bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryLoading 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

SummaryFull‐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

SummaryThe 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

SummaryAdvances 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

Summary 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

Summary 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

Summary 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

Summary 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

Summary 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

Summary 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

Summary 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

Summary 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

SummaryThe 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

SummaryArabinogalactan 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

SummaryProtein 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.