bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non‐host resistance in barley coleoptile cells
Authors: Kobayashi Yuhko , Kobayashi Issei , Funaki Yasuro , Fujimoto Shigeo , Takemoto Tsuyoshi , Kunoh Hitoshi
Published: March 2003

To show the involvement of microfilaments and microtubules in non‐host resistance of barley, partially dissected coleoptiles which had been inoculated with a non‐pathogen, Erysiphe pisi, were treated with several actin and tubulin inhibitors. If the coleoptiles were not treated with any of the inhibitors, the non‐pathogen always failed to penetrate the coleoptile cells. However, when coleoptiles were treated with actin or tubulin polymerization or depolymerization inhibitors, the non‐pathogen was able to penetrate successfully and to form haustoria in coleoptile cells of a non‐host plant, barley. Actin polymerization inhibitors, cytochalasins, were more effective in causing an increase in penetration efficiency of E. pisi than tubulin inhibitors. The effects of cytochalasins depended on the kind of cytochalasin; the strength of the actin depolymerizing activity correlated significantly with the efficiency of increasing the penetration of the non‐pathogen. When both actin and tubulin inhibitors were added simultaneously, the polarization of defense‐related responses, such as massive cytoplasmic aggregation, deposition of papillae and accumulation of autofluorescent compounds, at fungal penetration sites was suppressed. Actin inhibitors did not affect arrangement and stability of microtubules and vice versa, and a double treatment of coleoptile cells with both microfilament and microtubule inhibitors showed an additive effect in increasing the penetration efficiency of E. pisi. Furthermore, cytochalasin A treatment allowed other non‐pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non‐host barley cells. These results strongly suggest that microfilaments and microtubules might play important roles in the expression of non‐host resistance of barley.
Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis
Authors: Burkhardt Peter K. , Beyer Peter , Wünn Joachim , Klöti Andreas , Armstrong Gregory A. , Schledz Michael , von Lintig Johannes , Potrykus Ingo
Published: March 2003

Rice (Oryza sativa L.), the major food staple for more than two billion people, contains neither β‐carotene (provitamin A) nor C40 carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make β‐carotene into rice endosperm tissue. Investigation of the biochemical properties of immature rice endosperm using [14C]‐labelled substrates revealed the presence of geranyl geranyl diphosphate, the C20 general isoprenoid precursor necessary for C40 carotenoid biosynthesis. Phytoene synthase, which condenses two molecules of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for β‐carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil (Narcissus pseudonarcissus) under the control of either a constitutive or an endosperm‐specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a critical step in provitamin A biosynthesis in a non‐photosynthetic, carotenoid‐lacking plant tissue. These results have important implications for longterm prospects of overcoming worldwide vitamin A deficiency.
Style‐specific self‐compatible mutation caused by deletion of the S‐RNase gene in Japanese pear (Pyrus serotina)
Authors: Sassa Hidenori , Hirano Hisashi , Nishio Takeshi , Koba Takato
Published: March 2003

Monofactorial gametophytic self‐incompatibility in the family Rosaceae is controlled by the single multi‐allelic S‐locus. In Japanese pear, a naturally occurring self‐compatible mutant was found as a sport of a self‐incompatible cultivar with an S‐genotype of S2,S4. The S,4 allele has been shown to be non‐functional in the style of the mutant, but functional in pollen. The present study shows the S2‐RNase gene to be transcribed normally in the style of the mutant, but that the S4‐RNase gene transcript is not detectable. Furthermore, the S4‐RNase gene could not be detected in the mutant by genomic Southern blot, indicating that the mutant lacks the gene. The extent of deletion in the mutant was estimated to be more than 4 kbp, and spans the entire length of the S4‐RNase gene. These findings provide evidence that the S‐RNase gene is essential for the self‐incompatibility reaction in the style, but is not involved in controlling the self‐incompatibility behavior of pollen.
Light‐responsive elements of the tobacco PSI‐D gene are located both upstream and within the transcribed region
Authors: Yamamoto Yoshiharu Y. , Kondo Yukiko , Kato Akira , Tsuji Hideo , Obokata Junichi
Published: March 2003

