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The cox2 locus of the primitive angiosperm plant Acorus calamus: molecular structure, transcript processing and RNA editing.

Acorus calamus, or sweet flag, is a semiaquatic plant of uncertain taxonomic position. Molecular phylogenetic analysis using plastid rbcL sequences have suggested that Acorus calamus might be the most ancient surviving representative of the ancestral monocotyledonous plants. In order to provide molecular and phylogenetic data for the mitochondrial genetic system of Acorus, we have determined the structure of a mitochondrial locus, the cytochrome oxidase subunit II gene cox2. The Acorus cox2 gene harbors an unusually small group II intron, the smallest plant mitochondrial intron known to date. The transcript undergoes C-to-U RNA editing at eight sites. One of these sites is likely to play a dual functional role in both intron splicing and protein function. The 3' end of the mature transcript folds into a characteristic stem-loop structure that is presumably required for mitochondrial mRNA stability. Phylogenetic analysis of the cox2 sequence data, as well as the unusual intron structure, all support an evolutionarily isolated position for Acorus calamus.

Base Sequence↗

Analysis of the plant architecture via tree-structured statistical models: the hidden Markov tree models.

Plant architecture is the result of repetitions that occur through growth and branching processes. During plant ontogeny, changes in the morphological characteristics of plant entities are interpreted as the indirect translation of different physiological states of the meristems. Thus connected entities can exhibit either similar or very contrasted characteristics. We propose a statistical model to reveal and characterize homogeneous zones and transitions between zones within tree-structured data: the hidden Markov tree (HMT) model. This model leads to a clustering of the entities into classes sharing the same 'hidden state'. The application of the HMT model to two plant sets (apple trees and bush willows), measured at annual shoot scale, highlights ordered states defined by different morphological characteristics. The model provides a synthetic overview of state locations, pointing out homogeneous zones or ruptures. It also illustrates where within branching structures, and when during plant ontogeny, morphological changes occur. However, the labelling exhibits some patterns that cannot be described by the model parameters. Some of these limitations are addressed by two alternative HMT families.

Combretaceae↗

Controlling electron transfer in Acyl-CoA oxidases and dehydrogenases: a structural view.

Plants produce a unique peroxisomal short chain-specific acyl-CoA oxidase (ACX4) for beta-oxidation of lipids. The short chain-specific oxidase has little resemblance to other peroxisomal acyl-CoA oxidases but has an approximately 30% sequence identity to mitochondrial acyl-CoA dehydrogenases. Two biochemical features have been linked to structural properties by comparing the structures of short chain-specific Arabidopsis thaliana ACX4 with and without a substrate analogue bound in the active site to known acyl-CoA oxidases and dehydrogenase structures: (i) a solvent-accessible acyl binding pocket is not required for oxygen reactivity, and (ii) the oligomeric state plays a role in substrate pocket architecture but is not linked to oxygen reactivity. The structures indicate that the acyl-CoA oxidases may encapsulate the electrons for transfer to molecular oxygen by blocking the dehydrogenase substrate interaction site with structural extensions. A small binding pocket observed adjoining the flavin adenine dinucleotide N5 and C4a atoms could increase the number of productive encounters between flavin adenine dinucleotide and O2.

Acyl-CoA Dehydrogenase↗

Chemical constituents and antioxidant activity of Mallotus roxburghianus leaves.

Mallotus roxburghianus is used in the traditional medicine in North-Eastern India, but previously no work has been done on the identification of bioactive compounds. Two new compounds, 3-(1-C-beta-D-glucopyranosyl)-2,6-dihydroxy-5-methoxybenzoic acid (6) and 2,4,8,9,10-pentahydroxy-3,7-dimethoxyanthracene-6-O-beta-D-rhamnopyranoside (7) together with beta-sitosterol (1), stigmasterol (2), betulinic acid (3), 4-hydroxybenzoic acid (4), beta-sitosterol-beta-D-glucoside (5), and bergenin (8) were isolated and identified from the leaves of M. roxburghianus. The chloroform soluble portion of the alcoholic extract of leaf, and compounds 3, 6, 7, and 8 exhibited encouraging antioxidant activities.

Antioxidants↗

Structural determination of three new germacrane-type sesquiterpene alcohols from curdione by microbial transformation.

Five germacrane-type sesquiterpene alcohols obtained from curdione (1) by microbial biotransformation were isolated. Their structures were characterized as (2R)-2beta-hydroxycurdione (2), 1alpha, 10beta-epoxy-11-hydroxycurdione (3), (2S)-2alpha, 11-dihydroxycurdione (4), 11,15-dihydroxycurdione (5) and (3R)-3alpha-hydroxycurdione (6) based on the extensive NMR studies. Among them, 4, 5 and 6 are new compounds.

Alcohols↗

Lignans and secoiridoids from the root bark of Chionanthus virginicus L.: isolation, identification and HPLC analysis.

