AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana.

Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long-chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast-2-hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co-overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild-type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.

Towards doubling fibre yield for cotton in the semiarid agricultural area by increasing tolerance to drought, heat and salinity simultaneously.

Abiotic stresses such as extreme temperatures, water-deficit and salinity negatively affect plant growth and development, and cause significant yield losses. It was previously shown that co-overexpression of the Arabidopsis vacuolar pyrophosphatase gene AVP1 and the rice SUMO E3 ligase gene OsSIZ1 in Arabidopsis significantly increased tolerance to multiple abiotic stresses and led to increased seed yield for plants grown under single or multiple abiotic stress conditions. It was hypothesized that there might be synergistic effects between AVP1 overexpression and OsSIZ1 overexpression, which could lead to substantially increased yields if these two genes are co-overexpressed in real crops. To test this hypothesis, AVP1 and OsSIZ1 were co-overexpressed in cotton, and the impact of OsSIZ1/AVP1 co-overexpression on cotton's performance under normal growth and multiple stress conditions were analysed. It was found that OsSIZ1/AVP1 co-overexpressing plants performed significantly better than AVP1-overexpressing, OsSIZ1-overexpressing and wild-type cotton plants under single, as well as under multiple stress conditions in laboratory and field conditions. Two field studies showed that OsSIZ1/AVP1 co-overexpressing plants produced 133% and 81% more fibre than wild-type cotton in the dryland conditions of West Texas. This research illustrates that co-overexpression of AVP1 and OsSIZ1 is a viable strategy for engineering abiotic stress-tolerant crops and could substantially improve crop yields in low input or marginal environments, providing a solution for food security for countries in arid and semiarid regions of the world.

MAPK cascade gene family in Camellia sinensis: In-silico identification, expression profiles and regulatory network analysis.

BACKGROUND: Mitogen Activated Protein Kinase (MAPK) cascade is a fundamental pathway in organisms for signal transduction. Though it is well characterized in various plants, there is no systematic study of this cascade in tea. RESULT: In this study, 5 genes of Mitogen Activated Protein Kinase Kinase (MKK) and 16 genes of Mitogen Activated Protein Kinase (MPK) in Camellia sinensis were found through a genome-wide search taking Arabidopsis thaliana as the reference genome. Also, phylogenetic relationships along with structural analysis which includes gene structure, location as well as protein conserved motifs and domains, were systematically examined and further, predictions were validated by the results. The plant species taken for comparative study clearly displayed segmental duplication, which was a significant candidate for MAPK cascade expansion. Also, functional interaction was carried out in C. sinensis based on the orthologous genes in Arabidopsis. The expression profiles linked to various stress treatments revealed wide involvement of MAPK and MAPKK genes from Tea in response to various abiotic factors. In addition, the expression of these genes was analysed in various tissues. CONCLUSION: This study provides the targets for further comprehensive identification, functional study, and also contributed for a better understanding of the MAPK cascade regulatory network in C. sinensis.

Thermodynamics of ferredoxin binding to cyanobacterial nitrate reductase.

The role of the seven negatively charged amino acids of Synechocystis sp. PCC 6803 ferredoxin (Fd), i.e., Glu29, Glu30, Asp60, Asp65, Asp66, Glu92, and Glu93, predicted to form complex with nitrate reductase (NR), was investigated using site-directed mutagenesis and isothermal titration calorimetry (ITC). These experiments identified four Fd amino acids, i.e., Glu29, Asp60, Glu92, and Glu93, that are essential for the Fd binding and efficient electron transfer to the NR. ITC measurements showed that the most likely stoichiometry for the wild-type NR/wild-type Fd complex is 1:1, a Kd value 4.7 µM for the complex at low ionic strength residues and both the enthalpic and entropic components are associated with complex formation. ITC titrations of wild-type NR with four Fd variants, E29N, D60N, E92Q, and E93N demonstrated that the complex formation, although favorable, was less energetically favorable when compared to complex formation between the two wild-type proteins, suggesting that these negatively charged Fd residues at these positions are important for the effective and productive interaction with wild-type enzyme.

