bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxygen Requirement and Inhibition of C4Photosynthesis1
Authors: Maroco João P. , Ku Maurice S.B. , Lea Peter J. , Dever Louisa V. , Leegood Richard C. , Furbank Robert T. , Edwards Gerald E.
Published: July 2002

The basis for O2sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis(a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency.
Differential Expression of theS-Adenosyl-l-Methionine Synthase Genes during Pea Development1
Authors: Gómez-Gómez Lourdes , Carrasco Pedro
Published: July 2002

Two genes coding for S-adenosyl-l-methionine synthase (SAMS, EC 2.5.1.6) were previously isolated from pea (Pisum sativum) ovaries. Both SAMS genes were highly homologous throughout their coding regions but showed a certain degree of sequence divergence within the 5′ and the 3′ untranslated regions. These regions have been used as gene-specific probes to analyze the differential expression of SAMS1and SAMS2 genes in pea plants. The ribonuclease protection assay revealed different expression patterns for each individual gene. SAMS1 was strongly expressed in nearly all tissues, especially in roots. SAMS2 expression was weaker, reaching its highest level at the apex. Following pollination,SAMS1 was specifically up-regulated, whereasSAMS2 was expressed constitutively. The up-regulation ofSAMS1 during ovary development was also observed in unpollinated ovaries treated with auxins. In unpollinated ovaries an increase in SAMS1 expression was observed as a consequence of ethylene production associated with the emasculation process. In senescing ovaries both SAMS1 andSAMS2 genes showed increased expression. Ethylene treatment of unpollinated ovaries led to an increase in theSAMS1 mRNA level. However, SAMS2expression remained unchangeable after ethylene treatment, indicating that SAMS2 induction during ovary senescence was not ethylene dependent. SAMS mRNAs were localized by in situ hybridization at the endocarp of developing fruits and in the ovules of senescing ovaries. Our results indicate that the transcriptional regulation of SAMS genes is developmentally controlled in a specific way for each gene.
Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat1
Authors: Kumar Tewari Arun , Charan Tripathy Baishnab
Published: July 2002

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.
Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1
Authors: Gronwald John W. , Plaisance Kathryn L.
Published: July 2002

Two glutathioneS-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated,O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4′-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapid-equilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I θ-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.
Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa1
Authors: Hiltonen Thomas , Björkbacka Harry , Forsman Cecilia , Clarke Adrian K. , Samuelsson Göran
Published: July 2002

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. TheCoccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of theCoccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.
cis
-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development1
Authors: Emery Robert Joseph Neil , Leport Laurent , Barton Joanne Edith , Turner Neil Clifford , Atkins Craig Anthony
Published: July 2002

Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. Thecis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of correspondingtrans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantlycis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of thecis-CK. Since no isomerization of (trans)[2H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.
Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1
Authors: Ylstra Bauke , Garrido Dolores , Busscher Jacqueline , van Tunen Arjen J.
Published: July 2002

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petuniamonosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.
A Novel Nuclear Member of the Thioredoxin Superfamily
Authors: Laughner Beth J. , Sehnke Paul C. , Ferl Robert J.
Published: July 2002

We describe the isolation and characterization of a cDNA encoding maize (Zea mays L.) nucleoredoxin (NRX), a novel nuclear protein that is a member of the thioredoxin (TRX) superfamily. NRX is composed of three TRX-like modules arranged as direct repeats of the classic TRX domain. The first and third modules contain the amino acid sequence WCPPC, which indicates the potential for TRX oxidoreductase activity, and insulin reduction assays indicate that at least the third module possesses TRX enzymatic activity. The carboxy terminus of NRX is a non-TRX module that possesses C residues in the proper sequence context to form a Zn finger. Immunolocalization preferentially to the nucleus within developing maize kernels suggests a potential for directed alteration of the reduction state of transcription factors as part of the events and pathways that regulate gene transcription.
The Respiratory Burst and Electrolyte Leakage Induced by Sulfhydryl Blockers in Egeria densa Leaves Are Associated with H2O2 Production and Are Dependent on Ca2+ Influx
Authors: Marrè Maria Teresa , Amicucci Enrica , Zingarelli Luisa , Albergoni Francesco , Marrè Erasmo
Published: July 2002

