bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of agthi1, whose product is involved in biosynthesis of the thiamine precursor thiazole, in actinorhizal nodules of Alnus glutinosa
Authors: Ribeiro Ana , Praekelt Uta , Akkermans Antoon D.L. , Meacock Peter A. , van Kammen Ab , Bisseling Ton , Pawlowski Katharina
Published: March 2003

A cDNA clone, pAgthi1, encoding a homologue of yeast Thi4, which is involved in thiazole biosynthesis, was isolated from a library made from poly(A) RNA from actinorhizal nodules of Alnus glutinosa by differential screening with nodule and root cDNA, respectively. The corresponding gene, agthi1, was shown to be expressed at high levels in nodules and shoot tips of A. glutinosa, while having low expression levels in roots, flowers, and developing fruits. The function of AgThi1 was demonstrated by yeast complementation studies, in which AgThi1 was able to rescue a yeast thi4 mutant when fused to the yeast Thi4 signal peptide. In A. glutinosa nodules, high levels of agthi1 mRNA were detected in the infected cortical cells and in the pericycle of the nodule vascular system. A homologue of this cDNA, ara6/tz, was identified in Arabidopsis thaliana. ara6 maps in a region of chromosome 5 of Arabidopsis containing the tz locus which is consistent with the fact that ara6transcription is disturbed in two tz mutant lines. ara6/tz is expressed at high levels in chloroplast‐containing parenchymatic cells of leaves, inflorescence shoots and flowers of Arabidopsis, and at lower levels in the vascular system.
A transposon insertion in the Arabidopsis SSR16 gene causes an embryo‐defective lethal mutation
Authors: Tsugeki Ryuji , Kochieva Elena Z. , Fedoroff Nina V.
Published: March 2003

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co‐segregation of the mutant phenotype with the transposon‐borne drug‐resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3′ end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium, as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo‐defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.
Is E37, a major polypeptide of the inner membrane from plastid envelope, an S‐adenosyl methionine‐dependent methyltransferase?
Authors: Teyssier Emeline , Block Maryse A. , Douce Roland , Joyard Jacques
Published: March 2003

Using antibodies raised against E37, one of the major polypeptides of the inner membrane from the chloroplast envelope, it has been demonstrated that a single immunologically related polypeptide was present in total protein extracts from various higher plants (monocots and dicots), in photosynthetic and non‐photosynthetic tissues from young spinach plantlets, as well as in the cytoplasmic membrane from the cyanobacteria Synechococcus. This ubiquitous distribution of E37 strongly suggests that this protein plays an envelope‐specific function common to all types of plastids. Comparison of tobacco and spinach E37 amino acid sequences deduced from the corresponding cDNA demonstrates that consensus motifs for S‐adenosyl methionine‐dependent methyltransferases are located in both sequences. This hypothesis was confirmed using a biochemical approach. It was demonstrated that E37, together with two minor spinach chloroplast envelope polypeptides of 32 and 39 kDa, can be specifically photolabeled with [3H]‐S‐adenosyl methionine upon UV‐irradiation. Identification of E37 as a photolabeled polypeptide was established by immunoprecipitation. Furthermore, photolabeling of the three envelope polypeptides was specifically inhibited by very low concentration of S‐adenosyl homocysteine, thus providing evidence for the presence within these proteins of S‐adenosyl methionine‐ and S‐adenosyl homocysteine‐binding sites that were closely associated. Taken as a whole these results strongly suggest that E37 is an ubiquitous plastid envelope protein that probably has an S‐adenosyl methionine‐dependent methyltransferase activity. The 32 and 39 kDa envelope polypeptides probably have a similar methyltransferase activity.
The GURKE gene is required for normal organization of the apical region in the Arabidopsis embryo
Authors: Torres‐Ruiz Ramón A. , Lohner Andrea , Jürgens Gerd
Published: March 2003

Dicot plant embryos undergo a transition from radial to bilateral symmetry. In Arabidopsis, this change reflects patterning within the apical region, resulting in the formation of the cotyledon and shoot meristem primordia. Mutations in the GURKE gene give seedlings with highly reduced or no cotyledons. Both strong and weak gurke alleles confer this phenotypic variability although strong alleles often eliminate the entire apex and sometimes also part of the hypocotyl. The root and the root meristem as well as the radial pattern of concentric tissue layers are essentially normal. The mutant seedling phenotype can be traced back to the triangular/early‐heart stage of embryogenesis when abnormal cell divisions occur within the apical region such that no or only rudimentary cotyledon primordia are established. The postembryonic development of gurke seedlings was examined in culture. In weak alleles, apical growth gave rise to abnormal leaves and stem‐like structures and, eventually, abnormal flowers. In strong alleles, the apical region often failed to grow but occasionally produced fused leaf‐like structures with no dorso‐ventral polarity and a totally unorganized vascular system while no stems developed. The observations suggest that the GURKE gene is involved primarily in the organization of the apical region in the embryo and may also play a role during postembryonic development.
Characterization of anther‐expressed genes encoding a major class of extracellular oleosin‐like proteins in the pollen coat of Brassicaceae†
Authors: Ross Joanne H.E. , Murphy Denis J.
Published: March 2003

A large, heterogeneous, highly expressed gene family encoding oleosin‐like proteins is described in the Brassicaceae. íeven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE‐PCR and compared with other recently described anther‐specific oleosin‐like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3–5 mm B. napus buds, which contained microspores at the late‐vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C‐terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32–38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N‐terminal 15–20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal‐expressed oleosin‐like genes. This indicates the possibility of post‐translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin‐like proteins is synthesized in the tapeturn of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed‐specific intracellular oleosins, although their biological function may be different.
Importance of isoforms of starch‐branching enzyme in determining the structure of starch in pea leaves
Authors: Tomlinson Kim L. , Lloyd James R. , Smith Alison M.
Published: March 2003

The impact of a mutation at the r locus of peas (Pisum sativum L.) on the structure of starch in the leaf has been studied. The mutation specifically eliminates the A class of isoform of starch‐branching enzyme (SBE A) from the leaf, causing a 10‐fold reduction in the total activity of the enzyme. Gel‐permeation chromatography and thymol precipitation show that wild‐type leaf starch consists of polymers with the general characteristics of amylose and amylopectin, although amylose is only a very minor component of the starch. High‐performance anion exchange chromatography (HPAEC) of debranched amylopectin reveals that the distribution profile of branch lengths is strongly polymodal, and distinctly different from that of the amylopectin of storage starches. The mutation at the r locus results in the appearance of an amylopectin‐like glucan of low molecular weight in the starch. The absorbance of the iodine complex of the amylopectin and analysis by HPAEC both indicate that the mutation causes an increase in the average branch length of the amylopectin but does not affect the polymodal nature of the distribution of branch lengths. The extent to which these effects of the mutation are specifically due to the loss of SBE A is discussed. It is suggested that differences in properties between isoforms of SBE are not the main factors that determine the polymodal distribution of branch lengths in amylopectin.
Regulation of flavonol biosynthesis during anther and pistil development, and during pollen tube growth in Solanum tuberosum
Authors: van Eldik Gerben J. , Reijnen Wim H. , Ruiter René K. , van Herpen Marinus M.A. , Schrauwen Jan A.M. , Wullems George J.
Published: March 2003

The regulation of flavonol biosynthesis was studied in anthers and pistils of Solanum tuberosum. Flavonols are essential for functional pollen tube growth in a number of species. Flavonol accumulation in whole anthers started at the unicellular stage of pollen development and continued until pollen maturity. A cDNA clone encoding flavonol synthase (FLS) was isolated. Fls gene expression was detected in pistils, anthers, petals and ovaries, the organs in which flavonols are accumulating. Fls transcripts were present in unicellular and bicellular pollen, but not in mature pollen. The expression patterns of three genes encoding enzymes in the flavonoid biosynthetic pathway, chalcone synthase (chs), flavanone‐3‐hydroxylase and fls were analysed in developing anthers and pistils. Only chs transcripts accumulated concomitantly with the flavonols in anthers. In pistils of potato, pollen tube growth induced an increase in fls gene expression that, unlike the situation in pollinated pistils of petunia, did not result in an increased flavonol content. Flavonol biosynthesis in anthers is probably initiated by the expression of the chs gene, and flavonol accumulation in pistils upon pollen tube growth is not an universal phenomenon.
Induction of alternative oxidase synthesis by herbicides inhibiting branched‐chain amino acid synthesis
Authors: Aubert Serge , Bligny Richard , Day David A. , Whelan James , Douce Roland
Published: March 2003

Sycamore suspension cells (Acer pseudoplatanus L.) were incubated in the presence of sulfonylurea and imidazolinone herbicides. These inhibitors of acetolactate synthase (ALS), a key enzyme of branched‐chain amino acid synthesis, triggered a dramatic induction of the alternative oxidase (AOX). AOX activity increased in treated cells, eventually exceeding cytochrome (cyt) pathway activity. This induction of AOX activity was correlated with the accumulation of a 35 kDa AOX protein in isolated mitochondria, detected by Western blotting with a monoclonal antibody against Sauromatum guttatum AOX. It was preceded by the accumulation of putative 1.6 kb AOX mRNA, detected using an Aox cDNA probe from soybean. The metabolic perturbations induced by the herbicides rather than the herbicide molecules themselves were responsible for this induction of AOX. However, α‐oxobutyrate (one of the substrates of ALS) and its transamination product, α‐aminobutyrate, which accumulated after herbicide treatment, were not involved. The inhibition of branched‐chain amino acid synthesis was probably somehow responsible for the AOX induction since: (i) a mixture of those amino acids (leucine, isoleucine, valine) prevented AOX induction by ALS inhibitors; (ii) the herbicide Hoe 704, a potent inhibitor of acetolactate reducto‐isomerase (the enzyme following ALS in the branched‐chain amino acid pathway), also triggered AOX induction.
Gene targeting and instability of Agrobacterium T‐DNA loci in the plant genome
Authors: Risseeuw Eddy , Franke‐van Dijk Marry E.I. , Hooykaas Paul J.J.
Published: March 2003

