Transcriptional profiling of the leaves of near-isogenic rice lines with contrasting drought tolerance at the reproductive stage in response to water deficit.

BACKGROUND: Drought tolerance is a complex quantitative trait that involves the coordination of a vast array of genes belonging to different pathways. To identify genes related to the drought-tolerance pathway in rice, we carried out gene-expression profiling of the leaves of near-isogenic lines (NILs) with similar genetic backgrounds and different set of QTLs but contrasting drought tolerance levels in response to long-term drought-stress treatments. This work will help differentiate mechanisms of tolerance in contrasting NILs and accelerate molecular breeding programs to improve drought tolerance in this crop. RESULTS: The two pairs of rice NILs, developed at the International Rice Research Institute, along with the drought-susceptible parent, IR64, showed distinct gene-expression profiles in leaves under different water-deficit (WD) treatments. Drought tolerance in the highly drought-tolerant NIL (DTN), IR77298-14-1-2-B-10, could be attributed to the up-regulation of genes with calcium ion binding, transferase, hydrolase and transcription factor activities, whereas in the moderate DTN, IR77298-5-6-B-18, genes with transporter, catalytic and structural molecule activities were up-regulated under WD. In IR77298-14-1-2-B-10, the induced genes were characterized by the presence of regulatory motifs in their promoters, including TGGTTAGTACC and ([CT]AAC[GT]G){2}, which are specific to the TFIIIA and Myb transcription factors, respectively. In IR77298-5-6-B-18, promoters containing a GCAC[AG][ACGT][AT]TCCC[AG]A[ACGT]G[CT] motif, common to MADS(AP1), HD-ZIP, AP2 and YABBY, were induced, suggesting that these factors may play key roles in the regulation of drought tolerance in these two DTNs under severe WD. CONCLUSIONS: We report here that the two pairs of NILs with different levels of drought tolerance may elucidate potential mechanisms and pathways through transcriptome data from leaf tissue. The present study serves as a resource for marker discovery and provides detailed insight into the gene-expression profiles of rice leaves, including the main functional categories of drought-responsive genes and the genes that are involved in drought-tolerance mechanisms, to help breeders identify candidate genes (both up- and down-regulated) associated with drought tolerance and suitable targets for manipulating the drought-tolerance trait in rice.

Environmental Response and Genomic Regions Correlated with Rice Root Growth and Yield under Drought in the OryzaSNP Panel across Multiple Study Systems.

The rapid progress in rice genotyping must be matched by advances in phenotyping. A better understanding of genetic variation in rice for drought response, root traits, and practical methods for studying them are needed. In this study, the OryzaSNP set (20 diverse genotypes that have been genotyped for SNP markers) was phenotyped in a range of field and container studies to study the diversity of rice root growth and response to drought. Of the root traits measured across more than 20 root experiments, root dry weight showed the most stable genotypic performance across studies. The environment (E) component had the strongest effect on yield and root traits. We identified genomic regions correlated with root dry weight, percent deep roots, maximum root depth, and grain yield based on a correlation analysis with the phenotypes and aus, indica, or japonica introgression regions using the SNP data. Two genomic regions were identified as hot spots in which root traits and grain yield were co-located; on chromosome 1 (39.7-40.7 Mb) and on chromosome 8 (20.3-21.9 Mb). Across experiments, the soil type/ growth medium showed more correlations with plant growth than the container dimensions. Although the correlations among studies and genetic co-location of root traits from a range of study systems points to their potential utility to represent responses in field studies, the best correlations were observed when the two setups had some similar properties. Due to the co-location of the identified genomic regions (from introgression block analysis) with QTL for a number of previously reported root and drought traits, these regions are good candidates for detailed characterization to contribute to understanding rice improvement for response to drought. This study also highlights the utility of characterizing a small set of 20 genotypes for root growth, drought response, and related genomic regions.

Photosynthetic diffusional constraints affect yield in drought stressed rice cultivars during flowering.

