Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. To date, to our knowledge, no direct evidence exists for the involvement of RGPs in a particular biochemical process. The Arabidopsis (Arabidopsis thaliana) genome contains five RGP genes out of which RGP1 and RGP2 share the highest sequence identity. We characterized the native expression pattern of Arabidopsis RGP1 and RGP2 and used reverse genetics to investigate their respective functions. Although both genes are ubiquitously expressed, the highest levels are observed in actively growing tissues and in mature pollen, in particular. RGPs showed cytoplasmic and transient association with Golgi. In addition, both proteins colocalized in the same compartments and coimmunoprecipitated from plant cell extracts. Single-gene disruptions did not show any obvious morphological defects under greenhouse conditions, whereas the double-insertion mutant could not be recovered. We present evidence that the double mutant is lethal and demonstrate the critical role of RGPs, particularly in pollen development. Detailed analysis demonstrated that mutant pollen development is associated with abnormally enlarged vacuoles and a poorly defined inner cell wall layer, which consequently results in disintegration of the pollen structure during pollen mitosis I. Taken together, our results indicate that RGP1 and RGP2 are required during microspore development and pollen mitosis, either affecting cell division and/or vacuolar integrity.     

Exportin1 genes are essential for development and function of the gametophytes in Arabidopsis thaliana

Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recruited at the kinetochores during mitosis. Here we show that double mutants in Arabidopsis XPO1A and XPO1B are gametophytic defective. In homozygous–heterozygous plants, 50% of the ovules were arrested at different stages according to the parental genotype. Double-mutant female gametophytes of xpo1a-3/+; xpo1b-1/xpo1b-1 plants failed to undergo all the mitotic divisions or failed to complete embryo sac maturation. Double-mutant female gametophytes of xpo1a-3/xpo1a-3; xpo1b-1/+ plants had normal mitotic divisions and fertilization occurred; in most of these embryo sacs the endosperm started to divide but an embryo failed to develop. Distortions in male transmission correlated with the occurrence of smaller pollen grains, poor pollen germination, and shorter pollen tubes. Our results show that mitotic divisions are possible without XPO1 during the haploid phase, but that XPO1 is crucial for the maternal-to-embryonic transition.     

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.     

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate-limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1-/- ADC2-/-) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1-1 and adc2-2 had the ADC1-/- ADC2-/- genotype. In addition, some abnormal seeds were observed among the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- siliques. Viable offspring with the ADC1-/- ADC2-/- genotype could not be obtained from the ADC1+/- ADC2-/- and ADC1-/- ADC2+/- plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.     

The FORKED genes are essential for distal vein meeting in Arabidopsis

As in most dicotyledonous plants, the leaves and cotyledons of Arabidopsis have a closed, reticulate venation pattern. This pattern is proposed to be generated through canalization of the hormone auxin. We have identified two genes, FORKED 1 (FKD1) and FORKED 2 (FKD2), that are necessary for the closed venation pattern: mutations in either gene result in an open venation pattern that lacks distal meeting. In fkd1 leaves and cotyledons, the defect is first evident in the provascular tissue, such that the distal end of the newly forming vein does not connect to the previously formed, more distal vein. Plants doubly mutant for both genes have widespread defects in leaf venation, suggesting that the genes function in an overlapping manner at the distal junctions, but act redundantly throughout leaf veins. Expression of an auxin responsive reporter gene is reduced in fkd1 leaves, suggesting that FKD1 is necessary for the auxin response that directs vascular tissue development. The reduction in reporter gene expression and the fkd1 phenotype are relieved in the presence of auxin transport inhibition. The restoration of vein junctions in situations where auxin concentrations are increased indicates that distal vein junctions are sites of low auxin concentration and are particularly sensitive to reduced FKD1 and FKD2 activity.     

QQT proteins colocalize with microtubules and are essential for early embryo development in Arabidopsis

During Arabidopsis embryogenesis, the control of division between daughter cells is critical for pattern formation. Two embryo-defective (emb) mutant lines named quatre-quart (qqt) were characterized by forward and reverse genetics. The terminal arrest of qqt1 and qqt2 embryos was at the octant stage, just prior to the round of periclinal divisions that establishes the dermatogen stage . Homozygous embryos of a weaker allele of qqt1 were able to divide further, resulting in aberrant periclinal divisions. These phenotypic analyses support an essential role of the QQT proteins in the correct formation of the tangential divisions. That an important proportion of qqt1 embryos were arrested prior to the octant stage indicated a more general role in cell division. The analysis of QQT1 and QQT2 genes revealed that they belong to a small subgroup of the large family encoding ATP/GTP binding proteins, and are widely conserved among plants, vertebrates and Archaea. We showed that QQT1 and QQT2 proteins interact with each other in a yeast two-hybrid system, and that QQT1 and QQT2 tagged by distinct fluorescent probes colocalize with microtubules during mitosis, in agreement with their potential role in cell division and their mutant phenotype. We propose that QQT1 and QQT2 proteins participate in the organization of microtubules during cell division, and that this function is essential for the correct development of the early embryo.     

