Quantitative phosphoproteomic profiling of fiber differentiation and initiation in a fiberless mutant of cotton

Background

The cotton (Gossypium spp.) fiber cell is an important unicellular model for studying cell differentiation. There is evidence suggesting that phosphorylation is a critical post-translational modification involved in regulation of a wide range of cell activities. Nevertheless, the sites of phosphorylation in G. hirsutum and their regulatory roles in fiber cell initiation are largely unknown. In this study, we employed a mass spectrometry-based phosphoproteomics to conduct a global and site-specific phosphoproteome profiling between ovules of a fuzzless-lintless (fl) Upland cotton (G. hirsutum) mutant and its isogenic parental wild type (WT) at -3 and 0 days post-anthesis (DPA).

Results

A total of 830 phosphopeptides and 1,592 phosphorylation sites from 619 phosphoproteins were identified by iTRAQ (isobaric tags for relative and absolute quantitation). Of these, 76 phosphoproteins and 1,100 phosphorylation sites were identified for the first time after searching the P³DB public database using the BLAST program. Among the detected phosphopeptides, 69 were differentially expressed between the fl mutant and its WT in ovules at -3 and 0 DPA. An analysis using the Motif-X program uncovered 19 phosphorylation motifs, 8 of which were unique to cotton. A further metabolic pathway analysis revealed that the differentially phosphorylated proteins were involved in signal transduction, protein modification, carbohydrate metabolic processes, and cell cycle and cell proliferation.

Conclusions

Our phosphoproteomics-based research provides the first global overview of phosphorylation during cotton fiber initiation, and also offers a helpful dataset for elucidation of signaling networks in fiber development of G. hirsutum.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-466) contains supplementary material, which is available to authorized users.     

Protein expression changes during cotton fiber elongation in response to low temperature stress

Low temperature stress is one of the major abiotic stresses limiting the formation of cotton (Gossypium hirsutum L.) fiber qualities, especially fiber length. To investigate the molecular adaptation mechanisms of cotton fiber elongation to low temperature stress, two cotton cultivars, Kemian 1 (low temperature-tolerant) and Sumian 15 (low temperature-sensitive), were planted in the field at two sowing dates (25 April and 10 June). The two sowing dates resulted in different growing conditions and the main environmental difference between them was temperature, particularly the mean daily minimum temperature (MDTmin). When the sowing date was delayed, the MDTmin decreased from 26.9 °C (25 April) to 20.6 °C (10 June). Low temperature stress (MDTmin of 20.6 °C) shortened the fiber length significantly in two cultivars, but the decreased extent was larger in Sumian 15 than that in Kemian 1. Proteomic analysis of three developmental stages (10, 15 and 20 days post-anthesis [DPA]) showed that 37 spots changed significantly (p < 0.05) in abundance under low temperature stress and they were identified using mass spectrometry. These proteins were involved in malate metabolism, soluble sugar metabolism, cell wall loosening, cellulose synthesis, cytoskeleton, cellular response, and redox homeostasis. The results suggest that the enhancement of osmoticum maintenance, cell wall loosening, cell wall components biosynthesis, and cytoskeleton homeostasis plays important roles in the tolerance of cotton fibers to low temperature stress. Moreover, low levels of PEPCase, expansin, and ethylene signaling proteins may potentially lead to the low temperature sensitivity of Sumian 15 at the proteomic level.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80 kb (cgAUS1) and 1.85 kb (cgAUS2a, 2b), encoding for proteins of 68–69 kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9 kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS–PAGE and shot-gun type nanoUHPLC–ESI-MS/MS.     

Role of GhHDA5 in H3K9 deacetylation and fiber initiation in Gossypium hirsutum

Cotton fibers are single‐celled trichomes that initiate from the epidermal cells of the ovules at or before anthesis. Here, we identified that the histone deacetylase (HDAC ) activity is essential for proper cotton fiber initiation. We further identified 15 HDAC s homoeologs in each of the A‐ and D‐subgenomes of Gossypium hirsutum . Few of these HDAC homoeologs expressed preferentially during the early stages of fiber development [?1, 0 and 6 days post‐anthesis (DPA )]. Among them, GhHDA 5 expressed significantly at the time of fiber initiation (?1 and 0 DPA). The in vitro assay for HDAC activity indicated that GhHDA 5 primarily deacetylates H3K9 acetylation marks. Moreover, the reduced expression of GhHDA 5 also suppresses fiber initiation and lint yield in the RNA interference (RNA i) lines. The 0 DPA ovules of GhHDA 5 RNA i lines also showed alterations in reactive oxygen species homeostasis and elevated autophagic cell death in the developing fibers. The differentially expressed genes (DEG s) identified through RNA ‐seq of RNA i line (DEP 12) and their pathway analysis showed that GhHDA 5 modulates expression of many stress and development‐related genes involved in fiber development. The reduced expression of GhHDA 5 in the RNA i lines also resulted in H3K9 hyper‐acetylation on the promoter region of few DEG s assessed by chromatin immunoprecipitation assay. The positively co‐expressed genes with GhHDA 5 showed cumulative higher expression during fiber initiation, and gene ontology annotation suggests their involvement in fiber development. Furthermore, the predicted protein interaction network in the positively co‐expressed genes indicates HDA 5 modulates fiber initiation‐specific gene expression through a complex involving reported repressors.     

Validation of a novel in vitro assay using ultra performance liquid chromatography–mass spectrometry (UPLC/MS) to detect and quantify hydroxylated metabolites of BDE-99 in rat liver microsomes

The purpose of this study was to develop and validate an ultra performance liquid chromatography–mass spectrometry (UPLC/MS) method to investigate the hepatic oxidative metabolism of 2,2′,4,4′,5-pentabromodiphenyl ether (BDE-99), a widely used flame retardant and ubiquitous environmental contaminant. Hydroxylated metabolites were extracted using liquid-to-liquid extraction, resolved on a C₁₈ column with gradient elution and detected by mass spectrometry in single ion recording mode using electrospray negative ionization. The assay was validated for linearity, accuracy, precision, limit of quantification, range and recovery. Calibration curves were linear (R² ≥ 0.98) over a concentration range of 0.010–1.0 μM for 4-OH-2,2′,3,4′,5-pentabromodiphenyl ether (4-OH-BDE-90), 5′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (5′-OH-BDE-99) and 6′-OH-2,2′,4,4′,5-pentabromodiphenyl ether (6′-OH-BDE-99), and a concentration range of 0.0625–12.5 μM for 2,4,5-tribromophenol (2,4,5-TBP). Inter- and intra-day accuracy values ranged from −2.0% to 6.0% and from −7.7% to 7.3%, respectively, and inter- and intra-day precision values ranged from 2.0% to 8.5% and from 2.2% to 8.6% (n = 6), respectively. The limits of quantification were 0.010 μM for 4-OH-BDE-90, 5′-OH-BDE-99 and 6′-OH-BDE-99, and 0.0625 μM for 2,4,5-TBP. Recovery values ranged between 85 and 100% for the four analytes. The validated analytical method was applied to identify and quantify hydroxy BDE-99 metabolites formed in vitro. Incubation of BDE-99 with rat liver microsomes yielded 4-OH-BDE-90 and 6′-OH-BDE-99 as major metabolites and 5′-OH-BDE-99 and 2,4,5-TBP as minor metabolites. To our knowledge, this is the first validated UPLC/MS method to quantify hydroxylated metabolites of PBDEs without the need of derivatization.     

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR^−/−) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR^−/− mice fed MCD diet (FXR^−/−/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR^−/−/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR^−/−/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR^−/−/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.     

Homologous expression of γ-glutamylcysteine synthetase increases grain yield and tolerance of transgenic rice plants to environmental stresses

Various environmental stresses induce reactive oxygen species (ROS), causing deleterious effects on plant cells. Glutathione (GSH), a critical antioxidant, is used to combat ROS. GSH is produced by γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the Oryza sativa L. Japonica cv. Ilmi ECS (OsECS) gene, we generated transgenic rice plants overexpressing OsECS under the control of an inducible promoter (Rab21). When grown under saline conditions (100 mM) for 4 weeks, 2-independent transgenic (TGR1 and TGR2) rice plants remained bright green in comparison to control wild-type (WT) rice plants. TGR1 and TGR2 rice plants also showed a higher GSH/GSSG ratio than did WT rice plants in the presence of 100 mM NaCl, which led to enhanced redox homeostasis. TGR1 and TGR2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence when exposed to 10 μM methyl viologen (MV). Furthermore, the TGR1 and TGR2 rice seeds had approximately 1.5-fold higher germination rates in the presence of 200 mM salt. Under paddy field conditions, OsECS-overexpression in transgenic rice plants increased rice grain yield (TGW) and improved biomass. Overall, our results show that OsECS overexpression in transgenic rice increases tolerance and germination rate in the presence of abiotic stress by improving redox homeostasis via an enhanced GSH pool. Our findings suggest that increases in grain yield by OsECS overexpression could improve crop yields under natural environmental conditions.     

Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H⁺)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.