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Crotaoprostrin, a chalcone not yet known as a plant constituent, was isolated from the aerial parts of the Indian medicinal plant Crotalaria prostrata. The structures of the chalcone polyarvin and the partially hydrogenated naturally occurring derivatives crotaramin, crotaramosmin, and crotin were confirmed by chemical synthesis.
Methanolic extracts from Achyrocline satureioides (Dc.) Lam, Aristolochia macroura Gomez, Lithraea molleoides (Vell.) Engl., Schinus molle L., unlike those from Celtis spinosa Spreng, Chenopodium ambrosioides L., Petiveria alliacea L., and Plantago major L. showed cytotoxic activity against a human hepatocellular carcinoma cell line, Hep G2. Schinus molle L. was the most active (IC50=50+/-7 microg/ml). These results call for further studies of these extracts.
The compactness of plant chromosome and the structures of plant cell wall and cytoplasm pose a great resistance to fluorescence in situ hybridization (FISH), and consequently many new methods for improving spatial resolution are being exploited to overcome these problems. However, for plants with small chromosomes like rice and Brassica, there are still many difficulties. In this article a new and effective technique for preparation of extended DNA fibers (EDFs), using a series of treatments to prophase I chromosomes of Brassica oleracea PMCs, is presented. This technique allows longitudinal extension of the chromosomes 30-107 times longer than those of their metaphase counterparts. The length of the extended DNA fibers is between 89 microm and 273 microm, and the space resolution is 42.8-53.0 kb. Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from meiotic prophase I nuclei of B. olerecea. Through FISH to EDFs of pachytene chromosomes hybridized in situ with SRK (S-locus receptor kinase) and SPII (S-locus protein II) probes, for the first time we localized the accurate positions of S-locus and quantitatively analyzed the features of S genes in B. oleracea genome to show all S genes were single-copied. In addition, the length between two linked genes was measured to be about one micron. As a result, the highest space resolution which was about 4 kb was obtained.
Salicylic acid is an important regulator of induced plant resistance to pathogens. Consequently, the biosynthesis of salicylic acid and its regulation has received a lot of attention. Salicylic acid can be made from phenylalanine via cinnamic and benzoic acid. Recently, genetic studies in Arabidopsis have shown that salicylic acid is made in the chloroplast from isochorismate, a pathway that is known to operate in prokaryotes.
Two new polyprenylated benzophenones, a new polyprenylated phloroglucinol, and six new xanthone derivatives were isolated from the aerial parts of the Uzbekistan medicinal plant Hypericum scabrum. Their structures were elucidated on the basis of spectroscopic evidence. The isolated compounds showed moderate cytotoxicity for human tumor cells.
All cDNAs involved in carotenoid biosynthesis leading to lycopene in yellow petals of Gentiana lutea have been cloned from a cDNA library. They encode a geranylgeranyl pyrophosphate synthase, a phytoene synthase, a phytoene desaturase and a zeta-carotene desaturase. The indicated function of all cDNAs was established by heterologous complementation in Escherichia coli. The amino acid sequences deduced from the cDNAs were between 47.5% and 78.9% identical to those reported for the corresponding enzymes from other higher plants. Southern analysis suggested that the genes for each enzyme probably represent a small multi-gene family. Tissue-specific expression of the genes and expression during flower development was investigated. The expression of the phytoene synthase gene, psy, was enhanced in flowers but transcripts were not detected in stems and leaves by northern blotting. Transcripts of the genes for geranylgeranyl pyrophosphate (ggpps), phytoene desaturase (pds) and zeta-carotene desaturase (zds) were detected in flowers and leaves but not in stems. Analysis of the expression of psy and zds in petals revealed that levels of the transcripts were lowest in young buds and highest in fully open flowers, in parallel with the formation of carotenoids. Obviously, the transcription of these genes control the accumulation of carotenoids during flower development in G. lutea. For pds only a very slight increase of mRNA was found whereas the transcripts of ggpps decreased during flower development.
