The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection.

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

Gametophyte and embryonic ontogeny: understanding the reproductive calendar of Cypripedium japonicum Thunb. (Cypripedoideae, Orchidaceae), a lady's slipper orchid endemic to East Asia.

BACKGROUND: The genus Cypripedium L. is one of the five genera of the subfamily Cypripedioideae, members of which are commonly known as lady's slipper orchids. Cypripedium japonicum is a perennial herb native to East Asia, specifically China, Japan, and Korea. Due to its limited distribution, the species is included in the Endangered category of the IUCN Red List. RESULTS: We investigated gametophyte development, including complete embryogenesis, in C. japonicum. The complete reproductive cycle is presented based on our observations. Anther development begins under the soil, and meiosis of pollen mother cells begins 3 weeks before anthesis, possibly during early April. The megaspore mother cells develop just after pollination in early May and mature in mid-late June. The pattern of embryo sac formation is bisporic, and there are six nuclei: three forming the egg apparatus, two polar nuclei, and an antipodal cell in the mature embryo sac. Triple fertilization results in the endosperm nucleus, which degenerates when the proembryo reaches the eight-to-sixteen-cell stage. CONCLUSION: Our overall comparisons of the features of gametophyte and embryo development in C. japonicum suggest that previous reports on the embryology of Cypripedium are not sufficient for characterization of the entire genus. Based on the available information, a reproductive calendar showing the key reproductive events leading to embryo formation has been prepared.

Distribution of cell layers in floral organs of chrysanthemum analyzed with periclinal chimeras carrying a transgene encoding fluorescent protein.

KEY MESSAGE: A fluorescent protein visualized distributions of cell layers in floral organs of chrysanthemum using transgenic periclinal chimeras carrying a gene encoding a fluorescent compound. Plant meristems have three cell layers: the outermost layer (L1), the second layer (L2), and the inner layer (L3). The layers are maintained during development but there is limited knowledge of the details of cell layer patterns within floral organs. In this study, we visualized the distributions of cell layers in floral organs of chrysanthemum using periclinal chimeras carrying a gene encoding a fluorescent compound in the L1 or the L2/L3 layers. The L1 layer contributed most of the epidermal cells of organs including the receptacle, petal, anther, filament, style, stigma, and ovule. The transmitting tissue in the pistil and most of the internal area of the ovule were also derived from the L1. In crossing experiments, no progeny of the L1-chimeric plants showed fluorescence, indicating that the germ cells of chrysanthemum are not derived from the L1 layer. Since anthocyanin pigment is present only in the L1-derived epidermal cells of petals, L1-specific gene integration could be used to alter flower color in commercial cultivars, with a reduced risk of transgene flow from the transgenic chrysanthemums to wild relatives.

Meiosis, the master driver of gene duplication in higher plants?

Meiosis is a critical biological process for reproduction and genetic variation in higher plants. Gene duplication is a prominent feature of plant genomic architecture. Meiosis and gene duplication are of fundamental importance in unraveling the nature of genetics and evolution. The ideas and findings in this letter demonstrate a highly significant connection between meiosis and gene duplication, bring together these two disparate fields of study and highlight the importance of meiosis for understanding the evolutionary success of flowering plants. These insights and opinions open a new area of investigation and point to a significant way to illustrate the impact of duplicated genes on meiosis and fitness in higher plants, as well as their ultimate evolutionary, ecological, and agronomic impacts in light of challenges that have arisen due to global climate change. This study addresses novel ideas and viewpoints in plant developmental genomics and evolution.

Distinct Differentiation Characteristics of Endothelium Determine Its Ability to Form Pseudo-Embryos in Tomato Ovules.

