bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the PmSUC1 sucrose carrier gene from Plantago major L. is induced during seed development
Authors: Gahrtz Manfred , Schmelzer Elmon , Stolz Jürgen , Sauer Norbert
Published: March 2003

A cDNA clone of the plasma membrane sucrose‐H+sym‐ porter PmSUC1 from Plantago major L. has been isolated and expressed in Saccharomyces cerevisiae. The PmSUC1 protein was characterized in transgenic yeast and in proteoliposomes with an artificial proton‐motive‐force (pmf) generator. PmSUC1 catalyzes the active uptake of sucrose or maltose in the presence of pmf and is sensitive to uncouplers. Unlike the extremely pH‐dependent PmSUC2 sucrose‐H+ symporter, PmSUC1 is relatively insensitive to changes of the extracellular pH. In leaves and petioles of P. major, expression of PmSUC1 mRNA is restricted to the vascular system. The important new feature about PmSUC1 is that the highest mRNA levels are found in non‐vascular tissue of P. major flowers where the gene is transiently expressed during the early stages of seed development. In situ hybridization experiments show that PmSUC1 is expressed only in young ovules; the putative physiological role of PmSUC1 is discussed.
Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana
Authors: Dehesh Katayoon , Jones Aubrey , Knutzon Deborah S. , Voelker Toni A.
Published: March 2003

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl‐ACP thioesterase cDNA from C. hookeriana, designated Ch FatB2, has been identified, which, when expressed in Escherichia coli, provides thioesterase activity specific for 8:0‐ and 10:0‐ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1, another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0‐ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0‐ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.
Tissue‐specific expression of Arabidopsis AKT1 gene is consistent with a role in K+ nutrition
Authors: Lagarde Delphine , Basset Mireille , Lepetit Marc , Conejero Geneviève , Gaymard Frédéric , Astruc Suzette , Grignon Claude
Published: March 2003

AKT1, a putative inwardly directed K+ channel of Arabidopis, restores long‐term potassium uptake in a yeast mutant defective in K+ absorption. In this paper, the expression pattern of the gene encoding AKT1 is described. Northern blots indicate that AKT1 transcripts are preferentially accumulated in Arabidopsis roots. Owing to the difficulties in producing large quantities of Arabidopsis roots under hydroponic conditions, experiments were undertaken with Brassica napus, a related species. Potassium starvation experiments on B. napus plants show that changes in the K+ status of the organs do not modify AKT1 mRNA accumulation. Western blot analysis of B. napus proteins confirms the presence of AKT1 at the root plasma membrane. Tissue‐specific expression directed by the Arabidopsis AKT1 gene promoter was investigated by analysis of β‐glucuronidase (GUS) activity in transgenic Arabidopsis containing an AKT1‐GUS gene fusion. As determined by fluorimetric and histochemical tests, the AKT1 promoter directs preferential expression in the peripheral cell layers of root mature regions. The discrete activity found in leaves relates to leaf primordia and to small groups of cells, hydathodes, found on toothed margins of the Arabidopsis leaf lamina. These data are discussed with regard to a possible role of AKT1 in K+ nutrition of plants.
Nod factors and cytokinins induce similar cortical cell division, amyloplast deposition and MsEnod12A expression patterns in alfalfa roots
Authors: Bauer Petra , Ratet Pascal , Crespi Martin D. , Schultze Michael , Kondorosi Adam
Published: March 2003

