Enhancing trehalose biosynthesis improves yield potential in marker-free transgenic rice under drought, saline, and sodic conditions.

Edaphic factors such as salinity, sodicity, and drought adversely affect crop productivity, either alone or in combination. Despite soil sodicity being reported as an increasing problem worldwide, limited efforts have been made to address this issue. In the present study, we aimed to generate rice with tolerance to sodicity in conjunction with tolerance to salinity and drought. Using a fusion gene from E. coli coding for trehalose-6-phosphate synthase/phosphatase (TPSP) under the control of an ABA-inducible promoter, we generated marker-free, high-yielding transgenic rice (in the IR64 background) that can tolerate high pH (~9.9), high EC (~10.0 dS m-1), and severe drought (30-35% soil moisture content). The transgenic plants retained higher relative water content (RWC), chlorophyll content, K+/Na+ ratio, stomatal conductance, and photosynthetic efficiency compared to the wild-type under these stresses. Positive correlations between trehalose overproduction and high-yield parameters were observed under drought, saline, and sodic conditions. Metabolic profiling using GC-MS indicated that overproduction of trehalose in leaves differently modulated other metabolic switches, leading to significant changes in the levels of sugars, amino acids, and organic acids in transgenic plants under control and stress conditions. Our findings reveal a novel potential technological solution to tackle multiple stresses under changing climatic conditions.

Molecular cloning and characterization of salt overly sensitive gene promoter from Brassica juncea (BjSOS2).

Salt Overly Sensitive (SOS) pathway comprising SOS1, SOS2 and SOS3 genes has been recognized as the key mechanism controlling ion homeostasis under salinity stress. SOS2 component of this pathway encodes a serine/threonine protein kinase that together with SOS3 activates downstream Na(+)/H(+) antiporter SOS1, reestablishing cellular ion homeostasis under salinity stress. In the present study, we have found that the transcript levels of BjSOS2 are induced in response to various abiotic stresses. We have isolated a 713 bp promoter region of SOS2 gene from Brassica juncea to study the regulation of BjSOS2 under various abiotic stress conditions and further, to examine utility of the cloned upstream region in genetic engineering experiments. For this purpose, 713 bp BjSOS2 promoter:ß-glucuronidase (GUS) fusion construct, along with its two subsequent 5' deletion derivatives, D1 (443 bp) and D2 (209 bp), were stably transformed into B. juncea. Functional analysis of transgenic lines revealed significant increase in promoter activity under salinity, desiccation as well as abscisic acid (ABA) treatment which was consistent with increased transcript levels of GUS gene. BjSOS2 promoter possesses strong multi-stress inducible nature, suggesting its involvement in various aspects of stress signaling. Considering the fact that the simultaneous presence of multiple abiotic stress conditions under field conditions is a challenging threat to crop productivity, future studies may utilize the BjSOS2 promoter to drive stress-inducible expression of genes involved in imparting tolerance to multiple stresses.

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation.

Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO2 into organic acids for subsequent C4 or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC4PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers,which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.

Enhancing salt tolerance in a crop plant by overexpression of glyoxalase II.

Earlier we have shown the role of glyoxalase overexpression in conferring salinity tolerance in transgenic tobacco. We now demonstrate the feasibility of same in a crop like rice through overproduction of glyoxalase II. The rice glyoxalase II was cloned in pCAMBIA1304 and transformed into rice (Oryza sativa cv PB1) via Agrobacterium. The transgenic plants showed higher constitutive activity of glyoxalase II that increased further upon salt stress, reflecting the upregulation of endogenous glyoxalase II. The transgenic rice showed higher tolerance to toxic concentrations of methylglyoxal (MG) and NaCl. Compared with non-transgenics, transgenic plants at the T1 generation exhibited sustained growth and more favorable ion balance under salt stress conditions.

Constitutive overexpression of a stress-inducible small GTP-binding protein PgRab7 from Pennisetum glaucum enhances abiotic stress tolerance in transgenic tobacco.

The Rab GTPases are important components of endocytic network in plant cells. Endocytosis participates in the cell's reaction to extracellular stimuli by desensitizing, down-regulating or recycling receptors and membrane proteins. Rab7 is a small GTP-binding protein involved in intracellular vesicle trafficking from late endosome to the vacuole. We have isolated Rab7 cDNA from Pennisetum glaucum, a relatively drought-stress tolerant food grain crop grown commonly in India, during cDNA-subtractive hybridization of dehydration-stress treated plants. The PgRab7 ORF, encoding 207 aminoacids, was over-expressed in E. coli. The recombinant PgRab7 protein showed GTP-binding and GTPase activity. Transcript expression of PgRab7 gene was differentially up-regulated by different environmental stimuli such as cold, dehydration and NaCl and also by a plant hormone IAA. Overexpression of PgRab7 gene enhanced tolerance to NaCl and mannitol in transgenic tobacco. Transgenic plants also had increased alkaline phosphatase (ALP) activity. These results show that PgRab7 is a potential candidate gene for developing both salinity and dehydration tolerance in planta.

