bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for β‐amylase and on the accumulation of anthocyanin that are inducible by sugars
Authors: Mita Satoru , Murano Nobuhito , Akaike Mikiko , Nakamura Kenzo
Published: March 2003

We identified a mutant of Arabidopsis thaliana ecotype Col‐0 in which significantly reduced levels of expression of the gene for β‐amylase (ATβ‐Amy) were detected in leaves in response to high concentrations of sucrose, glucose or fructose. Genetic studies, including a cross with transgenic plants that harbored the ATβ‐Amy:GUS transgene with the promoter of ATβ‐Amy, indicated that this phenotype was caused by a recessive mutation, Iba1, that affected expression of ATβ‐Amy in trans. We also found a reduced level of sugar‐induced expression of ATβ‐Amy in the Landsberg erecta (Ler) ecotype compared with other ecotypes. This phenotype seemed to be due to a recessive trait, provisionally designated Iba2, that was linked to neither erecta nor Iba1. The Iba2 mutation also affected expression of ATβ‐Amy:GUS transgene. Accumulation of starch and sugars after treatment of leaves with sucrose was not affected in the Iba1 mutant and Ler plants. However, both Iba1 mutant and Ler plants accumulated low levels of anthocyanin in response to sucrose, results that suggested the existence of some genetic linkage between regulation of the expression of ATβ‐Amy and regulation of the accumulation of anthocyanin. Although the Iba1 and Iba2 mutations did not affect sugar‐inducible gene expression in general, the expression of sugar‐regulated genes other than the gene for β‐amylase was differentially affected in the Iba1 mutant and Ler plants. These results suggest that the sugar‐regulated expression of many genes in plants might be mediated by multiple signal‐transduction pathways.
Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana
Authors: Silverstone Aron L. , Chang Chien‐wei , Krol Elzbieta , Sun Tai‐ping
Published: March 2003

The GA1 gene of Arabidopsis thaliana encodes ent‐kaurene synthase A (KSA), which catalyzes the first committed step in the biosynthetic pathway of the plant hormone gibberellin (GA). Its location in the GA biosynthetic pathway has led to speculation that KSA regulation is one of the controlling steps. However, because KSA activity is so low that it is only measurable in Arabidopsis siliques, GA1 promoter‐GUS reporter gene fusions and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) were used to examine the expression pattern of GA1. The results from this study indicate that GA1 gene expression is highly regulated during growth and development, and it is restricted to specific cell types at the sites of expression. GA1 promoter activity is highest in rapidly growing tissues, e.g. shoot apices, root tips, developing flowers and seeds. It is also active in the vascular tissue of some non‐growing organs, such as expanded leaves, suggesting that these leaves may be a site of GA synthesis for transport to other organs. It was also found that the first one or two introns in the GA1 gene are required for proper expression. Because of the high degree of regulation, GA1 may act as a gatekeeper, controlling the flow of metabolites into the GA biosynthetic pathway, while the levels of specific bioactive GAs are controlled by other downstream steps.
Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi‐color fluorescence in situ hybridization with cosmid clones
Authors: Murata Minoru , Heslop‐Harrison J.S. , Motoyoshi Fusao
Published: March 2003

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In the ecotype Landsberg erecta, hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on one pair of metacentric chromosomes were much stronger than those on the acrocentric and the other pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija, had similar chromosomal distribution of the genes, but the hybridization signals on one pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi‐color FISH with cosmid clones and a centromeric 180 bp repeat as co‐probes. The metacentric chromosome 5 and its L arm carries the largest cluster of the genes, and the short arm of acrocentric chromosome 4 carries a small cluster in all three ecotypes. Chromosome 3 had another small cluster of 5S rRNA genes on its L arm. Chromosomes 1 and 2 had no 5S rDNA cluster, but they are morphologically distinguishable; chromosome 1 is metacentric and 2 acrocentric. Using the 5S rDNA as a probe, therefore, all chromosomes of A. thaliana could be identified by FISH. Chromosome 1 is large and metacentric; chromosome 2 is acrocentric carrying 18S‐5.8S‐25S rDNA clusters on its short arm; chromosome 3 is metacentric carrying a small cluster of 5S rDNA genes on its L arm; chromosome 4 is acrocentric carrying both 18S‐5.8S‐25S and 5S rDNAs on its short (L) arm; and chromosome 5 is metacentric carrying a large cluster of 5S rDNA on its L arm.
The plant 2‐Cys peroxiredoxin BAS1 is a nuclear‐encoded chloroplast protein: its expressional regulation, phylogenetic origin, and implications for its specific physiological function in plants
Authors: Baier Margarete , Dietz Karl‐Josef
Published: March 2003

