Monika Mahajan

Plant Small RNAs:Biogenesis, Mode of Action and Their Roles in Abiotic Stresses

Small RNAs (sRNAs) are 18-30 nt non-coding regulatory elements found in diverse organisms, which were initially identified as small double-stranded RNAs in Caenorhabditis elegans. With the development of new and improved technologies, sRNAs have also been identified and characterized in plant systems. Among them, micro RNAs (miRNAs) and small interfering RNAs (siRNAs) are found to be very important riboregulators in plants. Various types of sRNAs differ in their mode of biogenesis and in their function of gene regulation. sRNAs are involved in gene regulation at both transcriptional and post-transcriptional levels. They are known to regulate growth and development of plants. Furthermore, sRNAs especially plant miRNAs have been found to be involved in various stress responses, such as oxidative, mineral nutrient deficiency, dehydration, and even mechanical stimulus. Therefore, in the present review, we focus on the current understanding of biogenesis and regulatory mechanisms of plant sRNAs and their responses to various abiotic stresses.

Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set

Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS) is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) through post-transcriptional gene silencing (PTGS) of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25–93% reduction in flavonol (quercetin) content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin) was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA) at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless/less-seeded fruits via PTGS of FLS encoding gene in plants.

AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco

Highlight AtROS1 overexpression increases demethylation levels of promoters and coding regions of genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways to provide salt stress tolerance in transgenic tobacco.

Overexpression of a tea flavanone 3-hydroxylase gene confers tolerance to salt stress and Alternaria solani in transgenic tobacco

Flavan-3-ols are the major flavonoids present in tea (Camellia sinensis) leaves. These are known to have antioxidant and free radical scavenging properties in vitro. Flavanone 3-hydroxylase is considered to be an important enzyme of flavonoid pathway leading to accumulation of flavan-3-ols in tea. Expression analysis revealed the upregulation in transcript levels of C. sinensis flavanone 3-hydroxylase (CsF3H) encoding gene under salt stress. In this study, the biotechnological potential of CsF3H was evaluated by gene overexpression in tobacco (Nicotiana tabacum cv. Xanthi). Overexpression of CsF3H cDNA increased the content of flavan-3-ols in tobacco and conferred tolerance to salt stress and fungus Alternaria solani infection. Transgenic tobaccos were observed for increase in primary root length, number of lateral roots, chlorophyll content, antioxidant enzyme expression and their activities. Also, they showed lesser malondialdehyde content and electrolyte leakage compared to control tobacco plants. Further, transgenic plants produced higher degree of pectin methyl esterification via decreasing pectin methyl esterase (PME) activity in roots and leaves under unstressed and salt stressed conditions. The effect of flavan-3-ols on pectin methyl esterification under salt stressed conditions was further validated through in vitro experiments in which non-transgenic (wild) tobacco seedlings were exposed to salt stress in presence of flavan-3-ols, epicatechin and epigallocatechin. The in vitro exposed seedlings showed similar trend of increase in pectin methyl esterification through decreasing PME activity as observed in CsF3H transgenic lines. Taken together, overexpression of CsF3H provided tolerance to salt stress and fungus A. solani infection to transgenic tobacco through improved antioxidant system and enhanced pectin methyl esterification.

Comparative analysis of DNA methylation polymorphism in drought sensitive (HPKC2) and tolerant (HPK4) genotypes of horse Gram (Macrotyloma uniflorum).

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.

