bluuebunny/crossref_metadata_2025 huggingface dataset:
A wheat histone H3 promoter confers cell division‐dependent and ‐independent expression of the gus A gene in transgenic rice plants
Authors: Terada Rie , Nakayama Takuya , Iwabuchi Masaki , Shimamoto Ko
Published: March 2003

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to −1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (−1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division‐dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division‐independent detected in flower tissues including the anther wall and the pistil. In this study, replication‐dependent expression occurring in non‐dividing cells which undergo endoreduplication could not be discriminated from strict replication‐independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5′ sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, −908H3/GUS and −185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from −909 to −1711 contains the positive cis‐acting element(s) and that the proximal promoter region (up to −185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division‐dependent and ‐independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division‐dependent expression of plant genes is discussed.
The psaA operon pre‐mRNA of the Euglena gracilis chloroplast is processed into photosystem I and II mRNAs that accumulate differentially depending on the conditions of cell growth
Authors: Stevenson Jennifer K. , Hallick Richard B.
Published: March 2003

The chloroplast genome of Euglena gracilis contains a psaA operon which encodes a lysine tRNA gene, trnK; psaA and psaB photosystem I genes, and psbE, psbF, psbL and psbJ photosystem II genes. The pre‐mRNA of the psaA operon undergoes a complex processing pathway of 5′ and 3′ tRNA processing, splicing of 11 group II introns and one group II twintron, plus three intercistronic RNA cleavage events. The accumulated transcripts of the psaA operon have been characterized by Northern hybridization, S1 nuclease analysis and primer extension RNA sequencing. The mature 5′ end of the psaA—psaB—psbE—psbF—psbL—psbJ hexacistronic transcript lies 8 nt downstream of the trnK gene, and is the result of intercistronic trnK—psaA cleavage. Other intercistronic processing events occur between the psaA and psaB genes and the psaB and psbE genes. Processing at the latter site produces a dicistronic mRNA of PSI genes and a tetracistronic mRNA of PSII genes. The PSI dicistronic transcript is further processed to monocistronic psaA and psaB mRNAs. Secondary structural motifs within the intercistronic regions may be recognition sites for processing. The steady‐state levels of psaA operon mRNAs from Euglena grown under several different conditions have been determined. Accumulated transcripts from all growth conditions are spliced, and a proportion are also processed at the intercistronic sites. The products of intercistronic processing increase from heterotrophic dark‐ to heterotrophic light‐grown Euglena, and from heterotrophic light‐ to photoautotrophic light‐grown Euglena. The differential accumulation of psaA operon mRNAs may be a means of chloroplast gene regulation or, alternatively, a consequence of gene expression during chloroplast development.
Analysis of the cucumber malate synthase gene promoter by transient expression and gel retardation assays
Authors: Graham Ian A. , Baker Christopher J. , Leaver Christopher J.
Published: March 2003

Recently it has been demonstrated that the single‐copy malate synthase (MS) and isocitrate lyase (ICL) genes from cucumber are regulated by nutritional status in cucumber cell cultures. In this paper a new cucumber mesophyll protoplast transient expression system is described in which electroporated MS promoter—GUS reporter gene constructs exhibit the same pattern of expression as the endogenous MS gene. Both the electroporated MS—GUS constructs and the endogenous gene are expressed when protoplasts are cultured for 48 h on a non‐metabolizable carbon source such as mannitol or 3‐methylglucose, and repressed when cultured on a utilizable carbon source such as sucrose, glucose or fructose. A series of deletion mutants identified a region from position −248 to −125 relative to the start of transcription that is essential for expression of the MS—GUS construct under the different metabolic conditions. A 191 bp fragment spanning this region was fused, in both orientations, to the CaMV 35S core promoter. A pattern of metabolic regulation similar to that of the intact MS promoter was observed for these promoter fusion constructs which strongly suggests the presence of enhancer element(s) within this region. Comparison of the 191 bp region with other MS and ICL promoter sequences revealed a region of homology, designated RT. A gel retardation assay was used to assess binding of the 191 bp fragment to whole cell protein extracts from cell cultures expressing MS. Both the unlabelled 191 bp fragment and a synthetic oligonucleotide of RT compete specifically for the demonstrated binding activity.
ABA‐regulated promoter activity in stomatal guard cells
Authors: Taylor Jane E. , Renwick Kirstie F. , Webb Alex A.R. , McAinsh Martin R. , Furini Antonella , Bartels Dorothea , Quatrano Ralph S. , Marcotte William R. , Hetherington Alistair M.
Published: March 2003

