bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis of Costunolide, Dihydrocostunolide, and Leucodin. Demonstration of Cytochrome P450-Catalyzed Formation of the Lactone Ring Present in Sesquiterpene Lactones of Chicory
Authors: de Kraker Jan-Willem , Franssen Maurice C.R. , Joerink Maaike , de Groot Aede , Bouwmeester Harro J.
Published: July 2002

Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.
The Interaction of Light and Abscisic Acid in the Regulation of Plant Gene Expression
Authors: Weatherwax S. C. , Ong M. S. , Degenhardt J. , Bray E. A. , Tobin E. M.
Published: July 2002

Extended dark treatments of light-grown plants of both Lemna gibba and Arabidopsis thaliana resulted in substantial increases in abscisic acid (ABA) concentrations. The concentration of ABA could be negatively regulated by phytochrome action in Lemna. As has been noted in other species, ABA treatment reduced Lemna rbcS and Lhcb RNA levels, which are positively regulated by phytochrome in many species. In view of these observations, the possibility that phytochrome effects on gene expression may be mediated primarily by changes in ABA was tested using a transient assay in intact plants. The phytochrome responsiveness of the Lemna Lhcb2*1 promoter was still apparent in the presence of exogenous ABA. Additionally, when 2-bp mutations were introduced into this promoter so that phytochrome responsiveness was lost, a response to exogenous ABA was still present. We conclude that phytochrome- and ABA-response elements are separable in the Lhcb2*1 promoter. We tested whether the effects of ABA on RNA abundance could be inhibited by treatment with gibberellin and found no evidence for such an inhibition. We have also found that the ABA-responsive Em promoter of wheat can be negatively regulated by phytochrome action. It is likely that this regulation is mediated at least in part by phytochrome-induced changes in ABA levels. Our results demonstrate that it is essential to take into account that dark treatments and the phytochrome system can affect ABA levels when interpreting studies of light-regulated genes.
Expressed Sequence Tags of Chinese Cabbage Flower Bud cDNA
Authors: Lim C. O. , Kim H. Y. , Kim M. G. , Lee S. I. , Chung W. S. , Park S. H. , Hwang I. , Cho M. J.
Published: July 2002

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.
Phytochrome A Enhances the Promotion of Hypocotyl Growth Caused by Reductions in Levels of Phytochrome B in Its Far-Red-Light-Absorbing Form in Light-Grown Arabidopsis thaliana
Authors: Casal J. J.
Published: July 2002

We sought to determine if phytochrome B (phyB)-mediated responses to the red light (R)/far-red light (FR) ratio are affected by phytochrome A (phyA) activity in light-grown seedlings of Arabidopsis thaliana. Pulses of FR delayed into the dark period were less effective than end-of-day (EOD) FR in promoting hypocotyl growth over a given period in darkness. White light minus blue light interposed instead of darkness between the end of the white-light photoperiod and the FR pulse was sufficient to maintain responsivity to the decrease in phyB in FR-light-absorbing form in wild-type (WT) seedlings, but not in the phyA mutant. Compared with EOD R, hourly R+FR pulses provided throughout the night caused a stronger promotion of stem growth than a single EOD R+FR pulse in WT Arabidopsis, cucumber, mustard, sunflower, tobacco, and tomato, but not in phyA Arabidopsis or in the aurea mutant of tomato. WT seedlings of Arabidopsis responded to a range of high EOD R/FR ratios, whereas the phyA mutant required stronger reductions in the EOD R/FR ratio. In sunlight, phyA seedlings of Arabidopsis showed no response to the “early warning” signals of neighboring vegetation, and hypocotyl-growth promotion occurred at higher plant densities than in the WT. Thus, under a series of light conditions, the sensitivity or responsivity to reductions in the R/FR ratio were larger in WT than in phyA seedlings. A product of phyA is therefore proposed to enhance the hypocotyl-growth response to decreases in phyB in FR-light-absorbing form in light-grown seedlings.
Expansins in Deepwater Rice Internodes
Authors: Cho H. T. , Kende H.
Published: July 2002

