bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversible protein phosphorylation regulates jasmonic acid‐dependent and ‐independent wound signal transduction pathways in Arabidopsis thaliana
Authors: Rojo Enrique , Titarenko Elena , León José , Berger Susanne , Vancanneyt Guy , Sánchez‐Serrano José J.
Published: March 2003

Plants responses to mechanical injury are complex and include the induced expression of defence‐related genes. The phytohormone JA has been reported to mediate some of these responses. To elucidate further the signal transduction processes involved, the action of specific agonists and antagonists of known signalling effectors on the response of Arabidopsis thaliana plantlets to JA and wounding was investigated. The identification and characterization of a reversible protein phosphorylation step in a transduction pathway leading to JA‐induced gene transcription is reported. This phosphorylation event involved the opposing activities of a staurosporine‐sensitive protein kinase, negatively regulating the pathway, and a protein phosphatase, most probably of type 2 A, which activated JA‐responsive gene expression. JA activation via this pathway was blocked in the A. thaliana JA‐insensitive mutants jin1, jin4 and coi1, and by exogenous application of cycloheximide or auxins. Wound‐induced activation of JA‐responsive genes was also regulated by this protein phosphorylation step. An alternative wound signalling pathway, independent of JA, was also identified, leading to the transcriptional activation of a different set of genes. This JA‐independent pathway was also regulated by a protein phosphorylation switch, in which the protein kinase positively regulated the pathway while the protein phosphatase negatively regulated it. Moreover, a labile protein apparently repressed the expression of these genes. One of the genes analysed, JR3, had a complex pattern of expression, possibly because it was regulated via both of the wound signalling pathways identified. According to the function of an homologous gene, JR3 may be involved in feedback inhibition of the JA response.
Isolation of an Arabidopsis thaliana mutant in which accumulation of cucumber mosaic virus coat protein is delayed
Authors: Yoshii Motoyasu , Yoshioka Norimichi , Ishikawa Masayuki , Naito Satoshi
Published: March 2003

Cucumber mosaic virus (CMV) is known to systemically infect Arabidopsis thaliana ecotype Columbia plants. In order to identify the host factors involved in the multiplication of CMV, we isolated an A. thaliana mutant in which the accumulation of the coat protein (CP) of CMV in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in the mutant plants was caused by a single, nuclear and recessive mutation designated cum1‐1, which was located on chromosome IV. The cum1‐1 mutation did not affect the multiplication of tobacco mosaic virus, turnip crinkle virus or turnip yellow mosaic virus, which belong to different taxonomic groups from CMV. Accumulation of CMV CP in the inoculated leaves of cum1‐1 plants was also delayed either when CMV virion or CMV virion RNA was inoculated. On the other hand, when cum1‐1 and the wild‐type Col‐0 protoplasts were inoculated with CMV virion RNA by electroporation, the accumulations of CMV‐related RNAs and the coat protein were similar. These results suggest that the cum1‐1 mutation did not affect the uncoating of CMV virion and subsequent replication in an initially infected cell but affected the spreading of CMV within an infected leaf, possibly the cell‐to‐cell movement of CMV in a virus‐specific manner.
Production of yellow colour in flowers: redirection of flavonoid biosynthesis in Petunia
Authors: Davies Kevin M. , Bloor Stephen J. , Spiller Gayle B. , Deroles Simon C.
Published: March 2003

Chalcones are intermediates in the biosynthesis of all flavonoids. In addition, in some species they constitute the major yellow flower pigments. There are two types of chalcones, distinguished by the presence (6′‐hydroxychalcones) or absence (6′‐deoxychalcones) of a hydroxyl group at the 6′ position of the A‐ring. The 6′‐deoxychalcones are formed when the enzyme chalcone reductase (CHR) is active in conjunction with chalcone synthase (CHS). In Petunia, only 6′‐hydroxychalcones are synthesized, and except in the pollen of some genotypes, they are ephemeral intermediates in flavonoid metabolism. By introducing a CHR cDNA from Medicago sativa under the control of the 35S CaMV promoter into acyanic‐ or cyanic‐flowered lines of Petunia, flower colour was changed from either white to pale yellow or deep purple to pale purple, respectively. Lines were generated that accumulated up to 60% of their petal flavonoids as 6′‐deoxychalcones. Several different 6′‐deoxychalcones accumulated in the petals of the CHR transgenics. The structures of three of these were determined: one, butein 4‐O‐glucoside, is a novel plant chalcone. Another chalcone compound was identified in the pollen of the transgenics. The results show that the Petunia chalcone isomerase is unable to use 6′‐deoxychalcones as substrates so that 6′‐deoxychalcones are stable in Petunia petals, leaves and pollen, but some Petunia flavonoid enzymes can use 6′‐deoxychalcones as substrates to modify their structures. The introduction of CHR provides a method to redirect the flavonoid pathway into chalcone production, in order to modify flower colour or to reduce the biosynthesis of other flavonoid types.
Repair of genomic double‐strand breaks in somatic plant cells by one‐sided invasion of homologous sequences
Authors: Puchta Holger
Published: March 2003