Under nitrogen limitation, Rhizobium meliloti Nod factors induce cell divisions in the inner cortex of alfalfa roots in a still unknown way. These cell division clusters subsequently develop into symbiotically nitrogen‐fixing nodules. To study the involvement of plant signals in nodule initiation transgenic alfalfa carrying the promoter of the early nodulin gene MsEnod12A fused to the reporter gene gusA were generated. In untreated plants, low level GUS staining was only found in lateral root primordia and in front of the root apices. After inoculation with R. meliloti or after treatment with purified Nod factors, GUS activity was first induced in the cell division foci of the inner cortex. The GUS staining patterns in nodules and roots were in agreement with the activation of the endogenous MsEnod12A gene as revealed by reverse transcription‐PCR analysis, rendering the MsEnod12A‐gusA fusion a valuable novel marker for studying the onset of nodule and lateral root developmental processes. Treatment of roots with purified Nod factors and cytokinins induced similar patterns of cortical cell division, GUS staining and amyloplast accumulation while upon application of auxin transport inhibitors and auxins these patterns were different. Like the Nod factor responses, the cytokinin responses required photosynthesis and limiting combined nitrogen supply. Thus, cytokinins and Nod factors may share elements of their signal transduction pathways to the inner root cortex. A model on the possible involvement of cytokinins in coordinating plant metabolism with nodule initiation is proposed.
Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent‐kaurene synthase B from pumpkin (Cucurbita maxima L.)
Authors: Yamaguchi Shinjiro , Saito Tamio , Abe Hiroshi , Yamane Hisakazu , Murofushi Noboru , Kamiya Yuji
Published: March 2003

The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent‐kaurene. ent‐Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent‐kaurene by ent‐kaurene synthase B (KSB). A full‐length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.). Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening. The isolated full‐length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize [3H]CDP to [3H]ent‐kaurene. The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings. The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion‐diphosphate complex binding site. A putative transit peptide sequence that may target the translated product into the plastids is present in the N‐terminal region.
Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position‐dependent manner
Authors: van Hoof Ambro , Green Pamela J.
Published: March 2003

Premature termination of translation has often been associated with decreased mRNA accumulation in plants, but the affected step in gene expression has not been identified. To investigate this problem, the expression of wild‐type and mutant alleles of the bean phytohemagglutinin (PHA) gene has been examined in tobacco cells and transgenic plants. Measurement of mRNA decay rates in stably transformed cell lines demonstrated that premature nonsense codons markedly destabilized the mRNA. This decreased stability was also reflected by decreased accumulation of transcripts containing premature nonsense codons in transgenic plants. The positional dependence of the nonsense codon effect was evaluated by introducing premature nonsense codons at different distances from the PHA AUG start codon. Transcripts with nonsense codons about 20,40 or 60% of the way through the normal PHA coding region yielded highly unstable mRNAs, whereas a transcript with a nonsense codon at 80% was as stable as wild‐type. The ability to recognize and rapidly degrade certain transcripts with early nonsense codons could provide plant cells with a means to minimize the production of wasteful and possibly deleterious truncated proteins.
hrp gene‐dependent induction of hin1: a plant gene activated rapidly by both harpins and the avrPto gene‐mediated signal
Authors: Gopalan S. , Wei W. , He S.Y.
Published: March 2003

Two classes of bacterial genes are involved in the elicitation of the plant hypersensitive response (HR) in resistant plants: hrp genes and avr genes. hrp genes have been shown to be involved in the production and secretion of a new class of bacterial virulence/avirulence proteins, including harpin of Erwinia amylovora and harpinPss of Pseudomonas syringae. The ability of avr genes in the elicitation of the HR/resistance is dependent on functional hrp genes. The relationships between harpins and avr gene products are not known. This study investigates the plant genes induced by harpins and the effect of avr genes on the expression of such plant genes. A tobacco gene highly induced by harpins was isolated by a subtractive hybridization method. Induction of hin1 by P.s. pv. syringae 61 (Pss61) was found to be dependent on functional bacterial hrp genes. P. fluorescens (a saprophyte) or hrp mutants defective in the Hrp secretion pathway did not induce hin1 significantly. A hin1‐related gene in tomato cv. Rio Grande‐PtoR was found to be rapidly induced by P. s. pv. tomato T1 (a virulent bacterium on Rio Grande‐PtoR) containing the avrPto gene, which mediates the elicitation of the HR/resistance in a Pto plant resistance gene‐dependent manner. The induction of hin1 by bacteria correlates with production of harpins in planta. The putative open reading frame of hin1 encodes a novel protein of 221 amino acids. The data suggest that harpins and the avrPto‐mediated signal induce a common plant gene in the elicitation of the HR.
Characterization and mapping of Ds—GUS‐T‐DNA lines for targeted insertional mutagenesis
Authors: Smith D. , Yanai Y. , Liu Y.‐G. , Ishiguro S. , Okada K. , Shibata D. , Whittier R.F. , Fedoroff N.V.
Published: March 2003

The transposition patterns of the Ds—GUS transposon T‐DNA in 23 independent single‐copy lines have been characterized and the map positions of 10 of them on three of the five Arabidopsis chromosomes are reported. Using overexpressed Activator (Ac) elements as a transposase source, it was found that the primary determinant of transposition frequency is the insertion site of the Ac‐T‐DNA. Neither the structure of the transposon T‐DNA nor, in most cases, its insertion site have a significant effect on transposition frequency. Both the frequency and timing of transposition are influenced by the parent through which the transposon and transposase T‐DNAs are transmitted. Overall, nearly 75% of plants in which excision has occurred bear a reinserted element and very short‐range transpositions predominate, underlining the advantage of using mapped transposons for insertional mutagenesis.
Viral coat protein is targeted to, but does not gate, plasmodesmata during cell‐to‐cell movement of potato virus X
Authors: Oparka K.J. , Roberts A.G. , Roberts I.M. , Prior D.A.M. , Santa Cruz S.
Published: March 2003

The coat protein (CP) of potato virus X was localized immunocytochemically in infected leaves of susceptible Nicotiana species and shown to be targeted to the central cavity of plasmodesmata in virus‐infected cells. A viral deletion mutant, in which the CP gene was replaced with the gene for the green fluorescent protein (GFP), was restricted to single, inoculated cells. However, movement of the mutant virus was rescued on transgenic plants constitutively expressing the CP gene, and the CP was again targeted to plasmodesmata. The CP was not localized to plasmodesmata in uninfected transgenic plants and, in contrast to the plasmodesmata of PVX‐infected cells, the plasmodesmata of the transgenic plants did not allow the passage of 10 kDa fluorescent dextrans. We propose that the CP is not involved in plasmodesmal gating per se, but is necessary for transport of the viral RNA to, and possibly through, plasmodesmata.
The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE
Authors: Endrizzi Karin , Moussian Bernard , Haecker Achim , Levin Joshua Z. , Laux Thomas
Published: March 2003

The function of the SHOOT MERISTEMLESS (STM) gene in shoot and floral meristems throughout Arabidopsis development has been analyzed. The results show that STM plays a major role in maintaining shoot and floral meristems. In an allelic series of stm mutants the shoot meristem was either reduced or completely absent in mature embryos and mutant seedling cotyledons showed partial fusion, indicating that the STM gene affects embryonic shoot meristem development and spacing of cotyledons. Postembryonically, stm mutants initiated adventitious shoot development at a position corresponding to the shoot meristem in wild‐type. Repetitively initiated defective mutant shoot and floral meristems were consumed during primordia formation and typically terminated prematurely in fused ectopic primordia, indicating that STM is required for continuous shoot and floral meristem function.

Analogous defects were observed in stm embryonic and postembryonic development suggesting that similar mechanisms are employed in embryonic and postembryonic organ primordia initiation. Allelic combinations suggest different thresholds for STM requirement during plant development. STM requirement could not be bypassed by standard growth factor regimes or by shoot regeneration from calli. The results suggest that STM functions by preventing incorporation of cells in the meristem center into differentiating organ primordia and that this role can completely account for all defects observed in stm mutants.

Mutations in the WUSCHEL (WUS) and ZWILLE (ZLL) genes result in defective organization and premature termination of shoot meristems. Genetic interactions between STM, WUS and ZLL were analyzed and the results indicate that STM acts upstream of WUS and ZLL. Therefore, while STM appears to function in keeping central meristem cells undifferentiated, WUS and ZLL seem to be subsequently required for proper function of these cells.
Higher plant phosphoenolpyruvate carboxylase kinase is regulated at the level of translatable mRNA in response to light or a circadian rhythm
Authors: Hartwell James , Smith Lucy H. , Wilkins Malcolm B. , Jenkins Gareth I. , Nimmo Hugh G.
Published: March 2003

Phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to light in C3 and C4 plants and to a circadian oscillator in CAM plants. Increases in phosphoenolpyruvate carboxylase kinase activity require protein synthesis. This requirement has been analysed by quantifying translatable mRNA for this protein kinase using in vitro translation of isolated RNA followed by direct assay of kinase activity. In leaves of the CAM plant Bryophyllum (Kalanchoë) fedtschenkoi, in normal diurnal conditions, kinase mRNA was 20‐fold more abundant at night than in the day. In constant environmental conditions (continuous darkness, CO2‐free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. The appearance of kinase mRNA and activity was blocked by cordycepin and puromycin. In maize and barley, kinase mRNA increased in response to light. For all three plants, the phosphoenolpyruvate carboxylase kinase activity generated during in vitro translation was Ca2+‐independent. These results demonstrate that phosphoenolpyruvate carboxylase kinase activity is regulated at the level of translatable mRNA in C3, C4 and CAM plants.
A transcriptional activation domain of ATMYB2, a drought‐inducible Arabidopsis Myb‐related protein
Authors: Urao Takeshi , Noji Masa‐aki , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo
Published: March 2003

Trans‐activation activity of ATMYB2, a drought‐inducible Myb‐related protein in Arabidopsis thaliana, was analyzed using a transient assay of Arabidopsis leaf protoplasts. ATMYB2 activated the transcription of a reporter gene from the MYB‐binding site in a sequence‐specific manner. Deletion of the C‐terminal region of ATMYB2 reduced the trans‐activation of the reporter gene, indicating that the acidic region at the C‐terminus of ATMYB2 is required for transcriptional activation. The domain exchange analysis with the yeast GAL4 revealed that the C‐terminal acidic region of ATMYB2 contains a sufficient domain for trans‐activation. These results indicate that ATMYB2 acts as a transcriptional activator and that the C‐terminal acidic region of ATMYB2 can function as a transcriptional activation domain.
A plant in vitro system for the nuclear import of proteins
Authors: Merkle Thomas , Leclerc Denis , Marshallsay Christopher , Nagy Ferenc
Published: March 2003

This paper reports the development of an in vitro system that allows the direct assay of protein import into plant nuclei. In this assay the import of fluorescently labelled karyophilic protein substrates into nuclei isolated from evacuolated tobacco BY‐2 suspension cells is monitored. It is demonstrated that import of the fluorescently labelled peptide conjugates is rapid, saturable and nuclear localization signal (NLS)‐dependent. Exclusion of high molecular weight (70 kDa) dextran and substrates carrying mutated NLS sequences further underline the specificity of this system. Nuclear translocation of karyophilic import substrates in tobacco, similar to mammalian systems, is inhibited by the non‐hydrolysable GTP analogue GTP‐γ‐S. In contrast, protein uptake is not blocked by wheat germ agglutinin, N‐ethyl‐maleinimide and iodoacetic acid. Furthermore, it is shown that nuclear import of proteins is only partially inhibited by low temperature (0–4°C). The in vitro nuclear import assay does not depend on exogenously added ATP or cytosolic factors. However, a block of nuclear import with GTP‐γ‐S could be overcome by the addition of cytosolic extract, suggesting the dependence on cytosolic factors or proteins. These data indicate that the characteristics of nuclear protein import in plant and mammalian cells are similar, but may be, at least in some respects, also different from each other.
DNA fragmentation is regulated by ethylene during carpel senescence in Pisum sativum
Authors: Orzáez Diego , Granell Antonio
Published: March 2003

Treatment of the pea carpel with the ethylene action inhibitors, silver thiosulphate and 2,5‐norbornadiene, retarded its senescence and extended the growth time in which the unpollinated carpel was able to respond to gibberellins. Cells of the senescent carpel have characteristics of apoptotic cells, such as nuclei condensation and internucleosomal DNA fragmentation. Both DNA laddering and condensed nuclei can be prevented by the use of the ethylene action inhibitors, while ethylene treatment accelerates DNA fragmentation. It is postulated that ethylene produced by the flower after anthesis determines the fate of the ovary/ovule; in other words, if the ovary receives no additional stimulus (pollination or gibberellin treatment), it undergoes a senescence process, with some cells showing characteristics similar to those of programmed cell death already identified in animal systems.
Identification of new protein species among 33 different small GTP‐binding proteins encoded by cDNAs from Lotus japonicus, and expression of corresponding mRNAs in developing root nodules
Authors: Borg Søren , Brandstrup Birgitte , Jensen Trine Juul , Poulsen Carsten
Published: March 2003

In this study, 266 cDNA clones were isolated from a cDNA library made from mRNA of three‐week‐old root nodules of Lotus japonicus, employing a degenerate oligonucleotide probe that corresponds to a conserved region of small GTP binding (SMG) proteins. The clones were sorted into groups by cross hybridization and 3′ sequencing, and 33 contigs were sequenced in an orderly fashion. Twenty‐seven complete and six incomplete protein structures were deduced, which represent three subfamilies of the superfamily of signal transducing GTP‐binding proteins. The 33 proteins are divided into nine subclasses, of which seven belong to the Ypt/Rab subfamily, one subclass represents the Rho/Rac subfamily, and one subclass represents the Ran subfamily of small GTP‐binding proteins. The protein sequences were compared with related proteins from other plants, from mammals and other species, and discussed with respect to structure and function in different cellular processes. It is apparent that the number of genes encoding SMG proteins in plants must be quite large, since the large number of subclasses found in other eukaryotes is not fully represented in our analysis. Transcription patterns through root nodule development were analysed for 27 of the 33 cDNAs. Differential expression patterns may reflect whether the coded gene product is of importance for organ development. Most mRNAs appear to be constitutively expressed; however, a few unique mRNAs representing the subclasses Rab1, Rab2, Rab5, Rab7 and Rac show elevated levels in root nodules, and certain Rab7, Rab8 and Rab11 species are enriched in aerial parts of the plant. This suggests that most small GTPases have household functions, whereas a few may be required for specialized activities that are important for specialized cells.
Distinct response of Medicago suspension cultures and roots to Nod factors and chitin oligomers in the elicitation of defense‐related responses
Authors: Savouré Arnould , Sallaud Christophe , El‐Turk Joumana , Zuanazzi José , Ratet Pascal , Schultze Michael , Kondorosi Adam , Esnault Robert , Kondorosi Eva
Published: March 2003

The induction of plant defense‐related responses by chitin oligomers and the Rhizobium meliloti lipo‐chito‐oligosaccharide nodulation signals (Nod factors) in Medicago cell cultures and roots was investigated by following the expression of genes encoding enzymes of the isoflavonoid biosynthetic pathway, such as chalcone synthase, chalcone reductase, isoflavone reductase, as well as genes encoding a pathogenesis‐related protein and a peroxidase. In suspension‐cultured cells, all genes except the peroxidase gene were induced by both the R. meliloti Nod factor NodRm‐IV(C16:2,S) and chitin oligomers with a minimum of three sugar residues. However, activation of these genes was not elicited by the symbiotically inactive, desulfated NodRm‐IV(C16:2). Moreover, the cells were more sensitive to the chitin oligosaccharides than to the Nod factor. Analysis of flavonoids in Medicago microcallus cultures revealed differences between cells treated with N‐acetyl‐chitotetraose and those treated with Nod factor and demonstrated increased production of the phytoalexin medicarpin in the presence of Nod factor. In Medicago roots, none of the tested genes was activated by the N‐acetylchitotetraose, whereas the Nod factor at micro‐molar concentration enhanced transient expression of the isoflavonoid biosynthetic genes. The differential responses to Nod factors and chitin oligomers suggest that Medicago cells possess distinct perception systems for these related molecules.
The blue‐light receptor cryptochrome 1 shows functional dependence on phytochrome A or phytochrome B in Arabidopsis thaliana
Authors: Ahmad Margaret , Cashmore Anthony R.
Published: March 2003

Blue‐light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin‐type blue‐light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co‐action of the red/far‐red absorbing photoreceptor phytochrome with blue‐light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far‐red light pulses subsequent to blue‐light treatments. Furthermore, analysis of severely phytochrome‐deficient mutants showed that CRY1‐mediated blue‐light responses were considerably reduced, even though Western blots confirmed that levels of CRY1 photoreceptor are unaffected in these phytochrome‐deficient mutant backgrounds. It was concluded that CRY1‐mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB.
The LS locus of pea encodes the gibberellin biosynthesis enzyme ent‐kaurene synthase A
Authors: Ait‐Ali Tahar , Swain Stephen M. , Reid James B. , Sun Tai‐ping , Kamiya Yuji
Published: March 2003

