bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis
Authors: Avelange‐Macherel Marie‐Hélène , Joyard Jacques
Published: March 2003

A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed. COQ3 encodes a 3,4‐dihydroxy‐5‐hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p‐hydroxybenzoic acid. A full length cDNA encoding a homologue of DHHB‐methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant. The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant. The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35 360 Da. The predicted amino acid sequence contained all consensus regions for S‐adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB‐methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene. Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene. The NH2‐terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence. The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein. Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes. This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.
Significance of plasmalemma aquaporins for water‐transport in Arabidopsis thaliana
Authors: Kaldenhoff Ralf , Grote Karsten , Zhu Jian‐Jun , Zimmermann Ulrich
Published: March 2003

The plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti‐sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti‐sense lines have reduced steady‐state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti‐sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.
Rapid discrimination of sequences flanking and within T‐DNA insertions in the Arabidopsis genome
Authors: Ponce María Rosa , Quesada Víctor , Micol José Luis
Published: March 2003

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T‐DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T‐DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T‐DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T‐DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T‐DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T‐DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.
Enzymatic oxidations in the biosynthesis of complex alkaloids
Authors: Chou Wing‐Ming , Kutchan Toni M.
Published: March 2003

The biosynthesis of complex alkaloids in plants involves enzymes that, due to high substrate specificity, appear to have evolved solely for a role in secondary metabolism. At least one class of these enzymes, the oxidoreductases, catalyze transformations that are in some cases difficult to chemically mimick with an equivalent stereo‐ or regiospecificity and yield. Oxidoreductases are frequently catalyzing reactions that result in the formation of parent ring systems, thereby determining the class of alkaloid that a plant will produce. The oxidoreductases of alkaloid formation are a potential target for the biotechnological exploitation of medicinal plants in that they could be used for biomimetic syntheses of alkaloids. Analyzing the molecular genetics of alkaloid biosynthetic oxidations is requisite to eventual commercial application of these enzymes. To this end, a wealth of knowledge has been gained on the biochemistry of select monoterpenoid indole and isoquinoline biosynthetic pathways, and in recent years this has been complemented by molecular genetic analyses. As the nucleotide sequences of the oxidases of alkaloid synthesis become known, consensus sequences specific to select classes of enzymes can be identified. These consensus sequences will potentially facilitate the direct cloning of alkaloid biosynthetic genes without the need to purify the native enzyme for partial amino acid sequence determination or for antibody production prior to cDNA isolation. The current state of our knowledge of the biochemistry and molecular genetics of oxidases involved in alkaloid biosynthesis is reviewed herein.
High affinity RGD‐binding sites at the plasma membrane ofArabidopsis thalianalinks the cell wall
Authors: Canut Hervé , Carrasco Antoine , Galaud Jean‐Philippe , Cassan Catherine , Bouyssou Huguette , Vita Natalio , Ferrara Pascual , Pont‐Lezica Rafael
Published: March 2003

The heptapeptide Tyr‐Gly‐

Arg‐Gly‐Asp‐

Ser‐Pro containing the sequence

Arg‐Gly‐Asp

(RGD – the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr‐Gly‐Asp‐Gly‐Arg‐Ser‐Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD‐heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD‐heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 1 nM, and Kd2 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD‐binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane‐cell wall attachments.
Fine structure in cellulose microfibrils: NMR evidence from onion and quince
Authors: Ha Marie‐Ann , Apperley David C. , Evans Boyd W. , Huxham I. Max , Jardine W. Gordon , Viëtor Remco J. , Reis Danièle , Vian Brigitte , Jarvis Michael C.
Published: March 2003

It has been controversial for many years whether in the cellulose of higher plants, the microfibrils are aggregates of ‘elementary fibrils’, which have been suggested to be about 3.5 nm in diameter. Solid‐state NMR spectroscopy was used to examine two celluloses whose fibril diameters had been established by electron microscopy: onion (8–10 nm, but containing 40% of xyloglucan as well as cellulose) and quince (2 nm cellulose core). Both of these forms of cellulose contained crystalline units of similar size, as estimated from the ratio of surface to interior chains, and the time required for proton magnetisation to diffuse from the surface to the interior. It is suggested that the onion microfibrils must therefore be constructed from a number of cellulose subunits 2 nm in diameter, smaller than the ‘elementary fibrils’ envisaged previously. The size of these subunits would permit a hexagonal arrangement resembling the cellulose synthase complex.
Constituents of the tapetosomes and elaioplasts inBrassica campestristapetum and their degradation and retention during microsporogenesis
Authors: Ting Julie T. L. , Wu Sherry S. H. , Ratnayake Chandra , Huang Anthony H. C.
Published: March 2003

In

Brassica

anthers during microsporogenesis, the tapetum cells contain two abundant lipid‐rich organelles, the tapetosomes possessing oleosins and triacylglycerols (TAGs), and the elaioplasts having unique polypeptides and neutral esters.