Overexpression of the Rice SUMO E3 Ligase Gene OsSIZ1 in Cotton Enhances Drought and Heat Tolerance, and Substantially Improves Fiber Yields in the Field under Reduced Irrigation and Rainfed Conditions.

The Arabidopsis SUMO E3 ligase gene AtSIZ1 plays important roles in plant response to abiotic stresses as loss of function in AtSIZ1 leads to increased sensitivity to drought, heat and salt stresses. Overexpression of the AtSIZ1 rice homolog, OsSIZ1, leads to increased heat and drought tolerance in bentgrass, suggesting that the function of the E3 ligase SIZ1 is highly conserved in plants and it plays a critical role in abiotic stress responses. To test the possibility that the SUMO E3 ligase could be used to engineer drought- and heat-tolerant crops, the rice gene OsSIZ1 was overexpressed in cotton. We report here that overexpression of OsSIZ1 in cotton results in higher net photosynthesis and better growth than wild-type cotton under drought and thermal stresses in growth chamber and greenhouse conditions. Additionally, this tolerance to abiotic stresses was correlated with higher fiber yield in both controlled-environment and field trials carried out under reduced irrigation and rainfed conditions. These results suggest that OsSIZ1 is a viable candidate gene to improve crop yields under water-limited and rainfed agricultural production systems.

Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants.

The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na(+)/H(+)) antiporter that transports Na(+) into the vacuole and exports H(+) into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na(+)/H(+) antiporter that exports Na(+) to the extracellular space and imports H(+) into the plant cell. Plants rely on these enzymes either to keep Na(+) out of the cell or to sequester Na(+) into vacuoles to avoid the toxic level of Na(+) in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops.

Identification and Functional Analysis of microRNAs Involved in the Anther Development in Cotton Genic Male Sterile Line Yu98-8A.

Hybrid vigor contributes in a large way to the yield and quality of cotton (Gossypium hirsutum) fiber. Although microRNAs play essential regulatory roles in flower induction and development, it is still unclear if microRNAs are involved in male sterility, as the regulatory molecular mechanisms of male sterility in cotton need to be better defined. In this study, two independent small RNA libraries were constructed and sequenced from the young buds collected from the sporogenous cell formation to the meiosis stage of the male sterile line Yu98-8A and the near-isogenic line. Sequencing revealed 1588 and 1536 known microRNAs and 347 and 351 novel miRNAs from male sterile and male fertile libraries, respectively. MicroRNA expression profiles revealed that 49 conserved and 51 novel miRNAs were differentially expressed. Bioinformatic and degradome analysis indicated the regulatory complexity of microRNAs during flower induction and development. Further RT-qPCR and physiological analysis indicated that, among the different Kyoto Encyclopedia Gene and Genomes pathways, indole-3-acetic acid and gibberellic acid signaling transduction pathways may play pivotal regulatory functions in male sterility.

Pharmacological evaluation of hyperin for antihyperglycemic activity and effect on lipid profile in diabetic rats.

Antihyperglycemic potential of hyperin at 25 and 50 mg/kg doses for 30 days to streptozotocin induced diabetic rats has been reported. In oral glucose tolerance test, hyperin treated rats showed a significant reduction in blood glucose level after 120 min. It was found that hyperin exhibited dose dependent and significant antihyperglycemic activity in streptozotocin induced diabetic rats which were nearly similar with standard drug glybenclamide. Activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, glycogen phosphorylase, glycosylated haemoglobin and level of serum urea and creatinine were significantly decreased in hyperin supplemented diabetic rats, dose dependently. Activities of hexokinase and glycogen synthase were increased with augmentation in liver glycogen, insulin and haemoglobin content in hyperin treated diabetic rats. General hematological parameters did not show any significant change in hyperin treated diabetic rats hence it is safe at these doses. Histopathological studies showed significant morphological changes in pancreatic beta-cells of streptozotocin induced diabetic rats. A decreased number of secretory granules of beta-cells were observed in diabetic rats and these pathological abnormalities were normalized after treatment with hyperin and standard drug glybenclamide. Further, hyperin decreases significant in serum total cholesterol, triglyceride, low density lipoprotein, very low density lipoprotein levels coupled with elevation of high density lipoprotein in diabetic rats. These results suggest that hyperin has a pivotal role in blood glucose level in streptozotocin induced hyperglycemia by improving the function of pancreatic islets and increasing glycolysis and decreasing gluconeogenesis.

