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Tobacco's PR-1a gene is induced by pathogen attack or exogenous application of salicylic acid (SA). Nucleosome mapping and chromatin immunoprecipitation assay were used to delineate the histone modifications on the PR-1a promoter. However, the epigenetic modifications of the inducible promoter of the PR-1a gene are not fully understood yet.Southern approach was used to scan the promoter of PR-1a to identify presence of nucleosomes, ChIP assays were performed using anti-histones antibodies of repressive chromatin by di- methylated at H3K9 and H4K20 or active chromatin by acetylated H3K9/14 and H4K16 to find epigenetic malleability of nucleosome over core promoter in uninduced or induced state post SA treatment. Class I and II mammalian histone deacetylase (HDAC) inhibitor TSA treatment was used to enhance the expression of PR-1a by facilitating the histone acetylation post SA treatment. Here, we report correlated consequences of the epigenetic modifications correspond to disassembly of the nucleosome (spans from - 102 to + 55 bp, masks TATA and transcription initiation) and repressor complex from core promoter, eventually initiates the transcription of PR-1a gene post SA treatment. While active chromatin marks di and trimethylation of H3K4, acetylation of H3K9 and H4K16 are increased which are associated to the transcription initiation of PR-1a following SA treatment. However, in uninduced state constitutive expression of a negative regulator (SNI1) of AtPR1, suppresses AtPR1 expression by six-fold in Arabidopsis thaliana. Further, we report 50-to-1000-fold increased expression of AtPR1 in uninduced lsd1 mutant plants, up to threefold increased expression of AtPR1 in uninduced histone acetyl transferases (HATs) mutant plants, SNI1 dependent negative regulation of AtPR1, all together our results suggest that inactive state of PR-1a is indeed maintained by a repressive complex.The study aimed to reveal the mechanism of transcription initiation of tobacco PR-1a gene in presence or absence of SA. This is the first study that reports nucleosome and repressor complex over core promoter region maintains the inactivation of gene in uninduced state, and upon induction disassembling of both initiates the downstream gene activation process.

Tropical forests sequester six times higher carbon than that released by humans annually into the atmosphere. These biodiversity-rich tropical forests have high net primary productivity (NPP), which differs among constituent plant communities. Tropical moist deciduous forests occupy 179,335 km

We report the development of a Weibull based Long-Short-Term-Memory approach (W-LSTM) for the prediction of COVID-19 disease. The W-LSTM model developed in this study, performs better in terms of MSE, R2 and MAPE, as compared to the previously published models, including ARIMA, LSTM and their variations. Using W-LSTM model, we have predicted the beginning and end of the current cycle of COVID-19 in several countries. Performance of the model was validated as satisfactory in 82% of the 50 test countries, while asking for prediction for 10 days beyond the period of training. Accuracy of the above prediction with days beyond training was assessed in comparison with the MAPE that the model gave with cumulative global data. The model was applied to study correlation between the growth of infection and deaths, and a number of effectors that may influence the epidemic. The model identified age groups, trade with China, air traffic, country temperature and CoV-2 virus types as the likely effectors of infection and virulence leading to deaths. The predictors likely to promote or suppress the epidemic were identified. Some of the predictors had significant effect on the shape parameters of Weibull distribution. The model can function on cloud, take inputs in real time and handle large data country wise, at low costs to make predictions dynamically. Such predictions are highly valuable in guiding policy makers, administration and health. Interactive curves generated from the W-LSTM model can be seen at http://collaboration.coraltele.com/covid2/.

Litchi is a sub-tropical fruit crop with genotypes that bear fruits with variable seed size. Small seed size is a desirable trait in litchi, as it improves consumers’ preference and facilitates fruit processing. Seed specific transcriptome analysis was performed in two litchi genotypes with contrasting seed size to identify the genes associated with seed development. The transcriptomic sequence data from seeds at mid-development stages (16–28 days after anthesis) were de-novo assembled into 1,39,608 Trinity transcripts. Out of these, 6325 transcripts expressed differentially between the two contrasting genotypes. Several putative genes for salicylic acid, jasmonic acid and brassinosteriod pathways were down-regulated in seeds of the small-seeded litchi. The putative regulators of seed maturation and seed storage were down-regulated in the small-seeded genotype. Embryogenesis, cell expansion, seed size and stress related Trinity transcripts exhibited differential expression. Further studies on gene characterization will reveal the early regulators of seed size in litchi. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03098-8.

The present work investigates the applicability of a widespread bio-geochemical model (Biome BGC) to simulate monthly net primary productivity (NPP) and leaf area index (LAI) of Indian tropical deciduous forests. We simulated the monthly NPP and LAI of three plant functional types (PFTs) [dry mixed (DM), sal mixed (SM) and teak plantation (TP)] having distinct tree species compositions, canopy structure, different carbon assimilation rates and microclimate within a broad tropical deciduous forest during 2011–2012. The parameterization of 11 major eco-physiological parameters of Biome BGC was performed from in-situ physiological measurements gathered from 9 long-term ecological research plots in above three PFTs and PFT specific indices were developed. Bimodal trends, with highest peak in September during autumn and second peak in January during winter were observed for simulated monthly NPP in all three PFTs. Simulated NPP (gC/m2/year) values were 408.8 and 414.6; 376.8 and 392.9; and 327.5 and 338.2 during 2011 and 2012 in DM, SM and TP PFTs respectively. Observed NPP (gC/m2/year) values ranged between 463.4 and 493.1; 498.0 and 529.5; and 542.1 and 677.9 in 2012 in DM, SM and TP PFTs respectively. Biome BGC simulated NPP were in positive agreement with observed NPP in all PFTs (R2 = 0.92, 0.83 and 0.72 in DM, SM and TP respectively). In all PFTs Biome BGC led to an underestimation of LAI. The current investigation evaluated the operational application of Biome BGC in Indian tropical deciduous forest and opens scope for further improvement for LAI algorithms for better in-situ LAI simulation.

Purpose To relieve intracranial pressure and save patient inflicted with severe head injury, neurosurgeons restore cranial defects. These defects can be caused because of trauma or diseases (Osteomyelitis of bone) which are treated by cranioplasty, using the preserved bone of patient. In case of non-availability of bone, a cranial implant is generated using a biocompatible synthetic material, but this process is less accurate and time-consuming. Hence, this paper aims to present the use of rapid prototyping technology that allows the development of a more accurate patient-specific template and saves the surgery time. Design/methodology/approach A five-year-old girl patient having cranial defect was taken up for cranioplasty. CT (computed tomography) scans of the patient were used to generate 3D design of the implant suitable to conceal the defect on the left frontal portion using CAD/CAM (computer-aided design/ computer-aided manufacturing) software. The design was used for 3D printing to manufacture a base template, which was finally used to fabricate the actual implant using Simplex® P bone cement material to conceal the defect. Findings Surgery using Simplex® P implant was performed successfully on the patient, giving precise natural curvature to left frontal portion of the patient, decreasing surgery time by about 30 per cent. Originality/value The case demonstrates the development of a convenient, time-saving and aesthetically superior digital procedure to treat cranial defect in the absence of preserved bone flap using CT scan as input. 3D modelling and printing were deployed to produce an accurate template which was used to generate an implant using bone cement biocompatible material.

Tobacco’s PR-1a gene is induced by pathogen attack or exogenous application of Salicylic Acid (SA). However, the epigenetic modifications of the most important inducible promoter of the PR-1a gene are not understood clearly.Nucelosome mapping and chromatin immunoprecipitation assay were used to define the histone modification on the PR-1a promoter.Here, we report the epigenetic modifications over core promoter lead to disassembly of nucleosome (spans from −102 to +55 bp,masks TATA and transcription initiation) and repressor complex in induced state. ChIP assays demonstrate repressive chromatin of dimethylation at H3K9 and H4K20 of core promoter maintain uninduced state. While, active chromatin marks di and trimethylation of H3K4, acetylation of H3K9 and H4K16 are increased and lead the induction of PR-1a following SA treatment. TSA enhances expression of PR-1a by facilitating the histone acetylation, however increased expression of negative regulator (SNI1) of AtPR1, suppresses its expression in Arabidopsis thaliana’s mutants.Constitutive expression of AtPR1 in Histone Acetyl Transferases (HATs), LSD1, and SNI1 suggests that its inactive state is indeed maintained by a repressive complex and this strict regulation of pathogenesis related genes is conserved across species.SUMMARYHistone methylation and acetylation regulation of tobacco PR-1a promoter are significant for disassembly of the nucleosome and repressor proteins during induction.

Litchi is a sub-tropical fruit crop with contrasting genotypes bearing fruits with variable seed size. Small seed size is a desirable trait in litchi, as it improves consumer preference and facilitates fruit processing. Seed specific transcriptome analysis was performed in two litchi genotypes with contrasting seed size to identify the genes associated with seed development in litchi fruits. The transcriptomic data from seeds at mid- development stages (16 to 28 days after anthesis) were de-novo assembled into 1,39,608 Trinity transcripts. Out of these, 6,325 transcripts expressed differentially between the two contrasting genotypes. Several putative genes for salicylic acid, jasmonic acid and brassinosteriod pathways were down-regulated in the small-seeded litchi. The putative regulators of seed maturation and seed storage were down-regulated in the small-seeded genotype. Embryogenesis, cell expansion, seed size and stress related Trinity transcripts exhibited differential expression. Further studies can lead to identification and characterization of early regulators of seed size in litchi.

Transcriptome data are widely used for functional analysis of genes. De-novo assembly of transcriptome gives a large number of unigenes. A large proportion of them remain unannotated. Efficient computational methods are required for identifying genes and modeling those for regulatory and functional roles. Principal component analysis (PCA) was used in a novel approach to shortlist genes, independently of annotation in genome expression data, taking seed development inArabidopsis thalianaas a representative case. PCA was applied to published genome expression data from four lines of Arabidopsis, mutated in seed development. The PC separating all the developmental stages between a mutant and its respective wild type was selected for shortlisting genes as functionally more important. The shortlisted genes identified by PCA belong to a number of biological functions. The genes reported to give sensitivity to desiccation were identified in PCA analysis also in desiccation intolerant lines only. With respect to the network of 98 genes targeted by ABI3, a higher number of genes was identified as important in the mutants abi 3-5, fus 3-3 andlec 1-1 in comparison to abi 3-1. Ontological analysis and comparison with earlier studies suggest that PCA of genome expression data is useful for shortlisting functionally important genes.