CDeT6‐19 is an ABA‐regulated gene which has been isolated from Craterostigma plantagineum. The CDeT6‐19 gene promoter has been fused to the β‐glucuronidase reporter gene (GUS) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum. This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed‐specific, ABA‐regulated gene, Em, or the promoter from the ABA‐regulated CDeT27‐45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA‐regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA‐induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca2+ and other second messengers in ABA‐induced gene expression.
An Arabidopsis thaliana root‐specific kinase homolog is induced by dehydration, ABA, and NaCl
Authors: Hwang Inhwan , Goodman Howard M.
Published: March 2003

Genomic and cDNA clones have been isolated for an Arabidopsis thaliana gene, ARSK1, that encodes a protein with structural similarities to serine/threonine kinases. Expression of ARSK1 is root specific and is induced by exposing roots to air during growth or by treatment of roots with ABA or NaCl. ARSK1 gene expression in transgenic plants is confined to cells in the tissues of the root as measured by β‐glucuronidase (GUS) expression from an ARSK1 gene promoter—GUS gene construct. Transverse sections of the stained roots further defined the tissue‐specificity; high levels of expression in the epidermal, endoepidermal and cortex regions, but no or very little expression in the vascular system. Another feature of the expression pattern of the ARSK1 gene was a gradual increase in the expression level along the root with the highest level of expression in the region closest to the root meristem. These studies suggest that ARSK1 may have a role in the signal transduction pathway of osmotic stress.
Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis
Authors: Shirley Brenda W. , Kubasek William L. , Storz Gisela , Bruggemann Edward , Koornneef Maarten , Ausubel Frederick M. , Goodman Howard M.
Published: March 2003

Eleven loci that play a role in the synthesis of flavonoids in Arabidopsis are described. Mutations at these loci, collectively named transparent testa (tt), disrupt the synthesis of brown pigments in the seed coat (testa). Several of these loci (tt3, tt4, tt5 and ttg) are also required for the accumulation of purple anthocyanins in leaves and stems and one locus (ttg) plays additional roles in trichome and root hair development. Specific functions were previously assigned to tt1–7 and ttg. Here, the results of additional genetic, biochemical and molecular analyses of these mutants are described. Genetic map positions were determined for tt8, tt9 and tt10. Thin‐layer chromatography identified tissue‐ and locus‐specific differences in the flavonols and anthocyanidins synthesized by mutant and wild‐type plants. It was found that UV light reveals distinct differences in the floral tissues of tt3, tt4, tt5, tt6 and ttg, even though these tissues are indistinguishable under visible light. Evidence was also uncovered that tt8 and ttg specifically affect dihydroflavonol reductase gene expression. A summary of these and previously published results are incorporated into an overview of the genetics of flavonoid biosynthesis in Arabidopsis.
Genetic engineering of a quantitative trait: metabolic and genetic parameters influencing the accumulation of laurate in rapeseed
Authors: Voelker Toni A. , Hayes Thomas R. , Cranmer Ann M. , Turner Joann C. , Davies H. Maelor
Published: March 2003

Laurate can be produced in the seed reserve oil of Brassica napus (rapeseed) by the expression of an heterologous lauroyl acyl‐carrier protein thioesterase under the control of a napin seed‐storage protein promoter. Analysis of a large number of transgenic events, and their progeny after self‐pollination, shows that laurate can accumulate to nearly 60% of the triglyceride acyl groups. Up to 40 mol% laurate the phenotype is correlated positively with the number of thioesterase gene copies. The use of a tandem gene construct elevates the average laurate content. This effect correlates with an increased average number of T‐DNA insertions per event; no cis‐inactivation of tandem genes is apparent. Above 40 mol% laurate other factors apparently limit the phenotype. The expression timing conferred by the napin promoter is unlikely to be limiting, as it covers almost the entire period of oil deposition. A more significant limitation resides in the second acylation reaction of oil biosynthesis, as shown by the very low incorporation of laurate at the sn‐2 acyl group. The novel, high‐laurate oil is consequently rich in sn‐1,3‐dilauroyl triglycerides, but its unusual composition appears to pose no problems for mobilization during seed germination.
Distinct roles for light‐dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in higher plants
Authors: Runge Sönke , Sperling Ulrich , Frick Geneviève , Apel Klaus , Armstrong Gregory A.
Published: March 2003

