SLOW WALKER1, essential for gametogenesis in Arabidopsis, encodes a WD40 protein involved in 18S ribosomal RNA biogenesis

The progression of mitotic division cycles and synchronous development between and within the male and female reproductive organs are essential for plant sexual reproduction. Little is known about the genetic control of the progression of mitotic cycles of the haploid genome during gametogenesis in higher plants. Here, we report the phenotypic and molecular characterization of an Arabidopsis thaliana mutant, slow walker1 (swa1), in which the progression of the mitotic division cycles of the female gametophyte was disrupted. Confocal microscopy revealed that megagametophyte development was asynchronous in swa1, causing embryo sacs to arrest at two-, four-, or eight-nucleate stages within the same pistil. A delayed pollination experiment showed that a small fraction of the swa1 embryo sacs were able to develop into functional female gametophytes. The swa1 mutation also showed a slight reduction in penetrance through the male gametophyte, although the pollen grains were morphologically normal. Molecular analysis indicates that SWA1 encodes a protein with six WD40 repeats that is localized in the nucleolus in interphase cells. The SWA1 gene is expressed in cells undergoing active cell divisions, including functional megaspores and the female gametophytic cells. RNA interference results indicated that knockout of SWA1 inhibited root growth significantly and led to the accumulation of unprocessed 18S pre-rRNA. These data suggest that SWA1 most likely plays a role in rRNA biogenesis that is essential for the progression of the mitotic division cycles during gametogenesis in plants.     

Dbp9p, a member of the DEAD box protein family, exhibits DNA helicase activity

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins.     

Haplo-insufficiency of MPK3 in MPK6 mutant background uncovers a novel function of these two MAPKs in Arabidopsis ovule development

The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage-dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3(-/-) mpk6(+/-) plants are fertile. Genetic analysis revealed that the female sterility of mpk3(+/-) mpk6(-/-) plants is a sporophytic effect. In mpk3(+/-) mpk6(-/-) mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3(+/-) mpk6(-/-) ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development.     

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A₁ and A₂ are inhibited and delayed at site A₀. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.     

GAMETOPHYTIC FACTOR 1, Involved in Pre-mRNA Splicing, Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 (GFA1); a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homoiogs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogeneeis and embryogenesis in plant.     

Nuclear behavior is defective in the maize (Zea mays L.) lethal ovule2 female gametophyte

It has long been known that the maize lethal ovule2 mutation results in ovule abortion but has a much smaller effect on pollen development or function. The behavior of the nuclei, the microtubular cytoskeleton and other events were examined in normal and lo2 mutant female gametophytes in order to obtain an understanding the role of this gene in embryo sac formation. The effect of the lo2 mutation is manifested following meiosis. When the surviving single megaspore carries the mutant lo2 allele, often both the megaspore and its nucleus greatly enlarge, but the nucleus either fails to divide or divides only once or twice. Micronuclei are frequently present, nuclei are often clustered and the abundance and patterns of microtubules are abnormal in the mutant embryo sacs. The mutant female gametophytes are blocked at the one-, two- or four-nucleate stage. Nearly all the embryo sacs containing the lo2 allele fail to function as evidenced by the failure of transmission of closely linked loci. When mutant female gametophyte development is arrested, the immature embryo sac degenerates. This mutation appears to identify a gene that is essential in the female gametophyte for normal nuclear division and migration and the normal accompanying tubulin cytoskeleton behavior.     

SNAIL1 is essential for female gametogenesis in Arabidopsis thaliana

Two yeast Brix family members Ssf1 and Ssf2,involved in large ribosomal subunit synthesis, are essential for yeast cell viability and mating efficiency. Their putative homologs exist in the Arabidopsis genome; however, their role in plant development is unknown. Here, we show that Arabidopsis thaliana SNAIL1(At SNAIL1), a protein sharing high sequence identity with yeast Ssf1 and Ssf2, is critical to mitosis progression of female gametophyte development.The snail1 homozygous mutant was nonviable and its heterozygous mutant was semi-sterile with shorter siliques.The mutation in SNAIL1 led to absence of female transmission and reduced male transmission. Further phenotypic analysis showed that the synchronic development of female gametophyte in the snail1 heterozygous mutant was greatly impaired and the snail1 pollen tube growth, in vivo, was also compromised. Furthermore, SNAIL1 was a nucleolarlocalized protein with a putative role in protein synthesis.Our data suggest that SNAIL1 may function in ribosome biogenesis like Ssf1 and Ssf2 and plays an important role during megagametogenesis in Arabidopsis.     

Comprehensive mutational analysis of yeast DEXD/H box RNA helicases involved in large ribosomal subunit biogenesis

DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.     

A rice DEAD-box protein, OsRH36, can complement an Arabidopsis atrh36 mutant, but cannot functionally replace its yeast homolog Dbp8p

DEAD-box proteins comprise a large family and function in RNA metabolism. Although DEAD-box proteins are highly conserved among eukaryotes, little is known about the role of DEAD-box proteins in rice. In this study, we identified a rice DEAD-box protein, OsRH36, and demonstrated that OsRH36 and AtRH36 are functional orthologs. OsRH36 was expressed ubiquitously throughout the plant and the OsRH36-GFP fusion protein was localized to the nucleus and nucleolus. Furthermore, functional complementation tests among three homologous DEAD-box protein genes indicated that OsRH36 can restore segregation distortion in the Arabidopsis atrh36-1 mutant, but cannot complement the lethal phenotype in the yeast dbp8p mutant. Moreover, mutation of osrh36 also led to defective transmission of the osrh36 allele, as shown for the atrh36 mutants of Arabidopsis. Previously, we have shown that AtRH36 is involved at an early stage of rRNA maturation and is required for synchronous development of female gametophytes in Arabidopsis. Therefore, we suggest that OsRH36 is required for rRNA biogenesis and megagametogenesis in rice, consistent with the function of AtRH36 in Arabidopsis.     

Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1

In Arabidopsis thaliana, the female gametophyte is a highly polarized structure consisting of four cell types: one egg cell and two synergids, one central cell, and three antipodal cells. In this report, we describe the characterization of a novel female gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac. The eostre phenotype is caused by misexpression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac. It is known that BELL-KNAT proteins function as heterodimers whose activities are regulated by the Arabidopsis ovate family proteins (OFPs). We show that the phenotypic effect of BLH1 overexpression is dependent upon the class II knox gene KNAT3, suggesting that KNAT3 must be expressed and functional during megagametogenesis. Moreover, disruption of At OFP5, a known interactor of KNAT3 and BLH1, partially phenocopies the eostre mutation. Our study indicates that suppression of ectopic activity of BELL-KNOX TALE complexes, which might be mediated by At OFP5, is essential for normal development and cell specification in the Arabidopsis embryo sac. As eostre-1 embryo sacs also show nuclear migration abnormalities, this study suggests that a positional mechanism might be directing establishment of cell fates in early megagametophyte development.