Wound healing potential of flowers extracts of Woodfordia fruticosa Kurz.

Wound healing or repair is the body's natural process of regenerating dermal and epidermal tissue. Woodfordia fruticosa Kurz (Family: Lythraceae) is used traditionally in wound healing by the tribals of Chhattisgarh district. However, there is a paucity of scientific data in support. In this study, we evaluated antimicrobial activity of petroleum ether, chloroform, ethanolic and aqueous extracts against a diverse range of gram +ve and gram -ve bacteria along with pathogenic fungi. The wound healing activity of ethanolic extract was also evaluated at dose levels of 250 and 500 mg/kg body wt in rats by excision, incision and dead space wound healing models along with histopathology of wound area of skin. The ethanolic extract showed potent wound healing activity, as evident from the increase in the wound contraction and breaking strength in dose-dependent manner. Treatment with ethanolic extract (250 and 500 mg/kg body wt) showed significant dose-dependently decrease in epithelization period and scar area. Hydroxyproline, hexuronic acid and hexosamine contents, the important constituents of extracellular matrix of healing were also correlated with the observed healing pattern. During early wound healing phase, pro-inflammatory cytokines TNF-alpha, IL-6 and anti-inflammatory cytokine IL-10 levels were found to be upregulated by the ethanolic extract treatment. The ethanolic extract exhibited a strong and broad spectrum antimicrobial activity, as compared to other extracts. It showed very low Minimum inhibitory concentration (MIC) values and inhibited the growth of E. coli, Staphylococcus aureus and Candida albicans in concentration of 2.5 microg/disc. Thus, the results of the present study demonstrated the strong wound healing potential and antimicrobial activities of W. fruticosa, flowers, supporting the folklore use of the plant by the tribal people of Chhattisgarh district.

Achieving abiotic stress tolerance in plants through antioxidative defense mechanisms.

Climate change has increased the overall impact of abiotic stress conditions such as drought, salinity, and extreme temperatures on plants. Abiotic stress adversely affects the growth, development, crop yield, and productivity of plants. When plants are subjected to various environmental stress conditions, the balance between the production of reactive oxygen species and its detoxification through antioxidant mechanisms is disturbed. The extent of disturbance depends on the severity, intensity, and duration of abiotic stress. The equilibrium between the production and elimination of reactive oxygen species is maintained due to both enzymatic and non-enzymatic antioxidative defense mechanisms. Non-enzymatic antioxidants include both lipid-soluble (α-tocopherol and ß-carotene) and water-soluble (glutathione, ascorbate, etc.) antioxidants. Ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) are major enzymatic antioxidants that are essential for ROS homeostasis. In this review, we intend to discuss various antioxidative defense approaches used to improve abiotic stress tolerance in plants and the mechanism of action of the genes or enzymes involved.

Flavanones: A potential natural inhibitor of the ATP binding site of PknG of Mycobacterium tuberculosis.

Over the years, Mycobacterium tuberculosis has been one of the major causes of death worldwide. As several clinical isolates of the bacteria have developed drug resistance against the target sites of the current therapeutic agents, the development of a novel drug is the pressing priority. According to recent studies on Mycobacterium tuberculosis, ATP binding sites of Mycobacterium tuberculosis serine/threonine protein kinases (MTPKs) have been identified as the new promising drug target. Among the several other protein kinases (PKs), Protein kinase G (PknG) was selected for the study because of its crucial role in modulating bacterium's metabolism to survive in host macrophages. In this work, we have focused on the H37Rv strain of Mycobacterium tuberculosis. A list of 477 flavanones obtained from the PubChem database was docked one by one against the crystallized and refined structure of PknG by in-silico techniques. Initially, potential inhibitors were narrowed down by preliminary docking. Flavanones were then selected using binding energies ranging from -7.9 kcal.mol-1 to -10.8 kcal.mol-1. This was followed by drug-likeness prediction, redocking analysis, and molecular dynamics simulations. Here, we have used experimentally confirmed drug AX20017 as a reference to determine candidate compounds that can act as potential inhibitors for PknG. PubChem165506, PubChem242065, PubChem688859, PubChem101367767, PubChem3534982, and PubChem42607933 were identified as possible target site inhibitors for PknG with a desirable negative binding energy of -8.1, -8.3, -8.4, -8.8, -8.6 and -7.9 kcal.mol-1 respectively. Communicated by Ramaswamy H. Sarma.

