bluuebunny/crossref_metadata_2025 huggingface dataset:
Alfalfa NADH‐dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation
Authors: Vance Carroll P. , Miller Susan S. , Gregerson Robert G. , Samac Deborah A. , Robinson D. Lowell , Gantt J. Stephen
Published: March 2003

Glutamate synthase (GOGAT), a key enzyme in ammonia (NH4+) assimilation, occurs as two forms in plants: a ferredoxin‐dependent form (Fd‐GOGAT) and an NADH‐dependent form (NADH‐GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH‐GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH‐GOGAT to nearly identical positions. The transcribed region of this gene, 12 214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5′ from the translation initiation site confers nodule‐specific reporter gene activity when used in a chimeric β‐glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH‐GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH4+ assimilation, high levels of NADH‐GOGAT expression are strictly associated with effective nodules indicating that NADH‐GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd‐GOGAT PCR product showing greater than 85% identity to maize Fd‐GOGAT was isolated and used to investigate the contribution of this enzyme to NH4+ assimilation in nodules. Fd‐GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd‐GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.
RFLP and physical mapping with an rDNA‐specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4
Authors: Copenhaver Gregory P. , Pikaard Craig S.
Published: March 2003

Ribosomal RNA genes are organized in tandem arrays called nucleolus organizer regions (NORs). In a prior study, RFLP mapping on pulsed‐field gels placed NOR2 at the northern tip of Arabidopsis thaliana chromosome 2. New polymorphisms have allowed the other NOR, NOR4, to be mapped to the northern tip of chromosome 4. To map NOR‐associated loci, rDNA‐specific cleavage by I‐PpoI, an endonuclease with a 15 nucleotide recognition sequence involved in rDNA‐homing of a mobile, self‐splicing Group I intron in Physarum was exploited. I‐Ppo digestion of A. thaliana genomic DNA liberated two telomere‐containing fragments no larger than 13 kbp, and telomere polymorphisms identified using I‐PpoI co‐segregated with NOR2 and NOR4. Restriction mapping suggested that telomere‐proximal rRNA genes are oriented with their 5′ ends nearest the chromosome ends and their 3′ ends nearest the centromere. This orientation was confirmed using the polymerase chain reaction to clone one of the telomere—rDNA junctions, most likely the junction on chromosome 4. The telomeric repeats join the terminal rRNA gene downstream of its promoter, suggesting that this first gene is inactive. Subtelomeric repetitive DNAs are absent at the telomere—rDNA junction. Localization of NOR2, NOR4 and their associated telomeres, TEL2N and TEL4N, respectively, provides end points for the genetic and physical maps of chromosomes 2 and 4.
Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana
Authors: Adam Luc , Somerville Shauna C.
Published: March 2003

This paper reports on six Arabidopsis accessions that show resistance to a wild isolate of the powdery mildew pathogen, Erysiphe cichoracearum. Resistance at 7 days post‐inoculation in these accessions was characterized by limited fungal growth and sporadic development of chlorotic or necrotic lesions at inoculation sites. Three accessions, Wa‐1, Kas‐1 and SI‐0, were highly resistant, while the other accessions permitted some fungal growth and conidiation. Papilla formation was a frequent host response; however, cell death appeared to be neither a rapid nor a common response to infection. To determine the genetic basis of resistance, segregation analyses of progeny from crosses between each of the resistant accessions and Columbia (gl1), which is susceptible to the powdery mildew pathogen, were performed. For all accessions except SI‐0, resistance was conferred by a single locus. SI‐0 was unique in that two unlinked loci controlled the disease reaction phenotype. In accessions Wa‐1, Kas‐1, Stw‐0 and Su‐0, powdery mildew resistance was encoded by a semi‐dominant allele. However, susceptibility was dominant to resistance in accessions Te‐0 and SI‐0. Mapping studies revealed that powdery mildew resistances in Kas‐1, Wa‐1, Te‐0, Su‐0 and Stw‐0 were controlled by five independent loci. This study suggests that the Arabidopsis powdery mildew disease will be a suitable model system in which to investigate powdery mildew diseases.
Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2
Authors: Kim Byung Chul , Soh Moon Soo , Kang Bong Joong , Furuya Masaki , Nam Hong Gil
Published: March 2003

