Salt and drought stress-mitigating approaches in sugar beet (Beta vulgaris L.) to improve its performance and yield.

MAIN CONCLUSION: Although sugar beet is a salt- and drought-tolerant crop, high salinity, and water deprivation significantly reduce its yield and growth. Several reports have demonstrated stress tolerance enhancement through stress-mitigating strategies including the exogenous application of osmolytes or metabolites, nanoparticles, seed treatments, breeding salt/drought-tolerant varieties. These approaches would assist in achieving sustainable yields despite global climatic changes. Sugar beet (Beta vulgaris L.) is an economically vital crop for ~ 30% of world sugar production. They also provide essential raw materials for bioethanol, animal fodder, pulp, pectin, and functional food-related industries. Due to fewer irrigation water requirements and shorter regeneration time than sugarcane, beet cultivation is spreading to subtropical climates from temperate climates. However, beet varieties from different geographical locations display different stress tolerance levels. Although sugar beet can endure moderate exposure to various abiotic stresses, including high salinity and drought, prolonged exposure to salt and drought stress causes a significant decrease in crop yield and production. Hence, plant biologists and agronomists have devised several strategies to mitigate the stress-induced damage to sugar beet cultivation. Recently, several studies substantiated that the exogenous application of osmolytes or metabolite substances can help plants overcome injuries induced by salt or drought stress. Furthermore, these compounds likely elicit different physio-biochemical impacts, including improving nutrient/ionic homeostasis, photosynthetic efficiency, strengthening defense response, and water status improvement under various abiotic stress conditions. In the current review, we compiled different stress-mitigating agricultural strategies, prospects, and future experiments that can secure sustainable yields for sugar beets despite high saline or drought conditions.

Miracle Tree Moringa oleifera: Status of the Genetic Diversity, Breeding, In Vitro Propagation, and a Cogent Source of Commercial Functional Food and Non-Food Products.

Moringa oleifera Lam. (MO) is a fast-growing drought-resistant tree belonging to the family Moringaceae and native to the Indian subcontinent and cultivated and/or naturalized worldwide with a semi-arid climate. MO is also popularly known as a miracle tree for its repertoire of nutraceutical, pharmacological, and phytochemical properties. The MO germplasm is collected, conserved, and maintained by various institutions across the globe. Various morphological, biochemical, and molecular markers are used for determining the genetic diversity in MO accessions. A higher yield of leaves and pods is often desirable for making various products with commercial viability and amenable for trade in the international market. Therefore, breeding elite varieties adapted to local agroclimatic conditions and in vitro propagation are viable and sustainable approaches. Here, we provide a comprehensive overview of MO germplasm conservation and various markers that are employed for assessing the genetic diversity among them. Further, breeding and in vitro propagation of MO for various desirable agronomic traits are discussed. Finally, trade and commerce of various functional and biofortified foods and non-food products are enumerated albeit with a need for a rigorous and stringent toxicity evaluation.

Variations in Circadian Clock Organization & Function: A Journey from Ancient to Recent.

MAIN CONCLUSION: Circadian clock components exhibit structural variations in different plant systems, and functional variations during various abiotic stresses. These variations bear relevance for plant fitness and could be important evolutionarily. All organisms on earth have the innate ability to measure time as diurnal rhythms that occur due to the earth's rotations in a 24-h cycle. Circadian oscillations arising from the circadian clock abide by its fundamental properties of periodicity, entrainment, temperature compensation, and oscillator mechanism, which is central to its function. Despite the fact that a myriad of research in Arabidopsis thaliana illuminated many detailed aspects of the circadian clock, many more variations in clock components' organizations and functions remain to get deciphered. These variations are crucial for sustainability and adaptation in different plant systems in the varied environmental conditions in which they grow. Together with these variations, circadian clock functions differ drastically even during various abiotic and biotic stress conditions. The present review discusses variations in the organization of clock components and their role in different plant systems and abiotic stresses. We briefly introduce the clock components, entrainment, and rhythmicity, followed by the variants of the circadian clock in different plant types, starting from lower non-flowering plants, marine plants, dicots to the monocot crop plants. Furthermore, we discuss the interaction of the circadian clock with components of various abiotic stress pathways, such as temperature, light, water stress, salinity, and nutrient deficiency with implications for the reprogramming during these stresses. We also update on recent advances in clock regulations due to post-transcriptional, post-translation, non-coding, and micro-RNAs. Finally, we end this review by summarizing the points of applicability, a remark on the future perspectives, and the experiments that could clear major enigmas in this area of research.