Aim Orthodontic tooth movement is a pressing issue nowadays. An increased esthetic demand during orthodontic treatment has resulted in several alternative treatments. However, the need to avoid conventional fixed orthodontic prosthesis has led to the usage of computer-aided scanning, imaging, and printing technology along with the emergence of transparent dental aligners. The motive of this study is to present methodology of measurement of the stress applied by transparent dental aligners on human teeth using a strain gauge-based measurement device. Materials and methods Three dimensional (3D) scanner, 3D printer, thermoforming machine, strain gauge, data acquisition device, 3Shape Ortho Analyzer software were used. Results For a full-bridge Wheatstone bridge data acquisition system (DAQ), a standard aligner can strain a constantan-based strain gauge by nearly 2.5 × 10—4. This is based on the strain gauge factor of 2, input voltage 5 V for which a change in voltage of 2.5 mV was detected. Young's modulus for constantan strain gauge is given as 17.5 MPa; hence, this produced a stress of nearly 4.38 × 10—3 MPa. Conclusion This article describes an effective and convenient methodology for orthodontic treatment design for patients with crowding problem using computer-aided design (CAD) and computer-aided manufacturing (CAM) software and, thereafter, printing different stages of maxilla and mandible using fused deposition modeling (FDM) rapid prototyping technique. A transparent aligner is fabricated using thermoforming process, and the applied stresses on manipulated teeth by aligner can be evaluated using a strain gauge-based DAQ. Clinical significance This approach is expected to understand the efficacy of the thermoformed aligners for teeth movements by calculating applied forces and stresses. How to cite this article Bajaj D, Madhav I, Juneja M, Tuli R, Jindal P. Methodology for Stress Measurement by Transparent Dental Aligners using Strain Gauge. World J Dent 2018;9(1):13-18.

Carbon nanotubes (CNTs) hold tremendous potential due to their unique and modifiable properties. Their robust biological applications necessitate minimizing their cytotoxicity and increasing the solubilization. In the present manuscript, we have functionalized multiwalled carbon nanotubes (MWCNTs) using defect functionalization methodology to covalently bind carboxy and amino groups on their walls. This functionalization was reassured through fourier-transform infrared spectroscopy (FTIR), energy dispersive x-ray analysis (EDX), elemental and field emission scanning electron microscopy (FE-SEM) analysis. The observations demonstrated that addition of carboxy as well as amino groups on MWCNTs, besides enabling MWCNTs solubilization also significantly ameliorated the cytotoxicity and the oxidative stress in comparison to pristine MWCNTs. It is envisaged that changes in agglomeration of the functionalized MWCNTs and the acquired surface charge is the reason for the reduction of cytotoxicity. Zebra fish embryo model test system employed for in vivo analysis of the MWCNTs showed no significant toxicity on account of any nanoparticle tested pointing towards intrinsic mechanisms in place for deterring the damage in complex organisms. Overall, the observations besides pointing towards functionalized MWCNTs effectiveness towards weakening the toxicity of pristine MWCNTs also caution for extrapolating in vitro data to in vivo observations. The observations further lend credibility for exploiting the zebra fish as a model system for analyzing the effects of MWCNTs functionalization., Materials science; Chemistry; Environmental science; Biological sciences; Carbon nanotubes (CNTs); Multi walled carbon nanotubes (MWCNTs); Functionalized MWCNTs; MWCNT cytotoxicity

Indian tropical deciduous forest due to higher species richness, irregular stem densities and stem cover, multistoried canopy and microclimate, offers dynamism to the understanding of the spatial patters in aboveground biomass (AGB) and carbon (AGBC) within different plant functional types (PFTs). Field-based AGB and AGBC estimates are vital for carbon cycle study and provide inputs for data-deficit regional and global carbon and climate models. In this paper we generated the primary baseline data on tree composition, species richness (SR), AGB and AGBC gathered from nine 10,000 m 2 long-term ecological research (LTER) plots in three distinct PFTs {dry mixed (DM), sal mixed (SM) and teak plantation (TP)} within an Indian tropical deciduous forest along Himalayan foothills during 2009–2012. The functional relationship of PFT structural attributes i.e. SR, density, basal cover, leaf area index and litter fall on AGB was investigated. PCA was applied to study the relationship between PFT microclimate, structural attributes and diversity vs. AGB. A total of 22, 22 and 6 tree species were found in DM, SM and TP PFT, respectively. Shannon's diversity index was highest (2.46) in DM, whereas Simpson's dominance index was maximum (0.85) in TP. AGB (Mg ha −1 ) ranged from 290.82–455.99 among all PFTs. AGBC (Mg ha −1 ) ranged between 207.52–220.34, 215.58–228.87, and 125.94–141.18 in DM, SM and TP respectively. AGB observed strong positive correlation with tree density and cover. PCA revealed humidity and air temperature as the major microclimatic variables, leading to higher AGB.

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that infect a variety of cultivated (crop) and non-cultivated (weed) plants. The present study identified a novel begomovirus and satellites (alpha- and betasatellite) in Senna occidentalis (syn. Cassia occidentalis) showing leaf curl symptoms. The begomovirus shared a maximum sequence identity of 88.6 % with french bean leaf curl virus (JQ866297), whereas the alphasatellite and the betasatellite shared identities of 98 % and 90 % with ageratum yellow vein India alphasatellite (LK054802) and papaya leaf curl betasatellite (HM143906), respectively. No other begomovirus or satellites were detected in the suspected plants. We propose to name the virus "senna leaf curl virus" (SenLCuV).