By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana, two dominant photomorphogenic mutants, shy1‐1D and shy2‐1D, for two genetic loci designated as SHY1 and SHY2 (suppressor of hy2 mutation) have been isolated. Both of these non‐allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light‐independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1‐1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2‐1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion, and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2‐1D mutation is still light sensitive. Examination of red/far‐red light responses shows that the shy1‐1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far‐red light. The shy2‐1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far‐red light. Both mutations can also suppress the early‐flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis. This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants.
Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway
Authors: Lawton Kay A. , Friedrich Leslie , Hunt Michelle , Weymann Kris , Delaney Terrance , Kessmann Helmut , Staub Theodor , Ryals John
Published: March 2003

Benzothiadiazole (BTH) is a novel chemical activator of disease resistance in tobacco, wheat and other important agricultural plants. In this report, it is shown that BTH works by activating SAR in Arabidopsis thaliana. BTH‐treated plants were resistant to infection by turnip crinkle virus, Pseudomonas syringae pv ‘tomato’ DC3000 and Peronospora parasitica. Chemical treatment induced accumulation of mRNAs from the SAR‐associated genes, PR‐1, PR‐2 and PR‐5. BTH treatment induced both PR‐1 mRNA accumulation and resistance against P. parasitica in the ethylene response mutants, etr1 and ein2, and in the methyl jasmonate‐insensitive mutant, jar1, suggesting that BTH action is independent of these plant hormones. BTH treatment also induced both PR‐1 mRNA accumulation and P. parasitica resistance in transgenic Arabidopsis plants expressing the nahG gene, suggesting that BTH action does not require salicylic acid accumulation. However, because BTH‐treatment failed to induce either PR‐1 mRNA accumulation or P. parasitica resistance in the non‐inducible immunity mutant, nim1, it appears that BTH activates the SAR signal transduction pathway.
The Arabidopsis Athb‐10 (GLABRA2) is an HD‐Zip protein required for regulation of root hair development
Authors: Di Cristina Manlio , Sessa Giovanna , Dolan Liam , Linstead Paul , Baima Simona , Ruberti Ida , Morelli Giorgio
Published: March 2003

Homeodomain‐leucine zipper (HD‐Zip) proteins are putative transcription factors identified only in plants. Related Arabidopsis homeobox genes, isolated by virtue of sequence conservation within the helix‐3 region of the homeodomain, fall into four families based on sequence similarity. This paper reports the characterization of Athb‐10, a 747 amino acid protein belonging to the fourth HD‐ZIP family. The studies indicate that, although less conserved, the leucine zipper of Athb‐10 can functionally replace that of Athb‐2 in an in vitro DNA‐binding assay. Gene mapping experiments and sequence comparison analysis revealed that Athb‐10 corresponds to GLABRA2, a homeodomain protein involved in trichome development. The mRNA expression analysis revealed that Athb‐10/GLABRA2 is expressed not only in trichome‐bearing organs, but also in the root. The analysis of wild‐type and mutant plants showed that the Athb‐10/GLABRA2 gene expression in the aerial part of the plant and in the root is affected by mutations at the TTG locus. Morphological analysis of the gl2‐1 mutant revealed that the gene is necessary not only for local outgrowth of the trichome, but also for the regulation of root hair development in a subset of epidermal cells. Interestingly, the development of root hair cells in a position normally occupied by non‐hair cells is dependent upon the ethylene regime in which the gl2‐1 plants are grown. Sequence analysis of the gl2‐1 allele revealed that the mutant gene encodes a truncated protein that might still retain a partial activity responsible for the formation of aborted trichomes and for the ethylene‐dependent regulation of root hair formation.
Mutations in the AXR3 gene of Arabidopsis result in altered auxin response including ectopic expression from the SAUR‐AC1 promoter
Authors: Leyser H.M. Ottoline , Pickett F. Bryan , Dharmasiri Sunnethra , Estelle Mark
Published: March 2003

A new auxin response gene in Arabidopsis called AXR3 has been identified. This gene is defined by two semi‐dominant mutations which affect many auxin‐regulated developmental processes. Auxin has been shown to maintain apical dominance, inhibit root elongation, stimulate adventitious rooting, mediate root gravitropism, and stimulate transcription from the SAUR‐AC1 promoter. Mutant axr3 plants show enhanced apical dominance, reduced root elongation, increased adventitious rooting, no root gravitropism, and ectopic expression from the SAUR‐AC1 promoter. These phenotypes suggest an increased auxin response in the mutants. In support of this hypothesis, many of the phenotypes are partially restored to wild‐type by exogenous cytokinin, a treament that could restore a more wild‐type auxin to cytokinin ratio.
U‐rich tracts enhance 3′ splice site recognition in plant nuclei
Authors: Baynton Clair E. , Potthoff Sara J. , McCullough Andrew J. , Schuler Mary A.
Published: March 2003