In leaves of Egeria densa Planchon, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a temporary increase in nonmitochondrial respiration (ΔQO2) that is inhibited by diphenylene iodonium and quinacrine, two known inhibitors of the plasma membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388–394). In this paper we report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that induced by pathogen-derived elicitors in animal and plant cells: (a) NEM- and Ag+-induced ΔQO2 was associated with H2O2 production and both effects depended on the presence of external Ca2+; (b) Ca2+ influx was markedly increased by treatment with NEM; (c) the Ca2+channel blocker LaCl3 inhibited ΔQO2, electrolyte release, and membrane depolarization induced by the sulfhydryl reagents; and (d) LaCl3 also inhibited electrolyte leakage induced by the direct infiltration of the leaves with H2O2. These results suggest a model in which the interaction of sulfhydryl blockers with sulfhydryl groups of cell components would primarily induce an increase in the Ca2+ cytosolic concentration, followed by membrane depolarization and activation of a plasma membrane NADPH oxidase. This latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.
Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Carbamylation and 2-Carboxyarabinitol 1-Phosphate in Tobacco: Insights from Studies of Antisense Plants Containing Reduced Amounts of Rubisco Activase
Authors: Hammond Edward T. , Andrews T. John , Woodrow Ian E.
Published: July 2002

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m−2 s−1 was followed, the slow increase in CO2 assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO2 assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO2 assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.
Chloroplast-Avoidance Response Induced by High-Fluence Blue Light in Prothallial Cells of the Fern Adiantum capillus-veneris as Analyzed by Microbeam Irradiation1
Authors: Kagawa Takatoshi , Wada Masamitsu
Published: July 2002

Chloroplast movement was induced by partial cell illumination using a high-fluence blue microbeam in light-grown and dark-adapted prothallial cells of the fern Adiantum capillus-veneris. Chloroplasts inside the illuminated area moved out (high-fluence response [HFR]), whereas those outside moved toward the irradiated area (low-fluence response [LFR]), although they stopped moving when they reached the border. These results indicate that both HFR and LFR signals are generated by high-fluence blue light of the same area, and that an LFR signal can be transferred long-distance from the beam spot, although an HFR signal cannot. The lifetime of the HFR signal was calculated from the traces of chloroplast movement induced by a brief pulse from a high-fluence blue microbeam to be about 6 min. This is very short compared with that of the LFR (30–40 min; T. Kagawa, M. Wada [1994] J Plant Res 107: 389–398). These data indicate that the signal transduction pathways of the HFR and the LFR must be distinct.
Simultaneous Expression of NAD-Dependent Isocitrate Dehydrogenase and Other Krebs Cycle Genes after Nitrate Resupply to Short-Term Nitrogen-Starved Tobacco
Authors: Lancien Muriel , Ferrario-Méry Sylvie , Roux Yvette , Bismuth Evelyne , Masclaux Céline , Hirel Bertrand , Gadal Pierre , Hodges Michael
Published: July 2002

Mitochondrial NAD-dependent (IDH) and cytosolic NADP-dependent isocitrate dehydrogenases have been considered as candidates for the production of 2-oxoglutarate required by the glutamine synthetase/glutamate synthase cycle. The increase in IDH transcripts in leaf and root tissues, induced by nitrate or NH4+ resupply to short-term N-starved tobacco (Nicotiana tabacum) plants, suggested that this enzyme could play such a role. The leaf and root steady-state mRNA levels of citrate synthase, acotinase, IDH, and glutamine synthetase were found to respond similarly to nitrate, whereas those for cytosolic NADP-dependent isocitrate dehydrogenase and fumarase responded differently. This apparent coordination occurred only at the mRNA level, since activity and protein levels of certain corresponding enzymes were not altered. Roots and leaves were not affected to the same extent either by N starvation or nitrate addition, the roots showing smaller changes in N metabolite levels. After nitrate resupply, these organs showed different response kinetics with respect to mRNA and N metabolite levels, suggesting that under such conditions nitrate assimilation was preferentially carried out in the roots. The differential effects appeared to reflect the C/N status after N starvation, the response kinetics being associated with the nitrate assimilatory capacity of each organ, signaled either by nitrate status or by metabolite(s) associated with its metabolism.
Ascorbate Biosynthesis in Arabidopsis Cell Suspension Culture
Authors: Davey Mark W. , Gilot Christophe , Persiau Geert , Østergaard Jens , Han Yu , Bauw Guy C. , Van Montagu Marc C.
Published: July 2002

