bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel parsley 4CL1 cis‐element is required for developmentally regulated expression and protein‐DNA complex formation
Authors: Neustaedter David A. , Lee Stephen P. , Douglas Carl J.
Published: March 2003

The phenylpropanoid enzyme 4‐coumarate:coenzyme A ligase (4CL) participates in the biosynthesis of a wide range of secondary products with specialized function and tissue distribution in plants. The parsley 4CL1 promoter directs a complex tissue‐ and cell‐specific pattern of reporter gene expression in transgenic tobacco, consistent with the distribution of phenylpropanoid products and sites of 4CL expression in tobacco vegetative and floral organs. We generated mutants in a 4CL1 promoter element previously implicated as a site for protein–DNA complex formation to analyze its role in vivo4CL1 promoter surrounding the FP56 element is the site for formation of two large protein–DNA complexes, and that an intact FP56 element is required for formation of these complexes. Finally, the detergent deoxycholate was used to investigate the role of protein–protein interactions in FP56 complex formation. Our data suggest that the FP56 cis‐element plays a central role in transcriptional activation from the 4CL1 promoter, and that its role may be to nucleate formation of a large protein complex on the promoter.
Construction of tomato genomic DNA libraries in a binary‐BAC (BIBAC) vector
Authors: Hamilton Carol M.. , Frary Anne , Xu Yimin , Tanksley Steven D.. , Zhang Hong‐Bin
Published: March 2003

This is the first report of large insert genomic DNA libraries constructed in a binary‐BAC (BIBAC) vector. Genomic DNA libraries containing approximately 4.6 haploid nuclear genomic equivalents were constructed for Lycopersicon esculentum (cv. Mogeor) and Lycopersicon pennellii (LA716) in the BIBAC2 vector. The L. esculentum library has an average insert size of 125 kb and is comprised of 42 272 individual colonies stored as frozen cultures in a 384‐well format (108 plates). The L. pennellii library has an average insert size of 90 kb and is comprised of 53 760 individual clones (140 384‐well plates). In each of the libraries, it is estimated that 90% of the colonies contain genomic DNA inserts. The composition of the L. esculentum and L. pennellii libraries was determined by analyzing a series of randomly selected clones. The L. esculentum library was surveyed for clones containing chloroplast DNA (1.4%), mitochondrial DNA (0.012%) and repetitive DNA motifs. BIBAC clones that may contain a gene of interest can be identified from these libraries by colony hybridization with homologous or heterologous probes or by PCR pooling techniques. Once identified, BIBAC genomic DNA library clones are immediately suitable for Agrobacterium tumefaciens‐mediated plant transformation.
In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis
Authors: Campbell Paul , Braam Janet
Published: March 2003

Xyloglucan endotransglycosylases (XETs) are encoded by a gene family in Arabidopsis thaliana. These enzymes modify a major structural component of the plant cell wall, xyloglucan, and therefore may influence plant growth and development. We have produced four Arabidopsis XETs (TCH4, Meri‐5, EXGT and XTR9) using the baculovirus/insect cell system and compared their biochemical activities. TCH4, as previously demon‐ strated, and the other three proteins are capable of carrying out transglycosylation of xyloglucans. The Km for XLLGol acceptor oligosaccharide is in the range of 20–40 μm for all the XETs except XTR9, which has a Km of 5 μm and is significantly inhibited by high levels of XLLGol. All four enzymes are most active between pH 6.0 and 6.5. TCH4 and XTR9 have temperature optima of 18°C, whereas Meri‐5 and EXGT are most active at 28 and 37°C, respectively. Although the activity levels of three of the XETs are not influenced by the presence of fucose on the xyloglucan polymer, XTR9 has a clear preference for non‐fucosylated xyloglucan polymer. The four XETs show a marked preference for XLLGol over either XXFGol or XXXGol as acceptor oligosaccharide. All four XETs are glycosylated; however, only the activities of TCH4 and Meri‐5 are affected by the removal of the N‐glycan with PNGase F. These four enzymes most likely function solely as transglycosylases because xyloglucan endoglucanase activity was not apparent. Subtle differences in biochemical activities may influence the physiological functions of the distinct XETs in vivo.
Aquaporin Nt‐TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes
Authors: Gerbeau Patricia , Güçlü Josette , Ripoche Pierre , Maurel Christophe
Published: March 2003

