Shuqing Cao

Draft genome of the kiwifruit Actinidia chinensis

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1u2009Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage.

Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum

The WRKY transcription factors have been implicated in multiple biological processes in plants, especially in regulating defense against biotic and abiotic stresses. However, little information is available about the WRKYs in tomato (Solanum lycopersicum). The recent release of the whole-genome sequence of tomato allowed us to perform a genome-wide investigation for tomato WRKY proteins, and to compare these positively identified proteins with their orthologs in model plants, such as Arabidopsis and rice. In the present study, based on the recently released tomato whole-genome sequences, we identified 81 SlWRKY genes that were classified into three main groups, with the second group further divided into five subgroups. Depending on WRKY domains’ sequences derived from tomato, Arabidopsis and rice, construction of a phylogenetic tree demonstrated distinct clustering and unique gene expansion of WRKY genes among the three species. Genome mapping analysis revealed that tomato WRKY genes were enriched on several chromosomes, especially on chromosome 5, and 16xa0% of the family members were tandemly duplicated genes. The tomato WRKYs from each group were shown to share similar motif compositions. Furthermore, tomato WRKY genes showed distinct temporal and spatial expression patterns in different developmental processes and in response to various biotic and abiotic stresses. The expression of 18 selected tomato WRKY genes in response to drought and salt stresses and Pseudomonas syringae invasion, respectively, was validated by quantitative RT-PCR. Our results will provide a platform for functional identification and molecular breeding study of WRKY genes in tomato and probably other Solanaceae plants.

Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis

The transcription factor ZAT6 coordinately activates phytochelatin synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis. Cadmium (Cd) is an environmental pollutant with high toxicity to animals and plants. It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, the transcription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown. Here, we identified an Arabidopsis (Arabidopsis thaliana) Cd-resistant mutant xcd2-D (XVE system-induced cadmium-tolerance2) using a forward genetics approach. The mutant gene underlying xcd2-D mutation was revealed to encode a known zinc-finger transcription factor, ZAT6. Transgenic plants overexpressing ZAT6 showed significant increase of Cd tolerance, whereas loss of function of ZAT6 led to decreased Cd tolerance. Increased Cd accumulation and tolerance in ZAT6-overexpressing lines was GSH dependent and associated with Cd-activated synthesis of PC, which was correlated with coordinated activation of PC-synthesis related gene expression. By contrast, loss of function of ZAT6 reduced Cd accumulation and tolerance, which was accompanied by abolished PC synthesis and gene expression. Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1, and PCS2, but ZAT6 is capable of specifically binding to GSH1 promoter in vivo. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6. Taken together, our data provide evidence that ZAT6 coordinately activates PC synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis.

MAN3 gene regulates cadmium tolerance through the glutathione‐dependent pathway in Arabidopsis thaliana

Pollution of soil by the heavy metal cadmium (Cd) is a global environmental problem. The glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance. However, the regulation of this pathway is poorly understood. Here, we identified an Arabidopsis thaliana cadmium-tolerant dominant mutant xcd1-D (XVE system-induced cadmium-tolerance 1) and cloned XCD1 gene (previously called MAN3), which encodes an endo-β-mannanase. Overexpression of MAN3 led to enhanced Cd accumulation and tolerance, whereas loss-of-function of MAN3 resulted in decreased Cd accumulation and tolerance. In the presence of estradiol, enhanced Cd accumulation and tolerance in xcd1-D was associated with GSH-dependent, Cd-activated synthesis of PCs, which was correlated with coordinated activation of gene expression. Cd stress-induced expression of MAN3 and the consequently increased mannanase activity, led to increased mannose content in cell walls. Moreover, mannose treatment not only rescued the Cd-sensitive phenotype of the xcd1-2 mutant, but also improved the Cd tolerance of wild-type plants. Significantly, this mannose-mediated Cd accumulation and tolerance is dependent on GSH-dependent PC concentrations via coordinated control of expression of genes involved in PC synthesis. Our results suggest that MAN3 regulates the GSH-dependent PC synthesis pathway that contributes to Cd accumulation and tolerance in A. thaliana by coordinated control of gene expression.

RNAi-Directed Downregulation of Vacuolar H+-ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice

Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H+-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H+-ATPase activity and an enhancement of plasma membrane H+-ATPase activity, thereby increasing the concentrations of extracellular H+ and intracellular K+ and Na+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H+-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

Freezing sensitivity in the gigantea mutant of Arabidopsis is associated with sugar deficiency

The freezing sensitivity in the gi-3 mutant (an allele of the gigantea mutant) was associated with a constitutive reduction in soluble sugar content. Although sugar accumulation was evident in wild-type plants in response to cold treatment, the gi-3 mutant showed a constitutive reduction in soluble sugar content. There were no significant differences in the proline content and the transcript levels of cold-responsive gene RD29A and abscisic acid-responsive gene RAB18 between the wild type and the gi-3 mutant in response to cold treatment. These results suggest that freezing sensitivity in the gi-3 mutant is associated with sugar deficiency.