To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T‐DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the β‐glucuronidase (GUS) gene. Subsequently, the target lines were transformed with a replacement‐type T‐DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin‐resistance gene. For both Nicotiana species kanamycin‐resistant lines were selected which had lost the CD gene and the GUS activity. One tobacco line was the result of a precise gene targeting event. However, most other lines were selected due to a chromosomal deletion of the target locus. The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts. T‐DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable. A few lines were obtained in which the target locus had been deleted partially. Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies. We conclude that T‐DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue. The relationships between callus culture, genetic instability and the process of T‐DNA integration and deletion in the plant genome are discussed.
Abscisic acid and jasmonic acid activate wound‐inducible genes in potato through separate, organ‐specific signal transduction pathways
Authors: Dammann Christian , Rojo Enrique , Sánchez‐Serrano Jose J.
Published: March 2003

Mechanical damage to leaf tissue causes an increase in abscisic acid (ABA) which in turn activates the biosynthesis of jasmonic acid (JA). The resulting higher endogenous JA levels subsequently activate the expression of wound‐inducible genes. This study shows that JA induces the expression of different sets of genes in roots and leaves of potato plants. When roots of intact plants were treated with JA, high levels of proteinase inhibitor II (pin2), cathepsin D inhibitor, leucine aminopeptidase and threonine deaminase mRNAs accumulated in the systemic leaves. However, in the treated roots, very low, if any, expression of these genes could be detected. In contrast, a novel, root‐specific pin2 homologue accumulated in the JA‐treated root tissue which could not be detected in leaves, either systemic or those directly treated with JA. Application of okadaic acid and staurosporine revealed that a protein phosphorylation step is involved in the regulation of this differential response. In leaves, a protein phosphatase is required for the JA‐induced expression of pin2 and the other genes analysed. This phosphatase activity is not necessary for the JA‐induced expression of a pin2 homologue in roots, suggesting the existence of different transduction pathways for the JA signal in these organs. The requirement of a protein phosphatase activity for JA‐mediated gene induction has enabled identification of a JA‐independent pathway for ABA induction of pin2 and the other wound‐inducible genes. This alternative pathway involves a protein kinase, and appears to be selective for wound‐inducible genes. Our data suggest the presence of a complex, organ‐specific transduction network for regulating the effects of the plant hormones ABA and JA on gene expression upon wounding.
Transgene‐induced silencing of S‐locus genes and related genes in Brassica
Authors: Conner Joann A. , Tantikanjana Titima , Stein Joshua C. , Kandasamy M.K. , Nasrallah June B. , Nasrallah Mikhail E.
Published: March 2003

The S‐locus receptor kinase gene, SRK, and the S‐locus glycoprotein gene, SLG, are required for the self‐incompatibility response in Brassica. A transgenic approach was used to investigate the function of these genes. Several chimeric genes predicted either to interfere with SRK function in self‐incompatible hosts and possibly generate loss‐of‐function mutants, or to complement a defective SRK in a self‐compatible host and possibly generate gain‐of‐function plants, were designed. The transgenes led to a dramatic reduction in the expression of endogenous S‐locus and related genes, thus limiting the usefulness of transgenic approaches for the dissection of S‐locus gene function in Brassica. Several features of this ‘homology‐dependent gene silencing’ are described, including its tissue‐specific nature, the lack of a direct correlation between the severity of silencing and the extent of sequence similarity shared by the silenced gene and the transgene, its association with increased cytosine methylation of silenced endogenous genes in at least some cases, and its persistence in transgenic progenies that do not inherit the transgene. Most significantly, silencing of S genes in self‐incompatible host plants results in the breakdown of self‐incompatibility in the stigma, whereas a 75% reduction in S gene transcripts in anthers does not affect pollen phenotype.
Targeted misexpression of AGAMOUS in whorl 2 of Arabidopsis flowers
Authors: Jack Thomas , Sieburth Leslie , Meyerowitz Elliot
Published: March 2003