psaDb is a nuclear gene encoding the ferredoxin‐binding subunit of photosystem I in Nicotiana sylvestris. The organization of the light‐responsive cis‐elements of psaDb was studied using transgenic tobacco plants. Three types of psaDb chimeric constructs were created: (1) a 5′ upstream fragment of psaDb transcriptionally fused with the β‐glucuronidase (GUS) gene, and a series of its 5′ deletion derivatives, (2) the transcribed region of psaDb driven by the cauliflower mosaic virus (CaMV) 35S promoter, and (3) the 5′ terminal 35 bases (the entire leader, +1 to +23, and the initiation codon context, +24 to +35) of the psaDb mRNA translationally fused with a GUS reporter gene under the operation of the CaMV 35S promoter. Light‐responsiveness of these fusions in transgenic plants was examined by GUS assay and primer extension analysis. The results indicate that the light‐responsive elements (LRE) of psaDb are located both upstream (−170 to +24) and within (+1 to +861) the transcribed region. The internal LRE is utilized in etiolated seedlings but not in green leaves. The leader and initiation codon context construct (+1 to +35) did not show any light‐response under the conditions tested. Therefore, it is likely that a combination of the upstream and internal LREs generates the complex light‐responsive and tissue‐specific regulation of this gene. This study also revealed that psaDb has adjacent activator (−267 to −254) and repressor (−253 to −234) regions for basal transcriptional activity; the former contains the ACGT binding motif recognized by many plant bZIP proteins, and the latter has the R3 decamer motif found in several photosystem I‐related genes.
Post‐transcriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3′ flanking regions
Authors: Van Houdt Helena , Ingelbrecht Ivan , Van Montagu Marc , Depicker Ann
Published: March 2003

The transgenic tobacco plant GVCHS(320)‐1 harbours several T‐DNAs with the neomycin phosphotransferase II (nptll)‐encoding chimeric gene under control of the cauliflower mosaic virus 35S promoter (CaMV 35S). These T‐DNAs are distributed over two loci, named A and B. The primary transformant GVCHS(320)‐1 had substantially reduced steady‐state nptll transcript levels due to the activity of a post‐transcriptional silencing mechanism. Silencing of the nptll‐encoding transgenes in the progeny of GVCHS(320)‐1 requires the presence of the A locus that consists of two physically separated T‐DNAs. Although the B locus contains three T‐DNAs in the same orientation, it gives rise to high steady‐state nptll mRNA levels and NPTII protein levels even in homozygous condition. The B locus only becomes silenced in trans in the presence of a silencing locus. The data suggest that silencing of the nptll transgenes is reinforced by ageing. It is also suggested that silencing is correlated with a reduced NPTII protein/nptll mRNA ratio, which may be interpreted as the accumulation of unproductive nptll RNA molecules in silenced plants. The data further demonstrate that the silenced state is correlated with extensive C‐methylation of diagnostic sites in the 3′ three‐quarters of the coding sequence and in the 3′ region up to 1400 bp downstream of the polyadenylation signal.
Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves
Authors: Herbers Karin , Tacke Eckhardt , Hazirezaei Mohammad , Krause Klaus‐Peter , Melzer Michael , Rohde Wolfgang , Sonnewald Uwe
Published: March 2003

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem‐restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP‐glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis‐related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2‐week‐old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild‐type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.
Stress‐responsive accumulation of plastid chaperonin 60 during seedling development
Authors: Holland Neta , Belkind Anna , Holland Doron , Pick Uri , Edelman Marvin
Published: March 2003

Plastid chaperonin 60 (cpn60) is a chloroplast protein, presumed to assist in assembly and folding of plastid proteins. Although molecular chaperones often accumulate significantly in response to stress, this has never been demonstrated for cpn60. In this study, the accumulation of cpn60 in Nicotiana seedlings during their development was followed under different stress conditions. It was found that cpn60 accumulates markedly in developing seedlings in response to tentoxin and several other (but not all) stresses. Cpn60 accumulates only during a narrow period of seedling development. It is proposed that cpn60 accumulation under stress is developmentally regulated.
A robust method for detecting single‐nucleotide changes as polymorphic markers by PCR
Authors: Michaels Scott D. , Amasino Richard M.
Published: March 2003

Numerous techniques in plant molecular genetic analysis, such as mapping and positional cloning techniques, rely on the availability of molecular markers that can differentiate between alleles at a particular locus. PCR‐based cleaved amplified polymorphic sequences (CAPS) markers have been widely used as a means of rapidly and reliably detecting a single‐base change that creates a unique restriction site in one of a pair of alleles. However, the majority of single‐nucleotide changes do not create such sites and thus cannot be used to create CAPS markers. In this paper, a modification of the CAPS technique that allows detection of most single‐nucleotide changes by utilizing mismatched PCR primers is described. The mismatches in the PCR primers, in combination with the single‐nucleotide change, create a unique restriction site in one of the alleles.
Aberrant splicing of intron 1 leads to the heterogeneous 5′ UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content
Authors: Cai Xiu‐Ling , Wang Zong‐Yang , Xing Yan‐Yan , Zhang Jing‐Liu , Hong Meng‐Min
Published: March 2003