Loss-of-function mutations in the APX1 gene result in enhanced selenium tolerance in Arabidopsis thaliana.

It is generally recognized that excess selenium (Se) has a negative effect on the growth and development of plants. Numerous studies have identified key genes involved in selenium tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. In this study, we isolated an Arabidopsis selenium-resistant mutant from the mutant XVE pool lines because of its increased root growth and fresh weight in Se stress, and cloned the gene, which encodes the cytosolic ascorbate peroxidase (APX1). Two other APX1 gene knockout allelic lines were also selenium resistant, and the APX1-complementary COM1 restored the growth state of wild type under Se stress. In addition, these APX1 allelic lines accumulated more Se than did wild-type plants when subjected to Se stress. Further analysis revealed that the APX1-mediated Se tolerance was associated, at least in part, with the enhanced activities of antioxidant enzymes catalase, glutathione peroxidase and glutathione reductase. Moreover, enhanced Se resistance of the mutants was associated with glutathione (GSH), which had the higher expression level of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Our results provide genetic evidence indicating that loss-of-function of APX1 results in tolerance to Se stress.

Characterization of an Arabidopsis cadmium-resistant mutant cdr3-1D reveals a link between heavy metal resistance as well as seed development and flowering

A lot of studies have identified many key genes involved in heavy metal detoxification and tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. To gain insight into the regulatory mechanisms for heavy metal detoxification and tolerance, we performed a mutant screen for identifying Arabidopsis (Arabidopsis thaliana) cadmium (Cd)-resistant mutants. A Cd-resistant mutant cdr3-1D (cadmium-resistant) was isolated because of its increased root growth and fresh weight in Cd stress, and genetic analysis showed that cdr3-1D is a single dominant nuclear mutation. Compared with the wild type, the cdr3-1D mutant was more resistant to heavy metals Cd, Pb, and copper as well as hydrogen peroxide. Moreover, we also observed that seeds of the cdr3-1D mutant were larger than those of wild type, and that cdr3-1D displayed early flowering compared with wild type. A lower Cd/Pb content was detected in cdr3-1D plants than in wild-type plants when subjected to Cd/Pb treatment, which was associated, at least in part, with increase of expression of AtPDR8/AtPDR12, a pump excluding Cd/Pb and/or Cd/Pb-containing toxic compounds from the cytoplasm, respectively. In addition, enhanced Cd/Pb resistance of the cdr3-1D mutant was partially glutathione (GSH) dependent, which was related to increase of expression of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Taken together, our results provide genetic evidence indicating that CDR3 is involved in the regulation of heavy metal resistance as well as seed development and flowering.

The PSE1 gene modulates lead tolerance in Arabidopsis

Highlight PSE1 regulates Pb tolerance mainly through GSH-dependent phytochelatin synthesis by activating the expression of the genes involved in phytochelatin synthesis and at least partially through activating the expression of PDR12.

Cloning and Function Characterization of Two Dehydroascorbate Reductases from Kiwifruit (Actinidia chinensis L.)

Dehydroascorbate reductase (DHAR, EC 1.8.5.1) plays a critical role in the regeneration of l-ascorbic acid (AsA). To date, there is virtually no information on the molecular characteristics of DHAR in kiwifruit, an economically and nutritionally important horticultural crop with remarkably high AsA concentration. Here, we isolated two cDNAs encoding putative DHARs (designated as AcDHAR1 and AcDHAR2) from Actinidia chinensis cv. Hongyang. Both in silico and subcellular localization analyses demonstrated that AcDHAR1 and AcDHAR2 were targeted to cytosol and chloroplast, respectively. The recombinant AcDHAR1 and AcDHAR2 were expressed in Escherichia coli and purified using Ni-affinity chromatography. Enzymatic study shows both of them are thermostable and possess a relatively high affinity to dehydroascorbate with an optimal pH ranging from 6 to 8. In addition, transgenic Arabidopsis thaliana plants separately expressing either AcDHAR1 or AcDHAR2 were shown to have significantly increased AsA concentration and enhanced tolerance to salinity. The present study suggested that AcDHAR1 and AcDHAR2 may play a protective role in response to environmental stimuli in kiwifruit.