A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

High irradiance sensitive phenotype of <Emphasis Type="Italic">Arabidopsis hit2/xpo1a</Emphasis> mutant is caused in part by nuclear confinement of AtHsfA4a

In Arabidopsis, EXPORTIN1A (HIT2/XPO1A) and EXPORTIN1B (XPO1B) mediate the translocation of nuclear export sequence (NES)-bearing proteins from nucleus to cytoplasm. However, a mutation in HIT2/XPO1A but not in XPO1B induces sensitivity to high irradiance (HI). Arabidopsis thaliana heat stress elements A4a and A5 (AtHsfA4a and AtHsfA5) are involved in plant responses to HI and possess NESs; therefore, their nucleo-cytoplasmic partitioning was analyzed. In wild-type and xpo1b mutant cells, AtHsfA4a normally remained in the cytoplasm but became concentrated in the nucleus following exposure to HI, whereas AtHsfA5 was constitutively distributed in both cytoplasm and nucleus. However, in hit2/xpo1a mutant, AtHsfA4a and AtHsfA5 were always confined to the nucleus, regardless of the irradiance. Although AtHsfA4a can enhance the ability of plants to scavenge H₂O₂, and AtHsfA5 is a repressor of AtHsfA4a, athsfa5 but not athsfa4a mutant plants exhibited HI sensitivity. Additionally, athsfa4a plants expressing AtHsfA4aΔNES were sensitive to HI, but athsfa5 plants expressing AtHsfA5ΔNES were not. Meanwhile, hit2/athsfa4a double mutant was more tolerant to HI than hit2. These results indicate that both AtHsfA4a and AtHsfA5 were HIT2/XPO1A-specific substrates. Long-term accumulation of AtHsfA4a contributed to the hit2 HI-sensitive phenotype independent of the scavenging ability of H₂O₂, and the presence of AtHsfA5 could mitigate this adverse effect.     

Calcium chloride improves photosynthesis and water status in the C<Subscript>4</Subscript> succulent xerophyte <Emphasis Type="Italic">Haloxylon ammodendron</Emphasis> under water deficit

It is important to reveal the mechanism of plants coping with heat stress, which results in a severe retardation in crop growth and development. Although Synaptotagmin A (SYTA) regulates the cell endocytosis and the plasma membrane reparation of Arabidopsis, its roles in heat resistance are not well understood. In this study, we obtained the new finding that SYTA is related to the heat resistance of Arabidopsis. In the SYTA overexpression strains, the germination rate of the seeds and the survival rate of the seedlings improved after heat shock treatment, and their degree of membrane peroxidation was reduced. However, syta mutant showed the opposite results. Meanwhile, the expressions of some heat stress signal pathway genes were higher in SYTA overexpression strains than that in wild-type strains, and were lower in syta mutant strains. These results suggested that SYTA responded positively to heat shock and was involved in the heat stress signal pathway.     

Growth irradiance affects ureide accumulation and tolerance to photoinhibition in Eutrema salsugineum (Thellungiella salsuginea)

Plants are able to acclimate to their growth light environments by utilizing a number of short- and long-term mechanisms. One strategy is to prevent accumulation of excess reactive oxygen species that can lead to photoinhibition of photosynthesis. Ureides, generated from purine degradation, have been proposed as antioxidants and involved in certain abiotic stress responses. Eutrema salsugineum (Thellungiella salsuginea) is an extremophilic plant known to exhibit a high degree of tolerance to a variety of abiotic stresses that invariably generate reactive oxygen species. In the present study we have investigated the possible role of the ureide metabolic pathway during acclimation to growth irradiance and its conference of tolerance to photoinhibition in Eutrema. Ureide accumulation was greater under high light growth which also conferred tolerance to photoinhibition at low temperature as measured by the maximal quantum yield of PSII photochemistry. This may represent an adaptive plastic response contributing to the extreme tolerance exhibited by this plant. Our results would provide evidence that ureide accumulation may be involved in abiotic stress as another defence mechanism in response to oxidative stress.     

Overexpression of <Emphasis Type="Italic">Zm-HINT1</Emphasis> Confers Salt and Drought Tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Histidine triad nucleotide-binding protein 1 (HINT1) is highly conserved in many species and plays important roles in various biological processes. However, little is known about the responses of HINT1 to abiotic stress in plants. Salt and drought stress are major limiting factors for plant growth and development, and their negative effects on crop productivity may threaten the world’s food supply. Previously, we identified a maize gene, Zm-HINT1, which encodes a 138-amino-acid protein containing conserved domains including the HIT motif, helical regions, and β-strands. Here, we demonstrate that overexpression of Zm-HINT1 in Arabidopsis confers salt and drought tolerance to plants. Zm-HINT1 significantly regulated Na⁺ and K⁺ accumulation in plants under salt stress. The improve tolerance characteristics of Arabidopsis plants that were overexpressing Zm-HINT1 led to increased survival rates after salt and drought treatments. Compared with control plants, those plants that overexpressed Zm-HINT1 showed increased proline content and superoxide dismutase activity, as well as lower malondialdehyde and hydrogen peroxide accumulation under salt and drought treatments. The expression patterns of stress-responsive genes in Arabidopsis plants that overexpressed Zm-HINT1 significantly differed from those in control lines. Taken together, these results suggest that Zm-HINT1 has potential applications in breeding and genetic engineering strategies that are designed to produce new crop varieties with improved salt and drought tolerance.     

