bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of de novo purine biosynthesis genes in ureide‐producing legumes: induction of glutamine phosphoribosylpyrophosphate amidotransferase gene and characterization of its cDNA from soybean and Vigna
Authors: Kim Jong H. , Delauney Ashton J. , Verma Desh Pal S.
Published: March 2003

Soybean (Glycine max) and mothbean (Vigna aconitifolia) cDNA clones encoding glutamine phosphoribosylpyrophosphate amidotransferase (PRAT), the first enzyme of the de novo purine biosynthesis pathway, have been isolated from nodule cDNA libraries. The amino acid sequence deduced from soybean clone showed> 85% homology to the PRAT sequence of mothbean and 33–47% homology to those of bacteria, yeast, chicken, rat and human. The soybean clone encodes a protein with an N‐terminal sequence resembling a plastid‐targeting peptide. Downstream from this peptide is a sequence similar to the 11 amino acid propeptide found in the Bacillus subtilis, chicken, rat and human PRAT proteins. The mothbean cDNA, although lacking most of the plastid presequence, encodes the putative propeptide and efficiently complements purine auxotrophy in an Escherichia coli purF mutant. Both the soybean and mothbean clones encode characteristic cysteine residues that are known to be involved in the assembly of a [Fe‐S] cluster near the C‐terminus of this protein. Levels of PRAT mRNA in mothbean nodules were found to increase steadily as the nodules matured from 13 days to 23 days.

PRAT mRNA was not detectable in uninfected root tissue but a low level of transcript was detected in leaves. Treatment of uninfected root with l‐glutamine induced the PRAT mRNA transcript suggesting that glutamine produced as a result of assimilation of fixed nitrogen is funnelled into the de novo purine biosynthesis and controls the expression of this pathway in root nodules.
Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase
Authors: Strohm Michael , Jouanin Lise , Kunert Karl Josef , Pruvost Christophe , Polle Andrea , Foyer Christine Helen , Rennenberg Heinz
Published: March 2003

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase (gsh II) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15‐ to 60‐fold higher than those of wild‐type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild‐type plants. Precursor feeding experiments with cysteine and γ‐glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ‐glutamylcysteine, and (iii) regulation of the activities of both γ‐glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.
Co‐expression of native and introduced genes reveals cryptic regulation of HMG CoA reductase expression in Arabidopsis
Authors: Re Edward B. , Jones Daniel , Learned R. Marc
Published: March 2003

In eukaryotes, all isoprenoid compounds share a common precursor, mevalonic acid, whose synthesis is catalyzed by the enzyme 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase. As one step towards a better understanding of the role that this enzyme plays in coodinating isoprenoid biosynthesis in plants, Arabidopsis thaliana HMG CoA reductase was ectopically expressed in transgenic Arabidopsis plants. By using this molecular genetic approach, several novel and fundamental observations have been made regarding isoprenoid biosynthesis in Arabidopsis. First, it was demonstrated that the over‐expression of authentic Arabidopsis HMG CoA reductase is not sufficient to alter the bulk synthesis and accumulation of the abundant end products of the plant isoprenoid pathway. Second, active transcription of the transgene appears to co‐activate and deregulate expression of the native gene, resulting in a striking elevation of HMG CoA reductase mRNA levels. Finally, although very high levels of HMG CoA reductase mRNA were expressed in these transgenic plants, only modest increases in enzyme activity could be detected. Taken together, these data suggest that HMG CoA reductase expression is regulated at multiple levels in plants as well as animals, and they provide a foundation for elucidating the molecular mechanisms for mevalonate regulation in A.thaliana.
Engineered oryzacystatin‐I expressed in transgenic hairy roots confers resistance to Globodera pallida
Authors: Urwin Peter E. , Atkinson Howard J. , Waller David A. , McPherson Michael J.
Published: March 2003

