bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic expression and essential functions of Hes7 in somite segmentation
Authors: Bessho Yasumasa , Sakata Ryoichi , Komatsu Suguru , Shiota Kohei , Yamada Shuichi , Kageyama Ryoichiro
Published: July 2002

The basic helix–loop–helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Here, we found that Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anterior–posterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression oflunatic fringe and is essential for coordinated somite segmentation.
Evolution of the Neuropeptide Y Receptor Family: Gene and Chromosome Duplications Deduced from the Cloning and Mapping of the Five Receptor Subtype Genes in Pig
Authors: Wraith Amanda , Törnsten Anna , Chardon Patrick , Harbitz Ingrid , Chowdhary Bhanu P. , Andersson Leif , Lundin Lars-Gustav , Larhammar Dan
Published: July 2002

Neuropeptide Y (NPY) receptors mediate a variety of physiological responses including feeding and vasoconstriction. To investigate the evolutionary events that have generated this receptor family, we have sequenced and determined the chromosomal localizations of all five presently known mammalian NPY receptor subtype genes in the domestic pig, Sus scrofa (SSC). The orthologs of the Y1 and Y2 subtypes display high amino acid sequence identities between pig, human, and mouse (92%–94%), whereas the Y4, Y5, and y6 subtypes display lower identities (76%–87%). The lower identity of Y5 is due to high sequence divergence in the large third intracellular loop. TheNPY1R, NPY2R, and NPY5R receptor genes were localized to SSC8, the NPY4R to SSC14, andNPY6R to SSC2. Our comparisons strongly suggest that the tight cluster of NPY1R, NPY2R, and NPY5R on human chromosome 4 (HSA4) represents the ancestral configuration, whereas the porcine cluster has been split by two inversions on SSC8. These 3 genes, along with adjacent genes from 14 other gene families, form a cluster on HSA4 with extensive similarities to a cluster on HSA5, whereNPY6R and >13 other paralogs reside, as well as another large cluster on HSA10 that includes NPY4R. Thus, these gene families have expanded through large-scale duplications. The sequence comparisons show that the NPY receptor tripletNPY1R–NPY2R–NPY5R existed before these large-scale duplications.

[Sequence data for this article were deposited with the GenBank data library under accession nos.AF106081, PID g6457648 (for Pig Y1 sequence); accession nos. AF106082, PID g4249727 (for Pig Y2 sequence); accession no. AF227955 (for Pig Y4 sequence); accession nos. AF106083, PID g4249729 (for Pig Y5 sequence); accession no. AF227956 (for Pig Y6 sequence).]
Isolation of Lysophosphatidic Acid Phosphatase from Developing Peanut Cotyledons
Authors: Shekar Sunil , Tumaney Ajay W. , Rao T.J.V. Sreenivasa , Rajasekharan Ram
Published: July 2002

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [3H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min−1 mg−1. The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 ± 1.5 kD. The K  m values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 μm, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, orp-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.
Functional Interactions between Heterologously Expressed Starch-Branching Enzymes of Maize and the Glycogen Synthases of Brewer's Yeast
Authors: Seo Beom-seok , Kim Seungtaek , Scott M. Paul , Singletary George W. , Wong Kit-sum , James Martha G. , Myers Alan M.
Published: July 2002

Starch-branching enzymes (SBEs) catalyze the formation of α(1→6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native glycogen synthases (GSs) to produce significant quantities of α-glucan polymers. SBEIIa was phenotypically dominant to SBEIIb in terms of glucan structure. The specific branching enzyme present had a significant effect on the molecular weight of the product. From these data we suggest that SBEs and GSs work in a cyclically interdependent fashion, such that SBE action is needed for optimal GS activity; and GS, in turn, influences the further effects of SBE. Also, SBEIIa and SBEIIb appear to act before SBEI during polymer assembly in this heterologous system.
Biochemical Characterization of the aba2 and aba3 Mutants in Arabidopsis thaliana
Authors: Schwartz S. H. , Leon-Kloosterziel K. M. , Koornneef M. , Zeevaart JAD.
Published: July 2002

