Genome-wide Analysis of Alternative Splicing in An Inbred Cabbage (Brassica oleracea L.) Line 'HO' in Response to Heat Stress

Abstract

Introduction: High-throughput RNA sequencing (RNA-Seq) studies demonstrate that Alter-native Splicing (AS) is a widespread mechanism that enhances transcriptome diversity, particularly in plants exposed to environmental stress. In an attempt to determine the transcriptome and AS patterns of cabbage inbred line "HO" under Heat Stress (HS), RNA-Seq was carried out using HS-treated and con-trol samples. Genome-wide analysis indicated that AS is differentially regulated in response to HS. The number of AS events markedly increased in HS-treated samples compared to the control.

Conclusion: We identified 1,864 genes, including Heat shock transcription factor (Hsf) and heat shock protein (Hsp) genes, that exhibited >4-fold changes in expression upon exposure to HS. The enriched Gene Ontology (GO) terms of the 1,864 genes included 'response to stress/abiotic stimulus/chemical stimulus', among, which the genes most highly induced by HS encode small Hsps and Hsf proteins. The heat-induced genes also showed an increased number of AS events under HS conditions. In addi-tion, the distribution of AS types was altered under HS conditions, as the level of Intron Retention (IR) decreased, whereas other types of AS increased, under these conditions. Severe HS-induced AS was al-so observed in Hsfs and Hsps, which play crucial roles in regulating heat tolerance. Our results support the notion that AS of HS-related genes, such as HsfA2 and HsfB2a, are important for heat stress adapta-tion in cabbage.

Keywords: Alternative splicing; Cabbage; Heat shock; Heat shock protein; Heat shock transcription factor; Transcriptome.

Fig. (1)

Experimental design and data analysis. (a) HS treatment of heat tolerant inbred cabbage line ‘HO’. Arrows indicate the time of sample collection for total RNA extraction. (b, c) The work flow and algorithm used to detect AS and heat stress-responsive genes.

Fig. (2)

Schematic representation of alternative splicing. The figure illustrates the different types of alternative splicing: intron retention (IR), alternative 3′ splice site donor (A3SS), alternative 5′ splice site acceptor (A5SS), exon skipping (ES), and exon deletion (ED). Exons, introns, and constitutively/alternatively spliced regions are represented by boxes, black lines, and blue/orange lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. (3)

Distribution and number of predicted alternative splicing events in heat tolerant Brassica oleracea line ‘HO’ under control and heat stress conditions. (a, b) Total number of AS events and number of AS events based on AS type per a gene. (c, d) Pie charts showing the percentage of each splicing type per total number of AS events and the number of AS each type of AS events per gene. IR (intron retention), A5SS (alternative 5′splice site donor), A3SS (alternative 3′splice site acceptor), ES (exon skipping), and ED (exon deletion). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. (4)

GO biological process categories of the 1,864 heat-induced genes (a) and 3,555 heat-repressed genes (b). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. (5)

Validation of RNA-Seq data by q-PCR. (a) Expression levels of Hsps, small Hsps and (b) Hsfs genes. Pooled RNA from control and heat stress-treated samples was analyzed in triplicate for q-PCR. All data points mean ± SE of three independent experiments with different plants.

Fig. (6)

Distributions of predicted alternative splicing events of 1,864 heat-induced genes in heat tolerant Brassica oleracea line ‘HO’ under control and heat stress. (a & b) Number of AS events (left panel) and number of AS events based on AS type per gene (right panel). (c & d) Pie charts showing the percentage of each splicing type among total AS events (left panel) and the number of AS events based on AS type per gene (right panel). IR (intron retention), A5SS (alternative 5′ splice site donor), A3SS (alternative 3′ splice site acceptor), ES (exon skipping), and ED (exon deletion). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

Fig. (7)

Validation of alternative splicing events at the BoHsfA2 and BoHsfB2 loci. (a, d) Gene structures and transcripts of BoHsfA2 and BoHsfB2. Exons, introns, and constitutively (with no stress)/alternatively (with heat stress) spliced regions are represented by boxes, black lines, and blue/orange lines, respectively. (b, e) RT-PCR of BoHsfA2 and BoHsfB2. NS/HS indicates no stress/heat stress. PCR of samples under NS used 10-fold (×10) amounts of template compared with those under HS. (c, f) AS transcript patterns determined using MultiNA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)