Members of the major intrinsic protein (MIP) family, described in plants as water‐selective channels (aquaporins), can also transport small neutral solutes in other organisms. In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non‐electrolytes and evaluate the contribution of MIP homologues to such transport. PM and TP vesicles were purified from tobacco suspension cells by free‐flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped‐flow spectrofluorimetry. For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9 ± 9.6 × 10–6 and 1.0 ± 0.3 × 10–6 cm sec–1, respectively. Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process. This and the high channel‐mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport. A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt‐TIPa) was isolated from tobacco cells. Immunodetection of Nt‐TIPa in purified membrane fractions confirmed that the protein is localized in the TP. Functional expression of Nt‐TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol. These features could account for the transport selectivity profile determined in purified TP vesicles. These results support the idea that plant aquaporins have a dual function in water and solute transport. Because Nt‐TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.
Cloning and expression of a cDNA encoding betanidin 5‐O‐glucosyltransferase, a betanidin‐ and flavonoid‐specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae
Authors: Vogt Thomas , Grimm Rudi , Strack Dieter
Published: March 2003

Based on protein sequence data and RT–PCR, a full length cDNA encoding betanidin 5‐O‐glucosyltransferase (5‐GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br. (Aizoaceae). 5‐GT catalyses the transfer of glucose from UDP‐glucose to the 5‐hydroxyl group of the chromogenic betanidin. Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins. The 5‐GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae). The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa. The corresponding cDNA was expressed in Escherichia coli. The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D. bellidiformis cell suspension cultures. In addition to the natural substrate betanidin, ortho‐dihydroxylated flavonols and flavones were glycosylated preferentially at the B‐ring 4′‐hydroxyl group. 5‐GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed. The results are discussed in relation to molecular evolution of plant glucosyl‐ transferases.
Inter‐organellar crosstalk in higher plants: impaired chloroplast development affects mitochondrial gene and transcript levels
Authors: Hedtke Boris , Wagner Ina , Börner Thomas , Hess Wolfgang R.
Published: March 2003

Co‐ordination of gene expression between the three genomes present in plastids, mitochondria and nucleus is of crucial importance for plant cells. Previous studies revealed that in white leaves of the albostrians (Hordeum vulgare cv. Haisa) mutant, photosynthesis‐related plastid and nuclear genes are expressed only at an extremely low level. The plastids of this mutant lack ribosomes, photosynthetic activity and have only rudimentary membrane systems. Here we report on the expression of mitochondrial genes in albostrians barley. Steady‐state RNA levels of the mitochondrial genes encoding cytochrome oxidase or ATPase subunits, coxII, coxIII, atpA, atp6, atp9 and cob, were observed to be consistently elevated in the white leaves but not in roots. Investigation of mitochondrial DNA revealed an about three‐fold enhanced mitochondrial gene copy number in white compared to green leaf cells, but no differential amplification of mitochondrial genes. Analysis of plants in which the white albostrians plastids were combined with a new nuclear background showed that the enhanced transcript levels were a consequence of the impaired plastids and not of the nuclear albostrians allele. Furthermore, plants bleached by the carotenoid biosynthesis inhibitor norflurazon also showed an enhanced mitochondrial transcript level. These findings allow the conclusion that lack of chloroplast activity in an otherwise fully differentiated leaf leads to an increase in mitochondrial gene copy number and an elevated level of mitochondrial transcripts. Our results indicate an influence of plastids on the genetic apparatus of mitochondria in leaves but not in roots.
A glucocorticoid‐inducible transcription system causes severe growth defects in Arabidopsis and induces defense‐related genes
Authors: Kang Hong‐Gu , Fang Yiwen , Singh Karam B.
Published: March 2003

