Phytosphinganine Affects Plasmodesmata Permeability via Facilitating PDLP5-Stimulated Callose Accumulation in Arabidopsis.

Plant plasmodesmata (PDs) are specialized channels that enable communication between neighboring cells. The intercellular permeability of PDs, which affects plant development, defense, and responses to stimuli, must be tightly regulated. However, the lipid compositions of PD membrane and their impact on PD permeability remain elusive. Here, we report that the Arabidopsis sld1 sld2 double mutant, lacking sphingolipid long-chain base 8 desaturases 1 and 2, displayed decreased PD permeability due to a significant increase in callose accumulation. PD-located protein 5 (PDLP5) was significantly enriched in the leaf epidermal cells of sld1 sld2 and showed specific binding affinity to phytosphinganine (t18:0), suggesting that the enrichment of t18:0-based sphingolipids in sld1 sld2 PDs might facilitate the recruitment of PDLP5 proteins to PDs. The sld1 sld2 double mutant seedlings showed enhanced resistance to the fungal-wilt pathogen Verticillium dahlia and the bacterium Pseudomonas syringae pv. tomato DC3000, which could be fully rescued in sld1 sld2 pdlp5 triple mutant. Taken together, these results indicate that phytosphinganine might regulate PD functions and cell-to-cell communication by modifying the level of PDLP5 in PD membranes.

Notch signaling pathway regulates CD4+CD25+CD127dim/- regulatory T cells and T helper 17 cells function in gastric cancer patients.

Regulatory T cells (Tregs) and T helper 17 (Th17) cells contribute to cancer progression and prognosis. However, regulatory factors associated with Tregs-Th17 balance were not completely understood. We previously demonstrated an immune-modulatory capacity by Notch signaling inactivation to reverse Tregs-Th17 disequilibrium in chronic hepatitis C. Thus, the aim of current study was to assess the role of Notch signaling in modulation Tregs and Th17 cells function in gastric cancer (GC) patients. A total of 51 GC patients and 18 normal controls (NCs) were enrolled. Notch1 and Notch2 mRNA expressions were semiquantified by real-time polymerase chain reaction. Tregs/Th17 percentages, transcriptional factors, and cytokines production were investigated in response to the stimulation of Notch signaling inhibitor DAPT. Both Notch1 and Notch2 mRNA expressions were elevated in GC tissues and peripheral bloods in GC patients. CD4+CD25+CD127dim/- Tregs and Th17 cells percentage was also elevated in GC patients compared with in NCs. DAPT treatment did not affect frequency of either circulating Tregs or Th17 cells, however, reduced FoxP3/RORγt mRNA expression and interleukin (IL)-35/IL-17 production in purified CD4+ T cells from GC patients. Moreover, blockade of Notch signaling also inhibited the suppressive function of purified CD4+CD25+CD127dim/- Tregs from GC patients, which presented as elevation of cellular proliferation and IL-35 secretion. The current data further provided mechanism underlying Tregs-Th17 balance in GC patients. The link between Notch signaling and Th cells might lead to a new therapeutic target for GC patients.

Characterization of the Ubiquitin C-Terminal Hydrolase and Ubiquitin-Specific Protease Families in Rice (Oryza sativa).

The ubiquitin C-terminal hydrolase (UCH) and ubiquitin-specific processing protease (UBP) protein families both function in protein deubiquitination, playing important roles in a wide range of biological processes in animals, fungi, and plants. Little is known about the functions of these proteins in rice (Oryza sativa), and the numbers of genes reported for these families have not been consistent between different rice database resources. To further explore their functions, it is necessary to first clarify the basic molecular and biochemical nature of these two gene families. Using a database similarity search, we clarified the numbers of genes in these two families in the rice genome, examined the enzyme activities of their corresponding proteins, and characterized the expression patterns of all OsUCH and representative OsUBP genes. Five OsUCH and 44 OsUBP genes were identified in the rice genome, with four OsUCH proteins and 10 of 16 tested representative OsUBP proteins showing enzymatic activities. Two OsUCHs and five OsUBPs were found to be preferentially expressed in the early development of rice stamens. This work thus lays down a reliable bioinformatic foundation for future investigations of genes in these two families, particularly for exploring their potential roles in rice stamen development.

