Major Singh

Effect of water withdrawal on formation of free radical, proline accumulation and activities of antioxidant enzymes in ZAT12-transformed transgenic tomato plants

Water stress often leads to the accumulation of reactive oxygen species (ROS) and their excessive production alters the activities of enzymes involved in their removal. ZAT12 is a member of stress-responsive C(2)H(2) type Zinc Finger Protein (ZFP) reported to control the expression of several stress-activated genes in plants through ROS signaling. The ZAT12-transformed tomato lines (cv. H-86 variety Kashi Vishesh) when subjected to water withdrawal for 7, 14 and 21 days revealed significant and consistent changes in activities of enzymes SOD, CAT, APX, GR and POD paralleled with an increased proline levels. Unlike that in wild-type tomato, the leaf superoxide anion and hydrogen peroxide concentrations in the transformed tomato plants did not alter much, suggesting a well regulated formation of free radicals suppressing oxidative stress in the latter. Results suggest BcZAT12-transformed tomato lines ZT1, ZT2 and ZT6 to be better adapted to drought stress tolerance by accumulation of osmolyte proline and increased antioxidant response triggered by the ZAT12 gene. Therefore, the ZAT12-transformed tomato cv. H-86 lines will prove useful for higher yield of tomato crop in regions affected with severe drought stress.

Expression of rd29A::AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants

Transgenic tomato lines (cv. Kashi Vishesh) over-expressing AtDREB1A/CBF3 driven by stress-inducible rd29A promoter showed significantly higher activities of key antioxidant enzymes when exposed to water-deficit for 7, 14, and 21 days. Transgenic tomato plants exposed to water-deficit recorded lower levels of hydrogen peroxide and superoxide anion formation compared to the non-transgenic plants, suggesting alleviation of reactive oxygen species (ROS). A significant increase in activities of enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) was observed in response to the different durations of water-deficit conditions. In contrast, enzyme guaiacol peroxidase (POD) activity was lower in the transgenic lines and showed a negative correlation with ROS, ascorbic acid (AsA), and glutathione levels. The concentrations of AsA, glutathione and their reduced forms were higher in the transgenic plants and increased with ROS levels. These results indicate that AtDREB1A transgenic tomato lines are better adapted to water-deficit as they showed lower drought-induced oxidative stress due to activation of the antioxidant response.

The population genomics of begomoviruses: global scale population structure and gene flow

BackgroundThe rapidly growing availability of diverse full genome sequences from across the world is increasing the feasibility of studying the large-scale population processes that underly observable pattern of virus diversity. In particular, characterizing the genetic structure of virus populations could potentially reveal much about how factors such as geographical distributions, host ranges and gene flow between populations combine to produce the discontinuous patterns of genetic diversity that we perceive as distinct virus species. Among the richest and most diverse full genome datasets that are available is that for the dicotyledonous plant infecting genus, Begomovirus, in the Family Geminiviridae. The begomoviruses all share the same whitefly vector, are highly recombinogenic and are distributed throughout tropical and subtropical regions where they seriously threaten the food security of the worlds poorest people.ResultsWe focus here on using a model-based population genetic approach to identify the genetically distinct sub-populations within the global begomovirus meta-population. We demonstrate the existence of at least seven major sub-populations that can further be sub-divided into as many as thirty four significantly differentiated and genetically cohesive minor sub-populations. Using the population structure framework revealed in the present study, we further explored the extent of gene flow and recombination between genetic populations.ConclusionsAlthough geographical barriers are apparently the most significant underlying cause of the seven major population sub-divisions, within the framework of these sub-divisions, we explore patterns of gene flow to reveal that both host range differences and genetic barriers to recombination have probably been major contributors to the minor population sub-divisions that we have identified. We believe that the global Begomovirus population structure revealed here could facilitate population genetics studies into how central parameters of population genetics namely selection, recombination, mutation, gene flow, and genetic drift shape the global begomovirus diversity.

