bluuebunny/crossref_metadata_2025 huggingface dataset:
Sugar Regulation of Harvest-Related Genes in Asparagus
Authors: Davies K. M. , Seelye J. F. , Irving D. E. , Borst W. M. , Hurst P. L. , King G. A.
Published: July 2002

The signals controlling the abundance of transcripts up-regulated (pTIP27, pTIP31, and pTIP32) or down-regulated (pTIP20 and pTIP21) after harvest in asparagus (Asparagus officinalis L.) spears were examined. pTIP27 and pTIP31 are known to encode asparagine synthetase (AS) and a β-galactosidase (β-gal) homolog, respectively. The nucleotide sequences of pTIP20, pTIP21, and pTIP32 were determined, and they encode histone 3, histone 2B, and an unknown product, respectively. Changes in respiration, soluble sugars, and abundance of the five mRNAs were similar in the tips stored as 30-mm lengths or as part of 180-mm spears. We previously hypothesized that sugars may regulate the level of AS transcripts in asparagus tissue. Asparagus cell cultures were used to test the role of sugar status in regulating gene expression. Transcript abundance for AS, β-gal, and pTIP32 was low in cells in sugar-containing medium but increased within 12 h after transferring cells to a sugar-free medium. Histone 3 and histone 2B transcripts were, in general, abundant in cells on sugar-containing medium but declined in abundance when transferred to sugar-free medium. When cells were returned to sugar-containing medium the abundance of transcripts for histone 3 and histone 2B increased, whereas that for AS, β-gal, and pTIP32 decreased. Soluble sugar levels are known to decline rapidly in the tips of harvested spears. Metabolic regulation by sugar status may have a major influence on gene expression in asparagus spears and other tissues after harvest.
Biotin Synthase from Arabidopsis thaliana (cDNA Isolation and Characterization of Gene Expression)
Authors: Patton D. A. , Johnson M. , Ward E. R.
Published: July 2002

The full-length BIO2 cDNA from Arabidopsis thaliana was isolated using an expressed sequence tag that was homologous to the Escherichia coli biotin synthase gene (BioB). Comparisons of the deduced amino acid sequence from BIO2 with bacterial and yeast biotin synthase homologs revealed a high degree of sequence similarity. The amino terminus of the predicted BIO2 protein contains a stretch of hydrophobic residues similar in composition to transit peptide sequences. BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5-fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and biochemical stimuli.
Aspartic Acid 413 Is Important for the Normal Allosteric Functioning of ADP-Glucose Pyrophosphorylase
Authors: Greene T. W. , Woodbury R. L. , Okita T. W.
Published: July 2002

As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Greene, S.E. Chantler, M.L. Khan, G.F. Barry, J. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509–1513). One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to I2 vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wild-type recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706–24711). The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP.
Molecular Cloning of a Ripening-Specific Lipoxygenase and Its Expression during Wild-Type and Mutant Tomato Fruit Development
Authors: Kausch K. D. , Handa A. K.
Published: July 2002

A 94-kD protein that accumulates predominately in tomato (Lycopersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.
Changes in the Level and Activation State of the Plasma Membrane H+-ATPase during Aging of Red Beet Slices
Authors: Papini R. , De Michelis M. I.
Published: July 2002

The effect of aging on the plasma membrane (PM) H+-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity and the amount of immunodetectable H+-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H+-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H+-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H+-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H+-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H+-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H+-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities.
The Dark-Adaptation Response of the De-Etiolated Pea Mutant lip1 Is Modulated by External Signals and Endogenous Programs
Authors: Frances S. , Thompson W. F.
Published: July 2002

