Transcriptome analysis reveals differences in the mechanisms of fiber initiation and elongation between long- and short-fiber cotton (Gossypium hirsutum L.) lines

- Transcriptome analysis reveals differences in the mechanisms of fiber initiation and elongation between long- and short-fiber cotton ( Gossypium hirsutum L.) lines.
- Comparison of the LFG and SFG transcriptomes revealed a total of 3538 differentially expressed genes (DEGs).
- Notably, at all three developmental stages examined, two expression patterns, consistently downregulated (profile 0) and consistently upregulated (profile 7), were identified, and both were significantly enriched in the SFG and LFG.
- Functional annotation suggested that these DEGs, which included ERF1, TUA2, TUB1, and PER64, affect fiber elongation by participating in the ethylene response, microtubule synthesis, and/or the peroxidase (POD) catalytic pathway.
- Among all the cultivated species, upland cotton ( Gossypium hirsutum L.) is widely planted and ac- counts for more than 95% of the global cotton production..
- Identification of the genes involved in fiber initiation and elucidation of the fiber de- velopment mechanism are hard.
- When the expression of the R2R3 MYB transcription factor GhMYB109 was inhibited, fiber initiation was delayed, indicating that this gene is required in the initial development of fibers [15].
- Over- expression of GhMYB25 resulted in increases in the numbers of cotton fibers and leaf epidermal hairs, while gene silencing led to opposite results [16].
- By activating the function of the GhCaM7 gene, Ca 2+ plays key roles in promoting cotton fiber elongation, while ROS has an opposite effect in this path- way [23].
- In the present study, we observed seed phenotypes at the fiber initiation stage, measured the length of developing fibers between short- and long-fiber cotton lines, and confirmed the difference in fiber quality..
- Three ovules were randomly selected from each material, and the number of bulged cells within a unit area of 0.02 mm 2 (0.1 mm × 0.2 mm) in the middle of the surface was counted.
- Fiber elongation.
- The data presented in the table are the average values.
- Fiber developmental stage: The numbers in the table indicate the days post-anthesis (DPA).
- Numbers in the same color indicate that their corresponding materials and tissues were mixed equally according to RNA mass.
- The detailed calculation methods and parameter settings involved in the above ana- lysis are described in Additional file 1..
- All primer pairs used in the ana- lysis and their amplification efficiencies are shown in Additional file 2..
- The fiber phenotypes of the SFG Liao 1779) and LFG (601 LSC, J02–508) were different at the initiation, elongation and maturation stages.
- The average cell number of the four materials, from high to low, was in the order of Liao 1779, J02–508 and 601 LSC.
- i.e., the initial fiber numbers in the SFG were greater than those in the LFG..
- i Statistical results of the initial fiber number per unit area at 0 DPA.
- j Statistical results of the initial fiber number per unit area at 1 DPA.
- The height of the column is the average of three counts, and the error bars indicate ± S.D.
- The columns in the graph are arranged from large to small.
- The letters above the columns indicate the significance level of the initial fiber numbers of the four cotton materials.
- After fiber maturation, the FL of the four materials was measured.
- The difference be- tween the SFG and the LFG was extremely significant ac- cording to the results of the t-test.
- Phenotypic observations at each developmental stage suggest that significant differences in the final fiber.
- quality of the four materials were formed by the gradual accumulation of differences during fiber initiation and elongation.
- The ratio of clean reads to the total number of raw reads was 94.94 to 96.44% (Additional file 3), indicating that the proportion of low-quality data in the raw reads was very low.
- The gene expres- sion became increasingly dissimilar to that of the lint fiber initiation stage (SFG-0 and LFG-0) in the subse- quent two stages in both the SFG (SFG-5 and SFG-F10) and the LFG (LFG-5 and LFG-10) (Additional file 5)..
- the fibers and ovules in the previous two stages not sep- arating.
- Further confirming the relationship between the two groups at different developmental stages, the PCA showed that the same developmental stage of the four materials clustered together, indicating the difference be- tween developmental stages was greater than that be- tween materials (Additional file 6)..
- The expression patterns of the DEGs among three devel- opmental stages (0, 5, F10) (Additional file 7) in the SFG and LFG were analyzed (Fig.
- In the SFG, most DEGs were enriched in profile 0 (4974 genes, accounting for 26.00% of 19128 DEGs), followed by profile 6 (3576 genes, 18.70%) and profile 7 (2914 genes, 15.23.
