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

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.

Keywords: RNA-Sequencing; auxin; ethylene; flower pedicel abscission; leaf petiole abscission; oligonucleotide microarray; tomato (Solanum lycopersicum); transcriptome.

Figure 1

Effect of flower removal (A) or leaf deblading and ethylene treatment (B) on the kinetics of pedicel and petiole abscission, respectively, in tomato explants. Flowers and leaves were excised as indicated in the schematic illustrations. The debladed-leaf explants held in vials with water were prepared as previously described for the flower explants (Meir et al., 2010), and exposed to ethylene (10 μL L⁻¹ for 24 h). The percentage of accumulated pedicel or petiole abscission were monitored at the indicated time intervals following organ removal. The results are means of four replicates (n = 30 explants) ± SE.

Figure 2

Analysis of differentially expressed genes (DEG) in pooled samples of FAZ and LAZ. The number of transcripts indicated above the bars was obtained after performing the Illumina DEG analysis and dividing into groups. Total, transcripts expressed in both tissues regardless of the expression pattern; Group A, transcripts over-expressed in LAZ samples; Group B, transcripts over-expressed in FAZ samples; Group C, transcripts equally expressed in FAZ and LAZ samples; Group A1, transcripts exclusively expressed only in LAZ samples; Group B1, transcripts exclusively expressed only in FAZ samples. The detailed lists of genes in each category are summarized in Table S2.

Figure 3

Enrichment Gene Ontology (GO) terms in the FAZ (A) and LAZ (B). The enrichment analysis included 14,773 genes for the FAZ and 15,741 genes for the LAZ. The chart represents the top 10 GO listed in Tables S5, S6 in the categories of Molecular Function (blue), Biological Process (orange), and Cellular Components (green).

Figure 4

Comparison of frequencies of the GO “biological process” term of over-expressed transcripts in the LAZ (Group A) and FAZ (Group B). The bars represent the comparison of the occurrence frequencies of the GO “biological process” term in the GO annotations of 1135 and 2066 over-expressed transcripts in the FAZ and LAZ, respectively. The frequencies are given for the most abundant biological processes.

Figure 5

Comparison of frequencies of the GO “molecular function” term of over-expressed transcripts in the LAZ (Group A) and FAZ (Group B). The bars represent the comparison of the occurrence frequencies of the GO “molecular function” term in the GO annotations of 1135 and 2066 over-expressed transcripts in the FAZ and LAZ, respectively. The frequencies are given for the most abundant molecular functions.

Figure 6

Comparison of frequencies of the GO “cellular component” term of over-expressed transcripts in the LAZ (Group A) and FAZ (Group B). The bars represent the comparison of the occurrence frequencies of the GO “cellular component” term in the GO annotations of 1135 and 2066 over-expressed transcripts in the FAZ and LAZ, respectively. The frequencies are given for the most abundant cellular components.

Figure 7

Distribution of abscission-regulated transcription factor (TF) families over-expressed in the LAZ or in the FAZ (A), and exclusively expressed only in the LAZ or FAZ (B) during abscission. The changes in the abundance of 551 TF transcripts belonging to 20 families were determined in graph (A) for Group A (LAZ) and Group B (FAZ). The changes in the abundance of 141 TF transcripts belonging to 20 families were determined in graph (B) for Group A1 (LAZ) and Group B1 (FAZ). The Groups were classified according to the categories presented in Figure 2.

Figure 8

Validation by qPCR of differential expression patterns of selected genes in the FAZ and LAZ pooled samples following abscission induction. Expression levels were measured for tomato Aux/IAA24 (SlIAA24) (A), NPR1-like protein (B), Auxin response factor18 (SlARF18) (C), Gretchen Hagen3-15 (GH3-15) (D), SlMYB21 (E), Like Aux4 (SlLAX4) (F), LOB domain protein (G), Peroxidase1 (H), and 1-Aminocyclopropane-1-Carboxylate-Oxidase (LeACO) (I). The relative quantification of the gene expression level in the qPCR assay was determined by the comparative C_T method 2^−ΔΔCT, using ACTIN as a reference gene. The results are means of three biological replicates ± SD. The values presented on top of each bar indicate the expression levels derived from the RNA-Seq data. Transcript identities are indicated in the graphs by their gene ID. The qPCR and RNA-Seq analyses were performed with different samples taken from independent biological replicates of two separate experiments.