bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and expression of flavone synthase II from Gerbera hybrids
Authors: Martens Stefan , Forkmann Gert 
Published: March 2003

SummaryIn Gerbera hybrids, flavone synthesis is controlled by the locus Fns. The responsible enzyme, flavone synthase II, belongs to the NADPH‐dependent cytochrome P450 monooxygenases. From two different chemogenetic defined Gerbera lines with the dominant (fns + ·) or recessive (fns fns) alleles at the locus Fns, a cytochrome P450 fragment (CypDDd7a) was isolated using a differential display technique with upstream primers based on the conserved heme‐binding region of cytochrome P450 proteins. The full‐length cDNA (CYP93B2) which contained the open‐reading frame and part of the CypDDd7a sequence was isolated via 5′‐RACE and end‐to‐end PCR with gene specific primers. Northern blot analysis of total RNA of Gerbera hybrids indicated that the CYP93B2 gene was only transcribed in lines with the dominant allele fns +  and that the transcript levels during flower development are in agreement with the measured enzyme activity of FNS II and flavone accumulation. Microsomes from yeast cells expressing CYP93B2 catalysed the direct formation of [14C]‐flavones from the respective [14C]‐flavanones. Thus, CYP93B2 was shown to encode flavone synthase II. This is the first report of the isolation and expression of a functional FNS II cDNA clone from any species. The comparison of amino acid sequences revealed that CYP93B2 had 54% identity with the sequence of CYP93B1, which has recently been reported as a (2S)‐flavanone 2‐hydroxylase of Glycyrrhiza echinata L.
Distinct features of post‐transcriptional gene silencing by antisense transgenes in single copy and inverted T‐DNA repeat loci
Authors: Stam Maike , De Bruin Rob , Van Blokland Rik , Van Der Hoorn Renier A. L. , Mol Joseph N. M. , Kooter Jan M. 
Published: March 2003

SummaryThe application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as‐RNA) are expected to result in strong silencing but often there is no clear correlation between as‐RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post‐transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady‐state as‐RNA level, transcription level of the as‐transgenes, the repetitiveness and structure of the integrated T‐DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T‐DNA of which the as‐Chs gene is transcribed, although several‐fold lower than the endogenous Chs genes. As there are not sufficient as‐RNAs to degrade every mRNA, we speculate that silencing is induced by double‐stranded RNA. The second class contains two T‐DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as‐Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as‐Chs transgenes were proximal to the centre of the IR. Similar features have been described for co‐suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co‐suppression, either via ectopic DNA pairing or via dsRNA.
The soybean Eu3 gene encodes an Ni‐binding protein necessary for urease activity
Authors: Freyermuth Sharyn K. , Bacanamwo Methode , Polacco Joe C. 
Published: March 2003

SummaryMutation in Eu3 eliminates activity of both soybean ureases, the embryo‐specific (encoded by Eu1) and the tissue‐ubiquitous (encoded by Eu4). eu3‐e1 is a completely recessive null allele. Eu3‐e3 is a semi‐dominant specifying 0.1% wild‐type urease activity in the homozygous state and 5–10% as a heterozygote (
Meyer‐Bothling et al. 1987
). Antibodies to plant UreG, a homologue of the bacterial urease accessory protein, revealed a 32 kDa protein (p32) in embryos of the Eu3/Eu3 precursor genotype. p32 is identical to UreG by the criteria of size, antigenicity, and its ability to bind Ni2+, a trait expected from the deduced histidine‐rich N‐terminus of UreG. UreG was absent in eu3‐e1/eu3‐e1, and lack of UreG co‐segregated with eu3‐e1. Eu3‐e3 specified a UreG transcript which coded valine in place of alanine at residue 142 (A142V) confirming thatEu3 encodes UreG, which is renamed Eu3. Eu3 (A142V) retained Ni‐binding ability. Eu3 is directly involved in urease activation, since anti‐Eu3 (UreG) antibodies inhibited the in vitro activation of urease. Eu1 (embryo urease) and Eu3 accumulated in parallel in the developing embryo. The presence of Eu1 was not necessary for the high embryonic level of Eu3. However, the presence of Eu3 appeared to be important for accumulation of Eu1, perhaps by stabilizing it by Ni insertion. At the level of sensitivity employed Eu3 was detected in crude extracts of embryos but not non‐embryonic tissues which have 1/500th the embryo urease activity. Functional Eu3, however, is necessary for activation of the ubiquitous urease in non‐embryonic tissues.
Hyperpolarisation‐activated calcium currents found only in cells from the elongation zone of Arabidopsis thaliana roots
Authors: Kiegle Ed , Gilliham Matt , Haseloff Jim , Tester Mark 
Published: March 2003

