Author Contributions
Conceptualization, L.X. and J.C.; methodology, L.X., X.X.; software, L.X., W.L.; validation, L.X., X.X. and T.L.; formal analysis, L.X., W.L.; investigation, L.X., T.L.; resources, J.C.; data curation, L.X.; writing—original draft preparation, L.X.; writing—review and editing, Y.Y., J.C.; visualization, L.X.; supervision, J.C.; project administration, L.X., J.C.; funding acquisition, J.C. All authors have read and agreed to the published version of the manuscript.
Figure 1.
Identification of male fertility-related CCCH zinc-finger protein genes in B. campestris. (A) Expression analysis of CCCH zinc-finger protein genes in five pollen developmental stages of ‘Bcajh97-01A/B’ by RNA-seq. PMC, pollen mother cell stage; Tds, tetrads stage; UC, unicellular pollen stage; BC, bicellular pollen stage; TP, tricellular pollen stage. (B) Phylogenesis analysis of 11 male fertility-related CCCH zinc-finger proteins. (C) Distribution of the conserved domains of male fertility-related CCCH zinc-finger proteins.
Figure 1.
Identification of male fertility-related CCCH zinc-finger protein genes in B. campestris. (A) Expression analysis of CCCH zinc-finger protein genes in five pollen developmental stages of ‘Bcajh97-01A/B’ by RNA-seq. PMC, pollen mother cell stage; Tds, tetrads stage; UC, unicellular pollen stage; BC, bicellular pollen stage; TP, tricellular pollen stage. (B) Phylogenesis analysis of 11 male fertility-related CCCH zinc-finger proteins. (C) Distribution of the conserved domains of male fertility-related CCCH zinc-finger proteins.
Figure 2.
Expression analysis of BcMF30a and BcMF30c. Semi-RT-PCR (A) and qRT-PCR (B) analyses of BcMF30a and BcMF30c in five major tissues of ‘Bcajh97-01B’. R, root; Ste, stem; L, leaves; Inf, inflorescence; Si, silique. Semi-RT-PCR (C) and qRT-PCR (D) analyses of BcMF30a and BcMF30c in floral buds of ‘Bcajh97-01A/B’ at different developmental stages. PMC, pollen mother cell stage; Tds, tetrads stage; U, unicellular pollen stage; BC, bicellular pollen stage; TC, tricellular pollen stage. Ubiquitously expressed UBC10 was used as internal control. Promoter-GUS activities of BcMF30a (E) and BcMF30c (F) in inflorescences and germinated pollen. Bars = 2 mm and 50 μm. GUS: β-glucuronidase.
Figure 2.
Expression analysis of BcMF30a and BcMF30c. Semi-RT-PCR (A) and qRT-PCR (B) analyses of BcMF30a and BcMF30c in five major tissues of ‘Bcajh97-01B’. R, root; Ste, stem; L, leaves; Inf, inflorescence; Si, silique. Semi-RT-PCR (C) and qRT-PCR (D) analyses of BcMF30a and BcMF30c in floral buds of ‘Bcajh97-01A/B’ at different developmental stages. PMC, pollen mother cell stage; Tds, tetrads stage; U, unicellular pollen stage; BC, bicellular pollen stage; TC, tricellular pollen stage. Ubiquitously expressed UBC10 was used as internal control. Promoter-GUS activities of BcMF30a (E) and BcMF30c (F) in inflorescences and germinated pollen. Bars = 2 mm and 50 μm. GUS: β-glucuronidase.
Figure 3.
Transcriptional activation assays of BcMF30a and BcMF30c. (A) Schematic diagrams of plasmids used for transcriptional activation assays. GAL4 DNA-BD, GAL4 DNA-binding domain; GAL4-AD, GAL4 activation domain; BcMF30a and BcMF30c, the coding sequences of BcMF30a and BcMF30c. (B) The growth of transformed yeast on different yeast screening media.
Figure 3.
Transcriptional activation assays of BcMF30a and BcMF30c. (A) Schematic diagrams of plasmids used for transcriptional activation assays. GAL4 DNA-BD, GAL4 DNA-binding domain; GAL4-AD, GAL4 activation domain; BcMF30a and BcMF30c, the coding sequences of BcMF30a and BcMF30c. (B) The growth of transformed yeast on different yeast screening media.
