IDENTIFICATION OF A BAHD ACYLTRANSFERASE INVOLVED IN THE
ACETYLATION OF MONOLIGNOLS IN KENAF (Hibiscus cannabinus)
By
Sasha Annabel Peers

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Plant Biology
2012

ABSTRACT
IDENTIFICATION OF A BAHD ACYLTRANSFERASE INVOLVED IN THE
ACETYLATION OF MONOLIGNOLS IN KENAF (Hibiscus cannabinus)
By
Sasha Annabel Peers

Plant cell walls are the most abundant renewable resource on Earth and have been proposed as a
major feedstock for biofuel production. Lignin is a component of cell walls that provides support
and structural integrity to vascular tissues. These properties are also responsible for lignin being
the major obstacle for the processing of plant biomass to biofuels. Lignin structure can be altered
by acylation of lignin monomer units, namely, p-coumaryl alcohol, coniferyl alcohol, and sinapyl
alcohol. The significance of this phenomenon has not been established primarily because the
enzyme(s) responsible for the acylation of monolignols have not been identified. This study
sought to identify the enzyme(s) responsible for acetylation in phloem fiber lignin in kenaf
(Hibiscus cannabinus). Kenaf is a non-woody plant whose lignin has a high syringyl content and
extensive acetylation of syringyl units. This study reports on the identification of an enzyme
(AMT; acetyl monolignol transferase) that catalyzes the synthesis of sinapyl acetate using a
synthetic gene for a BAHD acyltransferase present in kenaf. AMT had a high activity with
sinapyl alcohol and very little activity with coniferyl alcohol, which is the expected selectivity
given the acetylation found in kenaf lignin. AMT was transformed into arabidopsis and seedlings
are being screened for AMT expression. The significance of lignin acylation is an intriguing
unanswered question in plant cell wall biology. Elucidating the enzymes involved in monolignol
acylation will provide a basis for uncovering their functional role in lignin structure and whether
changes to acylation levels in plants will prompt facile processing of biomass into biofuels.

	
  

Copyright by
SASHA ANNABEL PEERS
2012

	
  

This thesis is dedicated to my beloved family and friends.
I am forever grateful for your love and support.

	
  
iv	
  

ACKNOWLEDGMENTS

The author would like to thank the following people: Dr. Curtis Wilkerson for his guidance,
understanding, and financial support, Drs. Federica Brandizzi, Brad Day, and Amy Iezzoni for
their advice and understanding, Doug Whitten and Saunia Withers for their assistance on
experimental techniques, the Wilkerson lab for their positive energy and good humor, the Iezzoni
lab (and individuals previously listed above) for volunteering their time and effort during the
cherry pollination experiments, Dr. John Ralph and his lab members for providing the
chemically synthesized substrates used in the HPLC experiments, and lastly, the MSU Plant
Biology Department and the Great Lakes Bioenergy Research Center for their financial support.

	
  
v	
  

PREFACE

This thesis is presented in two chapters because it describes two areas of plant biotechnology –
fuel and food. First, “Identification of a BAHD Acyltransferase Involved in the Acetylation of
Monolignols in Kenaf (Hibiscus cannabinus)” was presented as the central project due to the
complete story it gave from start to finish. In short, lignin is a major obstacle in the conversion of
cellulose to fermentable sugars for ethanol production. In Chapter One, I present an enzyme
(acetyl monolignol transferase; AMT) that is involved in monolignol acylation in kenaf, a
modification that modification may improve sugar yields from bioenergy crops. Thus, the
purpose of this study was to identify and biochemically characterize AMT and overexpress AMT
in arabidopsis for an analysis of lignin structure and cell wall digestibility. Result from this study
will elucidate on the implications of altering lignin acylation in plants and reveal whether lignin
acylation can be utilized for facile processing of biomass into biofuels. Chapter Two, titled
“Characterization of the Sweet Cherry Pollination System”, was my initial project when I began
graduate school that involved an analysis of gametophytic self-incompatibility (GSI) in sweet
cherry in order to understand the genetic and molecular basis of this phenomenon. However, I
strongly pursued the AMT project because the pollination experiments were not successful for
the first two years and factors such as unexpected weather conditions and harvest frequency
made my time and resources very limited. Nevertheless, I was able to successfully perform the
laboratory pollinations, which resulted in an established laboratory pollination protocol and an
in-depth proteome analysis of cherry styles across different experimental conditions. These
studies have not been described in sweet cherry and as such, will provide a foundation for the
study of GSI in Prunus and further support the development of effective breeding programs.

	
  
vi	
  

TABLE OF CONTENTS

LIST OF TABLES....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
CHAPTER ONE: Identification of a BAHD Acyltransferase Involved in the Acetylation of
Monolignols in Kenaf (Hibiscus cannabinus) .................................................................................1
1.1 Introduction......................................................................................................................2
1.2 Materials and Methods...................................................................................................11
1.3 Results............................................................................................................................15
1.4 Discussion ......................................................................................................................23
1.5 Future Studies ................................................................................................................27
CHAPTER TWO: Characterization of the Sweet Cherry Pollination System ..............................29
2.1 Introduction....................................................................................................................30
2.2 Materials and Methods...................................................................................................34
2.3 Results............................................................................................................................37
2.4 Discussion ......................................................................................................................46
2.5 Future Studies ................................................................................................................49
APPENDICES ...............................................................................................................................51
APPENDIX A: Identification of an O-Methyltransferase Involved in Monolignol
Biosynthesis in Rice....................................................................................52
APPENDIX B: Proteome Analysis of Unpollinated and Pollinated Cherry Styles.............64
REFERENCES ..............................................................................................................................75

	
  
vii	
  

LIST OF TABLES

Table 1.1

Abundance Values for Transferase Family Proteins Identified in Kenaf ..............17

Table 1.2

Enzyme Activity for AMT with Various Substrate Combinations........................22

Table 1.3

Kinetic Parameters for AMT .................................................................................22

Table 2.1

Pollen Germination Percentage Values for Pollen Parents of Pollination
Experiments ...........................................................................................................37

Table A.1

Enzyme Activity for OsOMT and ATOMT1 with Various Caffeyl Substrates ....61

Table B.1

Proteomics Data for Cherry Pollination Experiments at 24 h................................65

	
  
viii	
  

LIST OF FIGURES

Figure 1.1

The Monolignol Biosynthetic Pathway ...................................................................4

Figure 1.2

Molecular Structure of Sinapyl Alcohol ..................................................................7

Figure 1.3

General Reaction Scheme for β-O-4’ and β-β’ Dimerization..................................8

Figure 1.4

Peptide Sequence for Kenaf AMT.........................................................................15

Figure 1.5

Peptide Sequence Alignment of Kenaf AMT and Arabidopsis Acetyltransferase 16

Figure 1.6

Heterologous Expression of AMT in E. coli .........................................................18

Figure 1.7

HPLC Analyses of AMT Activity Assays .............................................................20

Figure 1.8

Peptide Sequence Alignment of Kenaf AMT and Petunia PhCFAT.....................25

Figure 2.1

Genetic Representation for Competitive Interaction in the Solanaceae ................31

Figure 2.2

Genetic Representation for the “One-Allele-Match” Model in Tetraploid Sour
Cherry ....................................................................................................................32

Figure 2.3

Control and Pollinated Cherry Styles ....................................................................39

Figure 2.4

Pollen Grain Adhesion on Cherry Style and Pollen Germination .........................40

Figure 2.5

Pollen Germination on Cherry Style Surface.........................................................41

Figure 2.6

Pollen Tube Growth within Cherry Style ..............................................................42

Figure 2.7

Full Growth of Pollen Tube Down Cherry Style ...................................................44

Figure 2.8

Arrest of Pollen Tube Growth at Mid Stylar Region.............................................45

Figure A.1

Hypothetical Monolignol Biosynthetic Pathway in Grasses .................................54

Figure A.2

Peptide Sequence Alignment of Arabidopsis ATOMT1 and Rice OsOMT..........59

Figure A.3

Heterologous Expression of OsOMT and ATOMT1 in E. coli .............................60

	
  
ix	
  

CHAPTER ONE

Identification of a BAHD Acyltransferase Involved in the Acetylation of Monolignols in Kenaf
(Hibiscus cannabinus)

	
  

1	
  

1.1 INTRODUCTION
In recent years, plant cell walls have captured significant attention as being the most
abundant renewable energy resource on Earth. It has been reported that cell walls represent 70%
9

of the 170-200 x 10 tons of worldwide biomass production in land plants (1). Yet, humans only
utilize 2% of this cell wall biomass for the production of heat energy, timber, paper, and textiles.
Due to the considerable availability of plant biomass, plant cell walls have been proposed as a
major feedstock for the production of biofuels.
All plant cells contain a primary wall composed of polysaccharide-rich cellulose,
hemicelluloses, and pectin, and some also form a thickened secondary cell wall composed of
cellulose and hemicelluloses (1). During the formation of the secondary cell wall,
phenylpropanoid compounds are polymerized in the wall to form lignin. Lignin is a complex
phenolic polymer that provides structural integrity and support to water-conducting tissues.
Arising from its complex structure, lignin confers a hydrophobic and impenetrable barrier to land
plants, which not only supports vascular tissue but also aids in pathogen defense (2). It is because
of these characteristics that lignin has aided in the evolutionary adaptation of plants from aquatic
to terrestrial environments. However, due to its strength and prevalence in the cell wall, lignin
presents a major obstacle for the processing of plant biomass to biofuels.
In order to design more effective cell wall processing techniques, it is important to
understand the processes involved in the formation of cell wall components. All components of
the cell wall vary across plant species (1). Similarly, lignin amount and composition vary across
plant species and even among cell types (3). The classical mechanism for the biosynthesis of
lignin monomer units, also known as monolignols, involves a series of reactions that transform
the amino acid phenylalanine into one of three monolignols- hydroxyphenyl (H), guaiacyl (G) or

	
  

2	
  

syringyl (S) (Figure 1.1) (2). The phenylpropanoid pathway begins with the deamination of
phenylalanine to cinnamic acid by phenylalanine ammonia lyase (PAL), which is the first
committed step in phenylpropanoid biosynthesis (2). Cinnamic acid is then hydroxylated at the
4´-position of the aromatic ring by cinnamate 4-hydroxylase (C4H). The resulting p-coumaric
acid molecule is then activated (e.g. S-CoA) by 4-coumarate:CoA ligase (4CL), a key enzyme
that catalyzes the synthesis of p-coumaroyl CoA. At this point, monolignol biosynthesis begins
by the transesterification of p-coumaroyl CoA to a shikimic/quinic acid by hydroxycinnamoyl
CoA:shikimate hydroxycinnamoyl transferase (HCT) and subsequent hydroxylation of the
aromatic ring by p-coumaroyl shikimate 3´-hydroxylase (C3H). Shikimic/quinic acid is removed
by HCT to generate caffeoyl CoA. Caffeoyl CoA then undergoes successive O-methylation
reactions by caffeic acid O-methyltransferase (COMT) and caffeoyl CoA O-methyltransferase
(CCoAOMT), followed by hydroxylation via ferulate 5-hydroxylase (F5H). Monolignols are
then produced by reduction of hydroxycinnamyl CoA to hydroxycinnamaldehyde, followed by
conversion to an alcohol by cinnamate alcohol dehydrogenase (CAD).
Monolignols are synthesized in the cytoplasm and transported across the plasma
membrane to the apoplast via a poorly understood transport system. It has been suggested that
monolignol transport is an ATP-driven process and as such, it may involve ATP-binding cassette
(ABC) type transporters (4). However, the mechanism for monolignol transport remains unclear.
Once in the apoplast, monolignols undergo dehydrogenative polymerization, which begins by the
oxidation of monolignols by peroxidases and laccases to form monolignol radicals. Then, the
monolignol radicals are incorporated into the lignin polymer via radical coupling, wherein bond
formation between any two radicals is promoted by the single-electron oxidation of the
monolignols, resulting in formation of lignin throughout the cell wall (2,3).

	
  

3	
  

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Figure 1.1 The Monolignol Biosynthetic Pathway. The dark blue shaded portion of the pathway
represents the route that is predominantly observed in angiosperms for monolignol production.
The light blue shaded portions are routes that occur in some plant species but to a minor degree.
The white portions have not been observed to play a role in the production of monolignols.
Figure was adapted from Boerjan et al. (2003) (3). For interpretation of the references to color in
this and all other figures, the reader is referred to the electronic version of this thesis.

	
  

4	
  

Figure 1.1 (cont’d)
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Through numerous studies on mutants and transgenic plants, the major enzymes involved
in the biosynthesis of the three principal monolignols have been determined, as reviewed in (2)
and (3). However, there is evidence to suggest that lignin biosynthesis is not limited to these
three monomer units (5-12). Alternative monolignol precursors have been identified in the lignin
of mutants lacking CAD or COMT, both of which are key monolignol biosynthetic genes (2).
Down-regulation of the CAD gene in tobacco results in the incorporation of coniferaldehyde into
the lignin polymer (5). Similarly, CAD-deficient mutants in pine, maize, and arabidopsis
contained normal lignin levels; yet, the lignin composition was greatly altered from that of the
wild type. COMT-deficient mutants had 5-hydroxyconiferyl alcohol incorporated into their
lignin as a result of COMT deficiency (9,10). These instances demonstrate the plasticity of lignin
biosynthesis in that lignin can tolerate variation from the canonical structure, yet still confer the
primary function of strength and support to vascular tissue.

	
  

5	
  

Another example of lignin monomer diversity is illustrated by monolignols that undergo
modifications prior to incorporation into the lignin polymer. To date, one of the most widely
reported modifications to occur is that of monolignol acylation (13,14). This phenomenon has
been observed in many plant species, including p-coumarates in both C3 and C4 grasses (15-19);
p-hydroxybenzoates in hardwoods such as aspen, poplar, and willow (20-24); and acetates in
abaca, kenaf, palms, and sisal (25-28). Though many accounts of this phenomenon exist in a
number of plant species, the functional role of monolignol acylation is unknown.
The identification of acetate groups on lignin remained to be determined until methods
that preserved ester linkages were developed. These include nuclear magnetic resonance (NMR)
analysis and the “Derivatization Followed by Reductive Cleavage” (DFRC) method, which
cleaves ether linkages in the lignin polymer thereby releasing lignin monomers and dimers for
analysis (29). DFRC has been used for the analysis of γ-p-coumarates in the lignin of grasses
because the method does not disturb γ-ester groups on lignin (30). However, a major
disadvantage of DFRC is that it utilizes acetate-based reagents, which interfere with the analysis
of acetate groups on lignin. Additionally, lignin is commonly pre-acetylated prior to NMR
analysis for improved solubility and spectral dispersion (26). Thus, evidence for acetylated lignin
was not reported for quite some time.
Acetylated lignin was discovered in Hibiscus cannabinus, a non-woody plant commonly
known as kenaf, when NMR analysis demonstrated that its phloem (bast) fiber lignin was not
only rich in syringyl units, but also extensively acetylated (26). This discovery reinforced the
idea that monolignols are pre-acylated prior to incorporation into the lignin polymer. To further
analyze acetylation levels in kenaf, an alternative DFRC method was developed, called DFRC´,
wherein all acetate-based reagents were substituted with their propionate analogues (30). This

	
  

6	
  

new method has provided conclusive evidence that kenaf bast fibers are extensively acetylated,
particularly on syringyl units. In addition, the DFRC´ method has been utilized to analyze
acetylated lignin in other plant species such as abaca, sisal and hornbeam (28).
As referenced previously, the observation that kenaf bast fiber lignin contains a high
syringyl content was the initial motive for studying this plant system. NMR results for kenaf
lignin demonstrated that the molar syringaldehyde:vanillin ratio was 6.0, which supports prior
evidence for the high syringyl:guaiacyl ratio observed in hardwoods (26). In addition, it was
determined that over 50% of the lignin in kenaf bast fibers was acetylated and from this
proportion, about 95% of the acetylation was located on the γ-position of the carbon side chain
on syringyl units (Figure 1.2) (26). Though syringyl units were primarily acetylated, acetylation
was also observed on guaiacyl units. However, the degree of acetylation on guaiacyl units was
marginal when compared to that of syringyl units.
!
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Figure 1.2 Molecular Structure of Sinapyl Alcohol. The carbons of the side chain for sinapyl
alcohol are numbered in red.
There are many unexplored questions regarding monolignol acetylation. First, the
functional implications of acetylated lignin have not been demonstrated. It has been suggested
that lignin acylation may be a process involved in the regulation of lignin structure (31). Plants
with high levels of acylation, particularly acetylation, have a greater propensity to forming β-O4´ linkages rather than β-β´ linkages, which produces a more uncondensed lignin (Figure 1.3).
	
  

7	
  

The advantages of a predominantly β-O-4´ lignin structure are unclear in an evolutionary sense.
However, uncondensed lignin is desirable for the commercial purpose of processing biomass to
biofuels. Another hypothesis suggests that monolignol acetylation could be a mechanism
employed by plants to aid in drought tolerance since acetylation confers greater hydrophobicity
to the lignin polymer (28). However, this speculation does not explain why abaca, a plant species
whose lignin is highly acetylated, is not drought tolerant. Thus, the functional effects of this
particular acylation phenomenon remain poorly understood.
R

	
  

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Figure 1.3 General Reaction Scheme for β-O-4´ and β-β´ Dimerization. The β-O-4´ dimerization
process produces more linear structures. Monolignols linked via β-β´ dimerization have an
additional level of structural complexity, producing a more cross-linked lignin structure. Halfheaded arrows represent a single-electron transfer process, whereas full-headed arrows represent
a two-electron transfer process. Figure was adapted from Bonawitz and Chapple, (2010) (2).

	
  

8	
  

Figure 1.3 (cont’d)
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Secondly, the acetylated monolignols in lignin may indicate that there are alternate
biosynthetic pathways to monolignol biosynthesis (28). It is possible that the pre-acylation of
monolignol precursors occurs as a step in an alternative pathway that is distinct from the
canonical monolignol biosynthetic pathway. It is also possible that acylation occurs on
monolignols after they have been synthesized via the usual mechanism. In any case,
modifications to the traditional mechanism for monolignol biosynthesis cannot be excluded and
should be considered in all interpretations of lignin biosynthesis.

	
  

9	
  

Lastly, though analytical methods have aided in the identification of alternative lignin
structure in a number of plants, the enzyme(s) responsible for acylation modifications have not
been established. Recently, our group has identified OsPMT, a grass-specific enzyme that
catalyzes the synthesis of monolignol p-coumarates in rice (19). Identification of the enzymes
involved in acylation reactions is critical to understanding the mechanism of lignin acylation and
how such modifications can be utilized to improve plant processing for conversion into biofuels.
Additionally, manipulation of these enzymes in planta would provide a closer look into how
monolignol acylation relates to lignin structure and function.
The primary purpose of this study was to identify an enzyme responsible for the
acetylation of sinapyl alcohol in kenaf bast fiber lignin. A BAHD acyltransferase, herein called
acetyl monolignol transferase (AMT), from kenaf stem was found to catalyze the acetylation of
sinapyl alcohol to produce sinapyl acetate. AMT synthesized only trace amounts of coniferyl
acetate and did not produce p-coumaryl acetate, indicating AMT’s specificity for sinapyl alcohol.
These biochemical results support data from NMR studies on kenaf bast lignin, which have
shown extensive acetylation of sinapyl alcohol and negligible acetylation of coniferyl alcohol
(26,28,30,32). To date, there has been no evidence to support the presence of p-coumaryl
acetates in kenaf lignin (26,28,30,32).
Secondly, the purpose of this study was to produce AMT overexpression transgenic
plants to determine the effects of AMT expression on lignin structure and function. The findings
presented here will establish a basis for the study of acetylated lignin in kenaf and will also
contribute to understanding the mechanism of lignin acylation reactions. Overall, the objective is
to understand the mechanism of monolignol acylation and whether changes to acylation levels in
plants would prompt facile processing of plant biomass into biofuels.

	
  

10	
  

1.2 MATERIALS AND METHODS
Transcriptional Profiling of Kenaf cDNA Libraries
Plant material was provided by Dr. Ronald Hatfield at the U.S. Dairy Forage Research Center.
Saunia Withers at the Michigan State University (MSU) Department of Plant Biology performed
a total RNA extraction from kenaf bast fibers using TRIzol Reagent (Invitrogen, Carlsbad,
California), followed by cDNA synthesis using the Creator SMART cDNA Library Construction
Kit (Clontech, Mountain View, California) according to the manufacturer’s instructions. cDNA
was submitted to the MSU Genomics Core for Roche 454 sequencing using the 454 GSFLX
Titanium Sequencer. Contigs were aligned and compiled in a database, named Kenaf CLC,
created by Nick Thrower at the MSU Bioinformatics Core. The acetyl monolignol transferase
(AMT) gene candidate was selected based on three criteria: 1) classification as a BAHD
acyltransferase family protein, 2) annotation as an acetyltransferase according to The
Arabidopsis Information Resource (TAIR) and 3) relative abundance.

Gene Synthesis and Gateway Cloning
AMT from kenaf was submitted to GENEART (Carlsbad, California) to be chemically
synthesized with optimized codon usage for protein expression in Escherichia coli (E. coli).
Plasmid DNA for AMT was extracted from a culture grown from the bacterial stab provided by
GENEART. AMT was then cloned into the pDONR221 entry vector, followed by cloning into
the pDEST17 vector using Invitrogen Gateway system cloning technology. Cloning AMT into
the pDEST17 vector produced an expression clone with a N-terminal His6 tag to be used in
protein purification via immobilized metal affinity chromatography (IMAC). The His-tagged
clone was then transformed into BL21 pLysS chemically competent cells (Invitrogen).

	
  

11	
  

Heterologous Expression in E. coli
A one-liter bacterial culture containing the N-terminal His-tagged AMT expression clone was
grown at 37 °C to an A600 between 0.4 and 0.45. Protein expression was induced by the addition
of isopropyl-β-D-1-thiogalactopyranoside (IPTG; Roche Applied Technologies, Indianapolis,
Indiana). The culture was then grown for approximately 14 h at 18 °C, after which it was
centrifuged to obtain a pellet and stored at -80 °C. The culture pellet was resuspended in 20 mL
of binding buffer (20 mM Tris-HCl, pH 8, 0.5 M NaCl, and 1 mM 2-mercaptoethanol). The cells
were lysed using a French pressure cell press. Soluble and insoluble fractions were separated by
centrifugation at 50,000 x g for 30 min at 4 °C. The soluble fraction was syringe filtered prior to
storage and the insoluble portion was resuspended in 20 mM Tris-HCl, pH 8. Both fractions
were analyzed by SDS-PAGE to confirm that expression had taken place in the induced cultures.

Protein Purification by Fast Protein Liquid Chromatography (FPLC)
Five milliliters of soluble His-tagged AMT protein were loaded onto a 5 mL loop in preparation
for FPLC purification. First, the soluble protein was eluted through four stacked 5 mL Hi-Trap
desalting columns (GE Healthcare, Uppsala, Sweden) using binding buffer. Protein
concentration for each eluted fraction was determined using A280. Fractions with the highest
protein concentration were collected to yield 5 mL of protein. This sample was then loaded onto
a 1 mL IMAC Hi-Trap HP column (GE Healthcare) and eluted with buffer A (20 mM Tris-HCl,
pH 8, 0.5 M NaCl, 1 mM 2-mercaptoethanol, and 25 mM imidazole) and a gradient of buffer B
(20 mM Tris-HCl, pH 8, 0.5 M NaCl, 1 mM 2-mercaptoethanol, and 500 mM imidazole).
Purified fractions containing protein, as determined by UV absorbance peaks, were analyzed on
a SDS-PAGE gel. Protein bands were compared to the expected molecular weight for AMT (ca.

	
  

12	
  

49 kDa). A protein band having the expected molecular weight for AMT was cut from the SDSPAGE and analyzed by in-gel trypsin digestion and LC/MS/MS at the MSU Proteomics Core.
Peptides were identified from MS/MS fragment data by searching against Kenaf CLC and E.coli
databases using the program Mascot. AMT-containing fractions were collected and buffer
exchanged into 100 mM phosphate buffer, pH 6, containing one protease inhibitor tablet (Roche
Applied Science) using an Amicon 10K membrane filter (Millipore, Carrigtwohill, Ireland). The
enzyme was stored in 20 µL aliquots at -80 °C until use.

Enzyme Activity Assays
Enzyme activity assays were performed in 25 mM phosphate buffer, pH 5.8, 0.5 mM
dithiothreitol (DTT), 0.5 mM CoA thioester, 0.5 mM monolignol, and deionized water to a
volume of 100 µL. An AMT present reaction and no enzyme control were prepared for each CoA
thioester and monolignol combination. AMT reactions were initiated by adding 1 µg of AMT.
All reactions were incubated for 45 min and solubilized with an equal volume of methanol.
Protein was removed from the AMT reactions by using an Amicon 10K membrane filter. Ultra
Performance Liquid Chromatography (UPLC) was used to analyze the flow-through material at
280 nm and 340 nm. The eluting compounds were identified by comparison of their peak
retention times to standards for the CoA thioesters, monolignols and monolignol conjugates.

The monolignols (coniferyl and sinapyl alcohol) and monolignol conjugates (coniferyl pcoumarate, sinapyl p-coumarate, coniferyl caffeate, sinapyl caffeate, coniferyl acetate and
sinapyl acetate) were provided by Dr. John Ralph at the University of Wisconsin- Madison (33).
CoA thioester synthesis was performed by following a previously described method (34), which

	
  

13	
  

uses a C-terminal His-tagged 4-coumarate-ligase (4CL) from tobacco in the pCRT7/CT TOPO
vector (provided by Eran Pichersky, University of Michigan), the acid derivatives, coenzyme A
hydrate, and adenosine-5´-triphosphate (ATP). All chemicals for CoA thioester synthesis, as well
as acetyl CoA, were purchased from Sigma-Aldrich (St. Louis, Missouri). CoA thioester
concentration was quantified by using its maximum absorbance and extinction coefficient.

Enzyme Kinetics
Enzyme kinetics were performed in 25 mM phosphate buffer, pH 5.8, 0.5 mM DTT, 0.005 - 0.05
mM CoA thioester, 0.5 - 2 mM monolignol substrate, and deionized water to a volume of 100
µL. The reactions were initiated with purified AMT in 0.5X BSA (New England Biolabs,
Ipswich, Massachusetts). The amount of AMT added to each reaction set was variable and
dependent on the rate of product formation for that CoA thioester and monolignol combination.
Each 100 µL reaction was divided into 30 µL per time point. The reactions were run for 5, 10,
and 20 min and performed in triplicate. All reactions were solubilized with an equal volume of
methanol. The CoA thioester substrate used was acetyl CoA and the monolignol alcohol
substrates used were sinapyl alcohol and coniferyl alcohol. Peak areas were manually measured
from the product peaks corresponding to each enzyme reaction using Empower 2 Software
(Waters Corporation, Milford, Massachusetts). The peak areas (A280) were converted to
nMoles/sec/mg using extinction coefficients determined from standards. Kinetic parameters (Km
and Vmax) for each CoA thioester and monolignol substrate combination will be calculated using
a custom script from R64, version 2.15.2 (35).

	
  

14	
  

1.3 RESULTS
Identification of the Candidate Gene
The AMT candidate gene was selected based on three criteria. First, I sought a candidate gene
classified as a BAHD acyltransferase family protein. This classification was an important
consideration because BAHD acyltransferases constitute a super family of proteins that utilize
acyl CoA thioesters in acylation reactions (14). It was presumed that the AMT candidate would
be a BAHD acyltransferase due to its ability to acylate monolignols using acetyl CoA. An
analysis of AMT’s peptide sequence demonstrated that it had two conserved domains common to
all BAHD acyltransferases: the “HXXXD” and “DFGWG” motifs (Figure 1.4) (14).

MALLRPASLVFTVRRHDPELVVPSKPTPHECKTLSDIDDQDGHRFQIRGLHVYRCNASM
QGKDPVRVIREALAKALVFYYPFAGRIKEGPNRKLMVDCTGEGVLFIEADADVMLEEF
GGSLHPPFPCFKDLLCEPTGSNDLLNSPVLQIQVTRLKCGGFIFAHRFNHTMSDAVGLIQ
FMSAMGEIARGAVAPSIPPVWERHLLNARDPPLITCEHHEYDHATATNGTIMPTDNLVH
HSFFFGPTQISALKRLISDNVSCSTFDILTACVWRCRTIAMKLGPDEDVRLICIVNARYKF
NPPLPLGYYGNALGYPAALTTAGELSKKPLEYAVKLVKEAKAKATDEYMKSTADLMV
SRGRPNVNTVRSFLVSDLSRARFREVDFGWGKAEFGGPSNGTEIISFYIPSKNKEGKEGI
AVPVCLPASVMESFVKEINSTLADDEATGA
Figure 1.4 Peptide Sequence for Kenaf AMT. The AMT peptide sequence was 444 amino acids
in length and had an approximate molecular weight of 49 kDa. The HXXXD and DFGWG
motifs common to all BAHD acyltransferases are underlined and in bold.

Second, I sought a candidate gene annotated as an acetyltransferase according to TAIR. The
Kenaf CLC database utilizes TAIR to annotate proteins based on their closest homolog in
arabidopsis. AMT’s closest homolog in arabidopsis was an acetyltransferase gene (Figure 1.5),

	
  

15	
  

Alignment Report Report of 'Untitled.meg' - ClustalW (Slow/Accurate, Gonnet) : Monday, No
Alignment of 'Untitled.meg' - ClustalW (Slow/Accurate, Gonnet) : Monday, November
26, 2012 9:08 AM
26, 2012 9:08 AM
Majority
Majority
..............L.F.V.R..PEL..P.KPTP.E.K.LSDIDDQ.G.RFQI..
..............L.F.V.R..PEL..P.KPTP.E.K.LSDIDDQ.G.RFQI..
kenaf.pro ------MALLRPASLVFTVRRHDPELVVPSKPTPHECKTLSDIDDQDGHRFQIRG
kenaf.pro ------MALLRPASLVFTVRRHDPELVVPSKPTPHECKTLSDIDDQDGHRFQIRG
acetyl.pro MDHQVSLPQSTTTGLSFKVHRQQPELITPAKPTPRELKPLSDIDDQQGLRFQIPV
acetyl.pro MDHQVSLPQSTTTGLSFKVHRQQPELITPAKPTPRELKPLSDIDDQQGLRFQIPV

49
55

49
55

Majority
Majority
...YR.N.S.....PV.VI..ALA.ALV.YYPFAGR..E..NRKL.VDCTGEGVL
...YR.N.S.....PV.VI..ALA.ALV.YYPFAGR..E..NRKL.VDCTGEGVL
kenaf.pro LHVYRCNASMQGKDPVRVIREALAKALVFYYPFAGRIKEGPNRKLMVDCTGEGVL
kenaf.pro LHVYRCNASMQGKDPVRVIREALAKALVFYYPFAGRIKEGPNRKLMVDCTGEGVL
acetyl.pro IFFYRPNLSS-DLNPVQVIKKALADALVYYYPFAGRLRELSNRKLAVDCTGEGVL
acetyl.pro IFFYRPNLSS-DLNPVQVIKKALADALVYYYPFAGRLRELSNRKLAVDCTGEGVL

104
109

104
109

Majority
Majority
FIEA.ADV.L.E......L.PPFPC...LL....GS.D.LN.P.L..QVTRLKC.
FIEA.ADV.L.E......L.PPFPC...LL....GS.D.LN.P.L..QVTRLKC.
kenaf.pro FIEADADVMLEEFGGS--LHPPFPCFKDLLCEPTGSNDLLNSPVLQIQVTRLKCG
kenaf.pro FIEADADVMLEEFGGS--LHPPFPCFKDLLCEPTGSNDLLNSPVLQIQVTRLKCG
acetyl.pro FIEAEADVSLTELEEADALLPPFPCLDELLFDVEGSSDVLNTPLLLVQVTRLKCR
acetyl.pro FIEAEADVSLTELEEADALLPPFPCLDELLFDVEGSSDVLNTPLLLVQVTRLKCR

157
164

157
164

Majority
Majority
GFIFA.RFNHTM.D..GL..F.....E.A....APS.PPVW.RHLL.........
GFIFA.RFNHTM.D..GL..F.....E.A....APS.PPVW.RHLL.........
kenaf.pro GFIFAHRFNHTMSDAVGLIQFMSAMGEIARGAVAPSIPPVWERHLLNARD-PPLI
kenaf.pro GFIFAHRFNHTMSDAVGLIQFMSAMGEIARGAVAPSIPPVWERHLLNARD-PPLI
acetyl.pro GFIFALRFNHTMTDGAGLSLFLKSLCELAYRLHAPSVPPVWDRHLLTVSASEARV
acetyl.pro GFIFALRFNHTMTDGAGLSLFLKSLCELAYRLHAPSVPPVWDRHLLTVSASEARV

211
219

211
219

Majority
Majority
T..H.EY....A........D..V..SFFF....ISA...L.........F..L.
T..H.EY....A........D..V..SFFF....ISA...L.........F..L.
kenaf.pro TCEHHEYDHATATNGTIMPTDNLVHHSFFFGPTQISALKRLISDNVSCSTFDILT
kenaf.pro TCEHHEYDHATATNGTIMPTDNLVHHSFFFGPTQISALKRLISDNVSCSTFDILT
acetyl.pro THTHREYEDQVAID-AVDTGDPFVSRSFFFSAEEISAIRKLLPPDLHNTSFEALS
acetyl.pro THTHREYEDQVAID-AVDTGDPFVSRSFFFSAEEISAIRKLLPPDLHNTSFEALS

266
273

266
273

Majority
Majority
...WRCRTIA....P....RL.CI.N.R.K...PPL..GYYGN....PAA..TA.
...WRCRTIA....P....RL.CI.N.R.K...PPL..GYYGN....PAA..TA.
kenaf.pro ACVWRCRTIAMKLGPDEDVRLICIVNARYKFN-PPLPLGYYGNALGYPAALTTAG
kenaf.pro ACVWRCRTIAMKLGPDEDVRLICIVNARYKFN-PPLPLGYYGNALGYPAALTTAG
acetyl.pro SFLWRCRTIALNPDPNTEMRLTCIINSRSKLSNPPLSRGYYGNVFVIPAAIATAR
acetyl.pro SFLWRCRTIALNPDPNTEMRLTCIINSRSKLSNPPLSRGYYGNVFVIPAAIATAR

320
328

320
328

Majority
Majority
.L..KPLE.A..L..E.K...T..Y..S...LM..RGRP..........SDL...
.L..KPLE.A..L..E.K...T..Y..S...LM..RGRP..........SDL...
kenaf.pro ELSKKPLEYAVKLVKEAKAKATDEYMKSTADLMVSRGRPNVNTVRSFLVSDLSRA
kenaf.pro ELSKKPLEYAVKLVKEAKAKATDEYMKSTADLMVSRGRPNVNTVRSFLVSDLSRA
acetyl.pro DLMEKPLEFALRLIQETKSSVTEDYVRSVTALMATRGRPMFVAAGNYIISDLRHF
acetyl.pro DLMEKPLEFALRLIQETKSSVTEDYVRSVTALMATRGRPMFVAAGNYIISDLRHF

375
383

375
383

Majority
Majority
....VDFG.WGK...GG............SFY.P.KNK.G..G..V...LP...M
....VDFG.WGK...GG............SFY.P.KNK.G..G..V...LP...M
kenaf.pro RFREVDFG-WGKAEFGGPSNG----TEIISFYIPSKNKEGKEGIAVPVCLPASVM
kenaf.pro RFREVDFG-WGKAEFGGPSNG----TEIISFYIPSKNKEGKEGIAVPVCLPASVM
acetyl.pro DLGKVDFGPWGKPVYGGTAKAGIALFPGVSFYVPFKNKKGETGTVVAISLPVRAM
acetyl.pro DLGKVDFGPWGKPVYGGTAKAGIALFPGVSFYVPFKNKKGETGTVVAISLPVRAM

425
438

425
438

Majority
Majority
E.FV.E.N..L........
E.FV.E.N..L........
kenaf.pro ESFVKEINSTLADDEATGA
kenaf.pro ESFVKEINSTLADDEATGA
acetyl.pro ERFVAELNGVLIKRF
acetyl.pro ERFVAELNGVLIKRF

444
453

444
453

Figure 1.5 Peptide Sequence Alignment of Kenaf AMT and Arabidopsis Acetyltransferase. The
AMT candidate only had 49% similarity to the arabidopsis acetyltransferase. Sequences were
aligned using MegAlign (DNASTAR). Dots denote non-matching residues. Dashes signify gaps
inserted to obtain optimal sequence alignment. The HXXXD and DFGWG motifs are underlined.
	
  

16	
  

which functions in volatile production for plant defense (36). It is important to note that BAHD
acyltransferases are well known for serving functionally diverse roles in secondary metabolism,
regardless of their conserved domains and overall similarity. Thus, it was not likely that the
AMT candidate would have a similar function to the acetyltransferase in arabidopsis. Lastly, due
to the prevalence of acetylated syringyl units in kenaf lignin, the candidate gene was selected on
the basis of abundance (Table 1.1).
Table 1.1 Abundance Values for Transferase Family Proteins Identified in Kenaf
Representative Definition

Total Members

glycosyl transferase family 1 protein

1637

transferase family protein

751

HCT transferase family protein

485

transferase family protein

469

UDP-glucoronosyl/UDP-glucosyl transferase family protein

457

UTP-glucose-1-phosphate uridylyltransferase family protein

309

HCT transferase family protein

248

O-acetyltransferase family protein

232

transferase family protein

172

O-acetyltransferase family protein

144

protein arginine N-methyltransferase family protein

90

glycosyltransferase family 14 protein

76

galactosyltransferase family protein

76

HCT transferase family protein

73

glycosyltransferase family 14 protein

65

O-methyltransferase family 2 protein

60

O-acetyltransferase family protein

58

galactosyltransferase family protein

57

Note: The AMT candidate gene is indicated in bold/italics.

	
  

17	
  

Heterologous Expression in E. coli
Using a codon-optimized synthetic gene for kenaf AMT, a clone for a N-terminal His-tagged
AMT was produced and utilized for protein expression in E. coli. The protein was purified from
E. coli extracts using IMAC and analyzed by SDS-PAGE. Pre- and post-purification protein
samples demonstrated an induced protein in the 49 kDa range, as expected for AMT (Figure 1.6).
The un-induced controls did not show induction of a protein of similar size. SDS-PAGE analysis
demonstrated that AMT is mostly insoluble. However, protein expression was performed in a 1 L
culture to produce enough soluble protein for IMAC purification. Further analysis by
LC/MS/MS of tryptic fragments confirmed that the protein band was AMT.

T0
soluble

T0
insol.

18°C
soluble

18°C
insol.

IMAC
Fr34

IMAC
Fr35

IMAC
Fr36

kDa
75

50

Figure 1.6 Heterologous Expression of AMT in E. coli. SDS-PAGE analysis of soluble and
insoluble protein fractions for uninduced (T0) and induced (14 h) expression at 18 °C, and
IMAC fractions collected for AMT activity assays.

	
  

18	
  

Enzyme Activity Assays
AMT demonstrated a strong preference for sinapyl alcohol as the acyl acceptor. AMT produced
sinapyl acetate in a reaction containing 1 µg of AMT, 0.5 mM acetyl CoA, and 0.5 mM sinapyl
alcohol (Figure 1.7A). When using coniferyl alcohol, AMT produced only trace amounts of
coniferyl acetate (Figure 1.7B). AMT did not synthesize p-coumaryl acetate, suggesting that pcoumaryl alcohol was not a good substrate for AMT activity. Overall, the substrate specificity
observed in the AMT activity assays is similar to the acetylation specificity found in kenaf
lignin. When tested with other CoA thioester substrates, AMT demonstrated activity with pcoumaroyl CoA and caffeoyl CoA (Table 1.2). However, it is important to note that monolignol
p-coumarates and caffeates have not been observed in kenaf lignin (26,28,30,32).

Enzyme Kinetics
AMT substrate specificity was analyzed using acetyl CoA as the acyl donor and sinapyl alcohol
and coniferyl alcohol as the acyl acceptors. p-Coumaryl alcohol was not analyzed due to it being
a poor substrate for AMT activity. AMT had a strong affinity for sinapyl alcohol as the acyl
acceptor (Table 1.3). The activity observed with coniferyl alcohol and acetyl CoA was too small
to obtain the Km for these substrates. However, an estimated Vmax was calculated for saturating
coniferyl alcohol and varying acetyl CoA. Kinetic parameters were not obtained for varying
coniferyl alcohol and saturating acetyl CoA due to little activity with coniferyl alcohol. In these
experiments, high concentrations of coniferyl alcohol did not produce enough coniferyl acetate
to accurately detect Km or Vmax. Thus, AMT substrate specificity for coniferyl alcohol was only
examined under saturating monolignol conditions.

	
  

19	
  

@LM

A)

B

No enzyme

@LE
@LD

K:

@LB
BLA
BLM
A

!"#$%&' ($)* ()&!+ )&#,- .%&,-

BLE
BLD

J:

BLB
@KH
BLBB @LBB DLBB >LBB ELBB ?LBB MLBB NLBB ALBB GLBB @BLBB @@LBB
+AMT
<.=5'-6
@KE
;&%8)-1&%- I-=J 6.=&)! &!-'!$& E?%
B
@KD
@KB
7-8$#'-* (9 :6-#2 ;96'-%
/#$0-!' 1&%-2 3-4&5)'6
7-8$#' <-'"$*2 !"#$%&' ($)* ()&!+ )&#,- .%&,3&'- /#.='-*2
BKA
7-8$#' <-'"$* F32 >?@A>
>?@A>
@BCDECDB@D
/&,-2 @ $4 @
D2@G2@? /< :;CH&6'-#=
BKH
BKE
A

C

BKD
BKB
BKBB @KBB DKBB >KBB EKBB ?KBB HKBB LKBB AKBB IKBB @BKBB @@KBB
<.=5'-6
;&%8)-1&%- Assays. HPLC Chromatograms of AMT activity
Figure 1.7 HPLC Analyses of AMT ActivityBHI@@ 6.=&)! &!-'!$& E?%
assays with no enzyme or purified AMT (+AMT). A) acetyl CoA (A) and sinapyl alcohol (B) to
make sinapyl acetate (C) and B) (A) and coniferyl alcohol (D) to make coniferyl acetate (E).
7-8$#'-* (9 :6-#2 ;96'-%
/#$0-!' 1&%-2 3-4&5)'6
7-8$#' <-'"$*2 !"#$%&' ($)* ()&!+ )&#,- .%&,3&'- /#.='-*2
7-8$#' <-'"$* F32 >?@A>
>?@A>
@BCDECDB@D
/&,-2 @ $4 @
D2@A2?E /< :;CG&6'-#=
	
  
20	
  

!"#$%&' ($)* ()&!+ )&#,- .%&,Figure 1.7 (cont’d)

@KL

B)

No enzyme

@KE
@KD
D

J:

@KB
BKA
BKL
BKE

!"#$%&' ($)* ()&!+ )&#,- .%&,-

A

BKD
BKB
@KI
BKBB @KBB DKBB >KBB EKBB ?KBB LKBB MKBB AKBB NKBB @BKBB @@KBB
+AMT
<.=5'-6
@KE
;&%8)-1&%- H-=I !$=&)! &!-'!$& E?%

J:

@KD
D
@KB
7-8$#'-* (9 :6-#2 ;96'-%
/#$0-!' 1&%-2 3-4&5)'6
7-8$#' <-'"$*2 !"#$%&' ($)* ()&!+ )&#,- .%&,3&'- /#.='-*2
BKA
7-8$#' <-'"$* F32 >?@A>
>?@A>
@BCDECDB@D
/&,-2 @ $4 @
D2DB2BE /< :;CG&6'-#=
BKI

BKE

A

BKD
E

BKB
BKBB @KBB DKBB >KBB EKBB ?KBB IKBB LKBB AKBB GKBB @BKBB @@KBB
<.=5'-6
;&%8)-1&%- BIG@@ !$=&)! &!-'!$& E?%

	
  

21	
  
7-8$#'-* (9 :6-#2 ;96'-%
/#$0-!' 1&%-2 3-4&5)'6
7-8$#' <-'"$*2 !"#$%&' ($)* ()&!+ )&#,- .%&,3&'- /#.='-*2
7-8$#' <-'"$* F32 >?@A>
>?@A>
@BCDECDB@D
/&,-2 @ $4 @
D2@G2?@ /< :;CH&6'-#=

Table 1.2 Enzyme Activity for AMT with Various Substrate Combinations
Monolignol Substrate

CoA Thioester

AMT Activity

Sinapyl alcohol

Acetyl CoA

Yes

Sinapyl alcohol

p-Coumaroyl CoA

Yes

Sinapyl alcohol

Caffeoyl CoA

Yes

Sinapyl alcohol

Feruloyl CoA

Yes*

Coniferyl alcohol

Acetyl CoA

Yes*

Coniferyl alcohol

p-Coumaroyl CoA

Yes

Coniferyl alcohol

Caffeoyl CoA

Yes

Coniferyl alcohol

Feruloyl CoA

No

p-Coumaryl alcohol

Acetyl CoA

No

p-Coumaryl alcohol

p-Coumaroyl CoA

Yes*

p-Coumaryl alcohol

Caffeoyl CoA

No

p-Coumaryl alcohol

Feruloyl CoA

No

* Indicates that a negligible amount of product was formed, which suggests that the substrate
combination was a poor match for AMT activity.

Table 1.3 Kinetic Parameters for AMT
Varying

Saturating

Km

Vmax

Substrate

Substrate

(µM)

(nkat mg )

(s )

(mM s )

Acetyl CoA

197.2 ± 36.2

36.2 ± 2.3

1.77

8.98

Sinapyl alcohol

43.5 ± 7.5

30.0 ± 1.3

1.47

33.8

NA*

<8.5

NA

NA

Sinapyl
alcohol
Acetyl CoA
Acetyl CoA

Coniferyl
alcohol

Kcat
-1

-1

Kcat/Km
-1 -1

* NA indicates that the kinetic parameter was not obtained due to very little AMT activity.

	
  

22	
  

1.4 DISCUSSION
AMT is a BAHD acyltransferase in kenaf that is responsible for catalyzing the
acetylation of sinapyl alcohol to produce sinapyl acetate. It has been demonstrated that AMT has
a strong preference for sinapyl alcohol as the acyl acceptor and only marginal activity when
using coniferyl alcohol. p-Coumaryl alcohol was a poor substrate for AMT activity.
Interestingly, the substrate specificity observed in the AMT activity assays mimics the
acetylation observed in kenaf bast fiber lignin. Additionally, these observations support NMR
analyses of kenaf lignin, wherein only a trace amount of acetate groups were found on guaiacyl
units when compared to the amount of acetate groups on syringyl units (30).
AMT also demonstrated activity with the acyl donors p-coumaroyl CoA and caffeoyl
CoA. However, though this activity was observed in vitro, it is unlikely that these acylation
reactions take place in planta because monolignol p-coumarates and monolignol caffeates have
not been reported in kenaf lignin (26,28,30,32). It may be that p-coumarates have not been
detected in kenaf lignin due to the limited amount of p-coumaroyl CoA present in the cell. pCoumaroyl CoA is the central metabolite in the phenylpropanoid pathway leading to the
biosynthesis of not only monolignols but also a wide array of secondary metabolites, such as
flavonoids, coumarins, and tannins (37). Thus, the presence of p-coumaroyl CoA may be
transient in the cell and not abundant enough to produce monolignol p-coumarates. The same
argument can be made for caffeoyl CoA, which is quickly consumed by COMT and CCoAOMT
to drive the synthesis of coniferyl alcohol and sinapyl alcohol.
Del Rio et al. (2007) performed an analysis of naturally acetylated lignin on a large set of
plant species, including abaca, kenaf, and sisal (28). Lignin from both angiosperms and
gymnosperms was examined using the DFRC´ method and relative abundance values for total

	
  

23	
  

monolignol units, syringyl-to-guaiacyl (S/G) ratio, and acetylated monolignol units were
reported. It was demonstrated that native acetylation of monolignol units is a phenomenon that is
common among angiosperms. With some exceptions (i.e. bamboo and eucalyptus), monolignol
acetylation was predominantly detected on syringyl units, particularly at the γ-carbon of the side
chain. Kenaf demonstrated approximately 1.4 times as many total syringyl units than total
guaiacyl units. Of this proportion, guaiacyl units were significantly less acetylated than syringyl
units at 8.9% vs. 59%, respectively (28). These values demonstrate that acetylation of sinapyl
alcohol is strongly preferred in kenaf, even though the amount of total syringyl units and total
guaiacyl units detected was relatively similar. These observations strongly suggest that enzyme
preference for sinapyl alcohol may be the driving force behind the acetylation of syringyl units to
synthesize sinapyl acetates rather than coniferyl acetates.
Coniferyl acetate has been implicated in floral volatile biosynthesis in petunia because it
is used as substrate by isoeugenol synthase 1 (PhIGS1) to make isoeugenol, a major component
of floral scent in petunia. Dexter et al. (2007) (38) reported on a BAHD acyltransferase called
coniferyl alcohol acyltransferase (PhCFAT) that utilizes coniferyl alcohol and acetyl CoA to
produce coniferyl acetate in petunia petals. PhCFAT kinetics demonstrated a high affinity for
coniferyl alcohol and moderate affinity for sinapyl alcohol- 100% vs. 70%, respectively. To
determine whether AMT and PhCFAT were closely related, a protein BLAST was performed to
align the AMT and PhCFAT peptide sequences against one another (Figure 1.8). While both
enzymes had the HXXXD and DFGWG motifs conserved among all BAHD acyltransferases,
AMT and PhCFAT had a low peptide sequence identity (27%). Along with evidence of AMT’s
low enzyme rate with coniferyl alcohol, this suggests that AMT and PhCFAT have different
functions in the plant cell.

	
  

24	
  

Alignment Report Report of 'Untitled' - ClustalW (Slow/Accurate, Gonnet) : Thursday,
Alignment of 'Untitled' - ClustalW (Slow/Accurate, Gonnet) : Thursday,
November 1, 2012 3:27 PM
November 1, 2012 3:27 PM
Majority
..........F.V.....E.V....P..HE........D.............Y..
Majority
..........F.V.....E.V....P..HE........D.............Y..
Kenaf.pro
MALLRPASLVFTVRRHDPELVVPSKPTPHECKTLSDIDDQDGHRFQIRGLHVYRKenaf.pro
MALLRPASLVFTVRRHDPELVVPSKPTPHECKTLSDIDDQDGHRFQIRGLHVYRPetunia.pro -----MGNTDFHVTVKKKEVVAAVLPMHHEHWLPMSNLDLLLPPLDFGVFFCYKR
Petunia.pro -----MGNTDFHVTVKKKEVVAAVLPMHHEHWLPMSNLDLLLPPLDFGVFFCYKR

54
50

54
50

Majority
.......KD....I..ALA..LV..Y..AG.............C...GV.F..A.
Majority
.......KD....I..ALA..LV..Y..AG.............C...GV.F..A.
Kenaf.pro
CNASMQGKDPVRVIREALAKALVFYYPFAGRIKEGPNRKLMVDCTGEGVLFIEAD
Kenaf.pro
CNASMQGKDPVRVIREALAKALVFYYPFAGRIKEGPNRKLMVDCTGEGVLFIEAD 109
Petunia.pro SKINNDTKDDDETIKKALAETLVSFYALAGEVVFNSLGEPELLCNNRGVDFFHAY
Petunia.pro SKINNDTKDDDETIKKALAETLVSFYALAGEVVFNSLGEPELLCNNRGVDFFHAY 105

109
105

Majority
AD..L.......HP.......L..............VL..QVT.LKCGG......
Majority
AD..L.......HP.......L..............VL..QVT.LKCGG......
Kenaf.pro
ADVMLEEFGGSLHPPFPCFKDLLCEPTGSNDLLNSPVLQIQVTRLKCGGFIFAHR
Kenaf.pro
ADVMLEEFGGSLHPPFPCFKDLLCEPTGSNDLLNSPVLQIQVTRLKCGGFIFAHR 164
Petunia.pro ADIELNNLD-LYHPDVSVHEKLIPIKKHG-------VLSVQVTGLKCGGIVVGCT
Petunia.pro ADIELNNLD-LYHPDVSVHEKLIPIKKHG-------VLSVQVTGLKCGGIVVGCT 152

164
152

Majority
F.H...DA.....F..A...IAR.........P...R.LLN.R.PP.........
Majority
F.H...DA.....F..A...IAR.........P...R.LLN.R.PP.........
Kenaf.pro
FNHTMSDAVGLIQFMSAMGEIARG-AVAPSIPPVWERHLLNARDPPLITCEHHEY
Kenaf.pro
FNHTMSDAVGLIQFMSAMGEIARG-AVAPSIPPVWERHLLNARDPPLITCEHHEY 218
Petunia.pro FDHRVADAYSANMFLVAWAAIARKDNNINTVIPSFRRSLLNPRRPPQFDDSFIDS
Petunia.pro FDHRVADAYSANMFLVAWAAIARKDNNINTVIPSFRRSLLNPRRPPQFDDSFIDS 207

218
207

Majority
...........P.D.L...........I..L......N....S......A..W..
Majority
...........P.D.L...........I..L......N....S......A..W..
Kenaf.pro
DHATATNGTIMPTDNLVHHSFFFGPTQISALKRLISDN-VSCSTFDILTACVWRC
Kenaf.pro
DHATATNGTIMPTDNLVHHSFFFGPTQISALKRLISDN-VSCSTFDILTACVWRC 272
Petunia.pro TYVFLSSPPKQPNDVLTSRVYYINSQEINLLQSQATRNGSKRSKLECFSAFLWKT
Petunia.pro TYVFLSSPPKQPNDVLTSRVYYINSQEINLLQSQATRNGSKRSKLECFSAFLWKT 262

272
262

Majority
..............L...V....R...........Y.GN.L..P......G.L..
Majority
..............L...V....R...........Y.GN.L..P......G.L..
Kenaf.pro
RTIAMKLGPDEDVRLICIVN--ARYKFNPPLPLG-YYGNALGYPAALTTAGELSK
Kenaf.pro
RTIAMKLGPDEDVRLICIVN--ARYKFNPPLPLG-YYGNALGYPAALTTAGELSK 324
Petunia.pro -IAEGGIDDSKRCKLGIVVDGRQRLRHDSSTTMKNYFGNVLSVPYTEASVGQLKQ
Petunia.pro -IAEGGIDDSKRCKLGIVVDGRQRLRHDSSTTMKNYFGNVLSVPYTEASVGQLKQ 316

324
316

Majority
.PL.....LV.........E.........V...RP.............N......
Majority
.PL.....LV.........E.........V...RP.............N......
Kenaf.pro
KPLEYAVKLVKEAKAKATDEYMKSTADLMVSRGRP-----------NVNTVRSFL
Kenaf.pro
KPLEYAVKLVKEAKAKATDEYMKSTADLMVSRGRP-----------NVNTVRSFL 368
Petunia.pro TPLGKVADLVHTCLDNVANEHHFPSLIDWVELHRPRQAIVKVYCKDECNDEAAIV
Petunia.pro TPLGKVADLVHTCLDNVANEHHFPSLIDWVELHRPRQAIVKVYCKDECNDEAAIV 371

368
371

Majority
VS...R.....V.FGWG...FG....P..G........PS.NK.G..........
Majority
VS...R.....V.FGWG...FG....P..G........PS.NK.G..........
Kenaf.pro
VSDLSRARFREVDFGWGKAEFGG---PSNGTEIISFYIPSKNKEG-----KEGIA
Kenaf.pro
VSDLSRARFREVDFGWGKAEFGG---PSNGTEIISFYIPSKNKEG-----KEGIA 415
Petunia.pro VSSGLRFPLSQVNFGWGCPDFGSYIFPWGGQTGYVMPMPSPNKNGDWIVYMHLQK
Petunia.pro VSSGLRFPLSQVNFGWGCPDFGSYIFPWGGQTGYVMPMPSPNKNGDWIVYMHLQK 426

415
426

Majority
....L.................D..AT..
Majority
....L.................D..AT..
Kenaf.pro
VPVCLPASVMESFVKEINSTLADDEATGA
Kenaf.pro
VPVCLPASVMESFVKEINSTLADDEATGA
Petunia.pro KHLDLVETRAPHIFHPLTACYLDLTATY
Petunia.pro KHLDLVETRAPHIFHPLTACYLDLTATY

444
454

444
454

Figure 1.8 Peptide Sequence Alignment of Kenaf AMT and Petunia PhCFAT. Sequences were
aligned using MegAlign (DNASTAR). Dots denote non-matching residues. Dashes signify gaps
inserted to obtain optimal sequence alignment. The HXXXD and DFGWG motifs are underlined.

	
  

25	
  

To conclude, plants have evolved mechanisms for altering their lignin structure by the
utilization of monolignol substitutes or acylation modifications to monolignols. In some cases,
plants have substituted traditional monolignol precursors with upstream intermediates in the
monolignol biosynthetic pathway by down regulating biosynthetic enzymes. Additionally, in
many plants, monolignols can be pre-acylated prior to incorporation into the lignin polymer. In
both scenarios, the resulting lignin polymer is structurally different from the classical lignin
structure but may confer similar functional and morphological qualities to the plant.
The functional significance of lignin acylation is one of the most intriguing unanswered
questions in plant cell wall biology. Elucidating the mechanisms and enzyme(s) involved in
monolignol acylation will provide an excellent foundation for uncovering their role in lignin
structure and function. Kenaf represents a particularly fascinating plant system due to its high
syringyl content and extensive acetylation of syringyl units. The kenaf AMT enzyme is involved
in the acetylation of sinapyl alcohol. Future studies on AMT will detail the functional
consequences of this gene in planta. Ultimately, our objective is to understand the mechanism of
monolignol acylation and whether changes to acylation levels in plants would prompt facile
processing of plant biomass into biofuels.

	
  

26	
  

1.5 FUTURE STUDIES
AMT has been cloned into three plant expression vectors: pk2GW7, pk7YWG, and
pk7WGY (39). These vectors contain the 35S Cauliflower Mosaic Virus (CaMV) promoter and
are distinguished from one another in that pk7YWG has a N-terminal yellow fluorescence
protein (YFP) fusion tag, pk7WGY has a C-terminal YFP fusion tag, and pk2GW7 does not have
a YFP fusion tag. The three vectors were selected because it was not known whether the YFP tag
would interfere with AMT expression in planta. However, AMT was successfully expressed in a
N-terminal His-tagged expression vector, which suggests that expression would likely not be
interrupted with a N-terminal tag. The YFP-tagged vectors will be useful in analyzing transgenic
plant tissue for localization of AMT expression.
The AMT overexpression vectors were transformed into arabidopsis. Currently, I am in
the process of screening arabidopsis seedlings for AMT expression. Plants from this study will
be analyzed for: 1) lignin acetylation, 2) lignin composition, 3) cell wall digestibility, and 4)
physical and physiological features. The DFRC´ method will be utilized to detect acetylation
levels in lignin from AMT transgenic and wild type plants. Higher acetylation levels are
expected in the transgenic plants, due to AMT’s ability to catalyze acetylation reactions.
Particularly, acetylation is expected on syringyl units. Through lignin composition analysis, the
plants will be analyzed for syringyl content. Studies have shown that lignin in Arabidopsis
thaliana has a low S/G ratio (40,41), while kenaf lignin has a high S/G ratio (26,28,32). It would
be interesting to determine whether AMT expression in arabidopsis will yield a greater S/G ratio
when compared to wild type. A greater S/G ratio in AMT transgenic plants would support the
notion that AMT’s preference for sinapyl alcohol might be the driving force behind the

	
  

27	
  

formation of lignin with high syringyl content. This may have large implications for biofuels
since lignin with high syringyl content may produce uncondensed lignin that is easier to process.
The AMT transgenic plants will be analyzed for cell wall digestibility at the Great Lakes
Bioenergy Research Center (GLBRC) Cell Wall Analytical Facility to determine whether AMT
expression has the potential to modify lignin structure and generate plants with increased cell
wall digestibility. It may be that altering the level of acetylation on lignin may create a lignin
structure that provides greater access to cellulose; thus, allowing for increased sugar yields. In
addition, observing the physical and physiological characteristics of these plants could provide
clues into the functional role of AMT in planta. Particularly, I would like to know whether AMT
expression has an effect on stem strength or drought tolerance. If a reduction in stem strength is
observed, this may suggest that lignin acetylation generates lignin that is easier to break down for
access to cellulosic sugars. However, if stem strength is reduced to a significant degree, it may
have negative effects on plant structure. These studies will provide crucial information for the
implications of altering lignin acylation in plants. Furthermore, since lignin acetylation has been
associated with drought tolerance (28), it would be interesting to determine whether the AMT
transgenic plants are more drought tolerant than wild type. Overall, these future studies will shed
light on AMT’s functional role in kenaf and possibly other plant species.

	
  

28	
  

CHAPTER TWO

Characterization of the Sweet Cherry Pollination System

	
  

29	
  

2.1 INTRODUCTION
Self-incompatibility (SI) is a genetically controlled system for mate selection found in
angiosperms that prevents inbreeding by the rejection of self-fertilization. SI also promotes outcrossing between genetically unrelated plants of the same species, which generates and maintains
genetic diversity within the plant kingdom. This genetic diversity has allowed angiosperms to
thrive for millions of years and led to their dominance of most terrestrial ecosystems (42). In
gametophytic self-incompatibility (GSI), the SI reaction is determined by the haploid genotype
of the pollen and its interaction with the diploid tissue of the pistil. Pollen is rejected if there is a
match between the S-allele of the haploid pollen and one of the two S-alleles expressed in the
pistil. GSI is controlled by a single multi-allelic S-locus that includes at least two genes: a pistil-S
gene and a pollen-S gene, which control pistil and pollen specificity, respectively. The close
proximity of the pistil-S and pollen-S genes on the S-locus allows for these genes to co-segregate
as a single gene. Variants of the S-locus are referred to as a “haplotype” (43).
GSI is split into two systems, each of which is classified by its pistil-S determinant
molecule. In “Papaveraceae-type SI”, the pistil-S encodes a set of S-proteins, which are thought
to be involved as signaling molecules that aid in the recognition of pollen-S allele specificity
(44). The other system is generally referred to as S-RNase-based SI because the pistil-S encodes
a ribonuclease (S-RNase), which exerts its cytotoxicity on pollen with an identical S-allele. SRNase-based SI has also been coined “Solanaceae-type SI” because it was first discovered in P.
inflata and then extensively characterized in several members of the Solanaceae family,
including potato, tomato, and tobacco (45). In spite of this, members of the Rosaceae and
Plantaginaceae families also exhibit S-RNase-based SI, which demonstrates how “Solanaceaetype SI” is an inaccurate label for S-RNase-based SI.

	
  

30	
  

Genetic and molecular evidence for GSI in the Solanaceae and Prunus, a genus in the
Rosaceae, indicates that there are major differences between the SI mechanisms of these groups.
First, pollen grains containing two copies of the pollen-S allele give rise to different SI
phenotypes in the Solanaceae and Prunus due to a phenomenon called competitive interaction,
wherein breakdown of GSI occurs as a result of polyploidization. This phenomenon does not
confer self-compatibility in Prunus, whereas in the Solanaceae, self-compatibility has been
observed in species that have undergone genome duplication (46-48). Competitive interaction is
dictated by the S-genotype of the pollen, such that the presence of two distinct S-alleles
(heteroallelic) in the same pollen grain allows for full compatibility with a style of any S-allele
type. However, a pollen grain containing two of the same S-alleles (homoallelic) is rejected by a
style of the same S-genotype (Figure 2.1).
a. Solanaceae
1

2

1,1

1,2

2,2

Polyploidy

S1S1S2S2

S1S2

Figure 2.1 Genetic Representation for Competitive Interaction in the Solanaceae. A breakdown

b. Prunus

in pollen-part SI function confers self-compatibility in heteroallelic pollen. Conversely,
1,2 2,2
1
2
1,1 1,2 2,2
a,1 a,2 1,2 2,2
homoallelic pollen retains its self-incompatibility and as a result, is rejected by the style. Prunus
does not become self-compatible as a result of competitive interaction. This figure was adapted
from Hauck et al. (2006) (49).

	
  

Polyploidy

31	
  

First
mutation

Se
mut

Secondly, studies on competitive interaction have demonstrated that pollen-part

a. Solanaceae

mutations (PPM) in Prunus and the Solanaceae lead to two distinct outcomes in these groups.
1
2
1,1 1,2 2,2
PPM in Prunus confer self-compatibility, whereas PPM in the Solanaceae are predicted to be
pollen-lethal (43,47-50). Studies in Prunus found that the breakdown of SI in tetraploid sour
cherry was due to the accumulation of nonfunctional S-haplotypes in the pistil and pollen. These
Polyploidy
observations were used to develop the “one-allele-match” model, wherein any single functional

S-allele match between pollen and pistil would result in an incompatible reaction (Figure 2.2)
(49). In contrast, studies in Nicotiana alata have not been able to produce “true” pollen-S
S1S1S2S2
S1S2
mutants, which suggests that the pollen-S is necessary for pollen viability and survival (48).

b. Prunus
1

2

1,1

1,2

2,2

First
mutation

Polyploidy

S1S2

a,1 a,2 1,2 2,2

S1S1S2S2

a,c a,2 1,2 2,2

Second
mutation

SaS1S2S2

SaScS2S2

Figure 2.2 Genetic Representation for the “One-Allele-Match” Model in Tetraploid Sour
Cherry. Two nonfunctional S-haplotypes must be present in order for a breakdown in SI to occur.
Any single S-allele match between pollen and pistil confers self-incompatibility and
subsequently, rejection of pollen by the style. Lowercase letters in the pollen and style indicate
nonfunctional S-alleles. This figure was adapted from Hauck et al. (2006) (49).

	
  

32	
  

Lastly, there is evidence to suggest that non-S-specific factors are involved in the SI
mechanisms of Solanaceous species. HT-B, an asparagine-rich protein expressed in the style
(51), and the 120 kDa (120K) glycoprotein, an arabinogalactan protein in the style that is taken
up by pollen tubes (52,53), have been implicated in contributing to the SI response. RNA
interference (RNAi)-mediated suppression of HT-B and the 120K protein in Nicotiana and
Solanum chacoense both produced similar results in that S-specific pollen rejection was
disrupted (51,54-56). Interestingly, these factors have not been identified in Prunus.
This study reports on an analysis of pollination in sweet cherry (Prunus avium). Sweet
cherry was chosen as our model GSI system due to its regional availability and extensive
characterization of its S-RNases and SFBs (57-60). First, a laboratory pollination method was
established in order to determine an accurate timing of pollination in sweet cherry. Second, using
the laboratory pollination method, a deep proteome profile of un-pollinated (control) and
pollinated cherry style tissue was developed across different time points. It is known that the
pollen-S encodes a F-box protein (SFB) (43,50,61,62), which is a component of the SCF
complex that targets proteins for degradation via ubiquitination. Thus, a comprehensive analysis
of the sweet cherry proteome may help identify targets of ubiquitination by SFB. In addition, this
data set can be utilized in the identification of candidate genes involved in GSI. These studies
have not yet been described in sweet cherry and as such, the data presented here will provide
researchers with a framework for effective laboratory pollinations of sweet cherry and deep
proteome profiling data for the identification of S-specific and non-S-specific factors involved in
the SI response of Prunus.

	
  

33	
  

2.2 MATERIALS AND METHODS
Plant Material
Four sweet cherry varieties were used for this study: ‘Bing’ (S3S4), ‘Brooks’ (S1S9), ‘Emperor
Francis’ (EF; S3S4), and ‘NY54’ (NY; S2S6). All the plant material used in these experiments is
planted at the Clarksville Horticultural Experiment Station, Clarksville, Mich., and the
Southwest Michigan Research and Extension Center, Benton Harbor, Mich.

Pollen Collection
Cut branches were brought into the lab and forced indoors at room temperature. Open flowers
were removed to avoid cross-pollination from field pollinators. Pollen was collected from newly
opened flowers and allowed to dry for two days. After two days, the pollen was transferred into a
glass vial, sealed in a Drierite-filled bag, and stored at 4 °C until use.

Pollen Germination Assays
Two drops of germination media (10% sucrose, 100 ppm boric acid, 300 ppm calcium nitrate,
200 ppm magnesium sulfate, 100 ppm potassium nitrate, pH 7.3, autoclaved 25 min) were placed
onto a microscope slide using a Pasteur pipette. Two to three individual anthers were added to
the germination media and crushed with a glass rod to release pollen. Microscope slides were
placed on top of a Petri dish and into a plastic container filled with damp paper towels to control
humidity. Samples were incubated for 2 h in the sealed, humid container. Cover slips were
placed on the microscope slides and germination was observed using a compound microscope.
Percent germination was recorded for a subset of free pollen observed for each pollen variety.

	
  

34	
  

Pollination Assays
Pollinations were performed as described in Yamane et al. (2001) (63) with some modifications.
Flowers to be used for the pollination studies were emasculated and hand-pollinated when
receptive (24 h after emasculation). Pistils were pollinated in the lab with self- and non-self
pollen. The control pistils were not pollinated. Pistil collection took place after the indicated time
point (3, 6, and 12 h in 2011; 12 and 14 h in 2012). A sample of ten pistils per experiment was
collected and transferred to a fixing solution (1 chloroform: 3 (95%) ethanol: 1 glacial acetic
acid) for 24 h. The fixing solution was removed and the pistils were rinsed with 10N NaOH then
submerged completely in 10N NaOH for 24 h. After removal of NaOH, the pistils were rinsed 5
times with distilled water before being exposed to 0.1% aniline blue solution (in 33 mM K3PO4)
for 24 h. Pollen tubes were visualized using confocal laser scanning microscopy.

Pollen Tube Analysis via Confocal Laser Scanning Microscopy
An Olympus FluoView FV1000 confocal laser scanning microscope at the MSU Center for
Advanced Microscopy was used to analyze stained pollen tubes. Fluorescence images were
acquired using the “DAPI” DyeList selection. Differential Interference Contrast (DIC) was used
in combination with fluorescence imaging to acquire overlay images of the pollen tubes. Pollen
tubes were mounted onto microscope slides in 30% glycerol at the time of analysis. All pollen
tubes were analyzed within 1-week of initial staining.

Protein Extraction of Cherry Style Tissue
Pistils were ground to a fine powder in liquid nitrogen, enough to fill up to the 500 µL mark of a
1.5 mL microcentrifuge tube. Approximately 450 µL of 4X SDS buffer (50 mM Tris-HCl, pH

	
  

35	
  

6.8, 20% SDS, 40% glycerol, 0.1% bromophenol blue, and 2% beta-mercaptoethanol) was added
to cover the ground plant tissue and immediately vortexed until the plant material was fully
dissolved in SDS buffer. The samples were then sonicated for 15 min and stored at 4 °C until
use. The cherry protein samples were analyzed for degradation by SDS-PAGE, wherein a
standard of volumes was loaded on a SDS-PAGE gel to quantify protein abundance in each
sample. The protein extraction experiments were performed in duplicate to obtain two biological
replicates for proteome analyses.

Experimental LC/MS/MS
SDS-PAGE gels containing cherry style protein from control, non-self and self pollination
experiments were subjected to in-gel tryptic digest according to Shevchenko et al. (1996) (64)
with modifications, followed by LC/MS/MS at the MSU Proteomics Core. Data processing of
the LC/MS/MS peptide fragments was performed using BioWorks Browser, which generated a
Mascot Generic Format (MGF) text file utilized for peptide identification against the cherry
database. The results were loaded into Scaffold, v3.6.0 (Proteome Software), a tool that compiles
large amounts of proteomic data for visualization and analysis. Scaffold was utilized to filter the
proteomic data for the identification of post-translational modifications, such as phosphorylation
and ubiquitination, in cherry style protein.

	
  

36	
  

2.3 RESULTS
Pollen Germination Assays
The pollen germination experiments were performed over two harvest seasons. In 2011, the
pollen germination range was 20% – 35% for pollen collected from Brooks, EF, and NY54. In
2012, the pollen germination range was 11% – 37% for pollen collected from these same cherry
varieties. Pollen with a germination percentage of over 20% was used for the pollination
experiments. The cherry variety used for the pollen parent was dependent on the availability of
the maternal parent. In 2011, the pollination experiments were EF (S3S4) × EF (S3S4) and EF
(S3S4) × Brooks (S1S9). In 2012, the pollination experiments were EF (S3S4) × EF (S3S4), EF
(S3S4) × Brooks (S1S9), Bing (S3S4) × EF (S3S4), and Bing (S3S4) × Brooks (S1S9).

Table 2.1 Pollen Germination Percentage Values for Pollen Parents of Pollination Experiments
Pollen Parent

S-Genotype

Germination Percent

EF_1*

S3S4

25.0

S3S4

36.5

EF_2

S3S4

18.8

EF_3

S3S4

23.1

EF_4

S3S4

18.0

Brooks_1*

S1S9

24.5

S1S9

24.3

Brooks_2

S1S9

10.5

Brooks_3

S1S9

23.4

Note: Pollen parents were numbered to denote separate pollen grain collections. Shaded boxes
represent a pollen grain collection that was combined with the preceding pollen sample. The
asterisk denotes pollen parent samples that were used in the 2012 pollination experiments.

	
  

37	
  

Pollination and Pollen Tube Growth Analysis
In previous years, pollination experiments were not successful due to a complete lack of pollen
grains adhered to the stigma surface. The likely cause of this was that the cherry styles were
pollinated before full maturity; thus, affecting the production of exudates at the stigma surface
for pollen grain adhesion. In 2011 and 2012, cherry styles were prepared 24 h in advance and
examined for exudates production prior to beginning the pollination experiments. A comparative
analysis of stained control (unpollinated) and pollinated cherry styles confirmed that the
pollination experiments were successful (Figure 2.3). Stained cherry styles from the control
experiments revealed an absence of pollen grains on the stigma surface (Figure 2.3A – B). Pollen
grains and pollen tubes were identified on stained cherry styles from both incompatible and
compatible pollination experiments, indicating that pollen grain germination and pollen tube
growth had taken place. Figure 2.3C – D illustrates germinated pollen grains at the stigma
surface and pollen tubes growing from the pollen grains in cherry styles from an incompatible
pollination. Similar results were observed in cherry styles from compatible pollinations. The
pollen grains were circular and spread across the top of the stigma (Figures 2.4A and 2.5). Figure
2.4C – D shows the presence of pollen grains on the styles of two different pollination
experiments. These figures also demonstrate the absence and presence of pollen grain
germination, indicating that germination did not occur as a direct result of pollen grain adhesion
to the stigma. As such, it was critical to analyze a subset of cherry styles from all control and
pollination experiments to ensure that pollen grains had germinated. The pollen tubes appeared
thin and irregularly shaped in comparison to the stylar transmitting tissue, which was noticeably
thicker and less irregular in shape as demonstrated in Figure 2.6.

	
  

38	
  

A

B

C

D

Figure 2.3 Control and Pollinated Cherry Styles Illustration of the difference between control
(unpollinated) and pollinated cherry styles. A) Bing (S3S4) 24 h control, B) EF (S3S4) 24 h
control, C) 12 h Bing (S3S4) × EF (S3S4), and D) 24 h EF (S3S4) × EF (S3S4). For this and all
other confocal microscope figures, overlay images were produced from fluorescence and
Differential Interference Contrast (DIC) images. All scale bars are 200 µm.

	
  

39	
  

A

B

C

D

Figure 2.4 Pollen Grain Adhesion on Cherry Style and Pollen Germination Images show
adhesion of pollen grains on the stigma surface, pollen germination, and pollen tube growth
through the stigma surface. A) Pollen grains on the surface of a style from 12 h EF (S3S4) ×
Brooks (S1S9), B) Pollen tube growth was arrested mid style within 24 h EF (S3S4) × EF (S3S4),
C) Non-germinated pollen grains on a style in 12 h EF (S3S4) × NY (S2S6), and D) Germinated
pollen grains on a style in 12 h EF (S3S4) × EF (S3S4).
	
  

40	
  

A

B

C

D

E

F

Figure 2.5 Pollen Germination on Cherry Style Surface A-C) 24 h EF (S3S4) × NY (S2S6) and D-F) 12 h Bing (S3S4) × EF (S3S4).
	
  

41	
  

A

B

C

D

E

F

Figure 2.6 Pollen Tube Growth within Cherry Style A-C) 24 h EF (S3S4) × EF (S3S4) and D-F) 12 h Bing (S3S4) × Brooks (S3S4).
	