Rapid, reliable and meaningful estimates of leaf area index (LAI) are essential to functional characterization of forest ecosystems including biomass and primary productivity studies. Accurate LAI estimates of tropical deciduous forest are required in studies of regional and global change modeling. Tropical deciduous forest due to higher species richness, multiple species association, varied phenophases, irregular stem densities and basal cover, multistoried canopy architecture and different micro-climatic conditions offers dynamism to the understanding of the LAI dynamics of different PFTs in an ecosystem. This investigation reports a new indirect method for measurement of leaf area index (LAI) in a topical moist deciduous forest in Himalayan foothills using LAI-2000 Plant Canopy Analyzer. We measured the LAI in two seasons (summer; leaf senescence stage and post-monsoon; full green stage) in three (dry miscellaneous, sal mixed and teak plantations) plant functional types (PFT) in Katerniaghat Wildlife Sanctuary, India. Ground LAI values ranged between 2.41 and 6.89, 1.17 and 7.71, and 1.92 and 5.19 during post-monsoon season and 1.36–4.49, 0.67–3.1 and 0.37–1.83 during summer season in dry miscellaneous, sal mixed and teak plantation, respectively. We observed strong correlation between LAI and community structural parameters (tree density, basal cover and species richness), with maximum with annual litter fall (R2 > 0.8) and aboveground biomass (AGB) (R2 > 0.75). We provided equations relating LAI with AGB, which can be utilized in future studies for this region and can be reasonably extrapolated to other regions with suitable statistical extrapolations. However, the relations between LAI and other parameters can be further improved with incorporation of data from optimized and seasonal sampling. Our indirect method of LAI estimation using litter fall as a proxy, offers repetitive potential for LAI estimate in other PFTs with relatively time and cost-effective way, thereby generating quicker and reliable data for model run for regional and global change studies.

Thiamine deficiency is common in populations consuming polished rice as a major source of carbohydrates. Thiamine is required to synthesize thiamine pyrophosphate (TPP), an essential cofactor of enzymes of central metabolism. Its biosynthesis pathway has been partially elucidated and the effect of overexpression of a few genes such as thi1 and thiC, on thiamine accumulation in rice has been reported. Based on current knowledge, this review focuses on the potential of gene editing in metabolic engineering of thiamine biosynthesis pathway to improve thiamine in rice grains. Candidate genes, suitable for modification of the structural part to evolve more efficient versions of enzymes in the pathway, are discussed. For example, adjacent cysteine residues may be introduced in the catalytic domain of thi4 to improve the turn over activity of thiamine thiazole synthase 2. Motif specific editing to modify promoter regulatory regions of genes is discussed to modulate gene expression. Editing cis acting regulatory elements in promoter region can shift the expression of transporters and thiamine binding proteins to endosperm. This can enhance dietary availability of thiamine from rice grains. Differential transcriptomics on rice varieties with contrasting grain thiamine and functional genomic studies will identify more strategic targets for editing in future. Developing functionally enhanced foods by biofortification is a sustainable approach to make diets wholesome.

An inexpensive source of industrially useful enzymes is critical for their commercial production. We have produced an industrially valuable recombinant superoxide dismutase (SOD) in tobacco chloroplasts. A gene from Withania somnifera, encoding a highly stable Cu/Zn SOD, was cloned into a chloroplast transformation vector. It expressed the SOD in tobacco chloroplasts following transformation. The transplastomic plants accumulated the recombinant SOD at up to ∼9 % of the total soluble protein in leaves. The purified chloroplast-expressed recombinant SOD had an estimated specific activity of ∼4600 U/mg. Like the native enzyme, purified recombinant enzyme, prepared from tobacco leaves, was highly stable at high temperatures and tolerated a wide pH range, SDS, ethanol and protease treatment. The results establish the potential of chloroplast transformation for commercial production of recombinant SOD in plants.

Amplified fragment length polymorphism (AFLP) was used to assess genetic relatedness among 23 popular commercial cultivars of lychee (Litchi chinensis) and identify the markers associated with small-seeded cultivars. A combination of three primer pairs with high discriminatory ability was selected for resolving the identity of lychee cultivars. The marker system resolved the instances of synonymies and homonymies. The two cultivars, Dehrarose and Dehradun, whose names are often used interchangeably, could be discriminated by AFLP. A cultivar, Calcuttia (H), grown in the Punjab region was found genetically very similar to China (M) of Bihar region in India, suggesting that both could be the same clone. A number of cultivars with contrasting seed size were included in the study. The cultivars Bedana, Seedless Late, and China were positioned in one group, unlike the grouping previously reported based on morphological traits. The AFLP fingerprinting revealed five marker fragments exclusive to the genotypes, which bear fruit with small seed and large pulp.

In cereals, phytic acid (PA) or inositol hexakisphosphate (IP6) is a well-known phosphate storage compound as well as major chelator of important micronutrients (iron, zinc, calcium, etc.). Genes involved in the late phases of PA biosynthesis pathway are known in crops like maize, soybeans and barley but none have been reported from wheat. Our in silico analysis identified six wheat genes that might be involved in the biosynthesis of inositol phosphates. Four of the genes were inositol tetraphosphate kinases (TaITPK1, TaITPK2, TaITPK3, and TaITPK4), and the other two genes encode for inositol triphosphate kinase (TaIPK2) and inositol pentakisphosphate kinase (TaIPK1). Additionally, we identified a homolog of Zmlpa-1, an ABCC subclass multidrug resistance-associated transporter protein (TaMRP3) that is putatively involved in PA transport. Analyses of the mRNA expression levels of these seven genes showed that they are differentially expressed during seed development, and that some are preferentially expressed in aleurone tissue. These results suggest selective roles during PA biosynthesis, and that both lipid-independent and -dependent pathways are active in developing wheat grains. TaIPK1 and TaMRP3 were able to complement the yeast ScΔipk1 and ScΔycf1 mutants, respectively, providing evidence that the wheat genes have the expected biochemical functions. This is the first comprehensive study of the wheat genes involved in the late phase of PA biosynthesis. Knowledge generated from these studies could be utilized to develop strategies for generating low phyate wheat.

