bluuebunny/crossref_metadata_2025 huggingface dataset:
Pedicel Breakstrength and Cellulase Gene Expression during Tomato Flower Abscission
Authors: del Campillo E. , Bennett A. B.
Published: July 2002

Six cellulase genes were isolated from total RNA of the ethylene-treated tomato (Lycopersicon esculentum Mill.) flower abscission zone by reverse-transcription polymerase chain reaction using degenerate primers to conserved amino acid sequences from known plant cellulases. Four of the gene fragments are homologous to fruit pericarp cellulases. The other two are novel cellulase genes, referred to as Cel5 and Cel6. Breakstrength and cellulase gene expression were then analyzed in naturally abscising flowers and flower explants. In both naturally abscising flowers and flower explants induced to abscise in air or ethylene, both new cellulase mRNAs were correlated with flower shedding. Whereas the Cel5 mRNA increased in later stages of abscission, the Cel6 mRNA was present in nonabscising flowers and then decreased in the final stage of abscission. A third cellulase, Cel1, increased during the final stage of abscission in flower explants and yet did not increase during shedding in planta, although it was detectable at low levels in all abscission stages. Cel1 and Cel5 mRNA decreased 99% when indole-3-acetic acid was added during ethylene treatment, consistent with low levels of abscission (3%). In contrast, Cel6 mRNA increased slightly when indole-3-acetic acid was added. These results suggest that abscission is a multistep process involving both activated and repressed cellulase genes and that the relative importance of each cellulase in the process depends on the physiological conditions under which abscission takes place.
Two Genes Encode Highly Similar Chloroplastic NADP-Malic Enzymes in Flaveria (Implications for the Evolution of C4 Photosynthesis)
Authors: Marshall J. S. , Stubbs J. D. , Taylor W. C.
Published: July 2002

To gain an understanding of the molecular events underlying the evolution of C4 photosynthesis, we have undertaken a detailed study of the NADP-malic enzyme gene family in C4 and C3 species of Flaveria. Three genomic clones from the C4 species Flaveria bidentis were characterized and found to encode two highly similar chloroplastic forms of NADP-malic enzyme, termed ME1 and ME2. Genomic Southern blotting with gene-specific probes showed that both Me1 and Me2 are found in Flaveria trinervia (C4) and Flaveria pringlei (C3) as well as in F. bidentis. Northern blots demonstrated that Me1 expression in leaves parallels the degree of C4 photosynthesis in seven Flaveria species. Furthermore, whereas Me2 was expressed at a low level in both roots and leaves of F. bidentis, Me1 expression was seen only in leaves and was light-regulated. We discuss these results in the context of the evolution of C4 photosynthesis in Flaveria.
Novel Molecular Markers for Late Phases of the Growth Cycle of Arabidopsis thaliana Cell-Suspension Cultures Are Expressed during Organ Senescence
Authors: Callard D. , Axelos M. , Mazzolini L.
Published: July 2002

In an Arabidopsis thaliana T87-C3 cell-suspension culture, entry into the growth-arrest phase is rapidly followed by a loss of cell viability. Three cDNA clones, SRG1, SRG2, and SRG3, corresponding to genes with transcripts that accumulate during these late phases, were isolated by the mRNA differential display method. Amino acid sequence analysis shows that the putative SRG1 protein is a new member of the Fe(II)/ascorbate oxidase superfamily, and that SRG2 codes for a protein with significant homology to β-glucosidases. Significantly, all three SRG genes are expressed in senescing organs of Arabidopsis plants. Two previously characterized genes, SAG2 and SAG4, induced during natural senescence in Arabidopsis, were also found to be expressed in cell-suspension cultures and have expression kinetics similar to those observed for the SRG1 gene. Taken together these findings suggest that certain molecular events are common to both plant senescence and growth arrest in Arabidopsis cell suspensions. Both internucleosomal cleavage of nDNA and an apparent compaction of chromatin, two characteristic features of programmed cell death in animal cells, have been observed in Arabidopsis cell cultures at a stage corresponding to loss of cell viability.
Pressure Regulation of the Electrical Properties of Growing Arabidopsis thaliana L. Root Hairs
Authors: Lew R. R.
Published: July 2002

Actively growing Arabidopsis thaliana L. (Columbia wild type) root hairs were used to examine the interplay between cell turgor pressure and electrical properties of the cell: membrane potential, conductance, cell-to-cell coupling, and input resistance. Pressure was directly modulated using a pressure probe or indirectly by changing the extracellular osmolarity. Direct modulation of pressure in the range of 0 to about 15 x 105 Pa (normal turgor pressure was 6.8 [plus or minus] 2.0 x 105 Pa, n = 29) did not affect the membrane potential, conductance, coupling, or input resistance. Indirect modulation of turgor pressure by adding (hyperosmotic) or removing (hypo-osmotic) 200 mM mannitol/sorbitol affected the potential and conductance but not cell-to-cell coupling. Hypo-osmotic treatment depolarized the potential about 40 mV from an initial potential of about -190 mV and increased membrane conductance, consistent with an increase in anion efflux from the cell. Hyperosmotic treatment hyperpolarized the cell about 25 mV from the same initial potential and decreased conductance, consistent with a decline in cation influx. The results are likely due to the presence of an “osmo-sensor,” rather than a “turgor-sensor,” regulating the cell's response to osmotic stress.
The glossy1 Locus of Maize and an Epidermis-Specific cDNA from Kleinia odora Define a Class of Receptor-Like Proteins Required for the Normal Accumulation of Cuticular Waxes
Authors: Hansen J. D. , Pyee J. , Xia Y. , Wen T. J. , Robertson D. S. , Kolattukudy P. E. , Nikolau B. J. , Schnable P. S.
Published: July 2002

Mutations at the glossy1 (gl1) locus of maize (Zea mays L.) quantitatively and qualitatively affect the deposition of cuticular waxes on the surface of seedling leaves. The gl1 locus has been molecularly cloned by transposon tagging with the Mutator transposon system. The epi23 cDNA was isolated by subtractive hybridization as an epidermis-specific mRNA from Senecio odora (Kleinia odora). The deduced amino acid sequence of the GL1 and EPI23 proteins are very similar to each other and to two other plant proteins in which the sequences were deduced from their respective mRNAs. These are the Arabidopsis CER1 protein, which is involved in cuticular wax deposition on siliques, stems, and leaves of that plant, and the protein coded by the rice expressed sequence tag RICS2751A. All four proteins are predicted to be localized in a membrane via a common NH2-terminal domain, which consists of either five or seven membrane-spanning helices. The COOH-terminal portion of each of these proteins, although less conserved, is predicted to be a water-soluble, globular domain. These sequence similarities indicate that these plant orthologs may belong to a superfamily of membrane-bound receptors that have been extensively characterized from animals, including the HIV co-receptor fusin (also termed CXCR4).
Thermal Protection of the Oxygen-Evolving Machinery by PsbU, an Extrinsic Protein of Photosystem II, in Synechococcus species PCC 7002
Authors: Nishiyama Y. , Los D. A. , Hayashi H. , Murata N.
Published: July 2002

The evolution of oxygen is the reaction that is the most susceptible to heat in photosynthesis. We showed previously that, in the cyanobacterium Synechococcus sp. PCC 7002, some protein factors located on the thylakoid membranes are involved in the stabilization of this reaction against heat-induced inactivation, and we identified cytochrome c550 as one such factor (Y. Nishiyama, H. Hayashi, T. Watanabe, N. Murata [1994] Plant Physiol 105: 1313–1319). In the present study we purified another protein that appears to be essential for the stabilization of the oxygen-evolving machinery. The purified protein had an apparent molecular mass of 13 kD, and the gene encoding the 13-kD protein was cloned from Synechococcus sp. PCC 7002 and sequenced. The deduced amino acid sequence revealed that the protein was homologous to PsbU, an extrinsic protein of the photosystem II complex, which has been found in thermophilic species of cyanobacteria. Western analysis showed that the level of PsbU in thylakoid membranes was constant, regardless of the growth temperature. Our studies indicate that PsbU, a constituent of the photosystem II complex, protects the oxygen-evolving machinery against heat-induced inactivation.
Oxidative Burst and Hypoosmotic Stress in Tobacco Cell Suspensions
Authors: Cazalé Anne-Claire , Rouet-Mayer Marie-Aude , Barbier-Brygoo Hélène , Mathieu Yves , Laurière Christiane
Published: July 2002

Oxidative burst constitutes an early response in plant defense reactions toward pathogens, but active oxygen production may also be induced by other stimuli. The oxidative response of suspension-cultured tobacco (Nicotiana tabacum cv Xanthi) cells to hypoosmotic and mechanical stresses was characterized. The oxidase involved in the hypoosmotic stress response showed similarities by its NADPH dependence and its inhibition by iodonium diphenyl with the neutrophil NADPH oxidase. Activation of the oxidative response by hypoosmotic stress needed protein phosphorylation and anion effluxes, as well as opening of Ca2+ channels. Inhibition of the oxidative response impaired Cl− efflux, K+ efflux, and extracellular alkalinization, suggesting that the oxidative burst may play a role in ionic flux regulation. Active oxygen species also induced the cross-linking of a cell wall protein, homologous to a soybean (Glycine max L.) extensin, that may act as part of cell volume and turgor regulation through modification of the physical properties of the cell wall.
Uridine 5′-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia1
Authors: Santoso Djoko , Thornburg Robert
Published: July 2002

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5′-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase inN. plumbaginifolia cells.
Photoperiod Control of Gibberellin Levels and Flowering in Sorghum1
Authors: Lee In-Jung , Foster Kenneth R. , Morgan Page W.
Published: July 2002

Regulation of rhythmic peaks in levels of endogenous gibberellins (GAs) by photoperiod was studied in the short-day monocot sorghum (Sorghum bicolor [L.] Moench). Comparisons were made between three maturity (Ma) genotypes: 58M (Ma1Ma1,Ma2Ma2,phyB-1phyB-1, andMa4Ma4 [a phytochrome B null mutant]); 90M (Ma1Ma1,Ma2Ma2,phyB-2phyB-2, andMa4Ma4); and 100M (Ma1Ma1,Ma2Ma2,PHYBPHYB, andMa4Ma4). Plants were grown for 14 d under 10-, 14-, 16-, 18-, and 20-h photoperiods, and GA levels were assayed by gas chromatography-mass spectrometry every 3 h for 24 h. Under inductive 10-h photoperiods, the peak of GA20 and GA1 levels in 90M and 100M was shifted from midday, observed earlier with 12-h photoperiods, to an early morning peak, and flowering was hastened. In addition, the early morning peaks in levels of GA20 and GA1 in 58M under conditions allowing early flowering (10-, 12-, and 14-h photoperiods) were shifted to midday by noninductive (18- and 20-h) photoperiods, and flowering was delayed. These results are consistent with the possibility that the diurnal rhythm of GA levels plays a role in floral initiation and may be one way by which the absence of phytochrome B causes early flowering in 58M under most photoperiods.
Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells
Authors: Zimmermann Sabine , Talke Ina , Ehrhardt Thomas , Nast Gabriele , Müller-Röber Bernd
Published: July 2002

A cDNA encoding a novel, inwardly rectifying K+(K+in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K+in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda)cells, SKT1 was identified as a K+in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs+, when applied externally, inhibited SKT1-mediated K+incurrents half-maximally with an inhibitor concentration (IC50) of 105 μm. An almost identical high Cs+ sensitivity (IC50 = 90 μm) was found for the potato guard-cell K+in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K+inchannels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.
Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1
Authors: Pflugmacher Stephan , Sandermann Heinrich
Published: July 2002

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phylaChlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants.
Pathogen-Induced Changes in the Antioxidant Status of the Apoplast in Barley Leaves
Authors: Vanacker Hélène , Carver Tim L.W. , Foyer Christine H.
Published: July 2002

