bluuebunny/crossref_metadata_2025 huggingface dataset:
A bHLH transcription factor mediates organ, region and flower type specific signals on dihydroflavonol‐4‐reductase (dfr) gene expression in the inflorescence ofGerbera hybrida(Asteraceae)
Authors: Elomaa Paula , Mehto Merja , Kotilainen Mika , Helariutta Yrjö , Nevalainen Leena , Teeri Teemu H.
Published: March 2003

The angiosperm family Asteraceae is characterized by composite inflorescences, which are highly organized structures consisting of different types of flowers. In order to approach the control of floral organ differentiation in Asteraceae at molecular level, we are studying regulation of flavonoid biosynthesis in

Gerbera hybrida

. Dihydroflavonol‐4‐reductase (

dfr

) expression is regulated according to anthocyanin pigmentation patterns in all tested gerbera varieties at several anatomical levels. We have isolated a promoter for one of the

dfr

genes,

Pgdfr2

. Gerbera plants transgenic for a

Pgdfr2

‐

uidA

construct reveal that the activity of the

Pgdfr2

promoter from one variety follows the pigmentation in other varieties which have different color patterns. It is thus evident that the observed complex regulation of

dfr

expression occurs

in trans

. In order to identify the

trans

‐acting regulators, we isolated a cDNA (

gmyc1

) homologous to the previously characterized genes encoding bHLH‐type regulators of the anthocyanin pathway in plants. The expression of

gmyc1

in different varieties suggests that it has a major role in regulating

dfr

activity in corolla and carpel, but not in pappus and stamen. Specifically in gerbera, the identical patterns of

gmyc1

and

dfr

expression in corolla tissue suggest that GMYC1 also regulates

dfr

expression in a region and flower type specific manner. Our studies show that in gerbera GMYC1–

dfr

interaction is part of several developmental processes characteristic for Asteraceae (such as specification of flower types across the composite inflorescence), whereas in other processes (such as differentiation of sepal as pappus) other regulators control

dfr

expression to determine the spatial specificity.
Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites inArabidopsis thaliana
Authors: Lam Hon‐Ming , Hsieh Ming‐Hsiun , Coruzzi Gloria
Published: March 2003

In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, including

Arabidopsis thaliana

. To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis. In addition to the previously identified

ASN1

gene, we identified a novel class of asparagine synthetase genes in Arabidopsis (

ASN2

and

ASN3

) by functional complementation of a yeast asparagine auxotroph. The proteins encoded by the

ASN2/3

cDNAs contain a Pur‐F type glutamine‐binding triad suggesting that they, like

ASN1

, encode glutamine‐dependent asparagine synthetase isoenzymes. However, the ASN2/3 isoenyzmes form a novel dendritic group with monocot

AS

genes which is distinct from all other dicot

AS

genes including Arabidopsis

ASN1

. In addition to these distinctions in sequence, the

ASN1

and

ASN2

genes are reciprocally regulated by light and metabolites. Time‐course experiments reveal that light induces levels of

ASN2

mRNA while it represses levels of

ASN1

mRNA in a kinetically reciprocal fashion. Moreover, the levels of

ASN2

and

ASN1

mRNA are also reciprocally regulated by carbon and nitrogen metabolites. The distinct regulation of

ASN1

and

ASN2

genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.
Reduced inositol content and altered morphology in transgenic potato plants inhibited for 1D‐myo‐inositol 3‐phosphate synthase
Authors: Keller Ruth , Brearley Charles A. , Trethewey Richard N. , Müller‐Röber Bernd
Published: March 2003

Myo

‐inositol is a precursor of many plant metabolites, including polyols, cell wall components and phosphoinositides. The first committed step in the

de novo myo

‐inositol synthetic pathway is catalysed by the enzyme 1D‐

myo

‐inositol 3‐phosphate synthase (MIPS;
EC 5.5.1.4
), which converts D‐glucose 6‐phosphate to 1D‐

myo

‐inositol 3‐phosphate. Suppression of MIPS activity by an antisense RNA approach in transgenic potato (

Solanum tuberosum

L.) plants to below 20% of the wild‐type level in leaves resulted in strongly reduced levels of inositol, galactinol and raffinose (approximately 7%, 5% and 12%, respectively, of wild‐type values). In contrast, increases were observed for concentrations of hexose phosphates (up to 1.7‐fold), sucrose (twofold) and starch (two‐ to fourfold). Transgenic plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence and a decrease in overall tuber yield. These observations indicate a crucial role for

myo

‐inositol in plant physiology and development.
ATY3/GYPSYretrotransposon‐like sequence localizes to the centromeric regions of cereal chromosomes
Authors: Presting Gernot G. , Malysheva Ludmilla , Fuchs Jörg , Schubert Ingo
Published: March 2003

A 745 bp sequence (pSau3A9) located at the centromeres of several cereal species was isolated from a sorghum BAC library byJianget al. (1996,Proc. Natl Acad. Sci. USA, 93, 14210–14213). We have amplified a partially homologous 809 bp sequence from barley genomic DNA by PCR and localized it to the centromeres of barley, wheat and rye chromosomes by fluorescentin situhybridization (FISH). Sequence analysis showed this barley homolog ofpSau3A9to have high similarity to the integrase region of the polyprotein gene ofTy3/gypsygroup retrotransposons. Using this integrase sequence as a probe, several clones were isolated from a lambda library constructed of genomic barley DNA. One of the lambda clones contained coding regions for all five catalytic sites characteristic of the retrotransposon polyprotein. Two direct repeats flanking the polyprotein gene are homologous to the cereal centromeric sequence described byAragón‐Alcaideet al. (1996,Chromosoma, 105, 261–268) and may represent all or part of the long‐terminal repeats (LTRs). Different plasmid subclones containing various regions of the lambda clone were used in FISH to show that the entire polyprotein gene and upstream flanking sequences, including the presumed LTR, are present at barley centromeres. The preferential (or exclusive) localization of an apparently complete retroelement within the centromeric regions of several cereal species raises interesting questions about its role in karyotype evolution and centromere function.
Plants expressing thePtodisease resistance gene confer resistance to recombinant PVX containing the avirulence geneAvrPto
Authors: Tobias Christian M. , Oldroyd Giles E. D. , Chang Jeff H. , Staskawicz Brian J.
Published: March 2003

Elicitation of hypersensitive cell death and induction of plant disease resistance by

Pseudomonas syringae

pv.

tomato
(Pst) is dependent on activity of thePstHrp secretion system and the gene‐for‐gene interaction between the tomato resistance genePtoand the bacterial avirulence geneavrPto.AvrPtowas expressed transiently in resistant or susceptible plant lines via a potato virus X (PVX) vector. We found that while PVX is normally virulent on tomato, a PVX derivative expressingavrPtowas only capable of infecting plants lacking a functional Pto resistance pathway. Mutations in either thePtoorPrfgenes allowed systemic spread of the recombinant virus. These results indicate that recognition of AvrPto by Pto in resistant plant lines triggers a plant defense response that can confer resistance to a viral as well as a bacterial pathogen.
Regulation of a putative high‐affinity nitrate transporter (Nrt2;1At) in roots ofArabidopsis thaliana
Authors: Zhuo Degen , Okamoto Mamoru , Vidmar J. John , Glass Anthony D. M.
Published: March 2003

Putative high‐affinity nitrate (NO3–) transporter genes, designatedNrt2;1AtandNrt2;2At, were isolated fromArabidopsis thalianaby RT–PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30–73% identities with other eukaryotic highaffinity NO3–transporters. Both genes were induced by NO3–, butNrt2;1Atgene expression was not apparent in 2‐ and 5‐day‐old plants. By 10 days, and thereafter,Nrt2;1Atgene expression in roots was substantially higher than for theNrt2;2Atgene. RootNrt2;1Atexpression levels were strongly correlated with inducible high‐affinity13NO3–influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation ofNrt2;1Atexpression was mediated by NH4+, gln and other amino acids.
Potato StubSNF1 interacts with StubGAL83: a plant protein kinase complex with yeast and mammalian counterparts
Authors: Lakatos Lóránt , Klein Mathieu , Höfgen Rainer , Bánfalvi Zsófia
Published: March 2003

StubSNF1 is a potato cDNA that encodes a protein kinase similar to the yeast
SNF1
gene involved in transcriptional regulation of glucose‐repressible genes. The yeast SNF1 functions in a complex with GAL83/SIP1/SIP2 and SNF4 proteins. We have used StubSNF1 as bait in a yeast two‐hybrid system to screen for potato cDNAs encoding proteins that bind to StubSNF1. Three overlapping cDNAs, two different in size, were isolated. DNA sequence analysis revealed that they were orthologues of the yeast
GAL83/SIP1/SIP2
genes and their mammalian counterparts,
AMPK β
‐subunits. The direct interaction between the potato proteins StubGAL83 and StubSNF1 was shown by an
in vitro
binding assay. Southern and Northern hybridisations revealed that
StubGAL83
exists in a low copy number in the potato genome and is highly (but organ‐specifically) expressed in potato. In contrast,
StubSNF1
possesses low transcript levels in each organ, except in flowers where high amounts of StubSNF1 mRNA could be detected. We demonstrate here that StubGAL83 can also interact with yeast SNF4 in a yeast two‐hybrid system suggesting that plant SNF1 kinases may function in complexes similar to those detected in yeast and mammals.
Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts
Authors: Houba‐Hérin Nicole , Pethe Claude , D’Alayer Jacques , Laloue Michel
Published: March 2003

Cytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of the
N

6‐(isopent‐2‐enyl)‐side chain resulting in formation of adenine‐type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet. CKO activity is known to be inhibited by phenylureas, cytokinin agonists. We used 1‐(2‐azido‐6‐chloropyrid‐4‐yl)‐3‐(4‐[3H])phenylurea ([3H]‐azidoCPPU) to photolabel a glycosylated CKO from maize kernels. This enabled us to purify the enzyme. Peptide sequences were determined and the corresponding cDNA was cloned. The deduced amino acid sequence shares homology domains with FAD‐dependent oxidases. An original assay based on transient expression of the enzyme in moss protoplasts allowed the functionality of the recombinant enzyme to be demonstrated.
The helicase domain of the TMV replicase proteins induces the N‐mediated defence response in tobacco
Authors: Les Erickson F. , Holzberg Steve , Calderon‐Urrea Alejandro , Handley Vanessa , Axtell Michael , Corr Catherine , Baker Barbara
Published: March 2003

Tobacco mosaic virus (TMV) induces the hypersensitive response (HR) in tobacco plants containing the N gene. This defence response is characterized by cell death at the site of virus infection and inhibition of viral replication and movement. A previous study indicated that a portion of the TMV replicase containing a putative helicase domain is involved in HR induction. Here, this observation is confirmed and extended by showing that non‐viral expression of a 50 kDa TMV helicase fragment (p50) is sufficient to induce the N‐mediated HR in tobacco. Like the HR elicited by TMV infection, transgenic expression of p50 induces a temperature‐sensitive defence response. We demonstrate that recombinant p50 protein has ATPase activity, as suggested by the presence of conserved sequence motifs found in ATPase/helicase enzymes. A point mutation that alters one of these motifs abolishes ATPase activity in vitro but does not affect HR induction. These results suggest that features of the TMV helicase domain, independent of its enzymatic activity, are recognized by N‐containing tobacco to induce TMV resistance.
The Arabidopsis flowering‐time gene LUMINIDEPENDENS is expressed primarily in regions of cell proliferation and encodes a nuclear protein that regulates LEAFY expression
Authors: Aukerman Milo J.. , Lee Ilha , Weigel Detlef , Amasino Richard M..
Published: March 2003

Mutations in the LUMINIDEPENDENS ( LD) gene of Arabidopsis thaliana (L.) Heynh. (Arabidopsis) confer a late‐flowering phenotype, indicating that LD normally functions to promote the floral transition. RNA and protein blot analyses, along with the analysis of transgenic plants containing a fusion between a genomic fragment of LD and the reporter gene uidA ( GUS), indicate that LD is expressed primarily in apical proliferative regions of the shoot and root, including the shoot apical meristem and leaf primordia. Subcellular localization studies indicate that LD is a nuclear protein, consistent with its previously proposed transcriptional regulatory role. We have also found that in an apetala1 cauliflower ( ap1 cal) background the ld mutation converts the reproductive shoot apex to a more vegetative state, a phenotype that is similar to that seen for the leafy ( lfy) mutant. Furthermore, in situ hybridization analysis indicates that LFY levels are drastically reduced at the apex of ld ap1 cal plants after bolting. These data are consistent with the idea that at least one function of LD is to participate in the regulation of LFY.
FPF1 modulates the competence to flowering in Arabidopsis
Authors: Melzer Siegbert , Kampmann Grit , Chandler John , Apel Klaus
Published: March 2003

During the transition to flowering the FPF1 gene is expressed in the peripheral zone of apical meristems and in floral meristems of Arabidopsis. Constitutive expression of FPF1 causes early flowering in Arabidopsis under both long‐day and short‐day conditions and leads to a shortened juvenile phase as measured by the trichome distribution on the abaxial leaf surface. In the classical late flowering mutants, overexpression of FPF1 compensates partially for the late flowering phenotype, indicating that FPF1 acts downstream or in a parallel pathway to the mutated genes. The co‐overexpression of 35S::AP1 with 35S::FPF1 leads to a synergistic effect on the shortening of the time to flowering under short‐day conditions. The co‐overexpression of 35S::FPF1 and 35S::LFY, however, shows only an additive reduction of flowering time and the conversion of nearly every shoot meristem, except the inflorescence meristem, to a floral meristem under the same light conditions. In addition, the constitutive expression of FPF1 attenuates the severe lfy‐1 phenotype under short days and phenocopies to a great extent the lfy‐1 mutant grown under long‐day conditions. Thus, we assume that FPF1 modulates the competence to flowering of apical meristems.
Three 4‐coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms
Authors: Ehlting Jürgen , Büttner Daniela , Wang Qing , Douglas Carl J. , Somssich Imre E. , Kombrink Erich
Published: March 2003

The enzyme 4‐coumarate:CoA ligase (4CL) plays a key role in channelling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and specificities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to flavonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than flavonoids.
The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine‐rich repeat class of plant disease resistance genes
Authors: Wang Zi‐Xuan , Yano Masahiro , Yamanouchi Utako , Iwamoto Masao , Monna Lisa , Hayasaka Hiroshi , Katayose Yuichi , Sasaki Takuji
Published: March 2003

Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most serious diseases of rice. Here we describe the isolation and characterization of Pib, one of the rice blast resistance genes. The Pib gene was isolated by a map‐based cloning strategy. The deduced amino acid sequence of the Pib gene product contains a nucleotide binding site (NBS) and leucine‐rich repeats (LRRs); thus, Pib is a member of the NBS‐LRR class of plant disease resistance genes. Interestingly, a duplication of the kinase 1a, 2 and 3a motifs of the NBS region was found in the N‐terminal half of the Pib protein. In addition, eight cysteine residues are clustered in the middle of the LRRs, a feature which has not been reported for other R genes. Pib gene expression was induced upon altered environmental conditions, such as altered temperatures and darkness.
Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli
Authors: Kaiser Annette
Published: March 2003

The enzyme homospermidine synthase catalyzes the NAD+‐dependent conversion of 2 mol putrescine into homospermidine. Instead of putrescine, spermidine can substitute for the first putrescine moiety in plants, in which case diaminopropane instead of ammonia is released. The enzyme facilitates the formation of the ‘uncommon’ polyamine homospermidine which is an important precursor in the biosynthesis of pyrrolizidine alkaloids. The first plant homospermidine synthase was purified to apparent chemical homogenity from the root tissue culture Senecio vernalis (Asteraceae) (Böttcher et al. 1994, Can. J. Chem. 72, 80–85; Ober 1997, Dissertation). Four endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers against these peptides, a cDNA encoding homospermidine synthase was now cloned and characterized from Senecio vulgaris. The nucleotide sequence of the cloned cDNA revealed an open reading frame of 1155‐base pairs containing 385 amino acids with a predicted Mr of 44500. GenBank research revealed that the deduced amino acid sequence shows 59% identity to human deoxyhypusine synthase. The homospermidine synthase encoding cDNA was subcloned into the expression vector pet15b and overexpressed in E. coli. The recombinant enzyme formed upon expression catalyzed homospermidine synthesis.
An apospory‐specific genomic region is conserved between Buffelgrass (Cenchrus ciliaris L.) and Pennisetum squamulatum Fresen
Authors: Roche Dominique , Cong Peisheng , Chen Zhenbang , Hanna Wayne W. , Gustine David L. , Sherwood Robert T. , Ozias‐Akins Peggy
Published: March 2003

Twelve molecular markers linked to pseudogamous apospory, a form of gametophytic apomixis, were previously isolated from Pennisetum squamulatum Fresen. No recombination between these markers was found in a segregating population of 397 individuals (Ozias‐Akins et al. 1998, Proc. Natl Acad. Sci. USA, 95, 5127–5132). The objective of the present study was to test if these markers were also linked to the aposporous mode of reproduction in two small segregating populations of Cenchrus ciliaris (= Pennisetum ciliare (L.)Link), another apomictic grass species. Among 12 markers (sequence characterized amplified regions, SCARs), six were scored as dominant markers between aposporous and sexual C. ciliaris genotypes (presence/absence, respectively). Five were always linked to apospory and one showed a low level of recombination in 84 progenies. Restriction fragment length polymorphisms (RFLPs) were observed between sexual and apomictic phenotypes for three of the six remaining SCARs from P. squamulatum when used as probes. No recombination was observed in the F1 progenies. Preliminary data from megabase DNA analysis and sequencing in both species indicate that an apospory‐specific genomic region (ASGR) is highly conserved between the two species. Although C. ciliaris has a smaller genome size to P. squamulatum, a higher copy number for markers linked to apospory found in the former may impair the progress of positional cloning of gene(s) for apomixis in this species.
The liguleless2 gene of maize functions during the transition from the vegetative to the reproductive shoot apex
Authors: Walsh Justine , Freeling Michael
Published: March 2003

During a maize plant’s (Zea mays) development, the shoot apical meristem (SAM) generates an apex that proceeds through different phases: juvenile vegetative, adult vegetative and reproductive. During each phase the structures produced are distinguishable from structures produced during the other phases. In this paper, we demonstrate that the LIGULELESS2 (LG2) function is required for an accurate vegetative to reproductive phase transition. The maize gene liguleless2 (lg2) has been shown to encode a basic‐leucine zipper (bZIP) protein and to function in narrowing the region from which the ligule and auricle develop in a typical maize leaf. Here we show that lg2 mutant plants can have reduced long tassel branches, extra vegetative leaves and extra husk leaves when compared to wild‐type siblings. This indicates a role for the lg2 gene in the vegetative to reproductive phase transition of the shoot apex. We also discuss a potential role for the lg2 gene in general phase transition processes.
The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene
Authors: Zou Jitao , Wei Yangdou , Jako Colette , Kumar Arvind , Selvaraj Gopalan , Taylor David C.
Published: March 2003

