bluuebunny/crossref_metadata_2025 huggingface dataset:
The 5′ untranslated region of the At‐P5R gene is involved in both transcriptional and post‐transcriptional regulation
Authors: Hua Xue Jun , Van De Cotte Brigitte , Van Montagu Marc , Verbruggen Nathalie
Published: March 2003

The steady‐state level of transcripts coding for the pyrroline‐5‐carboxylate reductase of Arabidopsis (At‐P5R) increased under salt and heat stress, mainly because of an enhanced mRNA stability. However, the At‐P5R protein level was not induced, and its translation was inhibited at initiation stage and probably also at later stages. Replacement of the 5′ untranslated region (5′UTR) and β‐glucuronidase (gus) fusion analysis revealed that the first 92 bp region of the At‐P5R 5′UTR was sufficient to mediate transcript stabilization and translation inhibition during salt and heat stresses. Furthermore, the first 92 bp region of the At‐P5R 5′UTR was also involved in transcription efficiency in a promoter‐dependent manner. The results demonstrated that the stress regulation of At‐P5R is complex and involves the 5′UTR which acts at three levels, partly in opposing directions.
The role of calcium in ABA‐induced gene expression and stomatal movements
Authors: Webb Alex A. R. , Larman Mark G. , Montgomery Lucy T. , Taylor Jane E. , Hetherington Alistair M.
Published: March 2003

There is much interest in the transduction pathways by which abscisic acid (ABA) regulates stomatal movements (ABA‐turgor signalling) and by which this phytohormone regulates the pattern of gene expression in plant cells (ABA‐nuclear signalling). A number of second messengers have been identified in both the ABA‐turgor and ABA‐nuclear signalling pathways. A major challenge is to understand the architecture of ABA‐signalling pathways and to determine how the ABA signal is coupled to the appropriate response. We have investigated whether separate Ca2+‐dependent and ‐independent ABA‐signalling pathways are present in guard cells. Our data suggest that increases in [Ca2+]i are a common component of the guard cell ABA‐turgor and ABA‐nuclear signalling pathways. The effects of Ca2+ antagonists on ABA‐induced stomatal closure and the ABA‐responsive CDeT6‐19 gene promoter suggest that Ca2+ is involved in both ABA‐turgor signalling and ABA‐nuclear signalling in guard cells. However, the sensitivity of these pathways to alterations in the external calcium concentration differ, suggesting that the ABA‐nuclear and ABA‐turgor signalling pathways are not completely convergent. Our data suggest that whilst Ca2+‐independent signalling elements are present in the guard cell, they do not form a completely separate Ca2+‐independent ABA‐signalling pathway.
Identification of a cis‐regulatory element for L1 layer‐specific gene expression, which is targeted by an L1‐specific homeodomain protein
Authors: Abe Mitsutomo , Takahashi Taku , Komeda Yoshibumi
Published: March 2003

The Arabidopsis thaliana PROTODERMAL FACTOR1 (PDF1) gene encoding a putative extracellular proline‐rich protein is exclusively expressed in the L1 layer of shoot apices and the protoderm of organ primordia. In order to identify essential cis‐regulatory sequences required for the L1 layer‐specific expression, a series of 5′ deletions of the PDF1 promoter were fused to the β‐glucronidase (GUS) gene and introduced into Arabidopsis plants. Our analysis revealed that the minimum region necessary to confer L1‐specific expression of PDF1 is confined within a 260‐bp fragment upstream of the transcription start site. We identified an 8‐bp motif in this region that is conserved between promoter regions of all the L1‐specific genes so far cloned, and we designated it the L1 box. Electrophoretic mobility shift assays demonstrated that the L1‐specific homeodomain protein ATML1 can bind to the L1 box sequence in vitro. The GUS expression in transgenic plants disappeared when a mutation that abolishes binding of ATML1 was introduced into the PDF1 l1 box sequence of the construct. These results suggest that the L1 box plays a crucial role in the regulation of PDF1 expression in L1 cells and that ATML1 could cooperate to drive L1‐specific expression.
Identification of a novel pathogen‐responsive element in the promoter of the tobacco gene HSR203J, a molecular marker of the hypersensitive response
Authors: Pontier Dominique , Balagué Claudine , Bezombes‐Marion Isabelle , Tronchet Maurice , Deslandes Laurent , Roby Dominique
Published: March 2003