Notch Signaling Modulates the Balance of Regulatory T Cells and T Helper 17 Cells in Patients with Chronic Hepatitis C.

The imbalance of regulatory T cells (Tregs) and T helper 17 (Th17) cells contributes to the persistent hepatitis C virus (HCV) infection. However, modulatory factors associated with Tregs-Th17 balance were not fully elucidated. A recent study demonstrated an immunoregulatory strategy by inactivation of Notch signaling to reverse the disequilibrium of Tregs-Th17 cells in immune thrombocytopenia. Thus, the aim of this study was to assess the effect of Notch signaling in regulating the functions of Tregs and Th17 cells in chronic hepatitis C. A total of 46 patients with chronic hepatitis C and 17 normal controls (NCs) were enrolled. mRNA expressions of Notch1 and Notch2 were semiquantified by real-time reserve polymerase chain reaction. Percentages of Tregs-Th17, levels of key transcriptional factors, and cytokine productions were measured in response to treatment by DAPT, a γ-secretase inhibitor to suppress Notch signaling. We found that Notch1 and Notch2 mRNAs were significantly elevated in peripheral blood mononuclear cells from chronic hepatitis C patients compared with those from NCs. DAPT treatment reduced Th17 response by downregulation of RORγt expression and interleukin (IL)-17/IL-22 secretion. Tregs proportion, FoxP3 expression, and IL-10 production did not change significantly with DAPT treatment in chronic hepatitis C; however, blockage of Notch signaling inhibited the suppressive function of Tregs. Moreover, effective anti-HCV therapy not only reduced Notch1 and Notch2 expression but also decreased Tregs and Th17 proportions. The current data provided a novel mechanism underlying the modulation of Treg-Th17 balance. The link between Notch signaling and Th cells might lead to a new intervention for breaking immunotolerance of chronic HCV infection.

Association of polymorphisms of adiponectin gene promoter-11377C/G, glutathione peroxidase-1 gene C594T, and cigarette smoking in nonalcoholic fatty liver disease.

