Chunfeng Guan

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.

LchERF, a novel ethylene-responsive transcription factor from Lycium chinense, confers salt tolerance in transgenic tobacco

Key messageAn ERF gene,LchERF, was cloned fromL. chinensefor the first time. Overexpression ofLchERFconferred salt stress tolerance in transgenic tobacco lines during seed germination and vegetative growth.AbstractEthylene-responsive transcription factors (ERFs) play important roles in tolerance to biotic and abiotic stresses by regulating the expression of stress-responsive genes. Although the ERF proteins involved in defense responses against biotic stresses have been extensively documented, the mechanisms by which ERF subfamily genes regulate plant responses to abiotic stresses are largely unknown. In this study, a novel ethylene-responsive transcription factor, named LchERF, was isolated from Lycium chinense (a salinity-resistant plant). Analysis of the LchERF-deduced protein sequence showed that it had a typical AP2/ERF domain and belonged to the B-3 subgroup of the ERF subfamily. The expression of LchERF was found to be tissue specific in L. chinense under normal conditions. Upon treatment with NaCl, polyethylene glycol (PEG) or ethephon (ET), transcript levels of LchERF rapidly increased in L. chinense. Overexpression of LchERF conferred salt stress tolerance in transgenic tobacco during seed germination and vegetative growth. Compared with control lines, LchERF-overexpressing plants showed higher chlorophyll and proline contents, and were associated with lower H2O2 content under salt stress. Overall, our results demonstrate that LchERF might play an important role in the regulation of plant responses to abiotic stresses and mediate various physiological pathways that enhance salt stress tolerance in plants.

The glutathione synthesis may be regulated by cadmium-induced endogenous ethylene in Lycium chinense, and overexpression of an ethylene responsive transcription factor gene enhances tolerance to cadmium stress in tobacco

Glutathione (GSH) plays a pivotal role in heavy metal detoxification. Ethylene is one of the important plant hormones, which plays a critical role in triggering plant responses to different stresses such as cadmium (Cd) stress. Ethylene responsive transcription factor (ERF) belongs to one of the largest plant transcription factor families. ERF is known to be induced by ethylene and thus regulate multiple stress responses through the activation of stress-related genes. Until now, little has been done to explore the relationship among the accumulation of endogenous ethylene, ERF transcript levels and the GSH content in plants under Cd treatment and we will investigate these link. In this study, the gene transcript level of LchERF, LchGSH1 (gene responsible for the first-step GSH biosynthesis) and LchGSHS (gene responsible for the second-step GSH biosynthesis), endogenous ethylene accumulation, GSH content and Cd concentration in Lycium chinense with or without Cd stress treatment were studied. Furthermore, the transgenic tobacco expressing 35S::LchERF which grown under Cd stress condition was also investigated in this study. Our results showed that endogenous ethylene, LchERF, LchGSH1 and LchGSHS gene expression and GSH content can be induced by Cd treatment in L. chinense, however, reduced by co-treatment with 2-aminoethoxyvinlglycine (AVG), an inhibitor of ethylene biosynthesis. The transgenic tobacco expressing 35S::LchERF showed greater tolerance to Cd stress than non-transgenic plants. The expression of NtGSH1 and NtGSHS genes was increased in transgenic tobacco plants compared with non-transgenic plants, indicating that LchERF is associated with the expression level of GSH synthesis related genes in tobacco. Evidence was presented here that under Cd stress, GSH accumulation occurred at least partially via enhanced LchERF gene expression and the ethylene signal transduction pathways might be involved in this accumulation.

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.

Cloning of a cytosolic ascorbate peroxidase gene from Lycium chinense Mill. and enhanced salt tolerance by overexpressing in tobacco.

To evaluate the physiological importance of cytosolic ascorbate peroxidase (APX) in the reactive oxygen species (ROS)-scavenging system, a full-length cDNA clone, named LmAPX, encoding a cytosolic ascorbate peroxidase was isolated from Lycium chinense Mill. using homologous cloning, then the expression of LmAPX under salt stress was investigated. After sequencing and related analysis, the LmAPX cDNA sequence was 965 bp in length and had an open reading frame (ORF) of 750 bp coding for 250 amino acids. Furthermore, the LmAPX sequence was sub-cloned into prokaryotic expression vector pET28a and the recombinant proteins had a high expression level in Escherichia coli. Results from a southern blot analysis indicated that three inserts of this gene existed in the tobacco genome encoding LmAPX. Compared with the control plants (wild-type and empty vector control), the transgenic plants expressing the LmAPX gene exhibited lower amount of hydrogen peroxide (H2O2) and relatively higher values of ascorbate peroxidase activity, proline content, and net photosynthetic rate (Pn) under the same salt stress. These results suggested that overexpression of the LmAPX gene could decrease ROS production caused by salt stress and protect plants from oxidative stress.

