Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

An aquaporin gene from halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis>, <Emphasis Type="Italic">SpAQP1</Emphasis>, increases salt tolerance in transgenic tobacco

Key message

SpAQP1 was strongly induced by salt in an ABA-independent way, promoted seed germination and root growth in transgenic tobaccos and increased salt tolerance by increasing the activities of antioxidative enzymes.

Abstract

Aquaporin (AQP) plays crucial roles in the responses of plant to abiotic stresses such as drought, salt and cold. Compared to glycophytes, halophytes often have excellent salt and drought tolerances. To uncover the molecular mechanism of halophyte Sesuvium portulacastrum tolerance to salt, in this study, an AQP gene, SpAQP1, from S. portulacastrum was isolated and characterized. The amino acid sequence of SpAQP1 shared high homology with that of plant plasma membrane intrinsic proteins (PIPs) and contained the distinct molecular features of PIPs. In the phylogenic tree, SpAQP1 was evidently classified as the PIP2 subfamily. SpAQP1 is expressed in roots, stems and leaves, and was significantly induced by NaCl treatment and inhibited by abscisic acid (ABA) treatment. When heterologously expressed in yeast and tobacco, SpAQP1 enhanced the salt tolerance of yeast strains and tobacco plants and promoted seed germination and root growth under salt stress in transgenic plants. The activity of antioxidative enzymes including superoxide dismutase, peroxidase and catalase was increased in transgenic plants overexpressing SpAQP1. Taken together, our studies suggested that SpAQP1 functioned in the responses of S. portulacastrum to salt stress and could increase salt tolerance by enhancing the antioxidative activity of plants.

     

Acid phosphatase gene <Emphasis Type="Italic">GmHAD1</Emphasis> linked to low phosphorus tolerance in soybean,through fine mapping

Key message

Map-based cloning identified GmHAD1, a gene which encodes a HAD-like acid phosphatase, associated with soybean tolerance to low phosphorus stress.

Abstract

Phosphorus (P) deficiency in soils is a major limiting factor for crop growth worldwide. Plants may adapt to low phosphorus (LP) conditions via changes to root morphology, including the number, length, orientation, and branching of the principal root classes. To elucidate the genetic mechanisms for LP tolerance in soybean, quantitative trait loci (QTL) related to root morphology responses to LP were identified via hydroponic experiments. In total, we identified 14 major loci associated with these traits in a RIL population. The log-likelihood scores ranged from 2.81 to 7.43, explaining 4.23–13.98% of phenotypic variance. A major locus on chromosome 08, named qP8-2, was co-localized with an important P efficiency QTL (qPE8), containing phosphatase genes GmACP1 and GmACP2. Another major locus on chromosome 10 named qP10-2 explained 4.80–13.98% of the total phenotypic variance in root morphology. The qP10-2 contains GmHAD1, a gene which encodes an acid phosphatase. In the transgenic soybean hairy roots, GmHAD1 overexpression increased P efficiency by 8.4–16.5% relative to the control. Transgenic Arabidopsis plants had higher biomass than wild-type plants across both short- and long-term P reduction. These results suggest that GmHAD1, an acid phosphatase gene, improved the utilization of organic phosphate by soybean and Arabidopsis plants.

     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

Overexpression of soybean <Emphasis Type="Italic">GmERF9</Emphasis> enhances the tolerance to drought and cold in the transgenic tobacco

Ethylene response factors (ERFs) are widespread in plants, which are widely involved in plant response to biotic and abiotic stress. In this research, a soybean gene, GmERF9, was identified and the function was characterized. The results showed that GmERF9 contained a typical AP2/ERF binding domain and a putative nuclear localization signal sequence. The real-time fluorescence quantitative PCR (qPCR) revealed that the expression of GmERF9 could be induced by ethylene (ET), abscisic acid (ABA), drought, salt and cold stresses. GmERF9 protein could specifically bind to the GCC-box and activate the expression of the reporter gene in the yeast cells and tobacco leaves. Overexpression of GmERF9 enhanced the expression of pathogenesis-related (PR) genes, including PR1, PR2, Osmotin (PR5), and SAR8.2. Also, the overexpression of GmERF9 increased the accumulation of proline and soluble carbohydrate, and decreased the accumulation of malondialdehyde under drought and cold stresses in the transgenic tobacco compared to the wild type (WT) tobacco, which indicated that GmERF9 enhanced the tolerance to drought and cold stresses in the transgenic tobacco. In summary, the function of GmERF9 is involved in the response to environmental stresses for plants, which can be used as a candidate gene for genetic engineering of crops.     

