[Show abstract][Hide abstract] ABSTRACT: In this study, a comparative proteomic analysis was employed to identify fuzz fiber initiation-related proteins in wild-type diploid cotton (Gossypium arboreum L.) and its fuzzless mutant. Temporal changes in global proteomes were examined using 2-DE at five developmental time points for fuzz fiber initiation, and 71 differentially expressed protein species were identified by MS, 45 of which were preferentially accumulated in the wild-type. These proteins were assigned to several functional categories, mainly in cell response/signal transduction, redox homeostasis, protein metabolism and energy/carbohydrate metabolism. It was remarkable that more than ten key proteins with high-abundance were involved in gibberellic acid (GA) signaling and ROS scavenging, and increasing concentrations of active GAs and H2O2 were also detected approximately 5 dpa in wild type ovules. Furthermore, in vivo GA and H2O2 treatments of ovules inside young bolls showed that these compounds can synergistically promote fuzz fiber initiation. Our findings not only describe a dynamic protein network supporting fuzz initiation in diploid cotton fiber ovules, but also deepened our understanding of the molecular basis of cotton fiber initiation. Significance Our study reported the identification of differentially expressed proteins in wild-type diploid cotton (Gossypium arboreum L.) and its fuzzless mutant by comparative proteomic approach. In total, 71 protein species related to fuzz initiation were identified by MS. These proteins were assigned to several functional categories, mainly in energy/carbohydrate metabolism, protein metabolism, signal transduction, redox homeostasis etc. Importantly, a number of key proteins were found to be associated with GA signaling and ROS scavenging. In consistence with these findings, we detected the increase of GAs and H2O2 concentrations during fiber initiation, and our in vivo ovule experiments with GA and H2O2 injection and following microscopy observation of fuzz fiber initiation supported promoting effects of GA and H2O2 on cotton fiber initiation. These findings depicted a dynamic protein network supporting cotton fiber initiation in diploid cotton ovules. Our study is of major significance for understanding the molecular mechanisms controlling fuzz initiation and also provides a solid basis for further functional research of single nodes of this network in relation to cotton fiber initiation.
Journal of proteomics 03/2013; · 5.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In our previous study, we used a comparative proteomic approach based on 2-DE to profile dynamic proteomes of cotton fibers and found 235 protein spots differentially expressed during the elongation process ranging from 5 to 25 days post-anthesis (DPA). Of them, only 106 differentially expressed proteins (DEPs) were identified by mass spectrometry due to database limitations at the time. In the present work, we successfully identified the remaining 129 DEPs from the same experimental system using high-resolution mass spectrometry with an updated database. Bioinformatic analysis revealed that proteins involved in carbohydrate and protein metabolism, transport, and redox homeostasis are the most abundant, and glycolysis was found to be the most significantly regulated process during fiber elongation. Our high-confidence reference dataset, composed of 235 DEPs, provides a valuable resource for future studies on the molecular mechanism of cotton fiber elongation.
[Show abstract][Hide abstract] ABSTRACT: Two-dimensional gel electrophoresis (2-DE)-based proteomics approach was applied to extensively explore the molecular basis of plant development and environmental adaptation. These proteomics analyses revealed thousands of differentially expressed proteins (DEPs) closely related to different biological processes. However, little attention has been paid to how peptide mass fingerprinting (PMF) data generated by the approach can be directly utilized for the determination of protein phosphorylation. Here, we used the software tool FindMod to predict the peptides that might carry the phosphorylation modification by examining their PMF data for mass differences between the empirical and theoretical peptides and then identified phosphorylation sites using MALDI TOF/TOF according to predicted peptide data from these DEP spots in the 2-D gels. As a result, a total of 48 phosphorylation sites of 40 DEPs were successfully identified among 235 known DEPs previously revealed in the 2-D gels of elongating cotton fiber cells. The 40 phosphorylated DEPs, including important enzymes such as enolase, transketolase and UDP-L-rhamnose synthase, are presumed to participate in the functional regulation of numerous metabolic pathways, suggesting the reverse phosphorylation of these proteins might play important roles in elongating cotton fibers. The results also indicated that some different isoforms of the identical DEP revealed in our 2-DE-based proteomics analysis could be annotated by phosphorylation events. Taken together, as the first report of large-scale identification of phosphorylation sites in elongating cotton fiber cells, our study provides not only an excellent example of directly identifying phosphorylation sites from known DEPs on 2-D gels but also provides a valuable resource for future functional studies of phosphorylated proteins in this field.