In Arabidopsis, transcripts of the floral organ identity genes APETALA3, APETALA1, AGAMOUS, and PISTILLATA are expressed only in a subset of the floral organs. To test and extend present models for establishment of floral organ identity, we constructed a transgenic line that expresses the AGAMOUS gene under the control of the APETALA3 promoter (pAP3::AG). In wild‐type flowers, AGAMOUS is expressed in the third and fourth floral whorls, whereas APETALA3 is expressed at high levels in whorls two and three. Under the control of the APETALA3 promoter, AGAMOUS is misexpressed in the second whorl of the flower. The organs in the second whorl of pAP3::AG flowers either fail to develop or develop as stamens. When pAP3::AG is crossed to the homeotic mutants apetala1, apetala3, pistillata, agamous and superman, novel floral phenotypes result. In pAP3::AG agamous‐3 mutants, the flowers are indeterminate and consist of a repetition of sepals and stamens, rather than sepals and petals, as is observed in agamous single mutants; the indeterminacy demonstrates that AG is required in the fourth whorl to make the flower determinate.
The G‐box and H‐box in a 39 bp region of a French bean chalcone synthase promoter constitute a tissue‐specific regulatory element
Authors: Faktor Ouriel , Loake Gary , Dixon Richard A. , Lamb Chris J.
Published: March 2003

Two adjacent motifs, the G‐box and H‐box, in the bean chalcone synthase (CHS) gene promoter are essential for floral and root‐specific expression. To evaluate the cooperative role of these motifs as a tissue‐specific regulatory element, a synthetic 39 bp DNA fragment containing the two motifs was prepared. Polymers of this fragment were linked to minimal CHS15 promoter, or cauliflower mosaic virus 35S promoter, and fused to the β‐d‐glucuronidase (GUS) reporter gene. Measurements of GUS activity in transformed tobacco plants revealed that the 39 bp polymer confers upon both minimal promoters a high level of expression that follows the typical tissue‐specific expression pattern. Mutations in either the G‐box or H‐box motifs abolished expression, emphasizing the essential role of each motif in transcriptional activation. These results define an exclusive and cooperative element in the CHS15 promoter which contains the information necessary to establish the typical CHS15 tissue‐specific expression pattern. However, this element is present within the context of the native promoter, and it requires additional cis‐elements to modulate the level and temporal expression established during plant development.
A combinatorial role for exon, intron and splice site sequences in splicing in maize
Authors: Carle‐Urioste Jose C. , Brendel Volker , Walbot Virginia
Published: March 2003

Plant introns are typically AU‐rich or U‐rich, and this feature has been shown to be important for splicing. In maize, however, about 20% of the introns exceed 50% GC, and most of them are efficiently spliced. A series of constructs has been designed to analyze the cis requirements for splicing of the GC‐rich Bz2 maize intron and two other GC‐rich intron derivatives. By manipulating exon, intron and splice site sequences it is shown that exons can play an important role in intron definition: changes in exon sequences can increase splicing efficiency of a GC‐rich intron from 17% to 86%. The relative difference, or base compositional contrast, in GC and U content between exon and intron sequences in the vicinity of splice sites, rather than the absolute base‐content of the intron or exons, correlates with splicing efficiency. It is also shown that GC‐rich intron constructs that are poorly spliced can be partially rescued by an improved 3′ splice site.
Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana
Authors: Wymer Carol L. , Bibikova Tatiana N. , Gilroy Simon
Published: March 2003

In this study, confocal ratio analysis was used to image the relationship between cytoplasmic free calcium concentration ([Ca2+]c) and the development of root hairs of Arabidopsis thaliana. Although a localized change in [Ca2+]c that preceded or predicted the site of root hair initiation could not be detected, once initiated the majority of emerging root hairs showed an elevated [Ca2+]c (>1 μM) in their apical cytoplasm, compared with 100– 200 nM in the rest of the cell. These emerging root hairs then moved into a 3–5 h phase of sustained elongation during which they showed variable growth rates. Root hairs that were rapidly elongating exhibited a highly localized, elevated [Ca2+]c at the tip. Non‐growing root hairs did not exhibit the [Ca2+]c gradient. The rhd‐2 mutant, which is defective in sustained root hair growth, showed an altered [Ca2+]c distribution compared with wild‐type. These results implicate [Ca2+]c in regulating the tip growth process. Treatment of elongating wild‐type root hairs with the Ca2+ channel blocker verapamil (50 μM) caused dissipation of the elevated [Ca2+]c at the tip and cessation of growth, suggesting a requirement for Ca2+ channel activity at the root hair tip to maintain growth. Manganese treatment also preferentially quenched Indo‐1 fluorescence in the apical cytoplasm of the root hair. As manganese is thought to enter cells through Ca2+‐permeable channels, this result also suggests increased Ca2+ channel activity at the tip of the growing hair. Taken together, these data suggest that although Ca2+ does not trigger the initiation of root hairs, Ca2+ influx at the tip of the root hair leads to an elevated [Ca2+]c that may be required to sustain root hair elongation.
Expression of ACC oxidase promoter—GUS fusions in tomato and Nicotiana plumbaginifolia regulated by developmental and environmental stimuli
Authors: Blume Beatrix , Grierson Donald
Published: March 2003

