Rab7 GTPase-Mediated stress signaling enhances salinity tolerance in AlRabring7 tobacco transgenics by modulating physio-biochemical parameters.

The RING-type E3 ligases play a significant role in stress signaling, primarily through post-translational regulation. Ubiquitination is a crucial post-translational modification that regulates the turnover and activity of proteins. The overexpression of AlRabring7, RING-HC E3 Ub ligase in tobacco provides insights into the regulation of salinity and ABA signaling in transgenic tobacco. The seed germination potential of AlRabring7 transgenics was higher than WT, with NaCl and ABA treatments. The transgenics showed improved morpho-physio-biochemical parameters in response to salinity and ABA treatments. The photosynthetic pigments, soluble sugars, reducing sugars and proline increased in transgenics in response to NaCl and ABA treatments. The decreased ROS accumulation in transgenics on NaCl and ABA treatments can be co-related to improved activity of enzymatic and non-enzymatic antioxidants. The potential of transgenics to maintain ABA levels with ABA treatment, highlights the active participation of ABA feedback loop mechanism. Interestingly, the ability of AlRabring7 transgenics to upregulate Rab7 protein, suggests its role in facilitating vacuolar transport. Furthermore, the improved potassium accumulation and reduced sodium content indicate an efficient ion regulation mechanism in transgenic plants facilitating higher stomatal opening. The expression of downstream ion transporter (NbNHX1 and NbVHA1), ABA signaling (NbABI2 and NbABI5) and vesicle trafficking (NbMON1) responsive genes were upregulated with stress. The present study, reports that AlRabring7 participates in maintaining vacuolar transport, ion balance, ROS homeostasis, stomatal regulation through activation of Rab7 protein and regulation of downstream stress-responsive during stress. This emphasizes the potential of AlRabring7 gene for improved performance and resilience in challenging environments.

Deciphering the potential of Sargassum tenerrimum extract: metabolic profiling and pathway analysis of groundnut (Arachis hypogaea) in response to Sargassum extract and Sclerotium rolfsii.

Seaweed extracts have enormous potential as bio-stimulants and demonstrated increased growth and yield in different crops. The presence of physiologically active component stimulate plant stress signaling pathways, enhances growth and productivity, as well as serve as plant defense agents. The seaweed extracts can reduce the use of chemicals that harm the environment for disease management. In the present study, the Sargassum tenerrimum extract treatment was applied, alone and in combination with Sclerotium rolfsii, to Arachis hypogea, to study the differential metabolite expression. The majority of metabolites showed maximum accumulation with Sargassum extract-treated plants compared to fungus-treated plants. The different classes of metabolite compounds like sugars, carboxylic acids, polyols, showed integrated peaks in different treatments of plants. The sugars were higher in Sargassum extract and Sargassum extract + fungus treatments compared to control and fungus treatment, respectively. Interestingly, Sargassum extract + fungus treatment showed maximum accumulation of carboxylic acids. Pathway enrichment analysis showed regulation of different metabolites, highest impact with galactose metabolism pathway, identifying sucrose, myo-inositol, glycerol and fructose. The differential metabolite profiling and pathway analysis of groundnut in response to Sargassum extract and S. rolfsii help in understanding the groundnut- S. rolfsii interactions and the potential role of the Sargassum extract towards these interactions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01418-9.

Transcriptomic analysis towards identification of defence-responsive genes and pathways upon application of Sargassum seaweed extract on tomato plants infected with Macrophominaphaseolina.

