bluuebunny/crossref_metadata_2025 huggingface dataset:
Gibberellin promotes histone H1 kinase activity and the expression of cdc2 and cyclin genes during the induction of rapid growth in deepwater rice internodes
Authors: Sauter Margret , Mekhedov Sergei L. , Kende Hans
Published: March 2003

Partial submergence or treatment with either ethylene or gibberellin (GA) promotes rapid internodal growth in deepwater rice (Oryza sativa L.). Earlier work has shown that GA is the immediate hormonal signal for this growth response, which involves induction of the cell cycle at the G2/M phase transition and subsequent enhancement in the rate of DNA synthesis. In all eukaryotes, onset of mitosis is regulated by the p34cdc2/CDC28 protein kinase, whose activity is assayed by in vitro phosphorylation of histone H1. It was found that GA enhanced the activity of p34cdc2/CDC28‐like histone H1 kinase in the intercalary meristem of rice internodes. The enzyme activity showed a sharp peak that correlated with a decrease in the population of cells in the G2 phase during the first 4 h of GA treatment but not with changes in DNA synthesis. The level of histone H1 kinase activity increased again when cell division activity in the intercalary meristem is known to be high. The expression of two cdc2 homologs was examined. The mRNA level of one of these, cdc2Os‐2, was increased after 1 h of GA treatment, whereas the mRNA level of the other, cdc2Os‐1, was not affected. Two cDNAs, cycOs1 and cycOs2, which show high homology to cyclin cDNAs, were cloned from rice. They share 75.1% sequence identity at the amino acid level, and both of them are encoded by mRNAs of 1.6 kb. Expression of the two corresponding cyclin genes was enhanced by GA, and the time course of the induction was compatible with a role for both cyclins in regulating the G2/M phase transition. The cyclins were expressed in the intercalary meristem and the elongation zone of the internode, but the GA‐induced increase in transcript levels was restricted to the meristem only. The results support the hypothesis that induction of mitosis by GA is brought about by increased p34cdc2/CDC28 protein kinase activity, which may be the result of transcriptional activation of the cdc2Os‐2, cycOs1 and cycOs2 genes.
Identification of two MADS box genes that are expressed in the apical meristem of the long‐day plant Sinapis alba in transition to flowering
Authors: Menzel Gerhard , Apel Klaus , Melzer Siegbert
Published: March 2003

To understand the molecular mechanisms involved in the transition from vegetative to reproductive growth, the photoperiodic‐controlled induction of flowering in the long‐day plant Sinapis alba was used to screen for regulatory genes which are expressed upon induction in the apical meristem. By using the conserved MADS box‐encoding region of the organ identity gene AGAMOUS, the genes SaMADS A and SaMADS B were identified which are expressed in transition stages of mustard. RNA blot analysis has confirmed that the transcript levels of both genes are drastically increased shortly after the induction of flowering and that both genes are expressed earlier than the known MADS box genes. In situ hybridization studies have shown that the expression of the genes is restricted to the apical meristem of induced plants during early phases of reproductive development. The expression of SaMADS A is first detectable in the central zone of the meristem, a region where the earliest changes of an evoked meristem could be detected by classical physiological methods, suggesting that SaMADS A may have an important function during the transition to flowering.
Gametophyte‐specific transposition of the maize Ds element in transgenic tobacco
Authors: Firek Simon , Martin David J. , Roberts Michael R. , Sturgess Faye , Scott Rod , Draper John
Published: March 2003

To assess the potential advantages of a transposon‐tagging system based on gametophyte‐specific transposition a fusion between the anther‐specific Arabidopsis thaliana apg promoter and the maize Ac transposase gene was constructed and introduced into tobacco. The ability of this transposase source to activate Ds transposition in a developmentally controlled manner was monitored by crossing to plants harbouring the cell autonomous excision marker gene construct, Ds—SPT. A number of fully green, streptomycin‐resistant seedlings resulting from germinal transposition events were observed in the progeny of apg‐TPase x Ds—SPT F1 plants. Streptomycin‐resistant sectors were not observed in either F1 seedlings or F2 progeny, indicating a complete lack of somatic excision. Further crosses of apg—TPase sources to plants containing Ds—bar herbicide selection excision marker constructs gave reproducible gametophytic excision frequencies of up to 0.3%. Sequencing of Ds excision sites from F2 seedlings derived from single F1 plants revealed various sequence alterations in the original Ds insertion ‘footprint’ indicative of independent Ds excision events. Independent re‐insertion was confirmed by Southern analysis of F2 siblings. It is concluded that apg‐controlled Ac transposase expression activates male gametophyte‐specific Ds transposition.
Isolation and characterization of a cDNA that encodes maize uroporphyrinogen III methyltransferase, an enzyme involved in the synthesis of siroheme, which is a prosthetic group of nitrite reductase
Authors: Sakakibara Hitoshi , Takei Kentaro , Sugiyama Tatsuo
Published: March 2003

