Xiuqin Zhao

Drought-induced site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa L.)

An indica pyramiding line, DK151, and its recurrent parent, IR64, were evaluated under drought stress and non-stress conditions for three consecutive seasons. DK151 showed significantly improved tolerance to drought. The DNA methylation changes in DK151 and IR64 under drought stress and subsequent recovery were assessed using methylation-sensitive amplified polymorphism analysis. Our results indicate that drought-induced genome-wide DNA methylation changes accounted for ∼12.1% of the total site-specific methylation differences in the rice genome. This drought-induced DNA methylation pattern showed three interesting properties. The most important one was its genotypic specificity reflected by large differences in the detected DNA methylation/demethylation sites between DK151 and IR64, which result from introgressed genomic fragments in DK151. Second, most drought-induced methylation/demethylation sites were of two major types distinguished by their reversibility, including 70% of the sites at which drought-induced epigenetic changes were reversed to their original status after recovery, and 29% of sites at which the drought-induced DNA demethylation/methylation changes remain even after recovery. Third, the drought-induced DNA methylation alteration showed a significant level of developmental and tissue specificity. Together, these properties are expected to have contributed greatly to rice response and adaptation to drought stress. Thus, induced epigenetic changes in rice genome can be considered as a very important regulatory mechanism for rice plants to adapt to drought and possibly other environmental stresses.

Genome-wide temporal-spatial gene expression profiling of drought responsiveness in rice

BackgroundRice is highly sensitive to drought, and the effect of drought may vary with the different genotypes and development stages. Genome-wide gene expression profiling was used as the initial point to dissect molecular genetic mechanism of this complex trait and provide valuable information for the improvement of drought tolerance in rice. Affymetrix rice genome array containing 48,564 japonica and 1,260 indica sequences was used to analyze the gene expression pattern of rice exposed to drought stress. The transcriptome from leaf, root, and young panicle at three developmental stages was comparatively analyzed combined with bioinformatics exploring drought stress related cis-elements.ResultsThere were 5,284 genes detected to be differentially expressed under drought stress. Most of these genes were tissue- or stage-specific regulated by drought. The tissue-specific down-regulated genes showed distinct function categories as photosynthesis-related genes prevalent in leaf, and the genes involved in cell membrane biogenesis and cell wall modification over-presented in root and young panicle. In a drought environment, several genes, such as GA2ox, SAP15, and Chitinase III, were regulated in a reciprocal way in two tissues at the same development stage. A total of 261 transcription factor genes were detected to be differentially regulated by drought stress. Most of them were also regulated in a tissue- or stage-specific manner. A cis-element containing special CGCG box was identified to over-present in the upstream of 55 common induced genes, and it may be very important for rice plants responding to drought environment.ConclusionsGenome-wide gene expression profiling revealed that most of the drought differentially expressed genes (DEGs) were under temporal and spatial regulation, suggesting a crosstalk between various development cues and environmental stimuli. The identification of the differentially regulated DEGs, including TF genes and unique candidate cis-element for drought responsiveness, is a very useful resource for the functional dissection of the molecular mechanism in rice responding to environment stress.

Comparative Transcriptome Profiling of Chilling Stress Responsiveness in Two Contrasting Rice Genotypes

Rice is sensitive to chilling stress, especially at the seedling stage. To elucidate the molecular genetic mechanisms of chilling tolerance in rice, comprehensive gene expressions of two rice genotypes (chilling-tolerant LTH and chilling-sensitive IR29) with contrasting responses to chilling stress were comparatively analyzed. Results revealed a differential constitutive gene expression prior to stress and distinct global transcription reprogramming between the two rice genotypes under time-series chilling stress and subsequent recovery conditions. A set of genes with higher basal expression were identified in chilling-tolerant LTH compared with chilling-sensitive IR29, indicating their possible role in intrinsic tolerance to chilling stress. Under chilling stress, the major effect on gene expression was up-regulation in the chilling- tolerant genotype and strong repression in chilling-sensitive genotype. Early responses to chilling stress in both genotypes featured commonly up-regulated genes related to transcription regulation and signal transduction, while functional categories for late phase chilling regulated genes were diverse with a wide range of functional adaptations to continuous stress. Following the cessation of chilling treatments, there was quick and efficient reversion of gene expression in the chilling-tolerant genotype, while the chilling-sensitive genotype displayed considerably slower recovering capacity at the transcriptional level. In addition, the detection of differentially-regulated TF genes and enriched cis-elements demonstrated that multiple regulatory pathways, including CBF and MYBS3 regulons, were involved in chilling stress tolerance. A number of the chilling-regulated genes identified in this study were co-localized onto previously fine-mapped cold-tolerance-related QTLs, providing candidates for gene cloning and elucidation of molecular mechanisms responsible for chilling tolerance in rice.

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress

DNA methylation, one of the most important epigenetic phenomena, plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress. Consistent with visibly different phenotypes in response to salt stress, epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478 and salt-sensitive IR29. In addition, most tissue-specific DNA methylation loci were conserved, while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes. Strikingly, salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478. This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage, and helps to further elucidate the implications of DNA methylation in crop growth and development.