Spermidine synthase genes are essential for survival of Arabidopsis

The cellular polyamines putrescine, spermidine, and spermine are ubiquitous in nature and have been implicated in a wide range of growth and developmental processes. There is little information, however, on mutant plants or animals defective in the synthesis of polyamines. The Arabidopsis genome has two genes encoding spermidine synthase, SPDS1 and SPDS2. In this paper, we describe T-DNA insertion mutants of both of these genes. While each mutant allele shows normal growth, spds1-1 spds2-1 double-mutant seeds are abnormally shrunken and they have embryos that are arrested morphologically at the heart-torpedo transition stage. These seeds contain significantly reduced levels of spermidine and high levels of its precursor, putrescine. The embryo lethal phenotype of spds1-1 spds2-1 is complemented by the wild-type SPDS1 gene. In addition, we observed a nearly identical seed phenotype among an F2 seed population from the cross between the spds2-1 allele and SPDS1 RNA interference transgenic lines. These data provide the first genetic evidence indicating a critical role of the spermidine synthase in plant embryo development.     

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation

Among the >200 members of the leucine-rich repeat receptor kinase family in Arabidopsis thaliana, only a few have been functionally characterized. Here, we report a critical function in anther development for the SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SERK1) and SERK2 genes. Both SERK1 and SERK2 are expressed widely in locules until stage 6 anthers and are more concentrated in the tapetal cell layer later. Whereas serk1 and serk2 single insertion mutants did not show developmental phenotypes, serk1 serk2 double mutants were not able to produce seeds because of a lack of pollen development in mutant anthers. In young buds, double mutant anthers developed normally, but serk1 serk2 microsporangia produced more sporogenous cells that were unable to develop beyond meiosis. Furthermore, serk1 serk2 double mutants developed only three cell layers surrounding the sporogenous cell mass, whereas wild-type anthers developed four cell layers. Further confocal microscopic and molecular analyses showed that serk1 serk2 double mutant anthers lack development of the tapetal cell layer, which accounts for the microspore abortion and male sterility. Taken together, these findings demonstrate that the SERK1 and SERK2 receptor kinases function redundantly as an important control point for sporophytic development controlling male gametophyte production.     

The MYST histone acetyltransferases are essential for gametophyte development in Arabidopsis

Background  

Histone acetyltransferases (HATs) play critical roles in the regulation of chromatin structure and gene expression. Arabidopsis genome contains 12 HAT genes, but the biological functions of many of them are still unknown. In this work, we studied the evolutionary relationship and cellular functions of the two Arabidopsis HAT genes homologous to the MYST family members.     

Characterization of Arabidopsis decapping proteins AtDCP1 and AtDCP2, which are essential for post-embryonic development

Although decapping is an important process in eukaryotic mRNA turnover, little is known about this process in plants. Here, we identified Arabidopsis thaliana decapping proteins AtDCP1 and AtDCP2 and showed that (I) AtDCP2 is an active decapping enzyme, (II) AtDCP1 interacts with itself, (III) AtDCP1 and AtDCP2 are localized to cytoplasmic foci (putative Arabidopsis processing body), and (IV) AtDCP1 and AtDCP2 are essential for post-embryonic development. Our findings provide new insights into the role of decapping-dependent mRNA turnover.     

The BAM1/BAM2 receptor-like kinases are important regulators of Arabidopsis early anther development

Anther development involves the formation of several adjacent cell types required for normal male fertility. Only a few genes are known to be involved in early anther development, particularly in the establishment of these different cell layers. Arabidopsis thaliana BAM1 (for BARELY ANY MERISTEM) and BAM2 encode CLAVATA1-related Leu-rich repeat receptor-like kinases that appear to have redundant or overlapping functions. We characterized anther development in the bam1 bam2 flowers and found that bam1 bam2 anthers appear to be abnormal at a very early stage and lack the endothecium, middle, and tapetum layers. Analyses using molecular markers and cytological techniques of bam1 bam2 anthers revealed that cells interior to the epidermis acquire some characteristics of pollen mother cells (PMCs), suggesting defects in cell fate specification. The pollen mother-like cells degenerate before the completion of meiosis, suggesting that these cells are defective. In addition, the BAM1 and BAM2 expression pattern supports both an early role in promoting somatic cell fates and a subsequent function in the PMCs. Therefore, analysis of BAM1 and BAM2 revealed a cell-cell communication process important for early anther development, including aspects of cell division and differentiation. This finding may have implications for the evolution of multiple signaling pathways in specifying cell types for microsporogenesis.     