Abscisic acid (ABA)-deficient mutants in a variety of species have been identified by screening for precocious germination and a wilty phenotype. Mutants at two new loci, aba2 and aba3, have recently been isolated in Arabidopsis thaliana (L.) Heynh. (K.M. Leon-Kloosterziel, M. Alvarez-Gil, G.J. Ruijs, S.E. Jacobsen, N.E. Olszewski, S.H. Schwartz, J.A.D. Zeevaart, M. Koornneef [1996] Plant J 10: 655–661), and the biochemical characterization of these mutants is presented here. Protein extracts from aba2 and aba3 plants displayed a greatly reduced ability to convert xanthoxin to ABA relative to the wild type. The next putative intermediate in ABA synthesis, ABA-aldehyde, was efficiently converted to ABA by extracts from aba2 but not by extracts from aba3 plants. This indicates that the aba2 mutant is blocked in the conversion of xanthoxin to ABA-aldehyde and that aba3 is impaired in the conversion of ABA-aldehyde to ABA. Extracts from the aba3 mutant also lacked additional activities that require a molybdenum cofactor (Moco). Nitrate reductase utilizes a Moco but its activity was unaffected in extracts from aba3 plants. Moco hydroxylases in animals require a desulfo moiety of the cofactor. A sulfido ligand can be added to the Moco by treatment with Na2S and dithionite. Treatment of aba3 extracts with Na2S restored ABA-aldehyde oxidase activity. Therefore, the genetic lesion in aba3 appears to be in the introduction of S into the Moco.
Developmental Regulation of Pyrroline-5-Carboxylate Reductase Gene Expression in Arabidopsis
Authors: Hua X. J. , van de Cotte B. , Van Montagu M. , Verbruggen N.
Published: July 2002

At-P5R, a gene encoding the last enzyme of the proline (Pro) biosynthetic pathway in Arabidopsis thaliana, is developmentally regulated. To characterize the cis elements responsible for this developmental regulation, a series of 5[prime] deletions of the At-P5R promoter were transcriptionally fused to a β-glucuronidase (GUS)-coding region and transformed into Arabidopsis. The complete promoter of At-P5R directs strong GUS activity in root tips, the shoot meristem, guard cells, hydathodes, pollen grains, ovules, and developing seeds, all of which contain rapidly dividing cells and/or are undergoing changes in osmotic potential. This expression pattern is consistent with the function of Pro as an energy, nitrogen, and carbon source and as an osmoticum in response to dehydration. Promoters longer than 212 base pairs (bp) showed the same expression pattern, whereas those shorter than 143 bp did not direct any detectable GUS activity in any organs. This suggests that a 69-bp promoter region located between -212 and -143 bp is necessary to establish the tissue-specific expression of At-P5R during development. The Pro content measured in different organs suggests that, in addition to transcriptional control of the biosynthetic pathway, the transport of Pro may play a role in its distribution within Arabidopsis. Several aspects of the relationship between Pro metabolism and plant physiology are discussed.
cDNA Clones Encoding 1,3-β-Glucanase and a Fimbrin-Like Cytoskeletal Protein Are Induced by Al Toxicity in Wheat Roots
Authors: Cruz-Ortega R. , Cushman J. C. , Ownby J. D.
Published: July 2002

A cDNA library made from mRNA of Al-treated roots of an Al-sensitive wheat (Triticum aestivum cv Victory) cultivar was screened with a degenerate oligonucleotide probe derived from the partial amino acid sequence of the Al-induced protein TAI-18. Of seven clones that initially hybridized with the probe, one encoded a novel 1,3-β-glucanase having a calculated molecular weight of 46.3 and an isoelectric point of 6.0. Like the A6 1,3-β-glucanase gene products from Brassica napus and Arabidopsis thaliana, the predicted wheat protein had a C-terminal extension with three potential glycosylation sites. Northern analysis revealed that wheat 1,3-β-glucanase mRNA was up-regulated in Al-intoxicated roots, with highest expression after 12 h. The antibody to A6 1,3-β-glucanase from B. napus cross-reacted with a 56-kD protein that was induced after 24 h. A second partial cDNA clone showed similarity to genes encoding cytoskeletal fimbrin-like (actin-bundling) proteins. Although well studied in animals and fungi, fimbrins have not previously been described in plants. Fimbrin-like transcripts were up-regulated after 24 h of Al treatment in the Al-sensitive wheat cv Victory. In the Al-tolerant cv Atlas 66, fimbrin-like mRNA was up-regulated within 12 h by Al concentrations that did not inhibit root growth. Cellular stress associated with Al toxicity therefore causes up-regulation of a defense-related gene and a gene involved in the maintenance of cytoskeletal function.
Characterization and Functional Expression of a Ubiquitously Expressed Tomato Pectin Methylesterase
Authors: Gaffe J. , Tiznado M. E. , Handa A. K.
Published: July 2002