2‐Cys peroxiredoxins constitute a family of enzymes which catalyze the transfer of electrons from sulfhydryl residues to peroxides and are ubiquitously distributed among all organisms. This paper characterizes the higher plant 2‐Cys‐peroxiredoxin BAS1. (i) Escherichia coli over‐expressing BAS1 exhibit increased tolerance for alkyl hydroperoxides in vivo. This result substantiates the peroxiredoxin function of BAS1. (ii) BAS1 protein is associated with the soluble chloroplast fraction of mesophyll protoplasts. Import and processing of in vitro‐transcribed and cell‐free translated BAS1 protein into isolated chloroplasts provides conclusive evidence that the plant‐specific N‐terminal extension of bas1 encodes the chloroplast import signal which targets the pre‐form of BAS1 to the chloroplast stroma where it is cleaved to its mature size. (iii) Genomic analysis reveals that the targeting signal is encoded by a separate exon in Arabidopsis thaliana. (iv) The amino acid sequence of the BAS1 core protein of higher plants has a higher degree of similarity to open reading frames in the genome of the bluegreen algae Synechochystis PCC sp. 6803 and in the plastome of the red algae Porphyra purpurea than to any other nuclear‐encoded 2‐Cys peroxiredoxin. Therefore, it is tempting to speculate that the chloroplast import signal was added to an ancestor gene of endosymbiotic origin in the course of plant evolution. (v) The bas1 gene expression is regulated under the control of the cellular redox state which is in accordance with the anti‐oxidant function of the enzyme. While oxidative stressors increased expression only slightly, antioxidants such as reduced thiols strongly suppressed the transcript level. The implications of these findings are discussed with respect to the possible physiological functions of BAS1.
Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O‐xylosyltransferase from Phaseolus vulgaris
Authors: Martin Ruth C. , Mok Machteld C. , Mok David W.S.
Published: March 2003

Zeatin is rapidly metabolized to O‐xylosylzeatin in Phaseolus vulgaris seeds. The zeatin O‐xylosyltransferase mediating this conversion, a 50 kDa protein, occurs mainly in the endosperm, both in the cytoplasm and the nuclei. A monoclonal antibody specific to the enzyme was used to isolate cDNAs from an expression library derived from P. vulgaris seeds. Two highly homologous, full‐length cDNAs were isolated. The ORFs encode proteins of 69 and 67 kDa, respectively, with 90% homology at the amino acid level. cDNA‐encoded protein obtained from in vitro transcription/translation was processed to protein of 50 kDa by bean endosperm extract. Transgenic tobacco plants harboring the larger ORF under the control of the CaMV35S promoter were more sensitive to the auxin NAA than control plants. The symptoms included leaf chlorosis, restriction of root elongation, and eventual cessation of growth. The antigenic preprotein was processed, and labeled zeatin was converted to O‐xylosylzeatin in transgenic plants grown on NAA‐containing medium. Analyses of independently transformed families indicated that the presence of the transgene coincided with the increased auxin sensitivity and protein processing correlated with the manifestation of auxin‐induced damage. These results suggest that posttranslational processing regulates enzyme activity, and offer the possibility that cytokinin‐auxin balance may be affected by stimulation of cytokinin metabolic enzyme activity by auxin.
Topology and target interaction of the fusicoccin‐binding 14‐3‐3 homologs of Commelina communis
Authors: Oecking Claudia , Piotrowski Markus , Hagemeier Jens , Hagemann Klaus
Published: March 2003

Upon binding to a high‐affinity plasma membrane (PM) protein (a member of the 14‐3‐3 family of regulatory proteins), the fungal phytotoxin fusicoccin (FC) activates the H+‐ ATPase by hindering the inhibitory interaction of the enzyme’s C‐terminus with its catalytic site. Protease protection experiments carried out with sealed PM vesicles of different orientation proved that the FC‐binding site faces the cytoplasmic surface of the membrane. The in vivo induced activation of the H+‐ATPase by FC was retained during solubilization of PM proteins. Two‐dimensional gel systems combining a native separation of membrane protein complexes with a denaturing dimension as well as high‐performance anion‐exchange chromatography proved the existence of a labile ATPase:14‐3‐3 complex in plasma membranes. Stabilization of this complex could be achieved by FC treatment in vivo or in vitro. Mild proteolytic removal of the C‐terminal auto‐inhibitory domain of the H+ATPase liberated apparent hydrophobic 14‐3‐3 isoforms from the membrane in soluble form. During size exclusion chromatography of the proteolytically released proteins, co‐elution of 14‐3‐3 dimers, protein‐bound FC and the C‐terminus of the H+ATPase was observed. Moreover, the data suggest that 14‐3‐3 dimers themselves are not able to bind FC. Based on these results, it is proposed that the ‘FC receptor’ is represented by a labile complex between a 14‐3‐3 dimer and the H+‐ATPase whose formation is part of a mechanism regulating ATPase‐activity under physiological conditions. In our working model, binding of FC stabilizes this labile complex, thus leading to a strong and persistent activation of the H+‐ATPase in vivo. The possibility that the C‐terminus of the enzyme represents the binding domain for 14‐3‐3 homologs is discussed.
Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV)
Authors: Oparka K.J. , Prior D.A.M. , Cruz S. Santa , Padgett H.S , Beachy R.N.
Published: March 2003

Plasmodesmatal gating in epidermal cells of Nicotianatabacum was examined in expanding infection sites of tobacco mosaic virus (TMV) expressing a fusion between the viral movement protein and the green fluorescent protein (MP‐GFP). The infection sites were circular in profile and within 3 days post‐inoculation had developed a brightly fluorescent leading edge, giving them a characteristic ‘halo’ shape. Co‐localization of MP‐GFP with callose demonstrated that nearly all epidermal cell plasmodesmata were targeted with MP‐GFP. The fusion protein was located in the centre of the plasmodesmal pore, between paired callose platelets. Increase in plasmodesmatal size exclusion limit, as determined by the passage of microinjected 10 kDa Texas Red dextran, was restricted predominantly to cells within the fluorescent halo, and was virtually absent from cells in the centre of the expanding infection site. The plasmodesmata of these cells, however, remained fluorescently labelled with MP‐GFP. Injections outside the fluorescent infection site failed to show movement of dextran, while dextran injected into cells at the leading edge moved inwards towards the centre of the lesion but not outwards into cells lacking GFP. Leaf incisions through cells ahead of the infection front halted the advance of the virus, indicating that virus replication was absent in non‐fluorescent cells outside the infection site. The data provide the first demonstration that within an expanding infection site plasmodesmatal gating is under temporal control.
Genotype and environment interact to control dormancy and differential expression of the VIVIPAROUS 1 homologue in embryos of Avena fatua
Authors: Jones H.D. , Peters N.C.B. , Holdsworth M.J.
Published: March 2003

Embryo dormancy is a reversible developmental state during which germination is repressed. In this study, inbred lines of Avena fatua were used to analyse the influence of genotype and environment on the dormant phenotype, and on expression of the homologue of the maize transcription factor VIVIPAROUS 1 (afVP 1). The cDNA for afVP 1 was cloned from mature embryos. Analysis of the predicted protein sequence revealed a high degree of similarity to other VP 1/ABI 3‐related transcription factors, in particular in four regions previously shown to be highly conserved, including the BR2 region that has been shown to interact with several classes of sequence‐specific DNA binding proteins. The potential of imbibed mature embryos for dormancy was analysed and shown to be determined primarily by genotype and secondarily by previous environmental experience of the mature seed acting on embryo genotype. Under all conditions studied, expression of afVP 1 and the A. fatua homologue of Em (shown in maize to be regulated by VP 1 during embryo maturation) were positively correlated with the dormant phenotype, whereas expression of A. fatua AMY‐related RNAs was negatively correlated with dormancy (in barley AMY 6–4 has been shown to be repressed by VP 1). Expression of afVP 1 RNA was also shown in the dry seed to be positively correlated with the length of time required for seeds of the inbred lines to after‐ripen. These results suggest new functions for the VP 1 transcription factor family in the control of dormancy‐related processes in embryo cells of mature seeds, and the up‐regulation of afVP 1 and afEm RNAs in the dormant state suggests that they are regulated by a switching mechanism in the mature seed that shows some aspects of reversibility.
The major elderberry (Sambucus nigra) fruit protein is a lectin derived from a truncated type 2 ribosome‐inactivating protein
Authors: Van Damme Els J.M. , Roy Soma , Barre Annick , Rougé Pierre , Van Leuven Fred , Peumans Willy J.
Published: March 2003

The major protein of elderberry (Sambucus nigra L.) fruits is a lectin, called Sambucus nigra agglutinin IVf or SNAIVf. This lectin is composed of subunits that strongly resemble the B chain of the type 2 ribosome‐inactivating protein (RIP), called SNAVf, present in the same tissue. To corroborate the possible relationship between both proteins their corresponding cDNAs were cloned and compared. Alignment of the deduced amino acid sequences revealed that the cDNA encoding SNAIVf is almost identical to that of SNAVf except that its A chain is truncated. Northern blot analysis confirmed that the mRNA encoding SNAIVf is about 500 nucleotides shorter than the SNAVf mRNA. In addition, the occurrence of a truncated type 2 RIP gene was unambiguously demonstrated by the analysis of PCR amplified genomic sequences. These results not only demonstrate for the first time that a plant lectin is encoded by a truncated type 2 RIP gene but also address important questions with respect to the molecular evolution of RIP and lectins.
Localization of 14‐3‐3 proteins in the nuclei of arabidopsis and maize
Authors: Bihn Elizabeth A. , Paul Anna‐Lisa , Wang Steven W. , Erdos Gregory W. , Ferl Robert J.
Published: March 2003