B. campestris

, for its simplicity of possessing only the AA genome and one predominant oleosin of 45 kDa, was studied. In the developing anthers, the lipids and proteins of the tapetosomes and elaioplasts were concomitantly accumulated but selectively degraded or retained. Upon incubation of isolated tapetosomes in a pH‐5 medium, the predominant 45 kDa oleosin underwent selective enzymatic proteolysis to a 37 kDa fragment, which was not further hydrolyzed upon prolonged incubation. The unreacted 45 kDa oleosin was retained in the organelles, whereas the 37 kDa fragment was released to the exterior. The fragment would become the predominant 37 kDa polypeptide in the pollen coat. Isolated tapetosomes did not undergo hydrolysis of the TAGs upon incubation in media of diverse pHs. An alkaline lipase in the soluble fraction of the anther extract was presumed to be the enzyme that would hydrolyze the tapetosome TAGs, which disappeared in the anthers during development. The tapetum elaioplasts contained several unique polypeptides of 31–36 kDa. The gene encoding a 32 kDa polypeptide was cloned, and its deduced amino acid sequence was homologous to those of two proteins known to be present on the surface of fibrils in chromoplasts. Upon incubation of isolated elaioplasts in media of diverse pHs, the organelle polypeptides were degraded completely and most rapidly at pH 5, whereas the neutral esters remained unchanged; these neutral esters would become the major lipid components of the pollen coat. The findings show that the constituents of the two major tapetum organelles underwent very different paths of degradation, or modification, and transfer to the pollen surface.
Use of a proteome strategy for tagging proteins present at the plasma membrane
Authors: Santoni Véronique , Rouquié David , Doumas Patrick , Mansion Monique , Boutry Marc , Degand Hervé , Dupree Paul , Packman Len , Sherrier Janine , Prime Tracy , Bauw Guy , Posada Esmeralda , Rouzé Pierre , Dehais Patrice , Sahnoun Ilhem , Barlier Isabelle , Rossignol Michel
Published: March 2003

A plasma membrane (PM) fraction was purified from

Arabidopsis thaliana

using a standard procedure and analyzed by two‐dimensional (2D) gel electrophoresis. The proteins were classified according to their relative abundance in PM or cell membrane supernatant fractions. Eighty‐two of the 700 spots detected on the PM 2D gels were microsequenced. More than half showed sequence similarity to proteins of known function. Of these, all the spots in the PM‐specific and PM‐enriched fractions, together with half of the spots with similar abundance in PM fraction and supernatant, have previously been found at the PM, supporting the validity of this approach. Extrapolation from this analysis indicates that (i) approximately 550 polypeptides found at the PM could be resolved on 2D gels; (ii) that numerous proteins with multiple locations are found at the PM; and (iii) that approximately 80% of PM‐specific spots correspond to proteins with unknown function. Among the latter, half are represented by ESTs or cDNAs in databases. In this way, several unknown gene products were potentially localized to the PM. These data are discussed with respect to the efficiency of organelle proteome approaches to link systematically genomic data to genome expression. It is concluded that generalized proteomes can constitute a powerful resource, with future completion of

Arabidopsis

genome sequencing, for genome‐wide exploration of plant function.
TheEMB506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos
Authors: Albert Sylvie , Després Barbara , Guilleminot Jocelyne , Bechtold Nicole , Pelletier Georges , Delseny Michel , Devic Martine
Published: March 2003

The

EMB

506 gene of Arabidopsis, required for the normal development of the embryo beyond the globular stage, has been cloned. The gene encodes a protein of predicted size 35 kDa that contains five ankyrin (ANK) repeats within the C terminal moiety. ANK repeats are conserved domains of 33 amino acids involved in specific recognition of protein partners. The EMB 506 protein was detected at different stages of silique development but accumulated preferentially in the mature cauline leaves. The rescue of homozygous

emb

506 embryos by complementation with the wild‐type sequence cDNA demonstrated that the

emb

mutation is a consequence of the T‐DNA insertion and that integration and expression of the transgene occurred during gametogenesis and/or early embryo development. In addition to the drastic effect of the

emb

506 mutation during embryo development, complementation experiments revealed another effect of the gene:

emb

506 plants transformed with the wild‐type

EMB

506 sequence were able to produce viable seeds but showed a reduction of apical dominance and the presence of adventitious buds or bracts along the stem. This result supports the idea that genes essential for embryogenesis may also be required at other stages of the plant life cycle.
Molecular cloning and functional expression ofO‐methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis
Authors: Frick Susanne , Kutchan Toni M.
Published: March 2003

