bluuebunny/crossref_metadata_2025_split huggingface dataset:
FLP‐mediated recombination for use in hybrid plant production
Authors: Luo Hong , Lyznik L. Alexander , Gidoni David , Hodges Thomas K. 
Published: March 2003

SummaryWe have studied the feasibility in Arabidopsis of using a site‐specific recombination system FLP/FRT, from the 2 μm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site‐specific recombinase were crossed with plants transformed with a vector containing kanamycin‐resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self‐pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen‐ and tapetum‐specific gene, bcp1, flanked by FRT sites. Upon cross‐pollination of flowers on the same male‐sterile plants with pollen from FLP‐containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site‐specific recombinase can be used to restore fertility in male‐sterile plants, providing an alternative method for the production of hybrid seeds and plants.
Quantitative nature of the Prolamin‐box, ACGT and AACA motifs in a rice glutelin gene promoter: minimal cis‐element requirements for endosperm‐specific gene expression
Authors: Wu Chuan‐Yin , Washida Haruhiko , Onodera Yasuyuki , Harada Kyuya , Takaiwa Fumio 
Published: March 2003

SummaryThe −197 bp promoter of the rice seed storage protein gene, GluB‐1, is capable of conferring endosperm‐specific gene expression. This proximal 5′ flanking region contains four motifs, GCN4, AACA, ACGT and Prolamin‐box, which are conserved in many seed storage protein genes. We previously showed that multiple copies of GCN4 conferred endosperm expression pattern when fused to the −46 core promoter of CaMV 35S. In this paper we demonstrate, using a similar approach, that tandem repeated copies of any of the other three motifs are unable to direct expression in seeds as well as other tissues of transgenic rice plants. Mutational analysis of individual motifs in the −197 bp promoter resulted in remarkable reductions in promoter activity. These results indicate that the GCN4 motif acts as an essential element determining endosperm‐specific expression and that the AACA, ACGT and Prolamin‐box are involved in quantitative regulation of the GluB‐1 gene. A set of gain‐of‐function experiments using transgenic rice showed that either the Prolamin‐box or AACA, although often coupled with GCN4 in many genes, is insufficient to form a functional promoter unit with GCN4, whereas a combination of GCN4, AACA and ACGT motifs was found sufficient to confer a detectable level of endosperm expression. Taken together, our results provide direct insight into the importance of combinatorial interplay between cis‐elements in regulating the expression of seed storage protein genes.
SRL1: a new locus specific to the phyB‐signaling pathway in Arabidopsis
Authors: Huq Enamul , Kang Yurah , Halliday Karen J. , Qin Minmin , Quail Peter H. 
Published: March 2003

SummaryAs part of an effort to isolate new Arabidopsis mutants specifically defective in responsiveness to red light, we identified srl1 (short hypocotyl in red light) by screening an EMS‐mutagenized M2 population derived from a phytochrome B (phyB)‐overexpressor line (ABO). The srl1 mutant shows enhanced responsiveness to continuous red but not far‐red light, in both wild‐type and ABO backgrounds, consistent with involvement in the phyB‐signaling pathway but not that of phyA. The hypersensitive phenotype of srl1 is not due to overexpression of endogenous phyA or phyB, and the locus maps to the center of chromosome 2, distinct from any other known photomorphogenic mutants. srl1 seedlings display enhancement of several phyB‐mediated responses, including shorter hypocotyls, more expanded cotyledons, shorter petioles and modestly higher levels of CAB gene expression under red light than the wild type. Double mutant analyses show that the hypersensitive phenotype of srl1 is completely phyB‐dependent. The data suggest, therefore, that SRL1 may encode a negatively acting component specific to the phyB‐signaling pathway.
Constitutive expression of a putative high‐affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post‐transcriptional regulation by a reduced nitrogen source
Authors: Fraisier Vincent , Gojon Alain , Tillard Pascal , Daniel‐Vedele Françoise 
Published: March 2003

