bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified plant plasma membrane H+‐ATPase with improved transport coupling efficiency identified by mutant selection in yeast
Authors: Baunsgaard Lone , Venema Kees , Axelsen Kristian B. , Villalba José Manuel , Welling Annikki , Wollenweber Bernd , Palmgren Michael G.
Published: March 2003

Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H+‐ATPase). Random mutants of Arabidopsis H+‐ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H+‐ATPase (PMA1). A Trp‐874‐Leu substitution as well as a Trp‐874 to Lys‐935 deletion in the hydrophilic C‐terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp‐874‐Phe substitution in AHA1 was produced by site‐directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild‐type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient. These results provide evidence that Trp‐874 plays an important role in auto‐inhibition of the plant H+‐ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.
Identification of plant cytoskeletal, cell cycle‐related and polarity‐related proteins using Schizosaccharomyces pombe
Authors: Xia Guixian , Ramachandran Srinivasan , Hong Yan , Chan Yang‐Sun , Simanis Viesturs , Chua Nam‐Hai
Published: March 2003

The fission yeast Schizosaccharomyces pombe has been used to identify Arabidopsis thaliana proteins that may play a role in cell shape maintenance or cell cycle regulation. An Arabidopsis thaliana cDNA library was constructed in pREP5N vector under the control of the inducible nmt1 promoter and transformed into S. pombe. Expression of the A. thaliana sequences was induced and clones showing severe morphological changes were identified and analysed. Comparison of the sequences of the inserts with the sequence data bases revealed that several cDNAs encode proteins known to play a role in function of the cytoskeleton, the cell cycle and establishment of cell polarity. These include α‐1, α‐2, α‐6 and β‐6 tubulins, myosin heavy chain‐like protein, ubiquitin conjugating enzymes UBC9 (E2), 26S protease subunits, Ran‐binding protein, myb protein, PRL1 gene product and rho protein. Approximately 30% of the clones encode novel sequences. The results suggest that S. pombe phenotypic screening can be used to identify plant proteins involved in cell shape maintenance and regulation during cell cycle and development.
Virus‐mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells
Authors: Boevink P. , Santa Cruz S. , Hawes C. , Harris N. , Oparka K.J.
Published: March 2003

The green fluorescent protein (GFP) was targeted to the endoplasmic reticulum (ER) of living plant cells using a virus‐based expression system. The signal peptide from the storage protein, patatin was fused to the amino‐terminus of the GFP while the ER retention signal KDEL was fused to the carboxy‐terminus. The chimaeric gfp cDNA was inserted into a potato virus X‐based expression vector and in‐vitro transcripts, representing the recombinant viral genome, were inoculated on to Nicotiana benthamiana and N. clevelandii plants. In virus‐infected cells, the GFP was targeted both to the cortical ER and to a mobile system of ER elements which underwent streaming in the cytoplasm. In addition, a population of GFP‐containing inclusions was apparent. These inclusions were motile but remained closely associated with elements of the ER. Staining of the ER with membrane potential‐sensitive dyes confirmed that the GFP had been targeted to the ER. The utility of virus‐mediated delivery systems in studies of the plant endomembrane system is discussed.
Mechanisms of chilling‐induced oxidative stress injury and tolerance in developing maize seedlings: changes in antioxidant system, oxidation of proteins and lipids, and protease activities
Authors: Prasad Tottempudi K.
Published: March 2003

The mechanisms involved in chilling injury or tolerance in developing maize seedlings subjected to low temperature stress have been investigated. Although acclimation‐induced chilling tolerance was developmentally regulated, no tolerance was observed in non‐acclimated developing seedlings subjected to 7 days of 4°C stress. Consistent with previous results, chilling induced oxidative stress in the seedlings. Whereas acclimated seedlings had elevated levels (35–120%) of antioxidant enzymes, such as catalase, glutathione reductase and guaiacol peroxidase, non‐acclimated seedlings had lower levels of these enzyme activities during chilling conditions. The increase in the activity of mitochondrial catalase3 isozyme, a major anti‐oxidant enzyme in maize, was due to an increase in cat3 transcripts and CAT3 protein, as indicated by Northern and Western blot analyses. Besides, non‐acclimated seedlings had increased levels of oxidized proteins (two to threefold) and lipids (35–65%) during chilling and recovery compared with control or acclimated seedlings. Low‐temperature stress also inhibited two proteases, endopeptidase and aminopeptidase, by 35% in non‐acclimated seedlings. Since seedlings treated with prooxidants also promoted the oxidation of proteins and lipids and the inhibition of proteases, it was concluded that in vivo generated oxidative stress was likely responsible for affecting these processes. These results provide a correlative evidence to suggest that: (i) in non‐acclimated seedlings, chilling injury was partly due to the build up of reactive oxygen species (ROS) that promoted the oxidation of proteins and lipids and inhibited protease activity; (ii) in acclimated seedlings, chilling tolerance was partly due to an enhanced anti‐oxidant system that prevented the accumulation of ROS and therefore, prevented the oxidation of proteins and lipids and the inhibition of protease activities during 4°C stress and recovery.
The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time
Authors: Devlin Paul F. , Halliday Karen J. , Harberd Nicholas P. , Whitelam Garry C.
Published: March 2003