We have previously reported that three distinct patterns of waxy (Wx) gene transcript accumulation were present in 31 rice cultivars. The cultivars with high amylose content (group I) contain a 2.3 kb mature Wx mRNA, cultivars with intermediate amylose content (group II) produce both a 3.3 kb Wx pre‐mRNA, which contains intron 1, and the 2.3 kb Wx mature mRNA, and cultivars with no amylose (group III) accumulate only the 3.3 kb Wx pre‐mRNA. Analyses of the cDNAs reveals that four splice donor sites and three splice acceptor sites in intron 1 give rise to six splicing patterns in 2.3 kb Wx mRNA of group II cultivars. In addition, aberrant intron 1 excision causes either deletion of 4 or 5 nucleotides, or addition of 7 and 13 nucleotides at the junction of exon 1 and exon 2 of the 2.3 kb mRNA. In contrast, only one normal splicing pattern (one splice donor site and one splice acceptor site) was found in the 2.3 kb mRNA of group I cultivars. Nucleotide sequences of the Wx intron 1 in group I and group II cultivars differ by 16 individual bases. We suggest that these deletions or additions contribute to inefficient splicing of intron 1 from the 3.3 kb Wx pre‐mRNA, as well as an aberrant splicing of the Wx intron 1 to produce the 2.3 kb mRNA with a heterogeneous 5′ untranslated region (5′‐UTR). As a consequence, the total amount of translatable Wx mRNA, and therefore the Wx protein and amylose content, are reduced in the group II cultivars compared with the group I cultivars.
Guard cell cation channels are involved in Na+–induced stomatal closure in a halophyte
Authors: Véry Anne‐Aliénor , Robinson Michael F. , Mansfield Terry A. , Sanders Dale
Published: March 2003

The halophyte Aster tripolium, unlike well‐studied non‐halophytic species, partially closes its stomata in response to high Na+ concentrations. Since A. tripolium possesses no specific morphological adaptation to salinity, this stomatal response, preventing excessive accumulation of Na+ within the shoot via control of the transpiration rate, is probably a principal feature of its salt tolerance within the shoot. The ionic basis of the stomatal response to Na+ was studied in guard cell protoplasts from A. tripolium and from a non‐halophytic relative, Aster amellus, which exhibits classical stomatal opening on Na+. Patch‐clamp studies revealed that plasma membrane K+ channels (inward and outward rectifiers) of the halophytic and the non‐halophytic species are highly selective for K+ against Na+, and are very similar with respect to unitary conductance and direct sensitivity to Na+. On the other hand, both species possess a significant permeability to Na+ through non‐rectifying cation channels activated by low (physiological) external Ca2+ concentrations. Finally, it appeared that the differential stomatal response between the two species is achieved, at least in part, by a Na+‐sensing system in the halophyte which downregulates K+ uptake. Thus, increases in guard cell cytosolic Na+ concentration in A. tripolium but not in A. amellus, lead to a delayed (20–30 min) and dramatic deactivation of the K+ inward rectifier. This deactivation is probably mediated by an increase in cytosolic Ca2+ since buffering it abolishes the response. The possible role of K+ inward rectifiers in the response of A. tripolium’s stomata to Na+, suggested by patch‐clamp studies, was confirmed by experiments demonstrating that specific blockade of inward rectifying channels mimics Na+ effects on stomatal aperture, and renders aperture refractory to Na+.
Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis
Authors: Hirner Brigitte , Fischer Wolf N. , Rentsch Doris , Kwart Marion , Frommer Wolf B.
Published: March 2003

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H+‐amino acid co‐transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter‐GUS fusions shows that the genes have non‐overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem‐to‐phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.
Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall
Authors: Rhee Seung Y. , Somerville Chris R.
Published: March 2003

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell‐wall components in wild‐type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild‐type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild‐type. Enzymatic removal of callose from wild‐type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild‐type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild‐type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type‐specific pectin degradation to separate microspores.
Determination of intracellular Ca2+ in cells of intact wheat roots: loading of acetoxymethyl ester of Fluo‐3 under low temperature
Authors: Zhang Wen‐Hao , Rengel Zdenko , Kuo John
Published: March 2003