A novel <Emphasis Type="Italic">TaSST</Emphasis> gene from wheat contributes to enhanced resistance to salt stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

In this research, through the analyzing of the Triticum aestivum salt-tolerant mutant gene expression profile, under salt stress. A brand new gene with unknown functions induced by salt was cloned. The cloned gene was named Triticum aestivum salt stress protein (TaSST). GenBank accession number of TaSST is ACH97119. Quantitative polymerase chain reaction (qPCR) results exhibited that the expression TaSST was induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG). TaSST could improve salt tolerance of Arabidopsis-overexpressed TaSST. After salt stress, physiological indexes of transgenic Arabidopsis were better compared with WT (wild-type) plants. TaSST was mainly located in the cytomembrane. qPCR analyzed the expression levels of nine tolerance-related genes of Arabidopsis in TaSST-overexpressing Arabidopsis. Results showed that the expression levels of SOS3, SOS2, KIN2, and COR15a significantly increased, whereas the expression of the five other genes showed no obvious change. OsI_01272, the homologous gene of TaSST in rice, was interfered using RNA interference (RNAi) technique. RNAi plants became more sensitive to salt than control plants. Thus, we speculate that TaSST can improve plant salt tolerance.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

A soybean plastidic ATP/ADP transporter gene, <Emphasis Type="Italic">GmAATP</Emphasis>, is involved in carbohydrate metabolism in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic ATP/ADP transporter (AATP) imports adenosine triphosphate (ATP) from the cytosol into plastids, resulting in the increase of the ATP supply to facilitate anabolic synthesis in heterotrophic plastids of dicotyledonous plants. The regulatory role of GmAATP from soybean in increasing starch accumulation has not been investigated. In this study, a gene encoding the AATP protein, named GmAATP, was successfully isolated from soybean. Transient expression of GmAATP in Arabidopsis protoplasts and Nicotiana benthamiana leaf epidermal cells revealed the plastidic localization of GmAATP. Its expression was induced by exogenous sucrose treatment in soybean. The coding region of GmAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of GmAATP significantly increased the sucrose and starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III, and AtSSS IV), and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS, and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild type (WT). These findings suggest that GmAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis. All these results suggest that GmAATP could be used as a candidate gene for developing high starch-accumulating plants as alternative energy crops.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

A tomato plastidic ATP/ADP transporter gene <Emphasis Type="Italic">SlAATP</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

A plastidic ATP/ADP transporter (AATP) is responsible for importing ATP from the cytosol into plastids. Increasing the ATP supply is a potential way to facilitate anabolic synthesis in heterotrophic plastids of plants. In this work, a gene encoding the AATP protein, named SlAATP, was successfully isolated from tomato. Expression of SlAATP was induced by exogenous sucrose treatment in tomato. The coding region of SlAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of SlAATP significantly increased the starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of StAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AtAGPase), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild-type (WT). These findings suggest that SlAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of SlAATP expression might be used for increasing starch accumulation of plants in the future.     

Glutathione and transpiration as key factors conditioning oxidative stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to uranium

Although oxidative stress has been previously described in plants exposed to uranium (U), some uncertainty remains about the role of glutathione and tocopherol availability in the different responsiveness of plants to photo-oxidative damage. Moreover, in most cases, little consideration is given to the role of water transport in shoot heavy metal accumulation. Here, we investigated the effect of uranyl nitrate exposure (50 μM) on PSII and parameters involved in water transport (leaf transpiration and aquaporin gene expression) of Arabidopsis wild type (WT) and mutant plants that are deficient in tocopherol (vte1: null α/γ-tocopherol and vte4: null α-tocopherol) and glutathione biosynthesis (high content: cad1.3 and low content: cad2.1). We show how U exposure induced photosynthetic inhibition that entailed an electron sink/source imbalance that caused PSII photoinhibition in the mutants. The WT was the only line where U did not damage PSII. The increase in energy thermal dissipation observed in all the plants exposed to U did not avoid photo-oxidative damage of mutants. The maintenance of control of glutathione and malondialdehyde contents probed to be target points for the overcoming of photoinhibition in the WT. The relationship between leaf U content and leaf transpiration confirmed the relevance of water transport in heavy metals partitioning and accumulation in leaves, with the consequent implication of susceptibility to oxidative stress.     

Enhancement of starch content by constitutive expression of <Emphasis Type="Italic">GmTrxF</Emphasis> in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant carbohydrate metabolism biosynthesis. In this study, a gene encoding the TrxF protein, named GmTrxF, was isolated from soybean. The open reading frame (ORF) contained 540 nucleotides encoding 179 amino acids. The coding region of GmTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis. The starch content in GmTrxF expressing plants was increased by 57–109% compared to that in wild-type (WT). Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of GmTrxF up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that GmTrxF may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of GmTrxF expression might be used for increasing starch accumulation of plants in the future.     

Proteomics and functional analyses of <Emphasis Type="Italic">Arabidopsis</Emphasis> nitrilases involved in the defense response to microbial pathogens

Main conclusion

Proteomics and functional analyses of the Arabidopsis – Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.