Molecular Analysis of Late-Stage Fiber Development in Upland Cotton

Amanda Sooter, B.S.

A Thesis

Biotechnology

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

Master of Sciences

Approved

Kameswara Rao Kottapalli, PhD Chair of Committee

Paxton Payton, PhD

Susan San Francisco, PhD

Masoud Zabet-Moghaddam, PhD

Dominick Casadonte Interim Dean of the Graduate School

May, 2013

Texas Tech University, Amanda Sooter, May 2013

ACKNOWLEDGMENTS

The professional and technical expertise of several individuals has been invaluable in the achievement of this research project. I would like to thank Dr. Kameswara Rao Kottapalli and Dr. Paxton Payton for their knowledge of genomic and transcriptomic analyses as well as how all data generated relates to plant biology and biochemistry. Further, I am indebted to Dr. Susan San Francisco and Dr. Masoud Zabet-Moghaddam's skilled proficiency of proteomic analyses, especially when employing mass spectrometry for such purposes. The support of these four individuals has been instrumental in the completion of this thesis; I thank each and every one of you.

The United States Department of Agriculture Cropping Systems Research Laboratory in Lubbock, Texas, provided TM1 cotton for analysis grown in one of the greenhouses on the facility. Additionally, Marie Syapin was responsible for caring for the plants and the demanding process of collecting the fiber samples. Marie, this project would have been impossible without your hard-work.

A special thanks is needed for Illumina®, Inc. The optimization of next-generation sequencing protocols (especially when using a brand new instrument) necessitated some trial and error. The technical support of Illumina®, Inc. was incredibly generous and supportive of this research project.

As with all research projects, financial support of several organizations was crucial. This research was supported by grants from the Ogallala Aquifer Initiative, USDA-ARS CRIS 6208-21000-018-00D CI, and the Center for Biotechnology and Genomics of Texas Tech University.

I am appreciative of the learning opportunities afforded by both the United States Department of Agriculture and Texas Tech University's Center for Biotechnology and Genomics. The chance to utilize and gain experience in both next-generation sequencing and

Texas Tech University, Amanda Sooter, May 2013 mass-spectrometry is exceptional, and I look forward to using such techniques in my future research.

Finally, I would like to thank my two lab-mates, Miss Komal Ramesh Kunder and Mr. Abhishek Dass, for their unremitting teamwork and never-ending support and empathy.

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Texas Tech University, Amanda Sooter, May 2013

CONTENTS

ACKNOWLEDGMENTS ...... ii ABSTRACT ...... 1 LIST OF TABLES ...... 2 LIST OF FIGURES ...... 3 I. INTRODUCTION ...... 4 II. MATERIALS AND METHODS ...... 10 Overview ...... 10 Plant Material ...... 11 Transcriptomics ...... 12 RNA Extraction ...... 12 Illumina® TruSeq® RNA Library Preparation ...... 14 Normalization and Pooling of Libraries ...... 20 RNA Sequencing using Illumina® MiSeq ...... 20 Assembly of Sequencing Reads ...... 21 Creation of a Whole Cotton Fiber Transcriptome ...... 22 Creation of a Whole Cotton Fiber Proteome ...... 23 Proteomics ...... 23 Protein Extraction ...... 23 Protein Quantification ...... 24 Acetone Precipitation ...... 25 One-Dimensional (1D) Gel Electrophoresis (1DGE) ...... 25 In-Gel Tryptic Digestion ...... 26 Nano-LC-MS/MS ...... 26 Protein Identification ...... 28 Quantitative Proteomic Analysis ...... 28 IIII. RESULTS ...... 29 RNA Sequencing and Assembly ...... 29 Creation of Cotton Proteome Databases ...... 30 Gene Expression Profiling ...... 31 Protein Expression Profiling...... 32

Texas Tech University, Amanda Sooter, May 2013

Cell Wall Metabolism ...... 36 Cell Wall Modifications ...... 36 Pectinesterases ...... 36 Polygalacturonases and Pectin Lyases ...... 37 Cellulose Synthesis ...... 38 Major Carbohydrate Metabolism ...... 39 Sucrose Degradation ...... 39 Phenylpropanoids ...... 39 Signaling ...... 40 G Proteins ...... 41 Calcium Signaling ...... 42 14-3-3 Signaling Components ...... 44 GDSL Lipases ...... 45 Transcription Factors ...... 45 Chromatin Remodeling ...... 46 Miscellaneous ...... 47 Drought/Salt Responsive ...... 47 IV. DISCUSSION ...... 49 Protein Expression Profiling...... 49 Select Functional Categories and Their Implications in Secondary Cell Wall Biosynthesis ...... 50 Cell Wall Modifications ...... 50 Pectinesterases, Polygalacturonases, and Pectate Lyases ...... 50 Cellulose Synthesis ...... 52 Sucrose Degradation ...... 52 Phenylpropanoids ...... 53 G Proteins ...... 54 Calcium Signaling Components ...... 55 14-3-3 Signaling Components ...... 55 GDSL Lipases ...... 56 Chromatin Remodeling ...... 56 V. CONCLUSION ...... 57 VI. FUTURE WORK ...... 60 v

Texas Tech University, Amanda Sooter, May 2013

BIBLIOGRAPHY ...... 61 A. APPROACH TO PROTEOMIC ANALYSIS ...... 69 B. MASS SPECTROMETRY SET-UP ...... 71 C. EXPRESSION PATTERNS OF ALL DIFFERENTIALLY EXPRESSED TRANSCRIPTS ...... 73

Texas Tech University, Amanda Sooter, May 2013

ABSTRACT

Cotton is the world's most important textile and the number one value-added crop. It plays a crucial role in the economy of Texas – supporting close to 50,000 jobs and supplying $2 billion to the state economy. Its role is even more evident in the South Plains of Texas, which supplies approximately 10% of the world's cotton. Understanding molecular events associated with the developing fiber could provide candidate targets for genetic improvement. Such modifications could lead to substantial crop enhancements, in terms of either yield or fiber quality or both, signifying considerable economic ramifications for the industry. Late-stage (21 and 24 days post-anthesis, dpa) fiber samples of the Upland cotton cultivar were subjected to transcriptomic analysis using RNA Sequencing technology (Illumina MiSeq). Using de novo assembly in DNASTAR NGen software, the sequence reads with a quality score greater than 30 (9.2 million reads from 21 dpa and 7.5 million reads from 24 dpa) were assembled into 23942 contigs and 19750 contigs, respectively. RNA-Seq analysis using DNASTAR Array Star revealed 2928 differentially expressed transcripts. Differential expression was defined as those transcripts with 99% confidence and greater than 2.0-fold expression change between 21 and 24 dpa samples. Additionally, the proteomes from these two time-points were analyzed using nano liquid chromatography tandem mass spectrometry. The proteins were identified using a proteome created from transcriptomic data using The GPM search engine with a 1% false discovery rate. Proteins present in all replicates of each stage were annotated with the Mercator tool from Max Planck Institute. MapMan software was employed to visualize expression profiles at the pathway level of both transcriptomic and proteomic data. This mapping revealed up- regulation of pectinesterases and chromatin remodeling factors, such as DNA methyltransferases and histone deacetylases, and down-regulation of components of cell- signaling. Additionally, changes indicative of dormancy, prior to dehydration and final maturation appear to begin during this critical phase of fiber development. A complete analysis of this key transition in fiber development will be discussed.

Texas Tech University, Amanda Sooter, May 2013

LIST OF TABLES

2.1 Combinations of Adapter Indices for Illumina ® TruSeq Library Preparation ...... 18 2.2 Reagent Volumes used in Bradford Assay for Protein Quantification ...... 24 3.1 Statistics of Sequencing & Assembly Data ...... 29 3.2 Number of Proteins Identified...... 33 3.3 Cell Wall Modification Expression Levels ...... 32 3.4 Pectinesterase Expression Levels ...... 36 3.5 Polygalacturonase & Pectin Lyase Expression Levels ...... 37 3.6 Cellulose Synthesis Expression Levels ...... 38 3.7 Sucrose Degradation Expression Levels ...... 39 3.8 Phenylpropanoid Expression Levels ...... 40 3.9 G Protein Expression Levels ...... 41 3.10 Calcium Signaling Expression Levels ...... 43 3.11 14-3-3 Signaling Expression Levels ...... 44 3.12 GDSL Lipases Expression Levels ...... 45 3.13 Chromatin Remodeling Expression Levels ...... 46 3.14 DNA Methyltransferase & Histone Deacetylase Expression Levels ...... 47 3.15 Drought/Salt Responsive Element Expression Levels ...... 44 C.1 Table of Expression Patterns of All Differentially Expressed Transcripts ...... 74

Texas Tech University, Amanda Sooter, May 2013

LIST OF FIGURES

2.1 Research Approach ...... 11 2.2 TM1 bolls at 21 dpa (left) and 24 dpa (right)...... 12 2.3 Gradient Elution Method in Terms of % Solvent B ...... 27 3.1 Summary of the Fiber Transcriptome and Proteomes ...... 27 3.2 Differentially Expressed Transcripts ...... 31 3.3 Functional Annotation of Transcript Data Generated by the Mercator Tool ...... 34 3.4 Transcriptomic & Proteomic Data Mapped into Pathways via MapMan ...... 35 3.5 Cell Wall Modifications ...... 36 3.6 Pectinesterases ...... 37 3.7 Polygalacturonases & Pectin Lyases ...... 38 3.8 Cellulose Synthesis...... 39 3.9 Sucrose Degradation ...... 39 3.10 Phenylpropanoids ...... 40 3.11 Expression Trends of Various Components of Cell Signaling ...... 41 3.12 Expression Trends of Transcription Factors ...... 46 4.1 Relationship of UDP-Glucose Manufacturers and Cellulose Synthesis ...... 49 5.1 Summary of Interactions of Select Functional Categories of Transcripts and Their Implications in Secondary Cell Wall Biosynthesis ...... 55 A.1 Approach to Proteomic Analysis ...... 66 B.1 Diagram of Mass Spectrometry Set-Up ...... 68

Texas Tech University, Amanda Sooter, May 2013

CHAPTER I INTRODUCTION

Cotton is an economic staple in many countries worldwide. In the United States alone, the cotton industry generates annual business revenues in excess of $120 billion. In fact, the supplies and services alone needed to grow cotton amount to $5.3 billion yearly (Cotton Counts). The production and processing of U.S. grown cotton accounts for the employment of more than 440,000 Americans (National Cotton Women's Committee). This crop is a cornerstone of the food and fiber industries. Aside from its obvious popularity as a textile – approximately 75% of all U.S. cotton is used in apparel (National Cotton Council of America, 2013) – cotton is used for various food products, such as cooking oils, and is a large component of livestock feeds; cotton even composes 75% of each bill of American currency. Such considerations make cotton America’s number-one value added crop (Cotton Counts).

Cotton crops are grown in seventeen states, from Virginia to California, with a strong concentration in the Old South, in an area commonly referred to as the “Cotton Belt.” Over twelve million acres, or 19,000 square miles, boast cultivation of this cash crop, averaging fifteen million bales in production, the equivalent of 7.3 billion pounds annually (National Cotton Council of America, 2013). The United States supplies over 30% of the total world exports of cotton, without the inclusion of textile products (National Cotton Council of America, 2013).

Texas is the chief producing state, accounting for 35% of all American cotton. 50% of Texas cotton is grown in Congressional District 19 (locally referred to as the South Plains), home to Bailey, Borden, Castro, Cochran, Deaf Smith, Fisher, Floyd, Gaines, Garza, Hale, Hockley, Howard, Lamb, Lubbock, Lynn, Nolan, Parmer, Taylor, Terry, and Yoakum Counties, which is 18% of all U.S. cotton produced (National Cotton Council of America, 2013).

The most common variety of cotton grown (approximately 95% in the U.S. and 90% worldwide) is known as Upland cotton, Gossypium hirsutum. Upland cotton is native to Mexico and Central America (Cotton Program, Agricultural Marketing Service, 2001). It is

Texas Tech University, Amanda Sooter, May 2013 an allotetraploid species which is theorized to have developed approximately 1.5 million years ago as the result of genome wide duplications (Bowers, Chapman, Rong, & Paterson, 2003; Patterson, Bowers, & Chapman, 2004). It is G. hirsutum's high yield which makes this short fibered cotton species a favorite for production (Cotton Program, Agricultural Marketing Service, 2001).

In addition to its economic importance, the cotton fiber is an excellent research model to study cell wall growth and cellulose synthesis. This is primarily due to the fact that single fiber cells can easily be isolated. Further, the mature cotton fiber offers one of the most pure forms of cellulose, possessing more than 90% (w/w) crystalline cellulose (Haigler, Betancur, Stiff, & Tuttle, 2012).

The cotton fiber is a single-celled trichome, or fine, hair-like outgrowth, from the epidermis of the ovule. More than 20,000 fibers may develop semi-synchronously on each seed. Due to its paramount economic impact, the USDA developed a system to determine fiber quality. Fiber quality is determined by four main parameters: fiber length, length uniformity, strength, and micronaire. Fiber length is represented by the average length of the longer one- half of fibers, known as the upper half mean length. Both the variety and environmental factors can influence fiber length. Length uniformity is expressed as the ratio of the average length and the upper half mean length. It conveys evenness and strength to fabrics. Strength is another measure of fiber quality and is mainly affected by the variety planted. Fiber strength is mainly conferred by the secondary cell wall. The strength of the fiber is determined by placing a "beard" of parallel cotton fibers in two jaw clamps one-eighth inch apart. The force required to break the fibers is measured and reported in units of grams per tex. A tex unit is the weight in grams of 1,000 meters of cotton fiber. Finally, micronaire denotes the fineness and maturity of the fiber and is influenced by environmental factors (Cotton Program, Agricultural Marketing Service, 2001).

The development of the cotton fiber is a complex process. Essentially, the process involves each fiber elongating to its full length as a thin-walled tube. The cell wall thickens by depositing cellulose, leaving a hollow area in the center. After the growth of the fiber, the

Texas Tech University, Amanda Sooter, May 2013 living material dies, causing the fiber to collapse and twist about its own axis (Basra & Malik, 1984; Haigler et al., 2012).

Cotton fiber development is an approximately 50 day process and can be divided into four overlapping stages: initiation, elongation, cellulose biosynthesis, and maturation. Initiation begins several days before the flower opens, or anthesis, and lasts until approximately 3 days post-anthesis (dpa). Next, the fiber undergoes considerable elongation, growing up to 2 mm per day (Basra & Malik, 1984), or 2.5 cm total (Haigler et al., 2012). This process is also known as primary cell wall synthesis and lasts from 5 to 25 dpa. Secondary cell wall biosynthesis (or cellulose biosynthesis) occurs from 20 to 45 dpa. During this time, the secondary cell thickens principally through the deposition of almost pure cellulose. Finally, the fibers mature – the cells dehydrate and die, and the boll opens to expose the fluffy fiber (Basra & Malik, 1984; Haigler et al., 2012).

The financial magnitude of the cotton fiber is not lost to scientists and has spurred extensive research into the molecular mechanisms of its development. Stages of cotton fiber development are more than likely controlled by a few key regulatory elements, which result in changes at the transcriptome and proteome levels. A large amount of research has delved into individual genes and proteins and their roles in development (John & Crow, 1992; Li, Cai, Cheng, & Liu, 2002; Shimizu et al., 1997; Whittaker & Triplett, 1999; Zhu et al., 2011). There have also been attempts to characterize the transcriptome (Lacape et al., 2012; Xie, Sun, Stiller, & Zhang, 2011; Yuan, Tu, & Zhang, 2011; Zhu et al., 2011) and proteome (Du et al., 2013; Liu et al., 2012; Zheng et al., 2012; Yang, Bian, Yao, & Liu, 2008) of the developing cotton fiber using high throughput techniques.

Presently, the genome of Upland cotton has not been sequenced. However, the genome from the cotton species Gossypium raimondii, whose ancestor is thought to have contributed the D-genome to allotetraploid species, such as Gossypium hirsutum, was sequenced on Illumina HiSeq. 78.7 gigabytes of sequencing data was generated and estimated to have provided a 103.6 fold coverage of the genome. Sequencing reads were assembled into 41,307 contigs (K. Wang et al., 2012). In 2012, Lacape et al. employed EST pyrosequencing to contrast

Texas Tech University, Amanda Sooter, May 2013 transcript levels in 10 dpa (elongation) and 22 dpa (transition to secondary cell wall biosynthesis) to better understand molecular events governing fiber development. This approach was utilized for both Gossypium hirsutum (Upland cotton) and Gossypium barbadense (Pima) to compare the fiber qualities of the two species. The pyrosequencing generated 619,000 high quality reads which were assembled into 38,297 contigs and 7,775 singletons. 2,770 contigs were determined to be differentially expressed (>2-fold) between the two stages. The comparative digital gene expression revealed key genes, such as sucrose synthases and tubulins were up-regulated in the 22 dpa (Lacape et al., 2012). Though there is neither a complete genome nor a complete transcriptome of cotton available, there is a multitude of ESTs that have been produced through years of research. In 2011, there were 268,786 ESTs publicly available. Xie et al. (2011) created an interactive, fully downloadable database (http://www.leonxie.com) of all of the ESTs through a massive assembly project. The ESTs were assembled into a total of 28,432 unique contigs (25,371 were consensus contigs and 3,601 were singletons). Interestingly, cotton ESTs were found to show the most similarity to grape ESTs when compared with six model plants (Xie et al., 2011).

Even though a similar amount of genes is expressed in different tissues of the cotton plant, unique genes have been found to be expressed in the fiber (John & Crow, 1992). Therefore, transcriptomic analysis restricted to the fiber could provide better insight into the molecular landscape of the developing fiber. Yuan et al. (2011) provided a large-scale (10,979) set of ESTs specifically obtained from the developing fiber of Gossypium barbadense from -2 to 25 dpa. The ESTs were assembled into 1492 contigs and 4360 singletons (Yuan et al., 2011).

High-throughput proteomic techniques have also been applied to the study of cotton fiber development. Most comparative proteomic studies in the developing cotton fiber have focused on early fiber developmental stages as well as the rapid elongation phase. In early fiber studies in differentiating and initiating fibers, forty-six proteins identified using two- dimensional gel electrophoresis (2-DE) in conjunction with mass spectrometry were found to be differentially expressed between the two stages and had diverse functions, such as redox homeostasis, post-transcriptional and post-translational modulation, response to stress, and carbohydrate, protein, energy and sterol metabolism (Liu et al., 2012). 7

Texas Tech University, Amanda Sooter, May 2013

Yang et al. (2008) investigated the proteomes of elongating fibers and found processes such as energy metabolism, redox homeostasis, changes in the cytoskeleton, cellular responses, and protein turnover were key in this phase (Yang, et al., 2008). In elongating fibers of 10 dpa, 93 proteins were found to be preferentially expressed in wild-type as opposed to a fuzzless-lintless mutant. These proteins were implicated in nucleotide sugar metabolism as well as pectin biosynthesis. Further, cultured cells which were treated with ethylene or lignoceric acid (24:0) showed up-regulation of proteins and transcripts associated with the previously mentioned processes. These results suggest ethylene and lignoceric acid may promote elongation through activation of the pectin biosynthetic pathway (Pang et al., 2010). Zheng et al. (2012) investigated the effects of cold-temperature stress at the proteomic level of fiber at 10, 15, and 20 dpa in a low-temperature sensitive and low-temperature tolerant line. The fiber length was determined to be shortened in the event of low-temperature stress. Using 2-DE and mass spectrometry, 37 proteins were found to be differentially expressed and were involved in sugar metabolism, cytoskeleton, cell wall loosening, and redox homeostasis. Proteins associated with cell wall loosening, synthesis of cell wall components, and osmotic regulation exhibited enhanced levels of expression in the cold-tolerant line. Further, decreased levels of ethylene and expansin might be implicated in cold sensitivity (Zheng et al., 2012). In a recent continuation of a previous study, 2-DE with mass spectrometry was employed to identify 239 differentially expressed proteins between stages ranging from 5 to 25 dpa. Proteins with roles in the metabolism of proteins and carbohydrates as well as redox homeostasis were found to be the most abundant between the stages (Zhang, Yang, Zhang, & Liu, 2013).

This investigation seeks to provide a better understanding of the global overview of the molecular events that occur during cotton fiber development, specifically those associated with secondary cell wall biosynthesis. It is presented as a portion of a larger study involving six time-points which span early to late elongation to secondary cell wall biosynthesis – 3, 5, 11, 17, 21, and 24 dpa. This portion of the investigation aims to focus on the transition of fiber development from 21 and 24 dpa to reflect the events in the rapid secondary cell wall biosynthesis. These fibers underwent transcriptomic and proteomic analyses using RNA-

Texas Tech University, Amanda Sooter, May 2013 sequencing on Illumina MiSeq and mass spectrometry, respectively. Transcripts were assembled and analyzed for differential expression between the two stages. A whole fiber transcriptome and proteome were created by combining assembly data from all six stages. Although the proteomics data generated still needs further scrutiny, it is included at this time, simply to further strengthen the evidences provided by transcriptomic data. The differential gene expression profile of 21 and 24 dpa, along with their respective differential protein expression profiles are reported, lending valuable insight into genes and pathways in fiber development which could potentially be used to create higher quality and/or higher fiber yielding cotton germplasm.

Texas Tech University, Amanda Sooter, May 2013

CHAPTER II MATERIALS AND METHODS

Overview

Figure 2.1 describes the approach used to accomplish transcriptomic and proteomic analyses of the developing cotton fiber. Cotton fiber was obtained from TM1 cotton grown in glasshouse conditions, and the fibers were collected and ground to a fine powder for subsequent transcriptomic and proteomic analyses. For transcriptomic studies of the developing fiber, RNA was isolated from the ground fiber tissue, quantified, and checked for quality. cDNA libraries were created from the isolated RNA using Illumina TruSeq procedures. Sequencing reads from Illumina MiSeq were assembled first for individual replicates using NGen assembly software; assembled sequences underwent CAP3 assembly to create a reference transcriptome. This transcriptome was also employed to create a reference proteome. Assembled data underwent differential gene expression analysis using Array Star software with the QSeq application and functional annotation with the Mercator tool. Expression patterns and functional categorizations were used to map the transcriptomic data into pathways via MapMan.

Protein was extracted from the ground cotton tissue and quantified. 100 µg aliquots of protein were normalized to a volume of 15 µL using acetone precipitation then underwent one-directional gel electrophoresis (1DGE). Each lane was divided into twelve approximately equal pieces which were subsequently divided into smaller pieces. The proteins within the gel pieces were enzymatically digested with trypsin. The peptides were analyzed using nano-LC-MS/MS and identified through a search against the reference proteome previously created from sequencing data. Proteins identified were quantified based on the number of matched peptides then annotated with the Mercator tool. Protein expression patterns and functional annotation information were used to map the data to pathways via MapMan. The proteomic data was then used as a validation of transcriptomic data. A detailed discussion of each procedure follows.

Texas Tech University, Amanda Sooter, May 2013

Figure 2.1 - Research Approach

Plant Material

The Upland cotton cultivar TM1 was grown under glasshouse conditions at the USDA Cropping Systems Research Laboratory in Lubbock, Texas, in 2012. Cotton plants were grown in 2 liter pots in Sunshine Mix #1 potting soil (Sun Gro Horticulture, Bellevue, WA, USA) at 28°C during the day and 26°C during the night with an average daily relative humidity of 38% in the daytime and 29% at night. The plants were watered by drip irrigation (12 watering events/day) with Peters EXCEL 21-5-20 multipurpose no boron water soluble fertilizer (Everris, Summerville, SC, USA). Flowers were tagged on the day of anthesis. Five or more bolls were collected from the plants at two dates, 21 and 24 days post anthesis (dpa) (Figure 2.2).The fiber was separated from the bolls, immediately frozen in liquid nitrogen,

Texas Tech University, Amanda Sooter, May 2013 and stored at -80°C until use. The two developmental stages were chosen to reflect time- points in the rapid secondary cell wall synthesis stage of cotton fiber development.

Figure 2.2 - TM1 bolls at 21 dpa (left) and 24 dpa (right). The insets at the bottom right-hand corners show the fiber and ovules removed from the bolls.

Transcriptomics

RNA Extraction

Total cellular RNA was extracted from approximately 100 mg of fiber tissue using a modified hot borate method (Wan & Wilkins, 1994). RNA extraction was attempted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA); however, the procedure resulted in a low yield of RNA. In the more laborious Hot Borate method, the frozen fiber tissue was ground to a fine powder in a pre-cooled mortar and pestle and transferred to a glass homogenizer. The "Hot Borate," or XT Buffer, consisted of 0.2 M sodium borate decahydrate (Borax), pH 9.0; 30 mM ethylene glycol-bis(2-aminoethyl ether)- N, N'-tetraacetic acid (EGTA); 1% (w/v) sodium dodecyl sulfate; and 1% sodium deoxycholate. The Hot Borate buffer was supplemented with 2% (w/v) polyvinylpyrollidone (PVP)-40,000, 10 mM dithiothreotol, and 0.5% Nonidet-40 (NP-40). Preheated (80°C) XT

Texas Tech University, Amanda Sooter, May 2013

Buffer (2.5 ml) was added to each gram of the ground frozen cotton fiber tissue and homogenized for 2 minutes in a glass homogenizer. The homogenate was transferred to a 50 mL Nalgene Oakridge tube containing proteinase K at a ratio of 1.75 mg per 3.5 mL XT Buffer and incubated at 42°C for 1.5 hours with gentle shaking (100 rpm) on a rotary shaker. 2 M potassium chloride was added to the homogenate and incubated at 4°C for 1 hour. After centrifugation at 16,100 rcf (Eppendorf Centrifuge 5415R, Hauppauge, NY, USA) for 20 minutes at 4°C, the supernatant was filtered through a filtration column from the Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA). RNA was precipitated overnight at 4°C in 2 M lithium chloride. The precipitated RNA was collected by centrifugation at 4°C at 16,100 rcf for 20 minutes. The supernatant was discarded, and the pellet was washed three times in cold 2 M lithium chloride. The pellet was suspended in 500 µL of 10 mM Tris-HCl, pH 7.5, and clarified through centrifugation at 4°C at 16,100 rcf for 10 minutes. 50 µL of 2 M potassium acetate, pH 5.5, was added to the supernatant and incubated at 4°C for 15 minutes. Polysaccharides and insoluble materials were removed through centrifugation at 4°C at 16,100 rcf for 10 minutes.

From this point forward, the Spectrum Plant Total RNA kit was employed to facilitate ease of collection and cleanup of RNA. 540 µL of Binding Solution was added to the RNA-pellet and mixed by pipetting up and down 5 times. The mixture was added into a Binding Column, placed in a 2 mL microcentrifuge tube, and centrifuged at maximum speed for 1 minute to bind RNA to the column. Next, the column was washed three times: first with 500 µL of Wash Solution 1, then twice with 500 µL of Wash Solution 2. The column was dried through centrifugation at maximum speed for 1 minute and placed in a fresh 2 mL microcentrifuge tube. The RNA was eluted from the column by adding 45 µL of nuclease free water (GIBCO, Life Technologies, Grand Island, NY, USA) preheated to 56°C.

RNA quantity was estimated using NanoDrop quantification. RNA quality was determined through the use of the Agilent 2200 TapeStation using the R6K assay (Agilent, Santa Clara, CA, USA). 1 µL of each RNA sample was mixed with 4 µL of R6K Sample Buffer through gentle pipetting up and down 5 times. The solution was heat denatured at 72°C for 3 minutes with the PTC-200 Peltier Thermal Cycler (MJ Research, Reno, NV, USA), and placed on ice 13

Texas Tech University, Amanda Sooter, May 2013 for 2 minutes. The samples were collected at the bottoms of the 200 µL tubes through brief centrifugation and placed in the 2200 TapeStation. The samples were specified using the TapeStation Control Software, and the quality of the samples was analyzed through electrophoresis, giving an RNA Integrity Number equivalent (RINe) for each sample, based on the degradation (or lack thereof) of the RNA sample.

Illumina® TruSeq® RNA Library Preparation cDNA libraries were created from cotton fiber total RNA using Illumina's® TruSeq® RNA Sample Preparation Low Throughput Protocol. Two of the three replicates of RNA samples from each of the time points (21 and 24 dpa) with the highest RINe values determined by the 2200 TapeStation were pooled for a total of 4 µg of total RNA starting material.

(*All reagents used were from the TruSeq RNA Sample Prep Kit v1, (Illumina, San Diego, CA, USA) unless otherwise stated).

