Progression through meiosis I and meiosis II in Arabidopsis anthers is regulated by an A-type cyclin predominately expressed in prophase I

Abstract

Meiosis is often described as a special case of cell division since it differs from mitosis in having two nuclear divisions without an intervening S-phase. It will be of great interest to uncover what molecular mechanisms underlie these special features of meiosis. We previously reported that the tardy asynchronous meiosis (tam) mutant of Arabidopsis (Arabidopsis thaliana) is slower in cell cycle progression in male meiosis. Here we report that TAM encodes the A-type cyclin, CYCA1;2. The point mutation in tam replaced a conserved threonine with an isoleucine in the linker region between the alpha4 and alpha5 helices of the first cyclin fold. By studying the dynamics of a CYCA1;2-green fluorescent protein fusion protein under the control of the CYCA1;2 promoter, we found that the fusion protein was most abundant at pachytene, but was undetectable from late prophase I until telophase II. Nonetheless, cell cycle progression in tam was delayed in both pachytene and meiosis II. We conclude either that the CYCA1;2 produced in prophase I indirectly regulates meiosis II progression, or that a very low level of CYCA1;2 directly regulates meiosis II progression. Either of these scenarios is a deviation from the typical mode of action of mitotic cyclins in mitosis and meiosis I, in which each nuclear division is coupled with a peak of expression of mitotic cyclins.

Figure 1.

Complementation of tam with CYCA1;2. A, Genomic fragment that complemented the tam meiotic phenotype. White bars indicate noncoding regions, black bars indicate exons, and lines indicate introns. ATG and TGA, start and stop codons, respectively. C→T denotes position of the point mutation in tam. bp, Base pairs. B, Meiotic products. Left, Wild-type tetrads. Middle, Dyads in tam. Note the prominent median wall in the dyads. Right, Normal tetrads in a tam plant containing the DNA fragment illustrated in A. All plants were grown at 27°C. Scale bar = 10 μm.

Figure 2.

RT-PCR analysis of the CYCA1;2 transcript. A, RT-PCR amplification of CYCA1;2 and ACTIN8 (control) in five different organs. The amplifications for CYCA1;2 and ACTIN8 were in separate tubes, with 40 cycles for CYCA1;2 and 20 cycles for ACTIN8. A premix of solution for RT-PCR for each RNA sample was divided equally for CYCA1;2 and ACTIN8. B, In wild-type inflorescences, the CYCA1;2 transcript level was less than the DMC1 transcript level. The same RNA sample was used for both lanes, and the PCR was for 40 cycles. The amplifications for CYCA1;2 and ACTIN8 were in the same tube, and so were the amplifications for DMC1 and ACTIN8.

Figure 3.

CYCA1;2-GFP during male meiosis in wild type. Left, GFP signal. Middle, Corresponding bright-field images to the images on the left. Right, DAPI-staining of fixed samples showing the corresponding cell cycle stages of the fresh microsporocytes on the left. A to C, No GFP signal, late leptotene, or early zygotene (arrow). Three tapetal cells (arrowheads), two binucleate and one uninucleate, can be seen, consistent with the stage being leptotene or zygotene (Y. Wang et al., 2004). D to F, Weak GFP signal, late zygotene, or early pachytene. G to I, Strong GFP signal, pachytene. J to L, Weak GFP signal, diplotene. A nucleus presumably from an anther wall cell (not a tapetal cell) is also present (arrowhead). M to O, No GFP signal, prophase II. Three microsporocytes are shown in O. The two nuclei near the right margin are from a tapetal cell. P and R, GFP signal in telophase II microsporocytes. Arrows denote one of the nuclei. Cells in F and I were fixed and enzyme-treated for spreading chromosomes; cells in C, L, O, and R were fixed but not enzyme-treated. Scale bar = 20 μm.

Figure 4.

CYCA1;2-GFP is located in both the cytoplasm and nucleus. A, Unfixed microsporocyte showing the GFP signal. B, The differential interference contrast image corresponding to A. C, Spread chromosomes showing that the stage is pachytene. This cell and the cell in A were from separate medial anthers in the same bud. Arrows, Nucleus. Arrowheads, Nucleolus. Bar = 10 μm.

Figure 5.

Distribution of buds at different stages of meiosis. The frequency of buds at leptotene is defined as 1 in both wild type and tam, and the frequencies relative to that of leptotene were calculated for the other stages. The wild-type sample included 42 buds and the tam sample 59 buds. Lep, Leptotene; Zyg, zygotene; Pac, pachytene; Dip, early diplotene; Cyt, early cytokinesis. Dip to cyt corresponds to early diplotene to early tetrad stage in wild type, or to early diplotene to early dyad stage in tam.

Figure 6.

Durations of meiotic stages. Means ± ses are shown. Each mean ± se was derived from four to seven samples. Cyt2, early stage of the second cytokinesis observed only in tam. Cyt to Cyt2 corresponds essentially to the duration of the second nuclear division in tam male meiosis. Other abbreviations are the same as in Figure 5.