TPD (tapping panel dryness) is a complex physiological syndrome widely found in rubber tree (Hevea brasiliensis) plantations, which causes severe yield and crop losses in natural rubber-producing countries. The molecular mechanism underlying TPD is not known and there is presently no effective prevention or treatment for this serious disease. To investigate the molecular mechanism of TPD, we isolated and characterized genes for which the change of expression is associated with TPD. We report here the identification and characterization of a Myb transcription factor HbMyb1. HbMyb1 is expressed in leaves, barks, and latex of rubber trees, but its expression is significantly decreased in barks of TPD trees. Our results suggest that the expression of HbMyb1 is likely associated with TPD and that the function of HbMyb1 is associated with the integrity of bark tissue of rubber trees.
Many plants use day length as an environmental cue to ensure proper timing of the switch from vegetative to reproductive growth. Day-length sensing involves an interaction between the relative length of day and night, and endogenous rhythms that are controlled by the plant circadian clock. Thus, plants with defects in circadian regulation cannot properly regulate the timing of the floral transition. Here we describe the gene EARLY FLOWERING 4 (ELF4), which is involved in photoperiod perception and circadian regulation. ELF4 promotes clock accuracy and is required for sustained rhythms in the absence of daily light/dark cycles. elf4 mutants show attenuated expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), a gene that is thought to function as a central oscillator component. In addition, elf4 plants transiently show output rhythms with highly variable period lengths before becoming arrhythmic. Mutations in elf4 result in early flowering in non-inductive photoperiods, which is probably caused by elevated amounts of CONSTANS (CO), a gene that promotes floral induction.
The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses. Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK), phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms. In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid). Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions. In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.
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Laser capture microdissection (LCM) is a powerful system which allows the isolation of selectively targeted cells from a tissue section for the analysis of gene-expression profiles of individual cells. The technique has been successfully used for the isolation of specific mammalian cells, mainly cancer cells. However, LCM has never been reported to be applied to the gene expression analysis of plant cells. We used a modified LCM system and successfully applied it to target and isolate phloem cells of rice leaf tissue whose morphology is apparently different from the surrounding cells. Total RNA was extracted from microdissected (approximately 150) phloem cells and the isolated RNA was used for the construction of a cDNA library following the T7 RNA polymerase amplification. Sequence analysis of 413 randomly chosen clones from the library revealed that there was a high level of redundancy in the population and the clones could be subclassified into 124 different groups that contained related sequences. Approximately 37% of both the redundant population and the non-redundant subgroups had novel components while approximately 63% were either homologues to the known genes reported to be localized in phloem of different plant species, or were homologues to other known genes. In situ hybridization revealed that putative amino acid permease, one of the non-redundant clones, was specifically expressed in the phloem. The results proved the effectiveness of construction of a specialized cDNA library from the specific plant cells.
A new tetrahydroprotoberberine N-oxide alkaloid, (-)-cis-isocorypalmine N-oxide (1), together with two known compounds, 6-methoxydihydrosanguinarine (2) and norjuziphine (3), were isolated in continuing studies of the entire Formosan Corydalis tashiroi plant. The structures of these three compounds were determined through spectral analyses. In addition, compounds 1, 2, 3 and the seven alkaloids previously reported: (-)-cis-corydalmine N-oxide, (-)-trans-corydalmine N-oxide, (-)-trans-isocorypalmine N-oxide, scoulerine, protopine, oxysanguinarine and corydalmine, were found to possess antiplatelet aggregation activity.
By antimicrobial and cytotoxic-guided fractionation, a bioactive norquinone-methide triterpene, 15 alpha-hydroxypristimerin, was isolated from a South American medicinal plant, Maytenus scutioides. Its structure was determined on the basis of spectroscopic evidence. Successful chemical transformation of pristimerin to netzahualcoyene indicates that the 15-hydroxy compounds seems to be a possible percursor of 14(15)-ene-quinone-methide-triterpenoids in the biogenetic pathway.
