Brassinosteroid levels increase drastically prior to morphogenesis of tracheary elements.
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As the first step toward understanding the involvement of endogenous brassinosteroids (BRs) in cytodifferentiation, we analyzed biosynthetic activities of BRs in zinnia (Zinnia elegans L. cv Canary Bird) cells differentiating into tracheary elements. The results of feeding experiments suggested that both the early and late C6-oxidation pathways occur during tracheary element differentiation. Gas chromatography-mass spectrometry analysis revealed that five BRs, castasterone, typhasterol, 6-deoxocastasterone, 6-deoxotyphasterol, and 6-deoxoteasterone, actually existed in cultured zinnia cells and culture medium. Quantification of endogenous BRs in each stage of tracheary element differentiation by gas chromatography-mass spectrometry exhibited that they increased dramatically prior to the morphogenesis, which was consistent with the idea that BRs are necessary for the initiation of the final stage of tracheary element differentiation. Moreover, the proportion of each BR in culture medium was quite different from that in cells, suggesting that specific BRs are selectively secreted into medium and may function outside the cells. (+info)
Direct evidence of active and rapid nuclear degradation triggered by vacuole rupture during programmed cell death in Zinnia.
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Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active and rapid nuclear degradation is triggered by vacuole rupture. (+info)
CEVd-induced symptom modification as a response to a host-specific temperature-sensitive reaction.
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Natural selection of two new variants of citrus exocortis viroid (CEVd) was detected by observing tissues displaying both severe and mild symptoms from a single Gynura aurantiaca. The variants CEVd-S (severe) and CEVd-M (mild), differing by only five nucleotides confined to the pathogenic (P) domain, remained stable when propagated by rooted cuttings or from successive plants inoculated with tissue extracts or transcripts from cDNA clones. CEVd-S induces a very severe reaction in Gynura that is consistent throughout a range of environmental conditions. However, symptoms resulting from CEVd-M infection can vary from a nonsymptomatic condition to a severe reaction when grown at 40 degrees C. This differential response was confined to a single host, Gynura aurantiaca, and expressed under standard growing conditions. The distinct host responses induced by these variants could not be correlated with any changes in sequence or conformation of the dominant viroid variant, as predicted by molecular modeling. Therefore, the variable symptom expression appears to be associated with a specific temperature-sensitive response of Gynura aurantiaca. (+info)
Effects of elevated atmospheric CO2 concentration on leaf dark respiration of Xanthium strumarium in light and in darkness.
(20/487)
Leaf dark respiration (R) is an important component of plant carbon balance, but the effects of rising atmospheric CO(2) on leaf R during illumination are largely unknown. We studied the effects of elevated CO(2) on leaf R in light (R(L)) and in darkness (R(D)) in Xanthium strumarium at different developmental stages. Leaf R(L) was estimated by using the Kok method, whereas leaf R(D) was measured as the rate of CO(2) efflux at zero light. Leaf R(L) and R(D) were significantly higher at elevated than at ambient CO(2) throughout the growing period. Elevated CO(2) increased the ratio of leaf R(L) to net photosynthesis at saturated light (A(max)) when plants were young and also after flowering, but the ratio of leaf R(D) to A(max) was unaffected by CO(2) levels. Leaf R(N) was significantly higher at the beginning but significantly lower at the end of the growing period in elevated CO(2)-grown plants. The ratio of leaf R(L) to R(D) was used to estimate the effect of light on leaf R during the day. We found that light inhibited leaf R at both CO(2) concentrations but to a lesser degree for elevated (17-24%) than for ambient (29-35%) CO(2)-grown plants, presumably because elevated CO(2)-grown plants had a higher demand for energy and carbon skeletons than ambient CO(2)-grown plants in light. Our results suggest that using the CO(2) efflux rate, determined by shading leaves during the day, as a measure for leaf R is likely to underestimate carbon loss from elevated CO(2)-grown plants. (+info)
Pathogenicity of a natural recombinant associated with ageratum yellow vein disease: implications for geminivirus evolution and disease aetiology.
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Yellow vein disease of Ageratum conyzoides is caused by a viral DNA complex consisting of the genomic component (DNA A) of the monopartite begomovirus Ageratum yellow vein virus (AYVV, family: Geminiviridae) and a small satellite-like DNA beta component. AYVV DNA A is unable to induce symptoms in this host alone but can systemically infect A. conyzoides in which it accumulates to low levels. Here, we demonstrate that the yellow vein phenotype can also be produced by co-inoculating A. conyzoides with AYVV DNA A and recDNA-Abeta17, a naturally occurring recombinant of approximately the same size as DNA beta that contains sequences from both DNA A and DNA beta. Symptoms induced by DNA A and recDNA-Abeta17 in A. conyzoides and Nicotiana glutinosa are qualitatively similar to those associated with DNA A and DNA beta although milder. Recombination between DNA A and DNA beta to produce a chimera resembling recDNA-Abeta17 was observed after whitefly transmission of the disease in A. conyzoides. Hence, such recombination events are likely to occur frequently, implying that recombinants will normally be associated with this type of disease complex in the field. Possible implications of these findings for the evolution of begomoviruses and the aetiology of their diseases are discussed. (+info)
Secoisolariciresinol dehydrogenase purification, cloning, and functional expression. Implications for human health protection.
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Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the approximately 32-kDa NAD-dependent secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of approximately 32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, secoisolariciresinol and matairesinol, has been elucidated. Last, both secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called "mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers. (+info)
Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.
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Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction. (+info)
A novel dark-inducible protein, LeDI-2, and its involvement in root-specific secondary metabolism in Lithospermum erythrorhizon.
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Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities. (+info)