bluuebunny/crossref_metadata_2025 huggingface dataset:
ABA‐induced ion efflux in stomatal guard cells: multiple actions of ABA inside and outside the cell
Authors: MacRobbie E.A.C.
Published: March 2003

Abscisic acid (ABA) induces a transient stimulation of 86Rb+ efflux from isolated guard cells of Commelina communis L. The form of the efflux transients produced in suboptimal conditions (low concentrations of ABA and/or high external pH at which ABA will penetrate poorly) has been compared with the full transient. In suboptimal conditions the stimulation of efflux is both delayed and reduced. The pH‐dependence of the delay before initiation of the efflux transient suggests that a threshold internal concentration of ABA is required. However in suboptimal conditions even when the threshold internal concentration is reached and a transient is triggered, the degree of stimulation is reduced, an effect which also appears to depend on internal ABA. It is suggested that the differences reflect activation of different numbers of tonoplast ion channels for release of vacuolar ions. By contrast, the same end‐state seems to be reached in optimal and suboptimal conditions, but after different times. The relative efflux stimulation during the efflux transient tracks the declining ion content; both the peak and the end of the transient are reached at the same ion content, but at different times. It is suggested that this reflects an ABA‐induced change in the set‐point of a regulated ion channel which is sensitive to ion content, perhaps a stretch‐activated channel. This effect is independent of external concentration in the range 0.1–10 µM, and pH 6 and pH 8 are equally effective, suggesting an external site of action. Thus the results suggest multiple actions of ABA, involving both internal and external receptors. Regulation of both tonoplast ion channels by internal ABA, and of a regulated channel responsive to ion content by external ABA are suggested.
Subunit E of the vacuolar H+‐ATPase of Hordeum vulgare L.: cDNA cloning, expression and immunological analysis†
Authors: Dietz Karl‐Josef , Rudloff Susanne , Ageorges Agnès , Eckerskorn Christoph , Fischer Karsten , Arbinger Bettina
Published: March 2003

A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two‐dimensional gels. Amino acid sequences were determined from LysC endoproteinase‐peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H+‐ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26 228 g mol−1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free‐flow electrophoresis. Immunoprecipitation of newly synthesized 35S‐labelled membrane proteins with anti‐TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co‐purification of TpP 31 with the 55 kDa subunit of the H+‐ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H+‐ATPase.
The distribution of a spliceosome protein in cereal (Triticeae) interphase nuclei from cells with different metabolic activities and through the cell cycle
Authors: Glyn M.C.P. , Leitch A.R.
Published: March 2003

A monoclonal antibody (mAb) KSm2 and three human Sm sere, which detect the ‘D’ polypeptide in mammals and is associated with the U1, U2, U4/U6, U5, U7, U9–U12 small nuclear RNAs, have been used in Western blotting to show that the antigen is conserved in wheat (Triticum aestivum cv. Beaver). Immunocytochemistry to wheat and a barley (Hordeum vulgare) suspension culture using the mAb KSm2 has shown that the ‘D’ polypeptide occurs in interphase nuclei as speckles and foci outside the nucleoli and as tracks. Nucleoli usually had at least one focus at their periphery. Immunocytochemistry at the electron microscope level of resolution has shown that the signal occurs between chromatin axes and predominantly in the outer domain of the nucleus. Analysis of the barley suspension culture demonstrated that there was a significant increase in antigen through G1, S to G2. In the wheat meristematic cells at prophase only the foci remained, and at metaphase the distribution of foci was asymmetrical with foci occurring either at one pole or on the metaphase plate. At telophase the foci appeared to decrease in size and were incorporated non‐uniformly into the daughter nuclei because of their asymmetric distribution at metaphase. When wheat was grown at a range of temperatures, the root tip meristematic nuclei showed a number of different organization patterns. The average number of nucleoli increased and their size decreased. At the same time there was an increase in the average total area of foci as temperature increased from 4°, 10°, 25°, 34°, to 37°C. Thus the ‘D’ polypeptide distribution changes with metabolic activity and through the cell cycle.
The use of an alternative promoter in the Arabidopsis thaliana HMG1 gene generates an mRNA that encodes a novel 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase isoform with an extended N‐terminal region
Authors: Lumbreras Victoria , Campos Narciso , Boronat Albert
Published: March 2003

The enzyme 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGR, EC 1.1.1.34) catalyses the synthesis of mevalonate, the committed precursor of the great variety of isoprenoid compounds and derivatives synthesized in higher plants. It has previously been reported that Arabidopsis thaliana contains two differentially expressed genes, HMG1 and HMG2, that encode two HMGR isoforms (HMGR1 and HMGR2, respectively). This paper reports the characterization of a novel HMGR mRNA (HMGR1L mRNA) derived from the HMG1 gene. This mRNA is initiated 121 bp upstream from the transcription start site previously characterized. In contrast with the previously reported HMGR1 mRNA (HMGR1S mRNA), which is detected at high levels in all tissues of the plant, HMGR1L mRNA is present at relatively low levels and its expression is restricted mostly to seedlings, roots and inflorescences. HMGR1L and HMGR1S mRNAs are transcribed from alternative promoters. HMGR1L mRNA contains an in‐phase AUG start codon which allows the synthesis of a novel HMGR isoform (HMGR1L) having 50 additional amino acid residues at its N‐terminal end. Using an in vitro transcription‐translation system we have shown that HMGR1L is inserted into ER‐derived microsomes. It is thus unlikely that the extended N‐terminal region of HMGR1L might have a role in targeting the enzyme to plastids or mitochondria. These results support the previous proposal that the endoplasmic reticulum is the only cell compartment for the primary targeting of HMGR in Arabidopsis and reinforce the view that plant HMGR is under the control of complex mechanims operating at both transcriptional and post‐transcriptional levels.
Spatial structure of mitochondria and ER denotes changes in cell physiology of cultured tobacco protoplasts
Authors: Stickens Didier , Verbelen Jean‐Pierre
Published: March 2003

The structure of mitochondria and of the endoplasmic reticulum (ER) in mesophyll protoplasts and regenerated cells was studied in vivo using the dye DiOC6(3) (3,3′‐dihexyloxacarbocyanine iodide) and confocal laser scanning microscopy (CLSM). The relation to the cell’s physiology was investigated using a hormone‐based model system for elongation and division. The structure of the mitochondria and of their population depends on the status of the cell. In freshly isolated protoplasts small spherical mitochondria are clustered around the nucleus and the chloroplasts. During the first 4 days of culture they are transformed into long vermiform organelles which distribute evenly throughout the cytoplasm. In a medium containing only auxins, cells then enter a period of expansion. Their mitochondria retain the same structure but increase in quantity. In a medium with auxins and cytokinins cells start dividing. Their mitochondria typically become numerous and very small, and are distributed throughout the cytoplasm. Both types of organization were observed during weeks of ongoing expansion or division. The ER is always present as a network close to the cell membrane. In freshly isolated protoplasts a considerable part of the ER is clustered around the chloroplasts, the remaining part of the network has a reduced complexity and is partly broken. During subsequent protoplast culture the network grows into a complex web with fine meshes incorporating lots of plate‐like structures. This is the case in elongating cells as well as in dividing cells. Finally, the ER looks similar to the structure found in epidermal cells of the intact plant.
A rice bZIP protein, designated OSBZ8, is rapidly induced by abscisic acid
Authors: Nakagawa Hitoshi , Ohmiya Kunio , Hattori Tsukaho
Published: March 2003

A cDNA that encoded a bZIP protein, designated OSBZ8, was isolated from a rice embryo cDNA library by use of degenerate oligonucleotide probes that corresponded to the amino acid sequences conserved among the basic regions of plant G‐box‐binding factor‐type bZIP proteins (GBF). OSBZ8 was shown to have structural features typical of the GBF‐type bZIP proteins and to bind to G‐box and G‐box‐like sequences that include ABA‐responsive elements (ABREs) which have been functionally identified in the promoters of ABA‐inducible genes, such as Em, Osem and Rab16. The accumulation of OSBZ8 mRNA was induced by treatment with ABA of imbibed mature rice embryos, of young plant tissues and of suspension‐cultured cells. The accumulation of OSBZ8 mRNA in response to ABA preceded that of Osem and Rab16A mRNAs and was not inhibited by an inhibitor of protein synthesis, cycloheximide. By contrast, the induction of Osem and Rab16A was partially inhibited and almost completely inhibited, respectively, by cycloheximide. These results strongly suggest that OSBZ8 might be involved in the regulation of transcription by ABA in rice.
Carbon dioxide induces increases in guard cell cytosolic free calcium
Authors: Webb Alex A.R. , McAinsh Martin R. , Mansfield Terry A. , Hetherington Alistair M.
Published: March 2003

The hypothesis that increases in cytosolic free calcium ([Ca2+]i) are a component of the CO2 signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura‐2 fluorescence ratio photometry to measure changes in guard cell [Ca2+]i in response to challenge with 700 µl l−1 CO2. Elevated CO2 induced increases in guard cell [Ca2+]i which were similar to those previously reported in response to abscisic acid. [Ca2+]i returned to resting values following removal of the CO2 and further application of CO2 resulted in a second increase in [Ca2+]i. This demonstrated that the CO2‐induced increases in [Ca2+]i were stimulus dependent. Removal of extracellular calcium both prevented the CO2‐induced increase in [Ca2+]i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca2+ acts as a second messenger in the CO2 signal transduction pathway and that an increase in [Ca2+]i may be a requirement for the stomatal response to CO2.
A plant kinesin heavy chain‐like protein is a calmodulin‐binding protein
Authors: Reddy A.S.N. , Narasimhulu S.B. , Safadi F. , Golovkin M.
Published: March 2003

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin‐binding protein, PKCBP, was isolated from a potato expression library using protein‐protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and 35S‐labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C‐terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP‐ and microtubule‐binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N‐terminus and a putative coiled‐coil region in the middle. The calmodulin‐binding region was mapped to a stretch of 64 amino acid residues in the C‐terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide‐dependent manner. The kinesin heavy chain‐like calmodulin‐binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin‐binding domain. The presence of a calmodulin‐binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.
A benzothiadiazole derivative induces systemic acquired resistance in tobacco
Authors: Friedrich Leslie , Lawton Kay , Ruess Wilhelm , Masner Peter , Specker Nicole , Rella Manuela Gut , Meier Beatrice , Dincher Sandra , Staub Theodor , Uknes Scott , Métraux Jean‐Pierre , Kessmann Helmut , Ryals John
Published: March 2003

Systemic acquired resistance (SAR) is a pathogen‐induced disease resistance response in plants that is characterized by broad spectrum disease control and an associated coordinate expression of a set of SAR genes. Benzo(1,2,3)‐thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) is a novel synthetic chemical capable of inducing disease resistance in a number of dicotyledenous and monocotyledenous plant species. In this report, the response of tobacco plants to BTH treatment is characterized and the fact that it controls disease by activating SAR is demonstrated. BTH does not cause an accumulation of salicylic acid (SA), an intermediate in the SAR signal transduction pathway. As BTH also induces disease resistance and gene expression in transgenic plants expressing the nahG gene, it appears to activate the SAR signal transduction pathway at the site of or downstream of SA accumulation. BTH, SA and TMV induce the PR‐1a promoter using similar cis‐acting elements and gene expression is blocked by cycloheximide treatment. Thus, BTH induces SAR based on all of the physiological and biochemical criteria that define SAR in tobacco.
The developmentally regulated bZIP factor ROM1 modulates transcription from lectin and storage protein genes in bean embryos
Authors: Chern Maw‐Shenq , Eiben Hans Georg , Bustos Mauricio M.
Published: March 2003

Expression of the MAT genes DLEC2 and PHSβ, coding for phytohemagglutinin L‐subunit and β‐phaseolin, respectively, is coordinately induced in bean embryos at the onset of seed maturation and regulated at the level of transcription. Previously, it was reported that expression of DLEC2 and PHSβ genes is positively regulated by the acidic transcription activator PvALF. This article describes the regulator of MAT1 (ROM1), a basic/leucine‐zipper protein that binds to hybrid G box/C box motifs found on the DLEC2 and PHSβ seed enhancers. ROM1 repressed DLEC2‐ and PHSβ‐driven expression and antagonized transactivation of either promoter by PvALF, in a dosage‐dependent manner. By contrast, a PvALF/ROM1 hybrid protein activated transcription. Both repression by ROM1 and activation by the hybrid protein required the presence of intact G‐box/C‐box motifs. The abundance of ROM1 mRNA in embryos was highest during the cotyledon stage and decreased rapidly at the beginning of maturation, as phytohemagglutinin and phaseolin mRNAs were induced. The results demonstrate that ROM1 is a DNA‐binding site‐dependent repressor and point to its possible role as a negative modulator of PvALF‐activated transcription of MAT genes.
Transport of chimeric proteins that contain a carboxy‐terminal targeting signal into plant microbodies
Authors: Hayashi Makoto , Aoki Masahiro , Kato Akira , Kondo Maki , Nishimura Mikio
Published: March 2003

Malate synthase is a glyoxysome‐specific enzyme. The carboxy‐terminal tripeptide of the enzyme is Ser—Arg—Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy‐terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of β‐glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy‐terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy‐terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeprides with S, A or P at the −3 position, K or R at the −2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.
Cloning of PCP1, a member of a family of pollen coat protein (PCP) genes from Brassica oleracea encoding novel cysteine‐rich proteins involved in pollen‐stigma interactions
Authors: Stanchev Borislav S. , Doughty James , Scutt Charles P. , Dickinson Hugh , Croy Ronald R.D.
Published: March 2003

The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6–8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino‐acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT‐PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene (PCP1) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea, Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F2 segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine‐rich mature protein. The central part and C‐terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther‐specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S‐locus glycoproteins, but does not appear to be genetically linked to the S‐locus.
Specific expression of the AGL1 MADS‐box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development
Authors: Flanagan Catherine A. , Hu Yi , Ma Hong
Published: March 2003

Several members of the MADS‐box gene family have been shown to be important regulators of flower development, controlling such well‐studied early events as the formation of the floral meristem and the specification of floral organ identity. Other floral‐specific MADS‐box genes, of as yet unknown function, have been isolated by homology and are proposed to be part of a regulatory hierarchy controlling flower development. Some of these genes might regulate later aspects of flower development, such as development of individual floral organs, which is less well studied at the molecular level. This paper presents a detailed analysis of the expression pattern of one such gene from Arabidopsis, AGL1, using RNA in situ hybridization. It is found that AGL1 is specifically expressed in particular regions of the gynoecium and ovule, only during and after floral development stage 7. AGL1 expression at the tip of the growing carpel primordia, along the margins of the ovary valves in developing and mature gynoecia and in specific regions of developing and mature ovules provides important insights into the possible roles of AGL1. It is proposed that AGL1 may have regulatory functions in the structural definition and/or function of the valve margins, in axis maintenance during ovule development, in nutritional supply to the growing ovule and embryo sac, and in pollen tube guidance. In the floral homeotic mutants ag‐1, ap3‐3 and ap2‐2, AGL1 mRNA is expressed in an organ‐dependent manner, suggesting that AGL1 is a carpel‐specific gene and as such ultimately depends upon the carpel identity gene AG for proper gene expression.
Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis‐related expression under high light
Authors: Chamnongpol Sangpen , Willekens Hilde , Langebartels Christian , Van Montagu Marc , Inzé Dirk , Van Camp Wim
Published: March 2003

Transgenic tobacco deficient in either Cat1 (Cat1AS), Cat2 (Cat2AS), or both (CatGH) was generated through sense and antisense technology. Cat1AS, Cat2AS, and CatGH plants showed no visible phenotype when grown at low light (100 µmol m−2 sec−1. Under these conditions, deficiency in Cat1 and/or Cat2 did not lead to constitutive pathogenesis‐related (PR‐1) expression and did not potentiate PR‐1 induction by exogenous salicylic acid. This demonstrates that catalase suppression per se is not a sufficient signal for PR‐1 induction. In Cat1‐deficient plants exposed to higher light intensities (250–1000 µmol m−2 sec−1), PR‐1 expression was induced without pathogenic challenge and multiplication of Pseudomonas syringae pv. syringae was repressed. Yet, it is unlikely that Cat1 deficiency is mimicking the mode of action of salicylic acid in tobacco, because, concurrent with PR‐1 induction, Cat1 deficiency at high light provoked severe leaf damage, characterized by white necrotic lesions. Taken together, these results do not support the model that catalase inactivation is the key route by which salicylic acid induces PR defense responses in healthy tissue. However, because catalase deficiency is potentially lethal to leaves, catalase inactivation by salicylic acid could be of importance in the establishment of hypersensitive responses.
Isolation and characterization of rtcs, a maize mutant deficient in the formation of nodal roots
Authors: Hetz Winfried , Hochholdinger Frank , Schwall Michael , Feix Günter
Published: March 2003

The root system of maize consists of the primary root and a variable number of lateral seminal‐, crown‐ and brace roots. Except for the primary root and some minor roots forming at the mesocotyl, all other roots grow out of nodal regions, namely, the embryogenic scutellar node and the underground—as well as the lower above‐ground stem nodes. Besides their role in water and nutrient uptake, some of these roots (crown‐ and brace roots) are essential for the lodging resistance of the plants. This property of the crown roots has now been successfully used for screening a segregating F2 population of a cross between a flint inbred line and an En transposon line. Two allelic root‐deficient mutants have been isolated and have been designated rtcs‐1 and rtcs‐2 for their complete lack of formation of crown‐ and lateral seminal roots. They survive by the ability of the primary root to support the growth of the developing plant. The monogenic and recessive mutants appear to be affected in an early root‐forming function since no primordia are formed either in the case of embryo‐borne lateral seminal or stem‐derived crown roots. The Rtcs locus could be mapped to the short arm of chromosome 1 with the help of a co‐segregating RAPD marker. The effect of the mutation seems to be highly specific since no pleiotropic effects on other parts of the plants have been observed. The formation of adventitious roots can, however, still be induced in the mesocotyl region of the mutant.
An agarose gel electrophoresis method for the separation of arabinogalactan proteins
Authors: Stöhr Christine , Snogerup Lars , Pennell Roger I. , Kjellbom Per
Published: March 2003

A method for resolving plasma membrane associated arabinogalactan proteins (AGPs) has been developed. Plasma membranes purified by aqueous polymer two‐phase partitioning were first subjected to Triton X‐114 fractionation. The resulting water phase contained all detectable plasma membrane‐bound AGPs. Plasma membrane AGPs were then resolved in an SDS‐agarose gel electrophoresis system (SDS‐AGE). For separating plasma membrane AGP species of the same apparent molecular weight but with different net charge, a two‐dimensional electrophoresis system was used, utilizing isoelectric focusing in an immobilized pH gradient in the first dimension and SDS‐AGE in the second dimension. These methods enabled the separation of individual plasma membrane AGPs. In comparison, SDS‐PAGE methods left AGPs as unresolved high molecular‐weight smears. The methods described here may help to establish some basic features of AGPs, such as the number, organization, and protein and carbohydrate characteristics of plasma membrane AGPs, as well as the relationship between plasma membrane and extracellular AGPs.
A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70‐complexed and competent for flavinylation, membrane association and enzymatic activation
Authors: Al‐Babili Salim , Lintig Johannes v. , Haubruck Heinz , Beyer Peter
Published: March 2003

