Plastids undifferentiated, a nuclear mutation that disrupts plastid differentiation in Zea mays L

Photosynthetic development in any plant requires the intracellular co-ordination of chloroplast and nuclear gene expression programs. In this report, we investigate the role of a nuclear gene in photosynthetic development by examining C4 photosynthetic differentiation in a yellow mutant of maize (Zea mays L.). The plastids undifferentiated (pun) mutation disrupts plastid biogenesis in both bundle sheath and mesophyll cells, at an early developmental stage and in a light-independent manner. Chloroplast thylakoids are disrupted in the mutant and both membrane-associated and soluble chloroplast-encoded proteins accumulate at much reduced levels. The observed plastid morphology is consistent with a general defect in chloroplast biogenesis that is most likely exerted at the post-translational level. Despite aberrant chloroplast development, nuclear photosynthetic genes are expressed normally in pun mutants. Thus, neither functional chloroplasts nor the Pun gene product are required to establish nuclear photosynthetic gene expression patterns in maize.     

Two adjacent nuclear genes are required for functional complementation of a chloroplast trans-splicing mutant from Chlamydomonas reinhardtii

The chloroplast tscA gene from Chlamydomonas reinhardtii encodes a co-factor RNA that is involved in trans-splicing of exons 1 and 2 of the psaA mRNA encoding a core polypeptide of photosystem I. Here we provide molecular and genetic characterization of the trans-splicing mutant TR72, which is defective in the 3'-end processing of the tscA RNA and consequently defective in splicing exons 1 and 2 of the psaA mRNA. Using genomic complementation, two adjacent nuclear genes were identified, Rat1 and Rat2, that are able to restore the photosynthetic growth of mutant TR72. Restoration of the photosynthesis phenotype, however, was successful only with a DNA fragment containing both genes, while separate use of the two genes did not rescue the wild-type phenotype. This was further confirmed by using a set of 10 gene derivatives in complementation tests. The deduced amino acid sequence of Rat1 shows significant sequence homology to the conserved NAD+-binding domain of poly(ADP-ribose) polymerases of eukaryotic organisms. However, mutagenesis of conserved residues in this putative NAD+-binding domain did not reveal any effect on restoration efficiency. Immunodetection analyses with enriched fractions of chloroplast proteins indicated that Rat1 is associated with chloroplast membranes. Using the yeast three-hybrid system, we were able to demonstrate the specific binding of tscA RNA by the Rat1 polypeptide. We propose that the two nuclear factors Rat1 and Rat2 are involved in processing of chloroplast tscA RNA and in subsequent splicing of psaA exons 1 and 2.     

A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis

Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.     

EGY1 encodes a membrane-associated and ATP-independent metalloprotease that is required for chloroplast development

Chloroplast development requires coordinated expression of both nuclear- and chloroplast-encoded genes. To better understand the roles played by nuclear-encoded chloroplast proteins in chloroplast biogenesis, we isolated an Arabidopsis mutant, egy1-1, which has a dual phenotype, reduced chlorophyll accumulation and abnormal hypocotyl gravicurvature. Subsequent map-based cloning and DNA sequencing of the mutant gene revealed a 10-bp deletion in an EGY1 gene, which encodes a 59-kDa metalloprotease that contains eight trans-membrane domains at its C-terminus, and carries out beta-casein degradation in an ATP-independent manner. EGY1 protein accumulation varies between tissue types, being most prominent in leaf and stem tissues, and is responsive to light and ethylene. EGY1-GFP hybrid proteins are localized in the chloroplast. egy1 mutant chloroplasts had reduced granal thylakoids and poorly developed lamellae networks. Furthermore, the accumulation of chlorophyll a/b binding proteins of the light-harvesting complexes I and II (Lhca and Lhcb) are significantly decreased in three separate loss-of-function egy1 mutants. Taken together, these results suggest that EGY1 metalloprotease is required for chloroplast development and, hence, a defective EGY1 gene has pleiotropic effects both on chloroplast development and on ethylene-dependent gravitropism of light-grown hypocotyls.     

Characterization of a zebra mutant of rice with increased susceptibility to light stress

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.     

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.     

水稻黄绿叶调控基因YGL18的克隆与功能解析

叶色突变体往往伴随着叶绿素含量变化及叶绿体结构异常, 是研究叶绿体发育与光合作用相关基因功能的重要材料。该研究通过甲基磺酸乙酯(EMS)诱变籼稻(Oryza sativa subsp. indica)品种华占(HZ)获得黄绿叶突变体, 将其命名为ygl18 (yellow-green leaf 18)。与野生型相比, 黄绿叶突变体ygl18自三叶期起叶片开始变黄且程度不断加深, 同时伴随着光合速率与叶绿素含量下降, 且结实率、千粒重及有效穗数均显著降低。透射电镜观察结果显示, ygl18的叶绿体结构紊乱, 基质片层疏松, 发育受到抑制, 与叶片出现黄绿色表型一致。遗传分析表明, ygl18突变性状受1对隐性等位核基因控制, 这对等位基因位于水稻第3号染色体长臂标记InDel2和InDel3之间115.2 kb范围内。进一步研究发现该突变体表型是编码铁氧还蛋白FdC2的基因LOC_Os03g48040的5'UTR发生突变所致。通过CRISPR转基因实验验证了该基因对表型的控制作用。研究结果揭示了叶色调控网络的遗传基础, 可为今后选育高光效水稻品种提供新线索。     

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T‐DNA is responsible for the phenotype. To quickly map non‐annotated T‐DNA insertions, we developed a reliable, cost‐effective and easy method based on whole‐genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf ( http://genetics.umh.es/phenoleaf ) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal–distal cell cycle‐driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.     

The Arabidopsis repressor of light signaling SPA1 acts in the phloem to regulate seedling de-etiolation, leaf expansion and flowering time

Plants adjust their growth and development in response to the ambient light environment. These light responses involve systemic signals that coordinate differentiation of different tissues and organs. Here, we have investigated the function of the key repressor of photomorphogenesis SPA1 in different tissues of the plant by expressing GUS-SPA1 under the control of tissue-specific promoters in a spa mutant background. We show that SPA1 expression in the phloem vasculature is sufficient to rescue the spa1 mutant phenotype in dark-grown spa mutant seedlings. Expression of SPA1 in mesophyll, epidermis or root tissues of the seedling, by contrast, has no or only slight effects. In the leaf, SPA1 expression in both the phloem and the mesophyll is required for full complementation of the defect in leaf expansion. SPA1 in phloem and mesophyll tissues affected division and expansion of cells in the epidermal layer, indicating that SPA1 induces non-cell-autonomous responses also in the leaf. Photoperiodic flowering is exclusively controlled by SPA1 expression in the phloem, which is consistent with previous results showing that the direct substrate of the COP1/SPA complex, CONSTANS, also acts in the phloem. Taken together, our results highlight the importance of phloem vascular tissue in coordinating growth and development. Because the SPA1 protein itself is incapable of moving from cell to cell, we suggest that SPA1 regulates the activity of downstream component(s) of light signaling that subsequently act in a non-cell-autonomous manner. SPA1 action in the phloem may also result in mechanical stimuli that affect cell elongation and cell division in other tissues.