In-silico genome wide analysis of Mitogen activated protein kinase kinase kinase gene family in C. sinensis.

Mitogen activated protein kinase kinase kinase (MAPKKK) form the upstream component of MAPK cascade. It is well characterized in several plants such as Arabidopsis and rice however the knowledge about MAPKKKs in tea plant is largely unknown. In the present study, MAPKKK genes of tea were obtained through a genome wide search using Arabidopsis thaliana as the reference genome. Among 59 candidate MAPKKK genes in tea, 17 genes were MEKK-like, 31 genes were Raf-like and 11 genes were ZIK- like. Additionally, phylogenetic relationships were established along with structural analysis, which includes gene structure, its location as well as conserved motifs, cis-acting regulatory elements and functional domain signatures that were systematically examined. Also, on the basis of one orthologous gene found between tea and Arabidopsis, functional interaction was carried out in C. sinensis based on an Arabidopsis association model. The expressional profiles indicated major involvement of MAPKKK genes from tea in response to various abiotic stress factors. Taken together, this study provides the targets for additional inclusive identification, functional study, and provides comprehensive knowledge for a better understanding of the MAPKKK cascade regulatory network in C. sinensis.

NHX Gene Family in Camellia sinensis: In-silico Genome-Wide Identification, Expression Profiles, and Regulatory Network Analysis.

Salt stress affects the plant growth and productivity worldwide and NHX is one of those genes that are well known to improve salt tolerance in transgenic plants. It is well characterized in several plants, such as Arabidopsis thaliana and cotton; however, not much is known about NHXs in tea plant. In the present study, NHX genes of tea were obtained through a genome-wide search using A. thaliana as reference genome. Out of the 9 NHX genes in tea, 7 genes were localized in vacuole while the remaining 2 genes were localized in the endoplasmic reticulum (ER; CsNHX8) and plasma membrane (PM; CsNHX9), respectively. Furthermore, phylogenetic relationships along with structural analysis which includes gene structure, location, and protein-conserved motifs and domains were systematically examined and further, predictions were validated by the expression analysis. The dN/dS values show that the majority of tea NHX genes is subjected to strong purifying selection under the course of evolution. Also, functional interaction was carried out in Camellia sinensis based on the orthologous genes in A. thaliana. The expression profiles linked to various stress treatments revealed wide involvement of NHX genes from tea in response to various abiotic factors. This study provides the targets for further comprehensive identification, functional study, and also contributed for a better understanding of the NHX regulatory network in C. sinensis.

Melanin production by Pseudomonas sp. and in silico comparative analysis of tyrosinase gene sequences.

Background: Melanin finds enormous applications in different industries for its unique photoprotective and anti-oxidant properties. Due to its emerging demand, scientific researchers are putting efforts to unravel more microorganisms with a potential of producing melanin on large scale. Hence, the present study was aimed at the isolation of extracellular melanin producing microorganisms from lime quarries of Karnataka, India. Besides this, the tyrosinase gene governing melanin synthesis in different organisms were compared in silico to understand its evolutionary aspects. Material and methods: Melanin producing microorganisms were screened on tyrosine gelatin beef extract agar medium. Potential isolate was explored for submerged production of melanin in broth containing L-tyrosine. Melanin was characterized by UV-Vis spectroscopy, thin layer and high performance liquid chromatographic techniques. Antibacterial activity of melanin was performed by agar well assay. Comparative tyrosinase gene sequence analysis was performed by using Geneious 2021.1 trial version software. Results: Pseudomonas otitidis DDB2 was found to be potential for melanin production. No antibacterial activity was exerted by the melanin against tested pathogens. The in silico studies showed that the common central domain of tyrosinase protein sequence of selected Pseudomonas sps. exhibited 100% identity with the common central domain of Homo sapiens tyrosinase (NP_000363.1). Conclusions: Our study shows the production of melanin in good quantities by the isolate Pseudomonas otitidis DDB2 which can be explored for scale-up process. Since the melanin formed is of eumelanin type and the tyrosinase gene sequence of several Pseudomonas sp. showed relatedness to humans, this molecule may be further developed for sunscreen formulations.