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent‐kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent‐kaurene synthase A (KSA) and ent‐kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA‐responsive ls‐1 mutant of pea suggested that GA levels are reduced in a developmental‐ and organ‐specific manner due to reduced GA biosynthesis. Analysis of cell‐free enzyme preparations from WT and ls‐1 embryos at contact point reveals that ls‐1 reduces the activity of KSA but not KSB. To characterize the ls‐1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls‐1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls‐1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent‐kaurene in vitro. Based on the sequence of RT‐PCR products, three different truncated KSA proteins are predicted to exist in ls‐1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls‐1 allele is not null and is able to encode at least a partially functional KSA since a more severe ls allele has been identified. The ls‐1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.
Green‐fluorescent protein fusions for efficient characterization of nuclear targeting
Authors: Grebenok Robert J. , Pierson Elizabeth , Lambert Georgina M. , Gong Fang‐Cheng , Afonso Claudio L. , Haldeman‐Cahill Ruth , Carrington James C. , Galbraith David W.
Published: March 2003

The green‐fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co‐factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue‐specific expression of other popular reporter molecules, such as the β‐glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.
The green fluorescent protein as a marker to visualize plant mitochondria in vivo
Authors: Köhler Rainer H. , Zipfel Warren R. , Webb Watt W. , Hanson Maureen R.
Published: March 2003

To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two‐photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co‐localization with a mitochondrial‐specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three‐dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.
PcMYB1, a novel plant protein containing a DNA‐binding domain with one MYB repeat, interacts in vivo with a light‐regulatory promoter unit
Authors: Feldbrügge Michael , Sprenger Markus , Hahlbrock Klaus , Weisshaar Bernd
Published: March 2003

Light‐regulatory unit 1 (LRU1) is necessary for and sufficient to mediate light‐dependent activation of the chalcone synthase (CHS) minimal promoter in Petroselinum crispum. This composite promoter unit consists of at least two distinct cis‐acting elements, designated ACECHS and MRECHS, both of which are required for light induction. The ACGT‐containing element ACECHS interacts with common plant regulatory factors (CPRFs) which belong to the basic region/leucine zipper (bZIP) class of transcription factors. Here, we demonstrate that MRECHS, originally identified as an in vivo DNA footprint, is a MYB recognition element. This element possesses a functional core that is essential for light responsiveness and is specifically recognized by two distantly related MYB‐like proteins: MYB305 and the novel factor MYB1 from P. crispum. PcMYB1 was identified by both its specific binding to MRECHSin vitro and recognition of MRECHSin vivo. The deduced amino acid sequence revealed that PcMYB1 contains only one MYB‐like repeat. This portion of the protein constitutes the DNA‐binding domain. Mutational analysis of PcMYB1 in combination with sequence comparison suggests the presence of a helix‐turn‐helix structure containing a recognition helix that is sufficient for sequence‐specific binding. The structure of this distinct MYB‐like DNA‐binding domain appears to be conserved in proteins from all three eukaryotic phyla.
The phytochrome‐deficient pcd2 mutant of pea is unable to convert biliverdin IXα to 3(Z)‐phytochromobilin
Authors: Weller James L. , Terry Matthew J. , Reid James B. , Kendrick Richard E.
Published: March 2003