De novo characterization of the Lycium chinense Mill. leaf transcriptome and analysis of candidate genes involved in carotenoid biosynthesis

Lycium chinense Mill. (Chinese wolfberry), enriching in carotenoids, is an important Chinese herbal medicine. However, studies on the functional genomics research, especially the carotenoid biosynthesis and accumulation, are limited because of insufficiently available datasets. RNA-Seq was performed by the Illumina sequencing platform. Approximately 26 million clean reads were generated after filtering. Clean reads were assembled by SOAPdenovo and subsequently annotated. Among all 61,595 unigenes, 37,816 (61.39%), 25,266 (41.02%), and 17,598 (28.57%) unigenes were annotated in NCBI non-redundant protein, Swiss-Prot, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. A total of 16,073 and 11,394 unigenes were assigned to Gene Ontology and Cluster of Orthologous Group, respectively. Furthermore, the majority of genes encoding the enzymes in the carotenoid biosynthesis pathway were identified in the unigene datasets. We first found several genes related to L. chinense carotenoid biosynthesis. The expression levels and the biological functions of these genes involved in carotenoid biosynthesis in the leaf and the green ripening fruit were further confirmed by qPCR and high performance liquid chromatography (HPLC). In the present study, we first characterized the transcriptome of L. chinense leaf, which may provide useful data for functional genomics investigations in L. chinense in the future. And essential genes involved in the carotenoid biosynthesis pathway may contribute to elucidate the expression patterns in different stages of development and fruit ripening and the specific mechanisms of carotenoid biosynthesis/accumulation in L. chinense.

Salicylic acid treatment enhances expression of chalcone isomerase gene and accumulation of corresponding flavonoids during fruit maturation of Lycium chinense

Abstract Chalcone isomerase (CHI) is a key gene involved in flavonoid biosynthesis, and flavonoids are important active ingredients in goji berries. Goji berry is a fruit with multi-nutrients consumed all over the world. Up to now, expression patterns of CHI gene and the regulation of flavonoid accumulation in goji berries still remain unknown. In this work, a CHI gene was cloned from fruits of Lycium chinense and was given the name LcCHI. Its relative expression and accumulation of total corresponding flavonoids during fruit maturation were investigated. During the fruit maturation, the expression of LcCHI gene increased gradually, and the accumulation of total flavonoids showed an overall upward trend. This study also showed that the expression of LcCHI gene and the content of total flavonoids in the goji berries could be up-regulated by exogenous salicylic acid (SA) treatment. A reactive oxygen species-dependent signaling pathway was suggested to be involved in this process. This research showed that the expression of LcCHI gene was positively correlated with total flavonoid accumulation and the regulation of them by SA could be a potential approach to produce more desired and valuable flavonoids in goji berries.

Overexpression of AtchyB in Eustoma grandiflorum Shinn Enhances its Tolerance to High-Light Via Zeaxanthin Accumulation

Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection. To investigate the protective role of zeaxanthin and the xanthophyll cycle under high-light stress, we increased the capacity for their biosynthesis in Eustoma grandiflorum Shinn by overexpression of a gene (AtchyB) from Arabidopsis thaliana encoding β-carotene hydroxylase (BCH). This enzyme is involved in the conversion of β-carotene into zeaxanthin and plays an important role in the carotenoid biosynthetic pathway. Not only was the total carotenoid content of the transgenics enhanced (1.046- to 3.141-fold) but zeaxanthin biosynthesis was also faster and the compound was produced in larger quantities in transgenics (up to 3.344-fold) than in controls upon exposure to high-light stress. Additionally, a greater amount of xanthophyll cycle pigments (1.46- to 2.44-fold) was detected in the transgenics. Under high-light stress, untransformed controls showed obvious growth retardation, while transformants were more tolerant. The net addition of biomass in the transformants was more than that of non-transformants under high-light exposure. Furthermore, a new phenomenon was found: high-light stress induced an apparent periodical accumulation of biomass and zeaxanthin in transformants. Our results supplement data from previous research, and indicate that the periodic enhancement of zeaxanthin formation together with the periodic enlargement of the xanthophyll cycle pool contributes to long-term high-light stress protection and prevents plant damage.

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.

Positive feedback regulation of a Lycium chinense-derived VDE gene by drought-induced endogenous ABA, and over-expression of this VDE gene improve drought-induced photo-damage in Arabidopsis

Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress.