bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis
Authors: Kawaoka Akiyoshi , Kaothien Pulla , Yoshida Kazuya , Endo Saori , Yamada Keiko , Ebinuma Hiroyasu 
Published: March 2003

SummaryThe AC‐rich motif, Pal‐box, is an important cis‐acting element for gene expression involved in phenylpropanoid biosynthesis. A cDNA clone (Ntlim1) encoding a Pal‐box binding protein was isolated by Southwestern screening. The deduced amino acid sequence is highly similar to the members of the LIM protein family that contain a zinc finger motif. Moreover, Ntlim1 had a specific DNA binding ability and transiently activated the transcription of a β‐glucuronidase reporter gene driven by the Pal‐box sequence in tobacco protoplasts. The transgenic tobacco plants with antisense Ntlim1 showed low levels of transcripts from some key phenylpropanoid pathway genes such as phenylalanine ammonia‐lyase, hydroxycinnamate CoA ligase and cinnamyl alcohol dehydrogenase. Furthermore, a 27% reduction of lignin content was observed in the transgenic tobacco with antisense Ntlim1.
Salicylic acid is not required for Cf‐2‐ and Cf‐9‐dependent resistance of tomato to Cladosporium fulvum
Authors: Brading Penny A. , Hammond‐Kosack Kim E. , Parr Adrian , Jones Jonathan D. G. 
Published: March 2003

SummaryTomato leaves or cotyledons expressing the Cf‐2 or Cf‐9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene‐dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild‐type Cf0. Neither free nor conjugated salicylic acid accumulated in IF‐infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf‐9‐dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf‐2‐like response. SA also potentiated Cf‐9‐mediated necrosis in IF‐infiltrated wild‐type leaves. In contrast, the Cf‐2‐dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild‐type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF‐induced accumulation of three SA‐inducible defence‐related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm‐22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV‐inoculated N gene‐containing NahG tobacco plants.
Molecular characterization of a novel glucose‐6‐phosphate dehydrogenase from potato (Solanum tuberosum L.)
Authors: Wendt Urte Kristina , Wenderoth Irina , Tegeler Achim , Von Schaewen Antje 
Published: March 2003

SummaryWe describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox‐regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C‐terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein‐mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid‐localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.
Transgenic potato plants reveal the indispensable role of cystathionine β‐lyase in plant growth and development
Authors: Maimann Stefanie , Wagner Cornelia , Kreft Oliver , Zeh Michaela , Willmitzer Lothar , Höfgen Rainer , Hesse Holger 
Published: March 2003

SummaryMethionine (Met) is an essential amino acid that is often unavailable at sufficient dietary levels. In order to better understand Met pathway regulation, a cDNA encoding cystathionine β‐lyase (CbL; EC 4.4.1.8) has been cloned from Solanum tuberosum. An antisense construct of this gene was used to generate transgenic potato plants with reduced CbL levels. Transgenic plants exhibiting leaf CbL activity levels of up to 50% below wild‐type levels were obtained. Metabolite analysis revealed a reduction in Met levels in these CbL antisense plants, as well as remarkable increases in the pathway intermediates cystathionine, homoserine and cysteine. Unexpectedly, an increase in homocysteine was also observed. Levels of aspartate amino acid pathway intermediates (including aspartate, lysine and threonine) remained essentially unaffected. Neither transcript levels nor protein products of other pathway‐relevant genes were altered significantly in these plants. CbL antisense plants exhibited an altered phenotype characterized by a bushy growth habit, small light‐green leaves and small tubers. This phenotype could be alleviated upon Met supplementation, suggesting that low Met levels, rather than pathway intermediate accumulation, is responsible for the phenotypic effects of CbL transgene expression. These data unequivocally demonstrate the central role of CbL in Met biosynthesis, and, subsequently, in plant growth and development.
Maize ROP7 GTPase contains a unique, CaaX box‐independent plasma membrane targeting signal
Authors: Ivanchenko Maria , Vejlupkova Zuzana , Quatrano Ralph S. , Fowler John E. 
Published: March 2003

SummarySignals in the carboxy‐terminal hypervariable region (HVR) of Rho and Ras GTPases target these proteins to specific membrane compartments, where they function in signal transduction. ROP6 and ROP7 are closely related maize Rops (a plant‐specific Rho subgroup) that share HVR sequences divergent from other Rho HVRs. Both ROPs terminate in CAA, instead of the consensus C‐terminal CaaX motif required for membrane association of all characterized Ras and Rho GTPases. The ROP6/7 HVR contains two additional cysteines, potential sites for post‐translational modification that leads to membrane association; one is in an internal CaaX motif, which would be at the C‐terminus if the final intron in both genes were not removed. Transient expression of a GFP–ROP7 fusion revealed its near‐total association with the plasma membrane (PM). Furthermore, the ROP7 HVR is sufficient to target GFP to the PM. Surprisingly, the cysteine in the terminal CAA is not required for PM targeting of GFP–ROP7. In contrast, an internal HVR cysteine is essential for proper targeting of the fusion, and the cysteine in the internal CaaX is required for complete membrane association. Interestingly, this CaaX motif can also direct PM association when placed at the fusion C‐terminus by addition of an internal stop codon. Fractionation experiments confirm that maize ROPs associate with membranes in maize seedlings. Our analysis suggests that the ROP7 HVR directs PM localization by a mechanism independent of a C‐terminal CaaX motif; this mechanism may have evolved through addition of 3′ intron/exon sequences to aropprogenitor.
Cytokinin and gibberellin activate SaMADS A, a gene apparently involved in regulation of the floral transition in Sinapis alba
Authors: Bonhomme Françoise , Kurz Birgit , Melzer Siegbert , Bernier Georges , Jacqmard Annie 
Published: March 2003

SummaryIn plants of Sinapis alba induced to flower by one long day, the MADS box gene, SaMADS A, is expressed initially in the central corpus (L3 cells) of the shoot apical meristem (SAM), about 1.5–2 days before initiation of the first floral meristem. We have combined a physiological approach by testing the effects of three putative floral signals on SaMADS A expression in the SAM of S. alba plants with a transgenic approach using Arabidopsis thaliana plants. A single application of a low dose of a cytokinin or a gibberellin to the apex of vegetative S. alba plants is capable of mimicking perfectly the initial effect of the long day on SaMADS A transcription. A treatment combining the two hormones causes the same activation but seems to enhance the level of SaMADS A expression. A sucrose application to the apex of vegetative plants is, on the contrary, unable to activate SaMADS A expression. None of these chemicals, alone or combined, is capable of causing the floral shift at the SAM. Since the constitutive expression of SaMADS A leads to precocious flowering in A. thaliana and antisense expression of a fragment of the A. thaliana homologue AGL20 leads to a delay in flowering time, these results are consistent with SaMADS A activation being an intermediate event in a cytokinin‐ and/or gibberellin‐triggered signal transduction pathway that is involved in the regulation of floral transition in S. alba.
Construction of male and female PAC genomic libraries suitable for identification of Y‐chromosome‐specific clones from the liverwort, Marchantia polymorpha
Authors: Okada Sachiko , Fujisawa Masaki , Sone Takefumi , Nakayama Shigeki , Nishiyama Rie , Takenaka Mizuki , Yamaoka Shohei , Sakaida Megumi , Kono Kaoru , Takahama Masayoshi , Yamato Katsuyuki T. , Fukuzawa Hideya , Brennicke Axel , Ohyama Kanji 
Published: March 2003

SummaryUnlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1‐derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male‐specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male‐specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.
Tonoplast subcellular localization of maize cytochrome b5 reductases
Authors: Bagnaresi Paolo , Mazars‐Marty Daniele , Pupillo Paolo , Marty Francis , Briat Jean‐François 
Published: March 2003

SummaryPlant cytochrome b5 reductases (b5R) are assumed to be part of an ER‐associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b5) to ER‐associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem. J. 338, 499–505). Maize b5R isoforms are produced by a small multi‐gene family. The NFR cDNAs were shown to encode active b5Rs by heterologous expression in yeast. Both reductases, in addition to Fe3+‐chelates, efficiently reduced Cu2+‐chelates. Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots. Unexpectedly, maize b5Rs were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast‐enriched maize root fractions. Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b5R. This indicates that alternative ER‐associated electron donors for desaturases need to be sought, and that plant b5Rs may have previously unexpected functions.
The MLA6 coiled‐coil, NBS‐LRR protein confers AvrMla6‐dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat
Authors: Halterman Dennis , Zhou Fasong , Wei Fusheng , Wise Roger P. , Schulze‐Lefert Paul 
Published: March 2003

SummaryThe barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla‐RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full‐length CC‐NBS‐LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full‐length candidate cDNA, two single‐cell expression assays were used to demonstrate complementation of AvrMla6‐dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1‐independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC‐NBS‐LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.
Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate
Authors: Richardson Alan E. , Hadobas Paul A. , Hayes Julie E. 
Published: March 2003

SummaryPhosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild‐type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20‐fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.
The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily
Authors: Swiderski Michal R. , Innes Roger W. 
Published: March 2003

SummarySpecific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1. Previous work has shown that RPS5 encodes a member of the nucleotide binding site‐leucine rich repeat class of plant disease resistance genes. Here we report that PBS1 encodes a putative serine‐threonine kinase. Southern blot analysis revealed that the pbs1‐1 allele contained a deletion of the 3′ end of the PBS1 open reading frame. DNA sequence analysis of the pbs1‐2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases. The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants. Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function. The Pto kinase of tomato, which is required for specific resistance to P. syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain. These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins. We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition.
Phosphatidic acid activates a wound‐activated MAPK in Glycine max
Authors: Lee Sumin , Hirt Heribert , Lee Youngsook 
Published: March 2003

SummaryMany plant species demonstrate a systemic increase in phosphatidic acid (PA) levels after being wounded (Lee et al., 1997). To understand the role of PA in wound signal transduction, we investigated if PA can activate protein kinases in soybean (Glycine max L.). We found that a MAPK is activated in soybean seedlings in both wounded and neighboring unwounded leaves. The wound‐activated soybean kinase is specifically recognized by an antibody against the alfalfa MAPK, SIMK. When PA production is inhibited with n‐butanol, an inhibitor of phospholipase D, the wound‐induced activation of the MAPK is suppressed, suggesting that an elevation in PA levels is essential for its activation. Supporting this is the observation that exogenous PA activates the MAPK in suspension‐cultured soybean cells. Activation of the 49 kDa MAPK occurs almost exclusively by PA, as other lipids are unable to or can only weakly activate the kinase. PA‐induced activation of the MAPK is not a direct effect on the kinase but is mediated by upstream kinases. Our results suggest that PA acts as a second messenger in wound‐induced MAPK signaling in plants.
Amino acid permeases in developing seeds ofVicia fabaL.: expression precedes storage protein synthesis and is regulated by amino acid supply
Authors: Miranda Manoela , Borisjuk Ljudmilla , Tewes Annegret , Heim Ute , Sauer Norbert , Wobus Ulrich , Weber Hans 
Published: March 2003

SummaryFull length cDNAs encoding three amino acid permeases were isolated from seed‐specific libraries ofVicia faba. The predicted proteins VfAAP1, VfAAP3 and VfAAP4 share up to 66% identity among themselves. Functional characterization of VfAAP1 and VfAAP3 in a yeast mutant showed that these permeases transport a broad range of amino acids. However, VfAAP1 had a preference for cysteine and VfAAP3 for lysine and arginine.VfAAP1was highly expressed in cotyledons at early developmental stages and moderately in other sink tissues. Its peak of expression in cotyledons corresponded to the appearance of storage protein transcripts, suggesting that this transporter fulfills an important role in providing amino acids for storage protein biosynthesis.VfAAP3was expressed most abundantly in maternal tissues, that is in roots, stems, gynoecia, pods and seed coats at different developmental stages.VfAAP4transcripts could not be detected by northern hybridization.In situhybridization showed thatVfAAP1mRNA is distributed throughout cotyledon storage parenchyma cells, but could not be detected in the abaxial epidermal cell layer. It also accumulate in the chlorenchyma and thin‐walled parenchyma cells of seed coats.VfAAP1mRNA levels were lower in cotyledons cultured in the presence of glutamine, whereas expression of a vicilin storage protein gene was up‐regulated under similar conditions. Cysteine repressed the expression of theGUSreporter gene under control of theVfAAP1promoter, suggesting that this transporter is modulated at the transcriptional level. Regulation of amino acid transport in relation to storage protein accumulation is discussed.
A plastid envelope location of Arabidopsis ent‐kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway
Authors: Helliwell Chris A. , Sullivan James A. , Mould Ruth M. , Gray John C. , Peacock W. James , Dennis Elizabeth S. 
Published: March 2003

SummaryWe have used fusions of gibberellin biosynthesis enzymes to green fluorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 mono‐oxygenase and 2‐oxoglutarate‐dependent dioxygenase classes. We show that the N‐terminal pre‐sequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and ent‐kaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1–GFP fusion is also imported by isolated pea chloroplasts. The N‐terminal portion of the cytochrome P450 protein ent‐kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35S‐labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent‐kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid‐ and endoplasmic reticulum‐located steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.