During screening of ethylmethane sulphonate‐mutagenized pea (Pisum sativum L.) seedlings under far‐red light a mutant line, AF130, was isolated which showed a reduction in both red and far‐red light‐induced de‐etiolation responses. The photomorphogenic phenotype of AF130 results from a single recessive mutation which is not allelic with the previously described phytochrome chromophore biosynthesis mutant pcd1. This new mutant has been designated pcd2, for phytochrome chromophore deficient 2. Like pcd1, etiolated pcd2 seedlings are severely deficient in spectrally active phytochrome and contain wild‐type levels of phytochrome A apoprotein which is not substantially depleted by red light treatment. Etioplast preparations from pcd2 seedlings can metabolize heme to biliverdin (BV) IXα, but are unable to convert BV IXα to the phytochrome chromophore, phytochromobilin. The PCD1 and PCD2 genes therefore control consecutive steps in phytochromobilin synthesis. Despite a similarly severe impairment of photomorphogenic responses, pcd2 mutant seedlings do not display the strongly chlorotic phenotype of pcd1, suggesting that this characteristic of pcd1 does not result from phytochrome deficiency per se, but is a specific effect of the pcd1 mutation. A double mutant between pcd1 and pcd2 was constructed. This mutant is paler than pcd1 and less responsive to red light than either single mutant, but retains a strong response to blue light.
Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms‐0 to two powdery mildew diseases
Authors: Xiao Shunyuan , Ellwood Simon , Findlay Kim , Oliver Richard P. , Turner John G
Published: March 2003

Arabidopsis thaliana accession La‐er was susceptible, and accession Ms‐0 was resistant, to powdery mildew diseases caused by Erysiphe cruciferarum UEA1 and E. cichoracearum UCSC1. The resistance reaction phenotype of A. thaliana Ms‐0 to both pathogens was characterized, and the resistance loci were genetically mapped. Growth of E. cruciferarum UEA1 on Ms‐0 leaves was arrested after formation of the first appressorium: the underlying host epidermal cell collapsed, and occasionally there was necrosis of one or two host mesophyll cells. Growth of E. cichoracearum UCSC1 on Ms‐0 leaves was arrested after emergence of several germ tubes from the conidium, and there was necrosis of host mesophyll cells at the sites of infection. Examination of F2 progeny of a cross La‐er x Ms‐0 indicated that two independently‐segregating dominant loci were required for resistance to E. cruciferarum UEA1. One locus, named RPW6, was genetically mapped to chromosome 5, in a 5.6 cM interval flanked by pCITf16 and PI. The other locus, named RPW7, mapped to chromosome 3 in a 8.5 cM interval flanked by CDC2A and AFC1. Independent effects of RPW6 and RPW7 on E. cruciferarum UEA1 could be detected by quantitative measurements of growth of mycelium and production of conidia. Resistance to E. cichoracearum UCSC1 mapped to a single locus, named RPW8, at a location on chromosome 3 which we could not distinguish from RPW7. Evidently, RPW7 and RPW8 define either a complex resistance locus, or a common resistance gene with dual specificity.
Interaction of Arabidopsis kinesin‐like calmodulin‐binding protein with tubulin subunits: modulation by Ca2+‐calmodulin
Authors: Narasimhulu Soma B. , Kao Yu‐Lin , Reddy A.S.N.
Published: March 2003

Kinesin‐like calmodulin‐binding protein (KCBP) is a recently identified novel kinesin‐like protein that appears to be unique to and ubiquitous in plants. KCBP is distinct from all other known KLPs in having a calmodulin‐binding domain adjacent to its motor domain. We have used different regions of KCBP to study its interaction with tubulin subunits and the regulation of this interaction by Ca2+‐calmodulin. The results show that the carboxy‐terminal part of the KCBP, with or without calmodulin‐binding domain, binds to tubulin subunits and this binding is sensitive to nucleotides. In the presence of Ca2+‐calmodulin the motor with calmodulin‐binding domain does not bind to tubulin. This Ca2+‐calmodulin modulation is abolished in the presence of antibodies specific to the calmodulin‐binding domain of KCBP. Similar binding studies with the carboxy‐terminal part of KCBP lacking the calmodulinbinding domain show no effect of Ca2+‐calmodulin. These results indicate that Ca2+‐calmodulin modulates the interaction of KCBP with tubulin subunits and this modulation is due to the calmodulin‐binding domain in the KCBP. Calcium‐dependent calmodulin modulation of KCBP interaction with tubulin suggests regulation of KCBP function by calcium, the first such regulation of a kinesin heavy chain among all the known kinesin‐like proteins.
Splicing features in maize streak virus virion‐ and complementary‐sense gene expression
Authors: Wright E.A. , Heckel T. , Groenendijk J. , Davies J.W. , Boulton M.I.
Published: March 2003