Cell walls of deepwater rice (Oryza sativa L.) internodes undergo long-term extension (creep) when placed under tension in acidic buffers. This is indicative of the action of the cell wall-loosening protein expansin. Wall extension had a pH optimum of around 4.0 and was abolished by boiling. Acid-induced extension of boiled cell walls could be reconstituted by addition of salt-extracted rice or cucumber cell wall proteins. Cucumber expansin antibody recognized a single protein band of 24.5-kD apparent molecular mass on immunoblots of rice cell wall proteins. Expansins were partially purified by concanavalin A affinity chromatography and sulfopropyl (SP) cation-exchange chromatography. The latter yielded two peaks with extension activity (SP20 and SP29), and immunoblot analysis showed that both of these active fractions contained expansin of 24.5-kD molecular mass. The N-terminal amino acid sequence of SP20 expansin is identical to that deduced from the rice expansin cDNA Os-EXP1. The N-terminal amino acid sequence of SP29 expansin matches that deduced from the rice expansin cDNA Os-EXP2 in six of eight amino acids. Our results show that two expansins occur in the cell walls of rice internodes and that they may mediate acid-induced wall extension.
Molecular Cloning of Mannose-6-Phosphate Reductase and Its Developmental Expression in Celery
Authors: Everard J. D. , Cantini C. , Grumet R. , Plummer J. , Loescher W. H.
Published: July 2002

Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.
Effects of a Mechanical Stimulation on Localization of Annexin-Like Proteins in Bryonia dioica Internodes
Authors: Thonat C. , Mathieu C. , Crevecoeur M. , Penel C. , Gaspar T. , Boyer N.
Published: July 2002

Mechanical stimulation exerted by rubbing a young internode of Bryonia dioica plants inhibits its growth. Previous cellular and biochemical studies showed that this growth inhibition is associated with Ca2+ redistribution and profound modifications of plasma membrane characteristics. We extracted and purified Ca2+-dependent phospholipid-binding proteins from B. dioica internodes. Two main proteins, p33 and p35, and other minor bands were isolated and identified as annexin-like proteins because of their biochemical properties and their cross-reactions with antibodies against maize (Zea mays L.) annexins. Rabbit antiserum was obtained by injection of B. dioica p35. This antiserum was used for the immunocytolocalization of annexin-like proteins in internode parenchyma cells. It appeared that the distribution of annexin-like proteins was different before and 30 min after the mechanical stimulation. Western analysis of proteins in membrane fractions after separation by free-flow electrophoresis showed that p35 was present in most fractions, whereas p33 appeared mainly in plasmalemma-enriched fractions after the mechanical stimulation. It is hypothesized that a subcellular redistribution of these proteins might be involved in growth inhibition by mechanical stress.
Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Release across Nonvacuolar Membranes in Cauliflower
Authors: Muir S. R. , Sanders D.
Published: July 2002

Previous studies have indicated that the vacuole represents the major inositol 1,4,5-trisphosphate (InsP3)-mobilizable Ca2+ pool in higher plants. This finding is in contrast to animal cells, in which the endoplasmic reticulum and plasma membrane constitute the dominant InsP3-sensitive membranes. We used membrane vesicles prepared from cauliflower (Brassica oleracae L.) inflorescences that were separated on continuous sucrose gradients to demonstrate that cauliflower possesses at least two distinct membrane populations that are sensitive to InsP3. One of these membrane populations is nonvacuolar in origin and relies upon a Ca2+-ATPase to accumulate CA2+. In addition, we have shown that two polyclonal antibodies, raised against peptides corresponding to the animal type 1 InsP3 receptor, recognize immunologically related proteins in cauliflower, and that the distribution of immunoreactive proteins on a linear sucrose gradient reinforces the notion that cauliflower contains more than one membrane subtype that is sensitive to InsP3. To our knowledge, this is the first report describing an InsP3-sensitive Ca2+ store other than the vacuole in higher plant cells.
A Vacuole-Associated Annexin Protein, VCaB42, Correlates with the Expansion of Tobacco Cells
Authors: Seals D. F. , Randall S. K.
Published: July 2002

A Ca-dependent membrane-binding protein of the annexin family, VCaB42, has previously been shown to associate with vacuolar vesicles at physiological levels of Ca. In this study we used suspension-cultured cells of tobacco (Nicotiana tabacum BY-2) to show that VCaB42 is enriched 4.5-fold in intact vacuoles, whereas evacuolated protoplasts show a 12-fold reduction in VCaB42. VCaB42 distribution is thus comparable to that of the vacuole-associated H+-ATPase but is distinct from the endoplasmic reticulum-localized protein calnexin. Because VCaB42 is a vacuole-associated annexin, and given the putative function of annexins in vesicle fusion, we hypothesize a role for this protein in the vacuolation process of expanding cells. Consistent with this hypothesis, we show that VCaB42 levels correlate with age-associated and hormonally induced changes in cell volume in tobacco suspension cultures. The association of VCaB42 with vacuoles and its correlative pattern of expression relative to the expansion of cells is consistent with a possible role for VCaB42 in the early events of vacuole biogenesis.
Anion Channels and the Stimulation of Anthocyanin Accumulation by Blue Light in Arabidopsis Seedlings1
Authors: Noh Bosl , Spalding Edgar P.
Published: July 2002

Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels inArabidopsis thaliana. Here we show that 20 μm NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels ofPAL1, CHS, CHI, orDFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes, but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.
N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids1
Authors: Chapman Kent D. , Tripathy Swati , Venables Barney , Desouza Arland D.
Published: July 2002

Recently, the biosynthesis of an unusual membrane phospholipid,N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacumL.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120–126). Here we report that before induction of NAPE biosynthesis,N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [14C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [14C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species,N-lauroylethanolamine andN-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133–142) raises the possibility that a similar mechanism may operate in plant cells.
Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4Gene1
Authors: Williams Jacqueline , Phillips Andy L. , Gaskin Paul , Hedden Peter
Published: July 2002

cDNA corresponding to the GA4 gene ofArabidopsisthaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming thatGA4 encodes a GA 3β-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 μmcompared with 15 μm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3β-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA9 and GA6.
Metallothioneins 1 and 2 Have Distinct but Overlapping Expression Patterns in Arabidopsis
Authors: Garcı́a-Hernández Margarita , Murphy Angus , Taiz Lincoln
Published: July 2002

The spatial and temporal expression patterns of metallothionein (MT) isoforms MT1a andMT2a were investigated in vegetative and reproductive tissues of untreated and copper-treated Arabidopsis by in situ hybridization and by northern blotting. In control plants,MT1a mRNA was localized in leaf trichomes and in the vascular tissue in leaves, roots, flowers, and germinating embryos. In copper-treated plants, MT1a expression was also observed in the leaf mesophyll and in vascular tissue of developing siliques and seeds. In contrast, MT2a was expressed primarily in the trichomes of both untreated and copper-treated plants. In copper-treated plants, MT2a mRNA was also expressed in siliques. Northern-hybridization studies performed on developing seedlings and leaves showed temporal variations of MT1agene expression but not of MT2a expression. The possible implications of these findings for the cellular roles of MTs in plants are discussed.
Inhibition of the Gravitropic Response of Snapdragon Spikes by the Calcium-Channel Blocker Lanthanum Chloride
Authors: Friedman Haya , Meir Shimon , Rosenberger Ida , Halevy Abraham H. , Kaufman Peter B. , Philosoph-Hadas Sonia
Published: July 2002

The putative Ca2+-channel blocker LaCl3 prevented the gravitropic bending of cut snapdragon (Antirrhinum majus L.) spikes (S. Philosoph-Hadas, S. Meir, I. Rosenberger, A.H. Halevy [1996] Plant Physiol 110: 301–310) and inhibited stem curvature to a greater extent than vertical and horizontal stem elongation at the bending zone. This might indicate that LaCl3, which modulates cytosolic Ca2+, does not influence general stem-growth processes but may specifically affect other gravity-associated processes occurring at the stem-bending zone. Two such specific gravity-dependent events were found to occur in the bending zone of snapdragon spikes: sedimentation of starch-containing chloroplasts at the bottom of stem cortex cells, as seen in cross-sections, and establishment of an ethylene gradient across the stem. Our results show that the lateral sedimentation of chloroplasts associated with gravity sensing was prevented in cross-sections taken from the bending zone of LaCl3-treated and subsequently gravistimulated spikes and that LaCl3completely prevented the gravity-induced, asymmetric ethylene production established across the stem-bending zone. These data indicate that LaCl3 inhibits stem curvature of snapdragon spikes by preventing several gravity-dependent processes. Therefore, we propose that the gravitropic response of shoots could be mediated through a Ca2+-dependent pathway involving modulation of cytosolic Ca2+ at various stages.
Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen
Authors: Buitink Julia , Claessens Mireille M.A.E. , Hemminga Marcus A. , Hoekstra Folkert A.
Published: July 2002

Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativumL.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10−3 s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2Azz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2Azzmeasurements can provide an estimate of the optimum storage conditions.
Carbonic Anhydrase Activity and CO2-Transfer Resistance in Zn-Deficient Rice Leaves
Authors: Sasaki Haruto , Hirose Tatsuro , Watanabe Yoshito , Ohsugi Ryu
Published: July 2002

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativaL.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO2 fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO2-assimilation process increased from 10% to 21%. This change led to a reduced CO2concentration at the site of CO2 fixation, resulting in an increased gradient of CO2 between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO2 from the stomatal cavity to the site of CO2 fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.
Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves
Authors: Vander Peter , Vårum Kjell M. , Domard Alain , Eddine El Gueddari Nour , Moerschbacher Bruno M.
Published: July 2002

Chitin, a linear polysaccharide composed of (1→4)-linked 2-acetamido-2-deoxy-β-d-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1→4)-linked GlcNAc and 2-amino-2-deoxy-β-d-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1→4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partiallyN-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1→4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1→4)-linked GlcNAc with a degree of polymerization ≥ 7 strongly elicited POD activities but not PAL activities. PartiallyN-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.
Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic AcidO-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping1
Authors: Lapierre Catherine , Pollet Brigitte , Petit-Conil Michel , Toval Gabriel , Romero Javier , Pilate Gilles , Leplé Jean-Charles , Boerjan Wout , Ferret Valérie , De Nadai Véronique , Jouanin Lise
Published: July 2002

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acidO-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD orCOMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.
Temporal and Spatial Patterns of Accumulation of the Transcript of Myo-Inositol-1-Phosphate Synthase and Phytin-Containing Particles during Seed Development in Rice1
Authors: Yoshida Kaoru T. , Wada Tomikichi , Koyama Hiroshi , Mizobuchi-Fukuoka Ritsuko , Naito Satoshi
Published: July 2002

Myo-inositol-1-phosphate (I[1]P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis. Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds. We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants. Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers. In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA). Strong signals were detected in the scutellum and aleurone layer after 4 DAA. The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased. Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript. The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.
Antioxidative Defense System, Pigment Composition, and Photosynthetic Efficiency in Two Wheat Cultivars Subjected to Drought1
Authors: Loggini Barbara , Scartazza Andrea , Brugnoli Enrico , Navari-Izzo Flavia
Published: July 2002

We analyzed antioxidative defenses, photosynthesis, and pigments (especially xanthophyll-cycle components) in two wheat (Triticum durum Desf.) cultivars, Adamello and Ofanto, during dehydration and rehydration to determine the difference in their sensitivities to drought and to elucidate the role of different protective mechanisms against oxidative stress. Drought caused a more pronounced inhibition in growth and photosynthetic rates in the more sensitive cv Adamello compared with the relatively tolerant cv Ofanto. During dehydration the glutathione content decreased in both wheat cultivars, but only cv Adamello showed a significant increase in glutathione reductase and hydrogen peroxide-glutathione peroxidase activities. The activation states of two sulfhydryl-containing chloroplast enzymes, NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase, were maintained at control levels during dehydration and rehydration in both cultivars. This indicates that the defense systems involved are efficient in the protection of sulfhydryl groups against oxidation. Drought did not cause significant effects on lipid peroxidation. Upon dehydration, a decline in chlorophylla, lutein, neoxanthin, and β-carotene contents, and an increase in the pool of de-epoxidized xanthophyll-cycle components (i.e. zeaxanthin and antheraxanthin), were evident only in cv Adamello. Accordingly, after exposure to drought, cv Adamello showed a larger reduction in the actual photosystem II photochemical efficiency and a higher increase in nonradiative energy dissipation than cv Ofanto. Although differences in zeaxanthin content were not sufficient to explain the difference in drought tolerance between the two cultivars, zeaxanthin formation may be relevant in avoiding irreversible damage to photosystem II in the more sensitive cultivar.
Hypersensitive Cell Death and Papilla Formation in Barley Attacked by the Powdery Mildew Fungus Are Associated with Hydrogen Peroxide but Not with Salicylic Acid Accumulation1
Authors: Hückelhoven Ralph , Fodor József , Preis Christine , Kogel Karl-Heinz
Published: July 2002

We analyzed the pathogenesis-related generation of H2O2 using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp.hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death inMla12 and Mlg genotypes or after chemical activation by DCINA was associated with H2O2accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg andmlo5 genotypes and in DCINA-activated plants was paralleled by H2O2 accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H2O2 was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H2O2may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.
Long-Day Up-Regulation of a GAMYB Gene duringLolium temulentum Inflorescence Formation
Authors: Gocal Greg F.W. , Poole Andrew T. , Gubler Frank , Watts Robyn J. , Blundell Cheryl , King Rod W.
Published: July 2002

Long-day exposure of the grassLolium temulentum may regulate flowering via changes in gibberellin (GA) levels. Therefore, we have examined both GA levels and expression of a MYB transcription factor that is specific to the GA signal transduction pathway in monocots. ThisMYB gene from L. temulentum shows over 90% nucleotide identity with the barley and rice GAMYBgenes, and, like them, gibberellic acid (GA3) up-regulates its expression in the seed. Furthermore, cDNAs of both the barley andL. temulentum GAMYB show the same simple patterns of hybridization with digests of L. temulentum genomic DNA. Compared with vegetative shoot apices of L. temulentum, the in situ mRNA expression ofLtGAMYB does not change during the earliest steps of “floral” initiation at the apex. However, by 100 h (the double-ridge stage of flowering) its expression increased substantially and was highest in the terminal and lateral spikelet sites. Thereafter, expression declined overall but then increased within stamen primordia. Prior to increased LtGAMYBexpression, long-day exposure sufficient to induce flowering led to increased (5- to 20-fold) levels of GA1 and GA4in the leaf. Thus, increases first in GA level in the leaf followed by increased expression of LtGAMYB in the apex suggest important signaling and/or response roles in flowering.
Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels
Authors: Knutzon Deborah S. , Hayes Thomas R. , Wyrick Annette , Xiong Hui , Maelor Davies H. , Voelker Toni A.
Published: July 2002

Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 andsn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at thesn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels.
Supply of Fatty Acid Is One Limiting Factor in the Accumulation of Triacylglycerol in Developing Embryos1
Authors: Bao Xiaoming , Ohlrogge John
Published: July 2002