  

42	
  

Post-Pollination Time Course Analysis
It was crucial to determine an appropriate post-pollination harvest time that would allow for
analysis of fully-grown pollen tubes. Initially, pollinated cherry styles were collected at 3 and 6 h
post-pollination. However, these time ranges were not sufficient in length to allow for pollen
grain germination and subsequent pollen tube growth down the style. These cherry styles had
marginal or completely lacked pollen tube growth. In the rare case of pollen tube growth, the
pollen grains had begun to germinate but pollen tubes had not yet penetrated the stigma surface.
Cherry styles harvested at 12 and 24 h post-pollination revealed that these time ranges were
appropriate for detection of pollen tube growth (Figure 2.3C – D). Pollen tube growth in cherry
styles from the 12 h compatible and incompatible pollinations was measured at the lower 1/3 to
1/2 portion of the style. At 24 h, full pollen tube growth was observed in cherry styles from the
compatible pollination experiments only (Figure 2.7). Cherry styles from the 24 h incompatible
pollination experiments exhibited pollen tube growth; however, these pollen tubes did not grow
beyond half the length of the style (Figures 2.4B and 2.8C – D). This observation is commonly
referred to as the “abortion phenomenon”, wherein pollen tube growth is arrested approximately
halfway down the style in incompatible pollinations (65).

Proteomics Analysis of Unpollinated and Pollinated Cherry Styles
A representation of the proteomics data can be found in Appendix B. This subset of the
proteomics data, along with all other sets for these experiments, illustrates the number of spectra
detected within each pollination experiment (control, compatible, and incompatible) for each
identified protein. Identified proteins were annotated according to the cherry EST database,
which utilizes TAIR to annotate ESTs according to their closest homolog in arabidopsis.

	
  

43	
  

A

B

C

D

Figure 2.7 Full Growth of Pollen Tube Down Cherry Style A) Overlay image of pollen grains
on stigma surface and pollen tube growth in 24 h EF (S3S4) × Brooks (S1S9). Pollen tubes grew
down the entire length of the cherry style in 24 h EF (S3S4) × Brooks (S1S9) as shown by B)
fluorescence, C) DIC, and D) fluorescence-DIC overlay images.

	
  

44	
  

A

B

C

D

Figure 2.8 Arrest of Pollen Tube Growth at Mid Stylar Region Overlay images of pollen
grains on stigma surface and pollen tube growth in incompatible pollinations. A) 24 h Bing
(S3S4) × EF (S3S4) and B) 24 h EF (S3S4) × EF (S3S4). Pollen tube growth was arrested at mid
style in C) 24 h Bing (S3S4) × EF (S3S4) and B) 24 h EF (S3S4) × EF (S3S4).

	
  

45	
  

2.4 DISCUSSION
Interestingly, there were notable differences in pollen tube growth for the pollination
experiments performed in 2012. At 12 h, pollen tube growth for EF (S3S4) × EF (S3S4) was at
1/3 down the style, whereas in Bing (S3S4) × EF (S3S4), pollen tube growth was at 1/3 to 1/2
down the style. In pollen tubes for EF (S3S4) × Brooks (S1S9), growth was observed at 1/2 down
the style, while growth was observed at 3/4 down the style in Bing (S3S4) × Brooks (S1S9) pollen
tubes. The same Brooks and EF pollen samples were used in all the pollination experiments
performed. Thus, the differences observed in pollen tube growth for each time point experiment
cannot be largely attributed to inconsistencies across pollen samples. Also, the Bing pollination
experiments were performed days after the EF experiments, which would suggest that pollen
viability might have decreased during this time. However, longer pollen tube growth was
observed in the Bing experiments, indicating that pollen viability had not decreased significantly.
It may be that the Bing styles were more receptive than the EF styles, allowing pollen grains to
germinate and begin the growth process sooner in the Bing styles. This could explain why pollen
tubes grew longer in the Bing experiments, even though similar time points were used and pollen
viability may have been reduced. Nevertheless, it would be interesting to determine whether the
maternal parent may be more receptive to pollen from particular cherry varieties.
In depth proteome analysis gave interesting results from the control and experimental
pollinations that lend support to the S-RNase sequestration model. The sequestration model
suggests that S-RNases enter the pollen tube by endocytosis and undergo retrograde transport to
vacuoles. Studies on S-RNase uptake have demonstrated that S-RNases enter pollen tubes in a
genotype independent manner (66) and are sequestered within a vacuolar compartment in pollen

	
  

46	
  

tubes (65). It has also been shown that the S-RNase-containing compartment disrupts late in
incompatible pollinations (65). The observation that incompatible pollen tubes exhibit
compartment disruption suggests that S-RNases are able to exert their cytotoxic activity upon
release into the pollen cytosol. A similar mechanism has been found in the cytotoxin ricin from
the castor oil plant Ricinus communis, which enters the cell by endocytosis and is sorted to a
vacuole (65,67). It may be that the mechanism exhibited by ricin is a good model for S-RNase
compartmentalization in GSI. In previous years, the mono-ubiquitination of S4-RNase was
detected in a proteome analysis of control styles. This finding was fascinating because monoubiquitinated proteins in yeast are targeted to the vacuole instead of the proteasome (68). It may
be that S-RNase mono-ubiquitination may target S-RNases to the vacuole, wherein S-RNases are
sequestered until release into the pollen cytosol.
Other proteins annotated as “aerolysin pore-forming” and “porin” in the proteomics data
may be involved in the release of sequestered S-RNases in pollen tubes. The finding of an
aerolysin protein was interesting in that these pore-forming toxins are secreted as soluble
proteins and eventually become a transmembrane channel in the target cell (69). Spectra for the
aerolysin protein were 1.5X greater in incompatible styles than control and compatible styles. It
could be that an aerolysin protein is activated in incompatible pollinations to form pores for the
release of S-RNases into the pollen tube cytosol. A bioinformatic analysis of the “porin” protein
showed that it was specific to Prunus and the proteomics data demonstrated that it was twice as
abundant in incompatible styles than control styles. This raises the question of whether the porin
protein is specific to S-RNase release in Prunus. Overall, the candidate proteins presented here,
and others in the proteomics data, warrant further study for possible associations with S-specific
and non-S-specific mechanisms in Prunus.

	
  

47	
  

To conclude, it is clear that distinct SI mechanisms may exist between Prunus and the
Solanaceae. In Solanaceous species, competitive interaction confers self-compatibility and PPMs
are predicted to be pollen-lethal. Conversely, in Prunus, genome duplication does not confer
self-compatibility, whereas PPMs confer self-compatibility according to the “one-allele-match”
model. In order to gain insight on GSI in Prunus, this study provides an in-depth characterization
of the sweet cherry pollination system and a deep proteome analysis of cherry style tissue under
different experimental conditions. Previous work on this project has also generated an expressed
sequence tag (EST) database of cherry styles. Future studies in sweet cherry can utilize the data
presented here to establish an accurate timing for laboratory pollinations and explore the sweet
cherry proteome and transcriptome for the identification of S-specific and non-S-specific factors
involved in the SI response of Prunus.

	
  

48	
  

2.5 FUTURE STUDIES
The next step for characterization of the sweet cherry pollination system will involve cell
biological techniques to elucidate on S-RNase uptake and sequestration in sweet cherry. First, it
will be important to establish whether S-RNase uptake into pollen tubes occurs in a S-genotype
independent manner. Luu et al. (2000) utilized immunocytochemical labeling of pollen tubes
from Solanum chacoense and S-RNase antibodies to determine that S-RNases were present in the
cytosol of pollen tubes from compatible and incompatible crosses, demonstrating that S-RNase
uptake occurs in a S-genotype independent manner (66). The mechanism of S-RNase uptake has
not been described in sweet cherry and as such, it would be interesting to confirm whether SRNase uptake in sweet cherry is similar to what has been described in the Solanaceae.
Second, it is critical to find out what occurs to S-RNases once they have entered the
pollen tube. Goldraij et al. (2006) demonstrated that S-RNases were compartmentalized in a Sgenotype dependent manner within a vacuolar compartment in Nicotiana alata pollen tubes (65).
Using immunolabeling techniques and S-RNase antibodies, S-RNase compartmentalization was
shown to occur in compatible pollen tubes, while compartment breakdown was observed in
incompatible pollen tubes. These findings suggest that the S-RNase sequestration model may
explain the mechanism of GSI in the Solanaceae. However, the same argument cannot be made
for the mechanism of GSI in sweet cherry because S-RNase sequestration studies have not been
performed in this system. As such, it will be important to determine whether S-RNases remain
sequestered in the vacuole of pollen tubes in a S-genotype dependent manner.
Studies on S-RNase uptake and sequestration in sweet cherry can be performed by
immunolabeling techniques similar to those described above. Candidate genes can be identified
using the cherry EST database for the production of antibodies. These should include S-RNases

	
  

49	
  

of different S-genotypes and membrane proteins found in the plasma membrane and tonoplast,
such as “plasma membrane intrinsic protein” (PIP) and “tonoplast intrinsic protein” (TIP). The
nucleotide sequences for these genes can be codon-optimized and synthesized by GENEART for
Gateway cloning and subsequent heterologous expression in E. coli. Purified soluble protein
obtained from E. coli expression can be sent to GenScript (Piscataway, New Jersey) for custom
antibody production. Additionally, it would be beneficial to obtain an anti-callose antibody from
Biosupplies Australia for the identification of callose in the walls of pollen tubes.
The antibodies for S-RNases, callose, plasma membrane and tonoplast proteins, along
with confocal microscopy to detect immunofluorescence, can be utilized to analyze cherry style
tissue for determination of whether S-RNase uptake occurs in a S-genotype independent manner
and whether S-RNases are sequestered in a vacuolar compartment within compatible pollen
tubes. Cherry styles from different time points can be analyzed by immunofluorescence to obtain
a time course for S-RNase uptake and compartmentalization. Further, S-RNase compartment
breakdown might be observed in incompatible pollen tubes, if S-RNase compartmentalization is
similar to the mechanism described in the Solanaceae.
It may be that S-RNase uptake and sequestration in Prunus is similar to the Solanaceae.
However, there is evidence to suggest that different biochemical factors may be involved in the
recognition and rejection processes. HT-B, an asparagine-rich protein expressed in the style, has
been implicated in the SI response in Nicotiana and S. chacoense (51,54-56). Yet, HT-B has not
been identified in Prunus. Thus, it is important to determine the mechanism of S-RNase uptake
and sequestration in Prunus in order to carry out further analyses on the biochemical factors
involved in GSI. These future studies will have significant impacts on plant reproductive biology
in that they will help elucidate the mechanism of GSI in Prunus.

	
  

50	
  

APPENDICES

	
  

51	
  

APPENDIX A

Identification of an O-Methyltransferase Involved in Monolignol Biosynthesis in Rice

	
  

52	
  

A.1 INTRODUCTION
The significance of lignin acylation has not been established primarily due to not
knowing the enzyme(s) responsible for acylation of monolignols. This study addresses the
question of the role of lignin acylation by identifying the enzymes involved in acylation
reactions. Our laboratory has identified a p-coumaroyl monolignol transferase in Oryza sativa
(OsPMT) as an enzyme implicated in the p-coumaroylation of monolignols, a phenomenon that
is unique to grasses (17-19,70). OsPMT was most effective at producing sinapyl p-coumarate
and p-coumaryl p-coumarate. It has been proposed that OsPMT adds p-coumarates onto
monolignols after the reduction of hydroxycinnamyl intermediates by F5H and CAD.
Interestingly though, OsPMT co-expresses with C3H rather than F5H and CAD. Additionally,
while OsPMT has a higher affinity (lower Km) for sinapyl alcohol, OsPMT has a faster reaction
rate (higher Vmax) with p-coumaryl alcohol. This raises the question of whether the
transesterification of p-coumaroyl CoA by HCT is bypassed and p-coumaryl p-coumarate is used
as substrate for the synthesis of monolignols in grasses. It may be that caffeyl p-coumarate or pcoumaryl caffeate, the products of 3´-hydroxylation of p-coumaryl p-coumarate are the targets of
3´-O-methylation activity by an O-methyltransferase (OMT) to produce coniferyl p-coumarate
and p-coumaryl ferulate, respectively. Monolignols would then be produced by removal of the pcoumarate ester by OsPMT (Figure A.1).
This study reports on a grass-specific OMT found in O. sativa, herein called OsOMT,
which is co-expressed with several monolignol biosynthetic genes and catalyzes the synthesis of
coniferyl p-coumarate and p-coumaryl ferulate. The results show that OsOMT may be involved
in the 3´-O-methylation of monolignol conjugates in grasses. Furthermore, these observations
support the hypothesis that grasses may have an alternative pathway to lignin biosynthesis.

	
  

53	
  

A)

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"#
&'()*+,-./5,/()8)/

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&'()*+,-./
&'()*+,-,6%

Figure A.1 Hypothetical Monolignol Biosynthetic Pathway in Grasses. A, OsPMT effectively
"#
"#
utilizes p-coumaroyl CoA and p-coumaroyl alcohol to produce p-coumaryl p-coumarate. B, p-

"#

>7#
0"$=
Coumaryl p-coumarate is hypothesized to be the target of 3´-hydroxylation by C3H to produce
caffeyl p-coumarate or p-coumaryl caffeate. The reaction scheme presented here implicates the
"$%
"$%
#"
$%"
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synthesis of caffeyl p-coumarate. After C3H activity, the monolignol conjugates would then ;32,&./5,/()8)/
()234%-./5,/()8)/
7'8.9-):.()234%-./5,/()8)/
undergo 3´-O-methylation by an OMT to produce coniferyl p-coumarate or p-coumaryl ferulate.

?$=
?$=
"#
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Monolignols would then be produced by removal of the p-coumarate ester by OsPMT. The "#
"
"

original CoA thioester compounds and monolignol alcohols are highlighted in red and blue,
"
"
"
respectively. The dashed arrow represents a reaction that has not yet been tested using OsPMT.
"
"
"
0<#
00)1"$=
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Compounds that are not commonly observed in grass lignin are highlighted in grey. This figure
was adapted from Dr. John Ralph.
"#

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)*+,-./
)*+,-,6%

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"

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()234%-./5&'()*+,-,6% 7'8.9-):.()234%-./5&'()*+,-,6% ;32,&./5&'()*+,-,6%

54	
  

"#
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Figure A.1 (cont’d)
B)
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()234%-./5&'()*+,-,6% 7'8.9-):.()234%-./5&'()*+,-,6% ;32,&./5&'()*+,-,6%

55	
  

"#

A.2 MATERIALS AND METHODS
Rice Co-Expression Analysis
The Rice Oligonucleotide Array Database (www.ricearray.org) was utilized to identify OMT
gene candidates that co-expressed with monolignol biosynthetic genes, such as 4CL. Other genes
of interest were annotated as putative O-methyltransferases. Protein BLAST searches were
performed to identify OMT gene candidate homologs in arabidopsis.

Gene Synthesis and Gateway Cloning
OsOMT was submitted to GENEART for E. coli codon optimization and chemical synthesis.
Gateway cloning for OsOMT was performed as previously described (pg. 11) with one
modification. The His-tagged expression clone was transformed into BL21 chemically
competent cells (Invitrogen, Carlsbad, California). ATOMT1 was cloned into pDEST17 from
arabidopsis stem cDNA and transformed into BL21 pLysS chemically competent cells.

Heterologous Expression in E. coli
OsOMT protein expression was performed as described on pg. 12 with some modifications. One
100 mL culture was used to grow the His-tagged OsOMT clone. After IPTG induction, the
culture was grown for 4 h at 18 °C. ATOMT1 protein was expressed as described above;
however, ATOMT1 was grown for 6 h at 37 °C after IPTG induction.