The lip1 mutant of pea (Pisum sativum L.) exhibits a de-etiolated phenotype. When grown in darkness, lip1 plants have several characteristics normally associated only with light-grown plants. Young wild-type (WT) seedlings accumulate high levels of transcripts from plastid-related genes (such as those encoding chlorophyll a/b-binding proteins, ferredoxin, and the small subunit of Rubisco) only in the light. In contrast, regardless of the light conditions under which the plants are grown, young mutant seedlings accumulate transcript levels equal to or greater than those seen in light-grown WT seedlings of the same age. Under some conditions, light-grown lip1 seedlings failed to respond to dark treatment. The largest response to darkness observed in the mutant occurred when older seedlings were first grown under low-light conditions before transfer to darkness. The mutant's inability to respond to darkness is not due to a gross disturbance in the circadian clock. We conclude that environmental signals (light) and endogenous programs (developmental and circadian) regulate gene expression in both WT and mutant plants. However, mutant seedlings exhibit a developmentally regulated and exaggerated response to light. In addition, the effect of the mutation may be greatest during a brief period early in development.
Characterization of the Common Bean Uricase II and Its Expression in Organs Other than Nodules
Authors: Capote-Mainez N. , Sanchez F.
Published: July 2002

Uricase II is a purine metabolic enzyme highly induced in root nodules during the symbiosis established between legumes and bacteria of the genera Rhizobium and Bradyrhizobium. Here we describe the characterization of bean (Phaseolus vulgaris) nodule uricase II cDNA and show that uricase II is encoded by a single gene in the bean genome. This gene is also expressed in cotyledons, roots, and hypocotyls during bean seedling establishment, and an anti-uricase antibody recognizes the protein in different seedling organs. Uricase II has also been found in Leucaena leucocephala seedlings, suggesting that it participates during seedling establishment in legumes that do not transport ureides. A 50-kD polypeptide that is detected by the anti-uricase antibody is found in cotyledons during seedling development. This higher-molecular-mass form is also detected in developing roots and hypocotyls but not in nodules. In situ hybridization experiments in root seedlings showed uricase II transcripts in the metaxylem parenchyma cells and phloem fibers of the vascular system.
Uridine 5′-Diphosphate-Glucose Dehydrogenase from Soybean Nodules
Authors: Stewart Douglas C. , Copeland Les
Published: July 2002

A highly purified preparation of uridine 5′-diphosphate (UDP)-glucose (Glc) dehydrogenase (DH; EC 1.1.1.22) has been characterized from soybean (Glycinemax L.) nodules. The enzyme had native and subunit molecular masses of approximately 272 and 50 kD, respectively. UDP-Glc DH displayed typical hyperbolic substrate kinetics and hadKm values for UDP-Glc and NAD+of 0.05 and 0.12 mm, respectively. Thymidine 5′-diphosphate-Glc and UDP-galactose could replace UDP-Glc as the sugar nucleotide substrate to some extent, but the enzyme had no activity with NADP+. Soybean nodule UDP-Glc DH was labile in the absence of NAD+ and was inhibited by a heat-stable, low-molecular-mass solute in crude extracts of soybean nodules. UDP-Glc DH was also isolated from developing soybean seeds and shoots of 5-d-old wheat and canola seedlings and was shown to have similar affinities for UDP-Glc and NAD+ as those of the soybean nodule enzyme. UDP-Glc DH from all of these sources was most active in young, rapidly growing tissues.
Cloning of a Tobacco Apoplasmic Invertase Inhibitor1
Authors: Greiner Steffen , Krausgrill Silke , Rausch Thomas
Published: July 2002

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinantNt-inh1-encoded protein inhibits CWI from tobacco andChenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.
Induced β-Carotene Synthesis Driven by Triacylglycerol Deposition in the Unicellular AlgaDunaliella bardawil1
Authors: Rabbani Said , Beyer Peter , Lintig Johannes v. , Hugueney Philippe , Kleinig Hans
Published: July 2002

Under stress conditions such as high light intensity or nutrient starvation, cells of the unicellular alga Dunaliella bardawil overproduce β-carotene, which is accumulated in the plastids in newly formed triacylglycerol droplets. We report here that the formation of these sequestering structures and β-carotene are interdependent. When the synthesis of triacylglycerol is blocked, the overproduction of β-carotene is also inhibited. During overproduction of β-carotene no up-regulation of phytoene synthase or phytoene desaturase is observed on the transcriptional or translational level, whereas at the same time acetyl-CoA carboxylase, the key regulatory enzyme of acyl lipid biosynthesis, is increased, at least in its enzymatic activity. We conclude that under normal conditions the carotenogenic pathway is not maximally active and may be appreciably stimulated in the presence of sequestering structures, creating a plastid-localized sink for the end product of the carotenoid biosynthetic pathway.
In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite1
Authors: Jin Tie , Huppe Heather C. , Turpin David H.
Published: July 2002

