Elucidation of the molecular responses to waterlogging in Jatropha roots by transcriptome profiling

Abstract

Jatropha (Jatropha curcas) is a promising oil-seed crop for biodiesel production. However, the species is highly sensitive to waterlogging, which can result in stunted growth and yield loss. To date, the molecular mechanisms underlying the responses to waterlogging in Jatropha remain elusive. Here, the transcriptome adjustment of Jatropha roots to waterlogging was examined by high-throughput RNA-sequencing (RNA-seq). The results indicated that 24 h of waterlogging caused significant changes in mRNA abundance of 1968 genes. Comprehensive gene ontology and functional enrichment analysis of root transcriptome revealed that waterlogging promoted responses to hypoxia and anaerobic respiration. On the other hand, the stress inhibited carbohydrate synthesis, cell wall biogenesis, and growth. The results also highlighted the roles of ethylene, nitrate, and nitric oxide in waterlogging acclimation. In addition, transcriptome profiling identified 85 waterlogging-induced transcription factors including members of AP2/ERF, MYB, and WRKY families implying that reprogramming of gene expression is a vital mechanism for waterlogging acclimation. Comparative analysis of differentially regulated transcripts in response to waterlogging among Arabidopsis, gray poplar, Jatropha, and rice further revealed not only conserved but species-specific regulation. Our findings unraveled the molecular responses to waterlogging in Jatropha and provided new perspectives for developing a waterlogging tolerant cultivar in the future.

Keywords: ERFs; Jatropha curcas; RNA-seq; genomics; low oxygen; transcriptional control.

Figure 1

Functional classification of waterlogging-responsive DEGs. DEGs (1968 genes) were assigned into 35 functional bins using the Mercator annotation pipeline.

Figure 2

Waterlogging reprograming transcriptome of Jatropha roots. (A) Selected gene ontology (GO) categories (p-values calculated by GOHyperG) from up-regulated (Up) and down-regulated (Down) groups are shown. (B) PAGEMAN enrichment analysis with significant up/down-regulation (Wilcoxon rank sum test; p-value < 0.05).

Figure 3

Waterlogging caused differential expression of transcripts encoding proteins involved in energy, nitrate, and ethylene metabolisms. (A) Starch metabolism (Starch), glycolysis, and fermentation (B) Nitrate metabolism (C) Ethylene production and perception. A plus sign represents induction and a minus sign represents reduction. Numbers in brackets represent numbers of DEGs found in this analysis. Data can be found in Supplementary Table S1.

Figure 4

The group-VII ERFs of Jatropha. (A) Graphical representation of waterlogging-regulated transcription factors based on their assigned protein families. “Up” and “Down” represent up-regulation and down-regulation in this analysis. (B) Phylogenetic tree of group-VII ERF proteins. The full-length protein sequences were analyzed with a Neighbor-joining method. Numbers above branches represent the bootstrapped value from 1000 replicates. (C) Multiple sequence analysis of N-terminal sequences of group-VII ERFs. Bold letters indicate a conserved motif at the N-terminus initiated with Met₁-Cys₂, as identified by multiple sequence alignment. Asterisks and semi-colons indicate identical and conserved substitution, respectively.

Figure 5

Quantitative real-time PCR validation of transcriptome data for selected genes. Relative expression was normalized to the abundance of Ubiquitin (UBC; Jcr4S00238.120). Data represent mean ± SE (n = 3). ^*p-value < 0.01 (t-test).

Figure 6

Overview of transcriptome response for selected functional categories to waterlogging or submergence in Arabidopsis (ATH), Jatropha (JCA), gray poplar (PTR), and rice (OSA). PAGEMAN analysis of the gene expression data (|log₂ fold change| > 1; FDR < 0.05). Statistical analysis of over-represented functional bins was carried out using Fisher method. Z-scores indicate over/under representation (Numbers indicate z-scores; Green, over-represented; Red, under-represented). Data used to generate this figure can be found in Supplementary Table S5.

Figure 7

Response of OMCL clusters with genes involved in nitrogen metabolism and NO production to waterlogging. Yellow, Up-regulated DEGs; Blue, Down-regulated DEGs; White, Orthologs not differentially expressed. Numbers represent mean log₂ fold change values.