Guifeng Liu

Label-free Quantitative Proteomics Analysis of Etiolated Maize Seedling Leaves during Greening

To better understand light regulation of C4 plant maize development, we investigated dynamic proteomic differences between green seedlings (control), etiolated seedlings, and etiolated seedlings illuminated for 6 or 12 h using a label-free quantitative proteomics approach based on nanoscale ultraperformance liquid chromatography-ESI-MSE. Among more than 400 proteins identified, 73 were significantly altered during etiolated maize seedling greening. Of these 73 proteins, 25 were identified as membrane proteins that seldom had been identified with two-dimensional electrophoresis methods, indicating the power of our label-free method for membrane protein identification; 31 were related to light reactions of chlorophyll biosynthesis, photosynthesis, and photosynthetic carbon assimilation. The expression of photosystem II subunits was highly sensitive to light; most of them were not identified in etiolated maize seedlings but drastically increased upon light exposure, indicating that the complex process of biogenesis of the photosynthetic apparatus correlates with the transition from a dark-grown to a light-grown morphology. However, transcriptional analysis indicated that most transcripts encoding these proteins were not regulated by light. In contrast, the levels of mRNAs and proteins for enzymes involved in carbon assimilation were tightly regulated by light. Additionally phosphoenolpyruvate carboxykinase, the key enzyme of the phosphoenolpyruvate carboxykinase C4 pathway, was more tightly regulated by light than the key enzymes of the NADP-malic enzyme C4 pathway. Furthermore phosphoenolpyruvate carboxylase 1C, which was originally reported to be specifically expressed in roots, was also identified in this study; expression of this enzyme was more sensitive to light than its isoforms. Taken together, these results represent a comprehensive dynamic protein profile and light-regulated network of C4 plants for etiolated seedling greening and provide a basis for further study of the mechanism of gene function and regulation in light-induced development of C4 plants.

A novel bZIP gene from Tamarix hispida mediates physiological responses to salt stress in tobacco plants.

Basic leucine zipper proteins (bZIPs) are transcription factors that bind abscisic acid (ABA)-responsive elements (ABREs) and enable plants to withstand adverse environmental conditions. In the present study, a novel bZIP gene, ThbZIP1 was cloned from Tamarix hispida. Expression studies in T. hispida showed differential regulation of ThbZIP1 in response to treatment with NaCl, polyethylene glycol (PEG) 6000, NaHCO(3), and CdCl(2), suggesting that ThbZIP1 is involved in abiotic stress responses. To identify the physiological responses mediated by ThbZIP1, transgenic tobacco plants overexpressing exogenous ThbZIP1 were generated. Various physiological parameters related to salt stress were measured and compared between transgenic and wild type (WT) plants. Our results indicate that overexpression of ThbZIP1 can enhance the activity of both peroxidase (POD) and superoxide dismutase (SOD), and increase the content of soluble sugars and soluble proteins under salt stress conditions. These results suggest that ThbZIP1 contributes to salt tolerance by mediating signaling through multiple physiological pathways. Furthermore, ThbZIP1 confers stress tolerance to plants by enhancing reactive oxygen species (ROS) scavenging, facilitating the accumulation of compatible osmolytes, and inducing and/or enhancing the biosynthesis of soluble proteins.

Expression profiling of salinity-alkali stress responses by large-scale expressed sequence tag analysis in Tamarix hispid

Tamarix hispida, a woody halophyte, thrives in saline and saline-alkali soil. To better understand the gene expression profiles that manifest in response to saline-alkali stress, three cDNA libraries were constructed from leaf tissue of T.xa0hispida plants that were well watered and exposed to NaHCO3 for 24 and 52xa0h. A total of 9,447 high quality expressed sequence tags (ESTs) were obtained from the three libraries. These ESTs represent 3,945 unigenes, including 986 contigs and 2,959 singlets. The numbers of unigenes obtained from the three libraries were 1,752, 1,558 and 1,675, respectively. The EST analysis was performed to compare gene expression in the three cDNA libraries; the transcripts responsive to NaHCO3 were identified. The differentially expressed transcripts were identified. The up-regulation genes were involved in a variety function areas, such as stress-related proteins, hormone signaling transduction, antioxidative response, transcriptional regulators, protein synthesis and destination, ion homeostasis, photosynthesis and metabolism. The results indicated that the response to NaHCO3 in T.xa0hispida is a complex one, involving multiple physiological and metabolic pathways. Nine gene expression patterns were compared in response to NaHCO3 and NaCl using real time reverse transcription-polymerase chain reaction (RT-PCR). Gene expression trends were similar after a 24-h exposure to either NaCl or NaHCO3, however, great variability was found after a 52-h exposure, indicating that short-term responses to either salt may not be obviously different.

