxin and ethylene to the distal organs will influence the sensitivity of the AZ to
ethylene. In addition, ethylene can inhibit auxin transport, and auxin itself can stimulate
ethylene production (Beyer and Morgan, 1971; Beyer, 1973).

This leaf-explants abscission model can be applied to flower abscission induced
either by development cues or by exogenous ethylene (van Doom and Stead, 1997;
Taylor, 2001). Abscission of senescent leaves and flowers after fertilization is usually
accompanied with gradual auxin decline that coincides with gradual increase of ethylene
(Brown, 1997). The extent to which this model can be applied to other organs, such as
fruit, is still not clear. However, it is known that young developing fruit are a strong
source of auxin, which is transported basipetally across the AZ of the fruit pedicel, and
that loss of auxin transport is associated with fruit abscission (Drazeta et al., 2004; Else et

al., 2004).

2.2.2 Involvement of ethylene biosynthesis and perception in abscission

Early work has recognized that ethylene can promote organ abscission
(Rubinstein, 1965). Studies of mutants with deficiency in ethylene response have shaped
our current understanding of ethylene biosynthesis and perception on regulation of
abscission. Ethylene is synthesized from methionine through three enzymes: S-adenosyl-
methionine (AdoMet) synthase, l-aminocyclopropane ~1-carboxylic acid synthase
(ACS), and l-aminocyclopropane-l-carboxy1ic acid oxidase (ACO) (Yang and

Hoffrnann, 1984). Ethylene biosynthesis is stimulated by various stresses (Abeles, 1992),

flower senescence (Bulfler et al., 1980), fruit ripening (Yang and Hoffinann, 1980), and
auxin (Jone and Kende, 1979; Yu et al., 1979), while ethylene biosynthesis is inhibited by
aminoethoxy-vinylglycine (AVG) and aminooxyacetic acid (AOA) through blocking
activity of ACS (Yang and Hoffrnann, 1984). Both ACSs and ACOs are encoded by a
multigene family in plants (Vandenbussche et al., 2006). Increased expression of ACSs
or ACOs has been associated with the organ abscission among many plant species
(Mishra et al., 2008; dal Cin et al., 2005, Yuan et al., 2005; Murayama et al., 2006).
Transgenic melons with reduced expression of a fiuit ripening-related ACO revealed
decreased fruit abscission (Ayub et a1. 1996). Apple cultivars with ACSI-2, a
dysfunctional allele of ACSI, exhibited lower pre-harvest fruit drop than genotypes
carrying ACSI-1, a firnctional allele of A CS1 (Sato etal., 2004).

Many components of the ethylene signaling pathway have been identified in
various plant species, and a general model has been established based on studies in
Arabidopsis thaliana (Chen et al., 2005; Hall et al., 2007; Kendrick and Chang, 2008).
Ethylene is perceived by a family of membrane-bound receptors sharing homology with
prokaryotic histidine kinase receptors (Chang et al., 1993). Newly identified components,
RE VERSION-TO—EIHYLENE SENSITIVITY] (R TE 1)/GREEN—RIPE (GR), repress
ethylene response through E IHYLNE TRIPLE RESPONSE (E T R) receptors in the
membrane through an unknown mechanism. These receptors negatively regulate the
downstream ethylene response; in the absence of ethylene, receptors suppress the
downstream response through CONSTITUTIVE T RIPE RESPONSE 1 (CT RI ). Receptor
binding of ethylene inactivates CTRI, a negative regulator of the ethylene response,

which leads to activation of E THYLENE INSENSITIVE 2 (EIN2). In the nucleus, EIN2

10

activates EIN3 and EIN3-like 1 (EILI) that are controlled by the 26S proteasome-
dependent protein degradation pathway. EIN3 then activates ethylene responses through
binding to the EIN3-binding site (EBS) in the promoter of ETHYLENE RESPONSE
FACTOR 1 (ERF 1).

Although many genes are known to be involved in the ethylene signaling
pathway, only a small subset of these genes in Arabidopsis and tomato has been
investigated for their role in the timing of organ abscission. Gain-of-function mutation in
E TR] in Arabidopsis led to ethylene insensitivity and resulted in delayed senescence and
abscission of floral organs (Bleecker, et al., 1988; Bleecker and Patterson, 1997). The
ethylene receptor family in Arabidopsis is composed of five gene members and classified
into two subfamilies (Hua and Meyerowita, 1998). Loss-of-function receptor mutants
from any single member within the family did not show any phenotypic defects, while
loss-of-function of either triple etr] etr2 ein4 or quadruple etrl etr2 ein4 ers2 mutants led
to a constitutive ethylene response in the absence of ethylene (Hua and Meyerowita,
1998). However, the effects of these mutations on floral organ abscission have not been
investigated.

NE VER-RIPE (NR) encodes an ethylene receptor in tomato (Lanahan et al., 1994).
Semi-dominant nr mutants have defects in fruit ripening, flower senescence, and
abscission of flowers and fruit (Rick and Butler, 1956). In unfertilized tomato flowers,
frequency of flower abscission at pedicel was significantly lower in the nr mutants than
that in wild type plants (Lanahan et al., 1994). In addition, exogenous ethylene failed to
promote abscission of flower explants in nr mutants (Lanahan et al., 1994). In tomato,

the ethylene receptor family consists of six members and negatively regulates the

11

ethylene signaling pathway (Lashbrook et al., 1998). Transgenic plants with reduced
expression of LeETRI showed decreased plant size and delayed abscission of leaves and
flowers (Whitelaw et al., 2002). These results suggest that levels of ethylene receptors in
tomato possibly modulate the timing of organ abscission and fruit ripening.

Loss-of-function ein2 mutants in Arabidopsis completely blocked the ethylene
response and showed delayed senescence and abscission of floral organs (Ecker, 1995).
LeEILI , LeEILZ, and LeEIL3 in tomato are functionally redundant transcription factors
that act as positive regulators of ethylene signaling (Tieman et al., 2001). Reduced
expression of multiple LeEILs led to decreased leaf epinasty, delayed fruit ripening, and
delayed floral organ abscission (Tieman et al., 2001).

AZ differentiation among all the mutants mentioned above is indistinguishable
from that in wild type plants, suggesting that these genes do not play roles in AZ
formation. Presmnably, other signaling components are also involved in activation of
abscission. Interestingly, none of them showed complete blockage of floral organ
abscission, suggesting that these components in ethylene signaling are not essential for
activation of abscission.

2.2.3 Regulators in abscission through interaction with ethylene biosynthesis
Auxin

As mentioned above, the balance between ethylene and auxin is the predominant
effector of organ abscission (Addicott. 1982). Recent studies have shown that either an
auxin maxirna or an auxin gradient within the given cells is capable of determining
developmental reprogramming (Tanaka et al., 2006). Auxin is synthesized in both

tryptophan-dependent and tryptophan-independent pathways (Vanneste and Friml, 2009;

12

Woodward and Bartel, 2005). The unique feature of auxin as a signaling molecule is its
ability to move between cells in a directional manner. In plants, auxin transport is
achieved through both a long-distance source-to-sink pathway and short-distance cell-to—
cell polar transport (Merchant et al., 2002; Varmeste and Friml, 2009). In Arabidopsis,
the direction of polar auxin transport is mediated by PIN proteins (Billou et al., 2005;
Tanaka et al., 2006). Despite the diversity of cellular responses controlled by auxin, a
simple pathway fiom perception to transcriptional response has been established for
auxin signaling (V anneste and Friml, 2009). Briefly, the presence of auxin stabilizes
interactions between auxin receptors TRANSPORT INHIBITOR RESPONSE I (T IR] )
/A WON-BINDING F -BOX (AFB) and Aux/IAA transcriptional regulators, which trigger
ubiquitinylation of Aux/IAA. The proteolysis of Aux/IAA results in derepression of
A UMN RESPONSE FACTORS (ARFs), which activates downstream transcriptional
responses.

Recent transcriptome profiling analysis revealed that transcription levels of a few
Aux/IAA in Mirabilis jalapa were reduced during petiole and stem abscission induced by
auxin depletion (Meir et al., 2006). A few members of the ARF gene family were found
to regulate the timing of floral organs abscission in Arabidopsis (Ellis et al., 2005;
Okushima et al., 2005). ARFs encode proteins containing an amino-terminal domain that
binds to auxin response elements, an activation/repression domain. Single loss-of-
ftmction ar/2 mutants exhibited a slight delay in floral organ senescence and abscission,
whereas both arfl arfl double mutants and arfl arf7 arfl 9 triple mutants showed a
significant delay in abscission of floral organs. The expression of ACS2, ACS6, and

ACS8 was lower in flowers of an? mutants than that in wild type plants (Okushima et al.,

13

2005). These results point out the possibility that ARF2 regulates abscission through

promoting ethylene biosynthesis.

Jasmonates

Jasmonates (J As) are a group of plant hormones including jasmonic acid and its
metabolites, such as methyl JA (MeJA). Application of exogenous MeJA promotes
organ abscission in bean, citrus, and cherry tomato (Beno-Moualem et al., 2004;
Hartmond et al., 2000; Ueda et al., 1996). Acceleration of fruit abscission in citrus
induced by Me] A or a synthetic analog, coronatine, appears to result from increased
production of ethylene (Burns et al., 2003; Hartmond et al., 2000). Recent biochemical,
genetic, and molecular analyses have illustrated J A biosynthetic and signaling pathways
(Katsir et al., 2008). Briefly, JA is initially synthesized in chloroplasts from linolenic
acid and converted to 2-oxo-phytodienoic acid (OPDA) through lipoxygenase (LOX),
allene oxide synthase (AOS), and allene oxide cyclase (AOC). In peroxisomes, OPDA is
converted to various forms of JA by a few B-oxidation steps. Perception of IA is through
a simple signaling pathway. In essence, the presence of bioactive JA signals initiated
formation of coronatine-insensitive 1 (COIl)-JA-Jasmonate ZIM domain (J AZ) in which
JAZ proteins are degraded through the ubiquitin—26S proteasome pathway. Degradation
of JAZ proteins leads to the derepression of transcription of JA—responsive genes. Recent
genetic analyses regarding of cell separation in Arabidopsis revealed that abscission of
floral organs is delayed in JA-deficient mutant, aos (Ogawa et al., 2009), suggesting that

AOS promotes floral organ abscission. The aos ein2 double mutants showed more severe

14

delayed floral organ abscission, implying AOS presumably promote abscission through

interaction with ethylene signaling (Ogawa et al., 2009).

2.2.4 Ethylene-independent regulators in activation of abscission
An ethylene-independent signaling pathway

Genetic screens in Arabidopsis identified a mutant called inflorescence deficient
in abscission (ida) that fails to abscise sepals, petals, and stamens (Butenko et al., 2003).
IDA encodes a 77 amino acids protein with an amino-terminal signal peptide and a
carboxyl-terminal motif (EPIP) (Butenko et al., 2003). Scanning electron microscopy
(SEM) observation revealed that AZ differentiation in ida mutants is indistinguishable
from that in wild type plants (Butenko et al., 2003). Under exposure to exogenous
ethylene, the ida mutants showed ethylene responses similar as those in wild type with
the exception of floral organ abscission, suggesting that IDA regulates abscission in an
ethylene-independent manner or downstream of ethylene (Butenko et al., 2003). In
Arabidopsis, there are five additional IDA-LIKE (IDL) proteins that contain amino-
terminal peptides and conserved carboxyl-terminal EPIP (Butenko et al., 2003).
Overexpression of IDA and IDLs in Arabidopsis led to similar phenotypes including
premature floral organ abscission and silique dehiscence in addition to ectopic abscission
of inflorescence branches, cauline leaves, and pedicels (Stenvik et al., 2006; 2008). In
addition, expression of IDLs partially rescues ida phenotypes, suggesting functional
redundancy among these genes (Stenvik et al., 2008).

Stenvik and co-workers also revealed that EPIP domain of IDA and IBM can

substitute the function of IDA (Stenvik et al., 2008). Application of synthetic EPIP

15

peptides to the MS medium promoted premature abscission of flower explants from wild
type and induced abscission of flower explants from ida mutants (Stenvik et al., 2008).
These results suggest that IDA and IDLl can be recognized as signaling molecules to
regulate floral organ abscission.

In the search for potential targets of IDA, two groups have identified two leucine-
rich repeat (LRR) receptor-like protein kinases, HAESA (HAE) and HAESA-LIKEl
(HSLl), that presumably serve as receptors for IDA/IDLI proteins (Cho et al., 2008;
Stenvik et al., 2008). HAE belongs to a gene family with two additional paralogues,
HSLI and HSL2 (Jin et al., 2000; Cho et al., 2008). The hae hs12 double mutants fail to
shed their floral organs, and SEM revealed that AZ differentiation in hae hle is
indistinguishable from that in wild type plants (Cho et al., 2008). The hae hle double
mutants exhibit ethylene responses same as those in wild types with exception of floral
organ abscission (Cho et al., 2008). These evidences indicate that HAE and HSL2 are
required for floral organ abscission and redundantly regulate abscission in an ethylene-
independent manner. Genetic interactions among ida and hae hle revealed that IDA acts
upstream of HAE HSL2 or downstream of ethylene (Cho et al., 2008; Stenvik et al.,
2008)

In addition, Cho et a1 (2008) also found that a few components in the Mitogen-
Activated Protein (MAP) kinase cascade were involved in floral organ abscission in
Arabidopsis (Cho et al., 2008). In the canonical MAP Kinase (MAPK) signaling
cascade, a MAP kinase kinase kinase (MKKK) activates a MAP kinase kinase (MKK),
which in turn activates a MAP kinase (MPK). Majority of transgenic lines in

Arabidopsis with reduced expression of MKK4 and MKKS (MKK4-MKK5RNA1’) were

16

arrested in the cotyledon stages resulting from excessively clustered stomata (Wang et al.,
2007). The surviving MKK4-MKK5RNAi transgenic lines fail to abscise their floral
organs (Cho et al., 2008). SEM and petal break strength studies showed that AZ
differentiation in MKK4-MKK5RNAi transgenic lines was indistinguishable fi'om that in
wild type (Cho et al., 2008). Exogenous ethylene can not induce abscission of floral
organs in the MKK4-MKK5RNA1' transgenic lines, suggesting MKK4 MKKS regulate
abscission in an ethylene-independent way or downstream of ethylene. Consistent with
its roles in abscission, GUS reporter gene driven under MKK4 and MKK5 promoters was
expressed in the floral organs and the A25 of sepals, petals, and stamens (Cho et al.,
2008)

MKK4 and MKK5 have been shown to activate MPK3 and MPK6 in plant
defenses and firnction in stomata patterning (Wang et al., 2007). Single mpk3 or mpk6
loss-of-function did not show any phenotypic defect, while mpk3 mpk6 double mutants
were embryo lethal. To investigate functions of MPK3 and MPK6 on abscission, mutated
form of MPK3 (MPK3KR), converting a lysine to arginine, was transformed into mpk6
mutant (mpk6 MPK3”) (Cho et al., 2008). 10% of mpk6 MPK3” transgenic lines fail to
abscise their floral organs (Cho et al., 2008). Consistent with their role on abscission,
GUS reporter gene driven under MPK3 and MPK6 promoters was expressed in the floral
organs and the AZs of sepals, petals, and stamens (Cho et al., 2008). These results
suggest that MPK3 and MPK6 are positive regulators of floral organ abscission in
Arabidopsis

Genetic analyses revealed that MKK4 MKK5 act downstream of IDA, HAE HSL2

to regulate abscission. These studies have established a putative ethylene-independent

l7

signaling pathway on regulating activation of abscission from peptides (IDA and IDLs),
to receptors (HAE HSL2), to downstream cytoplasmic effectors (MKK4, MKK5, MPK3,

MPK6).

Chromatin modification

ACTIN RELATD PROTEINS (ARPs) are components of chromatin-remodeling
complexes (Olave et al., 2002). Arabidopsis contains nine ARP genes (McKinney et al.,
2002), and ARP4 and ARP7 have been shown to promote floral organ abscission in
Arabidopsis (Kandasamy et al., 2005a; 2005b). Although an arp7 null mutant is embryo-
lethal, transgenic plants with reduced expression of ARP7 revealed a severe delay in
floral organ abscission and other pleiotropic growth defects including dwarfed plants,
retarded root growth, altered flower development, and reduced fertility (Kandasamy et
al., 2005a) TheARP7-suppressed transgenic plants exhibited similar triple ethylene
responses as those in wild type, whereas exogenous ethylene did not promote floral
organs abscission in ARP7-suppressed transgenic plants (Kandasamy et al., 2005a).
These results suggest that ARP7 acts on organ abscission in an ethylene-independent
manner or downstream of ethylene perception. ARP4 is the closest paralog of ARP7, and
ARP4-suppressed transgenic plants also revealed pleiotropic defects including delayed
abscission of floral organs (Kandasamy et al., 2005b). These results suggest that ARP4
and ARP7 act similarly in regulating the timing of floral organ abscission. Considering
the pleiotropic effects of reduced expression of either ARP4 or ARP7, these genes appear
to regulate gene transcription on a global level and control various developmental events

including abscission.

18

MADS-box domain regulation

AGAMOUS-LIKE 15 (A GL1 5) encodes a MADS-domain transcriptional factor
(Fernandez et al., 2000). AGLI5 was preferentially expressed in developing embryos and
also expressed in the vegetative shoot apical meristems and at the base of floral organs
and cauline leaves (Fernandez et al., 2000). Loss-of-function ag115 mutants did not show
any visible phenotypic defects (Lehti-Shiu et al., 2005), however, overexpression of
AGL15 led to long-term maintenance of embryonic development, delayed flowering time,
fruit maturation, floral organ senescence, and abscission (Fernandez et al., 2000; Harding
et al., 2003). The 35S::AGL15 transgenic plants exhibited similar triple responses as
those in wild type plants under exposure of exogenous ethylene, and 35S::AGL15 etr]
double mutant showed additive effects including delayed senescence and abscission
(Fernandez et al., 2000). These results suggest that 35S::AGL15 inhibits abscission in an
ethylene-independent way or downstream of ethylene (Fernandez et al., 2000).
Considering the dramatic increase in tissue longevity among 35S::AGL15 transgenic
plants, AGLI5 possibly regulate organ abscission indirectly through maintaining or

enhancing juvenile state of young tissues (Fernandez et al., 2000).