A glucocorticoid‐inducible transcription system was employed to control the expression of AtEBP, an Arabidopsis transcription factor. A number of the transgenic AtEBP lines had developmental and growth defects when grown on dexamethasone (DEX), a strong synthetic glucocorticoid. However, these growth defects were not confined to the AtEBP lines but were observed with other transgenic lines that were generated using the same system, including empty vector lines. In about 25% of the AtEBP or empty vector transgenic lines, these growth defects were severe and in some cases led to death. As AtEBP has been linked to the plant defense response, the expression of specific defense‐related genes, including a number of pathogenesis‐related (PR) genes was also examined. PDF1.2, a plant defensin gene, was strongly induced in all transgenic lines examined following treatment with DEX, including empty vector lines that did not show any observable DEX‐induced growth defect. PR‐5 was induced to a lesser extent in all the lines, while the expression of PR‐1, PR‐2 and phenylalanine ammonia‐lyase 3 (PAL3) did not change significantly. While the induction of the AtEBP transgene and PDF1.2 had similar DEX concentration requirements, the kinetics of induction differed significantly, with the AtEBP transgene being induced within 1 h and PDF1.2 only being induced between 24 and 48 h. Although the molecular mechanisms underlying the growth defects and changes in gene expression remain to be determined, these changes appear to result from the glucocorticoid‐inducible system itself, and may therefore limit the usefulness of this system for controlling gene expression in Arabidopsis.
Homologues of yeast and bacterial rotenone‐insensitive NADH dehydrogenases in higher eukaryotes: two enzymes are present in potato mitochondria
Authors: Rasmusson Allan G. , Svensson Å. Staffan , Knoop Volker , Grohmann Lutz , Brennicke Axel
Published: March 2003

Two different cDNAs, homologous to genes for rotenone‐insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N‐terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino‐acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydro‐ genases, the NDB amino‐acid sequence contains a non‐conserved insert, which is similar to EF‐hand motifs for calcium binding. Phylogenetic analyses group the rotenone‐insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.
UPL1 and 2, two 405 kDa ubiquitin–protein ligases from Arabidopsis thaliana related to the HECT‐domain protein family
Authors: Bates Paul W. , Vierstra Richard D.
Published: March 2003

The ubiquitin/26S proteasome pathway is a major route for degrading abnormal and important short‐lived regulatory proteins in eukaryotes. Covalent attachment of ubiquitin, which triggers entry of target proteins into the pathway, is accomplished by an ATP‐dependent reaction cascade involving the sequential action of three enzymes, E1s, E2s and E3s. Although much of the substrate specificity of the pathway is determined by E3s (or ubiquitin–protein ligases, UPLs), little is known about these enzymes in plants and how they choose appropriate targets for ubiquitination. Here, we describe two 405 kDa E3s (UPL1 and 2) from Arabidopsis thaliana related to the HECT‐E3 family that is essential in yeast and animals. UPL1 and 2 are encoded by 13 kbp genes 26 cm apart on chromosome I, that are over 95% identical within both the introns and exons, suggesting that the two loci arose from a recent gene duplication. The C‐terminal HECT domain of UPL1 is necessary and sufficient to conjugate ubiquitin in vitro in a reaction that requires the positionally conserved cysteine within the HECT domain, E1, and an E2 of the UBC8 family. Given that HECT E3s help define target specificity of the ubiquitin conjugation, a continued characterization of UPL1 and 2 should be instrumental in understanding the functions of ubiquitin‐dependent protein turnover in plants and for identifying pathway substrates.
Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase
Authors: Peterbauer Thomas , Mucha Jan , Mayer Ulrike , Popp Marianne , Glössl Josef , Richter Andreas
Published: March 2003

Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT–PCR, followed by rapid amplification of cDNA ends (RACE–PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.
Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation
Authors: Willats William G. T. , Steele‐King Clare G. , Marcus Sue E. , Knox J. Paul
Published: March 2003

