ng Ji

A GSHS-like gene from Lycium chinense maybe regulated by cadmium-induced endogenous salicylic acid and overexpression of this gene enhances tolerance to cadmium stress in Arabidopsis

Key messageA GSHS gene,LcGSHS, was cloned fromL. chinensefor the first time. Evidence is presented here that endogenous SA accumulation maybe important for the regulation ofLcGSHSexpression level.AbstractGlutathione (GSH) plays a pivotal role in heavy metal detoxification. GSH synthetase (GSHS) catalyzes the rate-limiting step of GSH synthesis in plants. Salicylic acid (SA) is one of the important plant hormones, which plays a critical role in triggering plant responses to different stresses such as cadmium (Cd) stress. Until now, little has been done to explore the relationship among the accumulation of endogenous SA, GSHS transcript levels and the GSH content in plants under Cd treatment and we will investigate this link in this study. The chlorophyll content, transcripts level of LcGSHS gene, endogenous SA accumulation, GSH accumulation and Cd concentration in the leaves of Lycium chinense were studied under different treatment conditions. Endogenous SA, LcGSHS transcript expression and GSH content can be induced by Cd treatment in L. chinense, however, reduced by co-treatment with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of SA biosynthesis. Strong staining was observed in the leaves of Arabidopsis expressing ProLcGSHS::GUS under Cd stress and the staining was reduced by co-treatment with AIP. The transgenic Arabidopsis expressing ProLcGSHS::LcGSHS also showed greater tolerance to Cd stress than wild types. Evidence was presented here that under Cd stress, GSH accumulation occurred via enhanced LcGSHS gene expression and the SA signaling cascade was involved in this accumulation. Furthermore, the overexpression of LcGSHS in transgenic Arabidopsis resulted in greater tolerance to Cd stress than wild-type lines.

LcMKK, a novel group A mitogen-activated protein kinase kinase gene in Lycium chinense, confers dehydration and drought tolerance in transgenic tobacco via scavenging ROS and modulating expression of stress-responsive genes

The mitogen-activated protein kinase (MAPK) cascades have been previously implicated in signal transduction during plant responses to various environmental stresses. As the convergent point of the MAPK cascades, MAPKKs play paramount roles in amplifying, integrating, and channeling information between the extracellular stimuli and intracellular responses. However, the functional role of MAPKKs in Lycium chinense has never been explored. In this study, a novel MAPKK gene, LcMKK, in L. chinense belonging to group A MAPKKs was isolated and functionally characterized. The transcript level of LcMKK rapidly increased in L. chinense after drought treatments. Overexpression of LcMKK in tobacco conferred dehydration and drought tolerance. Under dehydration and drought conditions, the transgenic tobacco lines exhibited better water status, less accumulation of reactive oxygen species (ROS), higher levels of germination rate and antioxidant enzyme activity than the wild type. In addition, overexpression of LcMKK enhanced the expression of ROS-related and stress-responsive genes under drought conditions. Taken together, these data demonstrate that LcMKK acts as a positive regulator in dehydration/drought stress responses by either regulating ROS homeostasis through the activation of the cellular antioxidant defense system or modulating transcriptional levels of a variety of stress-associated genes.

A chimeric vacuolar Na+/H+ antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast

The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast.

Comparative transcriptome analysis of transcription factors in different maize varieties under salt stress conditions

Salt stress is a major environmental factor affecting plant growth and crop production worldwide, and the transcription factors (TFs) play crucial roles in plant response to salt stress. Identifying genes related to salt-tolerance contributes to salt-tolerant crop breeding. A comparative transcriptome analysis was carried out to investigate global gene expression of the entire TFs in two maize varieties with different salt-tolerant ability. Fifty-five TF families including 1283 TF genes were identified. Among them, 314 TF genes were differentially expressed in the two maize varieties under salt stress. 177 TF genes were detected with significantly higher expression levels in salt-tolerance variety compared with the salt-sensitive one. The differential expression of a set of TF families clearly demonstrated their important roles in salt tolerance. Further phylogenetic analysis and gene expression analysis of heat shock factors (HSFs) revealed that majority of these TFs were induced by salt stress, but different classes/subclasses had different response to salt stress. HSF class-B genes were detected with significantly higher expression levels in salt-tolerance variety compared with the salt-sensitive one under salt stress, which may result in different plants salt-tolerance ability. These results contribute to a better understanding of the complex mechanism of TFs in response to salt stress in maize and provide new sight for further research to perform systematic analysis of the TF families and to reveal their potential functions in the salt-tolerance for plants.

