Abstract
Cotton fibers are initiated from the epidermal cells of the ovule before or on the day of anthesis. Gossypium arboreum SMA-4 mutant contains recessive mutation (sma-4(ha)) and has the phenotypes of fibreless seeds and glabrous stems. In this study, fine mapping and alternative splicing analysis indicated a nucleotide substitution (AG → AC) at splicing site in a homeodomain-leucine zipper IV family gene (GaHD1) might cause gene A3S (Alternative 3′ splicing) mistake, suggested that GaHD1 was the candidate gene of sma-4(ha). Many genes related to the fiber initiation are identified to be differentially expressed in the mutant which could result in the blocked fiber initiation signals such as H2O2, or Ca in the mutant. Further comparative physiological analysis of H2O2 production and Ca2+ flux in the SMA-4 and wide type cotton confirmed that H2O2 and Ca were important fiber initiation signals and regulated by GaHD1. The in vitro ovule culture of the mutant with hormones recovered the fibered phenotype coupled with the restoration of these signals. Overexpressing of GaHD1 in Arabidopsis increased trichome densities on the sepal, leaf, and stem tissues while transient silencing of the GaHD1 gene in G. arboreum reduced the trichome densities. These phenotypes indicated that GaHD1 is the candidate gene of SMA-4 with a crucial role in acting upstream molecular switch of signal transductions for cotton trichome and fiber initiations.
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Acknowledgements
This work was jointly supported by the National Key R & D Program for Crop Breeding (2016YFD0101417), Natural Science Foundation of Zhejiang Province (LY17C060005), National Natural Science Foundation of China (Grant Nos. 31501349, 31301372, 31200909).
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JR and MD conceived and designed the entire research plans; MD and JR wrote the manuscript; MD, YC, and SH performed most of the experiments, HD, HZ, CS, and YJ provided technical assistance to MD; YC generated transgenic plants; JS did the NMT ion flux analysis; MD performed ovule culture experiment; MD, JR, YC, SH analyzed the data; JR and AP revised and polished the entire manuscripts.
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11103_2020_1000_MOESM1_ESM.pdf
Figure S1. Short Reads distributions adjacent to the mutation site. A: Reads normally spliced between the intron and exon junction site. (GT:117206718-AG:117206812) B: Short reads mapped to the introns indicate the intron retentions caused by the mutation of AG->AC (117206812); Several splicing reads indicate the splicing site has been changed to AG(117206840 and 117206879) due to the mutation. (PDF 196 kb)
11103_2020_1000_MOESM2_ESM.pdf
Figure S2. Heatmap of the FPKMs of overlapping downregulated genes in the SMA-4 mutant (MT) compared with the wild-type (WT) at three stages (−3 DPA, 0 DPA, and +3 DPA). (PDF 245 kb)
11103_2020_1000_MOESM3_ESM.pdf
Figure S3. Functional enrichment analysis of overlapping downregulated genes in SMA-4 mutant at three stages (−3 DPA, 0 DPA, and +3 DPA). A: Gene ontology (GO) analysis; B: KEGG pathway analysis. (PDF 454 kb)
11103_2020_1000_MOESM4_ESM.pdf
Figure S4. Heatmap of fold changes of important fiber development-related transcription factors in wild-type (WT) and SMA-4 mutant cotton. Colors indicate significant up- (red) and downregulated (blue) WT genes. (PDF 328 kb)
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Figure S5. GUS staining in Arabidopsis transformed with the GaHD1 promoter: GUS construct. The GUS activity signal was located primarily at the epidermal stem cells and trichomes (A, B), leaf edges and trichomes (C), and possibly the epidermal cells in seeds (D). (JPG 3067 kb)
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Ding, M., Cao, Y., He, S. et al. GaHD1, a candidate gene for the Gossypium arboreum SMA-4 mutant, promotes trichome and fiber initiation by cellular H2O2 and Ca2+ signals. Plant Mol Biol 103, 409–423 (2020). https://doi.org/10.1007/s11103-020-01000-3
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DOI: https://doi.org/10.1007/s11103-020-01000-3