Purification and Fragmentation of mRNA mRNA was isolated from total RNA by exploiting the poly-A tail specific to mRNA using poly-T oligo-attached magnetic beads. The 4 µg of total RNA was diluted to a volume of 50 µL using nuclease-free water in 200 µL PCR tubes. 50 µL of room temperature, fully resuspended RNA Purification Beads were added to each sample, and the entire volume was gently pipetted up and down 6 times to mix. The tubes were sealed and incubated in a thermal cycler for 65°C for 5 minutes, holding at 4°C with the lid heated to 100°C to facilitate binding of the mRNA to the poly-T magnetic beads. The tubes were allowed to sit at room temperature for 5 minutes then placed on a 96-well magnetic stand (Life Technologies, Carlsbad, CA, USA) for 5 minutes to separate mRNA from the solution. The entire supernatant was discarded from each well. The tubes were removed from the magnetic stand and washed with 200 µL of Bead Washing Buffer, and the entire volume was mixed by pipetting. The tubes were again placed on the magnetic stand for 5 minutes, and the supernatant (containing the majority of ribosomal and non-messenger RNA) was removed. 50 µL of thawed Elution Buffer, which was centrifuged for 5 seconds at 600xg, was pipetted to resuspend the beads. The samples were incubated in the thermal cycler for 2 minutes at 14

Texas Tech University, Amanda Sooter, May 2013

80°C with the heated lid option to facilitate the elution of mRNA (as well as contaminant rRNA due to non-specific binding) from the beads. 50 µL of thawed Bead Binding Buffer, which was centrifuged for 5 seconds at 600xg, was added to allow mRNA to specifically rebind the beads and restrict the non-specific binding of rRNA. The tubes were incubated at room temperature for 5 minutes, followed by 5 minutes on the magnetic stand at room temperature. The supernatant was removed, and the tubes were removed from the magnetic stand. The beads were washed by adding 200 µL of Bead Washing Buffer. The tubes were again placed on the magnetic stand for 5 minutes, and the supernatant was discarded, removing residual rRNA and other contaminants. The tubes were removed from the magnetic stand. Elute, Prime, Fragment Mix was added to each sample at a volume of 19.5 µL and mixed through gentle pipetting. This mix served to prime the mRNA with random hexamers and fragment the RNA. The samples were incubated for 8 minutes at 94°C with the lid heated to 100°C; the samples were removed when the temperature reached 4°C and centrifuged briefly.

First Strand cDNA Synthesis

The fragmented and primed mRNA was reverse transcribed into first strand cDNA using reverse transcriptase. The tubes were placed on the magnetic stand for 5 minutes, and 17 µL of the supernatant was transferred to a new tube. The thawed First Strand Master Mix was centrifuged for 5 seconds at 600xg. SuperScript II (Invitrogen, Life Technologies, Grand Island, NY, USA) was added at a ratio of 1 µL for every 9 µL of First Strand Master Mix. 8 µL of the First Strand Master Mix – SuperScript II mixture was added to each sample and mixed by pipetting. The tubes were sealed then incubated in the thermal cycler at 25°C for 10 minutes, 42°C for 50 minutes, 70°C for 15 minutes, with the lid heated to 100°C, and were removed to room temperature when the temperature reached 4°C.

Second Strand cDNA Synthesis

The RNA template was removed, and double-stranded cDNA was synthesized as follows. Thawed Second Strand Master Mix was centrifuged for 5 seconds at 600xg, added at a volume of 25 µL per sample and mixed by pipetting. The sealed tubes were incubated in the

Texas Tech University, Amanda Sooter, May 2013 thermal cycler for 1 hour at 16°C. After incubation, the tubes were removed and allowed to equilibrate to room temperature. 90 µL of well-dispersed, room temperature Agentcourt AMPure XP Magnetic Beads (Beckman Coulter Genomics, Danvers, MA, USA) were added to each sample and mixed by pipetting. The tubes were incubated at room temperature for 15 minutes and were then placed on the magnetic stand for 5 minutes. The supernatant was removed from each well. The beads were washed twice with 200 µL of freshly prepared 80% ethanol. The beads were allowed to dry for 5.5 minutes and removed from the magnetic stand. The thawed Resuspension Buffer was centrifuged for 5 seconds at 600xg, 52.5 µL was added to each sample and mixed by pipetting. The samples were incubated at room temperature for 2 minutes and again placed on the magnetic stand for 5 minutes. 50 µL of each sample was transferred to a new tube. As the protocol indicated the time was a "Safe Stopping Point," the samples were stored overnight at -20°C.

End Repair

The following day, the samples of double-stranded cDNA were removed from -20°C, thawed, and centrifuged briefly to collect the liquid at the bottom of the tubes. The overhangs which resulted from fragmentation were converted to blunt ends using the End Repair Mix, which possesses 3' to 5' exonuclease activity to remove 3' overhangs and polymerase activity to fill 5' overhangs. 10 µL of room temperature Resuspension Buffer was added to each tube as a substitution for the End Repair Control. This substitution was made in order to avoid decreasing coverage of the samples during sequencing. Next, End Repair Mix was added to the samples at a volume of 40 µL per sample, and the entire volume was mixed by pipetting. The tubes were sealed and placed in the thermal cycler and incubated for 30 minutes at 30°C. The samples were removed from the thermal cycler to room temperature. 160 µL of well- dispersed AMPure XP beads brought to room temperature were added to each sample, and the entire volume was mixed by pipetting thoroughly. The samples were incubated at room temperature for 15 minutes and an additional 5 minutes on the magnetic stand. Next, 255 µL of supernatant was removed from the tubes in two repetitions of 127.5 µL. While the tubes were left on the magnetic stand, a total of two 80% ethanol washes were performed as mentioned previously. The pellets were allowed to dry for 5.5 minutes and removed from the

Texas Tech University, Amanda Sooter, May 2013 magnetic stand. The pellets were resuspended in 17.5 µL of Resuspension Buffer, and samples were incubated at room temperature for 2 minutes with an additional 5 minutes on the magnetic plate. 15 µL of the supernatant was then transferred to a fresh 200 µL tube. The protocol offers a "Safe Stopping Point" after this step; however, the samples immediately underwent subsequent 3' adenylation, as detailed below.

Adenylation of 3' Ends

3' adenylation was completed in order to add a single adenosine to each 3' end of the double- stranded cDNA to prevent self-ligation of the cDNA blunt-ended fragments. 2.5 µL of Resuspension Buffer was added to each sample as a substitution for A-Tailing control, again to circumvent decreasing coverage in sequencing. Thawed A-Tailing Mix was added to each sample at a volume of 12.5 µL, and the entire volume was mixed by pipetting. The samples were incubated in a thermal cycler for 30 minutes at 37°C with the lid heated to 100°C and removed to room temperature when the temperature reached 4°C.

Adapter Ligation

The adenylated fragments then immediately underwent ligation of adaptor sequences. The adapter sequences contain a 5' thymine overhang which utilizes the 3' A overhang placed on the fragments during 3' adenylation and allows the sequences to hybridize to the flow cell during sequencing. The Illumina® TruSeq Low Throughput protocol allows up to 48 samples to be multiplexed together for sequencing through the use of a specific combination of adapter indices. Table 2.1 details the combinations of adapter indices to be used with the desired number of samples multiplexed.

The specific RNA Adapter Indices and Stop Ligation Buffer were thawed and centrifuged for 5 seconds at 600xg. As a substitution for Ligation control, room temperature Resuspension Buffer was added to each sample at a volume of 2.5 µL. Next, 2.5 µL of Ligation Mix (removed from -20°C immediately prior to use) and 2.5 µL of AR012 index was added to 21 dpa, and the same amount of AR006 index was added to 24 dpa. The entire volume was mixed by pipetting and incubated in a thermal cycler for 10 minutes at 30°C. The tubes were then removed to room temperature, and 5 µL of Stop Ligation Buffer was added to each well 17

Texas Tech University, Amanda Sooter, May 2013 and mixed by pipetting in order to inactivate ligation. 42 µL of well-dispersed, room temperature AMPure XP beads were added to each sample and mixed by pipetting. The samples were incubated at room temperature for 15 minutes with an additional 5 minutes on the magnetic stand. The supernatant was removed, and the magnetic beads were washed twice with 80% ethanol while the samples remained on the magnetic stand. The samples were allowed to air dry for 5.5 minutes and removed from the magnetic stand. 52.5 µL of Resuspension Buffer was added to each sample and mixed by pipetting. The tubes were incubated for 2 minutes at room temperature and placed on the magnetic stand to incubate for 5 minutes. 50 µL of supernatant was transferred to a fresh tube for an additional clean up. Well-dispersed, room temperature AMPure XP beads were added to each sample at a volume of 50 µL and mixed thoroughly by pipetting. The samples were incubated at room temperature for 15 minutes with an additional 5 minutes on the magnetic stand. The supernatant was removed, and the magnetic beads were washed twice with 80% ethanol while the samples remained on the magnetic stand. The samples were allowed to air dry for 5.5 minutes and removed from the magnetic stand. Each dried pellet was resuspended in 22.5 µL of Resuspension Buffer. The samples were incubated at room temperature for 15 minutes with an additional 5 minutes on the magnetic stand. 20 µL of supernatant was transferred to fresh 200 µL tubes.

Table 2.1- Combinations of Adapter Indices for Illumina ® TruSeq Library Preparation, Courtesy of the Illumina® TruSeq Library Preparation Guide

Plexity Option Set A Only Set B Only 2 1 AR006 and AR012 Not recommended 2 AR005 and AR019 Not recommended 3 1 AR002 and AR007 and AR001 and AR010 and AR019 AR020 2 AR005 and AR006 and AR003 and AR009 and AR015 AR025 3 2-plex options with any other AR008 and AR011 and adapter AR022 4 1 AR005 and AR006 and AR001 and AR008 and AR012 and AR019 AR010 and AR011 2 AR002 and AR004 and AR003 and AR009 and AR007 and AR016 AR022 and AR027 3 3-plex options with any other 3-plex options with any other adapter adapter 18

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PCR Enrichment of Fragments

Although the protocol again offered a "Safe Stopping Point" to store samples at -20°C for up to 7 days, the samples immediately underwent PCR enrichment to selectively enrich DNA fragments that have adapter molecules attached to both ends. PCR Master Mix and PCR Primer Cocktail were thawed then centrifuged at 600xg for 5 seconds. 5 µL of PCR Primer Cocktail was added to each sample, followed by 25 µL of PCR Master Mix, and the entire volume was mixed by pipetting. The samples were incubated with the following PCR program on the thermal cycler with the lid heated to 100°C: 98°C for 30 seconds; 15 cycles of 98°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds; 72°C for 5 minutes; 10°C until removed. The samples were then removed and underwent a final round of clean up. 50 µL of well-dispersed, room temperature AMPure XP beads were added to each sample and mixed by pipetting. The samples were incubated for 15 minutes at room temperature with an additional 5 minutes on the magnetic stand. 95 µL of supernatant was discarded, and the beads were washed twice with 80% ethanol. The samples were allowed to air dry for 5.5 minutes and removed from the magnetic stand. Each dried pellet was resuspended in 32.5 µL of Resuspension Buffer. The samples were incubated at room temperature for 15 minutes with an additional 5 minutes on the magnetic stand. 30 µL of supernatant was transferred to fresh 200 µL tubes, divided into ten 3 µL aliquots and stored at -80°C.

Library Validation and Quantification

The median fragment size of sample libraries was verified using Agilent 2200 TapeStation D1K Assay (Agilent, Santa Clara, CA, USA). 1 µL of each sample was mixed with 3 µL of D1K Sample Buffer by vortexing for 5 seconds. The tubes were spun down and loaded into the TapeStation along with 3 µL of D1K Ladder in the first well. The samples were analyzed to determine the median size of the fragments in the sample. Illumina® TruSeq desires a median size of 260 - 300 bp.

Libraries were quantified using Qubit® 2.0 Fluorometer (Invitrogen, Life Technologies, Grand Island, NY, USA). The double-stranded DNA High Sensitivity assay was employed. 1.2 mL Working Solution was prepared by diluting 6 µL of QubitTM reagent in 1194 µL

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QubitTM buffer (1:200 dilution). Standards were created by mixing 190 µL of Working Solution with 10 µL of each standard in separate, thin-wall, clear 0.5 mL PCR tubes (Invitrogen). 1 µL of sample library was diluted in 199 µL of Working Solution in triplicate. All tubes were vortexed for 2-3 seconds and incubated for 2 minutes. The tubes were inserted into the Qubit® 2.0 Fluorometer, first Standard 1, then Standard 2, followed by the sample tubes, and the values were read, using the on-instrument Dilution Calculator to determine the concentration of the original sample in ng/µL.

Normalization and Pooling of Libraries

The libraries were normalized to 10 nM and pooled for even coverage during subsequent sequencing. Using the average of the values measured using Qubit® 2.0 Fluorometer, the concentrations of the libraries were converted from ng/µL to nM using the following calculation:

Each library was then diluted to 10 nM using 10 mM Tris-HCl, pH 8.5, with 0.1% Tween-20. 10 µL of each library was pooled into a fresh tube and mixed by pipetting.

RNA Sequencing using Illumina® MiSeq

Fresh 0.2 N sodium hydroxide (NaOH) was prepared by adding 200 µL of 1.0 N NaOH to 800 µL nuclease free water in a microcentrifuge tube and inverted several times to mix. The pH of the solution was verified to be 12 or greater. The pooled library samples (10 nM) were diluted to 2 nM using 100 mM Tris-HCl, pH 8.5, with 0.1% Tween-20. The diluted, pooled libraries were then denatured to single strands by adding 5 µL of 0.2 N NaOH to 5 µL of the pooled libraries. The entire volume was mixed by pipetting and incubated at room temperature for 5 minutes. Pre-chilled HT1 buffer (supplied by Illumina®) was added to the denatured library at a volume of 990 µL to result in a library with a concentration of 20 pM. Finally, the denatured DNA was diluted to a final concentration of 18 pM by adding 60 µL of HT1 buffer to 540 µL of the 20 pM libraries. The denatured and diluted cDNA was stored on 20

Texas Tech University, Amanda Sooter, May 2013 ice until the MiSeq reagent cartridge was prepared. A sample sheet was created using the wizard-based interface of Illumina® Experiment Manager to generate FASTQ Only files. In the workflow selection, Other was selected as the category, with FASTQ only for the application. Specific settings, such as Paired End, 151 base pair reads, and no Adaptor Trimming were selected as run parameters. This sample sheet was uploaded to the MiSeq Controller Software, and the wizard-based interface was utilized to begin the run. The flow cell was rinsed with Milli-Q water, cleaned with an alcohol wipe, and loaded into the MiSeq. The PR2 bottle, supplied with the flow cell, and the waste bottle were loaded. The reagent cartridge, previously thawed in a 25°C water bath for at least one hour and inverted ten times, was loaded with 600 µL of the denatured and diluted 18 pM libraries. The sequencing run was performed after all parameters were checked.

Assembly of Sequencing Reads

The sequencing reads generated from MiSeq were assembled using SeqMan NGen from the DNASTAR Lasergene Genomics Suite (DNASTAR, Inc., Madison, WI, USA). The reads generated from MiSeq were converted to fastq format by unzipping the files. Reads were assembled by selecting Transcriptome Assembly in the Choose Project Type menu. De novo assembly was selected since there was no available fiber specific transcriptome at the time. The FASTQ files were then uploaded into the paired-end reads section, selecting Illumina > 50 nt for the Sequencing Technology employed. The 20 bp adapter sequence from library preparation was trimmed, and the following parameters were specified: minimum read length of 30 bp, minimum contig length of 500 bp, 90% match, and 21 mer. NGen software employs a de Bruijn graph algorithm for the assembly of short reads. After the assembly was completed in SeqMan NGen, the assembled sequences (in FASTA format) were reassembled into the Late Reference Transcriptome using the long read assembly CAP3 Assembly software (Huang & Madan, 1999). This software assembles long reads using an overlap-layout consensus (OLC) algorithm. In short, the CAP3 OLC algorithm functions as follows: regions of low quality reads at the 5' and 3' ends are removed, and overlapping reads are computed, with false overlaps removed. Afterwards, overlapping reads are assembled into contigs based on the overlap scores. Corrections are made to the contigs through the use

Texas Tech University, Amanda Sooter, May 2013 of forward-reverse constraints. Finally, a multiple sequence alignment is constructed to form consensus sequences (Huang & Madan, 1999).

Array Star version 5.1 from DNASTAR's Lasergene Genomics Suite was employed for normalization and gene quantification. Array Star is equipped with an application known as QSeq, which is fully integrated into the software for gene expression analysis. The four fastq files generated from sequencing in Illumina MiSeq (two files per time-point since it was paired-end sequencing) were uploaded into Array Star. In the Pre-Processing setup step, the default processing method, QSeq, and the default normalization method, RPKM (or reads per kilobase per million mapped reads), were elected in order to normalize the sequencing reads for a more accurate expression analysis (Mortazavi, Williams, McCue, Schaeffer, & Wold, 2008). The reads from each time-point were mapped to the Late Reference Transcriptome for differential gene expression analysis. The selected differentially expressed gene sequences were uploaded into the Mercator tool (MapMen Site of Analysis, Max Planck Institute for Molecular Plant Physiology, Munich, Germany, http://mapman.gabipd.org/ web/guest/app/mercator) for functional categorization of the gene sequences. The Mercator results, along with differential gene expression data obtained from RNA-Seq in Array Star, were used to map the genes into biological pathways using the freely available software, MapMan version 3.5.1R2 (Thimm, et al. 2004. ; MapMen Site of Analysis, Max Planck Institute for Molecular Plant Physiology, Munich, Germany, http://mapman.gabipd.org/ web/guest/mapman).

Creation of a Whole Cotton Fiber Transcriptome

As mentioned previously, the late stage cotton fiber development was part of a larger study to investigate the molecular mechanisms of cotton fiber development, including early (3 and 5 dpa) and middle (11 and 17 dpa) stages. The reads obtained by sequencing cDNA from all stages were assembled with SeqMan NGen in the same manner as mentioned above. Then, all stages, 3, 5, 11, 17, 21, and 24 dpa were assembled using CAP3 assembly software to create a whole cotton fiber transcriptome, that is, to our knowledge, the first of its kind.

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Creation of a Whole Cotton Fiber Proteome

The whole cotton fiber transcriptome resulting from the CAP3 Assembly was then used to create a whole cotton fiber proteome. Such a feat was accomplished through the utilization of a 6-frame translation Perl script developed in-house. Additionally, an alternative whole cotton proteome was created by combining the whole cotton fiber transcriptome, cotton ESTs (Udall et al., 2006), a recent hybrid assembly known as Cotton46 which contains approximately 4.4 million Sanger and 454 EST reads and includes 44,900 contigs assembled from several Gossypium species (Iowa State University, Brigham Young University, Georgia State University), and a cotton D-genome transcriptome (K. Wang et al., 2012). This assembly data was also assembled with CAP3 assembly software and translated with the same in-house Perl script. The resultant proteomes were used as reference databases for protein identification by The Global Proteome Machine GPM) search engine.

Proteomics

Protein Extraction

Total fiber protein was extracted using the phenol extraction protocol described by Kottapalli et al. (2008). Cotton fibers, which were previously separated from the ovules, were ground to a fine powder in liquid nitrogen. Approximately 200 mg of the homogenized tissue was transferred to a 2 mL microcentrifuge tube and gently mixed with 540 µL of Extraction Media [0.9 M Sucrose, 0.1 M Tris-HCl (pH 8.8), 10 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 0.4% (v/v) 2-mercaptoethanol, in Milli-Q water] and 540 µL Tris-buffered phenol (pH 8.8) at room temperature for 30 minutes using an inversion shaker. The samples were centrifuged for 30 minutes at 4°C at 16,100 rcf. The aqueous phase was transferred to a new tube and stored on ice. The bottom portion was re-extracted with 450 µL of Extraction Media and 450 µL of Tris-buffered phenol, mixed by inversion for 5 minutes, then centrifuged for 30 minutes at 16,100 rcf at 4°C. The aqueous phase was collected and combined with the portion collected from the first extraction. This liquid was divided into 2 equal portions in separate microcentrifuge tubes. Protein was precipitated by adding 5

Texas Tech University, Amanda Sooter, May 2013 volumes of 0.1 M ammonium acetate in methanol, vortexed briefly, and incubated overnight at -20°C.

The next day, samples were centrifuged for 30 minutes at 16,100 rcf at 4°C to collect precipitated proteins. The supernatant was discarded, and the pellet was washed by resuspending in 1 mL 0.1 M ammonium acetate in methanol by vortexing, sonication, and homogenization and incubated for 30 minutes at -20°C. The samples were then centrifuged for 20 minutes at 16,100 rcf at 4°C. This process of washing the protein pellet through resuspension, incubation, and centrifugation was repeated once for 0.1 M ammonium acetate in methanol, twice for 80% acetone, and once for 70% ethanol. The supernatant from the final wash was discarded, and the pellet was dried in an incubator set to 37°C. The protein was resuspended in 45 µL of LB-TT buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 18 mM Tris-HCl (pH 8.0-8.3), 14 mM Tris (8.0), 0.2% (v/v) Triton X-100, 1 EDTA-free proteinase inhibitor cocktail tablet, 50 mM dithiothreotol (added immediately prior to use)] and incubated for at least 1 hour. Once the protein was completely resuspended, it was stored at -80°C.

Protein Quantification

Protein was quantified using the Bradford assay. The samples were equilibrated to room temperature and vortexed briefly. The following volumes listed in Table 2.2 were added to 1.5 mL microcentrifuge tubes, briefly vortexed, and centrifuged for 5 seconds.

Table 2.2 - Reagent Volumes used in Bradford Assay for Protein Quantification Reagent Volume (µL) Protein Sample 6 LB-TT 54 Milli-Q Water 740 Protein Assay Dye (BioRad, Hercules, CA, USA) 200

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The samples were incubated at room temperature for 10 minutes, placed in optical cuvettes, and quantified using a DU 530 Life Science UV/VIS Spectrophotometer (Beckman Coulter, Brea, CA, USA). Considering the ingredients of LB-TT (including high molar urea and a few detergents), an LB-TT blank was used for a more accurate quantification, and protein concentrations were calculated using a bovine serum albumin (BSA) standard curve. Protein samples were then stored at -80°C in 100 µg aliquots.

Acetone Precipitation

Although all samples contained 100 µg of protein, there was a wide variation in the total volume of each sample. As a result, Pierce's protocol for acetone precipitation of proteins was used to normalize the volumes of all protein samples to 15 µL (Acetone precipitation of proteins, 2009). Four volumes of pre-chilled 80% acetone were added to each sample, vortexed briefly and incubated at -20°C for 60 minutes. The proteins were precipitated by centrifugation for 10 minutes at 13,000xg. The supernatant was removed, and the pellets were dried in a vacuum concentrator (Thermo, Waltham, MA, USA) until no liquid was visible in the tubes (approximately 5 minutes). The dried pellets were resuspended in 15 µL LB-TT.

One-Dimensional (1D) Gel Electrophoresis (1DGE)

100 µg of protein (the entire 15 µL) was mixed with 10 µL Loading Buffer (Fermentas, Thermo Scientific, Waltham, MA, USA), 2 µL 100 mM DTT, and 13 µL 10 mM Tris-HCl (pH 7.5). The samples were heat denatured for 10 minutes at 95°C then centrifuged for 1 minute at 13,000xg at 4°C. The supernatant was used for SDS polyacrylamide gel electrophoresis (SDS-PAGE) in commercially available Mini PROTEAN TGX 10% pre-cast gels (BioRad, Hercules, CA, USA). SDS-PAGE was conducted using a vertical electrophoresis unit (BioRad) at a constant voltage of 70V until the protein bands reached the end of gel line, or approximately 2 hours. The running buffer contained 250 mM Tris base, 0.92 M glycine, and 1% (w/v) sodium dodecyl sulfate. Molecular mass markers (5 µL of the commercially available EZ-RunTM Pre-Stained Rec Protein Ladder, Thermo, Waltham, MA, USA) were loaded in the lane adjacent to the samples. Gels were fixed with a solution

Texas Tech University, Amanda Sooter, May 2013 containing 50% methanol, 10% acetic acid, and 40% distilled water for 20 minutes, then stained overnight with a solution containing 10% phosphoric acid (85%), 10% ammonium sulfate, 20% methanol, and 0.12% Coomassie G-250.

In-Gel Tryptic Digestion

The proteins which underwent separation on 1DGE were subjected to tryptic digestion following the protocol detailed by Shevchenko, Tomas, Havlis, Olsen, and Mann (2006). Each sample lane was cut into 12 slices, and each slice was further cut into smaller pieces and placed in a well of a 96-well plate (Beckman-Coulter, Brea, CA, USA). The gel pieces were washed with 50% acetonitrile (ACN) in 100 mM ammonium bicarbonate (NH4HCO3) in 10 minute intervals to destain the gels. Reduction was performed by incubation for 1 hour at 56°C with 50 µL of 10 mM DTT. The gel pieces were washed with 50% ACN in Milli-Q water for 5 minutes at 37°C. The proteins were alkylated by adding 50 µL of 55 mM iodoacetamide (IA) and incubated at room temperature in the dark for 30 minutes. Again, the gel pieces were washed for 5 minutes with 50% ACN at 37°C then dehydrated by adding 50 µL of 100% ACN at 37°C for 5 minutes, or until the gel pieces turned white. The ACN was removed, and the gel pieces dried for 10 minutes at room temperature. The digestion was conducted by adding 30 µL of Trypsin solution (12.5 ng/µL in 25 mM NH4HCO3) with any additional volume of 25 mM NH4HCO3 necessary to completely cover the gel pieces and left for 18 hours at 37°C. Peptide extraction was performed by washing the gel pieces twice with 80 µL of 50% ACN, 0.1% formic acid in Milli-Q water, then once with 100% ACN. The extracted peptide solutions were dried in a vacuum concentrator (Thermo, Waltham, MA, USA) to zero-volume then stored at -20°C.

Nano-LC-MS/MS

The peptides obtained from the in-gel digestion were resuspended in 22 µL of 0.1% formic acid. 10 µL of each sample was then analyzed using nano-flow liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) using an LTQ-XL ion trap mass spectrometer (Thermo, CA, USA). Chromatographic separation of the peptides was performed using a Dionex nano-HPLC (Ultimate 3000) with a trapping column (C18, 3 µm, 100 Å, 75 µm 2 26

Texas Tech University, Amanda Sooter, May 2013 cm) followed by a reverse phase column (C18, 2 µm, 100 Å, 75 µm 15 cm, nanoViper). Peptides were first injected onto the trapping column at a rate of 5 µL/min. The column was equilibrated with 1% ACN, 0.1% formic acid in water and washed for 10 minutes with the same solvent at a flow rate of 300 nL/min. After washing, the trapping column was switched to the reverse-phase analytical column, and the bound peptides were eluted using solvents A (2% ACN, 0.1% formic acid in water) and B (98% ACN, 2% water, 0.1% formic acid). The gradient was kept constant for the first 10 minutes at 4% solvent B followed by a linear increase to 11% over 5 minutes. Solvent B was further increased to 40% in 35 minutes, then to 60% in 12 minutes, and to 95% in 3 minutes through three successive linear increases. Solvent B was maintained at 95% for 3 minutes. It was then rapidly decreased to 4% over 2 minutes and maintained at 4% for an additional 3 minutes to re-equilibrate the column. The gradient elution is depicted in Figure 2.3. The eluted peptides were directed into the nanospray ionization source of the LTQ-XL with a capillary voltage of 2 kV. The collected spectra were scanned over the mass range of 300-2000 atomic mass units. Data dependent scan settings were defined to choose the 6 most intense ions with dynamic exclusion list size of 100, exclusion duration of 30 seconds, repeat count of 2, and repeat duration of 15 seconds. MS/MS spectra were generated by collision-induced dissociation of the peptide ions at a normalized collision energy of 35%.

100 90 80

70 60 50 40 % B Solvent B % 30 20 10 0 0 10 20 30 40 50 60 70 80 Time (minutes)

Figure 2.3- Gradient Elution Method in Terms of % Solvent B

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Protein Identification

Protein identification was performed using peptide mass fingerprinting. The .raw spectra files generated through mass spectrometry were converted to .mzXML files through Mass Matrix conversion software (Case Western Reserve University, Cleveland, OH, USA). The .mzXML files were then uploaded into the GPM search engine (The Global Proteome Machine Organization, http://www.thegpm.org) and searched against the whole cotton fiber proteome created from RNA sequencing data as well as the whole cotton proteome created by combining the fiber transcriptome and other publicly available sequencing data. Simple, one- directory output was chosen, and reversed sequences were selected to be searched against as well. Carbamidomethylation of cysteine was selected as a complete modification, while oxidation of methionine (M) and tryptophan (W), deamidation of glutamine (Q) and aspartate (D), and dioxidation of methionine (M) and tryptophan (W) were all chosen as potential modifications. Further, the Ion Trap (4 Da) was selected as the method.

Quantitative Proteomic Analysis

For preliminary insight into the proteomics of cotton fiber development in 21 and 24 dpa, identified proteins from the GPM searches were compared to find those proteins present in each replicate through the use of the vertical lookup function in Excel. Their respective rI values, which indicate the number of peptides from each protein identified during mass spectrometric analysis were observed. The mean rI value for each protein in 21 dpa was compared to the respective value in 24 dpa replicates to gain insight into changes in expression across the two stages. The differential protein sequences were uploaded into Mercator tool (MapMen Site of Analysis, Max Planck Institute for Molecular Plant Physiology, Munich, Germany, http://mapman.gabipd.org/web/guest/app/mercator) in FASTA format and annotated. The resulting expression patterns and functional categorization data were used to map proteins into MapMan version 3.5.1R2 (Thimm et al., 2004.; MapMen Site of Analysis, Max Planck Institute for Molecular Plant Physiology, Munich, Germany, http://mapman.gabipd.org/web/guest/mapman) to provide proteomic insights into the molecular development of the cotton fiber in addition to the transcriptomic data obtained. 28

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CHAPTER III RESULTS

RNA Sequencing and Assembly

Transcriptomes of the two stages of cotton fiber development were studied by RNA- Sequencing using Illumina MiSeq sequencer. These two stages of development reflect time- points in the rapid cellulose biosynthesis stage of cotton fiber development. Detailed statistics of the two sequenced and assembled cDNA libraries are given in Table 3.1.

In the Illumina TruSeq RNA library preparation protocol, the adapters with index "barcodes" were added to each sample. After sequencing, the reads were de-multiplexed based on the index sequences and were assembled de novo in SeqMan NGen software based on the de Bruijn graph algorithm. For 21 dpa, 9.22 million reads were assembled into 23942 contigs, with 1964 (8.2%) contigs longer than 2000 bp, and the average length of all contigs was 1146 bp. Sequencing of 24 dpa cDNA resulted in 7.52 million reads which assembled into 19750 contigs. Of these, 1722 (8.7%) contigs were longer than 2000 bp, averaging 1138 bp per contig. Although the 21 dpa library generated considerably more reads than the 24 dpa library, 24 dpa sequencing data was just as high quality (evident in the similar Contig N50). This discrepancy is thought to be the result of differences in adapter ligation efficiencies in the TruSeq protocol as well as the fact that 21 dpa starting RNA was of a higher quality (high RINe) than 24 dpa RNA.

Table 3.1- Statistics of Sequencing & Assembly Data

Time-points 21 dpa 24 dpa # of Reads (Million) 9.22 7.52 # of Contigs 23942 19750 Contigs >2000 1964 1722 Contig N50 (bases) 1381 1394 Avg. Length Contigs (bases) 1146 1138 Avg. Length Assembled 141 139 Sequences (bases) Average Quality Score 35 35

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Contigs from both these stages were reassembled using CAP3 software based on overlap layout consensus algorithm to create a non-redundant "Late Fiber Development (LFD)” reference transcriptome with 31710 contigs. Furthermore, the LFD transcriptome was combined with the reference transcriptomes which were created from early and middle fiber development through the use of CAP3 assembly to create a non-redundant Cotton Fiber Transcriptome which contains 62,427 contigs.

Creation of Cotton Proteome Databases

The Cotton Fiber Transcriptome created from all six stages of cotton fiber development was then used to create proteome databases to be used for the identification of proteins using the data obtained from mass spectrometry. The tissue specific, in-house CBG1 Cotton Fiber Proteome resulted from the 6-frame translation of the Cotton Fiber Proteome using an in- house Perl script and contains 374,562 contigs. Additionally, a Global Cotton Proteome was created by combining RNA-Sequencing data in the Cotton Fiber Transcriptome with other publicly available cotton sequencing data using CAP3 assembly. The resulting assembly generated the Global Cotton Proteome in the same manner as described earlier. It contained 539,448 contigs. A summary of the creation of the fiber transcriptome and proteome is detailed in Figure 3.1.

Figure 3.1 - Summary of the Fiber Transcriptome and Proteomes

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Gene Expression Profiling

Using the Late Fiber Development (LFD) reference transcriptome in Array Star (DNASTAR Genome suite), the cDNA sequence reads from 21 and 24 dpa were analyzed for differential expression between the two fiber development stages. With fold change ≥ 2.0 and p < 0.01, 3602 transcripts were found to be significant and differentially expressed between 21 and 24 dpa. Among these, 2285 transcripts were found to be down-regulated, and 1317 were up- regulated (Figure 3.2). In general, transcripts associated with metabolism and cell signaling were down-regulated while those related to cell wall structural components were up- regulated. Figure 3.3 shows the functional categories of the genes generated by the Mercator tool (http://mapman.gabipd.org/web/guest/app/mercator). Closer examination of Bin 35, Not Assigned, revealed 35% of the 35% Not Assigned transcripts identified had no ontology. Figure 3.4 shows the MapMan overview of the changes in transcriptomic responses between 21 and 24 dpa. Cell signaling, cell wall metabolism, membrane and lipid metabolism, secondary metabolism showed distinctly different expression patterns than the two comparisons of early and middle fiber development (separate studies).

Figure 3.2 - Differentially Expressed Transcripts. Red indicates up-regulation; blue represents down-regulation. White indicates select transcripts p>0.01, greater than 2.0-fold change 31

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Protein Expression Profiling

For comparison of the whole proteomes of the two fiber stages, 100 µg of extracted protein were separated using 1-D gel electrophoresis (1DGE) and peptides were identified using nano liquid chromatography tandem mass spectrometry. Table 3.2 summarizes the number of proteins identified for each sample using a 1% FDR2 cutoff. When searching against the Global Cotton Proteome database, an average of 1370 proteins was identified for 21 dpa samples. This number increased to an average of 1500 proteins when searched against the tissue specific CBG Cotton Fiber Proteome. Proteomic samples from 24 dpa presented extensive challenges for mass spectrometric analysis. Approximately 120 proteins were identified after searching the database from two separate replicates; however, one replicate yielded 550 proteins after the database search. Again, there was an increase in the number of proteins identified when searching against the CBG Cotton Fiber Proteome to 140 proteins for samples with 120 proteins. Among the identified proteins, 45 were found to be differentially expressed between the two developmental stages, mapped into pathways using MapMan software, and are presented in conjunction with transcript data as shown in Figure 3.4. In most cases, the protein expression patterns were consistent with the expression patterns observed in the differentially expressed transcripts. Although protein expression was consistent with transcriptomic expression levels, the following data presented will focus solely on transcriptomic data due to the low numbers of proteins identified in 24 dpa samples.

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Table 3.2- Number of Proteins Identified

Stage # of Proteins Identified with 1% FDR % Global Transcript CBG Cotton Fiber Increase Assembly Proteome Proteome (539,448 Contigs) (374,562 Contigs) 21-1 1342 1534 12.5% 21-2 1502 1644 8.6% 21-3 1262 1327 4.9% 24-1 122 136 10.3% 24-2 550 606 10.2% 24-3 119 144 17.4%

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Figure 3.3 - Functional Annotation of Transcript Data Generated by the Mercator Tool

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Figure 3.4 - Transcriptomic & Proteomic Data Mapped into Pathways via MapMan. Boxes represent transcripts; triangles represent corresponding proteins. Red represents up-regulation of the transcript/protein; green shows down-regulation.

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Cell Wall Metabolism

Cell Wall Modifications

Transcripts associated with cell wall modification showed a general trend (80%) of down- regulation (Table 3.3, Figure 3.5). 75% of repressed genes were endoxyloglucan transferases.

Table 3.3 - Cell Wall Modification Expression Levels

ID Description Expression contig13424 (Touch 4); hydrolase, acting on glycosyl bonds / -4.2 DOWN xyloglucan: xyloglucosyl transferase contig15563 (XYLOGLUCAN ENDOTRANSGLYCOSYLASE 6) -2.7 DOWN contig30533 (ARABIDOPSIS THALIANA EXPANSIN A6) -0.8 DOWN contig2210 (XYLOGLUCAN ENDOTRANSGLYCOSYLASE 4) -0.7 DOWN contig27846 (ENDOXYLOGLUCAN TRANSFERASE A4 1.6 UP

Figure 3.5 - Cell Wall Modifications Pectinesterases

The transcripts from the pectinesterase family were shown to be highly up-regulated as shown in Table 3.4, Figure 3.6.

Table 3.4 - Pectinesterase Expression Levels

ID Description Expression contig10363 pectinesterase family protein -2.2 DOWN contig14912 pectin methylesterase -1.1 DOWN contig27511 pectinesterase family protein 1 UP contig26532 pectinesterase family protein 3.7 UP

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Table 3.4 Continued ID Description Expression contig29471 pectinesterase family protein 4.1 UP contig28522 pectinesterase family protein 5.2 UP contig23912 pectinesterase family protein 5.7 UP contig23374 pectinesterase family protein 6.4 UP contig24136 pectinesterase family protein 8.1 UP

Figure 3.6 - Pectinesterases Polygalacturonases and Pectin Lyases

The polygalacturonases and pectate lyases exhibited strong up-regulation at the transcriptomic level. Eight of eleven identified transcripts displayed this pattern (Table 3.5, Figure 3.7).

Table 3.5- Polygalacturonase & Pectin Lyase Expression Levels

ID Description Expression contig12590 BURP domain-containing protein -1.9 DOWN contig10380 pectate lyase family protein -1.3 DOWN contig12290 Lyase -1.2 DOWN contig3703 glycoside hydrolase family 28 protein / 1.9 UP polygalacturonase (pectinase) family protein contig30166 Polygalacturonase 3.4 UP contig30275 Polygalacturonase 4.4 UP contig26481 pectate lyase family protein 5.2 UP contig24311 PGA4 (POLYGALACTURONASE 4); 5.6 UP polygalacturonase contig24012 AT2G02720 pectate lyase family protein 5.9 UP contig24231 AT2G02720 pectate lyase family protein 6.7 UP contig26673 AT2G02720 pectate lyase family protein 7.2 UP 37

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Figure 3.7- Polygalacturonases & Pectin Lyases Cellulose Synthesis Table 3.6, Figure 3.8 details various genes involved in the synthesis of cellulose with their fold expression changes. Cellulose synthase isoforms were up-regulated; whereas, genes like irregular xylem cellulose synthase isoforms (IRX) were down-regulated.

Table 3.6 - Cellulose Synthesis Expression Levels

ID Description Expression contig9720 IRX1 (IRREGULAR XYLEM 1); cellulose -1.8 DOWN synthase contig13675 CSLD3 (CELLULOSE SYNTHASE-LIKE D3); -1.2 DOWN cellulose synthase/ transferase contig9540 IRX3 (IRREGULAR XYLEM 3); cellulose -1.1 DOWN synthase contig14480 CSLD3 (CELLULOSE SYNTHASE-LIKE D3); -1 DOWN cellulose synthase/ transferase contig18434 IRX6 -0.9 DOWN contig23899 CESA6 (CELLULOSE SYNTHASE 6); cellulose 0.6 UP synthase/ transferase contig40 ATGH9A1 (ARABIDOPSIS THALIANA 1.1 UP GLYCOSYL HYDROLASE 9A1); cellulase/ hydrolase contig28024 CESA2 (CELLULOSE SYNTHASE A2) 2.4 UP

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Figure 3.8 - Cellulose Synthesis Major Carbohydrate Metabolism

Sucrose Degradation

Genes associated with sucrose degradation like sucrose synthases and hexokinases, showed a general trend of repression (Table 3.7, Figure 3.9).

Table 3.7 - Sucrose Degradation Expression Levels

ID Description Expression contig21071 SUS6 (SUCROSE SYNTHASE 6); UDP- -1.6 DOWN glycosyltransferase/ sucrose synthase contig9760 SUS1 (SUCROSE SYNTHASE 1); UDP- -1 DOWN glycosyltransferase/ sucrose synthase contig17040 HXK3 | HXK3 (HEXOKINASE 3) -0.7 DOWN contig23703 HXK1 (HEXOKINASE 1) 1.1 UP

Figure 3.9- Sucrose Degradation Phenylpropanoids

Transcripts associated with phenylpropanoid metabolism exhibited general down-regulation. 80% of ten transcripts were down-regulated (Table 3.8, Figure 3.10).

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Table 3.8 - Phenylpropanoid Expression Levels

ID Description Expression contig17192 AT3G53260 | PAL2; phenylalanine ammonia- -3.1 DOWN lyase contig14682 4-coumarate--CoA ligase family protein -2.3 DOWN contig15492 CAD9 (CINNAMYL ALCOHOL -1.7 DOWN DEHYDROGENASE 9) contig12854 4CL1 (4-COUMARATE:COA LIGASE 1) -1.6 DOWN contig24324 AT4G34050 | caffeoyl-CoA 3-O- -1.5 DOWN methyltransferase, putative contig16072 AT2G37040 |pal1 (Phe ammonia lyase 1) -1.3 DOWN contig10371 AT4G34050 | caffeoyl-CoA 3-O- -1 DOWN methyltransferase, putative contig14517 OPCL1 (OPC-8:0 COA LIGASE1); 4-coumarate- -0.8 DOWN CoA ligase contig26336 AT3G53260 | PAL2; phenylalanine ammonia- 1.2 UP lyase contig31187 AT2G37040 |pal1 (Phe ammonia lyase 1) 1.6 UP

Figure 3.10 - Phenylpropanoids Signaling

There was a global down-regulation of signaling molecules. Of the 146 transcripts associated with signaling, 65% were down-regulated (Figure 3.11).

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10 Down-Regulated Transcripts 0 Up-Regulated -10 Transcripts

-20 Number of Transcripts Number -30

-40

Figure 3.11 - Expression Trends of Various Components of Cell Signaling G Proteins

G proteins exhibited both down and up-regulation as opposed to the general repression seen in most signaling molecules (Table 3.9).

Table 3.9 - G Protein Expression Levels

ID Description Expression contig20402 PRA1.H (PRENYLATED RAB ACCEPTOR 1.H) -3.6 DOWN contig18935 GTP binding / GTPase -2 DOWN contig15347 RabGAP/TBC domain-containing protein -2 DOWN contig14740 XLG3 (extra-large GTP-binding protein 3) -1.9 DOWN contig14040 ATGB2 (GTP-BINDING 2); GTP binding -1.4 DOWN contig15825 AtRABA6b (Arabidopsis Rab GTPase homolog A6b) -1.4 DOWN contig22410 ARAC1; GTP binding -1.4 DOWN contig12023 ARAC5 (RAC-LIKE GTP BINDING PROTEIN 5) -1.3 DOWN contig14294 GP ALPHA 1 (G PROTEIN ALPHA SUBUNIT 1) -1.1 DOWN contig15917 RAB6A; GTP binding -1.1 DOWN contig3709 RAB6A; GTP binding -1.1 DOWN contig3128 RHA1 (RAB HOMOLOG 1) -0.9 DOWN contig15433 ATSAR2 (ARABIDOPSIS THALIANA -0.9 DOWN SECRETION-ASSOCIATED RAS SUPER FAMILY 2)

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Table 3.9 Continued ID Description Expression contig18232 RID3 (ROOT INITIATION DEFECTIVE 3) -0.9 DOWN contig14610 Rho GDP-dissociation inhibitor family protein -0.9 DOWN contig17007 transducin family protein / WD-40 repeat family -0.6 DOWN protein contig27288 ATGB2 (GTP-BINDING 2) 0.6 UP contig24535 ATMIN7 (ARABIDOPSIS THALIANA HOPM 0.7 UP INTERACTOR 7) contig23751 RHD3 (ROOT HAIR DEFECTIVE 3) 0.8 UP contig31506 AGD4 (ARF-GAP domain 4) 1 UP contig28074 GTP binding / GTPase 1 UP contig27670 Ran-binding protein, putative 1 UP contig6607 guanylate-binding family protein 1 UP contig31474 RAB GTPase activator 1.1 UP contig31142 guanine nucleotide exchange family protein 1.1 UP contig27880 AtRABA1c (Arabidopsis Rab GTPase homolog A1c) 1.3 UP contig29401 RAB GTPase activator 1.4 UP contig24256 ATGB1 (ARABIDOPSIS THALIANA GTP- 1.4 UP BINDING PROTEIN 1) contig30858 Binding 1.7 UP contig29707 rac GTPase activating protein, putative 2.1 UP contig3509 AtRABH1e (Arabidopsis Rab GTPase homolog H1e) 2.3 UP contig27032 GTP binding / GTPase 2.7 UP contig31431 ROPGEF5 (ROP GUANINE NUCLEOTIDE 2.7 UP EXCHANGE FACTOR 5) contig29592 ATRABA1D (ARABIDOPSIS RAB GTPASE 2.8 UP HOMOLOG A1D) contig24724 rac GTPase activating protein, putative 2.9 UP contig28150 ROPGEF12 (RHO GUANYL-NUCLEOTIDE 4.6 UP EXCHANGE FACTOR 12)

Calcium Signaling

37 transcripts were associated with calcium signaling, and 29 (~78%) were shown to be down-regulated (Table 3.10).

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Table 3.10 - Calcium Signaling Expression Levels

ID Description Expression contig19071 PBP1 (PINOID-BINDING PROTEIN 1); calcium -3.9 DOWN ion binding contig15048 TCH2 (TOUCH 2); calcium ion binding -3.1 DOWN contig14652 calcium-transporting ATPase, plasma membrane- -2.5 DOWN type, putative contig13635 calmodulin-binding protein -2.1 DOWN contig10704 CAM1 (CALMODULIN 1); calcium ion binding -2.1 DOWN contig19227 iqd2 (IQ-domain 2); calmodulin binding -2 DOWN contig20425 calcium-transporting ATPase, plasma membrane- -1.6 DOWN type, putative contig15595 calmodulin-binding family protein -1.5 DOWN contig13165 CPK28; ATP binding / calcium ion binding / -1.4 DOWN calmodulin-dependent protein kinase contig19161 BON3 (BONZAI 3); calcium-dependent -1.4 DOWN phospholipid binding contig19664 calcium-binding protein, putative -1.4 DOWN contig20481 calmodulin-binding family protein -1.4 DOWN contig9815 CRT1 (CALRETICULIN 1); calcium ion binding / -1.3 DOWN unfolded protein binding contig14860 MSS3 (multicopy suppressors of snf4 deficiency in -1.2 DOWN yeast 3); calcium ion binding contig15220 CPK13; ATP binding / calcium ion binding / -1.2 DOWN calmodulin-dependent protein kinase contig18350 calcium-transporting ATPase, plasma membrane- -1.2 DOWN type, putative contig14975 VQ motif-containing protein -1.2 DOWN contig375 IQD14; calmodulin binding -1.1 DOWN contig29099 ATEHD1 (EPS15 HOMOLOGY DOMAIN 1); -1 DOWN GTP binding / GTPase/ calcium ion binding contig14988 ATCP1 (Ca2+-binding protein 1); calcium ion -1 DOWN binding contig19961 MSS3 (multicopy suppressors of snf4 deficiency in -1 DOWN yeast 3); calcium ion binding contig13461 AGD11 | AGD11 (ARF-GAP domain 11); calcium -1 DOWN ion binding contig17927 IQD6 (IQ-domain 6); calmodulin binding -0.9 DOWN contig10118 CRT1 (CALRETICULIN 1); calcium ion binding / -0.9 DOWN unfolded protein binding contig12852 iqd32 (IQ-domain 32); calmodulin binding -0.8 DOWN

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Table 3.10 Continued ID Description Expression contig20647 lipase class 3 family protein / calmodulin-binding -0.7 DOWN heat-shock protein, putative contig16612 CDPK6 (CALCIUM-DEPENDENT PROTEIN -0.7 DOWN KINASE 6) contig14909 PEPKR2 (Phosphoenolpyruvate carboxylase- -0.7 DOWN related kinase 2) contig21337 calcium ion binding -0.7 DOWN contig29800 calcium-binding protein, putative 0.9 UP contig25271 calcium-transporting ATPase 1 UP contig30062 ACA9 (AUTOINHIBITED CA(2+)-ATPASE 9) 1 UP contig25810 MSS3 (multicopy suppressors of snf4 deficiency in 1 UP yeast 3); calcium ion binding contig25667 phospholipid/glycerol acyltransferase family 1.3 UP protein contig4738 calcium-dependent protein kinase, putative / 1.3 UP CDPK, putative contig26597 calmodulin-binding protein 2.3 UP contig28350 ATEHD1 (EPS15 HOMOLOGY DOMAIN 1) 2.6 UP

14-3-3 Signaling Components

Although there were few transcripts identified as 14-3-3, both were down-regulated (Table 3.11).

Table 3.11 - 14-3-3 Signaling Expression Levels

ID Description Expression contig10581 GF14 PHI (GF14 PROTEIN PHI CHAIN) -1.1 DOWN contig10543 GRF2 (GENERAL REGULATORY FACTOR 2) -0.6 DOWN

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GDSL Lipases

There was a general repression (75%) of GDSL lipase transcripts (Table 3.12).

Table 3.12 - GDSL Lipases Expression Levels ID Description Expression contig17742 GDSL-motif lipase/hydrolase family protein -2 DOWN contig11960 GDSL-motif lipase/hydrolase family protein -1.2 DOWN contig9824 GDSL-motif lipase/hydrolase family protein -1.1 DOWN contig21925 GDSL-motif lipase/hydrolase family protein -0.9 DOWN contig9605 GDSL-motif lipase/hydrolase family protein -0.9 DOWN contig13437 GDSL-motif lipase/hydrolase family protein -0.9 DOWN contig27128 GDSL-motif lipase/hydrolase family protein 1.6 UP contig26956 GDSL-motif lipase/hydrolase family protein 4.5 UP

Transcription Factors

There was a global down-regulation of transcription factors with the exception of a few groups, such as the chromatin remodeling factors. 65% of over 200 transcription factors were repressed between 21 and 24 dpa (Figure 3.12).

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-5

-10

Number of TranscriptsNumber -15

-20

-25

Figure 3.12 - Expression Trends of Transcription Factors Chromatin Remodeling

There was considerable up-regulation of transcription factors involved in chromatin remodeling processes (84%) (Table 3.13). Furthermore, transcripts identified as DNA methyltransferases and histone deacetylases were identified (Table 3.14).

Table 3.13 - Chromatin Remodeling Expression Levels

ID Description Expression contig18024 ETL1; ATP binding / DNA binding / helicase -2.7 DOWN contig14058 transcription regulatory protein SNF2 -1.1 DOWN contig12101 SWIB complex BAF60b domain-containing -1 DOWN protein contig18195 CHR11 (CHROMATIN-REMODELING 0.6 UP PROTEIN 11) contig23987 SYD (SPLAYED); ATPase/ chromatin binding 0.6 UP contig21413 ATSWI3C (SWITCH/SUCROSE 0.7 UP NONFERMENTING 3C)

Texas Tech University, Amanda Sooter, May 2013 contig6956 transcription regulatory protein SNF2 0.8 UP contig29803 SYD (SPLAYED); ATPase/ chromatin binding 1.6 UP contig29095 transcription regulatory protein SNF2, putative 1.7 UP contig29274 transcription regulatory protein SNF2, putative 2 UP contig30557 transcription regulatory protein SNF2, putative 2.1 UP contig30735 SWI2 (SWITCH 2); ATP binding / DNA binding / 2.6 UP helicase contig31019 MOM (MORPHEUS MOLECULE) 2.6 UP contig27389 RGD3 (ROOT GROWTH DEFECTIVE 3) 2.7 UP

Table 3.14 - DNA Methyltransferase & Histone Deacetylase Expression Levels

ID Description Expression contig19645 DNMT2 (DNA METHYLTRANSFERASE-2) -1.7 DOWN contig29507 HOS15 (high expression of osmotically 1.5 UP responsive genes 15) contig24887 DRM1 (domains rearranged methylase 1) 1.7 UP contig29895 DMT2 (DNA METHYLTRANSFERASE 2) 3.1 UP contig30803 CMT2 (chromomethylase 2) 3.8 UP

Miscellaneous

Drought/Salt Responsive

There was a general up-regulation of transcripts associated with drought and salt stress (Table 3.15). Particularly, there was an up-regulation of dehydration-responsive family proteins; however, Mercator did not assign a more specific identifier.

Table 3.15 - Drought/Salt Responsive Element Expression Levels

ID Description Expression contig12812 response to water deprivation -2.6 DOWN contig14975 VQ motif-containing protein -1.2 DOWN contig21537 dehydration-responsive family protein -1 DOWN contig20541 dehydration-responsive protein-related 0.9 UP contig8083 dehydration-responsive protein-related 1.3 UP contig29430 dehydration-responsive protein-related 1.5 UP contig30181 dehydration-responsive family protein 2.6 UP contig29827 dehydration-responsive protein-related 3.7 UP

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Interestingly, storage proteins were found to be increasingly up-regulated from initiation (3 and 5 dpa) through secondary cell wall synthesis (21 and 24 dpa).

NOTES

1. Texas Tech University Center for Biotechnology and Genomics 2. False Discovery Rate

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CHAPTER IV DISCUSSION

Protein Expression Profiling

The number of proteins identified through mass spectrometry in 24 dpa samples was considerably less (on average 80% less) than the number of proteins in 21 dpa samples. This discrepancy may be explained based on the state of glycosylation of proteins in 24 dpa. Most proteins within the plant cell wall exist as glycoproteins (Northcote, 1989). Further, cell wall proteins known as arabinogalactan proteins (AGPs) were shown to be up-regulated. AGPs are a class of heavily glycosylated hydroxyproline rich glycoproteins. The carbohydrate moiety can account for 90-98% (w/w) of the protein's weight (Johnson, Jones, Bacis, & Schultz, 2003). Separation of proteins prior to mass spectrometric analysis employed C18 columns. The carbohydrate moieties could possibly have interacted with the C18 column and may have been retained on the column, preventing detection on the mass spectrometer. Enzymatic deglycosylation of 24 dpa proteins prior to GeLC and LC-MS/MS should reveal if heavy glycosylation of proteins at 24 dpa is interfering with detection. Additionally, there was approximately a 10% increase in the number of proteins identified using the CBG Cotton Fiber Proteome when compared to the Global Cotton Proteome. These findings further confirm a hypothesis put forth by the Association of Biomolecular Resource Facilities Proteome Informatics Research Group (iPRG). This group stipulated that refined, more specific protein databases could be created by using RNA-Seq data. A possible explanation as to why more proteins were identified using the CBG Cotton Fiber Proteome rather than the Global Cotton Proteome is that peptides searched against the Global Cotton Proteome mapped to more than one location, which resulted in lower scores associated with that particular hit. This increase also confirms the quality of RNA-Sequencing data and its subsequent assemblies.

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Select Functional Categories and Their Implications in Secondary Cell Wall Biosynthesis

Cell Wall Modifications

Transcripts associated with cell wall modifications, in general, exhibited repression. Further, 75% of these down-regulated transcripts were endoxyloglucan transferases. Xyloglucans comprise a large portion of plant cell walls. They serve as cross-links of cellulose fibrils (Bauer, Talmadge, Keegstra, & Albersheim, 1973). Loosening of the cell wall is important for elongation of the cell (Taiz, 1984). Endoxyloglucan transferases function by cleaving then reconnecting xyloglucan cross-links. This process is thought to play a large role in the loosening of the cell wall for elongation (Nishitani & Tominaga, 1992). It was recently discovered in tobacco cells that the integrity of the cell wall alters levels of endoxyloglucan transferases. Loosening of the cell wall resulted in a down-regulation of such molecules in order to restore cell wall structure (Nakagawa & Sakurai, 2001). Since the cell walls are preparing for thickening, such findings would be expected in cotton fibers undergoing secondary cell wall synthesis. Down-regulation of the endoxyloglucan transferases was contrary to findings by Shimizu et al. (1997) which stated that such levels remained constant during all stages of growth (Shimizu et al., 1997). However, such findings seem to be in accordance with cellular processes occurring during secondary cell wall synthesis: the cell is no longer lengthening, so endoxyloglucan transferases which aid in the process of loosening the cell wall would no longer be necessary.

Pectinesterases, Polygalacturonases, and Pectate Lyases

Pectins are complex carbohydrates which comprise up to 35% of primary plant cell walls. Although their presence is considerably less in the secondary cell wall, they still play an important role in structure (Buchanan, Gruissem, & Jones, 2000). Pectins are transported to the cell wall in a highly methyl-esterified form. After they are secreted into the cell wall, they can be modified by pectin esterases, also known as pectin methylesterases (PMEs). PMEs modify pectins through demethylesterification, resulting in the formation of acidified pectins and the release of methanol (Micheli, 2001). The consensus in the scientific community stipulates that pectin methylesterases within the cell wall can act on homogalacturonans 50

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(pectins) through two separate modes of action: randomly or linearly (Markoviě & Kohn, 1984). When PMEs act randomly, the deesterification of homogalacturonans results in the release of protons which promotes the activity of endopolygalacturonases, which leads to loosening of the cell wall (Moustacas et al., 1991). Conversely, the linear activity of PMEs on homogalacturonans leads to cell wall stiffening. The linear deesterification leads to blocks of free carboxylic acid groups which interact with calcium ions, forming a pectate gel (Goldberg, Morvan, Jauneau, & Jarvis, 1996). Such considerations may suggest PMEs in cotton fibers may employ a linear mode of action. In further support, pectin methyltransferases induced by auxins have been associated with elongation of the cell wall and the influx of water (Brian & Newcomb, 1954; Yoda, 1958), suggesting auxin-induced PMEs act through random deesterification to promote cell wall loosening. Auxins were also shown to be down-regulated.

The regions of polygalacturonic acid produced within the complex pectin molecule through the action of pectin methylesterases serve as the substrate for pectate lyases and polygalacturonases. These two classes of enzymes depolymerize the polygalacturonic acid (Fries, Ihrig, Brocklehurst, Shevchik, & Pickersgill, 2007; Yadav, Yadav, Yadav, & Yadav, 2009). Pectate lyase depolymerization activity depends on the presence of calcium ions (Marin-Rodriguez, 2002). This depolymerization of cell-wall polygalacturonides leads to the destruction of the integrity of plant tissues (Collmer & Keen, 1986) and has been implicated in the fruit ripening process in many different species (Markovic & Jörnvall, 1986; Seymour & Gross, 1996). Such an argument seems contradictory to the previous implications for pectin methylesterases – while PMEs seem to be strengthening the cell wall, the pectate lyases are breaking it down. This inconsistency resulted in further analysis of the transcriptomic data relating to the two classes. Although pectin methylesterases and pectate lyases both show high up-regulation of transcripts (greater than 5.0 fold in many cases), there were considerably less transcripts that mapped to the pectate lyases. PMEs had twice as many raw reads mapped, suggesting that although pectate lyases may be highly up-regulated, there is considerably more PMEs which may result in a compensatory mechanism and

Texas Tech University, Amanda Sooter, May 2013 ultimately leads to a strengthened secondary cell wall. Such discrepancies bring attention to the paradox of the relative fold change in transcriptomic analyses.