The biosynthesis ofl-ascorbic acid (l-AA) in an Arabidopsis (L.) Heynh. cell suspension culture was studied by quantifying the effects of incubation with a range of potential biosynthetic precursors, analogs, and inhibitors on the intracellular levels of reduced and oxidized forms of l-AA. Our results support the recently published biosynthetic pathway of l-AA froml-galactose (G.L. Wheeler, M.A. Jones, N. Smirnoff [1998] Nature 393: 365–369), but suggest that Arabidopsis cell suspension culture simultaneously contains two other routes leading tol-AA. The possible physiological significance of these alternate routes is discussed.
An Expansin Gene Expressed in Ripening Strawberry Fruit
Authors: Civello Pedro Marcos , Powell Ann L.T. , Sabehat Adnan , Bennett Alan B.
Published: July 2002

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria × ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.
Nitrite Reductase Mutants as an Approach to Understanding Nitrate Assimilation inChlamydomonas reinhardtii
Authors: Navarro Marı́a Teresa , Guerra Elena , Fernández Emilio , Galván Aurora
Published: July 2002

We constructed mutant strains lacking the nitrite reductase (NR) gene in Chlamydomonas reinhardtii. Two types of NR mutants were obtained, which either have or lack the high-affinity nitrate transporter (Nrt2;1, Nrt2;2, andNar2) genes. None of these mutants overexpressed nitrate assimilation gene transcripts nor NR activity in nitrogen-free medium, in contrast to NR mutants. This finding confirms the previous role proposed for NR on its own regulation (autoregulation) and on the other genes for nitrate assimilation in C. reinhardtii. In addition, the NR mutants were used to study nitrate transporters from nitrite excretion. At high CO2, only strains carrying the above high-affinity nitrate transporter genes excreted stoichiometric amounts of nitrite from 100 μm nitrate in the medium. A double mutant, deficient in both the high-affinity nitrate transporter genes and NR, excreted nitrite at high CO2 only when nitrate was present at mm concentrations. This suggests that there exists a low-affinity nitrate transporter that might correspond to the nitrate/nitrite transport system III. Moreover, under low CO2 conditions, the double mutant excreted nitrite from nitrate at micromolar concentrations by a transporter with the properties of the nitrate/nitrite transport system IV.
Iron Deficiency Decreases the Fe(III)-Chelate Reducing Activity of Leaf Protoplasts
Authors: González-Vallejo Elena B. , Morales Fermı́n , Cistué Luis , Abadı́a Anunciación , Abadı́a Javier
Published: July 2002

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.
Differentiation of Mucilage Secretory Cells of the Arabidopsis Seed Coat
Authors: Western Tamara L. , Skinner Debra J. , Haughn George W.
Published: July 2002

In some plant species, including Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type with a unique morphology and containing large quantities of polysaccharide mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under normal laboratory conditions. Thus, the Arabidopsis seed coat offers a unique system with which to use genetics to identify genes controlling cell morphogenesis and complex polysaccharide biosynthesis and secretion. As a first step in the application of this system, we have used microscopy to investigate the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis and the synthesis, secretion, and extrusion of mucilage. During seed coat development in Arabidopsis, the epidermal cells of the outer ovule integument grow and differentiate into cells that produce large quantities of mucilage between the primary cell wall and plasma membrane. Concurrent with mucilage production, the cytoplasm is shaped into a column in the center of the cell. Following mucilage secretion the cytoplasmic column is surrounded by a secondary cell wall to form a structure known as the columella. Thus, differentiation of the seed coat mucilage cells involves a highly regulated series of events including growth, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis.
Optical Coherence Microscopy. A Technology for Rapid, in Vivo, Non-Destructive Visualization of Plants and Plant Cells,
Authors: Hettinger James W. , Mattozzi Matthew de la Peña , Myers Whittier R. , Williams Mary E. , Reeves Aaron , Parsons Ronald L. , Haskell Richard C. , Petersen Daniel C. , Wang Ruye , Medford June I.
Published: July 2002

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 μm in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 × 5 × 10 μm) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a three-dimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.
Integrated Temporal Regulation of the Photorespiratory Pathway. Circadian Regulation of Two Arabidopsis Genes Encoding Serine Hydroxymethyltransferase
Authors: McClung C. Robertson , Hsu Meier , Painter Janet E. , Gagne Jennifer M. , Karlsberg Sharon D. , Salomé Patrice A.
Published: July 2002