- In the LFG, most DEGs were enriched in profile 3 (4665 genes, 22.43% of 20795 DEGs), followed by profile 0 (4491 genes, 21.60%) and profile 7 (3317 genes, 15.95%)..
- This finding is consistent with the trend exhibited by profile 0, indicating that DEGs enriched in profile 0 are involved in the regula- tion of fiber initiation.
- 4 Patterns of gene expression and GO enrichment of DEGs across three developmental stages in the SFG and LFG.
- a Patterns of gene expression across three developmental stages in the SFG and LFG inferred by Short Time-series Expression Miner (STEM) analysis.
- b GO enrichment analysis of three significant patterns in the SFG.
- c GO enrichment analysis of three significant patterns in the LFG.
- The significance of the most represented GO terms in each profile is indicated by the p value.
- Four hundred eighty-three more DEGs were enriched in profile 0 in the SFG than in the LFG (4974 vs.
- i.e., more genes were involved in fiber initiation in the SFG than in the LFG.
- Compared with profile 0, profile 7 had 402 fewer DEGs enriched in the SFG than in the LFG (2914 vs.
- i.e., more genes were involved in fiber elongation in the LFG than in the SFG.
- This finding was also consistent with the re- sult by which the FL of the LFG was greater than that of the SFG at 10 DPA (Fig.
- Most gene expression pat- terns during fiber development were similar between the SFG and LFG, but the main difference lied in the num- ber of genes and the function of key genes therein..
- In the SFG, genes within profile 0 were enriched mainly in the cellular macro- molecular metabolic process (GO:0044260, p = 1.05E-02), microtubule-based movement (GO:0007018, p = 1.52E-09), the microtubule-associated complex (GO:0005875, p = 9.45E-10), microtubule motor activity (GO:0003777, p = 1.37E-09), sequence-specific DNA binding (GO:.
- The genes within profile 7 were enriched mainly in the cell wall macromolecule metabolic process (GO:0044036, p = 8.28E-03), intracellu- lar signal transduction (GO:0035556, p = 5.85E-05), poly- meric cytoskeletal fibers (GO:0099513, p = 7.30E-07) and calmodulin binding (GO:0005516, p = 1.66E-03) (Fig.
- In the LFG, the significantly enriched terms in profiles 0 and 7 are roughly consistent with the corresponding pro- files in the SFG, with the exception of some specifically enriched terms, such as the oxidoreductase complex (GO:.
- A total of 196 genes were downregulated in the SFG or LFG and differentially expressed during the fiber initiation stage (Fig.
- There were 152 genes upregulated in the SFG or LFG and differentially expressed during the fiber elong- ation stage (Fig.
- The first type showed a regular pattern in which the expression levels of the DEGs were downregulated, and the expression levels were greater within LFG-0 than within SFG-0 (Fig.
- Therefore, the highly expressed genes in the LFG might have more promoting effects.
- Subsequent gene expression pattern analysis indicated that the DEGs involved in fiber initi- ation in the SFG were more than in the LFG (Fig.
- 2), which was positively correlated with the DEGs involved in fiber elongation in the LFG (Fig.
- Advances in the study of fiber development mechanisms by expression profile sequencing.
- a Venn diagram showing the number of DEGs shared between profile 0 in the SFG and LFG and LFG-0 vs SFG-0.
- b Venn diagram showing the number of DEGs shared between profile 7 in the SFG and LFG and LFG-F10 vs SFG-F10.
- compared the expression levels of 40,430 genes involved in the development of two cot- ton species and found that domestication altered the ex- pression of nearly a quarter of those genes [31].
- Later, the team used transcriptome sequencing to compare and analyze the transcription levels of multiple upland cot- ton cultivars and wild species and found that almost one-third of the genes are expressed in the fibers and that the expression of approximately 5000 genes has changed as a result of domestication [45]..
- i.e., too much or insufficient amounts will inhibit fiber elongation, which is one of the reasons for the differences in FL [32].
- In the present study, an ethylene response element binding factor ( ERF1 ) was identified, and its expression level significantly differed between the two groups (Fig..
- The KEGG enrichment results revealed that this gene plays a role in the ethylene regulatory network of.
- 6a, expression of the ERF1 gene (Gh_D11G0426) was gradually downregulated in both the SFG and LFG but was highly expressed in the LFG..
