bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell wall antibodies without immunization: generation and use of de‐esterified homogalacturonan block‐specific antibodies from a naive phage display library
Authors: Willats William G. T. , Gilmartin Philip M. , Mikkelsen Jørn Dalgaard , Knox J. Paul
Published: March 2003

Homogalacturonan (HG) is a multi‐functional pectic polysaccharide of primary cell walls involved in calcium cross‐linking and gel formation, and the regulation of ionic status and porosity of the cell wall matrix, and is a source of oligosaccharins functioning in development and defence. Phage display monoclonal antibodies with specificity for de‐esterified stretches (‘blocks’) of pectic HG have been isolated from a naive phage display library without the need for immunization of animals or conjugation of an oligosaccharide to protein. These antibodies, designated PAM1 and PAM2, bind specifically to de‐esterified and un‐substituted HG. Assays with a series of pectins de‐esterified by the action of plant or fungal pectin methyl esterases indicated that the antibodies were specific to de‐esterified blocks resulting from the blockwise action of plant pectin methyl esterases. Analysis of antibody binding to a series of oligogalacturonides indicated that optimal binding required in the region of 30 de‐esterified GalA residues. The recognition of such a large epitope by these antibodies allows the HG block architecture of primary cell walls to be identified and localized for the first time. Furthermore, we have demonstrated that monoclonal antibodies with high specificity and avidity to cell wall epitopes can be generated using a ‘single pot’ phage display approach.
The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW‐LIKE genes
Authors: Pysh Leonard D. , Wysocka‐Diller Joanna W. , Camilleri Christine , Bouchez David , Benfey Philip N.
Published: March 2003

Mutations at the SCARECROW (SCR) locus in Arabidopsis thaliana result in defective radial patterning in the root and shoot. The SCR gene product contains sequences which suggest that it is a transcription factor. A number of Arabidopsis Expressed Sequence Tags (ESTs) have been identified that encode gene products bearing remarkable similarity to SCR throughout their carboxyl‐termini, indicating that SCR is the prototype of a novel gene family. These ESTs have been designated SCARECROW‐LIKE (SCL). The gene products of the GIBBERELLIN‐INSENSITIVE (GAI) and the REPRESSOR of ga1–3 (RGA) loci show high structural and sequence similarity to SCR and the SCLs. Sequence analysis of the products of the GRAS (GAI, RGA, SCR) gene family indicates that they share a variable amino‐terminus and a highly conserved carboxyl‐terminus that contains five recognizable motifs. The SCLs have distinct patterns of expression, but all of those analyzed show expression in the root. One of them, SCL3, has a tissue‐specific pattern of expression in the root similar to SCR. The importance of the GRAS gene family in plant biology has been established by the functional analyses of SCR, GAI and RGA.
Two MAR DNA‐binding proteins of the pea nuclear matrix identify a new class of DNA‐binding proteins
Authors: Hatton Diane , Gray John C.
Published: March 2003

Four MAR‐binding proteins of 60, 65, 70 and 72 kDa have been detected by South‐Western blotting and isolated from pea nuclear matrices. Two cDNAs encoding the 60 and 65 kDa proteins (MARBP‐1 and MARBP‐2) were isolated from a pea leaf cDNA library by screening with a PCR product obtained using degenerate primers based on an amino acid sequence from the 60 kDa protein. The proteins of 560 and 550 amino acids are 86% identical and contain several KKD/E repeats near the C‐terminus. Escherichia coli‐expressed MARBP‐1 specifically binds A/T‐rich MAR DNA. The interaction of MARBP‐1/MARBP‐2 with MAR DNA involves novel DNA‐binding motifs. The MARBP‐1 and MARBP‐2 genes are expressed in a range of pea tissues and are encoded by genes at different loci. MARBP‐1 and MARBP‐2 are homologous to yeast nucleolar proteins Nop56p and Nop58p, which are involved in ribosome biogenesis, and to similar highly conserved proteins in other eukaryotes and in archaebacteria. MARBP‐1 and MARBP‐2 may have multifunctional roles in chromatin organisation and ribosome biogenesis.
Oxygen deprivation stimulates Ca2+‐mediated phosphorylation of mRNA cap‐binding protein eIF4E in maize roots
Authors: Manjunath Sivalinganna , Williams Alan J. , Bailey‐Serres Julia
Published: March 2003