SummaryCalcium currents across the plasma membrane of plant cells allow transduction of environmental signals as well as nutritive calcium uptake. Using transgenic Arabidopsis plants with cell‐specific expression of green fluorescent protein (GFP), we analyzed whole cell calcium currents in epidermal cells of the rapidly growing root apex, mature epidermal cells, cortical and epidermal cells from the elongation zone, and mature pericycle cells. In cells only from the rapidly growing root apex, a hyperpolarisation‐activated calcium current was identified. This current was irreversibly inhibited by 10 μm Al3+, as well as being inhibited by 1 m
m Co2+ and 100 μm verapamil. In no cells could a depolarisation‐activated current be attributed to calcium influx. In the growing root apex, the hyperpolarisation‐activated calcium current may function to allow constitutive uptake of calcium for rapid cell division and elongation.
Modes of interaction between the Arabidopsis Rab protein, Ara4, and its putative regulator molecules revealed by a yeast expression system
Authors: Ueda Takashi , Matsuda Noriyuki , Uchimiya Hirofumi , Nakano Akihiko 
Published: March 2003

SummaryAra4, a member of the Rab/Ypt GTPase family derived from Arabidopsis thaliana, causes severe growth inhibition when expressed in several yeast ypt mutants. Mutational analysis of ARA4 indicated that the Ara4 protein titrates at least three factors in yeast, including the GDP dissociation inhibitor (GDI). The coexpression of AtGDI1 (Arabidopsis GDI) suppressed the growth defect caused by Ara4 in yeast ypt1, suggesting that Ara4 and AtGDI1 interact in yeast to compensate for the titration of yeast GDI. We screened an Arabidopsis cDNA library for other suppressors that may also interact with Ara4 physiologically. A novel suppressor, SAY1, encoded a hydrophilic protein with two putative coiled‐coil regions, which showed partial similarity to the yeast Vps27 protein. To understand the structural requirements of Ara4 for interacting with these molecules, we examined whether AtGDI1 and SAY1 could suppress the growth defect of ypt1 caused by various mutant versions of ARA4. The results indicated that the interaction between Ara4 and AtGDI1 depends on the conserved C‐terminal Cys‐motif and Thr44 in the effector domain of Ara4. In contrast, neither of these motifs is necessary for the interaction between Say1 and Ara4. This approach provides a powerful method to dissect complex interactions between a GTPase and its regulators.
Transactivation properties of parsley proline‐rich bZIP transcription factors
Authors: Sprenger‐Haussels Markus , Weisshaar Bernd 
Published: March 2003

SummaryLight‐responsive chalcone synthase (CHS) gene activation requires LRUCHS, a light regulatory promoter unit including the MYB recognition element MRECHS and the ACGT‐containing element ACECHS. ACECHS is bound by the parsley basic region/leucine zipper (bZIP) factors CPRF1 and 4. Factors containing the bZIP domain exist in animals, plants and yeast, and recognize DNA sequence‐specifically after formation of homo‐ or heterodimers. To determine the potential role of CPRFs in the regulation of CHS promoter activity, we investigated the functions of distinct CPRF domains in a homologous co‐transfection system. The proline‐rich domains of CPRF1 and CPRF4 activate transcription, indicating that CPRF1 and CPRF4 have transactivating properties. Over‐expression of the CPRF1 bZIP domain caused a reduction of LRUCHS‐mediated light inducibility, and point mutations throughout ACECHS affected both responsiveness to UV‐containing white light and transactivation by CPRF1::VP16. The data suggest that a CPRF1‐containing bZIP heterodimer interacts with ACECHSin vivo. We discuss regulatory steps in light‐induced CHS transcription that may be influenced by CPRF1 and/or related bZIP factors.
AtRAD1, a plant homologue of human and yeast nucleotide excision repair endonucleases, is involved in dark repair of UV damages and recombination
Authors: Gallego Francesca , Fleck Oliver , Li Anatoliy , Wyrzykowska Joanna , Tinland Bruno 
Published: March 2003

SummaryPlants are unique in the obligatory nature of their exposure to sunlight and consequently to ultraviolet (UV) irradiation. However, our understanding of plant DNA repair processes lags far behind the current knowledge of repair mechanisms in microbes, yeast and mammals, especially concerning the universally conserved and versatile dark repair pathway called nucleotide excision repair (NER). Here we report the isolation and functional characterization of Arabidopsis thaliana AtRAD1, which encodes the plant homologue of Saccharomyces cerevisiae RAD1, Schizosaccharomyces pombe RAD16 and human XPF, endonucleolytic enzymes involved in DNA repair and recombination processes. Our results indicate that AtRAD1 is involved in the excision of UV‐induced damages, and allow us to assign, for the first time in plants, the dark repair of such DNA lesions to NER. The low efficiency of this repair mechanism, coupled to the fact that AtRAD1 is ubiquitously expressed including tissues that are not accessible to UV light, suggests that plant NER has other roles. Possible ‘UV‐independent’ functions of NER are discussed with respect to features that are particular to plants.
Degradation of Aux/IAA proteins is essential for normal auxin signalling
Authors: Worley Cathy K. , Zenser Nathan , Ramos Jason , Rouse Dean , Leyser Ottoline , Theologis Athanasios , Callis Judy 
Published: March 2003

SummaryThe growth substance auxin mediates many cellular processes, including division, elongation and differentiation. PSIAA6 is a member of the Aux/IAA family of short‐lived putative transcriptional regulators that share four conserved domains and whose mRNAs are rapidly induced in the presence of auxin. Here PSIAA6 was shown to serve as a dominant transferable degradation signal when present as a translational fusion with firefly luciferase (LUC), with an in vivo half‐life of 13.5 min in transgenic Arabidopsis seedlings. In a transient assay system in tobacco protoplasts using steady‐state differences as an indirect measure of protein half‐life, LUC fusions with full‐length PSIAA6 and IAA1, an Aux/IAA protein from Arabidopsis, resulted in protein accumulations that were 3.5 and 1.0%, respectively, of that with LUC alone. An N‐terminal region spanning conserved domain II of PSIAA6 containing amino acids 18–73 was shown to contain the necessary cis‐acting element to confer low protein accumulation onto LUC, while a fusion protein with PSIAA6 amino acids 71–179 had only a slight effect. Single amino acid substitutions of PSIAA6 in conserved domain II, equivalent to those found in two alleles of axr3, a gene that encodes Aux/IAA protein IAA17, resulted in a greater than 50‐fold increase in protein accumulation. Thus, the same mutations resulting in an altered auxin response phenotype increase Aux/IAA protein accumulation, providing a direct link between these two processes. In support of this model, transgenic plants engineered to over‐express IAA17 have an axr3‐like phenotype. Together, these data suggest that rapid degradation of Aux/IAA proteins is necessary for a normal auxin response.
Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single‐electrode voltage‐clamp study
Authors: Kurkdjian Armen , Bouteau François , Pennarun Anne‐Marie , Convert Monique , Cornel Daniel , Rona Jean‐Pierre , Bousquet Ulrike 
Published: March 2003

SummaryNod factor [NodRm‐IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well‐known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single‐electrode voltage‐clamp technique (dSEVC) shows that anion channel, K+ channel and H+‐ATPase pump currents are involved in young growing root hairs. The early Nod‐factor‐induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time‐dependent inward K+ current (IRKC). These two ion currents are then down‐regulated while the H+ pump is stimulated, allowing long‐term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod‐factor‐induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.
Activation of CDK‐activating kinase is dependent on interaction with H‐type cyclins in plants
Authors: Yamaguchi Masatoshi , Fabian Tanja , Sauter Margret , Bhalerao Rishikesh P. , Schrader Jarmo , Sandberg Göran , Umeda Masaaki , Uchimiya Hirofumi 
Published: March 2003

SummarycDNAs encoding cyclin H homologs were isolated from poplar (Populus tremulax tremuloides) and rice (Oryza sativa) plants, and were designated Pt;cycH;1 and Os;cycH;1, respectively. The deduced amino‐acid sequences showed 40–60% similarity to human cyclin H and Schizosaccharomyces pombe Mcs2, with higher similarity in the cyclin box region. While Pt;cycH;1 and Os;cycH;1 were expressed in all tissues examined, the transcripts accumulated abundantly in dividing cells. Expression of Os;cycH;1 was abundant in the S‐phase in partially synchronized suspension cells, and was induced by submergence in internodes of deepwater rice. A yeast two‐hybrid assay demonstrated that both Pt;CycH;1 and Os;CycH;1 were able to interact with rice R2 kinase, which is structurally and functionally similar to cyclin‐dependent kinase (CDK)‐activating kinase (CAK) of vertebrates. Moreover, an in vitro pull‐down assay showed that Os;CycH;1 specifically bound to R2 but not to other rice CDKs. When R2 was expressed in budding yeast CAK mutant, the suppression activity in terms of temperature‐sensitivity was enhanced by co‐expression with Os;cycH;1. Furthermore, in vitro kinase assay indicated that the kinase activities of R2 on CDKs and the carboxy‐terminal domain (CTD) of the largest subunit of RNA polymerase II were markedly elevated by binding to Os;CycH;1. Our results suggest that cyclin H is a regulatory subunit of CAK, which positively controls CDK‐ and CTD‐kinase activities in plant cells.
An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function
Authors: Koprek Thomas , McElroy David , Louwerse Jeanine , Williams‐Carrier Rosalind , Lemaux Peggy G. 
Published: March 2003

SummaryTo devise a method for function‐based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted‐repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds‐interrupted uidA reporter gene, were used to demonstrate high‐frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPase‐expressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and first intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds‐Ubi‐bar. In F1 plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F2 progeny derived from individual selfed F1 plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F3 plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F3 plants we observed two mutant phenotypes in which the transposed Ds elements co‐segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes.
Transient expression and heat‐stress‐inducedco‐aggregation of endogenous and heterologous small heat‐stress proteins in tobacco protoplasts
Authors: Kirschner Marc , Winkelhaus Sybille , Thierfelder Jörg M. , Nover Lutz 
Published: March 2003

SummaryHeat‐stress granules (HSG) are highly ordered, cytoplasmic chaperone complexes found in all heat‐stressed plant cells. We have developed an experimental system involving expression of cytosolic class I and class II small heat‐stress proteins (Hsps) of pea, Arabidopsis and tomato in tobacco protoplasts to study the structural prerequisites for the assembly of HSG or HSG‐like complexes. Class I and class II small Hsps formed class‐specific dodecamers of 210–280 kDa, which, upon heat stress, were incorporated into HSG complexes. Interestingly, class II dodecamers alone could form HSG‐like complexes (auto‐aggregation), whereas class I dodecamers could do so only in the presence of class II proteins (recruitment). By analysing C‐terminal deletion forms of Hsp17 class II, we obtained evidence that the intact C‐terminus is critical for the oligomerization state, for the heat‐stress‐induced auto‐aggregation and for recruitment of class I proteins. The class‐specific formation of dimers as a prerequisite for oligomerization was analysed by the yeast two‐hybrid system. In the presence of the endogenous (tobacco) set of heat‐stress‐induced proteins, all heterologous class I and class II proteins were incorporated into HSG complexes, whose ultrastructure was different from that of complexes formed by class I and class II proteins alone. Although other, more distantly related, members of the Hsp20 family, i.e. the plastidic pea Hsp21, the Drosophila Hsp23 and the mouse Hsp25, were well expressed in tobacco protoplasts and formed homo‐oligomers of 200–700 kDa, none of them could be recruited to HSG complexes.
Peroxisomal alanine : glyoxylate aminotransferase (AGT1) is a photorespiratory enzyme with multiple substrates in Arabidopsis thaliana
Authors: Liepman Aaron H. , Olsen Laura J. 
Published: March 2003

SummaryAt least two glyoxylate aminotransferases are hypothesized to participate in the steps of photorespiration located in peroxisomes. Until recently, however, genes encoding these enzymes had not been identified. We describe the isolation and characterization of an alanine : glyoxylate aminotransferase (AGT1, formerly AGT) cDNA from Arabidopsis thaliana. Southern blot analysis confirmed that Arabidopsis AGT1 is encoded by a single gene. Homologs of this class IV aminotransferase are also known in other plants, animals, and methylotrophic bacteria, suggesting an ancient evolutionary origin of this enzyme. AGT1 transcripts were present in all tissues of Arabidopsis, but were most abundant in green, leafy tissues. Purified, recombinant Arabidopsis AGT1 expressed in Escherichia coli catalyzed three transamination reactions using the following amino donor : acceptor combinations: alanine : glyoxylate, serine : glyoxylate, and serine : pyruvate. AGT1 had the highest specific activity with the serine : glyoxylate transamination, and apparent Km measurements indicate that this is the preferred in vivo reaction. In vitro import experiments and subcellular fractionations localized AGT1 to peroxisomes. Sequence analysis of the photorespiratory sat mutants revealed a single nucleotide substitution in the AGT1 gene from these plants. This transition mutation is predicted to result in a proline‐to‐leucine substitution at residue 251 of AGT1. When this mutation was engineered into the recombinant AGT1 protein, enzymatic activity using all three donor : acceptor pairs was abolished. We conclude that Arabidopsis AGT1 is a peroxisomal photorespiratory enzyme that catalyzes transamination reactions with multiple substrates.
Extensive developmental and metabolic alterations in cybrids Nicotiana tabacum (+ Hyoscyamus niger) are caused by complex nucleo‐cytoplasmic incompatibility
Authors: Zubko Mikhajlo K. , Zubko Elena I. , Ruban Alexander V. , Adler Klaus , Mock Hans‐Peter , Misera Simon , Gleba Yuri YU. , Grimm Bernhard 
Published: March 2003

SummaryThe genetic basis of multiple phenotypic alterations was studied in cell‐engineered cybrids Nicotiana tabacum (+ Hyoscyamus niger) combining the nuclear genome of N. tabacum, plastome of H. niger and recombinant mitochondria. The plants possess a complex, maternally inheritable syndrome of nucleo‐cytoplasmic incompatibility, severely affecting growth, metabolism and development. In vivo, the syndrome was manifested as: late germination of seeds; dramatic decrease of chlorophyll and carotenoids in cotyledons and leaves; altered morphology of cotyledons, leaves and flowers; and dwarfism. The leaf phenotype depended on light intensity. In ‘green flowers’ (an extreme phenotype), homeotic function B was downregulated. In vitro, the incompatibility syndrome was restricted to the pigment deficiency of cotyledons. Electron microscopy revealed perturbations in the differentiation of chloroplasts and palisade parenchyma cells in bleached leaves. The pigment deficiency accompanied by retarded growth is discussed as a result of plastome–genome incompatibility, whereas other features are likely to be due to nucleo‐mitochondrial incompatibilities.