The tobacco gene, HSR203J, which is specifically activated during the early steps of incompatible plant/pathogen interactions has been shown to be a molecular marker of the hypersensitive response (HR). It constitutes an ideal model for the identification of HR‐responsive cis‐regulatory elements. As a first step in the promoter dissection, deletion mutants of the 5′ flanking sequence of HSR203J fused to the GUS reporter gene were analyzed. Then, the construction and study of chimeric constructs containing HSR203J promoter fragments fused to a minimal promoter enabled us to identify a 28‐bp regulatory element located between −106 and −79 upstream of the transcription initiation site. This element has been shown to be necessary and sufficient for transcriptional activation in response to pathogen. It contains a 10‐bp palindrome followed by its unperfect repeat. The mutagenesis of these two sequence elements led to the identification of a 12‐bp motif termed HSRE (HSR203 responsive element) responsible for the marked induction of the HSR203J gene during the HR. Since this DNA region did not show any homology with known regulatory sequences, this 12 bp motif corresponds to a novel cis‐regulatory element.
An Arabidopsis inositol phospholipid kinase strongly expressed in procambial cells: Synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in insect cells by 5‐phosphorylation of precursors
Authors: Elge Stephan , Brearley Charles , Xia Hui‐Jun , Kehr Julia , Xue Hong‐Wei , Mueller‐Roeber Bernd
Published: March 2003

We have cloned a phosphatidylinositol‐4‐phosphate 5‐kinase (PIP5K) cDNA (AtP5K1) from Arabidopsis thaliana. By the application of cell permeabilization and short‐term nonequilibrium labelling we show that expression of AtP5K1 in Baculovirus‐infected insect (Spodoptera frugiperda) cells directs synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. The same phosphoinositides were produced by isolated whole‐cell membrane fractions of AtP5K1‐expressing insect cells. Their synthesis was not affected by adding defined precursor lipids, that is PtdIns(3)P, PtdIns(4)P, PtdIns(3,4)P2, or PtdIns(4,5)P2, in excess, indicating that substrates for the plant enzyme were not limiting in vivo. Enzymatic dissection of lipid headgroups revealed that AtP5K1‐directed synthesis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 proceeds via 5‐phosphory lation of precursors. Analysis of promoter‐reporter gene (β‐glucuronidase) fusions in transgenic plants revealed that expression of the AtP5K1 gene is strongest in vascular tissues of leaves, flowers, and roots, namely in cells of the lateral meristem, that is the procambium. Single‐cell sampling of sap from flower stem meristem tissue and neighbouring phloem cells, when coupled to reverse transcriptase – polymerase chain reaction, confirmed preferential expression of AtP5K1 in procambial tissue. We hypothesize that AtP5K1, like animal and yeast PIP5K, may be involved in the control of cell proliferation.
Knock‐out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis
Authors: Pesaresi Paolo , Varotto Claudio , Meurer Jörg , Jahns Peter , Salamini Francesco , Leister Dario
Published: March 2003

The prpl11–1 mutant of Arabidopsis thaliana was identified among a collection of T‐DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single‐copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11–1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild‐type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11–1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase‐binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.
Expression of Clarkia S‐linalool synthase in transgenic petunia plants results in the accumulation of S‐linalyl‐β‐d‐glucopyranoside
Authors: Lücker Joost , Bouwmeester Harro J. , Schwab Wilfried , Blaas Jan , Van Der Plas Linus H. W. , Verhoeven Harrie A.
Published: March 2003

Petunia hybrida W115 was transformed with a Clarkia breweri S‐linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S‐linalool produced by the plant in the various tissues is not present as free linalool, but was efficiently converted to non‐volatile S‐linalyl‐β‐d‐glucopyranoside by the action of endogenous glucosyltransferase. The results presented demonstrate that monoterpene production can be altered by genetic modification, and that the compounds produced can be converted by endogenous enzymatic activity.
A nuclear gene encoding mitochondrial Δ1‐pyrroline‐5‐carboxylate dehydrogenase and its potential role in protection from proline toxicity
Authors: Deuschle Karen , Funck Dietmar , Hellmann Hanjo , Däschner Klaus , Binder Stefan , Frommer Wolf B.
Published: March 2003