The enzyme ACC oxidase, catalysing the last step in the biosynthesis of the plant hormone ethylene, is encoded by a small multigene family in tomato, comprising three members, LEACO1, LEACO2 and LEACO3. LEACO1 is the major gene expressed during ripening, leaf senescence, and wounding (Barry et al., 1996). To investigate the transcriptional regulation of ACC oxidase gene expression, chimeric fusions between the β‐glucuronidase reporter gene and 97 bp of 5′ UTR plus 124, 396 and 1825 bp, respectively, of 5′ untranscribed LEACO1 sequence were constructed and introduced into Lycopersicon esculentum (Mill cv. Ailsa Craig) and Nicotiana plumbaginifolia. Analysis of transgenic tomatoes indicated that the region containing nucleotides −124 to +97 of the LEACO1 gene is sufficient to confer a marked increase in GUS activity during fruit ripening, albeit at very low levels. Fusion of 396 and 1825 bp of LEACO1 upstream sequence resulted in strong and specific induction of GUS expression in situations known to be accompanied by enhanced ethylene production. Reporter gene expression was similar to that of the endogenous LEACO1 gene, with major increases especially during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers. Analysis of transgenic N. plumbaginifolia plants confirmed the pattern of LEACO1 promoter activity detected in tomato leaves and flowers. Reporter gene expression was also induced following wounding, treatment with ethylene, and pathogen infection. Histochemical analysis illustrated localized GUS activity in the pericarp of ripening fruit, abscission zones of senescent petioles and unfertilized flowers, and at wound sites. These results demonstrate that ACC oxidase is regulated at the transcriptional level in a wide range of cell types at different developmental stages and in response to several external stimuli.
The maize actin‐depolymerizing factor, ZmADF3, redistributes to the growing tip of elongating root hairs and can be induced to translocate into the nucleus with actin
Authors: Jiang Chang‐Jie , Weeds Alan G. , Hussey Patrick J.
Published: March 2003

The maize actin depolymerizing factor, ZmADF3, binds G‐and F‐actin, and increases in vitro actin dynamics. Polyclonal antibodies have been raised against ZmADF3 and these detect a single band of approximately 17 kDa in all maize tissues examined, with the exception of pollen. In the development of root hairs, the distribution of ZmADF3 is related to actin reorganization. In the early stages of hair development, ZmADF3 is distributed throughout the cytoplasm. As the hair emerges and the microfilament bundles redirect to the outgrowth there is a simultaneous redistribution of ZmADF3 which now concentrates at the tip of the emerging hair and remains in this position as elongation proceeds. These observations show that ZmADF3 localizes to a region where actin is being remodelled during tip growth. After cytochalasin D treatment which disrupts actin filaments, short rods of ZmADF3 and actin appear in the nucleus suggesting that ZmADF3 may function by guiding actin to sites of actin polymerization.
Modulation of statolith mass and grouping in white clover (Trifolium repens) grown in 1‐g, microgravity and on the clinostat
Authors: Smith Jeffrey D. , Todd Paul , Staehelin L. Andrew
Published: March 2003

Current models of gravity perception in higher plants focus on the buoyant weight of starch‐filled amyloplasts as the initial gravity signal susceptor (statolith). However, no tests have yet determined if statolith mass is regulated to increase or decrease gravity stimulus to the plant. To this end, the root caps of white clover (Trifolium repens) grown in three gravity environments with three different levels of gravity stimulation have been examined: (i) 1‐g control with normal static gravistimulation, (ii) on a slow clinostat with constant gravistimulation, and (iii) in the stimulus‐free microgravity aboard the Space Shuttle. Seedlings were germinated and grown in the BioServe Fluid Processing Apparatus and root cap structure was examined at both light and electron microscopic levels, including three‐dimensional cell reconstruction from serial sections. Quantitative analysis of the electron micrographs demonstrated that the starch content of amyloplasts varied with seedling age but not gravity condition. It was also discovered that, unlike in starch storage amyloplasts, all of the starch granules of statolith amyloplasts were encompassed by a fine filamentous, ribosome‐excluding matrix. From light micrographic 3‐D cell reconstructions, the absolute volume, number, and positional relationships between amyloplasts showed (i) that individual amyloplast volume increased in microgravity but remained constant in seedlings grown for up to three days on the clinostat, (ii) the number of amyloplasts per cell remained unchanged in microgravity but decreased on the clinostat, and (iii) the three‐dimensional positions of amyloplasts were not random. Instead amyloplasts in microgravity were grouped near the cell centers while those from the clinostat appeared more dispersed. Taken together, these observations suggest that changing gravity stimulation can elicit feedback control over statolith mass by changing the size, number, and grouping of amyloplasts. These results support the starch ‐ statolith theory of graviperception in higher plants and add to current models with a new feedback control loop as a mechanism for modulation of statolith responsiveness to inertial acceleration.
Photosynthetic oxygen evolution within Sesbania rostrata stem nodules
Authors: James Euan K. , Minchin Frank R. , Oxborough Kevin , Cookson Alan , Baker Neil R. , Witty John F. , Crawford Robert M.M. , Sprent Janet I.
Published: March 2003