Regulators with unknown function

Genetic screening identified five delayed floral organ abscission (dabI, dab2,
dab3, dab4, dab5) mutants with delayed timing of floral organ abscission. These mutants
have been shown to be associated with five loci in Arabidopsis (Patterson and Bleecker,

2004). Microscopy studies revealed that AZ differentiation among these mutants is

19

indistinguishable from that in wild type plants. Rounding of AZ cells during the final cell
separation step was delayed in all five mutants, and irregular rounding of AZ cells was
observed only in the dabZ-I mutants. These observations were also associated with the
degree of petal break strength in the mutants. All five mutants displayed similar triple
responses as those in wild type under exogenous ethylene, suggesting DABs regulate
abscission either in an ethylene-independent manner or downstream of ethylene

(Patterson and Bleecker, 2004)

2.3 Separation of the Abscission Zone

Following activation, the next sequential step of abscission is indicated by the
loosening of primary cell walls and the dissolution of the middle larnella within the AZs
(Addicott, 1982; Osborne, 1989). Cytological studies have showed that AZ cells are
distinguished by increased endoplasmic reticulum and Golgi bodies, enriched starch
gains and microbodies during cell separation (Osborne, 1989). Following swelling and
dissolution of the middle larnella, cells within the AZ are rounded and separate from each
other. In addition, irregular cellulose microfibril arrangement has also been observed in
the cells within the AZ (Osborne, 1989). Either high turgor pressure or autolysis,
generated by the enlargement of separating cells at the time of separation, may contribute
to final rupture of the restraining vascular bundles not undergoing wall breakdown
(Sexton and Roberts, 1982).

During cell separation within the A23, loss of Ca2+, decreased methylated pectins,
and a lower wall pH have been observed (Stdsser et al., 1969; Poovaiah and Rasmussen,

1973; Addicott, 1982; Osborne, 1989). Increased activities of PG, cellulases, and

20

peroxidases have been observed at time of cell separation in many plant species
(Addicott, 1982; Osborne, 1989; Roberts and Sexton, 2002). Irnmunoanalysis on various
components of cell wall polysaccharides has demonstrated the dynamic events that occur
in cell wall remodeling during the induced abscission of leaf pedicel in Euphorbz'a
. Pulcherrima (Lee et al., 2008). High levels of UV-induced autofluorescence were
detected at the time of leaf separation, suggesting the accmnulation of polyphenolics
within AZ cells. A reduction in methylesterification of homogalacturonan (HG) and a
dramatic increase of de-esterification of HG were also detected within AZ cells at the
time of leaf abscission.

Based on the observation of cell wall modification in cell separation, it is not
surprising that many cell wall modifying and hydrolytic enzymes contribute to this
process. For example, polygalacturonases (PGs), beta-1, 4-endoglucanases (EGases), and
pectate lyases (PL) have been implicated in cell separation within the AZ (Cai and
Lashbrook; Laskowski et a1. 2006; Swarup et al., 2008; Leslie et al., 2007). In addition,
expansins and pathogenesis-related (PR) chitinases have also been associated with the
cell separation process (Belfield et al., 2005; Sampredro and Cosgove, 2005; Roberts et
al., 2002). Most studies on these genes related to organ abscission focused on expression
analysis using RT-PCR or GUS reporter gene approaches.

Despite the large number of cell wall modifying enzymes involved in abscission,
a very limited number of genes have been characterized in genetic analysis. Recent
genetic analysis revealed that three closely related Arabidopsis PGs, ARABIDOPSIS
DEHISCENCE ZONE PG] (ADPGI), ADPGZ and QUART ET 2 (QRTZ), contribute to

cell separation in anther and silique dehiscence, and in floral organ abscission (Ogawa et

21

al., 2009). Both single mutants of adpg] and adng showed reduced silique dehiscence,
whereas double mutants failed to dehiscence siliques (Ogawa et al., 2009). In addition,
both single mutant of adng and qrt2 showed delayed floral organ abscission, and adng
qrt2 double mutants exhibited slightly geater delay than either single mutant (Ogawa et
al., 2009). Furthermore, the adpg] adng qrt2 triple mutants exhibited delayed anther
dehiscence (Ogawa et al., 2009). Studies on flower explants also showed that T-DNA
insertion mutant of ADPGZ exhibited delayed floral organ abscission (Gonzalez-Carranza,
2007). Taken together, these results suggest partial functional redundancy among these
three genes. These results support the notion that ADPG] and ADPGZ are essential for
silique dehiscence, ADPGZ and QRTZ contribute to floral organ abscission, and all three
genes contribute to anther dehiscence. Many previous studies have implied that PGs play
very important roles in various cell separation events, and this study confirms the

importance of PGs in these events.

3. Hormonal Regulation of Organ Abscission in Agricultural Practices

In order to achieve maximum yield and optimized quality, regulation of organ
abscission has been widely used in various agicultural practices (Addicott, 1982).
Induced leaf abscission by chemical defoliation facilitates mechanical harvest in cotton
production, and chemical defoliation of young nursery trees prevents disease spread
during shipment (Addicott, 1982). Synthetic auxin compounds have been widely used in
many potted ornamental plants or cut flowers in order to delay abscission of flowers or
petals (Addicott, 1982). Many tree fruit species, such as apple, peach and citrus, retain

excess young fruits, and fruit thinning has become a necessary agicultural practice to

22

increase fruit size and quality and maintain consistency in annual bearing (Addicott,
1982) Application of ethylene releasing compounds, such as ethephon, can accelerate
dehiscence of shucks in many nut species during the harvest season (Addicott, 1982) On
the other hand, prevention of preharvest drop in many fleshy fruit species, like apple and
pear, can avoid yield loss (Addicott, 1982). In the following sections, I will address
various aspects of hormonal regulation in apple abscission including fi'uit thinning and

preharvest fruit drop.

3.1 Fruit thinning

Domesticated apple bears abundant flowers, which produce excess fi'uit that the
tree is unable to support. Many trees including apple naturally abscise some of their fruit
at an early stage of fruit development, a phenomena called ‘June drop’. Unlike mature
fruit, young fruit start to senesce only after they are already determined to drop
(Bangerth, 2000). Auxin transport to the AZ increases in young fi'uit shortly after
fertilization (Gruber and Bangerth, 1990), and ethylene production in young developing
fruit is very low (Blanpied, 1972, Miller et al., 1988). These data indicate that the leaf
explant model can not be applied to abscission of young fruit. It has been hypothesized
that abscission of young fruit is regulated by a hormonally-controlled dominance among
fruit and between fruit and shoot (Bangerth, 2000). Auxin transport from dominant
young fruit may repress transport from dominated fruit (Bangerth, 2000). Dominated
fruit may initiate abscission apparently in the absence of high levels of ethylene
production, perhaps reflecting a predominant role for auxin in mediating abscission of

young fruit (Bangerth, 2000).

23

In order to maximize crop value by optimizing marketable fruit size, yield, and
quality, post-harvest storage life, as well as to maintain consistent bearing, fruit thinning
is necessary and has been an established commercial practice all around the world
(Dennis, 2000). Fruit thinning can be done by hand or chemicals. However, hand
thinning is no longer practical in commercial apple production due to the high cost of
labor.

The most commonly used commercial thinning chemicals are NAA
(Naphthaleneacetic acid), 6-BA (6-Benzyladenine), and Sevin (Carbaryl) (Byers, 2003;
Wertheim, 1979;). NAA, a synthetic auxin, is a strong thinner and rate responsive; Sevin
is a relatively mild thinner and has a unique advantage as an insecticide; 6-BA, a
synthetic cytokinin, is also a mild thinner (Byers, 2003). Combinations among these
compounds have been shown very effective on fruit thinning (Byers et al., 2003;
Bukovac et al., 2008). Many factors affect the efficiency of chemical fi'uit thinning, for
example, genotypes, temperature, and application time (Byers et al., 2003).

The basis of selectivity in chemical fruit thinning is the presence of distinct vigor
among fi'uitlets. Chemical thinners intensify the natural competition among the fruitlets
and maximize the effectiveness. The mechanisms involved in fruit thinning are subject to
debate among physiologists. Dennis (2002) reviewed various explanations for the
thinning action of commonly used chemicals, especially for NAA, BA, and Sevin. These
proposed explanations include seed abortion and inhibition of seed development,
blockage of nutrient transport from leaf to fruit, reduction of sink strength of fruit,
reduced auxin synthesis by the seed, reduced auxin transport from fruit, elevation of

ethylene biosynthesis, and inhibition of photosynthesis (Dennis, 2002).

24

3.2 Preharvest fruit drop

Fruit abscission in advance of harvest (preharvest fruit drop) is a considerable
problem in commercial apple production, especially for McIntosh and its sports.
Preharvest fruit drop is also geatly influenced by various environmental factors and
cultural practices. Climacteric ethylene production associated with fruit ripening has
been implicated in preharvest abscission for some domesticated cultivars (Sun et al.,
2009; Walsh, 1977). MdACS] in domesticated apple is specifically expressed in the
ripening fruit and associated with climacteric ethylene accumulation (Harada et al., 1997;
Sunako et al., 1999). The dysfunctional allele, MdACSI-Z, exhibits low transcriptional
activity in ripening fruit associated with a transposon insertion in its promoter region
(Sunako et al., 1999). Apple cultivars homozygous for ACSI-2, associated with low
climacteric ethylene production, showed lower preharvest fi'uit drop than cultivars
carrying ACSI-I , the firnctional allele of ACSI (Costa et a1. 2005; Sato et al., 2004; Sun
et al., 2009). These results imply the importance of ethylene in preharvest fruit drop.

As shown for a leaf explant model, increased production of ethylene in fruit may
reduce basipetal transport of auxin to the pedicel AZ, at least in part by decreasing auxin
transport capacity (Beyer and Morgan, 1971; Riov and Goren, 1979; Suttle, 1988). In
some wild Malus species, natural fruit abscission is not correlated with a dramatic
increase in endogenous ethylene production, suggesting additional factors regulate fruit
abscission in these species (Sun et al., 2009).

Plant gowth regulators have been used in commercial practice for many years in

order to limit or reduce pre-harvest fruit drop (Edgerton, 1973; Wertheim, 197 3). Three

25

classes of compounds including NAA, AVG (aminoethoxyvinylglycine hydrochloride),
l-MCP (1-Methylcyclopropene) are currently available for commercial use (Greene,
1983; 2003; dal Cin et al., 2008; Yuan et al., 2008). NAA, a synthetic auxin, prevents
preharvest fruit drop within a short period after application, and its major side effect is to
accelerate fi'uit ripening (Yuan et al., 2005). AVG acts as an ethylene biosynthesis
inhibitor and l-MCP is an ethylene binding competitor. The major side effects of these
two compounds are inhibition of fruit ripening. Similar to chemical fruit thinners, factors
such as application time and weather influence the efficiency of these chemicals on

preventing preharvest drop (Greene, 2003).

3.3 Summary

Studies of fruit abscission in apple have mainly focused on the improved use of
synthetic chemicals to either promote or prevent abscission based on production goals,
but traits related to fruit abscission have not generally been targeted in breeding efforts.
Interestingly, the genus Malus includes many wild species that are anecdotally known to
retain mature fi'uit, especially the small-fruited species commonly referred to as crab
apples (Fiala, 1994). Publicly available germplasm resources (Hokanson et al., 2001;
Kresovich et al., 1995) will allow for efficient evaluation of important traits related to
fruit abscission. Genotypes can be developed as contrasting models to understand the
biological bases of these traits, and as tools in genetic analyses for mapping of genes that
influence these traits. In addition, genomics tools will allow for identification of genes
involved in abscission, which provides more insights into the mechanisms of fruit

abscission and actions of chemicals used to control fi'uit abscission.

26

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36

CHAPTER 2

Natural Variation in Fruit Abscission-Related Traits in Apple
(Malus)

Abstract

Abscission or retention of ripening fi'uit is a major component of seed dispersal
strategies and also has important implications for horticultural production. Abscission-
related traits have generally not been targeted in breeding efforts, and their genetic bases
remain mostly unknown. We evaluated 144 Malus accessions representing wild species,
domestic cultivars, and hybrids for abscission-related traits. We found that seasonal
timing of fruit abscission in wild species and hybrids showed a broad distribution similar
to that seen for domestic cultivars, and that internal ethylene concentration at the time of
abscission varied by over three orders of magritude. Wild species, domestic cultivars,
and hybrids all included representatives that showed abscission of fi'uit prior to
substantial production of ethylene, as well as accessions that retained fruit for a
sigrificant period of time following ethylene production. For all accessions that retained
fruit, fruit removal resulted in abscission of the pedicel, and exogenous ethylene
promoted abscission, suggesting that the abscission zone was functional. Our results
suggest important roles for mechanisms independent of fruit ethylene production in

abscission.

Key words: Malus, Abscission, Fruit ripening, Ethylene, Natural diversity

37

Introduction

During plant development, specific organs may undergo progammed separation
from the main plant body, a process called abscission (Osborne, 1989). Abscission plays
crucial roles in the health and reproductive success of plants. For example, shedding of
senescent leaves facilitates the recycling of mineral nutrients, abscission of floral organs
after pollination allows for a focus of energy on reproduction, and dropping of diseased
or infected organs reduces the spread of disease (Addicott, 1982). Abscission of ripening
fruits and mature seeds is an important process contributing to seed dispersal (Addicott,
1982)

Organ separation typically occurs in a pre-determined position, called the
abscission zone. The abscission zone may differentiate very early or relatively late in the
development of the organ, and is characterized by a few layers of small, densely
cytoplasmic cells, generally arranged transversely to the organ axis (Addicott, 1982;
Osborne, 1989; Osborne and Sargent, 1976a,b; Sexton and Roberts, 1982; Stosser et al.,
1969a,b; Webster, 1968). During initiation of abscission, these separation layer cells
expand and may divide. Subsequently, secretion of hydrolytic enzymes, increased
peroxidase activity, and loss of calcium and pectin from the wall of separation layer cells
presumably lead to the dissolution of the pectin-rich middle larnella, weakening the cell
wall and allowing disintegation of abscission zone tissues (Addicott, 1982; Morre, 1968;
Osborne, 1989; Rasmussen and Bukovac, 1969; Stosser et al., 1969b; Wittenbach and
Bukovac, 1975). Cells basal to the separation layers may undergo a process of trans-
differentiation to form a protective layer continuous with the periderrn of the stem

(Addicott, 1982). The vasculature, which passes through the separation layers, may not

38

always participate in abscission (Stosser et al., 1969a) thus provides a final connection to
the main plant body that can be broken by physical force.

Various environmental and developmental signals have been shown to induce
abscission by influencing the ratio between auxins and ethylene within the organ and
adjacent abscission zone cells (Addicott, 1982; Brown, 1997; Roberts et al., 2002; Taylor
and Whitelaw, 2001). According to a widely accepted model developed with leaf
explants, loss of basipetal flow of auxin through the abscission zone, for example during
leaf senescence, activates abscission by derepressing sensitivity of separation layer cells
to ethylene (Abeles, 1967; Abeles and Rubinstein 1964; Addicott, 1982; Addicott and
Lynch, 1951; Osborne, 1989; Rubinstein and Leopold, 1963; Sexton, 1995). Thus a
balance between auxin and ethylene signaling, rather than absolute levels of the
hormones, seems to be the predominant effector of abscission. The extent to which this
leaf model can be applied to other organs, such as fruit, is not clear. However, it is
known that developing fruit constitute a strong source of auxin, which is transported
basipetally across the separation layers of the fruit pedicel, and that loss of auxin
transport is associated with fruit abscission (Drazeta et al., 2004; Else et al., 2004). In
plants such as apple that naturally adjust crop load to meet physiological capacity, auxin
transport from dominant young fruit may repress transport from dominated fruit
(Bangerth, 2000). Dominated fruit may initiate abscission apparently in the absence of
high levels of ethylene production, perhaps reflecting a predominant role for auxin in
mediating abscission of young fruit (Bangerth, 2000).

The interactive contributions of auxin and ethylene signaling to abscission of

mature fruit have not been extensively studied. In many fruits, ripening is accompanied

39

by the production of sigrificant amounts of ethylene (Brady and Speirs, 1991; Reid,
1985). Transgenic melon expected to suppress expression of a film ripening-related ACC
OXIDASE gene, and thus accumulation of fruit ethylene, showed loss of abscission
(Ayub et al., 1996), and in domestic apple (Malus domestica Borkh.) blocking
climacteric ethylene production in the fi'uit through the use of the ethylene agonist 1-
methylcyclopropene was associated with delayed abscission (Sato et al., 2004). Walsh
(1977) observed that, for three domestic apple cultivars, abscission was preceded by the
accumulation of high levels of ethylene in the fruit. Although such experiments suggest
that fruit-produced ethylene can promote abscission, whether this ethylene acts directly or
indirectly, and the exact mechanism of this effect, remain unknown. Maturity in many
fi'uits that naturally abscise is not associated with high levels of ethylene production [e.g.,
sour cherry (Wittenbach and Bukovac, 1974)], suggesting either that low levels are
sufficient to promote abscission or that natural abscission can occur independently of
ethylene.

It is well known that exogenous ethylene accelerates abscission of ripening fruit
in a variety of fruit species, even those that do not produce high levels of endogenous
ethylene (Abeles et al., 1992; Brady and Speirs, 1991). As shown for a leaf explant
abscission model, increased levels of ethylene in the fruit may reduce basipetal transport
of auxin to the abscission zone, at least in part by decreasing auxin transport capacity
(Beyer and Morgan, 1971; Riov and Goren, 1979; Suttle, 1988). This mechanism may be
superimposed on the endogenous decrease in auxin synthesis in the fruit associated with
maturity, a phenomenon that may itself derepress ethylene generation (Abeles and

Rubinstein, 1964).

