Ectopic expression of DnaJ type-I protein homolog of Vigna aconitifolia (VaDJI) confers ABA insensitivity and multiple stress tolerance in transgenic tobacco plants

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5 min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants

Pathogen-induced AdDjSKI of the wild peanut, Arachisdiogoi, potentiates tolerance of multiple stresses in E. coli andtobacco

A gene encoding a serine-rich DnaJIII protein called AdDjSKI that has a 4Fe-4S cluster domain was found to be differentially upregulated in the wild peanut, Arachis diogoi in its resistance responses against the late leaf spot causing fungal pathogen Phaeoisariopsis personata when compared with the cultivated peanut, Arachis hypogaea. AdDjSKI is induced in multiple stress conditions in A. diogoi. Recombinant E. coli cells expressing AdDjSKI showed better growth kinetics when compared with vector control cells under salinity, osmotic, acidic and alkaline stress conditions. Overexpression of this type three J-protein potentiates not only abiotic stress tolerance in Nicotiana tabacum var. Samsun, but also enhances its disease resistance against the phytopathogenic fungi Phytophthora parasitica pv nicotianae and Sclerotinia sclerotiorum. In the present study we show transcriptional upregulation of APX, Mn-SOD and HSP70 under heat stress and increased transcripts of PR genes in response to fungal infection. This transmembrane-domain-containing J protein displays punctate localization in chloroplasts. AdDjSKI appears to ensure proper folding of proteins associated with the photosynthetic machinery under stress

Not Available

Not AvailableAbstract Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes ofNot Availabl

Not Available

Not AvailableMoth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.Not Availabl

Not Available

Not AvailableMoth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.Not Availabl

Not Available

Not AvailableMoth bean (Vigna aconitifolia (Jacq.) Marechal) is an important grain legume crop grown in rain fed areas of hot desert regions of Thar, India, under scorching sun rays with very little supplementation of water. An SSH cDNA library was generated from leaf tissues of V. aconitifolia var. RMO-40 exposed to an elevated temperature of 42 °C for 5 min to identify early-induced genes. A total of 488 unigenes (114 contigs and 374 singletons) were derived by cluster assembly and sequence alignment of 738 ESTs; out of 206 ESTs (28%) of unknown proteins, 160 ESTs (14%) were found to be novel to moth bean. Only 578 ESTs (78%) showed significant BLASTX similarity (<1 × 10–6) in the NCBI non-redundant database. Gene ontology functional classification terms were retrieved for 479 (65%) sequences, and 339 sequences were annotated with 165 EC codes and mapped to 68 different KEGG pathways. Four hundred and fifty-two ESTs were further annotated with InterProScan (IPS), and no IPS was assigned to 153 ESTs. In addition, the expression level of 27 ESTs in response to heat stress was evaluated through semiquantitative RT–PCR assay. Approximately 20 different signaling genes and 16 different transcription factors have been shown to be associated with heat stress in moth bean for the first time.Not Availabl

ROS detection in leaf epidermal cells by confocal microscopy and quantification of ROS in leaves using NBT staining.

<p>(A) Fluorescence levels in WT and transgenic plants before and after treatment with PEG as shown in confocal microscopy. Bright field images of WT and transgenic plants are also displayed. Stomata with water treatment. (a and b) WT; (c and d) representative transgenic line 7. Stomata after treatment with PEG. (e and f) WT; (g and h) transgenic line 7; (i and j) transgenic line 2; (k and l) transgenic line 4. The figures are representative confocal images of stomatal guard cells (n < 1000) with three biological repetitions (Bar-100μm). (B) Quantification of ROS production in cells after H₂DCFDA staining using ImageJ software. (C) Unstressed (untreated), WT, and transgenic leaves treated with 100mM NaCl as visualized after NBT staining. (D) Graphical representation of formazan content (μg mg^-1 dry weight) in leaves of unstressed, WT, and transgenic leaves after treatment with 100mM NaCl. All the experiments were performed in triplicates and data represented as mean ± SD (n = 3; biological replicates). Single leaf from each plant constitute one biological sample. Statistical analysis was performed with one-way ANOVA (*P<0.05, **P<0.001).</p

Relative gene expression of <i>AdLEA</i> in <i>A</i>. <i>diogoi</i> in response to various abiotic stresses.

<p><i>A</i>. <i>diogoi</i> samples were treated with extreme temperatures (high and low), polyethylene glycol, sorbitol, sodium chloride and methyl viologen and <i>AdLEA</i> expression profile was analyzed by qRT-PCR. Data plotted are the mean values ± SD from three independent experiments (n = 3; biological replicates). RNA from two trifoliate leaves of <i>A</i>. <i>diogoi</i> represents one biological sample. Statistical analysis was performed with one-way ANOVA (*P<0.05, **P<0.001).</p

Effect of oxidative stress on <i>AdLEA</i> transgenic plants.

<p>(A and C) WT and transgenic seedlings after growth on MS medium supplemented with 5 and 10μM MV respectively. (B and D) Recovery of seedlings on MV free medium. (E) Chlorophyll content (μg mg^-1 FW) in the seedlings after growth on MV supplemented medium. (F) Phenotypic differences in the leaf discs from WT and transgenic plant lines floated in dose-dependent concentrations (5 and 10μM) of MV. (G) Chlorophyll content (μg mg^-1 FW) in the leaf discs after treatment with MV. (H) Lipid peroxidation expressed as TBARS content (μmol g^-1 FW) in leaf discs after treatment with MV. All the experiments were performed in triplicates and data represented as mean ± SD (n = 3; biological replicates). 11 seedlings per plate constitute one biological sample for seedling assay. 15 leaf discs from a single leaf constitute one biological sample for disc senescence assay. Statistical analysis was performed with two-way ANOVA (*P<0.05, **P<0.001).</p

Effect of salinity stress on <i>AdLEA</i> transgenic plants.

<p>(A and C) WT and transgenic seedlings after growth on MS medium supplemented with 200mM and 300mM NaCl respectively. (B and D) Individual seedlings morphology after growth on 200mM and 300mM NaCl respectively, depicting differences in root length between the seedlings. (E) Phenotypic differences in the leaf discs from WT and transgenic plant lines floated in dose-dependent concentrations (200mM and 300mM) of NaCl. (F) Graphical representation of root lengths in NaCl-treated seedlings after 15d and 9d growth on 200mM and 300mM NaCl respectively. (G) <b>C</b>hlorophyll content (μg mg^-1 FW) in the leaf discs after 72 h of treatment with NaCl. (H) Lipid peroxidation expressed as TBARS content (μmol g^-1 FW) in leaf discs after 72 h of treatment with NaCl. All the experiments were performed in triplicates and data represented as mean ± SD (n = 3; biological replicates). 11 seedlings per plate constitute one biological sample for seedling assay. 15 leaf discs from a single leaf constitute one biological sample for disc senescence assay. Statistical analysis was performed with two-way ANOVA (*P<0.05, **P<0.001).</p