Prospects of improving flooding tolerance in lowland rice varieties by conventional breeding and genetic engineering

Flooding is a recurrent phenomenon in several lowland rice-growing areas in India and elsewhere. Even though rice is a reasonably flooding-tolerant crop, the annual loss incurred by farmers due to floods is large. There are excellent traditional rice types with high level flooding tolerance. Combining high level flooding tolerance to high grain yield through conventional breeding has been successful to a limited extent so far but there are enormous opportunities for the same. There are also hopes that flooding tolerance can be genetically engineered in rice using a transgenic approach. We take a look on the prospects for improvement of rice to flooding stress through conventional breeding and through plant genetic engineering

Production of high temperature tolerant transgenic plants through manipulation of membrane lipids

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Binary cloning vectors for efficient genetic transformation of rice plants

The availability of effective vector systems is a prerequisite for genetic manipulation of plants through recombinant DNA technology. We report here construction of a series of binary vectors that have cauliflower mosaic virus 35S promoter-driven genes encoding either resistance to hygromycin or phosphinothricin for selection of the transformants, and high strength constitutive promoters of either ubiquitin1 or actin1 genes for efficient expression of the transgenes. The efficacy of the constructs is tested in stably transformed Pusa Basmati 1 rice plants through β-glucuronidase reporter gene activity. Availability of vectors with variable promoters and selectable marker genes provides flexibility in stacking two genes. The vectors constructed in this study are suitable for both particle gun and Agrobacterium-based transformation protocols

Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing. RESULTS: Small RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions. CONCLUSIONS: We report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development

Experimentation in biology of plant abiotic stress responses

During the course of growth under natural field conditions, crop plants are exposed to a number of different abiotic stresses (such as water stress, temperature stress, salt stress, flooding stress, chemical stress and oxidative stress). These stresses exert adverse effects on metabolism, growth and yield of the crops. The intensity of the abiotic stresses is on the rise, implying that various possible solutions for mitigating the damage caused by such stresses must be combined for future increase in crop production. At the level of plant genetics, there are indications that it may be possible to improve plants against such stress factors. However, the practical success in this regard depends on how well we understand the biochemistry. physiology and molecular biology of the plant abiotic stress responses. The cellular response of plants to abiotic stresses is of complex nature involving simultaneous interplay of several mechanisms. Although there is a great deal of progress in cataloguing the biochemical reactions that are associated with plant abiotic stress responses, precise understanding of the defense reactions leading to acquisition of stress tolerance remains a challenge. A number of different experimental systems including lower and higher plants as well as microbes have been analyzed for examining the plant abiotic stress responses. The molecular analysis of the stress response has been carried out at the level of stress proteins, stress genes, stress promoters, trans-acting factors that bind to stress promoters and signal transduction components involved in mediation of stress responses. The functional relevance of the stress - associated genes is being tested in different trans-systems including yeast as well as higher plant species. In this article, we discuss selective features of experimentation in biology of plant abiotic stress responses

Plant Hsp100 proteins: structure, function and regulation

Hsp100/Clp family of proteins has been characterized to appreciable details in Escherichia coli, Saccharomyces cerevisiae and other such simpler biological species. In plants, yeast Hsp104-related protein was first identified in Oryza sativa. cDNA and genomic DNA clones encoding Hsp100 have been isolated and characterized from several plant species thus far. Detailed amino acid sequence analysis has revealed that Hsp100 members contain several conserved signatures. The signature sequences of various motifs of plant Hsp100 members are redefined by reducing the redundancy in this study. Based on in silico analysis, we find that a nucleotide sequence homologous to Phaseolus lunatus chloroplastic hsp100 is present in Arabidopsis genome. Yeast Hsp104 is implicated in the disaggregation of heat-inactivated proteins, thereby protecting cells during heat shock. Plant Hsp100 proteins have been shown to be functionally analogous to yeast Hsp104 by complementation studies. Hsp100 is proven to be critical for the acquisition of thermotolerance as shown by transgenic and mutation based analyses. There are indications that plant Hsp100 proteins interact and recruit components of translational machinery to specific mRNAs in order to enhance their translation. Studies on Arabidopsis thaliana, O. sativa and Zea mays reveal that besides heat stress, Hsp100 proteins are also developmentally regulated

Heat-tolerant basmati rice engineered by over-expression of hsp101

Rice is sensitive to high-temperature stress at almost all the stages of its growth and development. Considering the crucial role of heat shock protein 101 (Hsp101) in imparting thermotolerance to cells, we introduced Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T1 lines showed that the transgene segregated in a Mendelian fashion. We compared the survival of T2 transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants. The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress. This thermotolerance advantage appeared to be solely due to over-expression of Hsp101 as neither the expression of low-molecular-weight heat shock proteins (HSPs) nor of other members of Clp family proteins was altered in the transgenic rice. The production of high temperature tolerant transgenic rice cultivars would provide a stability advantage under supra-optimal temperature regime thereby improving its overall performance

Emerging trends in agricultural biotechnology research: use of abiotic stress-induced promoter to drive expression of a stress resistance gene in the transgenic system leads to high level stress tolerance associated with minimal negative effects on growth

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The emerging role of epitranscriptome in shaping stress responses in plants.

RNA modifications and editing changes constitute 'epitranscriptome' and are crucial in regulating the development and stress response in plants. Exploration of the epitranscriptome and associated machinery would facilitate the engineering of stress tolerance in crops. RNA editing and modifications post-transcriptionally decorate almost all classes of cellular RNAs, including tRNAs, rRNAs, snRNAs, lncRNAs and mRNAs, with more than 170 known modifications, among which m6A, Ψ, m5C, 8-OHG and C-to-U editing are the most abundant. Together, these modifications constitute the "epitranscriptome", and contribute to changes in several RNA attributes, thus providing an additional structural and functional diversification to the "cellular messages" and adding another layer of gene regulation in organisms, including plants. Numerous evidences suggest that RNA modifications have a widespread impact on plant development as well as in regulating the response of plants to abiotic and biotic stresses. High-throughput sequencing studies demonstrate that the landscapes of m6A, m5C, Am, Cm, C-to-U, U-to-G, and A-to-I editing are remarkably dynamic during stress conditions in plants. GO analysis of transcripts enriched in Ψ, m6A and m5C modifications have identified bonafide components of stress regulatory pathways. Furthermore, significant alterations in the expression pattern of genes encoding writers, readers, and erasers of certain modifications have been documented when plants are grown in challenging environments. Notably, manipulating the expression levels of a few components of RNA editing machinery markedly influenced the stress tolerance in plants. We provide updated information on the current understanding on the contribution of RNA modifications in shaping the stress responses in plants. Unraveling of the epitranscriptome has opened new avenues for designing crops with enhanced productivity and stress resilience in view of global climate change