Adventitious rooting of detached Arabidopsis thaliana leaves

Arabidopsis thaliana Heyhn is a model species in biochemical, physiological and molecular studies for which a plethora of mutants is available. This work aimed at developing a system for rooting of detached leaves, and evaluating time course of several relevant biochemical parameters during rooting assays with and without auxins. The rooting pattern was of the direct type (without callus formation) in all of the treatments and ecotypes analyzed and was rather stable. Considering the different parameters examined, peroxidase activity and contents of phenolic compounds and soluble sugars appeared as the most distinct biochemical markers of the rooting process in this system.     

Imaging photosynthesis in wounded leaves of Arabidopsis thaliana

Chlorophyll fluorescence imaging provides a non-invasive and non-destructive means with which to measure photosynthesis. This technique has been used, in combination with 14CO2 feeding, to study the spatial and temporal changes in source-sink relationships which occur in mechanically wounded leaves of Arabidopsis thaliana. Twenty-four hours after wounding, cells proximal to the wound margin showed a rapid induction of PhiII upon illumination (a measure of the efficiency of photosystem II photochemistry) whilst cells more distal to the wound margin exhibited a much slower induction of PhiII and a large, transient increase in NPQ (a measure of the rate constant for non-photochemical energy dissipation within the light-harvesting antenna). These results are indicative of an increase in sink strength in the vicinity of the wound and this was confirmed by the retention of 14C photosynthate in this region. It has been hypothesized that wound-induced cell wall (apoplastic) invertase (cwINV) activity plays a central role in generating localized increases in sink strength in stressed plant tissue and that hexose sugars generated by the sucrolytic activity of cwINV may act as a signal regulating gene expression. Enzyme activity measurements, quantitative RT-PCR, and T-DNA insertional mutagenesis have been used to determine that expression of AtcwINV1 is responsible for all induced cwINV activity in mechanically wounded leaves. Whilst inactivation of this gene abolished wound-induced cwINV activity, it did not affect localized alterations in source-sink relationships of wounded leaves or wound-regulated gene expression. The signals that may regulate source-sink relationships and signalling in wounded leaves are discussed.     

Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana

Abscisic acid (ABA) is a plant hormone involved in seed development and responses to various environmental stresses. Oxidation of abscisic aldehyde is the last step of ABA biosynthesis and is catalysed by aldehyde oxidase (EC 1.2.3.1). We have reported the occurrence of three isoforms of aldehyde oxidase, AOalpha, AObeta and AOgamma, in Arabidopsis thaliana seedlings, but none oxidized abscisic aldehyde. Here we report a new isoform, AOdelta, found in rosette leaf extracts, which efficiently oxidizes abscisic aldehyde. AO delta was specifically recognized by antibodies raised against a recombinant peptide encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3 and AAO4). Functionally expressed AAO3 protein in the yeast Pichia pastoris showed a substrate preference very similar to that of rosette AOdelta. These results indicate that AOdelta is encoded by AAO3. AOdelta produced in P. pastoris exhibited a very low Km value for abscisic aldehyde (0.51 microM), and the oxidation product was determined by gas chromatography-mass spectrometry to be ABA. Northern analysis showed that AAO3 mRNA is highly expressed in rosette leaves. When the rosette leaves were detached and exposed to dehydration, AAO3 mRNA expression increased rapidly within 3 h of the treatment. These results suggest that AOdelta, the AAO3 gene product, acts as an abscisic aldehyde oxidase in Arabidopsis rosette leaves.     

Polyphenolic compounds on leaves limit iron availability and affect growth of epiphytic bacteria

Polyphenolic compounds produced by plants can chelate iron, reducing its bioavailability to plant‐associated bacteria. In response to limited iron levels, most bacteria produce siderophores to acquire needed iron quantities. The amount of phenolic compounds detected in methanolic washings of leaves of different plant species varied greatly, being nearly sevenfold higher in Viburnum tinus than in Phaseolus vulgaris. In species with high levels of total phenolics (e.g. Pelargonium hortorum), tannin concentration of leaf washings was also high and accounted for up to 85% of total phenolics. Both stimulation of production of the siderophore pyoverdine in Pseudomonas syringae strain B728a and inhibition of growth of an isogenic mutant I‐1, deficient in pyoverdine production were associated with plants harbouring high levels of leaf surface phenolics. Levels of tannic acid sufficient to inhibit growth of the pyoverdine mutant in culture in an iron‐reversible fashion were similar to tannin levels found on leaves of plants such as P. hortorum. Additionally, the amount of pyoverdines produced by P. syringae and quantified in leaf washings from a variety of plants was directly related to the concentration of tannins released from the leaf, indicating that tannins were responsible for sequestering iron. Phenolic compounds, principally tannins, may thus play an important role in plant–microbe interactions.     