Pectin methylesterase (PME),a ubiquitous enzyme in plants, de-esterifies the methoxylated pectin in the plant cell wall. We have characterized a PME gene (designated as pmeu1) from tomato (Lycopersicon esculentum) with an expression that is higher in younger root, leaf, and fruit tissues than in older tissues. Hypocotyls and epicotyls show higher accumulation of pmeu1 transcripts compared with cotyledons. pmeu1 represents a single-copy gene in the tomato genome. Comparison of the deduced amino acid sequence of pmeu1 with other PME homologs showed that the N-terminal halves are highly variable, and the C-terminal halves are relatively conserved in plant PMEs. Constitutive expression of a fruit-specific PME antisense gene does not affect the level of pmeu1 transcripts in vegetative tissues but does lower the level of PMEU1 mRNA in developing tomato fruits. These results suggest that there exists developmentally regulated silencing of pmeu1 by a heterologous PME antisense gene. Expression of pmeu1 in tobacco (Nicotiana tabacum) under the control of the cauliflower mosaic virus 35S promoter caused up to a 4-fold increase in PME specific activity that was correlated with the accumulation of PMEU1 mRNA. In vitro transcription-translation analyses show that pmeu1 encodes a 64-kD polypeptide, whereas transgenic tobacco plants expressing pmeu1 accumulate a new 37-kD polypeptide, suggesting extensive posttranslational processing of PMEU1. These results are the first evidence, to our knowledge, of the functional characterization of a PME gene and the extensive modification of the encoded polypeptide.
Evidence that the Plant Host Synthesizes the Heme Moiety of Leghemoglobin in Root Nodules1
Authors: Santana Maria A. , Pihakaski-Maunsbach Kaarina , Sandal Niels , Marcker Kjeld A. , Smith Alison G.
Published: July 2002

Although it is well established that the plant host encodes and synthesizes the apoprotein for leghemoglobin in root nodules, the source of the heme moiety has been uncertain. We recently found that the transcript for coproporphyrinogen III oxidase, one of the later enzymes of heme synthesis, is highly elevated in soybean (Glycine maxL.) nodules compared with roots. In this study we measured enzyme activity and carried out western-blot analysis and in situ hybridization of mRNA to investigate the levels during nodulation of the plant-specific coproporphyrinogen oxidase and four other enzymes of the pathway in both soybean and pea (Pisum sativum L.). We compared them with the activity found in leaves and uninfected roots. Our results demonstrate that all of these enzymes are elevated in the infected cells of nodules. Because these are the same cells that express apoleghemoglobin, the data strongly support a role for the plant in the synthesis of the heme moiety of leghemoglobin.
Accumulation of a Clock-Regulated Transcript during Flower-Inductive Darkness in Pharbitis nil1
Authors: Sage-Ono Kimiyo , Ono Michiyuki , Harada Hiroshi , Kamada Hiroshi
Published: July 2002

To clarify the molecular basis of the photoperiodic induction of flowering in the short-day plantPharbitis nil cv Violet, we examined changes in the level of mRNA in cotyledons during the flower-inductive photoperiod using the technique of differential display by the polymerase chain reaction. A transcript that accumulated during the inductive dark period was identified and a cDNA corresponding to the transcript, designated PnC401 (P. nil C401), was isolated. RNA-blot hybridization verified that levels ofPnC401 mRNA fluctuated with a circadian rhythm, with maxima between 12 and 16 h after the beginning of the dark period) and minima of approximately 0. This oscillation continued even during an extended dark period but was damped under continuous light. Accumulation of PnC401 mRNA was reduced by a brief exposure to red light at the 8th h of the dark period (night-break treatment) or by exposure to far-red light at the end of the light period (end-of-day far-red treatment). These results suggest that fluctuations in levels of PnC401 mRNA are regulated by phytochrome(s) and a circadian clock and that they are associated with photoperiodic events that include induction of flowering.
Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds1
Authors: Rocha Façanha Arnoldo , de Meis Leopoldo
Published: July 2002

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H+-ATPase and H+-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to32Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanidep(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H+ pumps showed that the H+-ATPase was more active than H+-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H+-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.
Transport of Sterols to the Plasma Membrane of Leek Seedlings1
Authors: Moreau Patrick , Hartmann Marie-Andrée , Perret Anne-Marie , Sturbois-Balcerzak Bénédicte , Cassagne Claude
Published: July 2002

