bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of monoterpenoids in glandular trichomes of the catmint Nepeta racemosa
Authors: Clark Lawrence J. , Hamilton James G.C. , Chapman June V. , Rhodes Michael J.C. , Hallahan David L.
Published: March 2003

Plants within the Lamiaceae are characterized by their production of essential oils, largely composed of monoterpenoids. They also possess on their aerial surfaces different types of trichomes, including relatively large peltate glandular trichomes. Observation of leaves of the catmint Nepeta racemosa, using cryogenic scanning electron microscopy, showed that around 3000 peltate trichomes are present on a single expanded leaf. An approach has been developed for sampling directly from the subcuticular cavity of such trichomes, in order to characterize qualitatively and quantitatively their contents. Gland samples from Nepeta spp. were analysed by combined gas chromatography‐mass spectrometry. These analyses have confirmed that the monoterpenoids characteristic of these species (nepetalactones) accumulate within the subcuticular cavity of peltate glandular trichomes. Qualitative analysis showed that three different nepetalactone stereoisomers were accumulated in different proportions in peltate glands from individual N. racemosa plants. Quantitative analysis showed that individual peltate glands accumulate around 30 ng of nepetalactone by maturity, and that accumulation of nepetalactone probably occurs exclusively within this gland type.
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

Previous 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.
FISH studies reveal the molecular and chromosomal organization of individual telomere domains in tomato
Authors: Zhong Xiao‐Bo , Fransz Paul F. , Wennekes‐van Eden Jannie , Ramanna  , Kammen Ab van , Zabel Pim , Hans de Jong
Published: March 2003

The molecular and cytological organization of the telomeric repeat (TR) and the subtelomeric repeat (TGR1) of tomato were investigated by fluorescence in situ hybridization (FISH) techniques. Hybridization signals on extended DNA fibres, visualized as linear fluorescent arrays representing individual telomeres, unequivocally demonstrated the molecular co‐linear arrangement of both repeats. The majority of the telomeres consisted of a TR and a TGR1 region separated by a spacer. Microscopic measurements of the TR and TGR1 signals revealed high variation in length of both repeats, with maximum sizes of 223 and 1330 kb, respectively. A total of 27 different combinations of TR and TGR1 was detected, suggesting that all chromosome ends have their own unique telomere organization. The fluorescent tracks on the extended DNA fibres were subdivided into four classes: (i) TR–spacer–TGR1; (ii) TR–TGR1; (iii) only TR; (iv) only TGR1. FISH to pachytene chromosomes enabled some of the TR/TGR1 groups to be assigned to specific chromosome ends and to interstitial regions. These signals also provided evidence for a reversed order of the TR and TGR1 sites at the native chromosome ends, suggesting a backfolding telomere structure with the TGR1 repeats occupying the most terminal position of the chromosomes. The FISH signals on diakinesis chromosomes revealed that distal euchromatin areas and flanking telomeric heterochromatin remained highly decondensed around the chiasmata in the euchromatic chromosome areas. The rationale for the occurrence and distribution of the TR and TGR1 repeats on the tomato chromosomes are discussed.
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

An 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

This 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

An 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

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

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

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

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

The 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

An 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

A 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

Auxin 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.
Lipid peroxidation and the oxidative burst associated with infection of Capsicum annuum by Botrytis cinerea
Authors: Deighton Nigel , Muckenschnabel Ingo , Goodman Bernard A. , Williamson Brian
Published: March 2003

A combination of electron paramagnetic resonance (EPR) spectroscopy and analytical chemistry has been used to study the changes in free radical content, transition metal ion status and lipid peroxidation following inoculation of fruits of sweet pepper (Capsicum annuum) with Botrytis cinerea. EPR detected a high concentration of an unidentified free radical associated with the spreading lesion that extends into the surrounding, healthy tissues. In addition, the EPR‐detectable iron(III) was highest at the centre of the lesion, again displaying a gradient out into the surrounding tissues. Analyses for aldehydic products of lipid peroxidation were performed to assess the accumulation and potential of these compounds to contribute to the cell death associated with necrotrophic pathogens. In contrast to the spectrum of aldehydes typically observed within peroxidized biological samples, no accumulation of malondialdehyde nor n‐hexanal was observed. Instead, high levels of two hydroxyalkenals (4‐hydroxy‐2‐hexenal and 4‐hydroxy‐2‐nonenal) were detected at concentra‐ tions up to 4000 and 20 000 pmol g− 1, respectively, at the host–pathogen interface. These results are discussed in terms of the likely mechanisms of formation of these aldehydes.
Graft transmission of post‐transcriptional gene silencing: target specificity for RNA degradation is transmissible between silenced and non‐silenced plants, but not between silenced plants
Authors: Sonoda Shoji , Nishiguchi Masamichi
Published: March 2003

Using the grafting procedure, we examined the transmission of post‐transcriptional gene silencing (PTGS) in Nicotiana benthamiana which had been transformed with the coat protein gene, including the 3′ non‐translated region of the sweet potato feathery mottle potyvirus. Transmission of PTGS from silenced lines to non‐silenced ones was bidirectional, but occurred efficiently from root stocks to scions. The level of transgene methylation in non‐silenced scions grafted onto silenced root stocks was not increased. When grafted scions which had become silenced were removed from silenced root stocks and regrafted onto non‐silenced or vector‐transformed root stocks, PTGS was maintained. However, their progeny did not show PTGS. Previously we reported that our transgenic lines had different target specificities of PTGS for RNA degradation: one line recognized only the 3′ part of the transgene mRNA while others involved the whole transgene mRNA (
Sonoda et al. 1999
, Phytopathology, 89, 385–391). Using these lines, we showed that target specificity of PTGS induced in non‐silenced scions after grafting was determined by that in silenced root stocks. However, unexpectedly, target specificity of PTGS induced in silenced scions after grafting was not changed by grafting onto silenced root stocks showing different target specificity, indicating that the second PTGS from silenced root stocks was not superimposed to silenced scions.
Comparative genome analysis reveals extensive conservation of genome organisation for Arabidopsis thaliana and Capsella rubella
Authors: Acarkan Adile , Roßberg Mathias , Koch Marcus , Schmidt Renate
Published: March 2003

Genome colinearity has been studied for two closely related diploid species of the Brassicaceae family, Arabidopsis thaliana and Capsella rubella. Markers mapping to chromosome 4 of A. thaliana were found on two linkage groups in Capsella and colinear segments spanning more than 10 c
m were revealed. Detailed analysis of a 60 kbp region in A. thaliana and its counterpart in C. rubella showed virtually complete conservation of gene repertoire, order and orientation. The comparison of orthologous genes revealed very similar exon–intron structures and sequence identities of 90% or more were found for exon sequences. This extensive genome colinearity at the genetic and molecular level allows the efficient transfer of data from the well‐studied A. thaliana genome to other species in the Brassicaceae family, substantially facilitating genome analysis studies for species of this family.
Resistance to the plant PR‐5 protein osmotin in the model fungus Saccharomyces cerevisiae is mediated by the regulatory effects of SSD1 on cell wall composition
Authors: Ibeas José Ignacio , Yun Dae‐Jin , Damsz Barbara , Narasimhan Meena L. , Uesono Yukifumi , Ribas Juan Carlos , Lee Hyeseung , Hasegawa Paul M. , Bressan Ray A. , Pardo José M.
Published: March 2003

The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR‐5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1‐v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1‐d allele or a null ssd1Δ mutation displayed high sensitivity to osmotin. The SSD1‐v protein mediates osmotin resistance in a cell wall‐dependent manner. Deletion of SSD1‐v or SSD1‐d impeded sorting of the PIR proteins (osmotin‐resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post‐transcriptional regulation of gene expression. The sensitivity of ssd1Δ cells to osmotin was only partially suppressed by over‐accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall‐mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell‐wall morphology and lower levels of alkali‐insoluble cell‐wall glucans. Therefore SSD1 is an important regulator of fungal cell‐wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR‐5 proteins.
Integration of the FISH pachytene and genetic maps of Medicago truncatula
Authors: Kulikova Olga , Gualtieri Gustavo , Geurts René , Kim Dong‐Jin , Cook Douglas , Huguet Thierry , De Jong J. Hans , Fransz Paul F. , Bisseling Ton
Published: March 2003

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.
Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin‐treated oats
Authors: Yao Nan , Tada Yasuomi , Park Pyoyun , Nakayashiki Hitoshi , Tosa Yukio , Mayama Shigeyuki
Published: March 2003

Histological and cytological evidence of where and when apoptotic cells occur in Pc‐2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM‐TUNEL was positive in the non‐collapsed cells but not in the collapsed cells in the treated leaves. However, the EM‐TUNEL assay was positive in the nuclei of the non‐collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin‐treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin‐treated non‐collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM‐TUNEL assay indicated that EGTA co‐incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.
‘Caged cytoskeletons’: a rapid method for the isolation of microtubule‐associated proteins from synchronized plant suspension cells
Authors: McCutcheon S. , Hemsley R. J. , Jopson M. F. , Lloyd C. W.
Published: March 2003

In the cytoskeleton method for isolating microtubule‐associated proteins MAP65, DcKRP120‐1 and DcKRP120‐2, carrot cells are first converted to protoplasts but this method cannot be used to isolate mitotic MAPs as mitotic synchrony is eroded during lengthy cellulase treatment. Anti‐microtubule cycle blocks would also be unsuitable. We report here a method for overcoming these problems. Cellulase degradation of tobacco BY‐2 cells for only several minutes allows extraction of detergent‐soluble proteins, leaving synchronized ‘caged cytoskeletons’ for depolymerization and enabling affinity purification of MAPs on neurotubules. This rapid and simple method should be of general utility: it can be bulked up, avoids anti‐microtubule blocks, and is applicable to other cell suspensions. The effectiveness of the caged cytoskeleton method is demonstrated by comparing known MAPs (the 65 kDa structural MAPs and the kinesin‐related protein, TKRP125) in synchronized cells taken at the mitotic peak with those in unsynchronized cells.
Transactivation of BARNASE under the AtLTP1 promoter affects the basal pole of the embryo and shoot development of the adult plant in Arabidopsis
Authors: Baroux Célia , Blanvillain Robert , Moore Ian R. , Gallois Patrick
Published: March 2003

Genetically controlled expression of a toxin provides a tool to remove a specific structure and consequently study its role during a developmental process. The availability of many tissue‐specific promoters is a good argument for the development of such a strategy in plants. We have developed a conditional system for targeted toxin expression and demonstrated its use for generating embryo phenotypes that can bring valuable information about signalling during embryogenesis. The BARNASE gene was expressed in the Arabidopsis embryo under the control of two promoters, one from the cyclin AtCYCB1 gene and one from the AtLTP1 gene (LipidTransferProtein 1). One‐hundred percent seed abortion was obtained with the cyclin promoter. Surprisingly however, the embryos displayed a range of lethal phenotypes instead of a single arrested stage as expected from this promoter. We also show that BARNASE expression under the control of the AtLTP1 promoter affects the basal pole of the globular embryo. Together with reporter expression studies, this result suggests a role of the epidermis in controlling the development of the lower tier of the embryo. This defect was not embryo‐lethal and we show that the seedlings displayed a severe shoot phenotype correlated to epidermal defects. Therefore, the epidermis does not play an active role during organogenesis in seedlings but is important for the postgermination development of a viable plant.
Analysis of gene expression in Arabidopsis thaliana by array hybridization with genomic DNA fragments aligned along chromosomal regions
Authors: Hanano Shigeru , Amagai Masayuki , Kaneko Takakazu , Kuwata Chikara , Tsugane Taneaki , Sakurai Nozomu , Nakamura Yasukazu , Shibata Daisuke , Tabata Satoshi
Published: March 2003

The availability of the entire genomic sequence of the higher plant Arabidopsis thaliana prompted an analysis of chromosomal regions for gene expression with the use of high‐density DNA array filters spotted with genomic DNA fragments (genomic DNA array analysis). One TAC and nine P1 clones, each of which contains a genomic DNA insert of approximately 80 kb and was used for sequencing of chromosome 5, were arbitrarily selected for analysis. The total size of the genomic regions corresponding to these clones is 819 kb. A total of 339 DNA fragments (average size, 2.9 kb) that cover contiguously the 10 chromosomal regions was selected and spotted onto nylon filters. The filters were then subjected to hybridization with 33P‐labelled cDNA molecules that had been synthesized from polyadenylated RNA isolated from 3‐week‐old plants. Quantitative and reproducible measurement of hybridization signals allowed analysis of the transcription of all genes in the targeted regions that were expressed at a level above the limit of detection. The data revealed that the analysed chromosomal regions are rich in active genes, and that they also provided a basis for the identification of novel transcripts whose sequences are not represented in the expressed sequence tag (EST) database.
Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation
Authors: Merlot Sylvain , Mustilli Anna‐Chiara , Genty Bernard , North Helen , Lefebvre Valérie , Sotta Bruno , Vavasseur Alain , Giraudat Jérôme
Published: March 2003

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2–1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1–1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.
Intrinsic direct repeats generate consistent post‐transcriptional gene silencing in tobacco
Authors: Ma Chonglie , Mitra Amitava
Published: March 2003

It is well documented that transgenes with inverted repeats can efficiently trigger post‐transcriptional gene silencing (PTGS), presumably via a double stranded RNA induced by complementary sequences in their transcripts. We show here that transgenes with intrinsic direct repeats can also induce PTGS at a very high frequency (80–100%). A transgene with three or four repeats induced PTGS in almost 100% of the primary transformants, regardless of whether a strong (enhanced 35S promoter) or a relatively weak (chlorophyll a/b binding protein promoter) promoter was used. The PTGS induced by three or four repeats is consistently inherited in subsequent generations, and can inactivate homologous genes in trans. Based on the high frequency and consistent heritability, we propose that the intrinsic direct repeat within a transgene may act as a primary determinant of PTGS referred to as direct repeat‐induced PTGS (driPTGS). Silencing occurred in all five genes, in this and two previous reports, suggesting that driPTGS might be a universal gene silencing mechanism both in dicotyledonous tobacco plants and monocotyledonous rice cells. In addition, driPTGS may help dissect the gene silencing mechanism and generate silenced phenotypes useful for research and plant biotechnology products.
Searching limiting steps in the expression of chloroplast‐encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii
Authors: Eberhard Stephan , Drapier Dominique , Wollman Francis‐André
Published: March 2003

We performed a systematic investigation of the quantitative relationship between genome copy number, transcription, transcript abundance and synthesis of photosynthetic proteins in the chloroplast of the green algae Chlamydomonas reinhardtii grown either in mixotrophic or phototrophic conditions. The chloroplast gene copy number is lower in the latter condition and the half‐life and accumulation levels of most chloroplast transcripts are significantly reduced, although the relative rates of protein synthesis remain similar. Our study shows that, in most instances, chloroplast protein synthesis is poorly sensitive to changes in gene copy number or transcript abundance in the chloroplast. Treatment with 5‐fluoro‐2′‐deoxyuridine, that inhibits chloroplast DNA replication and decreases extensively the number of copies of the chloroplast genome, had limited effects on the abundance of most chloroplast transcripts and little if any effect on the rates of protein synthesis. When using rifampicin, that selectively inhibits chloroplast transcription, we found no direct correlation between the level of transcripts remaining in the chloroplast and the rates of chloroplast protein synthesis. For two chloroplast genes, a 90% decrease in the amount of transcript did not cause a drop in the rate of synthesis of the corresponding protein product. Overall, our results demonstrate that there is no gene dosage effect in the chloroplast and that transcript abundance is not limiting in the expression of chloroplast‐encoded protein.
Knock‐out of a putative transporter results in altered blue‐light signalling in Chlamydomonas
Authors: Dame Gregory , Gloeckner Gernot , Beck Christoph F.
Published: March 2003

Nitrogen starvation and blue light are the two environmental cues that control sexual differentiation in Chlamydomonas reinhardtii. Insertional mutagenesis was applied to generate mutants that still require nitrogen starvation as the initiating signal for gametogenesis but were no longer dependent on irradiation. In one mutant analysed, sequences adjacent to the site of insertion were cloned and used for the isolation of a genomic clone that, upon transformation, could complement the mutant phenotype. The gene identified (LRG6) encodes two mRNAs that appear to be the products of differential splicing. The two putative gene products derived from these mRNAs differ in their C‐terminal ends. Both predicted gene products exhibit multiple hydrophobic domains with α‐helical secondary structure typical for integral membrane proteins. These proteins may form pores, and may function as transporters of as‐yet unknown substrates. Since rendering the LRG6 gene non‐functional resulted in light‐independence of gamete formation, it is suggested that this transporter may inhibit signal flux from the photoreceptor to target genes – either directly by its activity or indirectly by serving as a scaffold for signalling proteins. Shutting off this transporter may be required for the activation of signal flux in this pathway. This concept is supported by the observed reduction in LRG6 mRNA levels during the first phase of gametic differentiation.
Arabidopsis thaliana root non‐selective cation channels mediate calcium uptake and are involved in growth
Authors: Demidchik Vadim , Bowen Helen C. , Maathuis Frans J.M. , Shabala Sergey N. , Tester Mark A. , White Philip J. , Davies Julia M.
Published: March 2003

Calcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage‐independent rapidly activating Ca2+‐permeable non‐selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium‐permeable NSCCs co‐existed with hyperpolarisation‐activated Ca2+ channels (HACCs), which activated 40–60 min after forming the whole‐cell configuration. HACCs activated at voltages <−130 to −150 mV, showed slow activation kinetics and were regulated by cytosolic Ca2+ ([Ca2+]cyt). Using aequorin‐expressing plants, a linear relationship between membrane potential (Vm) and resting [Ca2+]cyt was observed, indicating the involvement of NSCCs. Intact root 45Ca2+ influx was reduced by Gd3+ (NSCC blocker) but was verapamil and TEA+ insensitive. In the root elongation zone, both root net Ca2+ influx (measured by Ca2+‐selective vibrating microelectrode) and NSCC activity were increased compared to the mature epidermis, suggesting the involvement of NSCC in growth. A Ca2+ acquisition system based on NSCC and HACC co‐existence is proposed. In mature epidermal cells, NSCC‐mediated Ca2+ influx dominates whereas in specialised root cells (root hairs and elongation zone cells) where elevated [Ca2+]cyt activates HACCs, HACC‐mediated Ca2+ influx predominates.
The NPK1 mitogen‐activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin‐like protein
Authors: Ishikawa Masaki , Soyano Takashi , Nishihama Ryuichi , Machida Yasunori
Published: March 2003

The tobacco mitogen‐activated protein kinase kinase kinase NPK1 localizes to the equatorial region of phragmoplasts by interacting with kinesin‐like protein NACK1. This leads to activation of NPK1 kinase at late M phase, which is necessary for cell plate formation. Until now, its localization during interphase has not been reported. We investigated the subcellular localization of NPK1 in tobacco‐cultured BY‐2 cells at interphase using indirect immunofluorescence microscopy and fusion to green fluorescent protein (GFP). Fluorescence of anti‐NPK1 antibodies and GFP‐fused NPK1 were detected only in the nuclei of BY‐2 cells at interphase. Examination of the amino acid sequence of NPK1 showed that at the carboxyl‐terminal region in the regulatory domain, which contains the binding site of NACK1, NPK1 contained a cluster of basic amino acids that resemble a bipartite nuclear localization signal (NLS). Amino acid substitution mutations in the critical residues in putative NLS caused a marked reduction in nuclear localization of NPK1 in BY‐2 cells, indicating that this sequence is functional in tobacco BY‐2 cells. We also found that the 64‐amino acid sequence at the carboxyl terminus that contains NLS sequence is essential for interaction with NACK1, and that mutations in the NLS sequence prevented NPK1 from interacting with NACK1. Thus, the amino acid sequence at the carboxyl‐terminal region of NPK1 has dual functions for nuclear localization during interphase and binding NACK1 in M phase.
Evidence that the Arabidopsis nuclear gibberellin signalling protein GAI is not destabilised by gibberellin
Authors: Fleck Barbara , Harberd Nicholas P.
Published: March 2003

Plant growth is regulated by bioactive gibberellin (GA), although there is an unexplained diversity in the magnitude of the GA responses exhibited by different plant species. GA acts via a group of orthologous proteins known as the DELLA proteins. The Arabidopsis genome contains genes encoding five different DELLA proteins, the best known of which are GAI and RGA. The DELLA proteins are thought to act as repressors of GA‐regulated processes, whilst GA is thought to act as a negative regulator of DELLA protein function. Recent experiments have shown that GA induces rapid disappearance of nuclear RGA, SLR1 and SLN1 (DELLA proteins from rice and barley), suggesting that GA signalling and degradation of DELLA proteins are coupled. However, RGL1, another Arabidopsis DELLA protein, does not disappear from the nucleus in response to GA treatment. Here, we present evidence suggesting that GAI, like RGL1, is stable in response to GA treatment, and show that transgenic Arabidopsis plants containing constructs that enable high‐level expression of GAI exhibit a dwarf, GA non‐responsive phenotype. Thus, GAI appears to be less affected by GA than RGA, SLR1 or SLN1. We also show that neither of the two putative nuclear localisation signals contained in DELLA proteins are individually necessary for nuclear localisation of GAI. The various DELLA proteins have different properties, and we suggest that this functional diversity may explain, at least in part, why plant species differ widely in their GA response magnitudes.