Annotation and BAC/PAC localization of nonredundant ESTs from drought-stressed seedlings of anindica rice

To decipher the genes associated with drought stress response and to identify novel genes in rice, we utilized 1540 high-quality expressed sequence tags (ESTs) for functional annotation and mapping to rice genomic sequences. These ESTs were generated earlier by 3'-end single-pass sequencing of 2000 cDNA clones from normalized cDNA libraries constructed from drought-stressed seedlings of anindica rice. A rice UniGene set of 1025 transcripts was constructed from this collection through the BLASTN algorithm. Putative functions of 559 nonredundant ESTs were identified by BLAST similarity search against public databases. Putative functions were assigned at a stringency E value of 10-6 in BLASTN and BLASTX algorithms. To understand the gene structure and function further, we have utilized the publicly available finished and unfinished rice BAC/PAC (BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome) sequences for similarity search using the BLASTN algorithm. Further, 603 nonredundant ESTs have been mapped to BAC/PAC clones. BAC clones were assigned by a homology of above 95% identity along 90% of EST sequence length in the aligned region. In all, 700 ESTs showed rice EST hits in GenBank. Of the 325 novel ESTs, 128 were localized to BAC clones. In addition, 127 ESTs with identified putative functions but with no homology in IRGSP (International Rice Genome Sequencing Program) BAC/PAC sequences were mapped to the Chinese WGS (whole genome shotgun contigs) draft sequence of the rice genome. Functional annotation uncovered about a hundred candidate ESTs associated with abiotic stress in rice andArabidopsis that were previously reported based on microarray analysis and other studies. This study is a major effort in identifying genes associated with drought stress response and will serve as a resource to rice geneticists and molecular biologists

Identification of stress-responsive genes in an indica rice (Oryza sativa L.) using ESTs generated from drought-stressed seedlings

The impacts of drought on plant growth and development limit cereal crop production worldwide. Rice (Oryza sativa) productivity and production is severely affected due to recurrent droughts in almost all agroecological zones. With the advent of molecular and genomic technologies, emphasis is now placed on understanding the mechanisms of genetic control of the drought-stress response. In order to identify genes associated with water-stress response in rice, ESTs generated from a normalized cDNA library, constructed from drought-stressed leaf tissue of an indica cultivar, Nagina 22 were used. Analysis of 7794 cDNA sequences led to the identification of 5815 rice ESTs. Of these, 334 exhibited no significant sequence homology with any rice ESTs or full-length cDNAs in public databases, indicating that these transcripts are enriched during drought stress. Analysis of these 5815 ESTs led to the identification of 1677 unique sequences. To characterize this drought transcriptome further and to identify candidate genes associated with the drought-stress response, the rice data were compared with those for abiotic stress-induced sequences obtained from expression profiling studies in Arabidopsis, barley, maize, and rice. This comparative analysis identified 589 putative stress-responsive genes (SRGs) that are shared by these diverse plant species. Further, the identified leaf SRGs were compared to expression profiles for a drought-stressed rice panicle library to identify common sequences. Significantly, 125 genes were found to be expressed under drought stress in both tissues. The functional classification of these 125 genes showed that a majority of them are associated with cellular metabolism, signal transduction, and transcriptional regulation

Functional genomics of drought stress response in rice: transcript mapping of annotated unigenes of an indica rice (Oryza sativa L. cv. Nagina 22)

Rice being one of the widely cultivated cereals across diverse agroecological systems, is prone to high yield losses due to recurring droughts. In India, drought is a major constraint of rice production and accounts for as much as 15% of yield losses during some years. Conventional plant breeding techniques though cumbersome and time-consuming, have been immensely helpful in releasing drought-tolerant varieties. However, this is not adequate to cope up with the future demand for rice, as drought seems to spread to more regions and seasons across the country. Understanding the genes that govern rice plant architecture and response to drought stress is urgently needed to enhance breeding rice with improved drought tolerance. In order to identify genes associated with drought stress response and their temporal and spatial regulation, we took the genomic approach. By generating a large set of expressed sequence tags (ESTs) from cDNA libraries of drought-stressed seedlings and transcript profiling, we identified 589 genes presumed to be involved in drought stress. These 5814 ESTs are assembled into 2094 contigs and localized onto chromosome arms. We present here the physical map of the 2094 unigene set along with 589 annotated putative stress responsive genes of rice. Further, using ESTs, a few of drought quantitative trait loci (QTLs) have been dissected and putative candidate genes identified. This will be useful to rice researchers as ready reference source for breeding through developing candidate gene markers, molecular dissection of QTLs associated with drought stress and map-based cloning

Whole Genome Sequencing and Comparative Genomic Analysis Reveal Allelic Variations Unique to a Purple Colored Rice Landrace (Oryza sativa ssp. indica cv. Purpleputtu)