Light‐dependent NADPH:protochlorophyllide oxidoreductase (POR), a nuclear‐encoded plastid‐localized enzyme, catalyzes the photoreduction of protochlorophyllide (Pchlide) to chlorophyllide in higher plants, algae and cyanobacteria. Angiosperms require light for chlorophyll (Chl) biosynthesis and have recently been shown to contain two POR‐encoding genes, PorA and PorB, that are differentially regulated by light and developmental state. PorA expression rapidly becomes undetectable after illumination of etiolated seedlings, whereas PorB expression persists throughout greening and in adult plants. In order to study the in vivo functions of Arabidopsis POR A and POR B we have abolished the expression of PorA through the use of the phytochrome A‐mediated far‐red high irradiance response. Wild‐type seedlings grown in continuous far‐red light (cFR) display the morphology of white light (WL)‐grown seedlings, but contain only traces of Chl and do not green upon transfer to WL. This cFR‐induced greening defect correlates with the absence of PorA mRNA, the putative POR A protein, phototransformable Pchlide‐F655, and with strongly reduced POR enzymatic activity in plant extracts. In contrast, a cFR‐grown phyA mutant expresses the PorA gene, accumulates Chl and visibly greens in WL. Furthermore, constitutive overexpression of POR A in cFR‐grown transgenic Arabidopsis wild‐type seedlings restores Chl accumulation and WL‐induced greening. These data demonstrate that POR A is required for greening and that the availability of POR A limits Chl accumulation during growth in cFR. POR B apparently provides a means to sustain light‐dependent Chl biosynthesis in fully greened, mature plants in the absence of phototransformable Pchlide‐F655.
GCAC1 recognizes the pH gradient across the plasma membrane: a pH‐sensitive and ATP‐dependent anion channel links guard cell membrane potential to acid and energy metabolism
Authors: Schulz‐Lessdorf Barbara , Lohse Gabi , Hedrich Rainer
Published: March 2003

Ion channels in the plasma membrane of guard cells provide key mechanisms in signal perception and volume regulation during stomatal movement. Recent studies have suggested that the strongly voltage‐dependent, inactivating guard cell anion channel (GCAC1) acts as a sensor of the ambient extracellular CO2 concentration and as a target of modulation by nucleotides and Ca2+ ions. Applying the patch‐clamp technique it is demonstrated here that GCAC1 is activated by cytoplasmic ATP in a pH‐dependent manner.

When the apoplastic pH was buffered to 5.6 and the cytosolic pH dropped step‐wise from 7.8 to 5.6, the single‐channel activity increased as a function of proton concentration. This pH‐sensitivity is characterized by a titratable site with an apparent pK value around 6.9. While the steepness and direction of the transmembrane pH gradient did not affect the kinetics of activation, deactivation and fast inactivation of the whole‐cell anion current, the kinetics of slow inactivation and reactivation were strongly influenced. When at a constant intracellular proton concentration of pH 7.2 the external pH decreased from 7.2 to 5.6 the time constants of slow inactivation and the half‐times of reactivation increased two‐ and sevenfold, respectively.

The mechanism of nucleotide activation of GCAC1 was quantitatively analysed on the level of single‐channel events. Using inside‐out, cell‐free membrane patches, GCAC1 half‐activated around 0.4 mM ATP. The sigmoidal dose‐dependence of anion channel activation could be well fitted with an apparent Hill coefficient of 3.6. This behaviour might indicate that the activation process involves a strongly cooperative interaction of four ATP‐binding sites. Neither ATP nor its non‐hydrolysable analogue AMP‐PMP, which also activated GCAC1, altered the voltage‐dependent gating. AMP‐PMP stimulation and the insensitivity of GCAC1 towards the phosphatase inhibitor, okadaic acid, and the kinase inhibitors, staurosporine and H‐7, provided evidence that nucleotide binding rather than phosphorylation caused channel activation.

Since the time‐ and voltage‐dependent activity of GCAC1 is strongly modulated by ATP and protons, this channel is capable of sensing changes in the energy status, acid metabolism and the H+ ATPase activity of guard cells.
Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil‐body formation in seeds
Authors: Sarmiento Cristina , Ross Joanne H.E. , Herman Eliot , Murphy Denis J.
Published: March 2003

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T2 seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed‐specific and peaked at the mid‐late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS‐PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density‐gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10–1.14 g ml−1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane‐associated oleosins could be immunoprecipitated in the presence of protein A‐Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy‐immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti‐oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/ processing changes in the protein. Although oil‐body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil‐body size in desiccation‐tolerant seeds.
Exchanging sequence domains between S‐RNases from Nicotiana alata disrupts pollen recognition
Authors: Zurek Daniel M. , Mou Beiquan , Beecher Brian , McClure Bruce
Published: March 2003

In self‐incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S‐locus. Pollen tube growth is inhibited in the style when its single S‐allele matches either S‐allele present in the diploid pistil. Each S‐allele encodes an S‐RNase with a unique sequence. S‐RNases are secreted into the extracellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S‐RNases are the determinants of S‐allele specificity in the pistil, it is not known how allele‐specific information is encoded in the sequence. Therefore, we exchanged domains between S‐RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata SA2‐ and SC10‐RNases were exchanged. Among these nine constructs, the entire S‐RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S‐RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing SA2‐ and SC10‐RNase. However, none of the chimeric S‐RNases caused rejection of either SA2‐ or SC10‐pollen. We conclude that the recognition function of S‐RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S‐RNase is not localized to a specific domain.
Nuclear behavior is defective in the maize (Zea mays L.) lethal ovule2 female gametophyte
Authors: Sheridan William F. , Huang Bing‐Quan
Published: March 2003