An NADPH-dependent NO2−-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP+oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO2− reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO2− reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP+. NADP+ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO2− reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO2− reduction and (b) consume NADP+, releasing FNR from NADP+ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO2− reduction observed in vitro is capable of accounting for the observed rates of dark NO3−assimilation by C. reinhardtii.
Mutation of Residue Threonine-2 of the D2 Polypeptide and Its Effect on Photosystem II Function inChlamydomonas reinhardtii1
Authors: Andronis Christos , Kruse Olaf , Deák Zsuzsanna , Vass Imre , Diner Bruce A. , Nixon Peter J.
Published: July 2002

The D2 polypeptide of the photosystem II (PSII) complex in the green algaChlamydomonas reinhardtii is thought to be reversibly phosphorylated. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine-2 (Thr-2). We have investigated the role of D2 phosphorylation by constructing two mutants in which residue Thr-2 has been replaced by either alanine or serine. Both mutants grew photoautotrophically at wild-type rates, and noninvasive biophysical measurements, including the decay of chlorophyll fluorescence, the peak temperature of thermoluminescence bands, and rates of oxygen evolution, indicate little perturbation to electron transfer through the PSII complex. The susceptibility of mutant PSII to photoinactivation as measured by the light-induced loss of PSII activity in whole cells in the presence of the protein-synthesis inhibitors chloramphenicol or lincomycin was similar to that of wild type. These results indicate that phosphorylation at Thr-2 is not required for PSII function or for protection from photoinactivation. In control experiments the phosphorylation of D2 in wild-type C. reinhardtii was examined by 32P labeling in vivo and in vitro. No evidence for the phosphorylation of D2 in the wild type could be obtained. [14C]Acetate-labeling experiments in the presence of an inhibitor of cytoplasmic protein synthesis also failed to identify phosphorylated (D2.1) and nonphosphorylated (D2.2) forms of D2 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results suggest that the existence of D2 phosphorylation in C. reinhardtii is still in question.
Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells
Authors: Collings David A. , Asada Tetsuhiro , Allen Nina S. , Shibaoka Hiroh
Published: July 2002

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K+ and Ca2+ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.
Regulation of Ferulate-5-Hydroxylase Expression in Arabidopsis in the Context of Sinapate Ester Biosynthesis1
Authors: Ruegger Max , Meyer Knut , Cusumano Joanne C. , Chapple Clint
Published: July 2002

Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here we show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated thatF5H expression in leaves is dependent on sequences 3′ of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required forF5H transcript or sinapoylcholine accumulation in embryos.
Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1
Authors: Carrera Esther , Jackson Stephen D. , Prat Salomé
Published: July 2002

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potatoga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.
Cyanide-Resistant, ATP-Synthesis-Sustained, and Uncoupling-Protein-Sustained Respiration during Postharvest Ripening of Tomato Fruit1
Authors: Almeida Andrea Miyasaka , Jarmuszkiewicz Wieslawa , Khomsi Hamid , Arruda Paulo , Vercesi Anibal E. , Sluse Francis E.
Published: July 2002

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.
Cloning and Inactivation of Genes Encoding Ferredoxin- and NADH-Dependent Glutamate Synthases in the CyanobacteriumPlectonema boryanum. Imbalances in Nitrogen and Carbon Assimilations Caused by Deficiency of the Ferredoxin-Dependent Enzyme1
Authors: Okuhara Hiroaki , Matsumura Tomohiro , Fujita Yuichi , Hase Toshiharu
Published: July 2002