The process of 5′ and 3′ splice site definition in plant pre‐mRNA splicing differs from that in mammals and yeast. In mammals, splice sites are chosen by their complementarity to U1 snRNA surrounding the /GU at the 5′ splice site and by the strength of the pyrimidine tract preceding the AG/ at the 3′ splice site; in plants, the 3′ intron boundary is defined in a position‐dependent manner relative to AU‐rich elements within the intron. To determine if uridines are utilized to any extent in plant 3′ splice site recognition, uridines in the region preceding the normal (−1) 3′ splice site of pea rbcS3A intron 1 were replaced with adenosines. This mutant activates two cryptic 3′ splice sites (+62, +95) in the downstream exon, indicating that the uridines in the region immediately preceding the normal (−1) site are essential for recognition. Placement of different length uridine tracts upstream from the cryptic +62 site indicated that a cryptic exonic 3′ splice site containing 14 or 10 uridine tracts with a G at −4 can effectively outcompete the normal 3′ splice site containing an eight uridine tract with a U at −4. Substitutions at the −4 position demonstrated that the identity of the nucleotide at this position greatly affects 3′ splice site selection. It has been concluded that several factors affect competition between these 3′ splice sites. These factors include the position of the AU transition point, the strength of the uridine tract immediately preceding the 3′ terminal CAG/ and the identity of nucleotide −4.
Identification of mutants in metabolically regulated gene expression
Authors: Martin Thomas , Hellmann Hanjo , Schmidt Renate , Willmitzer Lothar , Frommer Wolf B.
Published: March 2003

Sucrose is the main transported form of assimilates, but, significantly, it also regulates a variety of processes such as photosynthesis and carbon or nitrogen storage. The effects of high sucrose levels are mediated directly by modulation of gene expression. The regulation of storage protein accumulation, here patatin from potato tubers, was used as a model system to study sucrose mediated signal transduction. The transcriptional regulation of patatin genes is conserved in transgenic Arabidopsis, as shown by the analysis of expression of two classes of patatin promoters fused to uidA. Two distinctly different patterns of gene expression were observed. In roots, class I promoter expression is strongly dependent on the exogenous supply of sugars. 3‐O‐methylglucose induction indicates that the sensor is located upstream of hexokinase. In contrast, the class II promoter is constitutively active in root tips and hydatodes. The progeny of a homozygous class I line was mutagenized with ethyl methane sulphonate and screened for signal transduction mutants using a non‐destructive screening system for GUS activity. Four mutants showing reduced sucrose responses (rsr) and two mutants with modified expression patterns (mep) regarding the root tip were identified. In backcross analyses, it was shown that rsr1‐1 carries a recessive trans mutation whereas rsr4‐1 seems to be a semi‐dominant trans mutation in sugar‐mediated gene regulation.
Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.
Authors: Neuhaus H.E. , Thom E. , Möhlmann T. , Steup M. , Kampfenkel K.
Published: March 2003

Recently, we have sequenced a cDNA clone from Arabidopsis thaliana L. encoding a novel putative ATP/ADP translocator (AATP1). Here, we demonstrate that the radioactively labeled AATP1 precursor protein, synthesized in vitro, is targeted to envelope membranes of isolated spinach chloroplasts. Antibodies raised against a synthetic peptide of AATP1 recognized a single polypeptide of about 62 kDa in chloroplast inner envelope preparations. The cDNA coding for the AATP1 protein was functionally expressed in Saccharomyces cerevisiae and Escherichia coli. In both expression systems, increased rates of ATP transport were observed after reconstitution of the extracted protein into proteoliposomes. To our knowledge, this is the first report on the functional expression of an intrinsic plant membrane protein in E. coli. To yield high rates of ATP transport, proteoliposomes had to be preloaded with ADP, indicating a counter‐exchange mode of transport. Carboxyatractyloside did not substantially interfere with ATP transport into proteoliposomes containing the plastidic ATP/ADP translocator. An apparent KM for ATP of 28 µM was determined which is similar to values reported for isolated plastids. The data presented here strongly support the conclusion that AATP1 represents a novel eukaryotic adenylate carrier and that it is identical with the so far unknown plastidic ATP/ADP translocator.
Cloning of a Ca2+‐ATPase gene and the role of cytosolic Ca2+ in the gibberellin‐dependent signaling pathway in aleurone cells
Authors: Chen Xiongfong , Chang Menchi , Wang Baiyang , Wu Ray
Published: March 2003

The ultimate goal of this investigation was to identify intermediary steps in the gibberellin (GA)‐dependent signaling pathway in rice aleurone cells. By using a differential display approach, a number of putative GA‐responsive genes were isolated. One of them, a GA‐responsive Ca2+‐ATPase gene, was identified and partially characterized. A genomic clone and a cDNA clone were isolated and sequenced. The deduced amino acid sequence showed that this protein resembles an endoplasmic reticulum membrane Ca2+‐ATPase. In a transient assay in rice aleurone cells, expression of the introduced Ca2+‐ATPase cDNA bypassed the GA requirement for stimulating the expression of a major target gene, the α‐amylase c gene (Osamy‐c). This result suggests that GA‐dependent expression of this Ca2+‐ATPase gene (OsCa‐atpase) plays an important role in the GA‐dependent signal‐transduction pathway. To investigate the possible involvement of other proteins and genes that may affect the intracellular Ca2+ level, compounds which can block different putative steps in the signal‐transduction pathway were introduced into rice aleurone cells, and then the level of the OsCa‐atpase transcript or the Osamy‐c transcript was monitored. In the presence of GA, the rice Ca2+‐ATPase and the Ca2+ channels appeared to co‐regulate the local concentration of cytosolic Ca2+. The release of Ca2+ from the internal stores to the cytoplasm was presumably initiated by inositol‐1,4,5‐triphosphate which reached a peak level within 25 min after GA induction. As a second messenger, Ca2+ binds to calmodulin (CaM), and the Ca2+/CaM complex regulates the cytosolic Ca2+ by affecting expression of the OsCa‐atpase. Finally, a working model is proposed for the GA‐dependent signaling pathway in aleurone cells.
Cooperation of two distinct cis‐acting elements is necessary for the S phase‐specific activation of the wheat histone H3 promoter
Authors: Ohtsubo Norihiro , Nakayama Takuya , Kaya Hidetaka , Terada Rie , Shimamoto Ko , Meshi Tetsuo , Iwabuchi Masaki
Published: March 2003

We have previously shown that the proximal promoter region (−185 to +57) of the wheat histone H3 gene (TH012) is sufficient for regulating S phase‐specific expression of a reporter GUS gene. To define the cis‐acting element(s) responsible for S phase‐specific expression, GUS fusion genes under the control of wild‐type or variously mutated H3 promoters were stably introduced into cultured rice Oc cells and their temporal expression was analyzed during the cell cycle by quantitative S1 analysis. The S phase‐specific expression of the full‐sized promoter (−1716 to +52) was significantly impaired by short internal deletions disrupting the type I element from −175 to −158 (CCACGTCACCaATCCGCG), composed of the Hex (CCACG‐TCA) and reverse‐oriented Oct (GATCCGCG) motifs. Moreover, the H3 proximal promoters (−184 to +52) harboring base‐substitution mutations in either or both of the Hex and Oct motifs could no longer activate gene expression during the S phase. These results indicate that the type I element is the first cis‐acting element identified responsible for the S phase‐specific expression of plant histone genes. Results also suggested the presence of a redundant cis‐acting element(s) responsible for S phase‐specific expression in the H3 far‐upstream region (−1716 to −185).
Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress
Authors: Hayashi Hidenori , Alia  , Mustardy Laszlo , Deshnium Patchraporn , Ida Miki , Murata Norio
Published: March 2003

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild‐type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild‐type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild‐type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild‐type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild‐type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.
Functional analysis of the Arabidopsis thaliana SBP‐box gene SPL3: a novel gene involved in the floral transition
Authors: Cardon Guillermo H. , Höhmann Susanne , Nettesheim Klaus , Saedler Heinz , Huijser Peter
Published: March 2003

The isolation and initial characterization of the Arabidopsis thaliana SPL3 gene are described. SPL3 belongs to a gene family encoding putative transcription factors characterized by a conserved DNA‐binding domain, the SBP domain. SPL3 transcription is developmentally regulated and is localized mainly in vegetative and inflorescence apical meristems, floral meristems and in leaf and floral organ primordia. SPL3 recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene AP1. Similarly to AP1, constitutive expression of SPL3 results in early flowering. However, constitutive expression of SPL3 in an ap1 mutant background showed that AP1 is not required for the early flowering phenotype of the SPL3 transgenic plants. The function of SPL3 during flowering as well as its possible functional redundancy are discussed.
Resistance to both cyst and root‐knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin
Authors: Urwin Peter E. , Lilley Catherine J. , McPherson Michael J. , Atkinson Howard J.
Published: March 2003