Global production of rice (Oryza sativa) grain is limited by water availability and the low 'leaf-level' photosynthetic capacity of many cultivars. Oryza sativa is extremely susceptible to water-deficits; therefore, predicted increases in the frequency and duration of drought events, combined with future rises in global temperatures and food demand, necessitate the development of more productive and drought tolerant cultivars. We investigated the underlying physiological, isotopic and morphological responses to water-deficit in seven common varieties of O. sativa, subjected to prolonged drought of varying intensities, for phenotyping purposes in open field conditions. Significant variation was observed in leaf-level photosynthesis rates (A) under both water treatments. Yield and A were influenced by the conductance of the mesophyll layer to CO2 (g(m)) and not by stomatal conductance (g(s)). Mesophyll conductance declined during drought to differing extents among the cultivars; those varieties that maintained g(m) during water-deficit sustained A and yield to a greater extent. However, the variety with the highest g(m) and yield under well-watered conditions (IR55419-04) was distinct from the most effective cultivar under drought (Vandana). Mesophyll conductance most effectively characterises the photosynthetic capacity and yield of O. sativa cultivars under both well-watered and water-deficit conditions; however, the desired attributes of high g(m) during optimal growth conditions and the capacity for g(m) to remain constant during water-deficit may be mutually exclusive. Nonetheless, future genetic and physiological studies aimed at enhancing O. sativa yield and drought stress tolerance should investigate the biochemistry and morphology of the interface between the sub-stomatal pore and mesophyll layer.

Transcriptome profiling of leaf elongation zone under drought in contrasting rice cultivars.

Inhibition of leaf elongation and expansion is one of the earliest responses of rice to water deficit. Despite this sensitivity, a great deal of genetic variation exists in the extant of leaf elongation rate (LER) reduction in response to declining soil moisture. We analyzed global gene expression in the leaf elongation zone under drought in two rice cultivars with disparate LER sensitivities to water stress. We found little overlap in gene regulation between the two varieties under moderate drought; however, the transcriptional response to severe drought was more conserved. In response to moderate drought, we found several genes related to secondary cell wall deposition that were down regulated in Moroberekan, an LER tolerant variety, but up-regulated in LER sensitive variety IR64.

Root attributes affecting water uptake of rice (Oryza sativa) under drought.

Lowland rice roots have a unique physiological response to drought because of their adaptation to flooded soil. Rice root attributes that facilitate growth under flooded conditions may affect rice response to drought, but the relative roles of root structural and functional characteristics for water uptake under drought in rice are not known. Morphological, anatomical, biochemical, and molecular attributes of soil-grown rice roots were measured to investigate the genotypic variability and genotype×environment interactions of water uptake under variable soil water regimes. Drought-resistant genotypes had the lowest night-time bleeding rates of sap from the root system in the field. Diurnal fluctuation predominated as the strongest source of variation for bleeding rates in the field and root hydraulic conductivity (Lpr) in the greenhouse, and was related to expression trends of various PIP and TIP aquaporins. Root anatomy was generally more responsive to drought treatments in drought-resistant genotypes. Suberization and compaction of sclerenchyma layer cells decreased under drought, whereas suberization of the endodermis increased, suggesting differential roles of these two cell layers for the retention of oxygen under flooded conditions (sclerenchyma layer) and retention of water under drought (endodermis). The results of this study point to the genetic variability in responsiveness to drought of rice roots in terms of morphology, anatomy, and function.

Comprehensive gene expression analysis of the NAC gene family under normal growth conditions, hormone treatment, and drought stress conditions in rice using near-isogenic lines (NILs) generated from crossing Aday Selection (drought tolerant) and IR64.

The NAC (NAM, ATAF1/2 and CUC2) genes are plant-specific transcriptional factors known to play diverse roles in various plant developmental processes. We describe the rice (Oryza sativa) OsNAC genes expression profiles (GEPs) under normal and water-deficit treatments (WDTs). The GEPs of the OsNAC genes were analyzed in 25 tissues covering the entire life cycle of Minghui 63. High expression levels of 17 genes were demonstrated in certain tissues under normal conditions suggesting that these genes may play important roles in specific organs. We determined that 16 genes were differentially expressed under at least 1 phytohormone (NAA, GA3, KT, SA, ABA, and JA) treatment. To investigate the GEPs in the root, leaf, and panicle of three rice genotypes [e.g., 2 near-isogenic lines (NILs) and IR64], we used two NILs from a common genetic combination backcross developed by Aday Selection and IR64. WDTs were applied using the fraction of transpirable soil water at severe, mild, and control conditions. Transcriptomic analysis using a 44K oligoarray from Agilent was performed on all the tissue samples. We identified common and specific genes in all tissues from the two NILs under both WDTs, and the majority of the OsNAC genes that were activated were in the drought-tolerant IR77298-14-1-2-B-10 line compared with the drought-susceptible IR77298-14-1-2-B-13 or IR64. In IR77298-14-1-2-B-10, seventeen genes were very specific in their expression levels. Approximately 70 % of the genes from subgroups SNAC and NAM/CUC3 were activated in the leaf, but 37 % genes from subgroup SND were inactivated in the root compared with the control under severe stress conditions. These results provide a useful reference for the cloning of candidate genes from the specific subgroup for further functional analysis.

