Mei-Liang Zhou

Improvement of drought and salt tolerance in Arabidopsis and Lotus corniculatus by overexpression of a novel DREB transcription factor from Populus euphratica.

A novel DREB (dehydration-responsive element binding) gene, designated PeDREB2a, was isolated from the desert-grown tree, Populus euphratica Oliv. PeDREB2a is classified into the A-5 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. Using semi-quantitative RT-PCR, we found that the PeDREB2a was greatly induced by drought, NaCl, low temperature, 1-naphthaleneacetic acid (NAA), 6-benzyl aminopurine (6-BA) and gibberellic acid (GA3) treatments in P. euphratica seedling. Yeast transactivity assay demonstrated that PeDREB2a gene encodes a transcription activator. Overexpression of PeDREB2a under the stress-inducible rd29A promotor in transgenic Arabidopsis and Lotus corniculatus forage plants resulted in enhanced tolerance to salt and drought stresses. The PeDREB2a overexpressing Arabidopsis lines showed higher root length and plant height and had elevated levels of soluble sugars and lower levels of malondialdehyde under stress conditions compared to control plants. The results revealed that PeDREB2a play an essential role as a DREB transcription factor in regulation of stress-responsive signaling in P. euphratica.

Plant Hairy Roots for Remediation of Aqueous Pollutants

Significant progress has been made in recent years in enhancing the ability of plants to tolerate, remove, and degrade pollutants. Plant root remediation of contaminated soils and groundwater shows great potential for future development due to its environmental compatibility and cost-effectiveness. Hairy roots are disease manifestations developed by plants that are wounded and infected by Agrobacterium rhizogenes. The application of transgenic hairy roots in phytoremediation has been suggested mainly because of their biochemical resemblance to the roots of the plant from which they are derived. The application of genetic engineering has greatly augmented removal rates of hazardous pollutants. In addition, the rhizospheric bacteria that live on or around plant hairy roots also lead to improved tolerance to normally phytotoxic chemicals and increased removal of pollutants. This paper provides a broad overview of the evidence supporting the suitability and prospects of hairy roots in phytoremediation of organic pollutants and heavy metals.

Genome-wide analysis of AP2/ERF family genes from Lotus corniculatus shows LcERF054 enhances salt tolerance

Lotus corniculatus is used in agriculture as a main forage plant. Members of the Apetala2/ethylene response factor (AP2/ERF) family play important roles in regulating gene expression in response to many forms of stress, including drought and salt. Here, starting from database of the L. corniculatus var. japonicus genome, we identified 127 AP2/ERF genes by insilico cloning method. The phylogeny, gene structures, and putative conserved motifs in L. corniculatus var. japonicus ERF proteins were analyzed. Based on the number of AP2/ERF domains and the function of the genes, 127 AP2/ERF genes from L. corniculatus var. japonicus were classified into five subfamilies named the AP2, dehydration-responsive element binding factor (DREB), ERF, RAV, and a soloist. Outside the AP2/ERF domain, many L. corniculatus var. japonicus-specific conserved motifs were detected. Expression profile analysis of AP2/ERF genes by quantitative real-time PCR revealed that 19 LcERF genes, including LcERF054 (KJ004728), were significantly induced by salt stress. The results showed that the LcERF054 gene encodes a nuclear transcription activator. Overexpression of LcERF054 in Arabidopsis enhanced the tolerances to salt stress, showed higher germination ratio of seeds, and had elevated levels of relative moisture contents, soluble sugars, proline, and lower levels of malondialdehyde under stress conditions compared to wild-type plants. The expression of hyperosmotic salinity response genes COR15A, LEA4-5, P5CS1, and RD29A was found to be elevated in the LcERF054-overexpressing Arabidopsis plants compared to wild type. These results revealed that the LcERF genes play important roles in L. corniculatus cv Leo under salt stress and that LcERFs are attractive engineering targets in applied efforts to improve abiotic stress tolerances in L. corniculatus cv Leo or other crops.

Genome-wide identification of genes involved in raffinose metabolism in Maize

The raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are synthesized by a set of distinct galactosyltransferases, which sequentially add galactose units to sucrose. The accumulation of RFOs in plant cells are closely associated with the responses to environmental factors, such as cold, heat and drought stresses. Systematic analysis of genes involved in the raffinose metabolism has not been reported to date. Searching the recently available working draft of the maize genome, six kinds of enzyme genes were speculated, which should encode all the enzymes involved in the raffinose metabolism in maize. Expression patterns of some related putative genes were analyzed. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as galactinol synthase (ZmGolS5, ZmGolS45 and ZmGolS37), raffinose synthase (ZmRS1, ZmRS2, ZmRS3 and ZmRS10), stachyose synthase (ZmRS8) and β-fructofuranosidase, are encoded by multiple gene members with different expression patterns. These results reveal the complexity of the raffinose metabolism and the existence of metabolic channels for diverse RFOs in maize and provide useful information for improving maize stress tolerance through genetic engineering.

Aldehyde dehydrogenase protein superfamily in maize

Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)+-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement.