SummaryThe NpNRT2.1 gene encodes a putative inducible component of the high‐affinity nitrate (NO3–) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3– supplied, NO3– contents were found to be remarkably similar in wild‐type and transgenic plants. Under specific conditions (growth on 10 m
m NO3–), the steady‐state NpNRT2.1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3– influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high‐affinity transport system. Both 15NO3– influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3– influx at both transcriptional and post‐transcriptional levels.
In situ analysis of RNA and protein expression in whole mounts facilitates detection of floral gene expression dynamics
Authors: Zachgo Sabine , Perbal Marie‐Christine , Saedler Heinz , Schwarz‐Sommer Zsuzsanna 
Published: March 2003

SummaryA three‐dimensional whole‐mount technique for detection of mRNA and protein expression patterns of floral regulatory genes in inflorescences from Antirrhinum majus is reported. This technique allows the observation of complex expression patterns in situ in developing flowers at different developmental stages initiated sequentially on the same inflorescence and labelled under the same conditions. Thereby, reconstruction from serial two‐dimensional sections can be circumvented. The technique was used to study early changes in the expression of DEFICIENS (DEF), a class B floral homeotic transcription factor. Whole‐mount analysis revealed that the order of appearance of DEF mRNA and protein expression in the floral primordium is opposite to the order of initiation of organ primordia. As a consequence, stamen primordia express the DEF gene prior to their initiation in whorl three, while petal primordia in the second whorl are morphologically distinct structures when second whorl DEF expression becomes established. This interesting feature was not readily detectable by previous analysis of serial sections. The particular usefulness of in situ analyses in whole mounts is further demonstrated in floral mutants with variable phenotypes and unpredictable sites of aberrant organ development.Keywords: whole mount, in situ hybridization, immunolocalization, Antirrhinum majus, flower development.
Diurnal changes in sucrose, nucleotides, starch synthesis and AGPS transcript in growing potato tubers that are suppressed by decreased expression of sucrose phosphate synthase
Authors: Geigenberger Peter , Stitt Mark 
Published: March 2003

SummarySucrose export from leaves is high during the day and lower at night, when it depends on starch remobilisation. We have investigated the consequences of diurnal changes of photoassimilate supply for starch synthesis and other metabolic processes in growing potato tubers. Sucrose, the levels of the transcripts for SUS and AGPS, the levels of key metabolites, and the rate of synthesis of starch and other major end products, including protein and cell wall polysaccharides, increased twofold or more between the start and end of the light period. The stimulation of starch synthesis was accompanied by an increase of UDPglucose and ADPglucose, whereas glycolytic intermediates remained unaltered, revealing that sucrose synthase and ADP‐glucose pyrophosphorylase are being co‐ordinately regulated. Sucrose synthase is stimulated via an increase of its substrates, UDP and sucrose. UDP increases due to an increase of the overall uridine nucleotide pool, and a decrease of the UTP/UDP ratio that occurs in parallel with a decrease of the ATP/ADP ratio and adenylate energy charge when biosynthetic fluxes are high at the end of the day. Within the time frame of the diurnal changes, the changes in the SUS and AGPS transcript levels do not lead to significant changes in the encoded enzymes. Transformants with a progressive decrease of sucrose phosphate synthase expression, where diurnal changes in leaf sugar levels were damped, exhibited a progressive attenuation of the diurnal changes of sucrose, nucleotide sugars and nucleotides, and fluxes in their tubers. It is concluded that metabolic processes in tubers are tightly linked to the momentary supply of sucrose.
LKP1 (LOV kelch protein 1): a factor involved in the regulation of flowering time in Arabidopsis
Authors: Kiyosue Tomohiro , Wada Masamitsu 
Published: March 2003