The cysteine proteinase inhibitor, oryzacystatin‐I (Oc‐I), and several engineered Oc‐I variants have been tested for efficacy in inhibiting growth and development of both the free‐living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida. To assist in the design of protein engineering experiments to improve the efficacy of Oc‐I, an alignment of 28 cystatins and a molecular model of Oc‐I were generated. Inhibitory activities (Ki) of wild‐type and variant forms of Oc‐I against both papain and the C. elegans cysteine proteinase, gcp‐1, were measured. For one variant, in which residue Asp86 was deleted (Oc‐IΔD86), the K,i was reduced by 13‐ to 14‐fold. LD50 studies to test the effect of Oc‐I and Oc‐IΔD86 against C. elegans showed the relative median potency of Oc‐IΔD86 to be 0.76 that of wild‐type Oc‐I. When expressed in tomato hairy roots both Oc‐I and Oc‐IΔD86 had a detrimental effect on growth and development of G. pallida. This effect was significantly greater on Oc‐IΔD86‐expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.
Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway
Authors: Fray Rupert G. , Wallace Andrew , Fraser Paul D. , Valero Daniel , Hedden Peter , Bramley Peter M. , Grierson Donald
Published: March 2003

Tomato plants transformed with a copy of the fruit‐expressed phytoene synthase cDNA under control of the CaMV 35S promoter showed ectopic production of carotenoids. High expressers were reduced in stature. The dwarf character was inherited with an inverse relationship between expression of phytoene synthase and plant height. Severely affected plants also showed reduced chlorophyll content in young leaves. These dwarfs showed a 30‐fold reduction in levels of gibberellin A1 (GA1) and growth was partially restored by treatment with exogenous GA3. Qualitative and quantitative changes in carotenoids were also found. It is proposed that the dwarf phenotype results from the over‐production of phytoene synthase, which converts geranylgeranyl diphosphate to phytoene and thereby diverts this intermediate away from the gibberellin (GA) and phytol biosynthetic pathways.
Specification of chimeric flowering shoots in wild‐type Arabidopsis
Authors: Hempel Frederick D. , Feldman Lewis J.
Published: March 2003

Within wild‐type Arabidopsis populations, a subset of the plants were found to have a single chimeric shoot on their primary shoot axes. The chimeric shoots were located below the lowest primary‐axis flower; and they exhibited features of both flowers and paraclades (lateral flowering shoots). Morphological analyses of chimeric shoots indicated that they developed from single primordia. In each chimeric shoot, the side furthest from the apical meristem was specified as ‘flower’—while the side closest to the meristem was specified as ‘paraclade’—suggesting that a stimulus from outside the apical meristem can directly induce primordia to develop as flowers. It is concluded that the development of the teratological chimeric shoots resulted from the overlap of the vegetative and floral specification processes within single primordia.
Expression of a single‐chain Fv antibody against abscisic acid creates a wilty phenotype in transgenic tobacco
Authors: Artsaenko Olga , Peisker Martin , zur Nieden Uta , Fiedler Ulrike , Weiler Elmar W. , Müntz Klaus , Conrad Udo
Published: March 2003

The plant hormone abscisic acid (ABA) participates in the control of several important physiological processes in plants such as stomata regulation, seed dormancy and stress tolerance. A new strategy was developed to study these phenomena by blocking abscisic acid with intracellularly expressed specific single‐chain variable fragment (scFv) antibodies. Here evidence is presented that the expression of single‐chain Fv antibodies against abscisic acid in the endoplasmic reticulum of transgenic tobacco cells leads to a wilty phenotype. Stomatal conductance is increased at high CO2 concentrations dependent on the level of antibody expression in leaves. Symptoms of abscisic acid deficiency were generated in the transformants although they have even higher levels of abscisic acid than wild‐type plants.
Molecular evolution of plant β‐glucan endohydrolases
Authors: Høj Peter B. , Fincher Geoffrey B.
Published: March 2003

The evolutionary relationships of two classes of plant β‐glucan endohydrolases have been examined by comparison of their substrate specificities, their three‐dimensional conformations and the structural features of their corresponding genes. These comparative studies provide compelling evidence that the (1→3)‐β‐glucanases and (1→3,1→4)‐β‐glucanases from higher plants share a common ancestry and, in all likelihood, that the (1→3,1→4)‐β‐glucanases diverged from the (1→3)‐β‐glucanases during the appearance of the graminaceous monocotyledons. The evolution of (1→3,1→4)‐β‐glucanases from (1→3)‐β‐glucanases does not appear to have invoked ‘modular’ mechanisms of change, such as those caused by exon shuffling or recombination. Instead, the shift in specificity has been acquired through a limited number of point mutations that have resulted in amino acid substitutions along the substrate‐binding cleft. This is consistent with current theories that the evolution of new enzymic activity is often achieved through duplication of the gene encoding an existing enzyme which is capable of performing the required chemistry, in this case the hydrolysis of a glycosidic linkage, followed by the mutational alteration and fine‐tuning of substrate specificity.