Leaves of two barley (Hordeum vulgare L.) isolines, Alg-R, which has the dominantMla1 allele conferring hypersensitive race-specific resistance to avirulent races of Blumeria graminis, and Alg-S, which has the recessive mla1 allele for susceptibility to attack, were inoculated with B. graminis f. sp. hordei. Total leaf and apoplastic antioxidants were measured 24 h after inoculation when maximum numbers of attacked cells showed hypersensitive death in Alg-R. Cytoplasmic contamination of the apoplastic extracts, judged by the marker enzyme glucose-6-phosphate dehydrogenase, was very low (less than 2%) even in inoculated plants. Dehydroascorbate, glutathione, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase were present in the apoplast. Inoculation had no effect on the total foliar ascorbate pool size or the redox state. The glutathione content of Alg-S leaves and apoplast decreased, whereas that of Alg-R leaves and apoplast increased after pathogen attack, but the redox state was unchanged in both cases. Large increases in foliar catalase activity were observed in Alg-S but not in Alg-R leaves. Pathogen-induced increases in the apoplastic antioxidant enzyme activities were observed. We conclude that sustained oxidation does not occur and that differential strategies of antioxidant response in Alg-S and Alg-R may contribute to pathogen sensitivity.
Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1
Authors: Ransom Wendy D. , Lao Pao-Chi , Gage Douglas A. , Boss Wendy F.
Published: July 2002

Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carotaL.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell.
Molecular and Biochemical Characterization of Cytosolic Phosphoglucomutase in Maize1
Authors: Manjunath Sivalinganna , Kenneth Lee Chien-Hsing , Winkle Patrick Van , Bailey-Serres Julia
Published: July 2002

Phosphoglucomutase (PGM) catalyzes the interconversion of glucose (Glc)-1- and Glc-6-phosphate in the synthesis and consumption of sucrose. We isolated two maize (Zea mays L.) cDNAs that encode PGM with 98.5% identity in their deduced amino acid sequence. Southern-blot analysis with genomic DNA from lines with different Pgm1 and Pgm2genotypes suggested that the cDNAs encode the two known cytosolic PGM isozymes, PGM1 and PGM2. The cytosolic PGMs of maize are distinct from a plastidic PGM of spinach (Spinacia oleracea). The deduced amino acid sequences of the cytosolic PGMs contain the conserved phosphate-transfer catalytic center and the metal-ion-binding site of known prokaryotic and eukaryotic PGMs. PGM mRNA was detectable by RNA-blot analysis in all tissues and organs examined except silk. A reduction in PGM mRNA accumulation was detected in roots deprived of O2 for 24 h, along with reduced synthesis of a PGM identified as a 67-kD phosphoprotein on two-dimensional gels. Therefore, PGM is not one of the so-called “anaerobic polypeptides.” Nevertheless, the specific activity of PGM was not significantly affected in roots deprived of O2 for 24 h. We propose that PGM is a stable protein and that existing levels are sufficient to maintain the flux of Glc-1-phosphate into glycolysis under O2 deprivation.
Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms1
Authors: Shannon Jack C. , Pien Fang-Mei , Cao Heping , Liu Kang-Chien
Published: July 2002

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.
Transcription from Heterologous rRNA Operon Promoters in Chloroplasts Reveals Requirement for Specific Activating Factors1
Authors: Sriraman Priya , Silhavy Daniel , Maliga Pal
Published: July 2002

The plastid rRNA (rrn) operon in chloroplasts of tobacco (Nicotiana tabacum), maize, and pea is transcribed by the plastid-encoded plastid RNA polymerase from a ς70-type promoter (P1). In contrast, therrn operon in spinach (Spinacia oleracea) and mustard chloroplasts is transcribed from the distinct Pc promoter, probably also by the plastid-encoded plastid RNA polymerase. Primer-extension analysis reported here indicates that in Arabidopsis both promoters may be active. To understand promoter selection in the plastid rrn operon in the different species, we have tested transcription from the spinach rrn promoter in transplastomic tobacco and from the tobacco rrn promoter in transplastomic Arabidopsis. Our data suggest that transcription of the rrn operon depends on species-specific factors that facilitate transcription initiation by the general transcription machinery.
Alterations in the Cytoskeleton Accompany Aluminum-Induced Growth Inhibition and Morphological Changes in Primary Roots of Maize1
Authors: Blancaflor Elison B. , Jones David L. , Gilroy Simon
Published: July 2002