A major function of phytochromes in light‐grown plants involves the perception of changes in the relative amounts of red and far‐red light (R:FR ratio) and the initiation of the shade‐avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end‐of‐day far‐red light (EOD FR) treatments. Phytochrome B‐deficient (phyB) mutants, which have a constitutive elongated‐petiole and early‐flowering phenotype, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild‐type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild‐type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.
Detection of single‐copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization
Authors: Fransz Paul F. , Stam Maike , Montijn Bob , Hoopen Rogier Ten , Wiegant Joop , Kooter Jan M. , Oud Oof , Nanninga Nanne
Published: March 2003

DNA sequences homologous to single‐copy genes were labelled with biotinylated dUTP or digoxygenin‐labelled dUTP and hybridized to chromosome spreads. The hybridization signals were visualized with fluorescent avidin‐ or antibody‐conjugates. This method allowed the detection of DNA targets on metaphase chromosomes as small as 1.4 kb. The hybridization signals were identified as fluorescent spots on both sister chromatids. Using an 18S rDNA probe as marker to identify chromosomes II and III it was possible to assign single‐copy genes to these chromosomes. In the line V30 the endogenous chalcone synthase gene (chsA) was mapped at the distal end of the short arm of chromosome 5. The cDNA probe for this single‐copy gene was 1.4 kb. In contrast, in the lines Mitchell and V26 chsA was localized at the distal end of the long arm of chromosome 3, suggesting that a chromosomal rearrangement had taken place. In a transformed Petunia uidA, transgenes were detected using a 2.7 kb probe. One transgene was mapped on one of the homologues of chromosome II proximal to the ribosomal genes. This homologue could be distinguished from the other by having the ribosomal genes at the distal end of the long arm. Using multicolour fluorescence in situ hybridization it was shown that it is possible to detect the endogenous chsA genes and both transgenes simultaneously.
Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize
Authors: Wirtz Uwe , Osborne Brian , Baker Barbara
Published: March 2003

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non‐autonomous Ac derivative, Dissociation (Ds), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep+) and non‐replicating (rep−) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep+) or mutant, inactive derivatives of these genes (rep−). Excision of Ds was detected only in those cell nuclei co‐transfected with the replicating Ds‐vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 × 105‐fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.
Description of 31 YAC contigs spanning the majority of Arabidopsis thaliana chromosome 5
Authors: Schmidt Renate , Love Karina , West Joanne , Lenehan Zoë , Dean Caroline
Published: March 2003

In order to generate a physical map of Arabidopsis thaliana chromosome 5, 142 molecular markers mapping to chromosome 5 have been used in colony hybridization experiments with four Arabidopsis, ecotype Columbia, yeast artificial chromosome (YAC) libraries. This resulted in 634 YAC clones being anchored on chromosome 5. Southern blot analysis confirmed their positioning and provided data, which along with knowledge of the sizes of all the YAC clones, enabled the clones to be arranged into 31 contigs. Genetic mapping of markers located within 29 of these contigs on the Landsberg erecta/Columbia recombinant inbred lines allowed positioning of the contigs along the chromosome. A high proportion of the YAC clones were found to contain chimaeric inserts. The availability of this YAC contig map will accelerate chromosome‐walking experiments, provide substrates for large‐scale genomic sequencing projects and facilitate the mapping of new probes to this chromosome.
Expression of a betaine aldehyde dehydrogenase gene in rice, a glycinebetaine nonaccumulator, and possible localization of its protein in peroxisomes
Authors: Nakamura Toshihide , Yokota Sadaki , Muramoto Yasunori , Tsutsui Kyoko , Oguri Yasuko , Fukui Kiichi , Takabe Tetsuko
Published: March 2003

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the plant biosynthetic pathway that leads to glycinebetaine. Rice plants (Oryza saliva L), albeit considered a typical non‐glycinebetaine accumulating species, have been found to express this enzyme at low levels. This observation evokes an interest in phylogenic evolution of the enzyme in the plant kingdom. It is reported here that rice plants possess the ability to take up exogenously added betaine aldehyde through the roots and convert it to glycinebetaine, resulting in an enhanced salt‐tolerance of the plants. A gene encoding a putative BADH from the rice genome was also cloned and sequenced. The gene was found to contain 14 introns, and the overall nucleotide sequence of the coding region is c. 78% identical to that of the barley BADH cDNA. Cloning of a partial BADH cDNA from rice was accomplished by reverse transcription‐polymerase chain reaction (RT‐PCR). The nucleotide sequence of the cloned fragment was found to be identical to the corresponding exon regions of the rice genomic BADH gene. The deduced amino acid sequences of rice and barley BADH both contain a C‐terminal tripeptide SKL, a signal known to target preproteins to microbodies. This localization was confirmed by an immuno‐gold labeling study of transgenic tobacco harboring barley cDNA, which showed BADH protein inside peroxisomes. Northern blot analysis revealed that the level of BADH mRNA is salt‐inducible.
A homolog of the Arabidopsis thaliana ERS gene is actively regulated in Rumex palustris upon flooding
Authors: Vriezen Wim H. , van Rijn Cynthia P.E. , Voesenek Laurentius A.C.J. , Mariani Celestina
Published: March 2003