40

Fruit abscission in advance of harvest (pre-harvest drop) is a considerable
production problem for many fruit crops, especially apple and pear (Pyrus communis L.)
In commercial apple, the timing of natural fi'uit drop, relative to the optimal commercial
harvest date, shows a geat degee of variability. Some cultivars, such as McIntosh, are
especially prone to preharvest drop. The genetic basis of this variability remains obscure.
Sato et al., (2004) found that several apple cultivars homozygous for the dysfirnctional
ACSI-2 allele of the ACC SYNTHASE I gene, which is important for climacteric ethylene
accumulation in apple (Costa et al., 2005; Harada et al., 2000), showed relatively low
degees of pre-harvest drop, and that homozygosity of ACSI-2 was associated with that
low preharvest drop in a small segegating population. This supports the
pharmacological evidence implicating climacteric ethylene in promoting abscission, and
suggests that ACSI allelotype is an important contributor. However, this study also
found that cultivars homozygous for the wild-type ACSI-1 allele nevertheless can show a
range of abscission behavior, ranging from nearly complete retention of fruit to nearly
complete pro-harvest drop, revealing the importance of additional factors (Sato et al.,
2004). This analysis was confounded by the fact that the optimal timing of commercial
harvest is not based solely on maturity indicators, but also on storage potential, which
may decline with a delay in harvest. Thus, varieties with geater capacity for storage may
be harvested at a more advanced stage of maturity. Accordingly, internal ethylene
concentration (IEC) of apple fruit has been found to vary dramatically among cultivars at
the optimal time of commercial harvest (Chu, 1988).

Although the relationships between the fruit ethylene production and abscission

have been documented for a few domestic apple cultivars (Walsh, 1977), there has been

41

no analysis of this phenomenon at a large scale or including wild apple species. In
tomato, variability in timing of abscission relative to climacteric ethylene production has
been recognized among species (Grumet et al., 1981). Although the genetic basis of
natural variation of this response in tomato has not been extensively studied, it is known
that ethylene-independent fi'uit retention in tomato can be indirectly conferred by loss of
function of JOINT LESS, a gene required for development of the abscission zone (Butler,
1936). The potential influences of ethylene sensitivity, controlled largely by the
availability of ethylene receptors (Klee, 2001 ), in abscission have not been extensively
studied in any film.

Retention of ripe fruit would be expected to confer considerable advantages for
current production regimes, and would be critical for potential mechanical harvesting of
apples. However, this trait has generally not been targeted in breeding efforts.
Interestingly, the genus Malus includes many wild species that are anecdotally known to
retain mature fi'uit, especially the small-fruited species commonly referred to as
crabapples (Fiala, 1994). A representation of Malus species and genotypes is maintained
at the USDA-ARS Plant Genetic Resources Unit in Geneva, NY. This reference
collection includes 28 wild Malus species and over 1000 M. domestica cultivars
originating fi'om throughout the northern hemisphere. A core subset considered to
represent the diversity of the entire collection is maintained at the Geneva site, allowing
for efficient evaluation of important traits relevant to industrial production (Hokanson et
al., 2001; Kresovich et al., 1995). To better characterize variability in endogenous timing
of fruit abscission, and help define the influence of fruit-produced ethylene in abscission

of mature fruit, we analyzed variation in abscission-related responses among these

42

accessions. Specifically, we assessed (1) seasonal timing of natural fruit abscission, (2)
endogenous ethylene production at the time of abscission, (3) pedicel abscission in

response to fruit removal, and (4) abscission of fruit in response to exogenous ethylene.

Material and Methods
Plant material

The Malus Gerrnplasm Collection is maintained at the United States Department
of Agriculture-Agicultural Research Service (ARS) Plant Genetic Resources Unit in
Geneva, NY. We targeted for evaluation a subset of accessions previously determined to
represent much of the diversity of the entire collection [the apple 'Core Collection'
(Hokanson et al., 2001; Kresovich et al., 1995)] as well as accessions previously noted by
USDA staff as exhibiting either premature fruit drop or fruit retention into the winter.
Plants were five to ten years old, budded on M7 or E7 semi-dwarfing rootstocks, and
managed in accordance with commercial practice only for insect or microbial pests. Only
healthy and vigorous trees were selected for evaluation. Species, hybrid and cultivar
nomenclature exactly followed ARS assignments.
Analysis of fruit abscission

For each accession, we determined the peak tinting of fruit abscission by counting
the number of naturally abscised fruit at two-week intervals, beginning August 27, 2006
when appreciable fi'uit abscission in some accessions was first noticed, and ending
November 10, 2006, when trees were mostly defoliated. Peak abscission was defined as
the observation date when at least 15% of fruit initially recorded for the accession had

abscised. In all cases, nearly all remaining fruit abscised by the subsequent observation

43

date (not shown). Accessions that showed less than 15% abscission of initial fruit at the
final observation date were defined as non-abscising. Of accessions classified as non-
abscising in 2006, all but six also showed less than 15% hit abscission when evaluated
at an equivalent date in 2007, with the remaining six accessions showing 50% or less
abscission in 2007. For each abscising accession, on the date defined as peak abscission,
20 fi'uit were selected that abscised when subjected to gentle force. All collected fi'uit
separated from the branch at the apparent pedicel/branch abscission zone, and showed
turgid, undamaged pedicels. On the final observation date, fi'uit from non-abscising
accessions were removed from the tree, leaving the pedicel attached to the fruit. Fruit
were maintained under laboratory conditions for 24 h before analyses.
Measurement of internal ethylene concentration (IEC), firmness and starch

All determinations of IEC, firmness, and starch were based on measurements of at
least five fruit of each accession. For IEC measurement, 1 ml of internal gas was
withdrawn from fruit submerged in water under a vacuum (Beyer and Morgan, 1970),
and analyzed by gas chromatogaphy using a Carle Series 400 AGC (Hach Co., Loveland,
CO) and certified ethylene standard (Matheson Gas Products, Chicago, IL). Flesh
firmness was determined using an Effigy FT-327 penetrometer (Effigy, Alfonsine, Italy)
with an ll-mm diameter probe. Starch content was evaluated by rating stain intensity
after dipping transverse sections into an iodide solution (5 mM potassium iodide, 17 mM
iodine), with a visual scale of 1 (intense staining; highest starch content) to 8 (no staining;
lowest starch content) using the Cornell Generic Starch Chart.

MdA CS1 genotyping

44

MdA CS1 allelotype was evaluated by the PCR using oligonucleotide primers
MdACSI-F (5'- GGTAATTGGAGTAATGAACTGAGCA-3') and MdA CSI-R (5'-
TCACTAT’ITGCTTGGACTGGG AAGT-3') that flank the transposon insertion found in
MdACSI-Z, as described by Sunako et al., (1999).

Evaluation of pedicel abscission and ethephon-promoted abscission

This experiment was carried out in early July, 2007 approximately 80 d after full
bloom for the standard cv. Gala. For each accession evaluated, 30 fruit were labeled on
each of two branches. On one of the two branches, the pedicel was severed midway
between the fruit and the branch to induce abscission. Abscission was monitored daily
by applying a gentle force on the defruited pedicel. On the remaining branch, marked
fi'uit were evaluated for natural abscission (control). A biological replicate, offset by two
days, was carried out using a separate branch, or branch of a separate tree. Abscission
zone morphology was evaluated on pedicels of fruit attached to the tree, without the aid
of microscopy.

Analysis of promotion of fi'uit abscission with ethephon was carried out in mid-
late September, 2007. A subset of non-abscising accessions identified in 2006 was
targeted for analysis. For each accession, branches with similar fi'uit load were tagged as
either experimental or control. For each of the experimental treatments, ethephon [(2-
chloroethylphosphonic acid), (600 ul/L active ingedient), Micro Flo, Memphis, TN,]
was applied with 0.1 % Silwet L-77 as a foliar spray. The control branch was treated
with 0.1 % Silwet L-77 only. The replicate treatment was offset by one day. Fruit IEC

was determined as described above. Fruit abscission was quantified by counting the

45

number of abscised fruit at defined intervals following treatment, and expressed as

percentage of initial number of fruit recorded for the accession.

Results
Seasonal timing of fruit abscission

To document variation in the seasonal timing of fruit abscission among wild
Malus species, Malus domestica cultivars, and hybrids, we examined 144 diverse
accessions at defined intervals during the period of natural fruit abscission. These
included 53 accessions representing 28 wild species, 61 Malus domestica cultivars, and
30 hybrids (Table 2.1 and not shown). We found that the seasonal timing of fruit
abscission was similarly and broadly distributed across observation dates for
representatives of wild Malus species, Malus domestica cultivars, and hybrids, and that
all three goups included non-abscising accessions. However, accessions showing
abscission at the earliest two observation dates were mainly Malus domestica cultivars
(25 of 33 accessions), whereas non-abscising accessions were predominantly wild Malus
species or hybrids (36 of 49 accessions) (Figure 2.1A). We then evaluated the potential
relationship between seasonal timing of fi'uit abscission and fi'uit size. We categorized
fruit into three classes: >100g, 30g-100g, and <30g, and identified representatives of each
fruit size class that exhibited abscission at each observation date. Small-finited
acCessions, nearly all wild species and hybrids, were overrepresented among the non-
abscising class (31 out of 49 accessions) (Figure 2.18). However, we observed 23 small-
fi'uited accessions that abscised, and 14 large-fruited accessions, nearly all domestic

cultivars, that did not abscise (Figure 2.13). This documents an association between an

46

Table 2.1 Number of wild speciesLdomestic cultivars, and hybrids used in this study
Species and cultivars No.

 

Mangustifolia
Masiatica
M.atrosanguinea
Mbaccata
Mbhutanica
Mcoronaria
Mhallz'ana
Mhupehensis
Mioensis
Mkirghisorum
Mmandshurica
Mmicromalus
Mprunifolia
Mrockii
Msz‘eboldii
Msieversii
Msylvestris
Mturesii

M. yunnanensis
M. x arnoldiana
M. x dawsoniana
M. x hartwigz'i

M. x magdeburgensis
M. x platycarpa
M. x robusta

M x scheideckeri
M. x soulardii

M. x sublobata
M. domestica
Hybrids 30

QHNh—IAI-dl—II—ir—tI—ANt—ANU’INF—‘uh—Br—iNWI—Bh—AMHAHWt—l

 

47

 

 

 

A 50

- M.domestica
[:1 Wild
- Hybrid

to

Cl

C

'5'

m

o

8

(Q

6

Z
- <30g
30-100g

40- - >100g

w

I:

O

"-1

m

m

a.)

8

N

0'

Z

 

 

 

8/27 9/ 8 9/22 10/7 10/22 NA

Harvest date
Figure 2.1. Frequency distribution of accessions evaluated for harvest date relative

to species (A) and fruit weight (B). NA, non-abscising

48

association between fi'uit retention and non-domestic, small-fi'uited genotypes, lack of
abscission among a small number of domestic cultivars, and the existence of alleles
specifying early season fruit drop among wild species.
Variation in fruit ethylene concentration at abscission

To help evaluate a potential role for fruit-produced ethylene in abscission, we
measured internal ethylene concentration (IEC) of fruit of each accession harvested at the
date of peak natural abscission. For each accession, readily abscising fruit were removed
from the tree, briefly allowed to equilibrate to the laboratory environment (Sfakiotakis
and Dilley, 1973), and assayed for IEC. We found that the IEC in abscising fi'uit among
different accessions varied by geater than three orders of magritude, from ~0.03 ul/L to
900 ul/L (Figure 2.2 and not shown). Multiple fruit from single accessions generally
showed low variability in IEC (standard error ~15% of mean values) suggesting the
observed variability reflected true tree-to-tree differences (not shown). Those accessions
showing the lowest IEC values in abscising fi'uit ($0.5 ul/L) included nine accessions
(eight domestic and one wild) that also exhibited high starch content, suggesting that the
ripening progam had not sigrificantly progessed in these accessions (Table 2.2). We
also evaluated IEC in fruit from non-abscising accessions. Unblemished fruit (were
removed from the tree in early November, allowed to briefly equilibrate to the laboratory
environment, and assayed for IEC under the same conditions as for naturally abscising
fruit. Surprisingly, the range of IEC from non-abscising fruit was similar to that observed
in abscising fruit (~0.07 ul/L to 580 ul/L), although accessions with non-abscising fruit
were underrepresented in the goup of accessions with highest IEC values (210 ul/L) (18

of 78 accessions) (Figure 2.2 and not shown). All non-abscising accessions with high

49

 

30

25-

20-

No. accessions
U.
l

 

|:l Abscising
1:1 Non-abscising

 

 

 

 

    

 

 

   

 

 

 

 

 

date (non-abscising accessions).

 

7 1-5

75310

10350

IEC range (pl/L)
Figure 2.2 Frequency distribution of accessions evaluated for internal ethylene
concentration (IEC) at peak harvest date (abscising accessions) or final sampling

50

50—100 100-900

high IEC values (_>_10 ul/L) showed relatively low starch content (starch index _>_6), and at
least a subset of these also exhibited low flesh firmness values (S~9O N) and fully
developed skin gound color (Table 2.2 )and not shown) suggesting that the ripening
progam was progessing. This subset included Malus prunifolia and Malus. x asiatz'ca
(= Malus prunifolia x Malus sieversii) (Luby, 2003), four domestic cultivars, and two
hybrids (Table 2.2).

It was previously reported that absence of significant preharvest drop among M.
domestica cultivars was associated with homozygosity of the dysfunctional MdACSI-Z
allele (Sato et al., 2004; see above). To further understand the variation in seasonal
timing of abscission and relationship between abscission and fruit IEC, we determined
the MdACSI allelotype for the studied accessions. The ACSI-1 allelotype was identified
in >70% of accessions, including 82 accession from wild species, 20 hybrids, and 37
domestic cultivars, whereas only ~9% of accessions (6 hybrids and 6 domestic cultivars)
exhibited the ACSI-Z/Z allelotype (Figure 2.3; not shown). We found that each allelic
goup (MdACSI-1/-I, -I/2, or -2/2) contained both abscising and non-abscising
accessions (Figure 2.3). The MdACS1-1/-I allelotype was overrepresented among
accessions showing the earliest natural abscission (August 27; 12 of 13 accessions),
whereas the MdA CSI-2/-2 allelotype was overrepresented among non-abscising
accessions (7 of 49 accessions) (Figure 2.3 and not shown). This suggests that MdACS]
allelotype is not only a possible determinant of the potential for preharvest drop, but also
for the non-abscising character.

To evaluate the relationship between timing of abscission and climacteric

associated ethylene production, and to determine if lack of capacity for climacteric

51

 

 

 

 

52

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8/27 9l8 9/22 1077 10i22 NA
Harvest date

 

 

 

Figure 2.3. Relationship between harvest date, IEC at abscission, and ACS]
allelotype for studied accessions.

53

ethylene production may have contributed to the low IEC in some accessions, we
measured IEC in harvested fi'uit after storage in the laboratory environment for 14 days
(Gussman et al., 1993; Sfakiotakis and Dilley, 1973). Of 14 abscising accessions
evaluated, 13 showed a striking (>500-fold) increase in IEC during this period (Table
2.2). These accessions were naturally abscising, revealing that climacteric ethylene
production followed rather than preceded natural fruit drop in these accessions.
Abscission in response to fruit removal and exogenous ethylene

These experiments identified 18 accessions that failed to abscise yet produced
high levels of ethylene in the fruit. These were not obviously distinguished from the
entire population in terms of A CS1 allelotype, IEC at harvest, or fruit weight (not shown).
All of the non-abscising accessions, including these, exhibited classical abscission zone
morphology. To evaluate the functionality of the abscission zone in these accessions, we
determined the abscission response of the fruit pedicel when hit was removed from
field-gown plants by cutting the pedicel halfway between the branch and fruit (Barlow,
1950). We previously observed that this treatment invariably resulted in abscission of the
remaining pedicel stub at the natural abscission zone within five to eight days, when
carried out with the Golden Delicious cultivar approximately 80 days following full
bloom (unpublished data). To interpret the results, we applied this analysis to nearly the
entire population. For the experiments reported here, a marked subset of fruit on each
tree was removed, and the timing of abscission of the remaining pedicel stubs was noted
at a daily interval.

