| Cotton is an important economic crop and is used as a major source of natural textile fiber and cottonseed oil. Cotton fibers are single-celled trichomes from individual epidermal cells on the outer intrgument of the ovules. Fiber development includes four distinct, but overlapping stages:initiation, elongation/primary cell wall (PCW) synthesis, secondary cell wall (SCW) deposition and maturation. Mutants are powerful tools to elucidate the molecular mechanisms of important genes associate with fiber development. In cotton, some important fiber mutants have been obtained. The XinWX is a cotton fiber mutant,which regarded to result in defective fiber initiation and the seeds were lintless and fuzzless. The XinFLM was found from XinWX which’s seeds were found with lint but no fuzz. Genetic analysis revealed that XinFLM was a dominant mutant from XinWX and they had only one different locus in the development of lint. In this study, we used XinWX and XinFLM as research materials. We performed a comparative analysis of transcriptional profile between XinWX and XinFLM during fiber initiation development using cDNA microarrays to identify some genes or pathways which play an important role in fiber initiation. The majory results are listed as following:1. XinWX and XinFLM showed no phenotypic differences except for the mature bolls. The seeds of XinFLM were found with lint but no fuzz, and XinWX were lintless and fuzzless. Scanning electron microscopy (SEM) showed there were no protrusions in the whole ovule and flanking surface in XinWX from-1 DPAto 1 DPA. In XinFLM, before the day of anthesis, no spherical protrusions were found on the whole ovule and’flanking surface, but on the day of anthesis, the whole ovule and flank surface was covered in many spherical protrusions. At 1 DPA, the numbers of protrusions in the ovule were much more, and some of which were began to elongate. These results indicated XinWX is a cotton fiber mutant, and spherical protrusions in XinFLM were began at 0 DPA.2. In order to explore the mechanism of fiber initiation, we employed cotton cDNA microarray which contains 29,184 probes to illustrate the variation of transcription profiling within and between XinWX and XinFLM at-1,0 and 1 DPA. The numbers of differentially expressed genes (DEGs) between adjacent time points during fiber initiation within and between the two lines were determined. The distribution of DEGs with a false discovery rate (FDR)< 0.05 and fold change> 2 showed that the level of transcriptional variation between adjacent time points in XinFLM was much higher than that in XinWX, indicating that much genes were involved in fiber initiation in XinFLM. The numbers of varied genes between XinWX and XinFLM were 834,357,1000, respectively from-1 to 1 DPA. We found that the number of DEGs in 0 DPA is less than that of-1 DPA and 1 DPA. Integrated with the results of SEM study, indicated that genes differentially expressed at 1 DPA may be involved in fiber elongation3. The GO annotation for all identified DEGs and enrichment analysis indicated that the oxidation-reduction processes, carbohydrate metabolic processes, lipid transport, hormone synthesis and metabolism related processes were enriched within and between XinWX and XinFLM. This implyed these pathways play critical role in cotton fiber initiation development. And then, we found many transcription factors involved in these pathways. Among them,12 ERFs have the same expression profiles with predominant expression in XinFLM at-1 or 0 DPA. QRT-PCR verified these results, suggested ERFs may have positive effect on fiber initiation. Expression analysis of hormone synthesis and metabolism related DEGs showed that AOC, which are key enzymes in JA biosynthesis, are co-upregulated in XinWX, suggesting a negative effect on progression of fiber initiation. We also found that all of the BR related genes in the array are co-upregulated in XinFLM. This is suggested that BR may play a critical role in cotton fiber initiation.4. Enrichment analysis showed the AOCs were co-upregulated in XinWX. To elaborated the relationship between AOCs and fiber initiation, we used the recently released cotton D genome sequence information as reference and combined with conserved domain of GhAOC which cloned before, and got four AOCs (Gorai.004G046300, Gorai.008Gl 85600, Gorai.004G046400, Gorai.004G046100) in G. raimondii. Except for GhAOC (corresponding to Gorai.004G046100), the other three AOCs in TM-1 were named as GhAOC2, GhAOC3, and GhAOC4, respectively, corresponding to Gorai.004G046300, Gorai.004G046400, and Gorai.008G185600. Expression patterns of the four AOCs in different tissues and organs of TM-1 showed that they were all expressed preferentially in fiber tissues, especially at -1 DPA. This suggested that AOC may involved in fiber initiation. To further confirm the AOCs role in fiber initiation stage, the expression levels during fiber initiation (from -3 to 3 DPA) using different fiber mutant lines were investigated. The QRT-PCR analysis showed that AOCs had similar expression profiles with higher expression levels in lintless-fuzzless than that in linted-fuzzless and linted-fuzzy, especially at -1 DPA. These results, in combination with expression data from different tissues and organs in TM-1, suggested that some up-regulation of AOCs was required for normal fiber initiation, but excessive overproduction of AOCs may lead to a suppressive effect.5. Previously reported genes encoding enzymes involved in JA biosynthesis and JA level exist feed-forward regulation. In order to investigate the relationship between expression of AOCs and JA in cotton, living and ovule in vitro were treated with different concentrations of JA. QRT-PCR showed the four AOCs had similar expression profile and were significantly up-regulated after JA treatment for 4h, and continued to 12h with a peak at 8 h of treatment. Indicating JA can significantly induce AOCs expression. We speculated that during fiber initiation, increased JA levels induced higher expression levels of AOCs, while the AOCs further regulated the dynamic equilibrium of JA. To determine whether JA biosynthesis was co-regulated during fiber initiation, seven genes that were differentially expressed in XinWX and XinFLM believed to be involved in JA biosynthesis were subjected to QRT-PCR analysis. The results showed that they all up-regulated in XinWX at-1 DPA, which is like with the expression profiles of AOCs. These results suggested that the JA biosynthesis pathway is up-regulated during fiber initiation in the fuzzless-lintless mutant, which results in overproduction of JA in XinWX compared to XinFLM. To confirm the effect of JA on fiber initiation,-2 DPA ovules were treated with JA in vitro. All the ovules treated with JA exhibited fewer fiber initiations than the controls. Many initiated fibers were broken in ovules after treatment with 0.1 μM JA. The fewer fiber initiations were detected after treatment with 0.5 μM JA, and no initiations were found after treatment with 2.5 μM JA. All of the results indicated that JA involved in fiber initiation, but overproduction of JA may suppress fiber development, and this inhibition has dosage effect. |
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