The anaphase‐promoting complex initiates zygote division in Arabidopsis through degradation of cyclin B1

As the start of a new life cycle, activation of the first division of the zygote is a critical event in both plants and animals. Because the zygote in plants is difficult to access, our understanding of how this process is achieved remains poor. Here we report genetic and cell biological analyses of the zygote‐arrest 1 (zyg1) mutant in Arabidopsis, which showed zygote‐lethal and over‐accumulation of cyclin B1 D‐box‐GUS in ovules. Map‐based cloning showed that ZYG1 encodes the anaphase‐promoting complex/cyclosome (APC/C) subunit 11 (APC11). Live‐cell imaging studies showed that APC11 is expressed in both egg and sperm cells, in zygotes and during early embryogenesis. Using a GFP‐APC11 fusion construct that fully complements zyg1, we showed that GFP‐APC11 expression persisted throughout the mitotic cell cycle, and localized to cell plates during cytokinesis. Expression of non‐degradable cyclin B1 in the zygote, or mutations of either APC1 or APC4, also led to a zyg1‐like phenotype. Biochemical studies showed that APC11 has self‐ubiquitination activity and is able to ubiquitinate cyclin B1 and promote degradation of cyclin B1. These results together suggest that APC/C‐mediated degradation of cyclin B1 in Arabidopsis is critical for initiating the first division of the zygote.     

ANAPHASE PROMOTING COMPLEX/CYCLOSOME‐mediated cyclin B1 degradation is critical for cell cycle synchronization in syncytial endosperms

Although it is known that in most angiosperms mitosis in early endosperm development is syncytial and synchronized,it is unclear how the synchronization is regulated.We showed previously that APC11,also named ZYG1,in Arabidopsis activates zygote division by interaction and degradation of cyclin B1.Here,we report that the mutation in APC11/ZYG1 led to unsynchronized mitosis and over-accumulation of cyclin B1-GUS in the endosperm.Mutations in two other APC subunits showed similar defects.Transgenic expression of stable cyclin B1 in the endosperm also caused unsynchronized mitosis.Further,downregulation of APC11 generated multi-nucleate somatic cells with unsynchronized mitotic division.Together,our results suggest that APC/C-mediated cyclin B1 degradation is critical for cell cycle synchronization.     

DEAD-BOX RNA HELICASE 27 regulates microRNA biogenesis,zygote division,and stem cell homeostasis

After double fertilization, zygotic embryogenesis initiates a new life cycle, and stem cell homeostasis in the shoot apical meristem (SAM) and root apical meristem (RAM) allows plants to produce new tissues and organs continuously. Here, we report that mutations in DEAD-BOX RNA HELICASE 27 (RH27) affect zygote division and stem cell homeostasis in Arabidopsis (Arabidopsis thaliana). The strong mutant allele rh27-1 caused a zygote-lethal phenotype, while the weak mutant allele rh27-2 led to minor defects in embryogenesis and severely compromised stem cell homeostasis in the SAM and RAM. RH27 is expressed in embryos from the zygote stage, and in both the SAM and RAM, and RH27 is a nucleus-localized protein. The expression levels of genes related to stem cell homeostasis were elevated in rh27-2 plants, alongside down-regulation of their regulatory microRNAs (miRNAs). Further analyses of rh27-2 plants revealed reduced levels of a large subset of miRNAs and their pri-miRNAs in shoot apices and root tips. In addition, biochemical studies showed that RH27 associates with pri-miRNAs and interacts with miRNA-biogenesis components, including DAWDLE, HYPONASTIC LEAVES 1, and SERRATE. Therefore, we propose that RH27 is a component of the microprocessor complex and is critical for zygote division and stem cell homeostasis.

As a new component of the microprocessor complex in Arabidopsis, DEAD-BOX RNA HELICASE 27 regulates the initiation of zygotic embryogenesis and stem cell homeostasis in the shoot and root meristems.     

EMBRYONIC FACTOR 19 encodes a pentatricopeptide repeat protein that is essential for the initiation of zygotic embryogenesis in Arabidopsis

Early embryogenesis is the most fundamental developmental process in biology.Screening of ethyl methanesulfonate(EMS)-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19(fac19)in which embryo development was arrested at the elongated zygote to octant stage.The number of endosperm nuclei decreased significantly in fac19 embryos.Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation,suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis.Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat(PPR)motifs.The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine.Crosses between FAC19/fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects,confirming the identity of the gene.This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.     

EMBRYONIC FACTOR 1 encodes an AMP deaminase and is essential for the zygote to embryo transition in Arabidopsis

Fusion of the egg and the sperm cells in plants produces a zygote that develops into an embryo. Screening of ethyl methanesulfonate-mutagenized populations of Arabidopsis led to the identification of EMBRYONIC FACTOR 1 (FAC1), a locus that gives a zygote-lethal phenotype when mutated. The FAC1 gene was identified by positional cloning and confirmed by a genetic complementation test against a T-DNA insertion allele. It encodes an AMP deaminase (AMPD) that is known in human and yeast to convert AMP to IMP to maintain the energy potential. Expression of FAC1 in a yeast AMPD mutant after removal of its N-terminal putative transmembrane domain complemented the mutant phenotype, suggesting a functional conservancy but a structural divergence through evolution. Although a low level of FAC1 expression was observed in all organs tested, using a reporter construct we observed a significantly increased FAC1 expression in the zygote, early embryo and endosperm. Furthermore, during somatic embryogenesis, a high level of FAC1 expression was observed in developing embryos including putative embryogenic cells. FAC1, therefore, represents one of the earliest expressed genes known in plants. It may act through AMP depletion to provide sufficient energy for the zygote to proceed through development.     

Ethylene as a potent inducer of sexual development

A novel and critical function of ethylene, a potent plant hormone, has been well documented in Dictyostelium, because it leads cells to the sexual development (macrocyst formation) by inducing zygote formation. Zygote formation (sexual cell fusion) and the subsequent nuclear fusion are the characteristic events occurring during macrocyst formation. A novel gene, zyg1 was found to be predominantly expressed during the sexual development, and its enforced expression actually induces zygote formation. As expected, the zygote inducer, ethylene enhances the expression of zyg1. Thus the function of ethylene has been verified at all of individual (macrocyst formation), cellular (zygote formation), and molecular levels (zyg1 expression). Based on our recent studies concerning the behavior and function of the zyg1 product (ZYG1 protein), the signal transduction pathways involved in zygote formation are proposed in this review.     

Identification of a human endonuclease complex reveals a link between tRNA splicing and pre-mRNA 3' end formation

tRNA splicing is a fundamental process required for cell growth and division. The first step in tRNA splicing is the removal of introns catalyzed in yeast by the tRNA splicing endonuclease. The enzyme responsible for intron removal in mammalian cells is unknown. We present the identification and characterization of the human tRNA splicing endonuclease. This enzyme consists of HsSen2, HsSen34, HsSen15, and HsSen54, homologs of the yeast tRNA endonuclease subunits. Additionally, we identified an alternatively spliced isoform of SEN2 that is part of a complex with unique RNA endonuclease activity. Surprisingly, both human endonuclease complexes are associated with pre-mRNA 3' end processing factors. Furthermore, siRNA-mediated depletion of SEN2 exhibited defects in maturation of both pre-tRNA and pre-mRNA. These findings demonstrate a link between pre-tRNA splicing and pre-mRNA 3' end formation, suggesting that the endonuclease subunits function in multiple RNA-processing events.     

Accumulation of pre-tRNA splicing ''2/3'' intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Three mitochondrial tRNA genes from Arabidopsis thaliana: evidence for the conversion of a tRNAPhe gene into a tRNATyr gene.

Three tRNA genes have been isolated from a genomic library of Arabidopsis thaliana: a tRNASer (GCU), a tRNATyr (GUA) and a tRNAGlu (UUC) genes. These genes are located closely on the same DNA fragment. The tRNASer and the tRNAGlu genes have both 99% sequence similarity with their mitochondrial counterparts from higher plants indicating that these three tRNA genes are mitochondrial. The tRNATyr gene shows a particular high sequence similarity with the mitochondrial tRNAPhe pseudogene from maize, and both genes are flanked by a tRNASer gene in the upstream region. Extensive sequence comparisons of the Arabidopsis thaliana mitochondrial sequence containing the three tRNA genes and the corresponding region from maize and soybean mitochondria have shown evidence that the tRNA Tyr gene has been generated from a mitochondrial tRNAPhe gene. The conversion was accomplished by three genetic events: a 4 base-pair deletion, a mutation and a recombination, which led to the transformation of the acceptor stem and the anticodon.     

Ethylene induces zygote formation through an enhanced expression of zyg1 in Dictyostelium mucoroides

We have previously demonstrated that a potent plant hormone, ethylene induces sexual development including zygote formation in Dictyostelium cells, and that a novel gene (zyg1) is also involved in zygote formation. Based on these findings, the present work was mainly designed to reveal (1) the precise relationship between the ethylene amount and zygote formation, and (2) the relation of in situ ethylene synthesis to zyg1 expression, using transformants that over- or under-produce ACC-oxidase (Dd-aco) involved in ethylene biosynthesis. ACO(OE) cells overexpressing Dd-aco gene overproduced ethylene and exhibited the augmented zygote formation. In contrast, ACO-RNAi cells, in which the expression of Dd-aco was suppressed by the RNAi method, showed a reduced level of ethylene production, thus resulting in inhibition of zygote formation. Importantly, the expression of zyg1 was affected by the amount of ethylene produced: Zyg1 expression was augmented in ACO(OE) cells, but was significantly suppressed in ACO-RNAi cells. In another experiment, we found that 1-methylcyclopropene (1-MCP), which is known to inhibit the function of ethylene by binding specifically to ethylene receptors, greatly suppresses zygote formation. These results indicate that ethylene is capable of inducing zygote formation through the expression of zyg1.     

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.     

The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate

Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gβ, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca²⁺ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.     

生长素在植物胚胎早期发育中的作用

有性生殖是有花植物的一个重要特征, 胚胎则是实现有性生殖和世代交替的重要载体。植物胚胎从双受精开始, 经历了合子极性建立、顶基轴形成、细胞层分化和器官形成等过程, 这些过程都受到生长素的调控。近年来的研究表明, 生长素在生物合成、极性运输和信号转导3个层面上调控胚胎的发育过程。该文以双子叶植物拟南芥(Arabidopsis thaliana)为例, 综述了生长素对胚胎早期发育过程, 包括合子极性和顶基轴建立、表皮原特化和对称模式转变、胚根原特化和根尖分生组织形成及茎尖分生组织形成等发育的调控机制。     

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.