In cell suspension cultures of the meadow rue

Thalictrum tuberosum

, biosynthesis of the anti‐microbial alkaloid berberine can be induced by addition of methyl jasmonate to the culture medium. The activities of the four methyltransferases involved in the formation of berberine from L‐tyrosine are increased in response to elicitor addition. Partial clones generated by RT–PCR with methyltransferase‐specific primers were used as hybridization probes to isolate four cDNAs encoding

O

‐methyltransferases from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate‐induced cell suspension cultures of

T. tuberosum

. RNA gel blot hybridization indicated that the transcripts for the methyltransferases accumulated in response to addition of methyl jasmonate to the cell culture medium. The cDNAs were functionally expressed in

Spodoptera frugiperda

Sf9 cells and were shown to have varying and broad substrate specificities. A difference of a single amino acid residue between two of the enzymes was sufficient to alter the substrate specificity. The four cDNAs were expressed either as four homodimers or as six heterodimers by co‐infection with all possible combinations of the four recombinant baculoviruses. These 10 isoforms thus produced displayed distinct substrate specificities and in some cases co‐infection with two different recombinant baculoviruses led to the

O

‐methylation of new substrates. The substrates that were

O

‐methylated varied in structural complexity from simple catechols to phenylpropanoids, tetrahydrobenzylisoquinoline, protoberberine and tetrahydrophenethylisoquinoline alkaloids, suggesting that some biosynthetic enzymes may be common to both phenylpropanoid and alkaloid anabolism.
Isoforms of acyl carrier protein involved in seed‐specific fatty acid synthesis
Authors: Suh Mi Chung , Schultz David J. , Ohlrogge John B.
Published: March 2003

Seeds of
Coriandrum sativum
(coriander) and
Thunbergia alata
(black‐eyed Susan vine) produce unusual monoenoic fatty acids which constitute over 80% of the total fatty acids of the seed oil. The initial step in the formation of these fatty acids is the desaturation of palmitoyl‐ACP (acyl carrier protein) at the D4 or D6 positions to produce D4‐hexadecenoic acid (16 : 1D4) or D6‐hexadecenoic acid (16 : 1D6), respectively. The involvement of specific forms of ACP in the production of these novel monoenoic fatty acids was studied. ACPs were partially purified from endosperm of coriander and
T. alata
and used to generate 3H‐ and 14C‐labelled palmitoyl‐ACP substrates. In competition assays with labelled palmitoyl‐ACP prepared from spinach (
Spinacia oleracea
), D4‐acyl‐ACP desaturase activity was two‐ to threefold higher with coriander ACP than with spinach ACP. Similarly, the
T. alata
D6 desaturase favoured
T. alata
ACP over spinach ACP. A cDNA clone,
Cs
‐ACP‐I, encoding ACP was isolated from a coriander endosperm cDNA library.
Cs
‐ACP‐I mRNA was predominantly expressed in endosperm rather than leaves. The
Cs
‐ACP‐I mature protein was expressed in
E. coli
and co‐migrated on SDS–PAGE with the most abundant ACP expressed in endosperm tissues. In
in vitro
D4‐palmitoyl‐ACP desaturase assays, the
Cs
‐ACP‐I expressed from
E. coli
was four‐ and 10‐fold more active than spinach ACP or
E. coli
ACP, respectively, in the synthesis of D4‐hexadecenoic acid from palmitoyl‐ACP. In contrast, D9‐stearoyl‐ACP desaturase activity from coriander endosperm did not discriminate strongly between different ACP species. These results indicate that individual ACP isoforms are specifically involved in the biosynthesis of unusual seed fatty acids and further suggest that expression of multiple ACP isoforms may participate in determining the products of fatty acid biosynthesis.
Post‐transcriptional gene silencing of chalcone synthase in transgenic petunias, cytosine methylation and epigenetic variation
Authors: O’Dell Michael , Metzlaff Michael , Flavell Richard B.
Published: March 2003

