bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)
Authors: Zrenner Rita , Salanoubat Marcel , Willmitzer Lothar , Sonnewald Uwe
Published: March 2003

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T‐type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber‐specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40‐fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.
Tyrosine phosphatase signalling in a lower plant: cell‐cycle and oxidative stress‐regulated expression of the Chlamydomonas eugametos VH‐PTP13 gene
Authors: Haring Michel A. , Siderius Marco , Jonak Claudia , Hirt Heribert , Walton Kevin M. , Musgrave Alan
Published: March 2003

The first evidence for tyrosine phosphatase signalling pathways in plants is presented by characterizing a putative protein tyrosine phosphatase gene from the unicellular green alga Chlamydomonas eugametos. This cDNA, referred to as VH‐PTP13, contains an open reading frame specifying a protein with a molecular weight of 30.3 kDa, that has significant homology with a distinct group of dual‐specificity phosphatases. The highest homology is found with CL‐100, a human stress‐response gene that regulates MAPkinase activity. The purified VH‐PTP13 protein expressed in E. coli had phosphatase activity and inactivated MAPkinases from alfalfa and tobacco. Non‐dividing C. eugametos gametes did not express the VH‐PTP13 gene whereas synchronously dividing vegetative cells only expressed VH‐PTP13 in the early G1‐phase of the cycle, implying a function there. When vegetative cells were subjected to oxidative stress, expression of the VH‐PTP13 gene was strongly induced, analogous to the human CL‐100 gene. Its potential role in plant signalling pathways is discussed.
Early fertilization events in the sexual and aposporous egg apparatus of Pennisetum ciliare (L.) Link
Authors: Vielle J.‐Ph. , Burson B.L. , Bashaw E.C. , Hussey M.A.
Published: March 2003

While detailed descriptions exist on the fine structural characteristics of double fertilization in sexual angiosperms, few studies have described fertilization in apomictic species. The results of this study with buffelgrass (Pennisetum ciliare (L.) Link syn=Cenchrus ciliaris L.) show that differences exist between the early fertilization events in sexual and apomictic (aposporous) female gametophytes. In unpollinated sexual female gametophytes, the egg cell did not show signs of parthenogenetic activity. In contrast, autonomous egg embryony was observed in unpollinated aposporous female gametophytes. Compared with the sexual genotype, degeneration of the synergids in the aposporous female gametophyte was precocious and rapid. During the first 4 h after pollination, the chalazal end of the sexually functional egg cell remained covered by the plasma membrane. In the aposporous embryo sac, a cell wall covered the plasma membrane of the egg cell several hours before penetration of the pollen tube into the female gametophyte.
Induction of ferritin synthesis in maize leaves by an iron‐mediated oxidative stress
Authors: Lobréaux Stéphane , Thoiron Séverine , Briat Jean‐François
Published: March 2003

Oxygen is essential for aerobic life, although reactive oxygen intermediates can be highly toxic to cells. Superoxide radicals and hydrogen peroxide become particularly harmful in vivo due to their reactivity with transition metals like iron, producing hydroxyl radicals, which are involved in DNA mutagenesis and lipid peroxidation. Control of the intracellular concentration of transition metals can prevent hydroxyl radical formation, protecting cells against oxidative damage. The iron storage protein ferritin plays an important role in these processes, and it has been shown that its synthesis is inducible by iron‐overload in plants. In this paper, the induction of ferritin synthesis in response to iron‐overload is investigated using de‐rooted maize plantlets. In this system, a rapid increase of iron concentration in the leaves is observed, leading to ferritin mRNA and subunit accumulation. Using the vp2 maize mutant, it is demonstrated that abscisic acid is not involved in this response, in contrast to previous results obtained using plants induced under hydroponic culture. In de‐rooted plantlets, ferritin transcript accumulation is inhibited when plants are co‐treated by iron and anti‐oxidant reagents like N‐acetyl cysteine or reduced glutathione. Furthermore, hydrogen peroxide treatment induced a dose‐dependent ferritin mRNA accumulation at a low concentration of iron. It can be concluded from these results that reactive oxygen intermediates are involved in the pathway leading to ferritin synthesis in response to iron in de‐rooted maize plantlets. Ferritin emerges, therefore, as an important component of the oxidative stress response in plants.
A molecular study of root‐knot nematode‐induced feeding sites
Authors: Van der Eycken Walter , de Almeida Engler Janice , Inzé Dirk , Van Montagu Marc , Gheysen Godelieve
Published: March 2003

In a compatible interaction, root‐knot nematodes (Meloidogyne) induce a sophisticated feeding site shortly after they have penetrated the plant root. The feeding site contains metabolically highly active giant cells. To gain insight into the molecular aspects that are typical for giant cells, a cDNA library from tomato roots infected with Meloidogyne incognita was differentially screened to find induced genes. Among the genes identified, two extensin genes (Lemmi8 and Lemmi11) and a Lea‐like gene (Lemmi9) were studied further.
Induction of systemic acquired resistance in cucumber by Pseudomonas syringae pv. syringae 61 HrpZPss protein
Authors: Strobel N.E. , Ji C. , Gopalan S. , Kuc J.A. , He S.Y.
Published: March 2003

Systemic acquired resistance (SAR) is an inducible plant defense response and is effective against a broad spectrum of pathogens. Biological induction of SAR usually follows plant cell death resulting from the plant hypersensitive response (HR) elicited by an avirulent pathogen or from disease necrosis caused by a virulent pathogen. The elicitation of the HR and disease necroses by pathogenic bacteria is controlled by hrp genes. Previously, it was shown that the Pseudomonas syringae 61 (Pss61) HrpZPss protein (formally harpinPss) elicited the HR in plants. In this study, it is shown that HrpZPss induced SAR in cucumber to diverse pathogens, including the anthracnose fungus (Colletotrichum lagenarium), tobacco necrosis virus and the bacterial angular leaf spot bacterium (P. s. pv. lachrymans). A hrpH mutant of Pss61, which is defective in the secretion of HrpZPss and, possibly, other protein elicitors, failed to elicit SAR. Pathogenesis‐related (PR) proteins, including peroxidase, β‐glucanase and chitinases, were induced in cucumber plants inoculated with Pss61, C. lagenarium or HrpZPss. The induction patterns of PR proteins by HrpZPss and Pss61 were the same, but were different from that induced by C. lagenarium. Interestingly, the hrpH mutant induced two of the three identified PR proteins, despite its failure to induce SAR. These results suggest that proteinaceous elicitors, such as HrpZPss, that traverse the bacterial Hrp secretion pathway are involved in the biological induction of SAR and that at least some PR proteins can be induced by bacterial factors that are not controlled by hrp genes.
Perception of free cutin monomers by plant cells
Authors: Schweizer Patrick , Felix Georg , Buchala Antony , Müller Celia , Métraux Jean‐Pierre
Published: March 2003

Enzymatic degradation of plant cuticles by fungal pathogens results in the release of free cutin monomers. The hypothesis that free cutin monomers are recognized by plant cells as endogenous stress‐related signals was tested in a model system consisting of cultured potato cells. Addition of cutin monomers in the micromolar range induced a transient alkalinization of the culture medium, similar to that observed with chitin or chitotetraose that served as positive control. The cutin monomers tested varied considerably in their potential to induce alkalinization, the most and least active compounds being cis‐9,10‐epoxy‐18‐hydroxystearic acid and palmitic acid, respectively. n,16‐dihydroxypalmitic acid (n = 8, 9 or 10) was found to be the major component of potato leaf cuticle and was among the most active cutin monomers. 9,10‐Dihydroxystearic acid, an analogue of the cutin monomer threo‐9,10,18‐trihydroxystearic acid, exhibited biological activity in a stereoselective manner, only the naturally occurring threo‐stereoisomer inducing a rapid and strong alkalinization response. Alkalinization of the culture medium was inhibited by addition of the protein‐kinase inhibitor K‐252a, and the onset of alkalinization was paralleled by changes in phosphorylation of specific proteins. The active cutin monomers also stimulated the production of the plant stress hormone ethylene and activated defence‐related genes at the mRNA level. The data provide evidence for a role of enzymatic breakdown products of plant cuticles as a new class of endogenous signal molecules.
An S‐locus‐related gene in Arabidopsis encodes a functional kinase and produces two classes of transcripts
Authors: Tobias Christian M. , Nasrallah June B.
Published: March 2003

ARK1 is a vegetatively expressed receptor protein kinase gene isolated from Arabidopsis thaliana based on its sequence similarity to Brassica genes involved in pollen‐stigma signaling and the self‐incompatibility response. This paper shows that the kinase domain of ARK1 auto‐phosphorylates on serine and threonine residues when expressed as a recombinant fusion protein. ARK1 produces a 2.9 kb transcript encoding a transmembrane receptor protein kinase and a 1.4 kb transcript encoding the receptor domain alone. Constitutive high‐level expression of ARK1 transcripts in transgenic Arabidopsis resulted in severe stunting and also disrupted normal cellular expansion and differentiation.
Isolation and developmental expression of male reproductive organ‐specific genes in a dioecious campion, Melandrium album (Silene latifolia)
Authors: Matsunaga Sachihiro , Kawano Shigeyuki , Takano Hiroyoshi , Uchida Hidenobu , Sakai Atsushi , Kuroiwa Tsuneyoshi
Published: March 2003

The dioecious campion Melandrium album (Silene latifolia) has sex chromosomes, and male and female plants form different unisexual flowers. The very youngest buds have both sex organs but the development of either a gynoecium in the male bud or stamens in the female bud is arrested as the bud matures. To examine the molecular mechanism of male‐specific differentiation, a cDNA library from male buds was differentially screened with cDNA from male and female buds. Four male reproductive organ‐specific cDNAs (corresponding to genes designated MROS1, MROS2, MROS3 and MROS4, respectively) were isolated. Northern blot analysis showed that the transcripts of MROS genes were expressed only in male reproductive organs. The transcripts of MROS3 and MROS4 accumulated in the early buds, while transcripts of MROS1 and MROS2 accumulated in the late buds and in the anthers of open flowers. The putative MROS2 protein has glycine‐rich regions, while the putative MROS3 protein exhibits no similarity to known proteins. Genomic Southern blot analysis showed that male and female genomes included MROS2 and MROS3 genes. Non‐radioactive in situ hybridization revealed the localization of the transcripts of MROS2 and MROS3 in male buds. MROS3 mRNA was distributed throughout the tapetum. After MROS2 mRNA had accumulated in the epidermis and the endothecium near the stomium, the accumulation of the mRNA increased significantly in the mature pollen. MROS2 seems to be a novel anther‐specific gene that functions in dehiscence of anthers and the maturation of pollen.
Fluorescent imaging of GUS activity and RT‐PCR analysis of gene expression in the shoot apical meristem
Authors: Fleming Andrew J. , Manzara Thianda , Gruissem Wilhelm , Kuhlemeier Cris
Published: March 2003

The use of promoter‐reporter gene constructs in transgenic plants is a powerful tool in the analysis of gene expression which can, however, be limited in the resolution of small structures, such as the apical meristem. This paper reports on a fluorescent imaging technique for the analysis of GUS reporter gene expression to cellular resolution in the apical meristem of tomato. Using this technique in combination with an RT‐PCR analysis of RBS gene‐specific transcript levels, it is shown that: 5′ upstream sequences of RBCS genes are sufficient to mimic the pattern of transcripts revealed by in situ hybridisation (no expression in the apical meristem, high expression in the initiated leaf primordia); the genes RBCS2, RBCS3A and RBCS3B are transcriptionally activated upon primordium initiation with transcripts for RBCS1 and RBCS3C accumulating later in leaf development; and that RBCS promoter activity cannot be induced in the apical meristem by light, an environmental signal which elevates RBCS transcript level in other aerial parts of the plant. These data provide a detailed picture of the complex transcriptional events occurring on leaf initiation and the establishment of the photosynthetic machinery; they describe two complementary techniques which allow the analysis of such complex events at the tissue and cellular level; and they characterize an in vivo assay system which can be used to analyse the factors involved in the initiation and maintenance of gene expression patterns in the apical meristem.
Induction of cdc2a and cyc1At expression in Arabidopsis thaliana during early phases of nematode‐induced feeding cell formation
Authors: Niebel Andreas , De Almeida Engler Janice , Hemerly Adriana , Ferreira Paulo , Inzé Dirk , Van Montagu Marc , Gheysen Godelieve
Published: March 2003

Root‐knot and cyst nematodes are plant parasites that induce large multinucleated feeding cells in the roots of their hosts. Cytological observations have shown that root‐knot nematodes induce giant cells by cycles of mitosis without cytokinesis whereas cyst nematodes provoke cell wall degradation leading to the formation of a large syncytium. This study was intended to characterize and compare the ability of both types of nematodes to induce progression through the cell cycle. For this purpose, the expression, upon nematode infection, of two cell cycle markers was followed: a marker for division competence, the cyclin‐dependent kinase cdc2a and a marker for the G2 phase, the mitotic cyclin cyc1At. For both types of nematodes, transcriptional activation of these markers was correlated with early phases of feeding cell development. Using molecular markers, it was thus possible to confirm and extend the observations of repeated mitosis in root‐knot nematode‐induced giant cells. Surprisingly, promoter activation of both cdc2a and cyc1At markers was also found upon cyst nematode infection, in feeding cells in which mitosis has not been clearly reported. Incorporation of tritiated thymidine in these syncytia confirms that they progress through the S phase of the cell cycle. One possibility is that cyst nematodes induce cycles of DNA endoreduplication shunting the M phase. Despite obvious differences in ontogeny, common molecular mechanisms, involving cycles of DNA endoreduplication and cdc2a and cyc1At expression, might thus be involved in the formation of a giant cell or a syncytium.
Molecular cloning of a carotenoid‐associated protein from Cucumis sativus corollas: homologous genes involved in carotenoid sequestration in chromoplasts
Authors: Vishnevetsky Michael , Ovadis Marianna , Itzhaki Hanan , Levy Maggie , Libal‐Weksler Yael , Adam Zach , Vainstein Alexander
Published: March 2003

Chromoplasts are carotenoid‐accumulating plastids found in the corollas and fruits of many higher plants. In most cases, the pigment in these plastids is accumulated with the aid of carotenoid‐associated proteins located within unique structures. This paper reports the isolation and characterization of the cDNA (CHRC) from Cucumis sativus corollas which encodes the chromoplast‐specific carotenoid‐associated protein CHRC. The transit peptide cleavage site was determined and, using a chloroplast uptake system, it is shown that CHRC can be post‐translationally targeted to these plastids where it is peripherally associated with thylakoids. Analysis of CHRC transcript level in Cucumis sativus revealed its temporal and tissue‐specific regulation: the transcript was detected only in corollas, where its level increased in parallel to flower development, peaking just before anthesis. CHRC shares significant homology (59%) with the gene coding for fibrillin — a protein in Capsicum annuum red fruits whose function is essentially identical to that of CHRC. A CHRC fragment including the potential active site of the protein was used as a probe in Northern blot analyses of floral and fruit tissues from various plants containing chromoplasts of different types: CHRC homologs of similar sizes were revealed in all cases. The existence of a group of homologous genes coding for chromoplast‐specific proteins which aid in the sequestration of carotenoids within specific structures is proposed.
Phloem‐specific expression of a plant homeobox gene during secondary phases of vascular development
Authors: Tornero Pablo , Conejero Vicente , Vera Pablo
Published: March 2003

This paper reports the isolation and characterization of a homeobox gene (VAHOX1) from Lycopersicon esculentum encoding a homeodomain protein that contains a leucine zipper motif. The bipartite homeodomain‐leucine zipper (HD‐Zip) motif has only been found in homeobox genes from dicotyledonous plants, indicating that this type of transcription factor regulates particular developmental processes in these plant species. Here, the genomic organization, sequence comparison and expression analysis of VAHOX1 are described. Transcriptional fusion of the 5′ promoter region of VAHOX1 with the reporter GUS gene (VAHOX1—GUS) and expression analysis of this construct in transgenic plants indicates that VAHOX1 is specifically expressed in the phloem during phases of secondary growth. Unlike other plant homeobox genes, VAHOX1 does not appear to be expressed in meristems, and the function of VAHOX1 is considered a likely candidate molecule that may participate in the regulation of the identity and/or activity of phloem tissues during secondary phases of vascular development.
Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme
Authors: Schmidt Ralf‐C. , Müller Axel , Hain Rüdiger , Bartling Dieter , Weiler Elmar W.
Published: March 2003

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole‐3‐acetonitrile to the plant growth hormone, indole‐3‐acetic acid in vitro. To probe the capacity of this enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV‐35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg−1 protein with indole3‐acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1‐[13C]‐indole‐3‐acetonitrile to 1‐[13C]‐indole‐3‐acetic acid in vivo as determined by HPLC/ GC‐MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole‐3‐acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole‐3‐acetonitrile, a strong auxin‐overproducing phenotype developed resulting in increased lateral root formation (at 10 µM indole‐3‐acetonitrile) or stunted shoot growth, excessive lateral root initiation, inhibition of root out‐growth and callus formation at the root/shoot interface (at 100 µM indole‐3‐acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole‐3‐acetonitrile to indole‐3‐acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high‐auxin phenotype upon transgenic plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.
Transcriptional activation of a maize α‐tubulin gene in mycorrhizal maize and transgenic tobacco plants
Authors: Bonfante Paola , Bergero Roberta , Uribe Xavier , Romera Carme , Rigau Joan , Puigdomenech Pere
Published: March 2003

Mycorrhizae are symbioses between soil fungi and roots, with substantial modifications of the cells of both partners. Thus, host cells colonized by mycorrhizal fungi may express otherwise inactive genes. Here it is demonstrated that two arbuscular mycorrhizal (AM) fungi, Glomus versiforme and Gigaspora margarita, cause the transcriptional activation of a gene coding for α‐tubulin in the colonized cells of maize and transgenic tobacco roots. Tobacco plants transformed with a construct containing the maize Tubα3 gene promoter fused upstream from the bacterial GUS gene showed an intense GUS activity only in meristematic regions. When colonized by G. margarita, GUS activity was detected in the cortical root cells containing fungal arbuscules. No GUS activity was found in root cells when transformants carrying Tubα1 promoter were used, nor when Tubα3 transformants were colonized by ericoid mycorrhizal fungi, leading to a non‐specific interaction. Activation of the Tubα3 appears to be specific to the gene and to the appropriate interaction. Further evidence that fully differentiated host tissues re‐activate the Tubα3 gene following colonization by AM fungi also comes from accumulation of the corresponding transcripts in maize root cells containing arbuscules.
Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development
Authors: Bachem Christian W.B. , Van Der Hoeven Rutger S. , De Bruijn Steef M. , Vreugdenhil Dick , Zabeau Marc , Visser Richard G.F.
Published: March 2003

Using a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLP†)‐derived technique for RNA fingerprinting (cDNA‐AFLP‡), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP‐glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA‐AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuberspecific transcript‐derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two lox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA‐AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA‐AFLP is a broadly applicable technology for identifying developmentally regulated genes.
Differential expression of a CAK (cdc2‐activating kinase)‐like protein kinase, cyclins and cdc2 genes from rice during the cell cycle and in response to gibberellin
Authors: Sauter Margret
Published: March 2003

Progress through the eukaryotic cell cycle is regulated by cyclin‐dependent cdc2 protein kinases. In rice (Oryza sativa L.), two cdc2 protein kinases, cdc2Os‐1 and cdc2Os‐2, and two cyclins, cycOs1 and cycOs2, have been described. In this study, we report on the cell‐cycle phase‐specific expression of these genes. Using partially synchronized suspension cells from rice, we found that cdc2Os‐1 was expressed constitutively throughout the cell cycle. The cdc2Os‐2 transcript level was elevated in G1 and S phase. The cycOs1 and cycOs2 transcripts increased steadily through G2 and dropped off rapidly in mitosis as is typical for mitotic cyclins. We hypothesize that the cdc2Os‐2 gene product acts in G1/S and that the growth‐promoting hormone gibberellin (GA) that induces expression of cdc2Os‐2, cycOs1 and cycOs2 in the intercalary meristem of deepwater rice internodes’ accelerates G1/S phase progression through increased expression of cdc2Os‐2 and G2/M phase progression through increased expression of the mitotic cyclins cycOs1 and cycOs2. The R2 gene from rice has 55% sequence identity to the cdc2‐activating kinase (CAK) family of protein kinases which have been shown to phosphorylate and thereby activate cdc2 protein kinases in animals and yeast. In partially synchronized suspension cells, R2 mRNA levels were elevated in G1 and S phase. In GA‐treated rice internodes, R2 transcript levels were elevated in the meristem and part of the elongation zone. These results are consistent with a role for R2 in regulating G1/S phase progression.
Pre‐activating wounding response in tobacco prior to high‐level ozone exposure prevents necrotic injury
Authors: Örvar Björn Lárus , McPherson Joan , Ellis Brian E.
Published: March 2003

In tobacco, both wounding and treatment with jasmonates prior to exposure of the tissue to high concentrations of ozone (250 to 500 p.p.b.) produce a dramatic decrease in ozone injury. A systemic pattern of increased ozone tolerance developed within 3–6 h after wounding and also after local application of jasmonates. Ozone treatment of transgenie (NahG) tobacco plants showed that the inability of these plants to accumulate salicylic acid is also accompanied by increased ozone tolerance. Expression of mRNA encoding the anti‐oxidant enzyme ascorbate peroxidase is upregulated by ozone challenge, wounding and by methyl jasmonate exposure within 3–4 h, while levels of carbonic anhydrase mRNA are simultaneously depressed following ozone exposure and methyl jasmonate treatment. The pattern of these results shows that the response to ozone challenge in tobacco involves signalling mechanisms similar to those induced in plants by other environmental stresses that generate reactive oxygen species.
Induction of 1‐aminocyclopropane‐1‐carboxylate oxidase mRNA by ethylene in mung bean hypocotyls: involvement of both protein phosphorylation and dephosphorylation in ethylene signaling
Authors: Kim Jeong Hoe , Kim Woo Taek , Kang Bin G. , Yang Shang Fa
Published: March 2003

Ethylene induced an increase in the level of 1‐aminocyclopropane‐1‐carboxylate (ACC) oxidase mRNA but suppressed the expression of ACC synthase transcript in mung bean hypocotyls. Induction of ACC oxidase transcript by ethylene was saturated at a low concentration (1–3 µl l−1) and detectable within 1 h after ethylene treatment; 2,5‐norbornadiene, an inhibitor of ethylene action, significantly reduced the basal level of ACC oxidase transcript present in intact mung bean hypocotyls. Treatment with the protein synthesis inhibitor cycloheximide completely inhibited the ethylene‐induced accumulation of ACC oxidase transcript, indicating that de novo protein synthesis is necessary for expression of the ethylene‐inducible ACC oxidase gene. ABA decreased the ethylene‐induced accumulation of ACC oxidase mRNA, but restored the ethylene‐suppressed level of ACC synthase (VR‐ACS1) transcript. The protein kinase inhibitor staurosporine effectively prevented ethylene‐induced ACC oxidase gene expression, whereas it substantially recovered the ethylene‐suppressed transcript level of ACC synthase, suggesting that protein phosphorylation plays a role in the induction of ACC oxidase and the suppression of ACC synthase by ethylene in mung bean hypocotyls. Okadaic acid, a potent inhibitor of protein phosphatase, did not affect the expression of ACC oxidase. However, addition of okadaic acid along with ethylene effectively blocked the ethylene‐induced accumulation of ACC oxidase transcript, while there was an increase in the level of ethylene‐suppressed ACC synthase transcript. These results indicate that protein dephosphorylation in addition to phosphorylation is necessary for the ethylene signaling that regulates the expression of these genes.
Transfer RNA‐mediated suppression of amber stop codons in transgenic Arabidopsis thaliana
Authors: Betzner Andreas S. , Oakes Marie P. , Huttner Eric
Published: March 2003

An artificial amber suppressor tRNALeu gene (supL.) was physically linked to a mutated gus reporter gene, p35S‐gus(amL), which was inactivated by an amber stop codon (amL). Upon introduction into Arabidopsis thaliana, the presence of the supL. gene was found to be correlated with cytotoxic effects observed during tissue culture and in mature plants. Those primary transformants that displayed cytotoxic symptoms were shown by X‐Gluc staining to express GUS as a result of amber stop codon suppression in vivo. Phenotypically normal lines were found by RT‐PCR to express supL. GUS activity above background level was barely detectable in these plants, indicating a low level expression of supL. However, the remaining suppressor activity was still sufficient to transactivate an amber‐mutated male sterility gene, pA9‐barnase(amL1) when combined within the same plant by crossing. The suppressor tRNALeu gene may thus be used in transgenic plants for gene transactivation.
Potato guard cells respond to drying soil by a complex change in the expression of genes related to carbon metabolism and turgor regulation
Authors: Kopka Joachim , Provart Nicholas J. , Müller‐Röber Bernd
Published: March 2003

Altering stomatal function by a guard cell‐targeted transgenic approach with the aim of increased stress tolerance and crop yield requires knowledge of the natural fluctuations of stomatal gene expression under stress conditions. We developed a fast method for the isolation of RNA from epidermal fragments of potato leaves (Solanum tuberosum L. cv. Désirée), demonstrated that this RNA preparation is highly enriched in guard cell transcripts and used this method to investigate the response of gene expression in guard cells to mild drought stress. Drought was applied in planta by withholding water over a period of 2–4 days. In the following work responses observed under these conditions are called ‘long‐term’ in contrast to immediate (short‐term) stomatal opening and closing responses to environmental stress. We observed both gene‐specific increases and decreases of steady‐state transcript levels. In particular, the mRNA levels of sucrose synthase and sucrose‐phosphate synthase were elevated 5.5‐fold and 1.4‐fold, respectively. In contrast, expression of an inwardly rectifying K+ channel from guard cells (kst1) and of a plasma membrane H+‐ATPase (pha2) was reduced to 26% and 36%, respectively, of the expression in watered controls. In addition, expression of vacuolar invertase, UDP‐glucose pyrophosphorylase, ADP‐glucose pyrophosphorylase (large subunit), cytosolic glyceraldehyde‐3‐phosphate dehydrogenase, a sucrose/H+ co‐transporter, and a novel isoform of phosphoenolpyruvate carboxylase were also reduced. Other genes exhibited unaltered expression. Compared with the response in whole leaves, the transcript levels of phosphoenolpyruvate carboxylase, vacuolar invertase, and cytosolic glyceraldehyde‐3‐phosphate dehydrogenase were regulated guard cell specifically. Most importantly, changes in steady‐state transcript levels were complete before the onset of a decrease in leaf water potential, when drought‐induced stomatal closure was already obvious. These data support the hypothesis that a systemic drought‐stress signal acts not only on short‐term stomatal movements but also on long‐term gene expression in guard cells. Such long‐term changes in gene expression might contribute to the fine‐tuning of guard cell responses to environmental stimuli.
Existence of three regulatory regions each containing a highly conserved motif in the promoter of plastid‐encoded RNA polymerase gene (rpoB)
Authors: Inada Hitoshi , Seki Motoaki , Morikawa Hiromichi , Nishimura Mitsuo , Iba Koh
Published: March 2003

The plastid gene rpoB encodes a plastid‐specific, DNA‐dependent RNA polymerase. A study of a barley mutant, albostrians, indicated that rpoB itself is transcribed by a nuclear‐encoded RNA polymerase. However, the molecular nature of the nuclear‐encoded RNA polymerase and mechanisms of the transcriptional regulation of rpoB expression have not been elucidated. Using an in vivo transient assay system for gene expression in plastids, the 5′‐flanking region of rpoB from Arabidopsis thaliana in plastids of cultured BY‐2 tobacco cells was analyzed. A series of deletion analyses of the 5′‐flanking region indicated that there were two positive and one negative regulatory regions for rpoB expression. Comparison of the 5′‐flanking sequences of rpoB among several plant species revealed the existence of three conserved motifs in these regions.
Identification of members of gene families in Arabidopsis thaliana by contig construction from partial cDNA sequences: 106 genes encoding 50 cytoplasmic ribosomal proteins
Authors: Cooke Richard , Raynal Monique , Laudié Michele , Delseny Michel
Published: March 2003

Partial cDNA sequencing to obtain expressed sequence tags (ESTs) has led to the identification of tags to about 8000 of the estimated 20 000 genes in Arabidopsis thaliana. This figure represents four to five times the number of complete coding sequences from this organism available in international databases. In contrast to mammals, many proteins are encoded by multigene families in A. thaliana. Using ribosomal protein gene families as an example, it is possible to construct relatively long sequences from overlapping ESTs which are of sufficiently high quality to be able to unambiguously identify tags to individual members of multigene families, even when the sequences are highly conserved. A total of 106 genes encoding 50 different cytoplasmic ribosomal protein types have been identified, most proteins being encoded by at least two and up to four genes. Coding sequences of members of individual gene families are almost always very highly conserved and derived amino acid sequences are almost, if not completely, identical in the vast majority of cases. Sequence divergence is observed in untranslated regions which allows the definition of gene‐specific probes. The method can be used to construct high‐quality tags to any protein.
Phosphorylation of nuclear proteins directs binding to salicylic acid‐responsive elements
Authors: Stange Claudia , Ramírez Ingrid , Gómez Isabel , Jordana Xavier , Holuigue Loreto
Published: March 2003

The cis‐located DNA sequence as‐1 (Activation Sequence‐1) from CaMV 35S promoter has been previously identified as an element that can confer inducibility by salicylic acid (SA) with immediate early kinetics. This sequence specifically binds to ASF‐1 (Activation Sequence Factor‐1), previously characterized in tobacco nuclear extracts. To assess whether modulation of ASF‐1 binding activity can explain the activation of the as‐1 sequence observed in vivo, we performed electrophoretic mobility shift assays using nuclear extracts from SA‐treated and water‐treated tobacco plants. Our results indicate that treatment of plants with SA increases ASF‐1 binding to as‐1 and to ocs, an as‐1‐like element from the Agrobacterium octopine synthase gene. In contrast, SA treatment has no effect on the binding of GT‐1 factor to its target light‐inducible box II element. Furthermore, treatment of nuclear extracts from SA‐treated plants with alkaline phosphatase decreases ASF‐1 binding to the as‐1 element. This can be reversed by pretreatment with 10 mM NaF. Accordingly, pretreatment of nuclear extracts from control water‐treated plants with ATP produces an increase in ASF‐1 binding activity similar to that observed with SA. This effect of ATP is reversed by treatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor. These results support the hypothesis that a nuclear protein kinase is involved in the immediate early events of transcriptional activation triggered by SA.
Use of Arabidopsis recombinant inbred lines reveals a monogenic and a novel digenic resistance mechanism to Xanthomonas campestris pv campestris
Authors: Buell C.R. , Somerville S.C.
Published: March 2003

Infiltration of the Arabidopsis thaliana accession Landsberg erecta (Ler) with Xanthomonas campestris pv campestris isolate 2D520 results in extensive necrosis and limited chlorosis within 5–6 days post‐inoculation (d.p.i.), which can lead to systemic necrosis within 23 d.p.i. In contrast, the accession Columbia (Col) remains asymptomatic after infiltration. Although both accessions support bacterial growth, 5–28‐fold more bacteria are present in Ler than in Col leaf tissue. Inheritance studies indicate that three independent, dominant or partially dominant, nuclear genes condition resistance to X. c. campestris 2D520. The major gene, termed RXC2, conditions monogenic resistance to X. c. campestris and was mapped to a 5.5 cM interval of chromosome V. Segregation data indicate that the locus RXC3 in conjunction with RXC4 confers digenic resistance to X. c. campestris. The combined action of RXC3 and RXC4 is correlated with a suppression of in planta bacterial levels and a suppression of symptoms relative to Ler. The RXC3 + RXC4‐mediated resistance is novel in that although the Col allele of RXC4 contributes positively to resistance, it is the Ler and not the Col allele of RXC3 that contributes positively to resistance. RXC3 was mapped to the bottom arm of chromosome V in a 2.7 cM interval within the major recognition gene complex MRC‐J, a cluster of genes involved in disease resistance. RXC4 was mapped to a 12 cM interval on chromosome II that also contains RXC1, a gene conferring tolerance to X. c. campestris.
Interaction of the maize and Arabidopsis kinase interaction domains with a subset of receptor‐like protein kinases: implications for transmembrane signaling in plants
Authors: Braun David M. , Stone Julie M. , Walker John C.
Published: March 2003

The kinase interaction (KI) domain of kinase‐associated protein phosphatase (KAPP) interacts with the phosphorylated form of an Arabidopsis thaliana receptor‐like protein kinase (RLK). The KI domain may recruit KAPP into an RLK‐initiated signaling complex. To examine additional roles that this domain may play in plant signal transduction, a search was conducted for other KI domain‐containing proteins. One gene was isolated which encodes a KI domain, the maize homolog of KAPP. To test whether the maize KI domain associates with other maize proteins, it was used as a probe in a protein–protein interaction cloning strategy. A new maize RLK, KI domain interacting kinase 1 (KIK1), was identified by its interaction with the maize KI domain. The maize KI domain and the KIK1 kinase domain association required phosphorylation of the kinase. This work establishes that the KI domain phosphorylation‐dependent signaling mechanism is present in both monocots and dicots. Additionally, it was determined that both the maize and Arabidopsis KI domains interact with several but not all of the active RLKs assayed. These multiple associations imply that KAPP may function in a number of RLK‐initiated signaling pathways.
Structure and subcellular localization of a small RNA‐binding protein from tobacco
Authors: Moriguchi Kazuki , Sugita Mamoru , Sugiura Masahiro
Published: March 2003

In this study, a cDNA encoding a small RNA‐binding protein was isolated from a Nicotiana sylvestris cDNA library. The predicted protein (RGP‐3) is 144 amino acid residues long, and contains a consensus sequence‐type RNA binding domain (CS‐RBD) of 83 amino acids and a short glycine‐rich region of 15 amino acids. RGP‐3 synthesized in Escherichia coli has high affinity for poly(U). Immunocytochemical analysis indicated that RGP‐3 is localized in the nucleoplasm, and that RGP‐1b, a related protein reported previously, is localized in the nucleolus. Possible roles of these proteins in pre‐mRNA or pre‐rRNA processing are discussed.
ZmEsr, a novel endosperm‐specific gene expressed in a restricted region around the maize embryo
Authors: Opsahl‐Ferstad Hilde‐Gunn , Deunff Erwan Le , Dumas Christian , Rogowsky Peter M.
Published: March 2003

A novel endosperm‐specific gene named Esr (embryo surrounding region) has been isolated by differential display between early developmental stages of maize endosperms and embryos. It is expressed in a restricted region of the endosperm, surrounding the entire embryo at early stages (4 to 7 days after pollination, DAP) and ever‐decreasing parts of the suspensor at subsequent stages. The expression starts at 4 DAP and is maintained until at least 28 DAP. A minimum of three Esr genes are present in the maize genome and at least two of them map to the short arm of chromosome 1 at position 56. The Esr genes contain no introns and show no significant nucleotide or amino acid sequence homologies to sequences in the databases. The open reading frames encode basic proteins of 14 kDa with presumptive signal peptides at their N‐termini followed by a hypervariable and a conserved region. The gene product may play a role in the nutrition of the developing embryo or in the establishment of a physical barrier between embryo and endosperm.
High‐efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T‐DNA 1′ promoter
Authors: Mengiste Tesfaye , Amedeo Paolo , Paszkowski Jerzy
Published: March 2003

Two selectable marker genes harbouring the bar coding region but differing in their promoters were compared in an Arabidopsis thaliana transformation assay using in planta infiltration with Agrobacterium tumefaciens. Surprisingly, in four Arabidopsis ecotypes examined, the 1′ promoter from the right T‐DNA was superior to the most commonly used 35S promoter of cauliflower mosaic virus (CaMV). The ecotype Wassilewskija gave the highest transformation frequencies, with an average of between 5.3 and 6.3 % of the seedlings subjected to the selection. This is approximately 30‐fold higher than previously reported results. Analysis of T‐DNA integration patterns in single transformed plants or pooled populations revealed independent T‐DNA integration events in each case. Results show that the 1′ promoter is an attractive alternative to the 35S promoter for the generation of T‐DNA insertion lines. The 1′ promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology‐mediated gene silencing.
An Arabidopsis mutant showing reduced feedback inhibition of photosynthesis
Authors: Van Oosten Jean‐Jacques M. , Gerbaud Alain , Huijser Casper , Dijkwel Paul P. , Chua Nam‐Hai , Smeekens Sjef C.M.
Published: March 2003

Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown. A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals. For this purpose, an in vivo reporter system was established consisting of the light‐ and sugar‐regulated plastocyanin promoter, fused to the luciferase coding sequence (PC—LUC construct). At the seedling stage, expression of the PC—LUC gene is repressed by sucrose, and a number of sucrose‐uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter. Three mutants have been characterized in more detail. The sugar analog 2‐deoxy‐d‐glucose (2DG) was used to repress whole plant photosynthesis, PC—LUC gene expression and total ribulose‐1,5‐bisphosphate activity. It was found that the sun6 mutation makes plants unresponsive to these 2DG‐induced effects. Moreover, unlike wild‐type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium. These findings suggest that the SUN6 gene is active in a hexose‐activated signal transduction pathway.
Map positions of 47 Arabidopsis sequences with sequence similarity to disease resistance genes
Authors: Botella Miguel A. , Coleman Mark J. , Hughes Douglas E. , Nishimura Marc T. , Jones Jonathan D.G. , Somerville Shauna C.
Published: March 2003

Map positions have been determined for 42 non‐redundant Arabidopsis expressed sequence tags (ESTs) showing similarity to disease resistance genes (R‐ESTs), and for three Pto‐like sequences that were amplified with degenerate primers. Employing a PCR‐based strategy, yeast artificial chromosome (YAC) clones containing the EST sequences were identified. Since many YACs have been mapped, the locations of the R‐ESTs could be inferred from the map positions of the YACs. R‐EST clones that exhibited ambiguous map positions were mapped as either cleavable amplifiable polymorphic sequence (CAPS) or restriction fragment length polymorphism (RFLP) markers using F8 (Ler x Col‐0) recombinant inbred (RI) lines. In all cases but two, the R‐ESTs and Pto‐like sequences mapped to single, unique locations. One R‐EST and one Pto‐like sequence each mapped to two locations. Thus, a total of 47 loci were identified in this study. Several R‐ESTs occur in clusters suggesting that they may have arisen via gene duplication events. Interestingly, several R‐ESTs map to regions containing genetically defined disease resistance genes. Thus, this collection of mapped R‐ESTs may expedite the isolation of disease resistance genes. As the cDNA sequencing projects have identified an estimated 63% of Arabidopsis genes, a very large number of R‐ESTs (∼95), and by inference disease resistance genes of the leucine‐rich repeat‐class probably occur in the Arabidopsis genome.