Purpleputtu (Oryza sativa ssp. indica cv. Purpleputtu) is a unique rice landrace from southern India that exhibits predominantly purple color. This study reports the underlying genetic complexity of the trait, associated domestication and de-domestication processes during its coevolution with present day cultivars. Along-with genome level allelic variations in the entire gene repertoire associated with the purple, red coloration of grain and other plant parts. Comparative genomic analysis using ‘a panel of 108 rice lines’ revealed a total of 3,200,951 variants including 67,774 unique variations in Purpleputtu (PP) genome. Multiple sequence alignment uncovered a 14 bp deletion in Rc (Red colored, a transcription factor of bHLH class) locus of PP, a key regulatory gene of anthocyanin biosynthetic pathway. Interestingly, this deletion in Rc gene is a characteristic feature of the present-day white pericarped rice cultivars. Phylogenetic analysis of Rc locus revealed a distinct clade showing proximity to the progenitor species Oryza rufipogon and O. nivara. In addition, PP genome exhibits a well conserved 4.5 Mbp region on chromosome 5 that harbors several loci associated with domestication of rice. Further, PP showed 1,387 unique when SNPs compared to 3,023 lines of rice (SNP-Seek database). The results indicate that PP genome is rich in allelic diversity and can serve as an excellent resource for rice breeding for a variety of agronomically important traits such as disease resistance, enhanced nutritional values, stress tolerance, and protection from harmful UV-B rays

Rice DREB1B promoter shows distinct stress-specific responses, and the overexpression of cDNA in tobacco confers improved abiotic and biotic stress tolerance

CBF/DREB (C-repeat binding factor/dehydration responsive element binding factor) family of transcription factors in plants is reported to be associated with regulation of gene expression under stress conditions. Here, we report the functional characterization of a DREB transcription factor, DREB1B gene from rice (Oryza sativa ssp. indica). The OsDREB1B gene was differentially regulated at the transcriptional level by osmotic stress, oxidative stress, salicylic acid, ABA, and cold. A 745 bp promoter region of OsDREB1B cDNA was fused to the β-glucuronidase (GUS) gene and introduced via Agrobacterium tumifaciens into the genome of Arabidopsis. Histochemical analysis of GUS expression in T2 transgenic Arabidopsis plants indicated that OsDREB1B shows stress-specific induction pattern in response to a variety of stresses like mannitol, NaCl, PEG, methyl viologen, cold, ABA, and salicylic acid. Leaf-order-dependent induction pattern of the promoter was observed in response to both cold and ABA stresses. Further, OsDREB1B cDNA was introduced into tobacco plants under the control of CaMV35S promoter to investigate the role of DREB1B product in plant stress response. Transgenic tobacco plants have shown improved seed germination, root growth, membrane stability, and 2, 2-diphenyl-1-pycrilhydrazil hydrate (DPPH) free radical scavenging activity under inhibitory concentrations of mannitol. Importantly, transgenic plants accumulated higher fresh weight under long-term osmotic stress, and also have shown retention of more water than the wild type during drought stress. Overexpression of OsDREB1B in tobacco also improved the oxidative and freezing stress tolerance of transgenic plants. In addition, tobacco plants constitutively expressing OsDREB1B have shown decreased sensitivity to tobacco streak virus infection. Constitutive expression of OsDREB1B in tobacco also induced the expression of PR genes in transgenic plants. The data obtained provide strong in vivo evidence that OsDREB1B is involved in both abiotic and biotic stress responses, and confers broad-spectrum stress tolerance to transgenic plants

Rice flavonoid pathway genes, OsDfr and OsAns, are induced by dehydration, high salt and ABA, and contain stress responsive promoter elements that interact with the transcription activator, OsC1-MYB

Molecular basis of regulation of abiotic stress responses and the flavonoid biosynthesis in rice was investigated. The role of the regulatory gene OsC1-Myb, encoding a MYB class of transcription activators in the stress-induced expression of the structural genes, OsDfr and OsAns, was analyzed. Northern analysis of shoot tissues of rice, Nagina 22, (Oryza sativa L. sub sp. indica) seedlings under dehydration stress or high salt or abscisic acid (ABA) showed a significant enhancement of transcript level and/or transcript stability of OsDfr and OsAns. Enhanced levels of the OsC1-myb transcript were also detected. The expression pattern of these three genes indicates that the stress responsive accumulation of OsDfr and OsAns transcripts is mediated by the transcription factor, OsC1-MYB. The 5' upstream region of the OsDfr and OsAns genes carry several regulatory domains, which share homology with some of the known stress responsive genes in plants. In addition, several putative myb and myc responsive domains were identified in the promoter region of the genes, OsDfr and OsAns. The recombinant OsC1-MYB protein binds in vitro to the myb responsive elements (MREs) in the OsDfr and OsAns promoters, suggesting that it is a potential transcription activator of stress-induced expression of structural genes of the flavonoid pathway

Transposable elements of maize

The maize Enhancer transposable element can be targeted to any locus and many unstable alleles have been isolated. Normally, the result is an autonomously controlled allele leading to the mutability expression that is coordinately inherited with this locus. In this study, six alleles at the c locus (c-m) autonomously controlled by En, gave rise to a modified expression (original pattern of mutability, coarse is modififed to fine) that is under the control of a factor, segregating independently. This factor modifies the coarse pattern to fine by delaying the early occurring mutation events (c→C) during kernel development leading to the appearance of relatively sharp colored spots. This factor shows En-like activity but is limited to the pattern differentiatio of any one of the progenitor c-m alleles. These source c-m alleles are not affected by the standard En. A model is presented to support the hypothesis that this factor is a modified En with an altered mutator (M) effect and thus delays the M effect on the coarse allele. This altered En allele can be considered as En-Malt