bluuebunny/crossref_metadata_2025 huggingface dataset:
N‐ and C‐terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP‐glucose pyrophosphorylase
Authors: Laughlin Mary J. , Chantler Sue Ellen , Okita Thomas W.
Published: March 2003

ADP‐glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues. Unlike the allosteric regulatory dependent properties of the leaf enzyme, the enzymes from non‐photosynthetic tissues exhibit varying levels of sensitivity to allosteric regulation, a behavior which may be an inherent property of the enzyme or a product of post‐translational modification. As partial proteolysis of the holoenzyme may account for the wide variation of allosteric regulatory behavior exhibited by enzymes from non‐photosynthetic tissues, small N‐ and C‐terminal peptide deletions were made on either the potato large and small subunit and co‐expressed with the counterpart wild‐type subunit in Escherichia coli. Removal of the putative carboxy‐terminal allosteric binding region from either subunit type results in an abolishment of enzyme formation indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation. Removal of a small 10 amino acid peptide from the N‐terminus of the small subunit increased its resistance to the allosteric inhibitor Pi as well as its sensitivity to heat treatment. Likewise, removal of the corresponding peptide (17 residues) at the N‐terminus of the large subunit also increased its resistance towards Pi inhibition but, in addition, increased its sensitivity to 3‐PGA activation. Deletion of an additional 11 residues reversed these changes in allosteric properties but at the expense of a reduced catalytic turnover rate. Combined, these results indicate that the N‐ and C‐terminal regions are essential for the proper catalytic and allosteric regulatory properties of the potato ADP‐glucose pyrophosphorylase. The possible significance of these results on the observed insensitivity to effector molecules by ADP‐glucose pyrophosphorylases from other non‐photosynthetic tissues is discussed.
Identification and Ds‐tagged isolation of a new gene at the Cf‐4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5
Authors: Takken Frank L. W. , Schipper Danny , Nijkamp H. John J. , Hille Jacques
Published: March 2003

Leaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2‐week‐old seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf‐9 and Cf‐2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine‐rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf‐4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon‐tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4‐specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf‐4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf‐4A.
Physical strain‐mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles
Authors: Fischer Karin , Schopfer Peter
Published: March 2003

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed‐back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.
Expression studies of Nrt2:1Np, a putative high‐affinity nitrate transporter: evidence for its role in nitrate uptake
Authors: Krapp Anne , Fraisier Vincent , Scheible Wolf‐Rüdiger , Quesada Alberto , Gojon Alain , Stitt Mark , Caboche Michel , Daniel‐Vedele Françoise
Published: March 2003

Expression of the gene Nrt2Np, which encodes a putative high‐affinity nitrate transporter of Nicotiana plumbaginifolia was studied under variable physiological conditions. Nrt2Np is rapidly induced by very low nitrate concentrations and repressed by reduced nitrogen metabolites. Furthermore, Nrt2Np is expressed in coordination with other genes involved in nitrate assimilation (Nia, Nii). A deficiency in nitrate reductase activity, which is accompanied by high internal nitrate concentration and low levels of nitrogen metabolites, e.g. glutamine, leads to an overexpression of Nrt2Np, showing that high nitrate concentration per se does not repress Nrt2Np expression. By investigating plants with altered nitrate uptake properties, we showed a correlation between Nrt2 mRNA accumulation and 15N nitrate influx rates, providing the first evidence that the expression of Nrt2 correlates with the rate of nitrate uptake. In situ hybridization revealed a tissue‐specific expression pattern. Nrt2Np mRNA accumulation is localized throughout all layers of the root tip, being highest in epidermal and endodermal cells. However, in mature root tissue, Nrt2 expression was detected mainly in the lateral root primordia and in the epidermis.
Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH
Authors: Miller Susan S. , Driscoll Brian T. , Gregerson Robert G. , Gantt J. Stephen , Vance Carroll P.
Published: March 2003

Malate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate ; malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C
4
metabolism, pH balance, stomatal and pulvinal movement, respiration, β‐oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule‐enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an

E. coliMdh– mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.
Amplicons of maize zein genes are conserved within genic but expanded and constricted in intergenic regions
Authors: Llaca Victor , Messing Joachim
Published: March 2003