The charcoal-rot caused by Macrophomina phaseolina is one of the major disease in many economically important crop plants including tomato. The molecular responses of the host plant against the M. phaseolina are poorly stated. In the present study, for the first time the molecular insight of tomato-Macrophomina interaction and Sargassum tenerrimum extract (SE) toward managing disease through RNA-seq approach is established. A total of 449 million high-quality reads (HQRs) were obtained and aligned to the tomato genome with an average mapping of 89.12%. The differentially expressed genes (DEGs) regulated across the different treatment pairs were identified. Several DEGs, such as receptor-like kinases (SlRLKs), transcription factors including SlWRKY70, SlGRAS4, SlERF4, SlERF25, pathogenesis related-1 (SlPR1), SlPR2, endochitinase and peroxidase were significantly up-regulated in SE + Macrophomina treated sample as compared to only Macrophomina treated sample. The crosstalk between salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) was a key factor to regulate resistance in tomato during SE + Macrophomina treatment. The KEGG pathway including plant hormone signal transduction, plant-pathogen interaction and mitogen-activated protein kinase (MAPK) signaling pathway were significantly enriched. The RNA-seq data were validated through qPCR using 12 disease-responsive genes and correlated significantly with R2 = 0.73. The present study suggests that SE act as an elicitor molecule and activate the defence-related pathways similar to PAMP-triggered immunity in tomato. The jasmonic acid (JA) mediated signaling pathway was identified as a key factor to induce resistance in tomato against Macrophomina infection. The present study depicts the beneficial effects of SE by regulating molecular mechanism towards defence responses in tomato against Macrophomina infection. The application of SE brings out new prospects to induce disease tolerance in the agricultural crops. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03565-4.

The Jatropha leaf curl Gujarat virus on infection in Jatropha regulates the sugar and tricarboxylic acid cycle metabolic pathways.

Jatropha, a popular biodiesel crop, suffers severe losses due to Jatropha leaf curl Gujarat virus (JLCuGV) infection in Gujarat (India). Metabolite profiling can help to understand the plant's innate immune response to geminivirus infection. Our study aims to compare metabolic profiles of an infected and healthy plant to unravel the changes in biochemical pathways on geminivirus infection in Jatropha. Gas chromatography-mass spectrometry (GC-MS) analysis was performed in healthy and infected tissue of Jatropha field plants which were identified to be infected with geminivirus. GC-MS analysis revealed that the metabolites like sugars, polyols, carboxylic acids, fatty acids, polyphenols, and amino acids were regulated on JLCuGV infection. The sugars (glucose, sucrose, and fructose) increased, while carboxylic acids (malic acid, citric acid and quinic acid) and polyols (galactinol, butanetriol, triethylene glycol, myo-inositol, erythritol) decreased remarkably in infected Jatropha tissue. All these metabolic variations indicated that sugar metabolism and tricarboxylic acid (TCA) cycle pathways are regulated as a defense response and a disease development response to geminivirus infection in Jatropha.

Expression of B. subtilis Phytase gene driven by fruit specific E8 promoter for enhanced minerals, metabolites and phytonutrient in cucumber fruit.

The fruit nutrigenomics is an interesting and important research area towards nutrition enhancement. The phytic acid is one of the major antinutrient compound, present in seeded fruits and crops. It hinders the absorption of iron (Fe), zinc (Zn), magnesium (Mg), potassium (K) and calcium (Ca), causing mineral deficiencies. In the present study, the BsPhy gene was overexpressed in the cucumber fruits using the tomato fruit specific E8 and constitutive CaMV 35S promoter. The E8 promoter imparted heterologous expression of GUS gene in cucumber fruits, furthermore, the fruit specific expression of E8 promoter with BsPhy gene was confirmed in transgenics (E8::BsPhy) using anti rabbit-phytase antibody. The physio-biochemical analysis of transgenics revealed, maximum phytase activity in E8::BsPhy cucumber fruits at 10 days after anthesis (DAA) compared to 35S::BsPhy and wild-type (WT) fruits. Consequently, E8::BsPhy fruits also showed increased amount of inorganic phosphorus (Pi), total phosphorus (P), minerals (Zn, Fe, Mg, K, Ca), total carotenoid and other macronutrients at 10 DAA compared to 35S::BsPhy fruits. The metabolite profiling of fruits (10 DAA) showed increased sugars, amino acids, sugar acids and polyols, in both E8::BsPhy and 35S::BsPhy transgenics suggesting higher phytate metabolism, compared to WT fruits. Interestingly, both the transgenic fruits showed higher fruit biomass and yield along with improved nutritional quality, which can be attributed to increased P and Zn contents in transgenic fruits, compared to WT fruits. Our findings reveal that the BsPhy gene enhances minerals and macronutrients in transgenic cucumber fruits making it nutritious and healthy.

Mechanism of high affinity potassium transporter (HKT) towards improved crop productivity in saline agricultural lands.