Five lignans, phillyrin (1), pinoresinol-beta-D-glucoside (2), pinoresinol di-beta-D-glucoside (3), phillyrin-2-O-beta-D-glucoside (6), phillyrin-6-O-beta-D-glucoside (7), and three secoiridoids, oleuropein (4), ligustroside (5) and angustifolioside B (8), have been isolated from the root bark of Chionanthus virginicus L. (Oleaceae), a raw material used in the commercial preparation of homeopathy tinctures. Compound 6 is a new lignan, and compounds 2, 3, 4, 5, 7 and 8 are described for the first time in the title plant. The structures of these compounds were elucidated by spectroscopic methods (NMR and HPLC-MS). An isocratic HPLC method for the rapid characterisation and quantification of the major constituents, 1 and 4, of the root bark is described and validated.

Chromatography, High Pressure Liquid↗

Diaryloxyacetate herbicides.

The discovery and investigation of a novel family of herbicides containing a diaryl acetal are described. The stability of the acetal limited herbicidal efficacy and recognizing that fact led to the design of analogs with commercial levels of post-emergence activity on broadleaf weeds. These compounds inhibited acetolactate synthase and in vitro activity data were used to guide target design. However, no members of this family provided a commercially valuable combination of herbicidal efficacy and crop selectivity.

Acetolactate Synthase↗

Sugars proportionately affect artemisinin production.

Little is known about the effect of sugars in controlling secondary metabolism. In this study, sugars alone or in combination with their analogs were used to investigate their role in the production of the antimalarial drug, artemisinin, in Artemisia annua L. seedlings. Compared to sucrose, a 200% increase in artemisinin by glucose was observed. Different ratios of fructose to glucose yielded artemisinin levels directly proportional to increases in relative glucose concentration. When the glucose analog, 3-O-methylglucose, was added with glucose, artemisinin production was dramatically decreased, but hexokinase activity was significantly increased compared to glucose alone. In contrast, neither mannose nor mannitol had any significant effect on artemisinin yield. In comparison with 30 g/l sucrose, artemisinin levels were significantly reduced by 80% in the presence of 27 g/l sucrose + 3 g/l palatinose, which cannot be transported into cells through the sucrose transporter. Together these results suggest that both monosaccharide and disaccharide sugars are likely acting not only as carbon sources but also as signals to affect the downstream production of artemisinin, and that the mechanism of these effects appears to be complex.

Artemisia annua↗

The role of the megagametophyte in maintaining loblolly pine (Pinus taeda L.) seedling arginase gene expression in vitro.

Following loblolly pine (Pinus taeda L.) seed germination, storage-protein breakdown in the megagametophyte and in the seedling results in a large increase in the seedling's free amino acid pool. A substantial portion of both the storage proteins and the amino acid pool is arginine, a very efficient nitrogen-storage compound. Free arginine is hydrolyzed in the seedling by the enzyme arginase (EC 3.5.3.1), which is under strong developmental control. At present, regulation of arginase in conifers is not well understood. Here we report the utilization of an in vitro culture system to address the separate impacts of the seedling and megagametophyte tissues on arginase enzyme activity, protein levels and patterns of gene expression. We also describe the generation of an anti-arginase antibody prepared from a histidine-tagged loblolly pine arginase fusion protein expressed in Escherichia coli. Our results indicate that arginase gene expression in the seedling is initiated by the seedling itself and then maintained or up-regulated by the megagametophyte. The contribution of storage-protein breakdown and the free amino acid pool, particularly arginine, in this regulation is also addressed.

Arginase↗

Characterization of a fertilization-induced and developmentally regulated plasma-membrane aquaporin expressed in reproductive tissues, in the wild potato Solanum chacoense Bitt.

Fertilization triggers a unique and complex developmental program leading to embryogenesis and seed set. Growth and differentiation are accompanied by rapid metabolic changes as well as massive cellular reorganization inside the fertilized ovule. Using differential display to isolate genes involved in pollen-pistil interactions and early fertilization events, we isolated from Solanum chacoense Bitt. a fertilization-induced plasma-membrane intrinsic protein of the PIP2 family that is predominantly expressed in pistil and anthers tissues. Major intrinsic proteins (MIPs) consist of a large family of highly conserved membrane-spanning proteins that are mainly represented in plants by the aquaporins. Aquaporins, mainly of the PIP and TIP type, have been shown in many species to serve as water channels. In S. chacoense the ScPIP2a mRNA is developmentally regulated during anthesis, with mRNA levels gradually decreasing as the pistil reaches maturity. In flowers, strongest expression was observed in elongating styles, in stamens, and transiently in ovules following fertilization. In styles, maximal expression levels correlated with phases of rapid style elongation and with the formation of epidermal papillae. ScPIP2a mRNA was also strongly expressed in developing fruit, consistent with a role in cell expansion during maturation and development.

