bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of components of the oxidative cross‐linking of glycoproteins and of callose synthesis in papillae formed during the interaction between non‐pathogenic strains ofXanthomonas campestris andFrench bean mesophyll cells
Authors: Brown Ian , Trethowan Jonathan , Kerry Maria , Mansfield John , Bolwell G. Paul
Published: March 2003

Immunogold labelling was used to probe the responses of mesophyll cells in French bean (

Phaseolus vulgaris
 L.) to anhrpAmutant ofXanthomonas campestrispv.vesicatoriaand a saprophytic strain ofX.c.The non‐pathogenic strains both caused localized alterations to the plant cell wall and formation of large papillae in adjacent cells. Immunocytochemistry showed the co‐localization, in the cell wall and paramural deposits, of anMr 42 000 proline‐rich glycoprotein with chitin‐binding activity (CBPRP) and the enzyme responsible for its immobilization, anMr 46 000 peroxidase. The CBPRP appeared to lose antigenicity after cross‐linking, and, unlike the peroxidase, was not detected consistently in the extracellular matrix that encapsulated bacteria onto the plant cell wall. The peroxidase may have a dual function in both the generation and utilization of H2O2 for cross‐linking of proteins and phenolics during the construction of papillae. A burst of H2O2 was detected 1–5 h after inoculation at reaction sites by histochemical staining with cerium chloride. Progressive expansion of papillae and cell‐wall alterations was, however, not associated with the maintenance of high levels of H2O2. Co‐localization of callose and anMr 65 000 polypeptide component of callose synthase was also demonstrated. Synthesis of callose appeared so rapid that the enzyme became embedded in the polysaccharide so that both were detected as integral to the developing papilla. Localized alterations to the cell wall and deposition of papillae were found to involve co‐ordinated synthetic and oxidative activities at microsites within responding cells, without activation of the hypersensitive reaction.
Cloning and characterization ofMS5fromArabidopsis: a gene critical in male meiosis
Authors: Glover Julie , Grelon Mathilde , Craig Stuart , Chaudhury Abdul , Dennis Elizabeth
Published: March 2003

In this paper, we describe the cloning of the

MS5

gene, a gene essential for male fertility in

Arabidopsis

. We previously defined the

MS5

locus by characterizing an EMS‐induced allele,

ms5–1

. We identified a new allele of

MS5

(

ms5–2

) that was T‐DNA‐generated and used the T‐DNA tag to clone the gene. Sequencing of mutant and wild‐type alleles together with complementation of the

ms5–1

mutant phenotype with a wild‐type genomic clone confirmed the identity of the gene. Differences between the phenotypes of the two mutant alleles could be attributed to differences in mutant gene structure. The semi‐dominant and dominant negative phenotypes of the

ms5–2

mutant probably result from production of a truncated polypeptide. An unknown locus in Landsberg erecta can counteract the dominant negative phenotype of

ms5–2

. Mutations in

MS5

cause the formation of ‘polyads’– tetrads with more than four pools of chromosomes after male meiosis. Similarities between the MS5 sequence and that of a number of proteins were found; two that may be significant were with a synaptonemal complex protein and with a regulatory subunit of a cyclin‐dependent kinase. The

MS5

gene is a member of a small gene family highly conserved amongst plant species.
Two different genes encode ferrochelatase in Arabidopsis: mapping, expression and subcellular targeting of the precursor proteins
Authors: Chow Keng See , Singh Davinder Pal , Walker Amanda R. , Smith Alison G.
Published: March 2003

Ferrochelatase is the last enzyme of haem biosynthesis. We have isolated 27 independent ferrochelatase cDNAs from