Characterization and Cytotoxicity of Pseudomonas Mediated Rhamnolipids Against Breast Cancer MDA-MB-231 Cell Line.

A biosurfactant producing bacterium was identified as Pseudomonas aeruginosa DNM50 based on molecular characterization (NCBI accession no. MK351591). Structural characterization using MALDI-TOF revealed the presence of 12 different congeners of rhamnolipid such as Rha-C8-C8:1, Rha-C10-C8:1, Rha-C10-C10, Rha-C10-C12:1, Rha-C16:1, Rha-C16, Rha-C17:1, Rha-Rha-C10:1-C10:1, Rha-Rha-C10-C12, Rha-Rha-C10-C8, Rha-Rha-C10-C8:1, and Rha-Rha-C8-C8. The radical scavenging activity of rhamnolipid (DNM50RL) was determined by 2, 3-diphenyl-1-picrylhydrazyl (DPPH) assay which showed an IC50 value of 101.8 µg/ ml. The cytotoxic activity was investigated against MDA-MB-231 breast cancer cell line by MTT (4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay which showed a very low IC50 of 0.05 µg/ ml at 72 h of treatment. Further, its activity was confirmed by resazurin and trypan blue assay with IC50 values of 0.01 µg/ml and 0.64 µg/ ml at 72 h of treatment, respectively. Thus, the DNM50RL would play a vital role in the treatment of breast cancer targeting inhibition of p38MAPK.

Do Social Connections and Digital Technologies Act as Social Cure During COVID-19?

Although COVID-19 pandemic has re-orientated humans to be more physically healthy and hygienic, it has also persuaded humans to create affiliations and experience a sense of belongingness through social networks and digital technologies. However, amidst these changes, experiences of COVID-19 patients and their perception of the outside world's attitudes toward them appears to be less attended in literature which formed the basis for the current study's objectives. Using qualitative methodology, the present study explored the experiences, perceptions and attitudes of patients and their care-givers' toward COVID-19. The thematic analysis emerged with four major themes. Psychological Experiences of People was generated prominently with sub-themes indicating the perceived experiences like fear of spreading diseases to others, and the need for psychological counseling. Attitude of others toward patients and caregivers revealed that family members and relatives played a major positive role on the patient's mental health, however, the neighbor's stigmatized attitude led to several undesired behaviors. Social Connectedness was another major theme derived from the study. Altruistic volunteers, a sub-theme of Social connectedness have indicated that amidst these negative factors, one can spread social harmony by motivating and supporting the victims with basic needs, financial support, hope and social empathy. Opinions of participants for digital technology through technological aids and preventive measures emphasized an overall positive attitude as it helped the society, in general to maintain social connections as well to curb the rate of COVID-19 cases.

Improving drought-, salinity-, and heat-tolerance in transgenic plants by co-overexpressing Arabidopsis vacuolar pyrophosphatase gene AVP1 and Larrea Rubisco activase gene RCA.

The severity and frequency of many abiotic stresses such as drought, salinity and heat, cause substantial crop losses worldwide, which poses a serious challenge in food security. To increase crop production, new approaches are needed. Previous research has shown that overexpression of the tonoplast H+ pyrophosphatase gene AVP1 leads to improved drought and salt tolerance in transgenic plants. Other research showed that overexpression of thermotolerant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene could maintain photosynthesis at higher temperatures, which contributes to higher heat tolerance in transgenic plants. In nature, abiotic stresses rarely come alone, instead these stresses often occur in various combinations. Therefore, it is desirable to make crops more tolerant to multiple stresses, which will likely lead to higher crop yield under various stress conditions. It is shown here that co-overexpression of the Arabidopsis gene AVP1 and the Larrea Rubisco activase gene RCA significantly increases drought, salinity and heat tolerance, resulting in higher biomass and seed yield than wild-type plants. AVP1/RCA co-overexpressing plants are as more drought- and salt-tolerant as AVP1-overexpressing plants, and as more heat-tolerant as RCA-overexpressing plants. More importantly, they produce higher seed yields than AVP1-overexpressing, RCA-overexpressing, and wild-type plants under combined drought and heat conditions.