Mapping genes essential for embryo development in Arabidopsis thaliana.

Summary We have previously isolated and characterized over 90 recessive mutants of Arabidopsis thaliana defective in embryo development. These emb mutants have been shown to differ in lethal phase, extent of abnormal development, and response in culture. We demonstrate in this report the value and efficiency of mapping emb genes relative to visible and molecular markers. Sixteen genes essential for embryo development were mapped relative to visible markers by analyzing progeny of selfed F₁ plants. Embryonic lethals are now the most common type of visible marker included on the linkage map of Arabidopsis. Backcrosses were used in several cases to orient genes relative to adjacent markers. Three genes were located to chromosome arms with telotrisomics by screening for a reduction in the percentage of aborted seeds produced by F₁ plants. A restriction fragment length polymorphism (RFLP) mapping strategy that utilizes pooled EMB/EMB F₂ plants was devised to increase the efficiency of mapping embryonic lethals relative to molecular markers. This strategy was tested by demonstrating that the biol locus of Arabidopsis is within 0.5 cM of an existing RFLP marker. Mapping embryonic lethals with both visible and molecular markers may therefore help to identify large numbers of genes with essential functions in Arabidopsis.     

An Arabidopsis homolog of RAPTOR/KOG1 is essential for early embryo development

The RAPTOR/KOG1 proteins are binding partners of the target of rapamycin (TOR) kinase that is present in all eucaryotes and plays a central role in the stimulation of cell growth and metabolism in response to nutrients. We show in this report that two genes are coding for RAPTOR/KOG1 homologs in the Arabidopsis and rice genomes. Disruption of the Arabidopsis AtRaptor1 gene leads to a very early arrest of embryo development whereas disruption of the AtRaptor2 gene, which is expressed at a lower level than AtRaptor1, has no visible effects on embryo and plant development. We also observed that mutations in the AtRaptor1 gene result in an earlier halt of embryo development than disruption of the AtTor gene.     

The late pollen actins are essential for normal male and female development in Arabidopsis

In angiosperms the late pollen actins (LPAs) are strongly expressed in mature pollen and pollen tubes and at much lower levels in ovules. Four Arabidopsis lines with homozygous knockout mutations in the four individual LPA genes displayed normal flowers, pollen, and seed set. However, when all four LPAs were silenced simultaneously with a single RNA interference (RNAi) construct targeting the 3′UTR of each mRNA, obvious reproductive defects were observed. Western analysis of various Late Pollen actin RNA interference (LPRi) epialleles showed total LPA protein and RNA expression levels were knocked down from 0% to 95% compared to wild-type levels. Reciprocal crosses with the RNAi lines demonstrated that lowered LPA expression was associated with defects in both male and female fertility. Strong epialleles showed significant reductions in normal silique and seed production and were nearly sterile. Dissection of the siliques from moderate LPRi epialleles revealed many unfertilized ovules, increased numbers of aborted seeds, and decreased numbers of healthy seeds. Microscopic analysis of LPRi pollen indicated that the pollen shape and size were normal, but pollen germinated poorly. While multiple LPA genes may have some functional redundancy, the combined expression of multiple LPA genes appears essential to normal male and female reproductive development.     

COPII genes SEC31A/B are essential for gametogenesis and interchangeable in pollen development in Arabidopsis

In eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A-mCherry and SEC31B-GFP exhibited a high co-localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a-1/+sec31b-3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.     

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.     

Expression of ASK1 during chick and early mouse development

Apoptosis signal-regulating kinase 1 (ASK1) is an important regulator of stress-induced cell death. ASK1 is activated by oxidative stress, TNF and endoplasmatic reticulum stress and activates the JNK- and p38-dependent intracellular death pathways. A number of studies have suggested that ASK1 may also have other roles in addition to its pro-apoptotic activity. Expression of ASK1 during early embryonic development has so far not been analyzed. We have identified and cloned chick ASK1 in a screen for FGF8 inducible genes in chick facial mesenchyme. Here we report the expression of chick ASK1 from the gastrulation stage (HH4) to day 4 of development, its expression in the developing inner organs and limbs, and we compare its expression to the expression of Ask1 during mouse development. Furthermore, we provide evidence that FGF signaling is required for ASK1 expression in chick nasal mesenchyme. In contrast, expression in the mouse nasal region was restricted to the epithelium and was independent of FGF signaling. Our analysis demonstrates that ASK1 has a spatially restricted and temporally dynamic expression pattern in both chick and mouse embryos, which includes conserved as well as species-specific expression domains.