The tropical wetland legume, Sesbania rostrata Brem. forms N2‐fixing nodules along its stem and on its roots after infection by Azorhizobium caulinodans. The N2‐fixing tissue is surrounded by a cortex of uninfected cells which, in the stem nodules (but not the root nodules), contain chloroplasts. The photosynthetic competence of these chloroplasts was assessed through a novel technique involving image analysis of chlorophyll a fluorescence. Calculation of the quantum efficiency of photosystem II (PS II) photochemistry from these images indicated that most of the chloroplasts with potential for non‐cyclic photosynthetic electron transport were concentrated within the mid‐ and inner‐cortex, close to the edge of the N2‐fixing tissue. PS II activity in the cortical cells was confirmed in vivo using O2‐specific microelectrodes which showed that the concentration of O2 (pO2) in the outer cortex could rise from less than 1% up to 23.4% upon increased irradiance of the nodule, but that the pO2 of the inner cortex and infected tissue remained less than 0.0025%. Nitrogenase activity of stem nodules, as measured using a flow‐through acetylene reduction assay (no H2 evolution was evident), showed a reversible increase of 28% upon exposure of the nodules to supplemental light. This increase resembled that obtained with stem nodules upon their exposure to an external pO2 of 40%.
Two different modes of early development and nitrogen assimilation in gymnosperm seedlings†
Authors: García‐Gutiérrez Angel , Dubois Frédéric , Cantón Francisco R. , Gallardo Fernando , Sangwan Rajbir S. , Cánovas Francisco M.
Published: March 2003

Light‐independent chloroplast development and expression of genes encoding chloroplast proteins occur in many but not all species of gymnosperms. Early development in maritime pine (Pinus pinaster) seedlings was strongly light‐independent, whereas Ginkgo biloba seedlings exhibited a typical angiosperm‐like morphogenesis with differentiated patterns in light and dark. In pine, chloroplast polypeptides were undetectable in the seed embryo and accumulated in cotyledons of both light‐ and dark‐grown plants in good correlation with light‐independent chlorophyll synthesis. In contrast, chlorophyll and chloroplast proteins were only detected in light‐grown ginkgo. Pine cytosolic glutamine synthetase (GS) and ferredoxin glutamate synthase (Fd‐GOGAT) were present at low levels in the seeds and accumulated at comparable amounts in light‐ and dark‐grown seedlings. Fd‐GOGAT was also barely detectable in the seeds of ginkgo and only accumulated in green plants with mature chloroplasts. In G. biloba seeds and etiolated plants only cytosolic GS was identified, while in light‐grown seedlings this molecular form was present at low abundance and choroplastic GS was the predominant isoenzyme. The above results have been confirmed by immunolocalization of GS protein in pine and ginkgo plantlets. In pine, GS was present in the peripheral cytoplasm of mesophyll cells and also in the phloem region of the vascular bundle. Immunocytochemical analysis showed that the labelling of mesophyll and phloem cells was only cytoplasmic. In developing ginkgo, GS antigens were present in the chloroplasts of mesophyll parenchyma cells of leaflets and green cotyledons. In contrast, a weak labelling of GS was observed in the parenchyma and phloem cells of non‐green cotyledons enclosed in the seed coat. Taking all this into account, our data indicate the existence of two different modes of GS and GOGAT regulation in gymnosperms in close correlation with the differential response of plants to light. Furthermore, the results suggest that glutamine and glutamate biosynthesis is confined to the chloroplast of mesophyll cells in species with light‐dependent chloroplast, development whereas compartmentation would be required in species with light‐independent plastid development.
The action potential in Chara: Ca2+ release from internal stores visualized by Mn2+‐induced quenching of fura‐dextran
Authors: Plieth Christoph , Sattelmacher Burkhard , Hansen Ulf‐Peter , Thiel Gerhard
Published: March 2003

The action potential (AP) in Chara is associated with a transient elevation in the concentration of cytoplasmic‐free Ca2+ ([Ca2+]cyt). The quenching properties of the fluorescent Ca2+ indicator dye fura‐dextran, in combination with Mn2+, was used to investigate whether this [Ca2+]cyt transient is due to Ca2+ release from internal stores or to Ca2+ influx across the plasma membrane. Adding Mn2+ to the external medium or pre‐injection of Mn2+ into the vacuole caused no perceivable quenching of the fura fluorescence, during an AP. This makes it unlikely that Ca2+ influx across the plasma membrane or the tonoplast contributes significantly to the [Ca2+]cyt transient in an excited cell. When cells were pre‐incubated in external solutions containing Mn2+ from 25 to 30 mM APs evoked a transient quenching of fura fluorescence in Mn2+‐free solutions. Under these conditions, the quenching must be attributed to an AP‐associated release of Mn2+ from internal stores. Based on the finding that exposing cells to millimolar concentrations of Mn2+ caused a progressive quenching of the fura fluorescence in non‐excited cells, it can be assumed that some Mn2+ enters the cells during pre‐incubation and is loaded into internal stores. During excitation, this stored Mn2+ is released together with Ca2+.
Arginine decarboxylase (polyamine synthesis) mutants of Arabidopsis thaliana exhibit altered root growth
Authors: Watson Mark B. , Emory Kimberly K. , Piatak Ruth Marie , Malmberg Russell L.
Published: March 2003