It has been demonstrated that 14‐3‐3 proteins are present in the nuclei of Arabidopsis thaliana and Zea mays cells using laser scanning confocal microscopy and immunocytochemistry with monoclonal antibodies against plant 14‐3‐3 proteins. Confirmation of nuclear localization provides insight into the range of functions normally attributed to 14‐3‐3 proteins, especially since the association of 14‐3‐3s with transcription factors is (thus far) a phenomenon unique to plants, and since 14‐3‐3 proteins do not possess a recognizable nuclear targeting sequence.
A role for the vacuole in auxin‐mediated control of cytosolic pH by Vicia mesophyll and guard cells
Authors: Frohnmeyer Hanns , Grabov Alexander , Blatt Michael R.
Published: March 2003

A role for cytosolic pH (pHi) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pHi are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pHi was measured in the presence of auxins and during cytosolic H+ loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pHi of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH‐sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H+ secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H+ secretion in the presence of auxin. BCECF fluorescence recording gave resting pHi values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pHi were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1‐naphthalene acetic acid and 3‐indoyl‐acetic acid, and not by the inactive (anti‐auxin) analogue 2‐naphthalene‐acetic acid. However, auxin treatments failed to evoke a change in pHi in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H+ buffering capacity of the protoplasts in the face of acid pHi loads imposed by adding Na+‐butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short‐term pHi homeostasis and generation of hormonally evoked H+ signalling in plant cells; they also conform with the view that the decrease in pHiper se is not a primary determinant in the stimulation of H+ secretion by auxin.
A bifunctional oleate 12‐hydroxylase: desaturase from Lesquerella fendleri
Authors: Broun Pierre , Boddupalli Sekhar , Somerville Chris
Published: March 2003

LFAH12, an oleate 12‐hydroxylase gene from Lesquerella fendleri (L.) was isolated on the basis of nucleotide sequence similarity to an oleate hydroxylase gene from Ricinus communis (L.). Transgenic Arabidopsis plants containing the Lesquerella gene under transcriptional control of the cauliflower mosaic virus 35S promoter accumulated ricinoleic, lesquerolic and densipolic acids in seeds, but not in leaves or roots. However, hydroxylase activity was detectable in crude extracts of vegetative tissues. The discrepancy between the presence of activity and the lack of hydroxy fatty acids suggests selective removal and breakdown of hydroxy fatty acids in vegetative organs. High levels of LFAH12 mRNA accumulation did not lead to correspondingly high levels of protein accumulation, suggesting that accumulation of the hydroxylase may be controlled post‐transcriptionally. Expression of the L. fendleri gene in transgenic plants of a fad2 mutant of Arabidopsis, which is deficient in cytoplasmic oleate Δ12 desaturase activity, resulted in partial suppression of the mutant phenotype in roots. Thus, unlike the hydroxylase from R. communis, the L. fendleri enzyme has both hydroxylase and desaturase activities. Fusion of the 5′ flanking region of the LFAH12 gene to the β‐glucuronidase coding sequence resulted in a high level of early seed‐specific expression of β‐glucuronidase activity in transgenic Arabidopsis plants.
DET1 represses a chloroplast blue light‐responsive promoter in a developmental and tissue‐specific manner in Arabidopsis thaliana
Authors: Christopher David A. , Hoffer Paul H.
Published: March 2003

The chloroplast psbD‐psbC loci, which encode the D2 and CP43 subunits of the photosystem II reaction center, respectively, are regulated by a blue light‐responsive promoter (BLRP). It has recently been shown in barley seedlings that activation of psbD‐psbC transcription by blue light involves inhibition of a protein kinase that represses the BLRP in the dark. To elucidate further the photosensory pathways regulating the psbD BLRP, the effects of three nuclear mutations on the expression of the BLRP in chloroplasts of Arabidopsis thaliana were examined. The mutants used included the det1–1 and det1–6 alleles for the nuclear protein DET1, involved in repressing photomorphogenesis, and the cry1 gene for the blue light photoreceptor, cryptochrome (CRY1), involved in hypocotyl elongation. The BLRP was not significantly expressed in cotyledons of light‐grown wild‐type seedlings, unlike the light‐responsive expression of the chloroplast, psbA and rbcL, and nuclear, Lhcb and Chs, genes. Analysis of the mutants revealed that DET1 represses transcription from the BLRP in a developmental and tissue‐specific manner, which is unique from the effects that DET1 has on other light‐regulated promoters. In addition, the cry1 mutation did not reduce the expression of the BLRP in response to blue light. This suggests that the BLRP is regulated by a different photosensory system relative to CRY1. A model is proposed involving blue light, DET1 and phytochrome in regulating transcription from the psbD BLRP.
Developmental expression and biochemical analysis of the Arabidopsis atao1 gene encoding an H2O2‐generating diamine oxidase
Authors: Møller Simon G. , McPherson Michael J.
Published: March 2003

