Phenolics and flavonoids from Polygonum posumbu and comparision of flavonoid compounds content in different tissues (leaves, stems and roots).

The growing global need for antioxidative phenolics and flavonoids for maintenance of human health resulted into search of new sustainable unexplored medicinal plants used by the traditional healers for various ailments. Many synthetic based products of phenolics and flavonoids have been used, however the demand of eco-friendly, natural herbal based products are increasing. As a result, the current study aims to explore traditional potential of Polygonum posumbu related to its phenolics and flavonoids. Optimization of extraction parameters were employed which includes: solvent selection (water, ethanol, methanol, acetone and ethyl acetate), ethanol composition (40-100%), solvent to sample ratio (30-70 ml/g), temperature (50-80 °C) and time (1-5 h). Under optimal conditions, total phenolics (TPC), total flavonoids (TFC), the extract yield (EY) and antioxidant activities of leaves extract were 162.79 ± 2.28 mg GAE/g, 56.57 ± 6.22 mg QE/g 27.96 ± 0.91%, and 27.34 ± 0.98 µg/ml respectively. Seven flavonoids were quantified in different tissues with significant (p ≤ 0.05) differences found in flavonoids contents in different parts of the plant. Highest concentration of flavonoids was observed in stems: (-)-epicatechin-53.19 ± 1.13 mg/g, myricetin-15.90 ± 0.13 mg/g, quercetin-50.66 ± 0.08 mg/g, luteolin-43.10 ± 0.47 mg/g, apigenin-16.73 ± 0.43 mg/g. Leaves and roots had the highest amount of genistein (05.06 ± 0.01 mg/g) and kaempferol (11.13 ± 0.06 mg/g) respectively. From the study it had been found that Polygonum posumbu possess a very good amount of phenolics and flavonoids and this study details first ever investigation on this plant in terms of phenolics and flavonoids. Therefore, this study enhanced the importance of this bioresource in functional food or nutraceutical industries.

Investigations on Regulation of MicroRNAs in Rice Reveal [Ca2+]cyt Signal Transduction Regulated MicroRNAs.

MicroRNAs (miRNAs) are critical components of the multidimensional regulatory networks in eukaryotic systems. Given their diverse spectrum of function, it is apparent that the transcription, processing, and activity of the miRNAs themselves, is very dynamically regulated. One of the most important and universally implicated signaling molecules is [Ca2+]cyt. It is known to regulate a plethora of developmental and metabolic processes in both plants and animals; however, its impact on the regulation of miRNA expression is relatively less explored. The current study employed a combination of internal and external calcium channel inhibitors to establishing that [Ca2+]cyt signatures actively regulate miRNA expression in rice. Involvement of [Ca2+]cyt in the regulation of miRNA expression was further confirmed by treatment with calcimycin, the calcium ionophore. Modulation of the cytosolic calcium levels was also found to regulate the drought-responsive expression as well as ABA-mediated response of miRNA genes in rice seedlings. The study further establishes the role of calmodulins and Calmodulin-binding Transcription Activators (CAMTAs) as important components of the signal transduction schema that regulates miRNA expression. Yeast one-hybrid assay established that OsCAMTA4 & 6 are involved in the transcriptional regulation of miR156a and miR167h. Thus, the study was able to establish that [Ca2+]cyt is actively involved in regulating the expression of miRNA genes both under control and stress conditions.

Investigation into the miRNA/5' isomiRNAs function and drought-mediated miRNA processing in rice.

MicroRNAs lie at the core of biological regulatory networks in plants. The recent discovery of isomiRs that are length variants of the annotated mature miRNAs has further unveiled the complexity of miRNome. Delineation of their functional relevance is critical to understand the complete functional spectrum of the miRNome. To apprehend the role of 5' isomiRs in rice, we performed a comprehensive analysis of the annotated miRNA pool using 8 deep-sequencing datasets from flag leaf and spikelet tissues from two cultivars of rice viz. N22 and PB1 grown under control and drought conditions. The products of the 5' start site variability termed as "5' isomiRs" were found to be widespread in all the datasets. It was possible to identify several 5' isomiRs that were highly distinct and abundant and supported by more than 90% of the tags that map in the region. Majority of miRNA/5' isomiR pair share similar tissue and drought-mediated expression dynamics. Analysis of the degradome data identified targets for several of these 5' isomiRs, thereby confirming their biological activity. Since the isomiRs are length variants at the 5' end, the target sites were found to be accordingly shifted as compared to the target site of the annotated miRNA. Further we also observed that drought affects the processing accuracy of several miRNAs across all tissues of both the cultivars leading to differential accumulation of 5' isomiR/miRNA pair.