Virulence and genotypic characterization of Listeria monocytogenes isolated from vegetable and soil samples

BackgroundListeria monocytogenes, a foodborne pathogen is ubiquitous to different environments including the agroecosystem. The organism poses serious public health problem. Therefore, an attempt has been made to gain further insight to their antibiotic susceptibility, serotypes and the virulence genes.ResultsOut of the 10 vegetables selected, 6 (brinjal, cauliflower, dolichos-bean, tomato, chappan-kaddu and chilli), 20 isolates (10%) tested positive for L. monocytogenes. The prevalence of the pathogen in the respective rhizosphere soil samples was 5%. Noticeably, L. monocytogenes was absent from only cabbage, broccoli, palak and cowpea, and also the respective rhizospheric soils. The 30 isolates + ve for pathogenicity, belonged to serogroup 4b, 4d or 4e, and all were positive for inlA, inlC, inlJ, plcA, prfA, actA, hlyA and iap gene except one (VC3) among the vegetable isolates that lacked the plcA gene. ERIC- and REP-PCR collectively revealed that isolates from vegetables and their respective rhizospheric soils had distinct PCR fingerprints.ConclusionsThe study demonstrates the prevalence of pathogenic L. monocytogenes in the selected agricultural farm samples. The increase in the number of strains resistant to ciprofloxacin and/or cefoxitin seems to pose serious public health consequences.

Engineering drought tolerant tomato plants over-expressing BcZAT12 gene encoding a C2H2 zinc finger transcription factor

Efficient genetic transformation of cotyledonary explants of tomato (Solanum lycopersicum, cv. H-86, Kashi vishesh) was obtained. Disarmed Agrobacterium tumifaciens strain GV 3101 was used in conjugation with binary vector pBinAR containing a construct consisting of the coding sequence of the BcZAT12 gene under the regulatory control of the stress inducible Bclea1a promoter. ZAT12 encodes a C₂H₂ zinc finger protein which confers multiple abiotic stress tolerance to plants. Integration of ZAT12 gene into nuclear genome of individual kanamycin resistant transformed T₀ tomato lines was confirmed by Southern blot hybridization with segregation analysis of T(1) plants showing Mendelian inheritance of the transgene. Expression of ZAT12 in drought-stressed transformed tomato lines was verified in T₂ generation plants using RT-PCR. Of the six transformed tomato lines (ZT1-ZT6) the transformants ZT1 and ZT5 showed maximum expression of BcZAT12 gene transcripts when exposed to 7 days drought stress. Analysis of relative water content (RWC), electrolyte leakage (EL), chlorophyll colour index (CCI), H₂O₂ level and catalase activity suggested that tomato BcZAT12 transformants ZT1 and ZT5 have significantly increased levels of drought tolerance. These results suggest that BcZAT12 transformed tomato cv. H-86 has real potential for molecular breeding programs aimed at augmenting yield of tomato in regions affected with drought stress.

Bacterial Community Structure in the Rhizosphere of a Cry1Ac Bt-Brinjal Crop and Comparison to Its Non-transgenic Counterpart in the Tropical Soil

To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4+-N, NO3−-N, and PO4−-P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes.

Development and bioassay of Cry1Ac-transgenic eggplant (Solanum melongena L.) resistant to shoot and fruit borer

Summary Eggplant (Solanum melongena L.) is an important fruit vegetable, commercially cultivated in the tropics and subtropics. However, the most serious constraint on eggplant production is damage caused by eggplant shoot and fruit borer (ESFB). The development of resistant transgenic plants, using an insecticidal crystal protein gene, is an available crop protection option. A Cry1Ac gene obtained from the National Research Center for Plant Biotechnology (NRCPB), New Delhi, India, was transformed via Agrobacterium-mediated gene transfer into an improved inbred line of eggplant (IVBL-9). Hypocotyls proved to be the most effective and suitable explants, with a transformation frequency of 17.3% and 2.9 shoots per explant. PCR and Southern blot analyses confirmed the presence of a single copy insertion of the Cry1Ac gene in seven independent transgenic plants. The insertion of a single copy of the gene was also confirmed by segregation analysis of T1 seed from T0 plants. ELISA analyses revealed the presence of the Cry1Ac protein, and quantitative estimates confirmed significant levels of Cry1Ac protein (2.46 – 4.33 ng ml–1 leaf extract) expressed in all seven transformed plants. High levels of expression of this insecticidal protein resulted in significant larval mortality and in the reduced growth of any surviving larva on transformed eggplant tissue.