To investigate the intracellular transport of sterols in etiolated leek (Allium porrumL.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3β-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3β-ol, 24-methyl-cholest-5-en-3β-ol, (24S)-24-ethylcholesta-5,22E-dien-3β-ol, and stigmasta-5,24(241)Z-dien-3β-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12°C) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.
Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley
Authors: Thompson Peter , Bowsher Caroline G. , Tobin Alyson K.
Published: July 2002

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgareL.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F1-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.
Opposite Effects on Spodoptera littoralis Larvae of High Expression Level of a Trypsin Proteinase Inhibitor in Transgenic Plants
Authors: De Leo Francesca , Bonadé-Bottino Michel A. , Ceci Luigi R. , Gallerani Raffaele , Jouanin Lise
Published: July 2002

This work illustrates potential adverse effects linked with the expression of proteinase inhibitor (PI) in plants used as a strategy to enhance pest resistance. Tobacco (Nicotiana tabacum L. cv Xanthi) and Arabidopsis [Heynh.] ecotype Wassilewskija) transgenic plants expressing the mustard trypsin PI 2 (MTI-2) at different levels were obtained. First-instar larvae of the Egyptian cotton worm (Spodoptera littoralis Boisd.) were fed on detached leaves of these plants. The high level of MTI-2 expression in leaves had deleterious effects on larvae, causing mortality and decreasing mean larval weight, and was correlated with a decrease in the leaf surface eaten. However, larvae fed leaves from plants expressing MTI-2 at the low expression level did not show increased mortality, but a net gain in weight and a faster development compared with control larvae. The low MTI-2 expression level also resulted in increased leaf damage. These observations are correlated with the differential expression of digestive proteinases in the larval gut; overexpression of existing proteinases on low-MTI-2-expression level plants and induction of new proteinases on high-MTI-2-expression level plants. These results emphasize the critical need for the development of a PI-based defense strategy for plants obtaining the appropriate PI-expression level relative to the pest's sensitivity threshold to that PI.
Carbohydrates in Individual Cells of Epidermis, Mesophyll, and Bundle Sheath in Barley Leaves with Changed Export or Photosynthetic Rate
Authors: Koroleva Olga A. , Farrar John F. , Deri Tomos A. , Pollock Christopher J.
Published: July 2002

Carbohydrate metabolism of barley (Hordeum vulgare) leaves induced to accumulate sucrose (Suc) and fructans was investigated at the single-cell level using single-cell sampling and analysis. Cooling of the root and shoot apical meristem of barley plants led to the accumulation of Suc and fructan in leaf tissue. Suc and fructan accumulated in both mesophyll and parenchymatous bundle-sheath (PBS) cells because of the reduced export of sugars from leaves under cooling and to increased photosynthesis under high photon fluence rates. The general trends of Suc and fructan accumulation were similar for mesophyll and PBS cells. The fructan-to-Suc ratio was higher for PBS cells than for mesophyll cells, suggesting that the threshold Suc concentration needed for the initiation of fructan synthesis was lower for PBS cells. Epidermal cells contained very low concentrations of sugar throughout the cooling experiment. The difference in Suc concentration between control and treated plants was much less if compared at the single-cell level rather than the whole-tissue level, suggesting that the vascular tissue contains a significant proportion of total leaf Suc. We discuss the importance of analyzing complex tissues at the resolution of individual cells to assign molecular mechanisms to phenomena observed at the whole-plant level.
Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants ofChlamydomonas reinhardtii
Authors: Dauvillée David , Colleoni Christophe , Shaw Eudean , Mouille Gregory , D'Hulst Christophe , Morell Matthew , Samuel Michael S. , Bouchet Brigitte , Gallant Daniel J. , Sinskey Anthony , Ball Steven
Published: July 2002

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants ofC. reinhardtii.
Subcellular Distribution and Tissue Expression of Phospholipase Dα, Dβ, and Dγ in Arabidopsis1
Authors: Fan Lu , Zheng Suqin , Cui Decai , Wang Xuemin
Published: July 2002

