The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in rice

The function of the novel gene MSP1 (MULTIPLE SPOROCYTE), which controls early sporogenic development, was elucidated by characterizing a retrotransposon-tagged mutation of rice. The MSP1 gene encoded a Leu-rich repeat receptor-like protein kinase. The msp1 mutation gave rise to an excessive number of both male and female sporocytes. In addition, the formation of anther wall layers was disordered and the tapetum layer was lost completely. Although the mutation never affected homologous chromosome pairing and chiasma maintenance, the development of pollen mother cells was arrested at various stages of meiotic prophase I, which resulted in complete male sterility. Meanwhile, plural megaspore mother cells in a mutant ovule generated several megaspores, underwent gametogenesis, and produced germinable seeds when fertilized with wild-type pollen despite disorganized female gametophytes. In situ expression of MSP1 was detected in surrounding cells of male and female sporocytes and some flower tissues, but never in the sporocytes themselves. These results suggest that the MSP1 product plays crucial roles in restricting the number of cells entering into male and female sporogenesis and in initiating anther wall formation in rice.     

Overexpression of TAPETUM DETERMINANT1 alters the cell fates in the Arabidopsis carpel and tapetum via genetic interaction with excess microsporocytes1/extra sporogenous cells

Previously, we reported that the TAPETUM DETERMINANT1 (TPD1) gene is required for specialization of tapetal cells in the Arabidopsis (Arabidopsis thaliana) anther. The tpd1 mutant is phenotypically identical to the excess microsporocytes1 (ems1)/extra sporogenous cells (exs) mutant. The TPD1 and EMS1/EXS genes may function in the same developmental pathway in the Arabidopsis anther. Here, we further report that overexpression of TPD1 alters the cell fates in the Arabidopsis carpel and tapetum. When TPD1 was expressed ectopically in the wild-type Arabidopsis carpel, the number of cells in the carpel increased significantly, showing that the ectopic expression of TPD1 protein could activate the cell division in the carpel. Furthermore, the genetic analysis showed that the activation of cell division in the transgenic carpel by TPD1 was dependent on EMS1/EXS, as it did not happen in the ems1/exs mutant. This result further suggests that TPD1 regulates cell fates in coordination with EMS1/EXS. Moreover, overexpression of TPD1 in tapetal cells also delayed the degeneration of tapetum. The TPD1 may function not only in the specialization of tapetal cells but also in the maintenance of tapetal cell fate.     

水稻msp1-4突变体的鉴定及其UDT1和GAMYB基因的表达分析

通过对粳稻‘9522’辐射诱变,得到一隐性雄性不育突变体msp1-4（MULTIPLE SPOROCYTE）,用遗传定位方法将该基因座位定位在分子标记WY-4和WY-8之间,相距0．8cM,物理距离247kb。测序分析证明这247kb区间中的MSP1基因的编码区在第758bp到767bp之间发生了10个碱基的缺失。形态学观察结果表明该突变体和已经报告过的msp1突变体的表型基本一致。为分析水稻其它与花药发育相关的基因在msp1-4中的表达变化,用半定量RT-PCR技术检测到影响绒毡层和花粉发育的重要基因UDT1和GAMYB的表达在突变体中比在野生型水稻中低,说明这2个基因可能位于MSP1基因的下游。     

Tapetum determinant1 is required for cell specialization in the Arabidopsis anther

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.     

The Mac1 Gene: Controlling the Commitment to the Meiotic Pathway in Maize

The switch from the vegetative to the reproductive pathway of development in flowering plants requires the commitment of the subepidermal cells of the ovules and anthers to enter the meiotic pathway. These cells, the hypodermal cells, either directly or indirectly form the archesporial cells that, in turn, differentiate into the megasporocytes and microsporocytes. We have isolated a recessive pleiotropic mutation that we have termed multiple archesporial cells1 (mac1) and located it to the short arm of chromosome 10. Its cytological phenotype suggests that this locus plays an important role in the switch of the hypodermal cells from the vegetative to the meiotic (sporogenous) pathway in maize ovules. During normal ovule development in maize, only a single hypodermal cell develops into an archesporial cell and this differentiates into the single megasporocyte. In mac1 mutant ovules several hypodermal cells develop into archesporial cells, and the resulting megasporocytes undergo a normal meiosis. More than one megaspore survives in the tetrad and more than one embryo sac is formed in each ovule. Ears on mutant plants show partial sterility resulting from abnormalities in megaspore differentiation and embryo sac formation. The sporophytic expression of this gene is therefore also important for normal female gametophyte development.     