Plant nematodes are major pests of agriculture. Transgenic plant technology has been developed based on the use of proteinase inhibitors as nematode anti‐feedants. The approach offers prospects for novel plant resistance and reduced use of environmentally damaging nematicides. A modified rice cystatin, Oc‐IΔD86, expressed as a transgene in Arabidopsis thaliana, has a profound effect on the size and fecundity of females for both Heterodera schachtii (beet‐cyst nematode) and Meloidogyne incognita (root‐knot nematode). No females of either species achieved the minimum size they require for egg production. Ingestion of Oc‐IΔD86 from the plant was correlated with loss of cysteine proteinase activity in the intestine thereby suppressing normal growth, as required of an effective anti‐feedant plant defence.
Post‐transcriptional β‐1,3‐glucanase gene silencing involves increased transcript turnover that is translation‐independent
Authors: Jacobs John J.M.R. , Litière Katia , Van Dijk Victor , Van Eldik Gerben J. , Van Montagu Marc , Cornelissen Marc
Published: March 2003

In tobacco line T17, basic β‐1,3‐glucanases are silenced when a β‐1,3‐glucanase (gn1) transgene of Nicotiana plumbaginifolia is present in a homozygous condition. Co‐suppression of endogenous and transgenic basic β‐1,3‐glucanases occurs via a post‐transcriptional process. This study shows that silencing correlates with a reduced stability of the transcripts encoded by gn1 and the co‐suppressed endogenous glucanase genes. The observed decreases in mRNA stability at least partially explain the reductions in mRNA level. Furthermore, the results show that the enhanced, silencing‐related turnover of gn1 mRNA does not require continued protein synthesis. The implications of these findings for the mechanism of gene silencing are discussed.
The maize gene empty pericarp‐2 is required for progression beyond early stages of embryogenesis
Authors: Scanlon Michael J. , Myers Alan M. , Schneeberger Richard G. , Freeling Michael
Published: March 2003

The defective kernel mutation empty pericarp2‐R (emp2‐R) causes retardation and subsequent abortion of maize kernel development. Analyses of genetic aneuploid kernels indicate that the embryo phenotype is not dependent on the endosperm genotype; the mutation conditions embryo defects even in the presence of a normal endosperm. Embryos reach an abnormal coleoptilar stage before aborting and disintegrating. The mutants form primary embryonic organs only; the scutellum and coleoptile develop, but no leaves are formed. Immunohisto‐localization studies utilized KNOX homeodomain proteins as markers of meristem formation and identity. These analyses indicate that the shoot meristem forms in emp2‐R mutant embryos, but does not mature to a tunica‐corpus shape. No evidence of leaf founder cell initialization was revealed in the mutant meristems. These data indicate that the emp2 gene may be required for embryogenic patterning beyond the coleoptilar stage of development.
Structural analysis of elements contributing to 5′ splice site selection in plant pre‐mRNA transcripts
Authors: Egoavil Cesar , Marton Heather A. , Baynton Clair E. , McCullough Andrew J. , Schuler Mary A.
Published: March 2003

In vivo analyses of the cis‐acting sequence requirements for pre‐mRNA splicing in tobacco nuclei have previously demonstrated that 5′ splice sites are selected by their position relative to AU‐rich sequences within plant introns and by their degree of complementarity to the 5′ end of U1 snRNA. To identify the specific nucleotide sequences promoting recognition of the 5′ exon‐intron boundary, multiple mutations have been introduced into a 22 nt AU‐rich block positioned between two competing 5′ splice sites in a derivative of pea rbcS3A intron 1. Transient expression of these mutant transcripts has delineated a 9 nt element positioned 30–38 nt downstream from the 5′ splice site whose sequence composition affects 5′ splice site choice. Uridine substitutions within this element do not alter 5′ splice selection patterns, and, in fact, enhance recognition of the upstream +1wt 5′ splice site slightly. Guanosine substitutions in this region, specifically creating an AG‐rich AAAGGAGAGGCAGA motif (mutations shown underlined), reduce recognition of the upstream +1wt 5′ splice site and enhance recognition of the downstream +106E 5′ splice site. Cytosine substitutions at homologous positions marginally reduce recognition of the upstream 5′ splice site. Additional mutations in this 9 nt element suggest that the AAAGGAGAGGCAGA motif acts as an specific 5′ exon‐defining element promoting recognition of downstream 5′ splice sites, while AU‐rich motifs promote recognition of upstream 5′ splice sites. Mutations in an adjacent AU‐rich region attenuate the effects of mutations in this short element but are not in themselves sufficient to alter 5′ splice site selection. It is concluded that specific elements on both sides of the exon‐intron boundary define the 5′ splice site in this plant transcript.
Calcium signalling in Arabidopsis thaliana responding to drought and salinity
Authors: Knight Heather , Trewavas Anthony J. , Knight Marc R.
Published: March 2003