Loading of Ca2+‐sensitive fluorescent probes into plant cells is an essential step to measuring activities of cytoplasmic free Ca2+ ions with a fluorescent imaging technique. A major barrier to the loading of the fluorescent probes into plant cells using the acetoxymethyl (AM) esters of the Ca2+‐sensitive dyes is the presence of cell‐wall associated esterases. These esterases hydrolyse the esterified form of the fluorescent probes, rendering the probes membrane‐impermeable. A novel non‐invasive loading protocol was described in this paper to load the Ca2+‐sensitive fluorescent probe Fluo‐3/AM ester into apical cells of intact wheat roots by incubating the roots in Fluo‐3/AM ester solution at 4°C for 2 h followed by 2‐h incubation in the dye‐free solution at 20°C. The incubation at low temperature inhibited extracellular hydrolysis of Fluo‐3/AM ester but had less effect on diffusion of membrane‐permeable Fluo‐3/AM ester across the plasma membrane, because hydrolysis of Fluo‐3/AM ester by extracellular esterases is a chemical process (high Q10), while diffusion of Fluo‐3/AM across the plasma membrane is a physical process (low Q10). The Fluo‐3/AM ester, accumulated in the root cells during the low temperature incubation, was then cleaved by intracellular esterases during the incubation at 20°C, releasing the membrane‐impermeable Ca2+‐sensitive Fluo‐3 in the cytoplasm. The root cells loaded with Fluo‐3 showed strong intracellular fluorescence under confocal microscopy. The fluorescence from the root cells was sensitive to the Ca2+ ionophore and hydrogen peroxide, indicating that the intracellular fluorescence was due to intracellular Ca2+ ions.
High‐efficiency cloning of Arabidopsis full‐length cDNA by biotinylated CAP trapper
Authors: Seki Motoaki , Carninci Piero , Nishiyama Yoko , Hayashizaki Yoshihide , Shinozaki Kazuo
Published: March 2003

Full‐length cDNAs are essential for functional analysis of plant genes. We constructed high‐content, full‐length cDNA libraries from Arabidopsis thaliana plants based on chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment, to select full‐length cDNA. More than 90% of the total clones obtained were of full length; recombinant clones were obtained with high efficiency (2.2 × 106/9 μg starting mRNA). Sequence analysis of 111 randomly picked clones indicated that 32 isolated cDNA groups were derived from novel genes in the A. thaliana genome.
Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling
Authors: Laudert Dietmar , Weiler Elmar W.
Published: March 2003

The analysis of allene oxide synthase (AOS) mRNA levels, of AOS polypeptide levels and specific enzymatic activities, as well as the quantitative determination of the levels of the octadecanoids cis‐12‐oxophytodienoic acid (cis‐OPDA) and JA following a number of treatments, has shown that AOS is a regulatory site in octadecanoid biosynthesis in A. thaliana. AOS activity, mRNA and polypeptide levels are increased in wounded leaves locally and systemically. The methyl esters of OPDA or JA (OPDAME, JAME) and coronatine, are strong inducers of AOS mRNA, polypeptide and enzymatic activity. Ethephon also induces AOS activity. Salicylic acid (SA) was an inducer of AOS activity while abscisic acid (ABA) had no effect. At the level of the octadecanoids, the consequences of induction of AOS by the different inducers were distinctly different, depending on the nature of the inducer. Wounding led to a strong, bi‐phasic accumulation of JA in wounded leaves and to a less pronounced increase in JA‐levels in systemic leaves. Levels of OPDA changed very little in wounded leaves and remained constant or even declined in systemic leaves. Ethephon treatment resulted in a strong, transient increase in JA‐levels kinetically coinciding with the second, more pronounced peak in wound‐induced JA. In SA‐treated leaves, the level of cis‐OPDA increased throughout the experimental period while there was no effect on JA levels during the first 24 h following treatment and only a slight accumulation after 48 h. Clearly, mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.
Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis
Authors: Rutledge Robert , Regan Sharon , Nicolas Olivier , Fobert Pierre , Côté Chantal , Bosnich Whynn , Kauffeldt Chris , Sunohara Glen , Séguin Armand , Stewart Don
Published: March 2003

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non‐flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS‐box genes. PCR cloning using degenerate primers targeted to the MADS‐box domain revealed the presence of over 27 MADS‐box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS‐box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS‐like cDNA clone called SAG1, whose conservation of intron position and tissue‐specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300‐million‐year‐old divergence of angiosperms and gymnosperms.
Differential expression of a senescence‐enhanced metallothionein gene inArabidopsisin response to isolates ofPeronospora parasiticaandPseudomonas syringae
Authors: Butt Adrian , Mousley Claudia , Morris Karl , Beynon Jim , Can Canan , Holub Eric , Greenberg Jean T. , Buchanan‐Wollaston Vicky
Published: March 2003

The metallothionein gene,

LSC54

, shows increased expression during leaf senescence in

Brassica napus

and

Arabidopsis thaliana

. A number of abiotic and biotic stresses have been shown to induce senescence‐like symptoms in plants and, to investigate this further, the promoter of the

LSC54

gene was cloned and fused to the GUS gene and transformed into

Arabidopsis

. The promoter was highly induced during leaf senescence and also in response to wounding; histochemical analysis indicated that this induction was localised to a few cells close to the wound site. The transgenic

Arabidopsis

tissue was infected with compatible and incompatible isolates of both the fungal biotroph,

Peronospora parasitica

and the bacterial necrotroph,

Pseudomonas syringae.