Flooding of maize seedlings causes O2 deprivation that leads to a global reduction in protein synthesis and selective translation of cytoplasmic mRNAs. Since selective translation in animal cells can involve the cap‐binding protein eIF4E, we characterized the distinct mRNA cap‐binding proteins eIF4E and eIFiso4E of maize. These proteins have 45% deduced amino acid sequence identity and are highly conserved at residues of eIF4E that function in intermolecular interactions in animals. Maize eIF4E is a phosphoprotein. O2 deprivation resulted in a decrease in the isoelectric point of eIF4E, consistent with additional phosphorylation. Modification of eIF4E was mimicked by treatment with caffeine under aerobic conditions and blocked by treatment with ruthenium red under O2 deprivation, implicating Ca2+ as a second messenger in eIF4E modification. In contrast, no isoelectric variants of eIFiso4E were detected. The possible role of cytosolic Ca2+ and pH in regulation of mRNA cap‐binding protein activity under O2 deprivation is discussed.
Emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA
Authors: Goshima Naoki , Mukai Toshihiro , Suemori Mamiko , Takahashi Misa , Caboche Michel , Morikawa Hiromichi
Published: March 2003

The emission of N2 and N2O from intact transgenic tobacco (clone 271) expressing antisense nitrite reductase (NiR) mRNA, and wild‐type plants grown aseptically, on NO3–, NO2– or NH4+ ‐containing medium was investigated. 15N contents of gas sampled from gas‐sealed pots, in which the plants were grown on 15N‐containing medium, were analyzed by gas chromato‐ graphy and mass spectrometry (GC–MS). No emission of N2 was detected in either of the gas samples from plant clone 271 or the wild‐type grown on NO3–‐containing medium. N2O emission from clone 271 grown on NO3–‐containing medium was detected, but not from the wild‐type plants. The N2O emission rate of clone 271 was 106 ng N2O mg–1 incorporated N week–1 and the N2O emission was inhibited by tungstate (a nitrate reductase inhibitor). No emission of N2O was found from clone 271 or wild‐type plants grown on medium containing NH4+. Emission of N2O also was detected from clone 271 grown on NO2–‐containing medium and its emission rate increased with increasing NO2– levels in plants. We speculate that NO3– is reduced to NO2– and that a part of NO2– is metabolized to N2O in clone 271.
The distinctive roles of five different ARC genes in the chloroplast division process in Arabidopsis
Authors: Marrison Joanne L. , Rutherford Stephen M. , Robertson Elizabeth J. , Lister Clare , Dean Caroline , Leech Rachel M.
Published: March 2003

ARC (accumulation and replication of chloroplasts) genes control different aspects of the chloroplast division process in higher plants. In order to establish the hierarchy of the ARC genes in the chloroplast division process and to provide evidence for their specific roles, double mutants were constructed between arc11, arc6, arc5, arc3 and arc1 in all combinations and phenotypically analysed. arc11 is a new nuclear recessive mutant with 29 chloroplasts compared with 120 in wild type. All the phenotypes of the double mutants are unambiguous. ARC1 down‐regulates proplastid division but is on a separate pathway from ARC3, ARC5, ARC6 and ARC11. ARC6 initiates both proplastid and chloroplast division. ARC3 controls the rate of chloroplast expansion and ARC11 the central positioning of the final division plane in chloroplast division. ARC5 facilitates separation of the two daughter chloroplasts. ARC5 maps to chromosome 3 and ARC11 and ARC6 map approximately 60 cM apart on chromosome 5.
AtAMT1 gene expression and NH4+ uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels
Authors: Rawat Suman R. , Silim Salim N. , Kronzucker Herbert J. , Siddiqi M. Yaeesh , Glass Anthony D. M.
Published: March 2003