Potassium Chloroaurate-Mediated In Vitro Synthesis of Gold Nanoparticles Improved Root Growth by Crosstalk with Sucrose and Nutrient-Dependent Auxin Homeostasis in Arabidopsis thaliana.

In a hydroponic system, potassium chloroaurate (KAuCl4) triggers the in vitro sucrose (Suc)-dependent formation of gold nanoparticles (AuNPs). AuNPs stimulate the growth of the root system, but their molecular mechanism has not been deciphered. The root system of Arabidopsis (Arabidopsis thaliana) exhibits developmental plasticity in response to the availability of various nutrients, Suc, and auxin. Here, we showed the roles of Suc, phosphorus (P), and nitrogen (N) in facilitating a AuNPs-mediated increase in root growth. Furthermore, the recuperating effects of KAuCl4 on the natural (IAA) auxin-mediated perturbation of the root system were demonstrated. Arabidopsis seedlings harboring the cell division marker CycB1;1::CDB-GUS provided evidence of the restoration efficacy of KAuCl4 on the IAA-mediated inhibitory effect on meristematic cell proliferation of the primary and lateral roots. Arabidopsis harboring synthetic auxin DR5rev::GFP exhibited a reinstating effect of KAuCl4 on IAA-mediated aberration in auxin subcellular localization in the root. KAuCl4 also exerted significant and differential recuperating effects on the IAA-mediated altered expression of the genes involved in auxin signaling and biosynthetic pathways in roots. Our results highlight the crosstalk between KAuCl4-mediated improved root growth and Suc and nutrient-dependent auxin homeostasis in Arabidopsis.

GWAS Reveals a Novel Candidate Gene CmoAP2/ERF in Pumpkin (Cucurbita moschata) Involved in Resistance to Powdery Mildew.

Pumpkin (Cucurbita moschata Duchesne ex Poir.) is a multipurpose cash crop rich in antioxidants, minerals, and vitamins; the seeds are also a good source of quality oils. However, pumpkin is susceptible to the fungus Podosphaera xanthii, an obligate biotrophic pathogen, which usually causes powdery mildew (PM) on both sides of the leaves and reduces photosynthesis. The fruits of infected plants are often smaller than usual and unpalatable. This study identified a novel gene that involves PM resistance in pumpkins through a genome-wide association study (GWAS). The allelic variation identified in the CmoCh3G009850 gene encoding for AP2-like ethylene-responsive transcription factor (CmoAP2/ERF) was proven to be involved in PM resistance. Validation of the GWAS data revealed six single nucleotide polymorphism (SNP) variations in the CmoAP2/ERF coding sequence between the resistant (IT 274039 [PMR]) and the susceptible (IT 278592 [PMS]). A polymorphic marker (dCAPS) was developed based on the allelic diversity to differentiate these two haplotypes. Genetic analysis in the segregating population derived from PMS and PMR parents provided evidence for an incomplete dominant gene-mediated PM resistance. Further, the qRT-PCR assay validated the elevated expression of CmoAP2/ERF during PM infection in the PMR compared with PMS. These results highlighted the pivotal role of CmoAP2/ERF in conferring resistance to PM and identifies it as a valuable molecular entity for breeding resistant pumpkin cultivars.

Development of SNP Markers for White Immature Fruit Skin Color in Cucumber (Cucumis sativus L.) Using QTL-seq and Marker Analyses.