The occurrence and function of the side chains occurring in the rhamnogalacturonan I domain of pectic poly‐ saccharides have been investigated during carrot cell development using monoclonal antibodies to defined epitopes of (1→4)‐β‐D‐galactan and (1→5)‐α‐L‐arabinan. Immunolocalization studies of carrot root apices indicated that cell walls in the central region of the meristem contained higher levels of (1→5)‐α‐arabinan than the cell walls of surrounding cells. In contrast (1→4)‐β‐galactan was absent from the cell walls of the central meristematic cells but appeared abundantly at a certain point during root cap cell differentiation and also appeared in cell walls of differentiating stele and cortical cells. This developmental pattern of epitope occurrence was also reflected in a suspension‐cultured carrot cell line that can be induced to switch from proliferation to elongation by altered culture conditions. (1→4)‐β‐galactan occurred at a low level in cell walls of proliferating cells but accumulated rapidly in cell walls following induction, before any visible cell elongation, while (1→5)‐α‐arabinan was present in cell walls of proliferating cells but was absent from cell walls of elongated cells. Immunochemical assays of the cultured cells confirmed the early appearance of (1→4)‐β‐galactan during the switch from cell proliferation to cell elongation. Anion‐exchange chromatography confirmed that (1→4)‐β‐galactan was attached to acidic pectic domains and also indicated that it was separate from a distinct homogalacturonan‐rich component. These results indicate that the neutral components of pectic polysaccharides may have important roles in plant cell development.
Reversible binding of the starch‐related R1 protein to the surface of transitory starch granules
Authors: Ritte Gerhard , Lorberth Ruth , Steup Martin
Published: March 2003

Intact starch granules were isolated from leaves of Solanum tuberosum L. (and from Pisum sativum L.), and the patterns of starch‐associated proteins were determined by SDS–PAGE. Depending on the pretreatment of the leaves the protein patterns varied: a 160 kDa compound was present in the starch‐associated protein fraction when the leaves were darkened and performed net starch degradation. However, following illumination (i.e. during net starch biosynthesis) the 160 kDa protein was recovered almost exclusively in a soluble state. The 160 kDa protein was identified to be the recently described starch‐related R1 protein. In in vitro assays recombinant R1 did bind to starch granules isolated from either illuminated or darkened leaves. However, binding to the latter was more effective. It is concluded that, depending upon the metabolic state of the cells, the starch granule surface changes and thereby affects binding of the R1 protein.
Stability determinants in the chloroplast psbB/T/H mRNAs of Chlamydomonas reinhardtii
Authors: Vaistij Fabián E. , Goldschmidt‐Clermont Michel , Wostrikoff Katia , Rochaix Jean‐David
Published: March 2003

The chloroplast gene psbB encodes the chlorophyll‐a binding protein P5 (CP47), one of the core subunits of photosystem II (PSII). The psbB mRNA and the downstream psbT and psbH transcripts fail to accumulate in the Chlamydomonas reinhardtii nuclear mutant 222E affected in the Mbb1 gene (
Monod et al. 1992
, Mol. Gen. Genet. 231, 449–459). By introducing chimeric genes consisting of sequences from psbB and the reporter gene aadA into the chloroplast, the target site of Mbb1 was mapped in the psbB 5′ untranslated region (UTR). Primer extension analysis indicates that the psbB RNA exists in a less abundant long form and a more abundant short form, with 5′ ends at positions −147 and −35 relative to the AUG initiation codon, respectively. The longer transcript is present both in the wild type (WT) and 222E mutant, but the shorter one accumulates only in the WT. Two putative stem‐loop structures in the longer 5′ UTR can be deleted individually without affecting psbB mRNA accumulation. Insertion of a poly G cassette in the long leader stabilizes a chimeric psbB transcript in the 222E mutant, suggesting the involvement of a 5′‐3′ exonuclease. We also show that psbH and psbT are transcribed from the upstream psbB gene promoter, and that the psbH mRNA has its own target sequence for Mbb1 function. We discuss the role of this nucleus‐encoded factor, required for specific chloroplast gene expression, in the assembly of the multi‐protein PSII complex.
Tissue‐specific induction of the mRNA for an extracellular invertase isoenzyme of tomato by brassinosteroids suggests a role for steroid hormones in assimilate partitioning
Authors: Goetz Marc , Godt Dietmute E. , Roitsch Thomas
Published: March 2003