Post‐transcriptional degradation of chalcone synthase RNA transcripts often occurs in petunia plants with additional copies of chalcone synthase genes. The cytosine methylation status of selected restriction endonuclease sites was studied in the chalcone synthase endogenous genes and transgenes of these plants. Methylation patterns varied between transformants and within a series of epigenetically related plants. There was heterogeneity in promoter transgene methylation patterns within and/or between cells, between leaves and flowers and between tandemly duplicated sequences. Thus methylation patterns are determined by more than the local DNA sequence or site of insertion. A site in the 5′ enhancer of the 35S promoter was methylated more frequently in white flowers than in purple flowers. An EcoRII site within the coding sequence of the endogenous ChsA genes is developmentally regulated, being frequently methylated in leaves but rarely in petals of wild‐type petunias or stable purple flowered transgenic plants. However, in petals of many transgenic plants, especially those that show extensive post transcriptional RNA degradation, the site is frequently methylated. Thus the transgenes influence developmental methylation of the endogenous genes. In the transgenes the EcoRII site is usually more frequently methylated in petals than in leaves. RNA degradation in petals is correlated in one set of epigenetically related transgenic plants with hypomethylation at the transgene EcoRII site in leaves. Possible relationships between the cytosine methylation patterns in vegetative and floral cells, epigenetic states of ChsA genes and the control of RNA transcript degradation are discussed.
Isolation, characterization and immunolocalization of a novel, modular tomato arabinogalactan‐protein corresponding to the LeAGP‐1 gene
Authors: Gao Minggeng , Kieliszewski Marcia J. , Lamport Derek T. A. , Showalter Allan M.
Published: March 2003

Arabinogalactan‐proteins (AGPs) are a family of hydroxyproline‐rich glycoproteins implicated to function in plant growth and development. This report focuses on a novel, modular AGP found in tomato, LeAGP‐1, which was predicted by DNA cloning and herein verified at the protein level as a major AGP component. LeAGP‐1 was isolated from tomato suspension‐cultured cells and verified to be an AGP by precipitation with (β‐D‐galactosyl)3 Yariv phenylglycoside and by amino acid composition analysis. Furthermore, LeAGP‐1 was determined to correspond to LeAGP‐1 clones based on three criteria: (1) amino acid composition identity, (2) amino acid sequence identity, and (3) specific immunoreactivity of glycosylated and deglycosylated LeAGP‐1 with an antibody developed against the highly basic subdomain predicted from LeAGP‐1 clones. The antibody was also used to immunolocalize LeAGP‐1 in tomato to the cell surface of suspension‐cultured cells, maturing metaxylem elements in young internodes and petioles, and stylar transmitting tissue cells. At the subcellular level, LeAGP‐1 immunolocalized to the cell walls of these particular cells as well as to intercellular spaces between stylar transmitting tissue cells. LeAGP‐1 now emerges as one of the most comprehensively studied AGPs in terms of (1) characterization at the genomic DNA, cDNA and protein levels, (2) known organ‐specific and developmentally regulated mRNA expression patterns, (3) development of an antibody against a unique, peptide subdomain which specifically recognizes LeAGP‐1 in its glycosylated and deglycosylated states, and (4) immunolocalization of a single, well‐defined AGP molecule at the tissue and subcellular levels.
A tobacco matrix attachment region reduces the loss of transgene expression in the progeny of transgenic tobacco plants
Authors: Ülker Bekir , Allen George C. , Thompson William F. , Spiker Steven , Weissinger Arthur K.
Published: March 2003

The RB7 matrix attachment region (MAR), when flanking a uidA (GUS) reporter gene, has been previously shown to increase uidA gene expression by 60‐fold in stably transformed tobacco suspension cell lines. We have now used the same co‐transformation procedure to determine the effect of flanking MARs on uidA gene expression in tobacco plants. The neomycin phosphotransferase selection gene and uidA reporter gene on separate plasmids were co‐transformed into seedlings by microprojectile bombardment. In primary transgenic plants, the average uidA expression in plants with MARs was twofold greater than in control plants without MARs, but there was no effect on variation of expression. GUS activity was not proportional to the number of integrated uidA transgenes over the entire range of copy numbers. However, in the lower part of the copy number range, MAR lines show a tendency for expression to increase with copy number. Transgene expression in backcross progenies of the MAR‐containing lines averaged threefold higher than in control progenies. MARs also reduced the loss of transgene expression in the BC1 generation. Sixty‐three per cent of the 21 MAR‐containing primary transformants, but only 20% of the 14 control primary transformants, produced backcross progenies in which no loss of transgene expression was observed. These observations are discussed in the context of homology‐dependent gene silencing.
Cryptochrome 1 controls tomato development in response to blue light
Authors: Ninu Luciano , Ahmad Margaret , Miarelli Carolina , Cashmore Anthony R. , Giuliano Giovanni
Published: March 2003

Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that bear a strong resemblance to photolyases, a rare class of flavoproteins that absorb light in the blue (B) and UV‐A regions of the spectrum and utilise it for photorepair of UV‐damaged DNA. In Arabidopsis, both CRY1 and CRY2 are implicated in numerous blue light‐dependent responses, including inhibition of hypocotyl elongation, leaf and cotyledon expansion, pigment biosynthesis, stem growth and internode elongation, control of flowering time and phototropism. No information about the in vivo function of CRY genes is available in other plant species. The tomato CRY1 gene (TCRY1) encodes a protein of 679 amino acids, which shows 78% identity and 88% similarity to Arabidopsis CRY1. In order to verify the in vivo function of TCRY1, we constructed antisense tomato plants using the C‐terminal portion of the gene. Partial repression of both mRNA and protein levels was observed in one of the transformants. The progeny from this transformant showed an elongated hypocotyl under blue but not under red light. This character co‐segregated with the transgene and was dependent on transgene dosage. An additional, partially elongated phenotype was observed in adult plants grown in the greenhouse under dim light and short days with no artificial illumination. This phenotype was suppressed by artificial illumination of both short and long photoperiods. The synthesis of anthocyanins under blue light was reduced in antisense seedlings. In contrast, carotenoid and chlorophyll levels and second positive phototropic curvature were essentially unaltered.
Arabidopsis thaliana proteins related to the yeast SIP and SNF4 interact with AKINα1, an SNF1‐like protein kinase
Authors: Bouly Jean‐Pierre , Gissot Lionel , Lessard Philippe , Kreis Martin , Thomas Martine
Published: March 2003

AKINα1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly conserved SNF1 family of protein kinases in eukaryotes. Recent data suggest that the plant SNF1‐related kinases (SnRK1 family) are key enzymes implicated in the regulation of carbohydrate and lipid metabolism. In Saccharomyces cerevisiae and mammals, the SNF1 and AMPKα protein kinases interact with two other families of proteins, namely SNF4/AMPKγ and SIP1/SIP2/GAL83/AMPKβ, to form active heterotrimeric complexes. In this paper, we describe the characterisation of three novel cDNAs. AKINβ1 and AKINβ2 encode proteins similar to SIP1, SIP2 and GAL83 and AKINγ codes for a protein showing similarity with SNF4. Using the two‐hybrid system, specific interactions have been shown between A. thaliana AKINβ1/β2, AKINγ and AKINγ as well as between the A. thaliana and S. cerevisiae subunits. Interestingly, AKINβ1, AKINβ2 and AKINγ mRNAs accumulate differentially in A. thaliana tissues and are modulated during development and under different growth conditions. These data suggest the presence in higher plants of a conserved heterotrimeric complex. Moreover, the differential transcription of different non‐catalytic subunits can constitute a first level of regulation of the SNF1‐like complex in plants.
Gibberellin 2‐oxidation and the SLN gene of Pisum sativum
Authors: Lester Diane R. , Ross John J. , Smith Jennifer J. , Elliott Robert C. , Reid James B.
Published: March 2003

Two cDNAs encoding gibberellin 2‐oxidases were isolated from maturing pea seeds. The first, PsGA2ox1, was isolated by activity screening of a Lambda‐ZAP cDNA library excised into phagemid form and expressed in Escherichia coli. The second, PsGA2ox2, was obtained initially as a PCR product using degenerate primers designed according to conserved regions of plant 2‐oxoglutarate‐dependent dioxygenases. E. coli heterologous expression products of PsGA2ox1 and PsGA2ox2 converted GA1 to GA8, as shown by HPLC‐radiocounting, and gas chromatography‐MS. PsGA2ox1 converted GA20 to GA29, but GA20 was a poor substrate for the PsGA2ox2 expression product. Furthermore, PsGA2ox1 converted GA29 to GA29‐catabolite at a low level of efficiency while PsGA2ox2 did not catalyse this step. A cDNA of PsGA2ox1 isolated from plants of genotype sln contained a single base deletion which was predicted to produce a truncated protein and gibberellin 2‐oxidase activity could not be demonstrated from this cDNA. A 10 bp size difference between the introns of the SLN and sln PsGA2ox1 genes was used to show co‐segregation between the SLN and sln phenotypes and the size of the PCR products. PsGA2ox1 transcripts were more abundant in cotyledons than in shoots, while the reverse was the case for PsGA2ox2. The expression patterns of the genes, together with the effects of the sln mutation, indicate that PsGA2ox1 plays a major role in GA20 deactivation in both shoots and maturing seeds, while the PsGA2ox2 gene might be important for GA1 deactivation in the shoot.
Cymbidium hybrida dihydroflavonol 4‐reductase does not efficiently reduce dihydrokaempferol to produce orange pelargonidin‐type anthocyanins
Authors: Johnson Eric T. , Yi Hankuil , Shin Byongchul , Oh Boung‐Jun , Cheong Hyeonsook , Choi Giltsu
Published: March 2003

