Harkamal Walia

Comparative Transcriptional Profiling of Two Contrasting Rice Genotypes under Salinity Stress during the Vegetative Growth Stage

Rice (Oryza sativa), a salt-sensitive species, has considerable genetic variation for salt tolerance within the cultivated gene pool. Two indica rice genotypes, FL478, a recombinant inbred line derived from a population developed for salinity tolerance studies, and IR29, the sensitive parent of the population, were selected for this study. We used the Affymetrix rice genome array containing 55,515 probe sets to explore the transcriptome of the salt-tolerant and salt-sensitive genotypes under control and salinity-stressed conditions during vegetative growth. Response of the sensitive genotype IR29 is characterized by induction of a relatively large number of probe sets compared to tolerant FL478. Salinity stress induced a number of genes involved in the flavonoid biosynthesis pathway in IR29 but not in FL478. Cell wall-related genes were responsive in both genotypes, suggesting cell wall restructuring is a general adaptive mechanism during salinity stress, although the two genotypes also had some differences. Additionally, the expression of genes mapping to the Saltol region of chromosome 1 were examined in both genotypes. Single-feature polymorphism analysis of expression data revealed that IR29 was the source of the Saltol region in FL478, contrary to expectation. This study provides a genome-wide transcriptional analysis of two well-characterized, genetically related rice genotypes differing in salinity tolerance during a gradually imposed salinity stress under greenhouse conditions.

Starch biosynthesis in cereal endosperm.

Stored starch generally consists of two d-glucose homopolymers, the linear polymer amylose and a highly branched glucan amylopectin that connects linear chains. Amylopectin structurally contributes to the crystalline organization of the starch granule in cereals. In the endosperm, amylopectin biosynthesis requires the proper execution of a coordinated series of enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), whereas amylose is synthesized by AGPase and granule-bound starch synthase (GBSS). It is highly possible that plastidial starch phosphorylase (Pho1) plays an important role in the formation of primers for starch biosynthesis in the endosperm. Recent advances in our understanding of the functions of individual enzyme isoforms have provided new insights into how linear polymer chains and branch linkages are synthesized in cereals. In particular, genetic analyses of a suite of mutants have formed the basis of a new model outlining the role of various enzyme isoforms in cereal starch production. In our current review, we summarize the recent research findings related to starch biosynthesis in cereal endosperm, with a particular focus on rice.

Cytokinin‐mediated source/sink modifications improve drought tolerance and increase grain yield in rice under water‐stress

Drought is the major environmental factor limiting crop productivity worldwide. We hypothesized that it is possible to enhance drought tolerance by delaying stress-induced senescence through the stress-induced synthesis of cytokinins in crop-plants. We generated transgenic rice (Oryza sativa) plants expressing an isopentenyltransferase (IPT) gene driven by P(SARK) , a stress- and maturation-induced promoter. Plants were tested for drought tolerance at two yield-sensitive developmental stages: pre- and post-anthesis. Under both treatments, the transgenic rice plants exhibited delayed response to stress with significantly higher grain yield (GY) when compared to wild-type plants. Gene expression analysis revealed a significant shift in expression of hormone-associated genes in the transgenic plants. During water-stress (WS), P(SARK)::IPT plants displayed increased expression of brassinosteroid-related genes and repression of jasmonate-related genes. Changes in hormone homeostasis were associated with resource(s) mobilization during stress. The transgenic plants displayed differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These changes and associated hormonal crosstalk resulted in the modification of source/sink relationships and a stronger sink capacity of the P(SARK)::IPT plants during WS. As a result, the transgenic plants had higher GY with improved quality (nutrients and starch content).

The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors.

We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence.

Towards Establishment of a Rice Stress Response Interactome

Rice (Oryza sativa) is a staple food for more than half the world and a model for studies of monocotyledonous species, which include cereal crops and candidate bioenergy grasses. A major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%–60% yield losses globally each year. To elucidate stress response signaling networks, we constructed an interactome of 100 proteins by yeast two-hybrid (Y2H) assays around key regulators of the rice biotic and abiotic stress responses. We validated the interactome using protein–protein interaction (PPI) assays, co-expression of transcripts, and phenotypic analyses. Using this interactome-guided prediction and phenotype validation, we identified ten novel regulators of stress tolerance, including two from protein classes not previously known to function in stress responses. Several lines of evidence support cross-talk between biotic and abiotic stress responses. The combination of focused interactome and systems analyses described here represents significant progress toward elucidating the molecular basis of traits of agronomic importance.

Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage

Rice yield is most sensitive to salinity stress imposed during the panicle initiation (PI) stage. In this study, we have focused on physiological and transcriptional responses of four rice genotypes exposed to salinity stress during PI. The genotypes selected included a pair of indicas (IR63731 and IR29) and a pair of japonica (Agami and M103) rice subspecies with contrasting salt tolerance. Physiological characterization showed that tolerant genotypes maintained a much lower shoot Na+ concentration relative to sensitive genotypes under salinity stress. Global gene expression analysis revealed a strikingly large number of genes which are induced by salinity stress in sensitive genotypes, IR29 and M103 relative to tolerant lines. We found 19 probe sets to be commonly induced in all four genotypes. We found several salinity modulated, ion homeostasis related genes from our analysis. We also studied the expression of SKC1, a cation transporter reported by others as a major source of variation in salt tolerance in rice. The transcript abundance of SKC1 did not change in response to salinity stress at PI stage in the shoot tissue of all four genotypes. However, we found the transcript abundance of SKC1 to be significantly higher in tolerant japonica Agami relative to sensitive japonica M103 under control and stressed conditions during PI stage.

Expression analysis of barley (Hordeum vulgare L.) during salinity stress

Barley (Hordeum vulgare L.) is a salt-tolerant crop species with considerable economic importance in salinity-affected arid and semiarid regions of the world. In this work, barley cultivar Morex was used for transcriptional profiling during salinity stress using a microarray containing ∼22,750 probe sets. The experiment was designed to target the early responses of genes to a salinity stress at seedling stage. We found a comparable number of probe sets up-regulated and down-regulated in response to salinity. The differentially expressed genes were broadly characterized using gene ontology and through expression-based hierarchical clustering to identify interesting features in the data. A prominent feature of the response to salinity was the induction of genes involved in jasmonic acid biosynthesis and genes known to respond to jasmonic acid treatment. A large number of abiotic stress (heat, drought, and low temperature) related genes were also found to be responsive to salinity stress. Our results also indicate osmoprotection to be an early response of barley under salinity stress. Additionally, we compared the results of our studies with two other reports characterizing gene expression of barley under salinity stress and found very few genes in common.

Protein abundances are more conserved than mRNA abundances across diverse taxa

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post‐transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large‐scale protein and mRNA expression data sets from seven different species: two bacteria, yeast, nematode, fly, human, and rice. We find that steady‐state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.

Dosage-dependent deregulation of an AGAMOUS-LIKE gene cluster contributes to interspecific incompatibility.

Postzygotic lethality of interspecies hybrids can result from differences in gene expression, copy number, or coding sequence and can be overcome by altering parental genome dosage. In crosses between Arabidopsis thaliana and A. arenosa, embryo arrest is associated with endosperm hyperproliferation and delayed development similar to paternal-excess interploidy crosses and polycomb-repressive complex (PRC) mutants. Failure is accompanied by parent-specific loss of gene silencing including the dysregulation of three genes suppressed by PRC. Increasing the maternal genome dosage rescues seed development and gene silencing. A gene set upregulated in the failing seed transcriptome encoded putative AGAMOUS-LIKE MADS domain transcription factors (AGL) that were expressed in normal early endosperm and were shown to interact in a previous yeast 2-hybrid analysis. Suppression of these AGLs expression upon cellularization required PRC. Preceding seed failure, expression of the PRC member FIS2 decreased concomitant with overexpression of the AGL cluster. Inactivating two members, AGL62 and AGL90, attenuated the postzygotic barrier between A. thaliana and A. arenosa. We present a model where dosage-sensitive loss of PRC function results in a dysregulated AGL network, which is detrimental for early seed development.

Root-Specific Transcript Profiling of Contrasting Rice Genotypes in Response to Salinity Stress

Elevated salinity imposes osmotic and ion toxicity stresses on living cells and requires a multitude of responses in order to enable plant survival. Building on earlier work profiling transcript levels in rice (Oryza sativa) shoots of FL478, a salt-tolerant indica recombinant inbred line, and IR29, a salt-sensitive cultivar, transcript levels were compared in roots of these two accessions as well as in the roots of two additional salt-tolerant indica genotypes, the landrace Pokkali and the recombinant inbred line IR63731. The aim of this study was to compare transcripts in the sensitive and the tolerant lines in order to identify genes likely to be involved in plant salinity tolerance, rather than in responses to salinity per se. Transcript profiles of several gene families with known links to salinity tolerance are described (e.g. HKTs, NHXs). The putative function of a set of genes identified through their salt responsiveness, transcript levels, and/or chromosomal location (i.e. underneath QTLs for salinity tolerance) is also discussed. Finally, the parental origin of the Saltol region in FL478 is further investigated. Overall, the dataset presented appears to be robust and it seems likely that this system could provide a reliable strategy for the discovery of novel genes involved in salinity tolerance.