bluuebunny/crossref_metadata_2025 huggingface dataset:
A pathogen‐ and salicylic acid‐induced WRKY DNA‐binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter
Authors: Yang Peizhen , Chen Chunhong , Wang Zeping , Fan Baofang , Chen Zhixiang
Published: March 2003

Using a simple oligo selection procedure, we have previously identified a tobacco sequence‐specific DNA‐binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)‐induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA‐binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis‐related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA‐binding activity. Pre‐treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA‐binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA‐binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense‐related genes during pathogen‐ and SA‐induced plant defense responses.
The expression of tobacco knotted1‐type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model
Authors: Nishimura Asuka , Tamaoki Masanori , Sato Yutaka , Matsuoka Makoto
Published: March 2003

We have isolated and characterized four tobacco homeobox genes, NTH1, NTH9, NTH20, NTH22 (Nicotiana tabacum homeobox) which belong to the class 1 knotted1‐type family of homeobox genes. Comparison of the inferred amino acid sequences of the ELK homeodomains of these genes and previously reported kn1‐type class 1 proteins has revealed that the four new tobacco genes belong to distinct subclasses, suggesting that each NTH gene may have distinct functions. Using in situ hybridization and by analysing the distribution of GUS activity in tobacco plants transformed with NTH promoter::GUS constructs, local‐ ized expression of the three NTH genes was observed in the shoot apical meristem (SAM). In the vegetative SAM, NTH1 and NTH15 showed overlapping expression in the corpus, NTH20 was expressed in the peripheral zone, and NTH9 was predominantly expressed in the rib zone. The expression patterns of the different NTH genes correspond to regions predicted by the cytohistological zonation model, suggesting that each NTH gene specifies the function of the SAM zone with which it is associated.
Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem
Authors: Rupp Hans‐Michael , Frank Markus , Werner Tomás , Strnad Miroslav , Schmülling Thomas
Published: March 2003

This study investigates the consequences of endo‐ genously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Tran‐ scriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature‐dependent increased cytokinin production in transgenic plants. Heat‐treated plants accumulated higher levels of unbound and bound zeatin‐type cyto‐ kinins, the latter being preferentially N‐conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin‐overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin‐overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.
Low‐pH‐mediated elevations in cytosolic calcium are inhibited by aluminium: a potential mechanism for aluminium toxicity
Authors: Plieth Christoph , Sattelmacher Burkhard , Hansen Ulf‐Peter , Knight Marc R.
Published: March 2003

Aluminium, the most abundant metal in the earth's crust, is highly toxic to most plant species. One of the prevailing dogmas is that aluminium exerts this effect by disrupting cellular calcium homeostasis. However, recent research gives strongly conflicting results: aluminium was shown to provoke either an increase or a decrease in cytosolic free calcium concentration ([Ca2+]c). To solve this question, we have adopted a novel approach: [Ca2+]c measurements in intact plant roots as opposed to isolated cells, and the correlative measurements of intracellular and external pH. The results obtained show that plant roots respond to low external pH by a sustained elevation in [Ca2+]c. In the presence of aluminium, this pH‐mediated elevation in [Ca2+]c does not occur, therefore any potential calcium‐mediated protection against low pH is likely to be irreversibly inhibited. The severity of the inhibitory effect of aluminium on [Ca2+]c depends on the concentration of external calcium, thus perhaps explaining why the effects of aluminium toxicity are ameliorated in calcium‐rich soils. It seems possible that a primary toxic effect of aluminium might be to impair calcium‐mediated plant defence responses against low pH.
Vacuolar processing enzyme is up‐regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions
Authors: Kinoshita Tetsu , Yamada Kenji , Hiraiwa Nagako , Kondo Maki , Nishimura Mikio , Hara‐Nishimura Ikuko
Published: March 2003

Vacuolar processing enzyme (VPE) has been shown to be responsible for maturation of various seed proteins in protein‐storage vacuoles. Arabidopsis has three VPE homologues; βVPE is specific to seeds and αVPE and γVPE are specific to vegetative organs. To investigate the activity of the vegetative VPE, we expressed the γVPE in a pep4 strain of the yeast Saccharomyces cerevisiae and found that γVPE has the ability to cleave the peptide bond at the carbonyl side of asparagine residues. An immunocytochemical analysis revealed the specific localization of the γVPE in the lytic vacuoles of Arabidopsis leaves that had been treated with wounding. These findings indicate that γVPE functions in the lytic vacuoles as the βVPE does in the protein‐storage vacuoles. The βVPE promoter was found to direct the expression of the β‐glucuronidase reporter gene in seeds and the root tip of transgenic Arabidopsis plants. On the other hand, both the αVPE and γVPE promoters directed the expression in senescent tissues, but not in young intact tissues. The mRNA levels of both αVPE and γVPE were increased in the primary leaves during senescence in parallel with the increase of the mRNA level of a senescence‐associated gene (SAG2). Treatment with wounding, ethylene and salicylic acid up‐regulated the expression of αVPE and γVPE, while jasmonate slightly up‐regulated the expression of γVPE. These gene expression patterns of the VPEs were associated with the accumulation of vacuolar proteins that are known to respond to these treatments. Taken together, the results suggest that vegetative VPE might regulate the activation of some functional proteins in the lytic vacuoles.
The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light‐dependent post‐transcriptional mechanism
Authors: Guo Hongwei , Duong Hien , Ma Nha , Lin Chentao
Published: March 2003

Cryptochrome 2 is a flavin‐type blue light receptor mediating floral induction in response to photoperiod and a blue light‐induced hypocotyl growth inhibition. cry2 is required for the elevated expression of the flowering‐time gene CO in response to long‐day photoperiods, but the molecular mechanism underlying the function of cry2 is not clear. The carboxyl domain of cry2 bears a basic bipartite nuclear localization signal, and the cry2 protein was co‐fractionated with the nucleus. Analysis of transgenic plants expressing a fusion protein of CRY2 and the reporter enzyme GUS (GUS–CRY2) indicated that the GUS–CRY2 fusion protein accumulated in the nucleus of transgenic plants grown in dark or light. The C‐terminal domain of cry2 that contains the basic bipartite nuclear localization signal was sufficient to confer nuclear localization of the fusion protein. Phenotypic analysis of transgenic plants expressing the fusion protein GUS–CRY2 demonstrated that GUS–CRY2 acts as a functional photoreceptor in vivo, mediating the blue light‐induced inhibition of hypocotyl elongation. These results strongly suggest that cry2 is a nuclear protein. Although no obvious light regulation was found for the nuclear compartmentation of GUS–CRY2 fusion protein, the abundance of GUS–CRY2 was regulated by blue light in a way similar to that of cry2.
The BANYULS gene encodes a DFR‐like protein and is a marker of early seed coat development
Authors: Devic Martine , Guilleminot Jocelyne , Debeaujon Isabelle , Bechtold Nicole , Bensaude Emmanuelle , Koornneef Maarten , Pelletier Georges , Delseny Michel
Published: March 2003

Mutations in the BANYULS (BAN) gene lead to precocious accumulation of anthocyanins in immature seed coat in Arabidopsis. The ban–1 allele has been isolated from a collection of T‐DNA transformants and found to be tagged by the integrative molecule. The sequencing of wild‐type and two independent mutant alleles confirmed the identity of the gene. Analysis of the full‐length cDNA sequence revealed an open reading frame encoding a 342 amino acid protein which shared strong similarities with DFR and other enzymes of the phenylpropanoid biosynthesis pathway. BAN expression was restricted to the endothelium of immature seeds at the pre‐globular to early globular stages of development as predicted from the maternal inheritance of the phenotype, and therefore represents a marker for early differentiation and development of the seed coat. BAN is probably involved in a metabolic channelling between the production of anthocyanins and pro‐anthocyanidins in the seed coat.
The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs element
Authors: Chen Wenqiong , Singh Karam B.
Published: March 2003

Glutathione S‐transferases (GSTs) are multi‐functional enzymes that play critical roles in a number of plant stress responses. Ocs elements are enhancer sequences found in some pathogen and GST promoters. We previously isolated an Arabidopsis GST gene, called GST6, which contained an ocs‐like element. Here we tested the role this putative ocs element plays in GST6 expression in response to auxin, salicylic acid (SA) and hydrogen peroxide (H2O2) using a transient assay and transgenic Arabidopsis. The requirement for the ocs element for basal and/or induced expression varied between the transient and transgenic studies, and in different plant tissues. The GST6 promoter showed the largest induction in roots following treatment with SA (84‐fold) or H2O2 (122‐fold), and the effect of mutating the ocs element was most pronounced in root tissue. However, in no case did mutation of the ocs element abolish GST6 expression, suggesting that other promoter element(s) are also important. Using the transient assay, other promoter element(s) critical for GST6 expression were identified that may also help mediate the activity of the ocs element. The finding that the ocs element plays a role in the transcriptional response of a plant GST gene to SA and H2O2 has potential significance for the plant defense response, where SA and H2O2 play critical roles.
Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene
Authors: Dong Hansong , Delaney Terrence P. , Bauer David W. , Beer Steven V.
Published: March 2003

Harpin, the product of the hrpN gene of Erwinia amylovora, elicits the hypersensitive response and disease resistance in many plants. Harpin and known inducers of systemic acquired resistance (SAR) were tested on five genotypes of Arabidopsis thaliana to assess the role of SAR in harpin‐induced resistance. In wild‐type plants, harpin elicited systemic resistance to Peronospora parasitica and Pseudomonas syringae pv. tomato, accompanied by induction of the SAR genes PR‐1 and PR‐2. However, in experiments with transgenic Arabidopsis plants containing the nahG gene which prevents accumulation of salicylic acid (SA), harpin neither elicited resistance nor activated SAR gene expression. Harpin also failed to activate SAR when applied to nim1 (non‐inducible immunity) mutants, which are defective in responding to SA and regulation of SAR. In contrast, mutants compromised in responsiveness to methyl jasmonate and ethylene developed the same resistance as did wild‐type plants. Thus, harpin elicits disease resistance through the NIM1‐mediated SAR signal transduction pathway in an SA‐dependent fashion. The site of action of harpin in the SAR regulatory pathway is upstream of SA.
The different pH optima and substrate specificities of extracellular and vacuolar invertases from plants are determined by a single amino‐acid substitution
Authors: Goetz Marc , Roitsch Thomas
Published: March 2003

Different plant invertase isoenzymes are characterized by a single amino‐acid difference in a conserved sequence, the WEC‐P/V‐D box. A proline residue is present in this sequence motif of extracellular invertase sequences, whereas a valine is found at the same position of vacuolar invertase sequences. The role of this distinct difference was studied by substituting the proline residue of extracellular invertase CIN1 from Chenopodium rubrum with a valine residue, by site‐directed muta‐ genesis. The mutated gene was heterologously expressed in an invertase‐deficient Saccharomyces cerevisiae strain. The single amino‐acid difference was shown to be the molecular basis for two enzymatic properties of invertase isoenzymes, for both the pH optimum and the substrate specificity. A proline in the WEC‐P/V‐D box determines the more acidic pH optimum and the higher cleavage rate of raffinose of extracellular invertases, compared to vacuolar invertases that have a valine residue at this position.
Glycosyl‐phosphatidylinositol‐anchor addition signals are processed in Nicotiana tabacum
Authors: Takos Adam M. , Dry Ian B. , Soole Kathleen L.
Published: March 2003

Recent studies have demonstrated the existence of glycosyl‐phosphatidylinositol (GPI)‐anchored proteins in higher plants. In this study we tested whether GPI‐addition signals from diverse evolutionary sources would function to link a GPI‐anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE′) fused with a tobacco secretion signal (PR‐1a) at the N‐terminus and either a yeast (GAS1), mammalian (Thy‐1) or putative plant (LeAGP‐1) GPI‐anchor addition signal at the C‐terminus. The yeast and plant C‐terminal signals were found to be capable of directing the addition of a GPI‐anchor to the endoglucanase protein (EGE′) as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol‐specific phospholipase C (PI‐PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE′ was fused to a truncated form of the LeAGP‐1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI‐anchor was not linked to the EGE′ protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI‐signals in plant cells and that there may be some similar preferences in GPI‐addition signal sequences for yeast and plant cells.
Premature termination codons destabilize ferredoxin‐1 mRNA when ferredoxin‐1 is translated
Authors: Petracek Marie E. , Nuygen Tuyen , Thompson William F. , Dickey Lynn F.
Published: March 2003

Ferredoxin‐1 (Fed‐1) mRNA is poorly translated in dark‐treated tobacco (Nicotiana tabacum) leaves, resulting in destabilization of Fed‐1 mRNA and a differential light/dark accumulation of the mRNA. Insertion of nonsense codons within the Fed‐1 coding sequence disrupts the light regulation of Fed‐1 mRNA abundance. Here we show that the nonsense codon effect results primarily from lowering the Fed‐1 mRNA stability in light‐treated leaf tissue and in rapidly growing tobacco cell cultures, but not in dark‐treated leaf tissue. These results suggest that nonsense codons trigger a decay pathway distinct from that seen for Fed‐1 mRNA in the dark. We propose that nonsense‐mediated decay of nonsense‐containing Fed‐1 mRNA occurs in light‐treated leaves and in non‐photosynthetic tobacco culture cells where Fed‐1 mRNA is being actively translated.
Cis‐elements and trans‐factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response
Authors: Guan Lingqiang M. , Zhao Jun , Scandalios John G.
Published: March 2003

The mechanisms by which the maize antioxidant Cat1 gene responds to abscisic acid (ABA) and osmotic stress have been investigated. Results show that during late embryogenesis, Cat1 expression in vivo is independent of endogenous ABA levels. However, exogenously applied ABA significantly enhances Cat1 expression. Transient assays using particle bombardment show that the proximal ABRE2 element on the Cat1 promoter is responsible for the induction of Cat1 expression by ABA. We further show that ABA induces the expression of Cat1 via the interaction between ABRE2 and one of its binding proteins, CBF1 (Cat1 binding factor 1). Using ABA‐deficient mutant embryos, we show that osmotic stress induces Cat1 expression through two alternate signal transduction pathways: an ABA signaling pathway leading to the interaction between the ABRE2 motif and CBF1, and a pathway via the interaction of ABRE2 and CBF2 (Cat1 binding factor 2) that is independent of ABA. The data presented clearly suggest that hydrogen peroxide (H2O2) plays an important intermediary role in the ABA signal transduction pathway leading to the induction of the Cat1 gene.
Intron‐tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium‐transformed plant cells
Authors: Ferrando Alejandro , Farràs Rosa , Jásik Ján , Schell Jeff , Koncz Csaba
Published: March 2003

Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T‐DNA‐mediated transformation, the detection of epitope‐tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope‐tagged proteins in Agrobacterium‐infected plant cells. We have developed a method for labelling plant coding sequences with intron‐tagged epitope‐coding domains that are not processed in Agrobacterium. Here we show that the expression of HA‐epitope‐tagged constructs encoding β‐glucuronidase and S‐phase kinase‐associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope‐tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium‐transformed plant cells.
Carotenoid biosynthesis during tomato fruit development: regulatory role of 1‐deoxy‐D‐xylulose 5‐phosphate synthase
Authors: Lois Luisa María , Rodríguez‐Concepción Manuel , Gallego Francesca , Campos Narciso , Boronat Albert
Published: March 2003

Plant isoprenoids represent a heterogeneous group of compounds which play essential roles not only in growth and development, but also in the interaction of plants with their environment. Higher plants contain two pathways for the biosynthesis of isoprenoids: the mevalonate pathway, located in the cytosol/endoplasmic reticulum, and the recently discovered mevalonate‐independent pathway (Rohmer pathway), located in the plastids. In order to evaluate the function of the Rohmer pathway in the regulation of the synthesis of plastidial isoprenoids, we have isolated a tomato cDNA encoding 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXS), the first enzyme of the pathway. We demonstrate in vivo activity and plastid targeting of plant DXS. Expression analysis of the tomato DXS gene indicates developmental and organ‐specific regulation of mRNA accumulation and a strong correlation with carotenoid synthesis during fruit development. 1‐Deoxy‐D‐xylulose feeding experiments, together with expression analysis of DXS and PSY1 (encoding the fruit‐specific isoform of phytoene synthase) in wild‐type and yellow flesh mutant fruits, indicate that DXS catalyses the first potentially regulatory step in carotenoid biosynthesis during early fruit ripening. Our results change the current view that PSY1 is the only regulatory enzyme in tomato fruit carotenogenesis, and point towards a coordinated role of both DXS and PSY1 in the control of fruit carotenoid synthesis.
Induction of dwarfism in transgenic Solanum dulcamara by over‐expression of a gibberellin 20‐oxidase cDNA from pumpkin
Authors: Curtis Ian S. , Ward Dennis A. , Thomas Stephen G. , Phillips Andrew L. , Davey Michael R. , Power J. Brian , Lowe Kenneth C. , Croker Stephen J. , Lewis Mervyn J. , Magness Simon L. , Hedden Peter
Published: March 2003

The gibberellin (GA) 20‐oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi‐dwarfs with smaller, deep‐green leaves and highly pigmented stems compared to the wild‐type. Such transformants flowered earlier than the wild‐type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA1 was reduced in leaves of the transformants to approximately 20% or less of that in the wild‐type and to about 40% or less in stems. The concentrations of other 13‐hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA17, which accumulated in the transformants due to 13‐hydroxylation of GA25. By contrast, the concentrations of non‐13‐hydroxylated GAs, GA4 and GA34, were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20‐oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild‐type, reflecting the feedback control of 20‐oxidase gene expression that serves as a homeostatic mechanism for GAs.
Cell‐type‐specific calcium responses to drought, salt and cold in the Arabidopsis root
Authors: Kiegle Edward , Moore Catherine A. , Haseloff Jim , Tester Mark A. , Knight Marc R.
Published: March 2003

Little is known about the signalling processes involved in the response of roots to abiotic stresses. The Arabidopsis root is a model system of root anatomy with a simple architecture and is amenable to genetic manipulation. Although it is known that the root responds to cold, drought and salt stress with increases in cytoplasmic free calcium, there is currently no information about the role(s) of the functionally diverse cell types that comprise the root. Transgenic Arabidopsis with enhancer‐trapped GAL4 expression in specific cell types was used to target the calcium reporting protein, aequorin, fused to a modified yellow fluorescent protein (YFP). The luminescence output of targeted aequorin enabled in vivo measurement of changes in cytosolic free calcium concentrations ([Ca2+]cyt) in specific cell types during acute cold, osmotic and salt stresses. In response to an acute cold stress, all cell types tested as well as plants constitutively expressing aequorin displayed rapid [Ca2+]cyt peaks. However, there were significant quantitative differences between different cell types in terms of their response to cold stress, osmotic stress (440 mM mannitol) and salt stress (220 mM NaCl), implying specific roles for certain cell types in the detection and/or response to these stimuli. In response to osmotic and salt stress, the endodermis and pericycle displayed prolonged oscillations in cytosolic calcium that were distinct from the responses of the other cell types tested. Targeted expression of aequorin circumvented the technical difficulties involved in fluorescent dye injection as well as the lack of cell specificity of constitutively expressed aequorin, and revealed a new level of complexity in root calcium signalling.
Developmentally and transgene regulated nuclear processing of primary transcripts of chalcone synthase A in petunia
Authors: Metzlaff Michael , O'dell Michael , Hellens Roger , Flavell Richard B.
Published: March 2003

The introduction of chalcone synthase A transgenes into petunia plants can result in degradation of chalcone synthase A RNAs and loss of chalcone synthase, a process called cosuppression or post‐transcriptional gene silencing. Here we show that the RNA degradation is associated with changes in premRNA processing, i.e. loss of tissue specificity in transcript cleavage patterns, accumulation of unspliced molecules, and use of template‐specific secondary poly(A) sites. These changes can also be observed at a lower level in leaves but not flowers of nontransgenic petunias. Based on this, a model is presented of how transgenes may disturb the carefully evolved, developmentally controlled post‐transcriptional regulation of chalcone synthase gene expression by influencing the survival rate of the endogenous and their own mRNA.
Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype
Authors: Wopereis Judith , Pajuelo Eloisa , Dazzo Frank B. , Jiang Qunyi , Gresshoff Peter M. , De Bruijn Frans J. , Stougaard Jens , Szczyglowski Krzysztof
Published: March 2003

Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus hypernodulating mutants, har1‐1, 2 and 3 that define a new locus, HYPERNODULATION ABERRANT ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral root initiation. At the cellular level these developmental alterations are associated with changes in the position and duration of root cell growth and result in a premature differentiation of har1‐1 mutant root. No significant differences between har1‐1 mutant and wild‐type plants were detected with respect to root growth responses to 1‐aminocyclopropane1‐carboxylic acid, the immediate precursor of ethylene, and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate root elongation of the har1‐1 mutant but not the wild‐type. After inoculation with Mesorhizobium loti, the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These observations implicate a role for the Har1 locus in both symbiotic and non‐symbiotic development of L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule number are integrated in an overall mechanism governing root growth and development.
An Ry‐mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y
Authors: Mestre Pere , Brigneti Gianinna , Baulcombe David C.
Published: March 2003

Ry confers extreme resistance to all strains of potato virus Y (PVY). To identify the elicitor of the Ry‐mediated resistance against PVY in potato, we expressed each of the PVY‐encoded proteins in leaves of PVY‐resistant (Ry) and ‐susceptible (ry) plants. For most of the proteins tested, there was no evident response. However, when the NIa proteinase was expressed in leaves of Ry plants, there was a hypersensitive response (HR). Proteinase active site mutants failed to induce the Ry‐mediated response. The HR was also induced by the NIa proteinase from pepper mottle virus (PepMoV), which has the same cleavage specificity as the PVY enzyme, but not by the tobacco etch virus (TEV) or the potato virus A (PVA) proteinases that cleave different peptide motifs. Based on these results, we propose that Ry‐mediated resistance requires the intact active site of the NIa proteinase. Although the structure of the active proteinase could have elicitor activity, it is possible that this proteinase releases an elicitor by cleavage of a host‐encoded protein. Alternatively, the proteinase could inactivate a negative regulator of the Ry‐mediated resistance response.
Cre‐lox site‐specific recombination between Arabidopsis and tobacco chromosomes
Authors: Koshinsky Heather A. , Lee Elsa , Ow David W.
Published: March 2003

To create hybrid chromosomes, we tested the Cre‐lox system to mediate recombination between Arabidopsis thaliana and Nicotiana tabacum chromosomes. Protoplasts of the two plants were fused to allow site‐specific recombination to join a promoter from tobacco to a hygromycin resistance coding‐region from Arabidopsis. The expected recombination junction was detected in hygromycin‐resistant calli. Analysis of one hybrid suspension cell line revealed the presence of markers corresponding to the north arm of Arabidopsis chromosome III, but not markers from other chromosome arms. However, these markers were not detected in regenerated plants. With a second hybrid cell line we obtained a single hygromycin‐resistant progeny from approximately 18 000 self‐fertilized seeds of one regenerated plant. Molecular analysis of this hybrid indicated that a small portion of the north arm of Arabidopsis chromosome V is present in the tobacco genome. However, neither the recombination junction nor Arabidopsis DNA was detected in tissue from the plant grown without selection or in the subsequent generation. Thus interspecies transfer of a chromosome arm between plant cells is possible, but maintenance of the hybrid chromosome in a plant is unlikely. The feasibility of site‐specific recombination between genomes of different species offers new possibilities for engineering hybrid chromosomes that may be maintained in cell culture.
Early steps in cold sensing by plant cells: the role of actin cytoskeleton and membrane fluidity
Authors: Örvar Björn Lárus , Sangwan Veena , Omann Franz , Dhindsa Rajinder S.
Published: March 2003

Many plants acquire freezing tolerance through cold acclimatization (CA), a prolonged exposure to low but non‐freezing temperatures at the onset of winter. CA is associated with gene expression that requires transient calcium influx into the cytosol. Alfalfa (Medicago sativa) cells treated with agents blocking this influx are unable to cold‐acclimatize. Conversely, chemical agents causing increased calcium influx induce cold acclimatization‐specific (cas) gene expression in alfalfa at 25°C. How low temperature triggers calcium influx is, however, unknown. We report here that induction of a CA‐specific gene (cas30), calcium influx and freezing tolerance at 4°C are all prevented by cell membrane fluidization, but, conversely, are induced at 25°C by membrane rigidification. cas30 expression and calcium influx at 4°C are also prevented by jasplakinolide (JK), an actin microfilament stabilizer, but induced at 25°C by the actin microfilament destabilizer cytochalasin D (CD). JK blocked the membrane rigidifier‐induced, but not the calcium channel agonist‐induced cas30 expression at 25°C. These findings indicate that cytoskeleton re‐organization is an integral component in low‐temperature signal transduction in alfalfa cell suspension cultures, serving as a link between membrane rigidification and calcium influx in CA.
Antisense‐inhibition of ADP‐glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreases starch but increases protein content and affects seed maturation
Authors: Weber Hans , Rolletschek Hardy , Heim Ute , Golombek Sabine , Gubatz Sabine , Wobus Ulrich
Published: March 2003

The small subunit of a Vicia faba ADP‐glucose pyrophosphorylase (AGP) cDNA was expressed in antisense orientation in Vicia narbonensis under the control of the seed‐specific legumin B4 promoter. From several independent transgenic lines both ADP‐glucose pyrophosphorylase AGP‐mRNA and AGP enzyme activity were reduced by up to 95% in the cotyledons during the mid‐ to late‐maturation phase. Starch was moderately decreased and sucrose was increased. In two of three lines, transcripts encoding the large subunit of AGP and the storage protein vicilin were increased, whereas legumin B‐mRNA was decreased. Transcripts of other storage‐associated genes were not altered. The cotyledons contained more protein and total nitrogen. Despite the reduction in starch, total carbon was not decreased and dry weight was unchanged. Compared to the wild type, transgenic seeds contained more water and accumulated dry weight during a longer period, and therefore had a prolonged seed‐filling period. Transgenic cotyledon cells of comparable age to the wild type were more highly vacuolated and contained smaller starch grains, indicating a delay in cellular differentiation. We conclude that a specific alteration in carbon metabolism can have pleiotropic effects on water and nitrogen content and induces temporal changes in seed development.
cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol
Authors: Hertzberg Magnus , Sievertzon Maria , Aspeborg Henrik , Nilsson Peter , Sandberg Göran , Lundeberg Joakim
Published: March 2003

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50–200 µg of total RNA and 1–2 µg of mRNA is required for each hybridisation, which is equivalent to 50–100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub‐mg amounts of plant tissue. Using 0.1 µg of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2–4 cell layers with a fresh weight of ∼0.5 mg.
Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress
Authors: Hong Suk‐Whan , Vierling Elizabeth
Published: March 2003

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report thatArabidopsis thalianaHsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid‐maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated fromArabidopsisT‐DNA mutant populations. One allele,hot1‐3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assayshot1‐3shows a similar, though somewhat more severe, phenotype to the previously described missense allelehot1‐1, revealing that thehot1‐1mutation is also close to null for protein activity. The second new mutant allele,hot1‐2,has an insertion in the promoter 101 bp 5′ to the putative TATA element. During heat stress thehot1‐2mutant produces normal levels of protein in hypocotyls and 10‐day‐old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. Thehot1‐2mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.
Silencing of a meristematic gene using geminivirus‐derived vectors
Authors: Peele Charles , Jordan Chad V. , Muangsan Nooduan , Turnage Michael , Egelkrout Erin , Eagle Patricia , Hanley‐Bowdoin Linda , Robertson Dominique
Published: March 2003

Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome‐mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana. Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down‐regulated throughout meristematic tissues. Our results demonstrate that geminivirus‐derived vectors can be used to study genes involved in meristem function in intact plants.
Establishment of gene‐trap and enhancer‐trap systems in the moss Physcomitrella patens
Authors: Hiwatashi Yuji , Nishiyama Tomoaki , Fujita Tomomichi , Hasebe Mitsuyasu
Published: March 2003

Because of its simple body plan and ease of gene knockout and allele replacement, the moss Physcomitrella patens is often used as a model system for studies in plant physiology and developmental biology. Gene‐trap and enhancer‐trap systems are useful techniques for cloning genes and enhancers that function in specific tissues or cells. Additionally, these systems are convenient for obtaining molecular markers specific for certain developmental processes. Elements for gene‐trap and enhancer‐trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene‐trap and 1073 enhancer‐trap lines were obtained from 5637 and 3726 transgenic lines, respectively. The expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene‐trap line YH209, which shows rhizoid‐specific expression, was obtained by 5′ and 3′ RACE. This gene was named PpGLU, and forms a clade with plant acidic α‐glucosidase genes. Thus, these gene‐trap and enhancer‐trap systems should prove useful to identify tissue‐ and cell‐specific genes in Physcomitrella.
Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley
Authors: Zhang Yuhua , Shewry Peter R. , Jones Huw , Barcelo Pilar , Lazzeri Paul A. , Halford Nigel G.
Published: March 2003

A chimaeric gene was constructed comprising a wheat high molecular weight glutenin subunit gene promoter, a 304‐bp sucrose non‐fermenting‐1‐related (SnRK1) protein kinase sequence in the antisense orientation, and the cauliflower mosaic virus 35S RNA gene terminator. Transgenic barley plants containing the antisense SnRK1 chimaeric gene were produced by particle bombardment of barley immature embryos with the aim of obtaining plants expressing the antisense SnRK1 sequence in the seeds. Despite the fact that the promoter was expected to be active only in seeds, two independent transgenic lines were found to fail to transmit the transgene to the T1 generation. These T0 plants had matured and died before this was discovered, but subsequently four other independent transgenic lines were found to be affected in the same way. Cytological analysis of the pollen grains in these lines showed that about 50% were normal but the rest had arrested at the binucleate stage of development, were small, pear‐shaped, contained little or no starch and were non‐functional. The presence of antisense SnRK1 transcripts was detected in the anthers of the four lines analyzed and a ubiquitin promoter/UidA (Gus) gene, one of the marker genes codelivered with the antisense gene, was found to be expressed only in the abnormal pollen. Expression analyses confirmed that SnRK1 is expressed in barley anthers and that expression of one class of SnRK1 transcripts (SnRK1b) was reduced in the abnormal lines. All of the abnormal lines showed approximately 50% seed set, and none of the transgenes were detected in the T1 generation.
Environmentally sensitive, SA‐dependent defense responses in the cpr22 mutant of Arabidopsis
Authors: Yoshioka Keiko , Kachroo Pradeep , Tsui Frank , Sharma Shashi B. , Shah Jyoti , Klessig Daniel F.
Published: March 2003