Some angiosperms are limited to a range of possible flower colors. This limitation can be due to the lack of an anthocyanin biosynthetic gene or to the substrate specificity of a key anthocyanin biosynthetic enzyme, dihydroflavonol 4‐reductase (DFR). Cymbidium hybrida orchid flowers primarily produce cyanidin‐type (pink to red) anthocyanins and lack the pelargonidin‐type (orange to brick‐red) anthocyanins. To investigate the underlying molecular mechanism of this flower color range, we cloned a Cymbidium DFR gene and transformed it into a DFR– petunia line. We found that the Cymbidium DFR did not efficiently reduce dihydrokaempferol (DHK), which is an essential step for pelargonidin production. Phylogenetic analysis of a number of DFR sequences indicate that the inability to catalyze DHK reduction has occurred at least twice during angiosperm evolution. Our results indicate that developing a pelargonidin‐type orange flower color in Cymbidium may require the transformation of a DFR gene that can efficiently catalyze DHK reduction.
MAP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response?
Authors: Cazalé Anne‐Claire , Droillard Marie‐Jo , Wilson Cathal , Heberle‐Bors Erwin , Barbier‐Brygoo Hélène , Laurière Christiane
Published: March 2003

Hypoosmotic stress activates a phosphorylation‐dependent oxidative burst. In‐gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen‐activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4‐like. Apigenin, a MAP kinase and cyclin‐dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst ( Cazaléet al. 1998
; Plant Physiol. 116, 659–669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6‐DMAP. However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst. Two‐dimensional in‐gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.
Accurate and high resolution in situ hybridization analysis of gene expression in secondary stem tissues
Authors: Regan Sharon , Bourquin Veronica , Tuominen Hannele , Sundberg Björn
Published: March 2003

Accurate in situ hybridization analysis in secondary stem tissues of plants has been hindered by specific characteristics of these tissues. First, secondary cell walls non‐specifically bind probes used for in situ hybridization thus preventing gene expression analysis in the lignified regions of the stem, such as the xylem. Second, the mRNA in the cambial meristem and its recent derivatives are prone to inadequate fixation when conventional techniques are used. Here we describe an in situ hybridization technique which uses fast freezing and freeze substitution to cryoimmobilize the mRNA followed by embedding in a methacrylate resin for high‐resolution analysis of gene expression. By using a transgenic poplar line harbouring rolC::uidA, rolC::iaaM, the gene expression pattern could be compared with histochemical GUS staining. This in situ hybridization technique results in superior preservation of cellular contents, retention of mRNA in all cell types in the poplar stem, a significant reduction of non‐specific binding to secondary cell walls and a resolution not previously possible in secondary tissues. This technique will be particularly valuable for the expression analysis of genes involved in xylogenesis and wood formation.
Isolation of a novel SUMO protein from tomato that suppresses EIX‐induced cell death
Authors: Hanania Uri , Furman‐Matarasso Noa , Ron Mily , Avni Adi
Published: March 2003

Challenging tomato or tobacco varieties with ethylene‐inducing xylanase (EIX) from the fungus Trichoderma viride causes rapid induction of plant defence responses leading to programmed cell death. Using the yeast two‐hybrid system, we isolated a novel protein, tomato small ubiquitin‐related modifier protein (T‐SUMO), which specifically interacts with EIX. T‐SUMO, a cytoplasmic protein, is a member of the ubiquitin‐like protein family. It shows homology to human protein sentrin/SUMO1, which suppresses tumour necrosis factor‐induced cell death. Transgenic plants that express T‐SUMO in the sense orientation suppress EIX induction of ethylene biosynthesis and cell death, while in the antisense orientation they enhance EIX‐induced ethylene biosynthesis. These results indicate that T‐SUMO is involved in mediating the signal generated by EIX that leads to induction of plant defence responses.
An abundant TIP expressed in mature highly vacuolated cells
Authors: Karlsson Maria , Johansson Ingela , Bush Max , McCann Maureen C. , Maurel Christophe , Larsson Christer , Kjellbom Per
Published: March 2003