A cDNA homologous to the ethylene‐response sensors (ERS/ETR1) from Arabidopsis thaliana was isolated from a Rumex palustris cDNA library. This cDNA, RP‐ERS1, was 2421 bp long and shared 66% nucleotide homology with ETR1 and ERS in their coding regions. The transcript level of RP‐ERS1 was actively regulated during the leaf‐elongation response of R. palustris upon flooding. RP‐ERS1 transcript levels increased after submergence, and also after exposure to high concentrations of ethylene and carbon dioxide and low concentrations of oxygen. These results suggest that R. palustris plants may respond to flooding stress by increasing the number of their ethylene receptors.
Intron‐mediated enhancement of transgene expression in maize is a nuclear, gene‐dependent process
Authors: Rethmeier Nikki , Seurinck Jef , Van Montagu Marc , Cornelissen Marc
Published: March 2003

In monocots, transgene expression can be stimulated by over two magnitudes by including an intron in the 5′ untranslated region (UTR). The underlying mechanism is presently unknown. Inclusion of the salT intron into the 5′ UTR of cat and bar genes stimulated expression of the first gene only, indicating that intron‐mediated enhancement of expression (IME) is gene‐dependent. Stimulation was associated with increased cat RNA levels, which did not result from a reduced cytoplasmic turnover and were not associated with increased translation. This implies that IME acts in the nucleus. Importantly, the cytoplasmic accumulation of spliced cat transcripts, even with IME, is less than that encoded by the intronless bar gene. As the cat and bar genes were flanked by identical regulatory signals, and the transcripts had a similar cytoplasmic stability, it may mean that IME rescues rather than stimulates gene expression.
The quiescent/colorless alleles of viviparous1 show that the conserved B3 domain of VP1 is not essential for ABA‐regulated gene expression in the seed
Authors: Carson Christian B. , Hattori Tsukaho , Rosenkrans Leonard , Vasil Vimla , Vasil Indra K. , Peterson Peter A. , McCarty Donald R.
Published: March 2003

A series of vp1 alleles distinguish at least two classes of maturation‐related genes that are regulated by the VP1 factor and abscisic acid (ABA). The intermediate vp1‐c821708 and vp1‐McW alleles have quiescent (non‐viviparous) anthocyanin‐deficient phenotypes while maintaining significant levels of maturation‐specific gene expression in the developing embryo. However, expression of the C1 regulatory gene of the anthocyanin pathway is not detected in these mutants. Reduced steady‐state levels of structurally altered VP1 proteins are detected in quiescent mutant embryos. The VP1‐McW protein sequence lacks the highly conserved region encoded by exons 3–5 of the Vp1 gene. A sensitive RT‐PCR assay was used to rule out significant amounts of intact transcripts in the vp1‐McW mutant that could account for the quiescent phenotype. In transient expression assays, the VP1‐McW protein and other mutants with a truncated B3 domain of VP1 retained a strong capacity to synergistically enhance ABA‐regulation of the Em‐GUS reporter gene; whereas transactivation of both Em‐GUS and C1‐sh‐GUS genes in the absence of hormone was strongly inhibited. These results indicate that the largest conserved region in VP1 homologs (B3) is critical for gene activation at low or insignificant ABA dosages; whereas the N‐terminal domain provides a key interface with ABA signaling pathways in the developing seed.
Transposon Display identifies individual transposable elements in high copy number lines
Authors: Va Dirk , De N , Broeck N , Maes Tamara , Sauer Matt , Zethof Jan , D Peter , Keukeleire E , D’hauw Mariëlla , Va Marc , Montagu N , Gerats Tom
Published: March 2003

The dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation‐mediated PCR. The resulting fragments are locus‐specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1‐tagged genes. Fragments that co‐segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.
Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism
Authors: Vo Klaus , Schwartzenberg  , Kruse Sven , Reski Ralf , Moffatt Barbara , Laloue Michel
Published: March 2003

Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G. was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain. The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa. Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome. The deduced amino acid sequence had a 52% identity with the human adenosine kinase. The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E. coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue. Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella.
Carrot DNA‐methyltransferase is encoded by two classes of genes with differing patterns of expression
Authors: Bernacchia Giovanni , Primo Antonella , Giorgetti Lucia , Pitto Letizia , Cella Rino
Published: March 2003

In the present study, the isolation and characterization of two distinct cDNAs that code for carrot DNA (cytosine‐5)‐methyltransferase (DNA‐METase) are reported. The screening of a cDNA library with a carrot genomic DNA fragment, previously obtained by PCR using degenerate primers, has led to the isolation of clones that belong to two distinct classes of genes (Met1 and Met2) which differ in sequence and size. Met1–5 and Met2–21 derived amino acid sequences are more than 85% identical for most of the polypeptide and completely diverge at the N‐terminus. The larger size of the Met2–21 cDNA is due to the presence of nearly perfect fivefold repeat of a 171 bp sequence present only once in the Met1–5 cDNA. Northern and in situ hybridization analyses with young carrot plants and somatic embryos indicate that both genes are maximally expressed in proliferating cells (suspension cells, meristems and leaf primordia), but differ quantitatively and spatially in their mode of expression. Polyclonal antibodies were raised in rabbit using fusion proteins corresponding to the regulatory and catalytic regions of the most highly expressed gene (Met1–5). In nuclear carrot extracts, both antibodies were found to recognize a band of about 200 kDa along with some additional bands of lower size. These results provide the first direct demonstration that DNA‐METases of a higher eukaryote are encoded by a gene family.
Gypsy‐like retrotransposons are widespread in the plant kingdom
Authors: Suoniemi Annu , Tanskanen Jaakko , Schulman Alan H.
Published: March 2003