Interestingly, all of the 122 accessions used in this experiment showed abscission

of pedicel stubs, with the median timing of abscission among accessions ranging from

54

Table 2.3. Accessions showing extremes of pedicel abscission

 

Accessions shoMnmlatively rapid and synchronous pedicel abscission

 

 

Days to Days to Days to
first 50% 95% Fruit ACSI
Accession Species Cultivar abscission abscission abscission weight (g) Allelotype
589765 angustifolia 3 4 4 12.3 ACSI -]/1 -I
589877 asiatica 4 4 5 48.4 ACSI-I/I-Z
136488 atrosanguinea 3 3 3 7.48 ACSI-I/I-I
588907 baccata Hirnalaica 3 3 3 l .82 ACSI -1/I -]
322713 baccata Mandshurica 3 3 4 5 .58 ACSI-I/I-I
483259 baccata Genvina 3 3 5 9.48 ACSI -1/1 -1
590062 bhutanica 3 4 5 1.48 ACS I -I/1 -1
589987 coronaria 3 3 4 20.9 ACSI-I/I-l
589983 coronaria 3 3 4 25.56 ACSI -1/I -]
588849 domestica Russian 4 5 6 22.05 ACSI -1/] -I
589478 domestica Novosibirski Swen 4 4 5 45.55 ACSI -I/1-1
589053 domestica Lady 4 5 6 75.92 ACSI -1/1 -I
588838 domestica Nova Easygro 4 5 6 106.96 ACSI-l/I-I
589913 domestica Dorsett Golden 5 6 6 1 10.28 ACSI -1/1 -I
589486 domestica Murray 4 4 5 175.46 ACSI -1/1 -I
588992 hybrid White Angel 3 4 4 2.48 ACSI-I/I-I
589819 hybrid PRI 2050-2 5 6 7 198.88 ACSI-Z/I-Z
589820 hybrid Paririe Fire 3 4 5 1.34 ACSI-I/I-I
589959 hybrid MA #8 3 4 4 1.96 ACSI -1/1 -I
589510 hybrid Garry 3 4 4 2.48 ACSI-I/I-I
588824 hybrid Almey 3 4 4 3.78 ACSI-I/I-I
589250 hybrid Red Jacket 3 3 4 7.96 ACSI -1/]-]
588870 hybrid Dolgo 3 3 4 13.86 ACSI-I/I-I
589775 hybrid PR1 2382-1 5 6 7 92.82 ACSI-I/I-l
588883 hybrid Demir 4 5 6 109.02 ACSI-I/I-I
590016 ioensis 3 4 5 14.56 ACSI -]/I-1
590004 ionesis 3 4 5 10.62 ACSI -1/1-I
613855 kirghisorum 3 4 5 42.96 ACSI-I/I-I
589832 prunifolia Xanthocarpa 3 3 4 4.28 ACSI-I/I-I
589421 rockii 3 4 5 2.8 ACSI-I/I-I
613932 sieboldii 5 5 6 0.44 ACSI-I/I-I
613806 sieboldii 3 3 3 0.44 ACSI -I/1 -I
594104 sieversii 4 5 5 26.84 ACSI -1/1 -I
589008 turesii 3 3 4 1.74 ACSI -1/] -]
588757 x hartwigii GMAL52 4 4 5 1.92 ACSI -]/1 -1
588959 x magdeburgensis 6 7 8 0.78 ACSI-I/I-I
589415 x platycarpc Hoopesii 3 3 3 34.54 ACS 1 -1/1 -1
588825 x robusta Robusta 5 3 3 4 3.14 ACSI-I/I-I
589383 x robusta Persicifolia 3 4 5 3.94 ACSI-I/I-I
589418 x scheideckt 3 3 4 3.1 ACSI -I/1 -]
588922 J: sublobata Yellow Autumn C 3 3 4 12.26 ACSI-I/I -1
589253 yunnanensi-s Carmine crab 4 5 6 0.81 ACSI -I/I -1

 

55

Table 2.3.Continued

 

Accessions showing relatively rapid and synchronous pedicel abscission

 

 

56

Days to Days to Days to
first 50 % 95% Fruit A CS]
Accession Species Cultivar abscission abscission abscission weight (g) Allelotype
589765 angustifolia 3 4 4 12.3 ACSI -1/1 -1
589877 asiatica 4 4 5 48.4 ACSI -1/1 -2
136488 atrosanguinea 3 3 3 7.48 ACSI -1/1 -]
58 8907 baccata Hirnalaica 3 3 3 1 .82 ACSI - 1/1-1
322713 baccata Mandshurica 3 3 4 5.58 ACSI -1/] -1
483259 baccata Genvina 3 3 5 9.48 ACSI -1/] -1
590062 bhutanica 3 4 5 1.48 ACSI -I/I -1
589987 coronaria 3 3 4 20.9 ACSI -1/1 -I
589983 coronaria 3 3 4 25.56 ACSI -1/I -]
588849 domestica Russian 4 5 6 22.05 ACS 1 -I /I -1
589478 domestica Novosibirski Swec 4 4 5 45.55 ACSI -]/I -]
589053 domestica Lady 4 5 6 75.92 ACSI -I /1 -I
588838 domestica Nova Easygro 4 5 6 106.96 ACSI -1/] -1
589913 domestica Dorsett Golden 5 6 6 1 10.28 ACSI -1/1 -1
589486 domestica Murray 4 4 5 17 5.46 A CS] -1 /1 -]
5 88992 hybrid White Angel 3 4 4 2.48 ACSI-I/I-I
589819 hybrid PR1 2050-2 5 6 7 198.88 ACSI-Z/l-Z
589820 hybrid Paririe Fire 3 4 5 1.34 ACSI -I/] -1
589959 hybrid MA #8 3 4 4 1.96 ACSI -I/1 -1
589510 hybrid Garry 3 4 4 2.48 ACSI -1/1 -]
588824 hybrid Almey 3 4 4 3 .78 ACSI -1/1 -I
589250 hybrid Red Jacket 3 3 4 7.96 ACSI -]/1 -]
588870 hybrid Dolgo 3 3 4 13.86 ACSI-I/I-I
589775 hybrid PRI 2382-1 5 6 7 92.82 ACSI-I/I-I

three to 17 days. We identified a subset of 42 accessions that showed relatively rapid and
synchronous abscission of all treated pedicels, beginning ~3 days after treatment, and
with >50% or >95% of pedicels abscising within 24 or 48 hours, respectively, thereafter
(Table 2.3). Abscission of pedicels for the remaining accessions was less rapid and less
synchronous, with a subset of 24 accessions showing abscission only after ~5 days, and
retention of >50% of pedicels for at least five days thereafter. The synchronous-abscising
subset was disproportionally lacking in domestic cultivars, large-fi'uited accessions,
and/or genotypes heterozygous or homozygous for ACSI-2 (p<0.05, Fisher's Exact Test;
Table 2.3). Neither the non-abscising accessions producing high levels of ethylene, nor
non-abscising accessions considered as a whole, were distinguished from the remainder
of accessions in terms of the abscission behavior of the pedicel following cutting.

As an alternative approach to analyze functionality of the abscission zone, we
subjected 24 non-abscising accessions to treatment with ethephon, which is metabolized
by plant tissues to produce free ethylene. Branches within the same tree were either
subject to a foliar spray, or used as mock-treated controls. Of these accessions, 15
showed abscission of >50% of fruit within 12 days of treatment, and all but three showed
abscission that was markedly geater than that of the control branches (Table 2.4). The
remaining three accessions showed abscission of <2 % of fruit after ethephon treatment;
however, fruit from these did not show substantial increases in IEC, suggesting that the

ethephon treatment was ineffective (Table 2.4).

57

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58

Discussion

In this study, variation in abscission-related traits was observed among Malus
accessions representing the breadth of genetic diversity found in domesticated varieties
and in the Malus genus. This information is useful both for understanding the underlying
biology of these traits and for the more applied goal of developing genotypes with
abscission characteristics better suited to modern apple production approaches. We
documented the range in seasonal timing of natural abscission for domestic and non-
domestic accessions, and the occurrence of accessions lacking fruit abscission. For
domestic cultivars, seasonal distribution of abscission is most likely influenced by the
broad range in seasonal timing of fruit maturation, a trait perhaps subject to selection
during domestication. However, we found a similar range of seasonal timing of
abscission among wild species. In this study, replicate observations of seasonal timing of
abscission could not be made for most accessions, because only a single specimen was
available during the duration of the study. Possibly, some tree-to-tree differences
observed were influenced by the physiological status of individual trees, rather than
strictly by genotype. However, none of the trees used in this study displayed visible
signs of stress. Neither did we observe separation of fruit from the tree independently of
the visible abscission zone, a phenomenon that has been connected with abscission
related to tissue damage (Walsh, 1977).

We also evaluated the tendency for natural abscission in relation to fruit size. It is
well known that many crabapple-type Malus genotypes retain fruit into the winter season.
Potentially, retention of ripened fruit in small-fruited genotypes is an adaptation that

facilitates access and seed dispersal by frugiverous birds (Harris et al., 2002). In contrast,

59

domestication of large-fruited genotypes may have favored those that readily dropped
ripe fruit, in order to facilitate collection from the naturally large trees. In this study, we
documented the anecdotal observations that lack of abscission is not absolutely coupled
to small fruit size. We identified 14 accessions, ten domestic cultivars and four hybrids,
that exhibited relatively large (>100 g) fruit and that did not show abscission. Thus,
alleles governing this trait may be readily exploited in the development of new
commercial varieties.

A trivial explanation for the lack of abscission identified in some accessions in
this study is that these genotypes may be adapted to longer growing seasons, and fail to
initiate the abscission process at the Geneva site before the onset of dormancy. While
this is possibly the case for some of the accessions, we noted that many of the non-
abscising accessions showed physiological characteristics that are typically associated
with ripening in domestic cultivars, including color development, loss of starch, and loss
of firmness, and/or also exhibited relatively high levels of internal ethylene (Table 2.2
and not shown). For these, abscission is apparently unlinked from ripening and/or
ethylene production.

Previous studies have shown that internal ethylene concentration can vary
strikingly among domestic varieties at the date of optimal commercial harvest, a
benchmark based both on fi'uit maturity and storage potential (e.g., Chu 1988). In this
study, we analyzed internal ethylene concentration at the time of abscission, or for non-
abscising accessions, at a time late in the season when plants were progressing into
dormancy. Blanpied (1972) observed substantial natural abscission in cv. Golden

Delicious and McIntosh preceding climacteric ethylene production, suggesting that for

60

these varieties, high levels of fruit ethylene are not required for abscission. There have
been no previous large-scale studies to examine genotypic differences in ethylene
production in relation to fruit abscission. Here, we found that internal ethylene content of
abscising fruit varied substantially among both domestic and non-domestic accessions.

We identified numerous accessions that showed natural abscission even when the
IEC was low. These included Marshall McIntosh, a skin color variant of McIntosh. In
our study, McIntosh showed only moderate starch content and relatively low flesh
firmness at abscission. This suggests that ripening was in progress, and supports both the
observation of Blanpied (1972) that this variety abscises in advance of significant
ethylene production, and anecdotal observations that this variety is prone to premature
abscission. We noted that this accession was distinguished among all of the evaluated
accessions by an extremely short pedicel, a trait that may lead to substantial physical
force on the abscission zone as the hit enlarges and becomes constrained by the branch
or neighboring fi'uit. In other accessions that showed abscission at low IEC, ripening had
apparently not significantly progressed, as evidenced by the high starch content.
Interestingly, eight of the nine accessions that showed abscission in advance of apparent
ripening are domesticated cultivars, and the ninth, M. x dawsoniana, is believed to have
domestica parentage, suggesting that abscission in advance of ripening may have been
subject to selection during domestication.

In contrast, we also identified accessions that did not show abscission, in spite of
high levels of ethylene in the fruit. A potential explanation is that the abscission process
was initiated, but was in an early stage, at the time of our measurements. However, this

appears unlikely, since our analysis was carried out at a time during the season when

61

trees were mostly defoliated and entering dormancy. In addition, none of the fruit from
non-abscising accessions appeared to be nearing abscission, because significant force was
required to remove the fi'uit at the apparent abscission zone when these were harvested
(not shown). These accessions, which include four domestic cultivars and two hybrids
with Golden Delicious parentage, are an attractive source of alleles conferring this trait
for breeding of commercial varieties.

It is interesting to speculate on the determinants that might govern the apparent
ethylene-independent retention of fruit seen in these accessions. All of the non-abscising
accessions examined in this study showed an adjacent enlargement and constriction at the
basal end of the pedicel, a characteristic of the abscission zone in cultivated apple. In
addition, our experiments showed that abscission could be induced either through
removal of the immature fruit, or, for the subset of the accessions analyzed, by exposure
to exogenous ethylene. This suggests that the natural fruit retention seen in these
accessions was not due to a homeotic absence of the abscission zone, as seen in tomato
mutants for the JOINT LESS gene (Mao et al., 2000). However, we cannot rule out the
possibility that one or more of these accessions show more subtle defects in abscission
zone filnction that precludes abscission of fruit under natural conditions. For example,
incomplete separation layer development has previously been shown as a mechanism
associated with loss of grain abscission in domesticated rice (Li et al. 2006). Anatomical
comparisons of accessions showing extremes of abscission habits identified here may
resolve this question. Another possibility is that the abscission process may be initiated,
but remain ultimately ineffective for pedicel breakage. Some genotypes may lack

effective production of one or more of the numerous enzymatic activities expected to be

62

required for cell wall disassembly. Here, comparative expression analyses of selected
abscission-associated genes among genotypes showing extremes in abscission behavior
may be informative. Alternatively, some abscission zone tissues might not fully
participate in abscission. For example, vascular tracheids and other cell types may have
highly modified secondary walls that likely present a challenge for cell wall disassembly.
These may persist after disintegration of other separation layer cell types, and effectively
retain the fruit until broken by physical force. Especially for Malus genotypes that have
small, lightweight fruits, even subtle variation in such cell types or numbers in the
abscission zone could predispose the fi'uit to drop or retention. To explore this, a detailed
study of abscission zone development among accessions is needed.

In the model plant Arabidopsis thaliana, a gene designated [DA is required for
floral organ abscission. Interestingly, in ida mutants, organ removal force ultimately
increases following a sharp decrease at the time of natural organ abscission (Butenko et a1.
2003). This identifies a potentially conserved mechanism that may act antagonistically to
cell disintegration, and in apple, may preclude efficient abscission in naturally non-
abscising accessions.

In conclusion, we documented diversity in fi'uit-abscission-related traits among
Malus accessions representing the breadth of genetic diversity seen in Malus. Our
findings suggest that important mechanism(s) independent of fruit ethylene production
act as determinants of natural abscission. Accessions showing phenotypic extremes in
abscission-related traits can be developed as contrasting models to understand the
biological bases of these traits, and as tools in genetic analyses for the mapping of genes

that influence these traits.

63

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67

CHAPTER 3
Molecular Analysis of Abscission Zone Development in Apple Fruit

Pedicels

Abstract

Organ abscission is a developmentally and environmentally regulated cell
separation process initiated in specialized tissues of the abscission zones (AZs).
Physiological studies of leaf abscission have suggested that the interaction between auxin
and ethylene signaling within the AZs regulates the initiation of cell separation, whereas
genetic analyses of floral organ abscission in Arabidopsis thaliana (Arabidopsis) have
identified an additional mechanism of abscission apparently unrelated to ethylene. In
fi'uit crops, precise regulation of fruit abscission is crucial to achieve maximum yield and
optimized market value. In this study, we identified transcriptional changes
accompanying the transition fiom competent-quiescent to activated AZs in the apple fi'uit
pedicel. The abscission-associated genes identified in this work contribute to our
understanding of fruit abscission, while suggesting a common molecular mechanism of

fruit abscission induced under various conditions.

Key words: apple, transcriptional profiling, ethylene, auxin

68

Introduction

Abscission is a process by which plants shed organs, such as leaves, flowers,
floral organs, or fruit, at a pre—determined position, called abscission zones (AZs)
(Addicott, 1982). The separation layers within the AZ are characterized by small,
densely cytoplasmic cells, generally arranged transversely to the organ axis (Addicott,
1982, Osborne, 1989; Sexton and Roberts, 1982). Organ abscission is generally
conceptualized as progressing through four steps (Patterson, 2001): (1) differentiation,
whereby morphologically distinct cell layers are formed in response to developmental
and/or environmental cues; (2) activation, characterized by the acquisition of the ability
of the separation layers to respond to abscission-promoting signals; (3) separation of cells
within abscission layers, resulting from the production of cell-wall modifying enzymes
and accompanied by dissolution of the pectin-rich middle larnella and expansion and
rounding of cells; (4) differentiation of protective layer (8) across the newly exposed
surface of the plant body.

Pioneering studies of abscission focused on the effects of various plant hormones,
especially ethylene and auxins, on differentiation and activation (Addicott, 1982; Sexton
and Roberts, 1982). These studies led to a widely accepted model whereby organ
abscission is conditioned by the balance between auxin and ethylene signaling, rather
than absolute levels of these hormones (Abeles, 1967; Abeles and Rubinstein, 1964;
Addicott, 1982; Osborne, 1989; Sexton, 1995). According to this model, auxin flow
across the AZ controls the sensitivity to ethylene; when auxin declines, the AZ becomes
more responsive to ethylene (Addicott, 1982; Sexton, 1995; Taylor and Whitelaw, 2001).

More recent genetic studies in the model plants Arabidopsis thaliana (Arabidopsis) and

69

Solanum lycopersicum (tomato) have identified genes involved in differentiation
(Hepworth et al., 2005; Mao et al., 2000; Mckim et al., 2008; Norberg et al., 2005;
Pinyopich et al., 2003) and components of auxin or ethylene signaling required for post-
differentiation step(s) (Bleecker, et al., 1988; Bleecker and Patterson, 1997; Chen et al.,
2002; Ellis et al., 2005; Lanahan et al., 1994; Okushima et al., 2005; Patterson and
Bleecker 2004; Tieman et al., 2001). Such studies have also clearly implicated
mechanism(s) of abscission acting independently of ethylene (Butenko et al., 2003; Cho

et al., 2008; Patterson and Bleecker 2004; Stenvik et al., 2006; 2008).

Outside of the model plants, there has been little work done to identify the genetic
components of abscission, especially those that function in early steps. In addition, there
have been few studies of genetic mechanisms of fi'uit abscission. In domestic apple
(Malus X domestica), precise control of mu abscission is critical for production. Similar
to many tree fruits, apple naturally bears an abundance of flowers and fruitlets. Although
many or most of the Mt abscise early in development, the resulting crop load is typically
still too great to allow the remaining fruit to attain marketable size or quality (Byers,
2003). In addition, high crop load can repress floral initiation, leading to the
phenomenon of biennial (alternate year) bearing (Byers, 2003). Consequently, flower or
fi'uit thinning by mechanical or chemical methods is generally required to reduce the final
crop load (Greene et al., 2003). An additional considerable problem in commercial apple
production is the abscission of mature fi'uit in advance of harvest (pre-harvest drop).
Identification of genetic components involved in apple fruit abscission therefore has both

fimdarnental and practical value. In this study, we approached this goal by characterizing

70

temporal changes in transcriptional profiles associated with abscission of the fruit pedicel

following fruit removal.

Material and methods
Plant materials and treatments

Experiments were carried out on 15-year-old apple (Malus domestica L. Borkh)
trees, cv. 'Golden Delicious' and 'Spur McIntosh', grown under natural environmental
conditions at the Horticultural Teaching Research Center in Michigan State University
(Holt, Michigan). All samples were immediately fiozen in liquid nitrogen and stored at -
80°C. Two biological replicates were offset by two days for the following three fruit
abscission induction experiments.