PLoS ONE 01/2013; 8(3):e58758. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The functional characterization of novel transcription factors identified by systematic analysis remains a major challenge due to insufficient data to interpret their specific roles in signaling networks. Here we present a DNA-binding sequence discovery method to in vitro identify a G-rich, 11-bp DNA-binding motif of a novel potential transcription factor AtYY1, a zinc finger protein in Arabidopsis, by using polymerase chain reaction-assisted in vitro selection and surface plasmon resonance analysis. Further mutational analysis of the conserved G bases of the potential motif confirmed that AtYY1 specifically bound to these conserved G sites. Additionally, genome-wide target gene analysis revealed that AtYY1 was involved in diverse cellular pathways, including glucose metabolism, photosynthesis, phototropism, and stress response.
[Show abstract][Hide abstract] ABSTRACT: An increasing number of microRNAs (miRNAs) have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miRNAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly identified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.
[Show abstract][Hide abstract] ABSTRACT: Plant annexins represent a multigene family involved in cellular elongation and development. A cDNA encoding a novel annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library and designated GhAnx1. This gene encodes a 316 amino acid protein with a theoretical molecular mass of 36.06 kDa and a theoretical pI of 6.19. At the amino acid level, it shares high sequence similarity and has evolutionary relationships with annexins from higher plants. The purified recombinant protein expressed in Escherichia coli was used to investigate its physicochemical properties. Circular dichroism spectrum analyses showed a positive peak rising to the maximum at 196 nm and a broad negative band rounding 215 nm, suggesting that the GhAnx1 protein was prominently α-helical. The fluorescence measurements indicated that it could bind to Ca(2+) in vitro. These results demonstrated that GhAnx1 was a typical annexin protein in cotton. A bioassay experiment was conducted to analyze its potential function and showed that E. coli cells expressing GhAnx1 were protected from tert-butyl hydroperoxide (tBH) stress, suggesting that it had a potential antioxidative role. Northern blot analyses revealed that GhAnx1 was highly expressed in fibers, especially during the elongation stage, suggesting that it might be important for fiber elongation.
[Show abstract][Hide abstract] ABSTRACT: Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H(2)O(2)) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H(2)O(2)-responsive proteins in the apoplast of rice seedling roots.
14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H(2)O(2) for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H(2)O(2) were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H(2)O(2) conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H(2)O(2)-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H(2)O(2) stress.
The present study is the first apoplast proteome investigation of plant seedlings in response to H(2)O(2) and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H(2)O(2) response.
PLoS ONE 01/2011; 6(2):e16723. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant microRNAs (miRNAs) have been shown to play critical roles in regulating gene expression at the post-transcriptional level. In this study, we employed high throughput sequencing combined with computational analysis to survey miRNAomes from the seedlings of rice under normal conditions and treatments of H(2)O(2) that result in oxidative stress. Comparison of the miRNAomes and subsequent northern blot analysis identified seven miRNA families differentially expressed under H(2)O(2) stress. Predicted and experimentally validated targets of these H(2)O(2)-responsive miRNAs are involved in different cellular responses and metabolic processes including transcriptional regulation, nutrient transport, auxin homeostasis, cell proliferation and programmed cell death. This indicates that diverse miRNAs form a complex regulatory network to coordinate plants' responses under oxidative stress. In addition, we also discovered 32 new miRNAs in the seedlings of rice. Interestingly, of these new miRNAs, miR3981 was originally found to be a putative exonic miRNA located in the exon of AK106348, suggesting that plants may also use some exons as an miRNA source. This study is the first genome-wide investigation of H(2)O(2)-regulated miRNAs in plants and broadens our perspectives on the important regulatory roles of miRNAs in plant oxidative stress and physiological adaption.
Nucleic Acids Research 11/2010; 39(7):2821-33. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The rice (Oryza sativa L.) metallothionein gene OsMT-I-4b has previously been identified as a type I MT gene. To elucidate the regulatory mechanism involved in its tissue specificity and abiotic induction, we isolated a 1 730 bp fragment of the OsMT-I-4b promoter region. Histochemical β-glucuronidase (GUS) staining indicated a precise spacial and temporal expression pattern in transgenic Arabidopsis. Higher GUS activity was detected in the roots and the buds of flower stigmas, and relatively lower GUS staining in the shoots was restricted to the trichomes and hydathodes of leaves. No activity was observed in the stems and seeds. Additionally, in the root of transgenic plants, the promoter activity was highly upregulated by various environmental signals, such as abscisic acid, drought, dark, and heavy metals including Cu²(+) , Zn²(+) , Pb²(+) and Al³(+) . Slight induction was observed in transgenic seedlings under salinity stress, or when treated with Co²(+) and Cd²(+) . Promoter analysis of 5'-deletions revealed that the region -583/-1 was sufficient to drive strong GUS expression in the roots but not in the shoots. Furthermore, deletion analysis indicated important promoter regions containing different metal-responsive cis-elements that were responsible for responding to different heavy metals. Collectively, these findings provided important insight into the transcriptional regulation mechanisms of the OsMT-I-4b promoter, and the results also gave us some implications for the potential application of this promoter in plant genetic engineering.
