Large-scale identification of leaf senescence-associated genes

Leaf senescence is a form of programmed cell death, and is believed to involve preferential expression of a specific set of "senescence-associated genes" (SAGs). To decipher the molecular mechanisms and the predicted complex network of regulatory pathways involved in the senescence program, we have carried out a large-scale gene identification study in a reference plant, Arabidopsis thaliana. Using suppression subtractive hybridization, we isolated approximately 800 cDNA clones representing SAGs expressed in senescing leaves. Differential expression was confirmed by Northern blot analysis for 130 non-redundant genes. Over 70 of the identified genes have not previously been shown to participate in the senescence process. SAG-encoded proteins are likely to participate in macromolecule degradation, detoxification of oxidative metabolites, induction of defense mechanisms, and signaling and regulatory events. Temporal expression profiles of selected genes displayed several distinct patterns, from expression at a very early stage, to the terminal phase of the senescence syndrome. Expression of some of the novel SAGs, in response to age, leaf detachment, darkness, and ethylene and cytokinin treatment was compared. The large repertoire of SAGs identified here provides global insights about regulatory, biochemical and cellular events occurring during leaf senescence.     

Rapid identification of potential drought tolerance genes from Solanum tuberosum by using a yeast functional screening method

Identification of major stress tolerance genes of a crop plant is important for the rapid development of its stress-tolerant cultivar. Here, we used a yeast functional screen method to identify potential drought-tolerance genes from a potato plant. A cDNA expression library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic stress conditions. The relative tolerances of the selected yeast transformants to multiple abiotic stresses were also studied. Specific potato cDNAs expressed in the tolerant yeast transformants were identified. Sixty-nine genes were found capable of enhancing hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated, 12 genes were selected, which could be most effective in imparting higher drought tolerance to potato with better survival in salt and high-temperature stresses. Orthologues of few genes identified here are previously known to increase osmotic stress tolerance of yeast and plants; however, specific studies are needed to confirm their role in the osmotic stress tolerance of potato.     

Identification of drought responsive proteins using gene ontology hierarchy

The availability of the complete genome sequences has facilitated access to essential information to identify proteins. The determination of Arabidopsis genome sequence has had a great impact to annotate data. The genome sequencing of Sorghum bicolor has been only recently completed and hither to the global response to abiotic stresses in this important crop remains largely unexplored. We used 2-D gel electrophoresis based proteomic approach refined with MALDI-TOF to analyze drought-stress response proteins in sorghum. Major changes in protein complement of sorghum were observed in hydroponic cultures at 96 hours under drought stress. Six most highly expressed proteins were excised for functional identification. Here, we developed a method to obtain functional distances between GO terms and analyzed distance values to allocate shortest path (SP) in GO hierarchy. The shortest paths for expressed proteins were noted for most informative common ancestor (MICA) terms, viz. binding, catalytic activity and primary metabolic process. We observed the expressed proteins belonged to the functional group of signal transduction mechanisms, carbohydrate transport and metabolism. These identified functions of proteins suggest a different mechanism of drought-stress tolerant in sorghum. The novel approach applied in this study may have great importance in further identifying proteins involved in abiotic and biotic stress conditions in crops.     

Allele mining for a drought responsive gene DRO1 determining root growth angle in donors of drought tolerance in rice (Oryza sativa L.)

Deeper Rooting 1 (DRO1) gene identified from a major QTL on chromosome 9 increases the root growth angle (RGA) and thus facilitates survival under drought and hence is an excellent candidate for rice improvement. Twenty-four major Indian upland and lowland genotypes including the ‘yield under drought’ (DTY) QTL donors were subjected to allele mining of DRO1 (3058 bp) using four pairs of overlapping primers. A total of 216 and 52 SNPs were identified across all genotypes in the gene and coding region (756 bp) respectively with transversions 3.6 fold more common than transitions in the gene and 2.5 times in the CDS. In 251 amino acid long protein, substitutions were found in 19 positions, wherein change in position 92 was the most frequent. Based on allele mining, the 24 genotypes can be classified into 16 primary structure variants ranging from complete functional allele (Satti, IR36 and DTY 3.1 donor, IR81896-B-B-195) to truncated non-functional alleles in PMK2, IR64, IR20 and Swarna. All the DTY donors, other than IR81896-B-B-195, and most of the upland drought tolerant cultivars (Nagina 22, Vandana and Dhagaddeshi) had accumulated 6–19 SNPs and 4–8 amino acid substitutions resulting in substantial differences in their protein structure. The expression analysis revealed that all the genotypes showed upregulation under drought stress though the degree of upregulation varied among genotypes. The information on structural variations in DRO1 gene will be very useful for the breeders, especially in the light of recent breeding programmes on improving drought tolerance using several DTY donors and upland accessions.Supplementary InformationThe online version of this article (10.1007/s12298-021-00950-2).     

Physiological insights into sulfate and selenium interaction to improve drought tolerance in mung bean

The present study involved two pot experiments to investigate the response of mung bean to the individual or combined SO₄²⁻ and selenate application under drought stress. A marked increment in biomass and NPK accumulation was recorded in mung bean seedlings fertilized with various SO₄²⁻ sources, except for CuSO₄. Compared to other SO₄²⁻ fertilizers, ZnSO₄ application resulted in the highest increase in growth attributes and shoot nutrient content. Further, the combined S and Se application (S + Se) significantly enhanced relative water content (16%), SPAD value (72%), photosynthetic rate (80%) and activities of catalase (79%), guaiacol peroxidase (53%) and superoxide dismutase (58%) in the leaves of water-stressed mung bean plants. Consequently, the grain yield of mung bean was markedly increased by 105% under water stress conditions. Furthermore, S + Se application considerably increased the concentrations of P (47%), K (75%), S (80%), Zn (160%), and Fe (15%) in mung bean seeds under drought stress conditions. These findings indicate that S + Se application potentially increases the nutritional quality of grain legumes by stimulating photosynthetic apparatus and antioxidative machinery under water deficit conditions. Our results could provide the basis for further experiments on cross-talk between S and Se regulatory pathways to improve the nutritional quality of food crops.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00992-6.     

Analysis of drought responsive proteins in wheat (Triticum durum) by 2D-PAGE and MALDI-TOF mass spectrometry

Drought is an abiotic stress that strongly influences plant growth, development and productivity. By proteomics study it is possible to identify the complex mechanism of water-stress response. The aim of this research was the analysis of wheat (Triticum durum) proteome changes under stress conditions by applying a severe water deficit treatment for 7 days. Stress-induced proteome changes were analyzed by two-dimensional gel electrophoresis in conjunction with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Thirty-six protein spots showed a reproducible significant change between control and stressed samples. The reasonable implications in drought response of the identified proteins were discussed. Results provide new insights that can lead to a better understanding of the molecular basis of drought-sensitivity in plants. Therefore, the obtained data could suggest the development of drought resistant wheat varieties, in order to improve agricultural production in dry regions.     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems. Received: 24 March 1997 / Accepted: 26 November 1997     

Sequence similarity based identification of abiotic stress responsive genes in chickpea

Chickpea (Cicer arietinum L.) is an important food legume crop, particularly for the arid regions including Indian subcontinent. Considering the detrimental effect of drought, temperature and salt stress on crop yield, efforts have been initiated in the direction of developing improved varieties and designing alternate strategies to sustain chickpea production in adverse environmental conditions. Identification of genes that confer abiotic stress tolerance in plants remains a challenge in contemporary plant breeding. The present study focused on the identification of abiotic stress responsive genes in chickpea based on sequence similarity approach exploiting known abiotic stress responsive genes from model crops or other plant species. Ten abiotic stress responsive genes identified in other plants were partially amplified from eight chickpea genotypes and their presence in chickpea was confirmed after sequencing the PCR products. These genes have been functionally validated and reported to play significant role in stress response in model plants like Arabidopsis, rice and other legume crops. Chickpea EST sequences available at NCBI EST database were used for the identification of abiotic stress responsive genes. A total of 8,536 unique coding long sequences were used for identification of chickpea homologues of these abiotic stress responsive genes by sequence similarity search (BLASTN and BLASTX). These genes can be further explored towards achieving the goal of developing superior chickpea varieties providing improved yields under stress conditions using modern molecular breeding approaches.     

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.     

Transient gene expression in electroporated bean cotyledon protoplasts

A protocol has been developed for the introduction of foreign DNA into cell protoplasts from immature bean cotyledons. The method yields high amounts of the reporter enzymes β-glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) when cognate genes are driven by the promoter and upstream sequences of a bean β-phas gene. Comparisons with expression in stable tobacco transformants indicate that transient assays can be used to investigate enhancer function. This techniques should be applicable to other gene systems as well.