Δ1‐pyrroline‐5‐carboxylate (P5C), an intermediate in biosynthesis and degradation of proline (Pro), is assumed to play a role in cell death in plants and animals. Toxicity of external Pro and P5C supply to Arabidopsis suggested that P5C dehydrogenase (P5CDH; EC 1.2.1.12) plays a crucial role in this process by degrading the toxic Pro catabolism intermediate P5C. Also in a Δput2 yeast mutant, lacking P5CDH, Pro led to growth inhibition and formation of reactive oxygen species (ROS). Complementation of the Δput2 mutant allowed identification of the Arabidopsis P5CDH gene. AtP5CDH is a single‐copy gene and the encoded protein was localized to the mitochondria. High homology of AtP5CDH to LuFIS1, an mRNA up‐regulated during susceptible pathogen attack in flax, suggested a role for P5CDH in inhibition of hypersensitive reactions. An Arabidopsis mutant (cpr5) displaying a constitutive pathogen response was found to be hypersensitive to external Pro. In agreement with a role in prevention of cell death, AtP5CDH was expressed at a basal level in all tissues analysed. The highest expression was found in flowers that are known to contain the highest Pro levels under normal conditions. External supply of Pro induced AtP5CDH expression, but much more slowly than Pro dehydrogenase (AtProDH) expression. Uncoupled induction of the AtProDH and AtP5CDH genes further supports the hypothesis that P5C levels have to be tightly controlled. These results indicate that, in addition to the well‐studied functions of Pro, for example in osmoregulation, the Pro metabolism intermediate P5C also serves as a regulator of cellular stress responses.
At‐HSP17.6A, encoding a small heat‐shock protein in Arabidopsis, can enhance osmotolerance upon overexpression
Authors: Sun Weining , Bernard Catherine , Van De Cotte Brigitte , Van Montagu Marc , Verbruggen Nathalie
Published: March 2003

Owing to their sessile lifestyle, it is crucial for plants to acquire stress tolerance. The function of heat‐shock proteins, including small heat‐shock proteins (smHSPs), in stress tolerance is not fully explored. To gain further knowledge about the smHSPs, the gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana (At‐HSP17.6A) was characterized. The At‐HSP17.6A expression was induced by heat and osmotic stress, as well as during seed development. Accumulation of At‐HSP17.6A proteins could be detected with heat and at a late stage of seed development, but not with osmotic stress, suggesting stress‐induced post‐transcriptional regulation of At‐HSP17.6A expression. Overproduction of At‐HSP17.6A could increase salt and drought tolerance in Arabidopsis. The chaperone activity of At‐HSP17.6A was demonstrated in vitro.
Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase
Authors: Zhang Xiao‐Ping , Sjöling Sara , Tanudji Marcel , Somogyi László , Andreu David , Eriksson L. E. Göran , Gräslund Astrid , Whelan James , Glaser Elzbieta
Published: March 2003

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position −2 or −3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1β precursor of ATP synthase (pF1β), was performed using a series of pF1β presequence mutants and mutant presequence peptides derived from the C‐terminal portion of the presequence. Replacement of −2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located −5 Arg or −15 Arg by Leu had only a low inhibitory effect. The C‐terminal portion of the pF1β presequence forms a helix–turn–helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C‐terminal pF1β presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1β presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the α‐MPP and β‐MPP subunits in close proximity to the H111XXE114H115X(116−190)E191 proteolytic active site on β‐MPP. Our results show for the first time that the amino acid at the −2 position, even if not an arginine, as well as structural properties of the C‐terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.
Tissue‐specific patterns of a maize Myb transcription factor are epigenetically regulated
Authors: Cocciolone Suzy M. , Chopra Surinder , Flint‐Garcia Sherry A. , McMullen Michael D. , Peterson Thomas
Published: March 2003

The maize p1 gene encodes a Myb‐homologous regulator of red pigment biosynthesis. To investigate the tissue‐specific regulation of the p1 gene, maize plants were transformed with constructs combining promoter and cDNA sequences of two alleles which differ in pigmentation patterns: P1‐wr (white pericarp/red cob) and P1‐rr (red pericarp/red cob). Surprisingly, all promoter/cDNA combinations produced transgenic plants with red pericarp and red cob (RR pattern), indicating that the P1‐wr promoter and encoded protein can function in pericarp. Some of the RR patterned transgenic plants produced progeny plants with white pericarp and red cob (WR pattern), and this switch in tissue‐specificity correlated with increased transgene methylation. A similar inverse correlation between pericarp pigmentation and DNA methylation was observed for certain natural p1 alleles, which have a gene structure characteristic of standard P1‐wr alleles, but which confer red pericarp pigmentation and are consistently less methylated than standard P1‐wr alleles. Although we cannot rule out the possible existence of tissue‐specific regulatory elements within the p1 non‐coding sequences or flanking regions, the data from transgenic and natural alleles suggest that the tissue‐specific pigmentation pattern characteristic of the P1‐wr phenotype is epigenetically controlled.
Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down‐regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants
Authors: Chabannes Matthieu , Barakate Abdellah , Lapierre Catherine , Marita Jane M. , Ralph John , Pean Michel , Danoun Saïda , Halpin Claire , Grima‐Pettenati Jacqueline , Boudet Alain Michel
Published: March 2003

