Comparative proteomic and biochemical analyses reveal different molecular events occurring in the process of fiber initiation between wild-type allotetraploid cotton and its fuzzless-lintless mutant

Abstract

To explore lint fiber initiation-related proteins in allotetraploid cotton (Gossypium hirsutum L.), a comparative proteomic analysis was performed between wild-type cotton (Xu-142) and its fuzzless-lintless mutant (Xu-142-fl) at five developmental time points for lint fiber initiation from -3 to +3 days post-anthesis (dpa). Using two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS) analyses, 91 differentially accumulated protein (DAP) species that are related to fiber initiation were successfully identified, of which 58 preferentially accumulated in the wild-type and 33 species in the fl mutant. These DAPs are involved in various cellular and metabolic processes, mainly including important energy/carbohydrate metabolism, redox homeostasis, amino acid and fatty acid biosynthesis, protein quality control, cytoskeleton dynamics, and anthocyanidin metabolism. Further physiological and biochemical experiments revealed dynamic changes in the carbohydrate flux and H2O2 levels in the cotton fiber initiation process. Compared with those in the fl mutant, the contents of glucose and fructose in wild-type ovules sharply increased after anthesis with a relatively higher rate of amino acid biosynthesis. The relative sugar starvation and lower rate of amino acid biosynthesis in the fl mutant ovules may impede the carbohydrate/energy supply and cell wall synthesis, which is consistent with the proteomic results. However, the H2O2 burst was only observed in the wild-type ovules on the day of anthesis. Cotton boll injection experiments in combination with electron microscope observation collectively indicated that H2O2 burst, which is negatively regulated by ascorbate peroxidases (APx), plays an important role in the fiber initiation process. Taken together, our study demonstrates a putative network of DAP species related to fiber initiation in cotton ovules and provides a foundation for future studies on the specific functions of these proteins in fiber development.

Fig 1. Scanning Electron Microscopy (SEM) images and representative 2-DE maps of the total proteins that were extracted from the cotton ovules of Xu-142 wild-type (wt) and its fiber-less mutant (fl).

(A) SEM images of the cotton ovular surfaces in the fiber initiation stages at -3 (a, f), -1 (b, g), 0 (c, h), +1 (d, i) and +3 (e, j) dpa from wt (a-e) and fl mutant (f-j). The scale bar is 30 μm for all of the images. (B) The representative 2-DE maps of the total proteins from +3 dpa cotton ovules of the wt and fl mutant. A total of 91 differentially accumulated protein spots (DAPs) were displayed on the gels. The spots that predominately accumulated in the wild-type (w1–w58) are presented on the “wt” gel, while those that preferentially accumulated in the mutant (m1–m33) are shown on the “fl” gel. (C) Representative differentially accumulated protein spots and their volume% value in the 2-DE gels of the wt and fl mutant cotton ovules. The volume% value (y-axes) of each spot was calculated by three independent experiments.

Fig 2. Functional categorization and isoform analyses of the 91 identified differentially accumulated proteins.

(A) Functional classification of the 91 identified differentially accumulated proteins. Abbreviations: EM, energy/carbohydrate metabolism; RH, redox homeostasis; AB, amino acid biosynthesis; PF, protein folding and stabilization; PS, protein synthesis; CT, cytoskeleton; DR, defense responses; FB, fatty acid biosynthesis; NM, nucleotide metabolism; AM, anthocyanidin metabolism; ST, signaling transduction; NT, nucleocytoplasmic transport; and U, unclassified proteins. (B) Venn diagram analysis of the 91 identified DAPs in this study and the 46 DAPs that were reported in Liu et al. (C) Possible isoforms as revealed by the 2-DE proteomic analyses and MS identification. Protein species due to the same protein accession number were mapped on the 2-DE gels together to show the possible isoforms. (D) Signal peptide of the GhsAPx protein. The possible mature sequence after signal peptide cleavage is shown in the black box. (E) GhsAPx-GFP fusion protein was located in the chloroplasts of cotton seedling leaf cells. (F) N-terminal sequencing of the spot w3 GhsAPx protein. The first six amino acids of w3 were “AASDPD”, which is identical to those of m7.

Fig 3. Carbohydrate metabolism is highly related to fiber initiation.

(A) Sucrose synthase (w58) differentially accumulated in the wt and fl mutant cotton ovules. The bars indicate the log-transformed values of the fold-change ratios of differentially accumulated proteins at five stages. (B) Carbohydrate contents as determined by an enzymatic method. The results are the mean values from three independent experiments. Statistical analyses were performed using student’s T test. Abbreviations: *, p<0.05; **, p<0.01.

Fig 4. Overview of the differently accumulated proteins that are associated with amino acid biosynthesis and protein turnover.

(A) Amino acid biosynthesis pathways indicating the enzymes that were identified as differentially accumulated in the wt and fl mutant cotton ovules. (B) Protein synthesis, folding and degradation pathways indicating the proteins that were identified as differentially accumulated in the wt and fl mutant cotton ovules. The bars indicate the log-transformed values of the fold-change ratios of differentially accumulated proteins at five stages, with those that accumulated in the wt charted in purple and those that accumulated in the fl mutant charted in blue.

Fig 5. Ascorbate-glutathione cycle is differentially regulated during the fiber initiation process in the wt and fl mutant cotton ovules.

(A) Ascorbate-glutathione cycle indicating the enzymes that were identified as differentially accumulated in wt and fl mutant cotton ovules. The bars indicate the log-transformed values of the fold-change ratios of differentially accumulated proteins at five stages, with those that accumulated in the wt charted in purple and those that accumulated in the fl mutant charted in blue. Abbreviations: SOD, superoxide dismutases; CAT, catalase; APx, ascorbate peroxidase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase. (B) Relative activities of APx isozymes with different subcellular locations in early-developmental cotton ovules. The enzyme activity of wt ovules at -3 dpa was normalized to 1. (C) Relative activity of DHAR in early-developmental cotton ovules. The enzyme activities of wt ovules at -3 dpa were normalized to 1. (D) The ratio of ascorbate/dehydroascorbate in the ovules of the wt and fl mutant. The results are the mean values from three independent experiments. The results of fl mutant were compared with the wt using student’s T test. Abbreviations: *, p<0.05; **, p<0.01; sAPx, chloroplast stromal APx; and cAPx, cytosolic APx.

Fig 6. H₂O₂ could promote cotton fiber initiation.

(A) H₂O₂ contents as determined in fertilized (solid lines) and unfertilized (dashed lines) cotton ovules of the wild-type and fl mutant. (B) Scanning electron micrographs of the ovules without or with fertilization blocked or the unfertilized ovules that were treated with 0.5 mM or 2.0 mM H₂O₂. The scale bar was 10 μm for all of the images. The number (C) and diameter (D) of the initiation bubbles on the surface of the ovules at 0 dpa were calculated. The results were compared with the unfertilized ovules using student’s T test. Abbreviations: *, p<0.05; **, p<0.01.

Fig 7. A putative network of differentially accumulated protein species relating to fiber initiation in cotton ovules.

Only the differentially accumulated proteins were mapped with line drawings of the abundance changes, which are depicted in purple histograms for the wild-type and blue for the mutant. The dotted line indicates that the pathway has not yet been confirmed in cotton. Detailed descriptions are given in the text. Abbreviations: PPP, pentose phosphate pathway.