The 78 101 base pair long sequence of a cluster of 22‐kDa alpha zein genes in the maize inbred BSSS53 was determined. Each zein gene is contained within a repeat unit that varies in length. If such a repeat, or amplicon, is aligned along the entire sequence, a 10.5‐fold sequence amplification is delineated. Because of insertions and deletions in intergenic regions, many of the zein genes are spaced over different distances. Only three out of 10 zein‐related sequences have an intact open reading frame, indicating an unusual large number of genes unable to contribute to the accumulation of normal‐size 22‐kDa zein proteins. It is proposed that the seven remaining zein‐related sequences be considered gene reserves because of their potential to be restored by gene conversion. Intergenic insertions in the cluster range from 1098 to 14 896 base pairs. Although they are composed of transposable element sequences, they also contain additional open reading frames, two of them showing homology to rice cDNA sequences. The average amplicon is 4423 base pairs long, with the sequence surrounding each zein gene more than 90 % conserved. Coincidently, the size of the amplicon is equivalent to the average gene density (one gene within 4640 bp) in the Arabidopsis thaliana genome, one of the smallest in plants. Successive steps of amplification and insertion of DNA might explain to a certain degree how genome size variation has been generated in plants.
Gibberellin‐photoaffinity labelling of two polypeptides in plant plasma membranes
Authors: Lovegrove Alison , Barratt D. H. Paul , Beale Michael H. , Hooley Richard
Published: March 2003

Two polypeptides of

M

r 68 kDa and 18 kDa were gibberellin (GA)‐photoaffinity labelled

in vitro

in plasma membrane preparations from oat (

Avena sativa

L.) aleurone and from leaves and stems of wild‐type and GA‐sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2‐dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pI form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi‐dwarf GA‐sensitivity mutant of sweet pea (

Lathyrus odoratus

L.),

lb

, showed reduced photoaffinity labelling of both polypeptides compared with the wild type,

Lb

. In the GA‐insensitive

Arabidopsis thaliana

mutant,

gai

, the level of labelling was the same as in wild type,

GAI

. This is the first report of GA‐binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.
Specific accumulation of GFP in a non‐acidic vacuolar compartment via a C‐terminal propeptide‐mediated sorting pathway
Authors: Di Sansebastiano Gian‐Pietro , Paris Nadine , Marc‐Martin Sophie , Neuhaus Jean‐Marc
Published: March 2003

The green fluorescent protein (GFP) from

Aequorea victoria

can be detected in living plant cells after transient transformation of protoplasts. Expression of the GFP can be used to monitor protein trafficking in a mixed cell population and also to study the different function and importance of organelles in different cell types. We developed a vacuolar form of GFP that was obtained by replacing the C‐terminal endoplasmic reticulum (ER)‐retention motif of mGFP5‐ER by the vacuolar targeting peptide of tobacco chitinase A. The vacuolar GFP was transported and accumulated in the vacuole as expected. However, we found two patterns of GFP accumulation after prolonged incubation (18–24 h) depending on the cell type. Most chloroplast‐rich protoplasts had a fluorescent large central vacuole. In contrast, most chloroplast‐poor protoplasts accumulated the GFP in one smaller vacuole but not in the large central vacuole, which was visible under a light microscope in the same cell. This differential accumulation reflected the existence of two different vacuolar compartments as described recently by immunolocalization of several vacuolar markers. We were able to characterize the vacuolar compartment to which GFP is specifically targeted as non‐acidic, since it did not accumulate neutral red while acidic vacuoles did not accumulate GFP.
A cytokinesis‐defective mutant ofArabidopsis(cyt1) characterized by embryonic lethality, incomplete cell walls, and excessive callose accumulation
Authors: Nickle Todd C. , Meinke David W.
Published: March 2003

The genetic control of cell division in eukaryotes has been addressed in part through the analysis of cytokinesis‐defective mutants. Two allelic mutants of

Arabidopsis

(

cyt1–1

and

cyt1–2

) altered in cytokinesis and cell‐wall architecture during embryogenesis are described in this report. Mutant embryos appear slightly abnormal at the heart stage and then expand to form a somewhat disorganized mass of enlarged cells with occasional incomplete walls. In contrast to the

keule and knolle

mutants of

Arabidopsis and

the

cyd

mutant of pea, which also exhibit defects in cytokinesis during embryogenesis,

cyt1

embryos cannot be rescued in culture, are desiccation‐intolerant at maturity, and produce cell walls with excessive callose as revealed through staining with the aniline blue fluorochrome, Sirofluor. Some

cyt1

defects can be partially phenocopied by treatment with the herbicide dichlobenil, which is thought to interfere with cellulose biosynthesis. The distribution of unesterified pectins in

cyt1

cell walls is also disrupted as revealed through immunocytochemical localization of JIM 5 antibodies. These features indicate that