Three phospholipase Ds (PLDs; EC 3.1.4.4) have been cloned from Arabidopsis, and they exhibit two distinct types of activities: polyphosphoinositide-requiring PLDβ and PLDγ, and polyphosphoinositide-independent PLDα. In subcellular fractions of Arabidopsis leaves, PLDα and PLDγ were both present in the plasma membrane, intracellular membranes, mitochondria, and clathrin-coated vesicles, but their relative levels differed in these fractions. In addition, PLDγ was detected in the nuclear fraction. In contrast, PLDβ was not detectable in any of the subcellular fractions. PLDα activity was higher in the metabolically more active organs such as flowers, siliques, and roots than in dry seeds and mature leaves, whereas the polyphosphoinositide-dependent PLD activity was greater in older, senescing leaves than in other organs. PLDβ mRNA accumulated at a lower level than the PLDα and PLDγ transcripts in most organs, and the expression pattern of the PLDβ mRNA also differed from that of PLDα and PLDγ in different organs. Collectively, these data demonstrated that PLDα, PLDβ, and PLDγ have different patterns of subcellular distribution and tissue expression in Arabidopsis. The present study also provides evidence for the presence of an additional PLD that is structurally more closely related to PLDγ than to the other two PLDs.
Brassinosteroid/Sterol Synthesis and Plant Growth as Affected bylka and lkb Mutations of Pea1
Authors: Nomura Takahito , Kitasaka Yukiko , Takatsuto Suguru , Reid James B. , Fukami Motohiro , Yokota Takao
Published: July 2002

The dwarf pea (Pisum sativum) mutants lka and lkb are brassinosteroid (BR) insensitive and deficient, respectively. The dwarf phenotype of the lkb mutant was rescued to wild type by exogenous application of brassinolide and its biosynthetic precursors. Gas chromatography-mass spectrometry analysis of the endogenous sterols in this mutant revealed that it accumulates 24-methylenecholesterol and isofucosterol but is deficient in their hydrogenated products, campesterol and sitosterol. Feeding experiments using 2H-labeled 24-methylenecholesterol indicated that the lkb mutant is unable to isomerize and/or reduce the Δ24(28) double bond. Dwarfism of thelkb mutant is, therefore, due to BR deficiency caused by blocked synthesis of campesterol from 24-methylenecholesterol. Thelkb mutation also disrupted sterol composition of the membranes, which, in contrast to those of the wild type, contained isofucosterol as the major sterol and lacked stigmasterol. Thelka mutant was not BR deficient, because it accumulated castasterone. Like some gibberellin-insensitive dwarf mutants, overproduction of castasterone in the lka mutant may be ascribed to the lack of a feedback control mechanism due to impaired perception/signal transduction of BRs. The possibility that castasterone is a biologically active BR is discussed.
Inhibition and Acclimation of Photosynthesis to Heat Stress Is Closely Correlated with Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase
Authors: Law R. David , Crafts-Brandner Steven J.
Published: July 2002

Increasing the leaf temperature of intact cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) plants caused a progressive decline in the light-saturated CO2-exchange rate (CER). CER was more sensitive to increased leaf temperature in wheat than in cotton, and both species demonstrated photosynthetic acclimation when leaf temperature was increased gradually. Inhibition of CER was not a consequence of stomatal closure, as indicated by a positive relationship between leaf temperature and transpiration. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is regulated by Rubisco activase, was closely correlated with temperature-induced changes in CER. Nonphotochemical chlorophyll fluorescence quenching increased with leaf temperature in a manner consistent with inhibited CER and Rubisco activation. Both nonphotochemical fluorescence quenching and Rubisco activation were more sensitive to heat stress than the maximum quantum yield of photochemistry of photosystem II. Heat stress led to decreased 3-phosphoglyceric acid content and increased ribulose-1,5-bisphosphate content, which is indicative of inhibited metabolite flow through Rubisco. We conclude that heat stress inhibited CER primarily by decreasing the activation state of Rubisco via inhibition of Rubisco activase. Although Rubisco activation was more closely correlated with CER than the maximum quantum yield of photochemistry of photosystem II, both processes could be acclimated to heat stress by gradually increasing the leaf temperature.
1-Aminocyclopropane-1-Carboxylate Oxidase Activity Limits Ethylene Biosynthesis in Rumex palustrisduring Submergence
Authors: Vriezen Wim H. , Hulzink Raymond , Mariani Celestina , Voesenek Laurentius A.C.J.
Published: July 2002

