Vascular plant one-zinc finger 1 (VOZ1) and VOZ2 negatively regulate phytochrome B-mediated seed germination in Arabidopsis

ABSTRACT

Seed germination is regulated by light. Phytochromes (Phys) act as red and far-red light photoreceptors to mediate seed germination. However, the mechanism of this process is not well understood. In this study, we found that the Arabidopsis thaliana mutants vascular plant one-zinc finger 1 (voz1) and voz2 showed higher seed germination percentage than wild type when PhyB was inactivated by far-red light. In wild type, VOZ1 and VOZ2 expression were downregulated after seed imbibition, repressed by PhyB, and upregulated by Phytochrome-interacting factor 1 (PIF1), a key negative regulator of seed germination. Red light irradiation and the voz1voz2 mutation caused increased expression of Gibberellin 3-oxidase 1 (GA3ox1), a gibberellin (GA) biosynthetic gene. We also found that VOZ2 is bound directly to the promoter of GA3ox1 in vitro and in vivo. Our findings suggest that VOZs play a negative role in PhyB-mediated seed germination, possibly by directly regulating GA3ox1 expression.     

AtAPOSTART1, an Arabidopsis thaliana PH-START domain protein involved in seed germination

The seed in the mature and dry state is metabolically inactive (quiescent) and is thus able to withstand extreme environmental conditions, such as drought and cold. Germination commences when the dry seed, shed from its parent plant, takes up water (imbibition) and ends when the root emerges through the seed coat. During seedling establishment, the reserves stored in the seed are metabolized, whereas the subsequent vegetative and reproductive growth is supported by photosynthesis. Here, we describe the functional characterization of the PH-START protein AtAPO1 (Arabidopsis thaliana APOSTART1), the putative homologue of PpAPO1 (Poa pratensis APOSTART1) in Arabidopsis thaliana. By using translational fusion of the AtAPO1 promoter to the uiaD gene and in situ hybridization analyses, we show that AtAPO1 is expressed in mature embryo sacs and developing embryos. The functional analysis of two at-apostart mutant alleles suggests that AtAPO1 is involved in the control of seed germination.     

An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

拟南芥——一把打开植物生命奥秘大门的钥匙

在过去的20年中,拟南芥作为模式植物广泛用于植物生命科学研究。历时10年的模式植物拟南芥的全基因组测序工作于2000年完成,通过测序获得的拟南芥基因组核苷酸序列全部公布在互联网上,有力地推动了植物生命科学研究向前发展。科学家提出的“2010计划”旨在通过全世界植物科学家的努力,到2010年能够尽可能多地了解拟南芥基因的功能。通过拟南芥研究所获得的信息将有助于人类对控制不同植物复杂生命活动机制的认识。     

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.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

PPF1 may suppress plant senescence via activating TFL1 in transgenic Arabidopsis plants

Senescence, a sequence of biochemical and physiological events, constitutes the final stage of development In higher plants and is modulated by a variety of environmental factors and intemal factors. PPF1 possesses an important biological function in plant development by controlling the Ca2 storage capacity within chloroplasts. Here we show that the expression of PPF1 might play a pivotal role in negatively regulating plant senescence as revealed by the regulation of overexpression and suppression of PPF1 on plant development. Moreover, TFL1, a key regulator in the floral repression pathway, was screened out as one of the downstream targets for PPF1 in the senescence-signaling pathway. Investigation of the senescence-related phenotypes in PPF1(-) tfl1-1 and PPF1( ) tfl1-1 double mutants confirmed and further highlighted the relation of PPF1 with TFL1 in tranegenic plants. The activation of TFL1 expression by PPF1 defines an important pathway possibly essential for the negative regulation of plant senescence in transgenic Arabidopsis.     

The participation of AtXPB1, the XPB/RAD25 homologue gene from Arabidopsis thaliana, in DNA repair and plant development

Nucleotide excision repair in Arabidopsis thaliana differs from other eukaryotes as it contains two paralogous copies of the corresponding XPB/RAD25 gene. In this work, the functional characterization of one copy, AtXPB1, is presented. The plant gene was able to partially complement the UV sensitivity of a yeast rad25 mutant strain, thus confirming its involvement in nucleotide excision repair. The biological role of AtXPB1 protein in A. thaliana was further ascertained by obtaining a homozygous mutant plant containing the AtXPB1 genomic sequence interrupted by a T-DNA insertion. The 3' end of the mutant gene is disrupted, generating the expression of a truncated mRNA molecule. Despite the normal morphology, the mutant plants presented developmental delay, lower seed viability and a loss of germination synchrony. These plants also manifested increased sensitivity to continuous exposure to the alkylating agent MMS, thus suggesting inefficient DNA damage removal. These results indicate that, although the duplication seems to be recent, the features described for the mutant plant imply some functional or timing expression divergence between the paralogous AtXPB genes. The AtXPB1 protein function in nucleotide excision repair is probably required for the removal of lesions during seed storage, germination and early plant development.     

The Arabidopsis AGC kinases NDR2/4/5 interact with MOB1A/1B and play important roles in pollen development and germination

Pollen formation and pollen tube growth are essential for the delivery of male gametes into the female embryo sac for double fertilization. Little is known about the mechanisms that regulate the late developmental process of pollen formation and pollen germination. In this study, we characterized a group of Arabidopsis AGC kinase proteins, NDR2/4/5, involved in pollen development and pollen germination. The NDR2/4/5 genes are mainly expressed in pollen grains at the late developmental stages and in pollen tubes. They function redundantly in pollen formation and pollen germination. At the tricellular stages, the ndr2 ndr4 ndr5 mutant pollen grains exhibit an abnormal accumulation of callose, precocious germination and burst in anthers, leading to a drastic reduction in fertilization and a reduced seed set. NDR2/4/5 proteins can interact with another group of proteins (MOB1A/1B) homologous to the MOB proteins from the Hippo signaling pathway in yeast and animals. The Arabidopsis mob1a mob1b mutant pollen grains also have a phenotype similar to that of ndr2 ndr4 ndr5 pollen grains. These results provide new evidence demonstrating that the Hippo signaling components are conserved in plants and play important roles in sexual plant reproduction.     

Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel

The role of heterotrimeric G proteins in pollen germination and tube growth was investigated using Arabidopsis thaliana plants in which the gene (GPA) encoding the G-protein a subunit (Galpha) was null or overexpressed. Pollen germination, free cytosolic calcium concentration ([Ca(2+)](cyt)) and Ca(2+) channel activity in the plasma membrane (PM) of pollen cells were investigated. Results showed that, compared with pollen grains of the wild type (ecotype Wassilewskija, ws), in vitro germinated pollen of Galpha null mutants (gpa1-1 and gpa1-2) had lower germination percentages and shorter pollen tubes, while pollen from Galpha overexpression lines (wGalpha and cGalpha) had higher germination percentages and longer pollen tubes. Compared with ws pollen cells, [Ca(2+)](cyt) was lower in gpa1-1 and gpa1-2 and higher in wGalpha and cGalpha. In whole-cell patch clamp recordings, a hyperpolarization-activated Ca(2+)-permeable conductance was identified in the PM of pollen protoplasts. The conductance was suppressed by trivalent cations but insensitive to organic blockers; its permeability to divalent cations was Ba(2+) > Ca(2+) > Mg(2+) > Sr(2+) > Mn(2+). The activity of the Ca(2+)-permeable channel conductance was down-regulated in pollen protoplasts of gpa1-1 and gpa1-2, and up-regulated in wGalpha and cGalpha. The results suggest that Galpha may participate in pollen germination through modulation of the hyperpolarization-activated Ca(2+) channel in the PM of pollen cells.     

A lysine-rich arabinogalactan protein in Arabidopsis is essential for plant growth and development, including cell division and expansion

Arabinogalactan proteins (AGPs), a family of hydroxyproline-rich glycoproteins, occur throughout the plant kingdom. The lysine-rich classical AGP subfamily in Arabidopsis consists of three members, AtAGP17, 18 and 19. In this study, AtAGP19 was examined in terms of its gene expression pattern and function. AtAGP19 mRNA was abundant in stems, with moderate levels in flowers and roots and low levels in leaves. AtAGP19 promoter-controlled GUS activity was high in the vasculature of leaves, roots, stems and flowers, as well as styles and siliques. A null T-DNA knockout mutant of AtAGP19 was obtained and compared to wild-type (WT) plants. The atagp19 mutant had: (i) smaller, rounder and flatter rosette leaves, (ii) lighter-green leaves containing less chlorophyll, (iii) delayed growth, (iv) shorter hypocotyls and inflorescence stems, and (v) fewer siliques and less seed production. Several abnormalities in cell size, number, shape and packing were also observed in the mutant. Complementation of this pleiotropic mutant with the WT AtAGP19 gene restored the WT phenotypes and confirmed that AtAGP19 functions in various aspects of plant growth and development, including cell division and expansion, leaf development and reproduction.     

植物生长调节物质对丰水梨花粉萌发和花粉管生长的影响

采用花粉液体培养法研究了植物生长调节物质对梨花粉萌发和花粉管生长的影响,结果表明：较低浓度的赤霉素、三十烷醇、吲哚乙酸及2,4-D均能促进花粉萌发和花粉管生长,而超过一定浓度时却起抑制作用,最适宜于花粉萌发和花粉管生长的赤霉素浓度为50～300mg／L,三十烷醇为3～100mg／L,吲哚乙酸为5～25mg／L,2,4-D为5～10mg／L。萘乙酸对花粉萌发和花粉管生长有抑制作用,抑制程度随培养基内其浓度的增大而加强。多效唑和脱落酸对花粉萌发有抑制作用,其抑制程度随浓度的上升而增强,但他们对花粉管生长却有促进怍用,其最适宜于花粉管生长的浓度分别为400mg／L和60mg／L,超过此浓度后,促进作用又有所下降,甚至出现抑制作用,如多效唑浓度达到1000mg／L时,能强烈地抑制花粉管生长。