Arabidopsis thaliana

by functional complementation of a yeast mutant. Twenty‐two of these cDNAs were similar to a previously isolated clone, AF3, and although they varied in length at the 5′ and 3′ ends, their nucleotide sequences were identical, indicating that they were derived from the same gene (ferrochelatase‐I). The remaining five cDNAs all encoded a separate ferrochelatase isoform (ferrochelatase‐II), which was 69% identical at the amino acid level to ferrochelatase‐I. Using RFLP analysis in recombinant inbred lines, the ferrochelatase‐I gene was mapped to chromosome V and that for ferrochelatase‐II to chromosome II. Northern analysis showed that both ferrochelatase genes are expressed in leaves, stems and flowers, and expression in the leaves is higher in the light than in the dark. However, in roots only ferrochelatase‐I transcripts were detected. High levels of sucrose stimulated expression of ferrochelatase‐I, but had no effect, or repressed slightly, the expression of the ferrochelatase‐II isoform. Import experiments into isolated chloroplasts and mitochondria showed that the ferrochelatase‐II gene encodes a precursor which is imported solely into the chloroplast, in contrast to ferrochelatase‐I which is targeted to both organelles. The significance of these results for haem biosynthesis and the production of haemoproteins, both within the plant cell and in different plant tissues, is discussed.
Characterization of a tobacco epoxide hydrolase gene induced during the resistance response to TMV
Authors: Guo Ailan , Durner Jörg , Klessig Daniel F.
Published: March 2003

A clone encoding a putative soluble epoxide hydrolase (EH‐1), an enzyme which converts epoxides to diols, was isolated by differential screening of a cDNA library prepared from tobacco mosaic virus (TMV)‐infected tobacco leaves. To confirm that EH‐1 encodes an epoxide hydrolase, the recombinant EH‐1 protein produced in bacteria was shown to have high epoxide hydrolase activity in vitro. Infection of resistant but not susceptible tobacco cultivars induced the accumulation of EH‐1 transcripts in both the inoculated and uninoculated, systemic leaves. EH‐1 expression was also induced in the inoculated and systemic tissues of TMV‐infected NahG plants, which are unable to accumulate salicylic acid (SA). However, EH‐1 expression in the inoculated leaves of NahG plants was delayed, whilst in the systemic leaves the induction was both later and weaker, compared to that observed in wild‐type plants. Furthermore, exogenously applied SA or its functional analog 2,6‐dichloroisonicotinic acid (INA) caused a rapid and transient accumulation of EH‐1 transcripts, whereas an inactive SA analog did not. Thus, the induction of EH‐1 gene expression appears to be regulated by both SA‐independent and SA‐dependent pathways. Since EH‐1 was expressed only in TMV‐resistant tobacco after infection, and the encoded enzyme is thought to help metabolize toxic compounds, we propose that EH‐1 may play a role in protection from oxidative damage associated with defense responses. It may also play a role in generating signals for activation of certain defense responses.
The glutathione‐deficient, cadmium‐sensitive mutant,cad2–1, ofArabidopsis thalianais deficient in γ‐glutamylcysteine synthetase
Authors: Cobbett Christopher S. , May Mike J. , Howden Ross , Rolls Barbara
Published: March 2003

This paper reports that the glutathione (GSH)‐deficient mutant,

cad2–1

, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, γ‐glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ‐glutamylcysteine.

In vitro

enzyme assays showed that the

cad2–1

mutant has 40% of wild‐type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The

CAD2

locus maps to chromosome 4 and is tightly linked to a gene,

GSHA

, identified by a previously isolated cDNA. A genomic clone of

GSHA

complements both the phenotypic and biochemical deficiencies of the

cad2–1

mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the

CAD2

gene encodes GCS. The

cad2–1

mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L‐buthionine‐[S,R] sulfoximine (BSO). Both root growth and GCS activity of the

cad2–1

mutant was less sensitive than the wild‐type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.
A flax transposon identified in two spontaneous mutant alleles of theL6rust resistance gene
Authors: Luck Joanne E. , Lawrence Greg J. , Finnegan E. Jean , Jones David A. , Ellis Jeff G.
Published: March 2003

Two spontaneous mutant alleles of the

L6

flax rust resistance gene,

l6‐X3A

and

l6‐X117

, contain the same transposable element designated

dLute

(defective

Linum usitatissimum

transposable element). The element is 314 bp long, 70% AT‐rich and, because it contains no extended open reading frame, is probably non‐autonomous. It has 14 bp imperfect terminal inverted repeats related to those in the

Ac

family of plant transposons and, like

Ac

, causes 8 bp target site duplications upon insertion. Multiple copies of

dLute

‐related sequences exist in the flax genome. Rust resistant revertants were recovered amongst the progeny of both mutants and reversion was associated with excision of

dLute

. Excision either restored the wild‐type

L6

sequence or was imprecise, leaving sequence alterations (‘footprints’) resulting in one to three amino acid alterations in the L6 protein. No phenotypic differences were discerned between plants containing the standard and revertant

