NADPH thioredoxin reductase C is localized in plastids of photosynthetic and nonphotosynthetic tissues and is involved in lateral root formation in Arabidopsis

Plastids are organelles present in photosynthetic and nonphotosynthetic plant tissues. While it is well known that thioredoxin-dependent redox regulation is essential for leaf chloroplast function, little is known of the redox regulation in plastids of nonphotosynthetic tissues, which cannot use light as a direct source of reducing power. Thus, the question remains whether redox regulation operates in nonphotosynthetic plastid function and how it is integrated with chloroplasts for plant growth. Here, we show that NADPH-thioredoxin reductase C (NTRC), previously reported as exclusive to green tissues, is also expressed in nonphotosynthetic tissues of Arabidopsis thaliana, where it is localized to plastids. Moreover, we show that NTRC is involved in maintaining the redox homeostasis of plastids also in nonphotosynthetic organs. To test the relationship between plastids of photosynthetic and nonphotosynthetic tissues, transgenic plants were obtained with redox homeostasis restituted exclusively in leaves or in roots, through the expression of NTRC under the control of organ-specific promoters in the ntrc mutant. Our results show that fully functional root amyloplasts are not sufficient for root, or leaf, growth, but fully functional chloroplasts are necessary and sufficient to support wild-type rates of root growth and lateral root formation.     

Isolation of precise plastid deletion mutants by homology-based excision: a resource for site-directed mutagenesis, multi-gene changes and high-throughput plastid transformation

We describe a simple and efficient homology-based excision method to delete plastid genes. The procedure allows one or more adjacent plastid genes to be deleted without the retention of a marker gene. We used aad A-based transformation to duplicate a 649 bp region of plastid DNA corresponding to the atp B promoter region. Efficient recombination between atp B repeats deletes the intervening foreign genes and 1984 bp of plastid DNA (co-ordinates 57 424–59 317) containing the rbc L gene. Only five foreign bases are present in Δ rbc L plants illustrating the precision of homology-based excision. Sequence analysis of non-functional rbc L-related sequences in Δ rbc L plants indicated an extra-plastidic origin. Mutant Δ rbc L plants were heterotrophic, pale-green and contained round plastids with reduced amounts of thylakoids. Restoration of autotrophy and leaf pigmentation following aad A-based transformation with the wild-type rbc L gene ruled out mutations in other genes. Excision and re-use of aadA shows that, despite the multiplicity of plastid genomes, homology-based excision ensures complete removal of functional aad A genes. Rescue of the Δ rbc L mutation and autotrophic growth stabilizes transgenic plastids in heteroplasmic transformants following antibiotic withdrawal, enhancing the overall efficiency of plastid transformation. Unlike the available set of homoplasmic knockout mutants in 25 plastid genes, the rbc L deletion mutant isolated here is readily transformed with the efficient aad A marker gene. This improvement in deletion design facilitates advanced studies that require the isolation of double mutants in distant plastid genes and the replacement of the deleted locus with site-directed mutant alleles and is not easily achieved using other methods.     

Increases in cell elongation,plastid compartment size and phytoene synthase activity underlie the phenotype of the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant of tomato

A characteristic trait of the high pigment-1 ( hp-1) mutant phenotype of tomato ( Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and phytoene synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of phytoene synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of phytoene synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.     

A plant DJ-1 homolog is essential for Arabidopsis thaliana chloroplast development

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development.     

Vir-115 gene product is required to stabilize D1 translation intermediates in chloroplasts

The nuclear gene mutant of barley, vir-115, shows a developmentally induced loss of D1 synthesis that results in inactivation of Photosystem II. Translation in plastids isolated from 1 h illuminated vir-115 seedlings is similar to wild type. In wild-type barley, illumination of plants for 16 to 72 h results in increased radiolabel incorporation into the D1 translation intermediates of 15–24 kDa. In contrast, these D1 translation intermediates were not observed in vir-115 plastids isolated from plants illuminated for 16–72 h. In addition, after 72 h of illumination, radiolabel incorporation into D1 was undetectable in vir-115 plastids. The level and distribution ofpsbA mRNA in membrane-associated polysomes was similar in wild-type and vir-115 mutant plastids isolated from plants illuminated for 16–72 h. Toeprint analysis showed similar levels of translation initiation complexes onpsbA mRNA in vir-115 and wild-type plastids. These results indicate that translation initiation and elongation of D1 is not significantly altered in the mutant plastids. Ribosome pausing onpsbA mRNA was observed in wild-type and vir-115 mutant plastids. Therefore, the absence of D1 translation intermediates in mutant plastids is not due to a lack of ribosome pausing onpsbA mRNA. Based on these results, it is proposed that vir-115 lacks or contains a modified nuclear-encoded gene product which normally stabilizes the D1 translation intermediates. In wild-type plastids, ribosome pausing and stabilization of D1 translation intermediates is proposed to facilitate assembly of cofactors such as chlorophyll will D1 allowing continued D1 synthesis and accumulation in mature chloroplasts.     

Rampant gene loss in the underground orchid Rhizanthella gardneri highlights evolutionary constraints on plastid genomes

Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ～110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.     

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.     

The plastid clpP gene may not be essential for plant cell viability

The plastid gene clpP is widely regarded as essential for chloroplast function and general plant cell survival. In this note we provide evidence that certain lines of non-photosynthetic maize (Zea mays) Black Mexican Sweet (BMS) suspension cells do not carry clpP in their plastid genomes. We also discuss several incidences in the literature where clpP is either missing or not expressed in other non-green cell lines and plants. We conclude that clpP is not required for general plant cell survival but instead may only be essential for the development and/or function of plastids with active gene expression.     

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.Communicated by R. Hagemann