Characterization of Transcription Regulatory Domains of OsMADS29: Identification of Proximal Auxin-Responsive Domains and a Strong Distal Negative Element.

OsMADS29 (M29) is a seed-specific MADS-box transcription factor involved in programmed cell death of nucellar tissue and maintaining auxin:cytokinin homeostasis. It affects embryo and endosperm development and starch filling during seed development in rice. Its expression seems to be tightly regulated by developmental, spatial, and temporal cues; however, cis- and trans-regulatory factors that affect its expression are largely unknown. In silico analysis of the 1.7 kb upstream regulatory region (URR) consisting of 1,290 bp promoter and 425 bp 5'-UTR regions revealed several auxin-responsive and seed-specific cis-regulatory elements distributed across the URR. In this study, the analysis of four URR deletions fused to a downstream ß-glucuronidase (GUS) reporter in transgenic rice has revealed the presence of several proximal positive elements and a strong distal negative element (NE). The promoter regions containing auxin-responsive elements responded positively to the exogenous application of auxins to transgenic seedlings. The proximal positive elements are capable of driving reporter expression in both vegetative and reproductive tissues. In contrast, the NE strongly suppresses reporter gene expression in both vegetative and reproductive tissues. In a transient onion peel assay system, the NE could reduce the efficacy of a 2x CaMV 35S promoter by ∼90%. Our results indicate the existence of a complex array of positive and negative regulatory regions along with auxin-responsive elements guiding the development-dependent and spatial expression of M29.

Activation of CDC48 and acetyltransferase encoding genes contributes to enhanced abiotic stress tolerance and improved productivity traits in rice.

World-wide crop productivity is highly impacted by various extreme environmental conditions. In the present investigation, activation tagged (AT) line A10-Ds-RFP6 of rice endowed with improved agronomic attributes was tested for its tolerance ability against drought and salinity stress conditions as well as identification of genes associated with these traits. Under both drought and salinity stress conditions, A10-Ds-RFP6 line exhibited increased seed germination rates and improved plant growth characteristics at seedling, vegetative and reproductive stages as compared to wild-type (WT) plants. Moreover, A10-Ds-RFP6 revealed effective antioxidant systems resulting in decreased accumulation of reactive oxygen species and delayed stress symptoms compared to WT plants. Reduced accumulation of malondialdehyde with concomitant increase in proline and soluble sugars in A10-Ds-RFP6 line further endorse its improved stress tolerance levels. Furthermore, A10-Ds-RFP6 disclosed enhanced plant water content, photosynthetic efficiency, stomatal conductance, water use efficiency and maximum quantum yield compared to WT plants. TAIL and qRT-PCR analyses of AT rice line revealed the integration site of Ds element in the genome and increased expression levels of CDC48 and acetyltransferase genes involved in various aspects of plant development and stress tolerance. As such, the promising AT line plausibly serve as a rare genetic resource for fortifying stress tolerance and productivity traits of elite rice cultivars.

Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties.

KEY MESSAGE: We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties. In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F1 seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F1 seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.

Expression of cold and drought regulatory protein (CcCDR) of pigeonpea imparts enhanced tolerance to major abiotic stresses in transgenic rice plants.

MAIN CONCLUSION: Transgenic rice expressing pigeonpea Cc CDR conferred high-level tolerance to different abiotic stresses. The multiple stress tolerance observed in CcCDR -transgenic lines is attributed to the modulation of ABA-dependent and-independent signalling-pathway genes. Stable transgenic plants expressing Cajanus cajan cold and drought regulatory protein encoding gene (CcCDR), under the control of CaMV35S and rd29A promoters, have been generated in indica rice. Different transgenic lines of CcCDR, when subjected to drought, salt, and cold stresses, exhibited higher seed germination, seedling survival rates, shoot length, root length, and enhanced plant biomass when compared with the untransformed control plants. Furthermore, transgenic plants disclosed higher leaf chlorophyll content, proline, reducing sugars, SOD, and catalase activities, besides lower levels of MDA. Localization studies revealed that the CcCDR-GFP fusion protein was mainly present in the nucleus of transformed cells of rice. The CcCDR transgenics were found hypersensitive to abscisic acid (ABA) and showed reduced seed germination rates as compared to that of control plants. When the transgenic plants were exposed to drought and salt stresses at vegetative and reproductive stages, they revealed larger panicles and higher number of filled grains compared to the untransformed control plants. Under similar stress conditions, the expression levels of P5CS, bZIP, DREB, OsLEA3, and CIPK genes, involved in ABA-dependent and-independent signal transduction pathways, were found higher in the transgenic plants than the control plants. The overall results amply demonstrate that the transgenic rice expressing CcCDR bestows high-level tolerance to drought, salt, and cold stress conditions. Accordingly, the CcCDR might be deployed as a promising candidate gene for improving the multiple stress tolerance of diverse crop plants.

Development of an inducible male-sterility system in rice through pollen-specific expression of l-ornithinase (argE) gene of E. coli.