Despite various efforts in identifying the genes governing the white immature fruit skin color in cucumber, the genetic basis of the white immature fruit skin color is not well known. In the present study, genetic analysis showed that a recessive gene confers the white immature fruit skin-color phenotype over the light-green color of a Korean slicer cucumber. High-throughput QTL-seq combined with bulked segregation analysis of two pools with the extreme phenotypes (white and light-green fruit skin color) in an F2 population identified two significant genomic regions harboring QTLs for white fruit skin color within the genomic region between 34.1 and 41.67 Mb on chromosome 3, and the genomic region between 12.2 and 12.7 Mb on chromosome 5. Further, nonsynonymous SNPs were identified with a significance of p < 0.05 within the QTL regions, resulting in eight homozygous variants within the QTL region on chromosome 3. SNP marker analysis uncovered the novel missense mutations in Chr3CG52930 and Chr3CG53640 genes and showed consistent results with the phenotype of light-green and white fruit skin-colored F2 plants. These two genes were located 0.5 Mb apart on chromosome 3, which are considered strong candidate genes. Altogether, this study laid a solid foundation for understanding the genetic basis and marker-assisted breeding of immature fruit skin color in cucumber.

Microbial Biosurfactant: A New Frontier for Sustainable Agriculture and Pharmaceutical Industries.

In the current scenario of changing climatic conditions and the rising global population, there is an urgent need to explore novel, efficient, and economical natural products for the benefit of humankind. Biosurfactants are one of the latest explored microbial synthesized biomolecules that have been used in numerous fields, including agriculture, pharmaceuticals, cosmetics, food processing, and environment-cleaning industries, as a source of raw materials, for the lubrication, wetting, foaming, emulsions formulations, and as stabilizing dispersions. The amphiphilic nature of biosurfactants have shown to be a great advantage, distributing themselves into two immiscible surfaces by reducing the interfacial surface tension and increasing the solubility of hydrophobic compounds. Furthermore, their eco-friendly nature, low or even no toxic nature, durability at higher temperatures, and ability to withstand a wide range of pH fluctuations make microbial surfactants preferable compared to their chemical counterparts. Additionally, biosurfactants can obviate the oxidation flow by eliciting antioxidant properties, antimicrobial and anticancer activities, and drug delivery systems, further broadening their applicability in the food and pharmaceutical industries. Nowadays, biosurfactants have been broadly utilized to improve the soil quality by improving the concentration of trace elements and have either been mixed with pesticides or applied singly on the plant surfaces for plant disease management. In the present review, we summarize the latest research on microbial synthesized biosurfactant compounds, the limiting factors of biosurfactant production, their application in improving soil quality and plant disease management, and their use as antioxidant or antimicrobial compounds in the pharmaceutical industries.

Salt and Drought Stress Responses in Cultivated Beets (Beta vulgaris L.) and Wild Beet (Beta maritima L.).

Cultivated beets, including leaf beets, garden beets, fodder beets, and sugar beets, which belong to the species Beta vulgaris L., are economically important edible crops that have been originated from a halophytic wild ancestor, Beta maritima L. (sea beet or wild beet). Salt and drought are major abiotic stresses, which limit crop growth and production and have been most studied in beets compared to other environmental stresses. Characteristically, beets are salt- and drought-tolerant crops; however, prolonged and persistent exposure to salt and drought stress results in a significant drop in beet productivity and yield. Hence, to harness the best benefits of beet cultivation, knowledge of stress-coping strategies, and stress-tolerant beet varieties, are prerequisites. In the current review, we have summarized morpho-physiological, biochemical, and molecular responses of sugar beet, fodder beet, red beet, chard (B. vulgaris L.), and their ancestor, wild beet (B. maritima L.) under salt and drought stresses. We have also described the beet genes and noncoding RNAs previously reported for their roles in salt and drought response/tolerance. The plant biologists and breeders can potentiate the utilization of these resources as prospective targets for developing crops with abiotic stress tolerance.

A Comprehensive Overview on the Production of Vaccines in Plant-Based Expression Systems and the Scope of Plant Biotechnology to Combat against SARS-CoV-2 Virus Pandemics.