L6

alleles.
Molecular characterization of the mutableflakedallele for flower variegation in the common morning glory
Authors: Habu Yoshiki , Hisatomi Yasuyo , Iida Shigeru
Published: March 2003

The mutable

flaked

(or

af

) lines of the common morning glory bear white flowers with colored flakes and sectors. The

af

allele shows incomplete dominance. Plants in the heterozygous state

A/af

bear lightly colored flowers with intensely colored flakes and occasionally with white sectors. We showed that the mutable

af

allele is caused by insertion of a new transposable element,

Tip100

, into the

CHS‐D

gene intron.

Tip100

is 3.9 kb long and belongs to the

Ac/Ds

family. Although the timing and frequency of the flower variegation vary in different lines, they carry an identical mutable allele. We also noticed that a

flaked

subline, bearing variegated flowers, carries a

Tip100

derivative,

Tip100–1

. The structure of

Tip100–1

contains an additional 48 bp terminal sequence as tandem repeats and its integration site is identical to that of

Tip100

. Another line, with stable white flowers, is a double mutant carrying two copies of

Tip100

in the

CHS‐D

gene. These results are discussed with regard to the variegated phenotypes of flowers in various mutable lines.
Diversity and similarity among recognition sequences of Dof transcription factors
Authors: Yanagisawa Shuichi , Schmidt Robert J.
Published: March 2003

Dof proteins are a family of transcription factors that share a unique DNA‐binding domain. Dof proteins were found recently in association with diverse promoters of plant‐specific genes, suggesting various roles of Dof proteins in plants. Through binding site selection experiments using randomly synthesized DNA, the recognition sequences of four maize Dof proteins were systematically analyzed. All selected oligonucleotides contained an AAAG sequence, suggesting that this sequence is the recognition core of Dof proteins. In fact, a single mutation in this sequence abolished binding of all four Dof proteins. Furthermore, the preference of each Dof protein for the sequence flanking the core motif was also analyzed using oligonucleotides containing a fixed AAAG and random flanking sequences. Similar, as well as distinct, flanking sequences were observed among the optimal binding sites. Changes in the flanking sequences did affect DNA‐binding of Dof proteins.
Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20‐oxidase genes
Authors: P Jeremy , Phillips . Coles andrew L. , Croker Stephen J. , García‐Lepe Raol , Lewis Mervyn J. , Hedden Peter
Published: March 2003

Gibberellin (GA) 20‐oxidase catalyses consecutive steps late in GA biosynthesis in plants. In
Arabidopsis
, the enzyme is encoded by a gene family of at least three members (
AtGA20ox1
,
AtGA20ox2
and
AtGA20ox3
) with differential patterns of expression. The genes are regulated by feedback from bioactive GAs, suggesting that the enzymes may be involved in regulating GA biosynthesis. To investigate this, we produced transgenic
Arabidopsis
expressing sense or antisense copies of each of the GA 20‐oxidase cDNAs. Over‐expression of any of the cDNAs gave rise to seedlings with elongated hypocotyls; the plants flowered earlier than controls in both long and short days and were 25% taller at maturity. GA analysis of the vegetative rosettes showed a two‐ to threefold increase in the level of GA4, indicating that GA 20‐oxidase normally limits bioactive GA levels. Plants expressing antisense copies of
AtGA20ox1
had short hypocotyls and reduced rates of stem elongation. This was reflected in reduced levels of GA4 in both rosettes and shoot tips. In short days, flowering was delayed and the reduction in the rate of stem elongation was greater. Antisense expression of A
tGA20ox2
had no apparent effects in long days, but stem growth in one transgenic line grown in short days was reduced by 20%. Expression of antisense copies of
AtGA20ox3
had no visible effect, except for one transgenic line that had short hypocotyls. These results demonstrate that GA levels and, hence, plant growth and development can be modified by manipulation of GA 20‐oxidase expression in transgenic plants.
A chromosomal paracentric inversion associated with T‐DNA integration in Arabidopsis
Authors: Laufs Patrick , Autran Daphné , Traas Jan
Published: March 2003

