Early Embryogenesis In Arabidopsis Research Articles

AtNSE1 and AtNSE3 are required for embryo pattern formation and maintenance of cell viability during Arabidopsis embryogenesis.

Embryogenesis is an essential process during seed development in higher plants. It has previously been shown that mutation of the Arabidopsis non-SMC element genes AtNSE1 or AtNSE3 leads to early embryo abortion, and their proteins can interact with each other directly. However, the crucial regions of these proteins in this interaction and how the proteins are cytologically involved in Arabidopsis embryo development are unknown. In this study, we found that the C-terminal including the Ring-like motif of AtNSE1 can interact with the N-terminal of AtNSE3, and only the Ring-like motif is essential for binding with three α motifs of AtNSE2 (homologous to AtMMS21). Using genetic assays and by analysing molecular markers of cell fate decisions (STM, WOX5, and WOX8) in mutant nse1 and nse3 embryos, we found that AtNSE1 and AtNSE3 work non-redundantly in early embryo development, and that differentiation of the apical meristem and the hypophysis fails in the mutants, which have disrupted auxin transportation and responses. However, the upper cells of the suspensor in the mutants seem to have proper embryo cell identity. Cytological examination showed that cell death occurred from the early embryo stage, and that vacuolar programmed cell death and necrosis in the nse1 and nse3 mutant embryos led to ovule abortion. Thus, AtNSE1 and AtNSE3 are essential for maintaining cell viability and growth during early embryogenesis. Our results improve our understanding of the functions of SMC5/6 complex in early embryogenesis in Arabidopsis.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAinvitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

Live-Cell Imaging and Optical Manipulation of Arabidopsis Early Embryogenesis

Intercellular communications are essential for cell proliferation and differentiation during plant embryogenesis. However, analysis of intercellular communications in living material in real time is difficult owing to the restricted accessibility of the embryo within the flower. We established a live-embryo imaging system to visualize cell division and cell fate specification in Arabidopsis thaliana from zygote division in real time. We generated a cell-division lineage tree for early embryogenesis in Arabidopsis. Lineage analysis showed that both the direction and time course of cell division between sister cells differed along the apical-basal or radial axes. Using the Arabidopsis kpl mutant, in which single-fertilization events are frequent, weshowed that endosperm development is not required for pattern formation during early embryogenesis. Optical manipulation demonstrated that damage to the embryo initial cell induces cell fate conversion of the suspensor cell to compensate for the disrupted embryo initial cell even after cell fate is specified.

Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical-basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.

LONO1 encoding a nucleoporin is required for embryogenesis and seed viability in Arabidopsis.

Early embryogenesis in Arabidopsis (Arabidopsis thaliana) is distinguished by a predictable pattern of cell divisions and is a good system for investigating mechanisms of developmental pattern formation. Here, we identified a gene called LONO1 (LNO1) in Arabidopsis in which mutations can abolish the first asymmetrical cell division of the zygote, alter planes and number of cell divisions in early embryogenesis, and eventually arrest embryo development. LNO1 is highly expressed in anthers of flower buds, stigma papilla of open flowers, and embryo and endosperm during early embryogenesis, which is correlated with its functions in reproductive development. The homozygous lno1-1 seed is not viable. LNO1, a homolog of the nucleoporin NUP214 in human (Homo sapiens) and Nup159 in yeast (Saccharomyces cerevisiae), encodes a nucleoporin protein containing phenylalanine-glycine repeats in Arabidopsis. We demonstrate that LNO1 can functionally complement the defect in the yeast temperature-sensitive nucleoporin mutant nup159. We show that LNO1 specifically interacts with the Arabidopsis DEAD-box helicase/ATPase LOS4 in the yeast two-hybrid assay. Furthermore, mutations in AtGLE1, an Arabidopsis homolog of the yeast Gle1 involved in the same poly(A) mRNA export pathway as Nup159, also result in seed abortion. Our results suggest that LNO1 is a component of the nuclear pore complex required for mature mRNA export from the nucleus to the cytoplasm, which makes LNO1 essential for embryogenesis and seed viability in Arabidopsis.

The Arabidopsis acbp1acbp2 double mutant lacking acyl‐CoA‐binding proteins ACBP1 and ACBP2 is embryo lethal

*In Arabidopsis thaliana, the amino acid sequences of membrane-associated acyl-CoA-binding proteins ACBP1 and ACBP2 are highly conserved. We have shown previously that, in developing seeds, ACBP1 accumulates in the cotyledonary cells of embryos and ACBP1 is proposed to be involved in lipid transfer. We show here by immunolocalization, using ACBP2-specific antibodies, that ACBP2 is also expressed in the embryos at various stages of seed development in Arabidopsis. *Phenotypic analyses of acbp1 and acbp2 single mutants revealed that knockout of either ACBP1 or ACBP2 alone did not affect their life cycle as both single mutants exhibited normal growth and development similar to the wild-type. However, the acbp1acbp2 double mutant was embryo lethal and was also defective in callus induction. *On lipid and acyl-CoA analyses, the siliques, but not the leaves, of the acbp1 mutant accumulated galactolipid monogalactosyldiacylglycerol and 18:0-CoA, but the levels of most polyunsaturated species of phospholipid, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, declined. *As recombinant ACBP1 and ACBP2 bind unsaturated phosphatidylcholine and acyl-CoA esters in vitro, we propose that ACBP1 and ACBP2 are essential in lipid transfer during early embryogenesis in Arabidopsis.

Identification and Functional Characterization of Inhibitor-3, a Regulatory Subunit of Protein Phosphatase 1 in Plants

Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase, and mediates diverse cellular processes in animal systems via the association of a catalytic subunit (PP1c) with multiple regulatory subunits that determine the catalytic activity, the subcellular localization, and the substrate specificity. However, no regulatory subunit of PP1 has been identified in plants so far. In this study, we identified inhibitor-3 (Inh3) as a regulatory subunit of PP1 and characterized a functional role of Inh3 in Vicia faba and Arabidopsis (Arabidopsis thaliana). We found Inh3 as one of the proteins interacting with PP1c using a yeast two-hybrid system. Biochemical analyses demonstrated that Arabidopsis Inh3 (AtInh3) bound to PP1c via the RVxF motif of AtInh3, a consensus PP1c-binding sequence both in vitro and in vivo. AtInh3 inhibited the PP1c phosphatase activity in the nanomolar range in vitro. AtInh3 was localized in both the nucleus and cytoplasm, and it colocalized with Arabidopsis PP1c in these compartments. Disruption mutants of AtINH3 delayed the progression of early embryogenesis, arrested embryo development at the globular stage, and eventually caused embryo lethality. Furthermore, reduction of AtINH3 expression by RNA interference led to a decrease in fertility. Transformation of the lethal mutant of inh3 with wild-type AtINH3 restored the phenotype, whereas that with the AtINH3 gene having a mutation in the RVxF motif did not. These results define Inh3 as a regulatory subunit of PP1 in plants and suggest that Inh3 plays a crucial role in early embryogenesis in Arabidopsis.

Arabidopsis GLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III.

Precise control of gene expression is critical for embryo development in both animals and plants. We report that Arabidopsis thaliana GLUTAMINE-RICH PROTEIN23 (GRP23) is a pentatricopeptide repeat (PPR) protein that functions as a potential regulator of gene expression during early embryogenesis in Arabidopsis. Loss-of-function mutations of GRP23 caused the arrest of early embryo development. The vast majority of the mutant embryos arrested before the 16-cell dermatogen stage, and none of the grp23 embryos reached the heart stage. In addition, 19% of the mutant embryos displayed aberrant cell division patterns. GRP23 encodes a polypeptide with a Leu zipper domain, nine PPRs at the N terminus, and a Gln-rich C-terminal domain with an unusual WQQ repeat. GRP23 is a nuclear protein that physically interacts with RNA polymerase II subunit III in both yeast and plant cells. GRP23 is expressed in developing embryos up to the heart stage, as revealed by beta-glucuronidase reporter gene expression and RNA in situ hybridization. Together, our data suggest that GRP23, by interaction with RNA polymerase II, likely functions as a transcriptional regulator essential for early embryogenesis in Arabidopsis.

Null mutation of AtCUL1 causes arrest in early embryogenesis in Arabidopsis.

The SCF (for SKP1, Cullin/CDC53, F-box protein) ubiquitin ligase targets a number of cell cycle regulators, transcription factors, and other proteins for degradation in yeast and mammalian cells. Recent genetic studies demonstrate that plant F-box proteins are involved in auxin responses, jasmonate signaling, flower morphogenesis, photocontrol of circadian clocks, and leaf senescence, implying a large spectrum of functions for the SCF pathway in plant development. Here, we present a molecular and functional characterization of plant cullins. The Arabidopsis genome contains 11 cullin-related genes. Complementation assays revealed that AtCUL1 but not AtCUL4 can functionally complement the yeast cdc53 mutant. Arabidopsis mutants containing transfer DNA (T-DNA) insertions in the AtCUL1 gene were shown to display an arrest in early embryogenesis. Consistently, both the transcript and the protein of the AtCUL1 gene were found to accumulate in embryos. The AtCUL1 protein localized mainly in the nucleus but also weakly in the cytoplasm during interphase and colocalized with the mitotic spindle in metaphase. Our results demonstrate a critical role for the SCF ubiquitin ligase in Arabidopsis embryogenesis.

Paternally inherited transgenes are down-regulated but retain low activity during early embryogenesis in Arabidopsis

We investigated the timing of transgene activation after fertilisation in Arabidopsis following crosses and using two transgenic promoters (from the AtCYCB1 and AtLTP1 genes). Using both a transactivation system and direct transcriptional fusion to drive β-glucuronidase reporter expression, reciprocal crosses showed a lack of expression of the paternal components. This is consistent with a lack of paternal genome activity previously reported during early seed development in Arabidopsis [Viella-Calzada et al. (2000) Nature 404, 91–94]. However, transactivation experiments of the BARNASE gene gave evidence that at least some paternal loci retain transcriptional activity, though at a low level, during early embryogenesis.

BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat.

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T-DNA-transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls (ban). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/ transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT (Transparent Testa) genes to two groups according to their epistatic relationship to ban. The flavonoid content of germinated ban and wild-type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild-type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis.

Embryo-defective mutants as tools to study essential functions and regulatory processes in plant embryo development

Plant embryo development depends on the coordinated expression of genes involved in biochemical processes, tissue differentiation, and developmental regulation. Descriptive studies of embryogenesis have provided a foundation for investigating the underlying mechanisms responsible for observed morphological changes. Late stages of embryogenesis have been examined in detail by analysis of abundant mRNAs produced in preparation for dormancy and germination. Genetic analyses of embryo-defective mutants have allowed a large number of genes with essential cellular and regulatory functions during early embryogenesis in Arabidopsis to be identified. The use of insertional mutants should now permit a greater number of these genes to be cloned and their roles in plant embryo development determined.