[Show abstract][Hide abstract] ABSTRACT: Ethylene-responsive factors (ERFs) are important regulators of plant gene expression. In this study, three novel ERF genes, GhERF2, GhERF3 and GhERF6, were isolated from cotton (Gossypium hirstum) using rapid amplification of cDNA ends-polymerase chain reaction. Transient expression analysis using GhERF-green fluorescent protein fusions showed that these three proteins were targeted to the nucleus. Fusion proteins consisting of GhERF2, GhERF3 or GhERF6 coupled to the GAL4 DNA binding domain strongly activated transcription in yeast. Furthermore, GhERF6 was shown to be able to bind specifically to GCC boxes using a particle bombardment assay in tobacco cells. Semi-quantitative reverse transcription-polymerase chain reaction revealed that GhERF2 and GhERF3 are constitutively expressed in all organs, while GhERF6 is only constitutively expressed in vegetative organs. When plants were treated with ethylene, abscisic acid, salt, cold and drought, the transcripts of GhERF2, GhERF3 and GhERF6 were rapidly induced to high levels. Promoter analysis also indicated that the 5' upstream regions of the three genes possess elements induced by these physiological and environmental factors. Collectively, our data suggest that GhERF2, GhERF3 and GhERF6 might function as positive trans-acting factors in the plant responses to ethylene, abscisic acid and other stresses and provide useful clues for further research into the mechanism of them in regulating cotton multiple stress responses.
[Show abstract][Hide abstract] ABSTRACT: Plants respond to abiotic stress through complex regulation of transcription, including both transcriptional activation and repression. Dehydration-responsive-element binding protein (DREB)-type transcription factors are well known to play important roles in adaptation to abiotic stress. The mechanisms by which DREB-type transcription factors activate stress-induced gene expression have been relatively well studied. However, little is known about how DREB-type transcriptional repressors modulate plant stress responses. In this study, we report the functional analysis of RAP2.1, a DREB-type transcriptional repressor.
RAP2.1 possesses an APETALA2 (AP2) domain that binds to dehydration-responsive elements (DREs) and an ERF-associated amphiphilic repression (EAR) motif, as the repression domain located at the C-terminus of the protein. Expression of RAP2.1 is strongly induced by drought and cold stress via an ABA-independent pathway. Arabidopsis plants overexpressing RAP2.1 show enhanced sensitivity to cold and drought stresses, while rap2.1-1 and rap2.1-2 T-DNA insertion alleles result in reduced sensitivity to these stresses. The reduced stress sensitivity of the plant containing the rap2.1 allele can be genetically complemented by the expression of RAP2.1, but not by the expression of EAR-motif-mutated RAP2.1. Furthermore, chromatin immunoprecipitation (ChIP) analysis has identified Responsive to desiccation/Cold-regulated (RD/COR) genes as downstream targets of RAP2.1 in vivo. Stress-induced expression of the RD/COR genes is repressed by overexpression of RAP2.1 and is increased in plants expressing the rap2.1 allele. In addition, RAP2.1 can negatively regulate its own expression by binding to DREs present in its own promoter. Our data suggest that RAP2.1 acts as a negative transcriptional regulator in defence responses to cold and drought stress in Arabidopsis.
A hypothetical model for the role of RAP2.1 in modulating plant responses to cold and drought is proposed in this study. It appears that RAP2.1 acts as a negative "subregulon" of DREB-type activators and is involved in the precise regulation of expression of stress-related genes, acting to keep stress responses under tight control.
[Show abstract][Hide abstract] ABSTRACT: In this study, we report isolation of a phosphatase gene designated GhHL1 from cotton and its functional characterization. GhHL1 transcripts were detected in all cotton tissues examined. Southern blotting analysis indicated that it exists in multiple-copies. Biochemical analysis showed that GhHL1 was Mg2+-dependent and cation-sensitive. Purified recombinant GhHL1 protein dephosphorylated both 3',5'-bisphosphate nucleotide and inositol 1,4-bisphosphate, demonstrating dual 3',5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. Overexpression of GhHL1 complemented yeast hal2 mutant and enhanced yeast growth under elevated NaCl or LiCl, showing a role in salt tolerance associated with ionic stress response. Taken together, these results show that GhHL1 is a functional and good candidate gene, which might be used to improve salt tolerance in plants.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) genes has been reported to play an important role in protecting host cells from oxidative injury in several model systems. A radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) known to have high catalytic activity was applied to mouse 3T3 fibroblasts to determine the protective effects of PHGPx against oxidative injury triggered by hydroperoxides such as hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide (t-BHP) and phosphatidylcholine hydroperoxide (PCOOH). We observed that preincubation of cells with RsPHGPx significantly increased cell viability, reduced levels of malondialdehyde (MDA), inhibited generation of reactive oxygen species (ROS), and maintained natural cell shapes after treatment with H(2)O(2), t-BHP or PCOOH, indicating that the exogenous RsPHGPx can act as an effective hydroperoxide-scavenger and may also protect target cells from oxidative damage. These results suggest the possibility for use of RsPHGPx as a therapeutic protectant.
[Show abstract][Hide abstract] ABSTRACT: A 1,482-bp promoter sequence of the cotton cellulose synthase gene (GhCesA4) was isolated from Chinese cultivar CRI12 of Gossypium hirsutum, and transcriptionally fused to a beta-glucuronidase (GUS) reporter gene for investigation of important regions controlling gene expression in transgenic tobacco plants. Histochemical staining showed that the full-length promoter directs efficient expression of the reporter gene in the roots, hypocotyls, vascular tissues of stems, trichomes, the central leaf veins, as well as in the anthers and pollen. Quantitative measurements of GUS activity demonstrated that higher expression levels were detected in the stems, fully expanded leaves, and styles of flowers. A series of 5' progressive deletions of the promoter revealed the presence of a negative regulatory region (-767 to -424) for promoter activity and a 247-bp fragment (-247 to -1) with the vascular tissue specificity of the basic transcription activity in the GhCesA4 promoter. Exposure of the transgenic tobacco to various abiotic stresses showed that the full-length construct predominantly responded to NAA, kinetin, and sugar. Furthermore, the NAA-response region was found to be located in the -1,482/-1204 fragment, while the element(s) for the sucrose-responsive expression may be present in the -247/-1 region in the GhCesA4 promoter. These findings will not only contribute to an explanation of the molecular mechanisms by which GhCesA4 participates in secondary cell wall morphogenesis and stress responses, but will also provide a good candidate for expression or accumulation of foreign genes of interest whose products are preferentially required in vascular tissues and are inducible under auxin treatment.
[Show abstract][Hide abstract] ABSTRACT: The entire encoding region for Momordica charantia phospholipid hydroperoxide glutathione peroxidase (McPHGPx) was cloned into pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The purified recombinant McPHGPx displayed GSH-dependent peroxidase activity towards phospholipid hydroperoxide, H2O2, and tert-butyl hydroperoxide and had the highest affinity with and catalytic efficiency for phospholipid hydroperoxide. The optimum temperature of the enzyme activity ranged from 40 to 50 degrees C, thus it is a thermostable enzyme compared to other PHGPx enzymes. Furthermore, McPHGPx expression in Saccharomyces cerevisiae PHGPx-deletion mutant rescued the susceptibilities to the oxidation-sensitive polyunsaturated fatty acid (linolenic acid), indicating its PHGPx complementation function in yeast. These results have well documented that McPHGPx functions as a PHGPx in vitro and in vivo and will be beneficial for further functional studies on plant PHGPx enzymes.
[Show abstract][Hide abstract] ABSTRACT: A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among approximately 1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development.
Journal of Proteome Research 10/2008; 7(11):4623-37. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.
[Show abstract][Hide abstract] ABSTRACT: A cDNA encoding one novel DRE-binding protein, GhDBP2, was isolated from cotton seedlings. It is classified into the A-6 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. Using semi-quantitative RT-PCR, we found that the GhDBP2 transcripts were greatly induced by drought, NaCl, low temperature and ABA treatments in cotton cotyledons. The DNA-binding properties of GhDBP2 were analyzed by electrophoretic mobility shift assay (EMSA), showing that GhDBP2 successfully binds to the previously characterized DRE cis-element as well as the promoter region of the LEA D113 gene. Consistent with its role as a DNA-binding protein, GhDBP2 is preferentially localized to the nucleus of onion epidermal cells. In addition, when GhDBP2 is transiently expressed in tobacco cells, it activates reporter gene expression driven by the LEA D113 promoter. Taken together, our results indicate that GhDBP2 is a DRE-binding transcriptional activator involved in activation of down-stream genes such as LEA D113 expression through interaction with the DRE element, in response to environmental stresses as well as ABA treatment.
Journal of Plant Physiology 03/2008; 165(2):214-23. · 2.70 Impact Factor