In Arabidopsis thaliana (ecotype Columbia) mutant line AS11, an EMS‐induced mutation at a locus on chromosome II results in a reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol, an altered seed fatty acid composition, and delayed seed development. A mutation has been identified in AS11 in a gene, which we designated as TAG1, that encodes a protein with an amino acid sequence which is similar to a recently reported mammalian DGAT, and, to a lesser extent, to acyl CoA:cholesterol acyltransferases. Molecular analysis revealed that the mutant allele in AS11 has a 147 bp insertion located at the central region of intron 2. At the RNA level, an 81 bp insertion composed entirely of an exon 2 repeat was found in the transcript. While the seed triacylglycerol content is reduced by the lesion in AS11, there is no apparent effect on sterol ester content in the mutant seed. The TAG1 cDNA was over‐expressed in yeast, and its activity as a microsomal DGAT confirmed. Therefore, the TAG1 locus encodes a diacylglycerol acyltransferase, and the insertion mutation in the TAG1 gene in mutant AS11 results in its altered lipid phenotype.
Cross‐talk between wound signalling pathways determines local versus systemic gene expression in Arabidopsis thaliana
Authors: Rojo Enrique , León José , Sánchez‐Serrano José J.
Published: March 2003

Plants react to mechanical damage by activating a set of genes, the products of which are thought to serve defensive functions. In solanaceous plants, cell wall‐derived oligosaccharides and the plant hormones jasmonic acid and ethylene participate in the signalling network for wound‐induced expression of proteinase inhibitors and other defence‐related genes, both in the locally damaged and in the systemic non‐damaged leaves. Here we show that in Arabidopsis thaliana, these signalling components interact in novel ways to activate distinct responses. In damaged tissues, oligosaccharides induce the expression of a specific set of wound‐responsive genes while repressing jasmonic acid‐responsive genes that are activated in the systemic tissues. The oligosaccharide‐mediated repression of the jasmonic acid‐dependent signalling pathway is exerted through the production and perception of ethylene in the locally damaged tissue. This cross‐talk between separate wound signalling pathways thus allows the set up of different responses in the damaged and the systemic tissues of plants reacting to injury.
Distinct osmo‐sensing protein kinase pathways are involved in signalling moderate and severe hyper‐osmotic stress
Authors: Munnik Teun , Ligterink Wilco , Meskiene Irute , Calderini Ornella , Beyerly John , Musgrave Alan , Hirt Heribert
Published: March 2003

Plant growth is severely affected by hyper‐osmotic salt conditions. Although a number of salt‐induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo‐sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper‐osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mm NaCl). In contrast, at high salt concentrations (above 750 mm NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen‐activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper‐osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca‐ tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra‐cellular compartmentation, suggesting an intra‐nuclear mechanism for the regulation of SIMK activity.
Transient expression of members of the germin‐like gene family in epidermal cells of wheat confers disease resistance
Authors: Schweizer Patrick , Christoffel Andreas , Dudler Robert
Published: March 2003

The wheat genome encodes a family of germin‐like proteins that differ with respect to regulation and tissue specificity of expression of the corresponding genes. While germin exhibits oxalate oxidase (E.C. 1.2.3.4.) activity, the germin‐like proteins (GLPs) have no known enzymatic activity. A role of oxalate oxidase in plant defence has been proposed, based on the capacity of the enzyme to produce H2O2, a reactive oxygen species. The role in defence of germin and other members of the germin‐like gene family was functionally assessed in a transient assay system based on particle bombardment of wheat leaves. Transient expression of the pathogen‐induced germin gf‐2.8 gene, but not of the constitutively expressed HvGLP1 gene, reduced the penetration efficiency of Blumeria (syn. Erysiphe) graminis f.sp. tritici, the causal agent of wheat powdery mildew, on transformed cells. Two engineered germin‐gf‐2.8 genes and the TaGLP2a gene, which all encoded proteins without oxalate oxidase activity, also reduced the penetration efficiency of the fungus, demonstrating that oxalate oxidase activity is not required for conferring enhanced resistance. Instead, activity tagging experi‐ments showed that in cells transiently expressing the germin gf‐2.8 gene, the transgene product became insolubilised at sites of attempted fungal penetration where localised production of H2O2 was observed. Thus, germin and GLPs may play a structural role in cell‐wall re‐enforcement during pathogen attack.
Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants
Authors: Pauly Markus , Albersheim Peter , Darvill Alan , York William S.
Published: March 2003

Cellulose and xyloglucan (XG) assemble to form the cellulose/XG network, which is considered to be the dominant load‐bearing structure in the growing cell walls of non‐graminaceous land plants. We have extended the most commonly accepted model for the macromolecular organization of XG in this network, based on the structural and quantitative analysis of three distinct XG fractions that can be differentially extracted from the cell walls isolated from etiolated pea stems. Approximately 8% of the dry weight of these cell walls consists of XG that can be solubilized by treatment of the walls with a XG‐specific endoglucanase (XEG). This material corresponds to an enzyme‐susceptible XG domain, proposed to form the cross‐links between cellulose microfibrils. Another 10% of the cell wall consists of XG that can be solubilized by concentrated KOH after XEG treatment. This material constitutes another XG domain, proposed to be closely associated with the surface of the cellulose microfibrils. An additional 3% of the cell wall consists of XG that can be solubilized only when the XEG‐ and KOH‐treated cell walls are treated with cellulase. This material constitutes a third XG domain, proposed to be entrapped within or between cellulose microfibrils. Analysis of the three fractions indicates that metabolism is essentially limited to the enzyme‐susceptible domain. These results support the hypothesis that enzyme‐catalyzed modification of XG cross‐links in the cellulose/XG network is required for the growth and development of the primary plant cell wall, and demonstrate that the structural consequences of these metabolic events can be analyzed in detail.
An efficient screen for reproductive pathways using mature seeds of monocots and dicots
Authors: Matzk Fritz , Meister Armin , Schubert Ingo
Published: March 2003

Seed samples of 32 species (obligate and facultative sexuals and apomicts of monocots and dicots) were investigated by flow cytometry to reveal the pathway of reproduction. Ten different pathways of seed formation could be reconstructed considering whether the female and/or male gametes were reduced or unreduced, the embryos arose via the zygotic or parthenogenetic route and the endosperm via the pseudogamous or autonomous route. The screen is suited to select sporophytic or gametophytic mutants in sexual species, to identify pure sexual or obligate apomictic genotypes from facultative apomictic species, and to analyze the inheritance of the individual reproductive processes. Corresponding unique results are presented for Arabidopsis, Arabis, Hypericum and Poa. The screen of mature seeds by flow cytometry yielded more information about the reproductive behavior of individual plants than any other available test, and is very useful both in basic research and plant breeding.
Movement of plant viruses is delayed in a β‐1,3‐glucanase‐deficient mutant showing a reduced plasmodesmatal size exclusion limit and enhanced callose deposition
Authors: Iglesias Victor Alejandro , Meins Frederick
Published: March 2003

Susceptibility to virus infection is decreased in a class I β‐1,3‐glucanase (GLU I)‐deficient mutant (TAG4.4) of tobacco generated by antisense transformation. TAG4.4 exhibited delayed intercellular trafficking via plasmodesmata of a tobamovirus (tobacco mosaic virus), of a potexvirus (recombinant potato virus X expressing GFP), and of the movement protein (MP) 3a of a cucumovirus (cucumber mosaic virus). Monitoring the cell‐to‐cell movement of dextrans and peptides by a novel biolistic method revealed that the plasmodesmatal size exclusion limit (SEL) of TAG4.4 was also reduced from 1.0 to 0.85 nm. Therefore, GLU I‐deficiency has a broad effect on plasmodesmatal movement, which is not limited to a particular virus type. Deposition of callose, a substrate for β‐1,3‐glucanases, was increased in TAG4.4 in response to 32°C treatment, treatment with the fungal elicitor xylanase, and wounding, suggesting that GLU I has an important function in regulating callose metabolism. Callose turnover is thought to regulate plasmodesmatal SEL. We propose that GLU I induction in response to infection may help promote MP‐driven virus spread by degrading callose.
Characterization of a nitrate‐permeable channel able to mediate sustained anion efflux in hypocotyl cells from Arabidopsis thaliana
Authors: Frachisse Jean‐Marie , Colcombet Jean , Guern Jean , Barbier‐Brygoo Hélène
Published: March 2003

We have characterized a new anionic current in Arabidopsis hypocotyl cells. This current, activated by membrane depolarization, has slow activation and deactivation kinetics in the 10 sec range. It presents many distinct properties from the rapid‐type anion current already described on the same membrane. The slow‐type channel is highly permeable to nitrate with a PNO3–/PCl– close to 20, but totally impermeable to sulphate. Activation of the channel requires cytosolic ATP and the slow current is partially inhibited by staurosporin, suggesting that channel regulation involves protein phosphorylation. The slow anion channel displays a unique pharmacological profile different from that of the rapid channel: the slow channel is inhibited by DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid) with an IC50 of 26 μM. The slow and rapid anion channels are probably dedicated to specific functions: the first is able to mediate sustained anion efflux, while the second is a good candidate to be involved in fast electrical signalling.
Disruption of the Arabidopsis photosystem I gene psaE1 affects photosynthesis and impairs growth
Authors: Varotto Claudio , Pesaresi Paolo , Meurer Jörg , Oelmüller Ralf , Steiner‐Lange Sabine , Salamini Francesco , Leister Dario
Published: March 2003