Different transgenic tobacco lines down‐regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down‐regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down‐regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.
Maize VP1 complements Arabidopsisabi3 and confers a novel ABA/auxin interaction in roots
Authors: Suzuki Masaharu , Kao Chien‐Yuan , Cocciolone Suzy , McCarty Donald R.
Published: March 2003

The maize Vp1 gene and abi3 gene of Arabidopsis are believed to be orthologs based on similarities of the mutant phenotypes and amino acid sequence conservation. Here we show that expression of VP1 driven by the 35S promoter can partially complement abi3–6, a deletion mutant allele of abi3. The visible phenotype of seed produced from VP1 expression in the abi3 mutant background is nearly indistinguishable from wild type. VP1 fully restores abscisic acid (ABA) sensitivity of abi3 during seed germination and suppresses the early flowering phenotype of abi3. The temporal regulation of C1‐β‐glucronidase (GUS) and chlorophyl a/b binding protein (cab3)‐GUS reporter genes in developing seeds of 35S‐VP1 lines were similar to wild type. On the other hand, two qualitative differences are observed between the 35S‐VP1 line and wild type. The levels of CRC and C1‐GUS expression are markedly lower in the seeds of 35S‐VP1 lines than in wild type suggesting incomplete complementation of gene activation functions. Similar to ectopic expression of ABI3 (Parcy et al., 1994), ectopic expression of VP1 in vegetative tissue enhances ABA inhibition of root growth. In addition, 35S‐VP1 confers strong ABA inducible expression of the normally seed‐specific cruciferin C (CRC) gene in leaves. In contrast, ectopic ABA induction of C1‐GUS is restricted to a localized region of the root elongation zone. The ABA‐dependent C1‐GUS expression expanded to a broader area in the root tissues treated with exogenous application of auxin. Interestingly, auxin‐induced lateral root formation is completely suppressed by ABA in 35S‐VP1 plants but not in wild type. These results indicate VP1 mediates a novel interaction between ABA and auxin signaling that results in developmental arrest and altered patterns of gene expression.
Novel ABA‐ and dehydration‐inducible aldehyde dehydrogenase genes isolated from the resurrection plant Craterostigma plantagineum and Arabidopsis thaliana
Authors: Kirch Hans‐Hubert , Nair Ambili , Bartels Dorothea
Published: March 2003

In order to identify genes that are critical for the ABA‐dependent stress response in the resurrection plant Craterostigma plantagineum, a gene was isolated with homology to class 3 variable substrate aldehyde dehydrogenases (ALDH). The C. plantagineum gene Cp‐ALDH constitutes a novel class of plant ALDHs. In a search for corresponding genes from Arabidopsis thaliana, Ath‐ALDH3 and Ath‐ALDH4 were isolated, showing 70% and 80% similarity to Cp‐ALDH. Phylogenetically, the Cp‐ and Ath‐ALDH3 and ‐ALDH4 proteins are closely related to aldehyde dehydrogenases from bacteria and mammalian species and are separated from known plant ALDHs and betaine‐aldehyde dehydrogenases (BADH). Cp‐ALDH transcript and polypeptide are up‐regulated in vegetative tissues and callus in response to dehydration or ABA‐treatment. Ath‐ALDH3 expression was induced in response to dehydration and ABA treatment, while Ath‐ALDH4 is constitutively expressed at a low level. Recombinant Cp‐ALDH protein oxidizes nonanal, propionaldehyde and acetaldehyde, with Km values of 2.2 µm, 0.27 mm and 3.23 mm, respectively, in an NAD‐dependent manner. Immunogold electron microscopy shows that Cp‐ALDH is localized in plastids.
Sub‐cellular localisation of GFP‐tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation
Authors: Criqui Marie Claire , Weingartner Magdalena , Capron Arnaud , Parmentier Yves , Shen Wen‐Hui , Heberle‐Bors Erwin , Bögre Laszlo , Genschik Pascal
Published: March 2003

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub‐cellular localisation of two different B1‐types and one A3‐type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP‐tagged fusion proteins in living tobacco cells. Both B1‐type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1‐type cyclins became degraded, as shown by time‐lapse images. A stable variant of cyclin B1;1‐GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase‐arrested cells after microtubule destabilising drug treatments. In contrast to the B1‐type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.
Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system
Authors: Gibon Yves , Vigeolas Helene , Tiessen Axel , Geigenberger Peter , Stitt Mark
Published: March 2003

Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol‐3‐phosphate dehydrogenase and glycerol‐3‐phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol‐3‐phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.
Constitutive expression of ETHYLENE‐RESPONSE‐FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi
Authors: Berrocal‐Lobo Marta , Molina Antonio , Solano Roberto
Published: March 2003

Infection of a plant by a pathogen induces a variety of defense responses that imply the action of several signaling molecules, including salicylic acid (SA), jasmonic acid (JA) and ethylene (E). Here we describe the role of ETHYLENE‐RESPONSE‐FACTOR1 (ERF1) as a regulator of ethylene responses after pathogen attack in Arabidopsis. The ERF1 transcript is induced on infection by Botrytis cinerea, and overexpression of ERF1 in Arabidopsis is sufficient to confer resistance to necrotrophic fungi such as B. cinerea and Plectosphaerella cucumerina. A positive co‐operation between E and SA pathways was observed in the plant response to P. cucumerina. Infection by Pseudomonas syringae tomato DC3000, however, does not affect ERF1 expression, and activation of ethylene responses by ERF1 overexpression in Arabidopsis plants reduces tolerance against this pathogen, suggesting negative crosstalk between E and SA signaling pathways, and demonstrating that positive and negative interactions between both pathways can be established depending on the type of pathogen.
Herbivore‐induced volatiles induce the emission of ethylene in neighboring lima bean plants
Authors: Arimura Gen‐ichiro , Ozawa Rika , Nishioka Takaaki , Boland Wilhelm , Koch Thomas , Kühnemann Frank , Takabayashi Junji
Published: March 2003

Herbivore attacks induce leaves to emit a specific blend of volatiles. Here we show that exposure to Tetranychus urticae‐induced volatiles, as well as T. urticae infestation and artificial wounding, activates the transcription of the genes involved in the biosynthesis of ethylene [S‐adenosylmethionine (SAM) synthetase and 1‐aminocyclopropane‐1‐carboxylic acid oxidase] and a gene involved in the biosynthesis of polyamines from SAM (SAM decarboxylase) in lima bean leaves. Moreover, exposure of leaves to any one of the seven major chemical components of T. urticae‐induced volatiles also induces expression of these genes. Furthermore, we found that, when lima bean plants were exposed to T. urticae‐induced volatiles, they emitted ethylene. Lima bean plants infested by T. urticae and artificially wounded plants also emitted ethylene. Endogenous polyamine levels were not increased in the exposed leaves or the infested leaves, suggesting that polyamine production from SAM was only slightly promoted at the metabolic levels present in the leaves. We found that jasmonate (JA) accumulated in leaves exposed to T. urticae‐induced volatiles, and that both JA and salicylate (SA) accumulated in leaves infested by T. urticae. These findings, as well as results of pharmacological analyses, suggest that, in leaves exposed to T. urticae‐induced volatiles, ethylene biosynthesis might be regulated by pathways involving JA and the ethylene positive feedback loop. They also suggest that ethylene biosynthesis might be regulated by signaling pathways involving JA, SA and ethylene in T. urticae‐infested leaves.
Light signalling mediated by phytochrome plays an important role in cold‐induced gene expression through the C‐repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana
Authors: Kim Hyoun‐Joung , Kim Yun‐Kyoung , Park Jin‐Young , Kim Jungmook
Published: March 2003

Low temperature induces a number of genes that encode the proteins promoting tolerance to freezing, mediated by ABA‐dependent and ABA–independent pathways in plants. The cis‐acting element called C/DRE is known to respond to low temperature independently of ABA action. To investigate the signalling and network of ABA‐independent pathways, the transgenic Arabidopsis plants were generated containing several copies of the C/DRE derived from cor15a gene with a minimal promoter fused to a GUS reporter gene. The transgenic plants containing four copies of the C/DRE (4C/DRE‐GUS) showed responsiveness to cold and drought treatments and were used for characterization of cold signalling and cross‐talk. Cold‐induced GUS expression was inhibited by okadaic acid at 1 nm, indicating that protein phosphatase 2A might act as a positive regulator. Light was shown to activate cold‐ and drought‐induced GUS expression. Photo‐reversibility of the GUS mRNA by red and far‐red light with concomitant cold treatment suggests a role of phytochrome as a photoreceptor in mediating light signalling to activate the cold‐induced gene expression through the C/DRE. Furthermore, GUS expression analysis in phyA or phyB or phyAphyB mutant backgrounds showed that phytochrome B is a primary photoreceptor responsible for the activation of cold‐stress signalling in response to light. Light enhanced the induction kinetics of CBF1, 2, and 3 encoding the cognate transcription factors, and cor15a, in a consecutive manner compared to the dark condition in the cold, suggesting that the connection point between cold and light signalling mediated by phytochrome is at a higher step than the expression of CBF genes.
Identification of a hard surface contact‐induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase, a novel fungal virulence factor
Authors: Kim Yeon‐Ki , Wang Yuhuan , Liu Zhi‐mei , Kolattukudy Pappachan E.
Published: March 2003

Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi. However, the molecular basis of this requirement is unknown. We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact. We report that one of the genes induced by hard‐surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases. chip6 expressed in E. coli catalyses glucosyl transfer from UDP‐glucose to cholesterol. Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation. The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase.
Fertilization in Arabidopsis thaliana wild type: Developmental stages and time course
Authors: Faure Jean‐Emmanuel , Rotman Nicolas , Fortuné Philippe , Dumas Christian
Published: March 2003

We describe some previously uncharacterised stages of fertilization in Arabidopsis thaliana and provide for the first time a precise time course of the fertilization process. We hand‐pollinated wild type pistils with wild type pollen (Columbia ecotype), fixed them at various times after pollination, and analysed 600 embryo sacs using Confocal Laser Scanning Microscopy. Degeneration of one of the synergid cells starts at 5 Hours After Pollination (HAP). Polarity of the egg changes rapidly after this synergid degeneration. Karyogamy is then detected by the presence of two nucleoli of different diameters in both the egg and central cell nuclei, 7–8 HAP. Within the next hour, first nuclear division takes place in the fertilized central cell and two nucleoli can then be seen transiently in each nucleus produced. In a second set of experiments, we hand‐pollinated wild type pistils with pollen from a transgenic promLAT52::EGFP line that expresses EGFP in its pollen vegetative cell. Release of the pollen tube contents into the synergid cell could be detected in living material. We show that the timing of synergid degeneration and pollen tube release correlate well, suggesting that either the synergid cell degenerates at the time of pollen tube discharge or very shortly before it. These observations and protocols constitute an important basis for the further phenotypic analysis of mutants affected in fertilization.
A role for nuclear localised proteasomes in mediating auxin action
Authors: R. Bahrami Ahmad ,   Ruth Bastow, Rolfe Stephen , Price Clive , Gray Julie E.
Published: March 2003

A number of important cellular events in animals and yeast are regulated by protein degradation, and it is becoming apparent that such regulated proteolysis is involved in many facets of plant physiology and development. We have investigated the role of protein degradation by proteasomes in plants using NtPSA1, a tobacco gene that is predominantly expressed in young developing tobacco tissues and has extensive homology to yeast and human α‐type proteasome subunit genes. The NtPSA1 cDNA was used to complement a lethal mutation of the yeast PRC1α subunit gene indicating that NtPSA1 encodes a functional proteasome subunit, and transient expression of an NtPSA1::GUS protein fusion in onion cells confirmed that the nuclear localisation signal that is present in the NtPSA1 peptide sequence is active in plant cells. Plants transformed with an antisense NtPSA1 gene had reduced levels of NtPSA1 mRNA and exhibited reduced apical dominance. In addition, these low NtPSA1 plants displayed several morphological defects associated with auxin resistance such as reduced stamen length, and showed increased tolerance to high concentrations of auxin. These results support a role for nuclear localised proteasomes in floral development and auxin responses.
Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis
Authors: Mudge Stephen R. , Rae Anne L. , Diatloff Eugene , Smith Frank W.
Published: March 2003

The completion of the Arabidopsis thaliana genome has revealed that there are nine members of the Pht1 family of phosphate transporters in this species. As a step towards identifying the role of this gene family in phosphorus nutrition, we have isolated the promoter regions from each of these genes, and fused them to the reporter genes β‐glucuronidase and/or green fluorescent protein. These chimeric genes have been introduced into A. thaliana, and reporter gene expression has been assayed in plants grown in soil containing high and low concentrations of inorganic phosphate (Pi). Four of these promoters were found to direct reporter gene expression in the root epidermis, and were induced under conditions of phosphate deprivation in a manner similar to previously characterised Pht1 genes. Other members of this family, however, showed expression in a range of shoot tissues and in pollen grains, which was confirmed by RT‐PCR. We also provide evidence that the root epidermally expressed genes are expressed most strongly in trichoblasts, the primary sites for uptake of Pi. These results suggest that this gene family plays a wider role in phosphate uptake and remobilisation throughout the plant than was previously believed.
The metal ion transporter IRT1 is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana
Authors: Varotto Claudio , Maiwald Daniela , Pesaresi Paolo , Jahns Peter , Salamini Francesco , Leister Dario
Published: March 2003

The mutants irt1‐1 and irt1‐2 of Arabidopsis thaliana were identified among a collection of T‐DNA‐tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutations responsible interfere with expression of IRT1, a nuclear gene that encodes the metal ion transporter IRT1. In irt1 mutants, photosensitivity and chlorophyll fluorescence parameters, as well as abundance and composition of the photosynthetic apparatus, are significantly altered. Additional effects of the mutation under greenhouse conditions, including chlorosis and a drastic reduction in growth rate and fertility, are compatible with a deficiency in iron transport. Propagation of irt1 plants on media supplemented with additional quantities of iron salts restores almost all aspects of wild‐type behaviour. The irt2‐1 mutant, which carries an En insertion in the highly homologous IRT2 gene of Arabidopsis thaliana, was identified by reverse genetics and shows no symptoms of iron deficiency. This, together with the finding that irt1‐1 can be complemented by 35S::IRT1 but not by 35S::IRT2, demonstrates that, although the products of the two genes are closely related, only AtIRT1 is required for iron homeostasis under physiological conditions.
Proof of C4 photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae)
Authors: Voznesenskaya Elena V. , Franceschi Vincent R. , Kiirats Olavi , Artyusheva Elena G. , Freitag Helmut , Edwards Gerald E.
Published: March 2003

Kranz anatomy, with its separation of elements of the C4 pathway between two cells, has been an accepted criterion for function of C4 photosynthesis in terrestrial plants. However, Bienertia cycloptera (Chenopodiaceae), which grows in salty depressions of Central Asian semi‐deserts, has unusual chlorenchyma, lacks Kranz anatomy, but has photosynthetic features of C4 plants. Its photosynthetic response to varying CO2 and O2 is typical of C4 plants having Kranz anatomy. Lack of night‐time CO2 fixation indicates it is not acquiring carbon by Crassulacean acid metabolism. This species exhibits an independent, novel solution to function of the C4 mechanism through spatial compartmentation of dimorphic chloroplasts, other organelles and photosynthetic enzymes in distinct positions within a single chlorenchyma cell. The chlorenchyma cells have a large, spherical central cytoplasmic compartment interconnected by cytoplasmic channels through the vacuole to the peripheral cytoplasm. This compartment is filled with mitochondria and granal chloroplasts, while the peripheral cytoplasm apparently lacks mitochondria and has grana‐deficient chloroplasts. Immunolocalization studies show enzymes compartmentalized selectively in the CC compartment, including Rubisco in chloroplasts, and NAD‐malic enzyme and glycine decarboxylase in mitochondria, whereas pyruvate, Pi dikinase of the C4 cycle is localized selectively in peripheral chloroplasts. Phosphoenolpyruvate carboxylase, a cytosolic C4 cycle enzyme, is enriched in the peripheral cytoplasm. Our results show Bienertia utilizes strict compartmentation of organelles and enzymes within a single cell to effectively mimic the spatial separation of Kranz anatomy, allowing it to function as a C4 plant having suppressed photorespiration; this raises interesting questions about evolution of C4 mechanisms.
Comprehensive transcript profiling of Pto‐ and Prf‐mediated host defense responses to infection by Pseudomonas syringae pv. tomato
Authors: Mysore Kirankumar S. , Crasta Oswald R. , Tuori Robert P. , Folkerts Otto , Swirsky Peter B. , Martin Gregory B.
Published: March 2003

The disease resistance gene Pto encodes a serine/threonine protein kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the effector protein AvrPto. Pto‐mediated resistance to bacterial speck disease also requires Prf, a protein with leucine‐rich repeats and a putative nucleotide‐binding site, although the role of Prf in the defense pathway is not known. We used GeneCalling, an open‐architecture, mRNA‐profiling technology, to identify genes that are either induced or suppressed in leaves 4 h after bacterial infection in the Pto‐ and Prf‐mediated tomato‐Pseudomonas(avrPto) interaction. Over 135 000 individual cDNA fragments representing an estimated 90% of the transcripts expressed in tomato leaves were examined and 432 differentially expressed genes were identified. The genes encode over 25 classes of proteins including 11 types of transcription factors and many signal transduction components. Differential expression of 91% of the genes required both Pto and Prf. Interestingly, differential expression of 32 genes did not require Pto but was dependent on Prf. Thus, our data support a role for Prf early in the Pto pathway and indicate that Prf can also function as an independent host recognition determinant of bacterial infection. Comprehensive expression profiling of the Pto‐mediated defense response allows the development of many new hypotheses about the molecular basis of resistance to bacterial speck disease.
Loss‐of‐function of a rice brassinosteroid biosynthetic enzyme, C‐6 oxidase, prevents the organized arrangement and polar elongation of cells in the leaves and stem
Authors: Hong Zhi , Ueguchi‐Tanaka Miyako , Shimizu‐Sato Sae , Inukai Yoshiaki , Fujioka Shozo , Shimada Yukihisa , Takatsuto Suguru , Agetsuma Masakazu , Yoshida Shigeo , Watanabe Yoshihisa , Uozu Sakurako , Kitano Hidemi , Ashikari Motoyuki , Matsuoka Makoto
Published: March 2003

Molecular genetic and physiological studies on brassinosteroid (BR)‐related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR‐deficient mutants of rice, BR‐deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C‐6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C‐6 oxidase gene, DWARF. Introduction of the wild‐type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.
Distinct regulation of CAB and PHYB gene expression by similar circadian clocks
Authors: Hall Anthony , Kozma‐Bognár László , Bastow Ruth M. , Nagy Ferenc , Millar Andrew J.
Published: March 2003

Phytochrome B (phyB) is a major phytochrome active in light‐grown plants. The circadian clock controls the expression of the PHYB gene. We have used the luciferase reporter gene (LUC) to monitor the rhythmic expression of PHYB in photoreceptor and clock‐associated mutant backgrounds. Surprisingly, we found that PHYB and CAB expression have different free‐running periods, indicating that separate circadian clocks control these genes. The effects of mutations show that the clocks share common components. This suggests that they are copies of the same clock mechanism in different locations, most likely in different cell layers. Furthermore, we show that phyB is required for a negative feedback loop that strongly antagonises the expression of PHYB. Compared to a system with only one clock, this regulatory complexity might allow the phase of peak expression for one clock‐controlled gene to alter, relative to other genes or to changing environmental conditions.
Over‐expression of an Arabidopsis gene encoding a glucosyltransferase of indole‐3‐acetic acid: phenotypic characterisation of transgenic lines
Authors: Jackson Rosamond G. , Kowalczyk Mariusz , Li Yi , Higgins Gillian , Ross Joe , Sandberg Göran , Bowles Dianna J.
Published: March 2003

An analysis of the multigene family of Group 1 glucosyltransferases (UGTs) of Arabidopsis thaliana revealed a gene, UGT84B1, whose recombinant product glucosylated indole‐3‐acetic acid (IAA) in vitro. Transgenic Arabidopsis plants constitutively over‐expressing UGT84B1 under the control of the CaMV 35S promoter have been constructed and their phenotype analysed. The transgenic lines displayed a number of changes that resembled those described previously in lines in which auxin levels were depleted. A root elongation assay was used as a measure of auxin sensitivity. A reduced sensitivity of the transgenic lines compared to wild‐type was observed when IAA was applied. In contrast, application of 2,4‐dichlorophenoxyacetic acid (2,4‐D), previously demonstrated not to be a substrate for UGT84B1, led to a wild‐type response. These data suggested that the catalytic specificity of the recombinant enzyme in vitro was maintained in planta. This was further confirmed when levels of IAA metabolites and conjugates were measured in extracts of the transgenic plants and 1‐O‐IAGlc was found to be elevated to approximately 50 pg mg−1 FW, compared to the trace levels characteristic of wild‐type plants. Surprisingly, in the same extracts, levels of free IAA were also found to have accumulated to some 70 pg mg−1 FW compared to approximately 15 pg mg−1 FW in extracts of wild‐type plants. Analysis of leaves at different developmental stages revealed the auxin gradient, typical of wild‐type plants, was not observed in the transgenic lines, with free IAA levels in the apex and youngest leaves at a lower level compared to wild‐type. In total, the data reveal that significant changes in auxin homeostasis can be caused by overproduction of an IAA‐conjugating enzyme.
Analysis of far‐red light‐regulated genome expression profiles of phytochrome A pathway mutants in Arabidopsis
Authors: Wang Haiyang , Ma Ligeng , Habashi Jessica , Li Jinming , Zhao Hongyu , Deng Xing W.
Published: March 2003

Phytochrome A (phyA) is the primary photoreceptor responsible for various far‐red (FR) light‐mediated responses. Previous studies have identified multiple phyA signaling mutants, including both positive and negative regulators of the phyA‐mediated responses. How these defined intermediates act to mediate FR light responses is largely unknown. Here a cDNA microarray was used to examine effects of those mutations on the far‐red light control of genome expression. Clustering analysis of the genome expression profiles supports the notion that phyA signaling may entail a network with multiple paths, controlling overlapping yet distinct sets of gene expression. FHY1, FAR1 and FHY3 most likely act upstream in the phyA signaling network, close to the phyA photoreceptor itself. FIN219, SPA1 and REP1 most likely act somewhere more downstream in the network and control the expression of smaller sets of genes. Further, this study also provides genomics evidence for the partial functional redundancy between FAR1 and FHY3. These two homologous proteins control the expression of a largely overlapping set of genes, and likely act closely together in the phyA‐mediated FR light responses.