Brassinosteroids (BRs) induce various growth responses when applied exogenously to plant tissues, and the analysis of biosynthetic mutants reveals an essential role for plant growth and development. Only a few BR‐regulated genes have been identified so far, and the corresponding gene products are assumed to be involved in cell elongation. The present study shows that BR growth responses are linked to the regulation of carbohydrate metabolism by induction of the mRNA for the key enzyme of an apoplastic phloem‐unloading pathway. Addition of BRs to autotrophic tomato suspension culture cells specifically elevates the activity of cell‐wall‐bound invertase, whereas the intracellular invertase activities were not affected. This enhanced enzyme activity was shown to correlate with the induction of the mRNA of extracellular invertase Lin6, whereas the mRNA levels of the other three extracellular invertase isoenzymes were not affected. The induction level induced by different BRs correlates with their growth‐promoting activity. The physiological significance of this regulation is further supported by the low concentrations and short incubation times required to induce Lin6 mRNA. This regulatory mechanism results in an elevated uptake of sucrose via the hexose monomers, and thus an increased supply of carbohydrates to the BR‐treated cells. Experiments with tomato seedlings showed that the localized BR‐dependent growth response of the hypocotyl elongation zone was accompanied by a specific induction of Lin6 mRNA that is restricted to the corresponding tissues. This study demonstrates a role of BRs in tissue‐specific source/sink regulation.
A plant virus vector for systemic expression of foreign genes in cereals
Authors: Choi Il‐Ryong , Stenger Drake C. , Morris T. Jack , French Roy
Published: March 2003

Inserts bearing the coding sequences of NPT II and β‐glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb‐CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18–30 days post‐inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT–PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS–NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.
Specific HR‐associated recognition of secreted proteins from Cladosporium fulvum occurs in both host and non‐host plants
Authors: Laugé Richard , Goodwin Paul H. , De Wit Pierre J. G. M. , Joosten Matthieu H. A. J.
Published: March 2003

The resistance of tomato (Lycopersicon esculentum) to the pathogenic fungus Cladosporium fulvum complies with the gene‐for‐gene concept. Host resistance is based on specific recognition of extracellular fungal proteins, resulting in a hypersensitive response (HR). Five proteins secreted by C. fulvum were purified and the encoding cDNA clone was obtained from two novel ones among them. Various tomato breeding lines and accessions of Lycopersicon pimpinellifolium were tested for their recognitional specificity by injection of the purified proteins or potato virus X‐based expression of the cDNA. We found that HR‐associated recognition of one or more of these proteins, in addition to recognition of the race‐specific elicitors AVR4 and AVR9 of C. fulvum, occurs among Lycopersicon species. Studies on the inheritance of this recognition confirmed that single dominant genes are involved. Furthermore, one of the extracellular proteins of C. fulvum is specifically recognized by Nicotiana paniculata, which is not a host for C. fulvum. These results indicate that plants have a highly effective surveillance system for the presence of ‘foreign’ proteins, which, together with the high mutation rate of pathogens, can explain the complex gene‐for‐gene relationships frequently observed in pathosystems.
Evidence that endo‐1,4‐β‐glucanases act on cellulose in suspension‐cultured poplar cells
Authors: Ohmiya Yasunori , Samejima Masahiro , Shiroishi Masahiro , Amano Yoshihiko , Kanda Takahisa , Sakai Fukumi , Hayashi Takahisa
Published: March 2003

Suspension‐cultured poplar (Populus alba) cells produce two distinct endo‐1,4‐β‐glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall‐bound endo‐1,4‐β‐glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo‐1,4‐β‐glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4‐dichlorophenoxyacetic acid (2,4‐D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello‐oligosaccharides. Cello‐oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello‐oligosaccharides by the extracellular enzyme by 90% and that by the wall‐bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo‐1,4‐β‐glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension‐cultured poplar cells.
Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6
Authors: Ichimura Kazuya , Mizoguchi Tsuyoshi , Yoshida Riichiro , Yuasa Takashi , Shinozaki Kazuo
Published: March 2003

