bluuebunny/crossref_metadata_2025 huggingface dataset:
The RCN1‐encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo
Authors: Deruère Jean , Jackson Karin , Garbers Christine , Söll Dieter , DeLong Alison
Published: March 2003

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine‐specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. TheRCN1gene encodes one of three A regulatory subunits inArabidopsis thaliana. A T‐DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have usedin vivoandin vitroassays to gauge the impact of thercn1mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts fromrcn1mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark‐grown, but not light‐grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild‐type seedlings mimics thercn1defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild‐type seedlings,RCN1mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots,RCN1is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light‐grown seedlings. Our results show that the wild‐typeRCN1‐encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.
Subcellular localization of ozone‐induced hydrogen peroxide production in birch (Betula pendula) leaf cells
Authors: Pellinen Riikka , Palva Tapio , Kangasjärvi Jaakko
Published: March 2003

The atmospheric air pollutant ozone (O3) is one of the environmental stresses that induce formation of reactive oxygen species (ROS) in plants. Previously, the toxicity of O3 has been believed to be a result of ROS formation from O3‐degradation. Recently, however, it has been shown that O3 induces active ROS production, which suggests that O3‐responses may be mechanistically similar to pathogen‐induced responses and that O3‐damage could be a result of deleterious firing by the ROS of pathways normally associated with the HR. The subcellular localization of O3‐induced H2O2 production was studied in birch (Betula pendula). O3 induced H2O2 accumulation first on the plasma membrane and cell wall. Experiments with inhibitors of possible sources for H2O2 in the cell wall suggested that both NADPH‐dependent superoxide synthase and the cell wall peroxidases are involved in this H2O2 production. The H2O2 production continued in the cytoplasm, mitochondria and peroxisomes when the O3‐exposure was over, but not in chloroplasts. The timing of mitochondrial H2O2 accumulation coincided with the first symptoms of visible damage and, at the same time, the mitochondria showed disintegration of the matrix. These responses may not be directly connected with defense against oxidative stress, but may rather indicate changes in oxidative balance within the cells that affect mitochondrial metabolism and the homeostasis of the whole cell, possibly leading into induction of programmed cell death.
Cloning and expression of flavone synthase II from Gerbera hybrids
Authors: Martens Stefan , Forkmann Gert
Published: March 2003

In 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

The 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

Mutation 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

Calcium 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

Ara4, 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

Light‐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

Plants 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

The 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

Nod 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

cDNAs 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

To 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

Heat‐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

At 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

The 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.
Differential regulation of the NO3– and NH4+ transporter genes AtNrt2.1 and AtAmt1.1 in Arabidopsis: relation with long‐distance and local controls by N status of the plant
Authors: Gansel Xavier , Muños Stéphane , Tillard Pascal , Gojon Alain
Published: March 2003

Regulation of root N uptake by whole‐plant signalling of N status was investigated at the molecular level in Arabidopsis thaliana plants through expression analysis of AtNrt2.1 and AtAmt1.1. These two genes encode starvation‐induced high‐affinity NO3– and NH4+ transporters, respectively. Split‐root experiments indicate that AtNrt2.1 expression is controlled by shoot‐to‐root signals of N demand. Together with 15NO3– influx, the steady‐state transcript level of this gene is increased in NO3–‐fed roots in response to N deprivation of another portion of the root system. Thus AtNrt2.1 is the first identified molecular target of the long‐distance signalling informing the roots of the whole plant's N status. In contrast, AtAmt1.1 expression is predominantly dependent on the local N status of the roots, as it is mostly stimulated in the portion of the root system directly experiencing N starvation. The same behaviour was found for NH4+ influx, suggesting that the NH4+ uptake system is much less efficient than the NO3– uptake system, to compensate for a spatial restriction of N availability. Other major differences were found between the regulations of AtNrt2.1 and AtAmt1.1 expression. AtNrt2.1 is strongly upregulated by moderate level of N limitation, while AtAmt1.1 transcript level is markedly increased only under severe N deficiency. Unlike AtNrt2.1, AtAmt1.1 expression is not stimulated in a nitrate reductase‐deficient mutant after transfer to NO3– as sole N source, indicating that NO3–per se acts as a signal repressing transcription of AtAmt1.1. These results reveal two fundamentally different types of mechanism involved in the feedback regulation of root N acquisition by the N status of the plant.
The shoot apical meristem restores its symplasmic organization during chilling‐induced release from dormancy
Authors: Rinne Päivi L. H. , Kaikuranta Päivi M. , Van Der Schoot Christiaan
Published: March 2003

The shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing‐tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3‐β‐d‐glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3‐β‐d‐glucanase, which was present in small spherosome‐like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3‐β‐d‐glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell–cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein‐tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation‐competent state, and implicate 1,3‐β‐d‐glucanase action at the plasmodesmata. Based on these findings we propose a model for ‘dormancy cycling’ which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.
Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis
Authors: Matzk Fritz , Meister Armin , Brutovská Renata , Schubert Ingo
Published: March 2003

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.
Characterization of the ethanol‐inducible alc gene‐expression system in Arabidopsis thaliana
Authors: Roslan Hairul A. , Salter Michael G. , Wood Chris D. , White Michael R. H. , Croft Kevan P. , Robson Frances , Coupland George , Doonan John , Laufs Patrick , Tomsett A. Brian , Caddick Mark X.
Published: March 2003

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene‐expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to β‐glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR‐mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose‐dependent, with negligible activity in the absence of the exogenous inducer for soil‐grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil‐grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.
Increasing maize seed methionine by mRNA stability
Authors: Lai Jinsheng , Messing Joachim
Published: March 2003

The amino acid methionine is a common protein building block that is also important in other cellular processes. Plants, unlike animals, synthesize methionine de novo and are thus a dietary source of this nutrient. A new approach for using maize as a source of nutrient methionine is described. Maize seeds, a major component of animal feeds, have variable levels of protein‐bound methionine. This variability is a result of post‐transcriptional regulation of the Dzs10 gene, which encodes a seed‐specific high‐methionine storage protein. Here we eliminate methionine variability by identifying and replacing the cis‐acting site for Dzs10 regulation using transgenic seeds. Interestingly, two different mechanisms affect mRNA accumulation, one dependent on and the other independent of the untranslated regions (UTRs) of Dzs10 RNA. Accumulation of chimeric Dzs10 mRNA was not reduced in hybrid crosses and was uncoupled from genomic imprinting by Dzr1, a regulator of Dzs10. Uniform high levels of Dzs10 protein were maintained over five backcross generations of the transgene. The increased level of methionine in these transgenic seeds allowed the formulation of a useful animal feed ration without the addition of synthetic methionine.
Recognition specificity of self‐incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa
Authors: Kimura Ryo , Sato Keiichi , Fujimoto Ryo , Nishio Takeshi
Published: March 2003

The determinants of recognition specificity of self‐incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S‐locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S‐locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans‐specific evolution of self‐incompatibility genes.
A role for arabinogalactan proteins in gibberellin‐induced α‐amylase production in barley aleurone cells
Authors: Suzuki Yoshihito , Kitagawa Mamiko , Knox J. Paul , Yamaguchi Isomaro
Published: March 2003

Arabinogalactan proteins (AGPs) are plant proteoglycans that have been implicated in plant growth and development. The possible involvement of AGPs in the action of gibberellin (GA), a class of plant hormones, was examined by applying β‐glucosyl Yariv reagent (β‐Glc)3Y, a synthetic phenyl glycoside that interacts selectively with AGPs, to barley aleurone protoplasts. Gibberellin induces transcription and secretion of α‐amylases in the protoplasts. Induction of α‐amylase was clearly inhibited by (β‐Glc)3Y but not by (α‐Gal)3Y, a negative control of the Yariv reagent that does not interact with AGPs. Transfection analysis, using an α‐amylase promoter–GUS fusion gene in the protoplasts, indicated that the transcriptional activation of the α‐amylase promoter was inhibited specifically by (β‐Glc)3Y. These observations are the first indication of an involvement of AGPs in a plant hormone function. The inhibitory effect of (β‐Glc)3Y was not observed when aleurone layers or half‐seed grains were used. This result, together with the fact that protoplasts do not have cell walls, suggests that the AGPs that function in α‐amylase induction reside at the plasma membrane. An aleurone‐specific AGP was detected by reversed‐phase HPLC, and supported the idea that an AGP may play an important role in aleurone‐specific events. The possible mechanism of AGP function in gibberellin‐induced α‐amylase production is discussed.
EL5, a rice N‐acetylchitooligosaccharide elicitor‐responsive RING‐H2 finger protein, is a ubiquitin ligase which functions in vitro in co‐operation with an elicitor‐responsive ubiquitin‐conjugating enzyme, OsUBC5b
Authors: Takai Ryota , Matsuda Noriyuki , Nakano Akihiko , Hasegawa Koji , Akimoto Chiharu , Shibuya Naoto , Minami Eiichi
Published: March 2003

EL5, a rice gene responsive to N‐acetylchitooligosaccharide elicitor, encodes a RING‐H2 finger protein with structural features common to the plant‐specific ATL family. We show that the fusion protein of EL5 with maltose binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin‐activating enzyme (E1), and the Ubc4/5 subfamily of the ubiquitin‐conjugating enzyme (E2). EL5 possesses the activity to catalyse the transfer of ubiquitin to the MBP moiety, and the RING‐H2 finger motif of EL5 is necessary for this activity. Thus, we concluded that EL5 represents a ubiquitin ligase (E3). We also show that two rice E2s (OsUBC5a, OsUBC5b) of the Ubc4/5 subfamily function as E2 which catalyses EL5‐mediated ubiquitination, and OsUBC5b was induced by elicitor, as well as EL5. These results strongly suggest that EL5 and OsUBC5b have roles in plant defense response through the turnover of protein(s) via the ubiquitin/proteasome system.
Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast
Authors: Franklin Scott , Ngo Binh , Efuet Ekem , Mayfield Stephen P.
Published: March 2003

