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 ∼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.Keywords: Cotton; Differentially expressed proteins; Dynamic changes; Fiber cell elongation; Network; Proteomic analysis;

Malate dehydrogenase(MDH) is a key enzyme that catalyzes the reversible oxidation of oxaloacetate to malate and plays an important role in the physiological processes of plant growth and development. However, cyto-solic malate dehydrogenase(cMDH), which is crucial for malate synthesis in the cytosol, still has not been extensively characterized in plants. Here, we isolated a cytosolic malate dehydrogenase gene, designated as GhcMDH1, from Gossypium hirsutum and characterized its possible molecular function in cotton fiber. The cloned cDNA of GhcMDH1 is 1520 base pairs in length, and has an open reading frame of 999 base pairs, encoding for 332 amino acid residues with an estimated molecular weight of 35580 and pI of 6.35. Sequence alignment showed that the de-duced amino acid sequence of GhcMDH1 protein shared a high similarity to other plant cMDHs. Confocal and im-munological analysis confirmed that GhcMDH1 protein was subcellularly localized to the cytosol. Quantitative real-time polymerase chain reaction(PCR) revealed that GhcMDH1 was constitutively expressed in all vegetative cotton tissues, with slightly lower levels in roots than stems and leaves. Interestingly, the transcripts of GhcMDH1 were detected in 5―25 d post anthesis(DPA) fibers and highly abundant at 15 DPA fibers. The total MDH activities and malate contents of cotton fibers were positively correlated with the fiber elongation rates, suggesting that GhcMDH1 may function in malate synthesis in fast fiber elongation. In agreement with this suspicion, the recombi-nant His-GhcMDH1 protein mainly drives the reaction towards malate generation in vitro. In conclusion, our mole-cular characterization of the GhcMDH1 gene provides valuable insights to further investigate the malate equilibrium in cotton fiber development.

The plant phospholipase D (PLD) plays versatile functions in multiple aspects of plant growth, development, and stress responses. However, until now, our knowledge concerning the PLD gene family members and their expression patterns in cotton has been limited. In this study, we performed for the first time the genome-wide analysis and expression profiling of PLD gene family in Gossypium arboretum, and finally, a total of 19 non-redundant PLD genes (GaPLDs) were identified. Based on the phylogenetic analysis, they were divided into six well-supported clades (α, β/γ, δ, ε, ζ and φ). Most of the GaPLD genes within the same clade showed the similar exon-intron organization and highly conserved motif structures. Additionally, the chromosomal distribution pattern revealed that GaPLD genes were unevenly distributed across 10 of the 13 cotton chromosomes. Segmental duplication is the major contributor to the expansion of GaPLD gene family and estimated to have occurred from 19.61 to 20.44 million years ago when a recent large-scale genome duplication occurred in cotton. Moreover, the expression profiling provides the functional divergence of GaPLD genes in cotton and provides some new light on the molecular mechanisms of GaPLDα1 and GaPLDδ2 in fiber development.

Cotton cytosolic pyruvate kinase GhPK6 is preferentially expressed in the late stage of fiber elongation process, transgenic experiments indicated that its expression level was negatively correlated to cell expansion rate.Pyruvate kinase (PK) plays vital regulatory roles in rapid cell growth in mammals. However, the function of PK in plant cell growth remains unclear. In allotetraploid upland cotton (Gossypium hirsutum L.), a total of 33 PK genes are encoded by the genome. Analysis of the transcriptome data indicated that only two cytosolic PK genes, GhPK6 and its duplicated gene GhPK26, are preferentially expressed in elongating cotton fiber cells. RT-qPCR and western blot analyses revealed that the expression of GhPK6 was negatively correlated with fiber elongation rate, which well explains the observed sharp increase of cytosolic PK activity at the end of fast fiber elongation process. Furthermore, virus-induced gene silencing of GhPK6 in cotton plants resulted in increased fiber cell elongation and reduced reactive oxygen species (ROS) accumulation. On the contrary, Arabidopsis plants ectopically expressing GhPK6 exhibited ROS-mediated growth inhibition, whereas the addition of ROS scavenging reagents could partly rescue this inhibition. These data collectively suggested that GhPK6 might play an important role in regulating cotton fiber elongation in a ROS-dependent inhibition manner.