Mitogen‐activated protein kinase (MAP kinase, MAPK) cascades play pivotal roles in signal transduction of extracellular stimuli, such as environmental stresses and growth regulators, in various organisms. Arabidopsis thaliana MAP kinases constitute a gene family, but stimulatory signals for each MAP kinase have not been elucidated. Here we show that environmental stresses such as low temperature, low humidity, hyper‐osmolarity, touch and wounding induce rapid and transient activation of the Arabidopsis MAP kinases ATMPK4 and ATMPK6. Activation of ATMPK4 and ATMPK6 was associated with tyrosine phosphorylation but not with the amounts of mRNA or protein. Kinetics during activation differ between these two MAP kinases. These results suggest that ATMPK4 and ATMPK6 are involved in distinct signal transduction pathways responding to these environmental stresses.
Genetic dissection of blue‐light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2
Authors: Weller James L. , Perrotta Gaetano , Schreuder Mariëlle E.L. , Van Tuinen Ageeth , Koornneef Maarten , Giuliano Giovanni , Kendrick Richard E.
Published: March 2003

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after γ‐ray mutagenesis of phyA phyB1 double‐mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL‐induced de‐etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non‐phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de‐etiolation, cry1 is active in older, WL‐grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant‐based characterization of cry1 in a plant species other than Arabidopsis.
A recessive mutation in the Arabidopsis SSI2 gene confers SA‐ and NPR1‐independent expression of PR genes and resistance against bacterial and oomycete pathogens
Authors: Shah Jyoti , Kachroo Pradeep , Nandi Ashis , Klessig Daniel F.
Published: March 2003

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)‐induced expression of pathogenesis‐related (PR) genes and systemic acquired resistance. However, loss‐of‐function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1‐independent pathway that currently remain to be elucidated. The ssi2‐1 mutant, identified in a genetic screen for suppressors of npr1‐5, affects signaling through the NPR1‐independent defense pathway(s). In comparison with the wild‐type (SSI2 NPR1) plants and the npr1‐5 mutant (SSI2 npr1‐5), the ssi2‐1 npr1‐5 double mutant and the ssi2‐1 NPR1 single mutant constitutively express PR genes [PR‐1, BGL2 (PR‐2) and PR‐5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2‐1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1‐dependent pathway. Analysis of ssi2‐1 NPR1 nahG and ssi2‐1 npr1‐5 nahG plants revealed that elevated SA levels were not essential for the ssi2‐1‐conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2‐1‐conferred phenotypes, including PR‐1 expression, and disease resistance. Based on these results, SSI2 or an SSI2‐generated signal appears to modulate signaling of an SA‐dependent, NPR1‐independent defense pathway, or an SA‐ and NPR1‐independent defense pathway.
Gastrodianin‐like mannose‐binding proteins: a novel class of plant proteins with antifungal properties
Authors: Wang Xiaochen , Bauw Guy , Van Damme Els J.M. , Peumans Willy J. , Chen Zhang‐Liang , Van Montagu Marc , Angenon Geert , Dillen Willy
Published: March 2003

The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle. In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms. From these corms, a protein fraction with in vitro activity against plant‐pathogenic fungi has previously been purified. Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported. Four isoforms that encoded two different mature proteins were identified at the cDNA level. Another isoform was detected in sequenced peptides. Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions. Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration. Gastrodianin was found to be homologous to monomeric mannose‐binding proteins of other orchids, of which at least one (Epipactis helleborine mannose‐binding protein) also displayed in vitro antifungal activity. This establishes the gastrodianin‐like proteins (GLIPs) as a novel class of antifungal proteins.
Arabidopsis UVH3 gene is a homolog of the Saccharomyces cerevisiae RAD2 and human XPG DNA repair genes
Authors: Liu Zongrang , Hall Jennifer D. , Mount David W.
Published: March 2003