The evolution of a new specificity has enabled a dramatic shift in the functional capabilities of the enzymes. (1→3)‐β‐Glucanases that play a major role, inter alia, in the protection of the plant against pathogenic microorganisms through their ability to hydrolyse the (1→3)‐β‐glucans of fungal cell walls, appear to have been recruited to generate (1→3,1→4)‐β‐glucanases, which quite specifically hydrolyse plant cell wall (1→3,1→4)‐β‐glucans in the graminaecous monocotyledons during normal wall metabolism. Thus, one class of β‐glucan endohydrolase can degrade β‐glucans in fungal walls, while the other hydrolyses structurally distinct β‐glucans of plant cell walls. Detailed information on the three‐dimensional structures of the enzymes and the identification of catalytic amino acids now present opportunities to explore the precise molecular and atomic details of substrate‐binding, catalytic mechanisms and the sequence of molecular events that resulted in the evolution of the substrate specificities of the two classes of enzyme.
Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: effect on CO2 assimilation and growth in low irradiance
Authors: Paul Matthew J. , Knight Jacqui S. , Habash Dimah , Parry Martin A.J. , Lawlor David W. , Barnes Simon A. , Loynes Andy , Gray John C.
Published: March 2003

To quantify the importance of the Calvin cycle enzyme phosphoribulokinase (PRK) in photosynthesis and to perturb photosynthesis without large direct reductions in leaf protein content, tobacco plants (Nicotiana tabacum L.) were transformed with an inverted cDNA encoding tobacco PRK. A population of plants expressing antisense RNA and a range of PRK activities from wild‐type to less than 5% of wild‐type were obtained. CO2 assimilation under the growing conditions (330 µmol photons m−2 sec−1, 350 µbar CO2, 25°C) was not inhibited until more than 85% of PRK activity had been removed. With reduction in PRK activity of between 85 and 95%, assimilation rates and amounts of chlorophyll compared with wild‐type were reduced by up to half. Decreased absorption of light by leaves with less chlorophyll accounte0d for only a small part of the reduction in assimilation rate. When PRK activity was below 15% of wild‐type, amounts of ribulose‐5‐phosphate, ribose‐5‐phosphate, ATP and fructose‐6‐phosphate were 1.5‐ to fivefold higher and levels of ribulose‐1,5‐bisphosphate, 3‐phosphoglyceric acid and ADP 1.5‐ to fourfold lower than in wild‐type. It is estimated that these changes maintained flux through PRK to realise the assimilation rates observed. A possible shift of control within the Calvin cycle towards fructose‐1,6‐bisphosphatase in plants with low PRK is discussed. Amounts of hexoses and starch in particular were reduced in plants expressing the lowest PRK activities; amounts of sucrose were little affected. Lower CO2 assimilation in plants with low PRK activity correlated with reduced relative growth rate of shoots and delayed flowering, but there was no effect on specific leaf area. It is concluded that (i) in wild‐type plants grown in constant low light, PRK has a flux‐control coefficient for CO2 assimilation of zero, and that even when amounts of PRK are reduced 20‐fold relative to wild‐type, altered amounts of metabolites compensate for much of the reduction in PRK protein; (ii) in plants where there is a 95% reduction in amounts of PRK, photosynthesis was reduced twofold without large changes in leaf protein content or leaf geometry.
Characterization of a maize G‐box binding factor that is induced by hypoxia
Authors: De Vetten Nick C. , Ferl Robert J.
Published: March 2003

G‐box cis‐acting DNA sequence elements are present in the promoter region of a number of signal‐inducible plant genes. In many cases this motif is essential for gene expression. Maize nuclear extracts contain a protein complex that binds specifically to the G‐box sequence. Previously, a protein called GF14 was described that is physically associated with the G‐box binding complex, but is not a DNA‐binding factor in and of itself. This paper reports the isolation of a cDNA encoding a maize G‐box binding factor (GBF). The deduced amino acid sequence indicates that maize GBF1 is a basic region‐leucine zipper protein. GBF1 binds to the G‐box element with specificity similar to that of the binding activity in nuclear extracts. Furthermore, maize GBF1 and the factor detected in nuclear extract are identical in their molecular weight and are immunologically related. GBF1 mRNA accumulates rapidly in hypoxically induced maize cells prior to the increase in Adh1 mRNA levels. Taken together with results that indicate that GBF1 binds to the hypoxia‐responsive promoter of maize Adh1, these observations suggest that GBF1 may be one of the factors involved in the activation of Adh1.
Site‐specific integration of DNA into wild‐type and mutant lox sites placed in the plant genome
Authors: Albert Henrik , Dale Emily C. , Lee Elsa , Ow David W.
Published: March 2003