Cellulose Synthesis

Although there were contradictory expression levels, there was up-regulation of transcripts of isoforms of cellulose synthase. Certain genes followed trends reported in previous studies, like Cellulose synthase A2 (CelA2), which was up-regulated, in concurrence with the findings of Pear, Kawagoe, Schreckengost, Delmer, and Stalker (1996). Only two cellulose synthase isoforms were identified; ten cellulose synthase isoforms have been identified in Arabidopsis (Doblin, Kurek, Jacob-Wilk, & Delmer, 2002), suggesting p < 0.01 may have been too stringent. Identification using p < 0.05 will be employed for further scrutiny. Cellulose synthase is a transmembrane enzyme complex which utilizes UDP-glucose as the substrate for glucan chain elongation, specifically the formation of cellulose (Pear, Kawagoe, Schreckengost, Delmer, & Stalker, 1996). Down-regulation of other enzymes identified as cellulose synthase or cellulose synthase-like may reflect changes at the level of the primary cell wall. Toward the end of elongation/onset of secondary cell wall synthesis, cellulose synthases responsible for the manufacture of cellulose in primary cell walls were down- regulated (Arpat et al., 2004). In a study comparing cellulose synthases in Arabidopsis and cotton, those responsible for deposition of cellulose in xylem tissue in Arabidopsis and those responsible for deposition in the primary cell wall were found to be phylogenetically similar but distinct from cellulose synthases associated with cellulose deposition in secondary cell walls (Betancur et al., 2010). The up-regulation of cellulose synthase isoforms suggests an increase in the formation of cellulose, the foremost component of the secondary cell wall; whereas, down-regulation of other cellulose synthases, specifically irregular xylem (IRX) isoforms suggests less cellulose deposition in the primary cell wall.

Sucrose Degradation

Transcripts associated with sucrose degradation showed a general pattern of down- regulation. Interestingly, this pattern differed from previously reported findings which stated that sucrose synthase levels remain relatively constant through the progression of fiber

Texas Tech University, Amanda Sooter, May 2013 development (Shimizu et al., 1997). Sucrose synthase is responsible for degrading sucrose into fructose and UDP-glucose (Chourey & Nelson, 1979). UDP-glucose is known as one of the most used substrates in cellulose synthesis. Upon further inspection of transcripts, there was an up-regulation of another enzyme responsible for synthesis of UDP-glucose. UDP- glucose pyrophosphorylase uses UTP and glucose-1-phosphate to create UDP-glucose and a pyrophosphate molecule (Kleczkowski, Geisler, Ciereszko, & Johansson, 2004). Further, hexokinase-1 was up-regulated. This enzyme is responsible for phosphorylating glucose, which could provide G1P for UDP production. Figure 4.1 represents the relationships between these enzymes and their roles in cellulose biosynthesis. Although sucrose synthase is repressed, another method of supplying UDP-glucose to cellulose synthase is providing this substrate, supporting the premise of increased cellulose deposition in the secondary cell wall.

Figure 4.1- Relationship of UDP-Glucose Manufacturers and Cellulose Synthase

Phenylpropanoids

The phenylpropanoid pathway is responsible for the biosynthesis of many plant cell wall phenolics, including lignins, lignans, flavonoids, cutins, and many others (Boerjan, Ralph, & Baucher, 2003; Anterola & Lewis, 2002). Cinnamyl alcohol dehydrogenase has been considered as a key regulatory component of phenylpropanoid synthesis because it catalyzes 53

Texas Tech University, Amanda Sooter, May 2013 the reduction of cinnamylaldehydes into cinnamyl alcohols. This is the final step in monolignol syntheis (Goffner et al., 1992). These monolignols can produce p- hydroxyphenyl, guaiacyl, and syringyl phenylpropanoid units. These can then be incorporated into lignin. Guaiacyl and syringyl units are the primary components of fiber lignin (Day et al., 2005). Most transcripts associated with the phenylpropanoid pathway were down-regulated. Further, CAD9 was shown to be down-regulated. Interestingly, this contradicts the findings of Fan et al. (2009) which showed up-regulation in cotton fibers during secondary cell wall synthesis using microarrays (Fan et al., 2009). These findings may suggest a down-regulation of lignin biosynthesis during the formation of the secondary cell wall. Such an argument can be further supported by the work of Hu et al. (1999) in transgenic aspen. The synthesis of lignin and cellulose were found to be compensatory. A 45% decrease in lignin resulted in the increase of cellulose by 15% (Hu et al., 1999).

G Proteins

G proteins are heterotrimeric proteins which are important components in signaling cascades. They function by integrating many signals and transducing them to downstream pathways (Studt, Humpf, & Tudzynski, 2013). G proteins consist of an α subunit (Gα) which is closely associated with two other subunits, (Gβγ). In mammals and fungi, Gα interacts with a G protein coupled receptor (GPCR) at the plasma membrane, which contains seven transmembrane alpha helices (Bohm, Gaudet, & Sigler, 1997). The binding of the cognate ligand to the GPCR results in a conformational change in the receptor which results in the exchange of GDP for GTP, activating the GPCR and causing its dissociation from the Gβγ subunits. Both the Gα subunit and Gβγ dimer can then bind and regulate their respective downstream effector molecules (Li, Wright, Krystofova, Park & Borkovich, 2007). Plant G proteins, on the other hand, are self-activating and do not interact with GPCRs (Urano et al., 2012). There are multitudes of targets in G protein signaling cascades (Wettschureck & Offermanns, 2005); therefore, there are many possible targets involved in G protein signaling. An in silico analysis of a Gα subunit in rice revealed its role in signaling after exposure to abiotic stressors, particularly salt, cold, drought stress (Yadav, Shukla, & Tuteja, 2013). A small GDP-binding protein known as ADP-ribosylation factor1 (Arf1) was found to 54

Texas Tech University, Amanda Sooter, May 2013 be pivotal in protein targeting in Arabidopsis. Its GDP-bound form (Arf1-GDP) is recruited to the Golgi where it functions in vesicle-mediated protein trafficking (Min et al., 2013). ADP-ribosylation factors were up-regulated in transcriptomic data. Interestingly, ROPGEFs (Rho of plant guanine nucleotide exchange factor) were found to be up-regulated as well. A recent study implicated ROPGEF4 in the initiation of a cell wall pattern. Its activity resulted in the local activation of ROP11 (a Rho GTPase) which lead to distinct patterning in the secondary cell walls of xylem cells as a result of local disassembly of cortical microtubules (Oda & Fukuda, 2012). G proteins have multiple targets in signaling, and their large variety adds even more targets. Some interesting subjects arising from the transcriptomic data provide insight into some roles for G proteins in secondary cell wall biosynthesis. A particular Gα subunit in rice was crucial in signaling resulting from drought, salt, and cold stress. Cotton fibers at 24 dpa are beginning to show an up-regulation of dehydration family proteins, G proteins may be effecting such signaling cascades. The up-regulation of ROFGEFs suggest their involvement in cell wall patterning in the secondary cell walls of cotton fiber.

Calcium Signaling Components

The majority of transcripts (78%) associated with calcium signaling were down-regulated. This finding is in accordance with the findings of Padmalatha et al. (2012), which ascertained that many transcripts representing calcium signal transduction pathways were down- regulated (Padmalatha et al., 2012). Hormones, light, stressors, and exposure to pathogens modulate calcium levels in plant cells (Harper, 2001; Knight & Knight, 2001). It was reported that, during elongation, calcium signaling components, such as calmodulin, were highly expressed (Gao et al., 2007). Such down-regulation of transcripts suggests that calcium signaling is repressed during secondary cell wall synthesis.

14-3-3 Signaling Components

14-3-3 transcripts were down-regulated. Previous findings have proposed that the 14-3-3 proteins in G. hirsutum may help regulate fiber cell elongation (Zhang et al., 2010; Zhu et al., 2011). In our group's as of yet unpublished data using the same techniques to investigate

Texas Tech University, Amanda Sooter, May 2013 elongation in 11 and 17 dpa, these 14-3-3 transcripts were found to be up-regulated. This expression pattern is in agreement with previous studies where 10 dpa fiber showed higher expression of 14-3-3 than 20 dpa (Lacape et al., 2012). Such findings may indicate a decreased necessity for 14-3-3 signaling in secondary cell wall biogenesis.

GDSL Lipases

GDSL lipases exhibited a general down-regulation. These enzymes are a relatively newly discovered class of enzymes with broad substrate specificity (Akoh, Lee, Liaw, Huang, & Shaw, 2004). Thus far, members of this class of lipolytic enzymes have been implicated in many important plant functions, including secondary metabolite synthesis, plant development, morphogenesis, and exposure to pathogens (Chepyshko, Lai, Huang, Liu, & Shaw, 2012). Presumably, the down-regulation of this particular class could possibly indicate the lessening of the necessity for such mechanisms in secondary cell wall synthesis. Down- regulation of GDSL lipases correlates with the general repression of flavonoids and phenylpropanoids.

Chromatin Remodeling

While most transcription factors were down-regulated, those associated with chromatin remodeling were up-regulated. Chromatin remodeling is an important event both in the repair of DNA, transcriptional activation and repression, as well as senescence or apoptosis of the cell (Wang, Allis, & Chi, 2007). In our study, we found several transcription factors involved with histone modifications, DNA methyltransferases and histone deacetylases, to be up- regulated. These types of modifications are most commonly associated with the condensation of chromatin into the transcriptionally inactive heterochromatin (Strahl & Allis, 2000). These findings lead us to speculate that up-regulation of these transcription factors will result in the formation of heterochromatin, leading the cotton fiber cell to a state of quiescence.

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CHAPTER V CONCLUSION

RNA Sequencing data generated from the transcriptomes of 21 and 24 dpa time-points in developing cotton fiber was used to create both transcriptome and proteome databases of cotton fiber in conjunction with transcriptomic data obtained from 3, 5, 11, and 17 dpa for future studies. The quality of such databases was indicated by various quality parameters of the short read assembly and further demonstrated by the increase in the number of proteins identified when using the CBG Cotton Fiber Proteome as opposed to a Global Cotton Proteome.

In terms of the biological relevance of findings, the data suggested that cotton fibers transitioning from 21 to 24 dpa, in brief, were re-allocating resources toward cellulose synthesis and strengthening of the cell wall and down-regulating many cellular processes, as indicated by repression of many signaling pathways and transcription factors. Up-regulation of the transcription factors associated with chromatin remodeling, specifically those associated with the formation of heterochromatin further support the previous stipulation. Cellulose synthesis was up-regulated, and transcripts thought to be involved with cellulose deposition in the primary cell wall were down-regulated. Though the enzyme most literature cites as responsible for the production of UDP-glucose (the substrate of cellulose synthase), sucrose synthase, was down-regulated, another enzyme with the same capabilities was up- regulated, UDP-glucose pyrophosphorylase. The up-regulation of cellulose synthesis can also be reflected in the down-regulation of a few pathways in secondary metabolism, specifically phenylpropanoids. The phenylpropanoids possess key regulatory elements of lignin, such as cinnamyl alcohol dehydrogenase (CAD) (Zhong & Ye, 2009). The down-regulation of this pathway suggests a down-regulation of lignin biosynthesis. The down-regulation of phenylpropanoids and flavonoids was further confirmed by the down-regulation of GDSL ligases, which serve as regulators of such pathways. Additionally, pectin methylesterases, pectate lyases, and polygalacturonases were up-regulated. The PMEs were implicated in the rigidifying of the cell wall through the formation of pectin gel by the deesterification of homogalactans. Endoxyloglucan transferases were down-regulated to further strengthen the

Texas Tech University, Amanda Sooter, May 2013 cell wall, considering the high expression of this class of enzymes in elongating fibers results in loosening of the cell wall. Most transcripts associated with signaling components and transcription factors were down-regulated. However, G proteins showed some up-regulation. This large family of proteins may result in signaling cascades which result in the patterning of the secondary cell wall as well as signal for the up-regulation of proteins responsive to dehydration stress. Considering the broad regulatory implications of so many signaling molecules, the fiber cells may be transitioning to a state of quiescence. This was further supported by up-regulation of chromatin remodeling factors, specifically DNA methyltransferases and histone deacetylases, suggesting certain portions of the DNA will be less accessible to transcriptional machinery (which has already been down-regulated), resulting in less transcriptional activity. Additionally, transcripts identified as dehydration family proteins were shown to be up-regulated. As cotton fibers mature, the living protoplasm of the cell decreases (Basra & Malik, 1984). This suggests there may be less free water within the cell. The fiber cells may be preparing for the next stage of development as well, dehydration and maturation of the fiber. In all, cellular resources are allocated to strengthening of the cell wall through cellulose synthesis and modification of pectins. The fiber cells begin to enter a quiescent state, indicated by down-regulation of many signaling components and transcription factors, with the exception of the chromatin remodeling factors. Figure 5.1 depicts these relationships.

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Figure 5.1 - Summary of Interactions of Select Functional Categories and Their Implications in Secondary Cell Wall Biosynthesis

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CHAPTER VI FUTURE WORK

Large amounts of transcriptomic data have been generated. Further scrutiny of the generated data is necessary to gain a better understanding of the molecular processes associated with the transition from 21 to 24 dpa in cotton fiber development. Proteomic data will be collected again for 24 dpa. Different techniques, such as enzymatic deglycosylation of protein extract prior to mass spectrometic analysis, will be employed to attempt to identify larger numbers of proteins than the numbers our research has identified at this time. A baseline of transcriptomic and proteomic data has been established for the developing TM1 cotton fiber ranging from 3 dpa to 24 dpa. This data will be used to investigate the effects of abiotic stress, such as drought, heat, and salt on the molecular development of the cotton fiber through comparison.

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APPENDIX A APPROACH TO PROTEOMIC ANALYSIS

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Figure A.1 – Approach to Proteomic Analysis

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APPENDIX B MASS SPECTROMETRY SET-UP

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Figure B.1 - Diagram of Mass Spectrometry Set-Up

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APPENDIX C TABLE OF EXPRESSION PATTERNS OF ALL DIFFERENTIALLY EXPRESSED TRANSCRIPTS

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Table C.1 - Table of Expression Patterns of All Differentially Expressed Transcripts

Functional Annotation ID Description Expression FLA9 (FASCICLIN-LIKE Cell Wall Protein - AGPs contig9301 ARABINOOGALACTAN 9) 1.2 UP

FLA9 (FASCICLIN-LIKE Cell Wall Protein - AGPs contig22464 ARABINOOGALACTAN 9) 1.1 UP leucine-rich repeat family protein / Cell Wall Protein - LRRs contig25146 extensin family protein 3.8 UP Pectin Esterases contig10363 pectinesterase family protein -2.2 DOWN Pectin Esterases contig14912 pectin methylesterase -1.1 DOWN Pectin Esterases contig27511 pectinesterase family protein 1 UP Pectin Esterases contig26532 pectinesterase family protein 3.7 UP Pectin Esterases contig29471 pectinesterase family protein 4.1 UP Pectin Esterases contig28522 pectinesterase family protein 5.2 UP Pectin Esterases contig23912 pectinesterase family protein 5.7 UP Pectin Esterases contig23374 pectinesterase family protein 6.4 UP Pectin Esterases contig24136 pectinesterase family protein 8.1 UP (Touch 4); hydrolase, acting on Cell Wall Modifications glycosyl bonds / contig13424 xyloglucan:xyloglucosyl transferase -4.2 DOWN

Cell Wall Modifications (XYLOGLUCAN contig15563 ENDOTRANSGLYCOSYLASE 6) -2.7 DOWN

Cell Wall Modifications (ARABIDOPSIS THALIANA EXPANSIN contig30533 A6) -0.8 DOWN

Cell Wall Modifications (XYLOGLUCAN contig2210 ENDOTRANSGLYCOSYLASE 4) -0.7 DOWN

Cell Wall Modifications contig27846 (ENDOXYLOGLUCAN TRANSFERASE A4 1.6 UP

Cellulose Synthesis IRX1 (IRREGULAR XYLEM 1); cellulose contig9720 synthase -1.8 DOWN

Cellulose Synthesis CSLD3 (CELLULOSE SYNTHASE-LIKE contig13675 D3); cellulose synthase/ transferase -1.2 DOWN

Cellulose Synthesis IRX3 (IRREGULAR XYLEM 3); cellulose contig9540 synthase -1.1 DOWN

Cellulose Synthesis CSLD3 (CELLULOSE SYNTHASE-LIKE contig14480 D3); cellulose synthase/ transferase -1 DOWN Cellulose Synthesis contig18434 IRX6 -0.9 DOWN

Cellulose Synthesis CESA6 (CELLULOSE SYNTHASE 6); contig23899 cellulose synthase/ transferase 0.6 UP 74

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Table C.1 Continued

Functional Annotation ID Description Expression ATGH9A1 (ARABIDOPSIS THALIANA Cellulose Synthesis GLYCOSYL HYDROLASE 9A1); cellulase/ contig40 hydrolase 1.1 UP Cellulose Synthesis contig28024 CESA2 (CELLULOSE SYNTHASE A2) 2.4 UP

Cell Wall Precursor RHM1 (RHAMNOSE BIOSYNTHESIS 1); Synthesis UDP-L-rhamnose synthase/ UDP- contig11530 glucose 4,6-dehydratase/ catalytic -1.2 DOWN

Cell Wall Precursor AUD1; UDP-glucuronate Synthesis decarboxylase/ catalytic/ dTDP- contig9936 glucose 4,6-dehydratase -1.1 DOWN

Cell Wall Precursor RHM1 (RHAMNOSE BIOSYNTHESIS 1); Synthesis UDP-L-rhamnose synthase/ UDP- contig14603 glucose 4,6-dehydratase/ catalytic -0.9 DOWN

Cell Wall Precursor GAE6 (UDP-D-GLUCURONATE 4- Synthesis EPIMERASE 6); UDP-glucuronate 4- contig19154 epimerase -0.8 DOWN

Cell Wall Precursor UGE1 (UDP-D-glucose/UDP-D- Synthesis galactose 4-epimerase 1); UDP- contig25775 glucose 4-epimerase 1 UP Cell Wall Degradation - AT5G20950 glycosyl hydrolase family beta 1,3 glucanases contig9482 3 protein 0.9 UP Cell Wall Degradation - beta 1,3 glucanases contig23933 glycosyl hydrolase family 3 protein 1.1 UP Cell Wall Degradation - AT5G20950 glycosyl hydrolase family beta 1,3 glucanases contig30732 3 protein 1.6 UP Cell Wall Degradation - ATFUC1 (alpha-L-fucosidase 1); alpha- mannan xylose contig14569 L-fucosidase -1.1 DOWN

Cell Wall Degradation - mannan xylose BXL2 (BETA-XYLOSIDASE 2); hydrolase, contig26223 hydrolyzing O-glycosyl compounds -0.9 DOWN Cell Wall Degradation - mannan xylose contig26608 glycosyl hydrolase family 5 protein 0.6 UP Cell Wall Degradation - Pectin Lyases & Polygalacturonases contig12590 BURP domain-containing protein -1.9 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression Cell Wall Degradation - Pectin Lyases & Polygalacturonases contig10380 pectate lyase family protein -1.3 DOWN Cell Wall Degradation - Pectin Lyases & Polygalacturonases contig12290 lyase -1.2 DOWN Cell Wall Degradation - glycoside hydrolase family 28 protein / Pectin Lyases & polygalacturonase (pectinase) family Polygalacturonases contig3703 protein 1.9 UP Cell Wall Degradation - Pectin Lyases & Polygalacturonases contig30166 polygalacturonase 3.4 UP Cell Wall Degradation - Pectin Lyases & Polygalacturonases contig30275 polygalacturonase 4.4 UP Cell Wall Degradation - Pectin Lyases & Polygalacturonases contig26481 pectate lyase family protein 5.2 UP Cell Wall Degradation - Pectin Lyases & PGA4 (POLYGALACTURONASE 4); Polygalacturonases contig24311 polygalacturonase 5.6 UP Cell Wall Degradation - Pectin Lyases & AT2G02720 pectate lyase family Polygalacturonases contig24012 protein 5.9 UP Cell Wall Degradation - Pectin Lyases & AT2G02720 pectate lyase family Polygalacturonases contig24231 protein 6.7 UP Cell Wall Degradation - Pectin Lyases & AT2G02720 pectate lyase family Polygalacturonases contig26673 protein 7.2 UP GPAT3 (GLYCEROL-3-PHOSPHATE Phospholipid Synthesis contig12568 ACYLTRANSFERASE 3); acyltransferase -2.6 DOWN

Phospholipid Synthesis contig4821 diacylglycerol kinase family protein -1.1 DOWN Phospholipid Synthesis contig21681 diacylglycerol kinase, putative -0.9 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression CLS (CARDIOLIPIN SYNTHASE); cardiolipin synthase/ Phospholipid Synthesis contig8541 phosphatidyltransferase 0.8 UP choline monooxygenase, putative Phospholipid Synthesis contig29491 (CMO-like) 1.1 UP AT3G11430 GPAT5 (GLYCEROL-3- Phospholipid Synthesis contig28328 PHOSPHATE ACYLTRANSFERASE 5) 2.2 UP AT3G11430 GPAT5 (GLYCEROL-3- Phospholipid Synthesis contig30168 PHOSPHATE ACYLTRANSFERASE 5) 3.4 UP cyclopropane fatty acid synthase, Phospholipid Synthesis contig6120 putative 3.4 UP MGD2; 1,2-diacylglycerol 3-beta- galactosyltransferase/ UDP- Glycolipid Synthesis contig18344 galactosyltransferase -1.1 DOWN AT2G43420 3-beta hydroxysteroid Metabolism (squalene, dehydrogenase/isomerase family steroids, etc.) contig7259 protein -1.4 DOWN Metabolism (squalene, steroids, etc.) contig5485 AT1G07380 ceramidase family protein -1.3 DOWN Metabolism (squalene, steroids, etc.) contig18964 AT1G07380 ceramidase family protein -1 DOWN Metabolism (squalene, AT3G07020 UDP-glucose:sterol steroids, etc.) contig11108 glucosyltransferase (UGT80A2) -0.8 DOWN AT2G43420 3-beta hydroxysteroid Metabolism (squalene, dehydrogenase/isomerase family steroids, etc.) contig26996 protein -0.8 DOWN Metabolism (squalene, steroids, etc.) contig11425 quinone reductase family protein -0.7 DOWN Metabolism (squalene, AT3G44830 lecithin:cholesterol steroids, etc.) contig26782 acyltransferase family protein 0.8 UP Metabolism (squalene, AT3G07020 UDP-glucose:sterol steroids, etc.) contig29727 glucosyltransferase (UGT80A2) 0.9 UP Metabolism (squalene, CAS1 (cycloartenol synthase 1); steroids, etc.) contig28147 cycloartenol synthase 0.9 UP Metabolism (squalene, steroids, etc.) contig25729 LOH2 (LAG ONE HOMOLOGUE 2) 1.4 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Metabolism (squalene, AT3G44830 lecithin:cholesterol steroids, etc.) contig18924 acyltransferase family protein 1.7 UP Metabolism (squalene, AT3G44830 lecithin:cholesterol steroids, etc.) contig29586 acyltransferase family protein 1.7 UP FAD2 (FATTY ACID DESATURASE 2); Metabolism - Fatty Acid delta12-fatty acid dehydrogenase/ Desaturation contig10883 omega-6 fatty acid desaturase -1 DOWN

Fatty Acid Synthesis & KCS10 (3-KETOACYL-COA SYNTHASE Elongation 10); acyltransferase/ catatic/ contig11163 transferase -1.2 DOWN

Fatty Acid Synthesis & KCS12 (3-KETOACYL-COA SYNTHASE Elongation 12); acyltransferase/ catatic/ contig16537 transferase -0.9 DOWN

Fatty Acid Synthesis & LACS1 | long-chain-fatty-acid--CoA Elongation ligase fami protein / long-chain acyl- contig22877 CoA synthetase fami protein -0.7 DOWN Fatty Acid Synthesis & Elongation contig23280 FATB (fatty acyl-ACP thioesterases B) 0.7 UP Fatty Acid Synthesis & LACS9 (LONG CHAIN ACYL-COA Elongation contig29458 SYNTHETASE 9 0.7 UP Fatty Acid Synthesis & KAS I | KAS I (3-KETOACYL-ACYL Elongation contig28525 CARRIER PROTEIN SYNTHASE I 0.8 UP Fatty Acid Synthesis & KCS20 (3-KETOACYL-COA SYNTHASE Elongation contig22944 20); fatty acid elongase 0.8 UP Fatty Acid Synthesis & Elongation contig31314 CAC3; acetyl-CoA carboxylase 1.4 UP

Fatty Acid Synthesis & Elongation KCS1 (3-KETOACYL-COA SYNTHASE 1); contig28750 acyltransferase/ fatty acid elongase 1.4 UP Fatty Acid Synthesis & acetyl-CoA synthetase, putative / Elongation contig26628 acetate-CoA ligase 1.6 UP

Fatty Acid Synthesis & ACC1 (ACETYL-COENZYME A Elongation CARBOXYLASE 1); acetyl-CoA contig23953 carboxylase 2.2 UP Degradation - Lipases contig18074 lipase class 3 family protein -1 DOWN Degradation - Lipases contig20335 lipase-related -0.8 DOWN Degradation - Lipases contig31577 lipase class 3 family protein 1.9 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Degradation - Lipases contig31184 triacylglycerol lipase 2.9 UP

Degradation - SRG3 (senescence-related gene 3); Lysophospholipases glycerophosphodiester contig11595 phosphodiesterase -2.2 DOWN Degradation - Lysophospholipases contig13905 PLDP1 (PHOSPHOLIPASE D P1) -1.2 DOWN

Degradation - SHV3 (SHAVEN 3); Lysophospholipases glycerophosphodiester contig10112 phosphodiesterase -0.9 DOWN Degradation - PLDALPHA1, PLD | PLDALPHA1 Lysophospholipases contig9825 (PHOSPHOLIPASE D ALPHA 1) -0.6 DOWN Degradation - Beta AT4G16760 ACX1 (ACYL-COA OXIDASE Oxidation contig11782 1); acyl-CoA oxidase -1.1 DOWN Degradation - Beta AT4G16760 ACX1 (ACYL-COA OXIDASE Oxidation contig11109 1); acyl-CoA oxidase -0.8 DOWN Degradation - Beta ACX4 (ACYL-COA OXIDASE 4); acyl-CoA Oxidation contig24505 oxidase 2.7 UP AT4G18240 ATSS4; transferase, Starch Synthesis contig20776 transferring glycosyl groups -1.8 DOWN Starch Synthesis contig9285 starch synthase, putative -1.5 DOWN Starch Synthesis contig19590 AtSS2 (starch synthase 2) -1.5 DOWN AT4G18240 ATSS4; transferase, Starch Synthesis contig16470 transferring glycosyl groups -1.3 DOWN

SBE2.2 (starch branching enzyme 2.2); Starch Synthesis contig5209 1,4-alpha-glucan branching enzyme -1.2 DOWN APL2 (ADPGLC-PPASE LARGE SUBUNIT); glucose-1-phosphate Starch Synthesis contig20427 adenylyltransferase -0.8 DOWN APL3; glucose-1-phosphate Starch Synthesis contig11272 adenylyltransferase -0.7 DOWN

Starch Synthesis contig10796 AT1G11720 ATSS3 (starch synthase 3) -0.7 DOWN AT4G18240 ATSS4; transferase, Starch Synthesis contig28591 transferring glycosyl groups 1.1 UP AT4G18240 ATSS4; transferase, Starch Synthesis contig29513 transferring glycosyl groups 1.8 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression

Starch Synthesis contig30811 AT1G11720 ATSS3 (starch synthase 3) 1.8 UP Sucrose Synthesis contig21831 sucrose-phosphatase 1 (SPP1) -1.8 DOWN

ATSPS1F (sucrose phosphate synthase Sucrose Synthesis contig27461 1F); sucrose-phosphate synthase 0.6 UP Starch Degradation contig22970 glucan phosphorylase, putative -0.7 DOWN Starch Degradation contig30984 BAM7 (BETA-AMYLASE 7) 2.3 UP

SUS6 (SUCROSE SYNTHASE 6); UDP- Sucrose Degradation contig21071 glycosyltransferase/ sucrose synthase -1.6 DOWN

SUS1 (SUCROSE SYNTHASE 1); UDP- Sucrose Degradation contig9760 glycosyltransferase/ sucrose synthase -1 DOWN Sucrose Degradation contig17040 HXK3 | HXK3 (HEXOKINASE 3) -0.7 DOWN Sucrose Degradation contig23703 HXK1 (HEXOKINASE 1) 1.1 UP AT3G30841 | 2,3-biphosphoglycerate- independent phosphoglycerate Glycolysis contig14485 mutase -1.2 DOWN

