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PubMed2002

Biochemical and molecular inhibition of plastidial carbonic anhydrase reduces the incorporation of acetate into lipids in cotton embryos and tobacco cell suspensions and leaves.

Two cDNAs encoding functional carbonic anhydrase (CA) enzymes were recently isolated from a non-photosynthetic, cotyledon library of cotton (Gossypium hirsutum) seedlings with putative plastid-targeting sequences (GenBank accession nos. AF132854 and AF132855). Relative CA transcript abundance and enzyme activity increased 9 and 15 times, respectively, in cotton embryos during the maximum period of reserve oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [(14)C]acetate incorporation into total lipids in cotton embryos in vivo, and in embryo plastids in vitro, suggesting a role for CA in plastid lipid biosynthesis. CA inhibitors did not affect acetyl-coenzyme A carboxylase activity or total storage protein synthesis. Similar results were obtained for two other plant systems: cell suspensions (and isolated plastids therefrom) of tobacco (Nicotiana tabacum), and chloroplasts isolated from leaves of transgenic CA antisense-suppressed tobacco plants (5% of wild-type CA activity). In addition, tobacco cell suspensions treated with the CA inhibitor ethoxyzolamide showed a substantial loss of CO(2) compared with controls. The rate of [(14)C]acetate incorporation into lipid in cell suspensions was reduced by limiting external [CO(2)] (scrubbed air), and this rate was further reduced in the presence of ethoxyzolamide. Together, these results indicate that a reduction of CA activity (biochemical or molecular inhibition) impacts the rate of plant lipid biosynthesis from acetate, perhaps by impairing the ability of CA to efficiently "trap" inorganic carbon inside plastids for utilization by acetyl-coenzyme A carboxylase and the fatty acid synthesis machinery.

Acetates↗
PubMed2002

The barley MLO modulator of defense and cell death is responsive to biotic and abiotic stress stimuli.

Lack of the barley (Hordeum vulgare) seven-transmembrane domain MLO protein confers resistance against the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). To broaden the basis for MLO structure/function studies, we sequenced additional mlo resistance alleles, two of which confer only partial resistance. Wild-type MLO dampens the cell wall-restricted hydrogen peroxide burst at points of attempted fungal penetration of the epidermal cell wall, and in subtending mesophyll cells, it suppresses a second oxidative burst and cell death. Although the Bgh-induced cell death in mlo plants is spatially and temporally separated from resistance, we show that the two processes are linked. Uninoculated mutant mlo plants exhibit spontaneous mesophyll cell death that appears to be part of accelerated leaf senescence. Mlo transcript abundance increases in response to Bgh, rice (Oryza sativa) blast, wounding, paraquat treatment, a wheat powdery mildew-derived carbohydrate elicitor, and during leaf senescence. This suggests a broad involvement of Mlo in cell death protection and in responses to biotic and abiotic stresses.

Alleles↗
PubMed2002

Down-regulation of tomato beta-galactosidase 4 results in decreased fruit softening.

Transcript abundance of the gene encoding beta-galactosidase II, a beta-galactosidase/exo-galactanase (EC 3.2.1.23) present during tomato (Lycopersicon esculentum) fruit ripening, was suppressed by expression of an antisense tomato beta-galactosidase 4 (TBG4) cDNA driven by the cauliflower mosaic virus 35S promoter. RNA gel-blot analysis was used to evaluate TBG4 mRNA levels in transgenic fruit. All of the antisense lines had attenuated TBG4 mRNA levels in turning stage fruit; however, TBG4 mRNA suppression was unstable, and mRNA levels varied in red-ripe fruit among the lines. Suppression of TBG4 mRNA levels in antisense fruit was correlated with a reduction in extractable exo-galactanase activity against a lupin galactan. All of the antisense lines had reduced free galactose levels at mature green stage 4, but levels comparable with controls during ripening. Total cell wall galactosyl contents in the antisense fruit were not significantly different from control fruit. Whole-fruit firmness was measured using a texture analyzer and the means of the peak force measurements for four of six antisense lines were significantly higher than control fruit. One antisense line had red-ripe fruit that were 40% firmer than controls. Fruit from this antisense line also had the lowest TBG4 mRNA and exo-galactanase levels and the highest wall galactosyl content during the early stages of ripening, implicating an involvement of this gene product in cell wall modification leading to fruit softening.

Biomechanical Phenomena↗
PubMed2003

N-acylethanolamine signaling in tobacco is mediated by a membrane-associated, high-affinity binding protein.

N-Acylethanolamines (NAEs) are fatty acid derivatives found as minor constituents of animal and plant tissues, and their levels increase 10- to 50-fold in tobacco (Nicotiana tabacum) leaves treated with fungal elicitors. Infiltration of tobacco leaves with submicromolar to micromolar concentrations of N-myristoylethanolamine (NAE 14:0) resulted in an increase in relative phenylalanine ammonia-lyase (PAL) transcript abundance within 8 h after infiltration, and this PAL activation was reduced after co-infiltration with cannabinoid receptor antagonists (AM 281 and SR 144528). A saturable, high-affinity specific binding activity for [(3)H]NAE 14:0 was identified in suspension-cultured tobacco cells and in microsomes from tobacco leaves (apparent K(d) of 74 and 35 nM, respectively); cannabinoid receptor antagonists reduced or eliminated specific [(3)H]NAE 14:0 binding, consistent with the physiological response. N-Oleoylethanolamine activated PAL2 expression in leaves and diminished [(3)H]NAE 14:0 binding in microsomes, whereas N-linoleoylethanolamine did not activate PAL2 expression in leaves, and did not affect [(3)H]NAE 14:0 binding in microsomes. The nonionic detergent dodecylmaltoside solubilized functional [(3)H]NAE 14:0-binding activity from tobacco microsomal membranes. The dodecylmaltoside-solubilized NAE-binding activity retained similar, but not identical, binding properties to the NAE-binding protein(s) in intact tobacco microsomes. Additionally, high-affinity saturable NAE-binding proteins were identified in microsomes isolated from Arabidopsis and Medicago truncatula tissues, indicating the general prevalence of these binding proteins in plant membranes. We propose that plants possess an NAE-signaling pathway with functional similarities to the "endocannabinoid" pathway of animal systems and that this pathway, in part, participates in xylanase elicitor perception in tobacco.

Cannabinoid Receptor Modulators↗
PubMed2003

Mapping the proteome of barrel medic (Medicago truncatula).

A survey of six organ-/tissue-specific proteomes of the model legume barrel medic (Medicago truncatula) was performed. Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from leaves, stems, roots, flowers, seed pods, and cell suspension cultures were obtained. Five hundred fifty-one proteins were excised and 304 proteins identified using peptide mass fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Nanoscale high-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry was used to validate marginal matrix-assisted laser desorption ionization time-of-flight mass spectrometry protein identifications. This dataset represents one of the most comprehensive plant proteome projects to date and provides a basis for future proteome comparison of genetic mutants, biotically and abiotically challenged plants, and/or environmentally challenged plants. Technical details concerning peptide mass fingerprinting, database queries, and protein identification success rates in the absence of a sequenced genome are reported and discussed. A summary of the identified proteins and their putative functions are presented. The tissue-specific expression of proteins and the levels of identified proteins are compared with their related transcript abundance as quantified through EST counting. It is estimated that approximately 50% of the proteins appear to be correlated with their corresponding mRNA levels.

Amino Acid Sequence↗
PubMed2003

Salicylic acid is an uncoupler and inhibitor of mitochondrial electron transport.

The effect of salicylic acid (SA) on respiration and mitochondrial function was examined in tobacco (Nicotiana tabacum) suspension cell cultures in the range of 0.01 to 5 mm. Cells rapidly accumulated SA up to 10-fold of the externally applied concentrations. At the lower concentrations, SA accumulation was transitory. When applied at 0.1 mm or less, SA stimulated respiration of whole cells and isolated mitochondria in the absence of added ADP, indicating uncoupling of respiration. However, at higher concentrations, respiration was severely inhibited. Measurements of ubiquinone redox poise in isolated mitochondria suggested that SA blocked electron flow from the substrate dehydrogenases to the ubiquinone pool. This inhibition could be at least partially reversed by re-isolating the mitochondria. Two active analogs of SA, benzoic acid and acetyl-SA, had the same effect as SA on isolated tobacco mitochondria, whereas the inactive p-hydroxybenzoic acid was without effect at the same concentration. SA induced an increase in Aox protein levels in cell suspensions, and this was correlated with an increase in Aox1 transcript abundance. However, when applied at 0.1 mM, this induction was transient and disappeared as SA levels in the cells declined. SA at 0.1 mM also increased the expression of other SA-responsive genes, and this induction was dependent on active mitochondria. The results indicate that SA is both an uncoupler and an inhibitor of mitochondrial electron transport and suggest that this underlies the induction of some genes by SA. The possible implications of this for the interpretation of SA action in plants are discussed.

Base Sequence↗
PubMed2004

Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism.

In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes.

Amino Acid Sequence↗
PubMed2004

Uncoupling the effects of abscisic acid on plant growth and water relations. Analysis of sto1/nced3, an abscisic acid-deficient but salt stress-tolerant mutant in Arabidopsis.

We have identified a T-DNA insertion mutation of Arabidopsis (ecotype C24), named sto1 (salt tolerant), that results in enhanced germination on both ionic (NaCl) and nonionic (sorbitol) hyperosmotic media. sto1 plants were more tolerant in vitro than wild type to Na(+) and K(+) both for germination and subsequent growth but were hypersensitive to Li(+). Postgermination growth of the sto1 plants on sorbitol was not improved. Analysis of the amino acid sequence revealed that STO1 encodes a 9-cis-epoxicarotenoid dioxygenase (similar to 9-cis-epoxicarotenoid dioxygenase GB:AAF26356 [Phaseolus vulgaris] and to NCED3 GB:AB020817 [Arabidopsis]), a key enzyme in the abscisic acid (ABA) biosynthetic pathway. STO1 transcript abundance was substantially reduced in mutant plants. Mutant sto1 plants were unable to accumulate ABA following a hyperosmotic stress, although their basal ABA level was only moderately altered. Either complementation of the sto1 with the native gene from the wild-type genome or supplementation of ABA to the growth medium restored the wild-type phenotype. Improved growth of sto1 mutant plants on NaCl, but not sorbitol, medium was associated with a reduction in both NaCl-induced expression of the ICK1 gene and ethylene accumulation. Osmotic adjustment of sto1 plants was substantially reduced compared to wild-type plants under conditions where sto1 plants grew faster. The sto1 mutation has revealed that reduced ABA can lead to more rapid growth during hyperionic stress by a signal pathway that apparently is at least partially independent of signals that mediate nonionic osmotic responses.

Abscisic Acid↗
PubMed2005

Conservation and divergence of circadian clock operation in a stress-inducible Crassulacean acid metabolism species reveals clock compensation against stress.

One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO(2) metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C(3) and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcript peak 3 h before subjective dusk. ELF3 transcripts appear in the evening, consistent with their role in gating light input to the circadian clock. Intriguingly, ZTL transcripts do not oscillate in Arabidopsis, but do in M. crystallinum. The transcript abundance of the clock-associated genes in M. crystallinum is largely unaffected by development and salt stress, revealing compensation of the central circadian clock against development and abiotic stress in addition to the well-known temperature compensation. Importantly, the clock in M. crystallinum is very similar to that in Arabidopsis, indicating that such a clock could control CAM without requiring additional components of the central oscillator or a novel CAM oscillator.

Circadian Rhythm↗
PubMed1994

Characterization and expression of two cDNAs encoding carbonic anhydrase in Arabidopsis thaliana.

Two distinct cDNA clones encoding carbonic anhydrase (CA) were isolated from an Arabidopsis thaliana lambda YES library. One of these clones, CA1, encodes a 36.1-kD polypeptide and is essentially the same as a previously reported Arabidopsis CA cDNA (C.A. Raines, P.R. Horsnell, C. Holder, J.C. Lloyd [1992] Plant Mol Biol 20: 1143-1148). Comparison of the derived amino acid sequence from this clone with other plant CAs suggests the presence of a chloroplastic transit peptide, which, when cleaved, would render a mature protein of 24.3 kD. The other identified clone, CA2, encodes a 28.3-kD polypeptide, which in addition to other residue changes, is 78 amino acids shorter at the N terminus than the primary product of CA1. The two cDNAs exhibit 76.9% sequence similarity at the DNA level and 84.6% identity between the predicted amino acid sequences. A polyclonal antibody generated against pea CA (N. Majeau, J.R. Coleman [1991] Plant Physiol 100: 1077-1078) hybridized to two protein bands (25 and 28 kD) from a total leaf extract and to only one band (25 kD) from a chloroplastic protein extract. The data suggest that the CA2 protein is an extrachloroplastic form of CA, presumably localized in the cytoplasm. Southern analysis indicated that CA1 and CA2 are encoded by different genes. Northern analysis of total leaf RNA resulted in hybridization of CA1- and CA2-derived probes to two transcripts of 1.47 and 1.2 kb, respectively. These data provide additional evidence that the CA2 clone is a full-length cDNA and that two transcribed CA genes are present in the Arabidopsis genome. Transcript levels of CA1 and CA2 decreased 70 and 20%, respectively, when mature plants were transferred to dark for 24 h. Seedlings germinated in the dark showed CA1 and CA2 transcript abundance levels of 4 and 22%, respectively, when compared with light-germinated seedlings. These data suggest that expression of CA1 is light regulated and dependent of leaf and/or chloroplast development. A possible role for cytoplasmic CA in the plant cell is discussed.

Amino Acid Sequence↗
PubMed2006

Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster.

Using a new Arabidopsis (Arabidopsis thaliana) mutant (Atnrt2.1-nrt2.2) we confirm that concomitant disruption of NRT2.1 and NRT2.2 reduces inducible high-affinity transport system (IHATS) by up to 80%, whereas the constitutive high-affinity transport system (CHATS) was reduced by 30%. Nitrate influx via the low-affinity transport system (LATS) was unaffected. Shoot-to-root ratios were significantly reduced compared to wild-type plants, the major effect being upon shoot growth. In another mutant uniquely disrupted in NRT2.1 (Atnrt2.1), IHATS was reduced by up to 72%, whereas neither the CHATS nor the LATS fluxes were significantly reduced. Disruption of NRT2.1 in Atnrt2.1 caused a consistent and significant reduction of shoot-to-root ratios. IHATS influx and shoot-to-root ratios were restored to wild-type values when Atnrt2.1-nrt2.2 was transformed with a NRT2.1 cDNA isolated from Arabidopsis. Disruption of NRT2.2 in Atnrt2.2 reduced IHATS by 19% and this reduction was statistically significant only at 6 h after resupply of nitrate to nitrogen-deprived plants. Atnrt2.2 showed no significant reduction of CHATS, LATS, or shoot-to-root ratios. These results define NRT2.1 as the major contributor to IHATS. Nevertheless, when maintained on agar containing 0.25 mm KNO(3) as the sole nitrogen source, Atnrt2.1-nrt2.2 consistently exhibited greater stress and growth reduction than Atnrt2.1. Evidence from real-time PCR revealed that NRT2.2 transcript abundance was increased almost 3-fold in Atnrt2.1. These findings suggest that NRT2.2 normally makes only a small contribution to IHATS, but when NRT2.1 is lost, this contribution increases, resulting in a partial compensation.

Agar↗
PubMed1995

Regulation of Photosynthesis during Leaf Development in RbcS Antisense DNA Mutants of Tobacco.

We have previously characterized RbcS antisense DNA mutants of tobacco that have drastic reductions in their content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). In this report we examine the impact of Rubisco loss on photosynthesis during tobacco (Nicotiana tabacum) leaf development. Photosynthetic capacities are depressed in the antisense leaves, but the patterns of change in photosynthetic rates during the development of these leaves are similar to those in wild-type plants: after attaining a maximum in young leaves, photosynthetic capacities undergo a prolonged senescence decline in older leaves. The alterations in photosynthetic capacities in both the wild type and mutant are closely correlated with changes in Rubisco activity and content. During wild-type leaf development, Rubisco accumulation is regulated by coordinate changes in RbcS and rbcL transcript accumulation, whereas in the antisense leaves, Rubisco content is a function of RbcS, but not rbcL, transcript abundance. This indicates that large subunit protein production is controlled posttranscriptionally in the mutants. The antisense leaves accumulate near-normal levels of chlorophyll and representative photosynthetic proteins throughout development, suggesting that photosynthetic gene expression is not feedback regulated by Rubisco abundance. Considered together, the data in this paper indicate that leaf developmental programs are generally insensitive to sharp reductions in Rubisco content and emphasize the metabolic plasticity of plant cells in achieving optimal photosynthetic rates.

Journal Article↗
PubMed1997

gamma-Aminobutyric acid stimulates ethylene biosynthesis in sunflower.

gamma-Aminobutyric acid (GABA), a nonprotein amino acid, is often accumulated in plants following environmental stimuli that can also cause ethylene production. We have investigated the relationship between GABA and ethylene production in excised sunflower (Helianthus annuus L.) tissues. Exogenous GABA causes up to a 14-fold increase in the ethylene production rate after about 12 h. Cotyledons fed with [14C]GABA did not release substantial amounts of radioactive ethylene despite its chemical similarity to 1-aminocyclopropane-1-carboxylic acid (ACC), indicating that GABA is not likely to be an alternative precursor for ethylene. GABA causes increases in ACC synthase mRNA accumulation, ACC levels, ACC oxidase mRNA levels, and in vitro ACC oxidase activity. In the presence of aminoethoxyvinylglycine or alpha-aminoisobutyric acid, GABA did not stimulate ethylene production. We therefore conclude that GABA stimulates ethylene biosynthesis mainly by promoting ACC synthase transcript abundance. Possible roles of GABA as a signal transducer are suggested.

Amino Acid Oxidoreductases↗
PubMed1997

Characterization and expression of caffeoyl-coenzyme A 3-O-methyltransferase proposed for the induced resistance response of Vitis vinifera L.

Cell-suspension cultures of Vitis vinifera L. cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h. CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species. The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl- and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification. Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid. Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine. The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown. Treatment of the cultured V. vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective. The data imply for the first time (to our knowledge) that the expression of phenyl-propanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR.

Amino Acid Sequence↗
PubMed1998

Expression of beta-amylase from alfalfa taproots.

Alfalfa (Medicago sativa L.) roots contain large quantities of beta-amylase, but little is known about its role in vivo. We studied this by isolating a beta-amylase cDNA and by examining signals that affect its expression. The beta-amylase cDNA encoded a 55.95-kD polypeptide with a deduced amino acid sequence showing high similarity to other plant beta-amylases. Starch concentrations, beta-amylase activities, and beta-amylase mRNA levels were measured in roots of alfalfa after defoliation, in suspension-cultured cells incubated in sucrose-rich or -deprived media, and in roots of cold-acclimated germ plasms. Starch levels, beta-amylase activities, and beta-amylase transcripts were reduced significantly in roots of defoliated plants and in sucrose-deprived cell cultures. beta-Amylase transcript was high in roots of intact plants but could not be detected 2 to 8 d after defoliation. beta-Amylase transcript levels increased in roots between September and October and then declined 10-fold in November and December after shoots were killed by frost. Alfalfa roots contain greater beta-amylase transcript levels compared with roots of sweetclover (Melilotus officinalis L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.). Southern analysis indicated that beta-amylase is present as a multigene family in alfalfa. Our results show no clear association between beta-amylase activity or transcript abundance and starch hydrolysis in alfalfa roots. The great abundance of beta-amylase and its unexpected patterns of gene expression and protein accumulation support our current belief that this protein serves a storage function in roots of this perennial species.