CYT1

plays an essential and unique role in plant growth and development and the establishment of normal cell‐wall architecture.
A 45 kDa protein isolated from the nodal walls ofChara corallinais localised to plasmodesmata
Authors: Blackman Leila M. , Gunning Brian E. S. , Overall Robyn L.
Published: March 2003

The cellular anatomy of the green alga,

Chara corallina,

was exploited to isolate putative plasmodesmataassociated proteins. In

C. corallina

, large internodal cells are symplastically connected via intervening nodal complexes of smaller cells which have plasmodesmata in their cell walls. Comparison of proteins extracted from walls with plasmodesmata (nodal complexes) with those from walls without plasmodesmata (external internodal walls) identified four putative plasmodesmata‐associated proteins. These putative plasmodesmata‐associated proteins were approximately 95, 45, 44 and 33 kDa. A monoclonal antibody (MAB45/22) was raised against the 45 kDa putative plasmodesmata‐associated protein (CPAP45). Using immunofluorescence, this antibody co‐localised with aniline blue induced fluorescence of callose in the source cell walls. MAB45/22 was localised to the plasmodesmata of

C. corallina

and, in particular, to the central cavity using immunogold cytochemistry. In contrast, a monoclonal antibody to callose specifically labelled the mouth of

C. corallina

plasmodesmata. MAB45/22 also labelled higher plant plasmodesmata.
Anions permeate and gate GCAC1, a voltage‐dependent guard cell anion channel
Authors: Dietrich Petra , Hedrich Rainer
Published: March 2003

GCAC1 is a strongly voltage‐dependent anion channel in the guard‐cell plasma membrane of

Vicia faba

. In patch–clamp experiments, we have investigated the permeation and gating properties of GCAC1 with respect to its anion dependence in the whole‐cell and excised‐patch configuration. The relative permeability followed the order SCN– > NO3– > Br– > Cl–, while the single‐channel conductances in symmetrical anionic solutions exhibited a nearly inverse sequence. The Cl– dependence of inward currents (Cl– release) is characterized by a maximum single‐channel conductance of 89 pS half‐saturating at 87 mM cytoplasmic chloride. In addition to this substrate saturation, anion release was also dependent on the external Cl– activity (

K

m = 16 mM). In the presence of SCN– and Cl–, the single‐channel conductance exhibited an anomalous mole‐fraction dependence, identifying GCAC1 as a multi‐ion single‐file pore. Using anions with increasing ionic size, a minimum pore diameter of 0.5 nm was assumed from their relative permeabilities. In line with an anion‐selective channel, a tenfold increase in the extracellular anion activity shifted the reversal potential by –59.8 mV. Simultaneously, the half‐activation potential shifted negatively by about 23 mV. A further analysis of the anion dependence revealed that extracellular rather than cytosolic anions affect the gating process of GCAC1. From anion substitution experiments, we conclude that anion concentration and species determines both permeation and gating of the plant anion channel GCAC1.
Molecular characterization of a plant FKBP12 that does not mediate action of FK506 and rapamycin
Authors: Xu Qiang , Liang Shuping , Kudla Jörg , Luan Sheng
Published: March 2003

Immuonosuppressive drugs FK506 and rapamycin block a number of signal transduction pathways in eukaryotic systems. The 12 kDa FK506 binding protein (FKBP12) mediates the action of both FK506 and rapamycin against their functional targets. In this report, we cloned, sequenced and characterized a gene encoding FKBP12 in

Vicia faba

(

Vf

FKBP12). While

Vf

FKBP12 is highly homologous to animal and yeast FKBP12, it does not mediate the action of FK506 and rapamycin. There are unique features in plant FKBP12 sequences that cause the variation in their function. One lies in the domain that is critical for interaction with calcineurin (CaN), the mammalian and yeast target of FKBP12‐FK506 complex. Protein–protein interaction assays revealed a low‐affinity and unstable

Vf

FKBP12‐FK506‐CaN ternary complex. In the genetic assay,

Vf

FKBP12 did not restore the sensitivity of yeast FKBP12 mutant to rapamycin or FK506, supporting that plant FKBP12‐ligand complexes are unable to block the function of the drug target. Also unique to plant FKBP12 proteins, a pair of cysteines is spatially adjacent to potentially form disulfide linkage. Treatment of

Vf

FKBP12 with reductant dithiothreitol (DTT) abolished the formation of

Vf

FKBP12‐FK506‐CaN ternary complex. Site‐directed mutagenesis to substitute one of the cysteines, Cys26, with Ser produced a similar effect as DTT treatment. These results indicate that an intramolecular disulfide bond is a novel structural feature required for the low–affinity interaction between plant FKBP12 and CaN. In conclusion, plant FKBP12 proteins have evolved structural changes that modify their protein‐protein interacting domains and cause loss of function against the drug targets.
Co‐ordinated expression of multiple enzymes in different subcellular compartments in plants
Authors: Dasgupta Santanu , Collins Glenn B. , Hunt Arthur G.
Published: March 2003

A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) NIa proteinase along with two other reporter genes (those encoding the

Escherichia coli

acetate kinase (ACK) and Tn9 chloramphenicol acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV NIa proteinase recognition sequence. In addition, in some instances, chloroplast targeting information (a transit peptide (TP) from a pea

rbcS

gene) was incorporated into the polyprotein. We found that the NIa proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP‐NIa‐ACK‐CAT or TP‐ACK‐CAT‐NIa failed to yield chloroplast‐localized ACK and CAT proteins, although the latter did give rise to a chloroplast‐localized ACK‐CAT polyprotein. These results indicate that the NIa proteinase acts

in cis

more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK‐NIa‐TP‐CAT and TP‐ACK‐NIa‐TP‐CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy for simultaneous engineering of multiple subcellular compartments in plants.
Mutations inArabidopsis thalianagenes involved in the tryptophan biosynthesis pathway affect root waving on tilted agar surfaces
Authors: Rutherford Robert , Gallois Patrick , Masson Patrick H.
Published: March 2003

Arabidopsis thaliana

roots grow in a wavy pattern upon a slanted surface. A novel mutation in the

anthranilate synthase α1

(

ASA1

) gene, named

trp5–2wvc1

, and mutations in the

tryptophan synthase α and β1

genes (

trp3–1

and

trp2–1

, respectively) confer a compressed root wave phenotype on tilted agar surfaces. When

trp5–2wvc1

seedlings are grown on media supplemented with anthranilate metabolites, their roots wave like wild type. Genetic and pharmacological experiments argue that the compressed root wave phenotypes of

trp5–2wvc1

,

trp2–1

and

trp3–1

seedlings are not due to reduced IAA biosynthetic potential, but rather to a deficiency in L‐tryptophan (L‐Trp), or in a L‐Trp derivative. Although the roots of 7‐day‐old seedlings possess higher concentrations of free L‐Trp than the shoot as a whole,

trp5–2wvc1

mutants show no detectable alteration in L‐Trp levels in either tissue type, suggesting that a very localized shortage of L‐Trp, or of a L‐Trp‐derived compound, is responsible for the observed phenotype.
Enhancement of the tolerance ofArabidopsisto high temperatures by genetic engineering of the synthesis of glycinebetaine
Authors: Alia  , Hayashi Hidenori , Sakamoto Atsushi , Murata Norio
Published: March 2003

Arabidopsis thaliana

was transformed with the

codA

gene for choline oxidase from

Arthrobacter globiformis

under control of the 35S RNA promoter of cauliflower mosaic virus. As a result, high levels of glycinebetaine accumulated in the seeds of transformed plants. Transformation with the

codA

gene significantly enhanced the tolerance to high temperatures during the imbibition and germination of seeds, as well as during growth of young seedlings. The extent of enhancement of the tolerance to high temperature was correlated with levels of choline oxidase expressed and of glycinebetine accumulated in the transformed plants. The induction of homologues of heat shock protein 70 at high temperature was less conspicuous in the transformed plants than in the wild‐type plants, suggesting that the transformation alleviated the high‐temperature stress.
In situmonitoring of DNA: the plant nuclear envelope allows passage of short DNA fragments
Authors: Gisel Andreas , Rothen Barbara , Iglesias Victor A. , Potrykus Ingo , Of Sautter Christ
Published: March 2003

We monitored the cellular localization of fluorescently labeled foreign DNA in living plant cells. After physical delivery of labeled DNA fragments to the cytoplasm, short fragments up to 1.5 kb in length were found equally distributed between the cytoplasm and nucleus after 60 min. In contrast, 2.5 kb DNA fragments did not appear inside the nucleus. Thus, foreign DNA can enter plant nuclei through the intact nuclear envelope, but the efficiency of this process declines with increasing size of fragment.
Identification of a dual‐specificity protein phosphatase that inactivates a MAP kinase fromArabidopsis
Authors: Gupta Rajeev , Huang Yafan , Kieber Joe , Luan Sheng
Published: March 2003