Retrotransposons propagate via an RNA intermediate which is then reverse‐transcribed and packaged into virus‐like particles. They are either copia‐ or gypsy‐like in coding domain order and sequence similarity, the gypsy‐like elements sharing their organization with the retroviruses but lacking retroviral envelope domains. Copia‐like retrotransposons, or at least their reverse transcriptase domains, appear broadly distributed in higher plants, but gypsy‐like elements have been reported only for scattered species. The authors have exploited the difference in domain order between these groups to amplify and clone segments bridging the reverse transcriptase – integrase region of specifically gypsy‐like retrotransposons. Species representative of the diversity of higher plants yielded products whose sequences establish that gypsy‐like transposons are dispersed throughout the plant genomes. This class of plant elements has been named romani retrotransposons. The presence of both types ubiquitously in the fungi, plants and animals support their existence as ancient distinct lineages and subsequent, vertical radiation.
Molecular cloning and functional heterologous expression of two alleles encoding (S)‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1), a new methyl jasmonate‐inducible cytochrome P‐450‐dependent mono‐oxygenase of benzylisoquinoline alkaloid biosynthesis
Authors: Pauli Hubert H. , Kutchan Toni M.
Published: March 2003

Alkaloids derived from the tetrahydrobenzylisoquinoline alkaloid (S)‐N‐methylcoclaurine represent a vast and varied structural array of physiologically active molecules. These compounds range from the dimeric bisbenzylisoquinolines, such as the muscle relaxant (+)‐tubocurarine, to the powerful anaesthetic opiate morphine, the anti‐microbial berberine and the anti‐microbial benzo[c]phenanthridine sanguinarine. The 3′‐hydroxylation of (S)‐N‐methylcoclaurine is a branch point that is the penultimate step in the biosynthesis of the central alkaloidal intermediate (S)‐reticuline. This study identified this enzyme as a cytochrome P‐450‐dependent mono‐oxygenase that has until now eluded attempts at identification using in vitro enzyme assays. Two alleles encoding this new enzyme (S)‐N‐methylcoclaurine 3′‐hydroxylase (CYP80B1) were isolated from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate‐induced cell‐suspension cultures of the California poppy Eschscholzia californica. Partial clones generated by RT–PCR with cytochrome P‐450‐specific primers were used as hybridization probes. RNA gel‐blot hybridization indicated that the transcripts for CYP80B1 accumulate in response to the addition of methyl jasmonate to the cell culture medium. Both alleles were functionally expressed in Saccharomyces cerevisiae and in Spodoptera frugiperda Sf9 cells in the presence and absence of the E. californica cytochrome P‐450 reductase. The enzyme was found to hydroxylate exclusively (S)‐N‐methylcoclaurine with a pH optimum of 7.5, temperature optimum of 35°C and a Km of 15 μm. In addition to the CYP80B1 alleles, another cytochrome P‐450 with an inducible transcript (CYP82B1) was isolated and expressed in the same manner, but was not found to be involved in alkaloid biosynthesis in this plant.
Identification of cat sequences required for intron‐dependent gene expression in maize cells
Authors: Rethmeier Nikki , Kramer Evelien , Va Marc , Montagu N , Cornelissen Marc
Published: March 2003

Transgene expression in maize cells changed from intron‐independent to intron‐dependent by an exact exchange of the bar coding region for that of cat. By deletion mapping an approximately 100 nucleotide sequence element at the 5′ end of the cat coding region was identified that, when inserted at the translation start site of the bar gene, impaired expression. Successive inclusion of the salT intron in the 5′ untranslated region (UTR) restored expression near to wild‐type bar expression levels. A chimeric gfp gene, but not nptII gene, behaved similarly. These observations are in agreement with the view that intron‐mediated enhancement of transgene expression does not enhance, but rather restores expression of an impaired gene.
Anoxia tolerance in tobacco roots: effect of overexpression of pyruvate decarboxylase
Authors: Tadege Million , Brändle Roland , Kuhlemeier Cris
Published: March 2003

