Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis

We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.     

拟南芥突变体tgd表型和分子生物学特性分析

主要研究1个由Ds插入所造成的大片段缺失拟南芥突变体tgd(ten gene deletion)．这个突变体来自于基因陷阱拟南芥突变体库．对这一突变体的后代卡那霉素抗性分离比分析和Southern杂交表明,只有1个Ds拷贝插入此突变体基因组中,但是Tail-PCR和随后用特异基因序列为引物的验证PCR证实在Ds插入过程中造成了30 kb基因片段的缺失．根据对拟南芥基因组序列的注释,这30 kb的序列中包含10个基因．这一多基因缺失突变体有多效性表型．整株表现为株型矮小,发育迟缓,根系不发达,极易失水而死,茎细弱,较短,花序不正常．其中,莲座叶的表型最为明显,突变体叶形较细,叶片较厚．为了研究这些基因在多效性表型产生中所发挥的功能,对来自ABRC种子库中所有基因的T-DNA插入突变体,即salk line的表型进行了分析,发现所有基因的T-DNA插入突变体均没有可见的表型．这一现象暗示,突变体tgd的表型是10个基因缺失的综合效应,但是10个基因的相互关系以及与tgd表型的相互关系仍有待于继续研究．     

CHLOROPLAST BIOGENESIS genes act cell and noncell autonomously in early chloroplast development

In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.     

A complement of ten essential and pleiotropic arabidopsis COP/DET/FUS genes is necessary for repression of photomorphogenesis in darkness.

Two genetic screens, one for mutations resulting in photomorphogenic development in darkness and the other for mutants with fusca phenotype, have thus far identified six pleiotropic Arabidopsis COP/DET/FUS genes. Here, we characterized representative mutants that define four additional pleiotropic photomorphogenic loci and a null mutant allele of the previously defined DET1 locus. Dark-grown seedlings homozygous for these recessive mutations exhibit short hypocotyls and expanded cotyledons and are lethal before reaching reproductive development. Dark-grown mutant seedlings also display characteristic photomorphogenic cellular differentiation and elevated expression of light-inducible genes. In addition, analyses of plastids from dark-grown mutants reveal partial chloroplast differentiation and absence of etioplast development. Root vascular bundle cells of light-grown mutant seedlings develop chloroplasts, suggesting that these FUS gene products are important for suppression of chloroplast differentiation in light-grown roots. Double-mutant analyses indicate that these pleiotropic cop/det/fus mutations are epistatic to mutations in phytochromes, a blue-light photoreceptor, and a downstream regulatory component, HY5. Therefore, there is a complement of at least 10 essential and pleiotropic Arabidopsis genes that are necessary for repression of photomorphogenic development.     

The Chloroplast Function Database: a large‐scale collection of Arabidopsis Ds/Spm‐ or T‐DNA‐tagged homozygous lines for nuclear‐encoded chloroplast proteins,and their systematic phenotype analysis

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post‐translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor–mutator) transposon‐ or T‐DNA‐tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein‐coding regions. We systematically observed 3‐week‐old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild‐type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear‐encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/ . The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.     

A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in arabidopsis chloroplasts

We recently characterized a novel heme biogenesis pathway required for heme c(i)' covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6. These mutants were functionally complemented with the corresponding wild type cDNAs. Using fluorescent protein reporters, we demonstrated that the CCB1, CCB2, CCB3, and CCB4 proteins are targeted to the chloroplast compartment of Arabidopsis. We have extended our study to the YGGT family, to which CCB3 belongs, by studying insertion mutants of two additional members of this family for which no mutants were previously characterized, and we showed that they are not functionally involved in the CCB system. Thus, we demonstrate the ubiquity of the CCB proteins in chloroplast heme c(i)' binding.     

Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance.     

Two P-type ATPases are required for copper delivery in Arabidopsis thaliana chloroplasts

Copper delivery to the thylakoid lumen protein plastocyanin and the stromal enzyme Cu/Zn superoxide dismutase in chloroplasts is required for photosynthesis and oxidative stress protection. The copper delivery system in chloroplasts was characterized by analyzing the function of copper transporter genes in Arabidopsis thaliana. Two mutant alleles were identified of a previously uncharacterized gene, PAA2 (for P-type ATPase of Arabidopsis), which is required for efficient photosynthetic electron transport. PAA2 encodes a copper-transporting P-type ATPase with sequence similarity to PAA1, which functions in copper transport in chloroplasts. Both proteins localized to the chloroplast, as indicated by fusions to green fluorescent protein. The PAA1 fusions were found in the chloroplast periphery, whereas PAA2 fusions were localized in thylakoid membranes. The phenotypes of paa1 and paa2 mutants indicated that the two transporters have distinct functions: whereas both transporters are required for copper delivery to plastocyanin, copper delivery to the stroma is inhibited only in paa1 but not in paa2. The effects of paa1 and paa2 on superoxide dismutase isoform expression levels suggest that stromal copper levels regulate expression of the nuclear genes IRON SUPEROXIDE DISMUTASE1 and COPPER/ZINC SUPEROXIDE DISMUTASE2. A paa1 paa2 double mutant was seedling-lethal, underscoring the importance of copper to photosynthesis. We propose that PAA1 and PAA2 function sequentially in copper transport over the envelope and thylakoid membrane, respectively.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Genetic and Mapping Analysis of Arabidopsis thaliana Male Sterile Mutant filament no elongation

To identify new genes important for anther development, we screened for male sterile mutants among a population of Arabidopsis ecotype Columbia (Col) mutagenized by T DNA insertion (provided by ARBC). A male sterile mutant line with normal vegetative and flora development but no seed yield was isolated from Salk_118481 line. T DNA insertion site identification showed that there were no T DNA sequences in the genome of the mutants. Genetic analysis indicated that the mutant was controlled by a single recessive nuclear gene named filament no elongation because the filament of the mutant remains very short at the 13-14 stage of anther development. The fne gene was mapped to a region of 97kb between the molecular makers MBD2 and MMG4 on chromosome 5 using map based cloning technique. No genes involved filament elongation were reported in this region, so we believe that FNE gene could be a new gene controlling filament elongation in Arabidopsis.     

Characterization of the class IV homeodomain-Leucine Zipper gene family in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome contains 16 genes belonging to the class IV homeodomain-Leucine zipper gene family. These include GLABRA2, ANTHOCYANINLESS2, FWA, ARABIDOPSIS THALIANA MERISTEM LAYER1 (ATML1), and PROTODERMAL FACTOR2 (PDF2). Our previous study revealed that atml1 pdf2 double mutants have severe defects in the shoot epidermal cell differentiation. Here, we have characterized additional members of this gene family, which we designated HOMEODOMAIN GLABROUS1 (HDG1) through HDG12. Analyses of transgenic Arabidopsis plants carrying the gene-specific promoter fused to the bacterial beta-glucuronidase reporter gene revealed that some of the promoters have high activities in the epidermal layer of the shoot apical meristem and developing shoot organs, while others are temporarily active during reproductive organ development. Expression profiles of highly conserved paralogous gene pairs within the family were found to be not necessarily overlapping. Analyses of T-DNA insertion mutants of these HDG genes revealed that all mutants except hdg11 alleles exhibit no abnormal phenotypes. hdg11 mutants show excess branching of the trichome. This phenotype is enhanced in hdg11 hdg12 double mutants. Double mutants were constructed for other paralogous gene pairs and genes within the same subfamily. However, novel phenotypes were observed only for hdg3 atml1 and hdg3 pdf2 mutants that both exhibited defects in cotyledon development. These observations suggest that some of the class IV homeodomain-Leucine zipper members act redundantly with other members of the family during various aspects of cell differentiation. DNA-binding sites were determined for two of the family members using polymerase chain reaction-assisted DNA selection from random oligonucleotides with their recombinant proteins. The binding sites were found to be similar to those previously identified for ATML1 and PDF2, which correspond to the pseudopalindromic sequence 5'-GCATTAAATGC-3' as the preferential binding site.