The mechanisms involved in regulating high‐affinity ammonium (NH4+) uptake and the expression of the AtAMT1 gene encoding a putative high‐affinity NH4+ transporter were investigated in the roots of Arabidopsis thaliana. Under conditions of steady‐state nitrogen (N) supply, transcript levels of the AtAMT1 gene and Vmax values for high‐affinity 13NH4+ influx were inversely correlated with levels of N provision. Following re‐supply of NH4NO3 to N‐starved plants, AtAMT1 mRNA levels and 13NH4+ influx declined rapidly but remained high when the conversion of NH4+ to glutamine (Gln) was blocked with methionine sulfoximine (MSX). This result demonstrates that end products of NH4+ assimilation, rather than NH4+ itself, are responsible for regulating AtAMT1 gene expression. Consistent with this hypothesis, AtAMT1 gene expression and NH4+ influx were suppressed by provision of Gln alone, or together with NH4NO3 plus MSX. Furthermore, AtAMT1 transcript levels and 13NH4+ influx were negatively correlated with root Gln concentrations, following re‐supply of N to N‐starved plants. In addition to this level of control, the data suggest that high cytoplasmic [NH4+] may inhibit NH4+ influx.
Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis
Authors: Roy Stéphane J. , Holdaway‐Clarke Terena L. , Hackett Grant R. , Kunkel Joseph G. , Lord Elizabeth M. , Hepler Peter K.
Published: March 2003

Cytoplasmic calcium concentration ([Ca2+]i) and extracellular calcium (Ca2+o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β‐D‐Glc)3). Growing pollen tubes were pressure injected with the ratio dye fura‐2 dextran and imaged after application of (β‐D‐Glc)3, which binds arabinogalactan proteins (AGPs). Application of (β‐D‐Glc)3 inhibited growth but not secretion. Ratiometric imaging of [Ca2+]i revealed an initial spread in the locus of the apical [Ca2+]i gradient and substantial elevations in basal [Ca2+]i followed by the establishment of new regions of elevated [Ca2+]i on the flanks of the tip region. Areas of elevated [Ca2+]i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca2+o influx at the tip of (β‐D‐Glc)3‐treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca2+]i, probably resulting from Ca2+o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.
Rhizobium Nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs
Authors: Cárdenas Luis , Feijó José A. , Kunkel Joseph G. , Sánchez Federico , Holdaway‐Clarke Terena , Hepler Peter K. , Quinto Carmen
Published: March 2003

Application of Nod factors to growing, responsive root hairs of the bean Phaseolus vulgaris induces marked changes in both the intracellular cytosolic free calcium (Ca2+) and in the influx of extracellular [Ca2+]. The intracellular [Ca2+], which has been measured by ratiometric imaging in cells microinjected with fura‐2‐dextran (70 kDa), elevates within 5 min from approximately 400 n
m to 1500 n
m in localised zones in the root hair apex. Of particular note is the observation that the elevated regions of [Ca2+] appear to shift position during short time intervals. Increases in and fluctuations of the intracellular [Ca2+] are also observed in the perinuclear region after 10–15 min treatment with Nod factors. The extracellular Ca2+ flux, detected with the non‐invasive, calcium specific vibrating electrode, is inwardly directed and also increases quickly in response to Nod factors from 13 pmol cm–2 s–1 to 28 pmol cm–2 s–1. Chitin‐oligomers, which are structurally similar but biologically inactive when compared to the active Nod factors, fail to elicit changes in either intracellular or extracellular Ca2+. The similar timing and location of the intracellular elevations and the increased extracellular influx provide support for the idea that Ca2+ participates in secretion and cell wall remodelling, which occur in anticipation of root hair deformation and curling.
CYP78A5 encodes a cytochrome P450 that marks the shoot apical meristem boundary in Arabidopsis
Authors: Zondlo Susan C. , Irish Vivian F.
Published: March 2003

The normal development of shoot structures depends on controlling the growth, proliferation and differentiation of cells derived from the shoot apical meristem. We have identified the CYP78A5 gene encoding a putative cytochrome P450 monooxygenase that is the first member of the CYP78 family from Arabidopsis. This gene is strongly expressed in the peripheral regions of the vegetative and reproductive shoot apical meristems, defining a boundary between the central meristematic zone and the developing organ primordia. In addition, CYP78A5 shows a dynamic pattern of expression during floral development. Overexpression of CYP78A5 affects multiple cell types, causing twisting and kinking of the stem and defects in floral development. To define the relationship of CYP78A5 to genes controlling meristem function, we examined CYP78A5 expression in plants mutant for SHOOT MERISTEMLESS, ZWILLE and ARGONAUTE, and have found that CYP78A5 expression is altered in these mutant backgrounds. We propose that CYP78A5 has a role in regulating directional growth in the peripheral region of the shoot apical meristem in response to cues established by genes regulating meristem function.
The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis
Authors: Stadler Ruth , Truernit Elisabeth , Gahrtz Manfred , Sauer Norbert
Published: March 2003