A copper amine oxidase encoding gene, atao1, has been isolated and characterized from Arabidopsis thaliana. Sequence analysis reveals that atao1 encodes a 668 amino acid polypeptide (ATAO1) with 48% identity to copper amine oxidases from pea and lentil. The promoter region of atao1 was transcriptionally fused with the reporter genes encoding β‐glucuronidase and modified green fluorescent protein. Analysis of transgenic Arabidopsis together with in situ hybridization of wild‐type plants reveals temporally and spatially discrete patterns of gene expression in lateral root cap cells, vascular tissue of roots, developing leaves, the hypocotyl, and in the style/stigmatal tissue. Enzyme activity assays show that ATAO1 preferentially oxidizes the aliphatic diamine putrescine with production of the corresponding aldehyde, ammonia and hydrogen peroxide, a recognized plant signal molecule and substrate for peroxidases. Histochemical analysis reveals that atao1 expression in developing tracheary elements precedes and overlaps with lignification and therefore is a good marker for vascular development. In both vascular tissue and the root cap, atao1 expression occurs in cells destined to undergo programmed cell death.
Summary
Authors: Baunsgaard Lone , Fuglsang Anja T. , Jahn Thomas , Korthout Henrie A. A. J. , De Boer Albertus H. , Palmgren Michael G.
Published: March 2003

The plasma membrane H+‐ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C‐terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H+‐ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H+‐ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C‐terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14–3‐3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14–3‐3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14–3‐3 protein binding motif in Raf‐1. The results demonstrate that the 14–3‐3 protein is an activator molecule of the H+‐ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H+‐ATPase.
A general method for gene isolation in tagging approaches: amplification of insertion mutagenised sites (AIMS)
Authors: Frey Monika , Stettner Cornelia , Gierl Alfons
Published: March 2003

A polymerase chain reaction (PCR) based procedure for the isolation of genes in transposon or T‐DNA tagging approaches has been developed. The method can be generally applied and allows the rapid isolation of putative gene sequences even in the presence of high numbers of insertion sequences. The technique has been used successfully for the isolation of the maize Bx1 gene tagged by a Mutator element.
Phage display selection can differentiate insecticidal activity of soybean cystatins
Authors: Koiwa Hisashi , Shade Richard E. , Zhu‐Salzman Keyan , Subramanian Lalitha , Murdock Larry L. , Nielsen S. Suzanne , Bressan Ray A. , Hasegawa Paul M.
Published: March 2003

Plant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain‐binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage‐displayed soyacystatins resulted in a 200–1000‐fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.
A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5′ splice site of the first intron
Authors: Isshiki Masayuki , Morino Kazuko , Nakajima Midori , Okagaki Ron J. , Wessler Susan R. , Izawa Takeshi , Shimamoto Ko
Published: March 2003

In cultivated rice two wild‐type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule‐bound starch synthase. The activity of Wxa is 10‐fold higher than that of Wxb at the level of both protein and mRNA.Wxb has a +1G to T mutation at the 5′ splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5′ splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild‐type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5′ splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.
FLD interacts with genes that affect different developmental phase transitions to regulate Arabidopsis shoot development
Authors: Chou Ming‐Lun , Yang Chang‐Hsien
Published: March 2003

A new fld mutant allele, fld‐2, which significantly delayed flowering, was isolated and characterized in Arabidopsis thaliana. Even under long‐day conditions after more than 100 days in the greenhouse, the majority of fld‐2 mutant plants had not bolted. In addition, mutant inflorescences produced more than 10 co‐florescences that were subtended by a high number of rosette‐like leaves before giving rise to flowers. The late‐flowering phenotype of the fld‐2 mutation could be partially overcome by both vernalization and GA treatment but it was not influenced by 5‐azaC treatment. Phenotypic analyses of double mutants indicated that fld‐2 is epistatic to early flowering mutants elf1, elf2 and elf3. In addition, fld‐2 could enhance vegetative characteristics in embryonic flower 1 (emf1) mutants by causing many small sessile leaves in fld‐2 emf1 double mutants. The relief of the terminal flower 1 (tfl1) mutant phenotype in fld‐2 tfl1 double mutants, and the enhancement of leafy (lfy) and apetala1 (ap1) mutant phenotypes in fld‐2 lfy and fld‐2 ap1 double mutants, suggest that FLD is also likely to be involved in the floral transition. Our results strongly suggest that the FLD gene plays a key role in regulating the reproductive competence of the shoot and results in different developmental phase transitions in Arabidopsis.
Cloning of the fructan biosynthesis pathway of Jerusalem artichoke
Authors: Van Der Meer Ingrid M. , Koops Andries J. , Hakkert Johanna C. , Van Tunen Arjen J.
Published: March 2003

To study the regulation of fructan synthesis in plants, we isolated two full‐size cDNA clones encoding the two enzymes responsible for fructan biosynthesis in Jerusalem artichoke (

Helianthus tuberosus

): 1‐sucrose:sucrose fructosyl transferase (1‐SST) and 1‐fructan:fructan fructosyl transferase (1‐FFT). Both enzymes have recently been purified to homogeneity from Jerusalem artichoke tubers (Koops and Jonker (1994)

J. Exp. Bot.

45, 1623–1631; Koops and Jonker (1996)

Plant Physiol.