MicroRNAs and Their Role in Plants During Abiotic Stresses

Abiotic stresses have been considered as the growth-limiting factors affecting plants. Nutrient deficiency, drought, salinity, cold, submergence, and hypoxia are some of the severe types of abiotic stresses. Interdisciplinary research has been carried out to find stress-regulating mechanisms. MicroRNAs (miRNAs) are the newly discovered, 18–24 nucleotides long molecule of the genome. They have been considered as the key players against plant stress. They have been identified in plants, animals, humans, and even microbes. miRNAs have been shown to regulate various stress-responsive genes, proteins and transcription factors, thus helping to counteract adverse conditions. Various stress-inducible miRNAs have been identified and well characterized. Most of these miRNAs have been conserved among plants. This conservative nature has become the basis of development of computational methods of miRNA identifications, in additional to the traditional cloning approach. Presence of computational strategy has further simplified the miRNA prediction. Using this approach various stress-responsive miRNAs have been predicted, annotated and functionally validated from cotton, grapes, rice, maize, and soyabean. This chapter reviews the expanding world of miRNAs, methods unveiling miRNAs from various organisms, and specifically stress-induced miRNAs.

Toxic Metals Accumulation, Tolerance and Homeostasis in Brassicaoilseed Species: Overview of Physiological, Biochemical and Molecular Mechanisms

Brassicaoilseed and related cruciferous crop species of economic importance are identified as metal hyper accumulator with high biomass. These species possess genetically inherited traits of metal hyper accumulation and tolerance. They have been reported to store metal in their upper ground part with the character of metal tolerance. These species has been adapted with various mechanisms to counter metal toxicity. This adaption attracted everyone to understand range of mechanism in these plants with relation to accumulation of metal ion and tolerance to nullify metal ion mediated toxicity. Toxic metal influenced the various physiological processes such as growth, photosynthesis, ion and water uptake and nitrate assimilation in plant. At the cellular level, they have been reported to cause damage including blocking functional groups of enzymes, denaturing or inactivating enzymes, disturbance in the function of polynucleotide, transport mechanism for nutrient ions and disrupting cell and organelle membrane integrity. These symptoms might occur due to interaction of biomolecules with excessive amount of toxic metals. In addition, toxic metal excess stimulates the formation of free radical and reactive oxygen species. Brassica species and related cruciferous crop evolved to survive and thrive in metal toxicity and adapt a range of mechanisms that may be involved in the detoxification and tolerance. Plant antioxidant system scavenged free radicals ion induced by toxic metal exposure. Such tolerance has also been related to increased level of antioxidant molecules and detoxifying enzymes in response to toxic metal ions. Plants are synthesizing a variety of metal chelating legends including phytochelatins, metallothioneins and organic acids. These legends ensure metal detoxification by complexation and vacuolar sequestration. A whole range of metal transporter families have been identified in plant that could play a key role in tolerance and metal homeostasis. These plants possess genes for resistance to toxic effects of a wide range of metals. Apart from tolerance to metal toxicity, these plants also have fine balance of metals that are regulated either by preventing or reducing the entry into the cell or through efflux mechanisms. So, Brassica utilized the mechanism of accumulation, translocation and uptake of toxic metal more efficiently for tolerance. Brassica has been proposed as a natural environmentally safe option to clean contaminated sites. These species are well adapted to a range of environmental conditions and suitable for phytoremediation due to adequate accumulation with highly regulated translocation and uptake of toxic metal. These species are likely source of genes for phytoremediation. These plants will be playing a key role in phytoremediation technology and can be used for remediation of polluted areas. The adaptation ability of Brassica to toxic metals can be utilized to understand the mechanism of tolerance to toxic metals and development of toxic metal restricted plants in metalliferous soil.

Gain of function mutation in tobacco MADS box promoter switch on the expression of flowering class B genes converting sepals to petals

One mutant transgenic line displaying homeotic conversion of sepals to petals with other phenotypic aberrations was selected and characterized at molecular level. The increased transcript level of gene encoding anthocyanidin synthase and petal specific class B genes, GLOBOSA and DEFECIENS in sepals of mutant line may be responsible for its homeotic conversion to petaloid organs. While characterizing this mutant line for locus identification, T-DNA was found to be inserted in 3′ untranslated region of promoter of class B MADS box gene, GLOBOSA. Here, CaMV 35S promoter of T-DNA might be deriving the expression of class B genes.