T‐DNA integration in the nuclear plant genome may lead to rearrangements of the plant target site. Here we present evidence for a chromosomal inversion of 26 cM bordered by two T‐DNAs in direct orientation, which is linked to the mgoun2 mutation. The integration sites of the T‐DNAs map at positions 80 and 106 of chromosome I and we show that each T‐DNA is bordered by plant sequences from positions 80 and 106, respectively. Although the T‐DNAs are physically distant, they are genetically closely linked. In addition, three markers located on the chromosome segment between the two T‐DNA integration sites show no recombination with the mgo2 mutation. We show that the inversion cannot be a consequence of a recombination event between the two T‐DNAs, but that the integration of the T‐DNAs and the inversion were two temporally linked events. T‐DNA integration mechanisms that could have led to this inversion are discussed.
Gravity‐induced reorientation of cortical microtubules observed in vivo
Authors: Himmelspach Regina , Wymer Carol L. , Lloyd Clive W. , Nick Peter
Published: March 2003

Cortical microtubules play an important role during morphogenesis by determining the direction of cellulose deposition. Although many triggers are known that can induce the reorientation of cortical plant microtubules, the reorientation mechanism has remained obscure. In our approach, we used gravitropic stimulation which is a strong trigger for microtubule reorientation in epidermal cells of maize coleoptiles. To visualize the gravitropically induced microtubule reorientation in living cells, we injected rhodamine‐conjugated tubulin into epidermal cells of intact maize coleoptiles that were exposed to gravitropic stimulation. From these in vivo observations, we propose a reorientation mechanism consisting of four different stages: (1) a transitional stage with randomly organized microtubules; (2) emergence of a few microtubules in a slightly oblique orientation; (3) co‐alignment: neighbouring microtubules adopt the oblique orientation resulting in parallel organized microtubules; and (4) the angle of these parallel, organized micro‐ tubules increases gradually. Thus, the overall reorienta‐ tion process could include selective stabilization/disassembly of microtubules (stage 2) as well as move‐ ment of individual microtubules (stages 3 and 4).
Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure
Authors: De Santis‐Maciossek Giovanna , Kofer Waltraud , Bock Armin , Schoch Siegrid , Maier Rainer M. , Wanner Gerhard , Rüdiger Wolfhart , Koop Hans‐Ulrich , Herrmann Reinhold G.
Published: March 2003

The plastid encoded RNA polymerase subunit genes rpoA
, B and C1 of tobacco were disrupted individually by PEG‐mediated plastid transformation. The resulting off‐white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo

–

material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild‐type. Western analysis demonstrates that plastids contain nuclear‐coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the ΔpH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo

–

plastids contain functional ribosomes. The detection of the plastid‐encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.
Profilin in Phaseolus vulgaris is encoded by two genes (only one expressed in root nodules) but multiple isoforms are generated in vivo by phosphorylation on tyrosine residues
Authors: Guillén Gabriel , Valdés‐López Víctor , Noguez Raúl , Olivares Juan , Rodríguez‐Zapata Luis Carlos , Pérez Héctor , Vidali Luis , Villanueva Marco A. , Sánchez Federico
Published: March 2003

Actin‐binding proteins such as profilins participate in the restructuration of the actin cytoskeleton in plant cells. Profilins are ubiquitous actin‐, polyproline‐, and inositol phospholipid‐binding proteins, which in plants are encoded by multigene families. By 2D‐PAGE and immunoblotting, we detected as much as five profilin isoforms in crude extracts from nodules of Phaseolus vulgaris. However, by immunoprecipitation and gel electrophoresis of in vitro translation products from nodule RNA, only the most basic isoform of those found in nodule extracts, was detected. Furthermore, a bean profilin cDNA probe hybridised to genomic DNA digested with different restriction enzymes, showed either a single or two bands. These data indicate that profilin in P. vulgaris is encoded by only two genes. In root nodules only one gene is expressed, and a single profilin transcript gives rise to multiple profilin isoforms by post‐translational modifications of the protein. By in vivo32P‐labelling and immunoprecipitation with both, antiprofilin and antiphosphotyrosine‐specific antibodies, we found that profilin is phosphorylated on tyrosine residues. Since chemical (TLC) and immunological analyses, as well as plant tyrosine phosphatase (AtPTP1) treatments of profilin indicated that tyrosine residues were phosphorylated, we concluded that tyrosine kinases must exist in plants. This finding will focus research on tyrosine kinases/tyrosine phosphatases that could participate in novel regulatory functions/pathways, involving not only this multifunctional cytoskeletal protein, but other plant proteins.
Molecular characterisation of a membrane‐bound galactosyltransferase of plant cell wall matrix polysaccharide biosynthesis
Authors: Edwards Mary E. , Dickson Cathryn A. , Chengappa Sumant , Sidebottom Christopher , Gidley Michael J. , Reid J. S. Grant
Published: March 2003