Incompatible isolates induced rapid cell death (the hypersensitive response) at the site of infection and, with both pathogens, early, localised expression of the GUS gene was observed. In contrast, relatively slow induction of the GUS gene was seen in the compatible interaction and this was correlated with the appearance of senescence‐like symptoms in the biotrophic interaction and cell death by necrosis that occurred in response to the necrotrophic pathogen. These results suggest that there are common steps in the signalling pathways that lead to cell death in the hypersensitive response, pathogen induced necrosis and senescence.
C6‐volatiles derived from the lipoxygenase pathway induce a subset of defense‐related genes
Authors: Bate Nicholas J. , Rothstein Steven J.
Published: March 2003

Six‐Carbon (C6‐) volatiles, including the aldehydes trans‐2‐hexenal, hexanal and cis‐3‐hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment of

Arabidopsis

seedlings with 10 μM concentrations of trans‐2‐hexenal induces several genes known to be involved in the plant’s defense response, including phenylpropanoid‐related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis‐related proteins PR‐1 or PR‐2, however, were not induced. Trans‐2‐hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX‐derived volatile. However, unlike MeJA, trans‐2‐hexenal did not induce hydroxymethylglutaryl‐coenzyme A reductase (HMGR) or thionin2–1. The inductive effect seemed to be limited to C6‐related volatiles, as C8‐, C9‐ and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans‐2‐hexenal quantitatively reduced wild‐type seed germination. Trans‐2‐hexenal also reduced the germination frequency of the MeJA resistant

Arabidopsis

mutant, jar1–1, supporting the notion that trans‐2‐hexenal and MeJA are recognized via different mechanisms. In addition, trans‐2‐hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10‐fold less effective than MeJA at inducing anthocyanin accumulation in

Arabidopsis

seedlings. These results suggest that C6‐volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.
A history of stress alters drought calcium signalling pathways inArabidopsis
Authors: Knight Heather , Brandt Stephan , Knight Marc R.
Published: March 2003

Environmental stresses commonly encountered by plants lead to rapid transient elevations in cytosolic free calcium concentration ([Ca2+]cyt) (

Bush 1995

;

Knight

et al

. 1991

). These cellular calcium (Ca2+) signals lead ultimately to the increased expression of stress‐responsive genes, including those encoding proteins of protective function (

Knight

et al

. 1996

;

Knight

et al

. 1997

). The kinetics and magnitude of the Ca2+ signal, or ‘calcium signature’, differ between different stimuli and are thought to contribute to the specificity of the end response (

Dolmetsch

et al

. 1997

;

McAinsh & Hetherington 1998

). We measured [Ca2+]cyt changes during treatment with mannitol (to mimic drought stress) in whole intact seedlings of

Arabidopsis thaliana

. The responses of plants which were previously exposed to osmotic and oxidative stresses were compared to those of control plants. We show here that osmotic stress‐induced Ca2+ responses can be markedly altered by previous encounters with either osmotic or oxidative stress. The nature of the alterations in Ca2+ response depends on the identity and severity of the previous stress: oxidative stress pre‐treatment reduced the mannitol‐induced [Ca2+]cyt response whereas osmotic stress pre‐treatment increased the [Ca2+]cyt response. Therefore, our data show that different combinations of environmental stress can produce novel Ca2+ signal outputs. These alterations are accompanied by corresponding changes in the patterns of osmotic stress‐induced gene expression and, in the case of osmotic stress pre‐treatment, the acquisition of stress‐tolerance. This suggests that altered Ca2+ responses encode a ‘memory’ of previous stress encounters and thus may perhaps be involved in acclimation to environmental stresses.
Iron homeostasis alteration in transgenic tobacco overexpressing ferritin
Authors: Van Wuytswinkel Olivier , Vansuyt Gérard , Grignon Nicole , Fourcroy Pierre , Briat Jean‐François
Published: March 2003

Intracellular iron concentration requires tight control and is regulated both at the uptake and storage levels. Our knowledge of the role that the iron‐storage protein ferritins play in plants is still very limited. Overexpression of this protein, either in the cytoplasm or the plastids of transgenic tobacco, was obtained by placing soybean ferritin cDNA cassettes under the control of the CAMV 35S promoter. The protein accumulated in 4‐ and 6‐day‐old seedlings and in leaves of 3‐week‐old plants but not in dry seeds or in 2‐day‐old seedlings, which is consistent with previous reports describing a post‐transcriptional control of ferritin amounts during the germination process. Overaccumulated ferritin in leaves was correctly assembled as 24‐mers. Transformants were more resistant to methylviologen toxicity, indicating that the transgenic ferritins were functional

in vivo.