Figure 4.
Subcellular localization of eGFP-BcMF30a and eGFP-BcMF30c fusion proteins in tobacco epidermal cells. (A) Constructs used for the subcellular localization analysis. (B) Both eGFP-BcMF30a and eGFP-BcMF30c were located in the nucleus and dispersed in the cytoplasm of tobacco epidermal cells. H2B is a marker protein of nucleus. Pictures represent white field images (Bright), epifluorescence (GFP and RFP) and merged images (Merge). Bar = 25 μm.
Figure 4.
Subcellular localization of eGFP-BcMF30a and eGFP-BcMF30c fusion proteins in tobacco epidermal cells. (A) Constructs used for the subcellular localization analysis. (B) Both eGFP-BcMF30a and eGFP-BcMF30c were located in the nucleus and dispersed in the cytoplasm of tobacco epidermal cells. H2B is a marker protein of nucleus. Pictures represent white field images (Bright), epifluorescence (GFP and RFP) and merged images (Merge). Bar = 25 μm.
Figure 5.
Construction of mutants by using the CRISPR/Cas9 system. (A) Genomic location of guide sequences (sgRNA-a, sgRNA-c, and sgRNA-ac) targeting BcMF30a and/or BcMF30c. (B) The gene editing of BcMF30a and BcMF30c in pCA-sgRNA-ac transgenic T0 lines. CRISPR/Cas9: complementation and clustered regularly interspaced short palindromic repeat/CRISPR-associated 9, sgRNA: single-guide RNA, sgRNA-a: sgRNA targeting BcMF30a, sgRNA-c: sgRNA targeting BcMF30c, sgRNA-ac: sgRNA targeting BcMF30a and BcMF30c.
Figure 5.
Construction of mutants by using the CRISPR/Cas9 system. (A) Genomic location of guide sequences (sgRNA-a, sgRNA-c, and sgRNA-ac) targeting BcMF30a and/or BcMF30c. (B) The gene editing of BcMF30a and BcMF30c in pCA-sgRNA-ac transgenic T0 lines. CRISPR/Cas9: complementation and clustered regularly interspaced short palindromic repeat/CRISPR-associated 9, sgRNA: single-guide RNA, sgRNA-a: sgRNA targeting BcMF30a, sgRNA-c: sgRNA targeting BcMF30c, sgRNA-ac: sgRNA targeting BcMF30a and BcMF30c.
Figure 6.
Quantitative detection and protein coding potential prediction of mutated BcMF30a and mutated BcMF30c in bcmf30a bcmf30c mutants. (A) Prediction of proteins coded by the mutated BcMF30a (bcmf30a) and mutated BcMF30c (bcmf30c). (B) The relative expression levels of bcmf30a and bcmf30c in the inflorescences of bcmf30a bcmf30c by qRT-PCR analysis.
Figure 6.
Quantitative detection and protein coding potential prediction of mutated BcMF30a and mutated BcMF30c in bcmf30a bcmf30c mutants. (A) Prediction of proteins coded by the mutated BcMF30a (bcmf30a) and mutated BcMF30c (bcmf30c). (B) The relative expression levels of bcmf30a and bcmf30c in the inflorescences of bcmf30a bcmf30c by qRT-PCR analysis.
Figure 7.
Cytological observation of mature pollen grains in bcmf30a bcmf30c mutants of B. campestris. Analysis of pollen abortion frequency in control, pCA-sgRNA-ac transgenic T0 lines (A), and bcmf30a bcmf30c mutants (B). The values are the mean ± SD. Alexander staining (C,H), SEM observation (D,E,I,J), and 4′,6-diamidino-2-phenylindole (DAPI) staining (F,G,K,L) of mature pollen grains in control plants and bcmf30a bcmf30c mutants. Bars = 50 μm in (C,H), 30 μm in (D,I), 5 μm in (E,J) and 20 μm in (F,G,K,L).
Figure 7.