Galactomannan biosynthesis in vitro is catalysed by membrane preparations from developing fenugreek seed endosperms. Two enzymes interact: a GDP‐mannose dependent (1→4)‐β‐d‐mannan synthase and a UDP‐galactose dependent (1→6)‐α‐d‐galactosyltransferase. The statistical distribution of galactosyl substituents along the mannan backbone, and the degree of galactose substitution of the primary product of galactomannan biosynthesis appear to be regulated by the specificity of the galactosyltransferase. We now report the detergent solubilisation of the fenugreek galactosyltransferase with retention of activity, the identification on gels of a putative 51 kDa galactosyltransferase protein, and the isolation, cloning and sequencing of the corresponding cDNA. The solubilised galactosyltransferase has an absolute requirement for added acceptor substrates. Beta‐(1→4)‐linked d‐manno‐oligosaccharides with chain lengths greater than or equal to 5 acted as acceptors, as did galactomannans of low to medium galactose‐substitution. The putative galactosyltransferase cDNA encodes a 51282 Da protein, with a single transmembrane alpha helix near the N terminus. We have also confirmed the identity of the galactosyltransferase by inserting the cDNA in frame into the genome of the methylotrophic yeast Pichia pastoris under the control of an AOX promoter and the yeast alpha secretion factor and observing the secretion of galactomannan α‐galactosyltransferase activity. Particularly high activities were observed when a truncated sequence, lacking the membrane‐spanning helix, was expressed.
Light regulation of assimilatory sulphate reduction in Arabidopsis thaliana
Authors: Kopriva Stanislav , Muheim Regula , Koprivova Anna , Trachsel Nadine , Catalano Cinzia , Suter Marianne , Brunold Christian
Published: March 2003

Adenosine 5′‐phosphosulphate reductase (APR) is considered to be a key enzyme of sulphate assimilation in higher plants. We analysed the diurnal fluctuations of total APR activity and protein accumulation together with the mRNA levels of three APR isoforms of Arabidopsis thaliana. The APR activity reached maximum values 4 h after light onset in both shoots and roots; the minimum activity was detected at the beginning of the night. During prolonged light, the activity remained stable and low in shoots, but followed the normal rhythm in roots. On the other hand, the activity decreased rapidly to undetectable levels within 24 h of prolonged darkness both in shoots and roots. Subsequent re‐illumination restored the activity to 50% in shoots and to 20% in roots within 8 h. The mRNA levels of all three APR isoforms showed a diurnal rhythm, with a maximum at 2 h after light onset. The variation of APR2 mRNA was more prominent compared to APR1 and APR3. 35SO42– feeding experiments showed that the incorporation of 35S into reduced sulphur compounds in vivo was significantly higher in light than in the dark. A strong increase of mRNA and protein accumulation as well as enzyme activity during the last 4 h of the dark period was observed, implying that light was not the only factor involved in APR regulation. Indeed, addition of 0.5% sucrose to the nutrient solution after 38 h of darkness led to a sevenfold increase of root APR activity over 6 h. We therefore conclude that changes in sugar concentrations are also involved in APR regulation.
A tobacco plasma membrane calmodulin‐binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants
Authors: Arazi Tzahi , Sunkar Ramanjulu , Kaplan Boaz , Fromm Hillel
Published: March 2003

All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal‐uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin‐binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K+ and to non‐selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two‐phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with reduced Ni2+ accumulation and enhanced Pb2+ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb2+. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.
An Arabidopsis gene encoding a chloroplast‐targeted β‐amylase
Authors: Lao Nga T. , Schoneveld Onard , Mould Ruth M. , Hibberd Julian M. , Gray John C. , Kavanagh Tony A.
Published: March 2003