110, 1167–1175) and their amino acid sequences have been partially determined. Using RT–PCR and primers based on these sequences, specific fragments of the genes were amplified from tubers of Jerusalem artichoke. These fragments were used as probes to isolate the cDNAs encoding 1‐SST and 1‐FFT from a tuber‐specific λZAP library. The deduced amino acid sequences of both cDNAs perfectly matched the sequences of the corresponding purified proteins. At the amino acid level, the cDNA sequences showed 61% homology to each other and 59% homology to tomato vacuolar invertase. Based on characteristics of the deduced amino acid sequence, the first 150 bp of both genes encode a putative vacuolar targeting signal. Southern blot hybridization revealed that both 1‐SST and 1‐FFT are likely to be encoded by single‐copy genes. Expression studies based on RNA blot analysis showed organ‐specific and developmental expression of both genes in growing tubers. Lower expression was detected in flowers and in stem. In other organs, including leaf, roots and dormant tubers, no expression could be detected. In tubers, the spatial and developmental expression correlates with the accumulation of fructans. Using the

1‐sst

and

1‐fft

cDNAs, chimeric genes were constructed driven by the CaMV 35S promoter. Analysis of transgenic petunia plants carrying these constructs showed that both cDNAs encode functional fructosyltransferase enzymes. Plants transformed with the 35S‐

1‐sst

construct accumulated the oligofructans 1‐kestose (GF2), 1,1‐nystose (GF3) and 1,1,1‐fructosylnystose (GF4). Plants transformed with the 35S‐

1‐fft

construct did not accumulate fructans, probably because of the absence of suitable substrates for 1‐FFT, i.e. fructans with a degree of polymerization ≥ 3 (GF2, GF3, etc.). Nevertheless, protein extracts from these transgenic plants were able to convert GF3, when added as a substrate, into fructans with a higher degree of polymerization. Progeny of crosses between a 35S‐

1‐sst

‐containing plant and a 35S‐

1‐fft‐

containing plant, showed accumulation of high‐molecular‐weight fructans in old, senescent leaves. Based on the comparison of the predicted amino acid sequences of

1‐sst

and

1‐fft

with those of other plant fructosyl transferase genes, we postulate that both plant fructan genes have evolved from plant invertase genes.
Multiple photoreceptors mediate the light‐induced reduction of GUS‐COP1 from Arabidopsis hypocotyl nuclei
Authors: Osterlund Mark T. , Deng Xing‐Wang
Published: March 2003

The subcellular localization of COP1, a key photomorphogenic repressor, is regulated by light in Arabidopsis seedlings. Photoreceptor loss‐of‐function mutants and dominant gain‐of‐function overexpression transgenes were both used to analyze the influences of the three photoreceptors, phyA, phyB, and CRY1, on the light‐regulated subcellular localization of COP1. Through a semiquantitative analysis of the nuclear abundance of GUS‐COP1 in the various genetic backgrounds, the specific roles of the individual photoreceptors have been established. The data suggest that multiple photoreceptors influence the light‐regulated subcellular localization of COP1 in white light. Under specific wavelengths of light, phyA, phyB, and CRY1 each play critical roles in mediating far‐red, red, and blue light signals, respectively. Our data also support an interdependency between CRY1 and the phytochromes in mediating the light‐regulated subcellular localization of COP1 and thus seedling development.
Quantitative analysis of the local rates of growth of dicot leaves at a high temporal and spatial resolution, using image sequence analysis
Authors: Schmundt Dominik , Stitt Mark , Jähne Bernd , Schurr Ulrich
Published: March 2003

A new technique is presented for quantitative mapping of dicot leaf growth at high spatial and temporal resolution, at a speed making online‐mapping feasible. Time lapse video sequences of growing leaves are captured by a personal computer (PC) with a frame‐grabber board and a standard CCD camera, and evaluated using algorithms that have been recently developed to analyse motion in dynamic image sequences. Growth can be detected at under 1% per hour, with a time resolution of minutes and a spatial resolution of a few millimeters. The new technique has been verified by comparing it with classical approaches to map integrated growth. Diurnal courses of leaf growth of

Ricinus communis

and tobacco are presented to demonstrate the localised character of growth in leaves. Expansion growth is restricted to the base of the leaf and is restricted to a few hours at the end of the night and the start of the day. The high resolution of the method is illustrated by mapping the responses to step‐changes in leaf turgor. A 3 bar turgor jump led to a rapid transient expansion over the entire length of the leaf that was partially reversed when the turgor was relaxed.
Lesion mimic mutants of rice with alterations in early signaling events of defense
Authors: Takahashi Akira , Kawasaki Tsutomu , Henmi Kenji , Shii Katsuhiko , Kodama Osamu , Satoh Hikaru , Shimamoto Ko
Published: March 2003