Wheat and its related genotypes show distinct distribution patterns for mineral nutrients in maternal and filial tissues in grains. X-ray-based imaging techniques are very informative to identify genotypes with contrasting tissue-specific localization of different elements. This can help in the selection of suitable genotypes for nutritional improvement of food grain crops. Understanding mineral localization in cereal grains is important for their nutritional improvement. Spatial distribution of mineral nutrients (Mg, P, S, K, Ca, Fe, Zn, Mn and Cu) was investigated between and within the maternal and filial tissues in grains of two wheat cultivars (Triticum aestivum Cv. WH291 and WL711), a landrace (T. aestivum L. IITR26) and a related wild species Aegilops kotschyi, using micro-proton-induced X-ray emission (µ-PIXE) and micro-X-ray fluorescence (µ-XRF). Aleurone and scutellum were major storage tissues for macro (P, K, Ca and Mg) as well as micro (Fe, Zn, Cu and Mn) nutrients. Distinct elemental distribution patterns were observed in each of the four genotypes. A. kotschyi, the wild relative of wheat and the landrace, T. aestivum L. IITR26, accumulated more Zn and Fe in scutellum and aleurone than the cultivated wheat varieties, WH291 and WL711. The landrace IITR26, accumulated far more S in grains, Mn in scutellum, aleurone and embryo region, Ca and Cu in aleurone and scutellum, and Mg, K and P in scutellum than the other genotypes. Unlike wheat, lower Mn and higher Fe, Cu and Zn concentrations were noticed in the pigment strand of A. kotschyi. Multivariate statistical analysis, performed on mineral distribution in major grain tissues (aleurone, scutellum, endosperm and embryo region) resolved the four genotypes into distinct clusters.

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less (initiator-containing) promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr (Initiator) elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS (transcription start site), and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas–mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3′ end of the target site abolished the cleavage activity completely. The mismatches at the 5′ end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3′ end. This approach provides a powerful method for genome engineering in plants.

Jatropha curcas L. (JCL) has been identified as a biodiesel plant globally. Efforts are underway to domesticate JCL for high seed yield. The plant has potential to grow on marginal/degraded/substandard lands to avoid competition with food crops, but little is known about its potential to reclaim degraded lands. At this study, several accessions of JCL were planted in 2005 on sodic soil to assess soil amelioration potential of the plant. After six years (2011) of plant growth, seed yield was not economically viable; however, soil properties improved significantly when compared to initial (0-year plantation) soil properties at 0–15 cm soil depth. Random soil samples were collected from 0 to 15 cm soil depth beneath and outside canopies of JCL with high, medium and poor growth in the year 2008 (3-year plantation) and 2011 (6-year plantation). Soil bulk density, pH, electrical conductivity (EC) and exchangeable sodium percentage (ESP) decreased and soil organic carbon (SOC), nitrogen (N), phosphorus (P), microbial biomass (MB-C, MB-N and MB-P) and enzyme activities (dehydrogenase, β-glucosidase and protease) increased significantly with effect of JCL plantation. Significant decrease in soil pH, EC and ESP has been noticed from 8.6 to 7.6, 1.29 to 0.98 dS m−1 and 20.7% to 13.8%, respectively. Similarly, soil fertility parameters like SOC, MB-C, dehydrogenase, β-glucosidase and protease increased significantly from 4.55 to 8.41 g kg−1, 98 to 352 μg g−1, 16.3 to 51.2 μg TPF g−1 h−1, 75.8 to 338.2 μg PNP g−1 h−1 and 43.7 to 163.2 μg Tyrosine g−1 h−1, respectively after 6 years of JCL cultivation on sodic soil. Changes in soil properties were significantly higher beneath the canopy than outside canopy. Soil sodicity parameters (bulk density, pH, EC and ESP) and fertility attributes (SOC, N, P, MB and enzymes) were significantly negatively and positively correlated with the height, biomass and litter fall of JCL, respectively. Furthermore, to test whether changes in soil properties are induced by test crop, changes were compared with Prosopis juliflora plantation of same age, which is generally planted for amelioration of sodic soils. The significant decrease in soil sodicity and increase in soil fertility conclude that JCL is equally good to reclaim the sodic soils.

Iron insufficiency is a worldwide problem in human diets. In cereals like wheat, the bran layer of the grains is an important source of iron. However, the dietary availability of iron in wheat flour is limited due to the loss of the iron-rich bran during milling and processing and the presence of anti-nutrients like phytic acid that keep iron strongly chelated in the grain. The present study investigated the localization of iron and phosphorus in grain tissues of wheat genotypes with contrasting grain iron content using synchrotron-based micro-X-ray fluorescence (micro-XRF) and micro-proton-induced X-ray emission (micro-PIXE). X-ray absorption near-edge spectroscopy (XANES) was employed to determine the proportion of divalent and trivalent forms of Fe in the grains. It revealed the abundance of oxygen, phosphorus, and sulphur in the local chemical environment of Fe in grains, as Fe-O-P-R and Fe-O-S-R coordination. Contrasting differences were noticed in tissue-specific relative localization of Fe, P, and S among the different genotypes, suggesting a possible effect of localization pattern on iron bioavailability. The current study reports the shift in iron distribution from maternal to filial tissues of grains during the evolution of wheat from its wild relatives to the present-day cultivated varieties, and thus suggests the value of detailed physical localization studies in varietal improvement programmes for food crops.