Plant survival during flooding relies on ethanolic fermentation for energy production. The available literature indicates that the first enzyme of the ethanolic fermentation pathway, pyruvate decarboxylase (PDC), is expressed at very low levels and is likely to be rate‐limiting during oxygen deprivation. The authors expressed high levels of bacterial PDC in tobacco to study the modulation of PDC activity in vivo, and assess its impact on the physiology of ethanolic fermentation and survival under oxygen stress. In contrast to leaves, wild‐type normoxic roots contained considerable PDC activity, and overexpression of the bacterial PDC caused only a moderate increase in acetaldehyde and ethanol production under anoxia compared to wild‐type roots. No significant lactate production could be measured at any time, making it unlikely that lactate‐induced acidification (LDH/PDC pH‐stat) triggers the onset of ethanol synthesis. Instead, the authors favour a model in which the flux through the pathway is regulated by substrate availability. The increased ethanolic flux in the transgenics compared to the wild‐type did not enhance anoxia tolerance. On the contrary, rapid utilisation of carbohydrate reserves enhanced premature cell death in the transgenics while replenishment of carbohydrates improved survival under anoxia.
A single nuclear locus is involved in both chloroplast RNAtrans‐splicing and 3′ end processing
Authors: Hahn Daniela , Nickelsen Jörg , Hackert Anke , Kück Ulrich
Published: March 2003

The complex maturation of the chloroplast

psaA

mRNA in

Chlamydomonas reinhardtii

requires the splicing of three separate exons in

trans

. At least 14 nuclear gene products and a small chloroplast RNA

(tscA)

seem to be involved in

psaA trans

‐splicing. To further elucidate the mechanisms that regulate

trans

‐splicing in

C. reinhardtii

, we have analyzed four different nuclear mutants; two affected in

trans

‐splicing of the

psaA

exons 1 and 2 (class C) and two blocked in both

psaA trans

‐splicing steps (class B). While mutants M18 (class C) and L118 (class B) have a mature chloroplast

tscA

RNA, no such transcripts could be detected in mutants TR72 (class C) and HN31 (class B). Northern hybridizations indicate that the absence of mature

tscA

transcripts in HN31 and TR72 results from a defect in the processing of a

tscA/chlN

precursor transcript. Since the

tscA

locus has been shown to mediate

psaA

exon 1 and 2

trans

‐splicing, the nucleus‐encoded factor affected in the class C mutant TR72 may be required for

tscA

3′ end maturation and only indirectly for

psaA

exon 1 and 2

trans

‐splicing. Moreover, further genetic analysis revealed that the nuclear HN31 locus is involved in both maturation of the

tscA/chlN

precursor and

trans

‐splicing of the

psaA

exons 2 and 3.
The control of source to sink carbon flux during tuber development in potato
Authors: Sweetlove Lee J. , Kossmann Jens , Riesmeier Jörg W. , Trethewey Richard N. , Hill Steven A.
Published: March 2003

We have used top‐down metabolic control analysis to investigate the control of carbon flux through potato (Solanum tuberosum) plants during tuberisation. The metabolism of the potato plant was divided into two blocks of reactions (the source and sink blocks) that communicate through the leaf apoplastic sucrose pool. Flux was measured as the transfer of 14C from CO2 to the tuber. Flux and apoplastic sucrose concentration were varied either by changing the light intensity or using transgenic manipulations that specifically affect the source or sink blocks, and elasticity coefficients were measured. We have provided evidence in support of our assumption that apoplastic sucrose is the only communicating metabolite between the source and sink blocks. The elasticity coefficients were used to calculate the flux control coefficients of the source and sink blocks, which were 0.8 and 0.2, respectively. This work suggests that the best strategy for the manipulation of tuber yield in potato will involve increases in photosynthetic capacity, rather than sink metabolism.
Jasmonate signaling mutants ofArabidopsisare susceptible to the soil fungusPythium irregulare
Authors: Staswick Paul E. , Yuen Gary Y. , Lehman Casey C.
Published: March 2003

Jasmonic acid has properties of a plant hormone, including the induction of specific genes associated with plant defense. We previously described

jar1‐1

, an

Arabidopsis

jasmonate response mutant that exhibits reduced sensitivity to methyl jasmonate. We have further characterized this mutant and two new alleles;

jar1‐2

from a gamma irradiated population, and

jar1‐4

from a T‐DNA mutant population. Seedling root growth in

jar1‐1

was equally insensitive to methyl jasmonate and jasmonic acid, indicating that the defect was not in the conversion of methyl jasmonate to the acid. None of the

jar1

mutants showed an altered sensitivity to auxin, cytokinin, or the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid, indicating that the lesion does not affect the general uptake or transport of hormones. A soil fungus,

Pythium irregulare

, was found to blight

jar1‐1

. Cultures of this organism caused the symptoms in all three

jar1

mutants but not in wild type, indicating that increased susceptibility was due to the lesion in the

JAR1

locus. A fatty acid desaturase triple mutant that is defective in the biosynthesis of jasmonic acid (J. Browse, Washington State University) was also susceptible, confirming that jasmonate is involved in resistance. The

jar1‐1

locus was mapped to the lower end of chromosome 2, about 11.4 cM from

as1 and

1.6 cM from

cer8

. These results establish that jasmonate signaling plays an important role in resistance to soil micro‐organisms in plants.
Cytoplasmic acidification and current influx follow growth pulses ofLilium longiflorumpollen tubes
Authors: Messerli Mark A. , Robinson Kenneth R.
Published: March 2003