Effect of heat-shock induced oxidative stress is suppressed in BcZAT12 expressing drought tolerant tomato

The transcription factor ZAT12 is a member of stress-responsive C2H2 type zinc finger protein (ZFP) reported to control the expression of stress-activated genes mediated via ROS in plants. BcZAT12-transformed tomato cv. H-86, var. Kashi vishesh (lines ZT1-ZT6) over-expressing the gene product is demonstrated herein to be tolerant to heat-shock (HS)-induced oxidative stress. Results reveal that the relative expression of ZAT12 as well as heat induced Hsp17.4 and Hsp21 gene transcripts increased in transgenic upon exposure to HS. The transformed tomato lines ZT1 and ZT5 had significantly lowered free radical formation, improved electrolyte leakage, relative water content and chlorophyll levels with an enhanced activities of antioxidant enzymes viz. superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase when exposed to HS. HS-induced oxidative stress by over-expression of the BcZAT12 gene transcripts in tomato as well as by largely enhancing the ROS-scavenging capacity and up regulation of Hsp transcripts. This enables the transgenic tomato plants to acquire a greater ability to counteract HS-induced oxidative stress, being endowed with more reduced antioxidant pools. The use of these HS-tolerant tomato lines could possibly be used for tomato cultivation in the areas affected by sudden temperature changes.

Transcription factors in abiotic stress tolerance

Abiotic stresses such as drought, high salinity, and extreme temperatures are common adverse environmental conditions that significantly reduce the crop productivity. Plants have the capability to sense and adjust to abiotic stresses, although the degree of adaptability to specific stresses varies from species to species. The adaptability to environmental stresses is controlled by either simple or complex cascades of molecular networks. Transcription factors (TFs) play vital regulatory roles in abiotic stress responses in plants by interacting with cis-elements present in the promoter region of various abiotic stress responsive genes. The identification and molecular tailoring of novel TFs involved in environmental stress responses have the potential to overcome a number of important limitations encountered in the generation of transgenic crop plants with superior yield under stress conditions. This opens an excellent opportunity to develop stress tolerant crops in future. This review summarizes the role of various transcription factors in crop improvement through transgenic technology.

Expression of ZAT12 transcripts in transgenic tomato under various abiotic stresses and modeling of ZAT12 protein in silico

ZAT12 a C2H2-zinc-finger protein is an abiotic stress-responsive transcription factor in plants having less information about their structure. Transcription analysis proved that ZAT12 transcripts over-expressed during drought, heat and salt stress conditions which led to an interest in 3-D structural studies of ZAT12in Brassica carinata. Over-expression of BcZAT12 in transformed tomato plants under abiotic stresses, suggest role of ZAT12 in conferring stress-tolerance in tomato. Sequence analysis of ZAT12 protein (Accession No. ABB55254.1) from B. carinata revealed it as a 161 amino acid long protein with short conserved motif 140LDLXL144 in C-terminal, a leucine rich L-Box with—14EXXAXCLXXL23 motif in N-terminal region and presence of two conserved Zinc-Finger motifs “CXXCXXXXXXXQALGGHXXXH” between positions 42–62 and 85–105. The two zinc finger motifs have presence of two conserved glutamic acid (Glu) and phenylalanine (Phe) residues. Two methionine (Met) residues at position 94 and 102 present in ZF-motif-2 were absent in ZF-motif-1. The 94Met and 97Ala in ZF-motif-2 were found to be replaced by serine (Ser) in ZF-motif-1. Homology and ab initio structural modeling of ZAT12 encoded BcZAT12 protein of B. carinata resulted in robust 3-D models and were evaluated for structural motifs, associated GO terms and protein-DNA interactions. The BcZAT12 protein model, was of good quality, reliable, stable and is deposited in PMDB database (PMDB ID: PM0078213). BcZAT12 is annotated as an intracellular protein having molecular function in Zn-binding which in turn regulates signal transduction/translation processes in response to abiotic stresses in plants. Results suggest BcZAT12 protein to interact directly with one strand of dsDNA via electrostatic and H-bonds.