RanGAP is required for post-meiotic mitosis in female gametophyte development in Arabidopsis thaliana

RanGAP is the GTPase-activating protein of the small GTPase Ran and is involved in nucleocytoplasmic transport in yeast and animals via the Ran cycle and in mitotic cell division. Arabidopsis thaliana has two copies of RanGAP, RanGAP1 and RanGAP2. To investigate the function of plant RanGAP, T-DNA insertional mutants were analysed. Arabidopsis plants with a null mutant of either RanGAP1 or RanGAP2 had no observable phenotype. Analysis of segregating progeny showed that double mutants in RanGAP1 and RanGAP2 are female gametophyte defective. Ovule clearing with differential interference contrast optics showed that mutant female gametophytes were arrested at interphase, predominantly after the first mitotic division following meiosis. In contrast, mutant pollen developed and functioned normally. These results show that the two RanGAPs are redundant and indispensable for female gametophyte development in Arabidopsis but dispensable for pollen development. Nuclear division arrest during a mitotic stage suggests a role for plant RanGAP in mitotic cell cycle progression during female gametophyte development.     

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.     

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.     

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.     

Arabidopsis thaliana PDX1.2 is critical for embryo development and heat shock tolerance

Main conclusion

PDX1.2 is expressed in the basal part of the globular-stage embryo, and plays critical roles in development, hypocotyl elongation, and stress response.

Abstract

The Arabidopsis thaliana PDX1.2 protein belongs to a small family of three members. While PDX1.1 and PDX1.3 have been extensively described and are well established to function in vitamin B₆ biosynthesis, the biological role of PDX1.2 still remains elusive. Here, we show that PDX1.2 is expressed early in embryo development, and that heat shock treatment causes a strong up-regulation of the gene. Using a combined genetic approach of T-DNA insertion lines and expression of artificial micro RNAs, we can show that PDX1.2 is critically required for embryo development, and for normal hypocotyl elongation. Plants with reduced PDX1.2 expression also display reduced primary root growth after heat shock treatments. The work overall provides a set of important new findings that give greater insights into the developmental role of PDX1.2 in plants.     

A lectin receptor-like kinase is required for pollen development in Arabidopsis

Lectin receptor-like kinases (Lectin RLKs) are a large family of receptor-like kinases with an extracellular legume lectin-like domain. There are approximately 45 such receptor kinases in Arabidopsis thaliana. Surprisingly, although receptor-like kinases in general are well investigated in Arabidopsis, relatively little is known about the functions of members of the Lectin RLK family. A number of studies implicated members of this family in various functions, such as disease resistance, stress responses, hormone signaling, and legume-rhizobium symbiosis. Our current work demonstrated that mutation in one Lectin RLK gene led to male sterility in Arabidopsis. The sterility was due to defects in pollen development. Pollen development proceeded normally in the mutant until anther stage 8. After that, all pollen grains deformed and collapsed. Mature pollen grains were much smaller than wild-type pollen grains, glued together, and totally collapsed. Therefore, the mutant was named sgc, standing for small, glued-together, and collapsed pollen mutant. The mutant phenotype appeared to be caused by an unidentified sporophytic defect due to the mutation. As revealed by analysis of the promoter-GUS transgenic plants and the gene expression analysis using RT-PCR, the gene showed an interesting temporal and spatial expression pattern: it had no or a low expression in young flowers (roughly before anther stage 6), reached a maximum level around stages 6-7, and then declined gradually to a very low level in young siliques. No expression was detected in microspores or pollen. Together, our data demonstrated that SGC Lectin RLK plays a critical role in pollen development.     

The ubiquitin-specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana

The ubiquitin/26S proteasome pathway is a major route for selectively degrading cytoplasmic and nuclear proteins in eukaryotes. In this pathway, chains of ubiquitins become attached to short-lived proteins, signalling recognition and breakdown of the modified protein by the 26S proteasome. During or following target degradation, the attached multi-ubiquitin chains are released and subsequently disassembled by ubiquitin-specific proteases (UBPs) to regenerate free ubiquitin monomers for re-use. Here, we describe Arabidopsis thaliana UBP14 that may participate in this recycling process. Its amino acid sequence is most similar to yeast UBP14 and its orthologues, human IsoT1-3 and Dictyostelium UbpA, and it can functionally replace yeast UBP14 in a ubp14Delta mutant. Like its orthologues, AtUBP14 can disassemble multi-ubiquitin chains linked internally via epsilon-amino isopeptide bonds using Lys48 and can process some, but not all, translational fusions of ubiquitin linked via alpha-amino peptide bonds. However, unlike its yeast and Dictyostelium orthologues, AtUBP14 is essential in Arabidopsis. T-DNA insertion mutations in the single gene that encodes AtUBP14 cause an embryonic lethal phenotype, with the homozygous embryos arresting at the globular stage. The arrested seeds have substantially increased levels of multi-ubiquitin chains, indicative of a defect in ubiquitin recycling. Taken together, the data demonstrate an essential role for the ubiquitin/26S proteasome pathway in general and for AtUBP14 in particular during early plant development.     

A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.     

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.     

EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis

Chloroplast biogenesis is tightly linked with embryogenesis and seedling development. A growing body of work has been done on the molecular mechanisms underlying chloroplast development; however, the molecular components involved in chloroplast biogenesis during embryogenesis remain largely uncharacterized. In this paper, we show that an Arabidopsis mutant carrying a T‐DNA insertion in a gene encoding a multiple membrane occupation and recognition nexus (MORN)‐containing protein exhibits severe defects during embryogenesis, producing abnormal embryos and thereby leading to a lethality of young seedlings. Genetic and microscopic studies reveal that the mutation is allelic to a previously designated Arabidopsis embryo‐defective 1211 mutant (emb1211). The emb1211 +/? mutant plants produce approximately 25% of white‐colored ovules with abnormal embryos since late globular stage when primary chloroplast biogenesis takes place, while the wild‐type plants produce all green ovules. Transmission electron microscopic analysis reveals the absence of normal chloroplast development, both in the mutant embryos and in the mutant seedlings, that contributes to the albinism. The EMB1211 gene is preferentially expressed in developing embryos as revealed in the EMB1211::GUS transgenic plants. Taken together, the data indicate that EMB1211 has an important role during embryogenesis and chloroplast biogenesis in Arabidopsis.