It has long been known that the maize lethal ovule2 mutation results in ovule abortion but has a much smaller effect on pollen development or function. The behavior of the nuclei, the microtubular cytoskeleton and other events were examined in normal and lo2 mutant female gametophytes in order to obtain an understanding the role of this gene in embryo sac formation. The effect of the lo2 mutation is manifested following meiosis. When the surviving single megaspore carries the mutant lo2 allele, often both the megaspore and its nucleus greatly enlarge, but the nucleus either fails to divide or divides only once or twice. Micronuclei are frequently present, nuclei are often clustered and the abundance and patterns of microtubules are abnormal in the mutant embryo sacs. The mutant female gametophytes are blocked at the one‐, two‐ or four‐nucleate stage. Nearly all the embryo sacs containing the lo2 allele fail to function as evidenced by the failure of transmission of closely linked loci. When mutant female gametophyte development is arrested, the immature embryo sac degenerates. This mutation appears to identify a gene that is essential in the female gametophyte for normal nuclear division and migration and the normal accompanying tubulin cytoskeleton behavior.
Azide‐sensitive thylakoid membrane insertion of chimeric cytochrome f polypeptides imported by isolated pea chloroplasts
Authors: Mould Ruth M. , Knight Jacqueline S. , Bogsch Erik , Gray John C.
Published: March 2003

Post‐translational integration of cytochrome f into thylakoid membranes was observed after import by isolated pea chloroplasts of a chimeric protein consisting of the presequence of the small subunit of ribulose 1,5‐bisphosphate carboxylase fused to the cytochrome f precursor. Import of a similar chimeric protein lacking the C‐terminal 33 amino acid residues resulted in a soluble cytochrome f protein in the thylakoid lumen, indicating that the C‐terminal region contains a stop‐transfer sequence for membrane integration. Azide inhibited the insertion of cytochrome f into the thylakoid membrane, whereas the ionophores nigericin and valinomycin had little effect on membrane insertion. The precursor of the 33 kDa protein, but not the 23 kDa protein, of the photosystem II oxygen‐evolving complex inhibited the thylakoid insertion of cytochrome f, suggesting competition for a component of the transport pathway. These experiments suggest that the post‐translational insertion of cytochrome f into the thylakoid membrane uses a SecA‐dependent pathway.
Correlation between activities of starch debranching enzyme and α‐polyglucan structure in endosperms of sugary‐1 mutants of rice
Authors: Nakamura Yasunori , Kubo Akiko , Shimamune Tomoyuki , Matsuda Toshiaki , Harada Kyuya , Satoh Hikaru
Published: March 2003

The biochemical lesion of the sugary‐1 mutation was examined in five different mutants of rice with varying phenotypes but with mutations at the same locus. The cells in the inner part of the endosperm of all mutants tested contained phytoglycogen instead of starch, while the cells located in the outer part of the endosperm tissue from some mutants were filled with numerous starch granules. The molecular size of phytoglycogen was markedly smaller than that of amylopectin as measured by Sephacryl S‐1000 chromatography. Analysis of the distribution of α‐1,4 chain lengths revealed that in phytoglycogen the number of A‐chains dramatically increased, while long B chains with DP ≥ 37 remarkably decreased or were almost absent, which resulted in the disappearance of the cluster structure. The results suggest that changes in the balance of enzymic activities induced by the mutations brought about a drastic alteration in polyglucan structure and the shape of the polyglucan granule. The greater the extent of phytoglycogen regions in su1 endosperm tissues became, the greater was the phytoglycogen content, and the greater the reduction in the activity of starch debranching enzyme, a type of enzyme referred to as R‐enzyme (RE), limit dextrinase or pullulanase. Immunoblot analysis showed that the reduction in RE activity was due to a decrease in the amount of RE protein, and that the reduction in RE was specific since proteins of starch‐branching enzymes I and IIa and ADP‐glucose pyrophosphorylase were not markedly affected by su1 mutations. The proportion of starch region to the whole endosperm tissue of various su1 mutants was correlated with the RE activity in these endosperms. The results strongly suggest that the reduction in RE activity is involved in the su1 phenotype and that the enzyme plays an essential role in determining the fine structure of the amylopectin molecule
Two TIP‐like genes encoding aquaporins are expressed in sunflower guard cells
Authors: Sarda X. , Tousch D. , Ferrare K. , Legrand E. , Dupuis J.M. , Casse‐Delbart F. , Lamaze T.
Published: March 2003

