Microgametogenesis

Abstract: Summary Callose (β-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.

Abstract: Polyploidy is an important evolutionary phenomenon but the mechanisms by which polyploidy arises still remain underexplored. There may be an environmental component to polyploidization. This study aimed to clarify how temperature may promote diploid gamete formation considered an essential element for sexual polyploidization. First of all, a detailed cytological analysis of microsporogenesis and microgametogenesis was performed to target precisely the key developmental stages which are the most sensitive to temperature. Then, heat-induced modifications in sporad and pollen characteristics were analysed through an exposition of high temperature gradient. Rosa plants are sensitive to high temperatures with a developmental sensitivity window limited to meiosis. Moreover, the range of efficient temperatures is actually narrow. 36 °C at early meiosis led to a decrease in pollen viability, pollen ectexine defects but especially the appearance of numerous diploid pollen grains. They resulted from dyads or triads mainly formed following heat-induced spindle misorientations in telophase II. A high temperature environment has the potential to increase gamete ploidy level. The high frequencies of diplogametes obtained at some extreme temperatures support the hypothesis that polyploidization events could have occurred in adverse conditions and suggest polyploidization facilitating in a global change context.

Abstract: 1. The technique for making autoradiographs with stripping film is described in detail. Methods for administering P32 to plants and for preparation of tissues for autoradiographs are given. 2. Two different methods, enzyme digestion and acid hydrolysis, which were used for separating RNA, DNA, and phosphoprotein in tissue sections, were compared and evaluated. Although either method is valid for quantitative work, precautions are necessary and certain limitations to the quantitative separation of RNA and DNA should be taken into account. More work is needed to properly characterize the products extracted during enzyme digestion and acid hydrolysis. 3. Incorporation of P32 into nuclei of lily anthers was studied by the autoradiographic method at all stages from premeiosis to mature pollen. A quantitative comparison of the amounts of P32 extracted by RNase, DNase, Feulgen hydrolysis and hot TCA showed that incorporation into DNA occurred at relatively short periods during interphase, namely: (1) at preleptotene before meiotic prophase in microsporocytes, (2) at late interphase preceding mitosis in the microspore and (3) during early to mid-interphase in the generative nucleus of the pollen grain (the tube nucleus incorporates P32 slowly during the same period but this P32 was not definitely identified as DNA phosphorus). Two successive mitoses occur in tapetal cells while the microsporocytes are in meiotic prophase. In the interphase preceding each division incorporation of P32 into DNA occurs, but following the second mitosis no further divisions or incorporation was detected. However, during this latter period incorporation into RNA and phosphoprotein reached the highest level detected at any stage. During the period of DNA synthesis, incorporation of P32 into RNA was low or absent, but in the interval between the cessation of DNA synthesis and prophase the incorporation of P32 into RNA of both nucleus and cytoplasm reached its peak. 4. Microphotometric determination of amounts of DNA (Feulgen stain) showed that the amount per nucleus remains constant in all stages, except for doubling during the periods when P32 is incorporated, and the reduction by half at each division of a nucleus. The 2C amount is present in sporogenous nuclei until preleptotene when values intermediate between 2C and 4C are found. By the beginning of leptotene 4C is present in each microsporocyte nucleus. The amount does not change through meiotic prophase. Each nucleus after the first meiotic division contains 2C and each nucleus of the quartet of spores has 1C. The amount per nucleus in microspores doubles toward the end of the long interphase prior to prophase. After the mitosis in the microspore, the generative nucleus attains the 2C level relatively early in interphase. The vegetative nucleus has 1C in the interphase immediately after mitosis, but could not be accurately measured at later stages. Tapetal nuclei have the 2C amount of DNA when the microsporocytes are in leptotene. The amount doubles while the microsporocytes are in zygotene and the first mitosis usually occurs while the microsporocytes are in early pachytene. Another doubling occurs during the relatively short interphase and a second mitosis occurs when the microsporocytes are in late pachytene or early diplotene. The 4C class was typical for tapetal nuclei at later stages, but this may, in some cases at least, result from fusion of nuclei in the multinucleate cells during or following the second mitosis. 5. The periods of DNA increase (measured microphotometrically) coincide very closely with the periods of incorporation of P32 into DNA. This finding supports the concept of the stability of the DNA molecule, and indicates that incorporation may be used as a criterion not only of synthesis of DNA, but also of increase in the amount of DNA per nucleus.

Abstract: Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter∷β-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.