The single‐stranded DNA geminiviruses produce transcripts from both strands (virion‐ and complementary‐sense) of a nuclear double‐stranded DNA molecule. In maize streak virus (MSV)‐infected maize plants, approximately 80% of the complementary‐sense transcripts produce the C1 protein, whilst the remaining 20% are spliced to remove a 92 nt intron and produce a C1:C2 fusion protein (Rep). Disruption of the complementary‐sense 3′ splice site abolished virus replication. The majority of the virion‐sense transcripts initiated one nucleotide upstream of the V1 (movement protein) gene and a minority a further 141 nucleotides upstream. A 76 nt intron, with features typical of plant introns, was identified within the V1 gene, upstream of the coat protein gene. Spliced and unspliced forms of each virion‐sense transcript were produced, but they differed in splicing efficiency. Approximately 50% of the major transcript and less than 10% of the minor transcript were processed. Mutagenesis of the consensus 5′ splice site in the V1 gene resulted in the use of alternative cryptic splice sites, confirming the importance of splicing for MSV infection. Spliced virion‐sense transcripts were also identified in tissue infected with the closely‐related Digitaria streak virus (DSV) but not with another subgroup I geminivirus, wheat dwarf virus. Collectively, the multiple transcript initiation sites and different splicing efficiencies suggest that splicing is an important feature in the regulation of both early and late gene expression in MSV and DSV.
A Cupheaβ‐ketoacyl‐ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases
Authors: Leonard Jeffrey M. , Knapp Steven J. , Slabaugh Mary B.
Published: March 2003

Acyl–acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8–C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a β‐ketoacyl‐ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II‐type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0‐ and 8:0‐ACP at a rate four‐ to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0‐ and 16:0‐ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain‐specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one‐third and one‐half, respectively. F2 progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4‐fold more 12:0 than the FatB2 parent, and the double‐transgenic progeny produced one‐quarter less 14:0 and one‐half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl‐ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.
Auxin transport inhibition precedes root nodule formation in white clover roots and is regulated by flavonoids and derivatives of chitin oligosaccharides
Authors: Mathesius Ulrike , Schlaman Helmi R. M. , Spaink Herman P. , Of Sautter Christ , Rolfe Barry G. , Djordjevic Michael A.
Published: March 2003

The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot‐inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non‐host rhizobia, lipo‐chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40–1 peptide or different flavonoids. The results show that clover‐nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot‐inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O‐acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot‐inoculation, but only after delivery by microtargeting. Non‐O‐acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40–1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.
Immunolocalization of a novel annexin‐like protein encoded by a stress and abscisic acid responsive gene in alfalfa
Authors: Kovács Izabella , Ayaydin Ferhan , Oberschall Attila , Ipacs Ildikó , Bottka Sándor , Pongor Sándor , Dudits Dénes , Tóth Éva Csordás
Published: March 2003

We report here on the isolation and characterization of a full‐length cDNA clone from alfalfa termed AnnMs2 encoding a 333 amino acid long polypeptide that shows 32–37% sequence identity with both mammalian and plant annexins, and has four tandem repeats. While other plant annexins exhibit a high level of sequence similarity to each other (up to 77% identity at amino acid level), AnnMs2 appears to be a distinct type of plant annexins. All the four endonexin folds contain the conserved eukaryotic motif within this alfalfa protein, but this element is considerably different in the second repeat. The AnnMs2 gene is expressed in various tissues of alfalfa with elevated mRNA accumulation in root and flower. This gene is activated in cells or tissues exposed to osmotic stress, abscisic acid (ABA) or water deficiency. The recombinant AnnMs2 protein is able to bind to phospholipid in the presence of Ca2+. Indirect immunofluorescence studies using affinity purified rabbit anti‐AnnMs2 peptide antibody show mainly nucleolar localization, but the protein sequence lacks the usual nuclear localization signal. The potential role of this novel annexin‐like protein in the basic and stress‐induced cellular functions is discussed.
Differential regulation of the tomato ETR gene family throughout plant development
Authors: Lashbrook Coralie C. , Tieman Denise M. , Klee Harry J.
Published: March 2003

Ethylene perception in plants is co‐ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two‐component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting ∼ 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two‐component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down‐regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue‐specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.