Double‐strand breaks (DSB) strongly enhance homologous recombination in the genome of somatic plant cells. To characterize the mechanism of homologous DSB repair in detail, Agrobacterium‐mediated targeting experiments were performed in tobacco, with repair constructs being homologous to either one or both ends of a transiently induced DSB. The recombination frequencies obtained were at least two orders of magnitude higher than without DSB induction and differed between the two T‐DNA by only one‐third. Thus, occurrence of homologies at both ends of the break is no prerequisite for recombination. The results demonstrate that most, if not all, reactions are initiated by one‐sided invasion of a homologous sequence. This indicates that the classical double‐strand break repair (DSBR) model originally proposed for meiotic recombination might be less appropriate for the description of homologous DSB repair in somatic plant cells than an integrative model based on a combination of the synthesis‐dependent strand annealing (SDSA) model and of a modified version of the one‐sided invasion (OSI) model of recombination. Direct consequences of this finding for the performance of gene targeting experiments, as well as possible implications for the stability of highly repetitive plant genomes, are discussed.
Lipochito‐oligosaccharides re‐initiate root hair tip growth in Vicia sativa with high calcium and spectrin‐like antigen at the tip
Authors: D Norbert C. A. , Ruijter  , Rook Martin B. , Bisseling Ton , Emons Anne Mie C.
Published: March 2003

Lipochito‐oligosaccharides, Nod factors secreted by Rhizobium bacteria, are signal molecules that induce deformation of root hairs of their host plant. A bioassay was used for deformation, and the cytological changes induced by specific lipochito‐oligosaccharides in root hairs of Vicia sativa L. (vetch), grown between glass slides, were examined. In the assay, root hairs of a particular developmental stage, those that were terminating growth, were susceptible to deformation. These hairs obtained characteristics of tip‐growing cells again: (i) a polar cytoplasmic organization and reverse fountain streaming, (ii) an accumulation of a spectrin‐like antigen at the tip, and (iii) a tip‐focused calcium gradient. Calcium gradients were visualized in Indo‐1 loaded root hairs with UV confocal microscopy and ratio‐imaging. The results show that hairs respond to the bacterial signal by recovering cytoplasmic polarity and exocytosis.
The Arabidopsis thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally equivalent for yeast complementation and are differentially expressed during plant development
Authors: Rodriguez Cecilia Monica , Freire Miguel Angel , Camilleri Christine , Robaglia Christophe
Published: March 2003

Two cDNAs (At.EIF4E1 and At.EIF4E2) encoding, respectively, the eukaryotic initiation factors eIF4E and eIF(iso)4E of Arabidopsis thaliana were isolated by complementation of a Saccharomyces cerevisiae conditional mutant. The deduced amino acid sequences of the proteins are homologous to those from monocotyledonous plants, yeast and mammals. The corresponding genes were identified in YAC clones mapping to chromosome IV (At.EIF4E1) and to chromosome V (At.EIF4E2). The yeast strain complemented by At.EIF4E2 grew poorly compared with an isogenic strain expressing At.EIF4E1. Northern and in situ hybridization analysis show that both Arabidopsis At.EIF4E1 and At.EIF4E2 mRNAs are differentially accumulated in plant tissues. The At.EIF4E1 mRNA is expressed in all tissues except in the cells of the specialization zone of the roots; the At.EIF4E2 mRNA is particularly abundant in floral organs and in young developing tissues. This work further demonstrates an association between a high level of EIF4E mRNAs and cell proliferation and suggests that the plant eIF4E isoforms may have distinct functions in cell development and metabolism.
Identification of cDNAS encoding plastid‐targeted glutathione peroxidase
Authors: Mullineaux Philip M. , Karpinski Stanislaw , Jiménez Ana , Cleary Suzanne P. , Robinson Colin , Creissen Gary P.
Published: March 2003

A cDNA was isolated from pea leaf RNA which encodes a phospholipid hydroperoxide glutathione peroxidase (PHGPX; E.C. 1.1.1.1.9). The N‐terminal section of this PHGPX encodes a recognisable chloroplast transit peptide. Efficient import in vitro of the pre‐PHGPX protein into the stroma of isolated pea chloroplasts confirmed that the PHGPX is a chloroplast‐located enzyme. The pea PHGPX has highly conserved homologues in Arabidopsis, citrus and Nicotiana sylvestris and the authors suggest that these proteins are also localised in the chloroplast and not in the cytosol as previously supposed.
The role of UDP‐glucose epimerase in carbohydrate metabolism of Arabidopsis
Authors: Dörmann Peter , Benning Christoph
Published: March 2003