The bacteriophage P1 Cre—lox site‐specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild‐type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild‐type and mutant lox sites were used in conjunction with two strategies for abolishing post‐integration Cre activity: (i) promoter displacement of a cre‐expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild‐type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild‐type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre—lox site‐specific recombination system is useful for the single‐copy integration of DNA into a chromosomal lox site.
Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes
Authors: Ni Min , Cui Decai , Einstein Jane , Narasimhulu Soma , Vergara Claudia E. , Gelvin Stanton B.
Published: March 2003

To investigate promoter strength and the tissue‐specific patterns of expression, chimeric promoters incorporating subdomains of mannopine synthase (mas2′) and octopine synthase (ocs) promoter and activator regions were affixed to a β‐glucuronidase reporter gene and the constructions introduced into tobacco plants. Addition of a trimer of the ocs upstream activating sequence (UAS) to a mas promoter/activator region resulted in highly elevated levels of GUS activity in all tissues examined. In leaf tissue, this chimeric promoter is approximately 156‐fold and 26‐fold stronger than are the CaMV 35S and the ‘enhanced’ double CaMV 35S promoters, respectively. Expression of GUS activity directed by the mas and ocs promoters/activators is limited to specific cell types. Addition of the ocs or mas UAS to the mas or ocs promoter/activator regions modulated these expression patterns. The addition of a trimer of the ocs UAS to the mas promoter/activator region resulted in a transcriptional control element that directed GUS expression in most cell types. In addition to the strong expression in transgenic tobacco plants, this novel promoter directed higher levels of GUS expression than did the CaMV 35S promoter in transiently transformed tobacco leaf discs and suspension culture cells, as well as in cassava and cowpea explants. It is proposed that the strong promoter containing a trimer of the ocs UAS affixed to the mas promoter/activator will be useful for the very high level constitutive expression of linked genes in a wide variety of plant species.
In vitro assembly of cellulose/xyloglucan networks: ultrastructural and molecular aspects
Authors: Whitney Sarah E.C. , Brigham Jennifer E. , Darke Arthur H. , Reid J.S. Grant , Gidley Michael J.
Published: March 2003

Features of the interaction between cellulose and xyloglucan have been studied using the cellulose‐producing bacterium Acetobacter aceti ssp. xylinum (ATCC 53524) and tamarind seed xyloglucan. Direct microscopic evidence is provided for the generation of cross‐bridges between cellulose ribbons produced in the presence of xyloglucan but not carboxymethyl‐cellulose. Cross‐bridge lengths are very similar to those observed for de‐pectinated onion cell walls. Similar cross‐bridge lengths are observed following mixing of isolated A. xylinum cellulose and xyloglucan, showing that network formation can be an abiotic process. The level of incorporation of xyloglucan in an actively growing system (ca. 38% of cellulose) is an order of magnitude higher than that observed in mixtures of isolated polymers and is comparable with cell wall levels.

NMR spectroscopy suggests that 80–85% of incorporated xyloglucan is segmentally rigid with the backbone adopting an extended ‘cellulosic’ conformation and probably aligned with cellulose chains. The remaining xyloglucan is more mobile and is assigned to cross‐bridges with, on average, a twisted backbone conformation. No evidence for specific involvement of side‐chain residues in binding is found, and the observation of cross‐bridges with a non‐fucosylated xyloglucan shows that fucose residues are not essential for network formation. Xyloglucan causes cellulose ribbons to become more amorphous and to have a decreased 1α/1β crystallite ratio without any significant alteration in ribbon diameter.

