bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a disease resistance locus in Arabidopsis that is functionally homologous to the RPG1 locus of soybean
Authors: Innes Roger W. , Bisgrove Sherryl R. , Smith Nadine M. , Bent Andrew F. , Staskawicz Brian J. , Liu Yi‐Chun
Published: March 2003

A new disease resistance locus in Arabidopsis, RPS3, was identified using a previously cloned avirulence gene from a non‐Arabidopsis pathogen. The avrB avirulence gene from the soybean pathogen Pseudomonas syringae pv. glycinea was transferred into a P. syringae pv. tomato strain that is virulent on Arabidopsis, and conversion to avirulence was assayed on Arabidopsis plants. The avrB gene had avirulence activity on most, but not all, Arabidopsis ecotypes. Of 53 ecotypes examined, 45 were resistant to a P. syringae pv. tomato strain carrying avrB, and eight were susceptible. The inheritance of this resistance was examined using crosses between the resistant ecotype Col‐0 and the susceptible ecotype Bla‐2. In F2 plants from this cross, the ratio of resistant:susceptible plants was approximately 3:1, indicating that resistance to P. syringae expressing avrB is determined by a single dominant locus in ecotype Col‐0, which we have designated RPS3. Using RFLP analysis, RPS3 was mapped to chromosome 3, adjacent to markers M583 and G4523, and ≤ 1 cM from another disease resistance locus, RPM1. In soybean, resistance to P. syringae strains that carry avrB is controlled by the locus RPG1. Thus, RPG1 and RPS3 both confer avrB‐specific disease resistance, suggesting that these genes may be homologs.
The carrot secreted glycoprotein gene EP1 is expressed in the epidermis and has sequence homology to Brassica S‐locus glycoproteins
Authors: van Engelen Fred A. , Hartog Marijke V. , Thomas Terry L. , Taylor Brian , Sturm Arnd , van Kammen Ab , de Vries Sacco C.
Published: March 2003

Non‐embryogenic carrot suspension cells secrete the EP1 glycoprotein. A cDNA clone encoding EP1 was isolated and sequenced. The EP1 sequence revealed a region of homology with Brassica S‐locus glycoprotein genes, an Arabidopsis S‐like gene and putative S‐like receptor protein kinases from maize and Arabidopsis. EP1 gene expression, analysed by in situ mRNA localization, was detected in cells located at the surface of the seedling: in the epidermis of the root, the hypocotyl and the cotyledons, in the root cap, and in a crescent of cells in the apical dome of the shoot. In developing seeds, expression was most pronounced in both the inner and outer integument epidermis.
Hydrogen peroxide and lignification
Authors: Olson P.D. , Varner J.E.
Published: March 2003

The production of hydrogen peroxide in plant tissue is demonstrated quickly with a simple histochemical test. The test solution, 50 mM potassium iodide in a 4% (w/v) potato starch suspension, is applied to the cut surface of the tissue to be tested. Hydrogen peroxide oxidizes iodide ions to iodine; the iodine is complexed by the starch to form a blue‐purple color. This test detects hydrogen peroxide production in cells undergoing lignification, i.e. tracheary elements and phloem fibers, and in some epidermal cells. In addition there is a rapid production of hydrogen peroxide in crushed cells. The test is negative under (i) anaerobic conditions and (ii) in the presence of catalase.
Hormonal content and sensitivity of transgenic tobacco and potato plants expressing single rol genes of Agrobacterium rhizogenes T‐DNA
Authors: Schmülling Thomas , Fladung Matthias , Grossmann Klaus , Schell Jeff
Published: March 2003

The expression of single rol genes of the TL‐DNA of Agrobacterium rhizogenes strain A4 in transgenic tobacco (Nicotiana tabacum L.) and potato (Solanum tuberosum L.) plants alters the internal concentrations of, and the sensitivity to, several plant hormones. The levels of immunoreactive cytokinins, abscisic acid, gibberellins and indole‐3‐acetic acid were analysed in tissues of the apical shoots, stems, leaves, roots and undifferentiated callus tissue. The addition of the dominant and morphogenetically active rolA, rolB, or rolC genes resulted in alterations in the content of several hormones. rolC overexpression in particular led to an up to fourfold increase in the content of isopentenyladenosine, dihydrozeatin riboside and trans‐zeatin riboside‐type cytokinins in potato plants. This increase correlated well with different levels of expression of the rolC gene in different transgenic plants. Furthermore it was shown that the dwarfism of P35s‐rolC transgenic tobacco and potato plants is correlated with a 28–60% reduction of gibberellic acid A1 concentration in apical shoots. Exogenous addition of gibberellic acid completely restored stem elongation in P35s‐rolC transgenic plants. Apical shoots of dwarf rolA transgenic tobacco plants also contained 22% less gibberellic acid A1 than control plants, but growth cannot be restored completely by exogenously added gibberellic acid. Similarly, the sensitivity of transgenic tobacco seedlings or callus tissues towards different phytohormone concentrations can be altered by the expression of single rol genes. The overexpression of the rolC gene in seedlings led to an altered response to auxins, cytokinins, abscisic acid, gibberellic acid and the ethylene precursor 1‐aminocyclopropane‐carboxylic acid. The overexpression of the rolB gene in tobacco calli led to necrosis at lower auxin concentrations than in the wild‐type, while other parameters of auxin action, like the induction of cell growth, remained unchanged.
The 20 kDa apoprotein of the CP24 complex of photosystem II: An alternative model to study import and intra‐organellar routing of nuclear‐encoded thylakoid proteins
Authors: Cai Daguang , Herrmann Reinhold G. , Klösgen Ralf Bernd
Published: March 2003

The 20 kDa polypeptide, the apoprotein of the chlorophyll a/b antenna complex CP24 associated with photosystem II, is a remote relative of light‐harvesting complex (LHC) apoproteins and thus a member of the extended cab gene family. LHC apoproteins are poly‐topic integral components of the thylakoid membrane with probably three transmembrane segments which originate in nuclear genes and are made in the cytosol as precursors. They possess exclusively stroma‐targeting transit peptides for import into the organelle and integrate into the thylakoid membrane via uncleaved hydrophobic domains of the mature protein. The CP24 apoprotein displays intriguing structural differences to LHC apoproteins with a potential impact on the routing and targeting processes during biogenesis. In particular, it lacks a pronounced second hydrophobic segment in the mature polypeptide chain found in LHCPs, and carries a transit peptide that is reminiscent of thylakoid‐targeting transit peptides. We have used in organello assays with isolated intact chloroplasts and the authentic precursor of the 20 kDa apoprotein from spinach, or appropriate chimaeric polypeptides consisting of a transit peptide and the mature part of various nuclear‐encoded thylakoid proteins of known location and targeting epitopes, in order to resolve the characteristics of its targeting properties, as well as to determine the contribution of the individual parts of the precursor molecule to its import and subsequent intra‐organellar routing. Our experiments demonstrate that the transit peptide of the CP24 apoprotein is required only for the import of the protein into the organelle. All subsequent steps, such as the integration of the protein into the thylakoid membrane, binding of chlorophyll, assembly into the CP24 complex and migration to the grana lamellae, still take place if the authentic transit peptide is replaced by a targeting signal of a nuclear‐encoded stromal protein.
The kinetics of cytological events during double fertilization in Zea mays L.
Authors: Mòl Rafal , Matthys‐Rochon Elisabeth , Dumas Christian
Published: March 2003

By using a clearing method, the process of double fertilization in Zea mays L. (line A 188) was analysed and the precise sequence of events was determined. The period from pollen tube arrival to gamete fusion was relatively short, possibly less than 1 h. The karyogamy was of premitotic type, and the time from the contact of male and female nuclei to the fusion of male and female nucleoli was about 5 h in the egg cell and 3 h in the central cell. In the central cell, the sperm nucleus fused with either one of the polar nuclei or the secondary nucleus, the latter being observed for the first time in maize. The zygote was in the resting period for 13–16 h before division commenced, changing the cell polarity during karyogamy and the resting period. The primary endosperm nucleus divided immediately after karyogamy was completed in the central cell. The embryo sacs with two‐celled proembryos contained four to eight endosperm nuclei. The timetable of fertilization events could be a standard for further studies on in vitro fertilization at the cytological and molecular levels.
Amplification and expression of the β‐glucuronidase gene in maize plants by vectors based on maize streak virus
Authors: Shen Wen‐Hui , Hohn Barbara
Published: March 2003

Maize streak virus (MSV) is a geminivirus infecting monocotyledonous plants. Its genome consists of one molecule of circular, single‐stranded DNA of 2.7 kb. The viral DNA can be efficiently introduced into maize plants by agroinfection which results in systemic infection. To explore the potential of MSV as a replicative gene vector, a reporter gene coding for β‐glucuronidase (GUS) was inserted into the non‐coding region of the viral genome. The resulting construct (MSV—GUS) of about 5.9 kb was still able to replicate in cells of maize plants although it was unable to induce viral symptoms. This replication led to a five to 10‐fold increase in the mean number of GUS‐positive spots per plant as compared with infections with the GUS gene without the MSV replicon. MSV—D—GUS, which differed from MSV—GUS by the deletion of genes V1 and V2 encoding a putative movement protein and the coat protein, respectively, also replicated and produced even more GUS‐positive spots. In both MSV—GUS‐ and MSV—D—GUS‐infected plants, the GUS‐positive spots were located mainly on the veins of leaves whose primodia had already developed at the time of inoculation and never on the leaves developing later. Thus, neither viral construct was able to move systemically, most probably because the DNAs were too large to be packaged.
The FUS3 gene of Arabidopsis thaliana is a regulator of gene expression during late embryogenesis
Authors: Bäumlein Helmut , Miséra Simon , Luerßen Hartmut , Kölle Kerstin , Horstmann Christian , Wobus Ulrich , Müller Andreas J.
Published: March 2003

The fus3 mutation of Arabidopsis thaliana affects several aspects of seed development. Mutant seeds are desiccation intolerant, viviparous and accumulate anthocyanins. Two major classes of storage proteins, the 12S cruciferins and the 2S albumins, are nearly absent, storage lipids are reduced and their composition is changed. The transcription of heterologous storage protein gene promoters in a fus3 genetic background is similarly affected. Our data suggest that the FUS3 gene is together with other genes like ABI3 and LEC1 central to the regulation of developmental processes during late embryogenesis.
The firefly luciferase gene as a non‐invasive reporter for Dendrobium transformation
Authors: Chia Tet‐Fatt , Chan Yang‐Sun , Chua Nam‐Hai
Published: March 2003

Here a screening method is described for transformed tissues and transgenic plants of Dendrobium (Orchidaceae) using the firefly luciferase gene (luc) as a combined marker/reporter gene. Protocorm‐likebodies (PLB) were bombarded with tungsten particles (1.3 µm) coated with plasmids carrying a 35S‐luc chimeric gene. Three weeks after bombardment 1 mM luciferin was added to the tissues and transformed cells were identified by virtue of their bioluminescence as monitored by low‐light video microscopy in combination with a real‐time photon imaging technique. Transformed tissues were excised, allowed to proliferate, and then subjected to a second round of screening. After three rounds of growth and screening, transformed Dendrobium tissues expressing luciferase were used to generate transgenic plants. Southern blot analysis of several transgenic lines confirmed the integration of the luciferase gene into the orchid genome. It is thought that this procedure can be used for transformation of not only orchids but other species as well.
Antisense RNA inhibition of Rubisco activase expression
Authors: Jiang Cai‐Zhong , Quick W. Paul , Alred Rhu , Kliebenstein Daniel , Rodermel Steven R.
Published: March 2003

Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO2 exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.
bZIP proteins bind to a palindromic sequence without an ACGT core located in a seed‐specific element of the pea lectin promoter
Authors: De Pater Sylvia , Katagiri Fumiaki , Kijne Jan , Chua Nam‐Hai
Published: March 2003

Previously, it has been shown that a trimer of a 22 bp fragment of the promoter of the seed‐specific pea lectin gene confers high expression in seed. Here it is reported that this fragment contains a binding site for the cloned basic domain/leucine zipper (bZIP) proteins TGA1a and Opaque‐2 (02). Gel shift assays, DNasel footprinting and methylation interference assays using purified TGA1a were performed to determine whether additional binding sites are present in the psl promoter. Within the 469 bp upstream region only one other TGAla binding site was found, which is much weaker than the one present in the 22 bp element. Both O2 and TGAla bound to the odd base palindromic C‐box sequence, ATGAGTCAT, present within the 22 bp fragment. The 22 bp fragment also contains the sequence CACGTA, which contains the ACGT core usually found in binding sites for bZIP proteins. However, this sequence did not significantly contribute to bZIP protein binding. The binding affinity of TGAla for the odd base palindromic sequence was low relative to a high‐affinity C‐box (ATGACGTCAT). By contrast, O2 strongly bound to the odd base C‐box; the affinity was comparable with that for high‐affinity G‐(GACACGTGTC) and C‐boxes. It is concluded that the presence of an ACGT core sequence is not a prerequisite for high‐affinity binding of O2.
Expression of a maize proteinase inhibitor gene is induced in response to wounding and fungal infection: systemic wound‐response of a monocot gene
Authors: Cordero Maria José , Raventós Dora , San Segundo Blanca
Published: March 2003

The isolation and characterization of cDNA and genomic clones encoding a proteinase inhibitor protein (MPI) in maize is reported. Accumulation of the MPI mRNA is induced in response to fungal infection in germinating maize embryos. The expression pattern of the MPI gene, in healthy and fungal infected maize tissues, was examined and compared with the expression pattern of a gene that codes for a pathogenesis‐related protein (the PRms protein) from maize. These two genes are induced by fungal infection, however different signals trigger their activation. Accumulation of the proteinase inhibitor mRNA is more a consequence of the wound produced by the penetration and colonization of the host tissues by the pathogen, than the result of a direct molecular recognition of the pathogen by the plant, as is the case for the induction of the PRms gene. Wounding, or treatment with abscisic acid or methyl jasmonate, stimulate MPI mRNA accumulation, but not PRms mRNA accumulation. Local and systemic induction of the MPI gene expression in response to wounding occurs in maize plants. To the authors' knowledge, this is the first example of a gene from a monocotyledonous species that clearly shows a systemic wound response. The possible functional implications for the existence of different signal transduction pathways that simultaneously activate a battery of defense mechanisms against potential pathogens are discussed.
Cloning and expression of soluble epoxide hydrolase from potato
Authors: Stapleton Andrew , Beetham Jeffrey K. , Pinot Franck , Garbarino Joan E. , Rockhold David R. , Friedman Mendel , Hammock Bruce D. , Belknap William R.
Published: March 2003

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.
The late‐flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis
Authors: Lee Ilha , Michaels Scott D. , Masshardt Amy S. , Amasino Richard M.
Published: March 2003

The late‐flowering phenotype of mutations in the LUMINIDEPENDENS (LD) gene and the late flowering caused by the naturally occurring dominant gene FRIGIDA (FRI) are suppressed in the Landsberg erecta (Ler) strain of Arabidopsis thaliana. This suppression is dependent on a locus on chromosome 5 designated FLC. Of the ecotypes tested, only the Ler strain contains the suppressor allele of FLC; ld mutations and FRI cause late flowering in the other genetic backgrounds. The allele at FLC also has a moderate effect on flowering time in the absence of FRI or ld mutations. The flowering time effects of FLC are gene dosage dependent.
Nod signal‐induced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications of the lipochitooligosaccharide
Authors: Felle Hubert H. , Kondorosi Éva , Kondorosi Ádám , Schultze Michael
Published: March 2003

Lipochitooligosaccharide Nod signals are important determinants of host specificity in the Rhizobium‐legume symbiosis. The most rapid response of plant cells to the R. meliloti Nod signal NodRm‐IV(C16:2,S) reported so far is the depolarization of the plasma membrane potential in alfalfa root hairs. In order to investigate whether this response may be part of a specific signal transduction cascade involved in the nodulation process, its specificity was studied with respect to host‐specific modifications of the lipochitooligosaccharide. Five different Nod factors displaying different degrees of activity in inducing root hair deformation or cortical cell divisions on alfalfa were tested. The ability of the Nod factors to elicit plasma membrane depolarization correlated well with their activity in the bioassays. Removal of the sulfate group (NodRm‐IV(C16:2)) led to inactivation of the Nod factor. An increase in the length of the chitooligosaccharide backbone (NodRm‐V(C16:2,S)) or saturation of the acyl chain (NodRm‐IV(C16:0,S)) resulted in severely reduced activity. In contrast, the O‐acetyl group at the non‐reducing terminus in NodRm‐IV(Ac,C16:2,S), which confers substantially higher activity in long‐term bioassays, did not enhance plasma membrane depolarization significantly in comparison with the non‐O‐acetylated factor. Thus, the rapid plasma membrane response is differentially sensitive to various structural motifs of the lipochitooligosaccharide. These data suggest that the different substituents modifying the basic Nod factor structure may have distinct functions, not all of them contributing to the interaction with a putative receptor in root hair cells. However, the overall specificity of the membrane depolarization for the cognate Nod factors raises the possibility that it is involved in a Nod signal transduction pathway.
The regulatory regions of the rice tungro bacilliform virus promoter and interacting nuclear factors in rice (Oryza sativa L.)
Authors: Yin Yanhai , Beachy Roger N.
Published: March 2003

The Rice Tungro Bacilliform Virus (RTBV) promoter confers phloem‐specific gene expression in transgenic rice plants. A series of promoter deletion mutants were fused with the Escherichia coliβ‐glucuronidase A (uidA) reporter gene and introduced into transgenic rice plants. The RTBV promoter confers substantially stronger expression in shoots than in roots. A fragment of the promoter comprising nucleotides −164 to +45 relative to the transcriptional start site contains sufficient information for phloem‐specific gene expression. Within this region, nucleotides −164 to −43 were essential for promoter function since deletion of this fragment dramatically reduced promoter activity. Gel‐retardation assays identified two groups of rice nuclear factors (RNFG1 and RNFG2) that bind to the −164 to +45 promoter fragment. Competition and DNasel footprinting experiments indicated that RNFG1 bound to nucleotides −3 to +8 (Box I) while RNFG2 bound to nucleotides −53 to −39 (Box II). Interactions between the two groups of factors were observed. In addition, we found differences in the binding of nuclear factors from shoots versus from roots, in agreement with the different activities of the promoter in these two organs. It is proposed that binding of RNFG1 and RNFG2 between nucleotides −164 to +45 is essential for the tissue‐specific expression of this promoter.
Recombinant stigmatic self‐incompatibility (S‐) protein elicits a Ca2+ transient in pollen of Papaver rhoeas
Authors: Franklin‐Tong Vernonica E. , Ride Jon P. , Franklin F. Christopher H.
Published: March 2003

Evidence for Ca2+ signalling in pollen during the self‐incompatibility (SI) response in Papaver rhoeas L. has been presented previously. However, it was not known whether the S‐protein alone could act as an elicitor of the response or whether the presence of other stigmatic components was required, since relatively crude stigmatic extracts had been used. The S1 gene has since been cloned and its product expressed in Escherichia coli has been shown to exhibit biological activity. In this paper it is reported that the recombinant protein (S1e) elicits a transient rise in [Ca2+]i in incompatible pollen. The Ca2+ signal appears indistinguishable from that elicited by S‐gene products partially purified from plant extracts in terms of both its timing and spatial distribution. Pollen tube growth is arrested directly after the rise in [Ca2+]i.

The results provide direct evidence that the S‐protein alone acts as an elicitor which triggers the Ca2+ signal for the pollen SI response. In addition, it is now clear that the recombinant S‐protein does not require several post‐translational processing events which take place in the plant to act as an elicitor. With respect to the spatial distribution of the Ca2+ transient, data are presented which correlate the localized rise in intracellular Ca2+ ([Ca2+]i) with the ‘nuclear complex’ and the endoplasmic reticulum which is associated with this region.
PvAlf, an embryo‐specific acidic transcriptional activator enhances gene expression from phaseolin and phytohemagglutinin promoters
Authors: Bobb Andrew J. , Eiben Hans Georg , Bustos Mauricio M.
Published: March 2003

Mutations in Vp1 and ABI3 genes of maize and Arabidopsis lead to drastic reductions in the synthesis of a subset of maturation‐specific products including seed storage proteins. Gene Phaseolus vulgarisABI3‐like factor (PvAlf), whose protein product is similar to the ABI3 and Vp1 proteins, has been cloned. Here, it is shown that PvAlf positively regulates phaseolin and phytohemagglutinin (PHA‐L) promoters in particle bombardment assays. PvAlf mRNA expression is embryo‐specific and temporally complex. PvAlf mRNA abundance is highest during two periods (9–14 and 22–35 days after flowering) that precede the onsets of seed maturation and seed abscission, respectively. Protein fusions with the DNA‐binding domain of the yeast transcriptional activator GAL4 demonstrated that the N‐terminal 243 amino acids of PvAlf function as a strong transcriptional activation domain in yeast (Saccharomyces cerevisiae) and plant cells. This domain consists of a central cluster rich in serine, threonine and proline (STP cluster) flanked by two negatively charged regions containing bulky hydrophobic residues similar to acidic activation domains of Vp1, the herpes simplex virus virion protein VP16 and transcription factors GCN4 and HAP4 from yeast. Together with the Vp1 proteins of maize and rice and ABI3, PvAlf constitutes a class (Vp1/ABI3‐like factors or VAlfs) of regulatory factors that are pivotal for the promotion of seed maturation and dormancy in angiosperms.
Induction of intrachromosomal homologous recombination in whole plants
Authors: Puchta Holger , Swoboda Peter , Hohn Barbara
Published: March 2003

The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β‐glucuronidase marker gene. Recombination frequencies were enhanced severalfold by DNA damaging agents like UV‐light or MMS (methyl methanesulfonate). Applying 3‐methoxybenzamide (3‐MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed.
Differential in vitro DNA binding activity to a promoter element of the gn1β‐1,3‐glucanase gene in hypersensitively reacting tobacco plants
Authors: Alonso Elena , De Carvalho Niebel Fernanda , Obregón Patricia , Gheysen Godelieve , Inzé Dirk , Van Montagu Marc , Castresana Carmen
Published: March 2003

In a hypersensitive reaction to pathogen infection, expression of the β‐1,3‐glucanase gn1 gene is induced in cells surrounding the necrotic lesions. The 5′‐flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response. Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the β‐glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression. Promoter sequences to −138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv. syringae inoculation. It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified. Among the binding sequences characterized, the promoter region extending from −250 to −217 bp contained the DNA motif ‐GGCGGC‐ found to be conserved in most if not all promoters of genes encoding pathogenesis‐related basic proteins. The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.
Altered regulation of tomato and tobacco pigmentation genes caused by the delila gene of Antirrhinum
Authors: Mooney Mark , Desnos Thierry , Harrison Kate , Jones Jonathan , Carpenter Rosemary , Coen Enrico
Published: March 2003

Delila (del), a regulatory gene of Antirrhinum, alters anthocyanin pigmentation when introduced into two Solanaceous species. In tomato, pigmentation in vegetative tissues is strongly increased while in tobacco, intensification of pigment is restricted to flowers. Although del transcripts are ubiquitous in the transgenic plants, transcript levels of host anthocyanin biosynthetic genes are only increased in pigmented regions. Constructs carrying the maize transposon Ac, inserted at the 3′ end of the 35S promoter prior to the start of translation of the del gene, give variegated leaves in tomato, suggesting that del acts cell‐autonomously and that it may be used as a phenotypic marker. In Arabidopsis, del has no strong phenotypic effects, suggesting that del may not be able to function effectively in all plant hosts.
Saturating the genetic map of Arabidopsis thaliana with embryonic mutations
Authors: Franzmann Linda H. , Yoon Elizabeth S. , Meinke David W.
Published: March 2003

One goal of the Arabidopsis genome project is to identify every gene with an essential function in growth and development. Towards that end, the results are reported here of a mapping project designed to enhance the linkage map of Arabidopsis and establish a valuable resource of mutations in essential genes with known map locations. Embryo‐defective (emb) mutations were chosen because they represent the most common heritable defect identified following mutagenesis in Arabidopsis. Multiple marker lines with easily scored phenotypes were constructed to facilitate mapping efforts. Recombination data were obtained for 169 mutants defective in embryo‐genesis. The chromosomal locations of 110 emb genes are presented in this report. Twenty‐six of these genes are tagged with T‐DNA. Nine other mutants isolated following seed transformation appear to contain chromosomal translocations. Another 31 mutant genes in the collectiohave been assigned to a linkage group but not yet placed on the map. Nineteen examples of duplicate alleles have also been found. This is consistent with the estimate that approximately 500 genes readily mutate to give an embryo‐defective phenotype in Arabidopsis. With continued progress, it may therefore be possible to approach saturation for this important class of mutations. Molecular cloning of these genes should be facilitated by identifying cDNAs and genomic sequences that map to similar locations.
Targeted disruption of the TGA3 locus in Arabidopsis thaliana
Authors: Miao Zhong‐He , Lam Eric
Published: March 2003

A major drawback to study gene functions in plant systems is the lack of an effective gene knockout strategy. With a large number of plant genes isolated and the accelerating pace by which this collection is growing, the need for their functional analyses at the whole plant level has become increasingly urgent. Here evidence is reported for the first successful disruption of a non‐selectable gene in Arabidopsis thaliana by creating a mutant of the TGA3 locus via targeted insertion of the bacterial neo gene conferring kanamycin (Km) resistance. A β‐glucuronidase (GUS) expression unit outside the region of homology was used as a screenable marker to distinguish homologous recombination events from those of ectopic insertions. PCR amplification coupled with Southern blot screening identified two putative homologous recombination events among 2580 Kmr calli. One callus line was subsequently isolated and the structure of the targeted TGA3 allele confirmed by Southern blot analyses. This study demonstrates the feasibility of targeting a non‐selectable locus in Arabidopsis. Combined with future improvements in negative selection strategies and efficient, transformation methodologies, gene replacement studies in plants could become a routine technique.
Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells
Authors: Felix Georg , Boller Thomas
Published: March 2003