The metabolic factors that determine oil yield in seeds are still not well understood. To begin to examine the limits on triacylglycerol (TAG) production, developingCuphea lanceolata, Ulmus carpinifolia, and Ulmus parvifolia embryos were incubated with factors whose availability might limit oil accumulation. The addition of glycerol or sucrose did not significantly influence the rate of TAG synthesis. However, the rate of 14C-TAG synthesis upon addition of 2.1 mm14C-decanoic acid (10:0) was approximately four times higher than the in vivo rate of TAG accumulation in C. lanceolata and two times higher than the in vivo rate in U. carpinifolia andU. parvifolia. In C. lanceolata embryos, the highest rate of 14C-TAG synthesis (14.3 nmol h−1 embryo−1) was achieved with the addition of 3.6 mm decanoic acid. 14C-Decanoic acid was incorporated equally well in all three acyl positions of TAG. The results suggest that C. lanceolata, U. carpinifolia, and U. parvifolia embryos have sufficient acyltransferase activities and glycerol-3-phosphate levels to support rates of TAG synthesis in excess of those found in vivo. Consequently, the amount of TAG synthesized in these oilseeds may be in part determined by the amount of fatty acid produced in plastids.
Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes Due to Light/Dark Treatment
Authors: Benková Eva , Witters Erwin , Van Dongen Walter , Kolar Jan , Motyka Václav , Brzobohatý Bretislav , Van Onckelen Harry A. , Machácková Ivana
Published: July 2002

Although cytokinins (CKs) affect a number of processes connected with chloroplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both species contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5′-monophosphate, zeatin riboside-5′-monophosphate, and dihydrozeatin riboside-5′-monophosphate), and N-glucosides (zeatin-N  9-glucoside, dihydrozeatin-N  9-glucoside, zeatin-N  7-glucoside, and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level ofN  9-glucosides of zeatin and dihydrozeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism.
Brassinosteroid-Insensitive Dwarf Mutants of Arabidopsis Accumulate Brassinosteroids
Authors: Noguchi Takahiro , Fujioka Shozo , Choe Sunghwa , Takatsuto Suguru , Yoshida Shigeo , Yuan Heng , Feldmann Kenneth A. , Tax Frans E.
Published: July 2002

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles ofBRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However,bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore,BRI1 is required for the homeostasis of endogenous BR levels.
Early Copper-Induced Leakage of K+ from Arabidopsis Seedlings Is Mediated by Ion Channels and Coupled to Citrate Efflux
Authors: Murphy Angus S. , Eisinger William R. , Shaff Jon E. , Kochian Leon V. , Taiz Lincoln
Published: July 2002

Copper tolerance among Arabidopsis ecotypes is inversely correlated with long-term K+ leakage and positively correlated with short-term K+ leakage (A. Murphy, L. Taiz [1997] New Phytol 136: 211–222). To probe the mechanism of the early phase of K+ efflux, we tested various channel blockers on copper and peroxide-induced K+efflux from seedling roots. The K+ channel blockers tetraethyl ammonium chloride and 4-aminopyridine (4-AP) both inhibited short-term copper-induced K+ efflux. In contrast, peroxide-induced K+ efflux was insensitive to both tetraethyl ammonium chloride and 4-AP. Copper-induced lipid peroxidation exhibited a lag time of 4 h, while peroxide-induced lipid peroxidation began immediately. These results suggest that short-term copper-induced K+ efflux is mediated by channels, while peroxide-induced K+ efflux represents leakage through nonspecific lesions in the lipid bilayer. Tracer studies with 86Rb+ confirmed that copper promotes K+ efflux rather than inhibiting K+uptake. Short-term K+ release is electroneutral, since electrophysiological measurements indicated that copper does not cause membrane depolarization. Short-term K+ efflux was accompanied by citrate release, and copper increased total citrate levels. Since citrate efflux was blocked by 4-AP, K+appears to serve as a counterion during copper-induced citrate efflux. As copper but not aluminum selectively induces citrate production and release, it is proposed that copper may inhibit a cytosolic form of aconitase.
Nucleoside Diphosphate Kinase Required for Coleoptile Elongation in Rice
Authors: Pan Ling , Kawai Maki , Yano Akira , Uchimiya Hirofumi
Published: July 2002