Glycophytic plants are susceptible to salinity and their growth is hampered in more than 40 mM of salt. Salinity not only affects crop yield but also limits available land for farming by decreasing its fertility. Presence of distinct traits in response to environmental conditions might result in evolutionary adaptations. A better understanding of salinity tolerance through a comprehensive study of how Na+ is transported will help in the development of plants with improved salinity tolerance and might lead to increased yield of crops growing in strenuous environment. Ion transporters play pivotal role in salt homeostasis and maintain low cytotoxic effect in the cell. High-affinity potassium transporters are the critical class of integral membrane proteins found in plants. It mainly functions to remove excess Na+ from the transpiration stream to prevent sodium toxicity in the salt-sensitive shoot and leaf tissues. However, there are large number of HKT proteins expressed in plants, and it is possible that these members perform in a wide range of functions. Understanding their mechanism and functions will aid in further manipulation and genetic transformation of different crops. This review focuses on current knowledge of ion selectivity and molecular mechanisms controlling HKT gene expression. The current review highlights the mechanism of different HKT transporters from different plant sources and how this knowledge could prove as a valuable tool to improve crop productivity.

AlRab7 from Aeluropus lagopoides ameliorates ion toxicity in transgenic tobacco by regulating hormone signaling and reactive oxygen species homeostasis.

The plants endomembrane system of the cellular compartments with its complex membrane trafficking network facilitates transport of macromolecules. The endomembrane dynamics are essential for maintaining basic and specific cellular functions including adaptation to the extracellular environment. The plant vacuole serves as a reservoir for nutrients and toxic metabolites and performs detoxification processes to maintain cellular homeostasis. The overexpression of AlRab7, a vesicle trafficking gene from Aeluropus lagopoides, improved germination and growth and reduced ionic and oxidative stress in transgenics. Moreover, the root and shoot of transgenic tobacco showed differential accumulation of phytohormone ABA and IAA with different ionic stresses. The improved growth (root and shoot length) can be co-related with higher IAA accumulation with NaCl stress. The low Na+ /K+ ratio with different NaCl stress treatments indicates better ion homeostasis in transgenics. Furthermore, the increased stomatal density and higher number of open stomata on both leaf surfaces in transgenics during NaCl stress suggest better gaseous exchange/functioning of guard cells. The maintained or increased superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase antioxidative enzyme activities suggest that an extensive reactive oxygen species (ROS) scavenging system was triggered to detoxify cellular ROS, which remained at low levels in transgenics during the different stress treatments. Our results suggest that the AlRab7 transgenic tobacco ameliorates ionic stress by facilitating differential and selective ion transport at vacuolar membrane regulating hormone signaling, ROS homeostasis, stomatal development, and movement.

Molecular cloning and characterization of high-affinity potassium transporter (AlHKT2;1) gene promoter from halophyte Aeluropus lagopoides.

HKT subfamily II functions as Na+- K+ co-transporter and prevents plants from salinity stress. A 760 bp promoter region of AlHKT2;1 was isolated, sequenced and cloned. The full length promoter D1, has many cis-regulatory elements like MYB, MBS, W box, ABRE etc. involved in abiotic stress responses. D1 and subsequent 5' deletions were cloned into pCAMBIA1301 and studied for its efficacy in stress conditions in heterologous system. Blue colour staining was observed in flower petals, anther lobe, and dehiscence slit of anther in T0 plants. The T1 seedlings showed staining in leaf veins, shoot vasculature and root except root tip. T1 seedlings were subjected to NaCl, KCl, NaCl + KCl and ABA stresses. GUS activity was quantified by 4-methylumbelliferyl glucuronide (4-MUG) assay under control and stress conditions. The smallest deletion- D4 also showed GUS expression but highest activity was observed in D2 as compared to full length promoter and other deletions. The electrophoretic mobility shift assay using stress-induced protein with different promoter deletions revealed more prominent binding in D2. These results suggest that AlHKT2;1 promoter is involved in abiotic stress response and deletion D2 might be sufficient to drive the stress-inducible expression of various genes involved in providing stress tolerance in plants.

Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors.

The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs (amiRNAs; C1/C4, C2/C3 and V1/V2) were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.