Respiratory acclimation in Arabidopsis thaliana leaves at low temperature

Acclimation of 25 degrees C-grown Arabidopsis thaliana at 5 degrees C resulted in a marked increase of leaf respiration in darkness (Rd) measured at 5 degrees C. Rd was particularly high in leaves developed at 5 degrees C. Leaf respiration (non-photorespiratory intracellular decarboxylation) in the light (Rl) also increased during cold acclimation, but less so than did Rd. The ratio Rd/Pt (Pt - true photosynthesis) was higher in more acclimated or cold-developed leaves, while the ratio Rl/Pt remained unchanged. In cold-acclimated leaves, Rl did not correlate with 3-phosphoglycerate and pyruvate nor with hexose phosphate pools in the cytosol. Rl in A. thaliana leaves was probably not limited by the substrate during cold acclimation. Under the conditions tested, Rd was more sensitive to low temperature stress than Rl.     

Cryptic chlorophyll breakdown in non-senescent green Arabidopsis thaliana leaves

Photosynthesis Research - Chlorophyll (Chl) breakdown is a diagnostic visual process of leaf senescence, which furnishes phyllobilins (PBs) by the PAO/phyllobilin pathway. As Chl breakdown disables...     

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.     

Role of acetyl-coenzyme A synthetase in leaves of Arabidopsis thaliana

Acetyl-coenzyme A synthetase (ACS) is a plastidic enzyme that forms acetyl-coenzyme A (acetyl-CoA) from acetate and coenzyme A using the energy from ATP. Traditionally it has been thought to be the major source for the production of acetyl-CoA destined for fatty acid formation. Recent work suggested that the accumulation of lipids in developing Arabidopsis seeds was more closely correlated with the expression of pyruvate dehydrogenase complex than with the expression of ACS, suggesting that most of the carbon for fatty acid formation in the plastids of seeds comes from pyruvate rather than from acetate. To explore the role of this enzyme, Arabidopsis plants with altered amounts of ACS were generated by overexpressing its cDNA in either the sense or the antisense configuration. The resulting plants had in vitro enzyme activities that ranged from about 5% to over 400% of wild-type levels. The rate of [1-14C]acetate conversion into fatty acids was closely related to the in vitro ACS activity, showing that the amount of enzyme clearly limited the capacity of leaves to convert exogenous acetate to fatty acids. There was, however, no relationship between the ACS level and the capacity of the plants to incorporate 14CO2 into 14C-labeled fatty acids. These data strongly support the idea that, although plants can convert acetate into fatty acids, relatively little carbon moves through this pathway under normal conditions.     

Spatio-temporal heterogeneity in Arabidopsis thaliana leaves under drought stress

Using chlorophyll (chl) fluorescence imaging, we studied the effect of mild (MiDS), moderate (MoDS) and severe (SDS) drought stress on photosystem II (PSII) photochemistry of 4-week-old Arabidopsis thaliana. Spatio-temporal heterogeneity in all chl fluorescence parameters was maintained throughout water stress. After exposure to drought stress, maximum quantum yield of PSII photochemistry (F(v)/F(m)) and quantum efficiency of PSII photochemistry (Φ(PSΙΙ)) decreased less in the proximal (base) than in the distal (tip) leaf. The chl fluorescence parameter F(v) /F(m) decreased less after MoDS than MiDS. Under MoDS, the antioxidant mechanism of A. thaliana leaves seemed to be sufficient in scavenging reactive oxygen species, as evident by the decreased lipid peroxidation, the more excitation energy dissipated by non-photochemical quenching (NPQ) and decreased excitation pressure (1-q(p)). Arabidopsis leaves appear to function normally under MoDS, but do not seem to have particular metabolic tolerance mechanisms under MiDS and SDS, as revealed by the level of lipid peroxidation and decreased quantum yield for dissipation after down-regulation in PSII (Φ(NPQ)), indicating that energy dissipation by down-regulation did not function and electron transport (ETR) was depressed. The simultaneous increased quantum yield of non-regulated energy dissipation (Φ(NO)) indicated that both the photochemical energy conversion and protective regulatory mechanism were insufficient. The non-uniform photosynthetic pattern under drought stress may reflect different zones of leaf anatomy and mesophyll development. The data demonstrate that the effect of different degrees of drought stress on A. thaliana leaves show spatio-temporal heterogeneity, implying that common single time point or single point leaf analyses are inadequate.     

Gravitropism in leaves of Arabidopsis thaliana (L.) Heynh

In higher plants, stems and roots show negative and positive gravitropism, respectively. However, current knowledge on the graviresponse of leaves is lacking. In this study, we analyzed the positioning and movement of rosette leaves of Arabidopsis thaliana under light and dark conditions. We found that the radial positioning of rosette leaves was not affected by the direction of gravity under continuous white light. In contrast, when plants were shifted to darkness, the leaves moved upwards, suggesting negative gravitropism. Analysis of the phosphoglucomutase and shoot gravitropism 2-1 mutants revealed that the sedimenting amyloplasts in the leaf petiole are important for gravity perception, as is the case in stems and roots. In addition, our detailed physiological analyses revealed a unique feature of leaf movement after the shift to darkness, i.e. movement could be divided into negative gravitropism and nastic movement. The orientation of rosette leaves is ascribed to a combination of these movements.     

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C₂₉, C₃₁ and C₃₃ alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4–5 weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

Metabolic recovery of Arabidopsis thaliana roots following cessation of oxidative stress

To cope with the various environmental stresses resulting in reactive oxygen species (ROS) production plant metabolism is known to be altered specifically under different stresses. After overcoming the stress the metabolism should be reconfigured to recover basal operation however knowledge concerning how this is achieved is cursory. To investigate the metabolic recovery of roots following oxidative stress, changes in metabolite abundance and carbon flow were analysed. Arabidopsis roots were treated by menadione to elicit oxidative stress. Roots were fed with ¹³C labelled glucose and the redistribution of isotope was determined in order to study carbon flow. The label redistribution through many pathways such as glycolysis, the tricarboxylic acid (TCA) cycle and amino acid metabolism were reduced under oxidative stress. After menadione removal many of the stress-related changes reverted back to basal levels. Decreases in amounts of hexose phosphates, malate, 2-oxoglutarate, glutamate and aspartate were fully recovered or even increased to above the control level. However, some metabolites such as pentose phosphates and citrate did not recover but maintained their levels or even increased further. The alteration in label redistribution largely correlated with that in metabolite abundance. Glycolytic carbon flow reverted to the control level only 18 h after menadione removal although the TCA cycle and some amino acids such as aspartate and glutamate took longer to recover. Taken together, plant root metabolism was demonstrated to be able to overcome menadione-induced oxidative stress with the differential time period required by independent pathways suggestive of the involvement of pathway specific regulatory processes.     

Role of growth regulators in the senescence of Arabidopsis thaliana leaves

A homozygous, dominant, C₂H₄-resistant line of Arabidopsis thaliana (L.) Heynh (cv. Columbia; er ) was selected from ethylmethylsulfonate-mutagenized seed, and used to test the role of C₂H₄ and other growth regulators in senescence of mature leaves. Chlorophyll (Chl) loss from disks excised from leaves of er was much slower than that from wild-type (WT) disks, whether they were held in the light or in the dark. C₂H₄ accelerated Che loss from WT disks but had no effect on the yellowing of mutant disks. C₂H₄ biosynthesis was higher in disks from the mutant plants, particularly in the light. In the dark, treatment with the cytokinin, 6-benzyladenine (BA), reduced Chl loss from wild-type disks, but had no effect on mutant disks. In the light, BA treatment stimulated chlorophyll breakdown in both wild type and mutant disks. Treatment with abscisic acid (ABA) stimulated chlorophyll loss in wild-type and mutant disks, whether they were held in the light or the dark. C₂H₄ production was stimulated in ABA-treated disks, but they still yellowed even when C₂H₄ production was inhibited by application of aminooxyacetic acid (AOA). These data indicate that C₂H₄ is only one of the factors involved in leaf senescence, and that the promotion of senescence by ABA is not mediated through its stimulation of C₂H₄ production.     

Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana

Multiple isoforms of -fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 sizeexclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a K _m for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 °C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.Abbreviations PEG polyethylene glycol - pI isoelectric point - PMSF phenylmethylsulphonyl fluoride We wish to acknowledge the support of the British/Swiss Joint Research Programme and the Sheffield University Research Support Fund. X.T. was in receipt of an Overseas Research Scholarship and a University of Sheffield Research Scholarship. We wish to thank Dr A. Moir for his help in N-terminal amino-acid sequencing.