Amino Acid Sequence↗
PubMed1999

Changed properties of the cytoplasmic matrix associated with desiccation tolerance of dried carrot somatic embryos. An In situ fourier transform infrared spectroscopic study

Abscisic acid-pretreated carrot (Daucus carota) somatic embryos survive dehydration upon slow drying, but fast drying leads to poor survival of the embryos. To determine whether the acquisition of desiccation tolerance is associated with changes in the physical stability of the cytoplasm, in situ Fourier transform infrared microspectroscopy was used. Although protein denaturation temperatures were similar in the embryos after slow or fast drying, the extent of the denaturation was greater after fast drying. Slowly dried embryos are in a glassy state at room temperature, and no clearly defined glassy matrix was observed in the rapidly dried embryos. At room temperature the average strength of hydrogen bonding was much weaker in the rapidly dried than in the slowly dried embryos. We interpreted the molecular packing to be "less tight" in the rapidly dried embryos. Whereas sucrose (Suc) is the major soluble carbohydrate after fast drying, upon slow drying the trisaccharide umbelliferose accumulates at the expense of Suc. The possibly protective role of umbelliferose was tested on protein and phospholipid model systems, using Suc as a reference. Both umbelliferose and Suc form a stable glass with drying: They depress the transition temperature of dry liposomal membranes equally well, they both prevent leakage from dry liposomes after rehydration, and they protect a polypeptide that is desiccation sensitive. The similar protection properties in model systems and the apparent interchangeability of both sugars in viable, dry somatic embryos suggest no special role of umbelliferose in the improved physical stability of the slowly dried embryos. Also, during slow drying LEA (late-embryogenesis abundant) transcripts are expressed. We suggest that LEA proteins embedded in the glassy matrix confer stability to these slowly dried embryos.

Journal Article↗
PubMed2000

Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint.

Monoterpene production in peppermint (Mentha x piperita L.) glandular trichomes is determined by the rate of biosynthesis, as determined by (14)CO(2) incorporation, and is restricted to leaves 12 to 20 d of age. Using oil glands isolated from peppermint leaves of different ages, in vitro assay of the eight sequential enzymes responsible for the biosynthesis of the principal monoterpene (-)-menthol indicated that all but one biosynthetic enzyme had a very similar developmental profile. Activities were highest in leaves 12 to 20 d of age, with a sharp peak centered at 15 d. The exception, (-)-menthone reductase, the last enzyme of the pathway, exhibited a later peak of activity, which was centered at approximately 21 d. The correlation between in vitro enzyme activity and the rate of biosynthesis measured in vivo suggests that monoterpene formation is controlled mainly by the coordinately regulated activity of the relevant biosynthetic enzymes. Developmental immunoblotting of limonene synthase, which catalyzes the committed step of the pathway, demonstrated a direct correlation between enzyme activity and enzyme protein, suggesting that the dynamic time course for the remaining pathway enzyme activities also reflects the corresponding protein levels. RNA-blot analyses indicated that the genes encoding enzymes of the early pathway steps are transcriptionally activated in a coordinated fashion, with a time course superimpossible with activity measurements and immunoblot data. These results demonstrating coincidental temporal changes in enzyme activities, enzyme protein level, and steady-state transcript abundances indicate that most of the monoterpene biosynthetic enzymes in peppermint are developmentally regulated at the level of gene expression.

Blotting, Northern↗
PubMed2000

Antisense suppression of 2-cysteine peroxiredoxin in Arabidopsis specifically enhances the activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism.

The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool.

Antioxidants↗