Analysis of transcriptional responses accompanying fi'uit removal utilized 'Golden
Delicious'. Immature fruit 60 days after fiill bloom were removed by serving the pedicel
with scissors. Abscission of pedicel stubs was monitored daily for six days following
fruit removal. For analysis of gene expression, the AZ samples were collected
immediately before fruit removal, and 1, 2, 3, and 4 days after fruit removal. Ten trees
were used for each replicate in a completely randomized design among the treated and
control experiments.

Analysis of transcriptional responses to the application of 6-Benzyl Aminopurine
(6-BA) utilized 'Spur McIntosh'. When the average fruit size was 10-12 mm, a
commercial 6-BA formulation at 200 ppm [Maxcel, active ingredient 1.9% BA, Valent
Biosciences, Libertyville, IL] was applied with 0.1% Silwet L-77 (Helena Chemical,
Collierville, TN) as a foliar spray until saturation. Controls received 0.1% Silwet L-77 in

water. 50 fruit clusters were labeled on each branch, and four branches were used per

71

replicate. For each fruit cluster, abscission of terminal and lateral fruit was recorded 6,
12, 18, and 24 days after application. For analysis of gene expression, the AZ samples
were collected immediately before application, and 2, 4, and 6 days after application.

Pre-harvest fruit drop experiments utilized 'Spur McIntosh'. For the treatment,
ethephon [(2-chloroethyl phosphonic acid), active ingredient 21.7%, Micro Flo,
Memphis, TN] at 300ppm was applied to the whole apple canopy with 0.1% Silwet L-77
two weeks in advance of the anticipated harvest date. Controls received 0.1% Silwet L-
77 in water. Three trees were used per replicate. Fruit abscission was recorded 2, 4, 5, 6,
and 8 days after application. Fruit samples used for ethylene measurement were collected
immediately before application, and 2, 4, and 5 days afler application. For internal
ethylene concentration (IEC) measurement, 1 mL of internal gas was withdrawn fi'om
fiuit through the calyx and analyzed by gas chromatography using a Carle Series 400
AGC (Hach Co., Loveland, CO) and certified ethylene standard (Matheson Gas Products,
Chicago, IL). For analysis of gene expression, the AZ samples were collected
immediately before application, and 2, 4, and 5 days after application.
Preparation of cDNA arrays, probe preparation, and hybridization

Total RNA was isolated independently from AZ segments collected fiom 'Golden
Delicious' before fruit removal, and 1, 2, 3, and 4 days following fi'uit removal, following
the protocol described by Hu et a1 (2002). Equivalent amounts of RNA from each sample
were pooled, and mRNA was purified using the Clontech mRNA Separator Kit
(Clontech, Mountainview, CA). cDNAs were synthesized using the ZAP-cDNA
Synthesis Kit (Stratagene, La Jolla, CA). cDNAs were size-fractionated by gel filtration

using Sephadex G-25 medium, cloned into the Uni-ZAP XR vector, and packaged into

72

ZAP-cDNA GigPackIII Gold cloning kit as specified by the manufacturer (Stratagene, La
Jolla, CA). Five thousand cDNA clones were selected from this library, amplified by
PCR using T3 and T7 oligonucleotide primers, and applied, in a 32-block format, to
UltraGAPSTM coated slides (Corning, Big Flats, NY) using an OminGrid robot
(Genernachines, San Carlos, CA) with Chipmaker pins (TeleChem, Sunnydale, CA).
After application, cDNAs were crosslinked to slides by exposure to UV radiation as
specified by the manufacturer.

For probe preparation, mRNA was purified from total RNA using the
PolyATract® mRNA Isolation System (Promega, Madsion, WI). Labeling and
hybridization were carried out according to Hedge et a1 (2002). Briefly, 1 ug of purified
mRNA was labeled with aminoallyl-dUTP (Sigma, St. Louis, MO) during reverse
transcription with oligo(dT) priming and Superscript II (Invitrogen, Carlsbad, CA).
Single-stranded cDNA was labeled with either Cy3- or Cy5-monoreactive dye
(Amersham, Piscataway, NJ) and hybridized to cDNA arrays at 42°C overnight in 30p]
hybridization buffer [50% formamide, 5x SSC (sodium chloride/sodium citrate buffer),
0.1% SDS (sodium dodecyl sulfate), containing 2 pg yeast tRNA]. Hybridized array
slides were scanned using an Affymetrix 428 array scanner (Affymetrix, Santa Clara,

CA) and analyzed with GenePix Pro 4.0 software (Axon Instruments, Union city, CA).

Experiment design and data analysis

Probes were prepared from AZ samples collected before fiuit removal, day 1, 2,
and mixed 3 and 4 days after fruit removal, and pedicels samples mixed at day 3 and 4
after fruit removals. RNA samples represented the pooled collections from 10 trees for

each time point. An interwoven loop with side-by—side replication design was used for

73

this experiment (Churchhill, 2002; Yang et al., 2002). Four data sets were derived for
each target sample, which included two biological and two technical replications.
Microarray data were analyzed using MAANOVA
(http://www.jax.org/staff/churchill/labsite/software), an add-on package implemented in
the statistical language R (http://www.r-project.org). Raw signal values were first
averaged for duplicated spots, then normalized using the ‘glowess’ on a per-slide basis.

To evaluate the sources of variability using ANOVA in MAANOVA as
previously described in Yang et al (2002), normalized signal values were fitted to a
mixed model—treating array with biological effects as random factors. For statistical
inference on differential expression of genes among treatments, the treatment variances
identified from the ANOVA test were further used for the pairwise comparisons between
the treatments using Student’s t-test with P-value and fold change cutoff at <0.05 and
>2.5-fold reSpectively. K-means clustering was performed on the normalized data using
CLUSTER (Eisen et al., 1998). Functional categorization of differentially-expressed
genes with E-value less than 1.0E-5 was assigned to the corresponding Arabidopsis
annotation in the Gene Ontology (GO) database in The Arabidopsis Information
Resources (TAIR) version 7 (www.mabidggsisxg). Otherwise, categories were
assigned based on annotation of BLASTx against non-redundant (nr) protein database in
NCBI (http://www.ncbi.nlm.nih.g9l/). EST assembly was done through EST clustering
program developed from Research Technology Support Facility (RTSF) at MSU
(http://wwwgenomicsmsu.edu).

Reverse Transcription (RT) - PCR analysis

74

For semi-quantitative RT-PCR analyses, single-stranded cDNAs were prepared
by reverse transcription with oligo(dT) priming and Superscript II. Gene-specific primers
were designed using Primer3 (http://primer3.sourceforge.net/webifphp), and cDNA
fragments were amplified by PCR using GoTaq DNA polymerase (Promega, Madsion,
WI). An apple EST corresponding to ELONGATION FACTOR la (Md_EF-1a) was
used as an internal control. Gene-specific primers used in these experiments are listed in
Table 3.2.

Results and discussion
Abscission kinetics of apple fruit pedicels

In Phaseolus vulgaris L. (dry bean), removal of the leaf blade induces formation
of the abscission zone in the lower pulvinus followed by abscission of the remaining
petiole (Rasmussen and Bukovac, 1969). This method has been widely used to study the
kinetics of leaf abscission in many plant species (Meir et al., 2006). In apple, our
preliminary experiments showed that independent of fruit development, fruit removal
through cutting in the middle of pedicel consistently induced abscission of remaining
pedicel stubs within a short period. We utilized this reproducible system of abscission
induction to study gene expression changes during pedicel abscission. To avoid ‘June
drop’, a period of natural fruit abscission occurring in June, we induced pedicel
abscission 60 days after full bloom (July) in ‘Golden Delicious’. Total accumulated
pedicel abscission reached 100% within six-days, the experimental period in the
treatment, while no fruit abscission was observed in the control experiment (Figure 3.1).
This experiment indicated that fruit removal led to activation and subsequent cell

separation within pedicel abscission zones.

75

Analysis of gene expression changes in abscission of fruit pedicels

To document gene expression changes in abscission of fruit pedicels, we
performed transcriptional profiling using a cDNA-based microarray representing genes
expressed both in the quiescent and activated AZs. A total of 146 ESTs representing 118
genes were found to be either up- or down- regulated during abscission of fruit pedicels
(Table 3.1). Among these 146 ESTs, the highest transcript frequency was seen in 14
ESTs, and expression patterns of these 14 ESTs during fruit pedicel abscission were
highly consistent (Figure 3.2). In addition, expression patterns of all other redundant
ESTs encoding the same gene were also consistent (data not shown). These results
support the reliability of this microarray experiment.

Functional classification of these 118 genes using GO Consortium biological
process revealed that 28% of total genes were classified as ‘unknown biological process’
and 21% of total genes had potential roles in response to stress (Figure 3.3). It is not
surprising, since the treatment resulted in wounding, that a high percent of differentially
expressed genes are grouped as stress response genes. Additionally, it is possible that
abscission process itself induces many stress-related genes. Support for the latter is that
percentage of genes in response to stress within differentially expressed genes exceeds
average of whole genome transcripts by two-fold in stamen AZ transcriptional profiling
experiment in Arabidopsis (Cai and Lashbrook, 2008). These 118 differentially
expressed ESTs were clustered into five groups based on the similarity of their temporal
responses during abscission of fi'uit pedicels (Figure 3.4). Up-regulated clusters G1, G2,

and G3 contain 22% (26 genes), 16% (19 genes), and 9% (11 genes) of total respectively,

76

and down-regulated G4 and G5 contain 29% (34 genes) and 23 % (28 genes) respectively
(Figure 3.4). Gene identities are annotated in Table 3.1.

To examine the reliability of temporal responses of these differentially expressed
genes identified in this microarray experiment, we chose six genes from four clusters for
semi-quantitative RT-PCR analysis. Orthologous of these genes from other species have
been implicated in organ abscission (Cai and Lashbrook, 2008). Genes encoding purple
acid phosphatase27 (PAP27) and proton-dependent oligopeptide transport (POT) from
cluster G1 revealed elevated expression in the pedicel AZs at day 1 after fi'uit removal,
and genes sharing homology with peroxidase and transcriptional factor NAC21, from
cluster GZ, showed increased expression at day 1, 2 and 3/4 (mixed AZ sample of day 3
and day 4 after treatment) after fi'uit removal (Figure 3.5). Also, pectate lyase-like fi'om
cluster G3 is up-regulated at day 2 and 3/4, and Aux/IAA from cluster G5 is down-
regulated during fruit pedicel abscission (Figure 3.5). Expression patterns of these six
genes in the pedicel AZs through semi-quantitative RT-PCR analysis were largely
V consistent with those observed in the microarray experiment (Figure 3.5). In addition, we
performed RT-PCR analysis on the adjacent pedicel samples harvested at the same time
as those in the A23 for these six genes. Surprisingly, these genes showed transcriptional
responses in the adjacent pedicel tissues similar to those in the AZs (Figure 3.5). This
implies that these genes are not specifically associated with activation and separation of
the fruit pedicel abscission zone.

Chemical fruit thinning
In domesticated apple cultivars, each flower cluster contains one king fruit and 3-

5 lateral fruit. Preliminary experiments showed that 6-BA promotes fruit thinning more

77

 

120

 

 

 

 

+ control
100 - —0— cutting
r; 80"
E
,3 60—
'92 40-
.0
<1
20‘
()4 cu c v - e

i i 31— 4
days after removing fi'uit

M10
0“

Figure 3.1 Effect of fruit removal on fruit pedicel abscission in apple. Fruit was
labeled individually at the midway of the pedicel in the control and cutting treatments.
Abscission of remaining pedicel or fi'uit was recorded at daily intervals and expressed as
a percentage of abscission. Values represent means from two replicates with 30 fruit per
treatment. Error bars indicate the standard error. The open circles represent the induced
pedicel abscission, and circles filled black indicate fiuit abscission in control.

78

 

@2111:

Standardized expression
DJ

 

 

 

 

2 _
1 _
0 n I .
0 1 2 3/4
days after treatment

Figure 3.2 Expression profiles of 14 redundant ESTs assembled in the same contig.
Standardized expression values are means of normalized absolute values divided by
10000. X-axis represents time courses afier fruit removal. Annotation of represented
EST (Md1_AO4) was shown in Table 3.1

79

Go Biological Process

Ea response to stress (11.76%)

I other metabolic process (14.4%)

CI response to abiotic or biotic stimulus (9.62%)
a other cellular processes (11.23%)

I unknown biological processes (14.43)
I other biological process(7.48%)

I developmental processes (4.27%)

:1 transport (3.74%)

I protein metabolism (3.20%)

I signal transduction (2.13%)

1:! cell organization and biogenesis (1.60
I transcription (1.60%)

 

I electron transport or energy pathways (1.06)

I no hits (13.3%)

Figure 3.3 Functional categorization of 118 differentially expressed genes based on
GO Biological Process. Functional categorization for genes sharing close homology
with proteins in Arabidopsis was performed using AGl (Arabidopsis Genome Initiative)
IDs in the TAIR website. Functional categorization of genes sharing close homology with
non-Arabidopsis proteins was manually classified.

80

efficiently in lateral fruit than in king fruit. In this experiment, total abscission of lateral
fruit induced by 6-BA reached approximately 95% 24 days after treatment, and that of
king fi'uit is 40% (Figure 3.6A). Total accumulated abscission of lateral fruit in control
and that of king fruit was only 8% (Figure 3.6A). The reason for the high percentage of
abscission in lateral fi'uit compared to the control is that the fruit thinning experiment
extended from late May through ‘June drop’. In order to know the temporal responses of
those differentially expressed genes during the fi'uit thinning process, we performed RT-
PCR analyses for some selected genes using the AZ samples collected from the lateral
fruit treated with 6-BA. We found that expression of Aux/IAA gradually decreased in the
pedicel AZs two days after treatment, whereas NAC showed significant expression at day
4 and 6 after treatment within the pedicel AZs (Figure 3.6B).

Although 6-BA has been used in apple fruit thinning for many years, its mode of
action remains largely unknown. It has been hypothesized that a correlative abscission
relationship is present among young fruitlets growing within the same cluster, in which
the dominant king fruit inhibits auxin export of dominated lateral fruit (Bangerth et al.,
2000). This hypothesis, however, remains untested. The similar temporal responses of
these selected genes during abscission induced by cutting and chemical the dominant
king fi'uit inhibits auxin export to dominated lateral fruit (Bangerth et al., 2000). This
hypothesis, however, remains untested. The similar temporal responses of these selected
genes during abscission induced by cutting and chemical thinners suggest a common
molecular pathway for fruit abscission induced by these two conditions.

Preharvest fruit drop

It has been shown that preharvest fruit drop is associated with ethylene production

81

 

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61(26) 62%
1.0- fl 1.0
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10 G5 (28) 0 l 2 3/4
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1.0-
0.1-
0.01 . . .
0 l 2 3/4
days afier treatment

Figure 3.4 Classification of 118 AZ differentially expressed transcripts into five
clusters with similar temporal responses in pedicel abscission. Standardized
expression values are means of normalized absolute expression values divided by 10000.
X-axis represents time courses after treatment. Number of genes in each cluster is shown
in the upper left corner of plots. Red bold lines represent average value of genes within
each cluster.

82

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83

in fruit (Sato et al., 2004). We induced premature fi'uit abscission in preharvest season
through application of the ethylene releasing compound, ethephon. Total accumulated
fruit abscission in ethephon treatment reached 100% with a peak at day 6 after
application, while fruit abscission in control experiment was only 5% (Figure 3.7A).
Internal ethylene concentration (IEC) in fruit harvested fiom the ethephon treatment
dramatically increased two days after application, while that in fruit from the control
treatment remained in the basal level for the duration of the experiment (Figure 3.7B).
We performed RT-PCR analysis for selected genes using RNAs from the fruit pedicel
AZs collected from ethephon treatment. Genes sharing homology with pectate lyase-like
(PLL) in Arabidopsis showed increased expression at day 2, 4, and 5 after ethephon
treatment, and expression of Aux/1AA decreased dramatically during fruit abscission
induced by ethephon (Figure 3.7C). The parallel expression patterns of these two genes
observed in pedicel abscission induced by cutting and ethephon treatments implicate at
least partially shared molecular mechanism in fi'uit abscission under these conditions.
Increased expression of PLL and decreased expression of auxin response factor
(ARF) in citrus was observed during the abscission of leaf explants induced by the
application of ethylene (Agusti et al., 2008). These results suggest that genes involved
either in fruit or leaf abscission and are induced by exogenous ethylene might be
conserved across organs and species. It has been assumed that preharvest fruit abscission
resulted fiom the increased production of ethylene in the fruit (Sato et al., 2004).
However, there are still no experiments to show if ethylene level within the pedicels AZs
will increase upon application of exogenous ethylene, or if this increase is from diffusion

from fruit or other organs. Application of a high concentration of ethephon did not lead

84

 

   
   
 
    

 

 

 

A 100 ' + LF-control
-0- LF-6-BA
+ KF-control
80 - vv~ KF-6-BA
8C: .
.5 6o-
E
0
_g 40 -
<
20 -
0 h l l l A
6 12 18 24
days after treatment
B Abscission Zone

 

ID AZO A22 A24 AZ6 putative identity

]

Md2H051‘—"' " " ' Aux/IAA

Md2Hll ‘ NAC domain protein

MdEF-l a elongation factor la

Figure 3.6 Effects of 6-BA application on fruit thinning (A) and expression profiles
of selected differentially expressed genes within the AZs in fruit abscission induced
by 6-BA (B). (A) Percentage of fruit abscission was plotted against time after treatment.
Lateral fruit and king fi'uit were labeled on each cluster separately, and mu abscission
was'recorded at day 6, 12, 18, and 24 after application in the both control and treatment.
Fruit abscission is presented as percentage of total abscission. LF (lateral fruit), KF (king
fruit) (B) Expression profiles of selected genes were performed through RT-PCR using
two biological replicates. AZO, A22, A24, and AZ6 represent AZ samples collected at
day 0, 2, 4, 6 from lateral fruit afier 6-BA treatment. Detailed annotation for each gene

can be found in Table 3.1. MdEF—la was used as internal control for the RT-PCR
analysis.

85

 

. contr I
100 :ethephon

80-
60-
40-
20-

M
O 2 . . f

2 4 5 6

 

0°.

B 1000

 

—o— contrtfil
-°- ethep on

100-

('1 2' 4 T
days after treatment

 

 

 

Abscission zone
C ID AZO A22 AZ4 AZ5 putative identity

Md2EO9 pectate lyase like

MdEF—la“ elongation factor la

Figure 3.7 Effects of ethephon application on fruit abscission prior to fruit
maturation(A), ethylene production in fruit (B), and expression profiles of selected
differential expressed genes within the AZs (C). (A) Percentage of fruit abscission was
plotted against time after ethephon treatment. Fruit abscission was recorded at day 2, 4,
5, 6, and 8 after application in the both control and treatment. Fruit abscission was
presented as the percentage of total abscission. Black circles represent the control and
the open circle represent the treatment. Error bars represent the standard error. (B)
Ethylene production in fruit was plotted against time after application. Internal ethylene
concentration (IEC) was measured on 10 fruit samples per replicate collected from the
control and treatments at day 0, 2, 4, and 5 after application. Error bars represent
standard error. (C) Expression profiles of these selected genes were analyzed through
RT-PCR using two biological replicates. AZO, AZ2, A24, and A25 represent AZ
samples collected at day 0, 2, 4, and 5 after ethephon treatment. Detailed annotation for
each clone can be seen in Table 3.1. MdEF-la was used as internal control for the RT-
PCR analysis.

 
 

86

to abscission of leaves, but leaves are the main organs to absorb the compound and
release ethylene. Our previous study on natural variation in fruit abscission in Malus
species showed there is non linear relationship between ethylene production in fi'uit and
fi'uit abscission (Sun et al., 2009). Taken together, there are presumably other factors that
regulate preharvest fruit drop.

Implications of differentially expressed genes in abscission

Genetic studies in Arabidopsis thaliana have identified both ethylene-dependent
and -independent pathways that regulate floral organ abscission (Binder and Patterson,
2009). In addition, Cai and Lashbrook (2008) used laser-capture microdisection (LCM)
system to collect enriched population of AZ cells, and they revealed potential new
abscission regulators involved in the natural abscission of stamens. In this study, we
identified a small subset of genes differentially expressed in the abscission of fruit
pedicels induced by fruit removal (Table 3.1). Some of these differentially expressed
genes showed similar temporal expression patterns as those homologous counterparts in
stamen AZ transcriptome profiling, suggesting a general molecular mechanism for organ
abscission across species.

Several genes involved in cell wall remodeling including peroxidase, extensin
precursor, and pectate lyase-like, were up-regulated during pedicel abscission induced by
fruit removal (Table 3.1). The repeating unit, Ser-Pro4 in extensin, a cell wall
hydroxyproline-rich glycoprotein, is highly conserved across dicots (Merkouropoulous
and Shirsat, 2003). For those 14 highly expressed ESTs shown in Figure 3.2, the contig
assembled from these 14 ESTs shared close homology with one of the extensin

precursors in Prunus Dulcis (Table 3.1; Garcia-Mas et al., 1992). The SPPPPX5-15fl

87

repeat in the extensin identified in this study is conserved when compared with that of
AtEXT4 in Arabidopsis (Table 3.3). GUS expression driven by the promoter of AtEXT 4
was observed in the A25 of sepals, petals, and stamens (Merkouropoulos et al., 2003). In
addition, AtEXT 4 showed increased expression during stamen abscission in Arabidopsis
(Cai and Lashbrook, 2008). Furthermore, this gene was also found to be induced by
various abiotic and biotic stresses in Arabidopsis ((Merkouropoulos et al., 1999). These
results suggest that extensin is not only associated with organ abscission but also stress
responses.

Many early studies showed that peroxidase activity is increased during abscission
of bean leaf and cherry fruit (Hall and Sexton, 1974; Poovaiah et al., 1973, 1974).
Peroxidases are encoded by a large gene family in Arabidopsis and involved in multiple
plant developmental and defensive events (Passardi et al., 2005; Cosio and Dunand,
2009). Starnen AZ transcriptome profiling showed that 14 peroxidases were up-regulated
during stamen abscission in Arabidopsis (Cai and Lashbrook, 2008). Leaf AZ
transcriptome analysis in Capsicum revealed that the level of hydrogen peroxide (H202)
was increased in leaf abscission (Sakamoto et al., 2008). These results suggest that
peroxidases presumably contribute to cell wall loosening in abscission through
controlling level of H202 in the abscission zones. Pectate lyase-like (PLL) genes are also
encoded by a large gene family in plants (Palusa et al., 2007). The apple gene
(Md_2E09) encoding PLL identified in this study shares close similarity with PLLI9 in
Arabidopsis, and regulatory region of this gene in Arabidopsis drove GUS expression in
the A28 of floral organs in abscission (Sun PhD thesis, Chapter 4). In addition, PLLI8,

phylogenetic closely related to PLLI9, was up- regulated during stamen abscission in

88

Arabidopsis (Cai and Lashbrook, 2008). Our study also showed that PLL has elevated
expression in the pedicels after fruit removal. These results indicate that this PLL in
apple is not specifically committed to fruit abscission.

Among up-regulated genes, some share close similarity with plant specific
transcriptional factors, NAC superfamily proteins (Table 3.1), with a consensus sequence
known as the NAC domain (petunia NAM and Arabidopsis ATAF 1 , ATAF2, and CUC2)
(Aida et al., 1997). Members of this gene family are expressed in various tissues and
organs along plant development, and some of them have functions on organ fusion and
2005). Stamen AZ transcriptome profiling also revealed that ANAC021 is up-regulated in
natural abscission of stamens (Cai and Lashbrook, 2008). Our study showed that
increased expression of an apple NAC gene (Md_ZHI I) was also observed in the pedicels
during pedicel abscission induced by fi'uit removal (Figure 3.5). These findings suggest
the potential involvement of apple NAC gene in organ abscission, but not specially
committed to activation of abscission zone separation (Ooka et al., 2003). One
differential expressed ESTs (Md_ZHI 1) shares close similarity with ARABIDOPSIS
NAC DOMAIN CONTAINING PROTEIN 21 (Table 3.1). ANAC021 (originally named
as NACI) is induced by auxin and is a target of microRNA 164 during lateral root
development (Xie et al., 2000, Guo et al., 2005). Stamen AZ transcriptome profiling also
revealed that ANAC021 is up-regulated in natural abscission of stamens (Cai and
Lashbrook, 2008). Our study showed that increased expression of an apple NAC gene
(Md_ZHI 1) was also observed in the pedicels during pedicel abscission induced by fruit
removal (Figure 3.5). These findings suggest the potential involvement of apple NAC

gene in organ abscission, but not specially committed to activation of abscission zone.

89

We observed that two Aux/IAA members showed reduced transcription in fruit
pedicel abscission (Table 3.1). The one, Md_2H05, homologous to IAA4 in Arabidopsis,
also decreased its expression during fruit abscission induced by 6-BA (fruit thinning) and
ethephon (preharvest drop) (Figure 3.6B and 3.7C). Aux/IAAS are encoded by a large
gene family in plants and are early auxin-responsive genes (Liscum and Reed, 2002).
Meir and co-authors showed that two Mj-Aux/IAA gene family members in Mirabilis
jalapa were down-regulated in the A23 during the abscission of petioles induced by
auxin depletion through deblading (Meir et al., 2006). A recent transcriptome profiling
analysis in citrus revealed that several auxin- response factors (ARF) were down-
regulated in abscission of citrus leaves 24 hours afier ethylene application (Agusti et al.,
2008). Consistent with earlier mentioned leaf explant model, down-regulation of auxin
signaling may promote organ abscission (Addicott, 1982).

Conclusion

In this study, we presented a transcriptional profile analysis of pedicel AZ and
identified a small population of differentially expressed genes within the pedicel AZs
during fruit abscission induced by fi'uit removal in 'Golden Delicious'. The differentially
expressed genes identified in this work provide a resource for further functional
characterization of genes associated with fruit abscission. This work also suggests a
potential common molecular mechanism for pedicel abscission induced by fruit removal,

chemical thirmers in early fruit developmental stages, and ethephon in preharvest season.

90

 

 

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94

Table 3.2. Primers used in RT-PCR analysis

1 B 1 O-For

1 B l O_Rev
1 GO4_For
1 GO4_Rev
2C03_For
2C03_Rev
2E09_For
2E09_Rev
2H05_For
2H05_Rev
2H1 1_For
2H 1 1_Rev
MdEF_For
MdEF_Rev

 

5' - GGTACACGAAACTCCAGC - 3'

5' - CCAGAAATCTCACTGGCTTGC - 3'
5'- GGTGGCTAGAAAGTTCACCGG -3'
5'- GCCAGCACTAAACTTGTGCACC -3'
5'- CTCAAAGGGACTTGGTAGC -3'

5'- CAAAGTCAGCATTGAAGGT -3'

5'- GCCATTGGTGGGAGTGCAG -3'

5'- GGCCACAAGCTCGATATGC -3'

5' - GATAAGCATGGATGGAGCTCC - 3'
5' - CCGAGCTGGATGTCTTCATG - 3'

5' - ATTG'I'I‘GGGCATACCGACATC - 3'
5'- GATAGCCGCCAAACCCAGC -3'

5'- AGGACGGACAGACCCGTGAG -3'
5'- GAGGAAGACGGAGGGGCTTG -3'

95

Table 3.3 Amino acid sequences of extensins in apple, almond, Arabidopsis.
Highlighted regions represent peptide repeat units

 

Contig2180 extensin precursor [Malus domestica]
MGKMVPSQVTSLVVTLLVAIVSLTLPSETSANYPYT

SPPPPASPPYHgg SPPPPSPSPPVHYSPPKHPYHEE SPPPPVYAPPKHPYHXE
SPPPPKKPYHXE SPPPPYPTPSKYSPPKHPYHXg SPPPPPKKPYHXE
SPPPPSPTPPKYSPPKHPYHX§ SPPPPPKKPYHXE SPPPPPPPVPTPSPPKKPYHXE
SPPPPPPVZE SPPPPVYSPPPPPKKPYKPPTPPAVHPPHGYIYSSPPPPHHY

 

 

 

 

 

Emle.65718 | extensin precursor [Prunus Dulcis]
MGKMGSSSVTSLVVTLLVAIVSLSLPSETSANYPYS

SPPPPVSPPYHXE SPPPPSPTPPVHYSPPKHPYHXE SPPPPVHYSPPKHPYHXE
SPPPPVYSPPKHPYHXE SPPPPSPSPPKHPYHZE SPPPPSPSPPKHPYHXE
SPPPPSPSPPKHPYHXE SPPPPSPSPPKHPYHXE SPPPPSPPKKPYHXE
SPPPPPSPTPPVYSPPKHPYHXE SPPPPSPPKKPYHXE SPPPPTPVYKPPVY
SPPPPPKKPYKPPTPPVHTAPPHPYIYSSPPPPHHY

 

 

 

 

 

 

 

 

reflNP_849895.1|Extensin 4 [Arabidopsis thalina]
MGAPMASFLVLAFSLAFVSQTTANYFYS

SPPPPVKHYSPPPVZE SPPPPVKHYSPPPVXE SPPPPVKHYSPPPVXE SPPPPVKYYSPPPVXE
SPPPPVXE SPPPPVKHYSPPPVXE SPPPPVKHYSPPPVXE SPPPPVKHYSPPPVXE
SPPPPVKHYSPPPSYTTLHHHRFTTHLLQSYTTLHHHRFTTHLLQLYTTPHHHPRSTTNTNLLLLRT
PLLTSTPTLPLQITTSSSLLDYLIVSNGVDDDTQENKESFGDIIKDTRSLTGELKRIH
MSKEKVSHFNVFSLFINTLL

 

96

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101

CHAPTER 4

Analysis of promoter activity of members of the PE CTA T E LYASE-LIKE (PLL)

gene family in cell separation and wall loosening in Arabidopsis

Abstract

Pectate lyases depolymerize pectins by catalyzing the eliminative cleavage of (l-
>4)-alpha-D-galacturonan. Pectate lyase-like (PLL) genes exist as large and complex
families in plants, but their cellular and organismal roles have not been well characterized.
As a first step to understand the functional diversity of PLL genes in plants, we
documented the spatial and temporal promoter activity of 23 of the 26 Arabidopsis PLL
genes throughout development. Numerous gene family members showed activity in
localized domains programmed for abscission, such as the abscission zones (AZs) of the
sepal, petal, and stamen, and seed, as well as the fruit dehiscence zone. Several other
members showed activity in cell types expected to facilitate separation, including the
endosperm layers during seed germination, and root endodermal and cortical layers
during lateral root emergence. Other PLL promoters were active in domains not
obviously programmed for separation, including the apparent vestigial AZs of the branch
and pedicel. These results suggest potential for unique and overlapping activity of PLL
genes, and provide guidance for analysis of individual gene function through reverse
genetics.

Key words: pectate lyase, GUS expression, cell separation, cell wall loosening,
Arabidopsis

102

Introduction

Pectins, a class of polysaccharides containing predominately 1, 4-linked a-D-
galacturonic acid (GalA) residues, are a major component of primary plant cell walls, and
within the wall form a matrix in which a network of cellulose and hemicellulose is
embedDED (Ridley et al., 2001; Schols and Vorgen, 2002; Willats et al., 2001). Pectins
not only contribute to the mechanical strength and physical properties of primary cell
walls (Jarvis, 1984; O’Neill et al., 2004; Wilson et al., 2000), but also function in
intercellular adhesion (Iwai et al., 2002; Jarvis et al., 2003; Knox, 1992), and can act as
signaling molecules in morphogenesis and pathogen defense (Ridley et al., 2001).

Plant growth and development is accompanied by dynamic remodeling of the cell
wall, which in turn requires modifications of the various cell wall components including
pectin. In accordance with the often complex structure of some pectins, an assortment of
pectinase activities modify or degrade these polymers. Pectinesterases target methyl-
esterified homogalacturonan (HG) yielding substrates for polygalacturonase (PG) and
pectate lyase (PL), which cleave the GalA backbone (Tucker and Seymour, 2002).
Rhamnogalacturonase (RGase) and rhamnogalacturonan lyase depolymerize branched
regions of rhamnogalacturonan (RG), whereas B-galactosidases and a-arabinosidases can
degrade the galactan/arabinan or arabinogalactan side chains (Tucker and Seymour,
2002). Pectate lyases (PLs, EC 4.2.2.2) have been most extensively studied in Erwinia
chrysanthemi, a major causal agent of sofi-rot diseases that affect a wide range of plant
species (Barras et al., 1994; Collmer and Keen, 1986; Kotoujansky, 1987). Their action
not only results in maceration of plant tissues but also can activate plant defense systems

(De Lorenzo et al., 1991; Fagard et al., 2007; Norrnan-Setterblad et al., 2000). Plant

103

PECTAT E LYASE—LIKE (PLL) genes encode proteins with strong amino acid sequence
homology with the PelC isoforrn of bacterial PLs (Marin-Rodriguez, et al., 2002) and
exist as large families in plants where studied. The abundance of PLL genes in plants,
including 26 in Arabidopsis, 12 in rice, and 22 in poplar, has arisen from multiple gene
duplication events (Palusa et al., 2007; Zhang, 2003), a process that may enhance
plasticity in adaptation to chang'ng environments (Lynch and Force, 2000). Theoretical
population genetics models predict that gene redundancy is evolutionarily stable only
when duplicated genes differ in some aspect of their function, suggesting that individual
members of large families such as PLL may have some unique function (Force, et al.,
1999)

Various analyses of PLL genes from plants revealed expression in a broad range
of organs including root, leaf, flower, pollen, filament, style, pistil, and ripening fi'uit
(Benitez-Burraco et al., 2003; Chourasia et al., 2006; Futamura et al., 2002; Marin-
Rodriguez et al., 2002; 2003; Mita et al., 2006; Palusa et al., 2007; Pua et al., 2001). PLL
genes in several studies also showed elevated expression in response to auxin, wounding,
and/or pathogen infection (Domingo et al., 1998; Laskowski et al., 2006; Milioni et al.,
2001; Palusa et al., 2007; Vogel et al., 2002). Transcriptional analysis in Arabidopsis
revealed that a small subset of PLL genes were up-regulated during stamen abscission
and in cortical cell separation during the emergence of the lateral root (Cai and
Lashbrook, 2008; Laskowski et al., 2006; Swarup et al., 2008). Reduced expression of
PL] in transgenic strawberry suggested a natural role in tissue softening during fruit

ripening (Jiménez-Bermfidez et al., 2002). All of these data implicate PLL genes in

104

various plant growth and development events including cell separation and wall
loosening.

During plant growth and develOpment, there are many events in which adjacent
cells separate in a coordinated manner (Roberts et al., 2002). Cell separation resulting in
organ abscission, or anther or fi'uit dehiscence, occurs in predetermined positions, called
abscission zones (A23) and dehiscence zones (DZs), respectively (Laskowski et al., 2006;
Leslie et al., 2007; Ostergaard et al., 2007). Intercellular space formed in leaves and
stems can result from restricted separation of cells at the tricellular regions (Jarvis, 1998).
Fruit ripening also involves limited cell separation, in which only middle larnella is
degraded, with tricellular junctions and plasmodesmata often remaining intact (Hallett et
al., 1992; Roy et al., 1994;). Targeted cell separation is also involved in the processes of
seed germination, lateral root emergence, and shedding of columella root cap cells
(Kuriyarna et al., 2007; Mollet et al., 2007; Wen et al., 2007). In this study, we examined
spatial-temporal expression patterns of promoters of PLL family members in the various
cell separation and wall loosening events that occur in Arabidopsis growth and

development.

Material and Methods
Plant growth conditions

Wild-type Arabidopsis thaliana (ecotype Col-0) was used for all experiments.
Standard growth conditions were l6-h-cool white fluorescent 1i ght/8-h-dark photoperiods
at 22°C. For growth in sterile culture, seeds were surface-sterilized and germinated on
one-half-strength Murashige and Skoog (MS) medium supplemented with lOmM MES

[2-(N-morpholino) ethanesulfonic acid] (Sigma, St. Louis, MO), pH adjusted to 5.7, and

105

solidified with 0.8% phytoagar (Invitrogen, Carlsbad, CA). For auxin treatment, 5-d-old
seedlings growing vertically on the above medium were transferred to medium
containing 1 uM indole-3-acetic acid (IAA) (Sigma, St. Louis, M0) for 24 h.
Phylogenetic analysis and protein domain identification

The peptide sequence of an enzymatically confirmed pectate lyase from banana,
Musa acuminata PLI (GenBank accession AF206319; Marin-Rodriguez, et al., 2003),
was utilized as a query in BLASTP (Basic Local Alignment Search Tool Protein)
analysis of Arabidopsis translated open reading frames [The Arabidopsis Information
Resource (TAIR) Version 7; http://www.arabidopsis.org], and closely related proteins
[Expect (E) value < 8E-3 6] were used for phylogenetic tree construction. Predicted
amino acid sequences were aligned using ClustalW2 (http://www.clustal.org) (Larkin et
a1 2007), and MEGA4 (Tamura et al. 2007) was used to construct the phylogenetic tree.
Annotated PLL protein domains were identified in the Pfam database
(http://pfamsangenacuk; Finn et al., 2008) as follows: PFO321 1, Pectate_lyase; PF00544,
Pec_lyase_C; PF09492, Pec_lyase; PF04431, Pec_lyase_N. Amino-terminal signal
peptides were defined by SignalP 3.0 (http://www.cbs.dtug_k§ervices/Sigrla_lfl) using a
maximum S-score larger than 0.95 and signal peptide probability greater than 0.80
(Emanuelsson et al., 2007). The number of ESTs for each gene in the NCBI Unigene
database was found at NCBI (http://www.ncbi.nlm.nih.g91/).
Transgenic plant construction

To engineer beta-glucuronidase (GUS) as a reporter for PLL promoter activity,
approximately 2 kb of genomic DNA, upstream from the start codon, was amplified by

PCR using gene-specific primers (Table 4.1) containing unique restriction sites, and

106

Table 4.1 Primers used for amplification of PLL promoters

 

At4g24780
At5g63180
Atl g67750
At3g27400
At4g13710
At3g24230
Atl g04680
At4g13210
At3g24670
At5g48900
At3g07010
At3g53 190
At5g09280
At4g22090

At1g30350
AT4g2208O
At2g02720
At3g54920
Atl gl4420
At5g15110
At3g01270
At3g55140
At3g09540

 

5' -AACCATGG'ITCTCT CT CT CT CT CACTIGG- 3'
5' -ATAAGC'ITCCACCTATCATTACCAACACC- 3'
5' -TTCCATGGATGAGTGAAGAGAGA AAGACA-3'
5' CGAAGCTTTAACACTGATAAATGAGTAGA— 3'
5' -AACCATGGTCTIGTCTCTCGAGAGGATT- 3'

5' -CGAAGCTITATCAATGTGTATCATGAAT— 3'

5' -ACCATGGTC'ITCTAAACATAGATTGAGA- 3'

5' -CGGAATICGCAAATGGCACTATAAACCAC- 3'
5' -AACCATGGTGAAGCT'ITCTICTTCTTC T- 3'

5' -AAGAATTCTGCACAAGAGACATAAAAGT— 3'
5' -AACCATGGTGTGCAAACAAAGGGAAAATG- 3'
5' -ATGAATTCACGAAAAATATAGCGTGACGG- 3'

5' -AACCATGGTGGAGAGGCAGAAGCTGAGCC- 3'
5' -AAGGTACCCACTTCAAGTCTTCGAAAGTA— 3'

5' -AACCATGGTG'ITGGTIGTI‘GTTAGAG'IT- 3'
5' -'ITGGTACCGCCGAAACAATAACCTC'ITI‘- 3'

5' -AACCATGGTG'ITGGATATATCAAAGCTCT- 3'
5' -AAGGTACCCAGGGTGCTTGTAAA'ITATGT- 3'

5' -AACCAT GGTG'ITCTT GCT CT GTTCT GTT- 3'

5' -AAGGTACCAAATCATGI I I ICCCGCCAA- 3'
5' -AACCATGGTGTGACAGCCATTG'I'IATGGC-3'
5' -AAGGTACCCTCGTAAG'ITCC'ITACCTATG3'

5' -AACCATGGTGCTGAAGAAAC'ITGTGATT- 3'
5' -GAAAGCTTAATCAGTAACTTTATI‘GACA- 3'

5' -AACCATGGI I I ICCGGCAAATCCGACTGA— 3'
5' -AAGGTACCAGTITA"I‘TCAGGTCATGTGT- 3'
5’- AACCATGGTAGTAATGTTGCA'ITIACIT—3 ’

5 ’- AAGGTACCATGAGGCAGCTGCCACCCTT-3 ’

5' -CGCCATGGTITCTI'GAAAATGTGATGCT- 3'
5' -AAGGATCCCCAAAGCCI I I IGCTGATA- 3'

5' -AACCATGGGG'I'TGGGTGGG'ITTATGGTT— 3'
5'-ATAAGCTTIATTCAATACTC'ITITCACG- 3'

5' -AACCATGG'ITGAATCAGCAGTGGTGAGA- 3'
5' -ATAAGCTTGATTGCGATCATCAAAAG'IT— 3'

5' -AACCATGGCGTTAGTGGCGGAI I 1'] GAC- 3'
5' -ACAAGCTITCAGAAGATACCACAATCGC- 3'

5' -TACCATGGI I IAAATATATTGCAAATGC- 3'
5' -ATAAGC'ITITAA'ITAC'ITGTATGATAAT- 3'

5' -GCCCATGGCTI‘TCI I I I'I I IGI I I ICAA- 3'
5' -CAGGATCCAAGATATATATAATAGTCTC -3'

5' -AACCATGGTT'I‘ATITGA'ITACCCCTITC- 3'
5' -TI'GGATCCTGTIGC'ITATCTGAGAAAGT- 3'

5' -AACCATGGCGTTCG'ITGTTATGCGACGT— 3'
5' -ACAAGCTITCTTGTACATACAGCAGAGA- 3'

5' GGCCATGGATCTAATT’I‘ATATAACAAATG- 3'
5' -ATAAGCTTCCATG'ITTAACATAC'ITC’IT— 3'

 

107

confirmed as lacking mutation by sequencing. These PCR products were cloned into the
pCAMBIA1305 vector (Cambia, Black Mountain, Australia) modified to contain an NcoI
site at the start codon of the GUS gene and multiple cloning sites replacing the
pCAMBIA Lac Z alpha and CaMV 3 5S regions. This resulted in PLLzGUS fusions that
preserve the authentic 5’ UTR and start codon position. Plasmid DNAs were introduced
into Agrobacterium tumefaciens strain GV3101 and transformed into wild-type plants
using the floral dipping method (Clough and Bent, 1998). Transgenic plants were
subjected to selection with the herbicide glufosinate (Basta; Beyer CropScience, Barmen,
Germany).
Histochemical GUS assay

Populations derived from at least four independently transformed transgenic lines
were analyzed for each PLLzGUS construction. Analysis utilized T2 plants from
transgenic lines segregating approximately 3:1 for herbicide resistance. For
histochemical GUS assays, plants or plant parts at various developmental stages were
immersed in GUS staining solution [0.5 mM X-gluc, 0.5% (v/v) Triton X-100, 50 mM
sodium phosphate buffer (pH adjusted to 7.2)] under vacuum infiltration for 5 min, and
incubated at 37°C for various lengths of time. After staining, tissues were incubated in
70% ethanol for several hours to remove chlorophyll. Staining was visualized using a
Nikon dissecting microscope equipped with a digital camera. Reproductive organs used
in this analysis were at stages 11 to 20 as described by Smyth et a1 (1990).
Expression analysis of AtGenExpress data

Microarray data were collected from the AtGenExpress Development data set

(Schmid et al., 2005; http://www.weigelworldorg/resources). Only results from wild-

108

type plants were included in the analysis. The logz-transforrned absolute signal values
were used in hierarchical clustering based on average linkage (Cluster version 3; Eisen et

al., 1998), and results were visualized in TreeView (Eisen et al., 1998).

Results
Phylogenetic Analysis

Based on peptide sequence homology with a known pectate lyase from banana
and annotated protein domains, the Arabidopsis genome encodes for 26 pectate-lyase-like
proteins. Neighbor-joining analysis partitioned these protein sequences into five
subfamilies (Figure 4.1). This genomic content and phylogenetic organization is
consistent with that previously reported for PLLs from multiple plant species including
Arabidopsis (Futamura et al., 2002; Palusa et al., 2007). All Arabidopsis PLLs exhibited
a recognizable Pec_lyase_C (Pfarn00544) domain (Y oder et al., 1993; Figure 4.1) and 23
of the proteins contain a probable amino-terminal signal peptide. The carboxyl-terminal
glycosyl-phosphatidylinositol (GPI) anchor previously identified in PMR6 (POWDERY
MILDEW RESISTANCE 6, also called PLL13) was not obviously present in other PLLs,
suggesting a specialized function of PMR6 associated with disease resistance (V ogel et a1,
2002) (Figure 4.1).
Estimation of PLL gene expression through analysis of public transcriptome data

As a first step to assess expression pattern of PLL genes in Arabidopsis, we
analyzed publicly available transcriptome data. Expressed sequence tags (ESTs) found in
public databanks corresponding to individual PLL gene family members were found to be

sourced from various tissues and stages across Arabidopsis growth and development, as

109

 

 

 

 

 

At4gl3210(PLL23) [2* ' 2
At3g24670(PLL22) mag-g 12
At5g48900(PLL21) 51
At3g07010(PLL20) 21%-E 89
Atlg04680(PLL26) g-E- 147

At3g24230(PLL24) 13* 0
At4g13710(PLL25) m-a—s—J I 10

At3g27400(PLL18) :E-g 4
At1g67750(PLLl6) mgr-E 63
At4g24780(PLL19) W 71
At5g63180(PLL15) 3+ 60

At5g04310(PLL12) W 9
At3g53l90(PLL17) 121+ 46
At3g54920(PLL13) 13%}— 61
At5g55720(PLL14) a§-== - 19

82 100 At2g02720(PLL9) 3* 5
100 Atlgl4420(PLL8) nags-s— H 16

At5g15110(PLLll) Eng-E- 2
At3g02170(PLL10) mega-E 34

61 At1g30350(PLL7) tug-=- 12

9 Atlgll920(PLL6) W 111

100 At4g22090(PLL5) W 0

At4g22080 (PLL4) W 4

At5g09280(PLL3) 3.: ]1v 7
At3g55140(PLL2) g-E :lv 23

At3g09540(PLL1) + 6

0.5 0.4 0.3 0.2 0.1 0.0 100aa

Figure 4.1. Phylogenetic tree of Arabidopsis PLL anrino acid sequences. Bootstrap
values are shown above nodes and indicate how consistently the data support the given
taxon bipartition. EST No. gives the numbers of ESTs for that gene currently found in
the NCBI database. The Pec_lyase_C domain is indicated as long green rectangles. The
amino-terminal signal peptide is represented by short yellow rectangles. The
Pec_lyase_N domain is indicated as blue rectangles. The GPI anchor in PMR6 is
indicated by a red rectangle. The scale bar at lower left shows the relative distance
between tree branches. “Images in this thesis/dissertation are printed in color”

110

well as various enviromnental conditions (not shown). Representational frequency of
ESTs for individual PLL genes was highly variable both across the gene family and
within subfamilies (Figure 4.1). Analysis of publicly available microarray data also
suggested that there was at least some unique developmental pattern of expression for
most PLL genes (Figure 4.2). A striking exception is the four members of subfamily II
(PLL8-PLLI I), which showed a similar expression pattern localized predominately to
pollen and stamens (Figure 4.2). Several genes (PLL3, 4, 6, 7, 12, and 24) were
transcriptionally silenced across most sampled tissues, whereas PLLZ was apparently
expressed ubiquitously, suggesting a very general function (Figure 4.2). These data are
generally consistent with the results of recent RT-PCR analysis of selected PLL genes
(Palusa et al., 2007) and support a collectively ubiquitous function for PLLs in growth
and development and the potential for functional specialization by many of these genes.
Analysis of PLL promoter activity

Approaches for analysis of gene expression can be difficult when applied to large
gene families, due to sequence homology among family members and/or inability to
resolve expression at the cellular level. Here, we utilized a histological reporter gene
(GUS) driven by individual PLL gene promoters. We engineered the GUS coding
sequence adjacent to ~2 kb of 5' UTR/promoter sequence, preserving the authentic start
codon of the PLL genes, and expressed the PLL:GUS fusions in transgenic Arabidopsis.
For each PLL gene, we analyzed at least four independent transgenic lines. GUS activity
patterns were generally consistent between independent lines, with the exception of three
genes (PLL6, PLLIZ, and PLL14) that showed weak and variable activity and were not

analyzed further. No GUS activity was observed in transgenic plants transformed with a

111

 

 

 

 

 

P--I
P-- 0
PL... I
P--
P--
P-..'
P-- 2
P-..
P--‘
P-J
P..- 4
P..-.0
P..-l
P-- 3
P-- 7
P-- 9
P..-.6
P-- 6
P--.3
P-- 4
P--.5
P-.. 2
P-- 5
P--
9 $5 ““8
0 l 10

Figure 4.2. Hierarchical clustering of RNA accumulation for 25 PLLs along
developmental stages based on public microarray data (AtGenExpress; Schmid et
al., 2005). Growth stages and plant parts were labeled according to the AtGenExpress.
Microarray signal value is indicated by color from yellow (lowest) to red (highest). PLL5
was not represented on the microarray used in this analysis. “Images in this
thesis/dissertation are printed in color”

112

promoterless GUS construction, or in non-transgenic plants. Based on the known roles
for pectins in cell adhesion and cell wall architecture, we focused our analysis on
developmental events associated with cell separation and cell wall remodeling.

PLL:GUS expression associated with cell separation

Floral orgmabscission zones.

Cell separation in Arabidopsis has been best characterized in the context of floral
organ abscission (Leslie et al., 2007). Arabidopsis exhibits abscission of sepals, petals,
and stamens following pollination. Abscission is conditioned by cell separation within
the AZ, a tightly localized region at the base of the floral organs (Patterson, 2001). We
analyzed developing flowers at various stages until Stage 18, when siliques began to
yellow (Smyth, 1990) (Figure 4.3). GUS activity within the AZs was observed for 18
PLL genes (Figure 4.3 and Table 4.2). For all of these genes, GUS expression was first
detected in the A23 of sepals, or petals, or stamens at Stage 16, which followed anthesis
by about two days and was marked by the withering of sepals and petals (Smyth et al.,
1990) (Figure 4.3). At Stage 17, which was marked by the abscission of sepals, petals
and stamens, stronger GUS activity was detected within the A23 of all three organ types.
Five PLL genes (PLL4, 5, 7, 20, and 23) showed markedly weaker GUS staining relative
to the remaining 13 genes (Figure 4.3 and not shown). For all genes, GUS expression in
the A23 was detectable but very weak at Stage 18 (not shown).

Fruit dehiscence zone and seed abscission zone
The Arabidopsis fi'uit consists of two valves separated by a replum. Mature fruits

dehisce due to cell separation in the so-called separation layer of cells that is distributed

113

 

Figure 4.3. Spatial and temporal PLL:GUS expression in the abscission zones of
sepal, petal, and stamen. Picture labels are given number of genes indicated in Figure
4.]. Each frame shows flowers at Stage 16 (left) and 17 (right). Arrowheads indicate
location of abscission zone of sepals, petals, and stamens. “Images in this
thesis/dissertation are printed in color”

114

along the valve margins (dehiscence zone; DZ) (Liljegren et al., 2004). Mature seeds are
released fi'om the funiculus, a stalk-like structure connecting seeds to the replum, at a site
referred to as the seed AZ (Pinyopich et al., 2003). We analyzed GUS activity during
development of flowers and fruit from Stage 16 through Stage 19, marked by valve
separation, and Stage 20, marked by seed abscission of seeds (Smyth, 1990) (Figure 4.4).
We found that 16 of the PLL promoters drove GUS expression within the apparent DZ of
developing siliques at the onset of stage 18, which proceeded separation of the valves by
approximately 24 h (Figure 4.4A and Table 4.2). GUS activity was first seen at the basal
and apical ends of siliques, where valve separation was initiated, and then became
established along the entire length of the fruit as valve separation progressed (Figure
4.4A and not shown). We also found that 16 PLL promoters drove GUS activity within
the apparent seed abscission zone (Figure 4.4B and Table 4.2). Activity was not seen
until Stage 20, concomitant with seed abscission.
Cell separation events associated with radicle emegence and lateral root initiation

During seed germination in Arabidopsis, the radicle penetrates a single
endosperm cell layer (Liu et al., 2005). GUS activity was observed in the endosperm cell
layer of l-d-old seedlings for PLLI6:GUS and PLL22:GUS transgenic plants (Figure 4.5
‘PLL22a, PLLI6a’). The stronger GUS activity was observed in the endosperm layers of
PLL22:GUS transgenic plants (Figure 4.5).

Arabidopsis lateral roots initiate from the pericycle cell layer and need to
penetrate the overlaying cortical and epidermal layers during emergence, a process that

requires separation of these cells (Laskowski et al., 1995). We analyzed GUS activity for

115

 

41'

  

.3... <

1N1~a\ I!

<-

1.! lulu.

 

%3.1

   

 

PLLs.-GUS expression in the dehiscence zone of siliques (A) and abscission

zone of mature seeds (B). Picture labels are given number of genes indicated in Figure

4.4.