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast‐encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon‐optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5′‐ and 3′‐UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non‐optimized GFP cassette (GFPncb), also under the control of the rbcL 5′‐ and 3′‐UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate ≈80‐fold more GFP than GFPncb‐transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5′‐ and 3′‐UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.
A dominant nuclear mutation in Chlamydomonas identifies a factor controlling chloroplast mRNA stability by acting on the coding region of the atpA transcript
Authors: Drapier Dominique , Girard‐Bascou Jacqueline , Stern David B. , Wollman Francis‐André
Published: March 2003

We have characterized a nuclear mutation, mda1‐ncc1, that affects mRNA stability for the atpA gene cluster in the chloroplast of Chlamydomonas. Unlike all nuclear mutations altering chloroplast gene expression described to date, mda1‐ncc1 is a dominant mutation that still allows accumulation of detectable amounts of atpA mRNAs. At variance with the subset of these mutations that affect mRNA stability through the 5′ UTR of a single chloroplast transcript, the mutated version of MDA1 acts on the coding region of the atpA message. We discuss the action of MDA1 in relation to the unusual pattern of expression of atpA that associates particularly short lived‐transcripts with a very high translational efficiency.
Constitutive gain‐of‐function mutants in a nucleotide binding site–leucine rich repeat protein encoded at the Rx locus of potato
Authors: Bendahmane Abdelhafid , Farnham Garry , Moffett Peter , Baulcombe David C.
Published: March 2003

Rx in potato encodes a protein with a nucleotide binding site (NBS) and leucine‐rich repeats (LRR) that confers resistance against Potato virus X. The NBS and LRR domains in Rx are present in many disease resistance proteins in plants and in regulators of apoptosis in animals. To investigate structure‐function relationships of NBS‐LRR proteins we exploited the potential of Rx to mediate a cell death response. With wild‐type Rx cell death is elicited only in the presence of the viral coat protein. However, following random mutagenesis of Rx, we identified mutants in which cell death is activated in the absence of viral coat protein. Out of 2500 Rx clones tested there were seven constitutive gain‐of‐function mutants carrying eight independent mutations. The mutations encoded changes in the LRR or in conserved RNBS‐D and MHD motifs of the NBS. Based on these findings we propose that there are inhibitory domains in the NBS and LRR. The constitutive gain‐of‐function phenotypes would be due to deletion or modification of these inhibitory domains. However activation of Rx is not simply release of negative regulation by the LRR and adjacent sequence because deleted forms of Rx that lack constitutive gain of function mutations are not active unless the protein is overexpressed.
Micrografting techniques for testing long‐distance signalling in Arabidopsis
Authors: Turnbull Colin G. N. , Booker Jon P. , Leyser H. M. Ottoline
Published: March 2003

Grafting in species other than Arabidopsis has generated persuasive evidence for long‐distance signals involved in many plant processes, including regulation of flowering time and shoot branching. Hitherto, such approaches in Arabidopsis have been hampered by the lack of suitable grafting techniques. Here, a range of micrografting methods for young Arabidopsis seedlings are described. The simplest configuration was a single‐hypocotyl graft, constructed with or without a supporting collar, allowing tests of root–shoot communication. More complex two‐shoot grafts were also constructed, enabling tests of shoot–shoot communication. Integrity of grafts and absence of adventitious roots on scions were assessed using plants constitutively expressing a GUS gene as one graft partner. Using the max1 (more axillary growth) and max3 increased branching mutants, it was shown that a wild‐type (WT) rootstock was able to inhibit rosette branching of mutant shoots. In two‐shoot grafts with max1 and WT shoots on a max1 rootstock, the mutant shoot branched profusely, but the WT one did not. In two‐shoot grafts with max1 and WT shoots on a WT rootstock, neither shoot exhibited increased branching. The results mirror those previously demonstrated in equivalent grafting experiments with the ramosus mutants in pea, and are consistent with the concept that a branching signal is capable of moving from root to shoot, but not from shoot to shoot. These grafting procedures will be valuable for revealing genes associated with many other long‐distance signalling pathways, including flowering, systemic resistance and abiotic stress responses.
The role of double‐strand break‐induced allelic homologous recombination in somatic plant cells
Authors: Gisler Brigitte , Salomon Siegfried , Puchta Holger
Published: March 2003

During meiosis, homologous recombination occurs between allelic sequences. To evaluate the biological significance of such a pathway in somatic cells, we used transgenic tobacco plants with a restriction site for the rare cutting endonuclease I‐SceI within a negative selectable marker gene. These plants were crossed with two tobacco lines containing, in allelic position, either a deletion or an insertion within the marker gene that rendered both marker gene and restriction site inactive. After the double‐strand break induction, we selected for repair events resulting in a loss of marker gene function. This loss was mostly due to deletions. We were also able to detect double strand break‐induced allelic recombination in which the break was repaired by a faithful copying process from the homologue carrying the shortened transgene. The estimated frequency indicates that homologous recombination in somatic cells between allelic sites appears to occur at the same order of magnitude as between ectopic sites, and is thus far too infrequent to act as major repair pathway. As somatic changes can be transferred to the germ line, the prevalence of intrachromatid rearrangements over allelic recombination might be an indirect prerequisite for the enhanced genome plasticity postulated for plants.