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS–PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So‐δTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity‐purified So‐δTIP‐specific peptide antibodies, we investigated the subcellular and tissue distribution of So‐δTIP. So‐δTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between δ‐TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So‐δTIP with that of more distantly related TIPs showed similarities with Arabidopsisγ‐TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So‐δTIP may be induced when the large vacuole is formed.
Defective splicing of the first nad4 intron is associated with lack of several complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant
Authors: Brangeon Judy , Sabar Mohammed , Gutierres Sophie , Combettes Bruno , Bove Jérôme , Gendy Cyrille , Chétrit Philippe , Des Francs‐Small Catherine Colas , Pla Magali , Vedel Fernand , De Paepe Rosine
Published: March 2003

In this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a specific mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the first intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two‐dimensional in organello mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or post‐translational steps in the synthesis of other mitochondrial proteins are affected. All of these defects co‐segregated with the abnormal phenotype in the offspring of a NMS1 × wild‐type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.
Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins
Authors: Zhang Shuqun , Liu Yidong , Klessig Daniel F.
Published: March 2003

Three protein kinases of 48, 44 and 40 kDa are activated at different stages in tobacco cells treated with fungal elicitins. Previously we demonstrated that the rapidly activated 48 kDa protein kinase is encoded by SIPK. Here we report that the elicitin‐activated 44 kDa kinase is encoded by WIPK. Activation of this kinase occurred 2–4 h after elicitin treatment and was preceded by dramatic increases in WIPK mRNA and protein levels. Studies using actinomycin D and cycloheximide demonstrated that de novo transcription and translation were required for this activation of the kinase activity. Strikingly, the kinetics of WIPK activation following elicitin treatment correlated with the onset of hypersensitive response (HR)‐like cell death. Moreover, staurosporine and K‐252a, two Ser/Thr protein kinase inhibitors that blocked WIPK activation, suppressed cell death. The timing for elicitin‐treated cells to commit to a death program correlated with the appearance of high levels of WIPK activity. These correlative data suggest that WIPK may play a role during HR development in tobacco. Interestingly, a fungal cell‐wall elicitor that does not cause cell death induced WIPK mRNA and protein to similar levels as those observed with the elicitins. However, no corresponding increase in WIPK activity was detected. Thus WIPK appears to be controlled at multiple levels.

Keywords: MAP kinases, WIPK, SIPK, elicitin, transcription, translation.
The DNA‐binding activity of Gal4 is inhibited by methylation of the Gal4 binding site in plant chromatin
Authors: Gälweiler Leo , Conlan R. Steven , Mader Patricia , Palme Klaus , Moore Ian
Published: March 2003

Derivatives of the Saccharomyces cerevisiae transcription factor Gal4 which act as effective transcription activators in yeast, Drosophila, mammalian cells and plant protoplasts are shown to direct expression from a GUS reporter construct when expressed in transgenic tobacco. However, in comparison to 35S‐GUS controls, Gal4‐mediated expression of the reporter gene was relatively weak and extremely variable. GUS expression was lost as plants matured and it was almost undetectable in most of their progeny. Gal4‐mediated gene expression could be restored by treating tissues with 5‐aza‐cytidine, implicating cytosine methylation in the loss of Gal4‐mediated expression. Restoration of reporter expression was not accompanied by an increase in steady‐state levels of the activator transcript. We propose that the DNA‐binding activity of Gal4 is sensitive to methylation of its binding site in plant chromatin. The Gal4‐DNA co‐crystal predicts that 5‐methylcytosine at either of the outer two positions of the binding site will effectively prevent Gal4 binding. We show that these positions become extensively methylated in transgenic plants and that methylation of Gal4‐binding sites interferes with Gal4 binding in vitro. These observations suggest that the Gal4 DNA‐binding domain is intrinsically sensitive to cytosine methylation and that, despite the success of Gal4‐based expression systems in yeast and Drosophila, Gal4 is not ideal for use in plant gene expression technology.
The characterization of differential calcium signalling in tobacco guard cells
Authors: Wood Nicola T. , Allan Andrew C. , Haley Ann , Viry‐Moussaïd Martine , Trewavas Anthony J.
Published: March 2003