Changes in cytosolic free calcium concentration ([Ca2+]cyt) in response to mannitol (drought) and salt treatments were detected in vivo in intact whole Arabidopsis seedlings. Transient elevations of [Ca2+]cyt to around 1.5 µM were observed, and these were substantially inhibited by pretreatment with the calcium‐channel blocker lanthanum and to a lesser extent, the calcium‐chelator EGTA. The expression of three genes, p5cs, which encodes Δ1‐pyrroline‐5‐carboxylate synthetase (P5CS), the first enzyme of the proline biosynthesis pathway, rab18 and Iti78 which both encode proteins of unknown function, was induced by mannitol and salt treatments. The induction of all three genes by mannitol was inhibited by pretreatment with lanthanum. Salt‐induced p5cs, but not rab18 and Iti78, expression was also inhibited by lanthanum. Induction of p5cs by mannitol was also inhibited by the calcium channel‐blockers gadolinium and verapamil and the calcium chelator EGTA, further suggesting the involvement of calcium signalling in this response. Mannitol induced greater levels of p5cs gene expression than an isoosmolar concentration of salt, at both relatively high and low concentrations. However, calcium transients were of a similar magnitude and duration in response to both mannitol and isoosmolar concentrations of salt, suggesting that a factor other than calcium is involved in the discrimination between drought and salinity signals in Arabidopsis. In order to gauge the involvement of the vacuole as an intracellular calcium store in the response of Arabidopsis to mannitol, [Ca2+]cyt was measured at the microdomain adjacent to the vacuolar membrane. The results obtained were consistent with a significant calcium release from the vacuole contributing to the overall mannitol‐induced [Ca2+]cyt response. Data obtained by using inhibitors of inositol signalling suggested that this release was occurring through IP3‐dependent calcium channels.
Ratio‐imaging of Ca2+i in the self‐incompatibility response in pollen tubes of Papaver rhoeas
Authors: Franklin‐Tong Vernonica E. , Hackett Grant , Hepler Peter K.
Published: March 2003

The data presented here describe ratio‐imaging of in intracellular free calcium (Ca2+i) during the self‐incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura‐2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial‐temporal alterations in Ca2+i, and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca2+]i) in the SI response.

Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip‐focused gradient of Ca2+bfi, with levels reaching 1–2 μM at the extreme apex of the pollen tube. Non‐growing pollen tubes did not exhibit this tip‐focused gradient. Basal levels of Ca2+i in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low‐level fluctuations +/− 50 nM observed. Challenge with incompatible S proteins resulted in S‐specific, rapid and dramatic alterations in [Ca2+]i within a few seconds of challenge. Increases in [Ca2+]i were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca2+]i in this region subsequently increased for several minutes, reaching> 1.5 μM. At the pollen tube tip, a diminution of the tip‐focused gradient was observed, which following some fluctuation, was reduced to basal levels within ∼1 min. Our data suggest that some of these alterations in [Ca2+]i might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca2+]i in the subapical/shank region are very rapid, because tip [Ca2+]i oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca2+]i at the pollen tube tip.
Gene identification with sequenced T‐DNA tags generated by transformation of Arabidopsis cell suspension
Authors: Mathur Jaideep , Szabados László , Schaefer Sabine , Grunenberg Britta , Lossow Andrea , Jonas‐Straube Esther , Schell Jeff , Koncz Csaba , Koncz‐Kálmán Zsuzsanna
Published: March 2003

A protocol for establishment and high‐frequency Agrobacterium‐mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T‐DNA tags. Thirty‐two self‐circularized T‐DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long‐range inverse polymerase chain reaction (LR‐iPCR). By bidirectional sequencing of the ends of T‐DNA‐linked plant DNA segments, nine T‐DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J‐domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen‐specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T‐DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.
Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis
Authors: Slabaugh Mary B. , Leonard Jeffrey M. , Knapp Steven J.
Published: March 2003