Plenty of proteomic studies were performed to characterize the allotetraploid upland cotton fiber elongation process, whereas little is known about the elongating diploid cotton fiber proteome.In this study, we used a two-dimensional electrophoresis-based comparative proteomic approach to profile dynamic proteomes of diploid Asian cotton ovules with attached fibers in the early stages of fiber elongation process. One-way ANOVA and Student-Newman-Keuls test were used to find the differentially displayed protein (DDP) spots.A total of 55 protein spots were found having different abundance ranging from 1 to 9 days post-anthesis (DPA) in a two-day interval. These 55 DDP spots were all successfully identified using high-resolution mass spectrometric analyses. Gene ontology analyses revealed that proteoforms involved in energy/carbohydrate metabolism, redox homeostasis, and protein metabolism are the most abundant. In addition, orthologues of the 13 DDP spots were also found in differential proteome of allotetraploid elongating cotton fibers, suggesting their possible essential roles in fiber elongation process.Our results not only revealed the dynamic proteome change of diploid Asian cotton fiber and ovule during early stages of fiber elongation process but also provided valuable resource for future studies on the molecular mechanism how the polyploidization improves the trait of fiber length.

MicroRNAs (miRNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of miRNAs have been identified in cotton fibers, the functions of miRNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis (DPA) and was subjected to high-throughput sequencing. A total of 95 known miRNAs were detected to be expressed in cotton seeds. The expression pattern of these identified miRNAs was profiled and 48 known miRNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel miRNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known miRNAs were successfully predicted and 900 expressed sequence tag (EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, miRNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5' RACE. This study is the first to show the regulatory network of miRNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these miRNAs in seed development.

•Proteome profiles of diploid cotton ovules were compared to those in fuzzless mutant.•Seventy-one protein species related to fuzz initiation were identified by MS analysis.•Increasing levels of active GAs and H2O2 were detected during fuzz initiation.•A protein network supporting fuzz initiation is depicted in diploid cotton ovules.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 described 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.Biological significanceOur study reported the identification of differentially expressed proteins in wild-type diploid cotton (G. 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.

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.

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 β-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.

Plant AP2/EREBP transcription factors play important roles in plant development and in plant responses to biotic and abiotic stresses. A novel gene for ethylene-responsive element binding protein (EREBP), designated GhERF5, which encodes a protein of 255 amino acids, was isolated by RACE-PCR from cotton (Gossypium hirsutum) seedlings. Sequence alignment revealed that GhERF5 contains a typical AP2/ERF domain, and belongs to the B3 subgroup of the ERF subfamily. Particle bombardment assay showed that GhERF5 functions as an in vivo transcription activator in tobacco cells, and it is located in the nuclei of onion epidermis cells. Semi-quantitative RT-PCR revealed that the expression of GhERF5 was highly and rapidly induced when plants were treated with exogenous ethylene, abscisic acid (ABA), salt, cold and drought. Promoter analysis indicated that there are conserved cis-acting elements induced by these stresses in the 5′-upstream region of the GhERF5 gene. These results suggest that the GhERF5 gene might play an important role in cotton response to ethylene, ABA and environmental stresses.

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°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.

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 ∼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.

Phosphoenolpyruvate carboxylase (PEPC) plays diverse physiological functions during plant development. In this study, a new phosphoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton (Gossypium hirsutum cv. zhongmian 35) by RACE-PCR. The cloned cDNA of GhPEPC2 is 3364 bp in length, and has an open reading frame of 2913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110.6 kD and pI of 5.56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs. Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome. The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cotton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers. In addition, the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km (83.6 μM) and lower Vmax (8.0 μmol min−1 mg−1) compared with the C3 PEPCs previously reported.

The 5' fragment (1 647 bp) of the cotton glucuronosyltransferase gene (GhGlcAT1) was transcriptionally fused to the β-glucuronidase (GUS) gene, and functionally analyzed for important regulatory regions controlling gene expression in transgenic tobacco plants. GUS activity analysis revealed that the full-length promoter drives efficient expression of the GUS gene in the root cap, seed coat, pollen grains and trichomes. Exposure of the transgenic tobacco to various abiotic stresses showed that the promoter was mainly responsive to the sugars (glucose and sucrose) as well as gibberellic acid. Progressive upstream deletion analyses of the promoter showed that the region from −281 to +30 bp is sufficient to drive strong GUS expression in the trichomes of shoot, suggesting that the 311 bp region contains all cis-elements needed for trichome-specific expression. Furthermore, deletion analysis also revealed that the essential cis-element(s) for sucrose induction might be located between −635 and −281 bp. In addition, sequence analysis of the regulatory region indicated several conserved motifs among which some were shared with previously reported seed-specific elements and sugar-responsive elements, while others were related with trichome expression. These findings indicate that a 1 647-bp fragment of the cotton GhGlcAT1 promoter contains specific transcription regulatory elements, and provide clues about the roles of GhGlcAT1 in cotton fiber development. Further analyses of these elements will help to elucidate the molecular mechanisms regulating the expression of the GhGlcAT1 gene during fiber elongation.

Previous investigations of plant responses to higher CO2 levels were mostly based on physiological measurements and biochemical assays. In this study, a proteomic approach was employed to investigate plant response to higher CO2 levels using rice as a model. Ten-day-old seedlings were progressively exposed to 760 ppm, 1140 ppm, and 1520 ppm CO2 concentrations for 24 h each. The net photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (E), and intercellular to ambient CO2 concentration ratio (Ci/Ca) were measured. Pn, Gs, and E showed a maximum increase at 1140 ppm CO2, but further exposure to 1520 ppm for 24 h resulted in down regulation of these. Proteins extracted from leaves were subjected to 2-DE analysis, and 57 spots showing differential expression patterns, as detected by profile analysis, were identified by MALDI-TOF/TOF-MS. Most of the proteins belonged to photosynthesis, carbon metabolism, and energy pathways. Several molecular chaperones and ascorbate peroxidase were also found to respond to higher CO2 levels. Concomitant with the down regulation of Pn and Gs, the levels of enzymes of the regeneration phase of the Calvin cycle were decreased. Correlations between the protein profiles and the photosynthetic measurements at the three CO2 levels were explored.

Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions. In this paper, Northern blot analysis was used to investigate the influence of lead stress on the expression patterns of 10 rice class I MT genes (OsMT-Is) in rice seedlings. With the exception of OsMT-I-3b, the data demonstrate dynamic changes of 9 OsMT-I transcripts in response to Pb2+ treatment in rice seedling roots. Of these genes, transcription of OsMT-I-1a, OsMT-I-1b, OsMT-I-2c, OsMT-I-4a, OSMT-I-4b and OsMT-I-4c increased significantly, while transcription of OsMT-I-2a and OsMT-I-3a increased marginally. In contrast, the expression of OsMT-I-2b was inhibited. Pb2+ induced the expression of 6 OsMT-I genes in seedling shoots, but had no obvious effects on the expression of OsMT-I-1a, OsMT-I-1b, OsMT-I-4a and OsMT-I-4b. All the 10 OsMT-Is had enhanced lead tolerance when heterologously expressed in lead-sensitive yeast mutant cells. These results provide an expression profile of the rice MT gene family in response to Pb2+ stress in rice seedlings and demonstrate increased lead tolerance in sensitive yeast mutant cells expressing OsMT-Is. This study lays a foundation for further analysis of the role of the rice MT gene family in respond to Pb2+ stress.

The transcription factors DREB1s/CBFs play important roles in the regulation of plant resistance to environmental stresses and are quite useful for generating transgenic plants tolerant to these stresses. In the present work, a cDNA encoding DREB1/CBF-like protein (GhDREB1L) from cotton was isolated, and its sequence features, DNA binding preference, and expression patterns of the transcripts were also characterized. GhDREB1L contained one conserved AP2/ERF domain and its amino acid sequence was similar to the DREB1/CBF group of the DREB family from other plants. The DNA-binding domain of GhDREB1L was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Electrophoretic mobility shift assay revealed that the purified GhDREB1L fusion protein had a specific binding activity with the previously characterized DRE element (core sequence, ACCGAC) and also with the DRE-like sequence (core sequence, GCCGAC) in the promoter of the dehydration-responsive late embryogenesis-abundant gene LEA D113. Semi-quantitative RT-PCR showed that GhDREB1L was induced in the cotton cotyledons by low temperature, as well as drought and NaCl treatments. These results suggested that the novel cotton GhDREB1L might play an important role in response to low temperature as well as drought and high salinity through binding to the DRE cis-element.

To gain a better understanding of the regulatory mechanism of plant metallothionein (MT) genes, a chimeric expression unit consisting of the β-glucuronidase (gusA) reporter gene under the control of a 1,324 bp fragment of the rice MT (ricMT) promoter was introduced into Arabidopsis via Agrobacterium tumefaciens. The strongest histochemical staining for GUS activity was observed in the cotyledons and hypocotyls of the transgenic seedlings and in the stigma, filaments and anthers of young and mature flowers, and especially in the wounded tissues of transgenic plants. In contrast, a relatively low level of reporter gene expression was seen in the young roots of transgenic seedlings and no GUS activity was detected in the stems, seeds and leaves, but GUS activity was observed in cotyledons and the first two true leaves. Promoter analysis of 5′ deletions further identified several important regions responsible for organ-specific expression including roots, flowers and wound induction, light and ABA, Cu and Zn responses. These findings demonstrate that a 1,324 bp fragment of the rice MT promoter performs a complicated transcriptional regulation with clearly functional regions in a model plant, and provide an important insight into the transcriptional regulation mechanisms that operate the temporal- and spatial-specific expression and stress responses of the rice MT gene. These results suggest that the ricMT promoter and its functional regions are potentially useful in genetic engineering of plants to express the desired genes whose products are preferentially needed in roots, flowers and wound induction.

In this study, the interaction between rice 14-3-3 protein and 1-aminocyclopropane carboxylic acid synthase (ACS) was observed in yeast cells using yeast two-hybrid assays. Given the fact that 14-3-3 proteins generally bind to their target proteins in a phosphorylation-dependent manner, a hypothesis regarding the regulatory role of 14-3-3 proteins in the activation of ACS is proposed in which 14-3-3 proteins may bind to the phosphorylated C-terminal tails of ACSs and help them to escape from their fated degradation when ethylene biosynthesis is needed. It is reasonable to believe that 14-3-3 protein may play an important role in regulating ACS activity.

The metal ion binding properties of a phytochelatin (PC) analogue, (Glu–Cys)4–Gly (named as EC4), have been studied by a divalent metal ion binding assay monitored by UV–visible spectroscopy, circular dichroism and NMR spectroscopy. Spectro- photometric titration with different divalent metal ions have revealed that the stiochoimetry of metal-bound EC4 was 1:1, and its metal binding affinities with different divalent metal ions in the order of Cd(II) > Cu(II) > Zn(II) > Pb(II) > Ni(II) > Co(II). UV–visible spectroscopic analysis of metal complexes indicated that four sulfur atoms in cysteine residues are attributable to ligand-to-metal charge transfer (LMCT) between divalent metal ions and EC4, and further confirmed by 1D H1 NMR study and Circular Dichroism. In addition, Circular Dichroism spectra of both free and metal-bound forms of EC4 revealed that metal coordination drives the nonapeptide chain to fold into a turned conformation. The comprehensive analysis of spectroscopic properties of the nonapeptide complexed with metal ions not only provides a fundamental description of the metal ion binding properties of PC analogue, but also shows a correlation between metal binding affinity of PC analogue and the induction activity of metal ions.

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.

Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this paper, a new ethylene response factor gene GhERF1 was isolated from cotton. The deduced amino acid sequence of GhERF1 contained an AP2/ERF domain, which shared high similarity with other reported AP2/ERF domains and was most closely related to the B3 subgroup of the ERF subfamily. The particle bombardment assay showed that GhERF1 functioned as an in vivo transcription activator in tobacco cells and was localized in the nuclei of onion epidermis cells. In addition, semi-quantitative RT-PCR revealed that GhERF1 accumulated highly and rapidly when plants were treated with exogenous ethylene, abscisic acid (ABA), high salinity, cold and drought. These results suggested that GhERF1 might be functionally important in acclimation of cotton to stress.

Abscisic acid (ABA) plays crucial roles in plant growth and development, as well as in response to various environmental stresses. To date, many regulatory genes involved in the ABA response network have been identified; however, their roles have remained to be fully elucidated. In this study, we identified AtYY1, an Arabidopsis homolog of the mammalian C2H2 zinc-finger transcription factor Yin Yang 1 (YY1), as a novel negative regulator of the ABA response. AtYY1 is a dual-function transcription factor with both repression and activation domains. The expression of AtYY1 was induced by ABA and stress conditions including high salt and dehydration. The yy1 mutant was more sensitive to ABA and NaCl than the wild-type, while overexpressing AtYY1 plants were less sensitive. AtYY1 loss also enhanced ABA-induced stomatal closing and drought resistance. Moreover, AtYY1 can bind the ABA REPRESSOR1 (ABR1) promoter and directly upregulate ABR1 expression, as well as negatively regulate ABA- and salt-responsive gene expression. Additional analysis indicated that ABA INSENSITIVE4 (ABI4) might positively regulate AtYY1 expression and that ABR1 can antagonize this regulation. Our findings provide direct evidence that AtYY1 is a novel negative regulator of the ABA response network and that the ABI4-AtYY1-ABR1 regulatory pathway may fine-tune ABA-responsive gene expression in Arabidopsis.

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.