CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane Confers Herbicide Tolerance.

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 - 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions.

Error-free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre-recombinase.

Precision genome editing by homology directed repair has tremendous potential for crop improvement. This study describes in planta homologous recombination mediated by CRISPR/Cas9 induced DNA double strand break in proximity to a single short (∼30 nt) homology arm. The efficiency of CRISPR/Cas9-mediated recombination between two loxP sites was compared with Cre (Cyclization recombination enzyme) and codon-optimized Cre-mediated site-specific recombination in sugarcane. A transgenic locus was generated with a selectable nptII coding sequence with terminator between two loxP sites located downstream of a constitutive promoter and acting as transcription block for the downstream promoter-less gusA coding sequence with terminator. Recombination between the two loxP sites resulted in deletion of the transcription block and restored gus activity. This transgenic locus provided an efficient screen for identification of recombination events in sugarcane callus following biolistic delivery of Cre, codon-optimized Cre, or the combination of sgRNA and Cas9 targeting the 5' loxP site. The Cre codon optimized for sugarcane displayed the highest efficiency in mediating the recombination that restored gus activity followed by cre and CRISPR/Cas9. Remarkably the short region of homology of the loxP site cleaved by Cas9 (30 nt)-mediated error-free recombination in all 21 events from three different experiments that were analyzed by Sanger sequencing consistent with homology directed repair. These findings will inform rational design of strategies for precision genome editing in plants.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

KEY MESSAGE: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Transgene Pyramiding of Salt Responsive Protein 3-1 (SaSRP3-1) and SaVHAc1 From Spartina alterniflora L. Enhances Salt Tolerance in Rice.

The transgenic technology using a single gene has been widely used for crop improvement. But the transgenic pyramiding of multiple genes, a promising alternative especially for enhancing complexly inherited abiotic stress tolerance, has received little attention. Here, we developed and evaluated transgenic rice lines with a single Salt Responsive Protein 3-1 (SaSRP3-1) gene as well as pyramids with two-genes SaSRP3-1 and Vacuolar H+-ATPase subunit c1 (SaVHAc1) derived from a halophyte grass Spartina alterniflora L. for salt tolerance at seedling, vegetative, and reproductive stages. The overexpression of this novel gene SaSRP3-1 resulted in significantly better growth of E. coli with the recombinant plasmid under 600 mM NaCl stress condition compared with the control. During early seedling and vegetative stages, the single gene and pyramided transgenic rice plants showed enhanced tolerance to salt stress with minimal wilting and drying symptoms, improved shoot and root growth, and significantly higher chlorophyll content, relative water content, and K+/Na+ ratio than the control plants. The salt stress screening during reproductive stage revealed that the transgenic plants with single gene and pyramids had better grain filling, whereas the pyramided plants showed significantly higher grain yield and higher grain weight compared to control plants. Our study demonstrated transgenic pyramiding as a viable approach to achieve higher level of salt tolerance in crop plants.

Genetic interaction involving photoperiod-responsive Hd1 promotes early flowering under long-day conditions in rice.

Although flowering in rice has been extensively investigated, few studies focused on genetic interactions. Flowering evaluation of two recombinant inbred line (RIL) populations involving photo-insensitive rice cultivars, Bengal and Cypress, and a weedy rice accession, PSRR-1, under natural long-day (LD) conditions, revealed six to ten quantitative trait loci (QTLs) and a major QTL interaction. In addition to the validation of several previously cloned genes using an introgression lines (IL) population of PSRR-1, a few novel QTLs were also discovered. Analysis of the marker profiles of the advanced backcross lines revealed that Hd1 allele of PSRR-1 was responsible for the photoperiodic response in the near-isogenic lines (NILs) developed in both cultivar backgrounds. Based on the phenotypic and genotypic data of the NILs, and NIL mapping population and the transcript abundance of key flowering pathway genes, we conclude that Hd1 and its interaction with a novel gene other than Ghd7 play an important role in controlling flowering under LD conditions. Our study demonstrates the important role of genetic interaction that regulates flowering time in rice and the need for further investigation to exploit it for breeding adaptable rice varieties.

Epigenetic regulation - contribution to herbicide resistance in weeds?