Seed oils of most Cuphea species contain > 90% medium chain (C8–C14) fatty acids. Thioesterases with specificity for these substrates are important determinants of the medium chain phenotype. The role of condensing enzymes, however, has not been investigated. cDNA clones encoding β‐ketoacyl‐acyl carrier protein (ACP) synthase (KAS) were isolated from C. wrightii, a C10/C12‐producing species. Deduced amino acid sequences of four unique clones were ∼ 60% identical to plant KAS I sequences and ∼ 75% identical to a distinct class of KAS sequences recently identified in castor and barley. A 46 kDa protein that was observed only in developing and mature seed was detected using antiserum directed against recombinant Cuphea KAS protein. The 46 kDa protein was abundant in developing seeds of six medium chain‐producing Cuphea species but barely detected in one long chain‐producing species. A 48 kDa protein identified immunologically as KAS I was expressed in both medium and long chain‐producing Cuphea species and was detected in all tissues tested. In in vitro assays, extracts from C. wrightii and C. viscosissima developing embryos were unable to extend fatty acid chains beyond C10 following treatment with 10 μm cerulenin, a potent inhibitor of KAS I. However, a C. viscosissima mutant, cpr‐1, whose seed oils are deficient in caprate relative to wild type, was impaired in extension of C8 to C10 in this assay and Western analysis revealed a specific deficiency in 46 kDa KAS in cpr‐1 embryos. These results implicate cerulenin‐resistant condensing activity in production of medium chain fatty acids in Cuphea.
Characterization of acyl‐ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola
Authors: Hawkins Deborah J. , Kridl Jean C.
Published: March 2003

Acyl‐acyl‐carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils. Acyl‐ACP thioesterases with specificity for each of the saturated acyl‐ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases. The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight. Acyl‐ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1‐ACP substrates, but had significant activity with 18:0 relative to that with 16:0‐ACP, suggesting a thioesterase might be involved in the production of stearate. Three distinct acyl‐ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class. When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1‐ACP showed relatively low activity with 16:0‐ACP as compared to 18:0‐ACP, similar to the substrate preferences shown by the crude seed extract. Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil. These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles.
Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response
Authors: He Zheng‐Hui , He Deze , Kohorn Bruce D.
Published: March 2003

Pathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. AnArabidopsiscell wall associated receptor kinase (Wak1) has an amino‐terminal domain that is tightly associated with the ECM, spans the plasma membrane and has a cytoplasmic protein kinase domain.Wak1expression is induced whenArabidopsisplants are infected with pathogen, or when the pathogen response is stimulated either by exogenous salicylate (SA) or its analog 2,2‐dichloroisonicotinic acid (INA). ThisWak1induction requires the positive regulator NPR1/NIM1. Thus Wak1 is a pathogen‐related (PR) protein. Expression of an antisense and a dominant negative allele ofWak1shows that induced expression ofWak1is needed for a plant to survive if stimulated by INA. Ectopic expression of the entire Wak1, or the kinase domain alone, can provide resistance to otherwise lethal SA levels. These experiments suggest that Wak1 expression and other PR proteins are protecting plants from detrimental effects incurred during the pathogen response. These results provide a direct link between a protein kinase that could mediate signals from the ECM, to the events that are precipitated by a pathogen infection.
A gene encoding 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase produces two transcripts: elucidation of a conserved response
Authors: Peck Scott C. , Kende Hans
Published: March 2003

Indole‐3‐acetic acid (IAA) promotes ethylene biosynthesis in stems of etiolated pea (Pisum sativum L.) seedlings by rapidly increasing the expression of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase mRNA and by enhancing the activity of the enzyme. Two cDNA clones encoding ACC synthase, Ps‐ACS1 and Ps‐ACS2, were isolated from a cDNA library prepared from the apical hooks of etiolated pea seedlings that had been treated with 100 μM IAA for 4 h. While studying the expression pattern of IAA‐induced ACC synthase mRNA, we observed that the probe for Ps‐ACS1 hybridized to two transcripts of 1.6 and 1.9 kb on RNA gel blots. The shorter transcript accumulated before the longer one did, indicating that it is not a degradation product of the latter. Because a similar observation, namely hybridization of one ACC synthase probe to two transcripts, has also been reported in other species, we investigated the relationship between the 1.6‐ and 1.9‐kb transcripts. DNA gel blot analysis using the entire cDNA as probe and RNA gel blot analysis using the 3′‐untranslated region as probe indicated that both transcripts are encoded by the same gene. Oligonucleotide‐directed RNase H mapping showed that the transcripts differ in the sequence of their 5′‐ends. Using 5′‐RACE to obtain the DNA sequence of the shorter transcript, we determined that the 1.6‐kb transcript (Ps‐ACS1b) begins within the second exon of the 1.9‐kb transcript (Ps‐ACS1a) and lacks the first 383 bases. Thus, Ps‐ACS1b does not encode a full‐length ACC synthase protein. Because the Ps‐ACS1b sequence is identical to that of Ps‐ACS1a, including proper splicing of the second intron, Ps‐ACS1b appears to result from the use of an alternative, internal promoter.
Isolation of putative plant transcriptional coactivators using a modified two‐hybrid system incorporating a GFP reporter gene
Authors: Cormack Robert S. , Hahlbrock Klaus , Somssich Imre E.
Published: March 2003