Overexpression of JcWRKY2 confers increased resistance towards Macrophomina phaseolina in transgenic tobacco.

WRKY proteins are plant-specific transcription factors (TFs), and form one of the largest families and are involved in plant development and responses to stress. The salicylic acid (SA) responsive WRKY family auto or cross-regulate the defence stress signalling pathways. In this study, we functionally validated the role of JcWRKY2 gene from biofuel crop Jatropha curcas towards improving resistance to tobacco transgenic against charcoal rot causing necrotrophic fungus, Macrophomina phaseolina. The microscopic studies revealed that JcWRKY2 participated in preventing the spread of infection in transgenic. The generation of H2O2 during M. phaseolina and combinatorial stress in transgenic induces the expression and activity of antioxidant enzymes. The transcript expression of SA biosynthetic (NtICS1) gene, pathogenesis-related (NtPR-10) gene and antioxidative enzymes (NtCAT1 and NtSOD) gene revealed that JcWRKY2 transgenic play a role in SA-mediated, antioxidative enzymes regulation during biotic challenges. The study highlights the potential of JcWRKY2 as an important regulator for plant biotic stress responses through the SA-dependent pathway.

The AlRabring7 E3-Ub-ligase mediates AlRab7 ubiquitination and improves ionic and oxidative stress tolerance in Saccharomyces cerevisiae.

The maintenance of ROS homeostasis, membrane biogenesis and recycling of molecules are common stress responses involving specific and complex regulatory network. Ubiquitination is an important and common mechanism which facilitates environmental adaptation in eukaryotes. In the present study we have cloned the AlRabring7, an E3-Ub-ligase, previously identified as AlRab7 interacting partner. The role of AlRabring7 for ubiquitinating AlRab7 and facilitating stress tolerance is analysed. The AlRabring7, with an open-reading frame of 702 bp encodes a protein of 233 amino acids, with RING-HC domain of 40 amino acids. In silico analysis shows that AlRabring7 is a C3HC4-type RING E3 Ub ligase. The protein - protein docking show interaction dynamics between AlRab7-AlRabring7-Ubiquitin proteins. The AlRab7 and AlRabring7 transcript showed up-regulation in response to different salts i.e: NaCl, KCl, CaCl2, NaCl + KCl, NaCl + CaCl2, imposing ionic as well as hyperosmotic stress, and also with oxidative stress by H2O2 treatment. Interestingly, the AlRabring7 showed early transcript expression with maximum expression in shoots on combinatorial stresses. The AlRab7 showed delayed and maximum expression with NaCl + CaCl2 stress treatment. The AlRab7 complements yeast ypt7Δ mutants and restored the fragmented vacuole. The in vitro ubiquitination assay revealed that AlRabring7 function as E3 ubiquitin ligase and mediates AlRab7 ubiquitination. Overexpression of AlRab7 and AlRabring7 independently and when co-transformed enhanced the growth of yeast cells during stress conditions. Further, the bimolecular fluorescence complementation assay shows the in planta interaction of the two proteins. Our results suggest that AlRab7 and AlRabring7 confers enhanced stress tolerance in yeast.

The JcWRKY tobacco transgenics showed improved photosynthetic efficiency and wax accumulation during salinity.

Salinity is one of the major factors negatively affecting crop productivity. WRKY transcription factors (TFs) are involved in salicylic acid (SA) mediated cellular reactive oxygen species homeostasis in response to different stresses, including salinity. Therefore, the effect of NaCl, NaCl + SA and SA treatments on different photosynthesis-related parameters and wax metabolites were studied in the Jatropha curcas WRKY (JcWRKY) overexpressing tobacco lines. JcWRKY transgenics showed improved photosynthesis rate, stomatal conductance, intercellular CO2 concentration/ambient CO2 concentration ratio (Ci/Ca ratio), electron transport rate (ETR), photosynthesis efficiency (Fv/Fm), photochemical quenching (qP), non-photochemical quenching (NPQ) and quantum yield of PSII electron transport (ΦPSII) in response to salinity stress, while exogenous SA application had subtle effect on these parameters. Alkane, the major constituent of wax showed maximum accumulation in transgenics exposed to NaCl. Other wax components like fatty alcohol, carboxylic acid and fatty acid were also higher in transgenics with NaCl + SA and SA treatments. Interestingly, the transgenics showed a higher number of open stomata in treated plants as compared to wild type (WT), indicating less perception of stress by the transgenics. Improved salinity tolerance in JcWRKY overexpressing tobacco transgenics is associated with photosynthetic efficiency and wax accumulation, mediated by efficient SA signalling. The transgenics showed differential regulation of genes related to photosynthesis (NtCab40, NtLhcb5 and NtRca1), wax accumulation (NtWIN1) and stomatal regulation (NtMUTE, NtMYB-like, NtNCED3-2 and NtPIF3). The present study indicates that JcWRKY is a potential TF facilitating improved photosynthesis with the wax metabolic co-ordination in transgenics during stress.

Deciphering hydrogen peroxide-induced signalling towards stress tolerance in plants.

Plants encounter a variety of adverse environmental conditions, such as high salinity, drought, extreme heat/cold and heavy metals contamination (abiotic stress) or attack of various pathogens (biotic stress). These detrimental environmental factors enhanced the ROS production such as singlet oxygen (1O2), superoxide (O2 •-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH•). ROS are highly reactive and directly target several cellular molecules and metabolites, which lead to severe cellular dysfunction. Plants respond to oxidative damages by activating antioxidant machinery to trigger signalling cascades for stress tolerance. H2O2 signalling balances the plant metabolism through cross-talk with other signals and plant hormones during growth, development and stress responses. H2O2 facilitates the regulation of different stress-responsive transcription factors (TFs) including NAC, Zinc finger, WRKY, ERF, MYB, DREB and bZIP as both upstream and downstream events during stress signalling. The present review focuses on the biological synthesis of the H2O2 and its effect on the upregulation of kinase genes and stress related TFs for imparting stress tolerance.

Functional Validation of JcWRKY2, a Group III Transcription Factor Toward Mitigating Salinity Stress in Transgenic Tobacco.

The plants being sessile cannot escape from the adverse environmental stresses, hence get negatively affected in terms of their growth and yield. Transcriptional control simultaneously regulate different cellular processes, minimizing the deleterious effects of these stresses. The salicylic acid (SA)-inducible WRKY family of transcription factors auto or crossregulate the stress signaling in response to abiotic and biotic stresses, facilitating enhanced stress tolerance. In this study, we characterized the group III WRKY gene, JcWRKY2 from ecological and economical valued shrub Jatropha curcas. The JcWRKY2 tobacco transgenics showed improved physiological growth parameters, elevated chlorophyll content, improved antioxidative activities, and increased endogenous SA with both salt and SA stress. Interestingly, the pretreatment with SA and hydrogen peroxide facilitated improved germination of transgenic seeds with salinity stress. The transgenics showed differential regulation of antioxidative enzymes, calcium/calmodulin, dehydrins, and phospholipase genes with salt and SA stress. The increased SA content in transgenics on stress treatments, enhanced the antioxidant capacity leading to reduced susceptibility to stresses. Thus, JcWRKY2 transgenics participate in SA-mediated, improved antioxidative status during salinity stress with reduced reactive oxygen species damage.

Development of fluorescent protein-based biosensing strains: A new tool for the detection of aromatic hydrocarbon pollutants in the environment.

The major sources for release of hydrocarbons into the environment include the effluents generated from chemical processing industries and ports. The introduction of such hazardous compounds into natural water bodies creates considerable disturbances in aquatic life and causes a threat to humans. Thus, it is essential to detect and quantify pollutants at various stages of the wastewater generation and treatment before they reach natural aquatic environments and contaminate them. This study reports the development of "biosensing strains" by cloning hydrocarbon recognizing promoter-operator and a reporter gene in bacterial strains for sensing the presence of pollutants at their lowest possible concentration. So far, various biosensing strains have been constructed with a fused promoter-operator region of the hydrocarbon degrading operons, but most of them use luxAB as a reporter gene. A novel approach in the present study aimed at constructing strains harboring two different fluorescent protein (FP)-based reporter genes for the quantification of multiple pollutants at a time. Two vectors were designed with a fusion of tbuT-gfp and phnR-cfp for the quantification of mono- and poly-aromatic hydrocarbons, respectively. The designed vectors were transformed into E. coli DH5α, and these strains were designated as E. coli DH5α 2296-gfp (containing pPROBE-Tbut-RBS-gfp-npt) and E. coli DH5α 2301-cfp (containing pPROBE-phn-RBS-cfp-npt). Both the developed recombinant strains were capable of successfully detecting mono- and poly-aromatic hydrocarbons in the range of 1-100 µM. The sensing capacity of recombinant strains was successfully validated with actual wastewater samples against available physico-chemical analytical techniques. The development of such recombinant microbial strains indicates the future for online contaminant detection, treatment quality monitoring and protection of aquatic flora and fauna.