A begomovirus and its associated alpha- and betasatellite were detected in tomato plants affected with leaf curl disease. Based on a nucleotide sequence identity of 99 %, this begomovirus was designated an isolate of cotton leaf curl Burewala virus (CLCuBuV). The alphasatellite exhibited 93 % sequence identity to cotton leaf curl Burewala alphasatellite (CLCuBuA) and is hence referred to here as a variant of CLCuBuA. The detected betasatellite was recombinant in nature and showed 70 % sequence identity to the known betasatellites. Inoculation of healthy tomato with CLCuBuV plus betasatellite, either in the presence or the absence of alphasatellite, led to typical leaf curling, while inoculation with CLCuBuV in the absence of betasatellite resulted in mild symptoms. This confirmed the role of the betasatellite in expression of disease symptoms. We propose to name the newly detected betasatellite tomato leaf curl Hajipur betasatellite (ToLCHJB).

Jatropha curcas has been widely planted without knowing its standard package of practice for optimizing the yield. Therefore, a standardized agro-technology of Jatropha is required. With this purpose, in this study an elite accession of Jatropha was planted at seven sites in India, covering a range of edapho-climatic conditions. Three experimental trials (spacing, pruning and irrigation and fertilizer) were carried out wherein its growth and yield performance were assessed for five years (2007-2012) at all the sites. The growth characters like plant height and number of branches showed significant variations among the sites and the effects of treatments were not uniform across the sites, indicating that site-specific package of practices should be followed instead of adopting the general recommendations. The seed yield was disappointing to recommend it randomly for large scale plantations on degraded lands in India. But results from the multi-location trials have shown good prospects at semi-arid (Bhavnagar) and drylands (Hyderabad); where plants in close spacing (2 x 2 m) produced significantly higher seed yield per unit area upto 1.4 t ha(-1) (Bhavnagar) in comparison to wide spacing. Pruning showed a negative effect on seed yield during initial five years. We found that the following prescription was sufficient to optimize the yield on India's degraded soils: irrigation at a 30 day interval, and for each planting hole 2 kg of Farm Yard Manure (organic manure) and nitrogen, phosphorous and potassium at 10 g, 20 g, and 10 g, respectively. (C) 2012 Elsevier Ltd. All rights reserved.

Litchi chinensis is a subtropical fruit crop, popular for its nutritional value and taste. Fruits with small seed size and thick aril are desirable in litchi. To gain molecular insight into gene expression that leads to the reduction in the size of seed in Litchi chinensis, transcriptomes of two genetically closely related genotypes, with contrasting seed size were compared in developing ovules. The cDNA library constructed from early developmental stages of ovules (0, 6, and 14 days after anthesis) of bold- and small-seeded litchi genotypes yielded 303,778,968 high quality paired-end reads. These were de-novo assembled into 1,19,939 transcripts with an average length of 865 bp. A total of 10,186 transcripts with contrast in expression were identified in developing ovules between the small- and large- seeded genotypes. A majority of these differences were present in ovules before anthesis, thus suggesting the role of maternal factors in seed development. A number of transcripts indicative of metabolic stress, expressed at higher level in the small seeded genotype. Several differentially expressed transcripts identified in such ovules showed homology with Arabidopsis genes associated with different stages of ovule development and embryogenesis.

A begomovirus and its associated betasatellites were amplified and sequenced from tobacco plants affected with leaf curl disease. The begomovirus was identified as a new strain of tomato leaf curl Pakistan virus (ToLCPKV), which is referred to here as ToLCPKV-India. A previously known betasatellite [tomato leaf curl Patna betasatellite (ToLCPaB)] and a new betasatellite were also found in leaf-curl-affected samples. The use of infectious clones of ToLCPKV-IN plus ToLCPaB for agroinoculation led to typical leaf curl, while ToLCPKV-IN together with the new betasatellite resulted in curling and chlorosis of leaves. Based on these disease symptoms, we propose to name the new betasatellite tobacco leaf chlorosis betasatellite (TbLChB).

Sterol glycosyltransferases (SGTs) catalyze the transfer of sugar molecules to diverse sterol molecules, leading to a change in their participation in cellular metabolism. Withania somnifera is a medicinal plant rich in sterols, sterol glycosides and steroidal lactones. Sterols and their modified counterparts are medicinally important and play a role in adaptation of the plant to stress conditions. We have identified 3 members of SGT gene family through RACE (Rapid Amplification of cDNA Ends) in addition to sgtl1 reported earlier. The amino acid sequence deduced from the ORF's showed homology (45-67 %) to the reported plant SGTs. The expression of the genes was differentially modulated in different organs in W. somnifera and in response to external stimuli. Salicylic acid and methyl jasmonate treatments showed up to 10 fold increase in the expression of sgt genes suggesting their role in defense. The level of expression increased in heat and cold stress indicating the role of sterol modifications in abiotic stress. One of the members, was expressed in E. coli and the enzyme assay showed that the crude enzyme glycosylated stigmasterol. W. somnifera expresses a family of sgt genes and there is a functional recruitment of these genes under stress conditions. The genes which are involved in sterol modification are important in view of medicinal value and understanding stress.