Uridine 5′‐diphospho‐glucose‐4‐epimerase (UDP‐Glc epimerase) catalyses the reversible epimerization of UDP‐galactose and UDP‐glucose. In contrast to bacteria and yeast, expression of the UDP‐Glc epimerase gene in Arabidopsis was found not to be induced by galactose. To elucidate the metabolic role of this enzyme, transgenic Arabidopsis plants expressing the respective cDNA in sense or antisense orientation were constructed, leading to a range of plant lines with different UDP‐Glc epimerase activities. No alterations in morphology were observed and the relative amounts of different galactose‐containing compounds were not affected if the plants were raised on soil. However, on agar plates in the presence of galactose, the growth of different lines was increasingly repressed with decreasing enzyme activity, and an increase in the UDP‐Gal content was observed in parallel, whereas the UDP‐Glc content was nearly constant. The amount of galactose in the cell wall was increased in plants with low UDP‐Glc epimerase activity grown on galactose, whereas the cellulose content in the leaves was not altered. Furthermore, starch determined at different times of the day was highly abundant in plants with low UDP‐Glc epimerase activity in the presence of galactose. It is proposed that low endogenous UDP‐Glc epimerase activity is responsible for the galactose toxicity of the wild‐type. Possible mechanisms by which the starch content might be modulated are discussed.
Disease resistance gene homologs correlate with disease resistance loci of Arabidopsis thaliana
Authors: Speulman Elly , Bouchez David , Holub Eric B. , Beynon Jim L.
Published: March 2003

The disease resistance genes RPS2 of Arabidopsis and N of tobacco, among other recently cloned resistance genes, share several conserved sequences. Degenerate oligonucleotide primers, based on conserved sequences in the nucleotide binding site (NBS) and a weak hydrophobic domain of RPS2 and N, were used to amplify homologous sequences from Arabidopsis thaliana. Amplification products were obtained that were similar in sequence to the disease resistance genes RPS2, RPM1, N and L6. The Arabidopsis CIC‐YAC library was used to identify the position of the disease resistance homologs on the Arabidopsis genome. Their map positions could be correlated with the disease resistance loci RPS5, RAC1, RPP9, CAR1, RPP7, RPW2, RPP1, RPP10, RPP14, RPP5, RPP4, RPS2, RPW6, HRT, RPS4, RPP8, RPP21, RPP22, RPP23, RPP24 and TTR1. This method was therefore not only successful in the identification of sequences located within gene clusters that are involved in disease resistance, but could also contribute to the cloning of disease resistance genes from Arabidopsis.
Molecular analysis of a second functional A1 gene (dihydroflavonol 4‐reductase) in Zea mays
Authors: Bernhardt Jürgen , Stich Karl , Schwarz‐Sommer Zsuzsanna , Saedler Heinz , Wienand Udo
Published: March 2003

Some genes involved in anthocyanin biosynthesis in Zea mays are duplicated and differentially expressed. From the analysis of the A1 gene (dihydroflavonol 4‐reductase), which is involved in this pathway, no molecular evidence for gene duplication was known to date. Isolation and analysis of A1 homologous genomic clones revealed the presence of a second A1 gene in maize and also two copies of the gene in Teosinte guerrero. The duplicated genes are structurally very similar and, at least in maize, the second gene is expressed.
The use of vibratome sections of cereal spikelets to study anther development and meiosis
Authors: Aragon‐Alcaide Luis , Beven Alison , Moore Graham , Shaw Peter
Published: March 2003

We have developed a method using vibratome sectioning coupled with confocal microscopy to analyse pre‐meiotic and meiotic events in developing wheat anthers. This report describes the application of fluorescence in situ hybridization to three‐dimensionally preserved tissue sections from intact florets. We show that excellent imaging of all the stages of meiosis up to telophase I can be achieved, and show centromere positions and pre‐meiotic homologous pairing both in meiocytes and tapetum cells. These clear results using intact tissue sections are contrasted with conventional spreading methods for analysis of meiosis in plants, which we show can produce artefactual or uninterpretable results.
Phosphatidate phosphatase from avocado (Persea americana) – purification, substrate specificity and possible metabolic implications for the Kennedy pathway and cell signalling in plants
Authors: Pearce Matthew L. , Slabas Antoni R.
Published: March 2003

Phosphatidate phosphatase (PAP; EC 3.1.3.4) is a pivotal enzyme in plant lipid metabolism positioned at a major branchpoint between the biosyntheses of phospholipids and triacylglycerols. The enzyme has been purified 7000‐fold from the microsomes of maturing Avocado fruit. The enzyme has a subunit molecular mass, as determined on SDS‐PAGE, of 49 kDa, and gel filtration studies revealed that it is monomeric. The enzyme was examined for the ability to hydrolyse sn‐1,2‐dioleoylglycerol‐3‐phosphate (PA), sn‐1‐oleoylglycerol‐3‐phosphate (LPA), sn‐2‐oleoylglycerol‐3‐phosphate (sn‐2‐LPA), and ceramide‐1‐phosphate. All substrates were used, but the apparent Vmax values for ceramide‐1‐phosphate and sn‐2‐LPA were considerably lower than for PA or LPA. A kinetic analysis of the purified enzyme was conducted using PA and LPA in Triton X‐100 mixed micelles according to the surface dilution kinetic model. The Vmax and interfacial Michaelis constant (KmB) were calculated for PA as 47 μmol min–1 mg–1 and 1.49 mol%, respectively. LPA was found to be a better substrate with a Vmax and KmB of 100 μmol min–1 mg–1 and 1.06 mol%, respectively. A detailed kinetic analysis of the effect of LPA on PA hydrolysis revealed LPA to be a potent competitive inhibitor of the reaction, with a Ki of 1.4 mol%. This possibly indicates that strict metabolic channelling is in operation in the Kennedy Pathway, with very low steady state concentrations of lyso‐phosphatidate with respect to phosphatidate in vivo.
Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination
Authors: West Christopher E. , Waterworth Wanda M. , Stephens Susan M. , Smith Craig P. , Bray Clifford M.
Published: March 2003