The psae1‐1 mutant of Arabidopsis was identified on the basis of a decrease in the effective quantum yield of photosystem II, among a collection of plants subjected to transposon tagging with the Enhancer element. The steady‐state redox level and the rate of re‐oxidation of P700 are significantly altered in psae1‐1 mutants. The responsible mutation was localised to psaE1, one of two Arabidopsis genes that encode subunit E of photosystem I. An additional mutant allele, psae1‐2, was identified by reverse genetics. In wild‐type plants, the psaE1 transcript is expressed at a higher level than psaE2 mRNA. In the mutants, however, the E1 transcript was barely detectable, and was expressed only in small groups of wild‐type cells resulting from somatic reversions. As a consequence, the amount of PsaE protein present in the mutant is significantly reduced. Concomitantly, the levels of other stromal photosystem I subunits (PsaC and PsaD) are also affected. Mutant plants showed a marked increase in light sensitivity and photoinhibition. Additional effects of the psae1 mutation include light green pigmentation, an increase in chlorophyll fluorescence and a decrease of approximately 50% in growth rate under greenhouse conditions.
The evolution of C4 plants: acquisition of cis‐regulatory sequences in the promoter of C4‐type pyruvate, orthophosphate dikinase gene
Authors: Nomura Mika , Sentoku Naoki , Nishimura Asuka , Lin Jenq‐Horng , Honda Chikako , Taniguchi Mitutaka , Ishida Yuji , Ohta Shozo , Komari Toshihiko , Miyao‐Tokutomi Mitsue , Kano‐Murakami Yuriko , Tajima Shigeyuki , Ku Maurice S. B. , Matsuoka Makoto
Published: March 2003

In a previous study, we identified the C4‐like pyruvate, orthophosphate dikinase gene (Pdk) in the C3 plant rice, with a similar structure to the C4‐type Pdk in the C4 plant maize. In order to elucidate the differences between C4‐type and C4‐like Pdk genes in C4 and C3 plants, we have produced chimeric constructs with the β‐glucuronidase (GUS) reporter gene under the control of the Pdk promoters. In transgenic rice, both rice and maize promoters directed GUS expression in photosynthetic organs in a light‐dependent manner. However, the maize promoter exhibited a much higher transcriptional activity than the rice promoter did. These results indicate that the rice C4‐like Pdk gene resembles the maize C4‐type Pdk gene in terms of regulation of expression. We also tested the activity of the rice promoter in transgenic maize. GUS activity was seen in both photosynthetic and non‐photosynthetic organs. Thus, the rice promoter does not confer a strict organ‐specific gene expression, as the maize promoter does. Moreover, the rice promoter directed GUS expression not only in mesophyll cells but also in bundle sheath cells, whereas the maize promoter directed expression only in mesophyll cells. Taken together, the results obtained from both transgenic maize and rice demonstrate that the rice and maize promoters differ not only quantitatively, but also qualitatively, in terms of their cell‐ and organ‐specificity. Experiments with swapped promoters using the rice and maize promoters further demonstrated that a limited sequence region from −330 to −76 of the maize promoter confers light‐regulated, high‐level expression to the rice promoter in maize mesophyll protoplasts. We conclude the gain of cis‐acting elements conferring high‐level expression and mesophyll cell specificity was necessary for establishment of a C4‐type Pdk gene during the course of evolution from C3 to C4 plants.
Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein
Authors: García‐Gómez Blanca I. , Campos Francisco , Hernández Magdalena , Covarrubias Alejandra A.
Published: March 2003

Two antigenically related glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell wall in response to water deficit in Phaseolus vulgaris. In this report, we show that p33 and p36 are able to adhere to leaf protoplasts, and that they bind to plasma membrane (PM) vesicles in a divalent cation‐dependent manner. Data from the partial amino acid sequence of the p33 and p36 proteins indicate that they contain repeats of the decapeptide POVYKPOVEK; therefore, they are related to proline‐rich proteins. Binding assays demonstrate that both proteins specifically bind to an 80 kDa PM protein. This binding is competed with a peptide that contains the RGD motif, as well as with fibronectin, which also includes this sequence, suggesting that the 80 kDa PM protein has an integrin‐like function whose natural ligands are p33 and p36. This is the first case where a PM ligand for a higher plant cell wall protein has been identified.
The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana
Authors: Takahashi Hideki , Watanabe‐Takahashi Akiko , Smith Frank W. , Blake‐Kalff Mechteld , Hawkesford Malcolm J. , Saito Kazuki
Published: March 2003

To investigate the uptake and long‐distance translocation of sulphate in plants, we have characterized three cell‐type‐specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high‐affinity sulphate transporter (Km for sulphate 3.6 ± 0.6 μm), whereas Sultr2;1 and Sultr2;2 encode low‐affinity sulphate transporters (Km for sulphate 0.41 ± 0.07 m
m and ≥ 1.2 m
m, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. β‐glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter–GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short‐term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate‐sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.
Replication of chloroplast, mitochondrial and nuclear DNA during growth of unirradiated and UVB‐irradiated Arabidopsis leaves
Authors: Draper Christopher K. , Hays John B.
Published: March 2003

Replication of Arabidopsis nuclear, mitochondrial and chloroplast DNA (ncDNA, mtDNA, cpDNA) was assayed by measuring respective changes in copies per leaf, employing quantitative PCR (QPCR) analysis with genome‐specific primer pairs. All three genomes showed parallel increases during growth of cotyledons and 5th leaves in planta, maintaining approximately 13 mtDNA copies and 280 cpDNA copies per haploid nuclear genome. Detached 5th leaves, which showed good growth and DNA replication on agar plates, were irradiated at (DNA‐effective) UV‐B fluences of 1.3–5.0 kJ m−2 and incubated under blue (photorepair‐active) plus gold light or gold light only. Under blue light, replication of all genomes after all UV fluences was approximately as efficient as replication in unirradiated leaves. UV‐irradiated leaves showed little growth under gold light only; 5 kJ m−2 stopped replication of all three genomes, 2.5 kJ m−2 stopped only cpDNA replication, and 1.3 kJ m−2 only delayed cpDNA replication. Immunoassays showed that 5 kJ m−2 induced about 1.2 cyclobutane pyrimidine dimers and 0.1 [6‐4]photoproducts per kbp of bulk DNA, and that both photoproducts were completely removed during 2–3 days under blue light, suggesting efficient photorepair of at least ncDNA and cpDNA. The evidence for efficient photorepair of organellar DNA contrasts with previous studies of irradiated 5‐day‐old seedlings, and with the apparent absence of Arabidopsis photolyases bearing transit peptides.
Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco
Authors: Kater Martin M. , Franken John , Van Aelst Adriaan , Angenent Gerco C.
Published: March 2003

Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over‐expressed the SUP gene in petunia and tobacco in a tissue‐specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over‐expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion.
The CYTOKININ‐HYPERSENSITIVE genes of Arabidopsis negatively regulate the cytokinin‐signaling pathway for cell division and chloroplast development
Authors: Kubo Minoru , Kakimoto Tatsuo
Published: March 2003

We isolated Arabidopsis thaliana mutants that respond more sensitively than the wild type to cytokinins. The calli produced from the mutants exhibit typical cytokinin responses, including rapid proliferation and chloroplast development in response to lower levels of cytokinins than in the wild type. The mutations are recessive and belong to two complementation groups designated ckh1 and ckh2 for cytokinin‐hypersensitive. CKH1 and CKH2 were mapped to the top of chromosome I and the middle of chromosome II, respectively. The cytokinin levels in these mutants were not increased. We speculate that the CKH1 and CKH2 gene products negatively regulate the signaling pathway leading from cytokinin perception to cell proliferation and chloroplast development.
The role of inorganic phosphate in the development of freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana
Authors: Hurry Vaughan , Strand Åsa , Furbank Robert , Stitt Mark
Published: March 2003

Low temperature inhibits sucrose synthesis, leading to a phosphate‐limitation of photosynthesis. We have used the Arabidopsis pho1‐2 and pho2‐1 mutants with decreased and increased shoot phosphate, respectively, to investigate whether low phosphate triggers cold acclimatization of photosynthetic carbon metabolism. Wild‐type Arabidopsis, pho1‐2 and pho2‐1 were grown at 23°C and transferred to 5°C to investigate acclimatization in pre‐existing leaves and in new leaves developing at 5°C. The development of frost tolerance and the accumulation of proline and sugars was unaltered or improved in pho1‐2, and impaired in pho2‐1. Sucrose phosphate synthase and cytoplasmic fructose‐1,6‐bisphosphatase activity and protein increase after transfer to 5°C. This increase was accentuated in pho1‐2 and attenuated in pho2‐1. RBCS and LHCB2 transcript levels decrease in pre‐formed wild‐type leaves after transfer to 5°C and recover in new leaves that develop at 5°C. The initial decrease was attenuated in pho1‐2, and accentuated in pho2‐1, where the recovery in new leaves was also suppressed. Rubisco activity increased in wild‐type leaves that developed at 5°C. This increase was accentuated in pho1‐2 and absent in pho2‐1. NADP‐glyceraldehyde‐3‐phosphate dehydrogenase, plastidic fructose‐1,6‐bisphosphatase and aldolase activity increase relative to phosphoglycerate kinase, transketolase and phosphoribulokinase in wild‐type leaves at 5°C. This shift was accentuated in pho1‐2 and reversed in pho2‐1. Transcript levels for COR genes increase transiently 1 day after transfer to 5°C but were very low in leaves that developed at 5°C in wild‐type Arabidopsis, pho1‐2 and pho2‐1. We conclude that low phosphate plays an important role in triggering cold acclimatization of leaves, leading in particular to an increase of Rubisco expression, changes in other Calvin cycle enzymes to minimize sequestration of phosphate in metabolites, and increased expression of sucrose biosynthesis enzymes.