Identification of rhizome-specific genes by genome-wide differential expression Analysis in Oryza longistaminata

BackgroundRhizomatousness is a key component of perenniality of many grasses that contribute to competitiveness and invasiveness of many noxious grass weeds, but can potentially be used to develop perennial cereal crops for sustainable farmers in hilly areas of tropical Asia. Oryza longistaminata, a perennial wild rice with strong rhizomes, has been used as the model species for genetic and molecular dissection of rhizome development and in breeding efforts to transfer rhizome-related traits into annual rice species. In this study, an effort was taken to get insights into the genes and molecular mechanisms underlying the rhizomatous trait in O. longistaminata by comparative analysis of the genome-wide tissue-specific gene expression patterns of five different tissues of O. longistaminata using the Affymetrix GeneChip Rice Genome Array.ResultsA total of 2,566 tissue-specific genes were identified in five different tissues of O. longistaminata, including 58 and 61 unique genes that were specifically expressed in the rhizome tips (RT) and internodes (RI), respectively. In addition, 162 genes were up-regulated and 261 genes were down-regulated in RT compared to the shoot tips. Six distinct cis-regulatory elements (CGACG, GCCGCC, GAGAC, AACGG, CATGCA, and TAAAG) were found to be significantly more abundant in the promoter regions of genes differentially expressed in RT than in the promoter regions of genes uniformly expressed in all other tissues. Many of the RT and/or RI specifically or differentially expressed genes were located in the QTL regions associated with rhizome expression, rhizome abundance and rhizome growth-related traits in O. longistaminata and thus are good candidate genes for these QTLs.ConclusionThe initiation and development of the rhizomatous trait in O. longistaminata are controlled by very complex gene networks involving several plant hormones and regulatory genes, different members of gene families showing tissue specificity and their regulated pathways. Auxin/IAA appears to act as a negative regulator in rhizome development, while GA acts as the activator in rhizome development. Co-localization of the genes specifically expressed in rhizome tips and rhizome internodes with the QTLs for rhizome traits identified a large set of candidate genes for rhizome initiation and development in rice for further confirmation.

Identification of functional candidate genes for drought tolerance in rice.

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs.

Comparative Metabolite Profiling of Two Rice Genotypes with Contrasting Salt Stress Tolerance at the Seedling Stage

Background Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level. Methods Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated. Results We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na+ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment. Conclusions Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress.

Genome-wide gene expression profiling of introgressed indica rice alleles associated with seedling cold tolerance improvement in a japonica rice background.

BackgroundRice in tropical and sub-tropical areas is often subjected to cold stress at the seedling stage, resulting in poor growth and yield loss. Although japonica rice is generally more cold tolerant (CT) than indica rice, there are several favorable alleles for CT exist in indica that can be used to enhance CT in rice with a japonica background. Genome-wide gene expression profiling is an efficient way to decipher the molecular genetic mechanisms of CT enhancement and to provide valuable information for CT improvement in rice molecular breeding. In this study, the transcriptome of the CT introgression line (IL) K354 and its recurrent parent C418 under cold stress were comparatively analyzed to explore the possible CT enhancement mechanisms of K354.ResultsA total of 3184 differentially expressed genes (DEGs), including 195 transcription factors, were identified in both lines under cold stress. About half of these DEGs were commonly regulated and involved in major cold responsive pathways associated with OsDREB1 and OsMyb4 regulons. K354-specific cold-induced genes were functionally related to stimulus response, cellular cell wall organization, and microtubule-based movement processes that may contribute to increase CT. A set of genes encoding membrane fluidity and defensive proteins were highly enriched only in K354, suggesting that they contribute to the inherent CT of K354. Candidate gene prediction based on introgressed regions in K354 revealed genotype-dependent CT enhancement mechanisms, associated with Sir2, OsFAD7, OsWAK112d, and programmed cell death (PCD) related genes, present in CT IL K354 but absent in its recurrent parent C418. In K354, a number of DEGs were co-localized onto introgressed segments associated with CT QTLs, providing a basis for gene cloning and elucidation of molecular mechanisms responsible for CT in rice.ConclusionsGenome-wide gene expression analysis revealed that genotype-specific cold induced genes and genes with higher basal expression in the CT genotype contribute jointly to CT improvement. The molecular genetic pathways of cold stress tolerance uncovered in this study, as well as the DEGs co-localized with CT-related QTLs, will serve as useful resources for further functional dissection of the molecular mechanisms of cold stress response in rice.

Complex molecular mechanisms underlying seedling salt tolerance in rice revealed by comparative transcriptome and metabolomic profiling

Highlight Comprehensive analyses of phenotypic, metabolic, and transcriptome data from two genotypes with contrasting salt tolerance provided a more complete picture of the molecular mechanisms underlying seedling tolerance in rice.

Comparative analysis of DNA methylation changes in two rice genotypes under salt stress and subsequent recovery.

DNA methylation, which is one of the best understood epigenetic phenomena, plays an important role in plant responses to environmental stimuli. The rice introgression line IL177-103 and its recurrent parent IR64, which show contrasting salt stress tolerance, were used to characterize DNA methylation changes under salt stress and subsequent recovery using methylation-sensitive amplified polymorphism (MSAP) analysis. The introgression line IL177-103 showed significantly improved tolerance to salinity, as represented by higher relative water content, endogenous abscisic acid content, activity of reactive oxygen species scavenging enzymes, and lower Na(+) concentration in shoots, compared with IR64. The MSAP results showed that less than 10.5% of detected DNA methylation sites were genotype specific, in line with their similar genetic background. Salt-induced DNA methylation changes in both genotypes were mostly detected in roots, and the major portion of the salt-induced DNA demethylation/methylation alterations remained even after recovery, implying their inheritance in the present generation. Furthermore, a few sites with stable DNA methylation differences were identified between salt-tolerant IL177-103 and salt-sensitive IR64, thus providing genotype-specific epigenetic markers. Collectively, these results provide valuable data for further dissection of the molecular mechanisms of salt-stress response and tolerance in rice.