Glycolysis contig5053 TPI (TRIOSEPHOSPHATE ISOMERASE) -1.1 DOWN UGP (UDP-glucose Glycolysis contig9476 pyrophosphorylase) -1 DOWN fructose-bisphosphate aldolase, Glycolysis contig12288 putative -1 DOWN glucose-6-phosphate isomerase, Glycolysis contig10876 cytosolic (PGIC) -0.9 DOWN AT1G12000 | pyrophosphate-- fructose-6-phosphate 1- phosphotransferase beta subunit, Glycolysis contig12432 putative -0.9 DOWN GAPC1 (GLYCERALDEHYDE-3- PHOSPHATE DEHYDROGENASE C Glycolysis contig160 SUBUNIT 1) -0.9 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression AT1G12000 | pyrophosphate-- fructose-6-phosphate 1- phosphotransferase beta subunit, Glycolysis contig12432 putative -0.9 DOWN UTP--glucose-1-phosphate Glycolysis contig9274 uridylyltransferase, putative -0.7 DOWN Glycolysis contig10059 pyruvate kinase, putative -0.6 DOWN AT3G30841 | 2,3-biphosphoglycerate- independent phosphoglycerate Glycolysis contig29036 mutase 0.6 UP UTP--glucose-1-phosphate Glycolysis contig4955 uridylyltransferase, putative 0.7 UP Oxidative Pentose G6PD4 (GLUCOSE-6-PHOSPHATE Phosphate contig21436 DEHYDROGENASE 4) -1.4 DOWN Oxidative Pentose Phosphate contig9943 emb2024 (embryo defective 2024) -0.8 DOWN ALDH10A9; 3-chloroallyl aldehyde Fermentation contig23178 dehydrogenase -0.6 DOWN ALDH2C4; 3-chloroallyl aldehyde Fermentation contig31434 dehydrogenase 2 UP Gluconeogenesis contig15851 MDH (MALATE DEHYDROGENASE) -1.1 DOWN Gluconeogenesis contig29550 ICL (ISOCITRATE LYASE) 1.1 UP TCA contig11381 aconitate hydratase, cytoplasmic -1.2 DOWN TCA contig12904 isocitrate dehydrogenase, putative -1.1 DOWN TCA contig15851 MDH (MALATE DEHYDROGENASE) -1.1 DOWN TCA contig25412 IAR4; oxidoreductase, 1.2 UP LPD1 (LIPOAMIDE DEHYDROGENASE TCA contig26615 1) 2 UP malate dehydrogenase, cytosolic, TCA contig9448 putative -1.2 DOWN malate dehydrogenase, cytosolic, TCA contig9791 putative -0.6 DOWN IIL1 (ISOPROPYL MALATE ISOMERASE TCA contig14303 LARGE SUBUNIT 1) 0.6 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression ACA7 (ALPHA CARBONIC ANHYDRASE TCA contig27726 7) 6.4 UP AtGolS1 (Arabidopsis thaliana Raffinose Metabolism contig25581 galactinol synthase 1) 4.3 UP AtSIP2 (Arabidopsis thaliana seed Raffinose Metabolism contig1192 imbibition 2) -0.7 DOWN AT1G78580 | ATTPS1 (TREHALOSE-6- Trehalose Metabolism contig15993 PHOSPHATE SYNTHASE) -1.7 DOWN AT1G78580 | ATTPS1 (TREHALOSE-6- Trehalose Metabolism contig16585 PHOSPHATE SYNTHASE) -1.1 DOWN ATTPS9; transferase, transferring Trehalose Metabolism contig28699 glycosyl groups 0.6 UP

IMPL1(MYO- Myoinositol contig16346 INOSITOLMONOPHOSPHATASELIKE1) -1 DOWN MIPS2(MYO-INOSITOL-1- Myoinositol contig23715 PHOSTPATESYNTHASE2) 0.9 UP MIPS2(MYO-INOSITOL-1- Myoinositol contig12330 PHOSTPATESYNTHASE2) 1.3 UP

IMPL1(MYO- Myoinositol contig31595 INOSITOLMONOPHOSPHATASELIKE1 1.3 UP Myoinositol contig31361 MIOX1(MYO-INISITOLOXYGENASE) 1.5 UP AT2G13680 | CALS5 (CALLOSE Callose contig21788 SYNTHASE 5) -1.8 DOWN Callose contig10206 ATGSL12 (glucan synthase-like 12) -1.3 DOWN Callose contig19992 ATGSL04 (glucan synthase-like 4) -1.2 DOWN Callose contig18889 ATGSL12 (glucan synthase-like 12) -0.9 DOWN AT4G03550 | ATGSL05 (GLUCAN Callose contig11433 SYNTHASE-LIKE 5) 0.7 UP AT2G13680 | CALS5 (CALLOSE Callose contig27478 SYNTHASE 5) 0.9 UP AT4G03550 | ATGSL05 (GLUCAN Callose contig15790 SYNTHASE-LIKE 5) 1 UP AT4G03550 | ATGSL05 (GLUCAN Callose contig28979 SYNTHASE-LIKE 5) 1.4 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression AT4G03550 | ATGSL05 (GLUCAN Callose contig5454 SYNTHASE-LIKE 5) 1.6 UP AT4G03550 | ATGSL05 (GLUCAN Callose contig24956 SYNTHASE-LIKE 5) 2.6 UP Other Sugars sugar isomerase (SIS) domain- Metabolism contig17830 containing protein -2.3 DOWN Other Sugars Metabolism contig19820 aldo/keto reductase family protein -1.6 DOWN Other Sugars haloacid dehalogenase-like hydrolase Metabolism contig13111 family protein -1.4 DOWN Other Sugars pfkB-type carbohydrate kinase family Metabolism contig14829 protein -0.7 DOWN Other Sugars haloacid dehalogenase-like hydrolase Metabolism contig2660 family protein -0.7 DOWN Other Sugars Metabolism contig28173 aldose reductase, putative 2.4 UP Waxes contig16571 AT5G57800 | CER3 (ECERIFERUM 3) -1.2 DOWN Waxes contig12315 AT5G57800 | CER3 (ECERIFERUM 3) -0.8 DOWN BETA-OHASE 1 (BETA-HYDROXYLASE Terpenes contig4751 1) -1.7 DOWN geranylgeranyl transferase type II beta Terpenes contig13450 subunit, putative -1.5 DOWN Terpenes contig16867 amine oxidase family -1.5 DOWN

AT1G76490 | HMG1 (HYDROXY Terpenes contig18504 METHYLGLUTARYL COA REDUCTASE 1) -1 DOWN GGR (geranylgeranyl reductase); Terpenes contig14124 farnesyltranstransferase -0.8 DOWN

AT1G76490 | HMG1 (HYDROXY Terpenes contig27421 METHYLGLUTARYL COA REDUCTASE 1) 1.1 UP PGGT-I; CAAX-protein Terpenes contig26974 geranylgeranyltransferase 1.2 UP Terpenes contig30319 CAS1 (cycloartenol synthase 1) 2.5 UP

N-Miscellaneous contig13884 strictosidine synthase family protein -1.8 DOWN Flavanoids contig11683 TT4 (TRANSPARENT TESTA 4) -3 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression AT3G55120 | TT5 (TRANSPARENT Flavanoids contig13040 TESTA 5) -2.9 DOWN Flavanoids contig266 BAN (BANYULS); oxidoreductase -2.3 DOWN AT1G75280 | isoflavone reductase, Flavanoids contig9361 putative -1.7 DOWN Flavanoids contig21023 cinnamoyl-CoA reductase-related -1.6 DOWN AT3G55120 | TT5 (TRANSPARENT Flavanoids contig31609 TESTA 5) -1.5 DOWN BEN1; binding / catalytic/ coenzyme Flavanoids contig12529 binding / oxidoreductase -1.4 DOWN AT1G75280 | isoflavone reductase, Flavanoids contig22574 putative -1.4 DOWN AT5G14700 | cinnamoyl-CoA Flavanoids contig17862 reductase-related -1.2 DOWN AT1G75280 | isoflavone reductase, Flavanoids contig9628 putative -1.2 DOWN AT5G14700 | cinnamoyl-CoA Flavanoids contig17341 reductase-related -1.1 DOWN AT1G75280 | isoflavone reductase, Flavanoids contig9299 putative -0.8 DOWN Flavanoids contig30240 PRR1 (PINORESINOL REDUCTASE 1) 1.7 UP Flavanoids contig27433 transferase 2.4 UP AT3G53260 | PAL2; phenylalanine Phenylpropanoids contig17192 ammonia-lyase -3.1 DOWN 4-coumarate--CoA ligase family Phenylpropanoids contig14682 protein -2.3 DOWN CAD9 (CINNAMYL ALCOHOL Phenylpropanoids contig15492 DEHYDROGENASE 9) -1.7 DOWN Phenylpropanoids contig12854 4CL1 (4-COUMARATE:COA LIGASE 1) -1.6 DOWN AT4G34050 | caffeoyl-CoA 3-O- Phenylpropanoids contig24324 methyltransferase, putative -1.5 DOWN AT2G37040 |pal1 (Phe ammonia lyase Phenylpropanoids contig16072 1) -1.3 DOWN AT4G34050 | caffeoyl-CoA 3-O- Phenylpropanoids contig10371 methyltransferase, putative -1 DOWN OPCL1 (OPC-8:0 COA LIGASE1); 4- Phenylpropanoids contig14517 coumarate-CoA ligase -0.8 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression AT3G53260 | PAL2; phenylalanine Phenylpropanoids contig26336 ammonia-lyase 1.2 UP AT2G37040 |pal1 (Phe ammonia lyase Phenylpropanoids contig31187 1) 1.6 UP Phenolics contig21134 L-ascorbate oxidase, putative -2 DOWN

Phenolics contig20406 AT2G40370 | LAC5 (laccase 5); laccase -1.8 DOWN

Phenolics contig17575 AT2G40370 | LAC5 (laccase 5); laccase -1.7 DOWN L-ascorbate oxidase/ copper ion Phenolics contig19871 binding / oxidoreductase -1 DOWN geranylgeranyl transferase type II beta Isoprenoids - Non MVA contig13450 subunit, putative -1.5 DOWN GGR (geranylgeranyl reductase); Isoprenoids - Non MVA contig14124 farnesyltranstransferase -0.8 DOWN PGGT-I; CAAX-protein geranylgeranyltransferase/ protein Isoprenoids - Non MVA contig26974 heterodimerization 1.2 UP Isoprenoids - Terpenoids contig30319 CAS1 (cycloartenol synthase 1) 2.5 UP Isoprenoids - Terpenoids contig17192 PAL2; phenylalanine ammonia-lyase -3.1 DOWN Isoprenoids - 4-coumarate--CoA ligase family Terpenoids contig14682 protein -2.3 DOWN Isoprenoids - CAD9 (CINNAMYL ALCOHOL Terpenoids contig15492 DEHYDROGENASE 9) -1.7 DOWN Isoprenoids - Terpenoids contig12854 4CL1 (4-COUMARATE:COA LIGASE 1) -1.6 DOWN Isoprenoids - caffeoyl-CoA 3-O-methyltransferase, Terpenoids contig24324 putative -1.5 DOWN Isoprenoids - Terpenoids contig16072 pal1 (Phe ammonia lyase 1 -1.3 DOWN Isoprenoids - caffeoyl-CoA 3-O-methyltransferase, Terpenoids contig10371 putative -1 DOWN Isoprenoids - OPCL1 (OPC-8:0 COA LIGASE1); 4- Terpenoids contig14517 coumarate-CoA ligase -0.8 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Isoprenoids - Terpenoids contig26336 PAL2; phenylalanine ammonia-lyase 1.2 UP Isoprenoids - Terpenoids contig31187 pal1 (Phe ammonia lyase 1 1.6 UP Glutathione/Abscorbate Metabolism contig9548 CB5-B (CYTOCHROME B5 ISOFORM B) -1.6 DOWN Glutathione/Abscorbate Metabolism contig9764 CB5-E (CYTOCHROME B5 ISOFORM E) -0.9 DOWN Glutathione/Abscorbate AT4G26850 | VTC2 (vitamin c Metabolism contig10637 defective 2) -0.6 DOWN Glutathione/Abscorbate AT4G26850 | VTC2 (vitamin c Metabolism contig25430 defective 2) 1.2 UP Tetrapyrrole Metabolism contig4190 flavodoxin family protein -1.5 DOWN Tetrapyrrole Metabolism contig16689 HEMA1; glutamyl-tRNA reductase -1.3 DOWN Tetrapyrrole Metabolism contig12885 COX10 (cytochrome c oxidase 10) -0.7 DOWN Electron Transport - NQR (NADPH:QUINONE Mitochondrial contig4114 OXIDOREDUCTASE) -1.3 DOWN Electron Transport - FRO1 (FROSTBITE1); NADH Mitochondrial contig5270 dehydrogenase (ubiquinone) -1.3 DOWN Electron Transport - GAMMA CA1 (GAMMA CARBONIC Mitochondrial contig11031 ANHYDRASE 1) -0.8 DOWN Electron Transport - ubiquinol-cytochrome C reductase Mitochondrial contig11984 UQCRX/QCR9-like family protein -0.8 DOWN Electron Transport - mitochondrial ATP synthase g subunit Mitochondrial contig12413 family protein -0.7 DOWN Electron Transport - ATPQ (ATP SYNTHASE D CHAIN, Mitochondrial contig137 MITOCHONDRIAL) -0.6 DOWN Electron Transport - Mitochondrial contig29352 cytochrome c oxidase subunit 2 2.3 UP Electron Transport - Mitochondrial contig28955 NADH dehydrogenase subunit 6 2.4 UP Electron Transport - COX1 | cytochrome c oxidase subunit Mitochondrial contig29070 1 2.5 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression ATP8 | Encodes subunit 8 of the Electron Transport - mitochondrial F(O) ATP synthase Mitochondrial contig30556 complex 2.5 UP Electron Transport - COX3 | Encodes cytochrome c oxidase Mitochondrial contig30636 subunit 3 3.2 UP TF - Chromatin ETL1; ATP binding / DNA binding / Remodeling contig18024 helicase -2.7 DOWN TF - Chromatin Remodeling contig14058 transcription regulatory protein SNF2 -1.1 DOWN TF - Chromatin SWIB complex BAF60b domain- Remodeling contig12101 containing protein -1 DOWN TF - Chromatin CHR11 (CHROMATIN-REMODELING Remodeling contig18195 PROTEIN 11) 0.6 UP TF - Chromatin SYD (SPLAYED); ATPase/ chromatin Remodeling contig23987 binding 0.6 UP TF - Chromatin ATSWI3C (SWITCH/SUCROSE Remodeling contig21413 NONFERMENTING 3C) 0.7 UP TF - Chromatin Remodeling contig6956 transcription regulatory protein SNF2 0.8 UP TF - Chromatin SYD (SPLAYED); ATPase/ chromatin Remodeling contig29803 binding 1.6 UP TF - Chromatin transcription regulatory protein SNF2, Remodeling contig29095 putative 1.7 UP TF - Chromatin transcription regulatory protein SNF2, Remodeling contig29274 putative 2 UP TF - Chromatin transcription regulatory protein SNF2, Remodeling contig30557 putative 2.1 UP TF - Chromatin SWI2 (SWITCH 2); ATP binding / DNA Remodeling contig30735 binding / helicase 2.6 UP TF - Chromatin Remodeling contig31019 MOM (MORPHEUS MOLECULE) 2.6 UP TF - Chromatin Remodeling contig27389 RGD3 (ROOT GROWTH DEFECTIVE 3) 2.7 UP TF - C3H Zinc Finger AT2G40140 | CZF1; transcription Family contig11590 factor -2.8 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression TF - C3H Zinc Finger AT2G40140 | CZF1; transcription Family contig11928 factor -1.8 DOWN TF - C3H Zinc Finger AT2G41900 zinc finger (CCCH-type) Family contig12263 family protein -1.2 DOWN TF - C3H Zinc Finger AT2G40140 | CZF1; transcription Family contig28770 factor -1.1 DOWN TF - C3H Zinc Finger AT2G41900 zinc finger (CCCH-type) Family contig14349 family protein -1 DOWN TF - C3H Zinc Finger AT2G41900 zinc finger (CCCH-type) Family contig12035 family protein -0.9 DOWN TF - C3H Zinc Finger Family contig27261 zinc finger (CCCH-type) family protein 1.7 UP TF - G2-Like family Transcription Factors, KAN2 (KANADI 2); DNA binding / GARP contig11294 transcription factor -1.3 DOWN TF - G2-Like family Transcription Factors, GARP contig20393 myb family transcription factor -1.2 DOWN TF - G2-Like family Transcription Factors, UNE16 (unfertilized embryo sac 16); GARP contig10264 transcription factor -0.8 DOWN TF - G2-Like family Transcription Factors, GARP contig19692 KAN (KANADI); transcription factor -0.6 DOWN TF - G2-Like family Transcription Factors, GPRI1 (GBF'S PRO-RICH REGION- GARP contig31115 INTERACTING FACTOR 1) 1.8 UP

AT5G48150 | Symbols: PAT1 | PAT1 TF - GRAS contig15859 (phytochrome a signal transduction 1) -2.2 DOWN

AT5G48150 | Symbols: PAT1 | PAT1 TF - GRAS contig15510 (phytochrome a signal transduction 1) -2.1 DOWN TF - GRAS contig14237 SCL1 (SCARECROW-LIKE 1) -1.4 DOWN AT5G52510 | scarecrow-like TF - GRAS contig16359 transcription factor 8 (SCL8) -1.4 DOWN scarecrow transcription factor family TF - GRAS contig14668 protein -1.3 DOWN 88

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression scarecrow transcription factor family TF - GRAS contig15816 protein -1.2 DOWN scarecrow transcription factor family TF - GRAS contig11472 protein -1 DOWN scarecrow transcription factor family TF - GRAS contig9062 protein -0.7 DOWN AT5G52510 | scarecrow-like TF - GRAS contig5131 transcription factor 8 (SCL8) -0.6 DOWN TF - MYB contig18414 AtMYB1 (myb domain protein 1) -2.5 DOWN TF - MYB contig12610 ATRL1 (ARABIDOPSIS RAD-LIKE 1) -2 DOWN TF - MYB contig17253 myb family transcription factor -1.7 DOWN AT3G13890 | ATMYB26 (MYB TF - MYB contig3730 DOMAIN PROTEIN 26) -1.4 DOWN TF - MYB contig21284 MYB43 (myb domain protein 43) -1.4 DOWN AT3G13890 | ATMYB26 (MYB TF - MYB contig17189 DOMAIN PROTEIN 26) -1.4 DOWN TF - MYB contig17724 MYB106 (myb domain protein 106) -1.4 DOWN TF - MYB contig10751 ATMYB5 (MYB DOMAIN PROTEIN 5) -1.3 DOWN

TF - MYB contig21396 MYB111 (MYB DOMAIN PROTEIN 111) -1.2 DOWN

TF - MYB contig14513 ATMYB94 (MYB DOMAIN PROTEIN 94) -1.2 DOWN TF - MYB contig14431 AtMYB93 (myb domain protein 93) -1.1 DOWN MYB4; DNA binding / transcription TF - MYB contig14907 factor -1 DOWN TRB2; DNA binding / double-stranded TF - MYB contig20761 telomeric DNA binding -1 DOWN TF - MYB contig11300 DNA binding / transcription factor -0.9 DOWN TF - MYB contig15179 myb family transcription factor -0.9 DOWN TF - MYB contig12145 MYB61 (MYB DOMAIN PROTEIN 61) -0.8 DOWN TF - MYB contig18120 myb family transcription factor -0.8 DOWN

TF - MYB contig20054 TKI1 (TSL-kinase interacting protein 1) -0.7 DOWN TF - MYB contig11695 MYB7 (MYB DOMAIN PROTEIN 7) -0.7 DOWN TF - MYB contig12163 myb family transcription factor -0.6 DOWN TF - MYB contig26053 DNA binding / transcription factor 0.6 UP TF - MYB contig27348 AtMYB70 (myb domain protein 70) 1.8 UP TF - MYB contig30351 DNA binding / transcription factor 2.9 UP

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression TF - ABI3VP1-related B3 contig18112 ABI3 (ABA INSENSITIVE 3) 1.1 UP CBF4 (C- REPEAT-BINDING TF - EREB contig20008 FACTOR 4) -4.8 DOWN AT1G19210 | AP2 domain-containing TF - EREB contig19395 transcription factor, putative -4.4 DOWN AT1G19210 | AP2 domain-containing TF - EREB contig19669 transcription factor, putative -2.7 DOWN TF - EREB contig19282 RAP2.1 (related to AP2 1) -2.6 DOWN AT1G19210 | AP2 domain-containing TF - EREB contig12308 transcription factor, putative -2.5 DOWN ERF5 (ETHYLENE RESPONSIVE TF - EREB contig12443 ELEMENT BINDING FACTOR 5) -2.1 DOWN CRF2 (CYTOKININ RESPONSE TF - EREB contig18684 FACTOR 2) -1.8 DOWN AP2 domain-containing transcription TF - EREB contig18616 factor, putative -0.7 DOWN AP2 domain-containing transcription TF - EREB contig27194 factor, putative 1 UP TF - EREB contig25587 AIL5 (AINTEGUMENTA-LIKE 5) 2.3 UP TF - Auxin Response Factors contig11864 ARF11 (AUXIN RESPONSE FACTOR 11) -0.9 DOWN TF - Auxin Response Factors contig14434 MP (MONOPTEROS) -0.9 DOWN TF - Auxin Response NPH4 (NON-PHOTOTROPHIC Factors contig3510 HYPOCOTYL) -0.7 DOWN TF - Auxin Response Factors contig23359 ARF2 (AUXIN RESPONSE FACTOR 2) 0.8 UP TF - Auxin Response Factors contig28491 ARF9 (AUXIN RESPONSE FACTOR 9) 1.3 UP TF - Auxin Response Factors contig30059 ARF2 (AUXIN RESPONSE FACTOR 2) 1.4 UP TF - Auxin Response Factors contig30024 ARF4 (AUXIN RESPONSE FACTOR 4) 3 UP TF - ARR (Arabidopsis AT3G57040 | Symbols: ARR9, ATRR4 Response Regulators) contig15090 | ARR9 (RESPONSE REGULATOR 9) -1.7 DOWN TF - ARR (Arabidopsis ARR14 (ARABIDOPSIS RESPONSE Response Regulators) contig1990 REGULATOR 14) -1.7 DOWN TF - ARR (Arabidopsis ARR2 (ARABIDOPSIS RESPONSE Response Regulators) contig18551 REGULATOR 2) -1.8 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression TF - ARR (Arabidopsis AT3G57040 | Symbols: ARR9, ATRR4 Response Regulators) contig23349 | ARR9 (RESPONSE REGULATOR 9) -0.8 DOWN TF - Basic Helix Loop Helix (bHLH) contig15995 SPT (SPATULA) -1.5 DOWN TF - Basic Helix Loop Helix (bHLH) contig20199 transcription factor -1.2 DOWN TF - Basic Helix Loop PIF3 (PHYTOCHROME Helix (bHLH) contig19397 INTERACTING FACTOR 3) -1.2 DOWN TF - Basic Helix Loop UNE12 (unfertilized embryo sac 12); Helix (bHLH) contig13042 DNA binding -1.2 DOWN TF - Basic Helix Loop transcription factor/ transcription Helix (bHLH) contig14186 regulator -1.1 DOWN TF - Basic Helix Loop Helix (bHLH) contig13191 transcription factor -0.9 DOWN TF - Basic Helix Loop transcription factor/ transcription Helix (bHLH) contig10603 regulator -0.7 DOWN TF - Basic Helix Loop transcription factor/ transcription Helix (bHLH) contig25857 regulator 0.8 UP TF - Basic Helix Loop basic helix-loop-helix (bHLH) family Helix (bHLH) contig30269 protein (bHLH096) 1.4 UP TF - Basic Helix Loop basic helix-loop-helix (bHLH) family Helix (bHLH) contig26616 protein 1.4 UP TF - Constans like Zinc AT3G07650 | COL9 (CONSTANS- Finger contig15988 LIKE 9) -1.2 DOWN TF - Constans like Zinc AT3G07650 | COL9 (CONSTANS- Finger contig16993 LIKE 9) -1.1 DOWN TF - Constans like Zinc PRR7 (PSEUDO-RESPONSE Finger contig31333 REGULATOR 7) 1 UP TF - Constans like Zinc Finger contig3008 zinc finger (B-box type) family protein 1.2 UP TF - Constans like Zinc APRR5 (ARABIDOPSIS PSEUDO- Finger contig30289 RESPONSE REGULATOR 5) 2.6 UP Dof-type zinc finger domain-containing TF - DOF like Zinc Finger contig20858 protein -1.3 DOWN Dof-type zinc finger domain-containing TF - DOF like Zinc Finger contig15824 protein -1.1 DOWN Dof-type zinc finger domain-containing TF - DOF like Zinc Finger contig16101 protein -0.9 DOWN 91

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Dof-type zinc finger domain-containing TF - DOF like Zinc Finger contig27710 protein -0.9 DOWN AT3G54810 | BME3 | zinc finger TF - GATA contig13916 (GATA type) family protein -3.1 DOWN TF - GATA contig22165 zinc finger (GATA type) family protein -2 DOWN AT3G54810 | BME3 | zinc finger TF - GATA contig14659 (GATA type) family protein -1.9 DOWN

TF - GATA contig18030 ZML1 (ZIM-LIKE 1); transcription factor -1.3 DOWN TF - GATA contig6123 zinc finger (GATA type) family protein 2 UP TF - C2H2 Zinc Finger contig15039 zinc finger (C2H2 type) family protein -4.1 DOWN AT1G27730 | STZ (salt tolerance zinc TF - C2H2 Zinc Finger contig21648 finger) -2.2 DOWN AT1G27730 | STZ (salt tolerance zinc TF - C2H2 Zinc Finger contig13312 finger) -2.2 DOWN AT1G27730 | STZ (salt tolerance zinc TF - C2H2 Zinc Finger contig14313 finger) -2.1 DOWN TF - C2H2 Zinc Finger contig18442 zinc finger (C2H2 type) family protein -1.9 DOWN AZF2 (ARABIDOPSIS ZINC-FINGER TF - C2H2 Zinc Finger contig13401 PROTEIN 2) -1.6 DOWN TF - C2H2 Zinc Finger contig17288 zinc finger (C2H2 type) family protein -1.6 DOWN TF - C2H2 Zinc Finger contig21647 NTT (NO TRANSMITTING TRACT) -1.5 DOWN TF - C2H2 Zinc Finger contig14737 SEU (seuss) -1.5 DOWN LRP1 (LATERAL ROOT PRIMORDIUM TF - C2H2 Zinc Finger contig19284 1) -1.2 DOWN TF - C2H2 Zinc Finger contig17134 zinc finger (CCCH-type) family protein -1.2 DOWN TF - C2H2 Zinc Finger contig22090 JKD (JACKDAW) -0.9 DOWN TF - C2H2 Zinc Finger contig11400 SEU (seuss) -0.8 DOWN AT1G27730 | STZ (salt tolerance zinc TF - C2H2 Zinc Finger contig21937 finger) -0.7 DOWN TF - C2H2 Zinc Finger contig30114 EMB2454 (embryo defective 2454) 1.4 UP AtIDD7 (Arabidopsis thaliana TF - C2H2 Zinc Finger contig27054 Indeterminate(ID)-Domain 7) 2.6 UP TF - C2H2 Zinc Finger contig29413 MGP (Magpie) 3.4 UP KNAT4 (KNOTTED1-LIKE HOMEOBOX TF - Homeobox contig11861 GENE 4) -2.5 DOWN ATHB6; DNA binding / protein binding TF - Homeobox contig19808 / sequence-specific -1.6 DOWN TF - Homeobox contig15717 HAT22; transcription factor -1.2 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression ATHB13; DNA binding / sequence- TF - Homeobox contig13213 specific DNA binding -1.1 DOWN ATHB-1 (ARABIDOPSIS THALIANA TF - Homeobox contig9123 HOMEOBOX 1) -0.7 DOWN TF - Homeobox contig27830 ANL2 (ANTHOCYANINLESS 2) 1.5 UP PRHA (PATHOGENESIS RELATED TF - Homeobox contig29500 HOMEODOMAIN PROTEIN A) 1.6 UP TF - Homeobox contig27466 ATHB-8 (HOMEOBOX GENE 8) 1.7 UP homeobox transcription factor, TF - Homeobox contig30140 putative 3.6 UP TCP family transcription factor, TF - TCP contig16611 putative -3.5 DOWN TCP family transcription factor, TF - TCP contig9759 putative -0.6 DOWN TF - TCP contig19604 AT-TCP20; transcription factor -1.3 DOWN TCP4 (TCP family transcription factor TF - TCP contig28911 4) 2.3 UP TF - WRKY contig16511 WRKY41 -4.6 DOWN TF - WRKY contig11341 WRKY40 -3.4 DOWN TF - WRKY contig16663 WRKY70 -1.7 DOWN TF - WRKY contig22230 WRKY74 -1.1 DOWN TF - WRKY contig21980 WRKY33 -0.9 DOWN TF - WRKY contig7916 WRKY21 2 UP BZIP34 | bZIP transcription factor TF - bZIP contig17570 family protein -0.9 DOWN bZIP transcription factor, putative TF - bZIP contig16039 (bZIP69) -1.4 DOWN TF - bZIP contig9529 GBF6 (G-BOX BINDING FACTOR 6) -1.4 DOWN TF - bZIP contig9923 GBF6 (G-BOX BINDING FACTOR 6) -1.3 DOWN TGA4 (TGACG MOTIF-BINDING TF - bZIP contig18842 FACTOR 4) -1.2 DOWN TF - bZIP contig20247 PAN (PERIANTHIA) -0.9 DOWN GBF4; DNA binding / sequence- TF - bZIP contig30777 specific DNA binding 1.4 UP TF - bZIP contig28826 bZIP transcription factor family protein 1.5 UP TF - CCAAT box NF-YC1 (NUCLEAR FACTOR Y, binding contig17936 SUBUNIT C1) -2 DOWN TF - CPP1-related contig21405 TCX2 (TESMIN/TSO1-LIKE CXC 2) -0.7 DOWN TF - Heat Shock Factor contig14184 HSF3 (HEAT SHOCK FACTOR 3) -0.8 DOWN 93

