Abstract
Male germline specification is a crucial step in double fertilization in flowering plants. Determining which genes are activated in the male germline, and how those genes are regulated, is essential to understand double fertilization. However, transcription activities in the male germline may be not easy to detect due to low-level expression of some genes and technical difficulties of isolating male germline cells. Here, through a series of gene reporter assays in Arabidopsis, we showed a weak male germline expression pattern of the SOLO DANCERS (SDS) gene, which was confirmed by RT-PCR. Compared to directly fusing the SDS sequence with GFP, adding the coding sequences of other genes such as CDKA1 can greatly enhance the detection of the male germline expression pattern of SDS. We found that SDS expression in the male germline is activated by a novel pathway that differs from the well-known DUO1 regulon. We also developed an SDS-based fluorescence reporter to analyze posttranscriptional regulation in the male germline. Our data suggest that stable gene products of CDKA1 and others may enhance the sensitivity of gene reporters, and the male germline may use diverse pathways to activate gene expression.
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Acknowledgements
We thank Dr. Haifeng Peng for critical reading of this manuscript, Professor Jikai Wen for providing total RNA extract of Schizosaccharomyces pombe, and Professor Gang Hao for his laboratory support. The project was financially supported by the Natural Science Foundation of China (No. 31000138) and the Natural Science Foundation of Guangdong Province (No. 9451064201003709).
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344_2019_10061_MOESM1_ESM.eps
Supplementary file1 (EPS 976 kb)—Fig. S1. The sds- mutant allele shows no significant difference of transmission with the wild-type allele. (A) The genomic structure of SDS and the insertion site of T-DNA in Salk_134177. LP, RP and LB1.3b are primers used to detect genotypes of F1 plants. Filled boxes indicate exons. The start codon and stop codon are indicated with vertical lines. T-DNA is inserted in the first exon. (B) Crossing male heterozygous sds+/- mutant with female wild-type Col-0 Arabidopsis to get F1 population. (C) The mutant sds- allele shows no significant difference of genetic transmission with the wild-type allele. The wild-type sds+ allele was amplified with the primer pair of LP and RP; The mutant sds- allele was amplified with the primer pair of LB1.3b and RP.
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Supplementary file2 (TIF 2565 kb)—Fig. S2. Cellular localization of CDKA1 protein in dividing generative cells of ProSDS:CDKA1-GFP transgenic plants. Representative pollen grains show GFP fluorescence (top) and DAPI staining results (bottom) at prophase (A), metaphase (B), telophase (c) and after division (D).
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Zhen, Y., Huang, J., Chen, X. et al. Detection of Weak Expression of SOLO DANCERS in the Male Germline Using CYCLIN-DEPENDENT KINASE A1 Coding Sequence. J Plant Growth Regul 39, 1236–1244 (2020). https://doi.org/10.1007/s00344-019-10061-8
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DOI: https://doi.org/10.1007/s00344-019-10061-8