Comparative analysis of root transcriptome profiles of two pairs of drought-tolerant and susceptible rice near-isogenic lines under different drought stress

Abstract

Background: Plant roots are important organs to uptake soil water and nutrients, perceiving and transducing of soil water deficit signals to shoot. The current knowledge of drought stress transcriptomes in rice are mostly relying on comparative studies of diverse genetic background under drought. A more reliable approach is to use near-isogenic lines (NILs) with a common genetic background but contrasting levels of resistance to drought stress under initial exposure to water deficit. Here, we examined two pairs of NILs in IR64 background with contrasting drought tolerance. We obtained gene expression profile in roots of rice NILs under different levels of drought stress help to identify genes and mechanisms involved in drought stress.

Results: Global gene expression analysis showed that about 55% of genes differentially expressed in roots of rice in response to drought stress treatments. The number of differentially expressed genes (DEGs) increased in NILs as the level of water deficits, increased from mild to severe condition, suggesting that more genes were affected by increasing drought stress. Gene onthology (GO) test and biological pathway analysis indicated that activated genes in the drought tolerant NILs IR77298-14-1-2-B-10 and IR77298-5-6-B-18 were mostly involved in secondary metabolism, amino acid metabolism, response to stimulus, defence response, transcription and signal transduction, and down-regulated genes were involved in photosynthesis and cell wall growth. We also observed gibberellic acid (GA) and auxin crosstalk modulating lateral root formation in the tolerant NILs.

Conclusions: Transcriptome analysis on two pairs of NILs with a common genetic background (~97%) showed distinctive differences in gene expression profiles and could be effective to unravel genes involved in drought tolerance. In comparison with the moderately tolerant NIL IR77298-5-6-B-18 and other susceptible NILs, the tolerant NIL IR77298-14-1-2-B-10 showed a greater number of DEGs for cell growth, hormone biosynthesis, cellular transports, amino acid metabolism, signalling, transcription factors and carbohydrate metabolism in response to drought stress treatments. Thus, different mechanisms are achieving tolerance in the two tolerant lines.

Figure 1

Confirmation of expression profiles by qRT-PCR experiment with 9 selected DEGs compared to microarray data in roots of rice NILs under 3 drought stress treatments with three biological replications for each stress condition. (A) Indicates comparison of microarray expression patterns of 9 selected DEGs from different functional categories with qRT-PCR data. In the histograms: × axis shows different rice genotypes used in this experiment i.e IR64 and four NILs including 10 = IR77298-14-1-2-B-10, 13 = IR77298-14-1-2-B-13, 11 = IR77298-5-6-B-11, and 18 = IR77298-5-6-B-18, severity of drought stress: FTSW0.2 indicates 20 percent of fraction of transpirable soil water which is considered as severe drought stress treatment, and FTSW0.5 indicates 50 percent of fraction of transpirable soil water which is considered as mild drought stress treatment., and Y axis representing fold changes in gene expression were transformed to log₂ scale, dotted lines indicate 1.5 fold or 1/1.5 fold, blue bar indicates the median of three qRT-PCR replicates, orange bar indicates the results of the median of three replications of microarray experiments. (B) Correlation analysis of the ratio of differentially expression level from microarray experiment to that from qRT-PCR at different drought stress treatments which are in good agreement with each other. The microarray data log₂-values (X-axis) were plotted against the qRT-PCR log₂-values (Y-axis). FTSW0.2 indicates 20 percent of fraction of transpirable soil water which is considered as severe drought stress treatment; FTSW0.5 indicates 50 percent of fraction of transpirable soil water which is considered as mild drought stress treatment and FTSW0.2+0.5 representing overall drought stress treatments.

Figure 2

Gene ontology enrichment analysis of genes specifically expressed in tolerant NILs of rice in response to water stress treatments in root tissue. This figure shows a colourful model of parametric analysis of gene set enrichment (PAGE) using agriGO web-based tool of tolerant NILs versus their susceptible counterparts in response to different drought stress treatments (0.2 and 0.5 FTSW) applied in this study. In the figure, the information includes: GO term, ontology including three GO categories namely biological process (P), molecular function (F) and cellular component (C), number of annotated genes in each GO term, GO description, a simple colourful model in which red colour system means up regulated and blue means down regulated, and adjusted P value (FDR), respectively. In this figure also genotype descriptions are: IR = IR64, 10 = IR77298-14-1-2-B-10, 13 = IR77298-14-1-2-B-13, 11 = IR77298-5-6-B-11, and 18 = IR77298-5-6-B-18 (A) specifically expressed DEGs in IR77298-14-1-2-B-10 at 0.2 FTSW, (B) specifically expressed DEGs in IR77298-5-6-B-18 at 0.2 FTSW, (C1) specifically activated DEGs in IR77298-14-1-2-B-10 at 0.5 FTSW, (C2) specifically repressed DEGs in IR77298-14-1-2-B-10 at 0.5 FTSW, (D) specifically expressed DEGs in IR77298-5-6-B-18 at 0.5 FTSW, The coloured blocks represent the level of expression of up-/down-regulation of each term at a certain drought stress. The yellow-to-red, cyan-to-blue and grayscale represent the term is activated, repressed and non-significant, respectively.

Figure 3

General view of genes specifically expressed in two tolerant lines compared to their susceptible counterparts under severe drought stress condition. In this figure: (A) indicates transcripts specifically expressed in IR77298-14-1-2-B-10, and (B) indicates transcripts specifically expressed in IR77298-5-6-B-18, from seven functional categories including cell growth, hormone biosynthesis, cellular transports, amino acid metabolism, reactive oxygen species (ROS), signaling and stress-regulated genes and carbohydrate metabolism. (C) and (D) indicate transcripts related to transcription factor families which are specifically expressed in two tolerant lines compared to their susceptible counterparts. Gene identifiers correspond to the each transcript are from MSU version 6.1 of Rice Oligoarray from Rice Genome Annotation Project (RGAP) 6.1 (http://rice.plantbiology.msu.edu/). A fold change ≥ 1.5 is shown in red (up-regulated), a fold change ≤ -1.5 is shown in green (down-regulated), and no change in black (FDR<0.05).