Plant acyl-acyl carrier protein thioesterases (TEs) terminate the acyl-acyl carrier protein track of fatty acid biosynthesis and play an essential role in determining the amount and composition of fatty acids entering the storage lipid pool. A combination of bioinformatics tools was used to predict a three-dimensional model for Arabidopsis FatB (AtFatB), which comprises a fold similar to that of Escherichia coli TEII, an enzyme that is functionally similar to plant TEs but lacks significant sequence similarity and displays different inhibitor sensitivity. The catalytic residues in AtFatB, Cys-264 and His-229, localize to the same region of the model as catalytic residues found in other enzymes with helix/multi-stranded sheet motifs (hot dog folds). Based on the model, we identified Asn-227 as a possible third member of the proposed papain-like catalytic triad. The conversion of Asn-227 to Ala resulted in a loss of detectable activity (>200-fold reduction), similar to the result seen for the equivalent mutation in papain. Mapping of plant TE specificity-affecting mutations onto the structural model showed that these mutations all cluster around the catalytic triad. Also, superposition of the crystallographically determined structures of the complexes of 4-hydroxybenzoyl-CoA TE with substrate and beta-hydroxydecanoyl thiol ester dehydrase with inhibitor onto the AtFatB model showed that the substrate and inhibitor localize to the same region as the AtFatB catalytic triad in their respective structures. Together these data corroborate the structural model and show that the hot dog fold is common to enzymes from both prokaryotes and eukaryotes and that this fold supports at least three different catalytic mechanisms.
The structure of the major plant polysaccharides contributing to dietary fiber, their organization within the plant cell wall, and the problems associated with their analysis are briefly reviewed. Alkaline, acidic, and detergent fractionation methods are described and considered in relation to their specificity. The analysis of pectin, often ignored as a component of dietary fiber, is examined in detail. The solubility of pectins from a number of common plant foods was determined, indicating that a considerable portion of pectin polyuronide is lost to many of the commonly used analytical procedures.
Large-scale, single pass sequencing and parallel gene expression analysis using DNA microarrays were employed for the comprehensive investigation of ripening in strawberry fruit. A total of 1701 cDNA clones (comprising 1100 strawberry ESTs and 601 unsequenced cDNAs) obtained from a strawberry (Fragariaxananassa) ripe fruit cDNA library were displayed on microarrays, and used for monitoring concurrent gene expression in receptacle and achene tissues. Analysis of expression ratios identified 66 out of the 259 (25%) achene-related clones and 80 out of 182 (44%) receptacle-related clones with more than a 4-fold difference in expression between the two tissue types. Half of the achene-associated genes putatively encode proteins with unknown function, and a large number of the remainder were proteins predicted to form part of the signal and regulation cascades related to achene maturation and acquisition of stress and desiccation tolerance. These included phosphatases, protein kinases, 14-3-3 proteins, transcription factors, and others. In the receptacle, key processes and novel genes that could be associated with ripening were identified. Genes putatively encoding proteins related to stress, the cell wall, DNA/RNA/protein, and primary metabolism were highly represented. Apart from providing a global observation on gene expression programmes and metabolic pathways in the developing strawberry, this study has made available a large database and unique information for gene discovery, promoter selection and markers for molecular breeding approaches.
Two N-acyl-homoserine lactone (acyl-HSL) synthase genes, lasI from Pseudomonas aeruginosa and yenI from Yersinia enterocolitica, were introduced into tobacco, individually and in combination. Liquid chromatograph-tandem mass spectrometry and thin-layer chromatography confirmed products of lasI and yenI activity in single and cotransformants. Cotransformants expressing plastid-localized LasI and YenI synthases produced the major acyl-HSLs for each synthase in all tissues tested. Total acyl-HSL signals accumulated in leaf tissue up to 3 pmol/mg of fresh weight, half as much in stem tissue, and approximately 10-fold less in root tissues. Acyl-HSLs were present in aqueous leaf washes from greenhouse-grown transgenic plants. Transgenic lines grown for 14 days under axenic conditions produced detectable levels of acyl-HSLs in root exudates. Ethyl acetate extractions of rhizosphere and nonrhizosphere soil from transgenically grown plants contained active acyl-HSLs, whereas plant-free soil or rhizosphere and nonrhizosphere soil from wild-type plants lacked detectable amounts of acyl-HSLs. This work shows that bioactive acyl-HSLs are exuded from leaves and roots and accumulate in the phytosphere of plants engineered to produce acyl-HSLs. These data further suggest that plants that are bioengineered to synthesize acyl-HSLs can foster beneficial plant-bacteria communications or deter deleterious interactions. Therefore, it is feasible to use bioengineered plants to supplement soils with specific acyl-HSLs to modulate bacterial phenotypes and plant-associated bacterial community structures.