The endothelium is an additional cell layer, differentiating from the inner epidermis of the ovule integument. In tomato (Solanum lycopersicum L.), after fertilization, the endothelium separates from integument and becomes an independent tissue developing next to the growing embryo sac. In the absence of fertilization, the endothelium may proliferate and form pseudo-embryo. However, the course of the reorganization of endothelium into pseudo-embryo in tomato ovules is poorly understood. We aimed to investigate specific features of endothelium differentiation and the role of the endothelium in the development of fertilized and unfertilized tomato ovules. The ovules of tomato plants ("YaLF" line), produced by vegetative growth plants of transgenic tomato line expressing the ac gene, encoding chitin-binding protein from Amaranthus caudatus L., were investigated using light and transmission electron microscopy. We showed that in the fertilized ovule of normally developing fruit and in the unfertilized ovule of parthenocarpic fruit, separation of the endothelium from integument occurs via programmed death of cells of the integumental parenchyma, adjacent to the endothelium. Endothelial cells in normally developing ovules change their structural and functional specialization from meristematic to secretory and back to meristematic, and proliferate until seeds fully mature. The secretory activity of the endothelium is necessary for the lysis of dying cells of the integument and provides the space for the growth of the new sporophyte. However, in ovules of parthenocarpic fruits, pseudo-embryo cells do not change their structural and functional organization and remain meristematic, no zone of lysis is formed, and pseudo-embryo cells undergo programmed cell death. Our data shows the key role of the endothelium as a protective and secretory tissue, needed for the normal development of ovules.

Cell fusion and nuclear fusion in plants.

Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall.

Gentian (Gentiana triflora) prevents transmission of apple latent spherical virus (ALSV) vector to progeny seeds.

MAIN CONCLUSION: Gentian plants ( Gentiana triflora ) severely restrict apple latent spherical virus (ALSV) invasion to the gametes (pollens and ovules) and block seed transmission to progeny plants. Early flowering of horticultural plants can be induced by infection of ALSV vector expressing Flowering Locus T (FT) gene. In the present study, flowering of gentian plants was induced by infection with an ALSV vector expressing a gentian FT gene and the patterns of seed transmission of ALSV in gentian were compared with those in apple and Nicotiana benthamiana. Infection of gentian progeny plants with ALSV was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and enzyme-linked immunosorbent assay (ELISA). ALSV was not transmitted to the progeny gentian plants, whereas small proportions of apple and N. benthamiana progeny plants are infected with ALSV. The in situ hybridization analyses indicated that ALSVs are not present in gentian pollen and ovules, but detected in most of gametes in apple and N. benthamiana. Collectively, these results suggest that seed transmission of ALSV is blocked in gentian plants through the unknown barriers present in their gametes. On the other hand, apple and N. benthamiana seem to minimize ALSV seed transmission by inhibiting viral propagation in embryos.

SUPPRESSOR OF FRIGIDA (SUF4) Supports Gamete Fusion via Regulating Arabidopsis EC1 Gene Expression.

The EGG CELL1 (EC1) gene family of Arabidopsis (Arabidopsis thaliana) comprises five members that are specifically expressed in the egg cell and redundantly control gamete fusion during double fertilization. We investigated the activity of all five EC1 promoters in promoter-deletion studies and identified SUF4 (SUPPRESSOR OF FRIGIDA4), a C2H2 transcription factor, as a direct regulator of the EC1 gene expression. In particular, we demonstrated that SUF4 binds to all five Arabidopsis EC1 promoters, thus regulating their expression. The down-regulation of SUF4 in homozygous suf4-1 ovules results in reduced EC1 expression and delayed sperm fusion, which can be rescued by expressing SUF4-ß-glucuronidase under the control of the SUF4 promoter. To identify more gene products able to regulate EC1 expression together with SUF4, we performed coexpression studies that led to the identification of MOM1 (MORPHEUS' MOLECULE1), a component of a silencing mechanism that is independent of DNA methylation marks. In mom1-3 ovules, both SUF4 and EC1 genes are down-regulated, and EC1 genes show higher levels of histone 3 lysine-9 acetylation, suggesting that MOM1 contributes to the regulation of SUF4 and EC1 gene expression.

Phosphorylation of SPOROCYTELESS/NOZZLE by the MPK3/6 Kinase Is Required for Anther Development.

