bluuebunny/crossref_metadata_2025 huggingface dataset:
An ordered Arabidopsis thaliana mitochondrial cDNA library on high‐density filters allows rapid systematic analysis of plant gene expression: a pilot study
Authors: Giegé Philippe , Konthur Zoltán , Walter Gerald , Brennicke Axel
Published: March 2003

The availability of the complete sequence of a genome allows a systematic analysis of its expression. Gene‐specific variations of transcription levels and phenomena such as transcript processing and RNA editing require large numbers of clones to be examined. For the completely sequenced mitochondrial genome of Arabidopsis thaliana we adapted robot technology to identify and characterize expressed genes. A cDNA library of about 50 000 clones was constructed, robot‐ordered into 384‐well microtitre plates and spotted onto high‐density filter membranes. These filters permit the isolation of large numbers of specific cDNA clones in a single hybridization step. The cox1, cox2 and cox3 genes were used to evaluate the feasibility and efficiency of this approach. A cluster of RNA editing sites observed outside the cox3 coding region identifies a novel reading frame orf95 in higher plants with significant similarity to a subunit of respiratory chain complex II.
Properties of proteins and the glassy matrix in maturation‐defective mutant seeds ofArabidopsis thaliana
Authors: Wolkers Willem F. , Alberda Mark , Koornneef Maarten , Léon‐Kloosterziel Karen M. , Hoekstra Folkert A.
Published: March 2003

In situ

Fourier transform infrared microspectroscopy was used to study the heat stability of proteins and hydrogen bonding interactions in dry maturation‐defective mutant seeds of

Arabidopsis thaliana

. α‐Helical, turn and β‐sheet conformations were the major protein secondary structures in all of these seeds. On heating, intermolecular extended β‐sheet structures, typical of protein denaturation, were formed in abscisic acid‐insensitive (

abi3

) and leafy cotyledon (

lec

) mutant seeds. Proteins in dry wild‐type seeds did not denature up to 150°C, but those in dry desiccation‐sensitive,

lec1–1

,

lec1–3

and

abi3–5

seeds did at 68, 89 and 87°C, respectively. In the desiccation‐tolerant

abi3–7

and

abi3–1

seeds, denaturation commenced above 120 and 135°C, respectively. Seeds of the

aba1–1 abi3–1

double mutant showed signs of denaturation already upon drying. The molecular packing in the seeds was studied by observing the shift in the position of the OH‐stretching vibration band with temperature. The maximal rate of change of this band with temperature was much higher in the desiccation‐sensitive

abi3–5

,

aba1–1 abi3–1

,

lec1–1

, and

lec1–3

mutant seeds than in the desiccation‐tolerant wild‐type,

abi3–1

,

abi3–7

, and

lec2–1

seeds. We interpret this to mean that the molecular packing density is higher in dry desiccation‐tolerant than in dry desiccation‐sensitive seeds, which is associated with a higher or lower protein denaturation temperature, respectively. The results are discussed in relation to the physiological and biochemical characteristics of these mutant seeds.
Functional complementation of yeast vma1Δ cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H+‐ATPase activity
Authors: Kim Woonbong , Wan Ching‐Yi , Wilkins Thea A.
Published: March 2003

The ability of a vacuolar H+‐ATPase (V‐ATPase) subunit homolog (subunit A) from plants to rescue the
vma
mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near‐identical isoforms of subunit A in mutant
vma1
Δ yeast cells successfully rescued the mutant
vma
phenotype, indicating that subunit A of plants and yeast have retained elements essential to V‐ATPases during the course of evolution. Although vacuoles become acidified, the plant–yeast hybrid holoenzyme only partially restored V‐ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N‐or C‐termini only slightly enhanced V‐ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant–yeast chimeric subunits are membrane‐bound. These results suggest that subunit A terminal domains contain structural information that impact V‐ATPase structure and function.
A 13‐bp cis‐regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors
Authors: Takeda Shin , Sugimoto Kazuhiko , Otsuki Hiroshi , Hirochika Hirohiko
Published: March 2003

