GhKNL1 controls fiber elongation and secondary cell wall synthesis by repressing its downstream genes in cotton (Gossypium hirsutum).

Cotton which produces natural fiber materials for the textile industry is one of the most important crops in the world. Class II KNOX proteins are often considered as transcription factors in regulating plant secondary cell wall (SCW) formation. However, the molecular mechanism of the KNOX transcription factor-regulated SCW synthesis in plants (especially in cotton) remains unclear in details so far. In this study, we show a cotton class II KNOX protein (GhKNL1) as a transcription repressor functioning in fiber development. The GhKNL1-silenced transgenic cotton produced longer fibers with thicker SCWs, whereas GhKNL1 dominant repression transgenic lines displayed the opposite fiber phenotype, compared with controls. Further experiments revealed that GhKNL1 could directly bind to promoters of GhCesA4-2/4-4/8-2 and GhMYB46 for modulating cellulose synthesis during fiber SCW development in cotton. On the other hand, GhKNL1 could also suppress expressions of GhEXPA2D/4A-1/4D-1/13A through binding to their promoters for regulating fiber elongation of cotton. Taken together, these data revealed GhKNL1 functions in fiber elongation and SCW formation by directly repressing expressions of its target genes related to cell elongation and cellulose synthesis. Thus, our data provide an effective clue for potentially improving fiber quality by genetic manipulation of GhKNL1 in cotton breeding.

Learning and memory impairment of mice caused by gaseous formaldehyde.

In order to study the e of formaldehyde exposure on learning and memory ability of mice. We used Kun Ming (KM) mice to demonstrate the neurotoxic effects of FA, and Balb/c mice to explore the neurobiological mechanism. The Morris water maze (MWM) test showed that the exposure of gaseous formaldehyde could cause spatial learning and memory impairment in mice. H & E staining showed that in the 3.0 mg/m3 formaldehyde exposed group, the arrangement of pyramidal cells in CA1 area of mouse hippocampus was loose and disordered, the cell morphology was swollen and deformed, and the apical dendrites were shortened or even disappeared. Biochemical indicators revealed high doses of FA exposure could cause oxidative damage in brain. Compared with the control group, there were significant differences in the levels of ROS, MDA, GSH and 8-OHDG in the 3.0 mg/m3 group (P < 0.01), also the monoamine neurotransmitters content and the content of TNF-α, IL-1ß and Caspase-3 (P < 0.01). Furthermore, the concentrations of cAMP, cGMP, NO and the activity of NOS in the cerebral cortex, hippocampus and brain stem after high doses of FA exposure were significantly different from those in the control group, indicating that FA exposure could interfere with the transduction of NO/cGMP signaling pathway. The results showed that FA could induce cognitive deficits and this extended investigation found that the toxicity of FA to the mouse nervous system is related to the NO/cGMP and cAMP signaling pathways.

Exposure to formaldehyde and diisononyl phthalate exacerbate neuroinflammation through NF-κB activation in a mouse asthma model.

Diisononyl phthalate (DINP) and formaldehyde both are associated with asthma and allergies. However, it is unclear about the adverse effect of DINP and formaldehyde exposure on the brain for asthma patients. Here, we determined the effect of DINP and/or formaldehyde exposure on neuroinflammation in brain by a murine asthma model and investigated the underlying mechanisms. Mice were exposed to formaldehyde and/or DINP and sensitization with ovalbumin. The results show that exposure to formaldehyde and/or DINP not only exacerbated allergic asthma-like symptoms, but also promoted neuroinflammation in brain. The incrassation of the airway wall and exacerbation of neuroinflammation were more obviously when mice were subjected to a combined exposure to DINP and formaldehyde. Exposure to DINP and/or formaldehyde enhances oxidative stress and the activation of NF-κB in the prefrontal cortex of mouse asthma model. Exposure to DINP and/or formaldehyde also induced an increase in IL-1ß, IL-17, and NGF. Blocking oxidative stress by administering melatonin or inhibiting NF-κB activation by treatment with Dehydroxymethylepoxyquinomicin effectively prevented increasing the levels IL-1ß, IL-17 and nerve growth factor. The data indicated that DINP and/or formaldehyde exposure promoted neuroinflammation in the brain through enhanced oxidative stress and activation of NF-κB in a mouse asthma model.

A R2R3-MYB transcription factor that is specifically expressed in cotton (Gossypium hirsutum) fibers affects secondary cell wall biosynthesis and deposition in transgenic Arabidopsis.