β‐Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that β‐amylases are not involved in the metabolism of chloroplast‐localized transitory leaf starch. However, we have identified a novel β‐amylase gene, designated ct‐Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct‐Bmy encodes a precursor protein which contains a typical N‐terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic β‐amylases of higher plants. Expression of the ct‐Bmy cDNA in E. coli confirmed that the encoded protein possesses β‐amylase activity. CT‐BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT‐BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT‐BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct‐Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.
Elicitor‐responsive, ethylene‐independent activation of GCC box‐mediated transcription that is regulated by both protein phosphorylation and dephosphorylation in cultured tobacco cells
Authors: Yamamoto Sumiko , Suzuki Kaoru , Shinshi Hideaki
Published: March 2003

In cultured XD6S tobacco cells, xylanase from Trichoderma viride (TvX) induced the expression of a luciferase reporter gene that was under the control of a GCC box, which is an 11 bp sequence (TAAGAGCCGCC) that is found in the 5′‐upstream region of pathogen‐responsive defence genes that include genes for class I basic chitinase. TvX‐induced biosynthesis of ethylene was not required for the TvX‐activated transcription. The TvX‐induced, GCC box‐mediated transcription of the reporter gene was completely blocked not only by staurosporine, an inhibitor of serine/threonine protein kinases, at 1 μM, but also by calyculin A, an inhibitor of protein phosphatases 1 and 2A, at 0.2 μM. It appeared also that protein synthesis de novo was required for the GCC box‐mediated transcription of the reporter gene. Accumulation of mRNAs for various ERFs (ethylene‐responsive transcription factors), which have been shown to bind specifically to the GCC box, was also induced by TvX prior to increases in the level of mRNA for a class I basic chitinase. In particular, the level of mRNA for EFR2 reached a maximum from 3 to 6 h, whereas levels of mRNAs for ERF3 and ERF4 were highest 0.5 h after the start of treatment of TvX and decreased thereafter. Moreover, induction of accumulation of the mRNA for ERF2 was inhibited by staurosporine and calyculin A. These results suggest that ERF2 might play a major role in TvX‐induced, GCC box‐mediated transcription of genes and that both protein kinase(s) and protein phosphatase(s) might be involved, as positive regulators, in the signal transduction pathway that leads to expression of ERF2 and subsequent GCC box‐mediated transcription of genes.
FALSIFLORA, the tomato orthologue of FLORICAULA and LEAFY, controls flowering time and floral meristem identity
Authors: Molinero‐Rosales Nuria , Jamilena Manuel , Zurita Sergio , Gómez Pedro , Capel Juan , Lozano Rafael
Published: March 2003

Characterization of the tomato falsiflora mutant shows that fa mutation mainly alters the development of the inflorescence resulting in the replacement of flowers by secondary shoots, but also produces a late‐flowering phenotype with an increased number of leaves below first and successive inflorescences. This pattern suggests that the FALSIFLORA (FA) locus regulates both floral meristem identity and flowering time in tomato in a similar way to the floral identity genes FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis. To analyse whether the fa phenotype is the result of a mutation in the tomato FLO/LFY gene, we have cloned and analysed the tomato FLO/LFY homologue (TOFL) in both wild‐type and fa plants following a candidate gene strategy. The wild‐type gene is predicted to encode a protein sharing 90% identity with NFL1 and ALF, the FLO/LFY‐like proteins in Nicotiana and Petunia, and about 80 and 70% identity with either FLO or LFY. In the fa mutant, however, the gene showed a 16 bp deletion that results in a frameshift mutation and in a truncated protein. The co‐segregation of this deletion with the fa phenotype in a total of 240 F2 plants analysed supports the idea that FA is the tomato orthologue to FLO and LFY. The gene is expressed in both vegetative and floral meristems, in leaf primordia and leaves, and in the four floral organs. The function of this gene in comparison with other FLO/LFY orthologues is analysed in tomato, a plant with a sympodial growth habit and a cymose inflorescence development.
Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development
Authors: Maucher Helmut , Hause Bettina , Feussner Ivo , Ziegler Jörg , Wasternack Claus
Published: March 2003

Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full‐length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co‐purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX‐derived 9‐ as well as 13‐hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na‐salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up‐regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.
Axillary meristem development in Arabidopsis thaliana
Authors: Grbić Vojislava , Bleecker Anthony B.
Published: March 2003

Axillary shoot apical meristems initiate post‐embryonically in the axils of leaves. Their developmental fate is a main determinant of the final plant body plan. In Arabidopsis, usually a single axillary meristem initiates in the leaf axil even though there is developmental potential for formation of multiple branches. While the wild‐type plants rarely form multiple branches in the leaf axil, tfl1‐2 plants regularly develop two or more branches in the axils of the rosette leaves. Axillary meristem formation in Arabidopsis occurs in two waves: an acropetal wave forms during plant vegetative development, and a basipetal wave forms during plant reproductive development. We report here the morphological and anatomical changes, and the STM expression pattern associated with the formation of axillary and accessory meristems during Arabidopsis vegetative development.
Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana
Authors: Oster Ulrike , Tanaka Ryouichi , Tanaka Ayumi , Rüdiger Wolfhart
Published: March 2003

Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets. The bulk of the Chls is found in the light‐harvesting antenna complexes of photosynthetic organisms. Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a. We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis. The recombinant enzyme produced in Escherichia coli catalyses an unusual two‐step oxygenase reaction that is the ‘missing link’ in the chlorophyll cycle of higher plants.
Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize
Authors: Yanagisawa Shuichi
Published: March 2003

Dof proteins are transcription factors that appear to be unique to plants. Maize Dof1 has been suggested to be a regulator for C4 photosynthetic phosphoenolpyruvate carboxylase (C4PEPC) gene expression. The present study demonstrates that Dof1 also enhances transcription from the promoters of both cytosolic orthophosphate dikinase (cyPPDK) genes and a non‐photosynthetic PEPC gene, which are not present in animals. Expression of Dof1‐specific antisense RNA or the DNA‐binding domain of Dof1 alone reduced the activities of these promoters in maize leaf protoplasts. Electrophoretic mobility shift assays revealed several Dof1‐binding sites in these promoters. The cyppdk1 promoter contained two Dof1‐binding sites, one of which was linked to the binding site of a plant bZIP protein. By using deleted or mutated cyppdk1 promoters, both Dof1‐binding sites were shown to be functional. Furthermore, Dof1 elevated the activities of the cyppdk and pepc promoters more strongly in greening protoplasts than in etiolated protoplasts, in accordance with the different activities of these promoters in two types of protoplasts. Another Dof protein of maize, Dof2, suppressed the activity of the C4pepc promoter but was able to activate certain other promoters. These results suggest that Dof proteins may play regulatory roles in multiple gene expressions associated with the plant‐specific pathway for carbon metabolism in maize. In addition, the primary characteristic of Dof proteins, i.e. different activities in distinct types of cells and opposite actions on promoters in different contexts, suggests the potential of Dof proteins to differentially regulate diverse promoters in a variety of plant tissues. Speculation raised by these results concerning the evolution of the C4pepc gene is also discussed.
Sucrose transport into barley seeds: molecular characterization of two transporters and implications for seed development and starch accumulation
Authors: Weschke Winfriede , Panitz Reinhard , Sauer Norbert , Wang Qing , Neubohn Birgit , Weber Hans , Wobus Ulrich
Published: March 2003

In order to understand sucrose transport in developing seeds of cereals at the molecular level, we cloned from a caryopses library two cDNAs encoding sucrose transporters, designated HvSUT1 and HvSUT2. Sucrose uptake activity was confirmed by heterologous expression in yeast. Both transporter genes are expressed in maternal as well as filial tissues. In a series of in situ hybridizations we analysed the cell type‐specific expression in developing seeds. HvSUT1 is preferentially expressed in caryopses in the cells of the nucellar projection and the endospermal transfer layer, which represent the sites of sucrose exchange between the maternal and the filial generation and are characterized by transfer cell formation. HvSUT2 is expressed in all sink and source tissues analysed and may have a general housekeeping role. The rapid induction of HvSUT1 gene expression in caryopses at approximately 5–6 days after fertilization coincides with increasing levels of sucrose as well as sucrose synthase mRNA and activity, and occurs immediately before the onset of rapid starch accumulation within the endosperm. Starch biosynthesis requires sucrose to be imported into the endosperm, as direct precursor for starch synthesis and to promote storage‐associated processes. We discuss the possible role of HvSUT1 as a control element for the endospermal sucrose concentration.
Functional, c‐myc‐tagged Cf‐9 resistance gene products are plasma‐membrane localized and glycosylated
Authors: Piedras Pedro , Rivas Susana , Dröge Swenja , Hillmer Stephan , Jones Jonathan D. G.
Published: March 2003

