Figure 2. Accumulation and localization of CDKA;1-YFP fusion protein during female and male gametophyte development.

(A-A^VI) Expression of a PRO_CDKA;1:CDKA;1-YFP construct completely rescues cdka;1 pollen resulting in wild-type-like pollen with one vegetative cell (arrowhead in A^V) and two sperm cells (triangle in A^V and A^VI, see also Figure 1). CDKA;1 is expressed throughout male gametophyte development but becomes restricted at anthesis to the two sperm cells as revealed by YFP accumulation. (B-B^VI) A hemizygous PRO_CDKA;1:CDKA;1-YFP^+/− allele in homozygous mutant cdka;1^−/− plants mimics heterozygous cdka;1^+/− mutant plants that produce pollen of which half resembles wild-type pollen but half comprises one vegetative cell and only one instead of two sperm cells at anthesis (see Figure 1). Continuous observation of the CDK-YFP fusion protein during male gametophyte development showed that the CDKA;1 protein concentration gradually decreased in 50% of pollen (marked by an asterisk) and that around the bicellular stage, clearly one pollen population can be identified that shows no or very little YFP fluorescence; occasionally, residual CDKA;1-YFP protein could be detected in the single sperm pollen at anthesis (red arrowhead in B^VI). A and B, Microspore mother cell; A^I and B^I, tetrads; A^II and B^II, monocellular stage; A^III and B^III, early bicellular stage; A^IV and B^IV, late bicellular stage; A^V and B^V, tricellular stage; A^VI and B^VI, anthesis. (C) Cartoon summarizing the decrease in CDKA;1 concentration as seen in B-B^VI. At the bicellular stage, CDKA;1 levels in mutant pollen (dashed yellow line) drop below an assumed threshold (dashed red line) for executing mitosis. Consistent with the disappearance of the YFP fluorescence at this stage, cdka;1 mutant pollen typically arrest before PMII. (D-D^VI) CDKA;1-YFP signal appears in all nuclei at all developmental stages of the developing embryo sac (MMC to FG7, arrowheads mark the nuclei in the embryo sac). At maturity, YFP fluorescence is only present in the gametic cells, the egg cell and the central cell (marked by a white triangle in D^VI). MMC, megaspore mother cell/microspore mother cell; MC, monocellular; BC, bicellular; TC, tricellular; FG, female gametophyte stage.