Although Al is one of the major factors limiting crop production, the mechanisms of toxicity remain unknown. The growth inhibition and swelling of roots associated with Al exposure suggest that the cytoskeleton may be a target of Al toxicity. Using indirect immunofluorescence microscopy, microtubules and microfilaments in maize (Zea maysL.) roots were visualized and changes in their organization and stability correlated with the symptoms of Al toxicity. Growth studies showed that the site of Al toxicity was associated with the elongation zone. Within this region, Al resulted in a reorganization of microtubules in the inner cortex. However, the orientation of microtubules in the outer cortex and epidermis remained unchanged even after chronic symptoms of toxicity were manifest. Auxin-induced reorientation and cold-induced depolymerization of microtubules in the outer cortex were blocked by Al pretreatment. These results suggest that Al increased the stability of microtubules in these cells. The stabilizing effect of Al in the outer cortex coincided with growth inhibition. Reoriented microfilaments were also observed in Al-treated roots, and Al pretreatment minimized cytochalasin B-induced microfilament fragmentation. These data show that reorganization and stabilization of the cytoskeleton are closely associated with Al toxicity in maize roots.
Inhibition of Wound-Induced Accumulation of Allene Oxide Synthase Transcripts in Flax Leaves by Aspirin and Salicylic Acid
Authors: Harms Karsten , Ramirez Ingrid , Peña-Cortés Hugo
Published: July 2002

Allene oxide synthase (AOS) mediates the conversion of lipoxygenase-derived fatty acid hydroperoxides to unstable allene epoxides, which supply the precursors for the synthesis of the phytohormone jasmonic acid (JA). In this study the characterization of AOS gene expression in flax (Linum usitatissimum) is reported. AOS was constitutively expressed in different organs of flax plants. Additionally, AOS gene expression was enhanced after mechanical wounding in both the directly damaged leaves and in the systemic tissue located distal to the treated leaves. This wound-induced accumulation of AOS required the de novo biosynthesis of other unknown proteins involved in the signaling pathway modulating wound-induced AOS gene expression. Furthermore, the wound-induced AOS mRNA accumulation was correlated with the increase in the levels of JA. Both JA and its precursor, 12-oxo-phytodienoic acid, activated AOS gene expression in a dose-dependent manner. Thus, JA could activate its own biosynthetic pathway in flax leaves. Moreover, neither salicylic acid (SA) nor aspirin influenced AOS enzymatic activity. It is interesting that pretreatment with SA or aspirin inhibited wound-induced accumulation of AOS transcripts. These results suggest that a potent inhibition of JA biosynthetic capacity in leaves can be affected by SA or aspirin at the level of AOS gene expression.
Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce
Authors: Bestwick Charles S. , Brown Ian R. , Mansfield John W.
Published: July 2002

Peroxidase activity was characterized in lettuce (Lactuca sativaL.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to anhrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s.pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H2O2 in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.
Directional Guidance of Nicotiana alataPollen Tubes in Vitro and on the Stigma
Authors: Lush W. Mary , Grieser Franz , Wolters-Arts Mieke
Published: July 2002

Pollen tubes navigate the route from stigma to ovule with great accuracy, but the cues that guide them along this route are not known. We reproduced the environment on the stigma of Nicotiana alata by immersing pollen in stigma exudate or oil close to an interface with an aqueous medium. The growth of pollen in this culture system mimicked growth on stigmas: pollen grains hydrated and germinated, and pollen tubes grew toward the aqueous medium. The rate-limiting step in pollen germination was the movement of water through the surrounding exudate or oil. By elimination of other potential guidance cues, we conclude that the directional supply of water probably determined the axis of polarity of pollen tubes and resulted in growth toward the interface. We propose that a gradient of water in exudate is a guidance cue for pollen tubes on the stigma and that the composition of the exudate must be such that it is permeable enough for pollen hydration to occur but not so permeable that the supply of water becomes nondirectional. Pollen tube penetration of the stigma may be the most frequently occurring hydrotropic response of higher plants.
Estimating the Excess Investment in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Leaves of Spring Wheat Grown under Elevated CO2
Authors: Theobald Julian C. , Mitchell Rowan A.C. , Parry Martin A.J. , Lawlor David W.
Published: July 2002

Wheat (Triticum aestivum L.) was grown under CO2 partial pressures of 36 and 70 Pa with two N-application regimes. Responses of photosynthesis to varying CO2 partial pressure were fitted to estimate the maximal carboxylation rate and the nonphotorespiratory respiration rate in flag and preceding leaves. The maximal carboxylation rate was proportional to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content, and the light-saturated photosynthetic rate at 70 Pa CO2 was proportional to the thylakoid ATP-synthase content. Potential photosynthetic rates at 70 Pa CO2 were calculated and compared with the observed values to estimate excess investment in Rubisco. The excess was greater in leaves grown with high N application than in those grown with low N application and declined as the leaves senesced. The fraction of Rubisco that was estimated to be in excess was strongly dependent on leaf N content, increasing from approximately 5% in leaves with 1 g N m−2 to approximately 40% in leaves with 2 g N m−2. Growth at elevated CO2 usually decreased the excess somewhat but only as a consequence of a general reduction in leaf N, since relationships between the amount of components and N content were unaffected by CO2. We conclude that there is scope for improving the N-use efficiency of C3 crop species under elevated CO2 conditions.
Tracheary Element Differentiation Uses a Novel Mechanism Coordinating Programmed Cell Death and Secondary Cell Wall Synthesis1
Authors: Groover Andrew , Jones Alan M.
Published: July 2002

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca2+ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca2+ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.
Arabidopsis ent-Kaurene Oxidase Catalyzes Three Steps of Gibberellin Biosynthesis
Authors: Helliwell Chris A. , Poole Andrew , James Peacock W. , Dennis Elizabeth S.
Published: July 2002

The Arabidopsis GA3cDNA was expressed in yeast (Saccharomyces cerevisiae) and the ability of the transformed yeast cells to metabolizeent-kaurene was tested. We show by full-scan gas chromatography-mass spectrometry that the transformed cells produceent-kaurenoic acid, and demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene toent-kaurenoic acid.
Proline Accumulation in Maize (Zea maysL.) Primary Roots at Low Water Potentials. II. Metabolic Source of Increased Proline Deposition in the Elongation Zone1
Authors: Verslues Paul E. , Sharp Robert E.
Published: July 2002

The proline (Pro) concentration increases greatly in the growing region of maize (Zea mays L.) primary roots at low water potentials (ψw), largely as a result of an increased net rate of Pro deposition. Labeled glutamate (Glu), ornithine (Orn), or Pro was supplied specifically to the root tip of intact seedlings in solution culture at high and low ψw to assess the relative importance of Pro synthesis, catabolism, utilization, and transport in root-tip Pro deposition. Labeling with [3H]Glu indicated that Pro synthesis from Glu did not increase substantially at low ψw and accounted for only a small fraction of the Pro deposition. Labeling with [14C]Orn showed that Pro synthesis from Orn also could not be a substantial contributor to Pro deposition. Labeling with [3H]Pro indicated that neither Pro catabolism nor utilization in the root tip was decreased at low ψw. Pro catabolism occurred at least as rapidly as Pro synthesis from Glu. There was, however, an increase in Pro uptake at low ψw, which suggests increased Pro transport. Taken together, the data indicate that increased transport of Pro to the root tip serves as the source of low-ψw-induced Pro accumulation. The possible significance of Pro catabolism in sustaining root growth at low ψw is also discussed.
Salicylic Acid Induces Rapid Inhibition of Mitochondrial Electron Transport and Oxidative Phosphorylation in Tobacco Cells1
Authors: Xie Zhixin , Chen Zhixiang
Published: July 2002

Salicylic acid (SA) is known to induce alternative pathway respiration by activating expression of the alternative oxidase gene. In the present study we report a rapid mode of action by SA on plant mitochondrial functions. SA at concentrations as low as 20 μm induced inhibition of both ATP synthesis and respiratory O2 uptake within minutes of incubation in tobacco (Nicotiana tabacum) cell cultures. Biologically active SA analogs capable of inducing pathogenesis-related genes and enhanced resistance also caused rapid inhibition of ATP synthesis and respiratory O2 uptake, whereas biologically inactive analogs did not. Inhibition of ATP synthesis and respiratory O2 uptake by SA was insensitive to the protein synthesis inhibitor cycloheximide, but was substantially reduced by the antioxidant N-acetylcysteine, suggesting a possible role for reactive oxygen species in the inhibition of mitochondrial functions. With exogenous NADH as the respiratory substrate, mitochondria isolated from SA-treated tobacco cell cultures were found to have normal capacities for both ATP synthesis and respiratory O2 uptake; direct incubation of isolated mitochondria with SA had no significant effect on these mitochondrial functions. These results indicate that (a) the respiration capacities of isolated mitochondria do not correspond to the in vivo respiration activities in SA-treated cell cultures and (b) the SA-induced inhibition of respiration in tobacco cell cultures may involve other components that are not present in isolated mitochondria. Given the recently demonstrated roles of mitochondria in plant disease resistance and animal apoptosis, this rapid inhibition by SA of mitochondrial functions may play a role in SA-mediated biological processes, including plant defense responses.
Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia coliViability under Heat and Cold Stress1
Authors: Soto Alvaro , Allona Isabel , Collada Carmen , Guevara Maria-Angeles , Casado Rosa , Rodriguez-Cerezo Emilio , Aragoncillo Cipriano , Gomez Luis
Published: July 2002

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress.
Tobacco O-Methyltransferases Involved in Phenylpropanoid Metabolism. The Different Caffeoyl-Coenzyme A/5-Hydroxyferuloyl-Coenzyme A 3/5-O-Methyltransferase and Caffeic Acid/5-Hydroxyferulic Acid 3/5-O-Methyltransferase Classes Have Distinct Substrate Specificities and Expression Patterns
Authors: Maury Stéphane , Geoffroy Pierrette , Legrand Michel
Published: July 2002

The biosynthesis of lignin monomers involves two methylation steps catalyzed by orthodiphenol-O-methyltransferases: caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferases (COMTs) and caffeoyl-coenzyme A (CoA)/5-hydroxyferuloyl-CoA 3/5-O-methyltransferases (CCoAOMTs). Two COMT classes (I and II) were already known to occur in tobacco (Nicotiana tabacum) and three distinct CCoAOMT classes have now been characterized. These three CCoAOMT classes displayed a maximum level of expression at different stages of stem development, in accordance with their involvement in the synthesis of lignin guaiacyl units. Expression profiles upon tobacco mosaic virus infection of tobacco leaves revealed a biphasic pattern of induction for COMT I, COMT II, and CCoAOMTs. The different isoforms were expressed inEscherichia coli and our results showed that CCoAOMTs and, more surprisingly, COMTs efficiently methylated hydroxycinnamoyl-CoA esters. COMT I was also active toward 5-hydroxyconiferyl alcohol, indicating that COMT I that catalyzes syringyl unit synthesis in planta may operate at the free acid, CoA ester, or alcohol levels. COMT II that is highly inducible by infection also accepted caffeoyl-CoA as a substrate, thus suggesting a role in ferulate derivative deposition in the walls of infected cells. Tobacco appears to possess an array of O-methyltransferase isoforms with variable efficiency toward the diverse planto-diphenolic substrates.
Rates of Sugar Uptake by Guard Cell Protoplasts ofPisum sativumL. Related to the Solute Requirement for Stomatal Opening
Authors: Ritte Gerhard , Rosenfeld Johanna , Rohrig Kerstin , Raschke Klaus
Published: July 2002