Ferritin overaccumulation in transgenic tobacco leaves leads to an illegitimate iron sequestration. As a consequence, these transgenic plants behave as iron deficient and activate iron transport systems as revealed by an increase in root ferric reductase activity and in leaf iron content.
Direct evidence for anthocyanidin synthase as a 2‐oxoglutarate‐dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma ofPerilla frutescens
Authors: Saito Kazuki , Kobayashi Mii , Gong Zhizhong , Tanaka Yoshikazu , Yamazaki Mami
Published: March 2003

Anthocyanidin synthase (ANS), an enzyme of the biosynthetic pathway to anthocyanin, has been postulated to catalyze the reaction(s) from the colorless leucoanthocyanidins to the colored anthocyanidins. Although cDNAs have been isolated that encode putative ANS, which exhibits significant similarities in amino acid sequence with members of a family of 2‐oxoglutarate‐dependent oxygenases, no biochemical evidence has been presented which identifies the actual reaction that is catalyzed by ANS. Here we show that anthocyanidins are formed

in vitro

through 2‐oxoglutarate‐dependent oxidation of leucoanthocyanidins catalyzed by the recombinant ANS and subsequent acid treatment. A cDNA encoding ANS was isolated from red and green formas of

Perilla frutescens

by differential display of mRNA. Recombinant ANS tagged with maltose‐binding‐protein (MBP) was purified, and the formation of anthocyanidins from leucoanthocyanidins was detected by the ANS‐catalyzed reaction in the presence of ferrous ion, 2‐oxoglutarate and ascorbate, being followed by acidification with HCl. Equimolar stoichiometry was confirmed for anthocyanidin formation and liberation of CO2 from 2‐oxoglutarate. The presumptive two‐copy gene of ANS was expressed in leaves and stems of the red forma of

P. frutescens

but not in the green forma plant. This corresponds to the accumulation pattern of anthocyanin. The mechanism of the reaction catalyzed by ANS is discussed in relation to the molecular evolution of a family of 2‐oxoglutarate‐dependent oxygenases.
Post‐transcriptional and developmental regulation of a CMS‐associated mitochondrial gene region by a nuclear restorer gene
Authors: Menassa Rima , L’Homme Yvan , Brown Gregory G.
Published: March 2003

Transcripts of the mitochondrial gene region
orf224/atp6
, which is associated with the Polima or
pol
cytoplasmic male sterility (CMS) of
Brassica napus
, differ among fertile, sterile and nuclear‐restored plants. We show here that the effects of the restorer gene
Rfp
on
orf224/atp6
transcripts varies among different floral organs. Relative to monocistronic
atp6
transcripts, levels of the dicistronic transcripts spanning
orf224
and
atp6
are dramatically reduced in petals, stamens and carpels, but not sepals, of restored flowers. In
pol
CMS plants, the relative levels of different
orf224/atp6
transcripts are similar among the floral organs. Analysis of guanylyltransferase‐labeled mtRNA indicates that only the dicistronic 2.2 and 1.9 kb
orf224/atp6
transcripts carry an initiator 5′ terminus; hence the 1.4 and 1.3 kb transcripts of restored plants, as well as the 1.1 kb
atp6
transcript common to all genotypes, are generated by RNA processing and not
de novo
initiation. Although steady‐state levels of dicistronic transcripts in flower buds are lower in restored than in sterile plants, run‐on transcription experiments show that these transcripts are synthesized at the same rate in both types of flowers. These findings imply that the restorer gene acts by conditioning the removal of sequences from the 5′ end of dicistronic transcripts in a developmentally regulated manner. Run‐on transcription experiments indicate that the single 1.1 kb
atp6
transcript of
nap
cytoplasm is also generated by removal of sequences from the 5′ end of a precursor. We suggest that specific endonucleolytic cleavage of a precursor RNA, followed by non‐specific 3′ to 5′ exonuclease action, may represent a common mechanism for tailoring transcripts in plant mitochondria.
Generation of enhancer trap lines in Arabidopsis and characterization of expression patterns in the inflorescence
Authors: Campisi Lauren , Yang Yingzhen , Yi Ying , Heilig Elizabeth , Herman Benjamin , Cassista A. Jon , Allen David W. , Xiang Hongjun , Jack Thomas
Published: March 2003

Eleven thousand, three hundred and seventy enhancer/promoter trap lines in
Arabidopsis
were generated via T‐DNA transformation utilizing the binary vector pD991 that contains a minimal promoter fused to the
uidA
reporter gene. Overall 31% of the lines generated exhibit a staining pattern in the inflorescence. Flanking DNA has been cloned from 15 lines exhibiting inflorescence staining patterns by either thermal asymmetric interlaced PCR (TAIL–PCR), inverse PCR (IPCR), or partial library construction. Seeds from these lines are available from the ABRC and NASC
Arabidopsis
stock centers and DNA pools are available from the ABRC.
Inhibition of protoporphyrinogen oxidase expression in Arabidopsis causes a lesion‐mimic phenotype that induces systemic acquired resistance
Authors: Molina Antonio , Volrath Sandy , Guyer Dave , Maleck Klaus , Ryals John , Ward Eric
Published: March 2003

We have used an antisense expression technology in
Arabidopsis
based on the yeast GAL4/UAS transactivation system (Guyer
et al
.
Genetics
1998; 149:633–639) to reduce levels of protoporphyrinogen IX oxidase (PPO), the last common enzyme of the biosynthesis of the haem group and chlorophyll. Plants expressing the antisense
PPO
gene presented growth alterations and their leaves showed necrotic lesions that appeared similar to lesions characteristic of the pathogen‐induced hypersensitive reaction, and seen in the so‐called lesion‐mimic mutants. Plants expressing the antisense gene also had high endogenous salicylic acid levels, constitutive expression of the
PR‐1
gene, and were resistant to
Peronospora parasitica
, consistent with the activation of systemic acquired resistance (SAR). Treatment of wild‐type plants with sublethal concentrations of herbicides that inhibit PPO also induced defence responses that conferred enhanced tolerance to
P. parasitica
. This effect was not observed in NahG and
nim1
plants, which are compromised in their ability to activate SAR. These results demonstrate that genetic or chemical disruption of a metabolic pathway can lead to the induction of a set of defence responses including activation of SAR.
Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2
Authors: Kleiner Oliver , Kircher Stefan , Harter Klaus , Batschauer Alfred
Published: March 2003

The cryptochrome blue light photoreceptor family of Arabidopsis thaliana consists of two members, CRY1 and CRY2 (PHH1). CRY2 contains a putative nuclear localization signal (NLS) within its C‐terminal region. We examined whether CRY2 is localized in the nucleus and whether the C‐terminal region of CRY2 is involved in nuclear targeting. Total cellular and nuclear protein extracts from Arabidopsis were subjected to immunoblot analysis with CRY2‐specific antibodies. Strong CRY2 signals were obtained in the nuclear fraction. Fusion proteins consisting of the green fluorescent protein (GFP) and different fragments of CRY2 were expressed in parsley protoplasts and the localization of the fusion proteins was determined by fluorescence and confocal laser scanning microscopy. GFP‐fusions containing the entire CRY2 protein or its C‐terminal region were found exclusively in the nucleus. We conclude from these results that CRY2 is localized in the nucleus and that nuclear localization is mediated by the C‐terminal region of CRY2.
Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement
Authors: Gao Minggeng , Showalter Allan M.
Published: March 2003

Arabinogalactan proteins (AGPs) are a family of highly glycosylated, hydroxyproline‐rich glycoproteins impli‐ cated in various aspects of plant growth and development. (β‐D‐glucosyl)3 and (β‐D‐galactosyl)3 Yariv phenylglycosides, commonly known as Yariv reagents, specifically bind AGPs in a non‐covalent manner. Here (β‐D‐galactosyl)3 Yariv reagent was added to Arabidopsis thaliana cell suspension cultures and determined to induce programmed cell death (PCD) by three criteria: (i) DNA fragmentation as detected by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) of DNA 3′‐OH groups; (ii) inter‐ nucleosomal DNA fragmentation as visualized by genomic Southern blotting; and (iii) structural changes characteristic of PCD including cytoplasmic shrinkage and condensation, chromatin condensation and nuclear membrane blebbing. These findings implicate AGP involvement in PCD in plants, presumably by perturbation of AGPs located at the plasma membrane–cell wall interface.
Cell‐cycle modulation of CK2 activity in tobacco BY‐2 cells†
Authors: Espunya M. Carme , Combettes Bruno , Dot Josep , Chaubet‐Gigot Nicole , Martínez M. Carmen
Published: March 2003

Protein kinase CK2 is an ubiquitous Ser/Thr kinase essential for cell growth. We have used the highly synchronizable tobacco BY‐2 cell line to investigate whether CK2 activity and expression are regulated in a cell cycle phase‐dependent manner in higher plants. Specific cDNA probes for tobacco CK2α and β subunits, respectively, and polyclonal antibodies recognising α and β subunits separately, were obtained to determine mRNA and protein levels of both subunits. Our results show that CK2 activity oscillates throughout the cell cycle, peaking at G1/S and M phases, due to a post‐translational regulation of the tetrameric enzyme. Additional levels of control of CK2 expression operate in relation to the proliferative state of the cells, including differential accumulation of α and β transcripts and post‐transcriptional regulation of protein levels (β subunit). Moreover, in vivo inhibition of CK2 activity corroborates the requirement of the functional CK2 to progress through the cell division cycle, and suggests that CK2 might play an important role at the G2/M checkpoint.
ACGT‐containing abscisic acid response element (ABRE) and coupling element 3 (CE3) are functionally equivalent
Authors: Hobo Tokunori , Asada Mihoko , Kowyama Yasuo , Hattori Tsukaho
Published: March 2003

