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. 2015 Oct 30:6:932.
doi: 10.3389/fpls.2015.00932. eCollection 2015.

De novo transcriptome profiling of cold-stressed siliques during pod filling stages in Indian mustard (Brassica juncea L.)

Somya Sinha  1 , Vivek K Raxwal  2 , Bharat Joshi  1 , Arun Jagannath  1 , Surekha Katiyar-Agarwal  3 , Shailendra Goel  1 , Amar Kumar  1 , Manu Agarwal  1
Affiliations

De novo transcriptome profiling of cold-stressed siliques during pod filling stages in Indian mustard (Brassica juncea L.)

Somya Sinha et al. Front Plant Sci. .

Abstract

Low temperature is a major abiotic stress that impedes plant growth and development. Brassica juncea is an economically important oil seed crop and is sensitive to freezing stress during pod filling subsequently leading to abortion of seeds. To understand the cold stress mediated global perturbations in gene expression, whole transcriptome of B. juncea siliques that were exposed to sub-optimal temperature was sequenced. Manually self-pollinated siliques at different stages of development were subjected to either short (6 h) or long (12 h) durations of chilling stress followed by construction of RNA-seq libraries and deep sequencing using Illumina's NGS platform. De-novo assembly of B. juncea transcriptome resulted in 133,641 transcripts, whose combined length was 117 Mb and N50 value was 1428 bp. We identified 13,342 differentially regulated transcripts by pair-wise comparison of 18 transcriptome libraries. Hierarchical clustering along with Spearman correlation analysis identified that the differentially expressed genes segregated in two major clusters representing early (5-15 DAP) and late stages (20-30 DAP) of silique development. Further analysis led to the discovery of sub-clusters having similar patterns of gene expression. Two of the sub-clusters (one each from the early and late stages) comprised of genes that were inducible by both the durations of cold stress. Comparison of transcripts from these clusters led to identification of 283 transcripts that were commonly induced by cold stress, and were referred to as "core cold-inducible" transcripts. Additionally, we found that 689 and 100 transcripts were specifically up-regulated by cold stress in early and late stages, respectively. We further explored the expression patterns of gene families encoding for transcription factors (TFs), transcription regulators (TRs) and kinases, and found that cold stress induced protein kinases only during early silique development. We validated the digital gene expression profiles of selected transcripts by qPCR and found a high degree of concordance between the two analyses. To our knowledge this is the first report of transcriptome sequencing of cold-stressed B. juncea siliques. The data generated in this study would be a valuable resource for not only understanding the cold stress signaling pathway but also for introducing cold hardiness in B. juncea.

Keywords: Brassica juncea; RNA-seq; cold stress; low temperature; silique; transcriptome.

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Figures

Figure 1
Figure 1
Flowchart representing computational pipeline employed for analysis of transcriptome sequenced from B. juncea siliques.
Figure 2
Figure 2
Size distribution and gene ontology analysis of the assembled transcripts. (A) Assembled transcripts were evaluated for their size distribution and the number of transcripts in each size range is presented. (B) Overlap in the distribution of annotated transcripts based on their databases hits. The LAST search was used to identify transcripts in NCBI nr database whose output was further annotated with Blast2GO for assigning gene ontology terms. PRIAM database and RPS blast were employed for identification of enzymes and conserved domains, respectively. (C) Gene ontology categorization of annotated B. juncea transcripts on the basis of cellular components, molecular functions and biological processes. The numbers of transcripts in the ten most enriched GO terms for each of the categories are shown.
Figure 3
Figure 3
Analysis of differentially expressed transcripts and their inter-relationships in B. juncea siliques. (A) Heat map of differentially expressed transcripts in control and cold stressed B. juncea siliques at different stages of development. Differentially expressed transcripts identified by pair-wise comparisons of all the 18 samples were clustered together on the basis of their expression values and heat map was plotted with TMM-normalized FPKM values. (B) Spearman correlation matrix of all the samples indicating extent of similarity between each possible pair. All the 13,342 transcripts exhibiting differential expression were clustered together and Spearman correlation matrix was plotted on TMM-normalized FPKM values. The key for sample labels should be interpreted as x DAP_y, where x denotes number of days; DAP represents days after pollination and y denotes either unstressed siliques –“C” or duration of cold stress in hours –“h”.
Figure 4
Figure 4
Detailed analysis of differentially expressed genes in early and late stages of siliques exposed to cold stress. Heat maps of differentially expressed transcripts in control and cold stressed B. juncea siliques at early (A) and late (C) stages of development. Differentially expressed transcripts identified by pair-wise comparisons of 9 samples of each stage were clustered together on the basis of their expression values and heat map was plotted with TMM-normalized FPKM values. Clusters of transcripts showing similar expression profile in early (B) and late (D) stages of silique development under control and stress conditions. The average expression of each cluster was plotted as dark blue line whereas gray lines represent expression of individual transcripts. The key for sample labels should be interpreted as x DAP_y, where x denotes number of days; DAP represents days after pollination and y denotes either unstressed siliques –“C” or duration of cold stress in hours –“h”.
Figure 5
Figure 5
Identification and validation of cold-inducible transcripts from early and late stages of silique development in B. juncea. (A) Transcripts of cluster 1 (early stage) and cluster 5 (late stage) were compared and candidates that were either specific or common to both the stages of silique development were identified. (B) Heat map showing relative abundance of selected cold-inducible transcripts belonging to the early, late and core subsets as determined by qPCR. (C) Scatter plot showing correlation between digital and qPCR expression profiles. Pearson correlation coefficient (R) was calculated between log2 fold change values of qPCR and digital expression. The key for sample labels should be interpreted as x DAP_y, where x denotes number of days; DAP represents days after pollination and y denotes either unstressed siliques –“C” or duration of cold stress in hours –“h”.
Figure 6
Figure 6
Expression profiling of B. juncea transcripts homologous to cold-stress signaling pathway components. (A) Heat map showing relative abundance of genes belonging to cold signaling pathway as determined by qPCR. (B) Scatter plot showing correlation between digital and qPCR expression profile. Pearson correlation coefficient (R) was calculated between log2 fold change values of qPCR and digital expression.
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