We confirm that there is not a standing pH gradient in the tips of lily pollen tubes, but show that there are pulses of H+ that occur during pulsatile growth. The [H+] increases in a zone at the tips of the tubes and travels rapidly as far as 60 μm down the shaft of the tube. The tip‐localized pH was found to drop to 6.0 during the largest pulses, from an average cytosolic level of 7.05 in tubes that had not yet begun to pulse. Correlation analysis indicates that the peaks of the pH pulses lag the peaks of the growth pulses by slightly more than 7.5 sec. Vibrating probe measurements reveal an influx of ionic current that peaks 7.5 sec after the peaks of the growth pulses. While this current may in part be H+ influx, we give evidence that K+ influx is also a component of the current pulses. The timing of the H+ and current pulses suggests that they may be involved in terminating the growth pulses.
The self‐incompatibility (S) locus inPetunia hybridais located on chromosome III in a region, syntenic for the Solanaceae
Authors: ten Hoopen Rogier , Harbord Robin M. , Maes Tamara , Nanninga Nanne , Robbins Timothy P.
Published: March 2003

Seven independent transformants were previously shown to carry T‐DNA inserts that are genetically linked to the self‐incompatibility (

S

)‐locus in

Petunia hybrida

. These T‐DNAs provided reliable targets for fluorescent

in situ

hybridisation (FISH) localisation. The T‐DNA loci were found to be distributed over the entire long arm of chromosome III. The most tightly linked T‐DNA loci were located in a subcentromeric position. Independent confirmation of this assignment of the

S

‐locus to chromosome III was obtained by RFLP analysis of a cDNA marker linked to the

S

‐locus of potato (CP100). Tight linkage was found between CP100 and a peroxidase isozyme locus previously mapped to chromosome III. The co‐localisation of CP100, peroxidase and the

S

‐locus in petunia reveals synteny around the

S

‐locus between four members of the Solanaceae family. The implications of a subcentromeric localisation of the

S

‐locus are discussed in relation to chromosome fragments associated with self‐compatible mutants.
Circadian dysfunction causes aberrant hypocotyl elongation patterns in Arabidopsis
Authors: Dowson‐Day Mandy J. , Millar Andrew J.
Published: March 2003

Many endogenous and environmental signals control seedling growth, including several phototransduction pathways. We demonstrate that the circadian clock controls the elongation of the Arabidopsis hypocotyl immediately upon germination. The pattern of hypocotyl elongation in constant light includes a daily growth arrest spanning subjective dawn and an interval of rapid growth at subjective dusk. Maximal hypocotyl growth coincides with the phase during which the cotyledons are raised, in the previously described rhythm of cotyledon movement. The rhythm of hypocotyl elongation was entrained by light–dark cycles applied to the imbibed seed and its period was shortened in the

toc1–1

mutant, indicating that it is controlled by a similar circadian system to other rhythmic markers. The daily growth arrest is abolished by the

early flowering 3
(elf3) mutation, suggesting that this defect may cause its long‐hypocotyl phenotype. Mutations that affect the circadian system can therefore cause gross morphological phenotypes, not because the wild‐type gene functions pleiotropically in several signalling pathways, but rather because the circadian clock exerts widespread control over plant physiology.
Comparative analysis ofShaplotypes with very similarSLGalleles inBrassica rapaandBrassica oleracea
Authors: Kusaba Makoto , Nishio Takeshi
Published: March 2003

Self‐incompatibility in

Brassica

is controlled by a single multi‐allelic locus (the

S

locus) which harbors at least two highly polymorphic genes,

SLG

and

SRK

. SRK is a putative transmembrane receptor kinase and its amino acid sequence of the extracellular domain of SRK (the S domain) exhibits high homology to that of SLG. The amino acid sequences of the SLGs of

S8

and

S46

haplotypes of

B. rapa

are very similar and those of

S23 and S29

haplotypes of

B. oleracea

were also found to be almost identical. In both cases, SLG and the S domain of SRK of the same haplotype were less similar. This seems to contradict the idea that SLG and SRK of the same haplotype have the same self‐recognition specificity. In the transmembrane‐kinase domain, the

SRK

alleles of the

S8

and

S46

haplotypes had almost identical nucleotide sequences in spite of their lower homology in the S domain. Such a cluster of nucleotide substitutions is probably due to recombination or related events, although recombination in the S locus is thought to be suppressed. Based on our observations, the recognition mechanism and the evolution of self‐incompatibility in

Brassica

are discussed.
Light regulates alternative splicing of hydroxypyruvate reductase in pumpkin
Authors: Mano Shoji , Hayashi Makoto , Nishimura Mikio
Published: March 2003

Hydroxypyruvate reductase (HPR) is a leaf peroxisomal enzyme that functions in the glycolate pathway of photorespiration in plants. We have obtained two highly similar cDNAs for pumpkin HPR (HPR1 and HPR2). It has been revealed that two HPR mRNAs might be produced by alternative splicing from a single type of pre‐mRNA. The HPR1 protein, but not the HPR2 protein, was found to have a targeting sequence into leaf peroxisomes at the C‐terminus, suggesting that alternative splicing controls the subcellular localization of the two HPR proteins. Immunoblot analysis and subcellular fractionation experiments showed that HPR1 and HPR2 proteins are localized in leaf peroxisomes and the cytosol, respectively. Moreover, indirect fluorescence microscopy and analyses of transgenic tobacco cultured cells and