Glutamate synthase (GOGAT) is a key enzyme in the assimilation of inorganic nitrogen in photosynthetic organisms. We found that, like higher plants, the facultative heterotrophic cyanobacterium Plectonema boryanum had ferredoxin (Fd)- and NADH-dependent GOGATs. The genes glsF, gltB, and gltDwere cloned, and structural analyses and target mutageneses demonstrated that glsF encoded Fd-GOGAT and thatgltB and gltD encoded the two subunits of NADH-GOGAT. All three mutants lacking one of the GOGAT genes were able to grow photosynthetically and heterotrophically. However, the Fd-GOGAT mutant exhibited a phenotype of marked nitrogen deficiency when grown under conditions of saturating illumination and CO2 supply. In these conditions the rate of the ammonia uptake from the culture medium was slower in the Fd-GOGAT mutant than in the wild type or in the NADH-GOGAT mutant, but no significant differences were found in the rate of the CO2 fixation-dependent O2 evolution among these strains. Our results suggest that, although both Fd- and NADH-GOGATs were operative in the cells growing in light, the contribution of Fd-GOGAT, which directly utilizes photoreducing power for the catalytic reaction, is essential for balancing photosynthetic nitrogen and carbon assimilation.
Cold-Induced Freezing Tolerance in Arabidopsis1
Authors: Wanner Leslie A. , Junttila Olavi
Published: July 2002

Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance.
Xanthophyll Cycle Pigment Localization and Dynamics during Exposure to Low Temperatures and Light Stress inVinca major1
Authors: Verhoeven Amy S. , Adams William W. , Demmig-Adams Barbara , Croce Roberta , Bassi Roberto
Published: July 2002

The distribution of xanthophyll cycle pigments (violaxanthin plus antheraxanthin plus zeaxanthin [VAZ]) among photosynthetic pigment-protein complexes was examined in Vinca majorbefore, during, and subsequent to a photoinhibitory treatment at low temperature. Four pigment-protein complexes were isolated: the core of photosystem (PS) II, the major light-harvesting complex (LHC) protein of PSII (LHCII), the minor light-harvesting proteins (CPs) of PSII (CP29, CP26, and CP24), and PSI with its LHC proteins (PSI-LHCI). In isolated thylakoids 80% of VAZ was bound to protein independently of the de-epoxidation state and was found in all complexes. Plants grown outside in natural sunlight had higher levels of VAZ (expressed per chlorophyll), compared with plants grown in low light in the laboratory, and the additional VAZ was mainly bound to the major LHCII complex, apparently in an acid-labile site. The extent of de-epoxidation of VAZ in high light and the rate of reconversion of Z plus A to V following 2.5 h of recovery were greatest in the free-pigment fraction and varied among the pigment-protein complexes. Photoinhibition caused increases in VAZ, particularly in low-light-acclimated leaves. The data suggest that the photoinhibitory treatment caused an enrichment in VAZ bound to the minor CPs caused by de novo synthesis of the pigments and/or a redistribution of VAZ from the major LHCII complex.
N-Acylethanolamines in Seeds. Quantification of Molecular Species and Their Degradation upon Imbibition1
Authors: Chapman Kent D. , Venables Barney , Markovic Robert , Blair Raymond W. , Bettinger Chris
Published: July 2002

N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 ± 89 ng g−1fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 ± 309 ng g−1 fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, withN-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g−1 fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35°C in 50 mm MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5,5′-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent Km of 65 μm and aVmax of 83 nmol min−1mg−1 protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of “endocannabinoids,” which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.
Differential Ion Accumulation and Ion Fluxes in the Mesophyll and Epidermis of Barley
Authors: Karley Alison J. , Leigh Roger A. , Sanders Dale
Published: July 2002

