Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development

Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1-3 and sqe1-4 plants have diminished stature and produce inviable seeds. The sqe1-3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1.     

A tumor suppressor homolog,AtPTEN1, is essential for pollen development in Arabidopsis

Although it is well known that Tyr phosphatases play a critical role in signal transduction in animal cells, little is understood of the functional significance of Tyr phosphatases in higher plants. Here, we describe the functional analysis of an Arabidopsis gene (AtPTEN1) that encodes a Tyr phosphatase closely related to PTEN, a tumor suppressor in animals. The recombinant AtPTEN1 protein, like its homologs in animals, is an active phosphatase that dephosphorylates phosphotyrosine and phosphatidylinositol substrates. RNA gel blot analysis and examination of promoter-reporter constructs in transgenic Arabidopsis plants revealed that the AtPTEN1 gene is expressed exclusively in pollen grains during the late stage of development. Suppression of AtPTEN1 gene expression by RNA interference caused pollen cell death after mitosis. We conclude that AtPTEN1 is a pollen-specific phosphatase and is essential for pollen development.     

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.     

SNAIL1 is essential for female gametogenesis in Arabidopsis thaliana

Two yeast Brix family members Ssf1 and Ssf2,involved in large ribosomal subunit synthesis, are essential for yeast cell viability and mating efficiency. Their putative homologs exist in the Arabidopsis genome; however, their role in plant development is unknown. Here, we show that Arabidopsis thaliana SNAIL1(At SNAIL1), a protein sharing high sequence identity with yeast Ssf1 and Ssf2, is critical to mitosis progression of female gametophyte development.The snail1 homozygous mutant was nonviable and its heterozygous mutant was semi-sterile with shorter siliques.The mutation in SNAIL1 led to absence of female transmission and reduced male transmission. Further phenotypic analysis showed that the synchronic development of female gametophyte in the snail1 heterozygous mutant was greatly impaired and the snail1 pollen tube growth, in vivo, was also compromised. Furthermore, SNAIL1 was a nucleolarlocalized protein with a putative role in protein synthesis.Our data suggest that SNAIL1 may function in ribosome biogenesis like Ssf1 and Ssf2 and plays an important role during megagametogenesis in Arabidopsis.     

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.     

An Arabidopsis thaliana plasma membrane proton pump is essential for pollen development

The plasma membrane proton pump (H(+)-ATPase) found in plants and fungi is a P-type ATPase with a polypeptide sequence, structure, and in vivo function similar to the mammalian sodium pump (Na(+), K(+)-ATPase). Despite its hypothetical importance for generating and maintaining the proton motive force that energizes the carriers and channels that underlie plant nutrition, genetic evidence for such a central function has not yet been reported. Using a reverse genetic approach for investigating each of the 11 isoforms in the Arabidopsis H(+)-ATPase (AHA) gene family, we found that one member, AHA3, is essential for pollen formation. A causative role for AHA3 in male gametogenesis was proven by complementation with a normal transgenic gene and rescue of the mutant phenotype back to wild type. We also investigated the requirement for phosphorylation of the penultimate threonine, which is found in most members of the AHA family and is thought to be involved in regulating catalytic activity. We demonstrated that a T948D mutant form of the AHA3 gene rescues the mutant phenotype in knockout AHA3 plants, but T948A does not, providing the first in planta evidence in support of the model in which phosphorylation of this amino acid is essential.     

Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana

In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.     

Phosphatidylglycerol is essential for the development of thylakoid membranes in Arabidopsis thaliana

Phosphatidylglycerol is a ubiquitous phospholipid in the biological membranes of many organisms. In plants, phosphatidylglycerol is mainly present in thylakoid membranes and has been suggested to play specific roles in photosynthesis. Here, we have isolated two T-DNA tagged lines of Arabidopsis thaliana that have a T-DNA insertion in the PGP1 gene encoding a phosphatidylglycerolphosphate synthase involved in the biosynthesis of phosphatidylglycerol. In homozygous plants of the T-DNA tagged lines, the PGP1 gene was completely disrupted. The growth of these knockout mutants was dependent on the presence of sucrose in the growth medium, and these plants had pale yellow-green leaves. The leaves of the mutants had remarkably large intercellular spaces due to the reduction in the number of mesophyll cells. The development of chloroplasts in the leaf cells was severely arrested in the mutants. Mesophyll cells with chloroplast particles are only found around vascular structures, whereas epidermal cells are enlarged but largely conserved. The content of phosphatidylglycerol in the mutants was reduced to 12% of that of the wild type. These results demonstrate that PGP1 plays a major role in the biosynthesis of phosphatidylglycerol in chloroplasts, and that phosphatidylglycerol is essential for the development of thylakoid membranes in A. thaliana.     

A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis

Complex I (the NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a complicated, multi-subunit, membranebound assembly and contains more than 40 different proteins in higher plants. In this paper, we characterize the Arabidopsis homologue (designated as AtCIB22) of the B22 subunit of eukaryotic mitochondrial Complex I. AtCIB22 is a single-copy gene and is highly conserved throughout eukaryotes. AtCIB22 protein is located in mitochondria and the AtCIB22 gene is widely expressed in different tissues. Mutant Arabidopsis plants with a disrupted AtCIB22 gene display pleiotropic phenotypes including shorter roots, smaller plants and delayed flowering. Stress analysis indicates that the AtCIB22 mutants’ seed germination and early seedling growth are severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis reveals that in moderate knockdown AtCIB22 mutants, genes including cell redox proteins and stress related proteins are significantly up-regulated, and that in severe knockdown AtCIB22 mutants, the alternative respiratory pathways including NDA1, NDB2, AOX1a and AtPUMP1 are remarkably elevated. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in Arabidopsis. Our findings also enhance our understanding about the physiological role of Complex I in plants.     