A cDNA coding for the carotenoid biosynthetic enzyme phytoene desaturase from Narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane‐bound populations of phytoene desaturase in the chromoplasts isolated from petals. The soluble form is enzymatically inactive and a constituent of a larger Hsp 70‐containing protein complex in the stroma, whereas the membrane‐bound form is functional. In vitro, the soluble form is able to associate on to/into protein‐free liposomal membranes made from chromoplast lipids, thereby gaining activity by binding added flavine adenine dinucleotide (FAD). Once bound to membranes, activated phytoene desaturase works independently of any added FAD, employing membrane‐bound electron acceptors. FAD, however, exerts no positive effect on the membrane‐association process. Its role is confined to enzymatic activation. Although carotenoid accumulation is strongly induced during flower development, only very low concentrations of phytoene desaturase transcripts are detectable, while the corresponding protein accumulates in low, but measurable amounts, appearing in soluble and membrane‐bound states. Post‐transcriptional mechanisms contribute significantly to carotenoid accumulation, as do factors determining the enzymatic activity of phytoene desaturase, for example by influencing the redox‐state of membrane‐bound electron acceptors.
Fungal pathogens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis‐related endo‐β‐1,3‐glucanases
Authors: Ham Kyung‐Sik , Wu Sheng‐Cheng , Darvill Alan G. , Albersheim Peter
Published: March 2003

Plant endo‐β‐1,3‐glucanases and chitinases inhibit the growth of some fungi and generate elicitor‐active oligosaccharides while depolymerizing polysaccharides of mycelial walls. Overexpression of the endo‐β‐1,3‐glucanases and/ or chitinases in transgenic plants provides, in some cases, increased protection against fungal pathogens. However, most of the phytopathogenic fungi that have been tested in vitro are resistant to endo‐β‐1,3‐glucanases and chitinases. Furthermore, some phytopathogenic fungi whose growth is inhibited by these enzymes are able to overcome the effect of these enzymes over a period of hours, indicating an ability of those fungi to adapt to the enzymes. Evidence is presented indicating that fungal pathogens secrete proteins that inhibit selective plant endo‐β‐1,3‐glucanases.A glucanase inhibitor protein (GIP‐1) has been purified to homogeneity from the culture fluid of the fungal pathogen of soybeans, Phytophthora sojae f. sp. glycines (Psg), and two basic pathogenesis‐related endo‐β‐1,3‐glucanases (EnGLsoy‐A and EnGLsoy‐B) have been purified from soybean seedlings. GIP‐1 inhibits EnGLsoy‐A but not EnGLsoy‐B. Moreover, GIP‐1 does not inhibit endo‐β‐1,3‐glucanases secreted by Psg itself nor does GIP‐1 inhibit PR‐2c, a pathogenesis‐related endo‐β‐1,3‐glucanase of tobacco. Evidence is presented that Psg secretes other GIPs that inhibit other endo‐β‐1,3‐glucanase(s) of soybean. Furthermore, GIP‐1 does not exhibit proteolytic activity but does appear to physically bind to EnGLsoy‐A. The results reported herein demonstrate specific interactions between gene products of the host and pathogen and establish the need to consider fungal proteins that inhibit plant endo‐β‐1,3‐glucanases when attempting to use the genes encoding endo‐β‐1,3‐glucanases to engineer resistance to fungi in transgenic plants.
Interactions between the ABI1 and the ectopically expressed ABI3 genes in controlling abscisic acid responses in Arabidopsis vegetative tissues
Authors: Parcy François , Giraudat Jérôme
Published: March 2003