Many pathogenic viral pandemics have caused threats to global health; the COVID-19 pandemic is the latest. Its transmission is growing exponentially all around the globe, putting constraints on the health system worldwide. A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causes this pandemic. Many candidate vaccines are available at this time for COVID-19, and there is a massive international race underway to procure as many vaccines as possible for each country. However, due to heavy global demand, there are strains in global vaccine production. The use of a plant biotechnology-based expression system for vaccine production also represents one part of this international effort, which is to develop plant-based heterologous expression systems, virus-like particles (VLPs)-vaccines, antiviral drugs, and a rapid supply of antigen-antibodies for detecting kits and plant origin bioactive compounds that boost the immunity and provide tolerance to fight against the virus infection. This review will look at the plant biotechnology platform that can provide the best fight against this global pandemic.

The Endophytic Microbiome as a Hotspot of Synergistic Interactions, with Prospects of Plant Growth Promotion.

The plant root is the primary site of interaction between plants and associated microorganisms and constitutes the main components of plant microbiomes that impact crop production. The endophytic bacteria in the root zone have an important role in plant growth promotion. Diverse microbial communities inhabit plant root tissues, and they directly or indirectly promote plant growth by inhibiting the growth of plant pathogens, producing various secondary metabolites. Mechanisms of plant growth promotion and response of root endophytic microorganisms for their survival and colonization in the host plants are the result of complex plant-microbe interactions. Endophytic microorganisms also assist the host to sustain different biotic and abiotic stresses. Better insights are emerging for the endophyte, such as host plant interactions due to advancements in 'omic' technologies, which facilitate the exploration of genes that are responsible for plant tissue colonization. Consequently, this is informative to envisage putative functions and metabolic processes crucial for endophytic adaptations. Detection of cell signaling molecules between host plants and identification of compounds synthesized by root endophytes are effective means for their utilization in the agriculture sector as biofertilizers. In addition, it is interesting that the endophytic microorganism colonization impacts the relative abundance of indigenous microbial communities and suppresses the deleterious microorganisms in plant tissues. Natural products released by endophytes act as biocontrol agents and inhibit pathogen growth. The symbiosis of endophytic bacteria and arbuscular mycorrhizal fungi (AMF) affects plant symbiotic signaling pathways and root colonization patterns and phytohormone synthesis. In this review, the potential of the root endophytic community, colonization, and role in the improvement of plant growth has been explained in the light of intricate plant-microbe interactions.

Identification of an Allelic Variant of the CsOr Gene Controlling Fruit Endocarp Color in Cucumber (Cucumis sativus L.) Using Genotyping-By-Sequencing (GBS) and Whole-Genome Sequencing.

The cucumber is a major vegetable crop around the world. Fruit flesh color is an important quality trait in cucumber and flesh color mainly depends on the relative content of ß-carotene in the fruits. The ß-carotene serves as a precursor of vitamin A, which has dietary benefits for human health. Cucumbers with orange flesh contain a higher amount of ß-carotene than white fruit flesh. Therefore, development of orange-fleshed cucumber varieties is gaining attention for improved nutritional benefits. In this study, we performed genotyping-by-sequencing (GBS) based on genetic mapping and whole-genome sequencing to identify the orange endocarp color gene in the cucumber breeding line, CS-B. Genetic mapping, genetic sequencing, and genetic segregation analyses showed that a single recessive gene (CsaV3_6G040750) encodes a chaperone DnaJ protein (DnaJ) protein at the Cucumis sativus(CsOr) locus was responsible for the orange endocarp phenotype in the CS-B line. The Or gene harbored point mutations T13G and T17C in the first exon of the coding region, resulting in serine to alanine at position 13 and isoleucine to threonine at position 17, respectively. CS-B line displayed increased ß-carotene content in the endocarp tissue, corresponding to elevated expression of CsOr gene at fruit developmental stages. Identifying novel missense mutations in the CsOr gene could provide new insights into the role of Or mechanism of action for orange fruit flesh in cucumber and serve as a valuable resource for developing ß-carotene-rich cucumbers varieties with increased nutritional benefits.

