Alternative splicing in tomato pollen in response to heat stress

Abstract

Alternative splicing (AS) is a key control mechanism influencing signal response cascades in different developmental stages and under stress conditions. In this study, we examined heat stress (HS)-induced AS in the heat sensitive pollen tissue of two tomato cultivars. To obtain the entire spectrum of HS-related AS, samples taken directly after HS and after recovery were combined and analysed by RNA-seq. For nearly 9,200 genes per cultivar, we observed at least one AS event under HS. In comparison to control, for one cultivar we observed 76% more genes with intron retention (IR) or exon skipping (ES) under HS. Furthermore, 2,343 genes had at least one transcript with IR or ES accumulated under HS in both cultivars. These genes are involved in biological processes like protein folding, gene expression and heat response. Transcriptome assembly of these genes revealed that most of the alternative spliced transcripts possess truncated coding sequences resulting in partial or total loss of functional domains. Moreover, 141 HS specific and 22 HS repressed transcripts were identified. Further on, we propose AS as layer of stress response regulating constitutively expressed genes under HS by isoform abundance.

Keywords: RNA-seq; alternative splicing; assembly; heat stress; tomato pollen; transcriptome.

Figure 1

AS events in pollen of different tomato cultivars exposed to HS. (A) Schematic representation for detection of AS events using reads witnessing a particular event. Detected events are IRs, ESs, AD sites and AA sites. (B and C) representing the number of genes showing particular AS events (IR, ES, AA and AD) under CO, HS or both conditions (Overlap) for the cultivars Moneymaker (B) and Red Setter (C).

Figure 2

Detection of IR and ES events accumulated or reduced under HS. (A and B) show the distribution of HS-dependent accumulated (log2 ratio > 0) and HS-dependent reduced (log2 ratio < 0) IR (light and dark blue) and ES (light and dark orange) events. The log2 ratio is based on normalized event frequencies between HS and CO samples in Moneymaker (A) and Red Setter (B). Given in C is the number of IR and ES solely detected under HS or CO condition for both Moneymaker and Red Setter.

Figure 3

Genes showing HS-dependent accumulation or reduction of AS in pollen. (A) and (B) display Venn diagrams representing the overlap of IR and ES containing genes showing HS-dependent accumulation (A) or reduction (B). Genes featuring IR (light and dark blue) and/or ES (light and dark orange) for the cultivars Moneymaker and Red Setter are shown. Numbers in white and bold lines indicate genes showing at least one common IR or ES event in both cultivars.

Figure 4

Enriched biological processes due to HS-dependent accumulation of AS. Enriched GO terms (biological process) within the 2,343 genes showing HS-dependent accumulation of AS. Character sizes are dependent on the adjusted P-values (Benjamini and Hochberg FDR correction with a < 0.01 significance level) of the enrichment analysis, whereby bigger character sizes indicate lower p-values. Coloring of the biological process terms is related to their affiliation to functional categories, namely heat response (orange); gene expression (red), biosynthetic processes (green) as well as transport and localization (blue).

Figure 5

CDS length alterations of transcripts due to AS under HS in pollen. Shown are changes in CDS length for HS-repressed (dark grey; left) and HS-specific transcripts (light grey; right) due to AS. Transcripts were classified in five categories with respect to the AS events observed: Only IR events (only IR); only ES events (only ES); IR and ES events (IR + ES); other AS events than IR and ES (others); transcripts perfectly spliced (perfectly). Numbers above the bars indicate the amount of transcripts assigned to the different categories.

Figure 6

New putative regulatory pathways via AS due to HSR in pollen. The model shows two stages of regulating HSR: I. Transcript levels may be altered by reduction (1) or induction (3) of expression.; II. Independent of expression (2). For II. the HSR is regulated by AS creating isoforms leading to reduced/altered functionality. The concept of this regulation for HS (2A; Solyc09g074680; Solyc11g069490) and CO (2B; Solyc09g064920; Solyc06g069370) dependent AS is shown. Gene models for the annotated ITAG2.4 isoform and the HS or CO specific isoforms are provided as well as the domain architecture of the translated protein.