Putrescine and polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase; the other with ornithine decarboxylase. The authors developed an in vivo screening strategy to identify mutants with low levels of arginine decarboxylase activity. The screen requires both a primary screen of the M2 generation and a secondary screen of the M3 generation. The method used was to screen 15 000 EMS‐mutagenized M2 seedlings for low levels of arginine decarboxylase (ADC) activity and identified seven mutants that fall into two complementation groups. These mutants have from 20% to 50% of wild‐type enzyme activity. Morphological alterations common among the mutants include increased levels of lateral root branching. The authors obtained a double mutant combining the alleles with the lowest activities from the two complementation groups; this has lower ADC enzyme activity and putrescine levels than either of the single mutants. The double mutant has highly kinked roots that form a tight cluster; it also has narrower leaves, sepals, and petals than either single mutant or wild‐type, and delayed flowering. These results suggest there may be more than one ADC gene in Arabidopsis, and that ADC and polyamine levels play roles in root meristem function and in lateral growth of leaf‐homolog organs.
Distinct patterns of pigment suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers
Authors: Que Qiudeng , Wang Huai‐Yu , Jorgensen Richard A.
Published: March 2003

A single‐copy sense Chalcone synthase (Chs) transgene driven by a strong promoter and producing a fully translatable transcript was converted to an allelic antisense Chs transgene by Cre‐lox‐mediated DNA recombination in petunia. The sense Chs allele suppressed flower pigmentation in a simple pattern determined by cells at the junctions between adjacent petals, as is typical of single‐copy sense Chs transgenes of this type, whereas the antisense Chs allele produced a different pattern of Chs suppression with white petal edges and reduced pigmentation throughout the petal limbs, as is typical of antisense Chs transgenes. In plants carrying a lox‐flanked Chs transgene, the presence of Cre protein can cause both sense‐specific and antisense‐specific patterns to be superimposed in the same flower, suggesting that sense and antisense suppression by single‐copy transgenes are mediated by different mechanisms or occur in different cellular or developmental compartments. The presence of Cre also causes the production of numerous, non‐clonal white spots, suggesting that the turnover state is not cell‐autonomous.
Chitinase in cucumber hypocotyls is induced by germinating fungal spores and by fungal elicitor in synergism with inducers of acquired resistance
Authors: Kästner Bärbel , Tenhaken Raimund , Kauss Heinrich
Published: March 2003

After root pretreatment with 2,6‐dichloroisonicotinic acid (DCIA or INA), hypocotyls of etiolated cucumber seedlings acquired resistance to infection by Colletotrichum lagenarium caused by the failure of the fungus to penetrate epidermal cell walls. The hypocotyls contained only low levels of class III chitinase and its mRNA prior to infection. This pathogenesis‐related (PR) gene was expressed strongly upon infection but only in resistant hypocotyls and soon after germination of the fungal spores. Chitinase was also induced early by an albino mutant strain of C. lagenarium that can barely penetrate the epidermis. Thus, early recognition of the fungus implies signal compounds able to pass, or being generated in, the hydrophobic epidermal surface. As the apoplastic chitinase accumulates timely at the site of a subsequent attack, it may contribute to disease resistance. The mechanism behind the enhanced responsiveness of epidermal cells was studied by gently abrading the cuticle of susceptible hypocotyls to allow permeation of a water‐soluble polymeric fungal elicitor. Induction of chitinase occurred only when the elicitor was applied simultaneously with a resistance inducer such as DCIA, salicylic acid (SA) or a benzothiadiazole (BTH). In addition, long‐term root pretreatment with DCIA conditioned the hypocotyls for enhanced elicitor responses. These results demonstrate that the above inducers of acquired resistance can affect expression of the cucumber chitinase gene not only as direct inducers. They can also act synergistically with fungal elicitors and, in addition, condition the hypocotyls in a developmental manner for potentiated elicitation.
Salicylic acid has a dual role in the activation of defence‐related genes in parsley
Authors: Thulke Oliver , Conrath Uwe
Published: March 2003