Various programs for genetic improvement in oil yield of the biofuel plant Jatropha curcas L. are currently in progress worldwide. In order to develop strategies for genetic improvement, it is important to estimate the degree of diversity at the genetic level among various genotypes of J. curcas. High-throughput sequencing of complexity-reduced nuclear genomic DNA of J. curcas coupled with computational analysis discovered 2,482 informative single nucleotide polymorphisms (SNPs). Genotyping of selective SNPs among 148 global collections of J. curcas lines and further diversity analysis through NTSYS-pc, DARwin and Structure 2.0 software revealed that a narrow level of genetic diversity existed among the indigenous genotypes as compared to the exotic genotypes of J. curcas. The level of marker informativeness along with distance-based and Bayesian clustering revealed grouping of the accession from Togo (Africa) with various Indian accessions at K = 4 and K = 5 values (where K represents the number of populations). The diverse accessions identified in the study will be of further use in genetic improvement of J. curcas through quantitative trait loci and association mapping.

The relationships among diversity, productivity and climate are complex, especially in tropical ecosystems; and are less studied. We studied here the bi- and tri-partite relationships between and among the plant diversity, productivity and climate variables (i.e., temperature, precipitation and moisture) in a tropical ecosystem in India by analyzing three forest types for the year 2010. Comparison of productivity (Carnegie–Ames–Stanford Approach model derived-net primary productivity, NPP) and climate condition with respect to 2001 showed significant increase in NPP and temperature and overall decrease in precipitation and moisture condition in last decade. The NPP for sal forest ranged from 579.4 to 1,142 for the year 2001 and 557.2 to 1231.6 g Cm−2 year−1 for the year 2010. The mean monthly temperature varied from 18 to 21.8°C and 26 to 27°C; and the annual precipitation varied from 15 to 45 and 32 to 76 cm/year for the year 2001 and 2010 respectively. Strong correlation was observed between monthly mean temperature and productivity during 2001, while a decrease was observed during the year 2010. The ecosystem has shown a trend of rapid drying in last decade, triggering more detail studies for understanding of the eco-climatology. In general, sal forest, a natural forest sowed higher diversity and productivity followed by mixed deciduous (MD) forest and teak plantation. Though, areas with higher NPP are well correlated with higher temperature, precipitation and moisture; they seem to be related to species density rather than diversity. However, there is a need to have year-on-year assessments in order to ascertain if this difference represents a real phenomenon rather than an artefact of sampling. The pilot study helps in understanding the complex relationships and is very useful in the fast changing climate.

The data on microclimate were collected between 2010 and 2011 in five forest communities (dry miscellaneous, sal mixed, lowland miscellaneous, teak and savannah) in a tropical moist deciduous forest in Katerniaghat Wildlife Sanctuary, Uttar Pradesh, India to compare how vegetation structure affects microclimate. Diurnal variations in microclimatic variables [photosynthetically active radiation (PAR) at forest understory level, air temperature, soil surface temperature, ambient CO2, air absolute humidity] were measured with LI-COR 840, LI-COR 191, LI-COR 190 SZ, LI-1400-101 and LI-1400-103 (LI-COR; Lincoln, NE, USA) at centre of three 0.5 ha plots in each forest community. The diurnal trend in microclimatic parameters showed wide variations among communities. PAR at forest floor ranged from 0.0024 to 1289.9 (μmol m−2s−1) in post-monsoon season and 0.0012 to 1877.3 (μmol m−2s−1) in mid-winter season. Among the five communities, the highest PAR value was observed in savannah and lowest in sal mixed forest. All the forest communities received maximum PAR at forest floor between 1000 and 1200 h. The ambient air temperature ranged from 19.15 to 26.69°C in post-monsoon season and 11.31 to 23.03°C in mid-winter season. Soil temperature ranged from 13.54 to 36.88°C in post-monsoon season and 6.39 to 29.17°C in mid-winter season. Ambient CO2 ranged from 372.16 to 899.14 μmol mol−1 in post-monsoon season and 396.65 to 699.65 μmol mol−1 in mid-winter season. In savannah ecosystem, diurnal trend of ambient CO2 was totally different from rest four communities. According to Canonical correspondence analysis, PAR and ambient CO2 are most important in establishment of forest community, among microclimatic variables.

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research. Electronic supplementary material The online version of this article (doi:10.1007/s10930-011-9373-6) contains supplementary material, which is available to authorized users.

De-embryonated cotyledon explants of peanut were co-cultivated under different conditions with Agrobacterium tumefaciens harbouring pIG121hm plasmid carrying intron-containing β-glucuronidase as a reporter while hygromycin phosphotransferase and neomycin phosphotransferase as selectable marker genes. Co-cultivation duration and temperature, various antioxidants and their concentrations, bacterial strains and explant characteristics (incised and non-incised) were examined either alone or in combinations for optimization of transient expression of the reporter gene. Up to 81% transformation was recorded when non-incised explants were co-cultivated with strain EHA101 for 5 days at 21°C on shoot induction medium containing 100 mg/L l-cysteine. Addition of the optimized concentration of augmentin (200 mg/L) along with cefotaxime (200 mg/L) to the shoot induction medium not only effectively eliminated bacterial growth, but also facilitated high frequency of shoot induction. The 40 mg/L hygromycin concentration prevented complete shoot regeneration of non-transgenic explants thus considered for the regeneration of transgenics. Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting. Survival rate of the grafted shoots was nearly 100% in glass-house conditions. The optimized protocol took around 3 months to generate healthy plants. Polymerase chain reaction, Southern blot hybridization, histochemical tests, segregation and hygromycin-leaf assays of selected transgenic plants showed integration of the transgene into peanut genome. No chimeras were noticed during the study.