The tobacco Tto1 is one of the few active LTR‐retrotransposons of plants, and its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. The expression of Tto1 RNA can also be activated by various stresses, including viral infection, wounding, and treatment with jasmonate, a signal molecule of plant defence responses. It is shown here that the Tto1 LTR promoter is responsible for a high level of expression in cultured tissues of transgenic tobacco plants. We demonstrate that a 13‐bp repeated motif (TGGTAGGTGAGAT) in the LTR functions as a cis‐regulatory element, which confers the responsiveness to tissue culture, wounding and methyl jasmonate. Fungal elicitors also activate the promoter containing multiple copies of the 13‐bp motif. Expression mediated by the 13‐bp motif is activated markedly by okadaic acid and moderately by K252a, so that both phosphorylation and dephosphorylation of proteins are possibly involved in the signalling pathways. Interestingly, the 13‐bp motif contains a conserved motif, Box L (also called AC‐I or H‐box like sequence) which has been shown to be involved in the expression of phenylpropanoid synthetic genes. Moreover, extended homologies are found between promoters of Tto1 and an asparagus defence gene, AoPR1, suggesting a possibility that the ancient insertion of an ancestral Tto1‐related retrotransposon has provided some of the promoter/regulatory sequences, including the 13‐bp motif‐related sequence, of the AoPR1 gene. Based on the structural and functional similarity between the two promoters, a possible evolutionary role of the regulatory sequences of LTR‐retrotransposons is discussed.
Non‐invasive quantitative detection and applications of non‐toxic, S65T‐type green fluorescent protein in living plants
Authors: Niwa Yasuo , Hirano Takanori , Yoshimoto Kohki , Shimizu Masanori , Kobayashi Hirokazu
Published: March 2003

Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100‐fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non‐invasive, quantitative detection system which is optimized for S65T‐type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non‐disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP‐positive cells can be quantitatively distinguished from non‐transformed cells under non‐selective conditions. This system will be useful in applications such as mutant screening, analysis of whole‐body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid‐ or mitochondria‐targeted sGFP(S65T). Etioplasts in dark‐grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.
Identification of three kinds of mutually related composite elements conferring S phase‐specific transcriptional activation
Authors: Taoka Ken‐ichiro , Kaya Hidetaka , Nakayama Takuya , Araki Takashi , Meshi Tetsuo , Iwabuchi Masaki
Published: March 2003

Conservation of the Oct motif (CGCGGATC) is a remarkable feature of plant histone gene promoters. Many of the Oct motifs are paired with a distinct motif, Hex, TCA or CCAAT‐box, constituting the type I element (CCACGTCANCGATCCGCG), type II element (TCACGCGGATC) and type III element (GATCCGCG‐N14‐ACCAATCA). To clarify the roles of these Oct‐containing composite elements (OCEs) in cell cycle‐dependent and tissue‐specific expression, we performed gain‐of‐function experiments with transgenic tobacco cell lines and plants harboring a derivative of the 35S core promoter/β‐glucuronidase fusion gene in which three or four copies of an OCE had been placed upstream. Although their activities were slightly different, results showed that each of the three types of OCEs could confer the ability to direct S phase‐specific expression on a heterologous promoter. In transgenic plants, the type I and III elements exhibited a similar activity, directing expression in meristematic tissues, whereas the activity of the type II element appeared to be restricted to young cotyledons and maturating guard cells. Mutational analyses demonstrated that the co‐operation of Oct with another module (Hex, TCA or CCAAT‐box) was absolutely required for both temporal and spatial regulation. Thus, OCEs play a pivotal role in regulation of the expression of plant histone genes.
Decreased expression of sucrose phosphate synthase strongly inhibits the water stress‐induced synthesis of sucrose in growing potato tubers
Authors: Geigenberger Peter , Reimholz Ralph , Deiting Uta , Sonnewald Uwe , Stitt Mark
Published: March 2003

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild‐type tubers. Antisense and co‐suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water‐stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short‐term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short‐term labelling experiments with intact tubers attached to well‐watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild‐type tubers. (ii) When wild‐type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild‐type tubers that were attached to water‐stressed wild‐type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry‐matter allocated to tubers in wild‐type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non‐stressed plants. However, SPS becomes a crucial factor in water‐stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.
A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii†
Authors: Fuhrmann Markus , Oertel Wolfgang , Hegemann Peter
Published: March 2003

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP‐fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble‐GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin–GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.
The gene encoding tobacco gibberellin 3β‐hydroxylase is expressed at the site of GA action during stem elongation and flower organ development
Authors: Itoh Hironori , Tanaka‐Ueguchi Miyako , Kawaide Hiroshi , Chen Xinbo , Kamiya Yuji , Matsuoka Makoto
Published: March 2003

Gibberellin 3β‐hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3β‐hydroxylase), which encodes a putative GA 3β‐hydroxylase, by RT‐PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA 3β‐hydroxylase. A high expression level of Nty was observed in shoot apices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter::GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. It is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3β‐hydroxylase expression in situ.
Molecular mechanisms underlying carotenogenesis in the chromoplast: multilevel regulation of carotenoid‐associated genes
Authors: Vishnevetsky Michael , Ovadis Marianna , Zuker Amir , Vainstein Alexander
Published: March 2003

In nature, carotenoid function and mode of action are highly determined by the neighboring protein and lipid molecules. Therefore an understanding of the proteins’ involvement in carotenoid sequestration would be of great help in elucidating carotenoid function in vivo. Based on a study of the expression of chromoplast‐specific carotenoid‐associated genes from cucumber corolla (CHRC and CHRD), a working model is presented wherein two major regulatory factors control carotenoid sequestration within the chromoplasts: (i) floral tissue‐specific transcriptional regulators of chromoplasto‐ genesis; and (ii) post‐transcriptional regulators related to the amount/type of sequestered carotenoids. This model is supported by the major role transcriptional regulation was found to play in the temporal and spatial expression of the CHRC gene, and by the fact that phytohormones such as gibberellic acid (GA3), abscisic acid and ethylene also acted as transcriptional regulators of CHRC expression. The primary response to GA3 was localized within the CHRC promoter to a 290 bp fragment. Furthermore, we demonstrated strong down‐regulation of CHRC expression at post‐transcriptional and translational/post‐translational levels resulting from inhibition of carotenoid biosynthesis, thus revealing a close link between carotenoid biosynthetic and sequestration machineries.
Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlase1 gene from ethylene‐treated Citrus fruit and its regulation during development
Authors: Jacob‐Wilk Debora , Holland Doron , Goldschmidt Eliezer E. , Riov Joseph , Eyal Yoram
Published: March 2003

We report on the isolation, functional expression and characterization of a cDNA encoding chlorophyllase, the enzyme catalyzing the first step in the chlorophyll breakdown pathway. The Chlase1 cDNA from Valencia Orange (Citrus sinensis cv. Valencia) was obtained by RT–PCR using degenerate primers based on the amino acid sequence of the previously purified protein. Chlase1 encodes a protein of 329 amino acids, including a sequence domain characterizing serine‐lipases and a putative chloroplast‐directing transit peptide. The Chlase1 gene encodes an active chlorophyllase enzyme which catalyzes the dephytylation of chlorophyll as shown by in vitro recombinant enzyme assays. Chlorophyllase expression at the transcript level in Valencia orange peel was found to be low and constitutive during natural fruit development without significant increase towards color‐break and ripening. However, ethylene treatment induced an increase in chlorophyllase transcript at all stages of development. An enhanced response to ethylene treatment was observed during the months of October and November, corresponding to the time of natural color‐break. The senescence‐delaying regulator gibberellin‐A3 (GA3) inhibited the effect of ethylene on chlorophyllase transcript accumulation. The data presented suggest that chlorophyllase may not be the regulator of chlorophyll breakdown during natural fruit ripening but is consistent with the notion that chlorophyll is gradually degraded during ripening due to a negative balance between synthesis and breakdown. According to this model, exogenous application of ethylene accelerates chlorophyll breakdown due to increased de novo synthesis of chlorophyllase. Further experi‐ mentation on the regulation and role of chlorophyllase in planta will be facilitated by the gene tools established in this work.
Over‐expression of a pepper plastid lipid‐associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress
Authors: Rey Pascal , Gillet Benjamin , Römer Susanne , Eymery Françoise , Massimino Jacqueline , Peltier Gilles , Kuntz Marcel
Published: March 2003

Proteins homologous to fibrillin, a pepper plastid lipid‐associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over‐expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 μmol photons m−2 sec−1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light‐dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild‐type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin‐related proteins fulfil an important function in plant development in relation to environmental constraints.
A resistance gene product of the nucleotide binding site –‐ leucine rich repeats class can form a complex with bacterial avirulence proteins in vivo
Authors: Leister R. Todd , Katagiri Fumiaki
Published: March 2003

Resistance (R) genes in plants mediate gene‐for‐gene disease resistance. The ligand‐receptor model, which explains the gene‐for‐gene specificity, predicts a physical interaction between an elicitor, which is directly or indirectly encoded by an avirulence (avr) gene in the pathogen, and the corresponding R gene product. The nucleotide binding site (NBS) – leucine rich repeats (LRR) class of R genes is the largest known class of R genes. Here we report that an NBS‐LRR R protein and its cognate Avr protein form a complex together in the plant cell. The Arabidopsis thaliana R genes RPS2 and RPM1 confer gene‐for‐gene disease resistance to strains of the phytopathogenic bacterium Pseudomonas syringae carrying the avr genes avrRpt2 and avrB, respectively. Using transient expression of these genes in Arabidopsis leaf mesophyll protoplasts, we first demonstrated that the protoplast system is appropriate for the investigation of the gene‐for‐gene recognition mechanism. Formation of an in vivo complex containing the RPS2 and AvrRpt2 proteins was demonstrated by co‐immunoprecipitation of the proteins following expression of the genes in protoplasts. This complex contained at least one additional plant protein of approximately 75 kDa. Unexpectedly, RPS2 also formed a complex with AvrB. We speculate that complex formation between AvrRpt2 and RPS2 is productive and leads to the elicitation of the resistance response, whilst complex formation between AvrB and RPS2 is unproductive and possibly competes with complex formation between AvrRpt2 and RPS2.
Homologues of a single resistance‐gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode
Authors: Van Der Vossen Edwin A. G. , Van Der Voort Jeroen N. A. M. Rouppe , Kanyuka Konstantin , Bendahmane Abdel , Sandbrink Hans , Baulcombe David C. , Bakker Jaap , Stiekema Willem J. , Klein‐Lankhorst Rene M.
Published: March 2003

The isolation of the nematode‐resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance‐gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R‐gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino‐acid identity) and belong to the leucine‐zipper, nucleotide‐binding site, leucine‐rich repeat (LZ‐NBS‐LRR)‐containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C‐terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.
Chaperone activity of tobacco HSP18, a small heat‐shock protein, is inhibited by ATP
Authors: Smýkal Petr , Mašín Jiří , Hrdý Ivan , Konopásek Ivo , Žárský Viktor
Published: March 2003

NtHSP18P (HSP18), a cytosolic class I small heat‐shock protein from tobacco pollen grains, was expressed in Escherichia coli. The viability of these cells was improved by 50% at 50°C, demonstrating its functionality in vivo. Purified recombinant protein formed 240 kDa HSP18 oligomers, irrespective of temperature. These oligomers interacted with the model substrate citrate synthase (CS) to form large complexes in a temperature‐dependent manner. Furthermore, HSP18 prevented thermally induced aggregation of CS at 45°C. The fluorescence probe bis‐ANS revealed the exposure of HSP18 hydrophobic surfaces at this temperature. Reactivation of chemically denatured CS was also significantly enhanced by HSP18. Surprisingly, HSP18 function was inhibited (in contrast to the related chaperone αβ‐crystallin and plant sHSPs studied so far) by the presence of ATP in a concentration‐dependent manner. The conformational changes of HSP18 imposed by ATP binding were indicated by the difference in the quenching of intrinsic tryptophan fluorescence, and implied more compact structure with ATP. Fluorescence measurements with bis‐ANS showed that the conformational shift of HSP18 is suppressed in the presence of ATP. Decreased chaperone activity of HSP18 in the presence of ATP is caused by the lower affinity of conformationally blocked HSP18 for the substrate, as demonstrated by a higher susceptibility of model substrate, malate dehydrogenase, to proteolytic cleavage. Our results suggest that the chaperone activity of some plant sHSPs could be regulated by the availability of ATP in the cytoplasm, which would provide a mechanism to monitor the cell environment, control biological activity of sHSPs, and coordinate it with other ATP‐dependent chaperones such as HSP70.
Differential expression and regulation of K+ channels in the maize coleoptile: molecular and biophysical analysis of cells isolated from cortex and vasculature
Authors: Bauer Claudia S. , Hoth Stefan , Haga Ken , Philippar Katrin , Aoki Naohiro , Hedrich Rainer
Published: March 2003

Recently, two K+ channel genes, ZMK1 and ZMK2, were isolated from maize coleoptiles. They are expressed in the cortex and vasculature, respectively. Expression in Xenopus oocytes characterized ZMK1 as an inwardly rectifying K+ channel activated by external acidification, while ZMK2 mediates voltage‐independent and proton‐inhibited K+ currents. In search of the related gene products in planta, we applied the patch–clamp technique to protoplasts isolated from the cortex and vasculature of Zea mays coleoptiles and mesocotyls. In the cortex, a 6–8 pS K+ channel gave rise to inwardly rectifying K+ currents. Like ZMK1, this channel was activated by apoplastic acidification. In contrast, protoplasts from vascular tissue expressing the sucrose transporter ZmSUT1 were dominated by largely voltage‐independent K+ currents with a single‐channel conductance of 22 pS. The pronounced sensitivity to the extracellular protons Ca2+, Cs+ and Ba2+ is reminiscent of ZMK2 properties in oocytes. Thus, the dominant K+ channels in cortex and vasculature most likely represent the gene products of ZMK1 and ZMK2. Our studies on the ZMK2‐like channels represent the first in planta analysis of a K+ channel that shares properties with the AKT3 K+ channel family.

Keywords: K+ channel, voltage‐independent, proton block, maize coleoptile.
FRL1 is required for petal and sepal development in Arabidopsis
Authors: Hase Yoshihiro , Tanaka Atsushi , Baba Tomohiro , Watanabe Hiroshi
Published: March 2003

A novel flower mutant, frl1 (frill 1) was isolated in Arabidopsis thaliana. The frl1 mutant has serrated petals and sepals but the other floral and vegetative organs appear to be normal. To analyse the role of the FRL1 gene, morphological, cytological and double mutant analyses were carried out. The frl1 flower had broader petals and sepals as compared with the wild‐type. The distal region of frl1 petals contained fewer epidermal cells but their size was variable and generally larger than that in the wild‐type. However, no significant difference was found in the basal region. Observations of the early petal development revealed that the morphology of the developing frl1 petal was normal until the middle of stage 9, but the frl1 phenotype became apparent in stages later than 10
. Furthermore, larger nuclei with varied sizes were observed in the distal region of frl1 petals, but not in this region in wild‐type petals. This strongly suggests that abnormal endo‐reduplication had occurred. These observations indicate that the frl1 mutation affects the number of cell divisions and the subsequent cell expansion during the late stage of petal lamina formation, and that FRL1 might be maintaining the mitotic state or suppressing the transition to the endo‐reduplication cycle. Double mutants with the homeotic mutants apetala3‐1 and agamous showed additive phenotypes. Ectopic petals in the third whorl of fr11 ag flowers were serrated, indicating that the FRL1 gene acts in petal and sepal development in an organ‐specific manner.
The hormonal regulation of axillary bud growth in Arabidopsis
Authors: Chatfield Steven P. , Stirnberg Petra , Forde Brian G. , Leyser Ottoline
Published: March 2003

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild‐type and hormone‐signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin‐mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin‐signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.
Size constraints for targeting post‐transcriptional gene silencing and for RNA‐directed methylation in Nicotiana benthamiana using a potato virus X vector
Authors: Thomas Carole L. , Jones Louise , Baulcombe David C. , Maule Andrew J.
Published: March 2003

Using a recombinant potato virus X (PVX) vector, we investigated the relationship between the length of RNA sequence identity with a transgene and the ability to promote post‐transcriptional gene silencing (PTGS) and transgene methylation. The lower size limit required for targeting reporter transgene mRNA de novo using PTGS was 23 nucleotides (nt) of complete identity, a size corresponding to that of small RNAs associated with PTGS in plants and RNA interference (RNAi) in animals. The size and sequence specificity were also explored for PTGS‐associated transgene methylation and for the targeting of the vector RNA. The PTGS‐competent short sequences resulted in similar patterns of methylation. In all cases, including specific sequences of 33 nt with or without symmetrical cytosine residues, the methylation was distributed throughout the transcribed region of the transgene. In contrast, short sequences lacking symmetrical cytosines were less efficient at promoting PTGS of the transgene mRNA. Short gfp sequences in the PVX vector provided as effective a target for the degradation of viral RNA as was found for PVX carrying the complete gfp cDNA. Short sequences were able to initiate PTGS of an endogenous gene, phyotene desaturase, although this occurred in the absence of DNA methylation. This experimental approach provides important insights into the relationship between short RNA sequences and PTGS.
Quantitative in vivo measurement of glutathione in Arabidopsis cells
Authors: Meyer Andreas J. , May Mike J. , Fricker Mark
Published: March 2003

A new, non‐destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH–bimane (GSB) conjugate. At low (10–100 µm) concentrations of MCB, labelling was mediated by a glutathione S‐transferase, which confers specificity for GSH. HPLC analysis of MCB‐labelled low molecular‐weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis–Menten kinetics with an apparent KM of 40 µm and Vmax of 470 µmol lcyt −1 min−1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of γ‐glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830–942 nmol g−1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane‐labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two‐photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 ± 3% cytoplasm was determined by manual segmentation, and a value of 37 ± 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 ± 0.3 and 3.2 ± 0.3 mm for cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 ± 0.5 and 3.5 ± 0.7 mm, respectively.
A new member of the Arabidopsis WRKY transcription factor family, AtWRKY6, is associated with both senescence‐ and defence‐related processes
Authors: Robatzek Silke , Somssich Imre E.
Published: March 2003

WRKY proteins constitute a large family of plant‐specific transcription factors whose precise functions have yet to be elucidated. Here we show that expression of one representative in Arabidopsis, AtWRKY6, is influenced by several external and internal signals often involved in triggering senescence processes and plant defence responses. Progressive 5′ deletions of the AtWRKY6 promoter allowed separation of defined regions responsible for the expression in distinct organs or upon pathogen challenge. Nuclear localization of AtWRKY6 was demonstrated; protein truncations and gain‐of‐function studies enabled delineation of a region harbouring a novel type of functional nuclear localization signal (NLS).
In vivo target promoter‐binding activities of a xenobiotic stress‐activated TGA factor
Authors: Johnson Christopher , Boden Erin , Desai Mihir , Pascuzzi Pete , Arias Jonathan
Published: March 2003

Xenobiotic chemicals induce the expression of nuclear detoxification genes. A full understanding of this protective response will require characterization of its transcriptional regulatory machinery. We describe here the use of a recently developed plant chromatin immunoprecipitation (ChIP) assay to define nuclear promoter targets of TGA1a, a tobacco basic/leucine zipper transcription factor whose activity is potentiated by herbicide‐induced xenobiotic stress. TGA1a selectively binds as‐1‐type cis‐elements, which regulate transcription of putative detoxification and defense genes. With ChIP, we show that endogenous TGA1a binds as‐1‐containing promoter sequences of two tobacco glutathione S‐transferase genes, GNT1 and GNT35. This binding activity is strongly enhanced by xenobiotic stress, as is expression of these genes. In contrast, TGA1a apparently does not bind in vivo to functional as‐1 elements in promoters of PR‐1a and PG13, genes whose expression is insensitive to this stimulus. The findings here thus discriminate between a number of possible functional promoter binding sites for a trans‐regulatory factor, within the context of a signal response pathway.
A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant–pathogen interaction incompatible
Authors: Belbahri Lassaad , Boucher Christian , Candresse Thierry , Nicole Michel , Ricci Pierre , Keller Harald
Published: March 2003

Plants activate disease resistance responses when they recognize pathogen‐derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro‐organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen‐inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1–PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.
Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds
Authors: Tierens Koenraad F. M.‐J. , Thomma Bart P. H. J. , Bari Rajendra P. , Garmier Marie , Eggermont Kristel , Brouwer Margreet , Penninckx Iris A. M. A. , Broekaert Willem F. , Cammue Bruno P. A.
Published: March 2003

An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild‐type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv. tomato and Peronospora parasitica. The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens. Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild‐type plants, but this induction was almost completely abolished in esa1. This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.
High free‐methionine and decreased lignin content result from a mutation in the Arabidopsis S‐adenosyl‐L‐methionine synthetase 3 gene
Authors: Shen Bo , Li Changjiang , Tarczynski Mitchell C.
Published: March 2003

As an approach to understand the regulation of methionine (Met) metabolism, Arabidopsis Met over‐accumulating mutants were isolated based on their resistance to selection by ethionine. One mutant, mto3, accumulated remarkably high levels of free Met – more than 200‐fold that observed for wild type – yet showed little or no difference in the concentrations of other protein amino‐acids, such as aspartate, threonine and lysine. Mutant plants did not show any visible growth differences compared with wild type, except a slight delay in germination. Genetic analysis indicated that the mto3 phenotype was caused by a single, recessive mutation. Positional cloning of this gene revealed that it was a novel S‐adenosylmethionine synthetase, SAMS3. A point mutation resulting in a single amino‐acid change in the ATP binding domain of SAMS3 was determined to be responsible for the mto3 phenotype. SAMS3 gene expression and total SAMS protein were not changed in mto3; however, both total SAMS activity and S‐adenosylmethionine (SAM) concentration were decreased in mto3 compared with wild type. Lignin, a major metabolic sink for SAM, was decreased by 22% in mto3 compared with wild type, presumably due to the reduced supply of SAM. These results suggest that SAMS3 has a different function(s) in one carbon metabolism relative to the other members of the SAMS gene family.
AtMYB21, a gene encoding a flower‐specific transcription factor, is regulated by COP1
Authors: Shin Byongchul , Choi Goh , Yi Hankuil , Yang Seungchan , Cho Insook , Kim Jonghyun , Lee Seunghee , Paek Nam‐Chon , Kim Jae‐Hong , Song Pill‐Soon , Choi Giltsu
Published: March 2003

Light is an important environmental signal that governs plant growth and development. One important light‐signalling component involved in plant light responses is COP1. The pleiotropic phenotypes of the cop1 mutant suggest that COP1 regulates not only photomorphogenesis, but also other developmental processes. We investigated the role of COP1 by identifying genes that are regulated by COP1. We report that AtMYB21, a gene encoding a flower‐specific transcription factor, is ectopically expressed in the cop1 mutant. Analysis shows that dark‐grown transgenic seedlings expressing AtMYB21‐GR fusion protein display some features of the cop1 mutant, including decreased hypocotyl cell expansion, open cotyledons in the dark, and seedling lethality in the presence of dexamethasone. Light‐grown adult transgenic plants expressing AtMYB21 have shorter stems, smaller and narrower leaves, narrower petals, and malformed carpels. In addition, we show that AtMYB21 directly regulates two genes that are also expressed more abundantly in the cop1 mutant. The results indicate that COP1 is required to repress the AtMYB21 gene in seedlings, and the pleiotropic phenotypes shown in the cop1 mutant are due to the combination of misregulation of genuine light‐signalling components and other tissue‐specific factors.
Barley stripe mosaic virus‐induced gene silencing in a monocot plant
Authors: Holzberg Steve , Brosio Paul , Gross Cynthia , Pogue Gregory P.
Published: March 2003

RNA silencing of endogenous plant genes can be achieved by virus‐mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus‐induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive‐sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the γb gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo‐bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo‐bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild‐type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo‐bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology‐dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA‐silencing mechanisms in monocot plant species.
Revival of a resurrection plant correlates with its antioxidant status
Authors: Kranner Ilse , Beckett Richard P. , Wornik Sabine , Zorn Margret , Pfeifhofer Hartwig W.
Published: March 2003

Myrothamnus flabellifolia, a short woody shrub from southern Africa, can survive severe desiccation of its vegetative organs. We studied mechanisms protecting this plant from oxidative damage during desiccation for 2 weeks, 4 and 8 months, and also during subsequent rehydration. This plant retains high concentrations of chlorophyll during desiccation, and these chlorophyll molecules are probably a source for potentially harmful singlet oxygen production. Desiccation triggered substantial increases in zeaxanthin and redox shifts of the antioxidants glutathione and ascorbate towards their oxidised forms. Simultaneously, the concentrations of violaxanthin, β‐carotene, ascorbate, α‐tocopherol, and glutathione reductase activity progressively decreased. Antheraxanthin, γ‐tocopherol, lutein, neoxanthin and glucose‐6‐phosphate dehydrogenase displayed less pronounced changes in response to desiccation. Even after 4 months of desiccation, Myrothamnus flabellifolia recovered rapidly upon rehydration. Re‐watering induced formation of ascorbate and glutathione, simultaneous reduction of their oxidised forms, and rapid production of α‐tocopherol and of various carotenoids. Only after 8 months of desiccation did the antioxidant system of M. flabellifolia break down; 3 weeks after the onset of rehydration, these plants abscised their leaves, but even then they were still able to recover and develop new ones. Ascorbate, β‐carotene and α‐tocopherol were totally depleted after 8 months of desiccation and did not recover upon rehydration; glutathione was partly maintained, but only in the oxidised form. We present a model demonstrating which parts of antioxidant pathways break down as oxidative stress becomes detrimental and we discuss some potential implications of our results for the genetic modification of crop plants to improve their drought tolerance.