We wished to determine whether the capacity of the sugar uptake mechanisms of guard cells of the Argenteum mutant of pea (Pisum sativum L.) sufficed to support a concurrent stomatal opening movement. Sugar uptake by guard cell protoplasts was determined by silicone-oil-filtering centrifugation. The protoplasts took up [14C]glucose, [14C]fructose, and [14C]sucrose (Suc), apparently in symport with protons. Mannose, galactose, and fructose competed with Glc for transport by a presumed hexose carrier. The uptake of Glc saturated with aK  m of 0.12 mm and aV  max of 19 fmol cell−1h−1. At external concentrations <1 mm, the uptake of Suc was slower than that of Glc. It exhibited a saturating component with a K  m varying between 0.25 and 0.8 mm and a V  max between 1 and 10 fmol cell−1 h−1, and at external concentrations >1 mm, a non-saturating component. At apoplastic sugar concentrations below 4 mm, sugar import was estimated to be mainly in the form of hexoses and too slow to support a simultaneous stomatal opening movement. If, however, during times of high photosynthesis and transpiration, the apoplastic Suc concentration rose and entered the range of non-saturating import, absorbed Suc could replace potassium malate as the osmoticum for the maintenance of stomatal opening.
AXR1 Acts after Lateral Bud Formation to Inhibit Lateral Bud Growth in Arabidopsis
Authors: Stirnberg Petra , Chatfield Steven P. , Leyser H.M. Ottoline
Published: July 2002

The AXR1 gene of Arabidopsis is required for many auxin responses. The highly branched shoot phenotype of mature axr1 mutant plants has been taken as genetic evidence for a role of auxin in the control of shoot branching. We compared the development of lateral shoots in wild-type Columbia and axr1-12 plants. In the wild type, the pattern of lateral shoot development depends on the developmental stage of the plant. During prolonged vegetative growth, axillary shoots arise and develop in a basal-apical sequence. After floral transition, axillary shoots arise rapidly along the primary shoot axis and grow out to form lateral inflorescences in an apical-basal sequence. For both patterns, the axr1mutation does not affect the timing of axillary meristem formation; however, subsequent lateral shoot development proceeds more rapidly inaxr1 plants. The outgrowth of lateral inflorescences from excised cauline nodes of wild-type plants is inhibited by apical auxin. axr1-12 nodes are resistant to this inhibition. These results provide evidence for common control of axillary growth in both patterns, and suggest a role for auxin during the late stages of axillary shoot development following the formation of the axillary bud and several axillary leaf primordia.
Stop-and-Go Movements of Plant Golgi Stacks Are Mediated by the Acto-Myosin System
Authors: Nebenführ Andreas , Gallagher Larry A. , Dunahay Terri G. , Frohlick Jennifer A. , Mazurkiewicz Anna M. , Meehl Janet B. , Staehelin L. Andrew
Published: July 2002

The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm. To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein α-1,2 mannosidase I. The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal α-1,2 mannosidases. Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots. Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks. In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random “wiggling.” Directed movement (maximal velocity 4.2 μm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion. In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior. We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by “stop signals” produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.
Wheat Granule-Bound Starch Synthase I and II Are Encoded by Separate Genes That Are Expressed in Different Tissues
Authors: Vrinten Patricia L. , Nakamura Toshiki
Published: July 2002

Studies of waxy mutations in wheat and other cereals have shown that null mutations in genes encoding granule-bound starch synthase I (GBSSI) result in amylose-free starch in endosperm and pollen grains, whereas starch in other tissues may contain amylose. We have isolated a cDNA from waxy wheat that encodes GBSSII, which is thought to be responsible for the elongation of amylose chains in non-storage tissues. The deduced amino acid sequences of wheat GBSSI and GBSSII were almost 66% identical, while those of wheat GBSSII and potato GBSSI were 72% identical. GBSSIIwas expressed in leaf, culm, and pericarp tissue, but transcripts were not detected in endosperm tissue. In contrast, GBSSIexpression was high in endosperm tissue. The expression ofGBSSII mRNA in pericarp tissue was similar at the midpoints of the day and night periods. The GBSSII genes were mapped to chromosomes 2AL, 2B, and 2D, whereasGBSSI genes are located on group 7 chromosomes. Gel-blot analysis indicated that genes related to GBSSII also occur in barley, rice, and maize. The possible role ofGBSSII in starch synthesis is discussed.
Regulation of Sulfate Assimilation by Nitrogen in Arabidopsis
Authors: Koprivova Anna , Suter Marianne , den Camp Roel Op , Brunold Christian , Kopriva Stanislav
Published: July 2002

Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5′-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mm each of NO3  −, NH4  +, or glutamine (Gln), or 1 mm  O-acetylserine (OAS). 35SO4  2− feeding showed that after addition of NH4  +, Gln, or OAS to nitrogen-starved plants, incorporation of 35S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.