A DREB gene from Limonium bicolor mediates molecular and physiological responses to copper stress in transgenic tobacco.

The DRE-binding (DREB) transcription factors play an important role in regulating stress-related genes. In the present study, a novel DREB gene (LbDREB) from Limonium bicolor was cloned. To characterize the function of DREB in heavy metal stress tolerance, LbDREB-transformed tobacco plants were generated and subjected to CuSO(4) stress. Analysis of the role of LbDREB in tolerance to copper stress in transgenic tobacco showed that overexpression of LbDREB increased the contents of soluble protein and proline, and elevated the ratio of K to Na under CuSO(4) stress. Moreover, overexpression of LbDREB can up-regulate some stress-related genes, including Cu/Zn superoxide dismutase (Cu/Zn SOD), peroxidases (PODs), late embryogenesis abundant (LEA), and lipid transfer proteins (LTP). These results suggest that LbDREB can enhance plant copper tolerance by up-regulating a series of stress-related genes, thereby mediating physiological processes associated with stress tolerance in plants.

A WRKY gene from Tamarix hispida, ThWRKY4, mediates abiotic stress responses by modulating reactive oxygen species and expression of stress-responsive genes

WRKY transcription factors are involved in various biological processes, such as development, metabolism and responses to stress. However, their exact roles in abiotic stress tolerance are largely unknown. Here, we demonstrated a working model for the function of a WRKY gene (ThWRKY4) from Tamarix hispida in the stress response. ThWRKY4 is highly induced by abscisic acid (ABA), salt and drought in the early period of stress (stress for 3, 6, or 9xa0h), which can be regulated by ABF (ABRE binding factors) and Dof (DNA binding with one finger), and also can be crossregulated by other WRKYs and autoregulated as well. Overexpression of ThWRKY4 conferred tolerance to salt, oxidative and ABA treatment in transgenic plants. ThWRKY4 can improve the tolerance to salt and ABA treatment by improving activities of superoxide dismutase and peroxidase, decreasing levels of O2− and H2O2, reducing electrolyte leakage, keeping the loss of chlorophyll, and protecting cells from death. Microarray analyses showed that overexpression of ThWRKY4 in Arabidopsis leads to 165 and 100 genes significantly up- and downregulated, respectively. Promoter scanning analysis revealed that ThWRKY4 regulates the gene expression via binding to W-box motifs present in their promoter regions. This study shows that ThWRKY4 functions as a transcription factor to positively modulate abiotic stress tolerances, and is involved in modulating reactive oxygen species.

Identification of genes responsive to salt stress on Tamarix hispida roots.

Plant roots are the primary site of perception and injury for salinity stress. In order to characterize the complexity of adaptation to salty environments in roots of Tamarix hispida, a woody halophyte, expressed sequence tag (EST) analysis was performed. Three cDNA libraries were generated from root tissues of T. hispida that were exposed to 0.4 M NaCl for 0 (control), 24 and 48 h. A total of 7726 ESTs were generated from the three libraries, and were assembled into 1142 contigs and 3026 singletons. EST analysis was performed to compare gene expression in the three cDNA libraries. Ninety redundant unique transcripts responsive to NaCl treatment were identified. Of them, 21 genes were novel or of unknown function while others were involved in the functional activities, such as ROS scavenging, lipid metabolism, osmolyte biosynthesis, signal transduction, transport, lignin synthesis and homeostasis. The genes, including those for metallothionein-like protein, polyubiquitin, hypothetical protein, and glycine-rich cell wall structural protein, were abundant in the libraries and showed obvious up-regulation after NaCl treatments, suggesting important roles in NaCl tolerance. The results of this study may contribute to our understanding of the molecular mechanism of salt tolerance in the roots of plants.

A novel vacuolar membrane H+-ATPase c subunit gene (ThVHAc1) from Tamarix hispida confers tolerance to several abiotic stresses in Saccharomyces cerevisiae

Plant vacuolar H+-ATPase (V-ATPase) plays an important role in response to different adverse environmental conditions. In the present study, we cloned and characterized a V-ATPase c subunit gene (ThVHAc1) from Tamarix hispida. The deduced ThVHAc1 amino acid sequence lacks a signal peptide and ThVHAc1 is a highly hydrophobic protein with four transmembrane regions. A transient expression assay showed that the ThVHAc1-GFP fusion protein is expressed on onion epidermal endomembrane cells. Real-time RT-PCR demonstrated that ThVHAc1 gene expression was induced by NaCl, NaHCO3, PEG and CdCl2 stress in T. hispida roots, stems and leaves. Exogenous application of abscisic acid (ABA) also stimulated ThVHAc1 transcript levels in the absence of stress, suggesting that ThVHAc1 is involved in ABA-dependent stress signaling pathway. Furthermore, the transgenic yeast expressing ThVHAc1 increased salt, drought, ultraviolet (UV), oxidative, heavy metal, cold and high temperature tolerance. Our results suggested that the ThVHAc1 gene from T. hispida serves a stress tolerance role in the species.