In barley (Hordeum vulgare L.) leaves, differential ion accumulation commonly results in inorganic phosphate (Pi) being confined to the mesophyll and Ca2+ to the epidermis, with preferential epidermal accumulation of Cl−, Na+, and some other ions. The pattern was confirmed in this study for major inorganic anions and cations by analysis of barley leaf protoplasts. The work focused on the extent to which differences in plasma membrane ion transport processes underlie these observations. Ion transport across the plasma membrane of barley epidermal and mesophyll protoplasts was investigated electrophysiologically (by microelectrode impalement and patch clamping) and radiometrically. Data from both approaches suggested that similar types of ion-selective channels and membrane transporters, which catalyze the transport of Ca2+, K+, Na+, and Pi, exist in the plasma membrane of the two cell types. In general, the simple presence or absence of ion transporters could not explain cell-type-specific differences in ion accumulation. However, patch-clamp data suggested that differential regulation of instantaneously activating ion channels in the plasma membrane could explain the preferential accumulation of Na+ in the epidermis.
Sodium-Dependent Nitrate Transport at the Plasma Membrane of Leaf Cells of the Marine Higher PlantZostera marinaL.
Authors: Garcı́a-Sánchez Marı́a J. , Jaime M. Paz , Ramos Alberto , Sanders Dale , Fernández JoséA.
Published: July 2002

NO3  −is present at micromolar concentrations in seawater and must be absorbed by marine plants against a steep electrochemical potential difference across the plasma membrane. We studied NO3  − transport in the marine angiospermZostera marina L. to address the question of how NO3  − uptake is energized. Electrophysiological studies demonstrated that micromolar concentrations of NO3  − induced depolarizations of the plasma membrane of leaf cells. Depolarizations showed saturation kinetics (K  m = 2.31 ± 0.78 μm NO3  −) and were enhanced in alkaline conditions. The addition of NO3  − did not affect the membrane potential in the absence of Na+, but depolarizations were restored when Na+ was resupplied. NO3  −-induced depolarizations at increasing Na+ concentrations showed saturation kinetics (K  m = 0.72 ± 0.18 mmNa+). Monensin, an ionophore that dissipates the Na+ electrochemical potential, inhibited NO3  −-evoked depolarizations by 85%, and NO3  − uptake (measured by depletion from the external medium) was stimulated by Na+ ions and by light. Our results strongly suggest that NO3  − uptake in Z. marina is mediated by a high-affinity Na+-symport system, which is described here (for the first time to our knowledge) in an angiosperm. Coupling the uptake of NO3  − to that of Na+ enables the steep inwardly-directed electrochemical potential for Na+to drive net accumulation of NO3  − within leaf cells.
Activation Tagging in Arabidopsis
Authors: Weigel Detlef , Ahn Ji Hoon , Blázquez Miguel A. , Borevitz Justin O. , Christensen Sioux K. , Fankhauser Christian , Ferrándiz Cristina , Kardailsky Igor , Malancharuvil Elizabeth J. , Neff Michael M. , Nguyen Jasmine Thuy , Sato Shusei , Wang Zhi-Yong , Xia Yiji , Dixon Richard A. , Harrison Maria J. , Lamb Chris J. , Yanofsky Martin F. , Chory Joanne
Published: July 2002

Activation tagging using T-DNA vectors that contain multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene has been applied to Arabidopsis plants. New activation-tagging vectors that confer resistance to the antibiotic kanamycin or the herbicide glufosinate have been used to generate several tens of thousands of transformed plants. From these, over 30 dominant mutants with various phenotypes have been isolated. Analysis of a subset of mutants has shown that overexpressed genes are almost always found immediately adjacent to the inserted CaMV 35S enhancers, at distances ranging from 380 bp to 3.6 kb. In at least one case, the CaMV 35S enhancers led primarily to an enhancement of the endogenous expression pattern rather than to constitutive ectopic expression, suggesting that the CaMV 35S enhancers used here act differently than the complete CaMV 35S promoter. This has important implications for the spectrum of genes that will be discovered by this method.
Immunolocalization of a Cysteine Protease in Vacuoles, Vesicles, and Symbiosomes of Pea Nodule Cells
Authors: Vincent Jason L. , Brewin Nicholas J.
Published: July 2002

PsCYP15A is a cysteine protease from pea (Pisum sativum L.). It was first recognized as an up-regulated transcript in wilted shoots and subsequently in root nodules containingRhizobium. Proteolytic activity of PsCYP15A in nodule extracts is now reported following immunopurification with polyclonal antiserum raised against recombinant antigen. Western-blot analysis indicated two forms of PsCYP15A, a pro-form (approximately 38 kD) and a mature form (approximately 30 kD). Both forms were present in most tissue samples, but only the mature form was isolated from cell-fractionated symbiosomes containing nitrogen-fixing bacteroids. Immunolabeling of nodule sections showed localization of PsCYP15A antigen in large vacuolar bodies, cytoplasmic vesicles, and the perisymbiont space. Immunolabeling of tissue sections from wilted shoots also indicated the presence of PsCYP15A in vacuoles and cytoplasmic vesicles. This protease may be involved in the adaptation to changes in cell turgor, both in wilted shoots and in nodule tissue. Additionally, the protease may be involved in protein turnover in the symbiosome compartment.
Purification and Characterization of Bifunctional Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase from Developing Soybean Seeds
Authors: Miron Daphna , Ben-Yaacov Sari , Reches Dalit , Schupper Avigail , Galili Gad
Published: July 2002

Both in mammals and plants, excess lysine (Lys) is catabolized via saccharopine into α-amino adipic semialdehyde and glutamate by two consecutive enzymes, Lys-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. To study the control of metabolite flux via this bifunctional enzyme, we have purified it from developing soybean (Glycine max) seeds. LKR activity of the bifunctional LKR/SDH possessed relatively high K  m for its substrates, Lys and α-ketoglutarate, suggesting that this activity may serve as a rate-limiting step in Lys catabolism. Despite their linkage, the LKR and SDH enzymes possessed significantly different pH optima, suggesting that SDH activity of the bifunctional enzyme may also be rate-limiting in vivo. We have previously shown that Arabidopsis plants contain both a bifunctional LKR/SDH and a monofunctional SDH enzymes (G. Tang, D. Miron, J.X. Zhu-Shimoni, G. Galili [1997] Plant Cell 9: 1–13). In the present study, we found no evidence for the presence of such a monofunctional SDH enzyme in soybean seeds. These results may provide a plausible regulatory explanation as to why various plant species accumulate different catabolic products of Lys.
Differential Interaction of Maize Root Ferredoxin:NADP+ Oxidoreductase with Photosynthetic and Non-Photosynthetic Ferredoxin Isoproteins
Authors: Onda Yayoi , Matsumura Tomohiro , Kimata-Ariga Yoko , Sakakibara Hitoshi , Sugiyama Tatsuo , Hase Toshiharu
Published: July 2002

In higher plants ferredoxin (Fd):NADP+ oxidoreductase (FNR) and Fd are each distributed in photosynthetic and non-photosynthetic organs as distinct isoproteins. We have cloned cDNAs for leaf FNR (L-FNR I and L-FNR II) and root FNR (R-FNR) from maize (Zea mays L.), and produced recombinant L-FNR I and R-FNR to study their enzymatic functions through kinetic and Fd-binding analyses. The K  m value obtained by assay for a diaphorase activity indicated that R-FNR had a 10-fold higher affinity for NADPH than L-FNR I. When we assayed for NADPH-cytochromec reductase activity using maize photosynthetic Fd (Fd I) and non-photosynthetic Fd (Fd III), the R-FNR showed a marked difference in affinity between these two Fd isoproteins; theK  m for Fd III was 3.0 μm and that for Fd I was 29 μm. Consistent with this, the dissociation constant for the R-FNR:Fd III complex was 10-fold smaller than that of the R-FNR:Fd I complex. This differential binding capacity was confirmed by an affinity chromatography of R-FNR on Fd-sepharose with stronger binding to Fd III. L-FNR I showed no such differential interaction with Fd I and Fd III. These data demonstrated that R-FNR has the ability to discriminate between these two types of Fds. We propose that the stronger interaction of R-FNR with Fd III is crucial for an efficient electron flux of NADPH-FNR-Fd cascade, thus supporting Fd-dependent metabolism in non-photosynthetic organs.
Exploiting Secondary Growth in Arabidopsis. Construction of Xylem and Bark cDNA Libraries and Cloning of Three Xylem Endopeptidases
Authors: Zhao Chengsong , Johnson Bobby J. , Kositsup Boonthida , Beers Eric P.
Published: July 2002