Protein Purification by FPLC
Five milliliters of soluble His-tagged OsOMT and ATOMT1 protein were used for FPLC
purification. FPLC purification was performed as described on pg. 12 with a minor change to the

	
  

56	
  

elution buffer for both protein purifications. The imidazole concentration of buffer A was
decreased to 20 mM. Protein bands resulting from each purified fraction were compared to the
expected molecular weight for OsOMT and ATOMT1 (ca. 40 kDa). Protein bands at about 40
kDa were extracted from the SDS-PAGE and analyzed by in-gel trypsin digestion and
LC/MS/MS. OsOMT peptides were identified against rice and E.coli databases using Mascot.
ATOMT1 peptides were identified using arabidopsis and E.coli databases. Fractions with the
proteins of interest were buffer exchanged into 100 mM phosphate buffer, pH 6 (with protease
inhibitors) using an Amicon 10K membrane filter. The enzymes were stored at -80 °C until use.

Enzyme Activity Assays
OsOMT and ATOMT1 activity assays were performed in 50 mM phosphate buffer, pH 6.8, 0.5
mM S-adenosyl methionine (SAM), 0.5 mM MgCl2, 0.5 mM caffeyl substrate, and deionized
water to a volume of 100 µL. Coupled enzyme assays using OsPMT and either OsOMT or
ATOMT1 were performed in 50 mM phosphate buffer, pH 6, 1 mM DTT, 0.5 mM SAM, 0.5
mM CoA thioester, 0.5 mM monolignol, and deionized water to a volume of 100 µL. Enzyme
activity assays were performed as previously described with a few modifications. All reactions
were incubated for 30 min, stopped with 2 µL of HCl, and then solubilized with a 1:1 addition of
100% methanol. OsPMT was used to synthesize caffeyl p-coumarate using caffeyl alcohol and pcoumaroyl CoA, and p-coumaryl caffeate was synthesized using p-coumaryl alcohol and
caffeoyl CoA. Caffeyl p-coumarate was also provided by Dr. John Ralph (33). CoA thioester
synthesis was performed as previously described on pg. 13.

	
  

57	
  

A.3 RESULTS
Identification of the Candidate Gene
Co-expression analysis was performed using the Rice Oligonucleotide Array Database to find an
OMT candidate gene that was co-expressed with several monolignol biosynthetic genes. 4CL-2
from rice (LOC_Os02g08100) was utilized as a query for co-expression analysis because it
encodes an isozyme that has been implicated in lignin formation (71). Thus, 4CL-2 was expected
to be co-expressed with other monolignol biosynthetic genes. The co-expression analysis
2

demonstrated a positive correlation (R = 0.88) between 4CL-2 and an OMT candidate gene
(LOC_Os08g06100). This candidate gene was annotated as a putative OMT in several grass
species within the Poaceae family, such as maize, miscanthus, and sorghum. A protein BLAST
of the OMT candidate against arabidopsis generated the most closely related gene with 58%
similarity as ATOMT1 (AT5G54160), which was annotated as a caffeate OMT (Figure A.2).

Heterologous Expression in E. coli
A His-tagged OsOMT clone was produced using a codon-optimized synthetic gene, whereas a
His-tagged ATOMT1 clone was synthesized from arabidopsis stem cDNA. These clones were
utilized for heterologous protein expression in E. coli. OsOMT and ATOMT1 were purified from
E. coli extracts using IMAC and analyzed by SDS-PAGE. Induced protein samples before and
after FPLC purification demonstrated protein bands at a molecular weight of approximately 40
kDa, as expected for both OsOMT and ATOMT1 (Figure A.3). The un-induced controls did not
show induction of the proteins of interest. SDS-PAGE analysis of the two proteins demonstrated
that they are very soluble, even under a short induction time. LC/MS/MS analysis confirmed that
the protein bands were in fact OsOMT and ATOMT1.

	
  

58	
  

Alignment Report Report of 'Untitled' - ClustalW (Slow/Accurate, Gonnet) : Monday, Novem
Alignment of 'Untitled' - ClustalW (Slow/Accurate, Gonnet) : Monday, November 26,
2012 10:32 AM
2012 10:32 AM
Majority
Majority
MGSTA..........D.EA...A.QLAS.S.LPM.LK.A.EL.LLE.........
MGSTA..........D.EA...A.QLAS.S.LPM.LK.A.EL.LLE.........
ATOMT1.pro MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPATOMT1.pro MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNGSPOsOMT.pro MGSTAADMAA--AA-DEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAG
OsOMT.pro MGSTAADMAA--AA-DEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAG

54
52

54
52

Majority
Majority
.........P.E.A.KLP.K.NP.A..M.DR.LRLL.SY.V..C...........
.........P.E.A.KLP.K.NP.A..M.DR.LRLL.SY.V..C...........
ATOMT1.pro -------MSPTEIASKLPTK-NPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVE
ATOMT1.pro -------MSPTEIASKLPTK-NPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVE
OsOMT.pro GGGKAALLTPAEVADKLPSKANPAAADMVDRMLRLLASYNVVRCEMEEGADGKLS
OsOMT.pro GGGKAALLTPAEVADKLPSKANPAAADMVDRMLRLLASYNVVRCEMEEGADGKLS

101
107

101
107

Majority
Majority
R.Y...PVCK.LT.NEDGVS.AAL.LMNQDKVLMESWY.LKDA.LDGGIPFNKAY
R.Y...PVCK.LT.NEDGVS.AAL.LMNQDKVLMESWY.LKDA.LDGGIPFNKAY
ATOMT1.pro RIYGLGPVCKYLTKNEDGVSIAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAY
ATOMT1.pro RIYGLGPVCKYLTKNEDGVSIAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAY
OsOMT.pro RRYAAAPVCKWLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAY
OsOMT.pro RRYAAAPVCKWLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAY

156
162

156
162

Majority
Majority
GM.AFEYHGTD.RFN.VFN.GM.NHS.I..KK.L..Y.GF......VDVGGG.GA
GM.AFEYHGTD.RFN.VFN.GM.NHS.I..KK.L..Y.GF......VDVGGG.GA
ATOMT1.pro GMSAFEYHGTDPRFNKVFNNGMSNHSTITMKKILETYKGFEGLTSLVDVGGGIGA
ATOMT1.pro GMSAFEYHGTDPRFNKVFNNGMSNHSTITMKKILETYKGFEGLTSLVDVGGGIGA
OsOMT.pro GMTAFEYHGTDARFNRVFNEGMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGA
OsOMT.pro GMTAFEYHGTDARFNRVFNEGMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGA

211
217

211
217

Majority
Majority
T....VS..P...GIN.DLPHVI..AP..PG.EHVGGDMF.SVP.G.DAI.MKWI
T....VS..P...GIN.DLPHVI..AP..PG.EHVGGDMF.SVP.G.DAI.MKWI
ATOMT1.pro TLKMIVSKYPNLKGINFDLPHVIEDAPSHPGIEHVGGDMFVSVPKG-DAIFMKWI
ATOMT1.pro TLKMIVSKYPNLKGINFDLPHVIEDAPSHPGIEHVGGDMFVSVPKG-DAIFMKWI
OsOMT.pro TVAAVVSRHPHIRGINYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKWI
OsOMT.pro TVAAVVSRHPHIRGINYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKWI

265
272

265
272

Majority
Majority
.HDWSDEHC...LKNCY..LPE.GKV...EC.LPE..D.......V.HVD.IMLA
.HDWSDEHC...LKNCY..LPE.GKV...EC.LPE..D.......V.HVD.IMLA
ATOMT1.pro CHDWSDEHCVKFLKNCYESLPEDGKVILAECILPETPDSSLSTKQVVHVDCIMLA
ATOMT1.pro CHDWSDEHCVKFLKNCYESLPEDGKVILAECILPETPDSSLSTKQVVHVDCIMLA
OsOMT.pro LHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMIMLA
OsOMT.pro LHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMIMLA

320
327

320
327

Majority
Majority
HNPGGKER.E.EF..LA.A.GF.G.K..........IE..K..
HNPGGKER.E.EF..LA.A.GF.G.K..........IE..K..
ATOMT1.pro HNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIELLKKL
ATOMT1.pro HNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIELLKKL
OsOMT.pro HNPGGKERYEREFRELARAAGFTGFKATYIYANAWAIEFTK
OsOMT.pro HNPGGKERYEREFRELARAAGFTGFKATYIYANAWAIEFTK

363
368

363
368

Figure A.2 Peptide Sequence Alignment of Arabidopsis ATOMT1 and Rice OsOMT. Ibrahim et
al. (1998) performed a comparative analysis of amino acid sequences from 26 plant OMTs to
determine that plant OMTs have five conserved regions (I-V) in their peptide sequences: region I
“LVDVGGGXG”, region II “GINFDLPHV”, region III “EHVGGDMF”, region IV “NGKVI”,
and region V “GGKERT” (72). Regions I and V are conserved among metal binding OMTs due
to their involvement in SAM and metal cation binding, respectively. The conserved motifs are
underlined in red. Sequences were aligned using MegAlign (DNASTAR). Dots denote residues
that do not match. Dashes signify gaps inserted to obtain optimal sequence alignment.

	
  

59	
  

T0
T0
18°C 18°C IMAC IMAC IMAC IMAC
Fr24
Fr25 Fr26
soluble insol. soluble insol. Fr23

A)
kDa
75

50

37

B)

T0
T0
37°C 37°C
soluble insol. soluble insol.

IMAC IMAC IMAC IMAC
Fr24 Fr25 Fr26 Fr27

kDa

75
50
37

Figure A.3 Heterologous Expression of OsOMT and ATOMT1 in E. coli. SDS-PAGE analysis
of soluble and insoluble protein fractions for A, OsOMT, un-induced (T0) and induced (4 h)
expression at 18 °C, and corresponding IMAC fractions, and B, ATOMT1, un-induced (T0) and
induced (6 h) expression at 37 °C, and corresponding IMAC fractions.

	
  

60	
  

Enzyme Activity Assays
In a previous study (73), it was determined that the OMT candidate gene catalyzed the 3´-Omethylation of caffeic acid. However, it had not been shown whether OsOMT had activity with
other caffeyl substrates found in the monolignol biosynthetic pathway, such as caffeyl alcohol,
caffeoyl CoA, and caffeyl aldehyde. HPLC analyses demonstrated that OsOMT catalyzes the 3´O-methylation of caffeyl alcohol and caffeyl aldehyde to produce coniferyl alcohol and coniferyl
aldehyde, respectively (Table A.1). OsOMT did not have activity with caffeoyl CoA, even in a
reaction containing twice the enzyme amount and incubation time. This observation strongly
suggests that not only is caffeoyl CoA a poor substrate for OsOMT activity but also, OsOMT
cannot be categorized as a CCoAOMT. ATOMT1 did not show similar activity as with OsOMT,
indicating that OsOMT and ATOMT1 have different functions in the plant cell.

Table A.1 Enzyme Activity for OsOMT and ATOMT1 with Various Caffeyl Substrates
Substrate

OsOMT Activity

ATOMT1 Activity

Caffeic acid

Yes

No

Caffeoyl CoA

No

No

Caffeyl aldehyde

Yes

No

Caffeyl alcohol

Yes

No

Caffeyl p-coumarate*

Yes

No

Caffeyl p-coumarate**

Yes

No

p-Coumaryl caffeate*

Yes

No

* Indicates that the monolignol conjugate was synthesized in a coupled enzyme assay containing
OsPMT and the appropriate CoA thioester and monolignol alcohol substrate.
** Indicates that the monolignol conjugate was chemically synthesized as a standard compound
and utilized in a single enzyme assay containing OsOMT and the proper reaction ingredients.

	
  

61	
  

OsPMT was used in a coupled enzyme assay to catalyze the synthesis of caffeyl pcoumarate and p-coumaryl caffeate. These reactions were coupled with the addition of OsOMT
and SAM to determine whether OsOMT could utilize the monolignol conjugates as substrate for
3´-O-methylation activity. It was determined that OsOMT does use caffeyl p-coumarate and pcoumaryl caffeate as substrates to synthesize coniferyl p-coumarate and p-coumaryl ferulate,
respectively. Nevertheless, it was important to determine whether activity with caffeyl pcoumarate to produce coniferyl p-coumarate was the result of OsOMT activity on the
monolignol conjugate and not caffeyl alcohol. Due to its substrate specificity with caffeyl
alcohol, OsOMT could have produced coniferyl alcohol from caffeyl alcohol, which would then
be utilized by OsPMT to synthesize caffeyl p-coumarate using the p-coumaroyl CoA present in
the reaction mixture. A chemically synthesized caffeyl p-coumarate substrate was utilized in a
similar reaction to determine that OsOMT does in fact have activity with caffeyl p-coumarate to
produce coniferyl p-coumarate.

	
  

62	
  

A.4 SIGNIFICANCE
Lignin has been classically described as a biopolymer of three distinct monomer units (H,
G, and S). However, as previously illustrated, many studies have identified that plants have the
ability to incorporate alternative monolignol precursors into their lignin. These alternative
monolignols include monolignols that are acylated prior to incorporation into the lignin polymer.
In particular, it has been proposed that grasses have an alternative pathway for monolignol
biosynthesis due to the prevalence of p-coumarate esters in their lignin. The purpose of this study
was to identify an enzyme that could potentially be involved in a novel acylation reaction in rice.
OsOMT is a grass-specific enzyme that catalyzes the synthesis of coniferyl p-coumarate and pcoumaryl ferulate. Co-expression analysis of 4CL-2 demonstrated a strong correlation with
OsOMT, which demonstrates a high probability that OsOMT is involved in lignin biosynthesis.
In addition, OsOMT is similar to putative OMT genes in other grass species within the Poaceae
family. All together, these findings suggest that an alternative pathway for the synthesis of
monolignols in grasses may exist. Furthermore, with the identification of OsOMT and future
studies on its biochemistry, it will enhance our understanding of acylation reactions in lignin and
the functional consequences of this phenomenon.

	
  

63	
  

APPENDIX B

Proteome Analysis of Unpollinated and Pollinated Cherry Styles

	
  

64	
  

The proteomics data displayed below (Table B.1) represents a single replicate of cherry style
protein extractions and experimental LC/MS/MS for the 24 h pollination experiments. The
individual cherry protein samples were labeled as follows: Bing (S3S4) × Brooks (S1S9) “BB24”,
control (unpollinated) styles “BC24”, and Bing (S3S4) × EF (S3S4) “BE24”. Since this data
subset contained 1507 identified proteins, only the first 100 proteins have been shown. Data in
the last three columns represent spectral counts, which are generally used as a measure of protein
abundance in pre-digested protein samples.