A heterocomplex of iron superoxide dismutases defends chloroplast nucleoids against oxidative stress and is essential for chloroplast development in Arabidopsis

There are three iron superoxide dismutases in Arabidopsis thaliana: FE SUPEROXIDE DISMUTASE1 (FSD1), FSD2, and FSD3. Their biological roles in chloroplast development are unknown. Here, we show that FSD2 and FSD3 play essential roles in early chloroplast development, whereas FSD1, which is found in the cytoplasm, does not. An fsd2-1 fsd3-1 double mutant had a severe albino phenotype on agar plates, whereas fsd2 and fsd3 single knockout mutants had pale green phenotypes. Chloroplast development was arrested in young seedlings of the double mutant. The mutant plants were highly sensitive to oxidative stress and developed increased levels of reactive oxygen species (ROS) during extended darkness. The FSD2 and FSD3 proteins formed a heteromeric protein complex in the chloroplast nucleoids. Furthermore, transgenic Arabidopsis plants overexpressing both the FSD2 and FSD3 genes showed greater tolerance to oxidative stress induced by methyl viologen than did the wild type or single FSD2- or FSD3-overexpressing lines. We propose that heteromeric FSD2 and FSD3 act as ROS scavengers in the maintenance of early chloroplast development by protecting the chloroplast nucleoids from ROS.     

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.     

The Drosophila homolog of Aut1 is essential for autophagy and development

The Drosophila homolog of yeast Aut1, CG6877/Draut1, is a ubiquitously expressed cytosolic protein. Draut1 loss of function was achieved by expression of an inverted repeat transgene inducing RNA interference. The effect is temperature-dependent and resembles an allelic series as described by Fortier, E. and Belote, J.M. (Genesis 26 (2000) 240-244). Draut1 loss of function larvae are unable to induce autophagy and heterophagy in fat body cells before pupariation and die during metamorphosis. To our knowledge, this is the first report of a multicellular animal lacking the function of a gene participating in the protein conjugation systems of autophagy.     

An Arabidopsis homolog of the bacterial peptidoglycan synthesis enzyme MurE has an essential role in chloroplast development

Enzymes encoded by bacterial MurE genes catalyze the ATP-dependent formation of uridine diphosphate- N -acetylmuramic acid-tripeptide in bacterial peptidoglycan biosynthesis. The Arabidopsis thaliana genome contains one gene with homology to the bacterial MurE : AtMurE . Under normal conditions AtMurE is expressed in leaves and flowers, but not in roots or stems. Sequence-based predictions and analyses of GFP fusions of the N terminus of AtMurE, as well as the full-length protein, suggest that AtMurE localizes to plastids. We identified three T-DNA-tagged and one Ds -tagged mutant alleles of AtMurE in A. thaliana . All four alleles show a white phenotype, and A. thaliana antisense AtMurE lines showed a pale-green phenotype. These results suggest that AtMurE is involved in chloroplast biogenesis. Cells of the mutants were inhibited in thylakoid membrane development. RT-PCR analysis of the mutant lines suggested that the expression of genes that depend on a multisubunit plastid-encoded RNA polymerase was decreased. To analyze the functional relationships between the MurE genes of cyanobacteria, the moss Physcomitrella patens and higher plants, a complementation assay was carried out with a P. patens ( Pp ) MurE knock-out line, which exhibits a small number of macrochloroplasts per cell. Although the Anabaena MurE, fused with the N-terminal region of PpMurE, complemented the macrochloroplast phenotype in P. patens , transformation with AtMurE did not complement this phenotype. These results suggest that AtMurE is functionally divergent from the bacterial and moss MurE proteins.     

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.     

Arabidopsis light-dependent protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development

During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.     

Chloroplast ribosome release factor 1 (AtcpRF1) is essential for chloroplast development

To study the functions of nuclear genes involved in chloroplast development, we systematically analyzed albino and pale green Arabidopsis thaliana mutants by use of the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study, we focused on one of these albino mutants, designated apg3-1 (for a lbino or p ale g reen mutant 3). A gene encoding a ribosome release factor 1 (RF1) homologue was disrupted by the insertion of a Ds transposon into the APG3 gene; a T-DNA insertion into the same gene caused a similar phenotype (apg3-2). The APG3 gene (At3g62910) has 15 exons and encodes a protein (422-aa) with a transit peptide that functions in targeting the protein to chloroplasts. The amino acid sequence of APG3 showed 40.6% homology with an RF1 of Escherichia coli, and complementation analysis using the E. coli rf1 mutant revealed that APG3 functions as an RF1 in E. coli, although complementation was not successful in the RF2-deficient (rf2) mutants of E. coli. These results indicate that the APG3 protein is an orthologue of E. coli RF1, and is essential for chloroplast translation machinery; it was accordingly named AtcpRF1. Since the chloroplasts of apg3-1 plants contained few internal thylakoid membranes, and chloroplast proteins related to photosynthesis were not detected by immunoblot analysis, AtcpRF1 is thought to be essential for chloroplast development. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.