Suspension‐cultured cells of Lycopersicon peruvianum L. reacted to the presence of mechanically damaged cells with a transient alkalinization of their culture medium. This response resembled the alkalinization observed after treatment with fungal signal molecules such as chitin fragments and ergosterol or after application of the protein phosphatase inhibitor calyculin A. When compounds implicated in wound signalling were tested, the 18 amino acid peptide systemin was found to be a potent inducer of the alkalinization response, with a half‐maximal activity at concentrations of ∼100 pM. The decrease in extracellular H+ was paralleled by an increase of K+, and induction of both ion fluxes was blocked by the protein kinase inhibitor K‐252a. Systemin also caused rapid increases in the activities of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase and phenylalanine ammonia‐lyase, two other responses commonly observed in cells treated with elicitors. The systemin analogue systemin‐Ala17, a reported systemin antagonist in the induction of proteinase inhibitors in tomato plants, provoked a much weaker alkalinization response and did not induce ACC synthase at all. When applied together with authentic systemin, this analogue antagonized induction of both responses, indicating that the perception system for systemin had very similar properties in the L. peruvianum cells as in tomato plants. In conclusion, suspension‐cultured L. peruvianum cells provide a convenient and highly sensitive system to study elements of wound response and, in particular, systemin perception.
Genetic dissection of the co‐expression of genes encoding the two isoforms of oleosins in the oil bodies of maize kernel
Authors: Lee Keunmyoung , Ratnayake Chandra , Huang Anthony H.C.
Published: March 2003

Oleosins are abundant structural proteins on the surface of intracellular oil bodies in seeds. In many maize (Zea mays L.) inbreds, there are three oleosins, OLE18, OLE17, and OLE16, termed according to their apparent molecular weight, which are present in the proportional amounts of about 1:1:2 in isolated oil bodies. In some inbreds, OLE18 and OLE17 occur as molecular weight variants with a molecular weight difference of 1000 or less. In inbreds CM555 and FR2, OLE18 and OLE17, respectively, are absent; the respective genes ole18 and ole17 in the inbreds are present, but are transcriptionally inactive. The F1 of CM555 x FR2 possesses both OLE18 and OLE17, as expected from the inheritance of ole18 and ole17 genes. In all inbreds examined, including CM555, FR2, and their F1 hybrids, and in both the diploidic embryos and triploidic aleurone layers, the quantity of OLE18 and/or OLE17 equals that of OLE16. Since OLE18 and OLE17 are close members of the high‐molecular weight (H) oleosin isoform whereas OLE16 belongs to the low‐molecular weight (L) oleosin isoform, the results indicate the presence of equal amounts of the H and L isoforms in the oil bodies. The equality suggests the occurrence of a hetero‐dimer, or ‐multimer, of the two isoforms. To probe the control mechanism for such a hetero‐mer formation, CM555, FR2, and their F1 hybrids, as well as Mo17 (a common inbred) have been used to analyze oleosin gene copy number, nuclear run‐off transcription activity, transcript amount, and oleosin protein content. It has been determined that equal levels of H and L oleosin isoforms accumulate in oil bodies irrespective of the dosage of functional H and L genes. Therefore, the control of equality is not the consequence of the competition among the active H isoform genes for transcriptional machineries, but occurs at the post‐transcriptional level, presumably at translation and/or through oleosin interaction (formation of hetero‐mer) and stability.
Induction and expression of seed‐specific promoters in Arabidopsis embryo‐defective mutants
Authors: Devic Martine , Albert Sylvie , Delseny Michel
Published: March 2003

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild‐type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1‐uidA chimeric gene, representative of the maturation programme and the Em 1‐uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em 1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.
The promoter of a H2O2‐inducible, Arabidopsis glutathione S‐transferase gene contains closely linked OBF‐ and OBP1‐binding sites
Authors: Chen Wenqiong , Chao Gerald , Singh Karam B.
Published: March 2003