The photorespiratory pathway is comprised of enzymes localized within three distinct cellular compartments: chloroplasts, peroxisomes, and mitochondria. Photorespiratory enzymes are encoded by nuclear genes, translated in the cytosol, and targeted into these distinct subcellular compartments. One likely means by which to regulate the expression of the genes encoding photorespiratory enzymes is coordinated temporal control. We have previously shown in Arabidopsis that a circadian clock regulates the expression of the nuclear genes encoding both chloroplastic (Rubisco small subunit and Rubisco activase) and peroxisomal (catalase) components of the photorespiratory pathway. To determine whether a circadian clock also regulates the expression of genes encoding mitochondrial components of the photorespiratory pathway, we characterized a family of Arabidopsisserine hydroxymethyltransferase (SHM) genes. We examined mRNA accumulation for two of these family members, including one probable photorespiratory gene (SHM1) and a second gene expressed maximally in roots (SHM4), and show that both exhibit circadian oscillations in mRNA abundance that are in phase with those described for other photorespiratory genes. In addition, we show that SHM1 mRNA accumulates in light-grown seedlings, although this response is probably an indirect consequence of the induction of photosynthesis and photorespiration by illumination.
Isolation of a cDNA Encoding a Granule-Bound 152-Kilodalton Starch-Branching Enzyme in Wheat
Authors: Båga Monica , Nair Ramesh B. , Repellin Anne , Scoles Graham J. , Chibbar Ravindra N.
Published: July 2002

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5′-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80–100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present inTriticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.
Acclimation of the Photosynthetic Machinery to High Temperature inChlamydomonas reinhardtiiRequires Synthesis de Novo of Proteins Encoded by the Nuclear and Chloroplast Genomes
Authors: Tanaka Yuji , Nishiyama Yoshitaka , Murata Norio
Published: July 2002

The mechanism responsible for the enhancement of the thermal stability of the oxygen-evolving machinery of photosystem II during acclimation of Chlamydomonas reinhardtii to high temperatures such as 35°C remains unknown. When cells that had been grown at 20°C were transferred to 35°C, the thermal stability of the oxygen-evolving machinery increased and within 8 h it was equivalent to that in cells grown initially at 35°C. Such enhancement of thermal stability was prevented by cycloheximide and by lincomycin, suggesting that the synthesis de novo of proteins encoded by both the nuclear and the chloroplast genome was required for this process. No increase in thermal stability was observed when cells that had been grown at 35°C were exposed to heat shock at 41°C, optimum conditions for the induction of the synthesis of homologs of three heat shock proteins (Hsps), namely, Hsp60, Hsp70, and Hsp22. Moreover, no synthesis of these homologs of Hsps was induced at 35°C. Thus it appears likely that Hsps are not involved in the enhancement of the thermal stability of the oxygen-evolving machinery.
Multisite Inhibition of Pinus pinea Isocitrate Lyase by Phosphate
Authors: Ranaldi Francesco , Vanni Paolo , Giachetti Eugenio
Published: July 2002

Our results show that the phosphate ion is a nonlinear competitive inhibitor of Pinus pinea isocitrate lyase. In addition, this compound induces a sigmoidal response of the enzyme, which usually exhibits standard Michaelis-Menten kinetics. This peculiar behavior ofP. pinea isocitrate lyase could be explained by a dimer (two-site) model, in which phosphate binds cooperatively, but the affinity of the vacant site for substrate (the magnesium-isocitrate complex) remains the same. As a result, the interaction of phosphate with free enzyme produces an inhibitor-enzyme-inhibitor species that is of significant importance in determining reaction rate; a possible regulatory role of the glyoxylate cycle by inorganic phosphate is suggested. The mode of phosphate inhibition is consistent with both the mechanism for magnesium ion activation of P. pineaisocitrate lyase and its site heterogeneity. Our results explain the cooperative effects observed by some authors in kinetic studies of isocitrate lyase carried out in phosphate buffers and also account for the higher K  m values determined by using such assay systems. Phosphate buffer should be avoided in performing isocitrate lyase kinetics.
Analysis of the Alternative Pathways for the β-Oxidation of Unsaturated Fatty Acids Using Transgenic Plants Synthesizing Polyhydroxyalkanoates in Peroxisomes
Authors: Allenbach Laure , Poirier Yves
Published: July 2002

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core β-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2,4-dienoyl-coenzyme A (CoA) reductase and Δ3-Δ2-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also calledR-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the β-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.
Arabidopsis Mutants Resistant to S(+)-β-Methyl-α, β-Diaminopropionic Acid, a Cycad-Derived Glutamate Receptor Agonist
Authors: Brenner Eric D. , Martinez-Barboza Nora , Clark Alexandra P. , Liang Quail S. , Stevenson Dennis W. , Coruzzi Gloria M.
Published: July 2002

