bluuebunny/crossref_metadata_2025 huggingface dataset:
Auxin regulation and spatial localization of an endo‐1,4‐β‐D‐glucanase and a xyloglucan endotransglycosylase in expanding tomato hypocotyls
Authors: Catalá Carmen , Rose Jocelyn K.C. , Bennett Alan B.
Published: March 2003

Xyloglucan, the primary hemicellulosic cell wall polysaccharide in dicotyledons, undergoes substantial modification during auxin‐stimulated cell expansion. To identify candidates for mediating xyloglucan turnover, the expression and auxin regulation of tomato Cel7 and LeEXT, genes encoding an endo‐1,4‐β‐glucanase (EGase) and a xyloglucan endotransglycosylase (XET), respectively, were examined. LeEXT mRNA was present primarily in elongating regions of the hypocotyl and was induced to higher levels by hormone treatments that elicited elongation of hypocotyl segments. Cel7 mRNA abundance was very low in both elongating and mature regions of the hypocotyl but was induced to accumulate to high levels in both hypocotyl regions by auxin application. Analysis of the time dependence of expression of Cel7 and LeEXT during auxin treatment suggested that induction of these genes is not required for rapid growth responses but may participate in the cell wall changes involved in sustained cell elongation. Localization of Cel7 and LeEXT mRNA by in situ hybridization revealed that both genes are expressed in outer cell layers of the hypocotyl. In untreated etiolated seedlings, LeEXT mRNA was detected in epidermal cells of the elongating region, a tissue considered to play a key role in auxin‐induced elongation. After auxin treatment, Cel7 and LeEXT mRNA showed an overlapping spatial distribution in the epidermis and outer cortical cell layers. We conclude that LeEXT and Cel7 exhibit both unique and overlapping patterns of expression and have the potential to act cooperatively in mediating cell wall disassembly associated with expansive growth.
Improved ballistic transient transformation conditions for tomato fruit allow identification of organ‐specific contributions of I‐box and G‐box to the RBCS2 promoter activity
Authors: Baum Katja , Gröning Birge , Meier Iris
Published: March 2003

An improved protocol for the ballistic transient transformation of developing tomato (Lycopersicon esculentum) fruits is reported, which allows high‐resolution cis‐analysis of fruit‐specific transcriptional activation. The tomato RBCS2 promoter fused to the firefly luciferase gene was used as a model system for this study. Osmotic treatment of fruit slices before, during and after particle bombardment, together with the optimization of bombardment conditions, resulted in a 100‐fold increase in RBCS2 promoter‐driven transient luciferase expression compared with previously reported protocols. Under these conditions, the transformed RBCS2 promoter was shown to be properly regulated in a developmental fashion. A cis‐analysis of the RBCS2 promoter was performed. A 37 bp domain is required for high‐level RBCS2 promoter activity both in leaves and young fruits. Two conserved sequence elements within this domain, an I‐box element (GATAAG) and a G‐box element (CACGTG), are necessary for its activity in leaves. In contrast, in young fruits, the G‐box is the single dominant cis‐acting element. These findings are discussed with respect to the proposed functions of G‐box and I‐box binding factors in regulating plant genes in different organs.
Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins
Authors: Fan Hua‐Ying , Hu Yi , Tudor Matthew , Ma Hong
Published: March 2003

MADS domain (for MCM1, AG, DEFA and SRF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled‐coil structure suggests a role in establishing a protein—protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C‐terminal region of AG in a yeast two‐hybrid selection, 156 clones that encode potential AG‐interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.
Comparative analysis of non‐random DNA repair following Ac transposon excision in maize and Arabidopsis
Authors: Rinehart Timothy A. , Dean Caroline , Weil Clifford F.
Published: March 2003

Ac/Ds transposable elements often leave short DNA rearrangements, or ‘footprints,’ at the sites where they excise. Previous studies at the maize waxy (wx) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end‐joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac‐containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis, including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5′ and 18 bp 3′ of the transposon. These studies demonstrate that transgenic Ac/Ds‐containing plants will be useful tools in dissecting plant DNA repair processes.
The effect of temperature on Agrobacterium tumefaciens‐mediated gene transfer to plants
Authors: Dillen Willy , De Clercq Janniek , Kapila Jyoti , Zambre Mukund , Van Montagu Marc , Angenon Geert
Published: March 2003

Agrobacterium tumefaciens is generally used to achieve genetic transformation of plants. The temperatures that have been used for infection with Agrobacterium in published transformation protocols differ widely and, to our knowledge, the effect of temperature on the efficiency of T‐DNA transfer to plants has not been investigated systematically. Agrobacterium tumefaciens strains harbouring a binary vector with the β‐glucuronidase (uidA) gene and either a nopaline‐, an octopine‐ or an agropine/ succinamopine‐type helper plasmid were tested in two transformation systems at temperatures between 15 and 29°C. One system involved cocultivation of Phaseolus acutifolius callus whereas in the other system Nicotiana tabacum leaves were vacuum‐infiltrated. In both situations, irrespective of the type of helper plasmid, the levels of transient uidA expression decreased notably when the temperature was raised above 22°C. Expression was low at 27°C and undetectable at 29°C. We anticipate that the efficiency of many published transformation protocols can be improved by reconsidering the factor of temperature.
A new model system for tomato genetics
Authors: Meissner Rafael , Jacobson Yuval , Melamed Sarah , Levyatuv Shai , Shalev Gil , Ashri Amram , Elkind Yonatan , Levy Avraham
Published: March 2003

The purpose of this study was to develop a model system for studying tomato genetics. Agronomic, genetic, and molecular data are presented which show that the miniature Lycopersicon esculentum cultivar, Micro‐Tom (Micro tomato), fulfills the requirements for such a model. It grows at high density (up to 1357 plants/m−2); it has a short life cycle (70–90 days from sowing to fruit ripening); and it can be transformed at frequencies of up to 80% through Agrobacterium‐mediated transformation of cotyledons. Moreover, it differs from standard tomato cultivars by only two major genes. Therefore, any mutation or transgene can be conveniently studied in Micro‐Tom’s background and, when needed, transferred into a standard background. We took advantage of Micro‐Tom’s features to improve the infrastructure for mutagenesis in tomato. A screening of 9000 M1 and 20 000 M2 EMS mutagenized plants is described. Mutants with altered pigmentation or modified shape of leaves, flowers and fruits were found. In addition, an enhancer trapping and a gene trapping system, based on the Ac/Ds maize transposable elements, were transformed into Micro‐Tom and found to be active. In summary, Micro‐Tom opens new prospects to achieve saturated mutagenesis in tomato, and facilitates the application of transposon‐based technologies such as gene tagging, trapping and knockout.
Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl
Authors: Gendreau Emmanuel , Höfte Herman , Grandjean Olivier , Brown Spencer , Traas Jan
Published: March 2003

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light‐grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light‐grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light‐ or dark‐grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark‐grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild‐type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light‐grown hypocotyls is not the consequence of a simple feed‐back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome‐controlled photomorphogenic program.
Upregulation of two ripening‐related genes from a non‐climacteric plant (pepper) in a transgenic climacteric plant (tomato)
Authors: Kuntz  , Chen  , Simkin  , Römer  , Shipton  , Drake  , Schuch  , Bramley
Published: March 2003

Activities of promoters from the capsanthin/capsorubin synthase and fibrillin genes, which are molecular markers for ripening in the non‐climacteric pepper fruits, have been studied in transgenic tomato plants that produce fruits of the climacteric type (characterized by an increase in respiration and ethylene production). The promoters of both genes were strongly upregulated during tomato fruit ripening in a manner similar to the induction of these genes in pepper fruits. Induction occurred at the mature green stage preceding ripening (a stage when ethylene production and respiration are known to rise in tomato fruits). Ethylene positively influenced the expression of both genes in tomato. Other plant growth regulators, namely abscisic acid, auxin and polyamines, did not alter gene expression. In contrast, water loss strongly induced both promoters. This dehydration‐mediated gene induction was inhibited by mitochondrial respiration inhibitors (mainly of the alternative oxidase). A slight positive effect with light, apparently not linked to normal photosynthesis but rather to photooxidative stress, was also observed. Taken together, the data indicate that activation of oxidase systems, leading to changes in the cellular redox balance, mediates the induction of both genes in tomato. Various cellular compartments are likely to be contributors to this process, which leads to the developmental regulation of nuclear genes encoding plastid‐located proteins.
Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells
Authors: Kunik Talya , Palanichelvam Karuppaiah , Czosnek Henryk , Citovsky Vitaly , Gafni Yedidya
Published: March 2003

The tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly‐transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem‐associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the β‐glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro‐injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant‐and insect‐cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)‐specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino‐terminus of the protein.
Phosphorylation mediates the nuclear targeting of the maize Rab17 protein
Authors: Jensen Anders B. , Goday Adela , Figueras Mercè , Jessop Alison C. , Pagès Montserrat
Published: March 2003

The maize abscisic acid‐responsive Rab17 protein localizes to the nucleus and cytoplasm in maize cells. In‐frame fusion of Rab17 to the reporter protein β‐glucuronidase (GUS) directed GUS to the nucleus and cytoplasm in transgenic Arabidopsis thaliana and in transiently transformed onion cells. Analysis of chimeric constructs identified one region between amino acid positions 66–96, which was necessary for targeting GUS to the nucleus. This region contains a serine cluster followed by a putative consensus site for protein kinase CK2 phosphorylation, and a stretch of basic amino acids resembling the simian virus 40 large T antigen‐type nuclear localization signal (NLS). Mutation of two basic amino acids in the putative NLS had a weak effect on nuclear targeting in the onion cell system and did not modify the percentage of nuclear fusion protein in the Arabidopsis cells. The mutation of three amino acids in the consensus site for CK2 recognition resulted in the absence of in vitro phosphorylated forms of Rab17 and in a strong decrease of GUS enzymatic activity in isolated nuclei of transgenic Arabidopsis. These results suggest that phosphorylation of Rab17 by protein kinase CK2 is the relevant step for its nuclear location, either by facilitating binding to specific proteins or as a direct part of the nuclear targeting apparatus.
BAC representation of two low‐copy regions of the genome of Arabidopsis thaliana
Authors: Bent Elisabeth , Johnson Samantha , Bancroft Ian
Published: March 2003

Two regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel‐purified DNA samples of YACs selected from the YAC‐based physical map of chromosome 4 were used to probe high‐density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty‐four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4‐fold redundant coverage, with a range of 2‐ to 22‐fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.
Ser6 in the maize actin‐depolymerizing factor, ZmADF3, is phosphorylated by a calcium‐stimulated protein kinase and is essential for the control of functional activity
Authors: Smertenko Andrei P. , Jiang Chang‐Jie , Simmons Nicola J. , Weeds Alan G. , Davies Dewi R. , Hussey Patrick J.
Published: March 2003

Maize actin‐depolymerizing factor, ZmADF, binds both G‐ and F‐actin and enhances in vitro actin dynamics. Evidence from studies on vertebrate ADF/cofilin supports the view that this class of protein responds to intracellular and extracellular signals and causes actin reorganization. As a test to determine whether such signal‐responsive pathways existed in plants, this study addressed the ability of maize ADF to be phosphorylated and the likely effects of such phosphorylation on its capacity to modulate actin dynamics. It is shown that maize ADF3 (ZmADF3) can be phosphorylated by a calcium‐stimulated protein kinase present in a 40–70% ammonium sulphate fraction of a plant cell extract. Phosphorylation is shown to be on Ser6, which is only one of nine amino acids that are fully conserved among the ADF/cofilin proteins across distantly related species. In addition, an analogue of phosphorylated ZmADF3 created by mutating Ser6 to Asp6 (zmadf3–4) does not bind G‐ or F‐actin and has little effect on the enhancement of actin dynamics. These results are discussed in context of the previously observed actin reorganization in root hair cells.
Acetyl‐CoA:benzylalcohol acetyltransferase – an enzyme involved in floral scent production in Clarkia breweri
Authors: Dudareva Natalia , D’Auria John C. , Nam Kyoung Hee , Raguso Robert A. , Pichersky Eran
Published: March 2003

Volatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators. They are also important flavor compounds in fruits. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California. The enzyme acetyl‐CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, and a cDNA encoding this enzyme has been isolated and characterized. The sequence of the 433‐residue BEAT protein does not show high similarity to any previously characterized protein, but a 35‐residue region from position 135–163 has significant similarity (42–56% identity) to several proteins known or suspected to use an acyl‐CoA substrate. E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.
The terminal O‐acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A‐dependent acyl transfer
Authors: St‐Pierre Benoit , Laflamme Pierre , Alarco Anne‐Marie , D Vincenzo , Luca E
Published: March 2003

The gene encoding acetyl CoA:deacetylvindoline 4‐O‐acetyltransferase (DAT) (EC 2.3.1.107) which catalyzes the last step in vindoline biosynthesis was isolated and characterized. The genomic clone encoded a 50 kDa polypeptide containing the sequences of nine tryptic fragments derived from the purified DAT heterodimer. However, cleavage of DAT protein to yield a heterodimer appears to be an artifact of the protein purification procedure, since the size of the protein (50 kDa) cross‐reacting with anti‐DAT antibody in seedlings and in leaves of various ages also corresponds to the size of the active recombinant enzyme. Studies with the intact plant and with developing seedlings showed that induction of DAT mRNA, protein accumulation and enzyme activity occurred preferentially in vindoline producing tissues such as leaves and cotyledons of light‐treated etiolated seedlings. The ORF of DAT showed significant sequence identity to 19 other plant genes, whose biochemical functions were mostly unknown. The Mr of ≈ 50 kDa, a HXXXDG triad, and a DFGWGKP consensus sequence are highly conserved among the 20 plant genes and these criteria may be useful to identify this type of acyltransferase. The involvement of some of these genes in epicuticular wax biosynthesis, fruit‐ripening and in benzoyltransfer reactions indicates that the plant kingdom contains a superfamily of multifunctional acyltransferases which operate by a reaction mechanism related to the ancient chloramphenicol O‐acetyltransferase and dihydrolipoyl acetyltransferase class of enzymes.
A mutant inLycopersicon esculentumMill. with highly reduced VA mycorrhizal colonization: isolation and preliminary characterisation
Authors: Barker S. J. , Stummer B. , Gao L. , Dispain I. , O’Connor P. J. , Smith S. E.
Published: March 2003

This paper reports the successful isolation and preliminary characterisation of a mutant of

Lycopersicon esculentum

Mill. with highly reduced vesicular‐arbuscular (VA) mycorrhizal colonization. The mutation is recessive and has been designated

rmc

. Colonization by

G. mosseae

is characterised by poor development of external mycelium and a few abnormal appressoria. Vesicles were never formed by this fungus in association with the mutant.

Gi. margarita

formed large amounts of external mycelium, complex branched structures and occasional auxiliary cells. Small amounts of internal colonization also occurred. Laser scanning confocal microscopy (LSCM) gave a clear picture of the differences in development of

G. intraradices

and

Gi. margarita

in mutant and wild‐type roots and confirmed that the fungus is restricted to the root surface of the mutants. The amenability of tomato for molecular genetic characterisation should enable us to map and clone the mutated gene, and thus identify one of the biochemical bases for inability to establish a normal mycorrhizal symbiosis. The mutant represents a key advance in molecular research on VA mycorrhizal symbiosis.
Cytosolic acidification but not auxin at physiological concentration is an activator of MAP kinases in tobacco cells
Authors: Tena Guillaume , Renaudin Jean‐Pierre
Published: March 2003

In higher plants, MAP kinase cascades are involved in the transduction of numerous stress‐related signals but much less is known about the effect of mitogenic signals. We have analysed MAP kinase activation in tobacco cells after treatment by auxin, a growth factor required at physiological concentrations for mitosis in plant cell cultures. From in‐gel assay of myelin basic protein kinase and from immunochemical detection of ERK related kinases, we show that the mitogenic effect of auxin, which was confirmed by the specific increase of several mRNAs species, did not rely on MAP kinase activation within the first 2 hours. These data contest previous results which could be due to the activation of MAP kinase by a signal other than auxin. In the second part of this study, we show that the treatment of the cells with high concentrations of various weak lipophilic acids such as auxin, in a non‐physiological concentration range, butyric or acetic acid is sufficient to activate transiently a MAP kinase. The data show that MAP kinase activation is the consequence of cytosolic acidification. Moreover, it is not sensitive to the protein kinase inhibitor staurosporine. These results suggest a functional role for cytosolic acidification as a second messenger mediating MAP kinase activation in the response of plant cells to various stresses.
BRANCHED SILKLESSmediates the transition from spikelet to floral meristem duringZea maysear development
Authors: Colombo Lucia , Marziani Giovanna , Masiero Simona , Wittich Peter E. , Schmidt Robert J. , Gorla Mirella Sari , Pè M. Enrico
Published: March 2003

The molecular and genetic control of inflorescence and flower development has been studied in great detail in model dicotyledonous plants such as

Arabidopsis

and

Antirrhinum

. In contrast, little is known about these important developmental steps in monocotyledonous species. Here we report the analysis of the

Zea mays

mutant

branched silkless1–2 (bd1–2)

, allelic to

bd1

, which we have used as a tool to study the transition from spikelet to floret development in maize. Floret development is blocked in the female inflorescence (the ear) of

bd1–2

plants, whereas florets develop almost normally in the male inflorescence (the tassel). Detailed phenotypic analyses indicate that in

bd1–2

mutants ear inflorescence formation initiates normally, however, the spikelet meristems do not proceed to form floret meristems. The ear spikelets, at anthesis, contain various numbers of spikelet‐like meristems and glume‐like structures. Furthermore, growth of branches from the base of the ear is often observed. Expression analyses show that the floral‐specific MADS box genes

Zea mays AGAMOUS1

(

ZAG1

), ZAG2 and

Zea mays MADS 2

(

ZMM2

) are not expressed in ear florets in

bd1–2

mutants, whereas their expression in tassel florets is similar to that of wild type. Taken together, these data indicate that the development from spikelet to floret meristem is differentially controlled in the ear and tassel in the monoecious grass species

Zea mays

, and that

BRANCHED SILKLESS

plays an important role in regulating the transition from spikelet meristem to floral meristem during the development of the female inflorescence of maize.
The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase
Authors: Nuccio Michael L. , Russell Brenda L. , Nolte Kurt D. , Rathinasabapathi Bala , Gage Douglas A. , Hanson Andrew D.
Published: March 2003

Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two‐step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a well‐established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline monooxygenase (CMO), a stromal enzyme with a Rieske‐type [2Fe‐2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet‐deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe‐2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post‐transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02– 0.05 μmol g–1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30‐fold. The choline precursors mono‐ and dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet‐deficient plants engineered with choline‐oxidizing genes, the size of the free choline pool and the metabolic flux to choline need to be increased to attain GlyBet levels as high as those in natural accumulators.
Correlation of defense gene induction defects with powdery mildew susceptibility inArabidopsisenhanced disease susceptibility mutants
Authors: Reuber T. Lynne , Plotnikova Julia M. , Dewdney Julia , Rogers Elizabeth E. , Wood William , Ausubel Frederick M.
Published: March 2003

We investigated the relative importance of specific

Arabidopsis thaliana

genes in conferring resistance to bacterial versus fungal pathogens. We first developed a pathosystem involving the infection of

Arabidopsis

accession Columbia with a virulent isolate of the obligate biotrophic fungal pathogen

Erysiphe orontii. E. orontii

elicited the accumulation of mRNAs corresponding to the defense‐related genes

PR1, BGL2 (PR2), PR5 and GST1

, but did not elicit production of the phytoalexin camalexin or the accumulation of defensin (

PDF1.2

) or thionin (

THI2.1

) mRNAs. We tested a set of 15 previously isolated

Arabidopsis

phytoalexin deficient

(pad),

non‐expresser of

PR (npr) and

enhanced disease susceptibility

(eds)

mutants that are more susceptible to

Pseudomonas syringae

for their susceptibility to

E. orontii.

Four of these mutants (

pad4–1, npr1–1, eds5–1 and

a double

npr1–1 eds5–1

mutant) as well as

Arabidopsis

lines carrying a

nahG

transgene exhibited enhanced susceptibility to

E. orontii

and reduced levels of

PR

gene expression

.

Comparison of the

PR

gene induction patterns in response to

E. orontii

in the various mutants and in the

nahG

transgenics suggests the existence of

NPR1

‐independent salicylate‐dependent and

NPR1

‐independent salicylate‐independent defense gene activation pathways. Eleven other

eds

and

pad

mutants did not show measurable enhanced susceptibility to

E. orontii

, suggesting that these mutants are defective in factors that are not important for the limitation of

E. orontii

growth.
High pressure freezing and freeze substitution reveal new aspects of fine structure and maintain protein antigenicity in barley aleurone cells
Authors: Lonsdale Jennifer E. , McDonald Kent L. , Jones Russell L.
Published: March 2003

High pressure freezing and freeze substitution (HPF‐FS) were used to prepare barley (

Hordeum vulgare

L. cv Himalaya) aleurone protoplasts for transmission electron microscopy (TEM). We show that HPF‐FS is superior to conventional chemical fixation and dehydration techniques for the preservation of cellular fine structure and antigenicity of proteins in barley aleurone protoplasts. HPF‐FS extracted fewer proteins from the cytosol and organelles of aleurone protoplasts and maintained the details of cellular structure. The cortical cytoskeleton, made up of microtubules, was observed for the first time by TEM in barley aleurone protoplasts prepared by HPF‐FS. Organelles such as protein storage vacuoles retained their proteinaceous contents, and other cellular organelles (including the Golgi apparatus, the nucleus and mitochondria) were also well preserved in protoplasts fixed by HPF‐FS. Antibodies to the vacuolar enzyme nuclease I, the tonoplast aquaporin α‐TIP and the glyoxysomal enzyme malate synthase showed that the antigenicity of organellar enzymes and membrane proteins was preserved in cells prepared by HPF‐FS. We conclude that HPF‐FS is superior to chemical fixation for the preparation of plant protoplasts for TEM and is the method of choice for the preservation of aleurone protoplasts for structural and immunochemical studies.
Analysis of alternative transcripts of the flaxL6rust resistance gene
Authors: Ayliffe Michael A. , Frost Donna V. , Finnegan E. Jean , Lawrence Gregory J. , Anderson Peter A. , Ellis Jeffery G.
Published: March 2003

The

L6

rust resistance gene from flax generates at least four transcript classes by alternative splicing of the third intron. The most abundant transcript class encodes a resistance protein containing domains that include nucleotide binding site motifs and a leucine‐rich repeat region (NBS‐LRR). The remaining three transcript classes encode truncated products which lack most of the C‐terminal part of the protein containing the leucine‐rich region (LRR). The four transcript classes occur in all plant organs examined and no induction of

L6

expression was observed following infection of resistant plants with an avirulent rust strain expressing the corresponding

A‐L6

avirulence gene. Flax plants transgenic for an intronless

L6

gene, incapable of encoding truncated resistance proteins by alternative splicing, expressed

L6

resistance indistiguishable from that of the wild‐type gene. Therefore, a definitive role for alternative transcripts and their predicted truncated products could not be assigned in the flax/flax rust system.
Mutations at therug4locus alter the carbon and nitrogen metabolism of pea plants through an effect on sucrose synthase
Authors: Craig Josephine , Barratt Paul , Tatge Helma , Déjardin Annabelle , Handley Linda , Gardner Christopher D. , Barber Lorraine , Wang Trevor , Hedley Cliff , Martin Cathie , Smith Alison M.
Published: March 2003

The biochemical and molecular basis of the wrinkled‐seeded phenotype of

rug4

mutants of pea (

Pisum sativum

L.) has been investigated. Mutant embryos have reduced starch contents and only 5% of the sucrose synthase activity of wild‐type embryos during development. Activities of other enzymes involved in the conversion of sucrose to starch are unaffected. A gene encoding an isoform of sucrose synthase expressed in the embryo co‐segregates with the

rug4

locus, and one of the three mutant alleles has been show to carry a point mutation in this gene that converts a highly conserved arginine residue to a lysine residue. It is highly likely that the reduced starch content of the mutant embryo is a direct consequence of the loss of sucrose synthase activity. The mutations reduce the activity of sucrose synthase in the testa and the leaf by 50% or less, but activity in

Rhizobium

‐infected root nodules is reduced by 85%. Although the nodules of mutant plants contain metabolically active bacteroids, the N content and δ15N values of these plants in the field indicate that, unlike wild‐type plants, they derive little of their N from N2 fixation via

Rhizobium

. Sucrose synthase thus appears to be essential for the supply of carbon for bacteroid metabolism and/or ammonia assimilation during nitrogen assimilation.
Glyoxalase I fromBrassica juncea: molecular cloning, regulation and its over‐expression confer tolerance in transgenic tobacco under stress
Authors: Veena  , Reddy Vanga S. , Sopory Sudhir K.
Published: March 2003

Despite its ubiquitous presence, the role of glyoxalase I has not been well investigated in plants. In order to find out its physiological functions, we have cloned and characterized a cDNA from

Brassica juncea

encoding glyoxalase I (

Gly I

) and made transgenic tobacco plants harbouring

Gly I

in both sense and antisense orientation. The transgenic nature of the plants was confirmed by Southern blotting, and the estimated number of genes inserted ranged from one to six. The transcript and protein levels of glyoxalase I were also monitored in transgenic plants. The expression of glyoxalase I in

B. juncea

was upregulated in response to salt, water and heavy metal stresses. In response to a high concentration of salt, the transcript level averaged threefold higher in 72 h, and an increase in the protein was also seen by immunoblotting. The transgenic plants over‐expressing glyoxalase I showed significant tolerance to methylglyoxal and high salt, as tested in detached leaf disc senescence assay. A comparison of plants expressing high and low levels of glyoxalase I showed that the tolerance to different salt concentrations was correlated with the degree of glyoxalase I expression. Our results suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions.
Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plantsin vivo
Authors: Hedtke Boris , Meixner Martin , Gillandt Sabine , Richter Ekkehard , Börner Thomas , Weihe Andreas
Published: March 2003

The recent identification of phage‐type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis and
in vitro
import experiments. To determine the subcellular localization of the phage‐type organellar RNA polymerases
in planta
, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 from
Arabidopsis thaliana
and RpoT from
Chenopodium album
were fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transform
A. thaliana
. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3‐GFP construct accumulated in chloroplasts of leaf cells and non‐green plastids (leucoplasts) of root cells. By demonstrating targeting
in planta
, the data add substantial evidence for the phage‐type RNA polymerases from
C. album
and
A. thaliana
to function in the transcriptional machinery of mitochondria and plastids.
Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plantNepenthes
Authors: Schulze Waltraud , Frommer Wolf B. , Ward John M.
Published: March 2003

Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In
Nepenthes
, insect‐derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in
Nepenthes
pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of
Nepenthes
. Expression of the ammonium transporter gene
NaAMT1
was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene,
NaAAP1
, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by
NaAAP1
may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene
NaNTR1
was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.
Microtubules regulate tip growth and orientation in root hairs ofArabidopsis thaliana
Authors: Bibikova Tatiana N. , Blancaflor Elison B. , Gilroy Simon
Published: March 2003

The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip‐focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of
Arabidopsis
. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip‐focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre‐treated with microtubule antagonists, using the caged‐calcium ionophore Br‐A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.
Sucrose transport into developing seeds of Pisum sativum L.
Authors: Tegeder Mechthild , Wang Xin‐Ding , Frommer Wolf B.. , Offler Christina E.. , Patrick John W..
Published: March 2003

The anatomy of developing pea seeds is characterized by transfer cells present in both coats and cotyledons at the maternal/filial interface. To determine the nature and cellular localization of sucrose transporters in pea seeds, a full‐length clone of a sucrose/H+ symporter ( PsSUT1) was isolated from a cotyledon cDNA library. Northern blot analyses of different organs showed that PsSUT1 is expressed in non‐seed tissues, including sucrose sinks and sources. Within developing seeds, transcripts of PsSUT1 and PsAHA1 genes were detected in all tissues, while transcripts of a sucrose binding protein ( GmSBP) were confined to cotyledon epidermal transfer cells. Signal intensities of PsSUT1 and PsAHA1 transcripts and protein products were most pronounced in the thin‐walled parenchyma cells of seed coats and epidermal transfer cells of cotyledons. For cotyledons, the highest transporter densities were localized to those portions of plasma membranes lining the wall ingrowth regions of epidermal transfer cells. Responses of [14C]sucrose influx to metabolic inhibitors indicated that proton‐coupled sucrose transport was operative in both seed coats and cotyledons. Cotyledon epidermal transfer cells were shown to support the highest sucrose flux. Maximal transport activity was found to account for the sucrose flux differences between seed tissues. Intercellular movement of the symplasmic tracer, 5‐(6)‐carboxyfluorescein (CF), demonstrated that symplasmic pathways interconnect the vascular tissues to thin‐walled parenchyma transfer cells of seed coats and, for cotyledons, epidermal transfer cells to storage parenchyma cells.
BELL1 and AGAMOUS genes promote ovule identity in Arabidopsis thaliana
Authors: Western Tamara L. , Haughn George W.
Published: March 2003

Molecular and genetic analyses have demonstrated that the Arabidopsis thaliana gene BELL1 (BEL1) is required for proper morphogenesis of the ovule integuments. Several lines of evidence suggest that BEL1 may act, at least in part, to repress the function of the organ identity gene AGAMOUS (AG) during ovule development. To study the relative roles of BEL1 and AG, plants homozygous for ag, bel1 or both were constructed in an ap2 mutant background where ovules form even in the absence of AG function. The loss of either BEL1 or AG led to a decrease in the number of mature ovules, accompanied by an increase in primordial outgrowths. These data suggest that BEL1 and AG gene products act early in ovule development in a partially redundant manner to direct ovule identity. Development of the abnormal integuments characteristic of the Bel1– mutant phenotype was found to be dependent on AG function. Finally, BEL1 appears to be required for embryo sac development independent of both other aspects of ovule morphogenesis and AG function. This study therefore suggests that both BEL1 and AG are required for several distinct aspects of ovule morphogenesis.
Interactions between introns via exon definition in plant pre‐mRNA splicing
Authors: Simpson Craig G. , Clark Gillian P. , Lyon Jackie M. , Watters Jenny , McQuade Clare , Brown John W. S.
Published: March 2003

Exon definition is a mechanism whereby splice sites are selected initially via interactions between splicing factors across an exon, prior to spliceosome assembly and intron removal. It occurs in the splicing of vertebrate pre‐mRNAs and, recently, evidence for exon definition and the role of exon sequences has been obtained in plant intron splicing. Here we demonstrate that interactions between plant introns influence splicing efficiency and that these interactions are consistent with an exon definition process. The splicing efficiency of a UA‐poor, inefficiently spliced intron (wheat amylase) increases 3.5‐ to 4.4‐fold when placed in tandem with a UA‐rich, well spliced, intron (legumin). Enhanced splicing is also observed with partial pea legumin intron sequences. However, mutation of splice sites in the partial UA‐rich intron sequences abolished the enhanced splicing effect such that intact splice sites at the 5′ and 3′ ends of the exon were required, thus pointing to exon definition. This was further supported by reducing the size of the intervening exon or replacing with a UA‐rich sequence which leads to loss of splicing of the UA‐poor intron. Finally, the results support UA‐rich sequences functioning early in the splicing process in plants.
A simple method to enrich an Agrobacterium‐transformed population for plants containing only T‐DNA sequences
Authors: Hanson Bill , Engler Dean , Moy York , Newman Bob , Ralston Ed , Gutterson Neal
Published: March 2003

A simple modification to standard binary vector design has been utilized to enrich an Agrobacterium‐transformed population for plants containing only T‐DNA sequences. A lethal gene was incorporated into the non‐T‐DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T‐DNA vectors (non‐T‐DNA lethal gene‐containing T‐DNA vectors). The lethal gene used here is a CaMV 35S‐barnase gene with an intron in the coding sequence (barnase‐INT); the screenable marker is a pMAS‐luciferase gene with an intron in the coding sequence (LUC‐int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T‐DNA vector or a control vector from which most of the barnase‐INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non‐T‐DNA sequences in the transgenic population using the NTL T‐DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the efficiency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar‐expressing tobacco line was trans‐ formed, indicating that barnase expression was responsible for the reduced frequency of incorporating non‐T‐DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase‐INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium‐transformed population for plants con‐ taining only T‐DNA sequences.