Arabidopsis thaliana

expressing fusion proteins with green fluorescent protein (GFP) revealed the different subcellular localizations of the two HPR proteins. Both mRNAs were induced developmentally and by light, but with quantitative differences. Almost equal amounts of the mRNAs were detected in pumpkin cotyledons grown in darkness, but treatment with light greatly enhanced the production of HPR2 mRNA. These findings indicate that light regulates alternative splicing of HPR mRNA, suggesting the presence of a novel mechanism of mRNA maturation, namely light‐regulated alternative splicing, in higher plants.
AnArabidopsismutant with deregulated ABA gene expression: implications for negative regulator function
Authors: Foster Randy , Chua Nam‐Hai
Published: March 2003

The physiological acclimation of plants to osmotic stresses involves a complex programme of gene regulation. In one signalling pathway, elevated levels of abscisic acid (ABA) activate a subset of stress genes. Because ABA responses lack a definable morphological phenotype, we have screened for mutants that exhibit deregulated ABA‐responsive gene expression. To monitor this ABA response, a line of

Arabidopsis thaliana

carrying a transgene composed of the ABA‐responsive

Arabidopsis kin2

promoter fused to the coding sequence for the firefly luciferase gene,

kin2

::

luc

, was generated. Patterns of ABA‐responsive luciferase activity were monitored by photon counting. In contrast to wild‐type plants which display a transient activation of

kin2

::

luc

, an ABA deregulated gene expression mutant (

ade1

) exhibits both sustained and enhanced levels of transgene activity. Levels of

kin2

,

cor47

and

rab18

expression in

ade1

plants are also enhanced and prolonged indicating that the molecular mechanism(s) altered in

ade1

plants affects the regulation of other ABA‐responsive genes. The mutant phenotype is specific for the ABA response as cold‐inducible

kin2

expression is unaltered in

ade1

plants. Genetic analyses indicate that the

ade1

mutant is a monogenic recessive trait. A role for negative regulator function in ABA signalling is discussed.
Map positions ofSFRgenes in relation to other freezing‐related genes ofArabidopsis thaliana
Authors: Thorlby Glenn , Veale Emma , Butcher Kevin , Warren Gareth
Published: March 2003

We determined the map positions of seven

SFR

genes and compared them to the positions of 51 genes suspected of involvement in freezing tolerance in

Arabidopsis thaliana

. The

SFR

genes were recognized by the freezing sensitivity of mutants; the others (including 14 whose map positions we have determined) were genes whose expression is induced by low temperature, genes involved in abscisic acid (ABA) biosynthesis and perception, and genes involved in tolerance of oxidative stress. The comparison of map positions indicated a limited set of potential identities, some of which were eliminated by further mapping or by an allelism test.
Functional analysis of a Golgi‐localized Kex2p‐like protease in tobacco suspension culture cells
Authors: Jiang Liwen , Rogers John C.
Published: March 2003

Kex2p is the prototype of a Golgi‐resident protease responsible for the processing of prohormones in yeast and mammalian cells. A Kex2p‐like pathway was shown to be responsible for processing the fungal KP6 protoxin in transgenic tobacco plants. We previously described a chimeric integral membrane reporter protein that traffics through Golgi to the lytic prevacuole where it was proteolytically processed. As a first step to isolate and clone the Kex2p‐like protease in plant cells, we designed and used a similar chimeric reporter protein containing Kex2 cleavage sites to assay the Kex2p‐like activity and to determine its substrate specificity in tobacco cells. Here we demonstrate that the Kex2 cleavage sites of the reporter were specifically processed by a protease activity with a substrate specificity characteristic of yeast Kex2p. This Kex2p‐like protease in tobacco cells is also a Golgi‐resident enzyme. Thus, the reporter protein provides a biochemical marker for studying protein traffic through the Golgi in plant cells. These results additionally should allow the design of synthetic substrates for use in biochemical purification of the plant enzyme.
The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia
Authors: Sessions Allen , Weigel Detlef , Yanofsky Martin F.
Published: March 2003

The Arabidopsis thaliana MERISTEM LAYER 1 (ATML1) gene is expressed in the epidermis of developing embryos and shoot meristems. To identify regulatory sequences necessary for epidermis‐specific expression, three fusions of overlapping ATML1 genomic sequences to the GUS reporter gene were introduced into Arabidopsis plants. All fusion genes conferred epidermis‐specific expression of both GUS mRNA and protein activity but varied in both the timing and relative levels of expression, suggesting partial redundancy of ATML1 regulatory elements. This study defines L1‐specific regulatory sequences that are sufficient to direct foreign gene expression in a layer‐specific manner.
Maize high chlorophyll fluorescent 60 mutation is caused by an Ac disruption of the gene encoding the chloroplast ribosomal small subunit protein 17
Authors: Schultes Neil P. , Sawers Ruairidh J. H. , Brutnell Thomas P. , Krueger Roger W.
Published: March 2003

The maize mutation high chlorophyll fluorescence 60‐muTable 1 (hcf60‐m1), generated through Activator (Ac) tagging, has insufficient photosynthetic electron transport. Here we show that the Hcf60 gene encodes a protein with substantial amino acid similarity to plant plastid and bacterial ribosomal small subunit protein 17 (RPS17) proteins. The lack of detectable HCF60 transcripts in mutant leaves, and insertion of the transposed Ac element 17 bp upstream of the start of translation in the mutated locus, suggest that little if any RPS17 is produced. The mutant phenotype is consistent with reduced plastid translation. Seedling lethal hcf60‐m1 plants display temperature and light‐dependent chlorophyll deficiencies, a depletion of plastid rRNA pools, and few high‐molecular‐weight polysomal complexes. Growth under moderate light conditions (27°C, 100 μE m−2 sec−1) allows for substantial chlorophyll accumulation in mutant leaves, yet the number of functional photosystem II complexes appears low. Nevertheless, the presence of a limited but intact C4 system indicates that some plastid translation occurs.