To investigate the signaling pathways through which defense responses are activated following pathogen infection, we have isolated and characterized the cpr22 mutant. This plant carries a semidominant, conditional lethal mutation that confers constitutive expression of the pathogenesis‐related (PR) genes PR‐1, PR‐2, PR‐5 and the defensin gene PDF1.2. cpr22 plants also display spontaneous lesion formation, elevated levels of salicylic acid (SA) and heightened resistance to Peronospora parasitica Emco5. The cpr22 locus was mapped to chromosome 2, approximately 2 cm telomeric to the AthB102 marker. By analyzing the progeny of crosses between cpr22 plants and either NahG transgenic plants or npr1 mutants, all of the cpr22‐associated phenotypes except PDF1.2 expression were found to be SA dependent. However, the SA signal transducer NPR1 was required only for constitutive PR‐1 expression. A cross between cpr22 and ndr1–1 mutants revealed that enhanced resistance to P. parasitica is mediated by an NDR1‐dependent pathway, while the other cpr22‐induced defenses are not. Crosses between either coi1–1 or etr1–1 mutants further demonstrated that constitutive PDF1.2 expression is mediated by a JA‐ and ethylene‐dependent pathway. Based on these results, the cpr22 mutation appears to induce its associated phenotypes by activating NPR1‐dependent and NPR1‐independent branches of the SA pathway, as well as an ethylene/JA signaling pathway. Interestingly, the SA‐dependent phenotypes, but not the SA‐independent phenotypes, are suppressed when cpr22 mutants are grown under high humidity.
Retracted: An ∼400 kDa membrane‐associated complex that contains one molecule of the resistance protein Cf‐4
Authors: Rivas Susana , Mucyn Tatiana , Van Den Burg Harrold A. , Vervoort Jacques , Jones Jonathan D. G.
Published: March 2003

Despite sharing more than 91% sequence identity, the tomato Cf‐4 and Cf‐9 proteins discriminate between two Cladosporium‐encoded avirulence determinants, Avr4 and Avr9. Comparative studies between Cf‐4 and Cf‐9 are thus of particular interest. To investigate Cf‐4 protein function in initiating defence signalling, we established transgenic tobacco lines and derived cell suspension cultures expressing c‐myc‐tagged Cf‐4. Cf‐4:myc encodes a membrane‐localized glycoprotein of approximately 145 kDa, which confers recognition of Avr4. Elicitation of Cf‐4:myc and Cf‐9:myc tobacco cell cultures with Avr4 and Avr9, respectively, triggered the synthesis of active oxygen species and MAP kinase activation. Additionally, an Agrobacterium‐mediated transient assay was used to express Cf‐4:myc and a newly engineered fusion protein Cf‐4:TAP. Both transiently expressed proteins were found to be functional in an in vivo assay, conferring a hypersensitive response (HR) to Avr4. Consistent with previous observations that Cf‐9 is present in a protein complex, gel filtration analysis of microsomal fractions solubilized with octylglucoside revealed that epitope‐tagged Cf‐4 proteins migrated at a molecular mass of 350–475 kDa. Using blue native gel electrophoresis, the molecular size was confirmed to be approximately 400 kDa. Significantly, this complex appeared to contain only one Cf‐4 molecule, supporting the idea that, as previously described for Cf‐9, additional glycoprotein partners participate with Cf‐4 in the perception of the Avr4 protein. Intriguingly, Cf proteins and Clavata2 (CLV2) of Arabidopsis are highly similar in structure, and the molecular mass of Cf‐4 and CLV complexes is also very similar (400 and 450 kDa, respectively). However, extensive characterization of the Cf‐4 complex revealed essentially identical characteristics to the Cf‐9 complex and significant differences from the CLV2 complex.
Early stages of seed development in Brassica napus: a seed coat‐specific cysteine proteinase associated with programmed cell death of the inner integument
Authors: Wan Lianglu , Xia Qun , Qiu Xiao , Selvaraj Gopalan
Published: March 2003

A maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identified a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post‐translational modifications. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows ≈72% identity to BnCysP1. It is, however, leaf senescence‐associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome‐deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post‐fertilization development of this important organ in rapeseed.