Mitogen‐activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an

Arabidopsis

gene encoding a dual‐specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (

Arabidopsis thaliana

dual‐specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual‐specificity protein phosphatases from all eukaryotes.
Organ identity genes and modified patterns of flower development inGerbera hybrida(Asteraceae)
Authors: Yu Deyue , Kotilainen Mika , Pöllänen Eija , Mehto Merja , Elomaa Paula , Helariutta Yrjö , Albert Victor A. , Teeri Teemu H.
Published: March 2003

We have used

Gerbera hybrida

(the cultivated ornamental, gerbera) to investigate the molecular basis of flower development in Asteraceae, a family of flowering plants that have heteromorphic flowers and specialized floral organs. Flowers of the same genotype may differ in a number of parameters, including sex expression, symmetry, sympetaly and pigmentation. In order to study the role of organ identity determination in these phenomena we isolated and functionally analysed six MADS box genes from gerbera; these were shown by phylogenetic analysis to be orthologous to well characterized regulatory genes described from

Arabidopsis

and

Antirrhinum

. Expression analysis suggests that the two gerbera

agamous

orthologues, the

globosa

orthologue and one of the

deficiens

orthologues may have functional equivalency to their counterparts, participating in the C and B functions, respectively. However, the function of a second

deficiens

orthologue appears unrelated to the B function, and that of a

squamosa

orthologue seems distinct from

squamosa

as well as from the A function. The induction patterns of gerbera MADS box genes conform spatiotemporally to the multi‐flowered, head‐like inflorescence typical of Asteraceae. Furthermore, gerbera plants transgenic for the newly isolated MADS box genes shed light onto the mechanistic basis for some floral characteristics that are typical for Asteraceae. We can conclude, therefore, that the pappus bristles are sepals highly modified for seed dispersal, and that organ abortion in the female marginal flowers is dependent upon organ identity and not organ position when position is homeotically altered.
Dissection of the ozone‐induced calcium signature
Authors: Clayton Helen , Knight Marc R. , Knight Heather , McAinsh Martin R. , Hetherington Alistair M.
Published: March 2003

The cellular responses of plants to numerous extracellular stimuli are mediated by transient elevations in the concentration of cytosolic free calcium ([Ca2+]cyt). We have addressed the question of how cells can use this apparently ubiquitous system to initiate so many specific and appropriate end responses. We show that the pollutant gas ozone elicits a biphasic Ca2+ response in intact Arabidopsis plants and a subsequent increase in expression of the gene encoding the antioxidant defence enzyme glutathione‐
S
‐transferase (GST). The second of the two [Ca2+]cyt peaks, but not the first, could be eliminated either by pre‐treatment of plants with lanthanum chloride, or by reducing the duration of ozone fumigation. Under these conditions, ozone‐induced
GST
expression was abolished. These data provide a functional dissection of the ozone Ca2+ signalling pathway and indicate that the second ozone‐induced [Ca2+]cyt peak provides the necessary information to direct expression of
GST
.
Rice ENOD40: isolation and expression analysis in rice and transgenic soybean root nodules
Authors: Kouchi Hiroshi , Takane Ken‐ichi , So Rollando B.. , Ladha Jagdish K.. , Reddy Pallavolu M..
Published: March 2003

The early nodulin ENOD40 has been proposed as playing a pivotal role in the organogenesis of legume root nodules. We have isolated the ENOD40 gene homologues ObENOD40 and OsENOD40 from the wild and cultivated rice genotypes Oryza brachyantha and Oryza sativa, respectively. Rice ENOD40s contain a sequence at the 5′ end (region I) for encoding an oligopeptide that is highly conserved in all legume ENOD40s. Furthermore, at the 3′ end (region II), the nucleotide sequence of rice ENOD40s exhibited a considerable homology to the corresponding region in legume ENOD40s. Among various organs of the rice plant, expression of OsENOD40 was detected only in stems. In situ hybridization studies revealed that, within the stem, transcription of OsENOD40 is confined to parenchyma cells surrounding the protoxylem during the early stages of development of lateral vascular bundles that conjoin an emerging leaf. Expression pattern of OsENOD40 promoter– GUS fusion in nodules developed on transgenic hairy roots of soybean was also found to be restricted to peripheral cells of nodule vascular bundles, thus evidencing that rice ENOD40 promoter activity is essentially the same as that of soybean ENOD40. Taken together, these results strongly suggest that OsENOD40 and legume ENOD40s share common, if not identical, functions in differentiation and/or function of vascular bundles.
The cell cycle dependent phosphorylation of histone H3 is correlated with the condensation of plant mitotic chromosomes
Authors: Houben Andreas , Wako Toshiyuki , Furushima‐Shimogawara Rieko , Presting Gernot , Künzel Gottfried , Schubert Ingo , Fukui Küchi
Published: March 2003

Mitotically dividing cells of Secale cereale, Hordeum vulgare and Vicia faba were studied by indirect immunofluorescence using an antibody recognizing phosphorylated histone H3. The study revealed the following features: (i) the H3 phosphorylation starts at prophase and ends at telophase in the pericentromeric chromatin, is associated with the condensation of mitotic chromosomes and is independent of the distribution of late replicating heterochromatin. (ii) Compared with other chromosome regions, the pericentromeric chromatin is histone H3 hyperphos‐ phorylated. (iii) The study of a semi‐dicentric chromo‐ some revealed that only at intact centromeres is the chromatin hyperphosphorylated at H3.
Signaling from the embryo conditions Vp1‐mediated repression of α‐amylase genes in the aleurone of developing maize seeds
Authors: Hoecker Ute , Vasil Indra K. , McCarty Donald R.
Published: March 2003

The VP1 transcription factor functions as both a repressor and an activator of gene expression in the developing aleurone. Vp1 activation of the anthocyanin pathway exhibits strict cell autonomy in aleurone. In contrast, Vp1‐mediated repression of hydrolase genes in aleurone cells during seed development is determined by a combination of cell autonomous and cell non‐autonomous signals. To analyze signaling between the embryo and aleurone during seed development, a T‐B3La chromosome translocation was used to create seed that has non‐concordant embryo and endosperm genotypes. We show that de‐repression of an Amy‐GUS reporter gene in developing vp1 mutant aleurone cells strongly depends on the presence of a viviparous embryo. Genetic ablation of the developing embryo in vp1 mutant and Vp1 seeds through the introduction of an early embryo mutation caused a similar enhancement of Amy‐GUS expression in the aleurone, suggesting that the quiescent embryo present in normal seed is a critical source of inhibitory signals. Analysis of an ABA deficient vp1 vp5 double mutant indicates that ABA synthesized in the embyro interacts additively with Vp1 to prevent precocious induction of α‐amylase genes in the aleurone of the developing seed. A lack of ABA synthesis, however, does not account for the strongly synergistic interaction between a viviparous vp1 embryo and mutant aleurone suggesting that a quiescent embyro is a source of other inhibitory signals.
Maize calreticulin localizes preferentially to plasmodesmata in root apex
Authors: Baluška František , Šamaj Jozef , Napier Richard , Volkmann Dieter
Published: March 2003

Using a polyclonal antibody raised against calreticulin purified and sequenced from maize, we performed an immunocytological study to characterize putative domain‐specific subcellular distributions of endoplasmic reticulum (ER)‐resident calreticulin in meristematic cells of maize root tip. At the light microscopy level, calreticulin was immunolocalized preferentially at cellular peripheries, in addition to nuclear envelopes and cytoplasmic structures. Punctate labelling at the longitudinal walls and continuous labelling at the transverse walls was characteristic. Immunogold electron microscopy revealed plasmodesmata as the most prominently labelled cell periphery structure. In order to further probe the ER‐domain‐specific distribution of maize calreticulin at plasmodesmata, root apices were exposed to mannitol‐induced osmotic stress. Plasmolysis was associated with prominent accumulations of calreticulin at callose‐enriched plasmodesmata and pit fields while the contracting protoplasts were depleted of calreticulin. In contrast, other ER‐resident proteins recognized by HDEL peptide and BiP antibodies localized exclusively to contracted protoplasts. This finding reveals that, in plasmolysed cells, calreticulin enriched ER domains at plasmodesmata and pit fields are depleted of other ER‐resident proteins containing the HDEL retention peptide.
Characterization of the unconventional myosin VIII in plant cells and its localization at the post‐cytokinetic cell wall
Authors: Reichelt Stefanie , Knight Alex E. , Hodge Tony P. , Baluska Frantisek , Samaj Jozef , Volkmann Dieter , Kendrick‐Jones John
Published: March 2003

Myosins are a large superfamily of motor proteins which, in association with actin, are involved in intra‐ cellular motile processes. In addition to the conventional myosins involved in muscle contractility, there is, in animal cells, a wide range of unconventional myosins implicated in membrane‐associated processes, such as vesicle transport and membrane dynamics. In plant cells, however, very little is known about myosins. We have raised an antibody to the recombinant tail region ofArabidopsis thalianamyosin 1 (a class VIII myosin) and used it in immunofluorescence and EM studies on root cells from cress and maize. The plant myosin VIII is found to be concentrated at newly formed cross walls at the stage in which the phragmoplast cytoskeleton has depolymerized and the new cell plate is beginning to mature. These walls are rich in plasmodesmata and we show that they are the regions where the longitudinal actin cables appear to attach. Myosin VIII appears to be localized in these plasmodesmata and we suggest that this protein is involved in maturation of the cell plate and the re‐establishment of cytoplasmic actin cables at sites of intercellular communication.
Rapid isolation of plant Ty1‐copia group retrotransposon LTR sequences for molecular marker studies
Authors: Pearce Stephen R. , Stuart‐Rogers Caroline , Knox Maggie R. , Kumar Amar , Ellis T. H. Noel , Flavell Andrew J.
Published: March 2003

The terminal sequences of long‐terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon‐based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR‐based method for the rapid isolation of LTR sequences of Ty1‐copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identified a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers specific for three pea LTRs have been used to reveal polymorphisms associated with the correspond‐ ing retrotransposons within the Pisum genus.
Analysis of mRNA stabilities during pollen development and in BY2 cells
Authors: Ylstra Bauke , McCormick Sheila
Published: March 2003

RNA stability is important in post‐transcriptional gene expression. However, its role during plant male gametophyte development has not been studied in detail, probably because of technical difficulties and because earlier work suggested that, in general, mRNAs expressed during late pollen development would be stable. We determined mRNA stabilities of 12 pollen‐expressed genes. We measured pollen mRNA decay during in planta development and in vitro maturation after transcriptional inhibition with ActinomycinD (ActD). Because five of these genes were also expressed in somatic cells we also measured RNA decay in BY2 cells after transcriptional inhibition with ActD. Ten of the mRNAs were highly stable during pollen development (t1/2 > 30 h), including all seven pollen‐specific mRNAs. However, two mRNAs showed a logarithmic decrease as pollen development proceeded, both in planta and in vitro. In fact, for one of these two, GUT8‐2b, the mRNA level decreased significantly within 3 h. Our results show that many pollen‐expressed mRNAs are indeed long‐lived but that at least some mRNAs undergo decay during pollen maturation. Thus, there is no overall cytoplasmic condition in pollen that stabilizes mRNAs and specific mRNA turnover mechanisms must exist. We detected reciprocal mRNA stabilities with two genes that are expressed in both BY2 cells and pollen. GUT15 mRNA, known to be unstable (t1/2 < 3 h) in BY2 cells, was highly stable in pollen. In contrast, GRP2 mRNA decayed in pollen but was highly stable in BY2 cells. Therefore, we conclude that mRNA stability can be tissue‐specific.
A rapid method for detection of plant gene transcripts from single epidermal, mesophyll and companion cells of intact leaves
Authors: Brandt Stephan , Kehr Julia , Walz Christina , Imlau Astrid , Willmitzer Lothar , Fisahn Joachim
Published: March 2003

A protocol for the detection of gene transcripts from single plant cells in living, undamaged plant tissue is described. Samples of leaf epidermal, mesophyll and companion cells were extracted by using glass microcapillaries and directly subjected to RT–PCR without any purification steps nor time consuming construction of cDNA libraries. The procedure is not restricted to surface cells or outer cell layers. Even cells from the central region of leaves could be harvested. For identification, companion cells were labelled by expression of the green fluorescent protein under control of a companion cell specific promoter. The described method is applicable to a wide range of plants and genes with different expression levels.
A heat shock transcription factor in pea is differentially controlled by heat and virus replication
Authors: Aranda Miguel A. , Escaler Margarita , Thomas Carole L. , Maule Andrew J.
Published: March 2003

Since some heat‐inducible genes [heat shock (hs) genes] can be induced by virus infection in pea [e.g. Hsp70;Aranda et al. 1996, Proc. Natl Acad. Sci. USA 93, 15289–15293], we have investigated the effect that heat and virus replication may have on the expression of a heat‐shock transcription factor gene (Hsf). We have characterized what appears to be the only member of the Hsf family in pea, PsHsfA. Similar to Hsp70, PsHsfA is heat‐inducible in vegetative and embryonic tissues, which is concordant with the presence of heat shock elements (HSEs) and stress responsive elements (STREs) on its promoter sequence. The expression of PsHsfA during virus replication was studied in pea cotyledons and leaves, and compared to that of Hsp70. In situ hybridization experiments showed that whereas Hsp70 is induced, there is no detectable increased accumulation of PsHsfA RNA associated with the replication of pea seed‐borne mosaic potyvirus (PSbMV). These experiments indicate that there is a selective control of virus‐induced hs gene expression, and suggest that different regulatory pathways control hs gene expression during heat shock and virus replication.
Light perception and the role of the xanthophyll cycle in blue‐light‐dependent chloroplast movements in Lemna trisulca L.
Authors: Tlałka Monika , Runquist Mårten , Fricker Mark
Published: March 2003

