Arabidopsis thaliana NOP10 is required for gametophyte formation

The female gametophyte is crucial for sexual reproduction of higher plants, yet little is known about the molecular mechanisms underlying its development. Here,we report that Arabidopsis thaliana NOP10(AtNOP10) is required for female gametophyte formation. AtNOP10 was expressed predominantly in the seedling and reproductive tissues, including anthers, pollen grains, and ovules.Mutations in AtNOP10 interrupted mitosis of the functional megaspore during early development and prevented polar nuclear fusion in the embryo sacs. AtNOP10 shares a high level of amino acid sequence similarity with Saccharomyces cerevisiae(yeast) NOP10(ScNOP10), an important component of the H/ACA small nucleolar ribonucleoprotein particles(H/ACA sno RNPs) implicated in 18 S r RNA synthesis and r RNA pseudouridylation. Heterologous expression of ScNOP10 complemented the mutant phenotype of Atnop10. Thus, AtNOP10 influences functional megaspore mitosis and polar nuclear fusion during gametophyte formation in Arabidopsis.     

Acetylglutamate kinase is required for both gametophyte function and embryo development in Arabidopsis thaliana

The specific functions of the genes encoding arginine biosynthesis enzymes in plants are not well characterized. We report the isolation and characterization of Arabidopsis thaliana N-acetylglutamate kinase(NAGK), which catalyzes the second step of arginine biosynthesis. NAGK is a plastid-localized protein and is expressed during most developmental processes in Arabidopsis. Heterologous expression of the Arabidopsis NAGK gene in a NAGK-deficient Escherichia coli strain fully restores bacterial growth on arginine-deficient medium. nagk mutant pollen tubes grow more slowly than wild type pollen tubes and the phenotype is restored by either specifically through complementation by NAGK in pollen, or exogenous supplementation of arginine. nagk female gametophytes are defective in micropylar pollen tube guidance due to the fact that female gametophyte cell fate specification was specifically affected. Expression of NAGK in synergid cells rescues the defect of nagk female gametophytes. Lossof-function of NAGK results in Arabidopsis embryos not developing beyond the four-celled embryo stage. The embryo-defective phenotype in nagk/NAGK plants cannot be rescued by watering nagk/NAGK plants with arginine or ornithine supplementation. In conclusion,our results reveal a novel role of NAGK and arginine in regulating gametophyte function and embryo development, and provide valuable insights into arginine transport during embryo development.     

MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana

The female gametophyte plays a central role in the sexual reproduction of angiosperms. We previously isolated the maa3 (magatama3) mutant of Arabidopsis thaliana, defective in development of the female gametophyte, micropylar pollen tube guidance, and preventing the attraction of multiple pollen tubes. We here observed that the nucleolus of polar nuclei is small, and that the fusion of polar nuclei often did not occur at the time of pollination. The MAA3 gene encodes a homolog of yeast Sen1 helicase, required for RNA metabolism. It is suggested that MAA3 may regulate RNA molecules responsible for nucleolar organization and pollen tube guidance.     

The MIDASIN and NOTCHLESS genes are essential for female gametophyte development in Arabidopsis thaliana

Female gametophyte development in Arabidopsis thaliana follows a well-defined program that involves many fundamental cellular processes. In this study, we report the involvement of the Arabidopsis thaliana MIDASIN1 (AtMDN1) gene during female gametogenesis through the phenotypic characterization of plants heterozygous for an insertional mdn1 mutant allele. The MDN1 yeast ortholog has previously been shown to encode a non-ribosomal protein involved in the maturation and assembly of the 60S ribosomal subunit. Heterozygous MDN1/mdn1 plants were semisterile and mdn1 allele transmission through the female gametophyte was severely affected. Development of mdn1 female gametophyte was considerably delayed compared to their wild-type siblings. However, delayed mdn1 female gametophytes were able to reach maturity and a delayed pollination experiment showed that a small proportion of the female gametophytes were functional. We also report that the Arabidopsis NOTCHLESS (AtNLE) gene is also required for female gametogenesis. The NLE protein has been previously shown to interact with MDN1 and to be also involved in 60S subunit biogenesis. The introduction of an AtNLE-RNA interference construct in Arabidopsis led to semisterility defects. Defective female gametophytes were mostly arrested at the one-nucleate (FG1) developmental stage. These data suggest that the activity of both AtMDN1 and AtNLE is essential for female gametogenesis progression.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization

Karyogamy, or nuclear fusion, is essential for sexual reproduction. In angiosperms, karyogamy occurs three times: twice during double fertilization of the egg cell and the central cell and once during female gametophyte development when the two polar nuclei fuse to form the diploid central cell nucleus. The molecular mechanisms controlling karyogamy are poorly understood. We have identified nine female gametophyte mutants in Arabidopsis (Arabidopsis thaliana), nuclear fusion defective1 (nfd1) to nfd9, that are defective in fusion of the polar nuclei. In the nfd1 to nfd6 mutants, failure of fusion of the polar nuclei is the only defect detected during megagametogenesis. nfd1 is also affected in karyogamy during double fertilization. Using transmission electron microscopy, we showed that nfd1 nuclei fail to undergo fusion of the outer nuclear membranes. nfd1 contains a T-DNA insertion in RPL21M that is predicted to encode the mitochondrial 50S ribosomal subunit L21, and a wild-type copy of this gene rescues the mutant phenotype. Consistent with the predicted function of this gene, an NFD1-green fluorescent protein fusion protein localizes to mitochondria and the NFD1/RPL21M gene is expressed throughout the plant. The nfd3, nfd4, nfd5, and nfd6 mutants also contain T-DNA insertions in genes predicted to encode proteins that localize to mitochondria, suggesting a role for this organelle in nuclear fusion.     

DRB2 is required for microRNA biogenesis in Arabidopsis thaliana

Background

The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants).

Principal Findings

Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants.

Conclusions/Significance

Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue.     

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.     

Arabidopsis thaliana GEX1 has dual functions in gametophyte development and early embryogenesis

GEX1 is a plasma membrane protein that is conserved among plant species, and has previously been shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immediately after fertilization and in the pollen vegetative cell. We functionally characterize GEX1 in Arabidopsis thaliana, and show that it is a versatile protein that performs functions during male and female gametophyte development, and during early embryogenesis. gex1-1/+ plants, which synthesize a truncated GEX1 mRNA encoding a protein lacking the predicted cytoplasmic domain, but still targeted to the plasma membrane, had embryos that arrested before the pre-globular stage. gex1-3/+ plants, carrying a null GEX1 allele, had defects during male and female gametophyte development, and during early embryogenesis. Using an antisense GEX1 transgenic line we demonstrate that the predicted GEX1 extracellular domain is sufficient and necessary for GEX1 function during the development of both gametophytes. The predicted cytoplasmic domain is necessary for correct early embryogenesis and mediates homodimer formation at the plasma membrane. We propose that dimerization of GEX1 in the zygote might be an upstream step in a signaling cascade regulating early embryogenesis.     

Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana

Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and γGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of γ-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.     

The Arabidopsis dynamin-related protein2 family is essential for gametophyte development

Clathrin-mediated membrane trafficking is critical for multiple stages of plant growth and development. One key component of clathrin-mediated trafficking in animals is dynamin, a polymerizing GTPase that plays both regulatory and mechanical roles. Other eukaryotes use various dynamin-related proteins (DRP) in clathrin-mediated trafficking. Plants are unique in the apparent involvement of both a family of classical dynamins (DRP2) and a family of dynamin-related proteins (DRP1) in clathrin-mediated membrane trafficking. Our analysis of drp2 insertional mutants demonstrates that, similar to the DRP1 family, the DRP2 family is essential for Arabidopsis thaliana development. Gametophytes lacking both DRP2A and DRP2B were inviable, arresting prior to the first mitotic division in both male and female gametogenesis. Mutant pollen displayed a variety of defects, including branched or irregular cell plates, altered Golgi morphology and ectopic callose deposition. Ectopic callose deposition was also visible in the pollen-lethal drp1c-1 mutant and appears to be a specific feature of pollen-defective mutants with impaired membrane trafficking. However, drp2ab pollen arrested at earlier stages in development than drp1c-1 pollen and did not accumulate excess plasma membrane or display other gross defects in plasma membrane morphology. Therefore, the DRP2 family, but not DRP1C, is necessary for cell cycle progression during early gametophyte development. This suggests a possible role for DRP2-dependent clathrin-mediated trafficking in the transduction of developmental signals in the gametophyte.