SunTIP7 and SunTIP20 are closely related sunflower cDNAs showing a deduced amino acid sequence homologous to proteins of the tonoplast intrinsic protein (TIP) family. Their expression in Xenopus oocytes caused a marked increase in osmotic water permeability (demonstrating that they are water channels) which was sensitive to mercury. In leaves, in situ hybridization revealed that both SunTIP7 and SunTIP20 mRNA accumulated in the guard cells. The possible involvement of SunTIPs in stomatal movement was examined by comparing the time course of transcript accumulation and leaf conductance during the daily cycle and following a water limitation. SunTIP7 mRNA fluctuations fitted changes occurring in leaf conductance. The transcript levels were markedly and systematically increased during stomatal closure. It is suggested that aquaporin SunTIP7 facilitates water exit associated with a decrease in guard cell volume. In the same conditions, the transcript level of SunTIP20 remained constant indicating that SunTIP genes are differentially regulated within the same cell.
AtLTP1 luciferase expression during carrot somatic embryogenesis
Authors: Toonen Marcel A.J. , Verhees John A. , Schmidt Ed D.L. , Van Kammen Ab , De Vries Sacco C.
Published: March 2003

The carrot (Daucus carota L.) EP2 gene encodes a Lipid Transfer Protein (LTP) which is expressed during protoderm formation in developing embryos. To develop a vital reporter system for gene expression during somatic embryo development a 1.1 kB fragment of the Arabidopsis thaliana LTP1 promoter was fused to the firefly luciferase (LUC) coding sequence. The AtLTP1 luciferase expression pattern in transformed carrot suspension cultures was identical to the expression pattern of the endogenous carrot EP2 gene. Cell tracking experiments revealed that all somatic embryos were derived from AtLTP1 luciferase expressing cell clusters. However, not all cell clusters that expressed the AtLTP1 luciferase reporter gene developed into a somatic embryo, suggesting that initiation of an embryogenic pathway in tissue culture does not always lead to development of a somatic embryo.
Drought‐ and wound‐induced expression in leaves of a gene encoding a chromoplast carotenoid‐associated protein
Authors: Chen Hsu‐Ching , Klein Annelyse , Xiang Minghui , Backhaus Ralph A. , Kuntz Marcel
Published: March 2003

A study has been carried out to investigate the regulation of the fibrillin (fib) gene, along with two carotenoid biosynthesis genes, namely those encoding geranylgeranyl pyrophosphate synthase (ggpps) and capsanthin‐capsorubin synthase (ccs) from bell pepper (Capsicum annuum), whose expression is greatly induced during fruit ripening. A homologous transient expression assay has shown that high expression of these genes in pepper fruit is regulated essentially at the transcriptional level. Transcription of ccs is mainly fruit‐specific and that of ggpps is highly induced in fruits. Expression of fib is more complex: it is induced not only by a developmental process in fruits but also, in pepper and tobacco leaves, by diverse environmental factors such as drought and mechanical wounding. The wound‐induced transcriptional activation of fib is light‐ and oxygen‐dependent. Evidence is provided for the involvement of superoxide anion production within plastids in the signalling pathway leading to induction of this nuclear gene by environmental stresses. Specific activation of this promoter in roots, but not in leaves, was also observed upon exogenous abscisic acid treatment. Drought or wounding also leads to the accumulation of the fibrillin polypeptide in leaves. Furthermore, a low level of fibrillin has also been detected in the leaves of non‐stressed plants. Taken together, our data suggest a general role for fibrillin in various plastid types and in response to environmental stresses, in addition to its function in assembly of carotenoid‐containing fibrils in chromoplasts.
A response‐regulator homologue possibly involved in nitrogen signal transduction mediated by cytokinin in maize
Authors: Sakakibara Hitoshi , Suzuki Megumi , Takei Kentaro , Deji Atsushi , Taniguchi Mitsutaka , Sugiyama Tatsuo
Published: March 2003

A cDNA clone, pZmCip1, encoding a maize (Zea mays) cytokinin‐inducible protein 1 was isolated utilizing the differential display technique, and studied using the expression of ZmCip1 in nitrogen‐starved maize plants. The cloned cDNA contained an open reading frame consisting of 157 amino acids with a predicted molecular mass of 16.7 kDa, which possesses similarity with the response‐regulators of bacterial two‐component signalling systems. In detached leaves, accumulation of ZmCip1 transcript by t‐zeatin was dose‐dependent in a range of 10–9 M to 10–7 M, and occurred within 30 min after treatment. The effect of t‐zeatin was replaceable by isopentenyladenosine or isopentenyl‐adenosine‐5′‐monophosphate. Pretreatment of detached leaves with cycloheximide did not inhibit the accumulation of the transcript. In whole plants, ZmCip1 transcript was transiently accumulated exclusively in leaves by supply of nitrate or ammonium ions to the roots, whereas the transcript was not accumulated in detached leaves by supply of the nitrogen nutrients. Both the cytokinin‐ and nitrate‐responsive accumulations of ZmCip1 transcript were accompanied by an increase in the immunotitratable protein. Isopentenyladenosine and/or its phosphorylated form(s) accumulated in roots 2 h after supply of nitrate to plants. These results, taken together, suggest that ZmCip1 is a primary response gene to cytokinins, and that it involves, at least in part, the nitrogen‐signal transduction mediated by cytokinin in maize.
Induced systemic resistance in wounded rice plants
Authors: Schweizer Patrick , Buchala Antony , Dudler Robert , Métraux Jean‐Pierre
Published: March 2003

Wounding of one leaf of young rice plants caused a strong and transient accumulation of non‐conjugated (–)‐JA, followed by induction of a number of pathogenesis‐related genes, in the treated leaf. The non‐treated leaf of wounded plants that emerged after the treatment was more resistant to challenge infection by the rice blast fungus Magnaporthe grisea (Hebert) Barr. The systemic leaf also showed a transient, but delayed accumulation of jasmonic acid. Unlike the wounded leaf, there was no accumulation of pathogenesis‐related mRNAs or proteins in the systemically protected leaf. Local application of jasmonic acid, the putative signal of the wound response, resulted in a similar degree of systemic disease resistance as wounding. The results suggest the operation of systemic, wound‐related signalling processes in rice that induce systemic disease resistance.
A YAC contig map of Arabidopsis thaliana chromosome 3
Authors: Camilleri Christine , Lafleuriel Jacqueline , Macadré Catherine , Varoquaux Fabrice , Parmentier Yves , Picard Georges , Caboche Michel , Bouchez David
Published: March 2003

We have constructed a YAC contig map of Arabidopsis thaliana chromosome 3. From an estimated total size of 25 Mb, about 21 Mb were covered by 148 clones arranged into nine YAC contigs, which represented most of the low‐copy regions of the chromosome. YAC clones were anchored with 259 molecular markers, including 111 for which linkage information was previously available. Most of the genetic map was included in the YAC coverage, and more than 60% of the genetic markers from the reference recombinant inbred line map were anchored, giving a high level of integration between the genetic and physical maps. The submetacentric structure of the chromosome was confirmed by physical data; 3R (the top arm of the linkage map) was about 12 Mb, and 3L (the bottom arm of the linkage map) was about 9 Mb. This YAC physical map will aid in chromosome walking experiments and provide a framework for large‐scale DNA sequencing of chromosome 3.
Association of γ‐tubulin with kinetochore/centromeric region of plant chromosomes
Authors: Binarová Pavla , Hause Bettina , Doležel Jaroslav , Dráber Pavel
Published: March 2003

Monoclonal antibodies raised against a phylogenetically conserved peptide from the C‐terminal domain of γ‐tubulin molecule were used for immunofluorescence detection of γ‐tubulin in acentriolar mitotic spindles of plant cells. The antibodies stained kinetochore fibres along their whole length, including the close vicinity of kinetochores. After microtubule disassembly by the antimicrotubular drugs amiprophos‐methyl, oryzalin and colchicine, γ‐tubulin was found on remnants of kinetochore fibres attached to chromosomes. In cells recovering from the amiprophos‐methyl treatment, γ‐tubulin was localized with the re‐growing kinetochore microtubule fibres nucleated or captured by kinetochore/centromeric regions. On isolated chromosomes, γ‐tubulin co‐localized with α‐tubulin in the kinetochore/centromeric region. The data presented suggest that in acentriolar higher plant cells γ‐tubulin might be directly or indirectly involved in modulation and/or stabilization of kinetochore–microtubule interactions.
Expression of the Arabidopsisabi1–1mutant allele inhibits proteinase inhibitor wound‐induction in tomato
Authors: Carrera Esther , Prat Salomé
Published: March 2003

Abscisic acid (ABA) is an essential component in the wound signalling cascade. Increased levels of endogenous ABA were observed after wounding and shown to be a requisite for wound‐induced expression of the proteinase inhibitor II genes. We have taken advantage of the dominant character of the Arabidopsis

abi1–1

mutation, to investigate whether ABI1 has a function in ABA signalling in response to wounding. Transgenic tomato plants carrying copies of either the wild‐type