Submergence strongly stimulates petiole elongation in Rumex palustris, and ethylene accumulation initiates and maintains this response in submerged tissues. cDNAs from R. palustris corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (RP-ACO1) were isolated from elongating petioles and used to study the expression of the corresponding gene. An increase inRP-ACO1 messenger was observed in the petioles and lamina of elongating leaves 2 h after the start of submergence. ACC oxidase enzyme activity was measured in homogenates of R. palustris shoots, and a relevant increase was observed within 12 h under water with a maximum after 24 h. We have shown previously that the ethylene production rate of submerged shoots does not increase significantly during the first 24 h of submergence (L.A.C.J. Voesenek, M. Banga, R.H. Thier, C.M. Mudde, F.M. Harren, G.W.M. Barendse, C.W.P.M. Blom [1993] Plant Physiol 103: 783–791), suggesting that under these conditions ACC oxidase activity is inhibited in vivo. We found evidence that this inhibition is caused by a reduction of oxygen levels. We hypothesize that an increased ACC oxidase enzyme concentration counterbalances the reduced enzyme activity caused by low oxygen concentration during submergence, thus sustaining ethylene production under these conditions. Therefore, ethylene biosynthesis seems to be limited at the level of ACC oxidase activity rather than by ACC synthase in R. palustrisduring submergence.
Solubilization and Partial Characterization of Homogalacturonan-Methyltransferase from Microsomal Membranes of Suspension-Cultured Tobacco Cells
Authors: Goubet Florence , Mohnen Debra
Published: July 2002

The transfer of a methyl group fromS-adenosyl-l-methionine onto the carboxyl group of α-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337–347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mm dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K  m forS-adenosyl-l-methionine of 18 μm, and an apparent V  max of 0.121 pkat mg−1 of protein. The apparentK  m for HGA and for pectin is 0.1 to 0.2 mg mL−1. Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.
Plant Succinic Semialdehyde Dehydrogenase. Cloning, Purification, Localization in Mitochondria, and Regulation by Adenine Nucleotides
Authors: Busch Karin B. , Fromm Hillel
Published: July 2002

Succinic semialdehyde dehydrogenase (SSADH) is one of three enzymes constituting the γ-aminobutyric acid shunt. We have cloned the cDNA for SSADH from Arabidopsis, which we designated SSADH1. SSADH1 cDNA encodes a protein of 528 amino acids (56 kD) with high similarity to SSADH fromEscherichia coli and human (>59% identity). A sequence similar to a mitochondrial protease cleavage site is present 33 amino acids from the N terminus, indicating that the mature mitochondrial protein may contain 495 amino acids (53 kD). The native recombinant enzyme and the plant mitochondrial protein have a tetrameric molecular mass of 197 kD. Fractionation of plant mitochondria revealed its localization in the matrix. The purified recombinant enzyme showed maximal activity at pH 9.0 to 9.5, was specific for succinic semialdehyde (K  0.5 = 15 μm), and exclusively used NAD+ as a cofactor (K  m = 130 ± 77 μm). NADH was a competitive inhibitor with respect to NAD+(K  i = 122 ± 86 μm). AMP, ADP, and ATP inhibited the activity of SSADH (K  i = 2.5–8 mm). The mechanism of inhibition was competitive for AMP, noncompetitive for ATP, and mixed competitive for ADP with respect to NAD+. Plant SSADH may be responsive to mitochondrial energy charge and reducing potential in controlling metabolism of γ-aminobutyric acid.
Pre-Anthesis Reserve Utilization for Protein and Carbohydrate Synthesis in Grains of Wheat
Authors: Gebbing Thomas , Schnyder Hans
Published: July 2002

We assessed the contribution of pre-anthesis reserve C to protein and carbohydrate deposition in grains of wheat (Triticum aestivum L.) using a new approach comprised of steady-state 13C/12C labeling and separation of the protein and carbohydrate fractions of mature grains. Experiments were performed with two spring wheat cultivars (Kadett and Star) grown with differential N fertilizer supply over 2 years. Pre-anthesis reserves contributed between 30% and 47% of the C in protein and 8% to 27% of the C in carbohydrates of grains. Partitioning of pre-anthesis C among the grain fractions was strongly dependent on the C/N (w/w) ratio in mobilized pre-anthesis biomass (r  2 = 0.92). There appeared to be no significant exchange of pre-anthesis C between amino acids and carbohydrates during redistribution. The mean apparent efficiency of mobilized carbohydrate-C use in grain filling (MECHO, estimated as the mass of pre-anthesis C deposited in grain carbohydrates per gram of pre-anthesis C mobilized from carbohydrates in vegetative plant parts) was 0.72, whereas that of protein-C (MEP) was 0.56. However, MEP and MECHO varied among treatments. MECHO increased with increasing contributions of water-soluble carbohydrates to total pre-anthesis carbohydrate mobilization. MEP decreased with increasing residence time of protein in vegetative biomass. Possible causes for variability of MEP and MECHO are discussed.
Functional Significance and Induction by Solar Radiation of Ultraviolet-Absorbing Sunscreens in Field-Grown Soybean Crops
Authors: Mazza Carlos A. , Boccalandro Hernán E. , Giordano Carla V. , Battista Daniela , Scopel Ana L. , Ballaré Carlos L.
Published: July 2002

