Structure and function of cytokinin oxidase/dehydrogenase genes of maize,rice,<Emphasis Type="Italic"> Arabidopsis</Emphasis> and other species

Cytokinin oxidases/dehydrogenases (CKX) catalyze the irreversible degradation of the cytokinins isopentenyladenine, zeatin, and their ribosides in a single enzymatic step by oxidative side chain cleavage. To date the sequences of 17 fully annotated CKX genes are known, including two prokaryotic genes. The CKX gene families of Arabidopsis thaliana and rice comprise seven and at least ten members, respectively. The main features of CKX genes and proteins are summarized in this review. Individual proteins differ in their catalytic properties, their subcellular localization and their expression domains. The evolutionary development of cytokinin-catabolizing gene families and the individual properties of their members indicate an important role for the fine-tuned control of catabolism to assure proper regulation of cytokinin functions. The use of CKX genes as a tool in studies of cytokinin biology and biotechnological applications is discussed.     

Determination of trehalose-6-phosphate in Arabidopsis seedlings by successive extractions followed by anion exchange chromatography-mass spectrometry

A method for the detection of trehalose-6-phosphate (T6P) in tissue of the model plant Arabidopsis thaliana is presented. Liquid-liquid extraction (LLE) and mixed mode solid-phase extraction (SPE) were used for sample pretreatment followed by anion exchange chromatography (AEC) coupled with electrospray ionization mass spectrometry (MS) for highly selective quantitative analysis. LLE of plant material was performed with chloroform/acetonitrile/water (3:7:16, v/v/v) followed by SPE with Oasis MAX material, which significantly reduced the complexity of the extracts. On-line coupling of MS with gradient AEC using a sodium hydroxide eluent was accomplished with a postcolumn ion suppressor. The method allows specific quantification of T6P with good linearity for spiked plant extracts, from 80 nM to 1.3 μM (r² > 0.98). The limit of detection in plant extracts was 40 nM. The recovery of the method was above 80% for relevant T6P levels. The method was applied to the determination of T6P in seedlings from four mutant A. thaliana lines (TRR1-4) resisting growth arrest caused by external supply of trehalose. Results reveal that T6P accumulation differed substantially in the four mutant lines and wild type (WT). It is concluded that the mutants circumvent the growth arrest observed in WT seedlings on 100 mM trehalose by different mechanisms.     

Solid-phase extraction of cytokinins from aqueous solutions with C18 cartridges and their use in a rapid purification procedure

Eleven cytokinins-including bases, ribosides, glucosides, and ribotides-were tested for their retention on C₁₈ cartridges that were washed with 40 mL of water or a dilute acid at pH 3. Cytokinins were then eluted with methanol and analyzed by high performance liquid chromatography (HPLC). All pure cytokinin were well retained when the cartridge was washed with water, but Z and (diH)Z were less well retained at pH 3. The ribotides required 80% methanol for elution. Cotton leaf tissue (500 mg dry wt) was spiked with cytokinins, extracted with 80% methanol, and the extract bulk purified with hexane, insoluble polyvinylpyrrolidone, and minicolumns (strong anion exchange, amino, and C₁₈ cartridges). Ribotides, added to leaf tissue, could not be recovered as ribotides; it was necessary to hydrolyze and purify them as ribosides. The cytokinins were separated and analyzed by HPLC on strong cation exchange and C₁₈ columns. Recoveries through the entire procedure averaged 70%.Cytokinin abbreviations (diH)Z Dihydrozeatin - (diH)Z dihydrozeatin riboside - (diH)[9R]Z trans-zeatin - Z t-zeatin riboside - [9R]Z t-zeatin-O-glucoside - (OG)Z t-zeatin riboside-O-glucoside - (OG)[9R]Z t-zeatin riboside-5-monophosphate - [9R-5P]Z N6(^2-isopentenyl)adenine - iP N6(^2-isopentenyl)adenosine - [9R]iP N6(^2-isopentenyl)adenosine-5-monophosphate-[9R-5P]iP     

Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils

AIM: To establish a rapid, improved soil environmental DNA extraction and purification protocol. METHODS AND RESULTS: Three different soil DNA isolation and four purification strategies were compared on different soil samples with variable rates of success. Bead beating extraction gave significantly higher DNA yields than microwave-based and liquid nitrogen grinding DNA extraction methods. The inclusion of soil washing prior to cell lysis decreased the amount of purification steps required. Although these soil types differed, polyvinylpolypyrrolidone (PVPP)-sepharose 2B column elution was sufficient for all three samples, yielding DNA pure enough for successful application in molecular studies. One soil sample retained 80% of the initial DNA after successful purification. CONCLUSIONS: Optimization of a purification protocol confirmed that only a combination of previously described methods proved sufficient in yielding pure environmental DNA from humic-rich soils. Total processing time for DNA extraction and subsequent purification from multiple samples was considerably more rapid than the previously described methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a new optimized soil DNA extraction and purification protocol that is suitable for different environmental sources that are rich in humic acid content.     