The Arabidopsis AtSUC1 protein has previously been characterized as a plasma membrane H+‐sucrose symporter. This paper describes the sites of AtSUC1 gene expression and AtSUC1 protein localization and assigns specific functions to this sucrose transporter in anther development and pollen tube growth. RNase protection assays revealed AtSUC1 expression exclusively in floral tissue, which was confirmed by analyses of AtSUC1 promoter‐β‐glucuronidase (GUS) plants. In situ hybridizations identified AtSUC1 expression in anther connective tissue, in funiculi and in fully developed pollen grains. Indirect immuno‐fluorescence analyses with anti‐AtSUC1 antiserum confirmed AtSUC1 protein localization in the connective tissue and funiculi. In mature pollen grains, however, despite high AtSUC1 mRNA levels no AtSUC1 protein was found. Only after pollination of stylar papillae was AtSUC1 protein detected inside the pollen and later inside the growing pollen tubes, suggesting a translation of pre‐existing AtSUC1 mRNA after pollination. Pollen germination analyses underlined the important role of sucrose for pollen tube growth. The data presented suggest a role of AtSUC1 in the controlled dehiscence of Arabidopsis anthers. It is postulated that an important function of AtSUC1 is the cell‐specific modulation of water potentials.
Regulation of spinach SNF1‐related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5′‐AMP
Authors: Sugden Chris , Crawford Russell M. , Halford Nigel G. , Hardie D. Grahame
Published: March 2003

Members of the SNF1‐related protein kinase‐1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP‐activated and yeast SNF1 protein kinases. Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine [equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)] within the ‘T loop’ between the conserved DFG and APE motifs. We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine. We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5′‐AMP, via binding to the substrate (i.e. the kinase). This is the first report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts. The possible physiological significance of these findings is discussed.
Modified expression of a carrot small heat shock protein gene, Hsp17.7, results in increased or decreased thermotolerance‡
Authors: Malik Mukesh K. , Slovin Janet P. , Hwang Cheol Ho , Zimmerman J. Lynn
Published: March 2003

We have determined that one small heat shock protein gene, encoding Hsp17.7, plays an important role in the ability of carrot cells and plants to survive thermal stress. Transgenic cells and regenerated plants were generated in which the carrotHsp17.7gene was either constitutively expressed (denoted CaS lines) or expressed as a heat inducible antisense RNA (denoted AH lines). Thermotolerance measurements demonstrated that CaS lines were more thermotolerant than vector controls and AH antisense lines were less thermo‐ tolerant than vector controls. RNA analysis demonstrated thatHsp17.7mRNA was detectable, but not abundant, prior to heat shock in CaS cells, but not in vector control cells. Conversely, RNA analysis of antisense cells showed that, after heat shock, the amounts of mRNA forHsp17.7was moderately less abundant in AH cells than in vector controls. Analysis of protein synthesis in CaS cells did not indicate substantial synthesis or accumulation of Hsp17.7, or any small Hsp, at 23°C. However, in the most thermotolerant line, protein synthesis was maintained at a higher rate than in other cell lines at a more extreme heat shock (42°C). In contrast, antisense AH cells showed reduced synthesis of many Hsp, large and small. These results suggest that theHsp17.7gene plays a critical, although as yet not understood, role in thermotolerance in carrot. This represents the first demonstration of the ability to both increase and decrease thermotolerance by the manipulation of expression of a single gene.
NRSA‐1: a resistance gene homolog expressed in roots of non‐host plants following parasitism by Striga asiatica (witchweed)
Authors: Gowda Bhavani S. , Riopel James L. , Timko Michael P.
Published: March 2003

