Zhigang Wei

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 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.

Comparative temporal analyses of the Pinus sylvestris L. var. mongolica litv. apical bud proteome from dormancy to growth

Bud dormancy in perennial plants adapts to environmental and seasonal changes. Bud dormancy is of ecological interest because it affects forest population growth characteristics and is of economical interest because it impacts wood production levels. To understand Pinus sylvestris L. var. mongolica litv. bud-dormancy and bud-burst mechanisms, we characterized the proteomes of their apical buds at the four critical stages that occur during the dormancy-to-growth transition. Ninety-six proteins with altered expression patterns were identified using NanoLC–ESI-MS/MS. The majority of these proteins (57%) are involved in metabolic and other cellular processes. For 28% of the proteins, a function could not be assigned. However, because their expression levels changed, they may be potential candidate bud development- or dormancy-related proteins. Of the 75 non-redundant bud proteins identified, ascorbate peroxidase, pathogenesis-related protein PR-10, and heat shock proteins dramatically increased during August and November, suggesting that they may involved in the initiation of bud dormancy. Conversely, S-adenosylmethionine synthetase, abscisic acid/stress-induced proteins, superoxide dismutase (SOD), caffeoyl-CoA O-methyltransferase, actin, and type IIIa membrane protein cp-wap13 had greater expression levels during April, suggesting that they may be involved in the initiation of bud dormancy-release. Cell division cycle protein 48 and eukaryotic initiation factors 4A-15 and 4A had greater expression levels during May, suggesting that they may regulate cell proliferate and differentiation in the shoot apical meristem. These observations provide insights into the molecular mechanisms that induce or break bud dormancy.

Genetic Linkage Maps of Betula platyphylla Suk Based on ISSR and AFLP Markers

Two separate genetic linkage maps for Chinese silver birch based on inter-simple sequence repeat (ISSR) and amplified fragment-length polymorphism (AFLP) were constructed by a pseudo-testcross mapping strategy. Eighty F1 progenies were obtained from the cross between two parental trees with desirable traits (the paternal one selected from ‘Qinghai’ and the maternal one from ‘Wangqing’). A total of 46 ISSR primers and 31 AFLP primers were employed to generate 102 ISSR and 355 AFLP polymorphic markers in the F1 progenies. About 5.7% of all the markers displayed high segregation distortion with a P value below 0.01 and such markers were not used for map constructions. The paternal map consisted of 137 loci, spread over 13 groups and spanned 694.2xa0cM at an average distance of 5.1xa0cM between the markers, while in the maternal map, 147 loci were distributed in 14 groups covering a map distance about 949.62xa0cM at an average distance of 6.5xa0cM. These initial maps can serve as the basis for developing a more detailed genetic map.

Differential expression of copper-zinc superoxide dismutase gene of Polygonum sibiricum leaves, stems and underground stems, subjected to high-salt stress.

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5′ untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3′ untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1–16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO3. The different mRNA levels’ expression of PS-CuZnSOD show the gene’s different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.

Analysis and identification of SCAR molecular markers associated with birch fiber length trait

The fiber length trait (FLT) of 538 individuals from nature birch population in Maorshan region, Heilongjang, China were measured, of which 100 individuals were selected as representative variety of correlated fragments screening with random amplified polymorphism DNA (RAPD) technique. In total of 20 RAPD primers were tested through multiple regression analysis between amplified strip and the character behaviors, and a correlative segment BFLR-16 was obtained. The correlation coefficient between BFLI-16 and FLT was 0.6144, with the significant level of 1%. BFLI-16 was then cloned, sequenced and transformed into SCAR marker. The percentage of identifying long fiber birches by this SCAR was more than 92. The result indicates that the SCAR markers has high specificity for the long fiber individuals and is highly linked with the gene controlling the character of fiber length, and its existence is significantly correlative with the increase in the fiber length.