An Insight into the Abiotic Stress Responses of Cultivated Beets (Beta vulgaris L.).

Cultivated beets (sugar beets, fodder beets, leaf beets, and garden beets) belonging to the species Beta vulgaris L. are important sources for many products such as sugar, bioethanol, animal feed, human nutrition, pulp residue, pectin extract, and molasses. Beta maritima L. (sea beet or wild beet) is a halophytic wild ancestor of all cultivated beets. With a requirement of less water and having shorter growth period than sugarcane, cultivated beets are preferentially spreading from temperate regions to subtropical countries. The beet cultivars display tolerance to several abiotic stresses such as salt, drought, cold, heat, and heavy metals. However, many environmental factors adversely influence growth, yield, and quality of beets. Hence, selection of stress-tolerant beet varieties and knowledge on the response mechanisms of beet cultivars to different abiotic stress factors are most required. The present review discusses morpho-physiological, biochemical, and molecular responses of cultivated beets (B. vulgaris L.) to different abiotic stresses including alkaline, cold, heat, heavy metals, and UV radiation. Additionally, we describe the beet genes reported for their involvement in response to these stress conditions.

Natural Genetic Resources from Diverse Plants to Improve Abiotic Stress Tolerance in Plants.

The current agricultural system is biased for the yield increase at the cost of biodiversity. However, due to the loss of precious genetic diversity during domestication and artificial selection, modern cultivars have lost the adaptability to cope with unfavorable environments. There are many reports on variations such as single nucleotide polymorphisms (SNPs) and indels in the stress-tolerant gene alleles that are associated with higher stress tolerance in wild progenitors, natural accessions, and extremophiles in comparison with domesticated crops or model plants. Therefore, to gain a better understanding of stress-tolerant traits in naturally stress-resistant plants, more comparative studies between the modern crops/model plants and crop progenitors/natural accessions/extremophiles are required. In this review, we discussed and summarized recent progress on natural variations associated with enhanced abiotic stress tolerance in various plants. By applying the recent biotechniques such as the CRISPR/Cas9 gene editing tool, natural genetic resources (i.e., stress-tolerant gene alleles) from diverse plants could be introduced to the modern crop in a non-genetically modified way to improve stress-tolerant traits.

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis.

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft-10 tsf-1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper-vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1-20 mutants. The terminal flower formed in tfl1-20 mutants converted to a hyper-vegetative shoot in ft-10 tsf-1 mutants. Grafting ft-10 tsf-1 or ft-10 tsf-1 tfl1-20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild-type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft-10 tsf-1 mutants the vasculature-specific misexpression of FT converted the hyper-vegetative shoot to a normal inflorescence, and in the ft-10 tsf-1 tfl1-20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.

Enhanced multiple stress tolerance in Arabidopsis by overexpression of the polar moss peptidyl prolyl isomerase FKBP12 gene.

KEY MESSAGE: PaFKBP12 overexpression in Arabidopsis resulted in stress tolerance to heat, ABA, drought, and salt stress, in addition to growth promotion under normal conditions. Polytrichastrum alpinum (alpine haircap moss) is one of polar organisms that can withstand the severe conditions of the Antarctic. In this study, we report the isolation of a peptidyl prolyl isomerase FKBP12 gene (PaFKBP12) from P. alpinum collected in the Antarctic and its functional implications in development and stress responses in plants. In P. alpinum, PaFKBP12 expression was induced by heat and ABA. Overexpression of PaFKBP12 in Arabidopsis increased the plant size, which appeared to result from increased rates of cell cycle. Under heat stress conditions, PaFKBP12-overexpressing lines (PaFKBP12-OE) showed better growth and survival than the wild type. PaFKBP12-OE also showed higher root elongation rates, better shoot growth and enhanced survival at higher concentrations of ABA in comparison to the wild type. In addition, PaFKBP12-OE were more tolerant to drought and salt stress than the wild type. All these phenotypes were accompanied with higher induction of the stress responsive genes in PaFKBP12-OE than in the wild type. Taken together, our findings revealed important functions of PaFKBP12 in plant development and abiotic stress responses.