ABI1

or the mutant

abi1–1

alleles were obtained and assayed for wound‐induction of the

pin2

or

LAP

genes. While normal levels of gene induction were observed in the transgenic

ABI1

plants, the

abi1–1

transformants displayed a severe wilty phenotype and reduced seed dormancy. Expression of the

abi1–1

dominant mutation blocked accumulation of the drought‐induced

TAS14

and

LE25

mRNAs in response to ABA, as well as ABA‐ and wound‐induced expression of the defense‐associated

pin2

and

LAP

transcripts. MeJA‐induction of the

pin2

and

LAP

mRNAs, on the contrary, was not affected in the

abi1–1

transformants. These results indicate that

abi1–1

inhibits wound‐induced expression of the

pin2

and

LAP

transcripts by blocking ABA‐induction of these genes. This implicates ABI1 in wound‐signalling and suggests that a common early ABA signalling pathway may function in the responses to wounding and water stress.
Towards functional characterisation of the members of theR2R3‐MYBgene family fromArabidopsis thaliana
Authors: Kranz Harald D. , Denekamp Marten , Greco Raffaella , Jin Hailing , Leyva Antonio , Meissner Ruth C. , Petroni Katia , Urzainqui Ana , Bevan Michael , Martin Cathie , Smeekens Sjef , Tonelli Chiara , Paz‐Ares Javier , Weisshaar Bernd
Published: March 2003

Transcription factors containing a conserved DNA‐binding domain similar to that of the proto‐oncogene

c‐myb

have been identified in nearly all eukaryotes. MYB‐related proteins from plants generally contain two related helix‐turn‐helix motifs, the R2 and R3 repeats. It was estimated that

Arabidopsis thaliana

contains more than 100

R2R3‐MYB

genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated

.

Sequence data for more than 90 different

A. thaliana R2R3‐MYB

genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of

R2R3‐MYB

genes in addition to the characteristic DNA‐binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the

A. thaliana

genome.

R2R3‐MYB

gene expression levels were determined under more than 20 different growth conditions including hormone treatment, infection with pathogens and various stress conditions.

MYB

genes are specifically expressed in different tissues and physiological conditions, indicating the potential for involvement in various regulatory processes. The sequence and expression data together with the map positions of nearly all

MYB

genes in

A. thaliana

provide a substantial basis for further studies of this important group of transcription factors.
Early post‐embryonic root formation is specifically affected in the maize mutantlrt1
Authors: Hochholdinger Frank , Feix Günter
Published: March 2003

The isolation and detailed characterisation of the maize mutant

lrt1

, which is completely deficient in the initiation of lateral roots at the primary and seminal lateral roots and of the crown roots at the coleoptilar node is described. The monogenic and recessive mutant was isolated from a segregating EMS mutagenised population, maps to the short arm of chromosome 2, and acts independently of the nodal root deficient

rtcs

locus. Histological analysis revealed that the mutation acts at a very early stage of root initiation, as indicated by the absence of primordia formation in the affected roots. At later stages of plant development lateral and crown root initiations recover leading to fertile plants. If grown in the dark, the mutant does not form an elongated mesocotyl, although the photomorphogenic response appears to be normal in the mutant. Furthermore, the wild‐type cannot be rescued from mutants by the application of auxin to germinating kernels. The gene impaired in

lrt1

seems to be of great importance for the general mechanism of early post‐embryonic root initiation, both from root and nodal tissues, since lateral and crown root initiation are both affected to the same extent and in the same transient time pattern.
NADH oxidase activity of soybean plasma membranes oscillates with a temperature compensated period of 24 min
Authors: Morré D. James , Morré Dorothy M.
Published: March 2003

The enzymatic activity of a hormone‐stimulated and growth‐related protein disulfide‐thiol oxidoreductase (NADH oxidase) with protein disulfide‐thiol interchange activity of etiolated hypocotyls of soybean oscillates with a period of about 24 min or 60 times per 24 h day. The oscillations were temperature compensated such that the period remained constant at about 24 min between 17 and 37°C, a temperature range over which enzymatic activity varied approximately fourfold (Q10 of 2). The oscillations were observed with intact tissue sections, with isolated plasma membrane vesicles, and with the detergent‐solubilized and partially purified enzyme. The oscillations were observed both with the oxidation of NADH and in the restoration of activity to scrambled ribonuclease used as a measure of the protein disulfide‐thiol interchange activity. The enzymatic activity which is located at the cell surface may represent a biochemical reaction with a potential function as an ultradian oscillator of circadian time keeping.
Low temperature regulation of theArabidopsisCBF family of AP2 transcriptional activators as an early step in cold‐inducedCORgene expression
Authors: Gilmour Sarah J. , Zarka Daniel G. , Stockinger Eric J. , Salazar Maite P. , Houghton Jaimie M. , Thomashow Michael F.
Published: March 2003

Cold‐induced expression of the

Arabidopsis COR

(cold‐regulated) genes is mediated by a DNA regulatory element termed the CRT (C‐repeat)/DRE (dehydration‐responsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic

Arabidopsis
 plants at non‐acclimating temperatures inducesCORgene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of theArabidopsisgenome along with genetic mapping experiments indicated that the threeCBFgenes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all threeCBFgenes increased within 15 min of transferring plants to low temperature, followed by accumulation ofCORgene transcripts at about 2 h.CBFtranscripts also accumulated rapidly in response to mechanical agitation. The promoter regions of theCBFgenes do not contain the CRT sequence, CCGAC, and overexpression ofCBF1did not have a detectable effect onCBF3transcript levels, suggesting that theCBFgene family is not subject to autoregulation. We propose that cold‐induced expression of CRT/DRE‐containingCORgenes involves a low temperaturestimulated signalling cascade in whichCBFgene induction is an early event.
Regulation of arbuscule formation by carbon in the plant
Authors: Blee Kristopher A. , Anderson Anne J.
Published: March 2003

Arbuscules are proposed to be the key site of interchange of carbon between root cells and the hyphae of arbuscular mycorrhizal (AM) fungi. This paper addresses how carbon availability is a driving force in regulating location and function of arbuscules in cortical cells. We discuss physical and biological limitations on arbuscule position. Altered expression, specifically in the arbusculated cell, of genes that govern sucrose hydrolysis may create a sink for sucrose in these cells. We propose a role for vacuolar invertase and cytoplasmic sucrose synthase in catalyzing the intracellular hydrolysis of sucrose, thus maintaining a gradient for symplastic influx of sucrose into the arbusculated cell and establishing a gradient for hexose efflux to the apoplast for fungal utilization. AM fungi may regulate hydrolysis of sucrose by stimulating the expression and activities of plant invertases by the production of plant hormones as well as through acidification of the arbuscular interface. We speculate that altered plant defense gene expression in arbusculated cells is consistent with regulation by sugar‐sensing mechanisms.
A male gametophyte‐specific monosaccharide transporter inArabidopsis
Authors: Truernit Elisabeth , Stadler Ruth , Baier Kerstin , Sauer Norbert
Published: March 2003

The

AtSTP2

gene (sugar transport protein 2) of

Arabidopsis thaliana

encodes a high affinity, low specificity monosaccharide carrier that can transport a number of hexoses and pentoses at similar rates. AtSTP2 has 12 putative transmembrane helices and a molecular mass of 55.0 kDa.

AtSTP2

expression was localized in

AtSTP2

promoter‐β‐glucuronidase (

GUS

)

Arabidopsis

plants showing

AtSTP2

‐driven GUS activity during pollen maturation and also in germinating pollen. Immunohistochemical studies with anti‐AtSTP2 antiserum as well as RNA

in situ

hybridization analyses modified these results and showed that

AtSTP2

expression is confined to the early stages of gametophyte development. Both

AtSTP2

mRNA and AtSTP2 protein are first seen at the time of beginning callose degradation and microspore release from the tetrades.

AtSTP2

mRNA and AtSTP2 protein are no longer detected after the mitotic divisions and the formation of the trinucleate gametophyte. No

AtSTP2

mRNA or AtSTP2 protein is seen in fully developed or germinating pollen. The putative role of AtSTP2 in the uptake of glucose units resulting from callose degradation during pollen maturation is discussed.
Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon‐cyclase is down‐regulated during ripening and is elevated in the mutantDelta
Authors: Ronen Gil , Cohen Merav , Zamir Dani , Hirschberg Joseph
Published: March 2003

The red colour of tomato (

Lycopersicon esculentum

) fruits is provided by the carotenoid pigment lycopene whose concentration increases dramatically during the ripening process. A single dominant gene,

Del

, in the tomato mutant

Delta

changes the fruit colour to orange as a result of accumulation of δ‐carotene at the expense of lycopene. The cDNA for lycopene ε‐cyclase (

CrtL‐e

), which converts lycopene to δ‐carotene, was cloned from tomato. The primary structure of CRTL‐E is 71% identical to the homologous polypeptide from

Arabidopsis

and 36% identical to the tomato lycopene β‐cyclase, CRTL‐B. The

CrtL‐e

gene was mapped to a single locus on chromosome 12 of the tomato linkage map. This locus co‐segregated with the

Del

gene. In the wild‐type tomato, the transcript level of

CrtL‐e

decreases at the ‘breaker' stage of ripening to a non‐detectable level in the ripe fruit. In contrast, it increases approximatley 30‐fold during fruit ripening in the

Delta

plants. The

Delta

mutation does not affect carotenoid composition nor the mRNA level of

CrtL‐e

in leaves and flowers. These results strongly suggest that the mutation

Del

is an allele of the gene for ε‐cyclase. Together with previous data, our results indicate that the primary mechanism that controls lycopene accumulation in tomato fruits is based on the differential regulation of expression of carotenoid biosynthesis genes. During fruit development, the mRNA levels for the lycopene‐producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase, while the mRNA levels of the genes for the lycopene β‐ and ε‐cyclases, which convert lycopene to either β‐ or δ‐carotene, respectively, decline and completely disappear.