Colorless phenylpropanoid derivatives are known to protect plants from ultraviolet (UV) radiation, but their photoregulation and physiological roles under field conditions have not been investigated in detail. Here we describe a fast method to estimate the degree of UV penetration into photosynthetic tissue, which is based on chlorophyll fluorescence imaging. In Arabidopsis this technique clearly separated the UV-hypersensitive transparent testa (tt) tt5 andtt6 mutants from the wild type (WT) andtt3, tt4, and tt7 mutants. In field-grown soybean (Glycine max), we found significant differences in UV penetration among cultivars with different levels of leaf phenolics, and between plants grown under contrasting levels of solar UV-B. The reduction in UV penetration induced by ambient UV-B had direct implications for DNA integrity in the underlying leaf tissue; thus, the number of cyclobutane pyrimidine dimers caused by a short exposure to solar UV-B was much larger in leaves with high UV transmittance than in leaves pretreated with solar UV-B to increase the content phenylpropanoids. Most of the phenylpropanoid response to solar UV in field-grown soybeans was induced by the UV-B component (λ ≤ 315 nm). Our results indicate that phenolic sunscreens in soybean are highly responsive to the wavelengths that are most affected by variations in ozone levels, and that they play an important role in UV protection in the field.
Expression of AtPRP3, a Proline-Rich Structural Cell Wall Protein from Arabidopsis, Is Regulated by Cell-Type-Specific Developmental Pathways Involved in Root Hair Formation
Authors: Bernhardt Christine , Tierney Mary L.
Published: July 2002

The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/β-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or α-naphthaleneacetic acid (α-NAA), compounds known to promote root hair formation. In the presence of 1-α-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or α-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced inttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs.
TheArabidopsis HKT1Gene Homolog Mediates Inward Na+ Currents inXenopus laevisOocytes and Na+ Uptake inSaccharomyces cerevisiae
Authors: Uozumi Nobuyuki , Kim Eugene J. , Rubio Francisco , Yamaguchi Takao , Muto Shoshi , Tsuboi Akio , Bakker Evert P. , Nakamura Tatsunosuke , Schroeder Julian I.
Published: July 2002

The Na+-K+ co-transporter HKT1, first isolated from wheat, mediates high-affinity K+ uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar.AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na+ uptake transporter inXenopus laevis oocytes, and the presence of external K+ did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na+, which correlates to the large inward Na+ currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K+ uptake when cultured in K+-limiting medium. However, expression ofAtHKT1 did rescue Escherichia colimutants carrying deletions in K+ transporters. The rescue was associated with a less than 2-fold stimulation of K+uptake into K+-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na+ and, to a small degree, K+ transport in heterologous expression systems.
Characterization and Expression of DNA Sequences Encoding Putative Type-II Metallothioneins in the Seagrass Posidonia oceanica
Authors: Giordani Tommaso , Natali Lucia , Maserti Bianca Elena , Taddei Sonia , Cavallini Andrea
Published: July 2002

Posidonia oceanica is a marine phanerogam, largely widespread in the Mediterranean sea, representing an important food substrate for many marine organisms. A progressive reduction ofP. oceanica meadows has been reported, due to anthropogenic coastal activity. Studying mechanisms by which this species responds to environmental stresses, three DNA sequences putatively encoding metallothioneins (MTs) have been isolated, by PCR. Two sequences, Pomt2a (accession no. AJ249603) andPomt2b (accession no. AJ249602), show high similarities with genes encoding type-II MTs and are interrupted by two and one intron, respectively. The third sequence, Pomt2c(accession no. AJ249604), is supposed to be a pseudogene, originated by retrotranscription of the Pomt2b mRNA. These sequences belong to a multigene family with at least five members. Northern hybridizations indicated that MT transcripts accumulation is constitutive and seasonally regulated. MT encoding RNAs increase after rhyzome harvesting and (at a lesser extent) after 15 d of cultivation in an aquarium. As for animal MTs, transcripts accumulation is observed also after exposure to trace metals such as copper and cadmium. In the case of copper, the effect depends on concentration. Finally, taking into consideration the great interest in studying the biogeochemical cycle of mercury in the Mediterranean basin and sinceP. oceanica is commonly considered a bioindicator of this metal, the effect of mercury treatments on the accumulation of MT transcripts has been analyzed: in only a few experiments a small increase in the level of transcripts was recorded, suggesting that MTs are not key elements in the mercury accumulation by this species.
Essential Role of Caffeoyl Coenzyme A O-Methyltransferase in Lignin Biosynthesis in Woody Poplar Plants
Authors: Zhong Ruiqin , Morrison W. Herbert , Himmelsbach David S. , Poole Farris L. , Ye Zheng-Hua
Published: July 2002