Continuous use of herbicides has resulted in the evolution of resistance to all major herbicide modes of action worldwide. Besides the well-documented cases of newly acquired resistance through genetic changes, epigenetic regulation may also contribute to herbicide resistance in weeds. Epigenetics involves processes that modify the expression of specific genetic elements without changes in the DNA sequence, and play an important role in re-programming gene expression. Epigenetic modifications can be induced spontaneously, genetically or environmentally. Stress-induced epigenetic changes are normally reverted soon after stress exposure, although in specific cases they can also be carried over multiple generations, thereby having a selective benefit. Here, we provide an overview of the basis of epigenetic regulation in plants and discuss the possible effect of epigenetic changes on herbicide resistance. The understanding of these epigenetic changes would add a new perspective to our knowledge of environmental and management stresses and their effects on the evolution of herbicide resistance in weeds. © 2017 Society of Chemical Industry.

Improvement of tissue culture, genetic transformation, and applications of biotechnology to Brassica.

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.

Expression Profiling Coupled with In-silico Mapping Identifies Candidate Genes for Reducing Aflatoxin Accumulation in Maize.

Aflatoxin, produced by Aspergillus flavus, is hazardous to health of humans and livestock. The lack of information about large effect QTL for resistance to aflatoxin accumulation is a major obstacle to employ marker-assisted selection for maize improvement. The understanding of resistance mechanisms of the host plant and the associated genes is necessary for improving resistance to A. flavus infection. A suppression subtraction hybridization (SSH) cDNA library was made using the developing kernels of Mp715 (resistant inbred) and B73 (susceptible inbred) and 480 randomly selected cDNA clones were sequenced to identify differentially expressed genes (DEGs) in response to A. flavus infection and map these clones onto the corn genome by in-silico mapping. A total of 267 unigenes were identified and majority of genes were related to metabolism, stress response, and disease resistance. Based on the reverse northern hybridization experiment, 26 DEGs were selected for semi-quantitative RT-PCR analysis in seven inbreds with variable resistance to aflatoxin accumulation at two time points after A. flavus inoculation. Most of these genes were highly expressed in resistant inbreds. Quantitative RT-PCR analysis validated upregulation of PR-4, DEAD-box RNA helicase, and leucine rich repeat family protein in resistant inbreds. Fifty-six unigenes, which were placed on linkage map through in-silico mapping, overlapped the QTL regions for resistance to aflatoxin accumulation identified in a mapping population derived from the cross between B73 and Mp715. Since majority of these mapped genes were related to disease resistance, stress response, and metabolism, these should be ideal candidates to investigate host pathogen interaction and to reduce aflatoxin accumulation in maize.

Ascribing Functions to Genes: Journey Towards Genetic Improvement of Rice Via Functional Genomics.

Rice, one of the most important cereal crops for mankind, feeds more than half the world population. Rice has been heralded as a model cereal owing to its small genome size, amenability to easy transformation, high synteny to other cereal crops and availability of complete genome sequence. Moreover, sequence wealth in rice is getting more refined and precise due to resequencing efforts. This humungous resource of sequence data has confronted research fraternity with a herculean challenge as well as an excellent opportunity to functionally validate expressed as well as regulatory portions of the genome. This will not only help us in understanding the genetic basis of plant architecture and physiology but would also steer us towards developing improved cultivars. No single technique can achieve such a mammoth task. Functional genomics through its diverse tools viz. loss and gain of function mutants, multifarious omics strategies like transcriptomics, proteomics, metabolomics and phenomics provide us with the necessary handle. A paradigm shift in technological advances in functional genomics strategies has been instrumental in generating considerable amount of information w.r.t functionality of rice genome. We now have several databases and online resources for functionally validated genes but despite that we are far from reaching the desired milestone of functionally characterizing each and every rice gene. There is an urgent need for a common platform, for information already available in rice, and collaborative efforts between researchers in a concerted manner as well as healthy public-private partnership, for genetic improvement of rice crop better able to handle the pressures of climate change and exponentially increasing population.

Ectopic expression of Pokkali phosphoglycerate kinase-2 (OsPGK2-P) improves yield in tobacco plants under salinity stress.