Two novel approaches for the study of Ca2+‐mediated signal transduction in stomatal guard cells are described. Stimulus‐induced changes in guard‐cell cytosolic Ca2+ ([Ca2+]cyt) were monitored using viable stomata in epidermal strips of a transgenic line of Nicotiana plumbaginifolia expressing aequorin (the proteinous luminescent reporter of Ca2+) and in a new transgenic line in which aequorin expression was targeted specifically to the guard cells. The results indicated that abscisic acid (ABA)‐induced stomatal closure was accompanied by increases in [Ca2+]cyt in epidermal strips. In addition to ABA, mechanical and low‐temperature signals directly affected stomatal behaviour, promoting rapid closure. Elevations of guard‐cell [Ca2+]cyt play a key role in the transduction of all three stimuli. However, there were striking differences in the magnitude and kinetics of the three responses. Studies using Ca2+ channel blockers and the Ca2+ chelator EGTA further suggested that mechanical and ABA signals primarily mobilize Ca2+ from intracellular store(s), whereas the influx of extracellular Ca2+ is a key component in the transduction of low‐temperature signals. These results illustrate an aspect of Ca2+ signalling whereby the specificity of the response is encoded by different spatial or kinetic Ca2+ elevations.
Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me‐JA but is dependent on MAPKK activity
Authors: Grant John J. , Yun Byung‐Wook , Loake Gary J.
Published: March 2003

Recognition of avirulent microbial pathogens activates an oxidative burst leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. A novel transgenic Arabidopsis line containing a gst1::luc transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of Pseudomonas syringae pv. tomato (Pst). Strong engagement of the oxidative burst was dependent on the presence of functional Pst hrpS and hrpA gene products. Experiments employing pharmacological agents suggested that at least two distinct sources, including an NADPH oxidase and a peroxidase‐type enzyme, contributed to the generation of redox cues. The analysis of gst1 and pal1 gene expression in nahG, coi1 and etr1 plants suggested that engagement of the oxidative burst and cognate redox signalling functioned independently of salicylic acid, methyl jasmonate and ethylene. In contrast, studies using a panel of protein kinase and phosphatase inhibitors and in‐gel kinase assays in these mutant backgrounds suggested that a 48 kDa mitogen‐activated protein kinase (MAPK) activity was required for the activation of gst1 and pal1 in response to redox cues. Thus the engagement of a bifurcating redox signalling pathway possessing a MAPK module may contribute both to the establishment of plant disease resistance, and to the development of cellular protectant mechanisms.
Genes determining pathogenicity to pea are clustered on a supernumerary chromosome in the fungal plant pathogen Nectria haematococca
Authors: Han Yinong , Liu Xiaoguang , Benny Ulla , Kistler H. Corby , VanEtten Hans D.
Published: March 2003

Three genes that contribute to the ability of the fungus Nectria haematococca to cause disease on pea plants have been identified. These pea pathogenicity (PEP) genes are within 25 kb of each other and are located on a supernumerary chromosome. Altogether, the PEP gene cluster contains six transcriptional units that are expressed during infection of pea tissue. The biochemical function of only one of the genes is known with certainty. This gene, PDA1, encodes a specific cytochrome P450 that confers resistance to pisatin, an antibiotic produced by pea plants. The three new PEP genes, in addition to PDA1, can independently increase the ability of the fungus to cause lesions on pea when added to an isolate lacking the supernumerary chromosome. Based on predicted amino acid sequences, functions for two of these three genes are hypothesized. The deduced amino acid sequence of another transcribed portion of the PEP cluster, as well as four other open reading frames in the cluster, have a high degree of similarity to known fungal transposases. Several of the features of the PEP cluster – a cluster of pathogenicity genes, the presence of transposable elements, and differences in codon usage and GC content from other portions of the genome – are shared by pathogenicity islands in pathogenic bacteria of plants and animals.
Members of the tomato LeEIL (EIN3‐like) gene family are functionally redundant and regulate ethylene responses throughout plant development
Authors: Tieman Denise M. , Ciardi Joseph A. , Taylor Mark G. , Klee Harry J.
Published: March 2003

The plant hormone ethylene regulates many aspects of growth, development and responses to the environment. The Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) protein is a nuclear‐localized component of the ethylene signal‐transduction pathway with DNA‐binding activity. Loss‐of‐function mutations in this protein result in ethylene insensitivity in Arabidopsis. To gain a better understanding of the ethylene signal‐transduction pathway in tomato, we have identified three homologs of the Arabidopsis EIN3 gene (LeEILs). Each of these genes complemented the ein3‐1 mutation in transgenic Arabidopsis, indicating that all are involved in ethylene signal transduction. Transgenic tomato plants with reduced expression of a single LeEIL gene did not exhibit significant changes in ethylene response; reduced expression of multiple tomato LeEIL genes was necessary to reduce ethylene sensitivity significantly. Reduced LeEIL expression affected all ethylene responses examined, including leaf epinasty, flower abscission, flower senescence and fruit ripening. Our results indicate that the LeEILs are functionally redundant and positive regulators of multiple ethylene responses throughout plant development.