Studies of the initial interactions of Striga asiatica with the non‐host plant species Tagetes erecta (marigold) established that parasite penetration through the root is arrested most frequently in the cortex. The arrest of parasite ingress is associated with browning and necrosis of root cortical cells flanking the invading endophyte and with increased intracellular wall appositions on the root cell walls directly adjacent to the plant‐parasite interface. Using a polymerase chain reaction‐based differential cDNA amplification strategy followed by 5′‐RACE, we have identified several gene products whose expression is induced in marigold roots during attempted parasitism by Striga. Among these was a 917 bp cDNA encoding a 221 amino acid protein with significant homology to proteins encoded by disease resistance genes from other plant species, including N, RPP5, L6 and M. This cDNA was subsequently used to isolate a nuclear gene, designated NRSA‐1, for non‐host resistance to Striga asiatica. NRSA‐1 is a member of a small gene family in marigold consisting of two to four members. RNA gel blot analysis showed that NRSA‐1 transcripts accumulate to high levels in roots near the site of Striga invasion within 120 h after parasite attachment, and appear at lower levels throughout the rest of the plant under Striga parasitism. NRSA‐1 expression is rapidly induced by treatment with jasmonic acid (JA), but not by mechanical wounding, treatment with salicylic acid, paraquat or ABA. A possible role for NRSA‐1 in the non‐host resistance mechanism is discussed.
Pseudomonas syringae pv tomato induces the expression of tomato EREBP‐like genes Pti4 and Pti5 independent of ethylene, salicylate and jasmonate
Authors: Thara Venkatappa K. , Tang Xiaoyan , Gu Yong Q. , Martin Gregory B. , Zhou Jian‐Min
Published: March 2003

The tomato genes Pti4 and Pti5 encode ethylene‐responsive element binding protein‐like transcription factors that bind to the GCC box, a conserved cis‐element in many defense‐related genes. The Pti proteins have previously been shown to interact with the tomato disease resistance protein Pto. Here we report that the expression of both Pti4 and Pti5 are induced by a virulent strain of Pseudomonas syringae pv tomato. The expression of Pti5 is further enhanced by the interaction of the Pto gene in tomato and the corresponding avrPto gene in the bacterium. The enhancement of Pti5 expression by Pto–avrPto interaction requires a functional Prf gene in the plant. Pti5 appears to be expressed specifically during biotic stresses, suggesting a specific role in plant defense. Pti4 and several EREBP‐like genes are induced by ethylene, salicylate and wounding. However, the Pseudomonas bacterium induced a wild‐type level of Pti4 and Pti5 transcripts in tomato plants carrying the nahG transgene, the Nr mutation, or the def1 mutation. In addition, the ethylene action inhibitor norbornadiene did not inhibit the induction of Pti4 and Pti5 either in the compatible or incompatible interactions. The results suggest that the Pseudomonas bacterium induces Pti4 and Pti5 expression through a pathway independent of salicylic acid, ethylene and jasmonic acid.
The SERRATE locus controls the formation of the early juvenile leaves and phase length in Arabidopsis
Authors: Clarke Jonathan H. , Tack Dimitri , Findlay Kim , Van Montagu Marc , Van Lijsebettens Mieke
Published: March 2003

The development of the shoot can be divided into a series of distinct developmental phases based on leaf character‐istics and inflorescence architecture. The relationship between phase length, defined by the number of organs produced, and the timing of the floral induction (V3‐I1 transition) is relatively ill defined. Characterization of the serrate mutant (CS3257; Arabidopsis Biological Research Center) revealed defects in both vegetative and inflores‐cence phase lengths, the timing of phase transitions, leaf number, the leaf initiation rate, and phyllotaxy. The timing of floral induction, however, is the same as in wild‐type in extended short days as well as in short days, whereas the flowering time response to photoperiod is unaffected. SERRATE is shown to be required for the development of early juvenile leaves (V1) and to promote late juvenile leaf development (V2), while suppressing adult leaf (V3) and inflorescence development (I1 and I2). The se mutation supports the hypothesis that the timing of floral induction is independent of vegetative and inflorescence phase lengths. The role of SERRATE in the regulation of phase length and leaf identity is discussed.
Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth
Authors: Espinosa‐Ruiz Ana , Bellés Jose M. , Serrano Ramón , Culiáñez‐Macià Francisco A.
Published: March 2003