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are the predominant neuroreceptors in the mammalian brain. Genes with high sequence similarity to animal iGluRs have been identified in Arabidopsis. To understand the role of Arabidopsis glutamate receptor-like (AtGLR) genes in plants, we have taken a pharmacological approach by examining the effects of BMAA [S(+)-β-methyl-α, β-diaminopropionic acid], a cycad-derived iGluR agonist, on Arabidopsis morphogenesis. When applied to Arabidopsis seedlings, BMAA caused a 2- to 3-fold increase in hypocotyl elongation and inhibited cotyledon opening during early seedling development. The effect of BMAA on hypocotyl elongation is light specific. Furthermore, BMAA effects on early morphogenesis of Arabidopsis can be reversed by the simultaneous application of glutamate, the native iGluR agonist in animals. To determine the targets of BMAA action in Arabidopsis, a genetic screen was devised to isolate Arabidopsis mutants with a BMAA insensitive morphology (bim). When grown in the light on BMAA,bim mutants exhibited short hypocotyls compared with wild type. bim mutants were grouped into three classes based on their morphology when grown in the dark in the absence of BMAA. Class-I bim mutants have a normal, etiolated morphology, similar to wild-type plants. Class-II bimmutants have shorter hypocotyls and closed cotyledons when grown in the dark. Class-III bim mutants have short hypocotyls and open cotyledons when grown in the dark, resembling the previously characterized constitutively photomorphogenic mutants (cop, det, fus, and shy). Further analysis of thebim mutants should help define whether plant-derived iGluR agonists target glutamate receptor signaling pathways in plants.
Molecular and Biochemical Analysis of a Madagascar Periwinkle Root-Specific Minovincinine-19-Hydroxy-O-Acetyltransferase
Authors: Laflamme Pierre , St-Pierre Benoit , De Luca Vincenzo
Published: July 2002

The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coliwere named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances.
Trehalose and Trehalase in Arabidopsis
Authors: Müller Joachim , Aeschbacher Roger A. , Wingler Astrid , Boller Thomas , Wiemken Andres
Published: July 2002

Trehalase is ubiquitous in higher plants. So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose. A putative trehalase gene,T19F6.15, has been identified in the genome sequencing effort in Arabidopsis. Here we show that this gene encodes a functional trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs. Furthermore, we present results on the distribution and activity of trehalase in Arabidopsis and we describe how inhibition of trehalase by validamycin A affects the plants response to exogenous trehalose (α-d-glucopyranosyl-[1, 1]-α-d-glucopyranoside). Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g−1 protein) and maturing siliques (approximately 250 nkat g−1 protein) and much lower in leaves, stems, and roots (less than 50 nkat g−1 protein). Inhibition of trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems. Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and trehalase may play a role in regulating the carbohydrate allocation in plants.
Biosynthesis of l-Ascorbic Acid and Conversion of Carbons 1 and 2 of l-Ascorbic Acid to Oxalic Acid Occurs within Individual Calcium Oxalate Crystal Idioblasts
Authors: Kostman Todd A. , Tarlyn Nathan M. , Loewus Frank A. , Franceschi Vincent R.
Published: July 2002

l-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various14C-labeled compounds and examined by micro-autoradiography for incorporation of 14C into calcium oxalate crystals. [14C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-14C]AsA also gave heavy labeling of crystals, whereas [6-14C]AsA gave no labeling. Labeled precursors of AsA (l-[1-14C]galactose;d-[1-14C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog,d-[1-14C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > l-galactose >d-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis viad-mannose and l-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments.
Iris Bulbs Express Type 1 and Type 2 Ribosome-Inactivating Proteins with Unusual Properties
Authors: Hao Qiang , Van Damme Els J.M. , Hause Bettina , Barre Annick , Chen Ying , Rougé Pierre , Peumans Willy J.
Published: July 2002

Two closely related lectins from bulbs of the Dutch iris (Iris hollandica var. Professor Blaauw) have been isolated and cloned. Both lectins, called Iris agglutinin b and Iris agglutinin r, possess N-glycosidase activity and share a high sequence similarity with previously described type 2 ribosome-inactivating proteins (RIP). However, these lectins show only 57% to 59% sequence identity to a previously characterized type 1 RIP from iris, called IRIP. The identification of the iris lectins as type 2 RIP provides unequivocal evidence for the simultaneous occurrence of type 1 and type 2 RIP in iris bulbs and allowed a detailed comparison of type 1 and type 2 RIP from a single plant, which provides further insight into the molecular evolution of RIP. Binding studies and docking experiments revealed that the lectins exhibit binding activity not only toward Gal/N-acetylgalactosamine, but also toward mannose, demonstrating for the first time that RIP-binding sites can accommodate mannose.
β-Aminobutyric Acid-Induced Protection of Arabidopsis against the Necrotrophic Fungus Botrytis cinerea
Authors: Zimmerli Laurent , Métraux Jean-Pierre , Mauch-Mani Brigitte
Published: July 2002