Time-Course Analysis of Levels of Indole-3-Acetic Acid and Expression of Auxin-Responsive GH3 Genes in Betula platyphylla

Indole-3-acetic acid (IAA) is a natural hormone that plays an important role in the essential physiological processes of plant growth, development, and environmental response. To maintain IAA homeostasis in plants, some members of the GH3 gene family may mediate the conjugation of amino acids to indole-3-acetic acid. In this study, nine birch BpGH3 genes were cloned, including three BpGH3.5 genes, three BpGH3.6 genes, two BpGH3.9 genes, and a single BpGH3.3 gene, that were likely to be auxin-responsive genes. Time-course expression analysis of these nine BpGH3 genes using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was conducted using birch plant tissues collected during a 1-year growth cycle. Further, we determined the endogenous free IAA content in birch during this period by using liquid chromatography tandem mass spectrometry. The results indicated that the IAA levels changed remarkably throughout the study period; the level remained relatively stable during the developmental stage from June 1 to July 1, and IAA was present in the birch leaves throughout the study period. The correlation between the expression of BpGH3 genes and IAA levels was analyzed using Pearson correlation analysis. The results revealed a significant negative correlation between the expressions of four BpGH3 genes, namely, BpGH3.3 (pu2009<u20090.05), BpGH3.5a (pu2009<u20090.01), BpGH3.5b (pu2009<u20090.01), and BpGH3.9a (pu2009<u20090.01) and the free IAA level, thereby suggesting that these genes might play roles in IAA regulation.

Linkage map construction and QTL analysis for Betula platyphylla Suk using RAPD, AFLP, ISSR and SSR

Abstract A linkage map for Betula platyphylla Suk was constructed based on RAPD, ISSR, AFLP and SSR markers by a pseudo-testcross mapping strategy. A F1 segregating population including 80 progenies was obtained from the cross between two superior trees selected from Qinghai and Wangqing provenance, respectively. The paternal map was constructed with 282 markers consisting of 14 major and 15 minor (5 triplets and 10 doublets) linkage groups and spanning 1131 cM at an average distance of 4.0 cM between adjacent markers. The maternal map has 277 markers consisting of 15 major and 8 minor (5 triplets and 3 doublets) groups covering 1288 cM at an average distance of 4.6 cM between adjacent markers. In the same pedigree we investigated association of genetic markers with seedling stem height and circumference. The composite interval mapping was used to detect the number of quantitative trait loci and their position on the genetic linkage maps. Three QTLs (one on the male map and two on the female map) were found explaining 13.4%, 17.5% and 18.8% of the trait variation, respectively.

Time-course analyses of abscisic acid level and the expression of genes involved in abscisic acid biosynthesis in the leaves of Betula platyphylla

Abscisic acid (ABA) is a plant hormone regulating the essential physiological processes of plants and plant responses to various environmental stresses. Recent studies revealed that the key enzymes involved in ABA biosynthesis were zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenases (NCED), short-chain dehydrogenase/reductase (SDR), and ABA-aldehyde oxidase (AAO). In this study, we cloned 12 unique genes potentially involved in ABA synthesis, including 5 of BpNCEDs, 1 of BpAAO, and 6 of BpZEPs from birch leaves. We analyzed the time-course expression of these 12 genes during the phase from May to August by real time reverse transcriptase-polymerase chain reaction (RT-PCR). Further, we determined birch endogenous ABA content during this period by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results indicated that ABA level significantly (P < 0.05) increased during the early stages of development and ABA was present in birch leaves throughout the studied period, suggesting that endogenous ABA is necessary for the normal growth and development of plants. The correlation between the expression of above-mentioned genes and ABA levels was analyzed by Pearson correlation analysis. The results revealed that the expression of three genes, namely, BpNCED4, BpNCED5 and BpZE6 significantly (Pxa0<xa00.05) correlated with the ABA level. Therefore, these three genes may play important roles in ABA biosynthesis.

Temporal expression of genes involved in the biosynthesis of gibberellins in birch ( Betula platyphylla )

Gibberellins (GAs) are a large family of endogenous plant growth regulators. Bioactive GAs influence nearly all processes during plant growth and development. In the present study, we cloned and identified 10 unique genes that are potentially involved in the biosynthesis of GAs, including one BpGGDP gene, two BpCPS genes, one BpKS gene, two BpKO genes, three BpGA20ox genes and one BpGA2ox gene. The temporal expression of these 10 genes in birch was analyzed during a one-year growth cycle by real time reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that their expression patterns were individually distinct throughout the one-year growth cycle. Three BpGA20ox genes, which are potential limiting factors of bioactive GA biosynthesis, displayed differential expression patterns during the course of the experiment. This study provides insights into the roles of genes involved in the biosynthesis of GAs during the early development of birch.