bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical and molecular characterization of a novel starch synthase from potato tubers
Authors: Edwards Anne , Marshall Jacqueline , Sidebottom Christopher , Visser Richard G.F. , Smith Alison M. , Martin Cathie 
Published: March 2003

An isoform of starch synthase from potato tubers which is present both in the stroma of the plastid and tightly bound to starch granules has been identified biochemically and a cDNA has been isolated. The protein encoded by the cDNA is 79.9 kDa and has a putative transit peptide and a distinct N‐terminal domain which is predicted to be highly flexible. It is similar in both amino acid sequence and predicted structure to the granule‐bound starch synthase II (GBSSII) of pea embryos. When expressed in Escherichia coli, the mature protein has starch synthase activity.The importance of the isoform has been assessed by biochemical measurements and antisense transformation experiments in which the amount of the isoform in the tuber is severely and specifically reduced. Both approaches indicate that the isoform contributes a maximum of 15% of the total starch synthase activity of the tuber. It is suggested that this isoform and the GBSSII of pea embryos represent a widely distributed class of isoforms of starch synthase. The contribution to total starch synthase activity of members of this class probably varies considerably from one type of storage organ to another.
Temporal and spatial expression patterns of PHYA and PHYB genes in Arabidopsis
Authors: Somers David E. , Quail Peter H. 
Published: March 2003

SummaryPhytochromes A and B have discrete photosensory functions in Arabidopsis. To determine whether differential temporal or spatial expression patterns of the PHYA and PHYB genes contribute to this phenomenon the expression of PHYA—GUS and PHYB—GUS reporter genes has been examined in transgenic Arabidopsis. Histochemical and quantitative biochemical analyses indicate that both transgenes are expressed extensively throughout the plant, including roots, shoots and flowers, during the entire life cycle, but with strong differences between the two in expression level and photoregulation, and more limited differences in spatial expression patterns. The data indicate that regulation is at the transcriptional level. In dry seed, PHYB—GUS is expressed throughout the embryo at three‐fold higher levels than PHYA—GUS, which is confined primarily to the embryonic root tip. By contrast, PHYA promoter activity, despite strong negative regulation in shoots by light, is consistently higher than PHYB (two‐ to 20‐fold) in both the light and dark in most tissues during all subsequent developmental phases, from seedling to mature adult. At the tissue level, most cells appear to express both transgenes at some level at all stages examined, with highest apparent activity in vascular tissue and root tips. With the notable exception of pollen, where high PHYB—GUS but not PHYA—GUS expression occurs, few major differences are observed in the quantitative spatial distribution pattern between the two transgenes. The strongly similar spatial and temporal expression patterns of PHYA—GUS and PHYB—GUS transgenes suggest that the differential photosensory activity of these two phytochromes occurs largely through differences in their (i) intrinsic biochemical activities, (ii) relative abundances, and/or (iii) independent and separate reaction partners, rather than through discrete, developmentally controlled expression patterns.
A general method to isolate genes tagged by a high copy number transposable element
Authors: Souer Erik , Quattrocchio Francesca , De Vetten Nick , Mol Joseph , Koes Ronald 
Published: March 2003

SummaryThe Petunia hybrida line W138 contains more than 200 copies of the transposable element dTph1. In W138 progeny these elements give rise to new unstable mutations at high frequency. With the aim of isolating these mutated genes a method was developed to isolate dTph1 flanking sequences unique for mutant plants. This method is based on differential screening of cloned inverse polymerase chain reaction (IPCR) products originating from the mutated plant. It directly yields a probe for the mutated gene which can be used to screen pre‐existing cDNA and genomic libraries. This method may be generally applicable to isolate genes tagged by other high copy number transposable elements, like Mutator (Mu) or Dissociation (Ds) in Zea mays.
The pef mutants of Arabidopsis thaliana define lesions early in the phytochrome signaling pathway
Authors: Ahmad Margaret , Cashmore Anthony R. 
Published: March 2003

SummaryIn a screen for early‐flowering mutants, a number of mutants that were early flowering under both short and long days were isolated. One such mutant, pef1, was selectively insensitive to both red and far‐red light in the inhibition of hypocotyl elongation response; a classic phytochrome phenotype mediated by both PHYA and PHYB. The pef1 mutant seedlings could not be phenotypically rescued by biliverdin, a precursor of the phytochrome chromophore, nor did they fail to complement any previously identified elongated hypocotyl (hy) mutants. Difference spectra and Western blot analysis showed normal concentrations of PHYA photoreceptor apoprotein, which appeared photochemically active. It was concluded that the pef1 mutant is defective in both PHYA‐ and PHYB‐ mediated signaling pathways, and may represent a lesion in an early step of the phytochrome signal transduction pathway. Additional pef mutants deficient specifically in PHYB‐mediated responses were also identified by this screen.
Cell‐to‐cell trafficking of cucumber mosaic virus movement protein:green fluorescent protein fusion produced by biolistic gene bombardment in tobacco
Authors: Itaya Asuka , Hickman Heather , Bao Yiming , Nelson Richard , Ding Biao 
Published: March 2003

SummaryPrevious micro‐injection studies showed that some recombinant viral movement proteins and plant proteins produced in and purified from Escherichia coli could traffic from cell to cell. However, the relevance of these findings obtained by micro‐injecting proteins produced in E. coli to the real functions of these proteins when produced in planta has been questioned. In this study, specific gene constructs were delivered by biolistic bombardment into tobacco (Nicotiana tabacum var Samsun) leaf epidermis for in planta production of the green fluorescent protein (GFP) and various fusions between the cucumber mosaic virus 3a movement protein (3a MP) and GFP. Free GFP remained in cells producing it. In contrast, 3a MP:GFP fusion protein moved from approximately half of the cells producing it into neighboring cells. The movement also occurred at 4°C. A mutant 3a MP:GFP was incapable of cell‐to‐cell movement in all cases. A 3a MP:GUS fusion protein produced in this manner also moved from cell to cell. Our data provide direct evidence that specific viral proteins produced in planta can be transported between cells. Furthermore, our data suggest that the CMV 3a MP contains a signal for transport. Our approach is simple and efficient and has many potential applications in studying plasmodesma‐mediated macromolecular transport.
An ABC‐transporter of Arabidopsis thaliana has both glutathione‐conjugate and chlorophyll catabolite transport activity
Authors: Tommasini Roberto , Vogt Esther , Fromenteau Myriam , Hörtensteiner Stefan , Matile Philippe , Amrhein Nikolaus , Martinoia Enrico 
Published: March 2003

SummaryAn ABC‐transporter of Arabidopsis thaliana exhibiting high sequence similarity to the human (MRP) and yeast (YCF) glutathione‐conjugate transporters has been analysed and used to complement a cadmium‐sensitive yeast mutant (DTY68) that also lacks glutathione‐conjugate transport activity. Comparison of the hydrophobicity plots of this A. thaliana MRP‐like protein with MRP and YCF demonstrates that the transmembrane domains are conserved, even at the N‐terminus where sequence identity is low. Cadmium resistance is partially restored in the complemented ycf mutant, and glutathione‐conjugate transport activity can be observed as well. The kinetic properties of the A. thaliana MRP‐like protein (AtMRP3) are very similar to those previously described for the vacuolar glutathione‐conjugate transporter of barley and mung bean. Furthermore, a hitherto undescribed ATP‐dependent transport activity could be correlated with the gene product, i.e. vesicles isolated from the complemented yeast, but not from DTY68 or the wild type, take up the chlorophyll catabolite Bn‐NCC‐. The results indicate that the product of the MRP‐like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.
Antisense expression of a sucrose non‐fermenting‐1‐related protein kinase sequence in potato results in decreased expression of sucrose synthase in tubers and loss of sucrose‐inducibility of sucrose synthase transcripts in leaves
Authors: Purcell Patrick C. , Smith Alison M. , Halford Nigel G. 
Published: March 2003

SummaryThis report describes the analysis of transgenic potato plants stably transformed with chimeric genes comprising either a patatin (tuber‐specific) or a ST‐LS1 (leaf‐ and stem‐specific) gene promoter and a potato sucrose non‐fermenting‐1 (SNF1)‐related protein kinase gene (PKIN1) sequence in the antisense orientation. Presence of the transgene was confirmed by Southern analysis. The transformants were screened using a peptide kinase activity assay for SNF1‐related activity and/or Northern blot analysis, and two independent transgenic lines from each transformation, PAT 1.3, PAT 1.10, LS 1.4 and LS 1.11, were selected. Antisense PKIN1 transcripts were detectable in all four of the selected lines, and measurements made using the specific peptide phosphorylation assay showed that SNF1‐related protein kinase activity had decreased in both PAT 1.3 and PAT 1.10 compared with controls. SNF1 regulates the expression of many genes encoding enzymes of carbohydrate metabolism in yeast. In order to investigate an analogous role for PKIN1, the activities of fructokinase, glucokinase, neutral and acid invertase and sucrose synthase in the tubers of PAT 1.3 and PAT 1.10 were compared with those in wild‐type controls. Sucrose synthase activity was decreased to 36% of wild‐type activity in tubers of PAT 1.10, and sucrose synthase transcript levels were decreased in tubers of both PAT 1.3 and PAT 1.10. Activities of the other enzymes were unaffected. Leaves of lines LS 1.4, LS 1.11 and controls were then excised and cultured on a medium containing 250 mM sucrose. This treatment induced sucrose synthase gene expression in the control leaves but not those of the transgenic lines. This finding is the first demonstration of a role for SNF1‐related protein kinases in the regulation of carbohydrate metabolism of higher plants.
Development of an AFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Ler/Cvi recombinant inbred line population
Authors: Alonso‐Blanco Carlos , Peeters Anton J. M. , Koornneef Maarten , Lister Clare , Dean Caroline , van den Bosch Natasja , Pot Jerina , Kuiper Martin T. R. 
Published: March 2003

SummaryAn amplified fragment polymorphism (AFLP) based linkage map has been generated for a new Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred line (RIL) population. A total of 321 molecular PCR based markers and the erecta mutation were mapped. AFLP markers were also analysed in the Landsberg erecta/Columbia (Ler/Col) RIL population (
Lister & Dean 1993) and 395 AFLP markers have been integrated into the previous Arabidopsis molecular map of 122 RFLPs, CAPSs and SSLPs. This enabled the evaluation of the efficiency and robustness of AFLP technology for linkage analyses in Arabidopsis. AFLP markers were found throughout the linkage map. The two RIL maps could be integrated through 49 common markers which all mapped at similar positions. Comparison of both maps led to the conclusion that segregating bands from a common parent can be compared between different populations, and that AFLP bands of similar molecular size, amplified with the same primer combination in two different ecotypes, are likely to correspond to the same locus. AFLPs were found clustering around the centromeric regions, and the authors have established the map position of the centromere of chromosome 3 by a quantitative analysis of AFLP bands using trisomic plants. AFLP markers were also used to estimate the polymorphism rate among the three ecotypes. The larger polymorphism rate found between Ler and Cvi compared to Ler and Col will mean that the new RIL population will provide a useful material to map DNA polymorphisms and quantitative trait loci.
TowardsArabidopsisgenome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays
Authors: Ruan Yijun , Gilmore James , Conner Timothy 
Published: March 2003

Summary cDNA microarrays containing 1443

Arabidopsis thaliana

genes were analyzed for expression profiles in major organs of

Arabidopsis

plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of

Arabidopsis

genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.
Genetic interactions of the Arabidopsis flowering time geneFCA,with genes regulating floral initiation
Authors: Page Tania , Macknight Richard , Yang Chang‐Hsien , Dean Caroline 
Published: March 2003

Summary The genes controlling the timing of the transition from vegetative to reproductive growth are likely candidates for regulators of genes initiating floral development. We have investigated the interaction of one particular gene controlling flowering time,

FCA

, with the meristem identity‐genes

TERMINAL FLOWER 1

(

TFL1

),

APETALA 1

(

AP1

) and

LEAFY

(

LFY

) and the floral repression gene

EMBRYONIC FLOWER 1

(

EMF1

). Double mutant combinations were generated and the phenotypes characterized. The influence of strong and intermediate

fca

mutant alleles on the phenotype conferred by a

35S‐LFY

transgene was also analysed. The results support a model where

FCA

function promotes flowering in multiple pathways, one leading to activation of

LFY

and

AP1

, and another acting in parallel with

LFY

and

AP1

. Only the latter pathway is predicted to be non‐functional in the intermediate

fca‐4

allele. The results are also consistent with

AP1

and

TFL1

negatively regulating

FCA

function. Combination of Columbia

fca

and

emf1

mutant alleles confirmed that

FCA

is required for the early flowering of

emf1. EMF1

and

FCA

are therefore likely to operate in different floral pathways.
Cell‐autonomous complementation ofmloresistance using a biolistic transient expression system
Authors: Shirasu Ken , Nielsen Kirsten , Piffanelli Pietro , Oliver Richard , Schulze‐Lefert Paul 
Published: March 2003

Summary The barley gene

Mlo

encodes a prototype of a novel class of plant proteins. In

mlo

mutants, absence of the 60 kDa wild‐type Mlo protein results in broad‐spectrum resistance to the powdery mildew fungus,

Erysiphe graminis

f. sp.

hordei

. To directly assess its function,

Mlo

was transiently expressed with a marker gene encoding a modified green fluorescent protein (GFP) in leaf epidermal cells of

mlo

resistant barley lines. Fungal inoculation of epidermal cells transfected with wild‐type

Mlo

led to haustorium formation and abundant sporulation. Therefore, expression of the wild‐type

Mlo

gene, in

mlo

resistant genotypes, is both necessary and sufficient to restore susceptibility to fungal attack. Complementation of

mlo

resistance alleles was restricted to single host cells, indicating a cell‐autonomous function for the wild‐type Mlo protein. We discuss our findings with respect to source–sink relationships of plants and biotrophic fungi and the potentially wide‐ranging use of the transient complementation assay to analyse host compatibility and defence in response to powdery mildew attack.
Digital mapping of bacterial artificial chromosomes by fluorescence in situ hybridization
Authors: Jackson Scott A. , Dong Fenggao , Jiang Jiming 
Published: March 2003

Summary The bacterial artificial chromosome (BAC) has become the most popular tool for cloning large DNA fragments. The inserts of most BAC clones average 100–200 kilobases (kb) and molecular characterization of such large DNA fragments is a major challenge. Here we report a simple and expedient technique for physical mapping of BAC inserts. Individual BAC molecules were immobilized on glass slides coated with Poly‐L‐lysine. The intact circular BAC molecules were visualized by fluorescence
in situ
hybridization using BAC DNA as a probe. The 7.4 kb BAC vector was extended to approximately 2.44 kb per micrometer. Digitally measured linear distances can be transformed into kilobases of DNA using the extension of BAC vector as a standard calibration. We mapped DNA fragments as small as 2 kb directly on circular BAC molecules. A rice BAC clone containing both tandem and dispersed repeats was analyzed using this technique. The distribution and organization of the different repeats within the BAC insert were efficiently determined. The results showed that this technique will be especially valuable for characterizing BAC clones that contain complex repetitive DNA sequences.
The TCP domain: a motif found in proteins regulating plant growth and development
Authors: Cubas Pilar , Lauter Nick , Doebley John , Coen Enrico 
Published: March 2003

SummaryThe cycloidea ( cyc) and teosinte branched 1 ( tb1) genes code for structurally related proteins implicated in the evolution of key morphological traits. However, the biochemical function of CYC and TB1 proteins remains to be demonstrated. To address this problem, we have analysed the predicted secondary structure of regions conserved between CYC and TB1, and looked for related proteins of known function. One of the conserved regions is predicted to form a non‐canonical basic‐Helix‐Loop‐Helix (bHLH) structure. This domain is also found in two rice DNA‐binding proteins, PCF1 and PCF2, where it has been shown to be involved in DNA‐binding and dimerization. This indicates that the conserved domain most probably defines a new family of transcription factors, which we have termed the TCP family after its first characterised members (TB1, CYC and PCFs). Other plant proteins of unknown function also belong to this family. We have studied two of these in Arabidopsis and have shown that they are expressed in rapidly growing floral primordia. This, together with the proposed involvement of cyc and tb1 in influencing meristem growth, suggests that many members of the TCP family may affect cell division. Some of these genes may have been recruited during plant evolution to generate new morphological traits.
Characterization of an Arabidopsis thaliana receptor‐like protein kinase gene activated by oxidative stress and pathogen attack
Authors: Czernic Pierre , Visser Botma , Sun Weining , Savouré Arnould , Deslandes Laurent , Marco Yves , Van Montagu Marc , Verbruggen Nathalie 
Published: March 2003

SummaryAn Arabidopsis thaliana cDNA clone that encodes a putative receptor‐like protein kinase gene (At‐RLK3) was characterized. The deduced 667‐amino acid protein consists of an amino‐terminal signal sequence, an extracellular domain, a single transmembrane domain, and a cytoplasmic domain with characteristics of serine/threonine protein kinase. Because of the original features of its extracellular domain, the At‐RLK3 protein is a member of a new class of receptor‐like protein kinases. The At‐RLK3 gene is present as a single copy within the Arabidopsis genome and its transcripts are detected in root, stem, leaf and flower. In cultured cells, the At‐RLK3 gene is activated upon oxidative stress and salicylic acid treatment. In plants, the gene appears to be differentially regulated during various plant–pathogen interactions: upon inoculation with strains of Pseudomonas syringae pv. tomato harboring or not, different avr genes, At‐RLK3 transcripts accumulate transiently at similar levels during both compatible and incompatible interactions. This gene is, however, preferentially expressed during the incompatible interaction induced by the soil‐borne vascular bacteria, Ralstonia solanacearum. The involvement of At‐RLK3 in signal transduction pathways during pathogen attack is discussed.
Transcriptional activator TGV mediates dexamethasone‐inducible and tetracycline‐inactivatable gene expression
Authors: Böhner Steffen , Lenk Ingo , Rieping Mechthild , Herold Michael , Gatz Christiane 
Published: March 2003

SummaryA chemically regulated gene expression system that can be switched on with dexamethasone and switched off with tetracycline was constructed. It is based on a transcriptional activator (TGV) that consists of the Tn10 encoded Tet repressor, the rat glucocorticoid receptor hormone binding domain and the transcriptional activation domain of Herpes simplex virion protein VP16. When stably expressed in transgenic tobacco plants, it mediates dexamethasone‐inducible transcription from a synthetic promoter (PTop10) consisting of seven tet operators upstream of a TATA‐box. Tetracycline interferes with induction by negatively regulating the DNA‐binding activity of the TetR moiety of TGV. The boundaries of the expression window of the TGV‐driven PTop10 reach from undetectable levels of the reporter enzyme β‐glucuronidase in the absence of dexa‐ methasone to induced levels reaching 15–20% of the Cauliflower Mosaic Virus 35S promoter (PCaMV35S). By modifying the sequence of PTop10, we generated a new target promoter (PTax) that is stably expressed over several generations and that can be activated to levels comparable to PCaMV35S, while yielding only slightly elevated background activities.
Dimerization and DNA binding of auxin response factors
Authors: Ulmasov Tim , Hagen Gretchen , Guilfoyle Tom J. 
Published: March 2003

SummaryAuxin response factors (ARFs) are transcription factors that bind with specificity to TGTCTC auxin response elements (AuxREs) found in promoters of primary/early auxin response genes. ARFs are encoded by a multi‐gene family, consisting of more than 10 genes. Ten ARFs have been analyzed by Northern analysis and were found to be expressed in all major plant organs and suspension culture cells of Arabidopsis. The predicted amino acid sequences indicate that the 10 ARFs contain a novel amino‐terminal DNA binding domain and a carboxyl‐terminal dimerization domain, with the exception of ARF3 which lacks this dimerization domain. All ARFs tested bind with specificity to the TGTCTC AuxRE, but there are subtle variations in the sequence requirements at positions 5 (T) and 6 (C) of the AuxRE. While the amino‐terminal domain of about 350 amino acids is sufficient for binding ARF1 to TGTCTC AuxREs, this domain is not sufficient for the binding of some other ARFs to palindromic AuxREs. Our results suggest that ARFs must form dimers on palindromic TGTCTC AuxREs to bind stably, and this dimerization may be facilitated by conserved motifs found in ARF carboxyl‐terminal domains. Dimerization in at least some cases may dictate which ARF(s) are targeted to AuxREs.