- This finding indicated that, compared with that in the SFG, the response to ethylene in the LFG was faster and more efficient, which may be the main reason for the longer fibers in 601 LSC and J02–508..
- Microtubules are an integral part of the cytoskeleton and are widespread throughout the cytoplasm.
- Microtubules, an essential structural component of fiber cells, participate in the maintenance of cell structure and form the cyto- skeleton together with microfilaments [50].
- com- pared with that of the control cells, the cell wall surface of these treated cells became wrinkled, and fiber elongation was significantly inhibited [51].
- In an in vitro ovule culture experiment, fiber production significantly decreased in the presence of sulfamethoxazole (a microtubule-disrupting agent) and significantly increased when treated with pacli- taxel (a microtubule-stabilizing agent).
- Comparative proteomic profiling between a short-fiber mutant ( Li1 ) and its wild type revealed that some cytoskeleton-related proteins in the Li1 mutant were significantly reduced and that the actin cytoskeleton structure was severely distorted.
- as a re- sult, vesicle trafficking was therefore blocked, suggesting that the short fibers of the Li1 mutant were associated with tubulin defects [54]..
- Both proteins were expressed at higher levels in the LFG than in the SFG, which provided adequate micro- tubule protein for fiber elongation.
- This finding also ex- plains why the fiber quality was better in the LFG (601 LSC and J02–508) than in the SFG lines..
- Overexpression of the GhCaM7 gene promoted fiber elongation, and the con- centration of ROS in the GhCaM7 -overexpressing line was greater than that in the wild type, indicating that ROS are key regulators involved in fiber elongation [23].
- detected a dramatic increase in ROS on the − 3 and − 2 DPA ovule surfaces in upland cotton fibreless mutants, in- dicating that homeostasis of ROS may play a key role in the regulatory mechanisms of cotton fiber development [56].
- PER64 (Gh_A02G1663) is a member of the POD family of enzymes.
- In the present study, the PER64 gene was highly expressed in the SFG (Fig.
- KEGG enrichment analysis revealed PER64 is involved in the phenylpropanoid biosynthesis process and catalyzes the synthesis of lignin from carbohydrates.
- Therefore, active POD in the SFG may be a reason leading to the inferiority in FL of and Liao 1779..
- The discussion of the genes with known functions above indicates that our RNA-seq results are reliable.
- In this study, differences in the fiber initiation and elong- ation stages between long- and short-fiber cotton lines were determined based on phenotypic observations.
- The developmental dynamics of the cotton fiber transcrip- tomes of these lines was the comparatively analyzed with RNA-seq.
- Gene expression pattern analysis of the DEGs between developmental stages revealed that profile 0 and profile 7 were enriched both in the SFG and LFG..
- Twenty-two genes were identified from profile 0 (related to fiber initiation), in which ERF1 was involved in ethylene metabolism and was expressed at a higher level in the LFG than in the SFG, which affected fiber initiation..
- Thirty-one genes were identified from profile 7 (related to fiber elongation), in which TUA2 and TUB1 were involved in microtubule synthesis and were expressed at a higher level in the LFG than in the SFG, while PER64 , which encoded a POD, was expressed at a higher level in the SFG.
- Therefore, the differen- tial expression of these genes may be the main reason leading to phenotypic differences in the fibers..
- The results of this study have increased the under- standing of relevant metabolic pathways involved in the process of fiber initiation and elongation.
- The dotted line in the figure indicates that FPKM = 1.
- R 2 : the square of the Pearson correlation coefficient.
- The meaning of each letter and number in the sample name is the same as that in Table 2 and Additional file 3.
- In the overall FPKM hierarchical clustering map, red indicates highly expressed genes, and blue indicates genes expressed at low levels.
- The meaning of each character and number in the sample name is the same as that in Additional file 4.
- The funders had no role in the design of the study, the collection, analysis, and interpretation of data, and in writing the manuscript..
- Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis.
- The HD-zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2.
- Comparative evolutionary and developmental dynamics of the cotton (Gossypium hirsutum) fiber transcriptome.
- Changes in the level of tubulin subunits during development of cotton (Gossypium hirsutum) fiber.
- Proteomic identification of differentially expressed proteins in the Ligon lintless mutant of upland cotton (Gossypium hirsutum L

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