A peptide transport gene (HvPTR1) expressed in the scutella of germinating barley grain has been cloned by an RT–PCR approach. Sequence analysis of the full length cDNA (2260 bp) revealed an open reading frame encoding a 579 amino acid protein of predicted molecular mass 63 kDa, which displayed 58% identity to the Arabidopsis thaliana peptide transporter AtPTR2‐B. Expression of HvPTR1 in Xenopus laevis oocytes conferred a 48‐fold increase in alanyl‐[14C]phenylalanine uptake relative to water injected oocytes, confirming the function of HvPTR1 as a peptide transporter. HvPTR1 expression was detectable only in the scutellum of the germinating barley grain, with no transcript found in roots, shoots or the embryo axis. Transcript levels increased rapidly from 6 to 24 h imbibition, correlating with the development of peptide transport activity in the barley scutellum. Peptide transport provides a significant source of organic nitrogen to the barley embryo for use in germination and growth processes associated with the early stages of seedling development. The temporal and spatial pattern of HvPTR1 expression is consistent with a central role for HvPTR1 in the transport of peptides in the germinating barley grain.
Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network†
Authors: Boevink Petra , Oparka Karl , Cruz Simon Santa , Martin Barry , Betteridge Alan , Hawes Chris
Published: March 2003

We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of

Nicotiana clevelandii

by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the

trans

‐membrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the

Arabidopsis

H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co‐localization of F‐actin in the GFP‐expressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N‐ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin‐directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time

in vivo

brefeldin A‐induced retrograde transport of Golgi membrane protein to the ER.
High‐resolution histographical mapping of glucose concentrations in developing cotyledons ofVicia fabain relation to mitotic activity and storage processes: glucose as a possible developmental trigger
Authors: Borisjuk Ljudmilla , Walenta Stefan , Weber Hans , Mueller‐Klieser Wolfgang , Wobus Ulrich
Published: March 2003

Previous studies provided evidence that the carbohydrate status triggers developmental processes in the growing cotyledons of

Vicia faba

. We describe here the high‐resolution mapping of glucose concentrations in tissue sections of developing faba bean cotyledons by quantitative bioluminescence and single‐photon imaging. Patterns of local glucose distributions are compared with tissue cell type, mitotic index and the distribution pattern of starch. During cotyledon differentiation, gradients in the glucose concentration emerge which are related to the particular cell type. Higher concentrations are found in non‐differentiated premature regions of the cotyledon whereas mature starch‐accumulating regions contain particularly low concentrations of glucose. In addition, glucose concentration is correlated to mitotic activity. The glucose distribution pattern is therefore related to the developmental gradient. Our data provide for the first time evidence for steep glucose gradients across developing plant embryos and favour the idea that sugar gradients may have morphogenic functions in developing cotyledons.
Higher plants possess two structurally different poly(ADP‐ribose) polymerases
Authors: Babiychuk Elena , Cottrill Phillippa B. , Storozhenko Sergei , Fuangthong Mayuree , Chen Yimin , O’Farrell Minnie K. , Van Montagu Marc , Inzé Dirk , Kushnir Sergei
Published: March 2003

One of the immediate reactions of the mammalian cell to many environmental stresses is a massive synthesis of poly(ADP‐ribose), catalyzed by poly(ADP‐ribose) polymerase (PARP). Most of the biological functions attributed to PARP are inferred from experimentation with mammalian cells. In plants, the biology of PARP may be more complicated and diverse than was previously thought. Two poly(ADP‐ribose) polymerase homologues were found in plants, the classical Zn‐finger‐containing polymerase (ZAP) and the structurally non‐classical PARP proteins (APP and NAP), which lack the characteristic N‐terminal Zn‐finger domain. By enzymatic and cytological experiments the recombinant APP protein was shown to be located in the nucleus and to possess DNA‐dependent poly(ADP‐ribose) polymerase activity in yeast. The nuclear localization was further confirmed by the analysis of transgenic tobacco plants that expressed a translational gene fusion between APP and the bacterial β‐glucuronidase. The app promoter was transcriptionally up‐regulated in cells pre‐determined to die because of deficiency in a DNA ligase I.
Tissue localization of expansins in deepwater rice
Authors: Cho Hyung‐Taeg , Kende Hans
Published: March 2003

Expansins are a family of proteins capable of inducing stress relaxation of isolated cell walls. In earlier studies, we showed that the expression of expansin genes in deepwater rice (