We have isolated two Arabidopsis thaliana genes, AtHAL3a and AtHAL3b, showing homology with HAL3, a yeast protein which regulates the cell cycle and tolerance to salt stress through inhibition of the PPZ1 type‐1 protein phosphatase. Expression of AtHAL3a in yeast hal3 mutants partially complements their LiCl sensitivity, suggesting possible conserved functions between both proteins. AtHAL3a and AtHAL3b are induced by salt stress and AtHAL3a is the most expressed in non‐stressed plants, particularly in seeds. In situ hybridization demonstrates enrichment of AtHAL3a mRNA in seed embryos and in the vascular phloem of different plant tissues. AtHAL3 proteins show striking homology with a group of proteins found in fungi, plants and animals and some homology with a large family of prokaryotic flavoproteins. Recombinant AtHAL3a protein purified from Escherichia coli was yellow because it contained a non‐covalently bound chromophore revealed as flavin mononucleotide. Trans‐ genic Arabidopsis plants, with gain of AtHAL3a function, show altered growth rates and improved tolerance to salt and osmotic stress.
The tomato I‐box binding factor LeMYBI is a member of a novel class of Myb‐like proteins
Authors: Rose Annkatrin , Meier Iris , Wienand Udo
Published: March 2003

The RBCS3A gene of tomato belongs to a small gene family consisting of five members. Although the RBCS1, RBCS2 and RBCS3A promoters contain closely related cis regulatory sequences, the expression patterns of the genes are different. Whereas the RBCS1 and RBCS2 genes are expressed in both leaves and young fruit, the RBCS3A promoter is highly active in leaves, but not in young fruit. This lack of transcription could be due to a mutation in the RBCS3A promoter creating the so‐called F‐box, a protein binding site located between the activating cis elements, the I‐box and G‐box. In order to identify proteins that bind to the RBCS3A I‐box/F‐box region, the yeast one‐hybrid system was used. One clone, LeMYBI was isolated which contains strong similarity to plant myb transcription factors. The encoded LeMYBI protein is at least 188 amino acids in length and contains two myb‐like domains located at the amino terminus and close to the carboxy terminus, separated by a negatively charged domain. The protein contains a SHAQKYF amino acid signature motif in the second myb‐like repeat, which is highly conserved in a number of recently identified plant myb‐related genes, thus defining a new class of plant DNA‐binding proteins. LeMYBI binds specifically to the I‐box sequence of the RBCS1, RBCS2 and RBCS3A promoters, therefore representing the first cloned I‐box binding factor. LeMYBI acts as a transcriptional activator in yeast and plants, and binds to the I‐box with a DNA‐binding domain located in the carboxyterminal domain.
The N‐terminal fragment of Arabidopsis photomorphogenic repressor COP1 maintains partial function and acts in a concentration‐dependent manner
Authors: Stoop‐Myer Chatanika , Torii Keiko U. , McNellis Timothy W. , Coleman Joseph E. , Deng Xing‐Wang
Published: March 2003

Arabidopsis seedlings exhibit distinct developmental patterns according to their light environment: photomorphogenesis in the light and etiolation or skotomorphogenesis in darkness. COP1 acts within the nucleus to repress photomorphogenesis in darkness, while light depletes COP1 from nucleus and abrogates this repression. COP1 contains three structural modules: a RING finger followed by a coiled‐coil domain, and a WD40 repeat domain at the C‐terminus. By introducing various domain deletion mutants of COP1 into cop1 null mutant backgrounds, we show that all three domains are essential for the function of COP1 in vivo. Interestingly, a fragment containing the N‐terminal 282 amino acids of COP1 (N282) with both the RING finger and coiled‐coil modules is sufficient to rescue the lethality of the cop1 null mutations at low expression level. However, high expression levels of the N282 fragment result in a phenocopy of the cop1 null mutation. The sensitivity of the seedling to levels of N282 could reflect the importance of the abundance of COP1 for the appropriate regulation of photomorpho‐ genic development.
Metabolic engineering of p‐hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor
Authors: Bak Søren , Olsen Carl Erik , Petersen Bent Larsen , Møller Birger Lindberg , Halkier Barbara Ann
Published: March 2003

Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P450 catalysing the conversion of tyrosine to p‐hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the production of high levels of the tyrosine‐derived glucosinolate p‐hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.
Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ‐line transformation
Authors: Mysore Kirankumar S. , Kumar C. T. Ranjith , Gelvin Stanton B.
Published: March 2003