. Leaf chlorophyll content under different growth conditions

17°C, 100 μE m−2 sec−1
27°C, 100 μE m−2 sec−1
Greenhouse

1st leaf
1st leaf
2nd leaf
1st leaf

WT
hcf60
WT
hcf60
WT
hcf60
WT
hcf60

Total chlorophyll
0.385 ± 0.42
>0.002 ±0.00017
1.11 ±0.15
0.79 ± 0.1
1.3 ±0.2
0.71 ± 0.17
0.89 ±0.077
0.026 ± 0.012

 μg mg−1 tissue
n = 4

n = 4

n= 7

n= 7

n= 7

n= 7

n = 4

n = 3

Total chlorophyll
100
>0.5
100
71
100
55
100
2.9

 % WT

Chlorophyll a/b
2.12 ±0.2
–
2.54 ±0.22
1.89 ±0.16
2.65 ±0.26
1.97 ±0.12
3.02 ±0.11
2.87 ±0.23
A membrane‐potential dependent ABC‐like transporter mediates the vacuolar uptake of rye flavone glucuronides: regulation of glucuronide uptake by glutathione and its conjugates
Authors: Klein Markus , Martinoia Enrico , Hoffmann‐Thoma Gudrun , Weissenböck Gottfried
Published: March 2003

In this paper we present results on the vacuolar uptake mechanism for two flavone glucuronides present in rye mesophyll vacuoles. In contrast to barley flavone glucosides ( Klein et al. (1996
) J. Biol. Chem. 271, 29666–29671), the flavones luteolin 7‐O‐diglucuronyl‐4′‐O‐glucuronide (R1) and luteolin 7‐O‐diglucuronide (R2) were taken up into vacuoles isolated from rye via a directly energised mechanism. Kinetic studies suggested that the vacuolar glucuronide transport system is constitutively expressed throughout rye primary leaf development. Competition experiments argued for the existence of a plant MRP‐like transporter for plant‐specific and non‐plant glucuronides such as β‐estradiol 17‐(β‐
d‐glucuronide) (E217G). The interaction of ATP‐dependent vacuolar glucuronide uptake with glutathione and its conjugates turned out to be complex: R1 transport was stimulated by dinitrobenzene‐GS and reduced glutathione but was inhibited by oxidized glutathione in a concentration‐dependent manner. In contrast, R2 uptake was not increased in the presence of reduced glutathione. Thus, the transport system for plant‐derived glucuronides differed from the characteristic stimulation of vacuolar E217G uptake by glutathione conjugates but not by reduced glutathione (
Klein et al. (1998
) J. Biol. Chem. 273, 262–270). Using tonoplast vesicles isolated with an artificial K+ gradient, we demonstrate for the first time for plant MRPs that the ATP‐dependent uptake of R1 is membrane‐potential dependent. We discuss the kinetic capacity of the ABC‐type glucuronide transporter to explain net vacuolar flavone glucuronide accumulation in planta during rye primary leaf development and the possibility of an interaction of potential substrates at both the substrate binding and allosteric sites of the MRP transporter regulating the activity towards a certain substrate.
Evidence that auxin promotes gibberellin A1 biosynthesis in pea
Authors: Ross John J. , O'neill Damian P. , Smith Jennifer J. , Kerckhoffs L. Huub J. , Elliott Robert C.
Published: March 2003

In shoots of the garden pea, the bioactive gibberellin (GA1) is synthesised from GA20, and the enzyme which catalyses this step (a GA 3‐oxidase –‐ PsGA3ox1) is encoded by Mendel's LE gene. It has been reported previously that decapitation of the shoot (excision of the apical bud) dramatically reduces the conversion of [3H]GA20 to [3H]GA1 in stems, and here we show that endogenous GA1 and PsGA3ox1 transcript levels are similarly reduced. We show also that these effects of decapitation are completely reversed by application of the auxin indole‐3‐acetic acid (IAA) to the ‘stump’ of decapitated plants. Gibberellin A20 is also converted to an inactive product, GA29, and this step is catalysed by a GA 2‐oxidase, PsGA2ox1. In contrast to PsGA3ox1, PsGA2ox1 transcript levels were increased by decapitation and reduced by IAA application. Decapitation and IAA treatment did not markedly affect the level of GA1 precursors. It is suggested that in intact pea plants, auxin from the apical bud moves into the elongating internodes where it (directly or indirectly) maintains PsGA3ox1 transcript levels and, consequently, GA1 biosynthesis.