Water uptake dynamics under progressive drought stress in diverse accessions of the OryzaSNP panel of rice (Oryza sativa).

In addition to characterising root architecture, evaluating root water uptake ability is important for understanding drought response. A series of three lysimeter studies were conducted using the OryzaSNP panel, which consists of 20 diverse rice (Oryza sativa L.) genotypes. Large genotypic differences in drought response were observed in this genotype panel in terms of plant growth and water uptake. Total water uptake and daily water uptake rates in the drought-stress treatment were correlated with root length density, especially at depths below 30cm. Patterns of water uptake among genotypes remained consistent throughout the stress treatments: genotypes that initially extracted more water were the same genotypes that extracted more water at the end of the study. These results suggest that response to drought by deep root growth, rather than a conservative soil water pattern, seems to be important for lowland rice. Genotypes in the O. sativa type aus group showed some of the greatest water uptake and root growth values. Since the OryzaSNP panel has been genotyped in detail with SNP markers, we expect that these results will be useful for understanding the genetics of rice root growth and function for water uptake in response to drought.

Water extraction and root traits in Oryza sativa × Oryza glaberrima introgression lines under different soil moisture regimes.

Drought is a major constraint to rainfed rice production in some parts of sub Saharan Africa (SSA) because of irregular and low rainfall. Improving root water uptake during progressive soil drying could contribute to terminal drought stress resistance. Two lysimeter experiments were conducted using 200 introgression lines (ILs) derived from Oryza sativa L.×Oryza glaberrima Steud. crosses (IR64/RAM54 and IR64/RAM90) to characterise water uptake and root traits under progressive soil drying and to assess genetic differences among these lines in relation to drought stress. The lysimeter facility setup and protocols for simultaneous monitoring of water uptake rates and leaf area are described. Significant genetic variability for water uptake and root traits among the ILs was observed under drought stress. Water uptake in the drought stress treatments was correlated with root length density (RLD) and root dry matter (RDM) at lower soil depths. The highest RLD values at depth were observed among the RAM90 ILs and genetic differences were observed between RAM90 and RAM54 populations, implying that accession RAM90 could be used as a source for improving drought avoidance in lowland rice. The ILs with greater water uptake identified in this study will be used in future research for improving drought resistance of rice in SSA.

Comparative transcriptome analysis of AP2/EREBP gene family under normal and hormone treatments, and under two drought stresses in NILs setup by Aday Selection and IR64.

The AP2/EREBP genes play various roles in developmental processes and in stress-related responses in plants. Genome-wide microarrays based on the gene expression profiles of the AP2/EREBP family were analyzed under conditions of normal growth and drought stress. The preferential expression of fifteen genes was observed in specific tissues, suggesting that these genes may play important roles in vegetative and reproductive stages of growth. A large number of redundant genes were differentially expressed following phytohormone treatments (NAA, GA3, KT, SA, JA, and ABA). To investigate the gene expression responses in the root, leaf, and panicle of three rice genotypes, two drought stress conditions were applied using the fraction of transpirable soil water (FTSW) under severe (0.2 FTSW), mild (0.5 FTSW), and control (1.0 FTSW) conditions. Following treatment, transcriptomic analysis using a 44-K oligoarray from Agilent was performed on all the tissue samples. We identified common and specific genes in all tissues from two near-isogenic lines, IR77298-14-1-2-B-10 (drought tolerant) and IR77298-14-1-2-B-13 (drought susceptible), under drought stress conditions. The majority of the genes that were activated in the IR77298-14-1-2-B-10 line were members of the AP2/EREBP gene family. Non-redundant genes (sixteen) were found in the drought-tolerant line, and four genes were selected as candidate novel reference genes because of their higher expression levels in IR77298-14-1-2-B-10. Most of the genes in the AP2, B3, and B5 subgroups were involved in the panicle under severe stress conditions, but genes from the B1 and B2 subgroups were down-regulated in the root. Of the four subfamilies, RAV exhibited the highest number of up-regulated genes (80%) in the panicle under severe stress conditions in the drought-tolerant line compared to Minghui 63 under normal conditions, and the gene structures of the RAV subfamily may be involved in the response to drought stress in the flowering stage. These results provide a useful reference for the cloning of candidate genes from the specific subgroup for further functional analysis.

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

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.