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo‐damage in strong blue light (SBL). The photo‐ receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue‐light photoreceptor in chloroplast movements was investigated. Time‐lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL‐dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis–trans isomerization of xanthophylls, thus photo‐isomerization may be a critical link in the signal transduction pathway.
Spatio‐temporal analysis of mitotic activity with a labile cyclin–GUS fusion protein
Authors: Colón‐Carmona Adán , You Ratha , Haimovitch‐Gal Tal , Doerner Peter
Published: March 2003

Plant growth responds rapidly to developmental and environmental signals, but the underlying changes in cell division activity are poorly understood. A labile cyclin‐GUS reporter was developed to facilitate the spatio‐temporal analysis of cell division patterns. The chimeric reporter protein is turned over every cell cycle and hence its histochemical activity accurately reports individual mitotic cells. Using Arabidopsis plants transformed with cyclin‐GUS, we visualized patterns of mitotic activity in wounded leaves which suggest a role for cell division in structural reinforcement.
Characterization of salicylic acid‐responsive, Arabidopsis Dof domain proteins: overexpression of OBP3 leads to growth defects
Authors: Kang Hong‐Gu , Singh Karam B.
Published: March 2003

Dof proteins are unique to plants and contain a single zinc finger DNA‐binding domain called the Dof domain. OBP1, an Arabidopsis Dof protein, was previously isolated through an interaction with OBF4, an ocs element‐binding protein. Two additional Dof proteins, called OBP2 and OBP3, were isolated through homologous screening. All three OBP proteins contain transcriptional activation domains in their C‐terminal region. While no significant differences were found between the OBP proteins in terms of their DNA and protein‐binding properties, tissue‐specific RNA expression patterns were found. The RNA expression levels of all three OBP proteins increased following treatment with auxin, salicylic acid (SA) or cycloheximide, although the level of induction varied among the different proteins and in the tissues tested. These results suggest that even though they have similar DNA binding and protein–protein interaction properties, the different OBP proteins are likely to have distinct functions in specific parts of the plant. There is a good correlation between the expression of the OBP proteins and the ocs element, a stress‐response element which is also induced by auxin, SA and cycloheximide. To begin to analyze the function of the OBP proteins, transgenic lines overexpressing OBP3 were generated. These plants showed a severe growth defect with altered root development and yellowish leaves. The severity of the growth defects correlated with OBP3 expression levels and in some cases led to death, suggesting that some Dof proteins play important roles in plant growth and development.
A bifunctional epimerase‐reductase acts downstream of the MUR1 gene product and completes the de novo synthesis of GDP‐L‐fucose in Arabidopsis
Authors: Bonin Christopher P. , Reiter Wolf‐Dieter
Published: March 2003

L‐Fucose is a monosaccharide found as a component of glycoproteins and cell wall polysaccharides in higher plants. The MUR1 gene of Arabidopsis thaliana encodes a GDP‐
d‐mannose 4,6‐dehydratase catalyzing the first step in the de novo synthesis of GDP‐
l‐fucose from GDP‐
d‐mannose (
Bonin et al. 1997
, Proc. Natl Acad. Sci. USA, 94, 2085–2090). Plant genes encoding the subsequent steps in
l‐fucose synthesis (3,5‐epimerization and 4‐reduction) have not been described previously. Based on sequence similarities to a bacterial gene involved in capsule synthesis we have cloned a gene from Arabidopsis, now designated GER1, which encodes a bifunctional 3,5‐epimerase‐4‐reductase in
l‐fucose synthesis. The combined action of the MUR1 and GER1 gene products converts GDP‐
d‐mannose to GDP‐
l‐fucose in vitro demonstrating that this entire nucleotide–sugar interconversion pathway could be reconstituted using plant genes expressed in Escherichia coli. In vitro assays indicated that the GER1 protein does not act as a GDP‐
d‐mannose 3,5‐epimerase, an enzymatic activity involved in the de novo synthesis of GDP‐
l‐galactose and
l‐ascorbic acid. Similarly,
l‐ascorbate levels in GER1 antisense plants were unchanged indicating that GDP‐
d‐mannose 3,5‐epimerase is encoded by a separate gene.