The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia  coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.
Regulation of Sulfur Nutrition in Wild-Type and Transgenic Poplar Over-Expressing γ-Glutamylcysteine Synthetase in the Cytosol as Affected by Atmospheric H2S
Authors: Herschbach Cornelia , van der Zalm Esther , Schneider Andrea , Jouanin Lise , De Kok Luit J. , Rennenberg Heinz
Published: July 2002

This study with poplar (Populus tremula ×Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing γ-glutamylcysteine (γ-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H2S. H2S taken up by the leaves increased cysteine, γ-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H2S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and γ-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H2S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.
Redox Control of ntcA Gene Expression in Synechocystis sp. PCC 6803. Nitrogen Availability and Electron Transport Regulate the Levels of the NtcA Protein
Authors: Alfonso Miguel , Perewoska Irène , Kirilovsky Diana
Published: July 2002

In this work we have studied the influence of the cellular redox status in the expression of the Synechocystis sp. PCC 6803 ntcA gene. Two different ntcAtranscripts with different 5′ ends were detected, depending on the different dark/light or nitrogen availability conditions. Accumulation of a 0.8-kb ntcA message was light and nitrogen dependent, whereas a longer 1.2-kb ntcA transcript was neither light nor nitrogen regulated. NtcA protein levels increased concomitantly with the accumulation of the 0.8-kb ntcAtranscript. The light-dependent accumulation of the ntcAgene and the NtcA protein was sensitive to electron transport inhibitors. In addition, Glc-grown Synechocystis sp. cells showed a similar ntcA expression pattern in darkness to that observed under illumination. These data suggested that electron transport, and not light per se may regulatentcA gene expression. Primer extension analysis, together with gel mobility-shift assays, demonstrated that in vitro, the Synechocystis sp. NtcA protein specifically bound to the putative promoter region from the light/nitrogen-dependentntcA transcript but not to that from the constitutive 1.2-kb ntcA mRNA. Band-shift experiments carried out in the presence of thiol oxidizing/modifiying agents and different reducing/oxidizing conditions suggested that NtcA binding to its own promoter was under a thiol-dependent redox mechanism. Our results suggest that the cellular redox status plays a central role in the autoregulatory mechanism of the NtcA protein.
Guard Cell Volume and Pressure Measured Concurrently by Confocal Microscopy and the Cell Pressure Probe,
Authors: Franks Peter J. , Buckley Thomas N. , Shope Joseph C. , Mott Keith A.
Published: July 2002

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 μm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.
Active Oxygen Produced during Selective Excitation of Photosystem I Is Damaging Not Only to Photosystem I, But Also to Photosystem II
Authors: Tjus Staffan Erling , Scheller Henrik Vibe , Andersson Bertil , Møller Birger Lindberg
Published: July 2002

With the aim to specifically study the molecular mechanisms behind photoinhibition of photosystem I, stacked spinach (Spinacia oleracea) thylakoids were irradiated at 4°C with far-red light (>715 nm) exciting photosystem I, but not photosystem II. Selective excitation of photosystem I by far-red light for 130 min resulted in a 40% inactivation of photosystem I. It is surprising that this treatment also caused up to 90% damage to photosystem II. This suggests that active oxygen produced at the reducing side of photosystem I is highly damaging to photosystem II. Only a small pool of the D1-protein was degraded. However, most of the D1-protein was modified to a slightly higher molecular mass, indicative of a damage-induced conformational change. The far-red illumination was also performed using destacked and randomized thylakoids in which the distance between the photosystems is shorter. Upon 130 min of illumination, photosystem I showed an approximate 40% inactivation as in stacked thylakoids. In contrast, photosystem II only showed 40% inactivation in destacked and randomized thylakoids, less than one-half of the inactivation observed using stacked thylakoids. In accordance with this, photosystem II, but not photosystem I is more protected from photoinhibition in destacked thylakoids. Addition of active oxygen scavengers during the far-red photosystem I illumination demonstrated superoxide to be a major cause of damage to photosystem I, whereas photosystem II was damaged mainly by superoxide and hydrogen peroxide.