Light Promotion of Hypocotyl Gravitropism of a Starch-Deficient Tobacco Mutant Correlates with Plastid Enlargement and Sedimentation1

Dark-grown hypocotyls of a starch-deficient mutant (NS458) of tobacco (Nicotiana sylvestris) lack amyloplasts and plastid sedimentation, and have severely reduced gravitropism. However, gravitropism improved dramatically when NS458 seedlings were grown in the light. To determine the extent of this improvement and whether mutant hypocotyls contain sedimented amyloplasts, gravitropic sensitivity (induction time and intermittent stimulation) and plastid size and position in the endodermis were measured in seedlings grown for 8 d in the light. Light-grown NS458 hypocotyls were gravitropic but were less sensitive than the wild type (WT). Starch occupied 10% of the volume of NS458 plastids grown in both the light and the dark, whereas WT plastids were essentially filled with starch in both treatments. Light increased plastid size twice as much in the mutant as in the WT. Plastids in light-grown NS458 were sedimented, presumably because of their larger size and greater total starch content. The induction by light of plastid sedimentation in NS458 provides new evidence for the role of plastid mass and sedimentation in stem gravitropic sensing. Because the mutant is not as sensitive as the WT, NS458 plastids may not have sufficient mass to provide full gravitropic sensitivity.

Plastid Ontogeny during Petal Development in Arabidopsis1

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Different sources of reduced carbon contribute to form three classes of terpenoid emitted by Quercus ilex L. leaves.

Quercus ilex L. leaves emit terpenes but do not have specialized structures for terpene storage. We exploited this unique feature to investigate terpene biosynthesis in intact leaves of Q. ilex. Light induction allowed us to distinguish three classes of terpenes: (i) a rapidly induced class including alpha-pinene; (ii) a more slowly induced class, including cis-beta-ocimene; and (iii) the most slowly induced class, including 3-methyl-3-buten-1-ol. Using 13C, we found that alpha-pinene and cis-beta-ocimene were labeled quickly and almost completely while there was a delay before label appeared in linalool and 3-methyl-3-buten-1-ol. The acetyl group of 3-methyl-3-buten-1-yl acetate was labeled quickly but label was limited to 20% of the moiety. It is suggested that the ocimene class of monoterpenes is made from one or more terpenes of the alpha-pinene class and that both classes are made entirely from reduced carbon pools inside the chloroplasts. Linalool and 3-methyl-3-buten-1-ol are made from a different pool of reduced carbon, possibly in nonphotosynthetic plastids. The acetyl group of the 3-methyl-3-buten-1-yl acetate is derived mostly from carbon that does not participate in photosynthetic reactions. Low humidity and prolonged exposure to light favored ocimenes emission and induced linalool emission. This may indicate conversion between terpene classes.

Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA.

A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

Componentes genéticos que afetam a via de direcionamento de proteínas organelares em Arabidopsis thaliana

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos.

Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus

The thiol tripeptides, glutathione (GSH) and homoglutathione (hGSH), perform multiple roles in legumes, including protection against toxicity of free radicals and heavy metals. The three genes involved in the synthesis of GSH and hGSH in the model legume, Lotus japonicus, have been fully characterized and appear to be present as single copies in the genome. The gamma-glutamylcysteine synthetase (gammaecs) gene was mapped on the long arm of chromosome 4 (70.0 centimorgans [cM]) and consists of 15 exons, whereas the glutathione synthetase (gshs) and homoglutathione synthetase (hgshs) genes were mapped on the long arm of chromosome 1 (81.3 cM) and found to be arranged in tandem, with a separation of approximately 8 kb. Both genes consist of 12 exons of exactly the same size (except exon 1, which is similar). Two types of transcripts were detected for the gshs gene, which putatively encode proteins localized in the plastids and cytosol. Promoter regions contain cis-acting regulatory elements that may be involved in the plant's response to light, hormones, and stress. Determination of transcript levels, enzyme activities, and thiol contents in nodules, roots, and leaves revealed that gammaecs and hgshs are expressed in all three plant organs, whereas gshs is significantly functional only in nodules. This strongly suggests an important role of GSH in the rhizobia-legume symbiosis.

