microRNAs (miRs) are a class of 21-24 nucleotide long non-coding RNAs responsible for regulating the expression of associated genes mainly by cleavage or translational inhibition of the target ...transcripts. With this characteristic of silencing, miRs act as an important component in regulation of plant responses in various stress conditions. In recent years, with drastic change in environmental and soil conditions different type of stresses have emerged as a major challenge for plants growth and productivity. The identification and profiling of miRs has itself been a challenge for research workers given their small size and large number of many probable sequences in the genome. Application of computational approaches has expedited the process of identification of miRs and their expression profiling in different conditions. The development of High-Throughput Sequencing (HTS) techniques has facilitated to gain access to the global profiles of the miRs for understanding their mode of action in plants. Introduction of various bioinformatics databases and tools have revolutionized the study of miRs and other small RNAs. This review focuses the role of bioinformatics approaches in the identification and study of the regulatory roles of plant miRs in the adaptive response to stresses.
Drought and water stress impose major limitations to crops, including Maize, as they affect the plant biology at multiple levels. Drought activates the cellular signalling machinery to maintain the ...osmotic and ROS homeostasis for controlling plant response and adaptation to stress. Molecular priming of seeds plays a significant role in imparting stress tolerance by helping plants to remember the stress, which improves their response when they encounter stress again.
In this study, we examined the effect of priming maize seeds with H2O2 and proline, individually or in combination, on response to drought stress. We investigated the role of molecular priming on the physiological, biochemical and molecular response of maize seedlings during drought stress.
We observed that seed-priming played a significant role in mediating stress tolerance of seedlings under drought stress as indicated by changes in growth, biochemical properties, pigment and osmolyte accumulation, antioxidant enzyme activities, gas exchange parameters and gene expression. Seed-priming resulted in reduced expression of specific miRNAs to increase target transcripts associated with synthesis of osmolytes and maintenance of ROS homeostasis for reducing potential damage to the cellular components.
Seed-priming induced changes in the growth, biochemical properties, pigment and osmolyte accumulation, antioxidant enzyme activities, gas exchange parameters and gene expression, though the response was dependent on the genotype, as well as concentration and combination of the priming agents.
•Seed-priming caused alterations in physiological, biochemical and molecular parameters of the drought stressed seedlings.•Response of drought stressed seedlings was dependent on the genotype, concentration and combination of the priming agents.•Seedlings grown from primed seeds showed relatively higher accumulation of ROS as well as the antioxidant enzymes activities.•Seed- priming lowered expression of specific miRNAs to increase transcripts related to osmolyte synthesis and ROS maintenance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Iris yellow spot virus (IYSV) causes severe damage and economic losses in onion production. Differential display-PCR was used to study changes in the gene expression of IYSV-infected onion plants. ...Representative up-regulated and down-regulated genes were selected for further study. Based on sequence analysis, the up-regulated genes were identified as retrotransposon protein, disease resistance-like proteins, chitinase, pathogenesis-related protein, cytochrome oxidase, cytochrome c, pentatricopeptide repeat-containing protein and pectin methylesterase. A DNA-binding transcriptional repressor protein gene was greatly down-regulated. . Most of the identified genes are known to play essential roles in plant defence systems, and are newly identified in onion sequences.
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BFBNIB, IZUM, KILJ, NMLJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of ...unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from
and a full capsid protein gene (770 nucleotides) of a
(tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a
, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Plant transformation technology offers ample opportunities for basic scientific and translational research. Several Agrobacterium-mediated plant transformation protocols are available, for ...transforming rice, through callus initiation and regeneration. The regularly used transformation procedures require time and skilled labor and are limited by the regeneration capabilities of the tissue. Here we describe a simple, robust and tissue culture-independent method for transformation of rice seeds using pCAMBIA-amiR820 as model construct. Plants obtained from the transformed seeds were selected on antibiotic media and tested for transgene integration and expression by molecular techniques. The transgenic seedlings thus produced include a mix of stable transformants and chimeras; however the first generation seeds contained stably integrated transgene.
Mature seeds of most of the higher plants harbor dormant embryos and go through the complex process of germination under favorable environmental conditions. The germination process involves dynamic ...physiological, cellular and metabolic events that are controlled by the interplay of several gene products and different phytohormones. The small non-coding RNAs comprise key regulatory modules in the process of seed dormancy and germination. Recent studies have implicated the small RNAs in plant growth in correlation with various plant physiological processes including hormone signaling and stress response. In this review we provide a brief overview of the regulation of seed germination or dormancy while emphasizing on the current understanding of the role of small RNAs in this regard. We have also highlighted specific examples of stress responsive small RNAs in seed germination and discussed their future potential.
The monopartite
Chili leaf curl virus
(ChiLCV) and its β-satellite (ChiLCB) have been found to co-exist in infected plants. The ability of βC1 protein to suppress RNA silencing was investigated using ...an in-house developed in-planta reversal of silencing assay, using
Nicotiana tabacum
lines harboring green fluorescent protein (GFP) silenced by short hairpin GFP (
Sh
GFP). Transient expression of recombinant βC1 complemented and increased the suppressor activity of ChiLCV coat protein (CP), and this was confirmed by molecular analysis. In silico analysis followed by a yeast two-hybrid screen-identified ChiLCV-CP as the interacting partner of the ChiLCB-βC1 protein. Subcellular localization through confocal analysis revealed that when βC1 and ChiLCV-CP were co-present, the fluorescence was localized in the cytoplasm indicating that nuclear localization of both proteins was obstructed. The cytoplasmic compartmentalization of the two viral suppressors of RNA silencing may be responsible for the enhanced suppression of the host gene silencing. This study presents evidence on the interaction of ChiLCV-CP and βC1 proteins and indicates that ChiLCB may support the ChiLCV in overcoming host gene silencing to cause Chili leaf curl disease.
Key points
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CP of ChiLCV and βC1 of ChiLCB contain RNA silencing suppression activity
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The RNA silencing suppression activity of ChiLCB-βC1 complements that of ChiLCV-CP
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There is a direct interaction between ChiLCB-βC1 and ChiLCV-CP
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CEKLJ, DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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