Analysis of Gene Expression Profile of Limonium bicolor under NaHCO3 Stress Using cDNA Microarray

Limonium bicolor, a halophytic species of flowering plant, thrives in saline-alkali soil, demonstrating that it has developed an efficient saline-alkali resistance system and has potential utility as a source of genetic determinants for saline-alkali tolerance. In this study, complementary DNA microarrays containing 1,240 clones of L. bicolor were constructed to have a better view of transcript expression in L. bicolor during saline-alkali (NaHCO3)-induced stress. We obtained transcript profiles of L. bicolor in response to NaHCO3 for 6, 24, and 48xa0h. A total of 149 transcripts were differentially regulated at least once under the conditions studied. Among these, at least six different patterns of transcript regulation could be distinguished. There were 111, 45, and 51 transcripts that were differentially regulated by NaHCO3 stress for 6, 24, and 48xa0h, respectively. Of these, nearly 35% were putative novel or functionally unknown genes, and the remainders were involved in a variety of functional areas such as defense, transport, metabolism, and transcription regulation. The microarray analysis demonstrated the complexity of, and differences in, gene expression patterns resulting from different NaHCO3 stress times. This study provides informative preliminary data and a starting point for more in-depth analyses of saline-alkali tolerance in L. bicolor.

Cloning of Ten Peroxidase (POD) Genes from Tamarix Hispida and Characterization of their Responses to Abiotic Stress

Plant peroxidases (PODs) have been ascribed a variety of biological functions, including hydrogen peroxide detoxification, lignin biosynthesis, hormonal signaling, and stress response. In the present study, ten POD genes, including three ascorbate peroxidases (class I PODs) and seven secretory peroxidases (class III PODs), were cloned from Tamarix hispida. The roles of the ten POD genes were addressed under different abiotic stress conditions, and gene expression profiles in roots, stems, and leaves were evaluated using real-time quantitative reverse-transcribed polymerase chain reaction. Our results showed that the relative abundance of the PODs was markedly different in roots, stems, and leaves, indicating that POD activity differs in these three organs. ThPOD1 and ThPOD8 were the most and least abundant, respectively, in all organs. The expression profiles in response to abiotic stresses were organ specific. All of the genes were highly induced by drought, salt, salt–alkaline, CdCl2, and abscisic acid (ABA) treatments in at least one organ. Five ThPOD genes were induced in roots, stems, and leaves under all of the studied stress conditions, indicating that they are closely associated with abiotic stress. Our results demonstrate that the ten plant peroxidases are all expressed in leaves, stems, and roots, that they are involved in different abiotic stress responses, and that they are controlled by an ABA-dependent stress signaling pathway.

Tamarix hispida metallothionein-like ThMT3, a reactive oxygen species scavenger, increases tolerance against Cd(2+), Zn(2+), Cu(2+), and NaCl in transgenic yeast.

A metallothionein-like gene, ThMT3, encoding a type 3 metallothionein, was isolated from a Tamarix hispida leaf cDNA library. Expression analysis revealed that mRNA of ThMT3 was upregulated by high salinity as well as by heavy metal ions, and that ThMT3 was predominantly expressed in the leaf. Transgenic yeast (Saccharomyces cerevisiae) expressing ThMT3 showed increased tolerance to Cd2+, Zn2+, Cu2+, and NaCl stress. Transgenic yeast also accumulated more Cd2+, Zn2+, and NaCl, but not Cu2+. Analysis of the expression of four genes (GLR1, GTT2, GSH1, and YCF1) that aid in transporting heavy metal (Cd2+) from the cytoplasm to the vacuole demonstrated that none of these genes were induced under Cd2+, Zn2+, Cu2+, and NaCl stress in ThMT3-transgenic yeast. H2O2 levels in transgenic yeast under such stress conditions were less than half those in control yeast under the same conditions. Three antioxidant genes (SOD1, CAT1, and GPX1) were specifically expressed under Cd2+, Zn2+, Cu2+, and NaCl stress in the transgenic yeast. Cd2+, Zn2+, and Cu2+ increased the expression levels of SOD1, CAT1, and GPX1, respectively, whereas NaCl induced the expression of SOD1 and GPX1.