Cytological observation of mature pollen grains in bcmf30a bcmf30c mutants of B. campestris. Analysis of pollen abortion frequency in control, pCA-sgRNA-ac transgenic T0 lines (A), and bcmf30a bcmf30c mutants (B). The values are the mean ± SD. Alexander staining (C,H), SEM observation (D,E,I,J), and 4′,6-diamidino-2-phenylindole (DAPI) staining (F,G,K,L) of mature pollen grains in control plants and bcmf30a bcmf30c mutants. Bars = 50 μm in (C,H), 30 μm in (D,I), 5 μm in (E,J) and 20 μm in (F,G,K,L).
Figure 8.
The semi-thin transverse sections of bcmf30a bcmf30c anthers. Semi-thin sections of anthers from the control plants (A–E) and bcmf30a bcmf30c (F–J) at the pollen mother cell stage (PMC, (A,F)), tetrad stage (Tetrad, (B,G)), uninucleate microspore stage (UNM, (C,H)), bicellular pollen stage (BC, (D,I)) and tricellular pollen stage (TC, (E,J)). BCP, bicellular pollen; MMC, microspore mother cell; Msp, microspore; P, pollen; dP, degenerated pollen; T, tapetum; TCP, tricellular pollen; Tds, tetrads. Bars = 25 μm.
Figure 8.
The semi-thin transverse sections of bcmf30a bcmf30c anthers. Semi-thin sections of anthers from the control plants (A–E) and bcmf30a bcmf30c (F–J) at the pollen mother cell stage (PMC, (A,F)), tetrad stage (Tetrad, (B,G)), uninucleate microspore stage (UNM, (C,H)), bicellular pollen stage (BC, (D,I)) and tricellular pollen stage (TC, (E,J)). BCP, bicellular pollen; MMC, microspore mother cell; Msp, microspore; P, pollen; dP, degenerated pollen; T, tapetum; TCP, tricellular pollen; Tds, tetrads. Bars = 25 μm.
Figure 9.
Transmission electron micrographs of pollen grains from bcmf30a bcmf30c mutants. Ultrastructure of pollen at different developmental stages from the control plants (A–C) and bcmf30a bcmf30c (D–G). (A,D) Late uninucleate stage; (B,E) bicellular pollen stage; (C,F,G) tricellular pollen stage. BCP, bicellular pollen; cBCP, collapsed BCP; GC, generative cell; Msp, microspore; cMsp, collapsed Msp; N, nucleus; TCP, tricellular pollen; cTCP, collapsed TCP; dTCP, degenerated TCP; V, Vacule; VN, vegetative nucleus. Bars = 5 μm.
Figure 9.
Transmission electron micrographs of pollen grains from bcmf30a bcmf30c mutants. Ultrastructure of pollen at different developmental stages from the control plants (A–C) and bcmf30a bcmf30c (D–G). (A,D) Late uninucleate stage; (B,E) bicellular pollen stage; (C,F,G) tricellular pollen stage. BCP, bicellular pollen; cBCP, collapsed BCP; GC, generative cell; Msp, microspore; cMsp, collapsed Msp; N, nucleus; TCP, tricellular pollen; cTCP, collapsed TCP; dTCP, degenerated TCP; V, Vacule; VN, vegetative nucleus. Bars = 5 μm.
Figure 10.
The in vitro and in vivo germination tests of bcmf30a bcmf30c mature pollen. (A) Pollen germination frequencies of bcmf30a bcmf30c pollen in vitro. Only viable pollen was counted for pollen germination rate statistics. The values are the mean ± SD. (B) Control and bcmf30a bcmf30c mature pollen germinated in vitro for 4 h. (C) Aniline blue staining of pollen tubes in pistils after self-pollination and cross-pollination for 12 h. The arrows indicated the pollen tubes and the asterisk indicated the end position of pollen tubes. Bars = 100 μm.
Figure 10.
The in vitro and in vivo germination tests of bcmf30a bcmf30c mature pollen. (A) Pollen germination frequencies of bcmf30a bcmf30c pollen in vitro. Only viable pollen was counted for pollen germination rate statistics. The values are the mean ± SD. (B) Control and bcmf30a bcmf30c mature pollen germinated in vitro for 4 h. (C) Aniline blue staining of pollen tubes in pistils after self-pollination and cross-pollination for 12 h. The arrows indicated the pollen tubes and the asterisk indicated the end position of pollen tubes. Bars = 100 μm.