To identify mechanisms of DNA repair in Arabidopsis thaliana, we have analyzed a mutant (uvh3) which exhibits increased sensitivity to ultraviolet (UV) light, H2O2 and ionizing radiation and displays a premature senescence phenotype. The uvh3 locus was mapped within chromosome III to the GL1 locus. A cosmid contig of the GL1 region was constructed, and individual cosmids were used to transform uvh3 mutant plants. Cosmid N9 was found to confer UV‐resistance, H2O2‐resistance and a normal senescence phenotype following transformation, indicating that the UVH3 gene is located on this cosmid and that all three phenotypes are due to the same mutation. Analysis of cosmid N9 sequences identified a gene showing strong similarity to two homologous repair genes, RAD2 (Saccharomyces cerevisiae) and XPG (human), which encode an endonuclease required for nucleotide excision repair of UV‐damage. The uvh3 mutant was shown to carry a nonsense mutation in the coding region of the AtRAD2/XPG gene, thus revealing that the UVH3 gene encodes the AtRAD2/XPG gene product. In humans, the homologous XPG protein is also involved in removal of oxygen‐damaged nucleotides by base excision repair. We discuss the possibility that the increased sensitivity of the uvh3 mutant to H2O2 and the premature senescence phenotype might result from failure to repair oxygen damage in plant tissues. Finally, we show that the AtRAD2/XPG gene is expressed at moderate levels in all plant tissues.
In vitro interactions between barley TALE homeodomain proteins suggest a role for protein–protein associations in the regulation of Knox gene function
Authors: Müller Judith , Wang Yamei , Franzen Rainer , Santi Luca , Salamini Francesco , Rohde Wolfgang
Published: March 2003

This paper describes two‐hybrid interactions amongst barley homeodomain proteins encoded by the Three Amino acid Loop Extension (TALE) superfamily. The class I KNOX protein BKN3 is shown to homodimerise and to associate with proteins encoded by the class I and II Knox genes BKn‐1 and BKn‐7. Furthermore, JUBEL1 and JUBEL2, two BELL1 homologous proteins, are identified and characterised as interacting partners of BKN3. Differences in the requirements of BKN3 derivatives for interactions with KNOX and JUBEL proteins imply the involvement of overlapping but slightly different domains. This set of results is an example for interactions amongst different classes of plant TALE homeodomain proteins, as previously described for related animal proteins. Apparently identical spatial and temporal expression patterns of BKn‐1, BKn‐3, BKn‐7, JuBel1 and JuBel2, as determined by in situ hybridisation, are compatible with possible interactions of their protein products in planta. Contradictory to the common model, that the transcriptional down‐regulation of certain class 1 Knox‐genes is the prerequisite for organ differentiation, transcripts of all five genes were, similar to Tkn1 and Tkn2/LeT6 of tomato, detected in incipient and immature leaves as well as in meristematic tissues. A characteristic phenotype is induced by the overexpression of JuBel2 in transgenic tobacco plants.
Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides
Authors: Kmiec Eric B. , Johnson Christina , May Gregory D.
Published: March 2003

Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direct gene‐conversion events in vitro through a process involving proteins from several DNA‐repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cell‐free extracts are competent to support oligonucleotide‐directed genetic repair. Using this system, we are studying Arabidopsis DNA‐repair mutants and the role of plant proteins in the DNA‐repair process. Here we describe a method for investigating mechanisms of plastid DNA‐repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucleotides designed to direct the conversion of mutations in antibiotic resistance genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and frameshift mutations directed by both types of modified oligonucleotides. This system enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA‐repair pathways.
Constitutive over‐expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis
Authors: Piao Hai Lan , Lim Jeong Hwa , Kim Soo Jin , Cheong Gang‐Won , Hwang Inhwan
Published: March 2003

GSK3/shaggy‐like protein kinases have been shown to play diverse roles in development and signal transduction pathways in various organisms. An Arabidopsis homologue of GSK3/shaggy‐like kinase, AtGSK1, has been shown to be involved in NaCl stress responses. In order to further clarify the role of AtGSK1 in NaCl stress responses in plants, we generated transgenic Arabidopsis plants that over‐expressed AtGSK1 mRNA. These plants showed enhanced resistance to NaCl stress when assayed either as whole plants or by measurement of root growth on NaCl plates. In addition, AtGSK1 transgenic plants in the absence of NaCl stress showed phenotypic changes, such as accumulation of anthocyanin, that were similar to those observed in wild‐type plants under NaCl stress. Transgenic plants accumulated 30–50% more Na+ than did wild‐type plants when subjected to NaCl stress, and Ca2+ content was increased by 15–30% in the transgenic plants regardless of the NaCl stress level. Northern blotting revealed that AtGSK1 over‐expression induced expression of the NaCl stress‐responsive genes AtCP1, RD29A and CHS1 in the absence of NaCl stress. In addition, AtCBL1 and AtCP1 were super‐induced in the NaCl‐stressed transgenic plants. Taken together, these results suggest that AtGSK1 is involved in the signal transduction pathway(s) of NaCl stress responses in Arabidopsis.
Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase
Authors: Koiwa Hisashi , D'Urzo Matilde Paino , Assfalg‐Machleidt Irmgard , Zhu‐Salzman Keyan , Shade Richard E. , An Haejung , Murdock Larry L. , Machleidt Werner , Bressan Ray A. , Hasegawa Paul M.
Published: March 2003