Functional validation of a novel isoform of Na+/H+ antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice.

Salt stress is an environmental factor that severely impairs plant growth and productivity. We have cloned a novel isoform of a vacuolar Na+/H+ antiporter from Pennisetum glaucum (PgNHX1) that contains 5 transmembrane domains in contrast to AtNHX1 and OsNHX1 which have 9 transmembrane domains. Recently we have shown that PgNHX1 could confer high level of salinity tolerance when overexpressed in Brassica juncea. Here,we report the functional validation of this antiporter in crop plant rice. Overexpression of PgNHX1 conferred high level of salinity tolerance in rice. Transgenic rice plants overexpressing PgNHX1 developed more extensive root system and completed their life cycle by setting flowers and seeds in the presence of 150 mM NaCl. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants for growing in high saline areas.

Characterization and functional validation of glyoxalase II from rice.

Glyoxalase II, one of the enzymes of the glyoxalase pathway, cDNA cloned from rice (OsglyII) consists of 1623 nucleotides with an open reading frame of 1010 bp encoding a polypeptide of 336 amino acids and an estimated isoelectric point of 8.08. The recombinant protein purified from Escherichia coli using Ni-NTA affinity chromatography showed molecular mass of approximately 37 kDa. Catalytic parameters of the protein were determined using S-D-lactoylglutathione as a thioester substrate. The K(m) (61 microM) and K(cat) (301 s(-1)) values were lower than those reported for Arabidopsis, human and yeast and showed pH optima at 7.2. The E. coli overexpressing OsglyII were able to grow on higher concentration of methylglyoxal. Transcript analysis in rice showed that OsglyII gene expression is stimulated within 15 min in response to various abiotic stresses as well as treatment with abscisic acid or salicylic acid. This multistress response of OsglyII gene documents its future utility in developing tolerance to various stresses in crop plants.

Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches.

Environmental constraints that include abiotic stress factors such as salt, drought, cold and extreme temperatures severely limit crop productivity. Improvement of crop plants with traits that confer tolerance to these stresses was practiced using traditional and modern breeding methods. Molecular breeding and genetic engineering contributed substantially to our understanding of the complexity of stress response. Mechanisms that operate signal perception, transduction and downstream regulatory factors are now being examined and an understanding of cellular pathways involved in abiotic stress responses provide valuable information on such responses. This review presents genomic-assisted methods which have helped to reveal complex regulatory networks controlling abiotic stress tolerance mechanisms by high-throughput expression profiling and gene inactivation techniques. Further, an account of stress-inducible regulatory genes which have been transferred into crop plants to enhance stress tolerance is discussed as possible modes of integrating information gained from functional genomics into knowledge-based breeding programs. In addition, we envision an integrative genomic and breeding approach to reveal developmental programs that enhance yield stability and improve grain quality under unfavorable environmental conditions of abiotic stresses.

Stress-inducible DREB2A transcription factor from Pennisetum glaucum is a phosphoprotein and its phosphorylation negatively regulates its DNA-binding activity.

Abiotic stress-mediated gene expression is regulated via different transcription factors of which drought-responsive element-binding (DREB) proteins play an important role. There are two types of DREBs. Presently, the function of DREB1 type protein is well studied; however, much less information is available for DREB2. In this study, a cDNA with an open reading frame of 332 amino acids, encoding the transcription activation factor DREB2A, was cloned from Pennisetum glaucum, a stress tolerant food grain crop. Phylogenetic tree revealed that PgDREB2A is more close to DREBs isolated from monocots, though it forms an independent branch. The PgDREB2A transcript was up-regulated in response to drought within 1 h of the treatment, whereas the induction was delayed in response to cold and salinity stress. However, during cold stress, the transcript was induced more as compared to drought and salinity. The recombinant PgDREB2A protein having a molecular mass of 36.6 kDa was purified using Ni-NTA affinity chromatography. Gel mobility shift assays using the purified protein and two cis elements of rd29A (responsive to dehydration 29A) gene promoter of Arabidopsis revealed that PgDREB2A binds to drought-responsive element (DRE) ACCGAC and not to GCCGAC. PgDREB2A is a phosphoprotein, which has not been reported earlier. The phosphorylation of PgDREB2A in vitro by P. glaucum total cell extract occurred at threonine residue(s). The phosphorylated PgDREB2A did not bind to the DREs. The present data indicate that stress induction of genes could occur via post-translational modification by phosphorylation of DREB2A.

Structural and functional analysis of a salt stress inducible gene encoding voltage dependent anion channel (VDAC) from pearl millet (Pennisetum glaucum).

We have cloned and characterized a gene encoding voltage-dependent anion channel from Pennisetum glaucum (PgVDAC). PgVDAC was identified while isolating genes that were differentially up-regulated following salt stress. The genomic organization of PgVDAC clone was well conserved compared to other plant VDAC genes in terms of number of introns, their position and phasing, however, the primary amino acid sequence of voltage dependent anion channel (VDAC) proteins did not show much conservation with other plant VDACs but their secondary and tertiary structures are well conserved as predicted by in silico structural and CD spectra analyses and results show it to be a typical membrane-spanning beta-barrel leading to the formation of pore in the membrane. The heterologous expression of PgVDAC protein in yeast strain lacking the endogenous mitochondrial VDAC gene could not functionally complement it as was also previously observed for the potato VDAC. Using real-time quantitative PCR analysis it was found that transcript expression profile of PgVDAC was quantitatively and kinetically up-regulated in response to salinity, desiccation, cold and exogenous application of salicylic acid (SA); however, there was no effect of exogenous application of abscisic acid (ABA) on its expression. Constitutive over-expression of PgVDAC appears to be deleterious in transgenic rice plant; however, low level of up-regulation imparted salinity stress adaptive response. A search for a more suitable inducible transgene system is currently under way to understand PgVDAC expression levels in plant development and its role in stress adaptation.

A novel isoform of ATPase c subunit from pearl millet that is differentially regulated in response to salinity and calcium.

Vacuolar ATPases help in maintaining the pH of the vacuoles and thereby play a crucial role in the functioning of vacuolar sodium-proton antiporter. Though the various subunits that make V(1) and V(0) sector have been reported in plants their regulation is not understood completely. We have cloned three different isoforms of vacuolar ATPase subunit c (VHA-c) from Pennisetum glaucum with homologies among themselves varying from 38% to approximately 73% at the nucleic acid level. Using real-time PCR approach we have shown that the three isoforms are regulated in a tissue-specific manner under salinity stress. While isoform III is constitutively expressed in roots and shoots and does not respond to stress, isoform I is upregulated under stress. Isoform II is expressed mainly in roots; however, under salinity stress its expression is downregulated in roots and upregulated in shoots. Tissue specific expression under salinity stress of isoform II was also seen after exogenous application of calcium. This study for the first time shows the presence of three isoforms of PgVHA-c and their differential regulation during plant development, and also under abiotic stress.

Transgenic tobacco overexpressing glyoxalase pathway enzymes grow and set viable seeds in zinc-spiked soils.

We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mm ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress.

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.

A modified cDNA subtraction to identify differentially expressed genes from plants with universal application to other eukaryotes.

We have designed a simple and efficient polymerase chain reaction (PCR)-based cDNA subtraction protocol for high-throughput cloning of differentially expressed genes from plants that can be applied to any experimental system and as an alternative to DNA chip technology. Sequence-independent PCR-amplifiable first-strand cDNA population was synthesized by priming oligo-dT primer with a defined 5' heel sequence and ligating another specified single-stranded oligonucleotide primer on the 3' ends of first-strand cDNAs by T4 RNA ligase. A biotin label was introduced into the sense strands of cDNA that must be subtracted by using 5' biotinylated forward primer during PCR amplification to immobilize the sense strand onto the streptavidin-linked paramagnetic beads. The unamplified first strand (antisense) of the interrogating cDNA population was hybridized with a large excess of amplified sense strands of control cDNA. We used magnetic bead technology for the efficient removal of common cDNA population after hybridization to reduce the complexity of the cDNA prior to PCR amplification for the enrichment and sequence abundance normalization of differentially expressed genes. Construction of a subtracted and normalized cDNA library efficiently eliminates common abundant cDNA messages and also increases the probability of identifying clones differentially expressed in low-abundance cDNA messages. We used this method to successfully isolate differentially expressed genes from Pennisetum seedlings in response to salinity stress. Sequence analysis of the selected clones showed homologies to genes that were reported previously and shown to be involved in plant stress adaptation.

Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione.

Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.

Cloning and characterization of a mitochondrial glyoxalase II from Brassica juncea that is upregulated by NaCl, Zn, and ABA.