Metabolomics-oriented isolation and structure elucidation of 37 compounds including two anthocyanins from Arabidopsis thaliana

In order to conduct metabolomic studies in a model plant for genome research, such as Arabidopsis thaliana (Arabidopsis), it is a prerequisite to obtain structural information for the isolated metabolites from the plant of interest. In this study, we isolated metabolites of Arabidopsis in a relatively non-targeted way, aiming at the construction of metabolite standards and chemotaxonomic comparison. Anthocyanins (5 and 7) called A8 and A10 were isolated and their structures were elucidated as cyanidin 3-O-[2-O-(β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-E-p-coumaroyl)-β-d-glucopyranoside]-5-O-[6-O-(malonyl)-β-d-glucopyranoside] and cyanidin 3-O-[2-O-(2-O-(E-sinapoyl)-β-d-xylopyranosyl)-6-O-(4-O-(β-d-glucopyranosyl)-E-p-coumaroyl)-β-d-glucopyranoside]-5-O-[β-d-glucopyranoside] from analyses of 1D NMR, 2D NMR (¹H NMR, NOE, ¹³C NMR, HMBC and HMQC), HRFABMS, FT-ESI-MS and GC-TOF-MS data. In addition, 35 known compounds, including six anthocyanins, eight flavonols, one nucleoside, one indole glucosinolate, four phenylpropanoids and a derivative, together with three indoles, one carotenoid, one apocarotenoid, three galactolipids, two chlorophyll derivatives, one steroid, one hydrocarbon, and two dicarboxylic acids, were also isolated and identified from their spectroscopic data.     

Sterol methyltransferase2: purification, properties, and inhibition

Expression of the Arabidopsis sterol methyltransferase2 (SMT2) cDNA in Escherichia coli yields a native protein, when purified to homogeneity, has the predicted molecular mass ca. 40 kDa on SDS-PAGE and recognizes native sterols synthesized by Arabidopsis with a Delta(24(25))-bond (cycloartenol; K(m) 35 microM and k(cat) 0.001s(-1)) and Delta(24(28))-bond (24(28)-methylenelophenol; K(m) 28 microM and k(cat) 0.01 s(-1)). Cycloartenol was converted to a single olefinic product-24(28)-methylenecycloartanol whereas 24(28)-methylenelophenol was converted to a mixture of three stereochemically related products with the Delta(24(28))Z-ethylidene, Delta(24(28))E-ethylidene, and Delta(25(27))-24 beta-ethyl side chains. Structural determinants essential to activity were the nucleophilic features at C-3 and C-24. The double bond position in the sterol substrate influenced catalytic efficiency according to the order: side chain, Delta(24(24))相似文献   

Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.     

Inflorescence abnormalities occur with overexpression of Arabidopsis lyrata FT in the fwa mutant of Arabidopsis thaliana

Arabidopsis thaliana is a quantitative long-day plant with the timing of the floral transition being regulated by both endogenous signals and multiple environmental factors. fwa is a late-flowering mutant, and this phenotype is due to ectopic FWA expression caused by hypomethylation at the FWA locus. The floral transition results in the activation of the floral development process, the key regulators being the floral meristem identity genes, AP1 (APETALA1) and LFY (LEAFY). In this study, we describe inflorescence abnormalities in plants overexpressing the Arabidopsis lyrata FT (AlFT) and A. thaliana FWA (AtFWA) genes simultaneously. The inflorescence abnormality phenotype was present in only a proportion of plants. All plants overexpressing both AlFT and AtFWA flowered earlier than fwa, suggesting that the inflorescence abnormality and earlier flowering time are caused independently. The inflorescence abnormality phenotype was similar to that of the double mutant of ap1 and lfy, and AP1 and LFY genes were down-regulated in the abnormal inflorescences. From these results, we suggest that not only does ectopic AtFWA expression inhibit AtFT/AlFT function to delay flowering but that overexpression of AtFWA and AlFT together inhibits AP1 and LFY function to produce abnormal inflorescences.     

The pleiotropic mutation dar1 affects plant architecture in Arabidopsis thaliana

Shoot architecture is shaped upon the organogenic activity of the shoot apical meristem (SAM). Such an activity relies on the balance between the maintenance of a population of undifferentiated cells in the centre of the SAM and the recruitment of organ founder cells at the periphery. A novel mutation in Arabidopsis thaliana, distorted architecture1 (dar1), is characterised by disturbed phyllotaxy of the inflorescence and consumption of the apical meristem late in development. SEM and light microscopy analyses of the dar1 SAM reveal an abnormal partitioning of meristematic domains, and mutations known to affect the SAM structure and function were found to interact with dar1. Moreover, the mutant shows an alteration of the root apical meristem (RAM) structure. Those observations support the hypothesis that DAR1 has a role in meristem maintenance and it is required for the normal development of Arabidopsis inflorescence during plant life.     

Endogenous isoprenoid and aromatic cytokinins in different plant parts of Cocos nucifera (L.)