Amino Acid Sequence↗

A putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 from cotton (Gossypium hirsutum L.) fibers.

A novel plant gene CFL1 was cloned from cotton (Gossypium hirsutum L.) fibers by expressed sequence tag (EST) database searching and 5'-RACE (rapid amplification of cDNA ends). This gene shows sequence homology with FKS1 which has been identified as the putative catalytic subunit of the yeast beta-1,3-glucan synthase. It encodes a protein (CFL1p) of 219 kDa with 13 deduced transmembrane helices and 2 large hydrophilic domains, one of which is at the N-terminus and the other in the internal region of the polypeptide. CFL1 displays 21% identity and 41% similarity to FKS1 at the amino acid level over its entire length, with 31% identity and 52% similarity for the hydrophilic central domain. Using RNA and protein blot analysis, CFL1 was found to be expressed at higher levels in cotton fibers during primary wall development. CFL1 also had a strong expression in young roots. Using a calmodulin (CaM)-gel overlay assay, the hydrophilic N-terminal domain of CFL1p was shown to bind to CaM, while the hydrophilic central domain did not. A putative CaM-binding domain, 16 amino acids long, was predicted in the hydrophilic N-terminal domain. Moreover, a product-entrapment assay demonstrated that a protein associated with an in vitro-synthesized callose pellet could be labeled by anti-CFL1 antibodies. Our finding suggests that CFL1 is a putative plant homolog of the yeast beta-1,3-glucan synthase subunit FKS1 and could be involved in callose synthesis.

Calmodulin-Binding Proteins↗

Identification of genes preferentially expressed during wood formation in Eucalyptus.

Wood is the most abundant biological resource on earth and it is also an important raw material for a major global industry with rapidly increasing demand. The genus Eucalyptus includes the most widely used tree species for industrial plantation, mainly for making pulp and paper. With the aim of identifying major genes involved in wood formation in Eucalyptus , we have developed a targeted approach of functional genomics based on the isolation of xylem preferentially expressed genes by subtractive PCR. Transcript profiling using cDNA arrays and analysis of variance (ANOVA) were used to identify differentially expressed ESTs between secondary xylem and leaves. Real-time RT-PCR was performed to confirm the differential expression of representative EST. Of 224 independent EST sequences obtained, 81% were preferentially expressed in xylem. One-third of the ESTs exhibiting homologies with proteins of known function fell into two main classes highlighting the importance of the auxin signalling through ubiquitin-dependent proteolysis on one hand, and of the enzymes involved in cell wall biosynthesis and remodelling, on the other. The functions of the genes represented by the remaining 61% of ESTs should be of great interest for future research. This systematic analysis of genes involved in wood formation in Eucalyptus provides valuable insights into the molecular mechanisms involved in secondary xylem differentiation as well as new candidate-genes for wood quality improvement.

DNA, Complementary↗

Recent developments in the field of plant steroid hormones.

Brassinolide and related brassinosteroids are a novel group of steroids which appear to be ubiquitous in plants. There is compelling evidence, particularly from recent genetic studies, that these steroids are essential for normal plant growth and development. Synthesis of brassinosteroids and aspects of their biochemistry are reviewed.

Brassinosteroids↗

Chlorophyll metabolism.

Since the 1970s, researchers have proposed several regulatory pathways governing chlorophyll metabolism, but only recently have the underlying molecular mechanisms been elucidated. The recent data indicate that such regulatory systems are more complex than originally anticipated. For instance, the pathways involve a series of protein-protein interactions, including complex formation, the dual localization of enzymes within chloroplasts, and a novel protein degradation mechanism that is triggered by pigments. Furthermore, several lines of evidence suggest that chlorophyll metabolism might not only significantly impact the assembly of photosynthetic machineries but also influence processes such as programmed cell death, the 'stay-green' phenomenon, and chloroplast-nucleus communication.

Chlorophyll↗

Secondary metabolites from Senecio burtonii (Compositae).

A cacalolide derivative named 4alpha-[2'-hydroxymethylacryloxy]-1beta-hydroxy-14-(5-->6) abeo eremophilan-12,8-olide and a shikimic acid derivative named (3'E)-(1alpha)-3-hydroxymethyl-4beta,5alpha-dimethoxycyclohex-2-enyloctadec-3'-enoate along with three known compounds, octacosan-1-ol, 3beta-hydroxyolean-12-en-28-oic acid and 3beta-acetoxyolean-12-en-28-oic acid were isolated from Senecio burtonii. Their structures and relative configurations were established on the basis of spectroscopic analysis.

Molecular Structure↗

Analysis of gene expression in rose petals using expressed sequence tags.

Single-pass sequences were obtained from the 5'-ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent.

Contig Mapping↗

Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp.

In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.

Acyclic Monoterpenes↗