KEY MESSAGE: Our results indicate that OsPGK2a-P gene is differentially regulated in contrasting rice cultivars under stress and its overexpression confers salt stress tolerance in transgenic tobacco. Phosphoglycerate kinase (PGK; EC = 2.7.2.3) plays a major role for ATP production during glycolysis and 1, 3-bisphosphoglycerate production to participate in the Calvin cycle for carbon fixation in plants. Whole genome analysis of rice reveals the presence of four PGK genes (OsPgks) on different chromosomes. Comparative expression analysis of OsPgks in rice revealed highest level of transcripts for OsPgk2 at most of its developmental stages. Detailed characterization of OsPgk2 transcript and protein showed that it is strongly induced by salinity stress in two contrasting genotypes of rice, i.e., cv IR64 (salt sensitive) and landrace Pokkali (salt tolerant). Ectopic expression of OsPgk2a-P (isolated from Pokkali) in transgenic tobacco improved its salinity stress tolerance by higher chlorophyll retention and enhanced proline accumulation, besides maintaining better ion homeostasis. Ectopically expressing OsPgk2a-P transgenic tobacco plants showed tall phenotype with more number of pods than wild-type plants. Therefore, OsPgk2a-P appears to be a potential candidate for increasing salinity stress tolerance and enhanced yield in crop plants.

Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass.

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.

Mapping of seed shattering loci provides insights into origin of weedy rice and rice domestication.

Seed shattering is an important trait that distinguishes crop cultivars from the wild and weedy species. The genetics of seed shattering was investigated in this study to provide insights into rice domestication and the evolution of weedy rice. Quantitative trait locus (QTL) analysis, conducted in 2 recombinant inbred populations involving 2 rice cultivars and a weedy rice accession of the southern United States, revealed 3-5 QTLs that controlled seed shattering with 38-45% of the total phenotypic variation. Two QTLs on chromosomes 4 and 10 were consistent in both populations. Both cultivar and weedy rice contributed alleles for increased seed shattering. Genetic backgrounds affected both QTL number and the magnitude of QTL effects. The major QTL qSH4 and a minor QTL qSH3 were validated in near-isogenic lines, with the former conferring a significantly higher degree of seed shattering than the latter. Although the major QTL qSH4 overlapped with the sh4, the presence of the nonshattering single nucleotide polymorphism allele in the weedy rice accession suggested involvement of a linked locus or an alternative molecular genetic mechanism. Overlapping of several QTLs with those from earlier studies indicated that weedy rice may have been derived from the wild species Oryza rufipogon. Natural hybridization of rice cultivars with the highly variable O. rufipogon present in different geographic regions might be responsible for the evolution of a wide range of phenotypic and genotypic variabilities seen in weedy rice populations worldwide.

Overexpression of an adenosine diphosphate-ribosylation factor gene from the halophytic grass Spartina alterniflora confers salinity and drought tolerance in transgenic Arabidopsis.

Adenosine diphosphate-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that play an important role in intracellular protein trafficking necessary for undertaking multiple physiological functions in plant growth and developmental processes. However, little is known about the mechanism of ARF functioning at the molecular level, as well as its involvement in abiotic stress tolerance. In this study, we demonstrated the direct involvement of an ARF gene SaARF from a grass halophyte Spartina alterniflora in abiotic stress adaptation for the first time. SaARF, which encodes a protein with predicted molecular mass of 21 kDa, revealed highest identity with ARF of Oryza sativa. The SaARF gene is transcriptionally regulated by salt, drought, cold, and ABA in the leaves and roots of S. alterniflora. Arabidopsis plants overexpressing SaARF showed improved seed germination and survival of seedlings under salinity stress. Similarly, SaARF transgenic Arabidopsis plants were more tolerant to drought stress, compared to wild-type plants, by maintaining chlorophyll synthesis, increasing osmolyte synthesis, and stabilizing membrane integrity. Oxidative damage due to moisture stress in transgenic Arabidopsis was also reduced possibly by activating antioxidant genes, AtSOD1 and AtCAT. Our results suggest that enhanced drought and salinity tolerance conferred by the SaARF gene may be due to its role in mediating multiple abiotic stress tolerance mechanisms.

A stress inducible SUMO conjugating enzyme gene (SaSce9) from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis.

BACKGROUND: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene 'SaSce9' from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. RESULTS: SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. CONCLUSIONS: These results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.

Histidine kinases in plants: cross talk between hormone and stress responses.

Two-component signaling pathways involve sensory histidine kinases (HK), histidine phosphotransfer proteins (HpT) and response regulators (RR). Recent advancements in genome sequencing projects for a number of plant species have established the TCS family to be multigenic one. In plants, HKs operate through the His-Asp phosphorelay and control many physiological and developmental processes throughout the lifecycle of plants. Despite the huge diversity reported for the structural features of the HKs, their functional redundancy has also been reported via mutant approach. Several sensory HKs having a CHASE domain, transmembrane domain(s), transmitter domain and receiver domain have been reported to be involved in cytokinin and ethylene signaling. On the other hand, there are also increasing evidences for some of the sensory HKs to be performing their role as osmosensor, clearly indicating toward a possible cross-talk between hormone and stress responsive cascades. In this review, we bring out the latest knowledge about the structure and functions of histidine kinases in cytokinin and ethylene signaling and their role(s) in development and the regulation of environmental stress responses.

Overexpression of a nascent polypeptide associated complex gene (SaßNAC) of Spartina alterniflora improves tolerance to salinity and drought in transgenic Arabidopsis.

Salinity and drought are the most important environmental constraints limiting crop growth and productivity. Here, we have characterized a gene 'SaßNAC' encoding the ß subunit of nascent polypeptide associated complex from a halophyte Spartina alterniflora and investigated its role toward abiotic stress regulation. Expression of SaßNAC was differentially regulated by abiotic stresses, including salinity, drought, cold, and ABA in leaves and roots of S. alterniflora. Constitutive over-expression of SaßNAC in Arabidopsis exhibited normal growth under non-stress conditions but enhanced tolerance to salt and drought stresses. Transgenic SaßNAC Arabidopsis retained more chlorophyll, proline, and showed improved ionic homeostasis with less damage under stress conditions compared to WT plants. This is a first report to demonstrate the involvement of ßNAC in imparting abiotic stress tolerance which might be due to protection of the newly synthesized polypeptides involved in various stress tolerance mechanisms from abiotic stress induced damage and inhibition of cell death in plant.

Salt stress induced variation in DNA methylation pattern and its influence on gene expression in contrasting rice genotypes.

BACKGROUND: Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant's responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. METHODOLOGY/PRINCIPAL FINDINGS: Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. CONCLUSIONS/SIGNIFICANCE: The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not "directed". The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process.

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging.

BACKGROUND: Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. RESULTS: We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. CONCLUSION: We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.

Evidence for directed evolution of larger size motif in Arabidopsis thaliana genome.

Transcription control of gene expression depends on a variety of interactions mediated by the core promoter region, sequence specific DNA-binding proteins, and their cognate promoter elements. The prominent group of cis acting elements in plants contains an ACGT core. The cis element with this core has been shown to be involved in abscisic acid, salicylic acid, and light response. In this study, genome-wide comparison of the frequency of occurrence of two ACGT elements without any spacers as well as those separated by spacers of different length was carried out. In the first step, the frequency of occurrence of the cis element sequences across the whole genome was determined by using BLAST tool. In another approach the spacer sequence was randomized before making the query. As expected, the sequence ACGTACGT had maximum occurrence in Arabidopsis thaliana genome. As we increased the spacer length, one nucleotide at a time, the probability of its occurrence in genome decreased. This trend continued until an unexpectedly sharp rise in frequency of (ACGT)N25(ACGT). The observation of higher probability of bigger size motif suggests its directed evolution in Arabidopsis thaliana genome.

Histidine kinase and response regulator genes as they relate to salinity tolerance in rice.

We have previously shown that Oryza sativa L. Pokkali maintains higher levels of transcripts under non-saline conditions, which are otherwise induced under salinity in the sensitive genotype-IR64. We wanted to test this hypothesis of differential gene regulation further, within the members of a given stress responsive gene family, which share significant structural and functional similarities. For this purpose, we chose to work on the two-component system (TCS family) which plays an important role in stress perception and signal transduction under hormonal, abiotic stress, light and developmental regulation. We present data to show that all members of TCS family, including sensory histidine kinases, phosphotransfer proteins and response regulators, are having differential transcript abundance (under both non-stress and salinity stress conditions) in contrasting rice genotypes. Further, under non-stress conditions, transcript abundance for all TCS members (except RR21) was found to be higher in the salt-tolerant genotype-Pokkali. TCS transcripts are otherwise induced by salinity stress to a relatively higher level in the sensitive cultivar IR64. A few of these members were also found to be localised within important salinity-related quantitative trait loci identified earlier. Based on the above findings, we propose that the TCS members may have a significant role in salinity tolerance in rice and can serve as useful 'candidate genes' for raising salinity-tolerant crop plants.