Although several nucleoside diphosphate (NDP) kinase genes have been cloned in plants, little is known about the functional significance of this enzyme during plant growth and development. We introduced a chimeric gene encoding an antisense RNA of NDP kinase under the control of the Arabidopsis heat shock protein HSP81-1 promoter into rice (Oryza sativaL.) plants using the Agrobacterium tumefacienstransformation system. The expression of antisense RNA down-regulated the accumulation of mRNA, resulting in reduced enzyme activity even under the standard growth temperature (25°C) in transgenic plants. Following heat shock treatment (37°C), NDP kinase activities in some transgenic rice plants were more reduced than those grown under 25°C. The comparison of the coleoptile growth under submersion showed that cell elongation process was inhibited in antisense NDP kinase transgenic plants, suggesting that an altered guanine nucleotide level may be responsible for the processes.
The Genepat-2, Which Induces Natural Parthenocarpy, Alters the Gibberellin Content in Unpollinated Tomato Ovaries
Authors: Fos Mariano , Nuez Fernando , Garcı́a-Martı́nez José L.
Published: July 2002

We investigated the role of gibberellins (GAs) in the effect of pat-2, a recessive mutation that induces facultative parthenocarpic fruit development in tomato (Lycopersicon esculentum Mill.) using near-isogenic lines with two different genetic backgrounds. Unpollinated wild-type Madrigal (MA/wt) and Cuarenteno (CU/wt) ovaries degenerated, but GA3 application induced parthenocarpic fruit growth. On the contrary, parthenocarpic growth of MA/pat-2 and CU/pat-2 fruits, which occurs in the absence of pollination and hormone application, was not affected by GA3. Pollinated MA/wt and parthenocarpic MA/pat-2 ovary development was negated by paclobutrazol, and this inhibitory effect was counteracted by GA3. The main GAs of the early-13-hydroxylation pathway (GA1, GA3, GA8, GA19, GA20, GA29, GA44, GA53, and, tentatively, GA81) and two GAs of the non-13-hydroxylation pathway (GA9 and GA34) were identified in MA/wt ovaries by gas chromatography-selected ion monitoring. GAs were quantified in unpollinated ovaries at flower bud, pre-anthesis, and anthesis. In unpollinated MA/pat-2 and CU/pat-2ovaries, the GA20 content was much higher (up to 160 times higher) and the GA19 content was lower than in the corresponding non-parthenocarpic ovaries. The application of an inhibitor of 2-oxoglutarate-dependent dioxygenases suggested that GA20 is not active per se. The pat-2mutation may increase GA 20-oxidase activity in unpollinated ovaries, leading to a higher synthesis of GA20, the precursor of an active GA.
Isolation of Arabidopsis Mutants Lacking Components of Acquired Thermotolerance
Authors: Burke John J. , O'Mahony Patrick J. , Oliver Melvin J.
Published: July 2002

Acquired thermotolerance is a complex physiological phenomenon that enables plants to survive normally lethal temperatures. This study characterizes the temperature sensitivity of Arabidopsis using a chlorophyll accumulation bioassay, describes a procedure for selection of acquired thermotolerance mutants, and provides the physiological characterization of one mutant (AtTS02) isolated by this procedure. Exposure of etiolated Arabidopsis seedlings to 48°C or 50°C for 30 min blocks subsequent chlorophyll accumulation and is eventually lethal. Arabidopsis seedlings can be protected against the effects of a 50°C, 30-min challenge by a 4-h pre-incubation at 38°C. By the use of the milder challenge, 44°C for 30 min, and protective pretreatment, mutants lacking components of the acquired thermotolerance system were isolated. Putative mutants isolated by this procedure exhibited chlorophyll accumulation levels (our measure of acquired thermotolerance) ranging from 10% to 98% of control seedling levels following pre-incubation at 38°C and challenge at 50°C. The induction temperatures for maximum acquired thermotolerance prior to a high temperature challenge were the same in AtTS02 and RLD seedlings, although the absolute level of chlorophyll accumulation was reduced in the mutant. Genetic analysis showed that the loss of acquired thermotolerance in AtTS02 was a recessive trait. The pattern of proteins synthesized at 25°C and 38°C in the RLD and AtTS02 revealed the reduction in the level of a 27-kD heat shock protein in AtTS02. Genetic analysis showed that the reduction of this protein level was correlated with the acquired thermotolerance phenotype.