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression TF - MADS box contig20678 AGL6 (AGAMOUS-LIKE 6) -1.1 DOWN TF - MADS box contig10558 SEP1 (SEPALLATA1) -0.8 DOWN TF - Trihelix contig14115 proline-rich family protein -1.7 DOWN ASIL1 (ARABIDOPSIS 6B- TF - Trihelix contig25062 INTERACTING PROTEIN 1-LIKE 1) 0.9 UP TF - Trihelix contig31664 transcription factor 0.9 UP ZLL (ZWILLE); translation initiation TF - Argonaute contig8878 factor 0.9 UP TF - Argonaute contig14483 AGO5 (ARGONAUTE 5) -0.8 DOWN ZLL (ZWILLE); translation initiation TF - Argonaute contig27290 factor 1.2 UP TF - AS2,Lateral Organ LBD21 (LOB DOMAIN-CONTAINING Boundaries contig17623 PROTEIN 21) -2.1 DOWN TF - AS2,Lateral Organ LBD15 (LOB DOMAIN-CONTAINING Boundaries contig16808 PROTEIN 15) -1.6 DOWN TF - Auxin/IAA contig16256 ATAUX2-11 (AUXIN INDUCIBLE 2-11) -3.8 DOWN TF - Auxin/IAA contig1332 IAA16; transcription factor -1.2 DOWN PAP1 (PHYTOCHROME- TF - Auxin/IAA contig10518 ASSOCIATED PROTEIN 1) -0.7 DOWN IAA11 (INDOLE-3-ACETIC ACID TF - Auxin/IAA contig10712 INDUCIBLE 11) -1.4 DOWN IAA29 (INDOLE-3-ACETIC ACID TF - Auxin/IAA contig14713 INDUCIBLE 29) -1.6 DOWN TF - Auxin/IAA contig286 IAA7 (INDOLE-3-ACETIC ACID 7) -0.7 DOWN VRN1 (REDUCED VERNALIZATION TF - B3 contig18686 RESPONSE 1) -0.7 DOWN TF - DNA DNMT2 (DNA Methyltransferases contig29895 METHYLTRANSFERASE-2) -1.7 DOWN TF - DNA DRM1 (domains rearranged methylase Methyltransferases contig24887 1) 1.7 UP TF - DNA Methyltransferases contig19645 DMT2 (DNA METHYLTRANSFERASE 2) 3.1 UP TF - DNA Methyltransferases contig30803 CMT2 (chromomethylase 2) 3.8 UP TF - ELF3 contig28556 ELF3 (EARLY FLOWERING 3) 2 UP TF - Global GTE4 (GLOBAL TRANSCRIPTION Transcription Factors contig11205 FACTOR GROUP E 4) -0.9 DOWN TF - Global DNA-binding bromodomain-containing Transcription Factors contig25396 protein -0.7 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression TF - Global GTB1; RNA binding / hydrolase, acting Transcription Factors contig31495 on ester bonds 1 UP TF - Global GTA2 (GLOBAL TRANSCRIPTION Transcription Factors contig27844 FACTOR GROUP A2) 1.7 UP TF - Global Transcription Factors contig10441 SPT16 (global transcription factor C 1.8 UP TF - Histone Acetyltransferases contig28665 MRG Family Protein -0.6 DOWN

TF - Histone HAC1 (HISTONE ACETYLTRANSFERASE Acetyltransferases contig31387 OF THE CBP FAMILY 1) 1.3 UP

TF - Histone HAC1 (HISTONE ACETYLTRANSFERASE Acetyltransferases contig6650 OF THE CBP FAMILY 1) 1.4 UP HOS15 (high expression of TF - HDA contig29507 osmotically responsive genes 15) 1.5 UP transcription factor jumonji (jmjC) TF - JUMONJI contig25438 domain-containing protein -2.3 DOWN transcription factor jumonji (jmj) family TF - JUMONJI contig31041 protein -1.6 DOWN transferase, transferring glycosyl TF - JUMONJI contig17787 groups 0.9 UP transcription factor jumonji (jmj) family TF - JUMONJI contig27506 protein 1.7 UP TF - JUMONJI contig21524 transcription factor 2.9 UP TF - Methyl binding domain contig29498 MBD8; methyl-CpG binding 1.4 UP TF - Nucleosome/chromatin HMGB3 (HIGH MOBILITY GROUP B assembly contig23557 3) -0.6 DOWN TF - PHD finger contig29722 PHD finger family protein 1.9 UP PHD finger transcription factor, TF - PHD finger contig29609 putative 3.2 UP CHR4 (CHROMATIN REMODELING TF - PHD finger contig29241 4) 4.4 UP PUB29 (PLANT U-BOX 29); ubiquitin- TF - PHOR1 contig17566 protein ligase -3.5 DOWN APRR5 (ARABIDOPSIS PSEUDO- TF - Psudo ARR contig30773 RESPONSE REGULATOR 5) 2.6 UP APRR2; transcription factor/ two- TF - Psudo ARR contig30289 component response regulator 2.7 UP 95

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression ATM (ATAXIA-TELANGIECTASIA TF - PWWP contig28456 MUTATED) 1.2 UP TF - PWWP contig31129 HUA2 (ENHANCER OF AG-4 2) 1.8 UP SUVH3 (SU(VAR)3-9 HOMOLOG 3); TF - SET contig16525 histone methyltransferase -1.6 DOWN TF - SET contig21102 SUVH6; methyl-CpG binding -1.6 DOWN TF - SET contig16854 SUVH3 (SU(VAR)3-9 HOMOLOG 3) -1.4 DOWN EFS (EARLY FLOWERING IN SHORT TF - SET contig28340 DAYS) 2.6 UP TF - SET contig30914 SUVH6; methyl-CpG binding 2.8 UP SDG2 (SET DOMAIN-CONTAINING TF - SET contig6998 PROTEIN 2) 3.7 UP ATPPC3 (PHOSPHOENOLPYRUVATE TF - Zinc finger CCHC contig12970 CARBOXYLASE 3) -1.1 DOWN AtHB31 (ARABIDOPSIS THALIANA TF - Zinc finger HD contig27592 HOMEOBOX PROTEIN 31) -1.1 DOWN TF - Zinc finger HD contig28592 MIF2 (MINI ZINC FINGER 2) -0.9 DOWN TF - Zinc finger HD contig26475 ATHB22 (HOMEOBOX PROTEIN 22) 1.7 UP AtHB33 (ARABIDOPSIS THALIANA TF - Zinc finger HD contig25250 HOMEOBOX PROTEIN 33) 2.2 UP Post-Translational Modifications contig20428 protein kinase, putative -3.4 DOWN Post-Translational Modifications contig18087 protein kinase, putative -3.2 DOWN Post-Translational CCR4 (ARABIDOPSIS THALIANA Modifications contig21206 CRINKLY4 RELATED 4) -2.8 DOWN Post-Translational Modifications contig17744 protein phosphotase 2C, putative -2.7 DOWN Post-Translational Modifications contig17626 protein phosphatase 2C, putative -2.3 DOWN Post-Translational Modifications contig7846 BRL3 (BRI1-LIKE 3) -2.3 DOWN Post-Translational Modifications contig13555 protein kinase, putative -2.3 DOWN Post-Translational TOPP6; protein serine/threonine Modifications contig16952 phosphatase -2.1 DOWN Post-Translational Modifications contig19583 ATPK3; -2 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression ATMPK2 (ARABIDOPSIS THALIANA Post-Translational MITOGEN-ACTIVATED PROTEIN Modifications contig13706 KINASE HOMOLOG 2) -1.9 DOWN Post-Translational leucine-rich repeat transmembrane Modifications contig16965 protein kinase, putative -1.9 DOWN Post-Translational RKF3 (RECEPTOR-LIKE KINASE IN IN Modifications contig22375 FLOWERS 3) -1.9 DOWN Post-Translational Modifications contig21227 BAM3 (BARELY ANY MERISTEM 3) -1.8 DOWN Post-Translational Modifications contig15110 ATP binding / kinase -1.8 DOWN Post-Translational Modifications contig5574 protein kinase family protein -1.7 DOWN Post-Translational Modifications contig15476 protein phosphatase 2C family protein -1.7 DOWN Post-Translational Modifications contig15807 protein kinase, putative -1.7 DOWN Post-Translational leucine-rich repeat transmembrane Modifications contig18300 protein kinase, putative -1.7 DOWN Post-Translational Modifications contig21884 MKK9 (MAP KINASE KINASE 9) -1.6 DOWN Post-Translational Modifications contig13849 protein phosphatase 2C, putative -1.6 DOWN Post-Translational Modifications contig12100 PHOT1 (PHOTOTROPIN 1) -1.6 DOWN Post-Translational NMT1 (MYRISTOYL-COA:PROTEIN N- Modifications contig16676 MYRISTOYLTRANSFERASE) -1.6 DOWN Post-Translational ATPEN2 (ARABIDOPSIS THALIANA Modifications contig21663 PTEN 2) -1.6 DOWN Post-Translational Modifications contig18107 APK2B (PROTEIN KINASE 2B) -1.6 DOWN Post-Translational Modifications contig22342 protein kinase, putative -1.6 DOWN Post-Translational Modifications contig19902 lectin protein kinase, putative -1.5 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression Post-Translational serine/threonine protein phosphatase Modifications contig1849 2C (PP2C6) -1.5 DOWN Post-Translational leucine-rich repeat transmembrane Modifications contig15338 protein kinase, putative -1.5 DOWN Post-Translational AtPPT1 (Arabidopsis thaliana Modifications contig17854 polyprenyltransferase 1) -1.4 DOWN Post-Translational Modifications contig18463 protein phosphatase 2C, putative -1.4 DOWN Post-Translational Modifications contig19712 protein kinase family protein -1.4 DOWN Post-Translational leucine-rich repeat transmembrane Modifications contig13836 protein kinase, putative -1.4 DOWN Post-Translational Modifications contig10345 lectin protein kinase family protein -1.3 DOWN Post-Translational Modifications contig19336 protein kinase, putative -1.3 DOWN Post-Translational Modifications contig12309 protein kinase family protein -1.3 DOWN Post-Translational Modifications contig12105 CKB1; protein kinase regulator -1.3 DOWN Post-Translational Modifications contig14214 protein phosphatase 2C, putative -1.3 DOWN Post-Translational Modifications contig10345 lectin protein kinase family protein -1.3 DOWN Post-Translational BRI1 (BRASSINOSTEROID INSENSITIVE Modifications contig19748 1) -1.3 DOWN Post-Translational PHS1 (PROPYZAMIDE-HYPERSENSITIVE Modifications contig11284 1) -1.2 DOWN Post-Translational ATB BETA; nucleotide binding / Modifications contig15822 protein phosphatase type 2A regulator -1.2 DOWN Post-Translational Modifications contig12920 protein kinase family protein -1.2 DOWN Post-Translational Modifications contig16313 CLV1 (CLAVATA 1) -1.2 DOWN Post-Translational Modifications contig20582 protein kinase family protein -1.1 DOWN

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued

Functional Annotation ID Description Expression Post-Translational SRF8 (STRUBBELIG-RECEPTOR FAMILY Modifications contig11359 8) -1.1 DOWN Post-Translational leucine-rich repeat transmembrane Modifications contig10407 protein kinase, putative -1.1 DOWN Post-Translational Modifications contig17270 protein kinase, putative -1.1 DOWN Post-Translational Modifications contig17376 protein kinase family protein -1.1 DOWN Post-Translational serine/threonine protein kinase, Modifications contig22170 putative -1 DOWN Post-Translational Modifications contig20135 protein phosphatase 2C, putative -1 DOWN Post-Translational Modifications contig18826 PID (PINOID) -1 DOWN Post-Translational Modifications contig11290 MAP3KA; ATP binding / kinase -1 DOWN Post-Translational Modifications contig12427 CKI1 (CASEIN KINASE I) -1 DOWN Post-Translational SNRK2.7 (SNF1-RELATED PROTEIN Modifications contig13943 KINASE 2.7) -1 DOWN Post-Translational CTR1 (CONSTITUTIVE TRIPLE Modifications contig17504 RESPONSE 1 -0.9 DOWN Post-Translational AFC1 (ARABIDOPSIS FUS3- Modifications contig14217 COMPLEMENTING GENE 1 -0.9 DOWN Post-Translational Modifications contig17719 protein kinase-related -0.9 DOWN Post-Translational Modifications contig20372 protein kinase, putative -0.9 DOWN Post-Translational Modifications contig15203 leucine-rich repeat family protein -0.9 DOWN Post-Translational Modifications contig16474 protein kinase family protein -0.9 DOWN Post-Translational Modifications contig30546 ATP binding / protein kinase -0.9 DOWN ATNEK1 (ARABIDOPSIS THALIANA Post-Translational NIMA-RELATED SERINE/THREONINE Modifications contig23978 KINASE 1) -0.8 DOWN 99

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Post-Translational peptide methionine sulfoxide Modifications contig13971 reductase family protein -0.8 DOWN Post-Translational CIPK9 (CBL-INTERACTING PROTEIN Modifications contig11139 KINASE 9) -0.8 DOWN Post-Translational CDKD1;3 (CYCLIN-DEPENDENT KINASE Modifications contig14435 D1;3) -0.8 DOWN Post-Translational Modifications contig13104 PP5.2 (PROTEIN PHOSPHATASE 5.2) -0.8 DOWN Post-Translational CKB4 (CASEIN KINASE II BETA SUBUNIT Modifications contig14204 4) -0.8 DOWN Post-Translational Modifications contig21466 protein kinase, putative -0.8 DOWN Post-Translational Modifications contig27130 protein kinase family protein -0.7 DOWN Post-Translational Modifications contig26077 CDC2 (CELL DIVISION CONTROL 2) -0.7 DOWN Post-Translational Modifications contig11401 casein kinase II alpha chain, putative -0.7 DOWN Post-Translational leucine-rich repeat transmembrane Modifications contig18779 protein kinase, putative -0.7 DOWN Post-Translational Modifications contig10596 CKI1 (CASEIN KINASE I) -0.6 DOWN Post-Translational Modifications contig14580 protein kinase family protein -0.6 DOWN Post-Translational ATMPK4 (ARABIDOPSIS THALIANA Modifications contig13300 MAP KINASE 4) -0.6 DOWN

Post-Translational ATB BETA; nucleotide binding / Modifications contig14255 protein phosphatase type 2A regulator -0.6 DOWN Post-Translational Modifications contig13767 protein phosphatase 2C, putative -0.6 DOWN Post-Translational Modifications contig28129 protein kinase family protein 0.6 UP Post-Translational AKIN10 (Arabidopsis SNF1 kinase Modifications contig24326 homolog 10) 0.6 UP Post-Translational Modifications contig17244 KEG (KEEP ON GOING) 0.7 UP

100

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Table C.1 Continued

Functional Annotation ID Description Expression Post-Translational Modifications contig31132 HAB1 (HOMOLOGY TO ABI1) 0.8 UP Post-Translational phosphatase/ protein tyrosine Modifications contig28198 phosphatase 0.8 UP Post-Translational Modifications contig25945 protein phosphatase 2C, putative 0.8 UP Post-Translational Modifications contig14097 zinc-binding protein, putative 1 UP Post-Translational Modifications contig25725 protein kinase, putative 1.1 UP Post-Translational Modifications contig21037 protein kinase family protein 1.1 UP Post-Translational Modifications contig24202 zinc-binding protein, putative 1.1 UP Post-Translational Modifications contig27372 CPK28 1.1 UP Post-Translational Modifications contig27052 protein kinase family protein 1.2 UP Post-Translational Modifications contig25844 galactolipase/ phospholipase 1.2 UP Post-Translational Modifications contig26603 protein kinase family protein 1.2 UP Post-Translational Modifications contig28637 protein kinase family protein 1.2 UP Post-Translational Modifications contig25472 protein kinase family protein 1.3 UP BSL3 | kelch repeat-containing Post-Translational serine/threonine phosphoesterase Modifications contig25717 family protein 1.3 UP Post-Translational Modifications contig29673 PID2 (PNOID) 1.3 UP Post-Translational Modifications contig28719 KEG (KEEP ON GOING) 1.5 UP Post-Translational Modifications contig27350 kinase 1.5 UP Post-Translational Modifications contig29252 CKB1; protein kinase regulator 1.6 UP 101

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Post-Translational leucine-rich repeat protein kinase, Modifications contig28792 putative 1.7 UP Post-Translational Modifications contig28671 protein kinase family protein 1.8 UP Post-Translational CPK6 (CALCIUM-DEPENDENT PROTEIN Modifications contig27857 KINASE 6) 2 UP Post-Translational Modifications contig27298 protein phosphatase 2C-related 2.1 UP Post-Translational CDKD1;1 (CYCLIN-DEPENDENT KINASE Modifications contig29194 D1;1) 2.1 UP Post-Translational Modifications contig30950 protein kinase, putative 2.3 UP Post-Translational Modifications contig25582 NAD+ ADP-ribosyltransferase 2.4 UP Post-Translational CIPK9 (CBL-INTERACTING PROTEIN Modifications contig26284 KINASE 9) 2.8 UP Post-Translational Modifications contig29548 KEG (KEEP ON GOING) 3.3 UP Post-Translational SNF4 (HOMOLOG OF YEAST SUCROSE Modifications contig30585 NONFERMENTING 4) 3.5 UP Protein Degradation General contig21715 AMC1 (METACASPASE 1) -3.1 DOWN Protein Degradation General contig4749 aspartyl protease family protein -2.2 DOWN Protein Degradation General contig18592 EGY2; metalloendopeptidase -1.3 DOWN Protein Degradation General contig13463 24 kDa vacuolar protein, putative -1.2 DOWN Protein Degradation AT1G01650 | aspartic-type General contig12113 endopeptidase/ peptidase -1.1 DOWN Protein Degradation pyrrolidone-carboxylate peptidase General contig18970 family protein -0.9 DOWN Protein Degradation General contig14616 AT2G14260 | PIP; aminopeptidase -0.8 DOWN Protein Degradation ATSTE24; endopeptidase/ General contig10689 metalloendopeptidase -0.7 DOWN

102

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation ATP-dependent protease La (LON) General contig17716 domain-containing protein -0.6 DOWN Protein Degradation General contig15848 prolyl oligopeptidase family protein -0.6 DOWN Protein Degradation General contig24394 AT2G14260 | PIP; aminopeptidase 0.8 UP Protein Degradation ATPREP2 (ARABIDOPSIS THALIANA General contig29847 PRESEQUENCE PROTEASE 2) 1.3 UP Protein Degradation AT1G01650 | aspartic-type General contig27123 endopeptidase/ peptidase 1.8 UP Protein Degradation Subtilases contig13330 subtilase family protein -0.8 DOWN Protein Degradation Subtilases contig20391 subtilase family protein -1.9 DOWN Protein Degradation Subtilases contig14218 ARA12; serine-type endopeptidase -1.1 DOWN Protein Degradation Subtilases contig31696 XSP1 (xylem serine peptidase 1) 1.3 UP Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig13892 KERNEL 1) -1.1 DOWN Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig15107 KERNEL 1) -0.8 DOWN Protein Degradation Cystein Proteases contig24786 cysteine protease inhibitor, putative 0.8 UP Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig25513 KERNEL 1) 1.3 UP Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig29806 KERNEL 1) 1.6 UP Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig31068 KERNEL 1) 2.2 UP Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig31069 KERNEL 1) 2.4 UP Protein Degradation AT1G55350 | DEK1 (DEFECTIVE Cystein Proteases contig28899 KERNEL 1) 3.5 UP Protein Degradation Aspartate Proteases contig4527 aspartyl protease family protein -1 DOWN

103

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation Aspartate Proteases contig643 aspartyl protease family protein 0.7 UP Protein Degradation Aspartate Proteases contig27619 aspartyl protease family protein 1.5 UP Protein Degradation Aspartate Proteases contig29608 aspartyl protease family protein 2.9 UP Protein Degradation Aspartate Proteases contig28256 aspartyl protease family protein 5.6 UP Protein Degradation Serine Proteases contig11055 LON1 (LON PROTEASE 1) -1.3 DOWN Protein Degradation Serine Proteases contig13246 LON1 (LON PROTEASE 1) -1.3 DOWN Protein Degradation ATRBL3 (ARABIDOPSIS RHOMBOID- Serine Proteases contig10784 LIKE PROTEIN 3) -0.9 DOWN Protein Degradation AT3G03380 | DegP7 (DegP protease Serine Proteases contig11152 7) -0.7 DOWN Protein Degradation scpl50 (serine carboxypeptidase-like Serine Proteases contig14838 50) -0.7 DOWN Protein Degradation AT3G03380 | DegP7 (DegP protease Serine Proteases contig26244 7) 1.1 UP Protein Degradation ATRBL2 (Arabidopsis thaliana Serine Proteases contig29823 Rhomboid-like 2) 1.1 UP Protein Degradation Metalloproteases contig17231 MMP (MATRIX METALLOPROTEINASE) -3.3 DOWN Protein Degradation Metalloproteases contig2795 AT4G23940 | FtsH protease, putative -1.4 DOWN Protein Degradation Metalloproteases contig21319 ftsh7 (FtsH protease 7) -1.2 DOWN Protein Degradation MAP1C (METHIONINE Metalloproteases contig17251 AMINOPEPTIDASE 1B) -1 DOWN Protein Degradation Metalloproteases contig19038 LON1 (LON PROTEASE 1) -0.9 DOWN Protein Degradation CAAX amino terminal protease family Metalloproteases contig21965 protein -0.9 DOWN Protein Degradation AT3G16290 | EMB2083 (embryo Metalloproteases contig15533 defective 2083) -0.9 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation ATG2; aminopeptidase/ Metalloproteases contig13098 metalloexopeptidase -0.7 DOWN Protein Degradation AT3G04340 | emb2458 (embryo Metalloproteases contig30476 defective 2458) 0.9 UP Protein Degradation Metalloproteases contig30219 AT4G23940 | FtsH protease, putative 1 UP Protein Degradation AT3G04340 | emb2458 (embryo Metalloproteases contig29549 defective 2458) 1.8 UP Protein Degradation AT3G16290 | EMB2083 (embryo Metalloproteases contig27061 defective 2083) 1.9 UP Protein Degradation AAA-type contig18506 AAA-type ATPase family protein -1.4 DOWN Protein Degradation AAA-type contig15964 AATP1 (AAA-ATPase 1) -1.2 DOWN Protein Degradation AAA-type contig11367 ERH3 (ECTOPIC ROOT HAIR 3) -0.7 DOWN Protein Degradation AAA-type contig11537 AAA-type ATPase family protein -0.6 DOWN Protein Degradation NSF (N-ethylmaleimide sensitive AAA-type contig24238 factor) 0.7 UP Protein Degradation AAA-type contig22978 cell division cycle protein 48, putative 0.8 UP Protein Degradation AAA-type contig25956 AAA-type ATPase family protein 1.2 UP Protein Degradation AAA-type contig30014 ATP binding 1.7 UP Protein Degradation AAA-type contig28768 ATP binding 1.8 UP Protein Degradation AAA-type contig30384 AAA-type ATPase family protein 1.8 UP Protein Degradation AAA-type contig28375 AAA-type ATPase family protein 2.3 UP Protein Degradation Ubiquitin contig18959 U-box domain-containing protein -2.8 DOWN Protein Degradation ATL2; protein binding / zinc ion Ubiquitin contig13013 binding -2.8 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig17333 family protein -2.6 DOWN Protein Degradation Ubiquitin contig18204 binding / ubiquitin-protein ligase -2 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig20565 family protein -2 DOWN Protein Degradation Ubiquitin contig21552 F-box family protein-related -2 DOWN Protein Degradation UBP17 (UBIQUITIN-SPECIFIC Ubiquitin contig18581 PROTEASE 17) -2 DOWN Protein Degradation kelch repeat-containing F-box family Ubiquitin contig20827 protein -1.9 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig21096 family protein -1.7 DOWN Protein Degradation ATL6; protein binding / zinc ion Ubiquitin contig20229 binding -1.7 DOWN Protein Degradation Ubiquitin contig17980 binding / ubiquitin-protein ligase -1.6 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig13483 family protein -1.6 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig12369 family protein -1.5 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig12622 family protein -1.5 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig19622 family protein -1.5 DOWN Protein Degradation PIT1 (pitchoun 1); protein binding / Ubiquitin contig22053 zinc ion binding -1.5 DOWN Protein Degradation Ubiquitin contig21632 BTB/POZ domain-containing protein -1.5 DOWN Protein Degradation Ubiquitin contig12179 ATUBC2-1; ubiquitin-protein ligase -1.4 DOWN Protein Degradation Ubiquitin contig20346 protein binding / zinc ion binding -1.4 DOWN Protein Degradation Ubiquitin contig16311 PUB26 (PLANT U-BOX 26) -1.4 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig14778 family protein -1.4 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig19723 family protein -1.4 DOWN Protein Degradation 26S proteasome regulatory subunit, Ubiquitin contig11510 putative -1.4 DOWN Protein Degradation Ubiquitin contig15775 protein binding / zinc ion binding -1.3 DOWN Protein Degradation BRH1 (BRASSINOSTEROID- Ubiquitin contig11213 RESPONSIVE RING-H2) -1.3 DOWN Protein Degradation Ubiquitin contig11201 F-box family protein -1.3 DOWN Protein Degradation Ubiquitin contig15661 F-box family protein (FBX6) -1.3 DOWN Protein Degradation kelch repeat-containing F-box family Ubiquitin contig20863 protein -1.3 DOWN Protein Degradation ATCUL1 (ARABIDOPSIS THALIANA Ubiquitin contig25825 CULLIN 1) -1.3 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig16767 family protein -1.2 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig20622 family protein -1.2 DOWN Protein Degradation Ubiquitin contig11536 RGLG2 (RING domain Ligase2) -1.2 DOWN Protein Degradation transcription factor jumonji (jmjC) Ubiquitin contig15371 domain-containing protein -1.2 DOWN Protein Degradation XERICO; protein binding / zinc ion Ubiquitin contig24481 binding -1.2 DOWN Protein Degradation SDIR1 (SALT- AND DROUGHT-INDUCED Ubiquitin contig14723 RING FINGER1) -1.2 DOWN Protein Degradation Ubiquitin contig20974 F-box family protein -1.2 DOWN Protein Degradation Ubiquitin contig10642 SGS domain-containing protein -1.2 DOWN Protein Degradation Ubiquitin contig13694 protein binding / zinc ion binding -1.1 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig12403 family protein -1.1 DOWN Protein Degradation XERICO; protein binding / zinc ion Ubiquitin contig11130 binding -1.1 DOWN Protein Degradation EDA40 (embryo sac development Ubiquitin contig20475 arrest 40) -1.1 DOWN Protein Degradation Ubiquitin contig17479 F-box family protein -1.1 DOWN Protein Degradation EID1 (EMPFINDLICHER IM Ubiquitin contig15161 DUNKELROTEN LICHT 1) -1.1 DOWN Protein Degradation Ubiquitin contig12258 ubiquitin family protein -1 DOWN Protein Degradation Ubiquitin contig12605 ubiquitin-conjugating enzyme, putative -1 DOWN Protein Degradation Ubiquitin contig21117 protein binding / zinc ion binding -1 DOWN Protein Degradation RHF2A (RING-H2 GROUP F2A); protein Ubiquitin contig16892 binding / zinc ion binding -1 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig16325 family protein -1 DOWN Protein Degradation ARI1 (ARIADNE); protein binding / zinc Ubiquitin contig13359 ion binding -1 DOWN Protein Degradation Ubiquitin contig20691 MAX2 (MORE AXILLARY BRANCHES 2) -1 DOWN Protein Degradation Ubiquitin contig17200 F-box family protein -1 DOWN Protein Degradation Ubiquitin contig11697 proteasome inhibitor-related -1 DOWN Protein Degradation Ubiquitin contig9508 UBQ10 (POLYUBIQUITIN 10) -0.9 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig12723 family protein -0.9 DOWN Protein Degradation armadillo/beta-catenin repeat family Ubiquitin contig21670 protein -0.9 DOWN Protein Degradation transcription factor jumonji (jmjC) Ubiquitin contig21382 domain-containing protein -0.9 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig5631 family protein -0.9 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig21017 family protein -0.9 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig20455 family protein -0.9 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig20861 family protein -0.9 DOWN Protein Degradation Ubiquitin contig16380 F-box family protein -0.9 DOWN Protein Degradation AtTLP7 (TUBBY LIKE PROTEIN 7); Ubiquitin contig14001 phosphoric diester hydrolase -0.9 DOWN protease-associated zinc finger Protein Degradation (C3HC4-type RING finger) family Ubiquitin contig10357 protein -0.8 DOWN Protein Degradation Ubiquitin contig21336 BTB/POZ domain-containing protein -0.8 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig901 family protein -0.7 DOWN Protein Degradation XERICO; protein binding / zinc ion Ubiquitin contig12735 binding -0.7 DOWN Protein Degradation Ubiquitin contig10172 protein binding / zinc ion binding -0.7 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig13299 family protein -0.7 DOWN Protein Degradation Ubiquitin contig14663 zinc finger protein-related -0.7 DOWN Protein Degradation kelch repeat-containing F-box family Ubiquitin contig30868 protein -0.7 DOWN Protein Degradation Ubiquitin contig10919 PBB1; endopeptidase -0.7 DOWN Protein Degradation Ubiquitin contig13 UBQ3 (POLYUBIQUITIN 3) -0.6 DOWN Protein Degradation Ubiquitin contig12325 ATUBA1; ubiquitin activating enzyme -0.6 DOWN Protein Degradation AT5G41700 | UBC8 (UBIQUITIN Ubiquitin contig18467 CONJUGATING ENZYME 8) -0.6 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression Protein Degradation AT5G41700 | UBC8 (UBIQUITIN Ubiquitin contig4888 CONJUGATING ENZYME 8) -0.6 DOWN Protein Degradation ubiquitin-conjugating enzyme, Ubiquitin contig9266 putative -0.6 DOWN Protein Degradation AT5G41700 | UBC8 (UBIQUITIN Ubiquitin contig9378 CONJUGATING ENZYME 8) -0.6 DOWN Protein Degradation AT5G41700 | UBC8 (UBIQUITIN Ubiquitin contig9593 CONJUGATING ENZYME 8) -0.6 DOWN Protein Degradation Ubiquitin contig20598 protein binding / zinc ion binding -0.6 DOWN Protein Degradation Ubiquitin contig6545 F-box family protein (FBX3) -0.6 DOWN Protein Degradation Ubiquitin contig15403 FBL17; ubiquitin-protein ligase -0.6 DOWN Protein Degradation ATCUL3 (ARABIDOPSIS THALIANA Ubiquitin contig11073 CULLIN 3) -0.6 DOWN Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig3293 family protein 0.6 UP Protein Degradation Ubiquitin contig28352 SKP2A 0.6 UP Protein Degradation CHIP (CARBOXYL TERMINUS OF Ubiquitin contig30567 HSC70-INTERACTING PROTEIN) 0.7 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig29676 family protein 0.7 UP Protein Degradation Ubiquitin contig25937 protein binding / zinc ion binding 0.7 UP Protein Degradation Ubiquitin contig26600 F-box family protein 0.7 UP Protein Degradation ATCUL3 (ARABIDOPSIS THALIANA Ubiquitin contig23402 CULLIN 3) 0.7 UP Protein Degradation UBP14 (UBIQUITIN-SPECIFIC Ubiquitin contig25860 PROTEASE 14) 0.7 UP Protein Degradation 20S proteasome alpha subunit B, Ubiquitin contig27072 putative 0.7 UP Protein Degradation Ubiquitin contig27562 IN2 (RPM1 INTERACTING PROTEIN 2) 0.8 UP