Identification of miRNA-mediated drought responsive multi-tiered regulatory network in drought tolerant rice, Nagina 22.

Comparative characterization of microRNA-mediated stress regulatory networks in contrasting rice cultivars is critical to decipher plant stress response. Consequently, a multi-level comparative analysis, using sRNA sequencing, degradome analysis, enzymatic and metabolite assays and metal ion analysis, in drought tolerant and sensitive rice cultivars was conducted. The study identified a group of miRNAs "Cultivar-specific drought responsive" (CSDR)-miRNAs (osa-miR159f, osa-miR1871, osa-miR398b, osa-miR408-3p, osa-miR2878-5p, osa-miR528-5p and osa-miR397a) that were up-regulated in the flag-leaves of tolerant cultivar, Nagina 22 (N22) and Vandana, but down-regulated in the sensitive cultivar, Pusa Basmati 1 (PB1) and IR64, during drought. Interestingly, CSDR-miRNAs target several copper-protein coding transcripts like plantacyanins, laccases and Copper/Zinc superoxide dismutases (Cu/Zn SODs) and are themselves found to be similarly induced under simulated copper-starvation in both N22 and PB1. Transcription factor OsSPL9, implicated in Cu-homeostasis also interacted with osa-miR408-3p and osa-miR528-5p promoters. Further, N22 flag leaves showed lower SOD activity, accumulated ROS and had a higher stomata closure. Interestingly, compared to PB1, internal Cu levels significantly decreased in the N22 flag-leaves, during drought. Thus, the study identifies the unique drought mediated dynamism and interplay of Cu and ROS homeostasis, in the flag leaves of drought tolerant rice, wherein CSDR-miRNAs play a pivotal role.

Identification of novel miRNAs from drought tolerant rice variety Nagina 22.

MicroRNAs regulate a spectrum of developmental and biochemical processes in plants and animals. Thus, knowledge of the entire miRNome is essential to understand the complete regulatory schema of any organism. The current study attempts to unravel yet undiscovered miRNA genes in rice. Analysis of small RNA libraries from various tissues of drought-tolerant 'aus' rice variety Nagina 22 (N22) identified 71 novel miRNAs. These were validated based on precursor hairpin structure, small RNA mapping pattern, 'star' sequence, conservation and identification of targets based on degradome data. While some novel miRNAs were conserved in other monocots and dicots, most appear to be lineage-specific. They were segregated into two different classes based on the closeness to the classical miRNA definition. Interestingly, evidence of a miRNA-like cleavage was found even for miRNAs that lie beyond the classical definition. Several novel miRNAs displayed tissue-enriched and/or drought responsive expression. Generation and analysis of the degradome data from N22 along with publicly available degradome identified several high confidence targets implicated in regulation of fundamental processes such as flowering and stress response. Thus, discovery of these novel miRNAs considerably expands the dimension of the miRNA-mediated regulation in rice.

Unique miRNome during anthesis in drought-tolerant indica rice var. Nagina 22.

MAIN CONCLUSION: Drought-tolerant rice variety, Nagina 22 (N22), has a unique spikelet miRNome during anthesis stage drought as well as transition from heading to anthesis. Molecular characterization of genetic diversity of rice is essential to understand the evolution and molecular basis of various agronomically important traits such as drought tolerance. miRNAs play an important role in regulating plant development as well as stress response such as drought. In this study, we characterized the yet unexplored dynamics of the spikelet miRNA population during developmental transition from 'heading' to 'anthesis' as well as anthesis stage drought stress in a drought-tolerant indica rice variety, N22. A significant proportion of miRNA population (~20 %) in N22 spikelets is modulated during transition from heading to anthesis indicating a unique miRNome at anthesis, a developmental stage highly sensitive to stress (drought/heat). Based on the analysis of degradome data, majority of differentially regulated miRNAs appear to regulate transcription factors, some of which are implicated in regulation of development and fertilization. Similarly, drought during anthesis leads to a global change in miRNA expression pattern including those which regulate ROS homeostasis. It was possible to identify several miRNAs that were not reported to be drought responsive in earlier studies. Interestingly, a significant proportion of the drought-regulated miRNAs co-localize within QTLs related to drought tolerance and associated traits. Comparison of the expression profiles between N22 and Pusa Basmati 1 (drought sensitive) identified miRNAs with variety-specific expression patterns during phase transition (miR164, miR396, miR812, and miR1881) as well as drought stress (miR1881) indicating an evolution of a distinct and variety-specific regulatory mechanism. The promoters of these miRNAs contain LREs (light-responsive elements) and are induced by dark treatment. It was also possible to identify 4 novel miRNAs including an intronic miRNA that was conserved in both rice varieties.