Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Ma Q

PROVIDER: S-EPMC4070576 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Ma Qifeng Q Wu Man M Pei Wenfeng W Li Haijing H Li Xingli X Zhang Jinfa J Yu Jiwen J Yu Shuxun S

BMC genomics 20140612

<h4>Background</h4>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 glo ...[more]

PMID: 24923550

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Project description:BackgroundCotton is the most essential textile crop worldwide, and phytohormones are critical for cotton fiber development. One example is the role of auxin in fiber initiation, but we know little molecular basis. MicroRNAs (miRNAs) have a significant function in cotton development; nevertheless their role in fiber initiation remains unclear. Here, exogenous IAA was applied to cotton plant before anthesis. Utilizing small RNA sequencing, the mechanism underlying miRNA-mediated regulation of fiber initiation under exogenous IAA treatment was investigated.ResultsWith exogenous IAA application, the endogenous IAA and GA contents of IAA treated (IT) ovules were higher than control (CK) ovules at the fiber initiation stage, while endogenous ABA content was lower in IT than CK. Using scanning electron microscopy, we found the fiber number and size were significantly promoted in IT at 0 DPA. Fiber quality analysis showed that fiber length, uniformity, strength, elongation, and micronaire of IT were higher than CK, though not statistically significant, while lint percent was significantly higher in IT. We generated six small RNA libraries using - 3, 0, and 3 DPA ovules of IT and CK, and identified 58 known miRNAs and 83 novel miRNAs together with the target genes. The differential expressed miRNAs number between IT and CK at - 3, 0, 3 DPA was 34, 16 and 24, respectively. Gene ontology and KEGG pathway enrichment analyses for the target genes of the miRNAs expressed in a differential manner showed that they were significantly enriched in 30 terms and 8 pathways. QRT-PCR for those identified miRNAs and the target genes related to phytohormones and fiber development was performed, and results suggested a potential role of these miRNAs in fiber initiation.ConclusionsThe exogenous IAA application affected the relative phytohormone contents in ovule and promoted fiber initiation in cotton. Identification and profiling of miRNAs and their targets at the fiber initiation stage provided insights for miRNAs' regulation function of fiber initiation. These findings not only shed light on the regulatory network of fiber growth but also offer clues for cotton fiber amelioration strategies in cotton.

Project description:We constructed cDNA library from 0-10 day post anthesis cotton fibers when fiber cells begin to initiate and elongate throughout this peroid. We randomly sequenced over 95,000 ESTs from this library, and with request of upland cotton ESTs from other laboratories, we acquired a gene pool of more than 30,000 UniESTs. The cotton UniESTs were then PCR-amplified and printed onto microarray. This array is comprised of about 29,000 high-quality cotton cDNAs (each sequence length>400bp, average length > 600bp) and external controls. Data for 8 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt0-Cy5 wt-3-Cy3 Set 1 (dye swap): wt-3-Cy3 wt0-Cy5 Set 2: wt0-Cy5 wt-3-Cy3 Set 2 (dye swap): wt-3-Cy3 wt0-Cy5 Set 3: wt0-Cy5 wt-3-Cy3 Set 3 (dye swap): wt-3-Cy3 wt0-Cy5 Set 4: wt0-Cy5 wt-3-Cy3 Set 4 (dye swap): wt-3-Cy3 wt0-Cy5 GSM209619, GSM209620, GSM209621, GSM209622, GSM209623, GSM209624, GSM209625, GSM209626, Data for 8 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+1-Cy5 wt-3-Cy3 Set 1 (dye swap): wt-3-Cy3 wt+1-Cy5 Set 2: wt+1-Cy5 wt-3-Cy3 Set 2 (dye swap): wt-3-Cy3 wt+1-Cy5 Set 3: wt+1-Cy5 wt-3-Cy3 Set 3 (dye swap): wt-3-Cy3 wt+1-Cy5 Set 4: wt+1-Cy5 wt-3-Cy3 Set 4 (dye swap): wt-3-Cy3 wt+1-Cy5 GSM209627, GSM209628, GSM209629, GSM209630, GSM209631, GSM209632, GSM209633, GSM209634, Data for 8 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+2-Cy5 wt-3-Cy3 Set 1 (dye swap): wt-3-Cy3 wt+2-Cy5 Set 2: wt+2-Cy5 wt-3-Cy3 Set 2 (dye swap): wt-3-Cy3 wt+2-Cy5 Set 3: wt+2-Cy5 wt-3-Cy3 Set 3 (dye swap): wt-3-Cy3 wt+2-Cy5 Set 4: wt+2-Cy5 wt-3-Cy3 Set 4 (dye swap): wt-3-Cy3 wt+2-Cy5 GSM209635, GSM209636, GSM209637, GSM209638, GSM209639, GSM209640, GSM209641, GSM209642, Data for 8 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: wt+3-Cy5 wt-3-Cy3 Set 1 (dye swap): wt-3-Cy3 wt+3-Cy5 Set 2: wt+3-Cy5 wt-3-Cy3 Set 2 (dye swap): wt-3-Cy3 wt+3-Cy5 Set 3: wt+3-Cy5 wt-3-Cy3 Set 3 (dye swap): wt-3-Cy3 wt+3-Cy5 Set 4: wt+3-Cy5 wt-3-Cy3 Set 4 (dye swap): wt-3-Cy3 wt+3-Cy5 GSM209643, GSM209644, GSM209645, GSM209646, GSM209647, GSM209648, GSM209649, GSM209650, Data for 6 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: fl+3-Cy5 wt-3-Cy3 Set 1 (dye swap): wt-3-Cy3 fl+3-Cy5 Set 2: fl+3-Cy5 wt-3-Cy3 Set 2 (dye swap): wt-3-Cy3 fl+3-Cy5 Set 3: fl+3-Cy5 wt-3-Cy3 Set 3 (dye swap): wt-3-Cy3 fl+3-Cy5 GSM209455, GSM209456, GSM209608, GSM209609, GSM209610, GSM209612, Data for 6 replicate experiments performed with biologically independent samples. Cy3 and Cy5 dyes were used as follows: Set 1: fl-3-Cy5 wt-3-Cy3 Set 1 (dye swap): wt-3-Cy3 fl-3-Cy5 Set 2: fl-3-Cy5 wt-3-Cy3 Set 2 (dye swap): wt-3-Cy3 fl-3-Cy5 Set 3: fl-3-Cy5 wt-3-Cy3 Set 3 (dye swap): wt-3-Cy3 fl-3-Cy5 GSM209613, GSM209614, GSM209615, GSM209616, GSM209617, GSM209618

Project description:BACKGROUND: Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points. RESULTS: Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene. CONCLUSIONS: The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Project description:Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Within the molecular scope of NCSLC, a complex landscape of dysregulated cellular signaling has emerged, defined largely by mutations in select mediators of signal transduction, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma (ALK) kinases. Consequently, these mutant kinases become constitutively activated and targets for chemotherapeutic intervention. Encouragingly, small molecule inhibitors of these pathways have shown promise in clinical trials or are approved for clinical use. However, many protein kinases are dysregulated in NSCLC without genetic mutations. To quantify differences in tumor cell signaling that are transparent to genomic methods, we established a super-SILAC internal standard derived from NSCLC cell lines grown in vitro and labeled with heavy lysine and arginine, and deployed them in a phosphoproteomic workflow. We identified 9019 and 8753 phosphorylation sites in two separate tumors. Relative quantification of phosphopeptide abundance between tumor samples allowed for the determination of specific hubs and pathways differing between each tumor. Sites downstream of Ras showed decreased inhibitory phosphorylation (Raf/Mek) and increased activating phosphorylation (Erk1/2) in one tumor versus another. In this way, we were able to quantitatively access oncogenic kinase signaling in primary human tumors.Through the use of quantitative proteomics, we demonstrated the feasibility and coverage that large scale mass spectrometry can leverage for understanding kinase networks in cancer. By incorporating Super-SILAC based quantitation into a typical pathology workflow, we were able to access and compare tumors from multiple patients in this analysis with high accuracy and dynamic range. We analyzed tumors from patients diagnosed with non-small cell lung cancer and were able to detect comprehensive phosphorylation networks relaying through known hubs of oncogenesis in lung cancer. We hereby show that it is possible to track changes to phosphorylation networks across multiple tumors, opening up the possibility that drug susceptibility and patient-specific stratification can be implemented downstream of classical pathology.

Dataset Information

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

Background

Results

Conclusions

Publications

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

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