The non-protein amino acid β-aminobutyric acid (BABA) protects numerous plants against various pathogens. Protection of Arabidopsis plants against virulent pathogens involves the potentiation of pathogen-specific defense responses. To extend the analysis of the mode of action of BABA to necrotrophs we evaluated the effect of this chemical on Arabidopsis plants infected with the gray mold fungusBotrytis cinerea. BABA-treated Arabidopsis were found to be less sensitive to two different strains of this pathogen. BABA protected mutants defective in the jasmonate and ethylene pathways, but was inactive in plants impaired in the systemic acquired resistance transduction pathway. Treatments with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, a functional analog of salicylic acid (SA), also markedly reduced the level of infection. Moreover, BABA potentiated mRNA accumulation of the SA-associated PR-1, but not the jasmonate/ethylene-dependent PDF1.2 gene. Thus, besides jasmonate/ethylene-dependent defense responses, SA-dependent signaling also contributes to restrict B. cinerea infection in Arabidopsis. Our results also suggest that SA-dependent signaling is down-regulated after infection by B. cinerea. The observed up-regulation of the PDF1.2 gene in mutants defective in the SA-dependent signaling pathway points to a cross-talk between SA- and jasmonate/ethylene-dependent signaling pathways during pathogen ingress.
Evidence for a Role of Salicylic Acid in the Oxidative Damage Generated by NaCl and Osmotic Stress in Arabidopsis Seedlings
Authors: Borsani Omar , Valpuesta Victoriano , Botella Miguel A.
Published: July 2002

Previous studies have shown that salicylic acid (SA) is an essential component of the plant resistance to pathogens. We now show that SA plays a role in the plant response to adverse environmental conditions, such as salt and osmotic stresses. We have studied the responses of wild-type Arabidopsis and an SA-deficient transgenic line expressing a salicylate hydroxylase (NahG) gene to different abiotic stress conditions. Wild-type plants germinated under moderate light conditions in media supplemented with 100 mmNaCl or 270 mm mannitol showed extensive necrosis in the shoot. In contrast, NahG plants germinated under the same conditions remained green and developed true leaves. The lack of necrosis observed in NahG seedlings under the same conditions suggests that SA potentiates the generation of reactive oxygen species in photosynthetic tissues during salt and osmotic stresses. This hypothesis is supported by the following observations. First, the herbicide methyl viologen, a generator of superoxide radical during photosynthesis, produced a necrotic phenotype only in wild-type plants. Second, the presence of reactive oxygen-scavenging compounds in the germination media reversed the wild-type necrotic phenotype seen under salt and osmotic stress. Third, a greater increase in the oxidized state of the glutathione pool under NaCl stress was observed in wild-type seedlings compared with NahG seedlings. Fourth, greater oxidative damage occurred in wild-type seedlings compared with NahG seedlings under NaCl stress as measured by lipid peroxidation. Our data support a model for SA potentiating the stress response of the germinating Arabidopsis seedling.
Cysteine Synthase Overexpression in Tobacco Confers Tolerance to Sulfur-Containing Environmental Pollutants
Authors: Noji Masaaki , Saito Maiko , Nakamura Michimi , Aono Mitsuko , Saji Hikaru , Saito Kazuki
Published: July 2002

Cysteine (Cys) synthase [O-acetyl-l-Ser(thiol)-lyase, EC 4.2.99.8; CSase] is responsible for the final step in biosynthesis of Cys. Transgenic tobacco (Nicotiana tabacum; F1) plants with enhanced CSase activities in the cytosol and in the chloroplasts were generated by cross-fertilization of two transformants expressing cytosolic CSase or chloroplastic CSase. The F1transgenic plants were highly tolerant to toxic sulfur dioxide and sulfite. Upon fumigation with 0.1 μL L−1 sulfur dioxide, the Cys and glutathione contents in leaves of F1 plants were increased significantly, but not in leaves of non-transformed control plants. Furthermore, the leaves of F1 plants exhibited the increased resistance to paraquat, a herbicide generating active oxygen species.