Overexpression of the rice gene OsSIZ1 in Arabidopsis improves drought-, heat-, and salt-tolerance simultaneously.

Sumoylation is one of the post translational modifications, which affects cellular processes in plants through conjugation of small ubiquitin like modifier (SUMO) to target substrate proteins. Response to various abiotic environmental stresses is one of the major cellular functions regulated by SUMO conjugation. SIZ1 is a SUMO E3 ligase, facilitating a vital step in the sumoylation pathway. In this report, it is demonstrated that over-expression of the rice gene OsSIZ1 in Arabidopsis leads to increased tolerance to multiple abiotic stresses. For example, OsSIZ1-overexpressing plants exhibited enhanced tolerance to salt, drought, and heat stresses, and generated greater seed yields under a variety of stress conditions. Furthermore, OsSIZ1-overexpressing plants were able to exclude sodium ions more efficiently when grown in saline soils and accumulate higher potassium ions as compared to wild-type plants. Further analysis revealed that OsSIZ1-overexpressing plants expressed higher transcript levels of P5CS, a gene involved in the biosynthesis of proline, under both salt and drought stress conditions. Therefore, proline here is acting as an osmoprotectant to alleviate damages caused by drought and salt stresses. These results demonstrate that the rice gene OsSIZ1 has a great potential to be used for improving crop's tolerance to several abiotic stresses.

Co-overexpression of AVP1 and PP2A-C5 in Arabidopsis makes plants tolerant to multiple abiotic stresses.

Abiotic stresses are major threats to agricultural production. Drought and salinity as two of the major abiotic stresses cause billions of losses in agricultural productivity worldwide each year. Thus, it is imperative to make crops more tolerant. Overexpression of AVP1 or PP2A-C5 was previously shown to increase drought and salt stress tolerance, respectively, in transgenic plants. In this study, the hypothesis that co-overexpression of AVP1 and PP2A-C5 would combine their respective benefits and further improve salt tolerance was tested. The two genes were inserted into the same T-DNA region of the binary vector and then introduced into the Arabidopsis genome through Agrobacterium-mediated transformation. Transgenic Arabidopsis plants expressing both AVP1 and PP2A-C5 at relatively high levels were identified and analyzed. These plants displayed enhanced tolerance to NaCl compared to either AVP1 or PP2A-C5 overexpressing plants. They also showed tolerance to other stresses such as KNO3 and LiCl at harmful concentrations, drought, and phosphorus deficiency at comparable levels with either AVP1 or PP2A-C5 overexpressing plants. This study demonstrates that introducing multiple genes in single T-DNA region is an effective approach to create transgenic plants with enhanced tolerance to multiple stresses.

The yield difference between wild-type cotton and transgenic cotton that expresses IPT depends on when water-deficit stress is applied.

Drought is the No. 1 factor that limits agricultural production in the world, thus, making crops more drought tolerant is a major goal in agriculture. Many genes with functions in abiotic stress tolerance were identified, and overexpression of these genes confers increased drought tolerance in transgenic plants. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis is one of them. Interestingly, when IPT-transgenic cotton was field-tested at two different sites, Texas and Arizona, different results were obtained. To explain this phenomenon, reduced irrigation experiments with different timing in applying water deficit stress were conducted. It was found that the timing of water deficit stress is critical for IPT-transgenic cotton to display its yield advantage over control plants (i.e. wild-type and segregated non-transgenic plants). If water deficit stress occurs before flowering (vegetative phase), IPT-transgenic cotton would outperform control plants; however, if water deficit stress occurs at or after flowering (reproductive phase), there would not be a yield difference between IPT-transgenic and control cotton plants. This result suggests that an early induction of IPT expression (before first flowering) is needed in order to realize the benefits of IPT-expression in transgenic plants that face water-deficit stress later in development.