Physical and ultrastructural basis of blue leaf iridescence in four Malaysian understory plants

Iridescent blue leaf coloration in four Malaysian rain forest understory plants, Diplazium tomentosum Bl. (Athyriaceae), Lindsaea lucida Bl. (Lindsaeaceae), Begonia pavonina Ridl. (Begoniaceae), and Phyllagathis rotundifolia Bl. (Melastomataceae) is caused by a physical effect, constructive interference of reflected blue light. The ultrastructural basis for this in D. tomentosum and L. lucida is multiple layers of cellulose microfibrils in the uppermost cell walls of the adaxial epidermis. The helicoidal arrangement of these fibrils is analogous to that which produces a similar color in arthropods. In B. pavonina and P. rotundifolia the blue-green coloration is caused by parallel lamellae in specialized plastids adjacent to the abaxial wall of the adaxial epidermis. The selective advantage of this color production, if any, is unknown.

Irradiance and spectral quality affect Asian tropical rain forest tree seedling development

Iridescent blue leaf coloration in four Malaysian rain forest understory plants, Diplazium tomentosum Bl. (Athyriaceae), Lindsaea lucida Bi. (Lindsaeaceae), Begonia pavonina Ridl. (Begoniaceae), and Phyllagathis rotundifolia Bl. (Melastoma- taceae) is caused by a physical effect, constructive interference of reflected blue light. The ultrastructural basis for this in D. tomentosum and L. lucida is multiple layers of cellulose microfibrils in the uppermost cell walls of the adaxial epidermis. The helicoidal arrangement of these fibrils is analogous to that which produces a similar color in arthropods. In B. pavonina and P. rotundifolia the blue-green coloration is caused by parallel lamellae in specialized plastids adjacent to the abaxial wall of the adaxial epidermis. The selective advantage of this color production, if any, is unknown.

Effects of copper on the photosynthesis of intact chloroplasts: interaction with manganese

Highly purified, intact chloroplasts were prepared from pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) following an identical procedure, and were used to investigate the cupric cation inhibition on the photosynthetic activity. In both species, copper inhibition showed a similar inhibitor concentration that decreases the enzyme activity by 50% (IC(50) approximately 1.8 microM) and did not depend on the internal or external phosphate (Pi) concentration, indicating that copper did not interact with the Pi translocator. Fluorescence analysis suggested that the presence of copper did not facilitate photoinhibition, because there were no changes in maximal fluorescence (F(m)) nor in basal fluorescence (F(o)) of copper-treated samples. The electron transport through the photosystem II (PSII) was also not affected (operating efficiency of PSII-F'v/F'm similar in all conditions). Yet, under Cu(2+) stress, the proportion of open PSII reaction centers was dramatically decreased, and the first quinone acceptor (Q(A)) reoxidation was fully inhibited, as demonstrated by the constant photochemical quenching (q(P)) along experiment time. The quantum yield of PSII electron transport (Phi(PSII)) was also clearly affected by copper, and therefore reduced the photochemistry efficiency. Manganese, when added simultaneously with copper, delayed the inhibition, as measured by oxygen evolution and chlorophyll fluorescence, but neither reversed the copper effect when added to copper-inhibited plastids, nor prevented the inhibition of the Hill activity of isolated copper-treated thylakoids. Our results suggest that manganese competed with copper to penetrate the chloroplast envelope. This competition seems to be specific because other divalent cations e.g. magnesium and calcium, did not interfere with the copper action in intact chloroplasts. All results do suggest that, under these conditions, the stroma proteins, such as the Calvin-Benson cycle enzymes or others are the most probable first target for the Cu(2+) action, resulting in the total inhibition of chloroplast photosynthesis and in the consequent unbalanced rate of production and consumption of the reducing power.