Germ cells are indispensable carriers of genetic information from one generation to the next. In contrast to the well-understood process in animals, information on the mechanism of germ cell initiation in plants is very limited. SPOROCYTELESS/NOZZLE was previously identified as an essential regulator of diploid germ cell (archesporial cell) differentiation in the stamens and ovules of Arabidopsis (Arabidopsis thaliana). Although SPOROCYTELESS (SPL) transcription is activated by the floral organ identity regulator AGAMOUS and epigenetically regulated by SET DOMAIN GROUP2, little is known about the regulation of the SPL protein. Here, we report that the protein kinases MPK3 and MPK6 can both interact with SPL in vitro and in vivo and can phosphorylate the SPL protein in vitro. In addition, phosphorylation of the SPL protein by MPK3/6 is required for SPL function in the Arabidopsis anther, as measured by its effect on archesporial cell differentiation. We further demonstrate that phosphorylation enhances SPL protein stability. This work not only uncovers the importance of SPL phosphorylation for its regulatory role in Arabidopsis anther development, but also supports the hypothesis that the regulation of precise spatiotemporal patterning of germ cell initiation and that differentiation is achieved progressively through multiple levels of regulation, including transcriptional and posttranslational modification.

RWP-RK domain-containing transcription factors control cell differentiation during female gametophyte development in Arabidopsis.

The formation of gametes is a prerequisite for any sexually reproducing organism in order to complete its life cycle. In plants, female gametes are formed in a multicellular tissue, the female gametophyte or embryo sac. Although the events leading to the formation of the female gametophyte have been morphologically characterized, the molecular control of embryo sac development remains elusive. We used single and double mutants as well as cell-specific marker lines to characterize a novel class of gene regulators in Arabidopsis thaliana, the RWP-RK domain-containing (RKD) transcription factors. Morphological and histological analyses were conducted using confocal laser scanning and differential interference contrast microscopy. Gene expression and transcriptome analyses were performed using quantitative reverse transcription-PCR and RNA sequencing, respectively. Our results showed that RKD genes are expressed during distinct stages of embryo sac development. Morphological analysis of the mutants revealed severe distortions in gametophyte polarity and cell differentiation. Transcriptome analysis revealed changes in the expression of several gametophyte-specific gene families (RKD2 and RKD3) and ovule development-specific genes (RKD3), and identified pleiotropic effects on phytohormone pathways (RKD5). Our data provide novel insight into the regulatory control of female gametophyte development. RKDs are involved in the control of cell differentiation and are required for normal gametophytic development.

Multiple and asymmetrical origin of polyploid dog rose hybrids (Rosa L. sect. Caninae (DC.) Ser.) involving unreduced gametes.

Background and Aims: Polyploidy and hybridization are important factors for generating diversity in plants. The species-rich dog roses ( Rosa sect. Caninae ) originated by allopolyploidy and are characterized by unbalanced meiosis producing polyploid egg cells (usually 4 x ) and haploid sperm cells (1 x ). In extant natural stands species hybridize spontaneously, but the extent of natural hybridization is unknown. The aim of the study was to document the frequency of reciprocal hybridization between the subsections Rubigineae and Caninae with special reference to the contribution of unreduced egg cells (5 x ) producing 6 x offspring after fertilization with reduced (1 x ) sperm cells. We tested whether hybrids arose by independent multiple events or via a single or few incidences followed by a subsequent spread of hybrids. Methods: Population genetics of 45 mixed stands of dog roses across central and south-eastern Europe were analysed using microsatellite markers and flow cytometry. Hybrids were recognized by the presence of diagnostic alleles and multivariate statistics were used to display the relationships between parental species and hybrids. Key Results: Among plants classified to subsect. Rubigineae , 32 % hybridogenic individuals were detected but only 8 % hybrids were found in plants assigned to subsect. Caninae . This bias between reciprocal crossings was accompanied by a higher ploidy level in Rubigineae hybrids, which originated more frequently by unreduced egg cells. Genetic patterns of hybrids were strongly geographically structured, supporting their independent origin. Conclusions: The biased crossing barriers between subsections are explained by the facilitated production of unreduced gametes in subsect. Rubigineae . Unreduced egg cells probably provide the highly homologous chromosome sets required for correct chromosome pairing in hybrids. Furthermore, the higher frequency of Rubigineae hybrids is probably influenced by abundance effects because the plants of subsect. Caninae are much more abundant and thus provide large quantities of pollen. Hybrids are formed spontaneously, leading to highly diverse mixed stands, which are insufficiently characterized by the actual taxonomy.

