Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase gene (<Emphasis Type="Italic">CsCSD1</Emphasis>) from <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Superoxide dismutase (SOD) proteins, which are widely present in the plant kingdom, play vital roles in response to abiotic stress. However, the functions of cucumber SOD genes in response to environmental stresses remain poorly understood. In this study, a SOD gene CsCSD1 was identified and functionally characterized from cucumber (Cucumis sativus). The CsCSD1 protein was successfully expressed in E. coli, and its overexpression significantly improved the tolerance of host E. coli cells to salinity stress. Besides, overexpression of CsCSD1 enhanced salinity tolerance during germination and seedling development in transgenic Arabidopsis plants. Further analyses showed that the SOD and CAT (catalase) activities of transgenic plants were significantly higher than those of wild-type (WT) plants under normal growth conditions as well as under NaCl treatment. In addition, the expression of stress-response genes RD22, RD29B and LEA4-5 was significantly elevated in transgenic plants. Our results demonstrate that the CsCSD1 gene functions in defense against salinity stress and may be important for molecular breeding of salt-tolerant plants.     

A Leucine-Rich Repeat Receptor-like Kinase from the Antarctic Moss <Emphasis Type="Italic">Pohlia nutans</Emphasis> Confers Salinity and ABA Stress Tolerance

Plant leucine-rich repeats receptor-like kinases (LRR-RLKs) play key roles in plant growth, development, and responses to environmental stresses. However, the functions of LRR-RLKs in bryophytes are still not well documented. Here, a putative LRR-RLK gene, PnLRR-RLK, was cloned and characterized from the Antarctic moss Pohlia nutans. Phylogenetic analysis revealed that PnLRR-RLK protein was clustered with the Arabidopsis thaliana LRR XI family proteins. Subcellular localization analysis of PnLRR-RLK revealed that it was mainly localized on plasma membrane. The expression of PnLRR-RLK was induced by mock high salinity, cold, drought, and exogenously supplied abscisic acid (ABA) and methyl jasmonate (MeJA). Meanwhile, the overexpression of PnLRR-RLK showed an increased tolerance of transgenic Arabidopsis to salt and ABA stresses than that of the wild type (WT) plants. Furthermore, the expression levels of several salt tolerance genes (AtHKT1, AtSOS3, AtP5CS1, and AtADH1) and an ABA negatively regulating gene AtABI1 were significantly increased in transgenic plants. Meanwhile, the expression levels of ABA biosynthesis genes (AtNCED3, AtABA1, and AtAAO3) and ABA early response genes (AtMYB2, AtRD22, AtRD29A, and AtDREB2A) were decreased in transgenic Arabidopsis after salt stress treatment. Therefore, these results suggested that PnLRR-RLK might involve in regulating salt stress-related and ABA-dependent signaling pathway, thereby contribute to the salinity tolerance of the Antarctic moss P. nutans.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

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.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.     

Three <Emphasis Type="Italic">P5CS</Emphasis> genes including a novel one from <Emphasis Type="Italic">Lilium regale</Emphasis> play distinct roles in osmotic,drought and salt stress tolerance

Proline accumulations in abiotically stressed plants is generally considered to benefit their stress tolerance. The Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) gene family, which encodes the rate-limiting enzyme in proline biosynthesis pathway, usually contains two duplicated genes in most plants. However, three P5CS genes including LrP5CS1, LrP5CS2 as well as a third one, LrP5CS3, were isolated from Lilium regale. LrP5CS3 is highly identical to LrP5CS1 in amino acid sequences, indicating they could come from a paralogous duplication. The phylogenetic tree suggested that the duplication of LrP5CS occurred independently after the divergence of Liliales and commelinoids. The expression of LrP5CS1 was strongly induced in leaves and roots both under drought and salinity, while that of LrP5CS3 was upregulated more moderately. LrP5CS2 stayed almost constitutive under stress. LrP5CS1 exhibited the highest activity after expressed in E. coli. Overexpression of LrP5CS genes conferred enhanced osmotic, drought and salt tolerance on transgenic Arabidopsis without negative effects in unstressed condition. Under salt stress, lines LrP5CS2 accumulated fewer proline than others, and lines LrP5CS1 grew better in root elongation. The roots of lines LrP5CS3 grew better than all others under unstressed condition and osmotic stress. Our study suggests that the three LrP5CS genes play distinct roles respectively in proline accumulation and abiotic stress tolerance.     