Oryza sativa

L.) is regulated by developmental, hormonal and environmental stimuli. Here, we describe the spatial distribution pattern of expansin transcripts and proteins in tissues and organs of deepwater rice using

in situ

mRNA hybridization and immunohistochemical analysis. Expansin transcripts and proteins are present at high levels in the growing internodal epidermis, which has thick cell walls and acts, therefore, as a growth‐limiting cell layer. Expansins are also concentrated in the differentiating vascular bundles of internodes. In the primary root, expansins are predominantly expressed in the tip region, particularly in the epidermis, differentiating vascular cylinder, and around the pericyle. Developing adventitious roots and lateral root primordia also contain high levels of expansin mRNA. In the shoot apex, expansin transcripts are abundant in the emerging leaf primordia. Our results indicate that expansins play an important role in the expansion and differentiation of plant tissues and organs.
Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores ofAdiantum capillus‐venerisL.
Authors: Uchida Kenko , Muramatsu Takamichi , Jamet Elisabeth , Furuya Masaki
Published: March 2003

In the present study, using a newly developed fluorescent differential display technique, we have carried out large‐scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern

Adiantum capillus‐veneris

L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo‐dT primer. The cDNA was then used as a template for PCR with the oligo‐dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 upregulated and four down‐regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up‐regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser‐(Pro)

n

repeats and showed homology with cell wall‐associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far‐red light and photo‐irreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.
Characterisation of the ethanol‐induciblealcgene expression system for transgenic plants
Authors: Salter Michael G. , Paine Jacqueline A. , Riddell Kay V. , Jepson Ian , Greenland Andrew J. , Caddick Mark X. , Tomsett A. Brian
Published: March 2003

We describe a chemically induced gene control mechanism for plants based on the ALCR transcription factor and

alcA

promoter of

Aspergillus nidulans

, which we have called the

alc

system. Ethanol, the chemical inducer, is not toxic at levels required for induction, and can be applied to the plants by spraying, root drenching and addition to liquid growth media. The

alc

system is very sensitive to ethanol and the induction is rapid; 0.01% (1.7 mM) ethanol in liquid growth media initiates chloramphenicol acetyl transferase (CAT) reporter gene expression within 4 h, with maximal expression occurring after 4 days. In the complete absence of ethanol, there is no detectable expression of CAT, nor do we observe induction in plants subjected to wound, cold or drought stress, or following treatment with either salicylic acid or methyl jasmonate. However, extreme anoxia resulting in elevated levels of alcohol dehydrogenase activity in both roots and leaves gave substantial induction of CAT in leaves but not in roots. We believe that the

alc

system will have broad utility in the exogenous control of plant gene expression in pure science and that it also has considerable potential in agriculture.
Activation of plant defense responses and sugar efflux by expression of pyruvate decarboxylase in potato leaves
Authors: Tadege Million , Bucher Marcel , Stähli Walter , Suter Marianne , Dupuis Isabelle , Kuhlemeier Cris
Published: March 2003

When plants are infected with avirulent pathogens, a selected group of plant cells rapidly die in a process commonly called the hypersensitive response (HR). Some mutations and overexpression of some unrelated genes mimic the HR lesion and associated defense responses. In all of these situations, a genetically programmed cell death pathway is activated wherein the cell actively participates in killing itself. Here we report a developmentally and environmentally regulated HR‐like cell death in potato leaves constitutively expressing bacterial pyruvate decarboxylase (PDC). Lesions first appeared on the tip of fully expanded source leaves. Lesion formation was accompanied by activation of multiple defense responses and resulted in a significant resistance to

Phytophthora infestans

. The transgenic plants showed a five‐ to 12‐fold increase in leaf tissue acetaldehyde and exported two‐ to 10‐fold higher amounts of sucrose compared to the wild‐type. When plants were grown at a higher temperature, both the lesion phenotype and sucrose export were restored to wild‐type situations. The reduced levels of acetaldehyde at the elevated temperature suggested that the interplay of acetaldehyde with environmental and physiological factors is the inducer of lesion development. We propose that sugar metabolism plays a crucial role in the execution of cell death programs in plants.
Cold‐regulated gene expression and freezing tolerance in an Arabidopsis thaliana mutant
Authors: Lee Hojoung , Xiong Liming , Ishitani Manabu , Stevenson Becky , Zhu Jian‐Kang
Published: March 2003

Low temperature is an important environmental factor influencing plant growth and development. In this study, we report the characterization of a genetic locus,

HOS2

, which is defined by three

Arabidopsis thaliana

mutants. The

hos2–1

,

hos2–2

and

hos2–3

mutations result in enhanced expression of

RD29A

and other stress genes under low temperature treatment. Gene expression in response to osmotic stress or ABA is not affected in the

hos2

mutants. Genetic analysis indicates that the

hos2

mutations are recessive and in a nuclear gene. Compared with the wild‐type plants, the

hos2–1

mutant plants are less capable of developing freezing tolerance when treated with low non‐freezing temperatures. However, the

hos2–1

mutation does not impair the vernalization response. These results indicate that

HOS2

is a negative regulator of low temperature signal transduction important for plant cold acclimation.
TheArabidopsis HAL2‐like gene family includes a novel sodium‐sensitive phosphatase
Authors: Gil‐Mascarell Rosario , López‐Coronado José M. , Bellés José M. , Serrano Ramón , Rodríguez Pedro L.
Published: March 2003

The yeast

HAL2

gene encodes a lithium‐ and sodium‐sensitive phosphatase that hydrolyses 3′‐phosphoadenosine‐5′‐phosphate (PAP). Salt toxicity in yeast results from Hal2 inhibition and accumulation of PAP, which inhibits sulphate assimilation and RNA processing. We have investigated whether the model plant