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) has recently been shown to participate in lignin biosynthesis in herbacious tobacco plants. Here, we demonstrate that CCoAOMT is essential in lignin biosynthesis in woody poplar (Populus tremula × Populus alba) plants. In poplar stems, CCoAOMT was found to be expressed in all lignifying cells including vessel elements and fibers as well as in xylem ray parenchyma cells. Repression of CCoAOMT expression by the antisense approach in transgenic poplar plants caused a significant decrease in total lignin content as detected by both Klason lignin assay and Fourier-transform infrared spectroscopy. The reduction in lignin content was the result of a decrease in both guaiacyl and syringyl lignins as determined by in-source pyrolysis mass spectrometry. Fourier-transform infrared spectroscopy indicated that the reduction in lignin content resulted in a less condensed and less cross-linked lignin structure in wood. Repression of CCoAOMT expression also led to coloration of wood and an elevation of wall-bound p-hydroxybenzoic acid. Taken together, these results indicate that CCoAOMT plays a dominant role in the methylation of the 3-hydroxyl group of caffeoyl CoA, and the CCoAOMT-mediated methylation reaction is essential to channel substrates for 5-methoxylation of hydroxycinnamates. They also suggest that antisense repression of CCoAOMT is an efficient means for genetic engineering of trees with low lignin content.
Purification and Characterization of Two Voltage-Dependent Anion Channel Isoforms from Plant Seeds
Authors: Abrecht Helge , Wattiez Ruddy , Ruysschaert Jean-Marie , Homblé Fabrice
Published: July 2002

Mitochondria were isolated from imbibed seeds of lentil (Lens culinaris) and Phaseolus  vulgaris. We copurified two voltage-dependent anion channel from detergent solubilized mitochondria in a single purification step using hydroxyapatite. The two isoforms from P. vulgaris were separated by chromatofocusing chromatography in 4m urea without any loss of channel activity. Channel activity of each isoform was characterized upon reconstitution into diphytanoyl phosphatidylcholine planar lipid bilayers. Both isoforms form large conductance channels that are slightly anion selective and display cation selective substates.
Association of Spectrin-Like Proteins with the Actin-Organized Aggregate of Endoplasmic Reticulum in the Spitzenkörper of Gravitropically Tip-Growing Plant Cells
Authors: Braun Markus
Published: July 2002

Spectrin-like epitopes were immunochemically detected and immunofluorescently localized in gravitropically tip-growing rhizoids and protonemata of characean algae. Antiserum against spectrin from chicken erythrocytes showed cross-reactivity with rhizoid proteins at molecular masses of about 170 and 195 kD. Confocal microscopy revealed a distinct spherical labeling of spectrin-like proteins in the apices of both cell types tightly associated with an apical actin array and a specific subdomain of endoplasmic reticulum (ER), the ER aggregate. The presence of spectrin-like epitopes, the ER aggregate, and the actin cytoskeleton are strictly correlated with active tip growth. Application of cytochalasin D and A23187 has shown that interfering with actin or with the calcium gradient, which cause the disintegration of the ER aggregate and abolish tip growth, inhibits labeling of spectrin-like proteins. At the beginning of the graviresponse in rhizoids the labeling of spectrin-like proteins remained in its symmetrical position at the cell tip, but was clearly displaced to the upper flank in gravistimulated protonemata. These findings support the hypothesis that a displacement of the Spitzenkörper is required for the negative gravitropic response in protonemata, but not for the positive gravitropic response in rhizoids. It is evident that the actin/spectrin system plays a role in maintaining the organization of the ER aggregate and represents an essential part in the mechanism of gravitropic tip growth.