We screened 93 lesion mimic mutants of rice for resistance to the blast fungus,
Magnaporthe grisea
, and found eight mutants that exhibited significant resistance to the fungus. We called these mutants
cdr
(cell death and resistance) and further analyzed three of them. Two mutations,
cdr1
and
cdr2
, were recessive and one,
Cdr3
, was dominant. Many small brownish lesions developed over the entire leaf of the mutants 20–50 days after sowing. TUNEL staining revealed that DNA fragmentation occurred in leaf blade cells of the homozygous
Cdr3
mutants. Autofluorescence and callose deposition were visible in leaf cells of these three mutants. Activation of two defense‐related genes,
PBZ1
and
PR1
, was observed in the leaves of the mutants; high expression of
PBZ1
was correlated with the lesion formation in the three mutants, whereas
PR1
was constitutively expressed in the
cdr2
and
Cdr3
mutants irrespective of the lesion formation. Levels of momilactone A, a major phytoalexin of rice, in these mutants were increased approximately 100–400‐fold relative to the wild‐type levels. Suspension‐cultured cells of the
cdr1
and
cdr2
but not
Cdr3
produced higher levels of H2O2 than the wild type when treated with calyculin A, an inhibitor of protein phosphatase 1. These results suggest that biochemical lesions of
cdr1
and
cdr2
lie in the early signaling steps leading to activation of the NADPH oxidase and that type‐1 protein phosphatase is operative in protein dephosphorylation involved in NADPH oxidase activation.
Developmental stage‐specific multi‐subunit plastid RNA polymerases (PEP) in wheat
Authors: Satoh Junko , Baba Kyoko , Nakahira Yoichi , Tsunoyama Yuichi , Shiina Takashi , Toyoshima Yoshinori
Published: March 2003

Most photosystem I and II plastid genes are transcribed by a plastid encoded Escherichia coli‐like RNA polymerase (PEP). In this study, we show that both promoter selectivity and light‐dependency of PEP change dramatically during development in wheat leaves. In the leaf tip, psbA and psbD promoter activities are light induced, whilst psbC, psbE and 16S rRNA promoters do not function efficiently irrespective of light conditions. In contrast to the leaf tip, in the basal portion all PEP promoters studied function in the dark as well as the light, except for psbD. Using in vitro transcription, we found that PEP in the illuminated leaf tip can initiate transcription from the –35 destructed psbA promoter, but the –35 element is essential for transcription in the basal portion. There is an extended –10 element in the psbA promoter, recognized by the PEP in the illuminated leaf tip or purified σ70‐type Escherichia coli RNA polymerase but not by the PEP in the leaf base. These results suggest that during wheat leaf development, PEP in the leaf base that is functional for most PEP promoters even in the dark is replaced by the light‐dependent PEP selectively transcribing the psbA and psbD promoters.
Phosphorylation of pea chloroplast acetyl‐CoA carboxylase
Authors: Savage Linda J. , Ohlrogge John B.
Published: March 2003

We have examined whether chloroplast acetyl‐CoA carboxylase is a phosphoprotein. Pea (Pisum sativum) chloroplasts were incubated in the presence of [γ‐33P]‐ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl‐CoA carboxylase. The β‐subunit of the carboxyltransferase was found to be labeled by33P. Phosphoamino acid analysis of the immunoprecipitated β‐subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of33P into carboxyltransferase β‐subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase‐agarose conjugate reducedin vitroacetyl‐CoA carboxylase activity by 67%. Furthermore, while acetyl‐CoA carboxylase activity and its carboxyltransferase half‐reaction were reduced in dephosphorylated extracts, the biotin carboxylase half‐reaction was not inhibited. The evidence presented here points to the carboxyltransferase β‐subunit of chloroplast acetyl‐CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.
Structural and functional analysis of the six regulatory particle triple‐A ATPase subunits from the Arabidopsis 26S proteasome
Authors: Fu Hongyong , Doelling Jed H. , Rubin David M. , Vierstra Richard D.
Published: March 2003

The 26S proteasome is a multi‐subunit ATP‐dependent protease responsible for degrading most short‐lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylin‐ drical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP‐dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, desig‐ nated RPT1–6 for regulatory particle triple‐A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub‐ units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3–6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non‐redundant functions.
Molecular and functional regulation of two NO3– uptake systems by N‐ and C‐status of Arabidopsis plants
Authors: Lejay Laurence , Tillard Pascal , Lepetit Marc , Olive Francesc Domingo , Filleur Sophie , Daniel‐Vedele Françoise , Gojon Alain
Published: March 2003