The present study reports the analyses of both isoprenoid and aromatic cytokinins in the coconut palm by combined high performance liquid chromatography and group specific enzyme immunoassays (HPLC-ELISA). The results showed that the isoprenoid cytokinins were several fold more abundant than the aromatic cytokinins in each of the plant parts analysed: immature inflorescence, shoot apical meristem (SAM), spear leaf and embryo. Within the isoprenoid cytokinins, the most abundant ones by type were the zeatin- (Z-), the isopentenyladenine- (iP-) and the dihydrozeatin- (DHZ-) type in decreasing order for most plant parts studied, and individually, zeatin riboside (ZR) or zeatin riboside-5-monophosphate (ZR5P) depending on the part. In the case of the iP-type cytokinins, the results showed that its 9-glucoside was the most abundant one in most parts. The isoprenoid cytokinin profiles in coconut showed a predominant pattern of 9-conjugation as a major metabolism route for these cytokinins. Analyses also showed the occurrence of the aromatic cytokinin 6-benzylaminopurine (BAP) and its riboside (BAPR), 9-glucoside (BAP9G), and nucleotide (BAPR5P). Their presence in coconut palm was unequivocally identified after permethylation by gas chromatography-mass spectrometry. They were more concentrated in the embryo and in the immature inflorescence than in the other two parts studied, however their concentration in each part was several times lower than that of isoprenoid cytokinins. All four were detected in each of the parts studied. The most abundant ones were BAPR and BAP9G in immature inflorescence; and BAPR in all of the other parts. When all cytokinins analysed are considered, differences between the plant parts studied were found. The zygotic embryos showed the highest content, double that in immature inflorescence, and five times more that in spear leaf and SAM. These differences are even greater when individual cytokinins are compared.     

The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions

The development of mitochondria and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting membranes. The aim of the present work was: (1) to enhance our understanding of the biochemical machinery of mitochondrial membranes and (2) to test the versatility of the procedure developed for the identification of the hydrophobic proteome of the chloroplast envelope [Molecular and Cellular Proteomics 2 (2003) 325-345]. A proteomic analysis was performed, to provide the most exhaustive view of the protein repertoire of these membranes. For this purpose, highly purified mitochondria were prepared from Arabidopsis cultured cells and membrane proteins were extracted. To get a more exhaustive array of membrane proteins from Arabidopsis mitochondria, from the most to the less hydrophobic ones, various extraction procedures (chloroform/methanol extraction, alkaline or saline treatments) were applied. LC-MS/MS analyses were then performed on each membrane subfraction, leading to the identification of more than 110 proteins. The identification of these proteins is discussed with respect to their mitochondrial localization, their physicochemical properties and their implications in the metabolism of mitochondria. In order to provide a new overview of the biochemical machinery of the plant mitochondria, proteins identified during this work were compared to the lists of proteins identified during previous proteomic analyses performed on plant and algae mitochondria (Arabidopsis, pea, Chlamydomonas, rice, etc.). A total of 502 proteins are listed. About 40% of the 114 proteins identified during this work were not identified during previous proteomic studies performed on mitochondria.     

Xylogalacturonan exists in cell walls from various tissues of Arabidopsis thaliana

Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH). Upon enzymatic treatment, XGA oligosaccharides were primarily produced from pectin extracts obtained from the young and mature leaves and to a lesser extent from those originating from the stem of A. thaliana. The oligosaccharide GalA(3)Xyl was predominantly formed from these pectin extracts. No XGA oligosaccharides were detected in digests of pectin extracts from the seeds and roots. A low number of XGA oligosaccharides was obtained from pectins of A. thaliana. This indicates a uniform distribution of xylose in XGA from A. thaliana. The predominant production of GalA(3)Xyl, as well as the release of linear GalA oligosaccharides pointed to a lower degree of xylose substitution in XGA from A. thaliana than in XGA from apple and potato. The estimated amount of XGA accounted for approximately 2.5%, 7% and 6% (w/w) of the total carbohydrate in the pectin fraction of the stem, young leaves and mature leaves, respectively.     

Expression and characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana

The present study reports the recombinant expression, purification, and partial characterization of a typical aspartic proteinase from Arabidopsis thaliana (AtAP A1). The cDNA encoding the precursor of AtAP A1 was expressed as a functional protein using the yeast Pichia pastoris. The mature form of the rAtAP A1 was found to be a heterodimeric glycosylated protein with a molecular mass of 47 kDa consisting of heavy and light chain components, approx. 32 and 16 kDa, respectively, linked by disulfide bonds. Glycosylation occurred via the plant specific insert in the light chain. The catalytic properties of the rAtAP A1 were similar to other plant aspartic proteinases with activity in acid pH range, maximal activity at pH 4.0, K_m of 44 μM, and k_cat of 55 s⁻¹ using a synthetic substrate. The enzyme was inhibited by pepstatin A.     

HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators

Phytochelatins (PCs) are nonprotein peptides with the general structure (γ-Glu-Cys)_n-Gly (PC_n), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (γ-Glu-Cys-Gly) and/or previously synthesized PC_n as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5′-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions.