bluuebunny/crossref_metadata_2025 huggingface dataset:
Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics
Authors: Jaramillo Javier López , Chueca Ana , Sahrawy Mariam , Gorgé Julio López
Published: March 2003

Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the AvaII site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli‐expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose‐1,6‐bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP‐malate dehydrogenase. Computer‐assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the α4‐helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N‐side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pI of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C‐side of the regulatory cysteines.
Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens
Authors: Girke Thomas , Schmidt Hermann , Zähringer Ulrich , Reski Ralf , Heinz Ernst
Published: March 2003

The moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel D5‐ and D6‐ desaturases are required. To isolate one of these, PCR‐based cloning was used, and resulted in the isolation of a full‐length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N‐terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b5‐related region and a C‐terminal sequence with low similarity (27% identity) to acyl‐lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ‐linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a D6‐desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC–MS. Only the transformed yeast cells were able to introduce a further double bond into the D6‐position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.
Low‐oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C3 plant
Authors: Moons Ann , Valcke Roland , Van Marc , Montagu
Published: March 2003

Pyruvate orthophosphate dikinase (PPDK) is known for its role in C4 photosynthesis but has no established function in C3 plants. Abscisic acid, PEG and submergence were found to markedly induce a protein of about 97 kDa, identified by microsequencing as PPDK, in rice roots (C3). The rice genome was found to contain two ppdk loci, osppdka and osppdkb. We isolated osppdka cDNA, which encodes a cytosolic rice PPDK isoform of 96.6 kDa, that corresponded to the ABA‐induced protein from roots. Western blot analysis showed a PPDK induction in roots of rice seedlings during gradual drying, cold, high salt and mannitol treatment, indicating a water deficit response. PPDK was also induced in the roots and sheath of submerged rice seedlings, and in etiolated rice seedlings exposed to an oxygen‐free N2 atmosphere, which indicated a low‐oxygen stress response. None of the stress treatments induced PPDK protein accumulation in the lamina of green rice seedlings. Ppdk transcripts were found to accumulate in roots of submerged seedlings, concomitant with the induction of alcohol dehydrogenase 1. Low‐oxygen stress triggered an increase in PPDK activity in roots and etiolated rice seedlings, accompanied by increases in phosphoenolpyruvate carboxylase and malate dehydrogenase activities. The results indicate that cytosolic PPDK is involved in a metabolic response to water deficit and low‐oxygen stress in rice, an anoxia‐tolerant species.
Post‐transcriptional silencing of chalcone synthase in petunia using a geminivirus‐based episomal vector
Authors: Atkinson Ross G. , Gleave Lara R. F. Bieleski andrew P. , Janssen Bart‐Jan , Morris Bret A. M.
Published: March 2003

A vector that produces DNA replicons (multicopy plant episomes) was constructed using elements of the geminivirus tobacco yellow dwarf virus (TYDV). All plant cells contain an integrated chromosomal T‐DNA copy of the TYDV elements that provides a template for the production of episomes in the cell nucleus. Transgenic Petunia hybrida plants containing a CaMV 35S promoter‐driven chalcone synthase A (ChsA) gene cloned into the episomal vector produced flowers with a white‐spotted phenotype at high frequency. The spots were found at random locations in the petals and occurred in corresponding positions in both the upper and lower epidermis, indicating that the spots were non‐clonal. The spotted phenotype was somatically stable and was inherited through meiosis. In white‐spotted flower tissue, steady‐state ChsA mRNA levels were downregulated but rates of RNA transcription were unaffected, suggesting that the phenotype resulted from post‐transcriptional gene silencing of the endogenous and episomal ChsA genes. Increases in both the frequency and extent of gene silencing in flowers correlated with increases in episome copy number in mature flowers, flower buds and young and fully expanded leaves. Relatively small increases in episome copy number (less than threefold) appeared sufficient to trigger the gene‐silenced phenotype.
A starch‐accumulating mutant ofArabidopsis thalianadeficient in a chloroplastic starch‐hydrolysing enzyme
Authors: Zeeman Samuel C. , Northrop Fred , Smith Alison M. , Rees Tom ap
Published: March 2003

The aim of this work was to identify enzymes that participate in the degradation of transitory starch in

Arabidopsis

. A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild‐type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo‐amylase (α‐amylase), β‐amylase, starch phosphorylase, maltase, pullulanase or D‐enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch‐hydrolysing activity, putatively identified as an endo‐amylase and present in wild‐type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.
UV‐B‐induced photomorphogenesis in Arabidopsis thaliana
Authors: Kim Byung Chul , Tennessen Daniel J. , Last Robert L.
Published: March 2003

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet‐B (UV‐B, 290–320 nm) radiation. In wild‐type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV‐B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV‐B, photomorphogenic responses of UV‐B sensitive mutants were indistinguishable from wild‐type plants at the low dose. This allowed us to examine UV‐B‐induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild‐type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV‐B. These results suggest that either phyA or phyB is required for UV‐B‐induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV‐B, while det2 was strongly affected by UV‐B irradiation. This suggests that COP1 and DET1 work downstream of the UV‐B signaling pathway.
Transcriptional and post‐transcriptional processes regulate gene expression in oxygen‐deprived roots of maize
Authors: Fennoy Sheila L. , Nong Thoa , Bailey‐Serres Julia
Published: March 2003

To investigate the regulation of gene expression in maize (Zea maysL.) in response to oxygen deprivation (flooding), we quantitated run‐on transcription in isolated nuclei, steady‐state mRNA accumulation and mRNA loading with ribosomes for seven genes that encode proteins synthesized predominantly in oxygen‐deprived roots (anaerobic polypeptides) and seven genes that encode proteins synthesized in aerobic roots (aerobic polypeptides). Run‐on transcription of anaerobic polypeptide genes was induced in response to oxygen deprivation and run‐on transcription of the aerobic polypeptide genes continued during the stress treatment. The increased accumulation of mRNAs that encode anaerobic polypeptides occurred concomitant with the induction of gene transcription and efficient association of these mRNAs with large polysomes. The steady‐state accumulation of aerobic polypeptide mRNAs was within twofold of aerobic levels and in a number of cases fewer ribosomes were loaded per transcript. These results demonstrate that selected synthesis of anaerobic polypeptides involves transcriptional as well as significant post‐transcriptional regulation of gene expression. The repression of synthesis of many aerobic polypeptides occurs without interruption of gene transcription and is due to translational regulation and possibly the sequestration of mRNAs on mRNPs. Ribosome loading patterns indicated that this translational control occurs at both initiation and post‐initiation phases in a message‐specific manner.
Thetitanmutants ofArabidopsisare disrupted in mitosis and cell cycle control during seed development
Authors: Liu Chun‐ming , Meinke David W.
Published: March 2003

We describe in this report a novel class of mutants that should facilitate the identification of genes required for progression through the mitotic cell cycle during seed development in angiosperms. Three non‐allelic

titan

(

ttn

) mutants with related but distinct phenotypes are characterized. The common feature among these mutants is that endosperm nuclei become greatly enlarged and highly polyploid. The mutant embryo is composed of a few giant cells in

ttn1

, several small cells in

ttn2

, and produces a normal plant in

ttn3

. Condensed chromosomes arrested at prophase of mitosis are found in the free nuclear endosperm of

ttn1

and

ttn2

seeds. Large mitotic figures with excessive numbers of chromosomes are visible in

ttn3

endosperm. The

ttn1

mutation appears to disrupt cytoskeletal organization because endosperm nuclei fail to migrate to the chalazal end of the seed. How double fertilization leads to the establishment of distinct patterns of mitosis and cytokinesis in the embryo and endosperm is a central question in plant reproductive biology. Molecular isolation of

TITAN

genes should help to answer this question, as well as related issues concerning cell cycle regulation, chromosome movement and endosperm identity in angiosperms.
A rapid method to screen for cell‐wall mutants using discriminant analysis of Fourier transform infrared spectra
Authors: Chen Limei , Carpita Nicholas C. , Reiter Wolf‐Dieter , Wilson Reginald H. , Jeffries Charles , McCann Maureen C.
Published: March 2003

We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild‐type and a previously defined set of cell‐wall mutants of

Arabidopsis

. Exploratory principal component analysis indicated that mutants deficient in different cell‐wall sugars can be distinguished from each other. Discrimination of cell‐wall mutants from wild‐type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild‐type was significantly out of the range of a wild‐type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography‐mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification.
Altered plastidic ATP/ADP‐transporter activity influences potato (Solanum tuberosumL.) tuber morphology, yield and composition of tuber starch
Authors: Tjaden Joachim , Möhlmann Torsten , Kampfenkel Karlheinz , Neuhaus Gudrun Henrichs andH. Ekkehard
Published: March 2003

The metabolic function of the plastidic ATP/ADP transporter (AATP) in heterotrophic plastids was examined in transgenic potato plants that exhibited increased or decreased amounts of the protein. Altered mRNA levels correlated with activities of the plastidic ATP/ADP transporter. Potato tubers with decreased plastidic ATP/ADP transporter activities exhibited reduced starch contents whereas sense lines accumulated increased amounts of tuber starch. Starch from wild‐type tubers had an amylose content of 18.8%, starch from antisense plants contained 11.5–18.0% amylose, whereas starch from sense plants had levels of 22.7–27.0%. The differences in physiological parameters were accompanied with altered tuber morphology. These changes are discussed with respect to the stromal ATP supply during starch biosynthesis.
Fatty acids and lysophospholipids as potential second messengers in auxin action. Rapid activation of phospholipase A2 activity by auxin in suspension‐cultured parsley and soybean cells
Authors: Holk Roland U. Paul andré , Scherer Günther F. E.
Published: March 2003

Activation of cytosolic phospholipase A2 is a typical signal transduction reaction in animal cells and occurs in plants in response to auxin, elicitors and wounding. Exogenously added fluorescent bis‐BODIPY‐phosphatidylcholine was taken up and hydrolysed by a cellular phospholipase A2. Rapid activation of a phospholipase A2 by auxin in suspension‐cultured parsley (

Petrosilenum crispum

L.) and soybean (

Glycine max

L.) cells was shown by detection and quantification of fluorescent reaction products of phospholipase A2. Hormone‐triggered fluorescent fatty acid accumulation could be detected as early as 5 min. Auxins at 2 μM or higher concentrations activated phospholipase A2 and fluorescent fatty acids accumulated 1.1‐ to threefold after 90–120 min, depending on the auxin concentration. Fluorescent lysolipid did not accumulate up to 150 μM auxin. Known inhibitors of phospholipase A2 inhibited hormone‐dependent fluorescent fatty acid accumulation in cell cultures and, previously, elongation growth in etiolated zucchini hypocotyl segments (

Scherer & Arnold (1997

)

Planta

202, 462–469). When lipids were labeled by [14C]‐choline and [14C]‐ethanolamine the corresponding lysophospholipids could be quantified in cell extracts. Radioactive lysophospholipids accumulated as rapidly as 1–2 min after auxin treatment but only at concentrations well above 100 μM auxin. We hypothesize that phospholipase A2 activation is an early intermediate step between receptor and downstream responses. We hypothesize that fatty acid(s) could be second messengers in several auxin functions, especially in cell elongation. Lysophospholipids seem to be indicators or second messengers for stress caused by high auxin concentrations or may have different auxin‐linked functions and are also known to accumulate during elicitor action.
A rice homolog of Cdk7/MO15 phosphorylates both cyclin‐dependent protein kinases and the carboxy‐terminal domain of RNA polymerase II
Authors: Yamaguchi Masatoshi , Umeda Masaaki , Uchimiya Hirofumi
Published: March 2003

The activation of cyclin‐dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T‐loop by a CDK‐activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When

R2

was overexpressed in a CAK‐deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti‐R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy‐terminal domain (CTD) of the largest subunit of RNA polymerase II of

Arabidopsis

. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T‐loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK‐ and CTD‐kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant

Arabidopsis

.
Plant responses to genotoxic stress are linked to an ABA/salinity signaling pathway
Authors: Albinsky Doris , Masson Jean E. , Bogucki Augustyn , Afsar Karin , Vass Imre , Nagy Ferenc , Paszkowski Jerzy
Published: March 2003

Cells have developed a complex network of reactions to avoid or reduce the deleterious consequences of DNA damage. Responses to genotoxic stress include activation of distinct stress signaling pathways, delay of cell cycle progression and induction of DNA repair. In contrast to other organisms, it is not known which signal transduction pathways sense genotoxic stress in plants. Here we describe an

Arabidopsis

mutant (

uvs66

) that appears to be affected in the perception of signals triggered by genotoxic treatments. The mutant

uvs66

was identified as hypersensitive to UV‐C and to the DNA‐damaging chemicals methyl methane sulfonate (MMS) and mitomycin C (MMC), but seems to perform light dependent repair, nucleotide excision repair and homologous recombinational repair as efficiently as the wild type. Exposure of

uvs66

plants to various environmental stresses revealed a normal response, with the exception of elevated salinity and abscisic acid (ABA). The hypersensitivity to NaCl and ABA is correlated with aberrant regulation of transcripts that are regulated by ABA (

RAB18

), or are induced by DNA damaging treatments (

AtRAD51

). The properties of the mutant

uvs66

suggest an unexpected link between ABA and/or salt stress mediated signals and genotoxic stress responses, and provide an important connection between the physiological and genetic responses of plants to abiotic stress factors.
Identification of genes expressed during earlyArabidopsiscarpel development by mRNA differential display: characterisation ofATCEL2, a novel endo‐1,4‐β‐D‐glucanase gene
Authors: Yung Mei‐Hing , Schaffer Robert , Putterill Jo
Published: March 2003

The floral homeotic gene

AGAMOUS
(AG) imparts carpel identity on the fourth whorl of floral organs in wild‐typeArabidopsisflowers. Less is known about the genes that regulate carpel patterning and differentiation. To identify candidate regulators, we screened for genes expressed in developing carpels. SinceArabidopsiscarpels are difficult to isolate, we used whole inflorescence apices of two floral homeotic mutants (pi and pi ag) and mRNA differential display, to identify carpel transcripts. Two of the resulting cDNA clones were shown to be expressed predominantly in flowers. They encodedAGL11,a MADS box transcription factor known to be expressed in the carpel and ovules, and a novelArabidopsisendo‐1,4‐β‐D‐glucanase (ATCEL2).In situhybridisation localised theATCEL2transcript to the developing septum and ovule primordia of young carpels.
Silencing of an aleurone‐specific gene in transgenic rice is caused by a rearranged transgene
Authors: Morino Kazuko , Olsen Odd‐Arne , Shimamoto Ko
Published: March 2003

In rice, silencing of the aleurone‐specific

Ltp2‐gus

transgene, causing easily detectable staining patterns on the grain surface, offers a convenient tool to study quantitative aspects of gene silencing in monocots. In this paper we analyzed phenotypes, occurrence, inheritance and environmental effects on the silencing. We also report on the cloning of transgenes, determination of their structure and analysis of transcripts from the transgene loci. The results show that various patterns of silencing appeared in the R2 generation at which most of the transgenes became homozygous and that they were inherited for five generations. In addition, silencing independently occurred in three generations and reversion to full expression was also found. Cloning of transgenes from a silenced L3.3 line demonstrated that this line carried two transgene loci: one carried an intact

Ltp2‐gus

gene and the other carried a rearranged transgene in which part of the

gus

gene was in the antisense orientation. Analysis of

gus

transcripts indicated that partial antisense RNA derived from the rearranged transgene was present in silenced lines and was polyadenylated but that it was absent in non‐silenced lines. RNA analyses suggested that the

Ltp2‐gus

silencing in the aleurone layer was post‐transcriptional and that it may be caused by interaction of partial antisense

gus

transcripts with normal sense transcripts. Possible involvement of antisense transcripts in post‐transcriptional silencing is discussed.
A minor form of starch branching enzyme in potato ( Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A
Authors: Jobling Stephen A.. , Schwall Gerhard P.. , Westcott Roger J.. , Sidebottom Christopher M.. , Debet Martine , Gidley Michael J.. , Jeffcoat Roger , Safford Richard
Published: March 2003

Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C‐terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.
Tobacco retinoblastoma‐related protein phosphorylated by a distinct cyclin‐dependent kinase complex with Cdc2/cyclin D in vitro
Authors: Nakagami Hirofumi , Sekine Masami , Murakami Hiroko , Shinmyo Atsuhiko
Published: March 2003

The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals. The growth‐suppressive activity of Rb is regulated by phosphorylation with cyclin‐dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase. We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb‐related protein, and show that this gene is expressed in all the organs examined at the mRNA level. We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two‐hybrid and in vitro binding assays. In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes. Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb‐related protein in vitro. Using immunoprecipitation with the anti‐cyclin D antibody, cyclin D can be found in a complex with Cdc2 in suspension‐cultured tobacco BY‐2 cells. These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb‐related protein by forming an active complex with cyclin D in plants.
The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid
Authors: Ephritikhine Geneviève , Fellner Martin , Vannini Candida , Lapous Danielle , Barbier‐Brygoo Hélène
Published: March 2003

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin‐resistant mutants. From a selection screen specifically developed to isolate auxin‐hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (× 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark‐green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (× 40) in ABA‐sensitivity of root elongation and, to a lesser extent, of ABA‐induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co‐segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild‐type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild‐type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous application of brassinosteroid, illustrating the pivotal importance of the BR‐related SAX1 gene.
Distribution of fungal endophyte genotypes in doubly infected host grasses
Authors: Wille Patrick A. , Aeschbacher Roger A. , Boller Thomas
Published: March 2003

Fungal endophytes of the genus Epichloë live intercellulary in above ground plant parts of many pooid ‘grasses of the temperate regions. The associations are characterized by single genotype entities since a given host individual normally contains a single endophyte genotype. They can persist over the life span of the hosts. This study examines whether two fungal genotypes can co‐exist within a host plant, and how fungal genotypes are distributed within a host in the case of double infections. We selected four Epichloë bromicola strains that we identified as unique genotypes through RAPD‘ analysis. Young Bromus erectus plants, derived from callus cultures, were artificially inoculated with all possible double‐strain mixtures of these fungal genotypes. For identification of fungal genotypes in planta, we designed genotype‐specific primer pairs that flanked size‐variable loci in the fungal genomes. Diagnostic PCR revealed that only one fungal genotype was present in most inoculated plants, but double infections were also observed with a frequency of 8% of all infected plants. Subsequent analyses of individual tillers of doubly infected plants revealed that, in a given tiller, both the leaf‐blade and the leaf‐sheath were colonized with only one endophyte genotype. Tillers without any detectable fungal DNA were also observed. Thus, co‐existence of multiple endophyte genotypes within a single host plant is governed by mutual exclusion at the tiller level.
Ethylene‐regulated gene expression in tomato fruit: characterization of novel ethylene‐responsive and ripening‐related genes isolated by differential display
Authors: Zegzouti Hicham , Jones Brian , Frasse Pierre , Marty Christel , Maitre Béatrice , Latché Alain , Pech Jean‐Claude , Bouzayen Mondher
Published: March 2003

Differential display was used to isolate early ethylene‐regulated genes from late immature green tomato fruit in order to obtain a broader understanding of the molecular basis by which ethylene coordinates the ripening process. Nineteen novel ethylene‐responsive (ER) cDNA clones were isolated that fell into three classes: (i) ethylene up‐regulated (ii) ethylene down‐regulated, and (iii) transiently induced. Expression analysis revealed that ethylene‐dependent changes in mRNA accumulation occurred rapidly (15 min) for most of the ER clones. The predicted proteins encoded by the ER genes are putatively involved in processes as diverse as primary metabolism, hormone signalling and stress responses. Although a number of the isolated ER clones correspond to genes already documented in other species, their responsiveness to ethylene is described here for the first time. Among the ER clones sharing high homology with regulatory genes, ER43, a putative GTP‐binding protein, and ER50, a CTR1‐like clone, are potentially involved in signal transduction. ER24 is homologous to the multi‐protein bridging factor MBF1 involved in transcriptional activation, and finally, two clones are homologous to genes involved in post‐transcriptional regulation: ER49, a putative translational elongation factor, and ER68, a mRNA helicase‐like gene. Six ER clones correspond to as yet unidentified genes. The expression studies indicated that all the ER genes are ripening‐regulated, and, depending on the clone, show changes in transcript accumulation either at the breaker, turning, or red stage. Analysis of transcript accumulation in different organs indicated a strong bias towards expression in the fruit for many of the clones. The potential roles for some of the ER clones in propagating the ethylene response and regulating fruit ripening are discussed.
Ecdysone agonist inducible transcription in transgenic tobacco plants
Authors: Martinez Alberto , Sparks Caroline , Hart Cliff A. , Thompson John , Jepson Ian
Published: March 2003

A novel chemical‐induced gene regulatory system for plants consisting of two molecular components is described. The first, or regulatory, cassette comprises a chimeric receptor composed of the hinge and ligand binding domains of the Heliothis virescens ecdysone receptor and the transactivation domain of the Herpes simplex VP16 protein fused to the DNA binding domain and transactivation of a mammalian glucocorticoid receptor. The second component, a reporter cassette, contains six copies of the glucocorticoid response element (GRE) fused to the minimal 35SCaMV promoter and β‐glucuronidase. The system uses a commercially available non‐steroidal ecdysone agonist, RH5992 (tebufenozide), as an inducer. Activation of gene expression is shown in both tobacco transient protoplasts and transgenic plants. The response is ligand dependent and is modulated by the change in minimal promoter context. The system is capable of inducing transgene activity up to 420‐fold corresponding to 150% of the activity observed with positive controls (35SCaMV:GUS).
The pre‐vacuolar t‐SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP
Authors: Bassham Diane C. , Raikhel Natasha V.
Published: March 2003

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans‐Golgi network are thought to fuse with a pre‐vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t‐SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to α‐SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with α‐SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre‐vacuolar compartment.
Downy mildew (Peronospora parasitica) resistance genes in Arabidopsis vary in functional requirements for NDR1, EDS1, NPR1 and salicylic acid accumulation
Authors: McDowell John M. , Cuzick Alayne , Can Canan , Beynon Jim , Dangl Jeffery L. , Holub Eric B.
Published: March 2003

To better understand the genetic requirements for R gene‐dependent defense activation in Arabidopsis, we tested the effect of several defense response mutants on resistance specified by eight RPP genes (for resistance to Peronospora parasitica) expressed in the Col‐0 background. In most cases, resistance was not suppressed by a mutation in the SAR regulatory gene NPR1 or by expression of the NahG transgene. Thus, salicylic acid accumulation and NPR1 function are not necessary for resistance mediated by these RPP genes. In addition, resistance conferred by two of these genes, RPP7 and RPP8, was not significantly suppressed by mutations in either EDS1 or NDR1. RPP7 resistance was also not compromised by mutations in EIN2, JAR1 or COI1 which affect ethylene or jasmonic acid signaling. Double mutants were therefore tested. RPP7 and RPP8 were weakly suppressed in an eds1‐2/ndr1‐1 background, suggesting that these RPP genes operate additively through EDS1, NDR1 and as‐yet‐undefined signaling components. RPP7 was not compromised in coi1/npr1 or coi1/NahG backgrounds. These observations suggest that RPP7 initiates resistance through a novel signaling pathway that functions independently of salicylic acid accumulation or jasmonic acid response components.
Conversion of compatible plant–pathogen interactions into incompatible interactions by expression of the Pseudomonas syringae pv. syringae 61 hrmA gene in transgenic tobacco plants
Authors: Shen Songhai , Li Qingshun , He Sheng‐Yang , Barker Kenneth R. , Li Debao , Hunt Arthur G.
Published: March 2003

The hrmA gene from Pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium P. syringae pv. tabaci in all examined tobacco cultivars. We expressed this gene in tobacco plants under the control of the tobacco Δ0.3 TobRB7 promoter, which is induced upon nematode infection in tobacco roots (
Opperman et al. 1994
, Science, 263, 221–223). A basal level of hrmA expression in leaves of transgenic plants activated the expression of pathogenesis‐related genes, and the transgenic plants exhibited high levels of resistance to multiple pathogens: tobacco vein mottling virus, tobacco etch virus, black shank fungus Phytophthora parasitica, and wild fire bacterium Pseudomonas syringae pv. tabaci. However, the hrmA transgenic plants were not significantly more resistant to root‐knot nematodes. Our results suggest a potential use of controlled low‐level expression of bacterial avr genes, such as hrmA, in plants to generate broad‐spectrum resistance to bacterial, fungal and viral pathogens.
Cell cycle function of a Medicago sativa A2‐type cyclin interacting with a PSTAIRE‐type cyclin‐dependent kinase and a retinoblastoma protein
Authors: Roudier François , Fedorova Elena , Györgyey Janos , Feher Attila , Brown Spencer , Kondorosi Adam , Kondorosi Eva
Published: March 2003

In plants multiple A‐type cyclins with distinct expression patterns have been isolated and classified into three subgroups (A1–A3), while in animal somatic cells a single type of cyclin A is required for cell‐cycle regulation from the S to M phases. We studied the function of an A2‐type cyclin from Medicago sativa (Medsa;cycA2) which, in contrast to animal and most plant A‐type cyclins, was expressed in all phases of the cell cycle. Using synchronized alfalfa cell cultures and anti‐Medsa;CycA2 polyclonal antibodies, we showed that while the mRNA level increased steadily from the late G1 to the G2‐M phase, the protein level after a rapid increase in S‐phase reached a plateau during the G2 phase. In the yeast two‐hybrid system, the Medsa;CycA2 protein interacted with the PSTAIRE‐motif‐containing cyclin‐dependent kinase Cdc2MsA and with the maize retinoblastoma protein. Unexpectedly, the CycA2‐associated kinase activity was biphasic: a first activity peak occurred in the S phase while the major one occurred during the G2/M transition, with no apparent dependence upon the actual levels of the Medsa;CycA2 and Cdc2MsA proteins. Immunohistological localization of the cyclin A2 protein by immunofluorescence and immunogold labelling revealed the presence of Medsa;CycA2 in the nucleus of the interphase and prophase cells, while it was undetectable thereafter during mitosis. Together these data suggest that Medsa;CycA2 plays a role both in the S phase and at the G2/M transition.
Development of clover yellow vein virus as an efficient, stable gene‐expression system for legume species
Authors: Masuta Chikara , Yamana Toshikazu , Tacahashi Yoko , Uyeda Ichiro , Sato Masanao , Ueda Shigenori , Matsumura Takeshi
Published: March 2003

A highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green fluorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC‐Pro on pClYVV to create three recombinant plasmids: pClYVV‐GFP, pClYVV‐GFP‐GS, and pClYVV‐GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green fluorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was confirmed by RT–PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and flowered early. As the GS gene, one of the nodulin genes for nitrogen fixation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.
A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid‐potentiated oxidative burst
Authors: Mur Luis A. J. , Brown Ian R. , Darby Robert M. , Bestwick Charles S. , Bi Yong‐Mei , Mansfield John W. , Draper John
Published: March 2003

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild‐type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S‐SH‐L). Challenge of 35S‐SH‐L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild‐type tobacco, both the onset of plant cell death and the induction of an active oxygen species‐responsive promoter (AoPR1‐GUS) were delayed following challenge of 35S‐SH‐L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild‐type tobacco, whereas in 35S‐SH‐L plants the onset of H2O2 accumulation was delayed by 2–3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1–2 h following inoculation in wild‐type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence‐gene induction, factors that together may determine the outcome of plant–pathogen interactions.
The spatial expression patterns of a phosphate transporter (MtPT1) from Medicago truncatula indicate a role in phosphate transport at the root/soil interface
Authors: Chiou Tzyy‐Jen , Liu Henry , Harrison Maria J.
Published: March 2003

The movement of phosphate from the soil into plant root cells is the first of many crucial transport events required to supply phosphorous (P) to cells throughout the plant. In addition to the ability to acquire phosphate from the soil, the majority of the vascular plants are able to form arbuscular mycorrhizal associations in which phosphate may be delivered to the cortex via a fungus. Previously, we cloned two phosphate transporter genes, MtPT1 and MtPT2 from Medicago truncatula roots. Complementation of a yeast phosphate transport mutant revealed that MtPT1 is a functional phosphate transporter and Northern analyses revealed that MtPT1 is expressed exclusively in roots (Liu et al., 1998, Mol. Plant‐Microbe Interact. 11, 14–22). Utilising an antibody specific for MtPT1, we have analysed the accumulation and spatial expression patterns of the MtPT1 transporter. MtPT1 transcript and protein levels show close correlation and increase dramatically in the roots in response to phosphate starvation. MtPT1 protein levels decrease in roots during development of a symbiosis with arbuscular mycorrhizal (AM) fungi, indicating that this transporter is not involved in symbiotic phosphate transport. Membrane fractionation and analysis of a MtPT1/GFP fusion protein revealed that MtPT1 is located in the plasma membrane, while in situ hybridisation and immunolocalisation demonstrate the presence of MtPT1 transcripts and protein in the epidermal cells and root hairs of M. truncatula roots. MtPT1 shows expression patterns consistent with a role specifically in the acquisition of phosphate from the soil and is distinct from the other phosphate transporter of this class described to date.
Different modes of de novo telomere formation by plant telomerases
Authors: Fitzgerald Matthew S. , Shakirov Eugene V. , Hood Elizabeth E. , McKnight Thomas D. , Shippen Dorothy E.
Published: March 2003

The telomerase reverse transcriptase can recognize broken chromosome ends and add new telomeres de novo in a reaction termed ‘chromosome healing’. Here we investigate new telomere formation in vitro by telomerases from a variety of flowering plant species. Comparing the electrophoretic mobilities and nucleotide sequences of the products, we uncovered three different modes of new telomere formation. The soybean telomerase, designated a Class I enzyme, only elongated DNA primers ending in telomeric nucleotides. Arabidopsis and maize telomerases, designated Class II enzymes, efficiently extended completely non‐telomeric sequences by positioning the 3′ terminus at a preferred site on the RNA template. Silene latifolia and sorghum telomerases constituted class III enzymes that elongated non‐telomeric DNA primers by annealing them at alternative sites on the RNA template. For all enzymes, errors were prevalent during synthesis of the first two repeats, likely reflecting lateral instability of the primer 3′ terminus on the template during the initial rounds of elongation. Class III telomerases, however, were five‐ to 13‐fold more error prone than class II, generating more mistakes in distal repeats added to the primers. This remarkable variability in enzyme–DNA interactions among plant telomerases does not reflect phylogenetic relationships, and therefore implies that the telomerase active site can evolve rapidly.
Dissection of RNA‐mediated ryegrass mosaic virus resistance in fertile transgenic perennial ryegrass (Lolium perenne L.)
Authors: Xu Jianping , Schubert Jörg , Altpeter Fredy
Published: March 2003

Ryegrass mosaic virus (RgMV) frequently reduces yield and persistence of perennial ryegrass. We introduced an untranslatable RgMV coat protein (RgMV‐CP) gene into perennial ryegrass using particle bombardment to explore the potential of RNA‐mediated virus resistance. Nine months after inoculation with the RgMV‐Bulgaria strain, the most resistant transgenic line showed no immuno‐detectable RgMV‐CP in all replications of the primary transformant and a sexual progeny. A significant influence of the transgenic line, virus strain and the period after inoculation on the RgMV level was observed. Typical examples of recovery resistance from initially susceptible plants were not identified. Molecular analysis revealed RgMV resistance operates by targeted RNA degradation resulting in post‐transcriptional transgene silencing (PTGS) along with inhibition of virus RNA replication and was not associated with a threshold of transgene expression. Suppression of PTGS by RgMV differed significantly in two progenies with identical RgMV‐CP transgene integration and a similar RgMV‐CP transgene methylation pattern, suggesting the presence of quantitative components controlling the stability of PTGS in perennial ryegrass. This study extends RNA‐mediated virus resistance, post transcriptional gene silencing and crop improvement by genetic engineering to an agronomically important perennial monocot.