Antarctic Moss Multiprotein Bridging Factor 1c Overexpression in Arabidopsis Resulted in Enhanced Tolerance to Salt Stress.

Polytrichastrum alpinum is one of the moss species that survives extreme conditions in the Antarctic. In order to explore the functional benefits of moss genetic resources, P. alpinum multiprotein-bridging factor 1c gene (PaMBF1c) was isolated and characterized. The deduced amino acid sequence of PaMBF1c comprises of a multiprotein-bridging factor (MBF1) domain and a helix-turn-helix (HTH) domain. PaMBF1c expression was induced by different abiotic stresses in P. alpinum, implying its roles in stress responses. We overexpressed PaMBF1c in Arabidopsis and analyzed the resulting phenotypes in comparison with wild type and/or Arabidopsis MBF1c (AtMBF1c) overexpressors. Overexpression of PaMBF1c in Arabidopsis resulted in enhanced tolerance to salt and osmotic stress, as well as to cold and heat stress. More specifically, enhanced salt tolerance was observed in PaMBF1c overexpressors in comparison to wild type but not clearly observable in AtMBF1c overexpressing lines. Thus, these results implicate the evolution of PaMBF1c under salt-enriched Antarctic soil. RNA-Seq profiling of NaCl-treated plants revealed that 10 salt-stress inducible genes were already up-regulated in PaMBF1c overexpressing plants even before NaCl treatment. Gene ontology enrichment analysis with salt up-regulated genes in each line uncovered that the terms lipid metabolic process, ion transport, and cellular amino acid biosynthetic process were significantly enriched in PaMBF1c overexpressors. Additionally, gene enrichment analysis with salt down-regulated genes in each line revealed that the enriched categories in wild type were not significantly overrepresented in PaMBF1c overexpressing lines. The up-regulation of several genes only in PaMBF1c overexpressing lines suggest that enhanced salt tolerance in PaMBF1c-OE might involve reactive oxygen species detoxification, maintenance of ATP homeostasis, and facilitation of Ca2+ signaling. Interestingly, many salt down-regulated ribosome- and translation-related genes were not down-regulated in PaMBF1c overexpressing lines under salt stress. These differentially regulated genes by PaMBF1c overexpression could contribute to the enhanced tolerance in PaMBF1c overexpressing lines under salt stress.

Molecular and physiological characterization of AtHIGD1 in Arabidopsis.

Flooding is a principal stress that limits plant productivity. The sensing of low oxygen levels (hypoxia) plays a critical role in the signaling pathway that functions in plants in flooded environments. In this study, to investigate hypoxia response mechanisms in Arabidopsis, we identified three hypoxia-related genes and subjected one of these genes, Arabidopsis thaliana HYPOXIA-INDUCED GENE DOMAIN 1 (AtHIGD1), to molecular characterization including gene expression analysis and intracellular localization of the encoded protein. AtHIGD1 was expressed in various organs but was preferentially expressed in developing siliques. Confocal microscopy of transgenic plants harboring eGFP-tagged AtHIGD1 indicated that AtHIGD1 is localized to mitochondria. Importantly, plants overexpressing AtHIGD1 exhibited increased resistance to hypoxia compared to wild type. Our results represent the first report of a biological function for an HIGD protein in plants and indicate that AtHIGD1 is a mitochondrial protein that plays an active role in mitigating the effects of hypoxia on plants.

Overexpression of a Barley Aquaporin Gene, HvPIP2;5 Confers Salt and Osmotic Stress Tolerance in Yeast and Plants.

We characterized an aquaporin gene HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, Δ 1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.

Cloning and functional characterization of a vacuolar Na+/H+ antiporter gene from mungbean (VrNHX1) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana.

Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.