In the present investigation, an inducible male-sterility system has been developed in the rice. In order to introduce inducible male-sterility, the coding region of l-ornithinase (argE) gene of E. coli was fused to the Oryza sativa indica pollen allergen (OSIPA) promoter sequence which is known to function specifically in the pollen grains. Transgenic plants were obtained with argE gene and the transgenic status of plants was confirmed by PCR and Southern blot analyses. RT-PCR analysis confirmed the tissue-specific expression of argE in the anthers of transgenic rice plants. Transgenic rice plants expressing argE, after application of N-acetyl-phosphinothricin (N-ac-PPT), became completely male-sterile owing to the pollen-specific expression of argE. However, argE-transgenic plants were found to be self fertile when N-ac-PPT was not applied. Normal fertile seeds were obtained from the cross pollination between male-sterile argE transgenics and untransformed control plants, indicating that the female fertility is not affected by the N-ac-PPT treatment. These results clearly suggest that the expression of argE gene affects only the male gametophyte but not the gynoecium development. Induction of complete male-sterility in the rice is a first of its kind, and moreover this male- sterility system does not require the deployment of any specific restorer line.

Current status of genetic engineering in cotton (Gossypium hirsutum L): an assessment.

Cotton is considered as the foremost commercially important fiber crop and is deemed as the backbone of the textile industry. The productivity of cotton crop, worldwide, is severely hampered by the occurrence of pests, weeds, pathogens apart from various environmental factors. Several beneficial agronomic traits, viz., early maturity, improved fiber quality, heat tolerance, etc. have been successfully incorporated into cotton varieties employing conventional hybridization and mutation breeding. Crop losses, due to biotic factors, are substantial and may be reduced through certain crop protection strategies. In recent years, pioneering success has been achieved through the adoption of modern biotechnological approaches. Genetically engineered cotton varieties, expressing Bacillus thuringiensis cry genes, proved to be highly successful in controlling the bollworm complex. Various other candidate genes responsible for resistance to insect pests and pathogens, tolerance to major abiotic stress factors such as temperature, drought and salinity, have been introduced into cotton via genetic engineering methods to enhance the agronomic performance of cotton cultivars. Furthermore, genes for improving the seed oil quality and fiber characteristics have been identified and introduced into cotton cultivars. This review provides a brief overview of the various advancements made in cotton through genetic engineering approaches.

Engineered Pichia pastoris for enhanced production of S-adenosylmethionine.

A genetically engineered strain of Pichia pastoris expressing S-adenosylmethionine synthetase gene from Saccharomyces cerevisiae under the control of AOX 1 promoter was developed. Induction of recombinant strain with 1% methanol resulted in the expression of SAM2 protein of ~ 42 kDa, whereas control GS115 showed no such band. Further, the recombinant strain showed 17-fold higher enzyme activity over control. Shake flask cultivation of engineered P. pastoris in BMGY medium supplemented with 1% L-methionine yielded 28 g/L wet cell weight and 0.6 g/L S-adenosylmethionine, whereas control (transformants with vector alone) with similar wet cell weight under identical conditions accumulated 0.018 g/L. The clone cultured in the bioreactor containing enriched methionine medium showed increased WCW (117 g/L) as compared to shake flask cultures and yielded 2.4 g/L S-adenosylmethionine. In spite of expression of SAM 2 gene up to 90 h, S-adenosylmethionine accumulation tended to plateau after 72 h, presumably because of the limited ATP available in the cells at stationery phase. The recombinant P pastoris seems promising as potential source for industrial production of S-adenosylmethionine.

Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.).

Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries.

Prediction of substrate-binding site and elucidation of catalytic residue of a phytase from Bacillus sp.

The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed ß-propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme.

Isolation and characterization of a pigeonpea cyclophilin (CcCYP) gene, and its over-expression in Arabidopsis confers multiple abiotic stress tolerance.

A full-length cDNA clone of pigeonpea (Cajanus cajan L.) encoding cyclophilin (CcCYP) has been isolated from the cDNA library of plants subjected to drought stress. Amino acid sequence of CcCYP disclosed similarity with that of single-domain cytosolic cyclophilins of various organisms. Expression profile of CcCYP in pigeonpea plants is strongly induced by different abiotic stresses, indicating its stress-responsive nature. Compared to the control plants, the transgenic Arabidopsis lines expressing CcCYP exhibited high-level tolerance against major abiotic stresses, viz., drought, salinity and extreme temperatures as evidenced by increased plant survival, biomass, chlorophyll content and profuse root growth. The CcCYP transgenics, compared to the controls, revealed enhanced peptidyl-propyl cis-trans isomerase (PPIase) activity under stressed conditions, owing to transcriptional activation of stress-related genes besides intrinsic chaperonic activity of the cyclophilin. The transgenic plants subjected to salt stress exhibited higher Na(+) ion accumulation in roots as compared to shoots, while a reverse trend was observed in the salt-stressed control plants, implicating the involvement of CcCYP in the maintenance of ion homeostasis. Expression pattern of CcCYP:GFP fusion protein confirmed the localization of CcCYP predominantly in the nucleus as revealed by intense green fluorescence. The overall results amply demonstrate the implicit role of CcCYP in conferring multiple abiotic stress tolerance at whole-plant level.

Characterization of expressed sequence tags (ESTs) of pigeonpea (Cajanus cajan L.) and functional validation of selected genes for abiotic stress tolerance in Arabidopsis thaliana.

Pigeonpea, a major grain legume crop with remarkable drought tolerance traits, has been used for the isolation of stress-responsive genes. Herein, we report generation of ESTs, transcript profiles of selected genes and validation of candidate genes obtained from the subtracted cDNA libraries of pigeonpea plants subjected to PEG/water-deficit stress conditions. Cluster analysis of 124 selected ESTs yielded 75 high-quality ESTs. Homology searches disclosed that 55 ESTs share significant similarity with the known/putative proteins or ESTs available in the databases. These ESTs were characterized and genes relevant to the specific physiological processes were identified. Of the 75 ESTs obtained from the cDNA libraries of drought-stressed plants, 20 ESTs proved to be unique to the pigeonpea. These sequences are envisaged to serve as a potential source of stress-inducible genes of the drought stress-response transcriptome, and hence may be used for deciphering the mechanism of drought tolerance of the pigeonpea. Expression profiles of selected genes revealed increased levels of m-RNA transcripts in pigeonpea plants subjected to different abiotic stresses. Transgenic Arabidopsis lines, expressing Cajanus cajan hybrid-proline-rich protein (CcHyPRP), C. cajan cyclophilin (CcCYP) and C. cajan cold and drought regulatory (CcCDR) genes, exhibited marked tolerance, increased plant biomass and enhanced photosynthetic rates under PEG/NaCl/cold/heat stress conditions. This study represents the first report dealing with the isolation of drought-specific ESTs, transcriptome analysis and functional validation of drought-responsive genes of the pigeonpea. These genes, as such, hold promise for engineering crop plants bestowed with tolerance to major abiotic stresses.

Expression of pigeonpea hybrid-proline-rich protein encoding gene (CcHyPRP) in yeast and Arabidopsis affords multiple abiotic stress tolerance.

A hybrid-proline-rich protein encoding gene (CcHyPRP) has been isolated and characterized, for the first time, from the subtracted cDNA library of pigeonpea (Cajanus cajan L.) plants subjected to drought stress. Functionality of CcHyPRP has been validated for abiotic stress tolerance using the heterologous yeast and Arabidopsis systems. The CcHyPRP contained a repetitive proline-rich (PR) N-terminal domain and a conserved eight cysteine motif (8CM) at the C-terminus. Southern analysis disclosed single-copy nature of CcHyPRP gene in the pigeonpea genome. Northern analysis revealed higher levels of CcHyPRP transcripts in PEG, NaCl, heat (42 degrees C), cold and ABA-treated plants compared with the weak signals observed in the untreated plants, suggesting stress-responsive nature of the CcHyPRP gene. In yeast, expression of CcHyPRP imparted marked tolerance against abiotic stresses exerted by PEG, high temperature, NaCl and LiCl. Transgenic Arabidopsis lines, expressing CcHyPRP under the control of CaMV35S and rd29A promoters, when subjected to PEG, mannitol, NaCl, LiCl and heat (42 degrees C) stress, developed into healthy plants with profuse root system and increased biomass in contrast to the weak-stunted wild-type plants. The CcHyPRP-transgenics driven by stress-inducible rd29A exhibited similar stress-tolerance as that of CaMV35S-lines without any negative effects on plant morphology, implying that stress-inducible promoters are preferable for production of stress tolerant transgenics. The overall results amply demonstrate the profound effect of CcHyPRP in bestowing multiple abiotic stress tolerance at cellular and whole plant levels. Accordingly, the multipotent CcHyPRP seems promising as a prime candidate gene to fortify crop plants with abiotic stress tolerance.

Structural and kinetic properties of Bacillus subtilis S-adenosylmethionine synthetase expressed in Escherichia coli.

S-adenosylmethionine (SAM) synthetase (EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine using l-methionine and ATP as substrates. SAM synthetase gene (metE) from Bacillus subtilis was cloned and over-expressed, for the first time, in the heterologus host Escherichia coli as an active enzyme. Size-exclusion chromatography (SEC) revealed a molecular weight of ~180 kDa, suggesting that the enzyme is a homotetramer stabilized by non-covalent interactions. SAM synthetase exhibited optimal activity at pH 8.0 and 45 degrees C with the requirement of divalent cation Mg(2+), and stimulated by the monovalent cation K(+). The enzyme followed sequential mechanism with a V(max) of 0.362 micromol/min/mg, and a K(m) of 920 microM and 260 microM for ATP and l-methionine, respectively. The urea-induced unfolding equilibrium of the recombinant enzyme revealed a multistate process, comprising partially unfolded tetramer, structural dimer, structural monomer and completely unfolded monomer, as evidenced by intrinsic and extrinsic fluorescence, circular dichroism (CD) and SEC. Absence of trimer in the SEC implicates that the enzyme is a dimer of dimer. Concordance between results of SEC and enzyme activity in the presence of urea amply establishes that tetramer alone with intersubunit active site(s) exhibits enzyme activity.