The Arabidopsis GPR1 Gene Negatively Affects Pollen Germination, Pollen Tube Growth, and Gametophyte Senescence.

Genes essential for gametophyte development and fertilization have been identified and studied in detail; however, genes that fine-tune these processes are largely unknown. Here, we characterized an unknown Arabidopsis gene, GTP-BINDING PROTEIN RELATED1 (GPR1). GPR1 is specifically expressed in ovule, pollen, and pollen tube. Enhanced green fluorescent protein-tagged GPR1 localizes to both nucleus and cytoplasm, and it also presents in punctate and ring-like structures. gpr1 mutants exhibit no defect in gametogenesis and seed setting, except that their pollen grains are pale in color. Scanning electron microscopy analyses revealed a normal patterned but thinner exine on gpr1 pollen surface. This may explain why gpr1 pollen grains are pale. We next examined whether GPR1 mutation affects post gametogenesis processes including pollen germination, pollen tube growth, and ovule senescence. We found that gpr1 pollen grains germinated earlier, and their pollen tubes elongated faster. Emasculation assay revealed that unfertilized gpr1 pistil expressed the senescence marker PBFN1:GUS (GUS: a reporter gene that encodes ß-glucuronidase) one-day earlier than the wild type pistil. Consistently, ovules and pollen grains of gpr1 mutants showed lower viability than those of the wild type at 4 to 5 days post anthesis. Together, these data suggest that GPR1 functions as a negative regulator of pollen germination, pollen tube growth, and gametophyte senescence to fine-tune the fertilization process.

[Analysis of the Gamete-Fusion Genes in the Haploid-inducing ZMS-P Maize Line].

This article is devoted to the study of the double fertilization mechanism in plants, in particular of the maize gamete membrane fusion genes. We detected and analyzed for the first time gamete-fusion genes in the maize genome. Using the BLAST program, we searched for the hap2 gene (generative cell specific 1 (gcs1)) homologs from Arabidopsis in the maize genome. The ZM_BFb0162K03 maize transcript was found, which had 67% identity to the Athap2 gene and contained a conserved region similar to the Athap2 gene fragment. In mRNA samples from the haploid-inducing and control maize lines, an PCR was conducted by using primers specific to the ZM_BFb0162K03 sequence fragment. Sequences of the PCR products from a fragment (1467 bp) of the Zm_hap2 gene of the haploid-inducing and the control maize lines were identical and also were identical to the maize sequences from the GenBank (ZM_BFb0162K03). PCR products (656 bp region of Zm_hap2) for the ZM_BFb0162K03 (1925 bp) maize sequence were observed for the cDNA of pollen grains, ovary, leaves, and roots of the haploid-inducing and control maize lines. Using the Blastx program, we found significant homology of the maize translated proteins to the GEX2, TET11, and TET12 proteins, involved in Arabidopsis gamete-fusion contacts.

Nuclear DNA level and life cycle of kelps: Evidence for sex-specific polyteny in Macrocystis (Laminariales, Phaeophyceae).

Giant kelp, Macrocystis pyrifera (Linnaeus) C. Agardh, is the subject of intense breeding studies for marine biomass production and conservation of natural resources. In this context, six gametophyte pairs and a sporophyte offspring of Macrocystis from South America were analyzed by flow cytometry. Minimum relative DNA content per cell (1C) was found in five males. Unexpectedly, nuclei of all female gametophytes contained approximately double the DNA content (2C) of males; the male gametophyte from one locality also contained 2C, likely a spontaneous natural diploid variant. The results illustrate a sex-specific difference in nuclear DNA content among Macrocystis gametophytes, with the chromosomes of the females in a polytenic condition. This correlates with significantly larger cell sizes in female gametophytes compared to males and resource allocation in oogamous reproduction. The results provide key information for the interpretation of DNA measurements in kelp life cycle stages and prompt further research on the regulation of the cell cycle, metabolic activity, sex determination, and sporophyte development.

Loading of the centromeric histone H3 variant during meiosis-how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.

Specific pools of endogenous peptides are present in gametophore, protonema, and protoplast cells of the moss Physcomitrella patens.

BACKGROUND: Protein degradation is a basic cell process that operates in general protein turnover or to produce bioactive peptides. However, very little is known about the qualitative and quantitative composition of a plant cell peptidome, the actual result of this degradation. In this study we comprehensively analyzed a plant cell peptidome and systematically analyzed the peptide generation process. RESULTS: We thoroughly analyzed native peptide pools of Physcomitrella patens moss in two developmental stages as well as in protoplasts. Peptidomic analysis was supplemented by transcriptional profiling and quantitative analysis of precursor proteins. In total, over 20,000 unique endogenous peptides, ranging in size from 5 to 78 amino acid residues, were identified. We showed that in both the protonema and protoplast states, plastid proteins served as the main source of peptides and that their major fraction formed outside of chloroplasts. However, in general, the composition of peptide pools was very different between these cell types. In gametophores, stress-related proteins, e.g., late embryogenesis abundant proteins, were among the most productive precursors. The Driselase-mediated protonema conversion to protoplasts led to a peptide generation "burst", with a several-fold increase in the number of components in the latter. Degradation of plastid proteins in protoplasts was accompanied by suppression of photosynthetic activity. CONCLUSION: We suggest that peptide pools in plant cells are not merely a product of waste protein degradation, but may serve as important functional components for plant metabolism. We assume that the peptide "burst" is a form of biotic stress response that might produce peptides with antimicrobial activity from originally functional proteins. Potential functions of peptides in different developmental stages are discussed.

Characterization of the Arabidopsis augmin complex uncovers its critical function in the assembly of the acentrosomal spindle and phragmoplast microtubule arrays.

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim γ-tubulin 3, plays a critical role in γ-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of γ-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.

Molecular characterization of the glauce mutant: a central cell-specific function is required for double fertilization in Arabidopsis.

Double fertilization of the egg cell and the central cell by two sperm cells, resulting in the formation of the embryo and the endosperm, respectively, is a defining characteristic of flowering plants. The Arabidopsis thaliana female gametophytic mutant glauce (glc) can exhibit embryo development without any endosperm. Here, we show that in glc mutant embryo sacs one sperm cell successfully fuses with the egg cell but the second sperm cell fails to fuse with the central cell, resulting in single fertilization. Complementation studies using genes from the glc deletion interval identified an unusual genomic locus having homology to BAHD (for BEAT, AHCT, HCBT, and DAT) acyl-transferases with dual transcription units and alternative splicing that could rescue the sterility defect of glc. Expression of these transcripts appears restricted to the central cell, and expression within the central cell is sufficient to restore fertility. We conclude that the central cell actively promotes its own fertilization by the sperm cell through a signaling mechanism involving products of At1g65450. Successful fertilization of the egg cell is not blocked in the glc mutant, suggesting that evolution of double fertilization in flowering plants involved acquisition of specific functions by the central cell to enable its role as a second female gamete.

Evidence for a role of Arabidopsis CDT1 proteins in gametophyte development and maintenance of genome integrity.

Meristems retain the ability to divide throughout the life cycle of plants, which can last for over 1000 years in some species. Furthermore, the germline is not laid down early during embryogenesis but originates from the meristematic cells relatively late during development. Thus, accurate cell cycle regulation is of utmost importance to avoid the accumulation of mutations during vegetative growth and reproduction. The Arabidopsis thaliana genome encodes two homologs of the replication licensing factor CDC10 Target1 (CDT1), and overexpression of CDT1a stimulates DNA replication. Here, we have investigated the respective functions of Arabidopsis CDT1a and CDT1b. We show that CDT1 proteins have partially redundant functions during gametophyte development and are required for the maintenance of genome integrity. Furthermore, CDT1-RNAi plants show endogenous DNA stress, are more tolerant than the wild type to DNA-damaging agents, and show constitutive induction of genes involved in DNA repair. This DNA stress response may be a direct consequence of reduced CDT1 accumulation on DNA repair or may relate to the ability of CDT1 proteins to form complexes with DNA polymerase ε, which functions in DNA replication and in DNA stress checkpoint activation. Taken together, our results provide evidence for a crucial role of Arabidopsis CDT1 proteins in genome stability.

Downregulation of the δ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis.

The mitochondrial ATP synthase (F(1)F(o) complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded F(o) part to the matrix-facing central stalk of F(1). We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.