Transcriptomics profiling of Indian mustard (Brassica juncea) under arsenate stress identifies key candidate genes and regulatory pathways

Abstract

Arsenic (As) is a non-essential element, a groundwater pollutant, whose uptake by plants produces toxic effects. The use of As-contaminated groundwater for irrigation can affect the crop productivity. Realizing the importance of the Brassica juncea as a crop plant in terms of oil-yield, there is a need to unravel mechanistic details of response to As stress and identify key functional genes and pathways. In this research, we studied time-dependent (4-96 h) transcriptome changes in roots and shoots of B. juncea under arsenate [As(V)] stress using Agilent platform. Among the whole transcriptome profiled genes, a total of 1,285 genes showed significant change in expression pattern upon As(V) exposure. The differentially expressed genes were categorized to various signaling pathways including hormones (jasmonate, abscisic acid, auxin, and ethylene) and kinases. Significant effects were also noticed on genes related to sulfur, nitrogen, CHO, and lipid metabolisms along with photosynthesis. Biochemical assays were conducted using specific inhibitors of glutathione and jasmonate biosynthesis, and kinases. The inhibitor studies revealed interconnection among sulfur metabolism, jasmonate, and kinase signaling pathways. In addition, various transposons also constituted a part of the altered transcriptome. Lastly, we profiled a set of key functional up- and down-regulated genes using real-time RT-PCR, which could act as an early indicators of the As stress.

Keywords: Brassica juncea; arsenic; microarray; phytohormones; transporters; transposons.

FIGURE 1

Differentially expressed (DE) genes of root (n = 3) and shoot (n = 2) dataset at different time points compared with the control root and shoot samples calculated by difference (A–C,F,G) in arsenic-exposed Brassica juncea seedlings. Scatter plot represents average intensity (x-axis) vs. log2 ratio (y-axis). Red and green colors define the up-regulated and down-regulated genes at each time point. Venn analysis of common and exclusively differential (up-regulated and down-regulated) genes in arsenic-exposed B. Juncea root (n = 3) (D,E) and shoot (n = 2) (H,I) samples.

FIGURE 2

Real time RT-PCR validation of microarray data of B. juncea roots at 4, 24, and 96 h (A,B); and of shoot at 24 and 96 h (C,D). The data of RT-PCR has been overlapped with that microarray to demonstrate expression patterns. The x-axis represents the expression of various genes in control conditions.

FIGURE 3

Metabolic pathway analysis of the DE genes based on KEGG database using SuperViewer online tool.

FIGURE 4

Expression profile of DE genes encoding transporters at various time points in root and shoot of arsenic-exposed B. juncea seedlings. Red and green represent up-regulated and down-regulated genes, respectively.

FIGURE 5

Expression profile of DE genes encoding proteins of redox (A), electron transport (B), and S-assimilation (C) at various time points in root and shoot of arsenic-exposed B. juncea seedlings. Red and green represent up-regulated and down-regulated genes, respectively.

FIGURE 6

Effect of Buthionine Sulfoximine (BSO), Ibuprofen (IBP), and Staurosporine (STS) on the activities of cysteine synthase (A) and γ-glutamylcysteine synthetase (C), and on the levels of cysteine (B) and glutathione (D) in control and arsenic-exposed B. juncea seedlings at 24 and 96 h. All values are means of triplicates ± SD. ANOVA significant at p ≤ 0.01. Different letters indicate significantly different values at a particular duration (DMRT, p ≤ 0.05).

FIGURE 7

Effect of IBP and STS on the log2-fold expression level of MAPK3 and OPR1 genes in arsenic-exposed B. juncea seedlings at 4 and 24 h. All values are means of triplicates ± SD.ANOVA significant at p ≤ 0.01. Different letters indicate significantly different values at a particular duration for a gene (DMRT, p ≤ 0.05).