SbMYB15 transcription factor mitigates cadmium and nickel stress in transgenic tobacco by limiting uptake and modulating antioxidative defence system.

Plants require different inorganic minerals in an appropriate amount for growth; however, imbalance can limit growth and productivity. Heavy metal accumulation causes toxicity and generates signalling crosstalk with reactive oxygen species (ROS), phytohormones, genes and transcription factors (TFs). The MYB (myeloblastoma) TFs participate in plant processes such as metabolism, development, cell fate, hormone pathways and responses to stresses. This is the first report towards characterisation of R2R3-type MYB TF, SbMYB15, from succulent halophyte Salicornia brachiata Roxb. for heavy metal tolerance. The SbMYB15 showed >5-fold increased transcript expression in the presence of CdCl2 and NiCl2•6H2O. The constitutive overexpression of SbMYB15 conferred cadmium and nickel tolerance in transgenic tobacco, with improved growth and chlorophyll content. Further, the transgenics showed reduced generation of reactive oxygen species (H2O2 and O2•-) as compared with the wild-type (WT) with both Cd2+ and Ni2+ stress. Transgenics also showed low uptake of heavy metal ions, increased scavenging activity of the antioxidative enzymes (CAT and SOD) and higher transcript expression of antioxidative genes (CAT1 and MnSOD). Thus, the present study signifies that SbMYB15 can be deployed for developing heavy metal tolerance in crop plants via genetic engineering.

AlNAC4 Transcription Factor From Halophyte Aeluropus lagopoides Mitigates Oxidative Stress by Maintaining ROS Homeostasis in Transgenic Tobacco.

NAC proteins are a large family of plant-specific transcription factors which regulate both ABA-dependent and -independent gene expression. These transcription factors participate in biotic and abiotic stress-response through intricate regulation at transcriptional, post-transcriptional and post-translational levels. In the present study, AlNAC4 transcription factor was isolated from a salt excreting halophyte Aeluropus lagopoides. The AlNAC4 has an open reading frame of 936 bp, encoding a protein of 312 amino acid, with an estimated molecular mass of 34.9 kDa. The AlNAC4 showed close homology to monocot NACs in the phylogenetic tree. In silico analysis revealed that AlNAC4 possess the characteristic A-E subdomains within the NAC domain. The AlNAC4 showed sixteen post-translational phosphorylation sites. The AlNAC4 transcript was significantly upregulated with dehydration and H2O2 treatments, showing its role in osmotic and oxidative stress, respectively. The recombinant protein showed binding to mono as well as tandem repeats of NAC recognition sequence (NACRS) of the erd1 promoter. This is the first report mentioning that overexpression of AlNAC4 improved oxidative stress tolerance in tobacco transgenics. The transgenics maintained ROS homeostasis during H2O2 treatment. The transgenics showed regulation of stress-responsive genes including CAT, SOD, LEA5, PLC3, ERD10B, THT1 and transcription factors like AP2, ZFP during oxidative stress. Key Message: The AlNAC4 transcription factor from recretohalophyte Aeluropus showed regulation with abiotic stresses and binding to NACRS elements of erd1 promoter. The AlNAC4 tobacco transgenics showed improved growth with oxidative stress.

The wheat TabZIP2 transcription factor is activated by the nutrient starvation-responsive SnRK3/CIPK protein kinase.

