De novo Transcriptome Sequencing and Development of Abscission Zone-Specific Microarray as a New Molecular Tool for Analysis of Tomato Organ Abscission.

Srivignesh Sundaresan,Shimon Meir,Sonia Philosoph-Hadas,Mark L Tucker,Joseph Riov,Raja Mugasimangalam,Nagesh A Kuravadi,Shoshana Salim,Bettina Kochanek

doi:10.3389/fpls.2015.01258

Abstract

Abscission of flower pedicels and leaf petioles of tomato (Solanum lycopersicum) can be induced by flower removal or leaf deblading, respectively, which leads to auxin depletion, resulting in increased sensitivity of the abscission zone (AZ) to ethylene. However, the molecular mechanisms that drive the acquisition of abscission competence and its modulation by auxin gradients are not yet known. We used RNA-Sequencing (RNA-Seq) to obtain a comprehensive transcriptome of tomato flower AZ (FAZ) and leaf AZ (LAZ) during abscission. RNA-Seq was performed on a pool of total RNA extracted from tomato FAZ and LAZ, at different abscission stages, followed by de novo assembly. The assembled clusters contained transcripts that are already known in the Solanaceae (SOL) genomics and NCBI databases, and over 8823 identified novel tomato transcripts of varying sizes. An AZ-specific microarray, encompassing the novel transcripts identified in this study and all known transcripts from the SOL genomics and NCBI databases, was constructed to study the abscission process. Multiple probes for longer genes and key AZ-specific genes, including antisense probes for all transcripts, make this array a unique tool for studying abscission with a comprehensive set of transcripts, and for mining for naturally occurring antisense transcripts. We focused on comparing the global transcriptomes generated from the FAZ and the LAZ to establish the divergences and similarities in their transcriptional networks, and particularly to characterize the processes and transcriptional regulators enriched in gene clusters that are differentially regulated in these two AZs. This study is the first attempt to analyze the global gene expression in different AZs in tomato by combining the RNA-Seq technique with oligonucleotide microarrays. Our AZ-specific microarray chip provides a cost-effective approach for expression profiling and robust analysis of multiple samples in a rapid succession.

Highlights

Abscission is a systematically regulated process in plant development, by which subtended organs, leaves, flowers, fruit, and seed, separate from the parent plant in response to various physiological cues
Based on these similar abscission percentages, we have chosen the indicated time points for RNA sampling from both abscission zone (AZ) for the RNA-Seq experiments, which correspond to steps 2–4 of the abscission process, namely acquisition of the competence of AZ cells to respond to abscission signals, execution of organ abscission, and beginning of formation of a protective layer
This study presents a de novo assembly of AZ-associated transcripts and provides valuable information about their functional annotation by using an integrated approach to enrich the transcriptome of S. lycopersicum flower AZ (FAZ) and leaf AZ (LAZ)

Summary

Introduction

Abscission is a systematically regulated process in plant development, by which subtended organs, leaves, flowers, fruit, and seed, separate from the parent plant in response to various physiological cues. This process is required to recycle nutrients for continuous growth, develop appropriate organs, survive diseases, and facilitate reproduction (Addicott, 1982; Sexton and Roberts, 1982; Roberts et al, 2002; Lewis et al, 2006). The abscission process is initiated or timed by changes in the auxin gradient across the AZ, and is acceltrated by ethylene (Roberts et al, 2002; McManus, 2008; Meir et al, 2010). During the late abscission stages, the cell wall and middle lamella are the major targets for degradation, which is operated by many cell wall modifiying enzymes, including polygalacturonases (PGs), xyloglucan endotransglucosylase/ hydrolase (XTH), β-1,4glucanase (cellulase, Cel), and expansins (EXP) (Lashbrook et al, 1994; del Campillo and Bennett, 1996; Cho and Cosgrove, 2000; Taylor and Whitelaw, 2001; Roberts et al, 2002; Ogawa et al, 2009; Meir et al, 2010)

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