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.     

Co-expression of <Emphasis Type="Italic">AtNHX1</Emphasis> and <Emphasis Type="Italic">TsVP</Emphasis> improves the salt tolerance of transgenic cotton and increases seed cotton yield in a saline field

Salinity is one of the major abiotic stressors affecting cotton production. The AtNHX1 gene from Arabidopsis thaliana and the TsVP gene from Thellungiella halophila?were co-expressed in cotton (cv. GK35) to improve its salt tolerance. Cotton with overexpressed AtNHX1-TsVP genes had higher emergence rates and higher dry matter accumulation under salt stress in the greenhouse and better emergence rates and survival rates in a saline field compared to the WT. More importantly, the cotton with overexpressed AtNHX1-TsVP genes had higher seed cotton yield in the saline field. The growth of transgenic cotton with overexpression of the AtNHX1-TsVP genes may be related to the accumulation of Na⁺, K⁺ and Ca²⁺ in leaves under salt stress. The accumulation of these cations could improve the ability to maintain ion homeostasis and osmotic potential in plant cells under salt stress, thereby conferring cells with higher relative water content and maintaining higher carbon assimilation capacity. These results reveal that overexpression of AtNHX1-TsVP significantly enhances the tolerance of transgenic cotton to high salinity compared to WT. This study aids efforts of breeding salt-tolerant cotton to achieve the strategy of “westward, eastward, northward” in Chinese cotton production.     

Ecophysiological response of native and invasive <Emphasis Type="Italic">Spartina</Emphasis> species to extreme temperature events in Mediterranean marshes

The recent IPCC WG2 5th Assessment Report (IPCC 2014), notes an increase in the frequency and duration of extreme climatic events, especially for the Mediterranean region. Together with climate change, the invasion of natural communities by non-indigenous species (NIS) constitutes a serious threat to biodiversity. One of these NIS is the American Spartina patens, now present in Western European marshes. The present study aims to understand the biochemical and photochemical responses of S. patens compared with S. maritima under extreme temperature events. Under normal and extreme heat conditions, S. patens had a higher photosynthetic efficiency (α), compared with cold wave events, where the native S. maritima was far more efficient. This reduced photosynthetic efficiency was mostly due to a decrease in the connectivity between photosystem II (PSII) antennae. This was accompanied by severe damage to the oxygen-evolving complex of PSII. On the other hand, S. patens oxygen evolving complexes (OECs) seem to be temperature insensitive. The light absorption capacity was maintained due to a higher net rate of reaction centre (RC) closure as a counteractive measure of the reduced number of RC, especially in S. maritima. The loss of connectivity between PSII antennae and damage in OECs under heat stress leads to a severe reduction in the maximum yield for photochemistry enhanced by the low probability of each absorbed quanta to produce electronic work. However, while S. maritima presents high energy losses under heat stress, S. patens developed efficient quenching mechanisms under thermal stress, through auroxanthin. In S. patens, cold wave-treated individuals also displayed a very active line of enzymatic defences for reactive oxygen species scavenging. In fact, only cold treated individuals of this species presented higher activities of anti-oxidant enzymes, revealing some degree of adaptation to this new environment. In contrast, in S. maritima the exposure to extreme heat periods led, in most cases, to a decrease in the enzymatic defences, leaving the cell prone to oxidative damage. In summary, S. patens appears to have a higher fitness for the incoming climatic scenarios, being more tolerant to heat stress, while S. maritima will have its photobiological fitness decreased. This will impose a shift in the salt marsh biodiversity, favouring the non-indigenous S. patens expansion.