KEY MESSAGE: The understanding of roles of bZIP factors in biological processes during plant development and under abiotic stresses requires the detailed mechanistic knowledge of behaviour of TFs. Basic leucine zipper (bZIP) transcription factors (TFs) play key roles in the regulation of grain development and plant responses to abiotic stresses. We investigated the role and molecular mechanisms of function of the TabZIP2 gene isolated from drought-stressed wheat plants. Molecular characterisation of TabZIP2 and derived protein included analyses of gene expression and its target promoter, and the influence of interacting partners on the target promoter activation. Two interacting partners of TabZIP2, the 14-3-3 protein, TaWIN1 and the bZIP transcription factor TaABI5L, were identified in a Y2H screen. We established that under elevated ABA levels the activity of TabZIP2 was negatively regulated by the TaWIN1 protein and positively regulated by the SnRK3/CIPK protein kinase WPK4, reported previously to be responsive to nutrient starvation. The physical interaction between the TaWIN1 and the WPK4 was detected. We also compared the influence of homo- and hetero-dimerisation of TabZIP2 and TaABI5L on DNA binding. TabZIP2 gene functional analyses were performed using drought-inducible overexpression of TabZIP2 in transgenic wheat. Transgenic plants grown under moderate drought during flowering, were smaller than control plants, and had fewer spikes and seeds per plant. However, a single seed weight was increased compared to single seed weights of control plants in three of four evaluated transgenic lines. The observed phenotypes of transgenic plants and the regulation of TabZIP2 activity by nutrient starvation-responsive WPK4, suggest that the TabZIP2 could be the part of a signalling pathway, which controls the rearrangement of carbohydrate and nutrient flows in plant organs in response to drought.

Ectopic Expression of JcWRKY Confers Enhanced Resistance in Transgenic Tobacco Against Macrophomina phaseolina.

Plants possess an innate immune system comprising of a complex network of closely regulated defense responses involving differential gene expression mediated by transcription factors (TFs). The WRKYs comprise of an important plant-specific TF family, which is involved in regulation of biotic and abiotic defenses. The overexpression of JcWRKY resulted in improved resistance in transgenic tobacco against Macrophomina phaseolina. The production of reactive oxygen species (ROS) and its detoxification through antioxidative system in the transgenics facilitates defense against Macrophomina. The enhanced catalase activity on Macrophomina infection limits the spread of infection. The transcript expression of antioxidative enzymes gene (CAT and SOD) and salicylic acid (SA) biosynthetic gene ICS1 showed upregulation during Macrophomina infection and combinatorial stress. The enhanced transcript of pathogenesis-related genes PR-1 indicates the accumulation of SA during different stresses. The PR-2 and PR-5 highlight the activation of defense responses comprising of activation of hydrolytic cleavage of glucanases and thaumatin-like proteins causing disruption of fungal cells. The ROS homeostasis in coordination with signaling molecules regulate the defense responses and inhibit fungal growth.

Chemical Derivatization of Metabolite Mass Profiling of the Recretohalophyte Aeluropus lagopoides Revealing Salt Stress Tolerance Mechanism.

Plants are the primary producers of food for human being. Their intracellular environment alternation is influenced by abiotic stress factors such as drought, heat and soil salinity. Aeluropus lagopoides is a strong halophyte that grows with ease under high saline muddy banks of creeks of Gujarat, India. To study the response of salinity on metabolite changes in Aeluropus, three treatments, i.e. control, salinity and recovery, were selected for both shoot and root tissue. The cytosolic metabolite state was analysed by molecular chemical derivatization gas chromatography mass profiling. During saline treatment, significant increase of compatible solutes in shoot and root tissue was observed as compared to control. Subsequently, metabolic concentration decreased under recovery conditions. The metabolites like amino acids, organic acids and polyols were significantly detected in both shoot and root of Aeluropus under salinity. The metabolites like proline, aspartic acid, glycine, succinic acid and glycolic acid were significantly upregulated under stress. The salicylic acid was found to play a role in maintaining the polyols level by its down-regulation during salinity. The principle component analysis of all detected metabolites in both shoot and root showed that metabolites expressed under salinity (component 1) were highly variable, while metabolites expressed under recovery (component 2) were comparatively less variable as compared to control. The evolved intracellular compartmentalization of amino acids, organic acids and polyols in A. lagopoides can be a hallmark to sustaining at high salinity stress.