Based on the findings that levels of xyloglucan incorporation, the presence and lengths of cross‐bridges, and the modification of cellulosic molecular organization are all similar to those found in plant cell walls, we suggest that A. aceti ssp. xylinum is a more useful model for primary plant cell walls and their assembly than has previously been appreciated.
Potato plants expressing antisense and sense S‐adenosylmethionine decarboxylase (SAMDC) transgenes show altered levels of polyamines and ethylene: antisense plants display abnormal phenotypes
Authors: Kumar Amar , Taylor Mark A. , Arif Siti A. Mad , Davies Howard V.
Published: March 2003

A molecular approach has been used to study the role of polyamines in plant development by manipulating the expression of the S‐adenosylmethionine decarboxylase (SAMDC) gene. SAMDC is a key enzyme involved in the biosynthesis of the polyamines spermidine and spermine and is also known to influence the rate of biosynthesis of ethylene. Previously, a cDNA clone of the SAMDC gene of potato has been isolated and characterized. This cDNA clone has been used to make antisense and sense SAMDC constructs under the control of the 35S CaMV or tetracycline‐inducible promoters. Agrobacterium‐mediated transformation has been used to produce transgenic potato plants with the engineered antisense and sense SAMDC genes in order to downregulate or overexpress the SAMDC transcript, respectively. Decreases or increases in the level of SAMDC transcript in the antisense and sense transgenic plants were observed, respectively. Antisense transgenic plants which expressed the engineered SAMDC gene constitutively under the control of the 35S CaMV promoter with a duplicated enhancer region showed a range of stunted phenotypes with highly branched stems, short internodes, small leaves and inhibited root growth. The abnormal characteristics of the antisense plants correlated with the altered levels of SAMDC transcript, SAMDC activity, polyamine content and rate of ethylene evolution. Attempts to produce sense transgenic plants with the 35S SAMDC sense construct were unsuccessful indicating that a constitutive overexpression of the engineered SAMDC is lethal to the plants. When antisense and sense SAMDC transgenes were expressed under the control of a tetracycline‐inducible promoter a number of transgenic plants were produced. In these antisense and sense plants, significant changes in the level of SAMDC transcript, SAMDC activity and free polyamine content were observed on tetracycline‐induction of detached leaves.
Phytochrome B mediates the photoperiodic control of tuber formation in potato
Authors: Jackson Stephen D. , Heyer Arnd , Dietze Jessyca , Prat Salomé
Published: March 2003

To determine whether phytochrome B is involved in the response of potato plants to photoperiod, a potato PHYB cDNA fragment was inserted in the antisense orientation behind the 35S CaMV promoter in Bin19 and this construct was transformed into Solanum tuberosum ssp. andigena plants which normally require short days for tuberization. Two independent transformants were obtained that had much lower levels of PHYB mRNA and protein, and which exhibited phenotypes characteristic of phyB mutants, for example, elongated stems and decreased chlorophyll content. The level of phyA, and of several phytochrome A‐controlled responses, was unaffected in these plants. The photoperiodic control of tuberization in these antisense PHYB plants was abolished, the plants tuberizing in short day, long day, or short day plus night break conditions. This result shows that phytochrome B is required for the photoperiodic control of tuberization in potato (Solanum tuberosum ssp. andigena) and that it regulates this developmental process by preventing tuber formation in non‐inductive photoperiods rather than by promoting tuberization in inductive photoperiods.
Analysis of phytochrome‐deficient yellow‐green‐2 and aurea mutants of tomato
Authors: van Tuinen Ageeth , Hanhart Corrie J. , Kerckhoffs L. Huub J. , Nagatani Akira , Boylan Margaret T. , Quail Peter H. , Kendrick Richard E. , Koornneef Maarten
Published: March 2003

Two alleles of the yellow‐green‐2 (yg‐2) and eight different alleles of the aurea (au) locus of tomato (Lycopersicon esculentum Mill.) were compared. All are characterized by a paler green colour compared with wild‐type (WT), an elongated hypocotyl in red light, and low or below detection limits of spectrophotometrically active phytochrome. Hypocotyl length was variable in white light, ranging from that of WT to more elongated. Immunochemical analysis revealed that etiolated seedlings of the yg‐2 mutant have approximately 25% of the WT level of phytochrome A protein (PHYA), whereas that of phytochrome B protein (PHYB) is normal. In this it resembles the au mutant. The au,yg‐2 double mutant has a more extreme chlorophyll deficiency than either parent. Since the yg‐2 and au mutants have a less severe phenotype at the adult stage, that is, are leaky, the additive effect can be explained by assuming that the mutants control two steps in the chromophore biosynthesis pathway. Combination, by crossing, of the yg‐2 and au mutants with a transgenic tomato line that overexpresses oat phytochrome A3 (PhyA‐3) essentially failed to restore the WT phenotype under white fluorescent light conditions, although under greenhouse conditions some evidence for increased sensitivity to light was observed. Immunochemically, oat PHYA‐3 protein is detectable in both the yg‐2,PhyA‐3 and au,PhyA‐3‘double’ mutants. Spectrophotometrical analysis, however, revealed that holophytochrome was undetectable in the yg‐2,PhyA‐3 and au,PhyA‐3‘double’ mutants. These results are compatible with both mutants being disturbed in phytochrome chromophore biosynthesis.
The ELONGATED gene of Arabidopsis acts independently of light and gibberellins in the control of elongation growth
Authors: Halliday Karen , Devlin Paul F. , Whitelam Garry C. , Hanhart Corrie , Koornneef Maarten
Published: March 2003