Two hairpin‐loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain‐like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin‐loop regions were constructed. Approximately 1010−1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin‐loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild‐type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pm) than parental scN, due to its increased association rate constant (2.6 ± 0.09 × 107 m−1sec−1). These results define residues in the first and second hairpin‐loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.
The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase‐like protein sinapoylglucose:choline sinapoyltransferase
Authors: Shirley Amber M. , McMichael Colleen M. , Chapple Clint
Published: March 2003

Serine carboxypeptidase‐like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism. The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions. Characterization of the Arabidopsis sng2 (sinapoylglucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism. The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae. Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, and have increased levels of choline and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT). Cloning of the SNG2 gene by a combination of map‐based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism.
Reduced expression of α‐tubulin genes in Arabidopsis thaliana specifically affects root growth and morphology, root hair development and root gravitropism
Authors: Bao Yiqun , Kost Benedikt , Chua Nam‐Hai
Published: March 2003

Different α‐tubulin cDNA sequences fused in an antisense orientation to a CaMV 35S promoter were introduced into Arabidopsis thaliana plants. Several independent transgenic lines that showed a moderate but clear reduction of α‐tubulin gene expression (TUA6/AS lines) were obtained and phenotypically characterized. Although no apparent abnormalities were detected in the aerial parts of TUA6/AS plants, root development was severely affected. Cells in TUA6/AS root tips were found to contain aberrant microtubular structures, to expand abnormally and to be unable to undergo regular cell division. These cellular defects caused a dramatic radial expansion of the root tip and inhibited root elongation. In addition, TUA6/AS roots displayed ectopic formation of root hairs, root hair branching and a reduced ability to respond to gravitropic challenges. Our results contribute to an improved understanding of the different roles microtubules play during root development and demonstrate that reverse genetics is a powerful tool to analyze cytoskeletal functions during plant organogenesis.
Inhibition of systemic onset of post‐transcriptional gene silencing by non‐toxic concentrations of cadmium
Authors: Ueki Shoko , Citovsky Vitaly
Published: March 2003

Post‐transcriptional gene silencing (PTGS) is an important mechanism for regulation of plant gene expression and virus–plant interactions. To better understand this process, the heavy metal cadmium was identified as a specific inhibitor in two different PTGS systems, constitutive and inducible. The pattern of cadmium‐induced inhibition of PTGS allowed several insights into PTGS development. First, cadmium treatment prevented only systemic but not local onset of PTGS, uncoupling between these two modes of PTGS. Second, non‐toxic, but not toxic, levels of cadmium inhibited PTGS, suggesting induction of a pathway that interferes with PTGS. Third, cadmium effects on PTGS closely paralleled those on the movement of tobamoviruses, suggesting that both processes may share common steps in their systemic transport pathways. Interestingly, these effects of cadmium do not represent a general property of toxic metal ions because two other such elements, that is zinc and aluminum, did not interfere with PTGS and viral systemic movement.
The role of auxin‐binding protein 1 in the expansion of tobacco leaf cells
Authors: Chen Jin‐Gui , Shimomura Shoji , Sitbon Folke , Sandberg Göran , Jones Alan M
Published: March 2003

Tobacco leaf was used to investigate the mechanism of action of auxin‐binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin‐inducible growth were each determined in control tobacco leaves and leaves over‐expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1‐induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1‐induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin‐inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY‐2) in culture displayed two dose‐dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]‐thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor.