Fig 1.
The expression patterns of DR5:GFP in the anther of yuc2yuc6.
(A-B) Comparison of the auxin reporter DR5:GFP signal in anthers of wild type (A) and yuc2yuc6 (B) at different developmental stages. Reporter signal in anthers observed in wild type from stages 9 to 12 is completely absent in yuc2yuc6. Bars = 100 μm.
Fig 2.
DII-VENUS for visualizing auxin-induced Aux/IAA degradation during pollen development.
(A) Fluorescence of proMSP1:mDII control and proMSP1:DII plants. MSP1 promoter (proMSP1) is a microspore-specific promoter. E: early, L: late. (B) Quantitative representation of the relative VENUS fluorescence intensity from nucleus of stage 10 and stage 11 pollen. The fluorescence intensity was measured using ImageJ software. Data shown in B represent mean with SD based on more than 20 pollen grains for each group (set as 1 for stage 11 pollen from proMSP1:DII). The symbol * indicates where the difference between proMSP1:DII and proMSP1:mDII at corresponding stage is significant in statistic test (p-value<0.05). Note that the fluorescence signals of DII in stage 10 and stage 11 were much weaker than that in mDII microspores. Bars = 7 μm.
Fig 3.
The yuc2yuc6 double mutants were defective in pollen development.
(A-D) Alexander staining of anthers of wild type (A), yuc2 (B), yuc6 (C) and yuc2yuc6 (D). Note that yuc2yuc6 lacked viable pollen. (E-P) Semi-thin cross sections of anthers from wild type (E-J) and yuc2yuc6 (K-P) were stained with toluidine blue. Anthers at (E and K) stage 7; (F and L) stage 8; (G and M) stage 9; (H and N) stage 10; (I and O) stage 11; (J and P) stage 12. A microspore or pollen showing reduced or completely invisible of cytoplasm we defined as degeneration state. Note that the degenerated microspores were evident in yuc2yuc6 from stage 10 to 12. (Q-Z) Ultra-structures of pollen grains from wild type (Q-U) and yuc2yuc6 (V-Z) plants. Microspores from anthers at stage 8 (Q and V); stage 9 (R and W); stage 10 (S and X); stage 11 (T and Y) were shown. Enlarged images from T and Y were shown in (U and Z). It is clear that the pollen wall pattern in yuc2yuc6 appears similar to that in WT. Most of the cytoplasm is degenerated in the microspore from stage 10 and 11 anthers of yuc2yuc6 (X and Y). T, Tapetum; Tds, tetrads; Mp, microspore; abMp, abnormal microspore; Pg, pollen grain; Dpg, degenerated pollen grain. Bars = 100μm (A-D), 20 μm (E-P), 1 μm (Q-Z).
Fig 4.
The early stages of male gametogenesis were defective in the yuc2yuc6 double mutants.
(A-J) DAPI staining of pollens from wild type (A-E) and yuc2yuc6 (F-J) at different developmental stages. Microspores from anthers at (A and F) stage 8; (B and G) stage 9; (C and H) stage 10 were shown. Some of the yuc2yuc6 microspores were degenerated and had little DAPI staining (H right). (D and I) pollens at the bicellular stage. Note that the nuclei of yuc2yuc6 were arrested at the unicellular state or degenerated and could not form two nuclei as in the wild type (I). (E and J) Pollen at the tricellular stage. The yuc2yuc6 pollen grains lacked genomic DNA (J) while WT pollen showed strong DAPI staining (E). (K) Quantitative analysis and comparison of pollen defects between wild type and yuc2yuc6 (n>600 for each stage). Uc, Unicellular pollen; Mn, microspore nucleus; Gc, generative cell; Vn, vegetative nucleus; Sc, sperm cell. Bc, Bicellular pollen; Tc, Tricellular pollen; De, Degenerated pollen. Bars = 5 μm.
Table 1.
Effects of mutations in YUC2 and YUC6 on male transmission frequency.
Fig 5.
Localized auxin production in tapetum did not rescue the pollen defects in yuc2yuc6 mutants.
(A) The green color indicates the expression pattern of the A9 promoter. Msp, Microsporocytes; Mp, microspores; Bc, Bicellular pollen; Tc, Tricellular pollen. The A9 promoter cloned from Arabidopsis is used to drive YUC2-GFP expression specifically in tapetum cells from stages 6 to 9 [61, 62]. (B) Morphology of adult shoots (Bars = 2cm). (C) Alexander staining (Bars = 100 μm) and DAPI staining (Bars = 10 μm) of ProA9:YUC2-GFP (yuc2yuc6) anthers and pollens. Note that the sterility phenotype and pollen defects were not rescued in ProA9:YUC2-GFP (yuc2yuc6) transgenic plants. TS, Tricellular Stage; BS, Bicellular Stage; US; Unicellular Stage. (D) In situ hybridization of GFP in ProA9:YUC2-GFP (yuc2yuc6) transgenic plants (Bars = 20 μm). (E) Fluorescence images (Bars = 100 μm) of the YUC2-GFP fusion protein in anthers from yuc2yuc6 transformed with ProA9:YUC2-GFP. In ProA9: YUC2-GFP anther, GFP is significantly expressed in tapetum cell of microsporocyte stage and early microspore stage.
Fig 6.
Localized auxin production in microsporocytes and microspores rescued the pollen defects in yuc2yuc6 mutants.
The green color indicates the expression pattern of the ARF17 (A) and LAT52 (B) promoter. The ARF17 promoter is known to be active in microsporocytes and microspores [63]. LAT52 is expressed in microspore and gametophyte specifically. Msp, Microsporocytes; Mp, microspores; Bc, Bicellular pollen; Tc, Tricellular pollen. Morphology of adult shoots (Bars = 2 cm), Alexander staining (Bars = 100 μm) of anthers and DAPI staining (Bars = 10 μm) of pollens from ProARF17:YUC2-GFP (yuc2yuc6) (A) and ProLAT52:YUC2-GFP (yuc2yuc6) (B) transgenic plants. Note that YUC2-GFP expression driven by ARF17 and LAT52 promoters are sufficient to rescue the yuc2yuc6 defects. In situ hybridization of GFP (Bars = 20 μm) and fluorescence images (Bars = 100 μm) of the YUC2-GFP fusion protein in anthers from yuc2yuc6 transformed with ProARF17:YUC2-GFP (A) and ProLAT52:YUC2-GFP (B). In ProARF17:YUC2-GFP (yuc2yuc6) anther, GFP is expressed in microsporocytes and in microspores at early stage (A). In ProLAT52:YUC2-GFP (yuc2yuc6) anther, GFP is significantly expressed in microspores at late stage and in bicellular pollens (B). TS, Tricellular Stage; BS, Bicellular Stage; US; Unicellular Stage.