A novel elongated mutant has been isolated from EMS‐mutagenized populations of the Arabidopsis thaliana ga4 mutant. After backcrossing with the Landsberg erecta (Ler) wild‐type (WT) followed by selling, the mutant phenotype was identified in the GA4 background. Seedlings of the mutant, which has been named elg (elongated), are characterized by elongated hypocotyls and petioles, leaves that are narrow and somewhat epinastic and early flowering. Allelism tests with the hy1–hy5 mutants indicate that elg is not allelic with any of these long‐hypocotyl mutants. From linkage analyses, the location of elg on chromosome 4, between cer2 and ap2 has been established. The pleiotropic phenotype of elg seedlings is suggestive of a disruption of phytochrome and/or gibberellin (GA) function. Although the elg mutant displays a light‐dependent long‐hypocotyl phenotype, elg seedlings retain a full range of photomorphogenic responses and the elg mutation acts additively with the photomorphogenic mutants phyB, hy1 and hy2. This suggests that ELG acts independently of phytochrome action. The elg mutation partially suppresses the effect of GA‐deficiency on elongation growth, and, although elg ga1 seedlings are more elongated than ga1 seedlings, both genotypes respond in the same way to applied GA. That applied GA and the elg mutation interact additively suggests that ELG acts independently of GA action.
Tissue and intra‐cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy
Authors: Bonfiglioli Roderick G. , Webb Daryl R. , Symons Robert H.
Published: March 2003

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.
Isolation of microtubule‐associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus
Authors: Chan Jordi , Rutten Twan , Lloyd Clive
Published: March 2003

A method for biochemically isolating microtubule‐associated proteins (MAPs) from the detergent‐extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self‐assemble. By adding it to rhodamine‐conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot‐affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti‐MAP 65, the nuclear periphery is also stained. Both the anti‐65 kDa and the anti‐120 kDa antibodies stain cortical MTs in detergent‐extracted, substrate‐attached plasma membrane disks (‘footprints’). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.
Tobacco plants epigenetically suppressed in phenylalanine ammonia‐lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus
Authors: Pallas Jacqueline A. , Paiva Nancy L. , Lamb Chris , Dixon Richard A.
Published: March 2003

The response of tobacco (Nicotiana tabacum L. cv. Xanthinc) plants, epigenetically suppressed for phenylalanine ammonia‐lyase (PAL) activity, was studied following infection by tobacco mosaic virus (TMV). These plants contain a bean PAL2 transgene in the sense orientation, and have reduced endogenous tobacco PAL mRNA and suppressed production of phenylpropanoid products. Lesions induced by TMV infection of PAL‐suppressed plants are markedly different in appearance from those induced on control plants that have lost the bean transgene through segregation, with a reduced deposition of phenofics. However, they develop at the same rate as on control tobacco, and pathogenesis‐related (PR) proteins are induced normally upon primary infection. The levels of free salicylic acid (SA) produced in primary inoculated leaves of PAL‐suppressed plants are approximately fourfold lower than in control plants after 84 h, and a similar reduction is observed in systemic leaves. PR proteins are not induced in systemic leaves of PAL‐suppressed plants, and secondary infection with TMV does not result in the restriction of lesion size and number seen in control plants undergoing systemic acquired resistance (SAR). In grafting experiments between wild‐type and PAL‐suppressed tobacco, the SAR response can be transmitted from a PAL‐suppressed root‐stock, but SAR is not observed if the scion is PAL‐suppressed. This indicates that, even if SA is the systemic signal for establishment of SAR, the amount of pre‐existing phenylpropanoid compounds in systemic leaves, or the ability to synthesize further phenylpropanoids in response to the systemic signal, may be important for the establishment of SAR. Treatment of PAL‐suppressed plants with dichloro‐isonicotinic acid (INA) induces PR protein expression and SAR against subsequent TMV infection. However, treatment with SA, while inducing PR proteins, only partially restores SAR, further suggesting that de novo synthesis of SA, and/or the presence or synthesis of other phenylpropanoids, is required for expression of resistance in systemic leaves.
Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS‐box genes
Authors: Davies Brendan , Di Rosa Alexandra , Eneva Tinka , Saedler Heinz , Sommer Hans
Published: March 2003

Floral organ identity is largely controlled by the spatially restricted expression of several MADS‐box genes. In Antirrhinum majus these organ identity genes include DEF, GLO and PLE. Single and double mutant analyses indicated that the type of organ found in a particular whorl is dependent on which combination of these genes is expressed there. This paper reports the ectopic expression of Antirrhinum organ identity genes, alone and in combinations, in transgenic tobacco. Although the phenotypes are broadly in agreement with the genetic predictions, several unexpected features are observed which provide information concerning the action of the organ identity genes. The presumed tobacco homologue of DEF, NTDEF, has been isolated and used to investigate the influence of ectopic expression of the Antirrhinum organ identity genes on the endogenous tobacco genes. Analysis of the spatial and temporal expression patterns of NTDEF and NTGLO reveals that the boundaries are not coincident and that differences exist in the regulatory mechanisms of the two genes concerning both induction and maintenance of gene expression. Evidence is provided which indicates that organ development is sensitive to the relative levels of organ identity gene expression. Expression of the organ identity genes outside the flower or inflorescence produced no effects, suggesting that additional factors are required to mediate their activity. These results demonstrate that heterologous genes can be used to predictably influence floral organ identity but also reveal the existence of unsuspected control mechanisms.
Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.)
Authors: Abel Gernot J.W. , Springer Franziska , Willmitzer Lothar , Kossmann Jens
Published: March 2003

Three isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber‐specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full‐length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non‐detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.
The cloning of two Arabidopsis genes belonging to a phosphate transporter family
Authors: Smith Frank W. , Ealing Paul M. , Dong Bei , Delhaize Emmanuel
Published: March 2003

Two genes, APT1 and APT2, with DNA sequences that exhibit significant sequence identity to yeast and fungal H+/orthophosphate co‐transporters, have been isolated from Arabidopsis thaliana. These genes are genetically linked and map to chromosome 5 between markers g4028 and m435. The genes encode almost identical 524 amino acid polypeptides and are predicted to contain 12 membrane‐spanning domains. Both APT1 and APT2 are predominantly expressed in A. thaliana root tissues. The level of expression of both genes in roots is regulated by the phosphorus status of the plant, being considerably enhanced when the plants were deprived of an external phosphate supply. The APT1 and APT2 polypeptides are likely to be associated with the membrane transport of phosphate within A. thaliana roots.
The structures of integration sites in transgenic rice
Authors: Takano Makoto , Egawa Hitomi , Ikeda Joh‐E , Wakasa Kyo
Published: March 2003

Extensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT‐rich region and/or at the repetitive sequence region, including a SAR‐like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.
Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships
Authors: Lacombe Eric , Hawkins Simon , Van Doorsselaere Jan , Piquemal Joël , Goffner Deborah , Poeydomenge Odile , Boudet Alain‐M. , Grima‐Pettenati Jacqueline
Published: March 2003

Cinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e. the first specific step in the synthesis of the lignin monomers. The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here. The identity of the EUCCR cDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coli. Sequence analysis revealed remarkable homologies with dihydroflavonol‐4‐reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway. Moreover, significant similarities were found with mammalian 3β‐hydroxysteroid dehydrogenase and bacterial UDP‐galactose‐4‐epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3β‐hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily. In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots. In agreement with the involvement of CCR in lignification, the CCR transcript was shown to be expressed in lignified organs, i.e. root and stem tissues, and was localized mainly in young differentiating xylem. On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins. This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content. This is particularly important for woody plants such as Eucalyptus which are used for pulp making.
Co‐ordinated induction of mRNAs for extracellular invertase and a glucose transporter in Chenopodium rubrum by cytokinins
Authors: Ehneß Rainer , Roitsch Thomas
Published: March 2003

Extracellular or cell wall invertase is regarded as crucial to supply sink tissues with carbohydrates via an apoplastic pathway. A cell wall invertase from Chenopodium rubrum was purified to homogeneity and the corresponding cDNA encoding CIN1 was identified via peptide sequences. The CIN1 mRNA was found to be highly induced by physiological concentrations of both adenine‐ and phenylurea‐derived cytokinins in suspension culture cells. This was paralleled both by a higher steady‐state protein level and a higher enzyme activity of the extracellular invertase. The cytokinin‐inducible accumulation of CIN1 mRNA in tissues of C. rubrum plants supports the physiological significance of this regulatory mechanism. In contrast to the extracellular sucrose cleaving enzyme, the mRNA levels of the two putative intracellular invertases CIN2 and CIN3 and of sucrose synthase were not elevated. In addition, it has been found that the accumulation of mRNA for one out of three hexose transporters present in the suspension culture cells is induced co‐ordinately with the mRNA for extracellular invertase by cytokinins. It has been shown that this regulatory mechanism results in higher uptake rates both for sucrose, via the hexose monomers, and for glucose. The increased level of both extracellular invertase and hexose transporters and the resulting higher carbohydrate supply are discussed with respect to the control of carbohydrate partitioning by plant hormones and the molecular basis for known physiological cytokinin responses such as the stimulation of cell division.
High mobility group proteins HMG‐1 and HMG‐I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter
Authors: Webster Carl I. , Packman Leonard C. , Pwee Keng‐Hock , Gray John C.
Published: March 2003

A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high‐level expression has been shown to interact with the high mobility group proteins HMG‐1 and HMG‐I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG‐1 protein of 154 amino acid residues containing a single HMG‐box and a C‐terminal acidic tail and an HMG‐I/Y‐like protein of 197 amino acid residues containing four AT‐hooks have been isolated and expressed in Escherichia coli to provide large amounts of full‐length proteins. DNase I footprinting identified eight binding sites for HMG‐I/Y and six binding sites for HMG‐1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T‐rich DNA, revealed that HMG‐I/Y binding was 400‐fold more sensitive than HMG‐1 binding. Binding‐site selection from a pool of random oligonucleotides indicated that HMG‐I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG‐1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.
Protein dephosphorylation mediates salicylic acid‐induced expression of PR‐1 genes in tobacco
Authors: Conrath Uwe , Silva Herman , Klessig Daniel F.
Published: March 2003

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis‐related (PR) proteins such as the acidic PR‐1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco ‐ elevated H2O2 levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA‐effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR‐1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA‐mediated induction of PR‐1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K‐252a and staurosporine induced PR‐1 gene expression. PR‐1 gene induction by K‐252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K‐252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR‐1 gene induction. The SA‐inducible acidic PR‐2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.
Cell‐cycle‐regulated transcription of A‐ and B‐type plant cyclin genes in synchronous cultures
Authors: Ito Masaki , Marie‐Claire Criqui , Sakabe Midori , Ohno Takeyoshi , Hata Shingo , Kouchi Hiroshi , Hashimoto Junji , Fukuda Hiroo , Komamine Atsushi , Watanabe Akira
Published: March 2003

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence of CYS is similar to that of A‐type cyclins, and CYM belongs to the group of B‐type cyclins. In a fashion similar to the pattern of expression seen for A‐type and B‐type cyclins in mammalian cells, CYS is expressed before CYM in C. roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2 and M phases of the cell cycle. Tobacco homologs of the two genes showed similar cell‐cycle dependent expression patterns in synchronous cultures of tobacco BY2 cells. In both systems, CYS was expressed much earlier in the cell cycle than most other plant A‐type cyclins, and hence CYS along with the soybean cyc1Gm can be classified into a distinct subclass. The activities of CYM and CYS promoters during the cell cycle were analyzed in stably transformed tobacco BY2 cells. Cyclin promoter sequences of 0.5 kb could confer the typical cell‐cycle‐dependent expression to the β‐glucuronidase (GUS) reporter gene: the CYS promoter directed S‐phase‐specific expression, whereas the CYM promoter drove M‐phase‐specific expression. These results indicate the important role of transcriptional regulation in the oscillations of cyclin mRNA levels during the cell cycle.