Secondary cell wall (SCW) is an important industrial raw material for pulping, papermaking, construction, lumbering, textiles and potentially for biofuel production. The process of SCW thickening of cotton fibers lays down the cellulose that will constitute the bulk (up to 96%) of the fiber at maturity. In this study, a gene encoding a MYB-domain protein was identified in cotton (Gossypium hirsutum) and designated as GhMYBL1. Quantitative real-time polymerase chain reaction (RT-PCR) analysis revealed that GhMYBL1 was specifically expressed in cotton fibers at the stage of secondary wall deposition. Further analysis indicated that this protein is a R2R3-MYB transcription factor, and is targeted to the cell nucleus. Overexpression of GhMYBL1 in Arabidopsis affected the formation of SCW in the stem xylem of the transgenic plants. The enhanced SCW thickening also occurred in the interfascicular fibers, xylary fibers and vessels of the GhMYBL1-overexpression transgenic plants. The expression of secondary wall-associated genes, such as CesA4, CesA7, CesA8, PAL1, F5H and 4CL1, were upregulated, and consequently, cellulose and lignin biosynthesis were enhanced in the GhMYBL1 transgenic plants. These data suggested that GhMYBL1 may participate in modulating the process of secondary wall biosynthesis and deposition of cotton fibers.

A fasciclin-like arabinogalactan protein, GhFLA1, is involved in fiber initiation and elongation of cotton.

Arabinogalactan proteins (AGPs) are involved in many aspects of plant development. In this study, biochemical and genetic approaches demonstrated that AGPs are abundant in developing fibers and may be involved in fiber initiation and elongation. To further investigate the role of AGPs during fiber development, a fasciclin-like arabinogalactan protein gene (GhFLA1) was identified in cotton (Gossypium hirsutum). Overexpression of GhFLA1 in cotton promoted fiber elongation, leading to an increase in fiber length. In contrast, suppression of GhFLA1 expression in cotton slowed down fiber initiation and elongation. As a result, the mature fibers of the transgenic plants were significantly shorter than those of the wild type. In addition, expression levels of GhFLAs and the genes related to primary cell wall biosynthesis were remarkably enhanced in the GhFLA1 overexpression transgenic fibers, whereas the transcripts of these genes were dramatically reduced in the fibers of GhFLA1 RNA interference plants. An immunostaining assay indicated that both AGP composition and primary cell wall composition were changed in the transgenic fibers. The levels of glucose, arabinose, and galactose were also altered in the primary cell wall of the transgenic fibers compared with those of the wild type. Together, our results suggested that GhFLA1 may function in fiber initiation and elongation by affecting AGP composition and the integrity of the primary cell wall matrix.

Cotton KNL1, encoding a class II KNOX transcription factor, is involved in regulation of fibre development.

In this study, the GhKNL1 (KNOTTED1-LIKE) gene, encoding a classical class II KNOX protein was identified in cotton (Gossypium hirsutum). GhKNL1 was preferentially expressed in developing fibres at the stage of secondary cell wall (SCW) biosynthesis. GhKNL1 was localized in the cell nucleus, and could interact with GhOFP4, as well as AtOFP1, AtOFP4, and AtMYB75. However, GhKNL1 lacked transcriptional activation activity. Dominant repression of GhKNL1 affected fibre development of cotton. The expression levels of genes related to fibre elongation and SCW biosynthesis were altered in transgenic fibres of cotton. As a result, transgenic cotton plants produced aberrant, shrunken, and collapsed fibre cells. Length and cell-wall thickness of fibres of transgenic cotton plants were significantly reduced compared with the wild type. Furthermore, overexpression and dominant repression of GhKNL1 in Arabidopsis resulted in a reduction in interfascicular fibre cell-wall thickening of basal stems of transgenic plants. Complementation revealed that GhKNL1 rescued the defective phenotype of Arabidopsis knat7 mutant in some extent. These data suggest that GhKNL1, as a transcription factor, participates in regulating fibre development of cotton.

GhHyPRP4, a cotton gene encoding putative hybrid proline-rich protein, is preferentially expressed in leaves and involved in plant response to cold stress.

Plant hybrid proline-rich proteins (HyPRPs) usually consist of an N-terminal signal peptide, a central proline-rich domain, and a conserved eight-cysteine motif C-terminal domain. In this study, one gene (designated as GhHyPRP4) encoding putative HyPRP was isolated from cotton cDNA library. Northern blot and quantitative reverse transcriptase-polymerase chain reaction analyses revealed that GhHyPRP4 was preferentially expressed in leaves. Under cold stress, GhHyPRP4 expression was significantly up-regulated in leaves of cotton seedlings. Using the genome walking approach, a promoter fragment of GhHyPRP4 gene was isolated from cotton genome. GUS (ß-glucuronidase) gene driven by GhHyPRP4 promoter was specifically expressed in leaves and cotyledons of the transgenic Arabidopsis thaliana. Furthermore, GUS expression in leaves was remarkably induced by cold stress. Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20°C for 60 h. These data suggested that GhHyPRP4 may be involved in plant response to cold stress during seedling development of cotton.

Two cotton Cys2/His2-type zinc-finger proteins, GhDi19-1 and GhDi19-2, are involved in plant response to salt/drought stress and abscisic acid signaling.

Cotton (Gossypium hirsutum) often encounters abiotic stress such as drought and high salinity during its development, and its productivity is significantly limited by those adverse factors. To investigate the molecular adaptation mechanisms of this plant species to abiotic stress, we identified two genes encoding Di19-like Cys2/His2 zinc-finger proteins in cotton. GFP fluorescence assay demonstrated that GhDi19-1 and GhDi19-2 are two nuclear-localized proteins. Quantitative RT-PCR and Northern blot analyses revealed that mRNA accumulation of both GhDi19-1 and GhDi19-2 was significantly promoted by salinity and drought. Expression of GUS gene driven by the GhDi19-1 and GhDi19-2 promoters, respectively, was intensively induced in cotyledons under NaCl and mannitol stresses. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis resulted in the seedlings displaying hypersensitivity to high salinity and abscisic acid (ABA). Seed germination and seedling growth of the transgenic Arabidopsis were dramatically inhibited by salinity and ABA, compared with wild type. In addition, expression levels of the ABA-responsive genes ABF3, ABF4, ABI5 and KIN1 were also remarkably altered in the transgenic plants under ABA treatment. Collectively, our results suggested that both GhDi19-1 and GhDi19-2 may be involved in response to salt/drought stress and ABA signaling during early stages of plant development.

Expression and localization of GhH6L, a putative classical arabinogalactan protein in cotton (Gossypium hirsutum).

Arabinogalactan proteins (AGPs) are a large family of highly glycosylated of hydroxyproline-rich glycoproteins that play important roles in plant growth, development, and signal transduction. A cDNA encoding a putative classical AGP named GhH6L was isolated from cotton fiber cDNA libraries, and the deduced protein contains 17 copies of repetitive motif of X-Y-proline-proline-proline (where X is serine or alanine and Y is threonine or serine). Northern blotting analysis and quantitative RT-PCR results showed that it was preferentially expressed in 10 days post-anthesis (dpa) fibers and was also developmentally regulated. A promoter fragment was isolated from cotton (Gossypium hirsutum) by genome walking PCR. Expression of beta-glucuronidase (GUS) gene under the GhH6L promoter was examined in the transgenic Arabidopsis plants; only petiole and pedicel were stained, no staining was detected in other tissues. Subcellular localization indicated that GhH6L was localized to the plasma membrane and in the cytoplasm. These data further our understanding of GhH6L as well as shed light on functional insight to GhH6L in cotton.

Characterization of 19 novel cotton FLA genes and their expression profiling in fiber development and in response to phytohormones and salt stress.

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), are usually involved in cell development in plants. To investigate the expression profiling as well as the role of FLA genes in fiber development, 19 GhFLA genes (cDNAs) were isolated from cotton (Gossypium hirsutum). Among them, 15 are predicted to be glycosylphosphatidylinositol anchored to the plasma membranes. The isolated cotton FLAs could be divided into four groups. Real-time quantitative reverse transcriptase polymerase chain reaction results indicated that the GhFLA genes are differentially expressed in cotton tissues. Three genes (GhFLA1/2/4) were specifically or predominantly expressed in 10 days post-anthesis fibers, and the transcripts of the other four genes (GhFLA6/14/15/18) were accumulated at relatively high levels in cotton fibers. Furthermore, expressions of the GhFLA genes are regulated in fiber development and in response to phytohormones and NaCl. The identification of cotton FLAs will facilitate the study of their roles in cotton fiber development and cell wall biogenesis.

Outer Membrane Lipoprotein Lip40 Modulates Adherence, Colonization, and Virulence of Actinobacillus pleuropneumoniae.

Bacterial lipoproteins are a set of membrane proteins with various functions; many of which are virulence factors of pathogenic bacteria. In the present study, we investigated the role of an outer membrane lipoprotein Lip40 in the pathogenesis of Actinobacillus pleuropneumoniae. A mutant strain (Δlip40) lacking Lip40 and a complemented strain (CΔlip40) were constructed. Δlip40 exhibited reduced adherence to the St. Jude porcine lung cells. The ability of the Δlip40 mutant to colonize the mouse lung tissues was significantly impaired compared to that of the wild type and complementation strains. Furthermore, an infection assay revealed that pigs infected with Δlip40 showed fewer clinical signs and lung lesions, indicating that Lip40 contributed to the development of porcine pleuropneumonia. Collectively, our data suggest that Lip40 is involved in the virulence of A. pleuropneumoniae.

Family-Wide Characterization of Histone Binding Abilities of PHD Domains of AL Proteins in Arabidopsis thaliana.

Alfin1-like (AL) is a family of proteins homologous to the alfalfa Alfin1 in plant and bears an Alfin domain and a PHD domain at their N- and C-terminus, respectively. There are 7 AL proteins in Arabidopsis, and the PHD domains of most AL proteins are reported to bind to histone H3K4me3. Here we reported gene cloning, protein expression and purification of the PHD domains of all the AL family proteins in Arabidopsis. We then systematically characterized their histone binding abilities by quantitative isothermal titration calorimetry and fluorescence polarization binding assays. Our binding results indicate that all the PHD domains of the AL proteins bind to the histone H3K4me3 peptide with varying methylation state preference and binding affinities. Our study presented here provides the foundation for further studies of the peptide state-specific recognition by PHD domains of AL proteins.

Overexpression of a cotton (Gossypium hirsutum) WRKY gene, GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants.

Soil salinity is one of the most serious threats in world agriculture, and often influences cotton growth and development, resulting in a significant loss in cotton crop yield. WRKY transcription factors are involved in plant response to high salinity stress, but little is known about the role of WRKY transcription factors in cotton so far. In this study, a member (GhWRKY34) of cotton WRKY family was functionally characterized. This protein containing a WRKY domain and a zinc-finger motif belongs to group III of cotton WRKY family. Subcellular localization assay indicated that GhWRKY34 is localized to the cell nucleus. Overexpression of GhWRKY34 in Arabidopsis enhanced the transgenic plant tolerance to salt stress. Several parameters (such as seed germination, green cotyledons, root length and chlorophyll content) in the GhWRKY34 transgenic lines were significantly higher than those in wild type under NaCl treatment. On the contrary, the GhWRKY34 transgenic plants exhibited a substantially lower ratio of Na(+)/K(+) in leaves and roots dealing with salt stress, compared with wild type. Growth status of the GhWRKY34 transgenic plants was much better than that of wild type under salt stress. Expressions of the stress-related genes were remarkably up-regulated in the transgenic plants under salt stress, compared with those in wild type. Based on the data presented in this study, we hypothesize that GhWRKY34 as a positive transcription regulator may function in plant response to high salinity stress through maintaining the Na(+)/K(+) homeostasis as well as activating the salt stress-related genes in cells.

GhMPK17, a cotton mitogen-activated protein kinase, is involved in plant response to high salinity and osmotic stresses and ABA signaling.

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H2O2 level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.

A cotton mitogen-activated protein kinase (GhMPK6) is involved in ABA-induced CAT1 expression and H(2)O(2) production.

The mitogen-activated protein kinase (MAPK) cascade is one of the major and evolutionally conserved signaling pathways and plays a pivotal role in the regulation of stress and developmental signals in plants. Here, we identified one gene, GhMPK6, encoding an MAPK protein in cotton. GFP fluorescence assay demonstrated that GhMAPK6 is a cytoplasm localized protein. Quantitative RT-PCR analysis revealed that mRNA accumulation of GhMPK6 was significantly promoted by abscisic acid (ABA). Overexpression of GhMPK6 gene in the T-DNA insertion mutant atmkk1 (SALK_015914) conferred a wild-type phenotype to the transgenic plants in response to ABA. Under ABA treatment, cotyledon greening/expansion in GhMPK6 transgenic lines and wild type was significantly inhibited, whereas the atmkk1 mutant showed a relatively high cotyledon greening/expansion ratio. Furthermore, CAT1 expression and H(2)O(2) levels in leaves of GhMPK6 transgenic lines and wild type were remarkably higher than those of atmkk1 mutant with ABA treatment. Collectively, our results suggested that GhMPK6 may play an important role in ABA-induced CAT1 expression and H(2)O(2) production.