A cDNA (1061 bp) Bj glyII was cloned from a mannitol induced library of Brassica juncea. It encoded a protein of 335 amino acids with a molecular weight of 36.52 kDa. The deduced amino acid sequence of the clone showed 92% and 56% identity with Pennisetum and rice glyoxalase II, respectively, and 30% identity was observed with the human glyoxalase II. Search for the identical residues revealed the presence of highly conserved THHHXDH domain which is involved in zinc binding. p-NN and pSORT analysis of this sequence revealed a N-terminal mitochondrial target peptide. The cDNA was cloned in pMAL and a fusion protein with MBP (78 kDa) was expressed in Escherichia coli. The recombinant protein was purified approximately sixfold by affinity purification on amylose column and showed its pH optima at 7.0. The K(m) was determined to be 120 microM using S-d-lactoylglutathione as substrate. The expression of Bj glyII under various abiotic stress conditions showed that it is upregulated by salinity, heavy metal stress, and ABA.

A pea chloroplast translation elongation factor that is regulated by abiotic factors.

We report the cloning and characterization of both the cDNA (tufA) and genomic clones encoding for a chloroplast translation elongation factor (EF-Tu) from pea. The analysis of the deduced amino acids of the cDNA clone reveals the presence of putative transit peptide sequence and four GTP binding domains and two EF-Tu signature motifs in the mature polypeptide region. Using in vivo immunostaining followed by confocal microscopy pea EF-Tu was localized to chloroplast. The steady state transcript level of pea tufA was high in leaves and not detectable in roots. The expression of this gene is stimulated by light. The differential expression of this gene in response to various abiotic stresses showed that it is down-regulated in response to salinity and ABA and up-regulated in response to low temperature and salicylic acid treatment. These results indicate that regulation of pea tufA may have an important role in plant adaptation to environmental stresses.

Genetic engineering of the glyoxalase pathway in tobacco leads to enhanced salinity tolerance.

The glyoxalase pathway involving glyoxalase I (gly I) and glyoxalase II (gly II) enzymes is required for glutathione-based detoxification of methylglyoxal. We had earlier indicated the potential of gly I as a probable candidate gene in conferring salinity tolerance. We report here that overexpression of gly I+II together confers improved salinity tolerance, thus offering another effective strategy for manipulating stress tolerance in crop plants. We have overexpressed the gly II gene either alone in untransformed plants or with gly I transgenic background. Both types of these transgenic plants stably expressed the foreign protein, and the enzyme activity was also higher. Compared with nontransformants, several independent gly II transgenic lines showed improved capability for tolerating exposure to high methylglyoxal and NaCl concentration and were able to grow, flower, and set normal viable seeds under continuous salinity stress conditions. Importantly, the double transgenic lines always showed a better response than either of the single gene-transformed lines and WT plants under salinity stress. Ionic measurements revealed higher accumulation of Na+ and K+ in old leaves and negligible accumulation of Na+ in seeds of transgenic lines as compared with the WT plants. Comparison of various growth parameters and seed production demonstrated that there is hardly any yield penalty in the double transgenics under nonstress conditions and that these plants suffered only 5% loss in total productivity when grown in 200 mM NaCl. These findings establish the potential of manipulation of the glyoxalase pathway for increased salinity tolerance without affecting yield in crop plants.

Isolation and characterization of a phospholipase C delta isoform from pea that is regulated by light in a tissue specific manner.

Phosphoinositide-specific phospholipases C (PLCs) play an important role in many cellular responses and are involved in the production of secondary messengers. We report the cloning and characterization of a cDNA encoding a PLC-delta from Pisum sativum (PsPLC). The amino acid sequence deduced from the cDNA sequence showed 75-80% identity to other plant PLCs and contained the characteristic X, Y and C2 domains. The genomic PLC clone from pea was also characterized and found to contain eight introns. The protein was expressed in Escherichia coli, but the recombinant product did not show any phosphoinositide (PI)- or phosphatidylinositol-4, 5-bisphosphate (PIP2)-specific activity, despite having all known residues required for such activity, and in spite of the fact that its C2 domain was shown to bind calcium. Under similar in vitro assay conditions the recombinant tobacco PLC used as a control showed calcium-dependent PI- and PIP2-specific activity. Though PsPLC did not show enzyme activity in vitro, and may represent an inactive form of PLC, such as those reported in some mammalian systems, analysis of the transcription of PsPLC showed that the gene is expressed in all pea tissues, and is regulated by light in a tissue-specific manner. Roots showed higher expression of PsPLC than shoots. A putative PsPLC promoter region (792 bp) was also cloned and found to contain root-specific and light-responsive cis elements, suggesting that this form of PLC may be involved in important functions in plants.