Arabidopsis thaliana

contains sodium‐sensitive PAP phosphatases. The

Arabidopsis HAL2

‐like gene family is composed of three members:

AtAHL

and

AtSAL2

, characterized in the present work, and the previously identified

AtSAL1

. The

AtAHL

and

AtSAL2

cDNAs complement the auxotrophy for methionine of the yeast

hal2

mutant and the recombinant proteins catalyse the conversion of PAP to AMP in a Mg2+‐dependent reaction sensitive to inhibition by Ca2+ and Li+. The PAP phosphatase activity of AtAHL is sensitive to physiological concentrations of Na+, whereas the activities of AtSAL1 and AtSAL2 are not. Another important difference is that AtAHL is very specific for PAP while AtSAL1 and AtSAL2 also act as inositol polyphosphate 1‐phosphatases. AtAHL constitutes a novel type of sodium‐sensitive PAP phosphatase which could act co‐ordinately with plant sulphotransferases and serve as target of salt toxicity in plants.
Specific checkpoints regulate plant cell cycle progression in response to oxidative stress
Authors: Reichheld Jean‐Philippe , Vernoux Teva , Lardon Filip , Van Montagu Marc , Inzé Dirk
Published: March 2003

The effects of oxidative stress on plant cell cycle progression were studied both in cell suspensions and
in planta
. Oxidative stress of variable severity was imposed by the addition of different concentrations of the methyl‐quinone, menadione, into the growth media. In cell suspensions, flow cytometry analyses demonstrated that low concentrations (20–50 μM) of menadione impaired the G1/S transition, slowed DNA replication, and delayed the entry into mitosis. Furthermore, cells in G1 were more sensitive to menadione‐mediated oxidative stress than cells in S phase. Cell cycle arrest was associated with an inhibition of the activity of cyclin‐dependent kinases, cell cycle gene expression, and a concomitant activation of stress genes. Menadione‐mediated oxidative stress was shown to have very similar effects on tobacco plants, suggesting that a general regulation mechanism takes place in plants. These results define an oxidative stress checkpoint pathway that modulates both the expression of the core cell cycle genes and oxidative defence genes. Redox sensing could be of key importance in controlling cell cycle progression in environmental stress conditions.
The HMG‐I/Y protein PF1 stimulates binding of the transcriptional activator GT‐2 to the PHYA gene promoter
Authors: Martínez‐García Jaime F.. , Quail Peter H..
Published: March 2003

The DNA‐binding proteins PF1 and GT‐2 are factors that bind to different functionally defined, positively acting cis‐elements in the PHYA genes of oat and rice, respectively. PF1 is an HMG‐I/Y protein, with its cognate cis‐element being an AT‐rich sequence, designated PE1, whereas GT‐2 is a transcriptional activator with twin DNA binding domains that recognize a triplet of GT‐boxes in a complex motif designated GTE. To further define the DNA‐binding activity of PF1 and to explore potential inter‐relationships between the two factors, we have performed a series of in vitro DNA‐binding experiments with both PE1 and GTE target sites. The data show that, consistent with its membership of the HMG‐I/Y protein family, PF1 can bend DNA when bound to PE1. In addition, PF1 can bind promiscuously, with varying affinity, to other AT‐containing motifs, including GTE. When co‐incubated with GT‐2, PF1 enhances the specific DNA‐binding activity of GT‐2 toward GTE, the first report of such activity for a plant HMG‐I/Y protein. This enhancement takes place without demonstrable physical contact between the two proteins, suggesting the possibility of a novel, indirect mechanism of recruitment involving DNA target‐site pre‐conditioning. The evidence indicates therefore that PF1 and GT‐2 do not perform functionally equivalent roles in positively regulating oat and rice PHYA gene expression. However, the data suggest the possibility that PF1 may act as an architectural factor, promiscuously recognizing a spectrum of AT‐containing elements in plant promoters, with the general function of catalyzing enhanced binding of conventional cognate transcriptional regulators to these elements via DNA bending.
A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana
Authors: Gómez‐Gómez Lourdes , Felix Georg , Boller Thomas
Published: March 2003

Peptides corresponding to the most conserved domain of eubacterial flagellin act as potent elicitors in cells of different plant species. In intact Arabidposis thaliana seedlings these peptides (flg22 and flg15) caused callose deposition, induction of genes coding for pathogenesis‐related proteins and a strong inhibition of growth. Half‐maximal growth inhibition occurred at peptide concentrations of approximately 100 nM. In contrast, peptides representing the corresponding flagellin domains of the plant‐associated bacteria A. tumefaciens and R. meliloti were inactive even at concentrations of 10 μM. With the exception of Ws‐0, all ecotypes of A. thaliana tested were sensitive to flg22. Crosses of Ws‐0 with the sensitive ecotypes Col‐0 and La‐er, respectively, resulted in sensitive F1 seedlings. In the F2 generation of both crosses, sensitivity segregated as a single trait with markers of chromosome 5 and a ratio of 3:1. Dominance of the locus sensing flagellin, termed FLS‐1, suggests that it encodes an element which is important for the perception of the flagellin signal.
Transfer of rps14 from the mitochondrion to the nucleus in maize implied integration within a gene encoding the iron–sulphur subunit of succinate dehydrogenase and expression by alternative splicing
Authors: Figueroa Pablo , Gómez Isabel , Holuigue Loreto , Araya Alejandro , Jordana Xavier
Published: March 2003

The maize mitochondrial genome does not contain a gene coding for ribosomal protein S14. In this paper we show that the functional rps14 gene was translocated to the nucleus and acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Transferred rps14 was found integrated between both exons of a gene encoding the iron–sulphur subunit of the respiratory complex II (sdh2). Sdh2 exon 1 and rps14 were separated by a typical plant nuclear intron that was spliced to give a mature poly(A)+ mRNA of 1.4 kb. This processed mRNA encoded a chimeric SDH2 (truncated)–RPS14 polypeptide, and we show that this chimeric polypeptide is targeted into isolated plant mitochondria, where it is proteolytically processed in a complex way. An alternative splicing event utilizing the same 5′ splice site and a different downstream 3′ splice site generated a second mature poly(A)+ mRNA of 1.3 kb that contained both sdh2 exons. This sdh2 transcript encoded an SDH2 polypeptide highly conserved compared with its homologues in other organisms, and it contained the three cysteine‐rich clusters that made up the three non‐heme iron–sulphur centres responsible for electron transport. To our knowledge, these results constitute the first evidence of alternative splicing playing a role in the expression and targeting of two mitochondrial proteins with different functions from the same gene.
Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola
Authors: Thomma Bart P. H. J. , Nelissen Inge , Eggermont Kristel , Broekaert Willem F.
Published: March 2003

The phytoalexin‐deficient Arabidopsis mutant pad3‐1, which is affected in the production of the indole‐type phytoalexin camalexin, has previously been shown not to display altered susceptibility to either the bacterium Pseudomonas syringae (Glazebrook & Ausubel 1994; Proc. Natl. Acad. Sci. USA, 91: 8955–8959) or the biotrophic fungi Peronospora parasitica (Glazebrook et al. 1997; Genetics, 146: 381–392) and Erysiphe orontii (Reuber et al. 1998; Plant J. 16: 473–485). We now show that this mutant is markedly more susceptible than its wild‐type parental line to infection by the necrotrophic fungus Alternaria brassicicola, but not to Botrytis cinerea. A strong camalexin response was elicited in wild‐type plants inoculated with either Alternaria brassicicola or Botrytis cinerea, whereas no camalexin could be detected in pad3‐1 challenged with these fungi. Hence, PAD3 appears to be a key determinant in resistance to at least A. brassicicola. The induction of salicylate‐dependent and jasmonate/ethylene‐dependent defense genes was not reduced in Alternaria‐challenged pad3‐1 plants compared to similarly treated wild‐type plants. Camalexin production could not be triggered by exogenous application of either salicylate, ethylene or jasmonate and was not, or not strongly, reduced in mutants with defects in perception of these defense‐related signal molecules. Camalexin‐production appears to be controlled by a pathway that exhibits little cross‐talk with salicylate‐, ethylene‐ and jasmonate‐dependent signalling events.
Ac as a tool for the functional genomics of rice
Authors: Enoki Hiroyuki , Izawa Takeshi , Kawahara Mihoko , Komatsu Mai , Koh Serry , Kyozuka Junko , Shimamoto Ko
Published: March 2003

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac‐flanking sequences revealed that 21 clones exhibited significant similarities with protein‐coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein‐coding rice genes. To examine the feasibility of PCR‐based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three‐dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.
Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves
Authors: Hugot Karine , Aimé Sébastien , Conrod Sandrine , Poupet Alain , Galiana Eric
Published: March 2003

During tobacco development, a transition state from susceptibility to resistance to fungal pathogen infection is observed. Leaves acquire resistance toPhytophthora parasisticawhen the plant becomes committed to flowering. The ability to develop resistance does not imply pathogen‐induced defence responses as for the onset of systemic acquired resistance (SAR). Throughout flowering growth, fungal establishment is restrained at two levels. The first level is the control of infection effectiveness. Using the salicylic acid non‐accumulating NahG plants, we demonstrate that this control does not require salicylic acid accumulation. The intercellular fluids (IFs) from tobacco leaves committed to flowering exhibit a cytotoxic activity on fungal zoospore cells based onin vitrogermination assays. Its accumulation is correlated to the control of infection effectiveness that occurs during flowering growth. The expression of this activity appears to constitute a developmental regulated mechanism that inhibits early steps of fungal pathogen installation. A second level of fungal growth control is the restriction of fungal hyphae expansion. In contrast to infection initiation, fungal hyphae spreading appears to be restricted by similar mechanisms induced during SAR as it is attested by the requirement of salicylic acid accumulation and by the correlating apoplastic accumulation of PR1 proteins. These results provide evidence for the activation of a set of at least two regulatory pathways during flowering growth. This activation leads to the induction of mechanisms which control fungal development by affecting the ability of the fungus to both infect and colonise plant tissues.
The ethylene‐inducible PK12 kinase mediates the phosphorylation of SR splicing factors
Authors: Savaldi‐Goldstein Sigal , Sessa Guido , Fluhr Robert
Published: March 2003

The tobacco PK12 is induced by the plant hormone ethylene and is a member of the LAMMER family of protein kinases. Members of this family contain in their C‐terminus a unique ‘EHLAMMERI/VLGPLP’ motif of unknown function, and are related to cyclin‐ and mitogen‐activated protein (MAP)‐dependent kinases. The animal members of this class play a role in differentiation. They phosphorylate and physically interact with serine/arginine‐rich (SR) splicing factors in vivo to alter their activity and the splicing of target mRNAs. SR proteins have been recently described in plants. The capability of PK12 LAMMER kinase to bind and phosphorylate SR proteins was tested in vitro by kinase and binding assays. The tobacco PK12 phosphorylated both animal and plant SR proteins and specifically interacted with the plant splicing factor atSRp34/SR1. In addition, by site‐directed mutagenesis, the LAMMER motif was found to be required for PK12 kinase activity but was not necessary for substrate binding. Consistent with a role in phosphorylation of splicing factors, PK12 was found to localize to the nucleus when transiently over‐expressed in suspension cells.
AtBI‐1, a plant homologue of Bax Inhibitor‐1, suppresses Bax‐induced cell death in yeast and is rapidly upregulated during wounding and pathogen challenge
Authors: Sanchez Pedro , De Torres Zabala Marta , Grant Murray
Published: March 2003

Extensive searches have so far failed to identify functional plant homologues of the mammalian apoptotic machinery. Here we report the isolation and characterisation of an Arabidopsis thaliana homologue of human Bax Inhibitor‐1, AtBI‐1, isolated during a differential screen of plants challenged with the phytopathogen Pseudomonas syringae. AtBI is a member of a small gene family in Arabidopsis, members of which display extensive amino acid identity to human BI‐1. AtBI‐1 is also functionally similar to BI‐1 in its ability to suppress the lethal phenotype in yeast conferred by expression of the mammalian proapoptotic protein, Bax. Expression of AtBI‐1 is rapidly upregulated in plants during wounding or pathogen challenge, suggesting a role in responses to biotic and abiotic stress. AtBI‐1 upregulation appears R gene independent and is not markedly affected by mutations required for specific classes of R genes. However, the accumulation of AtBI‐1 message is significantly reduced in coi1, in which defence responses to insects, pathogens and wounding are compromised.
A xylem‐specific cellulose synthase gene from aspen (Populus tremuloides) is responsive to mechanical stress
Authors: Wu Luguang , Joshi Chandrashekhar P. , Chiang Vincent L.
Published: March 2003

Angiosperm trees accumulate an elevated amount of highly crystalline cellulose with a concomitant decrease in lignin in the cell walls of tension‐stressed tissues. To investigate the molecular basis of this tree stress response, we cloned a full‐length cellulose synthase (PtCesA) cDNA from developing xylem of aspen (Populus tremuloides). About 90% sequence similarity was found between the predicted PtCesA and cotton GhCesA proteins. Northern blot and in situ hybridization analyses of PtCesA gene transcripts in various aspen tissues, and PtCesA gene promoter‐β‐glucuronidase (GUS) fusion analysis in transgenic tobacco, demonstrated conclusively that PtCesA expression is confined to developing xylem cells during normal plant growth. During mechanical stress induced by stem bending, GUS expression remained in xylem and was induced in developing phloem fibers undergoing tension stress, but was turned off in tissues undergoing compression on the opposite side of the bend. Our results suggest a unique role for PtCesA in cellulose biosynthesis in both tension‐stressed and normal tissues in aspen, and that the on/off control of PtCesA expression may be a part of a signaling mechanism triggering a stress‐related compensatory deposition of cellulose and lignin that is crucial to growth and development in trees.
In vivo targeting of a sunflower oil body protein in yeast secretory (sec) mutants
Authors: Beaudoin Frédéric , Wilkinson Barrie M. , Stirling Colin J. , Napier Johnathan A.
Published: March 2003

A sunflower oleosin was expressed in yeast to study the in vivo insertion of the protein into the endoplasmic reticulum (ER) and subsequent transfer to lipid bodies. The oleosin cDNA was expressed in a range of yeast secretory (sec) mutants to determine the precise targeting pathway of the oleosin to the ER. Subcellular fractionation experiments indicated that the signal recognition particle (SRP) is required for oleosin targeting to the ER and hence subsequent deposition on the lipid bodies in vivo. The expression of oleosin in a range of sec61 mutant alleles confirmed the role of the SEC61 translocon in insertion of oleosin into the ER membrane, as well as indicating an unusual substrate/translocon interaction for one particular allele (sec61‐3). Mistargeting of the oleosin due to impaired SRP function resulted in enhanced proteolysis of the plant protein in the transformed yeast, as determined by pulse–chase analysis. These data therefore provide the first in vivo evidence for the SRP‐dependent targeting of the oleosin to the ER, and the subsequent requirement for a functional SEC61 translocon to mediate the correct insertion of the protein into the membrane.