Germ‐line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens. We have recently identified several Arabidopsis ecotypes and T‐DNA‐tagged mutants that are recalcitrant to Agrobacterium‐mediated transformation of cut root segments. Some of these ecotypes and mutants are deficient in their ability to bind bacteria. Some are deficient in T‐DNA integration. We report here that using a germ‐line transformation protocol we transformed these ecotypes and mutants, including attachment‐ and integration‐defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild‐type plants. However, we could not transform otherwise highly susceptible Arabidopsis plants by germ‐line or root transformation using several vir and attachment‐deficient Agrobacterium mutants. These results indicate that certain plant factors important for transformation may exist in germ‐line tissue but may be lacking in some somatic cells.
Disruption of putative anion channel gene AtCLC‐a in Arabidopsis suggests a role in the regulation of nitrate content
Authors: Geelen Danny , Lurin Claire , Bouchez David , Frachisse Jean‐Marie , Lelièvre Françoise , Courtial Béatrice , Barbier‐Brygoo Hélène , Maurel Christophe
Published: March 2003

In animals and yeast, voltage‐dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plasma membrane excitability, and control of pH and membrane potential in intracellular compartments. To assess the function of CLCs in plants, we searched for CLC insertion mutants in a library of Arabidopsis lines transformed by Agrobacterium tumefaciens transferred DNA (T‐DNA). Using a polymerase chain reaction‐based screening procedure, an Arabidopsis line that carries a T‐DNA insertion within the C‐terminus of the AtCLC‐a coding sequence was identified. Progeny from this plant line, clca‐1, showed dramatically altered transcription of the AtCLC‐a gene. Plants homozygous for the clca‐1 mutation exhibited normal development and a morphology indistinguishable from the wild‐type. However, their capacity to accumulate nitrate under conditions of nitrate excess was reduced in roots and shoots, by approximately 50%, while chloride, sulphate and phosphate levels were similar to the wild‐type. In addition, the herbicide chlorate, an analogue of nitrate, induced a faster and more pronounced chlorosis in mutant plants. Hypersensitivity to chlorate as well as decreased nitrate levels co‐segregated with the T‐DNA insertion. They were found at various time points of the clca‐1 life cycle, supporting the idea that AtCLC‐a has a general role in the control of the nitrate status in Arabidopsis. Concordant with such a function, AtCLC‐a mRNA was found in roots and shoots, and its levels rapidly increased in both tissues upon addition of nitrate but not ammonium to the culture medium. The specificity of AtCLC‐a function with respect to nitrate is further supported by a similar free amino acid content in wild‐type and clca‐1 plants. Although the cellular localization of AtCLC‐a remains unclear, our results suggest that AtCLC‐a plays a role in controlling the intracellular nitrate status.
Understanding in vivo carbon precursor supply for fatty acid synthesis in leaf tissue
Authors: Bao Xiaoming , Focke Manfred , Pollard Mike , Ohlrogge John
Published: March 2003

The principal supply of carbon precursors for fatty acid synthesis in leaf tissue has been a much debated topic, with some experiments suggesting a direct supply from the C3 products of photosynthetic carbon fixation and colleagues suggesting the utilization of free acetate (for which concentrations in leaves in the range of 0.05–1.4 mM have been reported). To address this issue we first reassessed the in vivo rate of fatty acid synthesis using a new method, that of [13C]carbon dioxide labeling of intact Arabidopsis plants with the subsequent analysis of fatty acids by gas chromatography‐mass spectrometry (GC‐MS). This method gave an average value of 2.3 mmoles carbon atoms h−1 mg chlorophyll−1 for photosynthetic tissues. The method was extended by isotopic dilution analysis to measure the rate of fatty acid synthesis in the dark. There was negligible fatty acid synthesis (< 5% of the rate in the light) in the dark. In addition, the method allowed an estimate of the absolute rate of fatty acid degradation of about 4% of the total fatty acid content per day. With the in vivo rate of fatty acid synthesis in the light defined, if the bulk tissue acetate concentration available for fatty acid synthesis is 1 mM, this acetate pool can sustain fatty acid synthesis for approximately 60 min. When the leaves of Arabidopsis, barley and pea were given a 5 min pulse of [14C]carbon dioxide, the label rapidly appeared in fatty acids with a lag phase of less than 2–3 min. Continuous labeling with [14C]carbon dioxide, for up to 1 h, showed a similar result. Furthermore, 14C‐label in free acetate was less than 5% of that in fatty acids. In conclusion, these data suggest that either the bulk pool of acetate is not involved in fatty acid synthesis or the concentration of acetate must be less than 0.05 mM under strong illumination.
Disruption of an Arabidopsis cytoplasmic ribosomal protein S13‐homologous gene by transposon‐mediated mutagenesis causes aberrant growth and development
Authors: Ito Takuya , Kim Gyung‐Tae , Shinozaki Kazuo
Published: March 2003