Figure

Arrowheads indicate location of dehiscence zone of siliques and abscission zone of

seeds. “Images in this thesis/dissertation are printed in color”

4.].

116

all 23 PLL promoters in roots of seedlings growing on media supplemented with auxin,
which promotes lateral root initiation (Casimiro et al., 2003). Under these conditions, we
observed GUS activity for six PLL promoters in the apparent cortical and endodermal
layers of primary roots (Figure 4.5, Table 4.2). Activity for PLLI 6 and PLLZI was
restricted to cell layers directly overlaying new lateral root primordia, whereas activity
for the remaining four, PLLI9, 22, 23, and 26 was distributed evenly along the length of
root, including the regions of initiation (Figure 4.5).
Vestigial abscission zone of pedicelsand inflorescence brmcheLmd base of trichomes

Unlike many plants, Arabidopsis does not shed leaves, branches, or entire flowers
or fruit. Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) led
to abscission of inflorescence branches and pedicels, which suggested that ArabidOpsis
presumably has dormant and vestigial AZs at the base of these organs (Stenvik et al.,
2006). GUS activity was detected at the base of pedicels for two transgenic lines (Figure
4.5). Intensity of GUS staining at the base of pedicels from flowers at stage 17 (Figure
4.5, PLL15c, PLL24c) was stronger than that of flowers at stage 15 (Figure 4.5, PLLI5b,
PLL24b). GUS activity was also observed at the base of trichomes from rosette and
cauline leaves for four PLL genes (Figure 4.5, Table 4.2)
PLL:GUS expression associated with cell wall loosening events

Various analyses of PLLs in plants have shown that they were expressed in a
broad range of tissues (Marin-Rodriguez et al., 2002; Palusa et al., 2007). We further
carried out analysis of GUS expression driven under PLL promoters with seedlings at

different developmental stages and flowers from stage 11 (stigrnatic papillae appear) to

117

 
        

' 17.1.2411

Figure 4.5. PLL:GUS expression in the tissues involved in various cell separation
processes. Picture labels are as indicated for Figure 4.1. For transgenic line with more
than one picture being displayed, lower case letters were used to indicate different organs
at different developmental stages. Top row: PLL:GUS expression at the endoderrnis and
cortical cell layers during lateral root emergence. Middle row: PLL:GUS expression at
the base of trichome. Arrowheads indicate location of base of trichomes. Bottom row:
PLL22a and PLL16a are GUS expression in the endosperm cell layers during seed
germination. Arrows indicate location of endosperm cell layer. PLLI5b and PLL24b are
GUS expression at the base of pedicels of young flowers; PLLI5c and PLL24c are GUS
expression at the base of mature siliques. “Images in this thesis/dissertation are
printed in color”

118

stage 15 (stigma extends above long anthers) (Smyth etal., 1990).
Seedlings

GUS activity was detected in the l-d-old seedlings for 22 PLL transgenic lines
(Figure 4.6, top two rows). GUS staining within seedlings was restricted to hypocotyls,
cotyledons, root-hypococtyl junctions, roots, and root tips (Figure 4.6, Table 4.2). GUS
activity in the various regions of roots was detected within 12 transgenic lines (Figure 4.6,
Table 4.2), and PLLI3:GUS, PLL] 7:GUS, and PLL22:GUS additionally showed
expression in the root tips (Figure 4.6, Table 4.2). PLL25::GUS was uniquely
distinguished by a specific localization within the root tip (Figure 4.6, ‘PLL25’).

With the seedling differentiation, strong GUS activity was detected in the roots in
the 5-d-old seedlings for 12 PLL transgenic lines (Figure 4.6, bottom row). GUS
expression in various regions of roots in 5-d-old seedlings was similar as that in 1-d-old
seedlings. Robust expression of PLL13:GUS, PLL] 7:GUS, and PLL22:G US was limited
to the root apex region, including PLL22:GUS in the columella root cap (Figure 4.6).
PLL25:GUS was specifically expressed in the root apical meristems and root
differentiation zones (Figure 4.6 ‘PLL25a’).

Hydathodes

Hydathodes are highly specialized structures evolutionarily related to stomata,
and they are permanently Open pores to release water and solutes from xylem (Esau,
1977). Hydathodes in Arabidopsis are positioned at the leaf margins, and close to the
ending of xylem vessels. Evident GUS expression was observed in fully-open cotyledons

and rosette leaves for seven PLL transgenic lines (Figure 4.7A, Table 4.2). GUS activity

119

 

Figure 4.6 PLL:GUS expression in the seedlings. Picture labels are as in Figure 4.5.
Top two rows: GUS expression in the l-d-old seedlings; bottom row: GUS expression in
the root at 5-d-old seedlings. Arrows in the bottom row indicate the enlarged picture of
root apical meristem region. “Images in this thesis/dissertation are printed in color”

120

was only restricted to the hydathodes within cotyledons and rosette leaves (Figure 4.7).
sum

In Arabidopsis, stipules are present on the newly formed leaves and degenerate
with leaf expansion. GUS activity within shoot apex was observed for 19 PLL transgenic
lines (Figure 4.7B). Within the shoot apex, we observed strong GUS activity only in the
stipules of primordia and newly formed leaves (Figure 4.7B, Table 4.2). GUS expression
was evident in the first pair of true leaves in 6-d-old seedlings and in successive leaves
including cauline leaves in the older seedlings (Figure 4.7B, example PLLI9, PLL19a,
PLLI9b, PLL19c, PLLI9d). For all leaves, expression was the strongest in newly formed
leaves and decreased with leaf expansion concomitant with degeneration of stipules
(Figure 4.7B).
mm

GUS activity was detected in developing flowers at stage 15 for 23 PLL
transgenic lines (Figure 4.8A). GUS expression within flowers was restricted to stigma,
style, junction of anther and stamen filament, and stamen filament, pollen, and
developing seeds at stages fi'om 11 to 15 (Figure 4.8 A and B, Table 4.2). GUS activity
in styles was observed for 14 PLL:GUS transgenic lines, whereas GUS activity in stigma
was detected in other eight transgenic lines (Figure 4.8, Table 4.2). We observed evident
GUS staining in mature pollen for 13 PLL transgenic lines and at junction of stamen and
filament for 18 transgenic lines (Figure 4.8A, Table 4.2). We also observed that
PLLI:GUS was only expressed in the styles, and PLL10:GUS was only detected in the
mature pollen (Figure 4.8A). PLLI3:GUS was distinguished by its expression in the

developing seeds at stage 11 to 15 (Figure 4.8 A and B).

121

A
k \f
I’ll I; I'll.” I’H I'lrr'l‘ I‘ll/8‘ /-//:2. 171:.
' . r ‘ I " - . .

   

I‘/.l. I511
l'/.l. I8”
I l I 2211
l'l.l._’4u

 

Figure 4.7. PLL:GUS expression in the hydathodes (A) and stipules within shoot
apex (B). Picture labels were described as in the Figure 4.5. A. Top_ row: GUS
expression in the hydathodes of cotyledons; bottom row: GUS expression in the
hydathodes of rosette leaves. Arrowheads indicate the location of hydathodes (by). B.
Top two rows: GUS expression in the stipules of 6-d-old seedlings. Bottom row PLL19
is used as an example to show expression in the stipules at different stages of leaf
development. PLLI9a: stipules visible at the base of new formed leaf; PLLI9: enlarged
image of PLL19a within shoot apex; PLLI9b: weak stipule expression at the base of
expanding leaf; PLL19c: stipules at the base of rosette leaves at different stages of
development, PLL19d, single stipule detected at the base of expanded cauline leaves.
Arrowheads indicate the location of stipules. “Images in this thesis/dissertation are
printed in color”

 

 

Figure 4.8. PLL:GUS expression in the different parts of flower at stage 15 (A) and
stage (ll-14) (B). Picture labels were described as in the Figure 4.5. PLLIO as an
example showing expression in the pollen (enlarged picture), PLL25 as an example
showing expression in the junction between anther and filament (enlarged picture).
Arrowheads indicate stigma, style, pollen, junction between anther and filament, seeds,
and filament. “Images in this thesis/dissertation are printed in color”

123

Table 4.2. Summary result of Figure 4.3 to 4.8. “Images in this thesis/dissertation
are printed in color”

 

 

o—m-nxorxoooxo—va-nxo
u ~vambwm—v‘ —————— NNNNNNN
s 35533333333535333333333
Tissue o mmmmmmmmmmmmmmma—mmmmmmm
hypocotyl - I
hydathodes .- I I
R/Hjunctions -
"’0'. I III-II- I
roottlp -

I
seed coat I
Lateral root
stipules I — -
base of trichome
base of pedicel
base of branch
stigma
style
pollen
filament
An/F i junction
floral organs AZ
seeds AZ
silique DZ
developing seeds

 

 

124

Discussion
PLLs are abundantly and ubiquitously expressed throughout plant development

Pectate lyase is the primary virulence agent in the soft rot disease caused by
Erwinia sp. and has been extensively studied for its activity and function (Collmer and
Keen, 1986; Barras et al., 1994). Whole genome sequencing of plant species has
revealed that PLL5 are encoded by a large gene family, such as 26 in Arabidopsis, 12 in
rice, 22 in poplar (Palusa et al., 2007). Publicly available microarray data reveals that
PLL5 are expressed in various tissues and organs along plant development (Schmidt et al.,
2005; Ma et al., 2005; Zimmermann et al., 2004). Our results also show that the
promoter activity of PLL gene family members in Arabidopsis is observed during various
developmental events associated with cell separation and cell wall loosening (Figures
4.3-4.8).
PLL:GUS expression associated with cell separation

Cai and Lashbrook (2008) determined that the two PLL genes, PLLI8 and PLL23,
are developmentally regulated in the stamen abscission zone. Both PLL18 and PLL23
belong to the class of genes that were up-regulated between Stage 13 (anthesis) until at
least the end of Stage 15. The fact that the remaining PLL genes identified as abscission-
associated in our study were not identified by Cai and Lashbrook could be due to the
potential greater sensitivity of our approach or to the possibility that posttranscriptional
mechanisms play a predominant role in the regulation of their expression. The highly
overlapping promoter activity patterns within floral abscission zones suggest that these

genes could act in a highly redundant manner.

125

Overlapping GUS expression patterns observed with in the A23 of floral organs,
and seeds, and the DZs of siliques among multiple gene members suggest that the
function specialization related to cell separation presumably results from co-expression of
many PLL genes. Three closely related polygalacturonases, QUATER2 (QRT2),
ARABIDOPSIS DEHISCENCE ZONE POLGACTURONASEI (ADPGl) and ADPG2,
were partially redundantly expressed in the MS of floral organs and seeds, the DZs of
siliques and anthers (Ogawa et al., 2009). In addition, these members also exhibited
expression in other cell separation events, such as lateral root initiation and radicle
emergence. These results imply that the possibility of PLL5 participate in other cell
separation events.

Detection of GUS activity in the endosperm layer during radicle emergence for
two PLLs suggests that they are actively transcribed during the seed germination process.
Other cell wall modifying enzymes, such as PG, are reported to be expressed in the
endosperm region adjacent to the emerging radicles (Gonzalez-Carranza et al., 2007).
These results implied that these cell wall modifying enzymes presumably contribute to
the targeted cell separation occurring within the endosperm layer during radicle
emergence.

Increased transcriptional accumulation of PLL16 and PLL26 has been observed
during lateral root emergence induced by auxin (Laskowski et al., 2006, Swarup et al.,
2008). These results support that these six PLL5 were transcriptionally expressed in the
cortical and endodermal layers overlaying new lateral root primordia during lateral root
emergence induced by auxin, implying their contribution to this targeted cell separation

event.

126

Overexpression of IDA in Arabidopsis led to the abscission of pedicels and
inflorescence branches, suggesting the potential presence of these dormant A23 and the
capability of responding to some specific signals (Stenvik et al., 2006). Interestingly,
transgenic lines showing expression at the base (vestigial AZs) of pedicels and
inflorescence branches were not overlapping with those showing expression in the AZs of
floral organs (Table 4.2). These results support the hypothesis that there is evolutionary
function specialization regarding organ abscission among different members of PLL gene
family.

We observed that several PLL promoters drove GUS expression at the base of
trichomes (Figure 4.5). Other cell wall modifying enzymes, such as PGs and expansins,
have also shown expression at the base of trichome through a GUS reporter approach
(Gonzalez-Carranza et al., 2007; Cho and Cosgrove 2000). One possible explanation for
expression of these genes at the base of trichomes is that they may contribute to the cell
wall remodeling during initiation and emergence of trichomes from the leaf epidermis.
PLL:GUS expression associated with cell wall loosening events

We observed almost half of gene family members drove evident GUS expression
in various regions of roots (Figure 4.6). This result is largely supported by publicly
available microarray data and a comprehensive RT-PCR analysis which also show
abundant transcriptional expression in roots these genes (Palusa et al., 2007; Schmidt et
al., 2005). The majority of members with expression in roots also showed a robust level
of expression in the root elongation zones, additionally, three genes showed expression in
the root tips (Figure 4.6). PLL25:GUS was distinguished by its expression in root tips

and the root differentiation zone (Figure 4.6). These results suggest that PLL genes are

127

differentially expressed in various regions of roots and may contribute to cell wall
loosening events occurring in root elongation, differentiation, and radicle emergence.

We detected evident GUS expression in the hydathodes among several PLL:G US
transgenic lines (Figure 4.7A). Genes associated with cell separation, for example, IDA-
like4 (IDL4) showed expression in the stomata (Stenvik et al., 2008). The structural
features of hydathodes suggest their involvement in cell wall loosening or targeted cell
separation during formation of hydathodes (Roberts et al., 2002).

GUS activity patterns observed in the stipules suggested that these PLL5 were
temporally and spatially expressed in the stipules in a developmentally-dependent manner.
Gene members that drove GUS expression in the stipules are largely consistent with
members expressed in the shoot apex from the public microarray data analysis (Figure
4.7B and Figure 4.2). The function of stipules in Arabidopsis development remains
largely unknown. It has been shown that stipules are primary sites for high accumulation
of free-auxin, which is associated with vascular differentiation and leaf morphogenesis in
seedlings (Aloni et al., 2003; Cheng et al., 2007, Barkoulas et al., 2008). This
information implies that these PLLs may be auxin responsive and presumably contribute
to the degradation of stipules along leaf development in Arabidopsis.

Gene members that drove GUS expression in pollens are largely agreed with
those in transcriptiome analysis (Palusa et al., 2007). The first PLL gene reported in
higher plants is from pollen based on sequence homology with PelC in bacteria (Wing et
al., 1990). Multiple PLL: have been shown to be expressed in pollen in a wide range of
plants (Marin-Rodriguez et al., 2002). These data imply that PLLs presumably contribute

to the initial loosening of pollen cell wall to facilitate pollen tube emergence. Some

128

studies also showed that PLLs from tomato and tobacco were expressed in the styles and
pistils (Budelier et al., 1990; Wu et al., 1996). In our study, the majority of PLL gene
family members in Arabidopsis drove GUS expression either in stigma or styles (Table
4.2), suggesting that PLLs may participate in the degradation of stigmatic papillae or
softening of stylar tissue to facilitate pollen tube growth.
PLL:GUS expression in an overlapping and specializing manner

It is not known whether phylogenetically closely related paralogs in a large gene
family share similar expression patterns. Four phylogenetic closely-related paralogs in
subfamily II, PLL8-II, drove GUS expression in mature pollen (Figure 4.8, Table 4.2),
and transcriptome analysis showed that they are highly expressed in the stamens and
mature pollen (Figure 4.2). In addition, GUS expression patterns driven by these four
PLL promoters in other tissues were divergent (Table 4.2). We also observed that the
PLL5 used in this study drove GUS expression in a partially redundant and distinct
manner in various tissues and organs (Table 4.2). These results imply that each PLL gene
member has partially overlapping and specialized biological function during plant
development.
Implications

This analysis is the first report of comprehensive GUS expression analysis driven
by PLL promoters, and it is also the first step in determining the functions of PLLs in
plant development. Comprehensive expression analysis is useful for providing guidance
to reverse genetic screening in order to identify the phenotypic abnormality of gene-
specific mutants. For example, a loss-of-function mutant of PLL25 may have defects in

root differentiation, while mutants of PLLI3 may reveal the abnormality in seed

129

development. The overlapping expression observed in multiple PLLs will facilitate the

silencing of these genes simultaneously.

130

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CONCLUSION

Abscission or retention of ripening fi'uit is a major strategy of seed dispersal and also has
important implications for horticultural production. Abscission-related traits have generally not
been targeted in breeding efforts, and their genetic bases remain mostly unknown. As a first step
to elucidate the genetic bases of abscission related traits, we documented diversity in fi'uit-
abscission-related traits among Malus accessions representing the breadth of genetic diversity
seen in Malus. Our findings suggest that important mechanism(s) independent of hit ethylene
production act as determinants of natural abscission. Accessions showing phenotypic extremes
in abscission-related traits can be developed as contrasting models to understand the biological
bases of these traits, and as tools in genetic analyses for mapping genes that influence these traits.

In fruit crops, precise regulation of fruit abscission is crucial to achieve maximum yield
and optimized market value. We presented a transcriptional profile analysis and identified a
small population of differentially expressed genes within the pedicel AZs during pedicel
abscission induced by fruit removal in apple cv. 'Golden Delicious'. These differentially
expressed genes identified in this work provide a valuable resource for further firnctional
characterization of genes associate with fiuit abscission. This work also suggested a potential
common molecular mechanism on pedicel abscission induced by fruit removal, chemical
thinners in early fruit developmental stages, and ethephon in preharvest season.

As a first step to understand the fimctional diversity of PLL genes in plants, we
documented the spatial and temporal promoter activity of 23 of the 26 Arabidopsis PLL genes
throughout development. Our results suggest potential for unique and overlapping activity of
PLL genes, and provide guidance for analysis of individual gene function through reverse

genetics.

137

   

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