Maize csmd1 exhibits pre-meiotic somatic and post-meiotic microspore and somatic defects but sustains anther growth

Maize male reproductive development is complex and lengthy, and anther formation and pollen maturation are precisely and spatiotemporally regulated. Here, we document that callose, somatic, and microspore defect 1 (csmd1), a new male-sterile mutant, has both pre-meiotic somatic and post-meiotic gametophyte and somatic defects. Chromosome behavior and cell developmental events were monitored by nuclear staining viewed by bright field microscopy; cell dimensions were charted by Volocity analysis of confocal microscopy images. Aniline blue staining and quantitative assays were performed to record callose deposition, and expression of three callose synthase genes was measured by qRT-PCR. Despite numerous defects and unlike other maize male-sterile mutants that show growth arrest coincident with locular defects, csmd1 anther elongation is nearly normal. Pre-meiotically and during prophase I, there is excess callose surrounding the meiocytes. Post-meiotically csmd1 epidermal cells have impaired elongation but excess longitudinal divisions, and uninucleate microspores cease growth; the microspore nucleoli degrade followed by cytoplasmic vacuolization and haploid cell collapse. The single vascular bundle within csmd1 anthers senesces precociously, coordinate with microspore death. Although csmd1 anther locules contain only epidermal and endothecial cells at maturity, locules are oval rather than collapsed, indicating that these two cell types suffice to maintain an open channel within each locule. Our data indicate that csmd1 encodes a crucial factor important for normal anther development in both somatic and haploid cells, that excess callose deposition does not cause meiotic arrest, and that developing pollen is not required for continued maize anther growth.     

温光敏核不育水稻N28S无花粉败育的显微结构观察

采用石蜡切片和荧光显微技术观察了温光敏核不育水稻N28S无花粉败育过程中的显微结构变化,结果显示：N28S的小孢子母细胞形成后细胞质变得稀薄,一部分不能进行减数分裂,一部分减数分裂阻滞在细线期或胞质分裂异常,最终所有细胞液泡化解体消失。在此过程中,还观察到小孢子母细胞在细线期胼胝质壁不产生或提早消失,以及小孢子发育后期花药壁绒毡层的异常解体。认为N28S的无花粉败育是由小孢子母细胞的细胞质异常引起的,胼胝质壁和绒毡层的异常是结果而不是原因。     

The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map‐based cloning and sequence analysis, we identified a 1,459‐bp deletion in an adenosine triphosphate (ATP)‐binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very‐long‐chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.     

Molecular and Histochemical Analysis of a T-DNA Tagged Mutant of Arabidopsis Exhibiting Anther — Specific GUS Expression

An Arabidopsis thaliana mutant, exhibiting anther specific GUS expression, identified from a mutant population of Arabidopsis tagged with a promoterless β-glucuronidase (GUS), carries the T-DNA insertions at two distinct loci. We have been able to segregate the two inserts from each other by backcrossing with wild type plants. The insertion responsible for anther specific GUS expression in segregating population has been identified and confirmed to be in the upstream region of a putative peroxidase gene, AT2G24800. Here we report detailed histochemical and molecular characterization of the mutant Anth85, carrying a single insertion of T-DNA in the peroxidase gene. In Anth85, the GUS expression was observed in the anthers and rosette of the young seedlings. The expression of GUS in the anthers was restricted to the tapetum and microspores. The mutant has no developmental defects and the gene appears to be redundant for normal plant growth. Cloning of upstream region and detailed deletion study of upstream region in transgenic plants is likely to lead to the identification of anther specific promoter elements.     

Genetic control of male fertility in Arabidopsis thaliana: structural analysis of premeiotic developmental mutants

We have taken a mutational approach to identify genes important for male fertility in Arabidopsis thaliana and have isolated a number of nuclear male/ sterile mutants in which vegetative growth and female fertility are not altered. Here we describe detailed developmental analyses of four mutants, each of which defines a complementation group and has a distinct developmental end point. All four mutants represent premeiotic developmental lesions. In ms3, tapetum and middle layer hypertrophy result in the degeneration of microsporocytes. In ms4, microspore dyads persist for most of anther development as a result of impaired meiotic division. In ms5, degeneration occurs in all anther cells at an early stage of development. In ms15, both the tapetum and microsporocytes degenerate early in anther development. Each of these mutants had shorter filaments and a greater number of inflorescences than congenic male-fertile plants. The differences in the developmental phenotypes of these mutants, together with the non-allelic nature of the mutations indicate that four different genes important for pollen development, have been identified.     

Collection of Arabidopsis thalianaMorphological Insertion Mutants

A collection of transgenic Arabidopsis thalianaplants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12–18% of the total number of A. thalianagenes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.     

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization

 The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution. Received: 21 April 1998 / Revision accepted: 11 June 1998     

A High Throughput Genetic Screen Identifies New Early Meiotic Recombination Functions in Arabidopsis thaliana

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.     

Distinct contributions of β4GalNAcTA and β4GalNAcTB to <Emphasis Type="Italic">Drosophila</Emphasis> glycosphingolipid biosynthesis

Drosophila melanogaster has two β4-N-acetylgalactosaminyltransferases, β4GalNAcTA and β4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcβ,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that β4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the β4GalNAcT trisaccharide acceptor structure GlcNAcβ,3Manβ,4GlcβCer, are lethal. In contrast, flies doubly mutant for the β4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in β4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcβ,3Manβ,4GlcβCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the β4GalNAcT single mutants we show that β4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In β4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in β4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that β4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both β4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcβ,3Manβ,4GlcβCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.     

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium

We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (β-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait. Received: 26 October 1998 / Accepted: 8 January 1999     

Genetic analysis of leaf form mutants from the Arabidopsis Information Service collection.

Although a vast inventory of morphological mutants of Arabidopsis thaliana is available, only some have been used for genetic studies of leaf development. Such is the case with the Arabidopsis Information Service (AIS) Form Mutants collection, assembled by A. R. Kranz and currently stored at the Nottingham Arabidopsis Stock Centre, which includes a large number of mutant lines, most of which have been little studied. With the aim of contributing to the genetic dissection of leaf ontogeny, we have subjected 57 mutant lines isolated by others to genetic analysis; 47 of which were from the AIS collection. These are characterized by vegetative leaves of abnormal shape or size, and were chosen as candidates for mutations in genes required for leaf morphogenesis. The mutant phenotypes studied were shown to be inherited as single recessive Mendelian traits and were classified into 10 phenotypic classes. These mutant strains were found to fall into 37 complementation groups, 7 of which corresponded to known genes. Results of the phenotypic analysis and data on the genetic interactions of these mutants are presented, and their possible developmental defects discussed. Received: 28 October 1998 / Accepted: 21 February 1999     

温光敏核不育水稻N28S 无花粉败育的显微结构观察

采用石蜡切片和荧光显微技术观察了温光敏核不育水稻N28S 无花粉败育过程中的显微结构变化, 结果显示: N28S 的小孢子母细胞形成后细胞质变得稀薄, 一部分不能进行减数分裂, 一部分减数分裂阻滞在细线期或胞质分裂异常, 最终所有细胞液泡化解体消失。在此过程中, 还观察到小孢子母细胞在细线期胼胝质壁不产生或提早消失, 以及小孢子发育后期花药壁绒毡层的异常解体。认为N28S 的无花粉败育是由小孢子母细胞的细胞质异常引起的, 胼胝质壁和绒毡层的异常是结果而不是原因。     

Insertional mutagenesis in Arabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology

Summary We investigated the potential of the Agrobacterium tumefaciens T-DNA as an insertional mutagen in Arabidopsis thaliana. Arabidopsis lines transformed with different T-DNA vectors were generated using a leaf disc infection procedure adapted for efficient selection on either kanamycin or hygromycin medium. A standardized screening procedure was developed for the detection of recessive mutations in T2 populations of regenerated and/or transformed lines. Recessive mutations originating from the tissue culture procedure occurred at a low frequency — between 2% and 5%. Within 110 transformed lines that contained a total of about 150 T-DNA inserts, one recessive mutation, named pfl, cosegregated with a specific T-DNA copy. This pfl mutation mainly affected the morphology of the first seedling leaves under normal growth conditions and was mapped to chromosome 1. No recombination between the pfl locus and the kanamycin resistance marker on the T-DNA was detected when screening F2 and F3 populations of a mutant crossed to the wild type. The maximal genetic distance between the pfl locus and the kanamycin resistance gene, determined as 0.4±0.4 cMorgan, strongly suggests that the pfl mutation is induced by the insertion of the T-DNA. Our finding of one T-DNA-linked recessive mutation in 110 transgenic lines indicates that T-DNA can be used for mutagenization of the Arabidopsis genome under tissue culture conditions.     

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.     

Rice <Emphasis Type="Italic">Importin β1</Emphasis> gene affects pollen tube elongation

Importin β1 interacts with nuclear transport factors and mediates the import of nuclear proteins. We isolated a pollen-expressed gene, rice Importin β1 (OsImpβ1), from a T-DNA insertional population that was trapped by a promoterless β-glucuronidase (GUS) gene. The GUS reporter was expressed in the anthers and ovaries from early through mature developmental stages. Its expression was also observed in all floral organs. However, these patterns changed as the spikelet developed. T-DNA was inserted into the OsImpβ1 gene at 339 bp downstream from the translation initiation site. We obtained another T-DNA insertional allele by searching the flanking sequence tag database. In both lines, the wild-type and T-DNA-carrying progeny segregated at a ratio close to 1:1. The latter genotype was heterozygous (OsImpβ1/osimpβ1). Reciprocal crosses between WT and heterozygous plants demonstrated that the mutant alleles could not be transmitted through the male gametophyte. Close examination of the heterozygous anthers revealed that the mutant pollen matured normally. However, in vitro assays showed that tube elongation was hampered in the mutant grains. These results indicate that OsImpβ1 is specifically required for pollen tube elongation.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Searching for tagged male-sterile mutants of arabidopsis

Although many male-sterile mutants have been identified inArbidopsis thaliana, few of the corresponding genes have been cloned. In order to facilitate cloning of a male sterility gene, 23 of Feldmann's T-DNA-generated, reduced-fertility lines were screened to identify a tagged male-sterile mutation. Malesterile mutants were identified, as well as mutants that were both male and female sterile. Segregation of the kanamycin marker gene in the progeny of 15 of these lines was studied. Forty percent had functional T-DNAs (encoding resistance to kanamycin) inserted at a single locus, the remainder segregating for two or more functional T-DNA inserts. Linkage between T-DNA inserts and mutant phenotype was tested for six lines. In three of these lines, mutations were not linked to a T-DNA insert. In three lines, the mutation segregated with a T-DNA insert.