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Table C.1 Continued

Functional Annotation ID Description Expression Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig17705 family protein 0.8 UP Protein Degradation Ubiquitin contig31226 F-box family protein-related 0.8 UP Protein Degradation EBF1 (EIN3-BINDING F BOX PROTEIN Ubiquitin contig26357 1) 0.8 UP Protein Degradation BPM2 (BTB-POZ AND MATH DOMAIN Ubiquitin contig23798 2) 0.8 UP Protein Degradation Ubiquitin contig27882 PRT6 (PROTEOLYSIS 6) 0.9 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig29936 family protein 0.9 UP Protein Degradation Ubiquitin contig15185 binding 1 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig22515 family protein 1 UP Protein Degradation SKIP5 (SKP1/ASK-INTERACTING Ubiquitin contig28476 PROTEIN 5) 1 UP Protein Degradation UBP26 (UBIQUITIN-SPECIFIC Ubiquitin contig2146 PROTEASE 26) 1 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig27019 family protein 1.1 UP Protein Degradation armadillo/beta-catenin repeat Ubiquitin contig27214 protein-related 1.1 UP Protein Degradation Ubiquitin contig30157 PUB13 (PLANT U-BOX 13) 1.1 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig25552 family protein 1.1 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig8071 family protein 1.1 UP Protein Degradation armadillo/beta-catenin repeat Ubiquitin contig28538 protein-related 1.2 UP Protein Degradation AT1G55860 | UPL1 (UBIQUITIN- Ubiquitin contig27456 PROTEIN LIGASE 1) 1.3 UP Protein Degradation DRIP2 (DREB2A-INTERACTING Ubiquitin contig31482 PROTEIN 2) 1.3 UP

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Table C.1 Continued Functional Annotation ID Description Expression Protein Degradation Ubiquitin contig31044 ASK21 (ARABIDOPSIS SKP1-LIKE 21) 1.3 UP Protein Degradation kelch repeat-containing F-box family Ubiquitin contig29671 protein 1.3 UP Protein Degradation Ubiquitin contig31006 ATUBA2; ubiquitin activating enzyme 1.4 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig27692 family protein 1.4 UP Protein Degradation Ubiquitin contig23047 BTB/POZ domain-containing protein 1.4 UP Protein Degradation UBC25 (UBIQUITIN-CONJUGATING Ubiquitin contig24740 ENZYME 25) 1.5 UP Protein Degradation Ubiquitin contig29787 protein binding / zinc ion binding 1.5 UP Protein Degradation Ubiquitin contig30068 UPL4; ubiquitin-protein ligase 1.6 UP Protein Degradation AT1G70320 | UPL2 (UBIQUITIN- Ubiquitin contig30218 PROTEIN LIGASE 2) 1.6 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig25538 family protein 1.6 UP Protein Degradation Ubiquitin contig31064 F-box family protein 1.6 UP Protein Degradation AT4G38600 | KAK (KAKTUS); Ubiquitin contig25117 ubiquitin-protein ligase 1.7 UP Protein Degradation AT4G38600 | KAK (KAKTUS); Ubiquitin contig26985 ubiquitin-protein ligase 1.7 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig31110 family protein 1.8 UP Protein Degradation Ubiquitin contig29431 VIM1 (VARIANT IN METHYLATION 1) 1.9 UP Protein Degradation Ubiquitin contig31038 SAE2 (SUMO-ACTIVATING ENZYME 2) 2.1 UP Protein Degradation Ubiquitin contig23270 leucine-rich repeat family protein 2.2 UP Protein Degradation UBP8 (UBIQUITIN-SPECIFIC PROTEASE Ubiquitin contig27488 8) 2.2 UP

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Table C.1 Continued

Functional Annotation ID Description Expression Protein Degradation Ubiquitin contig28961 UPL6 (UBIQUITIN PROTEIN LIGASE 6) 2.4 UP Protein Degradation AT1G55860 | UPL1 (UBIQUITIN- Ubiquitin contig28569 PROTEIN LIGASE 1) 2.8 UP Protein Degradation kelch repeat-containing F-box family Ubiquitin contig31265 protein 2.9 UP Protein Degradation AT1G70320 | UPL2 (UBIQUITIN- Ubiquitin contig28602 PROTEIN LIGASE 2) 3 UP Protein Degradation UBC23 (UBIQUITIN-CONJUGATING Ubiquitin contig30154 ENZYME 23) 3.1 UP Protein Degradation AT1G70320 | UPL2 (UBIQUITIN- Ubiquitin contig30714 PROTEIN LIGASE 2) 3.2 UP Protein Degradation AT1G70320 | UPL2 (UBIQUITIN- Ubiquitin contig29682 PROTEIN LIGASE 2) 3.5 UP Protein Degradation AT1G55860 | UPL1 (UBIQUITIN- Ubiquitin contig28098 PROTEIN LIGASE 1) 3.5 UP Protein Degradation zinc finger (C3HC4-type RING finger) Ubiquitin contig31589 family protein 3.5 UP Protein Degradation AT1G70320 | UPL2 (UBIQUITIN- Ubiquitin contig28966 PROTEIN LIGASE 2) 4.4 UP Protein Degradation AT1G55860 | UPL1 (UBIQUITIN- Ubiquitin contig29864 PROTEIN LIGASE 1) 5.3 UP Protein Degradation AT1G70320 | UPL2 (UBIQUITIN- Ubiquitin contig31349 PROTEIN LIGASE 2) 6 UP Auxin Metabolism contig10135 BIG (BIG); binding -0.9 DOWN Auxin Metabolism contig30131 BIG (BIG); binding 3.1 UP GH3.3; indole-3-acetic acid amido Auxin Metabolism contig20936 synthetase -1.5 DOWN Auxin Metabolism contig12269 unknown protein -1.1 DOWN Auxin Metabolism contig17740 unknown protein -1.1 DOWN Auxin Metabolism contig9366 AILP1 -1 DOWN Auxin Metabolism contig13512 aldo/keto reductase family protein -1 DOWN Auxin Metabolism contig9247 auxin-responsive family protein -0.9 DOWN Auxin Metabolism contig21532 auxin-responsive protein, putative -0.8 DOWN DYL1 (DORMANCY-ASSOCIATED Auxin Metabolism contig4847 PROTEIN-LIKE 1) 0.7 UP Auxin Metabolism contig7126 unknown protein 0.7 UP 113

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression Auxin Metabolism contig27315 unknown protein 1.3 UP Auxin Metabolism contig29444 unknown protein 1.7 UP Abscisic Acid Metabolism contig17795 GRAM domain-containing protein -4.1 DOWN Abscisic Acid AAO4 (ARABIDOPSIS ALDEHYDE Metabolism contig19549 OXIDASE 4) -1.3 DOWN Abscisic Acid Metabolism contig18767 ATHVA22A -0.9 DOWN

Abscisic Acid NCED3 (NINE-CIS-EPOXYCAROTENOID Metabolism contig21730 DIOXYGENASE 3) -0.7 DOWN Abscisic Acid Metabolism contig15634 ABI3 (ABA INSENSITIVE 3) 1 UP Abscisic Acid Metabolism contig8660 ABA3 (ABA DEFICIENT 3) 1.1 UP Abscisic Acid Metabolism contig18112 ABI3 (ABA INSENSITIVE 3) 1.1 UP Abscisic Acid Metabolism contig29996 HVA22J (HVA22-LIKE PROTEIN J) 5.1 UP Brassinosteroid SMT2 (STEROL METHYLTRANSFERASE Metabolism contig11391 2) -1.6 DOWN Brassinosteroid Metabolism contig22334 ATTOP6B (topoisomerase 6 subunit B) -0.6 DOWN Brassinosteroid Metabolism contig28147 CAS1 (cycloartenol synthase 1) 0.9 UP ACS6 (1-AMINOCYCLOPROPANE-1- Ethylene Metabolism contig12055 CARBOXYLIC ACID (ACC) -3.5 DOWN ACS6 (1-AMINOCYCLOPROPANE-1- Ethylene Metabolism contig15616 CARBOXYLIC ACID -2.8 DOWN Ethylene Metabolism contig9429 F3H (FLAVANONE 3-HYDROXYLASE) -2.5 DOWN oxidoreductase, 2OG-Fe(II) oxygenase Ethylene Metabolism contig14812 family protein -2.1 DOWN MBF1C (MULTIPROTEIN BRIDGING Ethylene Metabolism contig20625 FACTOR 1C -1.8 DOWN

Ethylene Metabolism contig11187 ethylene-responsive protein, putative -1.6 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression EFE (ETHYLENE-FORMING ENZYME); 1- aminocyclopropane-1-carboxylate Ethylene Metabolism contig26735 oxidase -1.3 DOWN ACS6 (1-AMINOCYCLOPROPANE-1- Ethylene Metabolism contig16007 CARBOXYLIC ACID (ACC -1.2 DOWN Ethylene Metabolism contig21044 ethylene-responsive family protein -1.1 DOWN

Ethylene Metabolism contig15369 ethylene-responsive protein -related -1.1 DOWN

Ethylene Metabolism contig11243 ethylene-responsive protein, putative -0.9 DOWN Ethylene Metabolism contig9257 F3H (FLAVANONE 3-HYDROXYLASE) -0.8 DOWN oxidoreductase, 2OG-Fe(II) oxygenase Ethylene Metabolism contig30670 family protein 0.9 UP 2-oxoglutarate-dependent Ethylene Metabolism contig29356 dioxygenase, putative 1.2 UP Ethylene Metabolism contig29968 ethylene-responsive family protein 1.4 UP Ethylene Metabolism contig26685 AOP1; oxidoreductase 1.5 UP ETR1 (ETHYLENE RESPONSE 1); Ethylene Metabolism contig29400 ethylene binding 1.6 UP ATGA2OX1 (gibberellin 2-oxidase 1); Ethylene Metabolism contig28300 gibberellin 2-beta-dioxygenase 2.1 UP EFE (ETHYLENE-FORMING ENZYME); 1- aminocyclopropane-1-carboxylate Ethylene Metabolism contig25693 oxidase 2.7 UP Cytokinin Metabolism contig23465 CKX5 (CYTOKININ OXIDASE 5) -1.2 DOWN Cytokinin Metabolism contig17989 ATHK1 (histidine kinase 1) -0.9 DOWN Jasmonate Metabolism contig17324 lipoxygenase, putative -2.4 DOWN Jasmonate Metabolism contig9819 lipoxygenase family protein -1.6 DOWN Jasmonate Metabolism contig25660 AOC2 (ALLENE OXIDE CYCLASE 2) -1 DOWN Jasmonate Metabolism contig16786 AOS (ALLENE OXIDE SYNTHASE) 1.5 UP Jasmonate Metabolism contig23072 AT1G55020 | LOX1; lipoxygenase 1.8 UP Jasmonate Metabolism contig27828 AT1G55020 | LOX1; lipoxygenase 1.9 UP RK - DUF26 contig20822 protein kinase family protein -2 DOWN RK - DUF26 contig19058 kinase -1.5 DOWN RK - DUF26 contig19902 lectin protein kinase, putative -1.5 DOWN RK - DUF26 contig11666 protein kinase family protein -1.3 DOWN 115

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Table C.1 Continued Functional Annotation ID Description Expression RK - DUF26 contig18851 protein kinase family protein -1.1 DOWN RK - DUF26 contig16199 protein kinase family protein -1.1 DOWN serine/threonine protein kinase, RK - DUF26 contig22170 putative -1 DOWN leucine-rich repeat family protein / RK - DUF26 contig20009 protein kinase family protein -1 DOWN RK - DUF26 contig20094 kinase -1 DOWN RK - DUF26 contig18937 protein kinase family protein -1 DOWN RK - DUF26 contig18995 protein kinase family protein -0.9 DOWN RK - DUF26 contig19962 protein kinase family protein -0.8 DOWN RK - DUF26 contig19237 protein kinase family protein -0.8 DOWN RK - DUF26 contig17234 protein kinase family protein -0.7 DOWN RK - DUF26 contig18869 hypothetical protein 0.7 UP curculin-like (mannose-binding) lectin RK - DUF26 contig31644 family protein 1.6 UP RK - DUF26 contig29743 protein kinase family protein 1.8 UP curculin-like (mannose-binding) lectin RK - DUF26 contig30944 family protein 4.6 UP

leucine-rich repeat transmembrane RK - LRR contig16965 protein kinase, putative -1.9 DOWN CLV2 (clavata 2); protein binding / RK - LRR contig21899 receptor signaling protein -1.6 DOWN RK - LRR contig19058 kinase -1.5 DOWN

leucine-rich repeat transmembrane RK - LRR contig21773 protein kinase, putative -1.1 DOWN

leucine-rich repeat transmembrane RK - LRR contig17597 protein kinase, putative -1.1 DOWN RK - LRR contig2915 kinase -1 DOWN leucine-rich repeat family protein / RK - LRR contig17799 extensin family protein -0.9 DOWN RK - LRR contig14059 protein kinase family protein -0.7 DOWN PGIP1 (POLYGALACTURONASE RK - LRR contig18398 INHIBITING PROTEIN 1) -0.7 DOWN CLV2 (clavata 2); protein binding / RK - LRR contig16030 receptor signaling protein -0.7 DOWN 116

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression AtRLP51 (Receptor Like Protein 51); RK - LRR contig18218 protein binding -0.6 DOWN RK - LRR contig28386 leucine-rich repeat family protein 0.8 UP

leucine-rich repeat transmembrane RK - LRR contig7477 protein kinase, putative 0.9 UP RK - LRR contig23990 serine/threonine kinase 1.1 UP SERK1 (SOMATIC EMBRYOGENESIS RK - LRR contig29187 RECEPTOR-LIKE KINASE 1 1.5 UP leucine-rich repeat transmembrane RK - LRR contig26211 protein kinase, putative 1.6 UP RK - LRR contig31585 kinase 1.7 UP leucine-rich repeat transmembrane RK - LRR contig30923 protein kinase, putative 1.8 UP leucine-rich repeat transmembrane RK - LRR contig28153 protein kinase, putative 2.2 UP RK - LRR contig31650 disease resistance family protein 3.3 UP leucine-rich repeat family protein / RK - LRR contig25146 extensin family protein 3.8 UP RK - Catharanthus roseus-like RLK1 contig21205 protein kinase, putative -0.6 DOWN RK - Catharanthus roseus-like RLK2 contig15878 FER (FERONIA); kinase/ protein kinase -1.7 DOWN RK - Catharanthus roseus-like RLK3 contig17474 FER (FERONIA); kinase/ protein kinase -1.4 DOWN RK - S-locus RLK4 (RECEPTOR-LIKE PROTEIN KINASE glycoprotein like contig16093 4) -1.5 DOWN RK - Miscellaneous contig19058 kinase -1.5 DOWN RK - Miscellaneous contig18790 AtRLP4 (Receptor Like Protein 4) -1.2 DOWN leucine-rich repeat transmembrane RK - Miscellaneous contig18779 protein kinase, putative -0.7 DOWN RK - Miscellaneous contig14747 leucine-rich repeat protein-related -1.2 DOWN RK - Phosphoinositides contig9846 unknown -1.8 DOWN ATPH1 (ARABIDOPSIS THALIANA PLECKSTRIN HOMOLOGUE 1); RK - Phosphoinositides contig19146 phosphoinositide binding -1.6 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression 1-phosphatidylinositol-4-phosphate 5- RK - Phosphoinositides contig11971 kinase -1 DOWN

ATNUDX25 (ARABIDOPSIS THALIANA RK - Phosphoinositides contig29718 NUDIX HYDROLASE HOMOLOG 25) 1.1 UP phosphatidylinositol-4-phosphate 5- RK - Phosphoinositides contig28744 kinase family protein 1.6 UP binding / inositol or RK - Phosphoinositides contig29538 phosphatidylinositol kinase 1.8 UP 1-phosphatidylinositol-4-phosphate 5- RK - Phosphoinositides contig29224 kinase 2.3 UP ATPI4K ALPHA; 1-phosphatidylinositol RK - Phosphoinositides contig27930 4-kinase 2.4 UP phosphatidylinositol-4-phosphate 5- RK - Phosphoinositides contig28964 kinase family protein 2.9 UP ATM3 | thioredoxin M-type 3, Thioredoxins contig14865 chloroplast -2.9 DOWN Thioredoxins contig21959 thioredoxin family protein -2 DOWN Thioredoxins contig19939 ACHT2 | thioredoxin family protein -1.8 DOWN ATTRX1; oxidoreductase, acting on Thioredoxins contig18858 sulfur group of donors -0.7 DOWN ATPDIL1-1 (PDI-LIKE 1-1); protein Thioredoxins contig10584 disulfide isomerase -0.6 DOWN Glutaredoxins contig24656 glutaredoxin family protein 1.3 UP Glutaredoxins contig12826 glutaredoxin family protein -1.3 DOWN MSD1 (MANGANESE SUPEROXIDE Dismutases contig10394 DISMUTASE 1) -0.9 DOWN PBP1 (PINOID-BINDING PROTEIN 1); Calcium Signaling contig19071 calcium ion binding -3.9 DOWN

Calcium Signaling contig15048 TCH2 (TOUCH 2); calcium ion binding -3.1 DOWN calcium-transporting ATPase, plasma Calcium Signaling contig14652 membrane-type, putative -2.5 DOWN Calcium Signaling contig13635 calmodulin-binding protein -2.1 DOWN CAM1 (CALMODULIN 1); calcium ion Calcium Signaling contig10704 binding -2.1 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression iqd2 (IQ-domain 2); calmodulin Calcium Signaling contig19227 binding -2 DOWN calcium-transporting ATPase, plasma Calcium Signaling contig20425 membrane-type, putative -1.6 DOWN Calcium Signaling contig15595 calmodulin-binding family protein -1.5 DOWN CPK28; ATP binding / calcium ion binding / calmodulin-dependent Calcium Signaling contig13165 protein kinase -1.4 DOWN BON3 (BONZAI 3); calcium-dependent Calcium Signaling contig19161 phospholipid binding -1.4 DOWN Calcium Signaling contig19664 calcium-binding protein, putative -1.4 DOWN Calcium Signaling contig20481 calmodulin-binding family protein -1.4 DOWN CRT1 (CALRETICULIN 1); calcium ion Calcium Signaling contig9815 binding / unfolded protein binding -1.3 DOWN MSS3 (multicopy suppressors of snf4 deficiency in yeast 3); calcium ion Calcium Signaling contig14860 binding -1.2 DOWN CPK13; ATP binding / calcium ion binding / calmodulin-dependent Calcium Signaling contig15220 protein kinase -1.2 DOWN calcium-transporting ATPase, plasma Calcium Signaling contig18350 membrane-type, putative -1.2 DOWN Calcium Signaling contig14975 VQ motif-containing protein -1.2 DOWN Calcium Signaling contig375 IQD14; calmodulin binding -1.1 DOWN ATEHD1 (EPS15 HOMOLOGY DOMAIN 1); GTP binding / GTPase/ calcium ion Calcium Signaling contig29099 binding -1 DOWN ATCP1 (Ca2+-binding protein 1); Calcium Signaling contig14988 calcium ion binding -1 DOWN MSS3 (multicopy suppressors of snf4 deficiency in yeast 3); calcium ion Calcium Signaling contig19961 binding -1 DOWN AGD11 | AGD11 (ARF-GAP domain Calcium Signaling contig13461 11); calcium ion binding -1 DOWN IQD6 (IQ-domain 6); calmodulin Calcium Signaling contig17927 binding -0.9 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression CRT1 (CALRETICULIN 1); calcium ion Calcium Signaling contig10118 binding / unfolded protein binding -0.9 DOWN iqd32 (IQ-domain 32); calmodulin Calcium Signaling contig12852 binding -0.8 DOWN lipase class 3 family protein / calmodulin-binding heat-shock Calcium Signaling contig20647 protein, putative -0.7 DOWN CDPK6 (CALCIUM-DEPENDENT Calcium Signaling contig16612 PROTEIN KINASE 6) -0.7 DOWN PEPKR2 (Phosphoenolpyruvate Calcium Signaling contig14909 carboxylase-related kinase 2) -0.7 DOWN Calcium Signaling contig21337 calcium ion binding -0.7 DOWN Calcium Signaling contig29800 calcium-binding protein, putative 0.9 UP Calcium Signaling contig25271 calcium-transporting ATPase 1 UP ACA9 (AUTOINHIBITED CA(2+)-ATPASE Calcium Signaling contig30062 9) 1 UP MSS3 (multicopy suppressors of snf4 deficiency in yeast 3); calcium ion Calcium Signaling contig25810 binding 1 UP phospholipid/glycerol acyltransferase Calcium Signaling contig25667 family protein 1.3 UP calcium-dependent protein kinase, Calcium Signaling contig4738 putative / CDPK, putative 1.3 UP Calcium Signaling contig26597 calmodulin-binding protein 2.3 UP ATEHD1 (EPS15 HOMOLOGY DOMAIN Calcium Signaling contig28350 1) 2.6 UP PRA1.H (PRENYLATED RAB ACCEPTOR G Protein Signaling contig20402 1.H) -3.6 DOWN G Protein Signaling contig18935 GTP binding / GTPase -2 DOWN RabGAP/TBC domain-containing G Protein Signaling contig15347 protein -2 DOWN XLG3 (extra-large GTP-binding protein G Protein Signaling contig14740 3) -1.9 DOWN

G Protein Signaling contig14040 ATGB2 (GTP-BINDING 2); GTP binding -1.4 DOWN AtRABA6b (Arabidopsis Rab GTPase G Protein Signaling contig15825 homolog A6b) -1.4 DOWN 120

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Table C.1 Continued Functional Annotation ID Description Expression G Protein Signaling contig22410 ARAC1; GTP binding -1.4 DOWN ARAC5 (RAC-LIKE GTP BINDING G Protein Signaling contig12023 PROTEIN 5) -1.3 DOWN GP ALPHA 1 (G PROTEIN ALPHA G Protein Signaling contig14294 SUBUNIT 1) -1.1 DOWN G Protein Signaling contig15917 RAB6A; GTP binding -1.1 DOWN G Protein Signaling contig3709 RAB6A; GTP binding -1.1 DOWN G Protein Signaling contig3128 RHA1 (RAB HOMOLOG 1) -0.9 DOWN ATSAR2 (ARABIDOPSIS THALIANA SECRETION-ASSOCIATED RAS SUPER G Protein Signaling contig15433 FAMILY 2) -0.9 DOWN

G Protein Signaling contig18232 RID3 (ROOT INITIATION DEFECTIVE 3) -0.9 DOWN Rho GDP-dissociation inhibitor family G Protein Signaling contig14610 protein -0.9 DOWN transducin family protein / WD-40 G Protein Signaling contig17007 repeat family protein -0.6 DOWN G Protein Signaling contig27288 ATGB2 (GTP-BINDING 2) 0.6 UP ATMIN7 (ARABIDOPSIS THALIANA G Protein Signaling contig24535 HOPM INTERACTOR 7) 0.7 UP G Protein Signaling contig23751 RHD3 (ROOT HAIR DEFECTIVE 3) 0.8 UP G Protein Signaling contig31506 AGD4 (ARF-GAP domain 4) 1 UP G Protein Signaling contig28074 GTP binding / GTPase 1 UP G Protein Signaling contig27670 Ran-binding protein, putative 1 UP G Protein Signaling contig6607 guanylate-binding family protein 1 UP G Protein Signaling contig31474 RAB GTPase activator 1.1 UP guanine nucleotide exchange family G Protein Signaling contig31142 protein 1.1 UP AtRABA1c (Arabidopsis Rab GTPase G Protein Signaling contig27880 homolog A1c) 1.3 UP G Protein Signaling contig29401 RAB GTPase activator 1.4 UP ATGB1 (ARABIDOPSIS THALIANA GTP- G Protein Signaling contig24256 BINDING PROTEIN 1) 1.4 UP G Protein Signaling contig30858 binding 1.7 UP

G Protein Signaling contig29707 rac GTPase activating protein, putative 2.1 UP AtRABH1e (Arabidopsis Rab GTPase G Protein Signaling contig3509 homolog H1e) 2.3 UP 121

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression G Protein Signaling contig27032 GTP binding / GTPase 2.7 UP ROPGEF5 (ROP GUANINE NUCLEOTIDE G Protein Signaling contig31431 EXCHANGE FACTOR 5) 2.7 UP ATRABA1D (ARABIDOPSIS RAB GTPASE G Protein Signaling contig29592 HOMOLOG A1D) 2.8 UP

G Protein Signaling contig24724 rac GTPase activating protein, putative 2.9 UP ROPGEF12 (RHO GUANYL- G Protein Signaling contig28150 NUCLEOTIDE EXCHANGE FACTOR 12) 4.6 UP Sugar & N Physiology Signaling contig18981 EXO | EXO (EXORDIUM) -3.2 DOWN Sugar & N Physiology Signaling contig12675 EXO | EXO (EXORDIUM) -3 DOWN Sugar & N Physiology Signaling contig15474 EXL5 (EXORDIUM LIKE 5) -2.7 DOWN Sugar & N Physiology Signaling contig13669 EXO (EXORDIUM) -1.8 DOWN Sugar & N Physiology Signaling contig15604 EXL5 (EXORDIUM LIKE 5) -1.4 DOWN Sugar & N Physiology photoassimilate-responsive protein- Signaling contig9500 related -0.6 DOWN Sugar & N Physiology PDK (PYRUVATE DEHYDROGENASE Signaling contig26579 KINASE) 1.4 UP Sugar & N Physiology photoassimilate-responsive protein- Signaling contig27740 related 2.7 UP Sugar & N Physiology Signaling contig25793 EXL6 (EXORDIUM LIKE 6 3.7 UP NPY2 (NAKED PINS IN YUC MUTANTS Light Signaling contig14498 2) -1.5 DOWN Light Signaling contig22311 SPA1 (SUPPRESSOR OF PHYA-105 1) -1 DOWN Light Signaling contig21915 FRS5 (FAR1-related sequence 5) -1 DOWN ELIP1 (EARLY LIGHT-INDUCABLE Light Signaling contig16906 PROTEIN) -0.9 DOWN Light Signaling contig18917 PHYC (phytochrome defective c) -0.9 DOWN Light Signaling contig14244 PHYB (PHYTOCHROME B) -0.6 DOWN Light Signaling contig10918 FRS11 (FAR1-related sequence 11) -0.6 DOWN