We identified a Dissociation (Ds) transposon‐inserted Arabidopsis mutant of a gene (AtRPS13A) homologous to cytoplasmic ribosomal protein (RP) S13. We named our mutant pointed first leaf (pfl) 2 because of its similar phenotype to the pfl1 mutant of the RPS18 gene. This mutant caused multiple phenotypic changes, including aberrant leaf and trichome morphology, retarded root growth, and late flowering. Microscopic analysis showed that the first leaf blade of pfl2 contained a significantly reduced number of palisade cells, which suggests that the mutant phenotype was caused by reduced cell division. However, no phenotypic changes were observed during reproductive growth. In Arabidopsis, the RPS13 protein was encoded by a small expressed gene family including AtRPS13A. A pfl1 pfl2 double mutant showed no additive effect. These results suggest that RPS13 functions in quantitative and pleiotropic ways during growth and development, and that mutations at different kinds of RP gene loci are accumulatable without serious growth defects because they belong to small gene families.
Light‐induced nuclear import of phytochrome‐A:GFP fusion proteins is differentially regulated in transgenic tobacco and Arabidopsis
Authors: Kim Lana , Kircher Stefan , Toth Reka , Adam Eva , Schäfer Eberhard , Nagy Ferenc
Published: March 2003

Phytochromes (phy) are a family of photoreceptors that control various aspects of light‐dependent plant development. Phytochrome A (phyA) is responsible for the very low fluence response (VLFR) under inductive light conditions and for the high irradiance response (HIR) under continuous far‐red light. We have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco. The import is preceded by very fast, light‐induced formation of sequestered areas of phyA:GFP in the cytosol. Here we report that expression of the Arabidopsis phyA:GFP fusion protein in phyA‐deficient Arabidopsis plants complements the mutant phenotype. In these transgenic Arabidopsis lines, both light‐dependent cytosolic formation of sequestered areas of the phyA:GFP as well as VLFR or HIR‐mediated nuclear import of the fusion protein was observed. By contrast, light‐dependent nuclear import of the same fusion protein was induced only by continuous far‐red light (HIR) but not by pulses of far‐red light (VLFR) in transgenic tobacco. These results demonstrate that photoregulation of intracellular partitioning of the Arabidopsis phyA:GFP differs significantly in different genetic backgrounds.
Expression of the Fusarium resistance gene I‐2 colocalizes with the site of fungal containment
Authors: Mes Jurriaan J. , Van Doorn Aveline A. , Wijbrandi Jelle , Simons Guus , Cornelissen Ben J. C. , Haring Michel A.
Published: March 2003

The tomato resistance gene I‐2 is one of at least six members of a gene family that are expressed at low levels in the roots, stems and leaves of young tomato plants. Plants transformed with constructs containing a functional I‐2 promoter fused to the β‐glucuronidase (GUS) reporter gene were used in detailed expression studies. Highest GUS activity was found in stems of young tomato plants. Histochemical analysis revealed that the I‐2 promoter drives expression of the reporter gene in vascular tissue of fruits, leaves, stems and mature roots. In younger roots, expression was most abundant at the base of lateral root primordia. Microscopical analysis of young tomato plants revealed expression in tissue surrounding the xylem vessels. We show that in resistant plants, fungal growth into this region of the vascular tissue is prevented, suggesting a correlation with the I‐2‐mediated resistance response.
FLP‐mediated recombination for use in hybrid plant production
Authors: Luo Hong , Lyznik L. Alexander , Gidoni David , Hodges Thomas K.
Published: March 2003

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

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

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

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

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

Keywords: whole mount, in situ hybridization, immunolocalization, Antirrhinum majus, flower development.
Diurnal changes in sucrose, nucleotides, starch synthesis and AGPS transcript in growing potato tubers that are suppressed by decreased expression of sucrose phosphate synthase
Authors: Geigenberger Peter , Stitt Mark
Published: March 2003

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