Glutathione S‐transferases (GSTs) are a family of multi‐functional enzymes that play critical roles in the detoxification of xenobiotics and the protection of tissues against oxidative damage. GSTs are important enzymes in plant responses to a number of environmental stresses including herbicides and pathogen attack. Ocs elements are a group of related, 20 bp promoter elements which have been exploited by some plant pathogens to express genes in plants. Ocs elements have also been found to regulate the expression of a plant GST promoter. An Arabidopsis GST gene, called GST6 has been isolated. GST6 expression is under tissue‐specific control and is induced following treatment with auxin, salicylic acid and H2O2. The GST6 promoter contains a binding site for two Arabidopsis ocs element binding factors (OBF), that has some sequence homology to ocs element sequences. Interestingly, OBP1 (OBF binding protein), a DNA‐binding protein that was isolated by screening an Arabidopsis cDNA library with a labeled OBF protein as a probe, binds next to the OBF‐binding site on the GST6 promoter. OBP1 was able to significantly stimulate the binding of OBF proteins to the GST6 promoter, raising the possibility that interactions between the OBP1 and OBF proteins may be important for GST6 expression.
Li+‐regulated 1‐aminocyclopropane‐1‐carboxylate synthase gene expression in Arabidopsis thaliana
Authors: Liang Xiaowu , Shen Nancy F. , Theologis Athanasios
Published: March 2003

In Arabidopsis thaliana, 1‐aminocyclopropane‐1‐carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li+, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol‐phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)‐induced accumulation of the ACS2 mRNA. The effects of Li+ on the expression of ACS5 and ACS2 are specific, dose‐dependent, and can be reversed by Ca2+ and mimicked by the protein kinase inhibitor K‐252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5‐triphosphate (IP3)‐mediated Ca2+ mobilization. Since plants contain no Li+, the cation appears to unmask pre‐existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.
The elongation of amylose and amylopectin chains in isolated starch granules
Authors: Denyer Kay , Clarke Belinda , Hylton Christopher , Tatge Helma , Smith Alison M.
Published: March 2003

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule‐bound isoform of starch synthase ‐ GBSSI ‐ catalyses the synthesis of amylose in vivo, 14C from ADP[14C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of 14C into amylose. These compounds were rendered inactive by treatment of the extracts with α‐glucosidase, suggesting that they were malto‐oligosaccharides. Consistent with this idea, provision of pure malto‐oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of 14C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild‐type peas and lam mutant peas which lack GBSSI showed that this effect of malto‐oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.
Molecular cloning of a new receptor‐like kinase gene encoded at the Lr10 disease resistance locus of wheat
Authors: Feuillet Catherine , Schachermayr Gabriele , Keller Beat
Published: March 2003

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near‐isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor‐like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S‐domain glycoproteins and receptor‐like kinases were also found in LRK10, suggesting that LRK10 and S‐domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.
Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice
Authors: Meijer Annemarie H. , Scarpella Enrico , van Dijk Erwin L. , Qin Ling , Taal Anke J.C. , Rueb Saskia , Harrington Sandra E. , McCouch Susan R. , Schilperoort Rob A. , Hoge J. Harry C.
Published: March 2003

This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain—leucine zipper (HD‐Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two‐hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD‐Zip protein. This suggests that protein—protein interactions may also occur between different HD‐Zip proteins in rice, which would provide enormous versatility for generating specific gene‐control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA‐binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N‐terminal region of Oshox1, preceding the homeodomain.
Tryptophan decarboxylase is encoded by two autonomously regulated genes in Camptotheca acuminata which are differentially expressed during development and stress
Authors: López‐Meyer Melina , Nessler Craig L.
Published: March 2003

Camptothecin (CPT) is a valuable anti‐cancer monoterpene alkaloid produced by the Chinese tree Camptotheca acuminata. Tryptophan decarboxylase (TDC) supplies tryptamine for the indole moiety of CPT and its derivatives, and is considered a key step in monoterpene indole alkaloid biosynthesis as it links primary and secondary metabolism. This report describes the isolation and characterization of tdc1 and tdc2, two autonomously regulated TDC genes from Camptotheca. When expressed in Escherichia coli, the products of each gene could decarboxylate tryptophan, but were inactive against tyrosine, phenylalanine and 3,4‐dihydroxyphenylalanine (dopa). tdc1 was developmentally regulated, having its highest expression level in the apex, young stem and bark, tissues which also contain the highest levels of CPT. Expression of tdc1 also increased during seedling development and was correlated with alkaloid accumulation during germination. tdc2 expression was induced in Camptotheca leaf discs and cell suspension cultures treated with fungal elicitor or methyl jasmonate, treatments which did not affect tdc1 expression. Unlike tdc1, tdc2 expression was not detected in any unstressed Camptotheca tissues nor in developing seedlings. These data suggest that tdc1 may be part of a developmentally regulated chemical defense system in Camptotheca, while tdc2 serves as part of a defense system induced during pathogen challenge.