A full‐length cDNA clone, pZmSUMT1, encoding an S‐adenosyl‐l‐methionine‐dependent uroporphyrinogen III C‐methyltransferase (SUMT; EC 2.1.1.107) of maize was isolated from a root cDNA library. pZmSUMT1 had an insert of 1.7 kb and the amino acid sequence deduced from the open reading frame of the cDNA was similar to that of SUMT from various bacteria and also to the SUMT catalytic region of siroheme synthase (cysG) from Escherichia coli. Overproduction of ZmSUMT1 in a cysG mutant of E. coli eliminated the requirement of the strain for cysteine. Transcripts for ZmSUMT1 accumulated rapidly in both roots and leaves in response to the addition of nitrate to the culture medium. The effects of biochemical inhibitors on the nitrate‐dependent induction of the gene for ZmSUMT1 coincided with the effects on the genes for other nitrate‐assimilatory enzymes, nitrate reductase and nitrite reductase. An import experiment in vitro suggested that the gene product might be located in plastids. The results together indicate that ZmSUMT1 might be involved in the synthesis of siroheme, a prosthetic group of nitrite reductase, and that the expression of its gene is coregulated with that of other nitrate‐assimilatory genes.
Characterization of proteins that interact with the GTP‐bound form of the regulatory GTPase Ran in Arabidopsis
Authors: Haizel Thomas , Merkle Thomas , Pay Aniko , Fejes Erzsebet , Nagy Ferenc
Published: March 2003

Ran, a small soluble GTP‐binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells. Genes encoding Ran‐like proteins have been isolated from different higher plant species. Overexpression of plant Ran cDNAs, similarly to their mammalian/yeast homologues, suppresses the phenotype of the pim46‐1 cell cycle mutant in yeast cells. The mammalian/yeast Ran proteins have been shown to interact with a battery of Ran‐binding proteins, including the guanidine nucleotide exchange factor RCC1, the GTPase‐activating Ran‐GAP, nucleoporins and other Ran‐binding proteins (RanBPs) specific for Ran‐GTP. Here, the characterization of the first Ran‐binding proteins from higher plants is reported. The yeast two‐hybrid system was used to isolate cDNA clones encoding proteins of approximately 28 kDa (At‐RanBP1a, At‐RanBP1b) that interact with the GTP‐bound forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana. The deduced amino acid sequences of the At‐RanBP1s display high similarity (60%) to mammalian/yeast RanBP1 proteins and contain the characteristic Ran‐binding domains. Furthermore, interaction of the plant Ran and RanBP1 proteins, is shown to require the acidic C‐terminal domain (‐DEDDDL) of Ran proteins in addition to the presence of an intact Ran‐binding domain. In whole cell extracts, the GST‐RanBP1a fusion protein binds specifically to GTP‐Ran and will not interact with Rab/Ypt‐type small GTP‐binding proteins. Finally, in good agreement with their proposed biological function, the At‐Ran and the At‐RanBP genes are expressed coordinately and show the highest level of expression in meristematic tissues.
The C‐terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER
Authors: Gomord Véronique , Denmat Lise‐Anne , Fitchette‐Lainé Anne‐Catherine , Satiat‐Jeunemaitre Béatrice , Hawes Chris , Faye Loïc
Published: March 2003

Proteins are co‐translationally transferred into the endo‐plasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C‐terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C‐terminal extension was fused to the vacuolar or extracellular (Δpro) forms of sporamin. The resulting SpoHDEL or ΔproHDEL, as well as Spo and Δpro, were expressed at high levels in transgenic tobacco cells (Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or Δpro induces accumulation of these sporamin forms in a compartment that co‐purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or ΔproHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone‐bound proteins that escape the ER to the plant lysosomal compartment for degradation.
Novel ‘dot‐blot’ assays for glycosyltransferases and glycosylhydrolases: optimization for xyloglucan endotransglycosylase (XET) activity
Authors: Fry Stephen C.
Published: March 2003

A novel dot‐blot method is described for the rapid, semi‐quantitative assay of xyloglucan endotransglycosylase (XET) activity. Test paper was prepared by impregnating Whatman No. 1 filter paper with xyloglucan (2.5 g m−2 paper) plus a sulphorhodamine conjugate of the xyloglucan oligosaccharide, XLLG (1 µmol m−2). Spots of putative enzyme solution were applied to the test paper, which was incubated under humid conditions. XET‐catalysed transglycosylation produced a xyloglucan‐sulphorhodamine conjugate. The test paper was washed to remove unreacted oligosaccharide, leaving fluorescent spots of the xyloglucan‐sulphorhodamine reaction product hydrogen‐bonded to the paper. The assay is suitable for testing crude plant extracts (including Arabidopsis stems squashed directly on to the test paper). The test paper is also suitable for tissue‐printing of plant specimens and for preparation of zymograms after non‐denaturing gel electrophoresis. Tissue prints indicated that extractable XET activity tends to be concentrated in the outer tissues and sometimes in or near the vascular bundles. The method reported can be quantified by fluorimetry and can potentially be extended to the assay of other transglycosylases (including polysaccharide synthases, fructosyl‐transferase and dextransucrase) and hydrolases.
Efficient gene targeting in the moss Physcomitrella patens
Authors: Schaefer Didier G. , Zrÿd Jean‐Pierre
Published: March 2003

The moss Physcomitrella patens is used as a genetic model system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. Transformation experiments designed to target three single‐copy genomic loci were performed to determine the efficiency of gene targeting in this plant. Mean transformation rates were 10‐fold higher with the targeting vectors and molecular evidence for the integration of exogenous DNA into each targeted locus by homologous recombination is provided. The efficiency of gene targeting determined in these experiments is above 90%, which is in the range of that observed in yeast and several orders of magnitude higher than previous reports of gene targeting in plants. Thus, gene knock‐out and allele replacement approaches are directly accessible to study plant development in the moss Physcomitrella patens. Moreover, efficient gene targeting has so far only been observed in lower eukaryotes such as protozoa, yeasts and filamentous fungi, and, as shown here the first example from the plant kingdom is a haplobiontic moss. This suggests a possible correlation between efficient gene targeting and haplo‐phase in eukaryotes.
An Arabidopsis thaliana cDNA complementing a hamster apoptosis suppressor mutant
Authors: Gallois Patrick , Makishima Tomoko , Hecht Valérie , Despres Barbara , Laudié Michele , Nishimoto Takeharu , Cooke Richard
Published: March 2003

Programmed cell death or apoptosis is a process in which unwanted cells are eliminated during growth and development. In mammals, several genes have been identified whose products are necessary to prevent entry into the apoptotic process. We have isolated a clone from an Arabidopsis thaliana cDNA library whose predicted translation product shows highly significant similarity to the mammalian defender against apoptotic death 1 (DAD1) protein. Transformation of the mutant hamster tsBN7 cells, which undergo apoptosis at restrictive temperature, demonstrates that the plant protein is as efficient as human DAD1 in rescuing these hamster cells from apoptosis. In contrast to mammals, Southern hybridisation and genomic data indicate that there are probably two genes in Arabidopsis thaliana. Northern blot analysis shows that AtDAD transcripts are present in all tissues examined, although the abundance of the transcripts is reduced in siliques during the maturation and desiccation phase of the seed. This is the first experimental proof that a homologue of an animal gene involved in apoptosis exists in plants and the first demonstration of complementation of a vertebrate mutant by a plant cDNA. Our results suggest that this process of suppression of apoptosis has been conserved in animals and plants.
Yeast 5‐aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco
Authors: Zavgorodnyaya Anna , Papenbrock Jutta , Grimm Bernhard
Published: March 2003

Synthesis of the tetrapyrrole precursor 5‐aminolevulinate (ALA) in plants starts with glutamate and is a tRNA‐dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA‐S) in mitochondria. A gene encoding a fusion protein of yeast ALA‐S with an amino‐terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild‐type tobacco and a chlorophyll‐deficient transgenic line expressing glutamate 1‐semi‐aldehyde aminotransferase (GSA‐AT) antisense RNA. Expression of ALA‐S in the GSA‐AT antisense transgenic line provided green‐pigmented co‐transformants similar to wild‐type in chlorophyll content, while transformants derived from wild‐type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA‐S to the ALA supply for chlorophyll synthesis. ALA‐S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA‐S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA‐AT. Steady‐state RNA and protein levels and, consequently, the enzyme activity of GSA‐AT were reduced in plants expressing ALA‐S. In analogy to the light‐dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end‐products is proposed, which co‐ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA‐AT gene expression. The genetically engineered tobacco plants containing the yeast ALA‐S activity demonstrate functional complementation of the catalytic activity of the plant ALA‐synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.
ACL5: an Arabidopsis gene required for internodal elongation after flowering
Authors: Hanzawa Yoshie , Takahashi Taku , Komeda Yoshibumi
Published: March 2003

In rosette plants, the formation of initial flowers is closely linked to the lengthening of internodes (bolting). In order to clarify the molecular basis of bolting, mutants with reduced lengths of internodes were screened. This paper presents the identification and characterization of recessive mutations in ACAULIS5 (ACL5), a gene required for internodal growth in Arabidopsis thaliana. Unlike previously described mutants with reduced size of organs, the acl5 mutant has a severe defect that is restricted to the process of cell elongation after transition to the reproductive stage and shows no phenotype before floral induction. The results of RNA blot hybridizations showed that the acl5 mutation causes a striking reduction in the transcript levels of genes encoding the tonoplast intrinsic protein (γ‐TIP) and the endoxyloglucan transferase (EXGT‐A1), both of which have recently been suggested to be important for cell elongation. Furthermore, our morphological study indicates that the mutation also causes proliferative arrest of the apical inflorescence meristem. These results strongly suggest that, during the reproductive phase, the wild‐type ACL5 gene product has a critical function not only in the control of elongation growth of organs but also in the continued maintenance of the proliferative activity of flower‐producing meristems.
Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter
Authors: Smith Frank W. , Hawkesford Malcolm J. , Ealing Paul M. , Clarkson David T. , Vanden Berg Peter J. , Belcher Ann R. , Warrilow Andrew G.S.
Published: March 2003

A cDNA encoding a high‐affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (Mr = 72 550), which is predicted to have 12 membrane‐spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The Km for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH‐dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co‐transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur‐starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re‐supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O‐acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O‐acetylserine acting as a positive regulator.
Transposon sequences drive tissue‐specific expression of the maize regulatory gene R‐s
Authors: May Bruce P. , Dellaporta Stephen L.
Published: March 2003

The R family of genes in maize encodes transcription factors that activate anthocyanin synthesis. The R‐r complex at the r locus contains two components, of which one, the aleurone‐pigmenting R‐s subcomplex, is an unusual promoter flanked by two head‐to‐head, nearly identical coding regions. The central region of the promoter, designated σ, contains inverted terminal repeats and subterminal repeats, both of which are features of the CACTA transposon family. Transcription from the R‐s promoter was strongest in aleurone but transcripts had a surprisingly variable initiation site and splicing pattern. Microprojectile bombardment indicated that the sequences introduced by transposition act as an enhancer of transcription in aleurone but not in coleoptile or mesocotyl tissue. Moreover, the region containing the subterminal repeats was able to activate expression when fused to a minimal promoter, suggesting that a factor that binds to the repeats may be responsible. Regions downstream of σ encode a leader containing three ORFs, two of which are variably removed by splicing. The results presented here show that transposons can mediate the rearrangement of genes and confer new splicing and expression patterns. In the case of the r complex, this has resulted in a new phenotype, the pigmentation of aleurone cells.
Transgenic analysis of tomato endo‐β‐1,4‐glucanase gene function. Role of cel1 in floral abscission
Authors: Lashbrook Coralie C. , Giovannoni James J. , Hall Bradford D. , Fischer Robert L. , Bennett Alan B.
Published: March 2003

Endo‐β‐1,4‐glucanase (EGase) expression in ripening tomato fruit and abscising tomato flowers reflects the overlapping accumulation of structurally divergent gene transcripts, including EGase1 (Cel1) and EGase2 (Cel2). Transgenic plants in which cel1 expression was specifically inhibited were evaluated in order to assess the potential role(s) played by Cel1 during fruit ripening and floral abscission. The constitutive expression of an antisense cel1 gene driven by the 35S promoter of cauliflower mosaic virus resulted in the reduction of Cel1 mRNA to trace levels in both ripening fruit and abscising abscission zones of transgenic tomato plants. Transgenic fruit in which cel1 expression was reduced to less than 0.1% of wild‐type levels exhibited normal growth and softening behaviour. Transgenic floral abscission zones expressing 5–7% of wild‐type levels of Cel1 mRNA exhibited an incidence of abscission reduced by up to one third. The incomplete loss of abscission ability by transgenic flowers may indicate that the contribution of Cel1 activity is insufficient to account for all floral abscission and attests to the presence in tomato abscission zones of additional cell wall hydrolase activities, including Cel2.
Two bZIP proteins from Antirrhinum flowers preferentially bind a hybrid C‐box/G‐box motif and help to define a new sub‐family of bZIP transcription factors
Authors: Martínez‐García Jaime F. , Moyano Enriqueta , Alcocer Marcos J. C. , Martin Cathie
Published: March 2003

Two genes encoding bZIP proteins are expressed in flowers of Antirrhinum majus, predominantly in vascular tissues, carpels and anthers. The sequences of cDNA clones encoding these proteins show them to belong to a distinct sub‐family of bZIP proteins which also includes LIP19 from rice and MLIPI5 and OBF1 from maize. The sub‐family is characterized by the inclusion of very small proteins consisting of essentially a basic domain and a long leucine zipper. Members also have a conserved upstream open reading frame (uORF) in their 5′ leader sequences, implying a common mode of post‐transcriptional control. In vitro, the Antirrhinum bZIP proteins preferentially bind to a novel hybrid C‐box/G‐box motif which is found in the promoters of some plant histone genes and of some nuclear‐encoded genes with plastidial protein products. Expression of the bZIP proteins in transgenic tobacco under control of the CaMV 35S promoter supports the view that they can regulate expression of genes which contain the preferred target motif within their regulatory sequences, although both enhancement and repression of transcript levels of target genes were observed, indicating that the bZIP proteins probably interact with other factors to modulate transcription in different ways, as has been observed for the small MAF family of bZIP proteins in vertebrates.
Low temperature signal transduction during cold acclimation: protein phosphatase 2A as an early target for cold‐inactivation
Authors: Monroy Antonio F. , Sangwan Veena , Dhindsa Rajinder S.
Published: March 2003

The authors have previously shown that cold acclimation and cold acclimation‐specific (cas) gene expression in alfalfa require cold‐triggered calcium influx and phosphorylation of specific pre‐existing proteins. In this study, the authors used the expression of cas15 gene as an end‐point marker to examine the role of protein phosphorylation in low temperature signal transduction in alfalfa cells. Whereas the protein kinase inhibitor staurosporine prevented the cold induction of cas15, the protein phosphatase inhibitor okadaic acid induced the cas15 at 25°C. Upon exposure of cells to cold, total cellular protein phosphatase activity rapidly declined by 30% but all of this decline could be attributed to an almost complete inhibition of protein phosphatase 2A (PP2A). This cold‐inactivation of PP2A was found to be mediated by calcium influx and could be reproduced at 25°C by treating the cells with the calcium ionophore A23187 or with the calcium channel agonist Bay K8644. On exposure of cells to cold, the transcript and protein levels of the PP2A catalytic subunit (PP2Ac) did not decline, but the binding of anti‐PP2Ac antibody to native PP2Ac increased, indicating a demasking of the PP2Ac epitope. The possible mechanisms by which cold might inhibit PP2A are discussed.
Molecular cloning and functional analysis of the Arabidopsis thaliana DNA ligase I homologue
Authors: Taylor Richard M. , Hamer Marcus J. , Rosamond John , Bray Clifford M.
Published: March 2003

A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach. The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pI) of 8.20. Alignment of the A. thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae showed good sequence homology (42–45% identity, 61–66% similarity), particularly around the active site. Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species. Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S. cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase. The level of the DNA ligase transcript was not increased in A. thaliana seedlings in response to DNA damage induced by a period of enhanced UV‐B irradiation. However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells.
Targeting and modification of prokaryotic cell–cell junctions by tobacco mosaic virus cell‐to‐cell movement protein
Authors: Heinlein Manfred , Wood Malcolm R. , Thiel Teresa , Beachy Roger N.
Published: March 2003

The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell‐to‐cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.
Homology modelling and molecular dynamics aided analysis of ligand complexes demonstrates functional properties of lipid‐transfer proteins encoded by the barley low‐temperature‐inducible gene family, blt4
Authors: Keresztessy Zsolt , Hughes Monica A.
Published: March 2003

The homology modelling technique was used to predict the tertiary structures of three members of the low‐temperature‐inducible barley vegetative shoot epidermal lipid‐transfer protein (LTP) family, BLT4, on the basis of the X‐ray crystallographically determined three‐dimensional structure of a maize seedling LTP. Differences between the maize LTP and the BLT4 family include amino acid substitutions around the entrance and inside the predicted hydrophobic binding tunnels of these proteins. Because of the deletion of the loop region corresponding to Val60–Gly62 of the maize LTP from all three BLT4 LTPs, their internal hydrophobic tunnels are longer. Molecular dynamics modelling shows that BLT4.9 can accommodate hexadecanoic acid in its binding tunnel in similar conformation to the maize LTP. However, modelled cis,cis‐9,12‐octadecandienoic acid had a more favourable interaction with the BLT4.9 LTP than with the maize protein. Di‐cis,cis‐9,12‐octadecandienoyl phosphatidylglycerol and di‐cis,cis‐9,12‐octadecandienoyl phosphatidylcholine were modelled in the BLT4.9 structure with the fatty acyl group at position 1 embedded in the binding tunnel and the group at position 2 located on the solvent accessible surface of the protein. The results of the modelling suggest that the phospholipid headgroup can form hydrogen and salt bridges with polar and charged residues outside the binding tunnel and the exposed hydrocarbon chain interacts with hydrophobic amino acids on the surface. These results are consistent with the proposal that BLT4 LTPs have a lipid‐transfer function associated with frost acclimation in barley.
Brown‐midrib maize (bm1) – a mutation affecting the cinnamyl alcohol dehydrogenase gene
Authors: Halpin Claire , Holt Karen , Chojecki Jan , Oliver Duncan , Chabbert Brigitte , Monties Bernard , Edwards Keith , Barakate Abdellah , Foxon Geoffrey A.
Published: March 2003

Brown‐midrib (bm) mutants of maize have modified lignin of reddish‐brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co‐segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.
Molecular characterisation of a novel plant homeobox gene expressed in the maturing xylem zone ofPopulus tremula× tremuloides
Authors: Hertzberg Magnus , Of Olsson Ol
Published: March 2003

In trees, wood is produced from the vascular cambium. However, little is known about how differentiation of the cambial derivatives is controlled at the molecular level. Hybrid aspen,Populus tremula × tremuloides, is a fast‐growing, wood‐forming plant from which an efficient transformation and regeneration protocol has been developed. We use this species as a model to study xylem cell differentiation. In a cross‐section of the stem, cells in the cambial region at different stages of development can be seen in a radial distribution. We dissected out the cambial region and constructed a cDNA‐library from this tissue. The library was screened with a degenerated primer mix complementary to the third helix region of the homeobox. A number of cDNAs hybridising to the probe were isolated, purified, cloned and sequenced. Characterisation of the expression pattern of two such homeobox cDNAs revealed that one of the cDNAs, denotedPttHB1, was specifically expressed in the xylem maturation zone, indicating a role in the regulation of secondary wall formation, while the other, denotedPttHB2was expressed in differentiating xylem and phloem cells. Structurally, the two hybrid aspen homeobox genes are homologous to each other, but no close relationship could be identified between these genes and any other homeobox sequence described thus far, in plants or other eukaryotes. We therefore conclude that thePttHB1andPttHB2genes belong to a novel hitherto undescribed sequence class of homeobox genes. We designate this as PALE.
cDNA cloning, gene expression and subcellular localization of anthocyanin 5‐aromatic acyltransferase fromGentiana triflora
Authors: Fujiwara Hiroyuki , Tanaka Yoshikazu , Yonekura‐Sakakibara Keiko , Fukuchi‐Mizutani Masako , Nakao Masahiro , Fukui Yuko , Yamaguchi Masaatsu , Ashikari Toshihiko , Kusumi Takaaki
Published: March 2003

Acylation of anthocyanins with hydroxycinnamic acid derivatives is one of the most important and less understood modification reactions during anthocyanin biosynthesis. Anthocyanin aromatic acyltransferase catalyses the transfer of hydroxycinnamic acid moieties from their CoA esters to the glycosyl groups of anthocyanins. A full‐length cDNA encoding the anthocyanin 5‐aromatic acyltransferase (5AT) (
EC 2.3.1.153
) that acylates the glucose bound at the 5‐position of anthocyanidin 3,5‐diglucoside was isolated from petals of

Gentiana triflora

on the basis of the amino acid sequence of the purified enzyme. The isolated full‐length cDNA had an open reading frame of 469 amino acids and the calculated molecular weight was 52 736. The deduced amino acid sequence contains consensus motifs that are conserved among the putative acyl CoA‐mediated acyltransferases, and this indicates that 5AT is a member of a proposed superfamily of multifunctional acyltransferases (

St‐Pierre

et al

. (1998

)

Plant J.

14, 703–713). The cDNA was expressed in

Escherichia coli

and yeast, and confirmed to encode 5AT. The enzymatic characteristics of the recombinant 5AT were consistent with those of the native gentian 5AT. Immunoblot analysis using specific antibodies to 5AT showed that the 5AT protein is present in petals, but not in sepals, stems or leaves of

G. triflora

. RNA blot analysis showed that the

5AT

gene is expressed only in petals and that its expression is temporally regulated during flower development coordinately with other anthocyanin biosynthetic genes. Immunohistochemical analysis demonstrated that the 5AT protein is specifically expressed in the outer epidermal cells of gentian petals and that it is localized mainly in the cytosol.
Transgenic plants changed in carbon allocation pattern display a shift in diurnal growth pattern
Authors: Kehr Julia , Hustiak Frauke , Walz Christina , Willmitzer Lothar , Fisahn Joachim
Published: March 2003

Photosynthesis, partitioning of carbohydrates and growth have to be highly orchestrated to enable an efficient performance of plants. To study the diurnal relationships between carbon distribution and growth, we analysed transgenic potato plants with altered carbon allocation patterns. To modify carbohydrate supply of growing sinks, we used plants that accumulated starch as a consequence of inhibition in triose‐phosphate export from chloroplasts and plants that were genetically inhibited in starch production. Carbon assimilation was analysed by gas exchange and single cell analysis of source leaves. Export was determined by microanalysis of phloem exudates and internodal growth rates were measured by displacement transducers. Gas exchange measurements showed similar assimilation rates in the wild‐type and transgenic plants during the light period. Sugar analysis of phloem exudates and epidermal cells revealed a severe shift of sucrose concentrations in the individual plant lines. Moreover, epidermal cells turned out to be a potential storage site for carbohydrates in potato. Finally, we could demonstrate that changing the diurnal rhythm of carbon allocation results in a change in the diurnal growth pattern.
Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment
Authors: Hibberd Julian M. , Linley Philip J. , Khan Muhammad S. , Gray John C.
Published: March 2003

The green fluorescent protein gene (

gfp

) is a widely used reporter in both animals and plants. Fusions between the plastid

rrn

promoter or the

Escherichia coli trc

promoter and the

gfp

coding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of the

arc

6 mutant of

Arabidopsis thaliana

2–4 days after bombardment. The fusion of the

gfp

coding region to the bacterial

trc

promoter demonstrated that a bacterial promoter is active in chloroplasts

in vivo

. GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non‐photosynthetic plastids in a range of higher plants.
Floral dip: a simplified method forAgrobacterium‐mediated transformation ofArabidopsis thaliana
Authors: Clough Steven J. , Bent Andrew F.
Published: March 2003

The

Agrobacterium

vacuum infiltration method has made it possible to transform

Arabidopsis thaliana

without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor‐intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing

Agrobacterium tumefaciens

, 5% sucrose and 500 microliters per litre of surfactant Silwet L‐77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and

Agrobacterium

could be applied to plants at a range of cell densities. Repeated application of

Agrobacterium

improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high‐throughput transformation of

Arabidopsis

for efforts such as T‐DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone
Authors: Gubler Frank , Raventos Dora , Keys Margaret , Watts Robyn , Mundy John , Jacobsen John V.
Published: March 2003

GAMYB is an MYB transcription factor which is expressed in cereal aleurone cells in response to gibberellin (GA). HvGAMYB binds to the TAACAAA box of a high‐pI α‐amylase gene promoter and transcriptionally activates its expression. In this study, we examined the role of HvGAMYB in activating expression of other GA‐regulated genes encoding hydrolytic enzymes. In transient expression experiments, HvGAMYB transactivated expression of reporter genes fused to a low‐pI α‐amylase gene promoter, an EII(1–3,1–4)‐β‐glucanase gene promoter and a cathepsin B‐like protease promoter. HvGAMYB DNA binding specificity was determined using a PCR‐based random site selection using HvGAMYB fusion protein isolated fromE. coli. The deduced consensus closely resembled gibberellin response elements in α‐amylase promoters. Functional analysis of HvGAMYB by transient expression of C terminal HvGAMYB deletions in barley aleurone cells identified two transcriptional activation domains (TADs) which function in transcriptional regulation of both high‐ and low‐pI α‐amylase promoters. The same TADs were identified using a heterologous yeast expression system. Together, these results indicate that HvGAMYB has two TADs. These domains are C‐terminal to its DNA‐binding domain.
Self‐processing 2A‐polyproteins – a system for co‐ordinate expression of multiple proteins in transgenic plants
Authors: Halpin Claire , Cooke Susan E. , Barakate Abdellah , Amrani Abdel El , Ryan Martin D.
Published: March 2003

Achieving co‐ordinate, high‐level and stable expression of multiple transgenes in plants is currently difficult. Expression levels are notoriously variable and influenced by factors that act independently on transgenes at different genetic loci. Instability of expression due to loss, re‐arrangement or silencing of transgenes may occur, and is exacerbated by increasing numbers of transgenic loci and repeated use of homologous sequences. Even linking two or more genes within a T‐DNA does not necessarily result in co‐ordinate expression. Linking proteins in a single open reading frame – a polyprotein – is a strategy for co‐ordinate expression used by many viruses. After translation, polyproteins are processed into constituent polypeptides, usually by proteinases encoded within the polyprotein itself. However, in foot‐and‐mouth disease virus (FMDV), a sequence (2A) of just 16–20 amino acids appears to have the unique capability to mediate cleavage at its own C‐terminus by an apparently enzyme‐independent, novel type of reaction. This sequence can also mediate cleavage in a heterologous protein context in a range of eukaryotic expression systems. We have constructed a plasmid in which the 2A sequence is inserted between the reporter genes chloramphenicol acetyltransferase (CAT) and β‐glucuronidase (GUS), maintaining a single open reading frame. Here we report that expression of this construct in wheatgerm lysate and transgenic plants results in efficient cleavage of the polyprotein and co‐ordinate expression of active CAT and GUS. Self‐processing polyproteins using the FMDV 2A sequence could therefore provide a system for ensuring coordinated, stable expression of multiple introduced proteins in plant cells.
Phosphorylation‐dependent interactions between enzymes of plant metabolism and 14‐3‐3 proteins
Authors: Moorhead Greg , Douglas Pauline , Cotelle Valérie , Harthill Jean , Morrice Nick , Meek Sarah , Deiting Uta , Stitt Mark , Scarabel Marie , Aitken Alastair , MacKintosh Carol
Published: March 2003

Far‐Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)‐labelled 14‐3‐3 proteins. Binding to DIG‐14‐3‐3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14‐3‐3‐binding phosphopeptides, indicating that the 14‐3‐3 proteins bind to phosphorylated sites. The proteins that bound to the DIG‐14‐3‐3s were also immunoprecipitated from extracts with anti‐14‐3‐3 antibodies, demonstrating that they were bound to endogenous plant 14‐3‐3 proteins. 14‐3‐3‐binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14‐3‐3 proteins and specific elution with a 14‐3‐3‐binding phosphopeptide. Purified 14‐3‐3‐binding proteins included sucrose–phosphate synthase, trehalose‐6‐phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium‐dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14‐3‐3 proteins. In contrast to the phosphorylated NR‐14‐3‐3 complex which is activated by dissociation with 14‐3‐3‐binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14‐3‐3‐binding phosphopeptide and the phosphopeptide‐inhibited activity was reactivated by adding excess 14‐3‐3 proteins. Thus, 14‐3‐3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14‐3‐3‐binding status of defined target proteins change in response to extracellular stimuli.
Glycine and serine catabolism in non‐photosynthetic higher plant cells: their role in C1 metabolism
Authors: Mouillon Jean‐Marie , Aubert Serge , Bourguignon Jacques , Gout Elisabeth , Douce Roland , Rébeillé Fabrice
Published: March 2003

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C‐labelled substrates, we studied the catabolism of each of these amino acids in non‐photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra‐ hydrofolate (CH2‐THF) produced during this reaction did not equilibrate with the overall CH2‐THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two‐thirds) involved serine‐to‐glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC–SHMT enzymatic system, which is therefore required in all plant tissues.