Light Signaling contig29041 binding / catalytic/ coenzyme binding 1.5 UP

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Table C.1 Continued Functional Annotation ID Description Expression Light Signaling contig28344 ENP (ENHANCER OF PINOID) 1.6 UP Light Signaling contig28038 COP1-interacting protein-related 1.8 UP

Light Signaling contig28151 CIP1 (COP1-INTERACTIVE PROTEIN 1) 2.8 UP Light Signaling contig28200 CSN6A 4.1 UP MAP Kinase Signaling contig9788 AT3G13530 | MAPKKK7 -2 DOWN MAP Kinase Signaling contig16920 ATMPK19; MAP kinase -1.8 DOWN MAP Kinase Signaling contig14256 protein kinase, putative -1.7 DOWN ATMPK4 (ARABIDOPSIS THALIANA MAP Kinase Signaling contig3319 MAP KINASE 4) -1.2 DOWN MAP Kinase Signaling contig16261 MAPKKK5 -0.9 DOWN MAP Kinase Signaling contig30867 AT3G13530 | MAPKKK7 1.2 UP 14-3-3 Signaling contig10581 GF14 PHI (GF14 PROTEIN PHI CHAIN) -1.1 DOWN GRF2 (GENERAL REGULATORY FACTOR 14-3-3 Signaling contig10543 2) -0.6 DOWN DNAJ heat shock N-terminal domain- Heat Response contig13106 containing protein -3.5 DOWN Heat Response contig16222 DNAJ heat shock protein, putative -1.2 DOWN DNAJ heat shock N-terminal domain- Heat Response contig16860 containing protein -1.1 DOWN HSC70-1 (HEAT SHOCK COGNATE Heat Response contig23250 PROTEIN 70-1) -1 DOWN Heat Response contig3052 AT-HSFB4 -0.9 DOWN DNAJ heat shock N-terminal domain- Heat Response contig20123 containing protein -0.9 DOWN Heat Response contig20546 ATERDJ3A; oxidoreductase -0.8 DOWN DNAJ heat shock N-terminal domain- Heat Response contig18547 containing protein -0.8 DOWN Heat Response contig19 HSP70 (heat shock protein 70) -0.6 DOWN HSP81-3; ATP binding / unfolded Heat Response contig22993 protein binding 1 UP Heat Response contig8459 CPHSC70-2EAT SHOCK PROTEIN 70-2 1.3 UP Heat Response contig31321 DNAJ heat shock protein, putative 1.3 UP

Heat Response contig28561 CLPB4 (CASEIN LYTIC PROTEINASE B4) 1.4 UP DNAJ heat shock N-terminal domain- Heat Response contig26394 containing protein 1.7 UP Heat Response contig29593 GRV2 (GRAVITROPISM DEFECTIVE 2) 1.7 UP 123

Texas Tech University, Amanda Sooter, May 2013

Table C.1 Continued Functional Annotation ID Description Expression DNAJ heat shock N-terminal domain- Heat Response contig30620 containing protein 1.7 UP ATHSP90.1 (HEAT SHOCK PROTEIN Heat Response contig29956 90.1) 1.9 UP Heat Response contig30816 GRV2 (GRAVITROPISM DEFECTIVE 2) 2.5 UP SRC2 (SOYBEAN GENE REGULATED BY Cold Response contig15983 COLD-2) -1.4 DOWN

Cold Response contig12528 cold-shock DNA-binding family protein -0.6 DOWN universal stress protein (USP) family Cold Response contig22932 protein 1 UP universal stress protein (USP) family Cold Response contig22846 protein 1 UP HOS15 (high expression of osmotically Cold Response contig29507 responsive genes 15) 1.5 UP Drought/Salt Response contig12812 response to water deprivation -2.6 DOWN Drought/Salt Response contig14975 VQ motif-containing protein -1.2 DOWN

Drought/Salt Response contig21537 dehydration-responsive family protein -1 DOWN dehydration-responsive protein- Drought/Salt Response contig20541 related 0.9 UP dehydration-responsive protein- Drought/Salt Response contig8083 related 1.3 UP dehydration-responsive protein- Drought/Salt Response contig29430 related 1.5 UP

Drought/Salt Response contig30181 dehydration-responsive family protein 2.6 UP dehydration-responsive protein- Drought/Salt Response contig29827 related 3.7 UP NADK2; NAD+ kinase/ calmodulin Light Response contig3751 binding -2.5 DOWN Unspecified Cellular AT3G62020 | GLP10 (GERMIN-LIKE Response contig14200 PROTEIN 10) -2.2 DOWN Unspecified Cellular Response contig720 GLP7 (GERMIN-LIKE PROTEIN 7) -1 DOWN Unspecified Cellular universal stress protein (USP) family Response contig22846 protein 1 UP

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Table C.1 Continued Functional Annotation ID Description Expression Unspecified Cellular Response contig28217 SAH7 3.6 UP Unspecified Cellular AT3G62020 | GLP10 (GERMIN-LIKE Response contig25883 PROTEIN 10) 5.4 UP Unspecified Cellular pollen Ole e 1 allergen and extensin Response contig25528 family protein 6.3 UP

Cell Division contig19990 RBR1 (RETINOBLASTOMA-RELATED 1) -2.2 DOWN structural maintenance of chromosomes (SMC) family protein Cell Division contig4166 (MSS2) -1.9 DOWN ribosome recycling factor family Cell Division contig17462 protein -1.5 DOWN DNAJ heat shock N-terminal domain- Cell Division contig11723 containing protein -1.1 DOWN Cell Division contig15933 AAR2 protein family -0.9 DOWN APC8 (ANAPHASE-PROMOTING Cell Division contig22218 COMPLEX SUBUNIT 8) -0.8 DOWN regulator of chromosome Cell Division contig11387 condensation (RCC1) family protein -0.7 DOWN SCD1 (STOMATAL CYTOKINESIS- Cell Division contig14601 DEFECTIVE 1) 0.9 UP Cell Division contig28165 UVR8 (UVB-RESISTANCE 8) 1.5 UP Cell Division contig28960 MSL2 (MscS-LIKE 2) 1.5 UP

Cell Division contig23860 cell division cycle protein 48, putative 1.6 UP structural maintenance of Cell Division contig27340 chromosomes (SMC) family protein 2.2 UP Cell Division contig29193 FTSZ1-1; protein binding 2.6 UP CKS1 (CYCLIN-DEPENDENT KINASE- Cell Cycle contig15478 SUBUNIT 1) -1.6 DOWN ICK5; cyclin binding / cyclin-dependent Cell Cycle contig11790 protein -1.4 DOWN CKS1 (CYCLIN-DEPENDENT KINASE- Cell Cycle contig10422 SUBUNIT 1) -1.4 DOWN CYC1BAT; cyclin-dependent protein Cell Cycle contig21113 kinase -1 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression peptidyl-prolyl cis-trans isomerase Cell Cycle contig11631 cyclophilin-type family protein -0.9 DOWN Cell Cycle contig20812 RAD9 -0.8 DOWN Cell Cycle contig21768 CYCD1;1 (CYCLIN D1;1) -0.6 DOWN peptidyl-prolyl cis-trans isomerase, Cell Cycle contig14071 putative -0.6 DOWN peptidyl-prolyl cis-trans isomerase Cell Cycle contig25059 cyclophilin-type family protein 0.7 UP FKBP15-2; FK506 binding / peptidyl- Cell Cycle contig23573 prolyl cis-trans isomerase 1 UP peptidyl-prolyl cis-trans isomerase Cell Cycle contig22828 cyclophilin-type family protein 1.2 UP peptidyl-prolyl cis-trans isomerase, Cell Cycle contig24195 putative 1.6 UP Cell Cycle contig29200 CYCA1;1 (Cyclin A1;1) 2 UP Cell Cycle contig28132 CYP59 (CYCLOPHILIN 59) 2.9 UP SPL9 (SQUAMOSA PROMOTER Development contig20375 BINDING PROTEIN-LIKE 9) -1.2 DOWN SPL9 (SQUAMOSA PROMOTER Development contig22189 BINDING PROTEIN-LIKE 9) -1 DOWN SDP1 (SUGAR-DEPENDENT1); Development contig27055 triacylglycerol lipase 0.9 UP Development contig9222 PAP85; nutrient reservoir 1 UP TOR (TARGET OF RAPAMYCIN); 1- Development contig29315 phosphatidylinositol-3-kinase 1.1 UP Development contig22511 PAP85; nutrient reservoir 1.4 UP Development contig9233 cupin family protein 1.5 UP Development contig9214 PAP85; nutrient reservoir 1.5 UP Development contig22586 PAP85; nutrient reservoir 1.7 UP Development contig22460 PAP85; nutrient reservoir 1.8 UP Development contig22469 PAP85; nutrient reservoir 1.8 UP CRA1 (CRUCIFERINA); nutrient Development contig104 reservoir 2 UP AT2S3; lipid binding / nutrient Development contig22461 reservoir 2.3 UP

Cell Organization contig19990 RBR1 (RETINOBLASTOMA-RELATED 1) -2.2 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression ATEXO70D3 (exocyst subunit EXO70 Cell Organization contig20913 family protein D3); protein binding -2.1 DOWN Cell Organization contig16568 plastid-lipid associated protein PAP -2 DOWN ORP1A (OSBP(OXYSTEROL BINDING Cell Organization contig17477 PROTEIN)-RELATED PROTEIN 1A) -1.9 DOWN SYP121 (SYNTAXIN OF PLANTS 121); Cell Organization contig14942 SNAP receptor/ protein anchor -1.8 DOWN Cell Organization contig18511 ankyrin repeat family protein -1.6 DOWN Cell Organization contig12724 ABIL1 (Abi-1-like 1) -1.6 DOWN Cell Organization contig10935 coatomer beta subunit, putative -1.5 DOWN Cell Organization contig19823 ATPP2-A1; carbohydrate binding -1.4 DOWN Cell Organization contig21094 PEX19-2 -1.4 DOWN ATEXO70G1 (exocyst subunit EXO70 Cell Organization contig10950 family protein G1) -1.4 DOWN TUB1; GTP binding / GTPase/ Cell Organization contig9446 structural molecule -1.3 DOWN CAD1 (constitutively activated cell Cell Organization contig18570 death 1) -1.3 DOWN Cell Organization contig20013 myosin heavy chain-related -1.2 DOWN phragmoplast-associated kinesin- Cell Organization contig18569 related protein 2 (PAKRP2) -1.2 DOWN Cell Organization contig15991 AtPP2-A12 (Phloem protein 2-A12) -1.2 DOWN ADF1 (ACTIN DEPOLYMERIZING Cell Organization contig9885 FACTOR 1) -1.2 DOWN ATARP6; structural constituent of Cell Organization contig20553 cytoskeleton -1.2 DOWN Cell Organization contig8665 kinesin motor protein-related -1.1 DOWN Cell Organization contig11897 PRF3 (PROFILIN 3); actin binding -1.1 DOWN Cell Organization contig12110 kinesin motor protein-related -1.1 DOWN Cell Organization contig20036 dynein light chain, putative -1.1 DOWN SYP121 (SYNTAXIN OF PLANTS 121); Cell Organization contig12687 SNAP receptor -1.1 DOWN Cell Organization contig9217 TUB6 (BETA-6 TUBULIN) -1 DOWN ATK2 (ARABIDOPSIS THALIANA Cell Organization contig20163 KINESIN 2) -1 DOWN Cell Organization contig12603 PIR121; transcription activator -1 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression TUA6; structural constituent of Cell Organization contig9279 cytoskeleton -1 DOWN Cell Organization contig19264 FIB (FIBRILLIN); structural molecule -1 DOWN Cell Organization contig22401 ATP binding / microtubule motor -1 DOWN SYP32 (SYNTAXIN OF PLANTS 32); Cell Organization contig11192 SNAP receptor -1 DOWN Cell Organization contig16441 ankyrin repeat family protein -0.9 DOWN U2A' (U2 small nuclear Cell Organization contig12012 ribonucleoprotein A) -0.9 DOWN TUB8; structural constituent of Cell Organization contig9704 cytoskeleton -0.9 DOWN chromosome-associated kinesin- Cell Organization contig17537 related -0.9 DOWN Cell Organization contig15014 XIE; motor/ protein binding -0.9 DOWN STT3B (staurosporin and temperature Cell Organization contig817 sensitive 3-like b) -0.9 DOWN Cell Organization contig18466 ACD1 (ACCELERATED CELL DEATH 1) -0.9 DOWN Cell Organization contig19764 ankyrin repeat family protein -0.8 DOWN TUB1; GTP binding / GTPase/ Cell Organization contig26023 structural molecule -0.8 DOWN Cell Organization contig13269 AtPP2-B1 (Phloem protein 2-B1) -0.8 DOWN Cell Organization contig25785 tubulin folding cofactor B -0.8 DOWN

Cell Organization contig12236 coatomer gamma-2 subunit, putative -0.8 DOWN TUA6; structural constituent of Cell Organization contig9554 cytoskeleton -0.7 DOWN TUB8; structural constituent of Cell Organization contig9263 cytoskeleton -0.7 DOWN TUA2; structural constituent of Cell Organization contig9203 cytoskeleton -0.7 DOWN KINESIN-13A; ATP binding / Cell Organization contig9412 microtubule motor -0.7 DOWN Cell Organization contig9954 CbbY protein-related -0.7 DOWN coatomer protein complex, subunit Cell Organization contig11634 alpha, putative -0.7 DOWN Cell Organization contig9857 clathrin heavy chain, putative -0.6 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression

Cell Organization contig26055 tubulin-specific chaperone C-related 0.6 UP KINESIN-13A; ATP binding / Cell Organization contig23764 microtubule motor 0.7 UP Cell Organization contig26251 ankyrin repeat family protein 0.7 UP Cell Organization contig26569 myosin heavy chain-related 0.7 UP VLN4 (ARABIDOPSIS THALIANA VILLIN Cell Organization contig23227 4) 0.7 UP coatomer protein complex, subunit Cell Organization contig21897 beta 2 (beta prime) 0.7 UP Cell Organization contig24833 VAM3; SNAP receptor 0.7 UP MRH2 (MORPHOGENESIS OF ROOT Cell Organization contig26531 HAIR 2) 0.8 UP Cell Organization contig26731 adaptin family protein 0.8 UP Cell Organization contig31250 AKRP (ANKYRIN REPEAT PROTEIN) 0.9 UP ZCF125; ATP binding / microtubule Cell Organization contig28987 motor 0.9 UP Cell Organization contig28607 kinesin motor family protein 1 UP Cell Organization contig17290 epsilon-adaptin, putative 1 UP Cell Organization contig25911 ACD1 (ACCELERATED CELL DEATH 1) 1.1 UP Cell Organization contig24233 PIR121; transcription activator 1.2 UP ATEXO70D1 (exocyst subunit EXO70 Cell Organization contig26946 family protein D1) 1.2 UP Cell Organization contig29032 kinesin motor family protein 1.3 UP armadillo/beta-catenin repeat family Cell Organization contig27477 protein 1.4 UP Cell Organization contig23705 clathrin heavy chain, putative 1.4 UP Cell Organization contig30672 kinesin light chain-related 1.5 UP Cell Organization contig30271 KEG (KEEP ON GOING) 1.5 UP Cell Organization contig29410 heat shock protein binding 1.6 UP Cell Organization contig21610 clathrin heavy chain, putative 1.7 UP Cell Organization contig29483 clathrin heavy chain, putative 1.7 UP tryptophan synthase, alpha subunit, Cell Organization contig4889 putative 1.9 UP Cell Organization contig29506 XIK; motor/ protein binding 1.9 UP MOR1 (MICROTUBULE Cell Organization contig15358 ORGANIZATION 1) 1.9 UP 129

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Table C.1 Continued Functional Annotation ID Description Expression Cell Organization contig30256 ATFIM1; actin binding 1.9 UP Cell Organization contig29635 kinesin motor protein-related 2 UP Cell Organization contig28930 ankyrin repeat family protein 2 UP Cell Organization contig27464 SCAR2 (SCAR HOMOLOG 2) 2.3 UP Cell Organization contig26092 kinesin motor protein-related 2.3 UP Cell Organization contig27616 MYA2 (ARABIDOPSIS MYOSIN 2) 2.4 UP Cell Organization contig27256 epsilon-adaptin, putative 2.5 UP ATMAP70-5 (microtubule-associated Cell Organization contig30180 proteins 70-5) 2.8 UP Cell Organization contig29869 clathrin heavy chain, putative 2.9 UP ACT3 (actin 3); structural constituent Cell Organization contig23965 of cytoskeleton 3.4 UP ADF8 (ACTIN DEPOLYMERIZING Cell Organization contig25219 FACTOR 8) 4.7 UP DWF4 (DWARF 4); steroid 22-alpha Cytochrome p450 contig2945 hydroxylase -2.8 DOWN TT7 (TRANSPARENT TESTA 7); Cytochrome p450 contig220 flavonoid 3'-monooxygenase -2.4 DOWN CYP714A1; electron carrier/ heme Cytochrome p450 contig18763 binding -1.9 DOWN CYP72A9; electron carrier/ heme Cytochrome p450 contig19344 binding -1.5 DOWN Cytochrome p450 contig23750 CYP96A5; electron carrier -1.4 DOWN CYP94D2; electron carrier/ heme Cytochrome p450 contig20487 binding -1.3 DOWN CYP706A6; electron carrier/ heme Cytochrome p450 contig22185 binding -1.3 DOWN CYP76C2; electron carrier/ heme Cytochrome p450 contig10105 binding -1 DOWN CYP86B1; electron carrier/ heme Cytochrome p450 contig10032 binding 0.7 UP CPD (CONSTITUTIVE Cytochrome p450 contig29067 PHOTOMORPHOGENIC DWARF) 1.2 UP CYP86B1; electron carrier/ heme Cytochrome p450 contig22801 binding 1.3 UP CYP82C4; electron carrier/ heme Cytochrome p450 contig27091 binding 1.6 UP

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Table C.1 Continued Functional Annotation ID Description Expression FAH1 (FERULIC ACID 5-HYDROXYLASE Cytochrome p450 contig22635 1) 1.9 UP Cytochrome p450 contig28661 TT7 (TRANSPARENT TESTA 7) 2 UP CYP82C4; electron carrier/ heme Cytochrome p450 contig27399 binding 2.3 UP BGAL1 (Beta galactosidase 1); beta- Glucosidases contig10793 galactosidase 0.8 UP Glucosidases contig18695 glycosyl hydrolase family 38 protein 0.8 UP BGAL5 (beta-galactosidase 5); beta- Glucosidases contig27761 galactosidase 1.4 UP

Glucosidases contig29092 glycoside hydrolase family 47 protein 1.5 UP Glucosidases contig16755 BGLU41 (BETA GLUCOSIDASE 41) 1.6 UP Glucosidases contig23353 BGAL13; beta-galactosidase 5.1 UP Alcohol Dehydrogenases contig22892 alcohol dehydrogenase, putative -1.2 DOWN Alcohol Dehydrogenases contig17580 alcohol dehydrogenase, putative -1.5 DOWN Peroxidases contig10502 peroxidase, putative -1.7 DOWN Peroxidases contig16705 cationic peroxidase, putative -1.2 DOWN RCI3 (RARE COLD INDUCIBLE GENE 3); Peroxidases contig6087 peroxidase -1.2 DOWN Peroxidases contig13593 peroxidase, putative -0.9 DOWN Peroxidases contig11844 peroxidase, putative -0.7 DOWN PRXR1; electron carrier/ heme Peroxidases contig22506 binding / peroxidase 1.5 UP

Peroxidases contig29820 peroxidase 12 (PER12) (P12) (PRXR6) 2.7 UP Phosphatases contig19170 phosphatase -3.9 DOWN AT1G07990 | SIT4 phosphatase- Phosphatases contig13200 associated family protein -1.8 DOWN AT1G09870 | histidine acid Phosphatases contig13619 phosphatase family protein -1.8 DOWN ATEYA (Arabidopsis thaliana EYES Phosphatases contig15060 ABSENT homolog) -1.7 DOWN acid phosphatase survival protein Phosphatases contig18493 SurE, putative -1 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression AT1G07990 | SIT4 phosphatase- Phosphatases contig7256 associated family protein -0.8 DOWN AT1G07990 | SIT4 phosphatase- Phosphatases contig28909 associated family protein 1.1 UP AT1G09870 | histidine acid Phosphatases contig26831 phosphatase family protein 1.2 UP calcineurin-like phosphoesterase Phosphatases contig5455 family protein 2.6 UP Oxidases contig16172 AT-AER (alkenal reductase) -3.1 DOWN Oxidases contig16670 AT4G22010 | sks4 (SKU5 Similar 4) -1.3 DOWN oxidoreductase, 2OG-Fe(II) oxygenase Oxidases contig11173 family protein -0.9 DOWN Oxidases contig30840 FLD (FLOWERING LOCUS D) 1 UP Oxidases contig26169 AT4G22010 | sks4 (SKU5 Similar 4) 1 UP Oxidases contig30958 LDL3 (LSD1-LIKE3) 2 UP Oxidases contig22793 sks11 (SKU5 Similar 11) 6.3 UP UDP Glycosyltransferases contig20386 glycosyltransferase family protein 2 -3 DOWN UDP UGT72B3 (UDP-GLUCOSYL Glycosyltransferases contig22029 TRANSFERASE 72B3) -2.6 DOWN

UDP PARVUS (PARVUS); polygalacturonate Glycosyltransferases contig19733 4-alpha-galacturonosyltransferase -2.5 DOWN UDP Glycosyltransferases contig16784 glycosyltransferase family protein -2.3 DOWN UDP Glycosyltransferases contig16974 exostosin family protein -2.3 DOWN

UDP PARVUS (PARVUS); polygalacturonate Glycosyltransferases contig18448 4-alpha-galacturonosyltransferase -1.9 DOWN UDP XXT5 (XYLOGLUCAN Glycosyltransferases contig12509 XYLOSYLTRANSFERASE 5) -1.3 DOWN

UDP GAUT12 Glycosyltransferases contig15291 (GALACTURONOSYLTRANSFERASE 12) -1.3 DOWN UDP Glycosyltransferases contig16879 glycosyltransferase family protein 2 -1.2 DOWN

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Table C.1 Continued Functional Annotation ID Description Expression UDP MUR3 (MURUS 3); catalytic/ Glycosyltransferases contig19781 transferase -1.1 DOWN UDP alpha 1,4-glycosyltransferase family Glycosyltransferases contig21830 protein -1 DOWN UDP alpha 1,4-glycosyltransferase family Glycosyltransferases contig19497 protein -1 DOWN GAUT12 UDP (GALACTURONOSYLTRANSFERASE Glycosyltransferases contig12229 12) -1 DOWN UDP Glycosyltransferases contig13137 IRX14 (irregular xylem 14); transferase -0.7 DOWN UDP Glycosyltransferases contig20538 exostosin family protein -0.7 DOWN UDP transferase, transferring glycosyl Glycosyltransferases contig3579 groups -0.7 DOWN QUA1 (QUASIMODO 1); UDP polygalacturonate 4-alpha- Glycosyltransferases contig11600 galacturonosyltransferase -0.6 DOWN UDP Glycosyltransferases contig15402 exostosin family protein 0.7 UP UDP transferase, transferring glycosyl Glycosyltransferases contig27521 groups 2.6 UP UDP UGT73B5 (UDP-glucosyl transferase Glycosyltransferases contig25767 73B5) 2.6 UP

O-methyltransferases contig117 ATOMT1 (O-METHYLTRANSFERASE 1) -0.9 DOWN FAD-binding domain-containing Nitrilases contig13982 protein -2.5 DOWN AtFAAH (Arabidopsis thaliana fatty Nitrilases contig18490 acid amide hydrolase) -1.6 DOWN AtFAAH (Arabidopsis thaliana fatty Nitrilases contig14689 acid amide hydrolase) -1.4 DOWN

Nitrilases contig16657 glycosyl transferase family 17 protein -0.6 DOWN FAD-binding domain-containing Nitrilases contig17043 protein -0.6 DOWN Nitrilases contig18027 aminoacylase, putative -0.6 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression aminoacylase, putative / N-acyl-L- Nitrilases contig2579 amino-acid amidohydrolase 0.6 UP tropinone reductase, putative / Nitrilases contig30852 tropine dehydrogenase 1.2 UP

Glutathione S- ATGSTF8 (ARABIDOPSIS THALIANA Transferases contig201 GLUTATHIONE S-TRANSFERASE PHI 8) -0.6 DOWN Glutathione S- ATGSTU8 (GLUTATHIONE S- Transferases contig24133 TRANSFERASE TAU 8) 1.6 UP

Glutathione S- glutathione S-transferase C-terminal Transferases contig28879 domain-containing protein 1.7 UP GDSL-motif lipase/hydrolase family GDSL Lipases contig17742 protein -2 DOWN GDSL-motif lipase/hydrolase family GDSL Lipases contig11960 protein -1.2 DOWN GDSL-motif lipase/hydrolase family GDSL Lipases contig9824 protein -1.1 DOWN GDSL-motif lipase/hydrolase family GDSL Lipases contig21925 protein -0.9 DOWN GDSL-motif lipase/hydrolase family GDSL Lipases contig9605 protein -0.9 DOWN GDSL-motif lipase/hydrolase family GDSL Lipases contig13437 protein -0.9 DOWN GDSL-motif lipase/hydrolase family GDSL Lipases contig27128 protein 1.6 UP GDSL-motif lipase/hydrolase family GDSL Lipases contig26956 protein 4.5 UP Beta 1,3 Glucan Hydrolases contig13037 glycosyl hydrolase family 17 protein -1.8 DOWN Beta 1,3 Glucan Hydrolases contig6666 glycosyl hydrolase family 17 protein -1.7 DOWN Beta 1,3 Glucan Hydrolases contig19648 glycosyl hydrolase family 17 protein -1.4 DOWN Beta 1,3 Glucan PDCB3 (PLASMODESMATA Hydrolases contig13787 CALLOSE-BINDING PROTEIN 3) -1.3 DOWN Beta 1,3 Glucan Hydrolases contig10658 glycosyl hydrolase family 17 protein -1 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression Beta 1,3 Glucan Hydrolases contig29739 glycosyl hydrolase family 17 protein -0.8 DOWN Beta 1,3 Glucan Hydrolases contig22020 glycosyl hydrolase family 17 protein -0.7 DOWN Beta 1,3 Glucan Hydrolases contig23533 glycosyl hydrolase family 17 protein 0.8 UP Beta 1,3 Glucan Hydrolases contig29832 glycosyl hydrolase family 17 protein 3.2 UP Protein Targeting IMPA-2 (IMPORTIN ALPHA ISOFORM Nucleus contig9524 2) -1.2 DOWN Protein Targeting NTF2B (NUCLEAR TRANSPORT FACTOR Nucleus contig11448 2B) -1 DOWN Protein Targeting nuclear transport factor 2 (NTF2), Nucleus contig20732 putative -0.7 DOWN Protein Targeting Nucleus contig24978 importin beta-2, putative 1.1 UP Protein Targeting Nucleus contig30761 nucleoporin family protein 1.3 UP Protein Targeting Nucleus contig25713 nucleoporin family protein 1.3 UP Protein Targeting Nucleus contig28835 binding / protein transporter 2 UP Protein Targeting Golgi Apparatus contig13565 protein binding / zinc ion binding -1 DOWN Protein Targeting Golgi STT3B (staurosporin and temperature Apparatus contig817 sensitive 3-like b) -0.9 DOWN Protein Targeting Golgi Apparatus contig27217 transport protein, putative 0.8 UP Protein Targeting Vacuole contig16925 VCL1 (VACUOLELESS 1) 0.6 UP Protein Targeting ATVSR3 (ARABIDOPSIS THALIANA Vacuole contig7276 VACULOLAR SORTING RECEPTOR 3) 0.9 UP Protein Targeting BETA-VPE (BETA VACUOLAR Vacuole contig25746 PROCESSING ENZYME) 1.8 UP Protein Targeting Plasma Membrane contig577 ATSLY1; protein transporter -0.9 DOWN

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Table C.1 Continued

Functional Annotation ID Description Expression Protein Targeting TIC21 (TRANSLOCON AT INNER Chloroplast contig21538 MEMBRANE OF CHLOROPLASTS 21) -1.6 DOWN Protein Targeting PLSP1 (plastidic type I signal peptidase Chloroplast contig17542 1) -0.8 DOWN TOC132 (MULTIMERIC TRANSLOCON Protein Targeting COMPLEX IN THE OUTER ENVELOPE Chloroplast contig25041 MEMBRANE 132) 0.8 UP HCF106; proton motive force Protein Targeting dependent protein transmembrane Chloroplast contig31448 transporter 1.2 UP ATTIM23-2; P-P-bond-hydrolysis- Protein Targeting driven protein transmembrane Mitochondria contig19429 transporter -1 DOWN Protein Targeting Mitochondria contig16163 metaxin-related -1.2 DOWN mitochondrial import inner membrane Protein Targeting translocase subunit Mitochondria contig25838 Tim17/Tim22/Tim23 family protein 1.3 UP Protein Targeting PEX14; protein binding / protein Peroxisomes contig18093 transporter -0.8 DOWN

Abbreviations

TF – Transcription Factor

RK – Receptor Kinase

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