The mac1 mutation alters the developmental fate of the hypodermal cells and their cellular progeny in the maize anther.

In angiosperm ovules and anthers, the hypodermal cell layer provides the progenitors of meiocytes. We have previously reported that the multiple archesporial cells1 (mac1) mutation identifies a gene that plays an important role in the switch of the hypodermal cells from the vegetative pathway to the meiotic (sporogenous) pathway in maize ovules. Here we report that the mac1 mutation alters the developmental fate of the hypodermal cells of the maize anther. In a normal anther a hypodermal cell divides periclinally with the inner cell giving rise to the sporogenous archesporial cells while the outer cell, together with adjacent cells, forms the primary parietal layer. The cells of the parietal layer then undergo two cycles of periclinal divisions to give rise to three wall layers. In mac1 anthers the primary parietal layer usually fails to divide periclinally so that the three wall layers do not form, while the archesporial cells divide excessively and most fail to form microsporocytes. The centrally located mutant microsporocytes are abnormal in appearance and in callose distribution and they fail to proceed through meiosis. These failures in development and function appear to reflect the failure of mac1 gene function in the hypodermal cells and their cellular progeny.     

The Magnaporthe grisea snodprot1 homolog, MSP1, is required for virulence

Secreted proteins play central roles in plant-microbe interactions acting as signals, toxins, and effectors. One important group of small secreted proteins is the snodprot1 family, members of which have demonstrated phytotoxic properties. A split-marker transformation system was applied for gene deletion of the snodprot1 homolog, MSP1, in the rice blast fungus Magnaporthe grisea. msp1 mutants were phenotypically indistinguishable from wild type and elaborated apparently normal appressoria. However, the deletion mutants were greatly reduced in virulence primarily due to impaired growth in planta. Western blot analysis showed that the protein was secreted and not associated with the fungal cell wall. When purified MSP1 protein was applied to wounded leaf tissue, no apparent phytotoxic effects were noted. This is the first report to the authors' knowledge that directly implicates a snodprot1 protein as a virulence factor.     

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana . The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.     

雄性不育与可育楸树花发育的细胞学比较研究

楸树（Catalpa bungei C.A.Meyer.）属紫葳科(Bignoniaceae)梓树属(Catalpa),落叶乔木,是我国特有的珍贵优质用材树种。本文用石蜡切片法对可育株和雄性不育株楸树的大、小孢子发生及雌、雄配子体发育过程进行了详细地比较观察。结果表明：可育株和不育株楸树雌蕊的发育基本相同,胚珠倒生,薄珠心,单珠被,胚囊发育为蓼型。可育株雄蕊花药四室,药隔薄壁组织发达;异型绒粘层,由药壁绒粘层和药隔绒粘层组成;花药壁表皮细胞在小孢子母细胞减数分裂前后开始径向伸长加厚,直到花药开裂并不降解,这可能与花药开裂有关;成熟花粉为四合花粉。雄性不育株花药的早期发育到次生造胞细胞时期与可育雄蕊的相同,小孢子母细胞减数分裂前绒毡层发育不充分;四分体时期,绒毡层细胞高度液泡化,细胞质稀薄,已提前降解,小孢子四分体因绒毡层结构和功能异常而不能正常发育,因此楸树雄性不育为结构型雄性不育。     

Selection of stable mutants from cultured rice anthers treated with ethyl methane sulfonic acid

To increase the frequency of stable mutants from cultured anthers of rice, the effects of EMS treatment on callus induction, plant regeneration and mutant induction were investigated according to the timing of treatment after anther inoculation on the medium. The frequency of callus induction was highest in anthers treated with 0.5% EMS 10 days after culture. Anthers treated directly at the initiation of culture exhibited a very low callus induction level, and the such calluses exhibited a poor plant regeneration capacity. The frequency of regeneration of green plants was significantly decreased by EMS treatments immediately after anther inoculation as compared with control. The frequencies of stable mutants were 20.7% and 12.0% in EMS treatments at 10 and 20 days, but unstable mutants were 43.1% and 52.6%, respectively. A total of 14 stable mutants, semidwarf mutants (4 lines), grain-shape mutants (2 lines) and glabrous mutants (8 lines) were selected from doubled haploid lines of the A₂ generation. The frequencies of callus induction, green plant regeneration and stable mutants were maximal in anthers treated with 0.5% EMS 10 days after culture.