Table B.1 Proteomics Data for Cherry Pollination Experiments at 24 h
#
1

Identified Proteins
Chain E, Leech-Derived Tryptase InhibitorTRYPSIN
COMPLEX

BB24

BC24

BE24

204

327

239

225

206

210

238

143

251

177

125

223

146

119

182

149

117

174

139

120

139

| Symbols: ATMETS, ATMS1, ATCIMS | ATCIMS
2

(COBALAMIN-INDEPENDENT METHIONINE
SYNTHASE) | chr5:5935773-5939197 FORWARD
| Symbols: LOS2 | LOS2 (Low expression of osmotically

3

responsive genes 1); phosphopyruvate hydratase |
chr2:15328160-15330865 REVERSE

4

| Symbols: GAPC-2 | GAPC-2; glyceraldehyde-3-phosphate
dehydrogenase | chr1:4608462-4610491 REVERSE
| Symbols: PR3, PR-3, CHI-B, B-CHI, ATHCHIB |

5

ATHCHIB (BASIC CHITINASE); chitinase |
chr3:3962508-3963952 REVERSE

6
7

	
  

| Symbols: | ATP synthase beta chain 2, mitochondrial |
chr5:2825740-2828353 FORWARD
| Symbols: UBQ6 | UBQ6 (ubiquitin 6); protein binding |
chr2:19351771-19352244 FORWARD

65	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

102

127

96

118

89

118

105

79

122

86

105

113

84

85

100

80

85

96

92

55

100

95

44

97

90

55

90

65

86

71

85

42

85

| Symbols: BIP2, BIP | BIP (LUMINAL BINDING
8

PROTEIN); ATP binding | chr5:16824925-16827708
REVERSE
| Symbols: ANNAT2 | ANNAT2 (ANNEXIN

9

ARABIDOPSIS 2); calcium ion binding / calciumdependent phospholipid binding | chr5:25991141-25992780
FORWARD
| Symbols: ACT11 | ACT11 (ACTIN-11); structural

10

constituent of cytoskeleton | chr3:3858122-3859615
FORWARD

11
12
13

| Symbols: | fructose-bisphosphate aldolase, putative |
chr5:963388-964981 REVERSE
| Symbols: MEE6, CS1, APX1 | APX1 (ASCORBATE
PEROXIDASE 1) | chr1:2438002-2439432 FORWARD
| Symbols: BIP1 | BIP1; ATP binding | chr5:1054066910543278 REVERSE
| Symbols: | UTP--glucose-1-phosphate uridylyltransferase,

14

putative / UDP-glucose pyrophosphorylase, putative /
UGPase, putative | chr5:5696957-5700847 REVERSE

15
16

| Symbols: AAC1 | AAC1 (ADP/ATP CARRIER 1);
binding | chr3:2605712-2607036 REVERSE
| Symbols: | elongation factor 1-alpha / EF-1-alpha |
chr5:24306452-24307901 FORWARD
| Symbols: AT1G56075.1, LOS1 | LOS1 (Low expression of

17

osmotically responsive genes 1); translation elongation
factor/ translation factor, nucleic acid binding |
chr1:20971910-20974742 REVERSE

18

	
  

keratin 1 [Homo sapiens]

66	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

19

polyphenyl oxidase 2 precursor

82

46

82

20

aerolysin pore forming?

62

58

83

59

81

60

47

81

62

53

50

66

62

49

46

36

61

51

26

64

56

56

27

62

50

40

54

47

40

57

48

37

58

60

35

48

52

43

47

21
22
23
24
25
26

| Symbols: | heat shock protein 81-4 (HSP81-4) |
chr5:22694828-22697293 REVERSE
| Symbols: ROC3 | ROC3 (rotamase CyP 3); peptidyl-prolyl
cis-trans isomerase | chr2:7207944-7208465 FORWARD
| Symbols: | glycosyl hydrolase family 17 protein |
chr4:9200310-9201457 REVERSE
dehydrin cor29
| Symbols: SHD | SHD (SHEPHERD); ATP binding |
chr4:12551912-12555861 REVERSE
| Symbols: MLP423 | MLP423 (MLP-LIKE PROTEIN 423)
| chr1:8500642-8501447 REVERSE
| Symbols: RCI1, GRF3 | GRF3 (GENERAL

27

REGULATORY FACTOR 3); protein phosphorylated
amino acid binding | chr5:15427507-15428515 FORWARD

28

| Symbols: HSP70 | HSP70 (heat shock protein 70); ATP
binding | chr3:3991494-3993696 REVERSE
| Symbols: ATPDIL2-1, UNE5, MEE30 | ATPDIL2-

29

1/MEE30/UNE5 (PDI-LIKE 2-1); thiol-disulfide exchange
intermediate | chr2:19488573-19490753 FORWARD
| Symbols: BG3 | BG3 (BETA-1,3-GLUCANASE 3);

30

hydrolase, hydrolyzing O-glycosyl compounds |
chr3:21192895-21194024 REVERSE

31
32

	
  

| Symbols: ATP1 | ATPase subunit 1 | chrM:302166-303689
REVERSE
| Symbols: | pollen Ole e 1 allergen and extensin family
protein | chr2:14641377-14642676 REVERSE

67	
  

Table B.1 (cont’d)
#
33
34

Identified Proteins
| Symbols: | malate dehydrogenase, cytosolic, putative |
chr1:1189417-1191266 REVERSE
| Symbols: TUB1 | TUB1 (tubulin beta-1 chain); structural
molecule | chr1:28455039-28457263 REVERSE

BB24

BC24

BE24

50

35

56

38

51

51

51

43

46

55

36

48

34

46

58

46

50

40

45

38

53

61

22

47

44

54

32

28

44

46

36

38

42

| Symbols: TPX1 | TPX1 (THIOREDOXIN-DEPENDENT
35

PEROXIDASE 1); antioxidant | chr1:24563187-24564416
REVERSE

36

| Symbols: CRT1 | CRT1 (CALRETICULIN 1); calcium ion
binding | chr1:21093687-21096295 REVERSE
| Symbols: ATC4H, C4H, CYP73A5 |
ATC4H/C4H/CYP73A5 (CINNAMATE 4-

37

HYDROXYLASE, CINNAMATE-4-HYDROXYLASE);
trans-cinnamate 4-monooxygenase | chr2:1300093813002760 REVERSE

38
39
40

| Symbols: | glycosyl hydrolase family 3 protein |
chr1:29354690-29357762 REVERSE
| Symbols: | cytosol aminopeptidase family protein |
chr4:15046595-15049310 REVERSE
unnamed protein product [Homo sapiens]
| Symbols: ATPDIL1-1 | ATPDIL1-1 (PDI-LIKE 1-1);

41

protein disulfide isomerase | chr1:7645756-7648684
FORWARD
| Symbols: GR-RBP7, GRP7, CCR2, ATGRP7 | ATGRP7

42

(COLD, CIRCADIAN RHYTHM, AND RNA BINDING
2); RNA binding | chr2:9272557-9273396 REVERSE

43

	
  

| Symbols: | isoflavone reductase, putative | chr4:1826601818267598 REVERSE

68	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

31

38

44

30

42

40

23

32

62

58

17

35

36

25

46

38

23

43

30

33

37

30

29

41

28

29

42

19

33

47

44

15

40

| Symbols: SAM-2, MAT2 | MAT2/SAM-2 (S44

adenosylmethionine synthetase 2) | chr4:796298-797479
REVERSE

45
46
47
48

| Symbols: ACT2/7, ACT7 | ACT7 (actin 7) | chr5:30528103054221 FORWARD
| Symbols: | cysteine protease inhibitor, putative / cystatin,
putative | chr5:19303822-19304190 REVERSE
| Symbols: SHD | SHD (SHEPHERD); ATP binding |
chr4:12551912-12555861 REVERSE
| Symbols: | glycosyl hydrolase family 3 protein |
chr1:29354690-29357762 REVERSE
| Symbols: TPI, ATCTIMC | ATCTIMC (CYTOSOLIC

49

TRIOSE PHOSPHATE ISOMERASE); triose-phosphate
isomerase | chr3:20564771-20567055 FORWARD
| Symbols: | succinyl-CoA ligase (GDP-forming) beta-

50

chain, mitochondrial, putative / succinyl-CoA synthetase,
beta chain, putative / SCS-beta, putative | chr2:88126558814939 FORWARD

51
52
53

| Symbols: CSD1 | CSD1 (COPPER/ZINC SUPEROXIDE
DISMUTASE 1) | chr1:2827703-2829056 FORWARD
| Symbols: | Ras-related GTP-binding family protein |
chr5:24124676-24126275 REVERSE
| Symbols: | lipid-associated family protein |
chr4:18432944-18433575 FORWARD
| Symbols: ATARFA1E | ATARFA1E (ADP-

54

RIBOSYLATION FACTOR A1E); GTP binding /
phospholipase activator/ protein binding | chr3:2306326223064520 FORWARD

	
  

69	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

47

16

35

56

2

40

38

23

34

33

21

41

38

24

31

22

27

44

39

30

23

28

28

36

36

30

25

29

25

36

| Symbols: ATGUS3 | ATGUS3 (ARABIDOPSIS
55

THALIANA GLUCURONIDASE 3); beta-glucuronidase |
chr5:13253142-13255948 REVERSE

56
57

No hits found
| Symbols: | extracellular dermal glycoprotein, putative /
EDGP, putative | chr1:787143-788444 FORWARD
| Symbols: | ATP synthase delta chain, mitochondrial,

58

putative / H(+)-transporting two-sector ATPase, delta
(OSCP) subunit, putative | chr5:4310561-4311944
REVERSE
| Symbols: SUS1, ASUS1, ATSUS1 | SUS1 (SUCROSE

59

SYNTHASE 1); UDP-glycosyltransferase/ sucrose synthase
| chr5:7050601-7054034 REVERSE
| Symbols: ATPQ | ATPQ (ATP SYNTHASE D CHAIN,
MITOCHONDRIAL); hydrogen ion transporting ATP

60

synthase, rotational mechanism / hydrogen ion transporting
ATPase, rotational mechanism | chr3:19407667-19409097
FORWARD

61
62

epidermal cytokeratin 2 [Homo sapiens]
| Symbols: | 40S ribosomal protein S7 (RPS7A) |
chr1:18063522-18064603 REVERSE
| Symbols: ATAVP3, AVP-3, AVP1 | AVP1 (vacuolar-type

63

H+-pumping pyrophosphatase 1); ATPase | chr1:53991105402180 FORWARD
| Symbols: | phosphoglucomutase, cytoplasmic, putative /

64

glucose phosphomutase, putative | chr1:8219935-8224175
FORWARD

	
  

70	
  

Table B.1 (cont’d)
#
65

Identified Proteins
| Symbols: ACLB-2 | ACLB-2 (ATP-citrate lyase B-2) |
chr5:20072274-20075421 FORWARD

BB24

BC24

BE24

35

31

24

38

21

31

20

38

31

31

21

37

28

21

38

31

23

33

18

34

34

27

31

27

19

42

23

36

26

21

31

10

42

38

17

27

| Symbols: | isocitrate dehydrogenase, putative / NADP+
66

isocitrate dehydrogenase, putative | chr1:2454275124545524 FORWARD

67

| Symbols: | malate dehydrogenase (NAD), mitochondrial |
chr1:19858634-19860470 REVERSE
| Symbols: MTLPD1 | dihydrolipoamide dehydrogenase 1,

68

mitochondrial / lipoamide dehydrogenase 1 (MTLPD1) |
chr1:17721101-17722810 REVERSE

69

| Symbols: AHA5 | AHA5 (ARABIDOPSIS H(+)-ATPASE
5); ATPase | chr2:10422602-10426810 FORWARD
| Symbols: GST29, ATGSTU18 | ATGSTU18

70

(GLUTATHIONE S-TRANSFERASE 29); glutathione
transferase | chr1:3395740-3396808 REVERSE

71

putative allergen Pru p 1.02
| Symbols: PCK1, PEPCK | PCK1/PEPCK

72

(PHOSPHOENOLPYRUVATE CARBOXYKINASE 1);
ATP binding / phosphoenolpyruvate carboxykinase (ATP) |
chr4:17802968-17806326 REVERSE

73

polyphenyl oxidase 2 precursor
| Symbols: PLDALPHA2 | PLDALPHA2

74

(PHOSPHLIPASE D ALPHA 2); phospholipase D |
chr1:19587609-19590220 REVERSE

75
76

	
  

| Symbols: | band 7 family protein | chr1:2629759526298813 REVERSE
keratin 9 [Homo sapiens]

71	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

25

21

36

30

25

26

22

21

38

25

27

29

18

37

25

16

31

32

25

18

36

26

30

22

22

22

34

| Symbols: LHCB5 | LHCB5 (LIGHT HARVESTING
77

COMPLEX OF PHOTOSYSTEM II 5); chlorophyll binding
| chr4:6408196-6409492 FORWARD
| Symbols: EMB1395, MEE58, SAHH, SAHH1, HOG1 |

78

HOG1 (HOMOLOGY-DEPENDENT GENE SILENCING
1); adenosylhomocysteinase | chr4:8054926-8056671
FORWARD

79

| Symbols: | leucine-rich repeat family protein |
chr3:7280936-7282033 FORWARD
| Symbols: PHGPX, LSC803, ATGPX6 | ATGPX6

80

(GLUTATHIONE PEROXIDASE 6); glutathione
peroxidase | chr4:7010015-7011324 REVERSE
| Symbols: | Identical to Uncharacterized protein
At5g48480 [Arabidopsis Thaliana] (GB:Q9LV66); similar

81

to unknown [Populus trichocarpa] (GB:ABK95611.1);
contains domain SSF54593 (SSF54593) | chr5:1966204019662884 FORWARD

82
83

| Symbols: TUA2 | TUA2 (tubulin alpha-2 chain) |
chr1:18521405-18523397 FORWARD
| Symbols: | porin, putative | chr3:85761-87619
FORWARD
| Symbols: ATAVP3, AVP-3, AVP1 | AVP1 (vacuolar-type

84

H+-pumping pyrophosphatase 1); ATPase | chr1:53991105402180 FORWARD
| Symbols: | uridylate kinase / uridine monophosphate

85

kinase / UMP kinase (PYR6) | chr5:9276662-9278094
FORWARD

	
  

72	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

33

17

27

33

15

29

25

27

24

23

24

27

19

17

38

13

24

37

18

29

25

18

17

36

19

25

27

30

19

22

| Symbols: PIP2D, PIP2;5 | PIP2;5/PIP2D (plasma
86

membrane intrinsic protein 2;5); water channel |
chr3:20313095-20314716 FORWARD
| Symbols: SAHH2 | SAHH2 (S-ADENOSYL-L-

87

HOMOCYSTEINE (SAH) HYDROLASE 2);
adenosylhomocysteinase | chr3:8588020-8589678
REVERSE

88
89

No hits found
| Symbols: | elongation factor 1-alpha / EF-1-alpha |
chr5:24306452-24307901 FORWARD
| Symbols: | sorbitol dehydrogenase, putative / L-iditol 2-

90

dehydrogenase, putative | chr5:21129046-21130510
FORWARD
| Symbols: NDPK1 | NDPK1 (nucleoside diphosphate

91

kinase 1); ATP binding / nucleoside diphosphate kinase |
chr4:5923421-5924363 FORWARD
| Symbols: ATGSR1 | ATGSR1 (Arabidopsis thaliana

92

glutamine synthase clone R1); glutamate-ammonia ligase |
chr5:14950804-14952886 REVERSE

93

aerolysin pore forming?
| Symbols: ATTRX H1, ATTRX1 | ATTRX1 (Arabidopsis

94

thaliana thioredoxin H-type 1); thiol-disulfide exchange
intermediate | chr3:18962104-18962936 REVERSE
| Symbols: PGK1 | PGK1 (PHOSPHOGLYCERATE

95

KINASE 1); phosphoglycerate kinase | chr3:40611344063147 REVERSE

	
  

73	
  

Table B.1 (cont’d)
#

Identified Proteins

BB24

BC24

BE24

24

21

25

25

15

29

23

24

22

17

22

29

17

21

30

| Symbols: PLD, PLDALPHA1 | PLDALPHA1
96

(PHOSPHOLIPASE D ALPHA 1); phospholipase D |
chr3:5330842-5333481 FORWARD
| Symbols: GF14 PHI, GRF4 | GRF4 (GENERAL

97

REGULATORY FACTOR 4); protein phosphorylated
amino acid binding | chr1:12867242-12868492 FORWARD

98
99
100

	
  

| Symbols: | aspartyl protease family protein |
chr3:20151269-20153577 REVERSE
No hits found
| Symbols: | trypsin and protease inhibitor family protein /
Kunitz family protein | chr1:6149336-6149926 FORWARD

74	
  

REFERENCES

	
  

75	
  

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