Systemic acquired resistance is an inducible plant defence state, the activation of which depends mostly on the accumulation of salicylic acid (SA). During the past several years, it has been demonstrated that pretreatment of cultured parsley cells with SA potentiates the elicitation of several defence responses that are local in whole plants, including the accumulation of phenylpropanoid products. Here it is reported that while anionic peroxidase and mannitol dehydrogenase encoding genes are directly responsive to SA, pretreating parsley cells with SA not only enhances elicitation of the phenylpropanoid genes phenylalanine ammonia‐lyase and 4‐coumarate:CoA ligase but also of genes for PR‐10 and a hydroxyproline‐rich glycoprotein. Enhanced induction of these genes was seen at low levels of endogenous free SA. Enhancement of phenylalanine ammonia‐lyase gene activation was proportional to the length of SA pretreatment. Furthermore, the ability of SA analogues to both potentiate elicited and directly induce defence gene activation correlated with their biological activity to promote plant disease resistance. In summary, these results emphasize that SA has at least a dual role in plant defence gene activation.
Psl: a novel Spm‐like transposable element from Petunia hybrida
Authors: Snowden Kimberley C. , Napoli Carolyn A.
Published: March 2003

The identification of a spontaneous mutable Hf1 allele in Petunia hybrida provided an opportunity to isolate and characterize a novel transposable element. This 9.9 kb element has features in common with members of the Spm family, such as homologous terminal inverted repeats and a 3 bp target site direct duplication within the Hf1 gene. The element is named Petunia Spm‐like (Psl). The footprints left by excising elements have been isolated from several germinal revertants and sequence analysis shows similarities to those left by other Spm family members. Southern analysis shows that the transposon is present at low copy number in the genome of different inbred lines and species of Petunia. The germinal excision frequency of Psl was 21–33% in outcross populations. The element appears to be very mobile somatically in the inbred line V26, with 38% of plants from an inbred population showing new Psl‐hybridizing bands by Southern analysis. The high somatic and germinal excision frequency demonstrated by Psl suggests that this element may have utility for gene tagging in petunia.
A novel tomato gene that rapidly responds to wound‐ and pathogen‐related signals
Authors: O'Donnell Philip J. , Truesdale Mark R. , Calvert Caroline M. , Dorans Alison , Roberts Michael R. , Bowles Dianna J.
Published: March 2003

The expression of a novel defence‐related gene from tomato which responds rapidly to wound‐ and pathogen‐related signals has been characterised. The gene, which encodes a protein with homology to glucosyl transferase enzymes, is expressed within 15 min of mechanical damage to tomato leaves, and responds to signals which differ from those on the systemin/jasmonic acid pathway typical of well‐characterised wound‐induced genes of tomato. Furthermore, expression of the gene is also rapidly and specifically induced during a resistance response elicited by the application of Avr9 avirulence peptide to tomato plants carrying the corresponding Cf9 resistance gene. Whilst expression can also be induced by the application of exogenous salicylic acid and related analogues to tomato plants, several lines of evidence suggest that elevated salicylic acid is not a causal signal in planta during either the wound or pathogen resistance response.
An efficient screening for terminal deletions and translocations of barley chromosomes added to common wheat
Authors: Schubert Ingo , Shi Fang , Fuchs Jörg , Endo Takashi R.
Published: March 2003

As a prerequisite to determine physical gene distances in barley chromosomes by deletion mapping, a reliable, fast and inexpensive approach was developed to detect terminal deletions and translocations in individual barley chromosomes added to the chromosome complement of common wheat. A refined fluorescence in situ hybridization (FISH) technique subsequent to N‐banding made it possible to detect subtelomeric repeat sequences (HvT01) on all 14 chromosome arms of barley. Some chromosome arms could be distinguished individually based on the number of FISH signals or the intensity of terminal FISH signals. This allowed the detection and selection of deletions and translocations of barley chromosomes (exemplified by 7H and 4HL), which occurred in the progeny of the wheat lines containing a pair of individual barley chromosomes (or telosomes) and a single so‐called gametocidal chromosome (2C) of Aegilops cylindrica. This chromosome is known to cause chromosomal breakage in the gametes in which it is absent. Terminal deletions and translocations in barley chromosomes were easily recognized in metaphase and even in interphase nuclei by a decrease in the number of FISH signals specific to the subtelomeric repeat. These aberrations were verified by genomic in situ hybridization. The same approach can be applied to select deletions and translocations of other barley chromosomes in wheat lines that are monosomic for the Ae. cylindrica chromosome 2C.
Differential gene expression in Arabidopsis monitored using cDNA arrays
Authors: Desprez Thierry , Amselem Joëlle , Caboche Michel , Höfte Herman
Published: March 2003

Large numbers of genes are being discovered on a daily basis for a variety of organisms including Arabidopsis thaliana. To obtain more functional information on these genes, efficient expression monitoring methods need to be developed. In this report we studied the steady‐state mRNA levels of over 800 Arabidopsis genes in parallel using high‐density arrays of partially sequenced cDNA. The technology is simple and robust and reliably permits the detection of down to twofold variation in mRNA levels. The detection limit lies below 0.01% of the total mRNA population. The comparison of the profiles obtained for light‐grown and dark‐grown seedlings revealed significant variations in mRNA levels for about 16% of the cDNA investigated. This technology not only provides new functional information on anonymous genes, and thus may guide reverse‐genetics approaches, but also constitutes a powerful tool for global gene expression studies, with many potential applications in plant biology.