| Fiber elongation is the key determinant of fiber quality and output in cotton. Although expression profiling and functional genomics provide some data, the mechanism of fiber development is still not well understood. Here, two genes encoding calcium binding proteins, GhCaM7and GhAnn2were isolated based on their high expression level relative to other GhCaMs and GhAnns, respectively, in fiber cells at the fast elongation stage. Using transgenic and cell biological technology, we had an elaboration on their roles in fiber development. The main results are as follow:1. GhCaM7(TC232366) is expressed to high levels in developing fiber cells.Bioinformatics searches of the DFCI Cotton Gene Index revealed7GhCaM family members coding4predicted GhCaM isoforms in upland cotton. RT-PCR showed that three members were found to be expressed at relatively high levels in fiber cells, especially in the phase of fiber fast elongation. qRT-PCR analysis revealed that TC232366{GhCaM7) had the highest expression level among these three GhCaM genes. Scanning ion-selective electrode analysis showed that the profile of Ca2+influx at the fiber cell tip was similar to the expression profile of GhCaM7. The expression level of GhCaM7in0DPA ovules was lower in the fuzzless/lintless mutants compared with the normal upland cotton lines YZ1and Xuzhou142.2. GhCaM7plays positive role in early fiber elongation.Overexpression of GhCaM7promotes early but not late elongation of fiber cells, whereas GhCaM7suppression by RNAi delays fiber initiation and inhibits fiber elongation. In vitro ovule culture assay showed that when the ovules were cultured in vitro for10days, fiber cells of the GhCaM7overexpression lines were significantly longer than wild-type fiber cells. However, fiber elongation was inhibited significantly in GhCaM7RNAi lines. Moreover, pharmacological experiments revealed that fiber elongation was inhibited by trifluoperazine (TFP, CaM antagonist) in a dose-dependent manner. The data suggested that GhCaM7plays positive roles in fiber early elongation.3. GhCaM7is a positive regulator of ROS during early fiber elongation.H2O2and Ca2+starvation treatments on culturing ovules showed that ROS also plays positive roles in early fiber elongation. By2’,7’-DCFDA staining and imaging and H2O2quantification we found there was more ROS accumulation in GhCaM7OE young fiber cells than that of wild type and RNAi lines, which was also supported by in vitro ovule culture assays, such as H2O2, DPI, TFP treatments on wild type and GhCaM7transgenic ovules. Thus, GhCaM7is a positive regulator of ROS during early fiber elongation and GhCaM7is likely to act upstream of ROS production.4. ROS also has a regulatory effect on the expression of GhCaM7.Scanning ion-selective electrode analysis showed that when treated with20μM H2O2, there was an increasing rate of Ca2+influx at the fiber cell tip. Ovule culture assays showed that ROS could also promote Ca2+release from intracellular calcium pools. According to qRT-PCR analysis, we found the expression of GhCaM7was induced by exogenous20μM H2O2under both normal condition and the treatment of exogenous Ca2+starvation. When there was no exogenous calcium, H2O2could promote the expression level of GhCaM7and fiber elongation, while in the presence of2-APB (calcium pools release channel blocker), H2O2had a negligible effect on the expression level of GhCaM7and fiber elongation. From the results above, we concluded that H2O2could trigger Ca2+signal by increasing Ca2+influx from the extracellular environment, or promoting Ca2+release from intracellular calcium pools. We speculate that this might lead to the Ca2+signal being sensed by GhCaM7and the expression level of GhCaM7being up-regulated.5. GhCaM7Plays a Positive Role in IAA-induced Early Fiber ElongationQRT-PCR analysis showed that the expression of GhCaM7was induced by IAA, consistent with a role for GhCaM7in IAA-induced fiber elongation. Ovule culture assay was then performed to further analyse the relationship between GhCaM7and auxin. When the ovules were cultured in BT medium with NPA or without IAA, there was no significant difference in fiber length among ovules of GhCaM7overexpresser, RNAi and wild-type. Moreover, when the ovules were cultured in BT medium with TFP, H2O2application would not promote fiber elongation well. Therefore, GhCaM7plays a positive role in IAA-induced early fiber elongation.6. GhCaM7and IAA coordinately regulate the production of ROS.Ovules cultural assay showed IAA promotes early fiber elongation partly by inducing ROS production. When ovules were treated with IAA, their ROS level increased, but the ROS signal disappeared when20μM TFP was also applied to the medium. Addition of5μM IAA to the TFP-containing medium was not able to recover the ROS signal very well, indicating that TFP could partly abolish IAA-induced ROS production, further suggesting that CaM plays a positive role in IAA-induced ROS production. In order to confirm this result, GhCaM7transgenic ovules (-1DPA) were collected and treated for5h in BT medium with different concentration of IAA and then2’7’-DCFDA fluorescence dye was loaded. There was no detectable difference in ROS fluorescence between GhCaM7transgenic and wild type-1DPA ovules when treated with5μM NPA, an auxin transport inhibitor. When treated with20μM IAA, GhCaM7overexpresser ovules showed much stronger fluorescence, and RNAi lines showed much weaker fluorescence, than wild type. These results show that GhCaM7and IAA coordinately regulate the production of ROS which plays a positive role in early fiber development.7. GhAnn2is preferentially expressed in fast elongating cotton fibersBioinformatics analysis suggested GhAnn2might come from D genome. Then all the annexins from D genome were found out. RT-PCR and qRT-PCR showed7of14D-genome annexins expressed in fibers. Due to the relatively high expression levels in fibers at the fast elongating stage, GhAnn2/Gorai.007G060900would be a good candidate for studying the role of annexin in fiber elongation.8. RNAi silencing of GhAnn2results in shorter and thinner fibersWe found the development of GhAnn2RNAi fiber cells was significantly inhibited. The relative expression levels of several fiber elongation related genes were decreased both in5DPA and10DPA fibers of GhAnn2RNAi lines compared to the wild-type, especially the GhEXPA1gene, indicating that GhAnn2expression is required for normal fiber elongation. Paraffin histological sections showed that the fiber wall thickness showed that the fiber cell wall of GhAnn2RNAi lines were significantly thinner than that of wild type. Genes associated with the deposition of the secondary cell wall cellulose were found to be down-regulated in both15and20DPA fibers of GhAnn2RNAi lines, which is consistent with the reduced cell wall thickness phenotypes.9. RNAi silencing of GhAnn2decreases Ca2+influx at the apex of fiber cellsScanning ion-selective electrode technique was used to measure the Ca2+influx at the fiber cell apex from the extracellular into intracellular environment. The result showed that the Ca2+influx at the apex of GhAnn2RNAi fiber cells was decreased compared to that of wild type. Ovule culture assay showed that GhAnn2fibers of RNAi lines were more sensitive to La3+(calcium channel inhibitor), consistent with the decreased Ca2+influx from the apex of GhAnn2RNAi fibers.10. RNAi silencing of GhAnn2down-regulates expression of Ca2+sensors in elongating fiber cellsCa2+influx can contribute to the generation of Ca2+signals which can be sensed by Ca2+sensors. The expression levels of2CaMs,4CMLs,4CCaMKs and2CBLs were down-regulated in GhAnn2RNAi fibers compared to the wild type, especially regarding to the two CaMs and CBLs. The data provided further evidence that GhAnn2may have an effect on Ca2+influx and signaling during fiber development. |
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