Dual hybrid interacting screening in yeast led to the identification of two proteins from Arabidopsis both exhibiting sequence similarity to a family of transcriptional coactivators from a diverse range of organisms. Their discovery constitutes the first description of such plant proteins. A modified yeast two‐hybrid approach utilising the green fluorescent protein (GFP) of Aequora victoria was developed and used to clone one of the putative plant transcriptional coactivators from an Arabidopsis cDNA library. The two proteins, designated KIWI and KELP, can associate both hetero‐ and homomerically and their genes were cloned and mapped on the Arabidopsis genome. Both proteins are believed to play a role in gene activation during pathogen defence and plant development. The involvement of these proteins in general plant transcription as well as the advantages of using GFP as a reporter gene for detecting protein–protein interactions are discussed.
Cell‐to‐cell transport of proteins: requirement for unfolding and characterization of binding to a putative plasmodesmal receptor
Authors: Kragler Friedrich , Monzer Jan , Shash Khalid , Xoconostle‐Cázares Beatriz , Lucas William J.
Published: March 2003

Plasmodesmata and the nuclear pore complex (NPC) mediate the selective trafficking of proteins and protein‐nucleic acid complexes. The events underlying the translocation of endogenous and viral proteins through plasmodesmata were investigated to further explore the parallels between these cell‐to‐cell and intracellular communication systems. Studies performed with crosslinked KNOTTED1 (KN1) revealed that a conformational change is required for the cell‐to‐cell movement of this protein. Microinjection of gold‐conjugated KN1 established that, as with the NPC, a combination of protein unfolding and microchannel dilation appears to be involved in protein translocation. However, during this process the extent of microchannel dilation is much less than observed for the NPC, which may reflect a physical limitation imposed by the cell wall. Co‐injection of KN1‐gold with unbound KN1 or cucumber mosaic virus movement protein (CMV‐MP) established that the KN1‐gold probe is highly effective at blocking plasmodesmal transport of KN1 and CMV‐MP. This result provided the foundation for competition experiments which demonstrated that KN1 and the viral movement proteins of CMV and tobacco mosaic virus likely utilize a common receptor in the pathway for cell‐to‐cell transport of proteins. A combination of biochemical fractionation methods, an

in vitro

binding assay founded on the high affinity between KN1‐gold and the putative common plasmodesmal receptor, and microinjection techniques were used to isolate plasmodesmal constituents involved in cell‐to‐cell transport. A model describing the steps involved in protein transport through plasmodesmata is presented.
Cell marking inArabidopsis thaliana andits application to patch–clamp studies
Authors: Maathuis Frans J. M. , May Sean T. , Graham Neil S. , Bowen Helen C. , Jelitto Till C. , Trimmer Paul , Bennett Malcolm J. , Sanders Dale , White Philip J.
Published: March 2003

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane‐patch voltage‐clamp (patch–clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch–clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch–clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of

Arabidopsis

roots. Transgenic

Arabidopsis

were generated in which the expression of green fluorescent protein (GFP) from

Aequoria victoria

was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non‐fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch–clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic

Arabidopsis

expressing GFP in a cell‐specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch–clamp studies.
Generation of co‐dominant PCR‐based markers by duplex analysis on high resolution gels
Authors: Hauser Marie‐Theres , Adhami Farzaneh , Dorner Maria , Fuchs Esther , Glössl Josef
Published: March 2003

Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high‐performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready‐to‐use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single‐nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co‐dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36

Arabidopsis thaliana

loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (

n

= 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co‐dominant PCR‐based molecular markers for fine‐scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.