Trehalose Metabolism-Related Genes in Maize

Maize is a cereal crop that is grown widely throughout the world in a range of agro-ecological environments. Trehalose is a nonreducing disaccharide of glucose that has been associated with tolerance to different stress conditions, including salt and drought. Bioinformatic analysis of genes involved in trehalose biosynthesis and degradation in maize has not been reported to date. Through systematic analysis, 1 degradation-related and 36 trehalose biosynthesis-related genes were identified. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs, isolated from other plant species such as Arabidopsis and rice, were revealed. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to salt stress, jasmonic acid, and abscisic acid treatment were analyzed. Microarray data demonstrated that some of the genes show differential, organ-specific expression patterns in the 60 different developmental stages of maize. It was discovered that some of the key enzymes such as hexokinase, trehalose-6-phosphate synthase, and trehalose-6-phosphate phosphatase are encoded by multiple gene members with different expression patterns. The results highlight the complexity of trehalose metabolism and provide useful information for improving maize stress tolerance through genetic engineering.

Nicotianamine synthase gene family as central components in heavy metal and phytohormone response in maize

Nicotianamine (NA) is an important divalent metal chelator and the main precursor of phytosiderophores. NA is synthesized from S-adenosylmethionine in a process catalyzed by nicotianamine synthase (NAS). In this study, a set of structural and phylogenetic analyses have been applied to identify the maize NAS genes based on the maize genome sequence release. Ten maize NAS genes have been mapped; seven of them have not been reported to date. Phylogenetic analysis and expression pattern from microarray data led to their classification into two different orthologous groups. C-terminal fusion of ZmNAS3 with GFP was found in the cytoplasm of Arabidopsis leaf protoplast. Expression analysis by reverse transcription polymerase chain reaction revealed ZmNAS genes are responsive to heavy metal ions (Ni, Fe, Cu, Mn, Zn, and Cd), and all 10 ZmNAS genes were only observed in the root tissue except of ZmNAS6. The promoter of ZmNAS genes was analyzed for the presence of different cis-element response to all kinds of phytohormones and environment stresses. We found that the ZmNAS gene expression of maize seedlings was regulated by jasmonic acid, abscisic acid, and salicylic acid. Microarray data demonstrated that the ZmNAS genes show differential, organ-specific expression patterns in the maize developmental steps. The integrated comparative analysis can improve our current view of ZmNAS genes and facilitate the functional characterization of individual members.

Overexpression of a Lotus corniculatus AP2/ERF transcription factor gene, LcERF080, enhances tolerance to salt stress in transgenic Arabidopsis

The APETALA2/ethylene-responsive element binding factors (AP2/ERF) play central roles in the stress response in plants. In this study, we identified and isolated a novel salt stress-related gene, LcERF080, that encodes an AP2/ERF protein in Lotus corniculatus cultivar Leo. LcERF080 was classified into the B-4 group of the ERF subfamily based on multiple sequence alignment and phylogenetic characterization. Expression of LcERF080 was strongly induced by salt, abscisic acid, 1-aminocyclopropane-1-carboxylic acid, methyl jasmonate, and salicylic acid stresses. Subcellular localization assay confirmed that LcERF080 is a nuclear protein. LcERF080 overexpression in Arabidopsis resulted in pleiotropic phenotypes with a higher seed germination rate and transgenic plants with enhanced tolerance to salt stress. Further, under stress conditions, the transgenic lines exhibited elevated levels of soluble sugars and proline as well as relative moisture contents but a lower malondialdehyde content than in control plants. The expression levels of hyperosmotic salinity response genes COR15A, RD22, and P5CS1 were found to be elevated in the LcERF080-overexpressing Arabidopsis plants compared to the wild-type plants. These results reveal that LcERF080 is involved in the responses of plants to salt stress.

Characterization of Genes Involved in Isoprenoid Diphosphate Biosynthesis in Maize

Terpenoids are the largest class of secondary metabolites with indispensable functions for plant growth and development. Maize is a very important cereal crop that is grown widely throughout the world. However, many genes and gene family members involved in terpenoid biosynthesis are still unknown in maize. Through systematic analysis, 43 isoprenoid diphosphate biosynthesis-related genes were identified. These genes are members of 15 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Using a comprehensive approach, the intron/exon structures and their responses to jasmonic acid treatment were investigated. Meta-analysis revealed that some of the genes show differential, organ-specific expression patterns in the 60 different developmental stages of maize. It was found that all 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway genes present the same expression patterns which were highly expressed in leaves and showed the lowest expression in seed. All investigated MEP pathway enzymes are localized in plastids by transient assay using Arabidopsis cells. In addition, some of the enzymes, such as acetyl-CoA C-acetyltransferase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns, and both homomeric and heteromeric geranyl diphosphate synthases exist in maize. The results reveal the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in maize.

Identification of JAZ1-MYC2 Complex in Lotus corniculatus

Lotus corniculatus is exposed to many forms of biotic stresses, including pathogen and herbivore attack. Jasmonates (JAs) are lipid-based signaling molecules that play important roles in mediating secondary metabolite biosynthesis and plant defense responses. Here, we first characterized LcJAZ1 and LcMYC2, two key components of the JAs signaling pathway. Both LcJAZ1 and LcMYC2 mRNA accumulated quickly in response to JAs. Subcellular localization analysis showed that both LcJAZ1 and LcMYC2 are nuclear proteins. In addition, LcJAZ1 protein was degraded after JAs treatment, but LcMYC2 is not. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays demonstrated that LcJAZ1 interacts with LcMYC2 and AtMYC2. In addition, LcMYC2 interacts with AtJAZ1. The Arabidopsis protoplast transactivation assay revealed that LcJAZ1 acts as repressors of LcMYC2- or AtMYC2-mediated transcription. Our data indicate that the conserved JAZ-MYC complex contributes to the modulation of the JAs signaling pathway.