BACKGROUND: The number of studies on adiponectin, GPx-1 gene polymorphisms, and nonalcoholic fatty liver disease (NAFLD) susceptibility is increasing, but none have investigated the effect of cigarette smoking in combination with the gene polymorphisms on the susceptibility to NAFLD. In order to understand the distribution of adiponectin and GPx-1 in the local population, to explore the possible association of cigarette smoking with adiponectin and GPx-1 gene polymorphisms in the pathogenesis of NAFLD, we conducted this research, examining the distribution of polymorphisms of adiponectin and GPx-1 in NAFLD patients and healthy controls, analyzing the association between these polymorphisms and cigarette smoking. METHODS: Two hundred nonalcoholic simple fatty liver (NAFL), 200 nonalcoholic steatohepatitis (NASH), and 200 nonalcoholic fatty hepatic cirrhosis (NAFHC) cases from the First Affiliated Hospital of Xinxiang Medical College in China from February 2011 to November 2014 were selected for this study, and 200 healthy individuals as a control group. No significant difference among the four groups in age, sex, ethnicity, and birthplace was observed. The genetic polymorphisms of adiponectin gene promoter-11377C/G and GPx-1 gene C594T were analyzed using polymerase chain reaction-restriction fragment length polymorphisms in peripheral blood leukocytes of the above-mentioned cases. The interaction between the two mutants and the gene-environment association of the genotypes with cigarette smoking were analyzed. RESULTS: The frequencies of adiponectin gene promoter-11377C/G(CG), -11377C/G (GG), GPx-1 gene C594T (CT) and C594T (TT) were 24.50%, 26.00%, 24.00%, and 25.50% in the NAFL group, 34.50%, 37.00%, 35.00%, and 36.00% in the NASH group, 42.00%, 46.00%, 43.50%, and 45.50% in the NAFHC group, and 14.00%, 14.50%, 13.00%, and 14.00% in the control group, respectively. Statistical tests showed a significant difference in the frequencies among each group (p < 0.01). The risk of NAFLD significantly increased in patients with adiponectin gene promoter-11377C/G (CG) genotype [odds ratio (OR)NAFL = 2.5278; ORNASH = 6.1823; ORNAFHC = 17.8570), in those with -11377C/G (GG) genotype (ORNAFL = 2.5900; ORNASH = 6.4017; ORNAFHC = 18.9023), in those with GPx-1 gene C594T (CT) genotype (ORNAFL = 2.6687; ORNASH = 6.7772; ORNAFHC = 22.2063), and in those with C594T (TT) genotype (ORNAFL = 2.6330; ORNASH = 6.4729; ORNAFHC = 21.5682). Combined analysis of the polymorphisms showed that percentages of adiponectin gene promoter -11377C/G (GG)/GPx-1 gene C594T (TT) in the NAFL, the NASH, NAFHC, and control groups was 7.00%, 13.50%, 21.00%, and 2.00%, respectively (p < 0.01). The people who carried the adiponectin gene promoter -11377C/G (GG)/GPx-1 gene C594T (TT) had a high risk of NAFLD (ORNAFL = 7.2800; ORNASH = 41.2941; ORNAFHC = 363.9724), and statistical analysis suggested a positive association between -11377C/G (GG) and C594T (TT) in increasing the risk of NAFLD (γ2NAFL = 2.2071, γ4 NAFL = 2.0773; γ2 NASH = 2.1084; γ4NASH = 2.0543; γ2 NAFHC = 2.1387; γ4NAFHC = 2.0004). Likewise, there were also positive association in the pathogenesis of NAFLD between -11377C/G (CG) and C594T (TT), -11377C/G (CG) and C594T (CT), -11377C/G (GG), and C594T (TT) (CT).The frequencies of smoking index (SI) ≤ 400 and SI > 400 were 22.50% and 26.50% in the NAFL group, 29.00% and 40.50% in the NASH group, 34.00% and 51.50% in the NAFHC group, and 15.50% and 12.00% in the control group, respectively. Statistical tests showed a significant difference in the frequencies among each group (all p < 0.01). The risk of NAFLD significantly increased in patients with SI ≤ 400 (ORNAFL = 2.0636; ORNASH = 4.4474; ORNAFH C = 10.9677) and in those with SI > 400 (ORNAFL = 3.1393; ORNASH = 8.0225; ORNAFHC = 21.4583), and statistical analysis suggested a positive association between cigarette smoking and -11377C/G (CG), -11377C/G (GG), C594T (CT), and C594T (TT) in increasing the risk of NAFLD (all γ > 1). CONCLUSION: Adiponectin gene promoter -11377C/G (CG), -11377C/G (GG), GPx-1 gene C594T (CT), C594T (TT), and cigarette smoking are risk factors in NAFLD, and the significant association between genetic polymorphisms of -11377C/G, C594T, and cigarette smoking amplify the risk of NAFLD.

Interaction of Polymorphisms of Resistin Gene Promoter -420C/G, Glutathione Peroxidase -1 Gene Pro198Leu and Cigarette Smoking in Nonalcoholic Fatty Liver Disease.

BACKGROUND: Many studies have suggested that cigarette smoking and polymorphisms of resistin and glutathione peroxidase-1 (GPx-1) genes are closely correlated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, few reports have investigated these associations with respect to NAFLD susceptibility. We, therefore, examined the distribution of polymorphisms in GPx-1 and resistin genes in NAFLD patients and healthy controls and analyzed the relationship between these polymorphisms and smoking status. METHODS: Nine hundred NAFLD patients and 900 healthy controls were selected, and the genetic polymorphisms of resistin gene promoter-420C/G and GPx-1 gene Pro198Leu were analyzed by polymorphism-polymerase chain reaction (PCR) in DNA extracted from peripheral blood leukocytes. Interactions between the two mutants and the gene-environment interaction with cigarette smoking were also analyzed. RESULTS: Genotype frequencies of -420C/G (GG) and Pro198Leu (LL) were significantly higher in NAFLD cases (49.56% and 50.11%, respectively) compared with healthy controls (23.67% and 24.22%, respectively) (P = 0.0069; P = 0.0072). Moreover, the risk of NAFLD with -420C/G (GG) was significantly higher than in controls (odds ratio [OR] =3.1685, 95% confidence interval (CI) =1.9366-5.2073). Individuals carrying Pro198Leu (LL) had a high risk of NAFLD (OR = 3.1424, 95% CI = 1.7951-5.2367). Combined analysis of the polymorphisms showed that the -420C/G (GG)/Pro198Leu (LL) genotype was significantly more common in the NAFLD group than in the control group (39.44% vs. 12.78%, respectively, P = 0.0054), while individuals with -420C/G (GG)/Pro198Leu (LL) had a high risk of NAFLD (OR = 5.0357, 95% CI = 3.1852-7.8106). Moreover, the cigarette smoking rate in the NAFLD group was significantly higher than in the control group (OR = 1.8990, P = 0.0083 in the smoking index (SI) ≤400 subgroup; OR = 5.0937, P = 0.0051 in the SI >400 subgroup), and statistical analysis suggested a positive interaction between cigarette smoking and -420C/G (GG) (γ = 5.6018 in the SI ≤400 subgroup; γ = 4.4770 in the SI >400 subgroup) and Pro198Leu (LL) (γ = 5.7715 in the SI ≤400 subgroup; γ = 4.5985 in the SI >400 subgroup) in increasing the risk of NAFLD. CONCLUSION: NAFLD risk factors include -420C/G (GG), Pro198Leu (LL) and cigarette smoking, and these three factors have a significant additive effect on NAFLD risk.

Targeted Lipidomics Studies Reveal that Linolenic Acid Promotes Cotton Fiber Elongation by Activating Phosphatidylinositol and Phosphatidylinositol Monophosphate Biosynthesis.

The membrane lipids from fast-elongating wild-type cotton (Gossypium hirsutum) fibers at 10 days post-anthesis, wild-type ovules with fiber cells removed, and ovules from the fuzzless-lintless mutant harvested at the same age, were extracted, separated, and quantified. Fiber cells contained significantly higher amounts of phosphatidylinositol (PI) than both ovule samples with PI 34:3 being the most predominant species. The genes encoding fatty acid desaturases (Δ(15)GhFAD), PI synthase (PIS) and PI kinase (PIK) were expressed in a fiber-preferential manner. Further analysis of phosphatidylinositol monophosphate (PIP) indicated that elongating fibers contained four- to five-fold higher amounts of PIP 34:3 than the ovules. Exogenously applied linolenic acid (C18:3), soybean L-α-PI, and PIPs containing PIP 34:3 promoted significant fiber growth, whereas a liver PI lacking the C18:3 moiety, linoleic acid, and PIP 36:2 were completely ineffective. The growth inhibitory effects of carbenoxolone, 5-hydroxytryptamine, and wortmannin were reverted by C18:3, PI, or PIP, respectively, suggesting that PIP signaling is essential for fiber cell growth. Furthermore, cotton plants expressing virus-induced gene-silencing constructs that specifically suppressed GhΔ(15)FAD, GhPIS, or GhPIK expression, resulted in significantly short-fibered phenotypes. Our data provide the basis for in-depth studies on the roles of PI and PIP in mediating cotton fiber growth.

Quantitative proteomics and transcriptomics reveal key metabolic processes associated with cotton fiber initiation.

An iTRAQ-based proteomics of ovules from the upland cotton species Gossypium hirsutum and its fuzzless-lintless mutant was performed, and finally 2729 proteins that preferentially accumulated at anthesis in wild-type ovules were identified. We confirmed that the gene expression levels of 2005 among these proteins also increased by performing an RNA sequencing transcriptomics. Expression of proteins involved in carboxylic acid metabolism, small-molecule metabolic processes, hormone regulation, and lipid metabolism was significantly enhanced in wild-type ovules. Quantitative real-time PCR verified the increased expression of 26 genes involved in these processes. Cotton 3-hydroxyacyl-CoA dehydratase (GhPAS2) catalyzing the third reaction of very long-chain fatty acid (VLCFA) biosynthesis, accumulated at anthesis in wild-type ovules. Heterogeneous expression of GhPAS2 restored viability to the Saccharomyces cerevisiae haploid psh1-deletion strain deficient in PAS2 activity. Application of VLCFA biosynthesis inhibitor acetochlor (2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide; ACE) and gibberellic acid to the unfertilized cotton ovules significantly suppressed fiber cell protrusion. In this study, the profiling of gene expression at both transcriptome and proteome levels provides new insights into cotton fiber cell initiation. BIOLOGICAL SIGNIFICANCE: Cotton fiber initiation determines the ultimate number of fibers per ovule, thereby determining fiber yield. In total, 2729 proteins were preferentially accumulated in wild-type ovules at anthesis. The most up-regulated proteins were assigned to carboxylic acid metabolism, small-molecule metabolic processes, hormone regulation, and lipid metabolism. In consistence with these findings, we characterized GhPAS2 gene coding for the enzyme that catalyzes VLCFA production. VLCFA biosynthesis inhibitor, acetochlor, was shown to significantly suppress fiber initiation. This study provides a genome-scale transcriptomic and proteomic characterization of fiber initial cells, laying a solid basis for further investigation of the molecular processes governing fiber cell development.

Correlations between polymorphisms of extracellular superoxide dismutase, aldehyde dehydrogenase-2 genes, as well as drinking behavior and pancreatic cancer.

OBJECTIVE: To investigate the correlation between drinking behavior combined with polymorphisms of extracellular superoxide dismutase (EC-SOD) and aldehyde dehydrogenase-2 (ALDH2) genes and pancreatic cancer. METHODS: The genetic polymorphisms of EC-SOD and ALDH2 were analyzed by polymerase chain reaction restriction fragment length polymorphism in the peripheral blood leukocytes obtained from 680 pancreatic cancer cases and 680 non-cancer controls. Subsequently the frequency of genotype was compared between the pancreatic cancer patients and the healthy controls.The relationship of drinking with pancreatic cancer was analyzed. RESULTS: The frequencies of EC-SOD (C/G) and ALDH2 variant genotypes were 37.35% and 68.82% respectively in the pancreatic cancer cases, and were significantly higher than those in the healthy controls (21.03% and 44.56%, all P<0.01). People who carried EC-SOD (C/G) (OR=2.24, 95% CI= 1.81-4.03, P<0.01) or ALDH2 variant genotypes (OR=2.75, 95% CI=1.92-4.47, P<0.01) had a high risk to develop pancreatic cancer. Those who carried EC-SOD (C/G) genotype combined with ALDH2 variant genotype had a high risk for pancreatic cancer (29.56% vs. 6.76%, OR=7.69, 95% CI=3.58-10.51, P<0.01). The drinking rate of the pancreatic cancer group (64.12%) was significantly higher than that of the control group (40.15%; OR=2.66, 95% CI=1.30-4.42, P<0.01). An interaction between drinking and EC-SOD (C/G)/ALDH2 variant genotypes increased the risk of occurrence of pancreatic cancer (OR=25.00, 95% CI= 11.87-35.64, P<0.01). CONCLUSION: EC-SOD (C/G), ALDH2 variant genotypes and drinking might be the risk factors of pancreatic cancer.

How cotton fibers elongate: a tale of linear cell-growth mode.

Cotton fibers (cotton lint) are single-celled trichomes that differentiate from the ovule epidermis. Unidirectional and fast-growing cells generally expand at the dome-shaped apical zone (tip-growth mode); however, previous studies suggest that elongating fiber cells expand via a diffuse-growth mode. Tip-localized Ca(2+) gradient and active secretary vesicle trafficking are two important phenomena of tip-growth. Recently, a high Ca(2+) gradient is found in the cytoplasm of fast-elongating cotton fiber cells near the growing tip. Several protein coding genes participating in vesicle coating and transport are highly expressed in elongating fiber cells. Taken together with the observation that ethylene acts as a positive regulator for cotton fiber and several Arabidopsis tissues that are known to elongate via tip growth prompted us to propose a linear-growth mode for similar cell types.

Clinical study on the treatment of gastroesophageal reflux by acupuncture.

OBJECTIVE: To explore the clinical efficacy and safety of acupuncture in treating gastroesophageal reflux (GER). METHODS: Sixty patients with confirmed diagnosis of GER were randomly assigned to two groups. The 30 patients in the treatment group were treated with acupuncture at acupoints Zhongwan (CV 12), bilateral Zusanli (ST36), Sanyinjiao (SP6), and Neiguan (PC6), once a day, for 1 week as a therapeutic course, with interval of 2-3 days between courses; the 30 patients in the control group were administered orally with omeprazole 20 mg twice a day and 20 mg mosapride thrice a day. The treatment in both group lasted 6 weeks. Patients' symptoms and times of reflux attacking were recorded, the 24-h intraesophageal acid/bile reflux were monitored, and the endoscopic feature of esophageal mucous membrane was graded and scored at three time points, i.e., pre-treatment (T0), immediately after ending the treatment course (T1) and 4 weeks after it (T2). Besides, the adverse reactions were also observed. RESULTS: Compared with those detected at T0, 24-h intraesophageal pH and bile reflux, endoscopic grading score and symptom score were all decreased significantly at T1 in both groups similarly (P<0.01), showing insignificant difference between groups (P>0.05). These indices were reversed at T2 to high level in the control group (P<0.05), but the reversion did not occur in the treatment group (P>0.05). No serious adverse reaction was found during the therapeutic period. CONCLUSION: Acupuncture can effectively inhibit the intraesophageal acid and bile reflux in GER patients to alleviate patients' symptoms with good safety and is well accepted by patients.

Comparative proteomics indicates that biosynthesis of pectic precursors is important for cotton fiber and Arabidopsis root hair elongation.

The quality of cotton fiber is determined by its final length and strength, which is a function of primary and secondary cell wall deposition. Using a comparative proteomics approach, we identified 104 proteins from cotton ovules 10 days postanthesis with 93 preferentially accumulated in the wild type and 11 accumulated in the fuzzless-lintless mutant. Bioinformatics analysis indicated that nucleotide sugar metabolism was the most significantly up-regulated biochemical process during fiber elongation. Seven protein spots potentially involved in pectic cell wall polysaccharide biosynthesis were specifically accumulated in wild-type samples at both the protein and transcript levels. Protein and mRNA expression of these genes increased when either ethylene or lignoceric acid (C24:0) was added to the culture medium, suggesting that these compounds may promote fiber elongation by modulating the production of cell wall polymers. Quantitative analysis revealed that fiber primary cell walls contained significantly higher amounts of pectin, whereas more hemicellulose was found in ovule samples. Significant fiber growth was observed when UDP-L-rhamnose, UDP-D-galacturonic acid, or UDP-D-glucuronic acid, all of which were readily incorporated into the pectin fraction of cell wall preparations, was added to the ovule culture medium. The short root hairs of Arabidopsis uer1-1 and gae6-1 mutants were complemented either by genetic transformation of the respective cotton cDNA or by adding a specific pectin precursor to the growth medium. When two pectin precursors, produced by either UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase 4-reductase or by UDP-D-glucose dehydrogenase and UDP-D-glucuronic acid 4-epimerase successively, were used in the chemical complementation assay, wild-type root hair lengths were observed in both cut1 and ein2-5 Arabidopsis seedlings, which showed defects in C24:0 biosynthesis or ethylene signaling, respectively. Our results suggest that ethylene and C24:0 may promote cotton fiber and Arabidopsis root hair growth by activating the pectin biosynthesis network, especially UDP-L-rhamnose and UDP-D-galacturonic acid synthesis.

A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities.

To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

Characterization of two cotton cDNAs encoding trans-2-enoyl-CoA reductase reveals a putative novel NADPH-binding motif.

Very long chain fatty acids are important components of plant lipids, suberins, and cuticular waxes. Trans-2-enoyl-CoA reductase (ECR) catalyses the fourth reaction of fatty acid elongation, which is NADPH dependent. In the present study, the expression of two cotton ECR (GhECR) genes revealed by quantitative RT-PCR analysis was up-regulated during cotton fibre elongation. GhECR1 and 2 each contain open reading frames of 933 bp in length, both encoding proteins consisting of 310 amino acid residues. GhECRs show 32% identity to Saccharomyces cerevisiae Tsc13p at the deduced amino acid level, and the GhECR genes were able to restore the viability of the S. cerevisiae haploid tsc13-deletion strain. A putative non-classical NADPH-binding site in GhECR was predicted by an empirical approach. Site-directed mutagenesis in combination with gas chromatography-mass spectrometry analysis suggests that G(5X)IPXG presents a putative novel NADPH-binding motif of the plant ECR family. The data suggest that both GhECR genes encode functional enzymes harbouring non-classical NADPH-binding sites at their C-termini, and are involved in fatty acid elongation during cotton fibre development.

Molecular cloning, expression profiling, and yeast complementation of 19 beta-tubulin cDNAs from developing cotton ovules.

Microtubules are a major structural component of the cytoskeleton and participate in cell division, intracellular transport, and cell morphogenesis. In the present study, 795 cotton tubulin expressed sequence tags were analysed and 19 beta-tubulin genes (TUB) cloned from a cotton cDNA library. Among the group, 12 cotton TUBs (GhTUBs) are reported for the first time here. Transcription profiling revealed that nine GhTUBs were highly expressed in elongating fibre cells as compared with fuzzless-lintless mutant ovules. Treating cultured wild-type cotton ovules with exogenous phytohormones showed that individual genes can be induced by different agents. Gibberellin induced expression of GhTUB1 and GhTUB3, ethylene induced expression of GhTUB5, GhTUB9, and GhTUB12, brassinosteroids induced expression of GhTUB1, GhTUB3, GhTUB9, and GhTUB12, and lignoceric acid induced expression of GhTUB1, GhTUB3, and GhTUB12. When GhTUBs were transformed into the Saccharomyces cerevisiae inviable mutant, tub2, which is deficient in beta-tubulin, one ovule-specific and eight of nine fibre-preferential GhTUBs rescued this lethality. This study suggests that the proteins encoded by cotton GhTUBs are involved during cotton fibre development.

The ascorbate peroxidase regulated by H(2)O(2) and ethylene is involved in cotton fiber cell elongation by modulating ROS homeostasis.

Ascorbate peroxidase (APX) is a reactive oxygen species (ROSs) scavenging enzyme involved in regulation of intracellular ROS levels by reduction of H(2)O(2) to water using ascorbate as an electron donor. In New Phytologist 2007; 175:462-71, we identified a cotton cytosolic APX1 (GhAPX1) that was significantly accumulated during the fast fiber-cell elongation period, through a proteomics approach. Both the transcript levels of GhAPX1 and the total APX activity were highly induced in response to in vitro applied H(2)O(2) or ethylene. Further analysis showed that ethylene promoted H(2)O(2) production 1 day after it was included in the culture medium, suggesting that H(2)O(2) induced cell elongation processes may be placed downstream of the ethylene signal transduction pathway. In this addendum, quantitative real-time RT-PCR showed that only cytosolic APX1, not other cotton APX genes including a second cytosolic APX2, a glyoxysomal and a stromal APXs, was up-regulated during fiber cell elongating. Exogenous H(2)O(2) was found to induce ethylene production if wild-type cotton ovules were cultured for a longer period of time, implying that there was a feedback regulatory mechanism from H(2)O(2) to ethylene biosynthesis in modulating cotton fiber development.

Saturated very-long-chain fatty acids promote cotton fiber and Arabidopsis cell elongation by activating ethylene biosynthesis.

Fatty acids are essential for membrane biosynthesis in all organisms and serve as signaling molecules in many animals. Here, we found that saturated very-long-chain fatty acids (VLCFAs; C20:0 to C30:0) exogenously applied in ovule culture medium significantly promoted cotton (Gossypium hirsutum) fiber cell elongation, whereas acetochlor (2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide; ACE), which inhibits VLCFA biosynthesis, abolished fiber growth. This inhibition was overcome by lignoceric acid (C24:0). Elongating fibers contained significantly higher amounts of VLCFAs than those of wild-type or fuzzless-lintless mutant ovules. Ethylene nullified inhibition by ACE, whereas C24:0 was inactive in the presence of the ethylene biosynthesis inhibitor (l-[2-aminoethoxyvinyl]-glycine), indicating that VLCFAs may act upstream of ethylene. C24:0 induced a rapid and significant increase in ACO (for 1-aminocyclopropane-1-carboxylic acid oxidase) transcript levels that resulted in substantial ethylene production. C24:0 also promoted Ser palmitoyltransferase expression at a later stage, resulting in increased sphingolipid biosynthesis. Application of C24:0 not only stimulated Arabidopsis thaliana root cell growth but also complemented the cut1 phenotype. Transgenic expression of Gh KCS13/CER6, encoding the cotton 3-ketoacyl-CoA synthase, in the cut1 background produced similar results. Promotion of Arabidopsis stem elongation was accompanied by increased ACO transcript levels. Thus, VLCFAs may be involved in maximizing the extensibility of cotton fibers and multiple Arabidopsis cell types, possibly by activating ethylene biosynthesis.

A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell development.

Reactive oxygen species (ROS) play important roles in multiple physiological processes such as cellular signalling and stress responses, whereas, the hydrogen peroxide (H(2)O(2)) scavenging enzyme ascorbate peroxidase (APX) participates in the regulation of intracellular ROS levels. Here, a cotton (Gossypium hirsutum) cytosolic APX1 (GhAPX1) was identified to be highly accumulated during cotton fibre elongation by proteomic analysis. GhAPX1 cDNA contained an open reading frame of 753-bp encoding a protein of 250 amino acid residues. When GhAPX1 was expressed in Escherichia coli, the purified GhAPX1 was a dimer consisting of two identical subunits with a molecular mass of 28 kDa. GhAPX1 showed the highest substrate specificity for ascorbate. Quantitative real-time polymerase chain reaction (PCR) analyses showed that GhAPX1 was highly expressed in wild-type 5-d postanthesis fibres with much lower transcript levels in the fuzzless-lintless mutant ovules. Treating in vitro cultured wild-type cotton ovules with exogenous H(2)O(2) or ethylene induced the expression of GhAPX1 and hence increased total APX activity proportionally, followed by extended fibre cell elongation. These data suggest that GhAPX1 expression is upregulated in response to an increase in cellular H(2)O(2) and ethylene. GhAPX1 encodes a functional enzyme that is involved in hydrogen peroxide homeostasis during cotton fibre development.

Genetic and biochemical studies in yeast reveal that the cotton fibre-specific GhCER6 gene functions in fatty acid elongation.

3-ketoacyl-CoA synthase catalyses the initial condensation reaction during fatty acid elongation using malonyl-CoA and long-chain acyl-CoA as substrates. Previously, it was reported that several genes encoding putative cotton 3-ketoacyl-CoA synthases were significantly up-regulated during early cotton fibre development. In this study, GhCER6 cDNA that contains an open reading frame of 1479 bp, encoding a protein of 492 amino acid residues homologous to the Arabidopsis condensing enzyme CER6, was isolated and cloned. In situ hybridization results demonstrated that GhCER6 mRNA was detected only in the elongating wild-type cotton fibre cells. When GhCER6 was transformed to the Saccharomyces cerevisiae elo3 deletion mutation strain that was deficient in the production of 26-carbon fatty acids and displayed a very slow-growth phenotype, the mutant cells were found to divide similarly compared with those of the wild-type cells. Further, heterologous expression of GhCER6 restored the viability of the S. cerevisiae haploid elo2 and elo3 double-deletion strain. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that GhCER6 was enzymatically active since the yeast elo2 and elo3 double-deletion mutant expressing the cotton gene produced very-long-chain fatty acids that are essential for cell growth. The results suggest that GhCER6 encodes a functional 3-ketoacyl-CoA synthase.

Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation.

Upland cotton (Gossypium hirsutum) produces the most widely used natural fibers, yet the regulatory mechanisms governing fiber cell elongation are not well understood. Through sequencing of a cotton fiber cDNA library and subsequent microarray analysis, we found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation. The 1-Aminocyclopropane-1-Carboxylic Acid Oxidase1-3 (ACO1-3) genes responsible for ethylene production were expressed at significantly higher levels during this growth stage. The amount of ethylene released from cultured ovules correlated with ACO expression and the rate of fiber growth. Exogenously applied ethylene promoted robust fiber cell expansion, whereas its biosynthetic inhibitor l-(2-aminoethoxyvinyl)-glycine (AVG) specifically suppressed fiber growth. The brassinosteroid (BR) biosynthetic pathway was modestly upregulated during this growth stage, and treatment with BR or its biosynthetic inhibitor brassinazole (BRZ) also promoted or inhibited, respectively, fiber growth. However, the effect of ethylene treatment was much stronger than that of BR, and the inhibitory effect of BRZ on fiber cells could be overcome by ethylene, but the AVG effect was much less reversed by BR. These results indicate that ethylene plays a major role in promoting cotton fiber elongation. Furthermore, ethylene may promote cell elongation by increasing the expression of sucrose synthase, tubulin, and expansin genes.