Characterization of lycopene β-cyclase gene from Lycium chinense conferring salt tolerance by increasing carotenoids synthesis and oxidative stress resistance in tobacco

Salt stress is a principal cause for yield reduction in crops. In plants, carotenoids are involved in abiotic stress resistance, while the underlying mechanisms for that have not been identified. In this study, the lycopene β-cyclase (LclycB) gene of Lycium chinense was identified to be related to salt stress and was found to have the highest expression level in leaves through quantitative RT-PCR. The coding region of this gene was obtained through reverse transcription-PCR. The function of LclycB was confirmed in vitro with an Escherichia coli expression system. To study the function of LclycB, it was transformed into tobacco and maintained till T2 generation. Four-week old transgenic plants treated with 150xa0mM NaCl showed enhanced tolerance to oxidation and less reactive oxygen species (ROS) accumulation. Eight-week old transgenic plants treated with NaCl showed better growth than the control with higher photosystem activity and total antioxidants activity. The detection of photosynthetic pigments demonstrated that the transgenic plants retained higher contents of chlorophyll and carotenoids than the control under normal and salt-treated conditions. Furthermore, the thylakoid structure of chloroplast was notably disorganized in WT leaves compare to that of transformants by electron microscopy. Although carotenoid is the precursor of abscisic acid (ABA),the ABA content and stomatal conductance reflect nearly no differences between transgenic and control plants. These results demonstrated that the salt tolerance of LclycB might be ascribed to the enhanced carotenoid content for its ROS scavenging ability, photoprotection and membrane stabilization.

LcBiP, a endoplasmic reticulum chaperone binding protein gene from Lycium chinense, confers cadmium tolerance in transgenic tobacco

Cadmium (Cd) accumulation is very toxic to plants. The presence of Cd may lead to excessive production of reactive oxygen species (ROS), and then cause inhibition of plant growth. The endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which has been shown to function as a sensor of alterations in the ER environment. BiP overexpression in plants was shown to increase drought tolerance through inhibition of ROS accumulation. Due to the above relationships, it is likely that there may be a link between Cd stress tolerance, ROS accumulation and the BiP transcript expression in plants. In this study, a BiP gene, LcBiP, from L. chinense was isolated and characterized. Overexpression of LcBiP in tobacco conferred Cd tolerance. Under Cd stress conditions, the transgenic tobacco lines exhibited better chlorophyll retention, less accumulation of ROS, longer root length, more glutathione (GSH) content, and less antioxidant enzyme activity than the wild type. These data demonstrated that LcBiP act as a positive regulator in Cd stress tolerance. It is hypothesized that the improved Cd tolerance of the transgenic tobacco plants may be due to the enhanced ROS scavenging capacity. The enhancement of GSH content might contribute to this ROS scavenging capacity in the transgenic plants. However, the underlying mechanism for BiP‐mediated increase in Cd stress tolerance need to be further clarified.

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K+ and Na+ in Arabidopsis root cell

While the role of the vacuolar NHX Na+/H+ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na+ and K+ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na+/H+ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na+ and K+ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na+ into root cell vacuoles and also promoted high cytosolic and vacuolar K+ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na+ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na+ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na+ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na+ sequestration in the vacuole and K+ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.

Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco

Flavonoids, as plant secondary metabolites, are widespread throughout the plant kingdom and involved in many physiological and biochemical processes. Drought resistance is attributed to flavonoids with respect to protective functions in the cell wall and membranes. The flavanone 3-hydroxylase (F3H) gene which encodes flavanone 3-hydroxylase, is essential in flavonoids biosynthetic pathway. Lycium chinense (L.xa0chinense) is a deciduous woody perennial halophyte that grows under a large variety of environmental conditions and survives under extreme drought stress. A novel cDNA sequence coding a F3H gene in Lycium chinense (LcF3H, GenBank: KJ636468.1) was isolated. The open reading frame of LcF3H comprised 1101xa0bp encoding a polypeptide of 366 amino acids with a molecular weight of about 42xa0kDa and an isoelectric point of 5.32. The deduced LcF3H protein showed high identities with other plant F3Hs, and the conserved motifs were found in LcF3H at similar positions like other F3Hs. The recombinant protein converted naringen into dihydrokaempferol inxa0vitro. Since studies have shown that amongst flavonoids, flavan-3-ols (catechin and epicatechin) have direct free radical scavenging activity to maintain the normal physiological function of cells inxa0vivo, these data support the possible relationship between the oxidative damage and the regulation of LcF3H gene expression in L.xa0chinense under drought stress. In order to better understand the biotechnological potential of LcF3H, gene overexpression was conducted in tobacco. The content of flavan-3-ols and the tolerance to drought stress were increased in LcF3H overexpressing tobacco. Analysis of transgenic tobacco lines also showed that antioxidant enzyme activities were increased meanwhile the malondialdehyde (MDA) content and the content of H2O2 were reduced comparing to nontransformed tobacco plants. Furthermore, the photosynthesis rate was less decreased in the transgenetic plants. These results suggest that LcF3H plays a role in enhancing drought tolerance in L.xa0chinense, and its overexpression increases tolerance to drought stress by improving the antioxidant system in tobacco.

Overexpression of lycopene ε-cyclase gene from lycium chinense confers tolerance to chilling stress in Arabidopsis thaliana.

Lutein plays an important role in protecting the photosynthetic apparatus from photodamage and eliminating ROS to render normal physiological function of cells. As a rate-limiting step for lutein synthesis in plants, lycopene ε-cyclase catalyzes lycopene to δ-carotene. We cloned a lycopene ε-cyclase gene (Lcε-LYC) from Lycium chinense (L. chinense), a deciduous woody perennial halophyte growing in various environmental conditions. The Lcε-LYC gene has an ORF of 1569bp encoding a protein of 522 aa. The deduced amino acid sequence of Lcε-LYC gene has higher homology with LycEs in other plants, such as Nicotiana tabacum and Solanum tuberosum. When L. chinense was exposed to chilling stress, relative expression of Lcε-LYC increased. To study the protective role of Lcε-LYC against chilling stress, we overexpressed the Lcε-LYC gene in Arabidopsis thaliana. Lcε-LYC overexpression led to an increase of lutein accumulation in transgenic A. thaliana, and the content of lutein decreased when transgenics were under cold conditions. In addition, the transgenic plants under chilling stress displayed higher activities of superoxide dismutase (SOD) and peroxidase (POD) and less H2O2 and malondialdehyde (MDA) than the control. Moreover, the photosynthesis rate, photosystem II activity (Fv/fm), and Non-photochemical quenching (NPQ) also increased in the transgenetic plants. On the whole, overexpression of Lcε-LYC ameliorates photoinhibition and photooxidation, and decreases the sensitivity of photosynthesis to chilling stress in transgenic plants.

Cloning and Expression Analysis of 9-cis-Epoxycarotenoid Dioxygenase Gene 1 Involved in Fruit Maturation and Abiotic Stress Response in Lycium chinense

Abscisic acid (ABA) plays a crucial role in plant adaptations to environmental stress, growth, and development, such as seed dormancy and germination. 9-cis-epoxycarotenoid dioxygenase (NCED) is a rate-limiting enzyme in regulation of ABA biosynthesis in plants. To understand the potential role of NCED in fruit ripening and stress tolerance, a NCED gene (LcNCED1) was cloned from the leaves of Lycium chinense. LcNCED1 has an ORF of 1824xa0bp, which encodes a peptide of 607 amino acids. The deduced amino acid sequence of the LcNCED1 protein shares high identity with other NCEDs. Tissue distribution analysis reveals that LcNCED1 is abundantly expressed in leaves, stems, and flowers. In fruits, the expression level of LcNCED1 is in accordance with the accumulation of ABA. In addition, ABA accumulation in leaves was associated with enhanced expression of LcNCED1 induced strongly by abiotic stresses (drought, salt, and CdCl2). Collectively, our results indicated that LcNCED1 might play a key role in the regulation of fruit ripeness and abiotic stress adaption in L. chinense possibly through regulation of ABA biosynthesis.