The K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter <Emphasis Type="Italic">AhNHX1</Emphasis> improved tobacco tolerance to NaCl stress by enhancing K<Superscript>+</Superscript> retention

High salinity is the one of important factors limiting plant growth and crop production. Many NHX-type antiporters have been reported to catalyze K⁺/H⁺ exchange to mediate salt stress. This study shows that an NHX gene from Arachis hypogaea L. has an important role in K⁺ uptake and transport, which affects K⁺ accumulation and plant salt tolerance. When overexpressing AhNHX1, the growth of tobacco seedlings is improved with longer roots and a higher fresh weight than the wild type (WT) under NaCl treatment. Meanwhile, when exposed to NaCl stress, the transgenic seedlings had higher K⁺/H⁺ antiporter activity and their roots got more K⁺ uptake. NaCl stress could induce higher K⁺ accumulation in the roots, stems, and leaves of transgenic tobacco seedlings but not Na⁺ accumulation, thus, leading to a higher K⁺/Na⁺ ratio in the transgenic seedlings. Additionally, the AKT1, HAK1, SKOR, and KEA genes, which are involved in K⁺ uptake or transport, were induced by NaCl stress and kept higher expression levels in transgenic seedlings than in WT seedlings. The H⁺-ATPase and H⁺-PPase activities were also higher in transgenic seedlings than in the WT seedlings under NaCl stress. Simultaneously, overexpression of AhNHX1 increased the relative distribution of K⁺ in the aerial parts of the seedlings under NaCl stress. These results showed that AhNHX1 catalyzed the K⁺/H⁺ antiporter and enhanced tobacco tolerance to salt stress by increasing K⁺ uptake and transport.     

Overexpression of <Emphasis Type="Italic">Populus tomentosa</Emphasis> cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants

Reactive oxygen species (ROS) play key roles in plants and are regulated by several ROS-scavenging enzymes. Ascorbate peroxidase (APX), which catalyzes the reduction of hydrogen peroxide to water, a vital part of ROS formation, plays a significant role in higher plants. In this study, a cytosolic APX gene from Populus tomentosa, named PcAPX, was identified and characterized. Recombinant PcAPX had a calculated mass of 33.24 kD and showed high activity towards ascorbic acid (ASA) and hydrogen peroxide (H₂O₂). Real-time PCR analysis showed that APX mRNA expression levels were higher in leaves than roots or stems of P. tomentosa. Compared with wild-type, transgenic tobacco plants overexpressing PcAPX showed no significant difference in morphology under normal conditions. However, the transgenic plants were more resistant to drought, salt and oxidative stress conditions, as shown by decreased levels of malondialdehyde and increased levels of chlorophyll. Moreover, decreased H₂O₂ levels, increased ASA consumption, an increase in the NADP to NADPH ratio, and higher APX activity in the transgenic plants suggested an increased ability to eliminate ROS. These data suggest that PcAPX overexpression in transgenic tobacco plants can enhance tolerance to drought, salt and oxidative stress. Therefore, APX has a crucial role in abiotic stress tolerance in plants.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

Cloning <Emphasis Type="Italic">PIP</Emphasis> genes in drought-tolerant vetiver grass and responses of transgenic <Emphasis Type="Italic">VzPIP2;1</Emphasis> soybean plants to water stress

Vetiver grass [Vetiveria zizanioides (L.) Nash] displays comprehensive abiotic stress tolerance closely related to fine maintenance of plant water relation mediated by plasma membrane intrinsic proteins (PIPs). Two open reading frame sequences of PIPs (867 and 873 bp) were cloned from vetiver grass and named as VzPIP1;1 and VzPIP2;1, respectively. Expression of green fluorescent protein revealed only subcellular localization of VzPIP2;1 in the plasma membrane. Agrobacterium tumefaciens mediated transgenic (VzPIP2;1) soybean plants had a higher water content in above-ground parts under sufficient water supply through enhancing transpiration as compared to the non-transgenic plants but displayed a more severe drought injury because of a lower photosynthesis and a higher transpiration rate. However, A. rhizogenes mediated transgenic soybean plants kept a higher water content in above-ground parts by improving root water transport and kept a more effective photosynthesis under normal and drought conditions.     

Over-expression of the <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> harpin-encoding gene <Emphasis Type="Italic">hrpZm</Emphasis> confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T₂–T₄ generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.