The Cf‐9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf‐9 function, we tagged the Cf‐9 protein with a triple myc epitope at either the amino‐ or carboxy‐terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf‐9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c‐myc antibodies, c‐myc : Cf‐9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf‐9 protein is highly glycosylated. Plasma membranes were isolated using two‐phase partitioning, and c‐myc : Cf‐9 was enriched in these fractions, indicating that Cf‐9 is a plasma membrane protein. This was confirmed by silver‐enhanced immunogold labelling of tobacco protoplasts carrying the amino‐terminal c‐myc tag; a higher labelling density was observed on the surface of protoplasts derived from c‐myc : Cf‐9 tobacco compared to untransformed control. The presence of Cf‐9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.
A new type of mutation in the plant photoreceptor phytochrome B causes loss of photoreversibility and an extremely enhanced light sensitivity
Authors: Kretsch Thomas , Poppe Christoph , Schäfer Eberhard
Published: March 2003

Photoreversibility, reversion of the inductive effect of a brief red light pulse by a subsequent far‐red light pulse, is a property of photoresponses regulated by the plant photoreceptor phytochrome B (phyB). We screened for mutants with impaired photoreversibility to gain better insight into the phyB‐specific signalling cascade. The phenotype of the mutant described is caused by a single amino acid exchange in a phyB subdomain that is highly conserved in all phytochromes but whose functional significance was unknown. The mutated phyB showed a slower dark reversion but no major alterations in its spectral properties. In addition to its loss of photoreversibility, the mutant also exhibited a hypersensitivity towards continuous red‐light irradiation and an altered phenotype of adult plants under short‐day conditions.
Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis
Authors: Rottmann William H. , Meilan Richard , Sheppard Lorraine A. , Brunner Amy M. , Skinner Jeffrey S. , Ma Caiping , Cheng Shuping , Jouanin Lise , Pilate Gilles , Strauss Steven H.
Published: March 2003

PTLF, thePopulus trichocarpahomolog ofLEAFY(LFY) andFLORICAULA, was cloned to assess its function in a dioecious tree species.In situhybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of thePTLFcDNA inArabidopsisaccelerated flowering, only one of the many tested transgenic lines ofPopulusflowered precociously. The majority of trees within a population of 3‐year‐old transgenic hybridPopuluslines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely amongPopulusgenotypes, giving consistent early flowering in only a single maleP. tremula × P. tremuloideshybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.
Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease
Authors: Chen Meng , Choi YangDo , Voytas Daniel F. , Rodermel Steve
Published: March 2003

Variegated plants have green‐ and white‐sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non‐pigmented plastids that lack organized lamellar structures. Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned. We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities). Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane‐associated events. Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis. We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity. These include two putative ATP‐binding sites. VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype. One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors. We conclude that redundant activities may be a general mechanism to explain nuclear gene‐induced plant variegations.
Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione
Authors: Harms Karsten , Von Ballmoos Peter , Brunold Christian , Höfgen Rainer , Hesse Holger
Published: March 2003

The coding sequence of the wild‐type, cys‐sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1.30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5′‐signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20‐fold higher SAT activity than those prepared from wild‐type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O‐acetylserine(thiol)‐lyase, the enzyme which converts O‐acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur‐containing compounds can be enhanced by metabolic engineering.
Transgenic tobacco plants co‐expressing Agrobacterium iaa and ipt genes have wild‐type hormone levels but display both auxin‐ and cytokinin‐overproducing phenotypes
Authors: Eklöf Staffan , Åstot Crister , Sitbon Folke , Moritz Thomas , Olsson Olof , Sandberg Göran
Published: March 2003

Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole‐3‐acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside‐5′‐monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild‐type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly ± associated with IAA and cytokinin overproduction, and observed in the iaaËipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.