ACGT‐containing ABA response elements (ABREs) have been functionally identified in the promoters of various genes. In addition, single copies of ABRE have been found to require a cis‐acting, coupling element to achieve ABA induction. A coupling element 3 (CE3) sequence, originally identified as such in the barley HVA1 promoter, is found approximately 30 bp downstream of motif A (ACGT‐containing ABRE) in the promoter of the Osem gene. The relationship between these two elements was further defined by linker‐scan analyses of a 55 bp fragment of the Osem promoter, which is sufficient for ABA‐responsiveness and VP1 activation. The analyses revealed that both motif A and CE3 sequence were required not only for ABA‐responsiveness but also for VP1 activation. Since the sequences of motif A and CE3 were found to be similar, motif‐exchange experiments were carried out. The experiments demonstrated that motif A and CE3 were interchangeable by each other with respect to both ABA and VP1 regulation. In addition, both sequences were shown to be recognized by a VP1‐interacting, ABA‐responsive bZIP factor TRAB1. These results indicate that ACGT‐containing ABREs and CE3 are functionally equivalent cis‐acting elements. Furthermore, TRAB1 was shown to bind two other non‐ACGT ABREs. Based on these results, all these ABREs including CE3 are proposed to be categorized into a single class of cis‐acting elements.
Natural allelic variation identifies new genes in the Arabidopsis circadian system
Authors: Swarup Kamal , Alonso‐Blanco Carlos , Lynn James R. , Michaels Scott D. , Amasino Richard M. , Koornneef Maarten , Millar Andrew J.
Published: March 2003

We have analysed the circadian rhythm of Arabidopsis thaliana leaf movements in the accession Cvi from the Cape Verde Islands, and in the commonly used laboratory strains Columbia (Col) and Landsberg (erecta) (Ler), which originated in Northern Europe. The parental lines have similar rhythmic periods, but the progeny of crosses among them reveal extensive variation for this trait. An analysis of 48 Ler/Cvi recombinant inbred lines (RILs) and a further 30 Ler/Col RILs allowed us to locate four putative quantitative trait loci (QTLs) that control the period of the circadian clock. Near‐isogenic lines (NILs) that contain a QTL in a small, defined chromo‐ somal region allowed us to confirm the phenotypic effect and to map the positions of three period QTLs, designated ESPRESSO, NON TROPPO and RALENTANDO. Quantitative trait loci at the locations of RALENTANDO and of a fourth QTL, ANDANTE, were identified in both Ler/Cvi and Ler/Col RIL populations. Some QTLs for circadian period are closely linked to loci that control flowering time, including FLC. We show that flc mutations shorten the circadian period such that the known allelic variation in the MADS‐box gene FLC can account for the ANDANTE QTL. The QTLs ESPRESSO and RALENTANDO identify new genes that regulate the Arabidopsis circadian system in nature, one of which may be the flowering‐time gene GIGANTEA.
Unsaturated fatty acids inhibit MP2C, a protein phosphatase 2C involved in the wound‐induced MAP kinase pathway regulation
Authors: Baudouin Emmanuel , Meskiene Irute , Hirt Heribert
Published: March 2003

When mechanically injured, plants develop multiple defense systems including the activation of specific genes. These responses are triggered by a complex network of signalling events that include Ca2+ fluxes, the production of free fatty acids from membrane lipids, as well as the activation of mitogen‐activated protein kinases (MAPK). In the present paper, we address the question of the regulation of the MAPK pathway by wound‐induced Ca2+ and fatty acid signals. We report that MP2C, a serine/threonine protein phosphatase 2C from alfalfa involved in MAPK pathway inactivation, is inhibited specifically in vitro by long‐carbon‐chain polyunsaturated fatty acids, and α‐linolenic acid, the primary product of the octadecanoid pathway, was found to be the most potent inhibitor. Ca2+ also inhibits MP2C, but only at high concentrations, and other divalent cations show similar inhibitory effect, making it unlikely that Ca2+ is involved in the regulation of MP2C in vivo. Overall, our data suggest that cross‐talk between wound‐induced MAPK and octadecanoid pathways may occur at the level of protein phosphatase 2C and linolenic acid.