SummaryIn plants, light is not only an energy source but also a very important signal that modulates development and differentiation. Here, we report a putative photo‐regulatory factor sequence in LKP1 (LOV kelch protein 1). LKP1 cDNA encodes a protein of 610 amino acids and with a molecular weight of 65 905 with an LOV domain and kelch repeats. LOV domains are present in a number of sensor proteins involved in the detection of light, oxygen or voltage. The LKP1 LOV is very similar to the LOV domains in NPH1, a plasma membrane‐associated blue light receptor kinase that regulates phototropism (Huala, E., Oeller, P.W., Liscum, E., Han, I‐S., Larsen, E. & Briggs, W.R. (1997) Science, 278, 2120–2123). LKP1 mRNA accumulates in roots, stems, flowers and siliques. It is most abundant in leaves, and least abundant in seeds. Transgenic plants with a β‐glucuronidase (GUS) reporter gene driven by a 1.5 kb LKP1 promoter display strong GUS activity in leaves. Transgenic plants with a 35S::LKP1 cDNA gene overexpress LKP1 mRNA. These plants have elongated hypocotyls and petioles with elongated cells, and exhibit distinct cotyledon movement during the day. Expression of 35S::LKP1 in transgenic Arabidopsis promotes late flowering in plants grown under long‐day, but not under short‐day conditions. Vernalization does not affect the late flowering phenotype of the 35S::LKP1 plants. Transgenic plants possessing the 35S::GFP‐LKP1 construct also have long hypocotyles and petioles, and a late flowering phenotype, suggesting that the GFP‐LKP1 fusion protein is active. The GFP‐associated fluorescence in 35S::GFP‐LKP1 plants is observed in nuclei and cytosol, indicating that LKP1 is a new nucleo‐cytoplasmic factor that influences flowering time in the long day pathway of Arabidopsis.
Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells
Authors: Bouvier Florence , Suire Claude , D'Harlingue Alain , Backhaus Ralph A. , Camara Bilal 
Published: March 2003

SummaryThe nature of isoprenoids synthesized in plants is primarily determined by the specificity of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non‐secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy‐xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non‐green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.
Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato
Authors: Weller James L. , Schreuder Mariëlle E.L. , Smith Harry , Koornneef Maarten , Kendrick Richard E. 
Published: March 2003

SummaryThe role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white‐light‐grown wild‐type or phyA‐deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de‐etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high‐irradiance R, and, surprisingly, that the phyA‐dependent low‐irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far‐red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low‐irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.
Indole‐3‐glycerol phosphate, a branchpoint of indole‐3‐acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana
Authors: Ouyang Jian , Shao Xiang , Li Jiayang 
Published: March 2003

SummaryThe phytohormone indole‐3‐acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp‐independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole‐3‐glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2‐1 and trp3‐1. Antisense transgenic plants display some auxin deficient‐like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild‐type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3‐1 and trp2‐1 plants. These results suggest that indole‐3‐glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp‐independent IAA de novo biosynthetic pathway.
A new hyperrecombinogenic mutant of Nicotiana tabacum
Authors: Gorbunova Vera , Avivi‐Ragolski Naomi , Shalev Gil , Kovalchuk Igor , Abbo Shahal , Hohn Barbara , Levy Avraham A. 
Published: March 2003

SummaryWe have isolated a hyperrecombinogenic Nicotiana tabacum mutant. The mutation, Hyrec, is dominant and segregates in a Mendelian fashion. In the mutant, the level of mitotic recombination between homologous chromosomes is increased by more than three orders of magnitude. Recombination between extrachromosomal substrates is increased six‐ to ninefold, and intrachromosomal recombination is not affected. Hyrec plants were found to perform non‐homologous end joining as efficiently as the wild type, ruling out the possibility that the increase in homologous recombination is due to a defect in end joining. In addition, Hyrec plants show significant resistance to gamma‐irradiation, whereas UV resistance is not different from the wild type. This suggests that homologous recombination can be strongly up‐regulated in plants. Moreover, Hyrec constitutes a novel type of mutation: no similar mutant was reported in plants and hyperrecombinogenic mutants from other organisms usually show sensitivity to DNA damaging agents. We discuss the insight that this mutant provides into understanding the mechanisms of recombination plus the potential application for gene targeting in plants.
Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4‐reductase
Authors: Johnson Eric T. , Ryu Sunhyo , Yi Hankuil , Shin Byongchul , Cheong Hyeonsook , Choi Giltsu 
Published: March 2003

SummaryMany plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4‐reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin‐type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate‐binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.
Maize protein kinase CK2: regulation and functionality of three β regulatory subunits
Authors: Riera Marta , Peracchia Giovanna , De Nadal Eulàlia , Ariño Joaquín , Pagès Montserrat 
Published: March 2003

SummaryBiochemical and crystallographic data suggest that, in contrast with other organisms, the active maize protein kinase CK2 might be composed simply of a catalytic polypeptide (CK2α), thus lacking CK2β regulatory subunits. To investigate the existence and functionality of CK2β regulatory subunits in Zea mays, we have screened a maize cDNA library using different approaches and have isolated three full‐length cDNAs encoding CK2β regulatory subunits (CK2β‐1, CK2β‐2 and CK2β‐3) and a cDNA coding for a novel CK2α catalytic subunit, CK2α‐3. The pattern of expression of all these α/β subunits has been studied in different organs and developmental stages using specific probes for each isoform, and indicates that while CK2α subunits are constitutive, CK2β subunits are expressed differentially during embryo development. The yeast two‐hybrid system and pull‐down assays have been used to study specific interactions between the different subunits. While CK2α subunits are unable to self‐associate, preferential interactions between α/β isoforms and β/β isoforms can be predicted. Furthermore, we show that maize CK2α/β subunits assemble into a structural tetrameric complex which has very similar properties to those described in other organisms, and that expression of maize CK2β subunits in yeast allows the rescue of the phenotypic defects associated to the lack of CK2 function, thus demonstrating the functionality of maize CK2β regulatory subunits.
Hyperosmotic stress rapidly generates lyso‐phosphatidic acid in Chlamydomonas
Authors: Meijer Harold J.G. , Arisz Steven A. , Van Himbergen John A.J. , Musgrave Alan , Munnik Teun 
Published: March 2003

SummaryPlant cells are continuously exposed to environmental stresses such as hyper‐osmolarity, and have to respond in order to survive. When 32P‐labelled Chlamydomonas moewusii cells were challenged with NaCl, the formation of a new radiolabelled phospholipid was stimulated, which was barely detectable before stimulation. The phospholipid was identified as lyso‐phosphatidic acid (LPA), and was the only lyso‐phospholipid to be accumulated. The increase in LPA was dose‐ and time‐dependent. When other osmotically active compounds were used, the formation of LPA was also induced with similar kinetics, although salts were better inducers than non‐salts. At least part of the LPA was generated by phospholipase A2 (PLA2) hydrolysing phosphatidic acid (PA). This claim is based on PA formation preceding LPA production, and PLA2 inhibitors decreasing the accumulation of LPA and promoting the conversion of PA to diacylglycerol pyrophosphate. The latter is another metabolic derivative of PA that is implicated in cell signalling. The involvement of multiple lipid‐signalling pathways in hyperosmotic stress responses is discussed.
A critical role for disproportionating enzyme in starch breakdown is revealed by a knock‐out mutation in Arabidopsis
Authors: Critchley Joanna H. , Zeeman Samuel C. , Takaha Takeshi , Smith Alison M. , Smith Steven M. 
Published: March 2003

SummaryDisproportionating enzyme (d‐enzyme) is a plastidial α‐1,4‐glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T‐DNA insertion. While d‐enzyme activity is eliminated in the homozygous dpe1–1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1–1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1–1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de‐starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto‐oligosaccharides occurs in dpe1–1 but not in wild type. These data show that d‐enzyme is required for malto‐oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1–1 suggests that malto‐oligosaccharides affect an enzyme that attacks the starch granule, or that d‐enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1–1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto‐oligosaccharides generated during the dark period. We conclude that the primary function of d‐enzyme is in starch degradation.