Four microsatellite-enriched genomic libraries for CA(15), GA(15), AAG(8) and ATG(8) repeats and transcriptome sequences of five cDNA libraries of Gossypium herbaceum were explored to develop simple sequence repeat (SSR) markers. A total of 428 unique clones from repeat enriched genomic libraries were mined for 584 genomic SSRs (gSSRs). In addition, 99,780 unigenes from transcriptome sequencing were explored for 8,900 SSR containing sequences with 12,471 expressed SSRs. The present study adds 1,970 expressed SSRs and 263 gSSRs to the public domain for the use of genetic studies of cotton. When 150 gSSRs and 50 expressed SSRs were tested on a panel of four species of cotton, 68 gSSRs and 12 expressed SSRs revealed polymorphism. These 200 SSRs were further deployed on 15 genotypes of levant cotton for the genetic diversity assessment. This is the first report on the successful use of repeat enriched genomic library and expressed sequence database for microsatellite markers development in G. herbaceum.

Plastid transformation vectors are used for high-level expression of industrially important recombinant proteins in plants. In the present study, new vectors for plastid transformation were developed. One of these vectors targets transgenes at a new site in the chloroplast genome. Intergenic regions of trnfM-trnG, ndhB-trnL and rrn16-trnV were selected as sites for transgene insertion. Tobacco chloroplast was successfully transformed with designed vectors, and the transplastomic plants accumulated recombinant protein as high as 5–6% of total soluble protein which remained localized in the chloroplasts. Although the vectors were designed using the plastid genome of Nicotiana tabacum, flanking regions used in two vectors show a high level of homology with chloroplast genomes of other plant species, thus it might be possible to use them for the transformation of a wider range of plant species.

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg(-1)) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95 °C, respectively. At 95 °C, thermal inactivation rate constant (K (d)) of the enzyme was 2.46 × 10(-3) min(-1) and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5-11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.

Sterols are important components of cell membranes, hormones, signalling molecules and defense-related biotic and abiotic chemicals. Sterol glycosyltransferases (SGTs) are enzymes involved in sterol modifications and play an important role in metabolic plasticity during adaptive responses. The enzymes are classified as a subset of family 1 glycosyltransferases due to the presence of a signature motif in their primary sequence. These enzymes follow a compulsory order sequential mechanism forming a ternary complex. The diverse applications of sterol glycosides, like cytotoxic and apoptotic activity, anticancer activity, medicinal values, anti-stress roles and anti-insect and antibacterial properties, draws attention towards their synthesis mechanisms. Many secondary metabolites are derived from sterol pathways, which are important in defense mechanisms against pathogens. SGTs in plants are involved in changed sensitivity to stress hormones and their agrochemical analogs and changed tolerance to biotic and abiotic stresses. SGTs that glycosylate steroidal hormones, such as brassinosteroids, function as growth and development regulators in plants. In terms of metabolic roles, it can be said that SGTs occupy important position in plant metabolism and may offer future tools for crop improvement.

Introduction Withania somnifera (Ashwagandha) is a high-value Ayurvedic medicinal plant and an important constituent of several dietary supplements. In order to substantiate the health claims, the herb has drawn considerable scientific attention. Objective The objective of the study was to investigate the alterations in primary and secondary metabolites of W. somnifera fruits during its maturity using NMR spectroscopy. Methodology Fruits at different stages of development from one week after fertilisation until maturity, classified in seven developmental stages, were analysed by a combined use of one- and two-dimensional NMR experiments. Results Seventeen metabolites were characterised and quantified from non-polar and polar extracts of different fruit development stages of W. somnifera. The principal component analysis of polar metabolites at different stages could be grossly classified into three metabolic phases, viz. initial phase, developmental phase and maturation phase. Conclusion Qualitative and quantitative analysis of metabolites in W. somnifera fruits indicated specific stages when fruits can be harvested for obtaining substantial bioactive ingredients for desirable pharmacological activity. This study potentially provides a complementary tool for quality control of herbal medicinal products when W. somnifera fruits are used. Copyright © 2011 John Wiley & Sons, Ltd.

A high yielding elite plant of Jatropha curcas was established under aseptic condition from field-grown nodal explants. Shoots were proliferated in MS medium supplemented with 0.5 mg dm(-3) benzyladenine and 0.1 mg dm(-3) indolebutyric acid along with 10 mg dm(-3) adenine sulphate and a combination of 15 mg dm(-3) each of L-glutamine and L-arginine. However, within 15-20 d of culture incubation, tissue browning/necrosis leading to poor plant regeneration in vitro was observed. A set of different antioxidants, namely, reduced glutathione, ascorbic acid, tocopherol and cysteine were used in the medium individually and in combination to solve the problem of tissue browning and necrosis. The addition of antioxidants proved beneficial for the growth of the shoots. The optimum medium comprised of 25 mg dm(-3) reduced glutathione and 10 mg dm(-3) ascorbic acid, where proliferating shoots having highest leaf canopy area, remained fresh, green and regenerative up to 40 d of culture incubation without any subculture. The activities of antioxidant enzymes, such as superoxide dismutase was higher in control shoots, indicating that tissue browning/necrosis was associated with oxidative stress which was further supported by higher contents of H(2)O(2) and phenolics in control shoots compared to the other treatments. Similarly glutathione reductase, ascorbate peroxidase and guiacol peroxidase was higher in treated shoots than control indicating that these shoots have developed antioxidant enzymatic protective system which determine the ability to survive in oxidative stress and up regulation of these enzymes would help to reduce the built up of reactive oxygen species. (C) 2010 Elsevier Ltd. All rights reserved.