Root NO

3

–

uptake and expression of two root NO

3

–

transporter genes (

Nrt2;1

and Nrt1) were investigated in response to changes in the N‐ or C‐status of hydroponically grown Arabidopsis thaliana plants. Expression of Nrt2;1 is up‐regulated by NO

3

–

starvation in wild‐type plants and by N‐limitation in a nitrate reductase (NR) deficient mutant transferred to NO

3

–

as sole N source. These observations show that expression of Nrt2;1 is under feedback repression by N‐metabolites resulting from NO

3

–

reduction. Expression of Nrt1 is not subject to such a repression. However, Nrt1 is over‐expressed in the NR mutant even under N‐sufficient conditions (growth on NH

4

NO

3

medium), suggesting that expression of this gene is affected by the presence of active NR, but not by N‐status of the plant. Root

15

NO

3

–

influx is markedly increased in the NR mutant as compared to the wild‐type. Nevertheless, both genotypes have similar net

15

NO

3

–

uptake rates due to a much larger

14

NO

3

–

efflux in the mutant than in the wild‐type. Expressions of Nrt2;1 and Nrt1 are diurnally regulated in photosynthetically active A. thaliana plants. Both increase during the light period and decrease in the first hours of the dark period. Sucrose supply prevents the inhibition of Nrt2;1 and Nrt1 expressions in the dark. In all conditions investigated, Nrt2;1 expression is strongly correlated with root

15

NO

3

–

influx at 0.2 m

m

external concentration. In contrast, changes in the Nrt1 mRNA level are not always associated with similar changes in the activities of high‐ or low‐affinity NO

3

–

transport systems.
Hormonal regulation of a cysteine proteinase gene, EPB‐1, in barley aleurone layers: cis‐ and trans‐acting elements involved in the co‐ordinated gene expression regulated by gibberellins and abscisic acid
Authors: Cercós Manuel , Gómez‐Cadenas Aurelio , Ho Tuan‐Hua David
Published: March 2003

The synthesis of EPB, a cysteine proteinase responsible for the degradation of seed endosperm storage proteins in barley (Hordeum vulgare), is induced by gibberellins (GA) and repressed by abscisic acid (ABA). The EPB gene family consists of two very similar members, EPB‐1 and EPB‐2, with the former being more highly induced by GA. We have functionally characterized the cis‐acting elements in the EPB‐1 promoter and determined that a gibberellin response element (GARE), a pyrimidine box and an upstream element are necessary for GA induction. By comparison with the promoters of α‐amylase genes, which are also induced by GA, we suggest that GARE is coupled with the upstream element and the pyrimidine box to form a GA response complex. In addition, we have shown that the 3′‐untranslated/untranscribed region of the EPB‐1 gene is required for a low background expression in the absence of GA. Constitutive expression of a transcription factor, GAMyb, in the absence of GA leads to the transactivation of EPB‐1 expression in a dosage dependent manner with the highest level comparable to that in fully GA‐induced tissue. Co‐expression of a truncated version of GAMyb containing only the DNA binding domain blocks the GA‐induction of EPB‐1, further supporting the role of GAMyb in the regulation of gene expression. Although ABA is very effective in blocking the GA induction of EPB‐1, it has no effect on the GAMyb‐mediated expression of EPB‐1. We suggest that ABA acts upstream of the formation of functional GAMyb which co‐ordinates the hormonal regulation of a diverse group of genes in cereal aleurone layers, including those encoding EPB and α‐amylases.
Antisense inhibition of the GDP‐mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence
Authors: Keller Ruth , Renz Franziska Springer andreas , Kossmann Jens
Published: March 2003

GDP‐mannose pyrophosphorylase (GMPase, EC 2.7.7.22) catalyses the synthesis of GDP‐D‐mannose and represents the first committed step in the formation of all guanosin‐containing sugar nucleotides found in plants which are precursors for cell wall biosynthesis and, probably more important, the synthesis of ascorbate. A full‐length cDNA encoding GMPase from S. tuberosum was isolated. Transgenic potato plants were generated in which the GMPase cDNA was introduced in antisense orientation to the 35S promoter. Transformants with reduced GMPase activity were selected. Transgenic plants were indistinguishable from the wild‐type when held under tissue culture conditions, however, a major change was seen 10 weeks after transfer into soil. Transgenic plants showed dark spots on leaf veins and stems with this phenotype developing from the bottom to the top of the plant. In case of the line with the strongest reduction, all aerial parts finally dried out after 3 months in soil, in contrast to the wild‐type plants which did not start to senesce at this time. This coincides with a reduction of ascorbate contents in the transgenic plants, which is in agreement with the recently proposed pathway of ascorbate biosynthesis. Furthermore, leaf cell walls of the transgenic potato plants had mannose contents that were reduced to 30–50% of the wild‐type levels, whereas the composition of tuber cell walls was unchanged. The glycosylation pattern of proteins was unaffected by GMPase inhibition, as studied by affinoblot analysis.