In Arabidopsis, the abscisic acid (ABA)‐Insensitive ABI1 and ABI3 genes have been proposed to act in separate ABA signalling cascades. Recessive mutations in ABI3 alter various physiological processes during seed development, whereas the dominant abi1 mutation inhibits ABA responses largely in vegetative tissues. The seed‐specific ABI3 gene was ectopically expressed in the vegetative tissues of transgenic Arabidopsis plants carrying a transcriptional fusion between the CaMV 35S promoter and the ABI3 cDNA. Genetic interactions between the ectopically expressed ABI3 and endogenous ABI1 genes were investigated by monitoring diverse ABA responses in vegetative tissues. Ectopic expression of ABI3 conferred to plantlets the ability to accumulate the seed‐specific At2S3 and AtEm1 mRNAs in response to ABA, and the abi1 mutation inhibited this ABI3‐dependent induction of AtEm1 by ABA. Furthermore, ectopic expression of ABI3 also influenced ABI1‐dependent responses that occur in wild‐type vegetative tissues. Expression of ABI3 increased ABA induction of the Rab18 mRNA and ABA inhibition of root growth, and both responses were sensitive to the abi1 mutation in the presence as in the absence of ABI3. Finally, although ABI3 is thought to be a transcription activator and stomatal regulation is not known to involve transcriptional events, the ectopically expressed ABI3 gene suppressed the effect of the abi1 mutation on stomatal regulation. The present data demonstrate that ABI1 and ABI3 genetically interact in controlling diverse ABA responses in transgenic vegetative tissues. The possibility that the endogenous ABI1 and ABI3 genes may similarly act in a common ABA signalling pathway in seed is discussed with previous phenotypic studies of the abi1 and abi3 mutants.
Tam3 produces a suppressible allele of the DAG locus of Antirrhinum majus similar to Mu‐suppressible alleles of maize
Authors: Chatterjee Manash , Martin Cathie
Published: March 2003

Tam3 from Antirrhinum majus belongs to the Ac/Ds family of transposable elements. An allele of the DAG locus of Antirrhinum (dag::Tam3), which is required for chloroplast development and leaf palisade differentiation, has been generated by Tam3 insertion into the untranslated leader sequence of the gene. This allele gives rise to a cold‐sensitive phenotype, where mutant tissue containing wild‐type revertant somatic sectors is observed in the leaves of plants grown at 15°C, while leaves of plants grown at 25°C appear near wild‐type. The temperature sensitivity of dag::Tam3 results from expression of the DAG locus responding to the activity of the transposable element, the transposition of which is very sensitive to growing temperature. Genetic suppression of Tam3 transposition, using the STABILISER locus, also results in suppression of the dag mutant phenotype. dag::Tam3 represents a Tam3‐suppressible allele similar to those described for Mu transposons in maize. Suppression of the dag mutant phenotype in response to element inactivation appears to result from use of an alternative promoter at the 3′ end of the Tam3 element. The production of suppressible alleles by an Ac‐like element is discussed in relation to the mutagenic potential of plant transposons in producing complex genetic diversity.
A promoter trap for Chlamydomonas reinhardtii: development of a gene cloning method using 5′ RACE‐based probes
Authors: Haring Michel A. , Beck Christoph F.
Published: March 2003

A promoterless radial spoke protein RSP3 gene has been used to identify promoter regions in the genome of Chlamydomonas reinhardtii. The acceptor strain pf‐14 arg7 was transformed with a linearized vector containing the ARG 7.8 gene as a selection marker and a promoterless RSP3 gene. The frequency at which the motility was restored in transformants varied from 2–3%. Several of these were motile only in ammonium‐free medium, indicating that the procedure could be used to select inducible promoters. Transformation of nitrogen‐starved cells produced about twice as many transformants which were only motile in ammonium‐free medium. Since one of the tagging vectors contained an RSP3 gene with a hybridization flag in its 3′ untranslated region, it was possible to estimate the size of the new RSP3 transcripts in transformants. The results suggested that in most cases a hybrid RNA was generated consisting of the tagged gene transcript and reporter gene RNA. By 5′ RACE, these parts of the new transcripts were amplified and it was shown that the generated DNA fragments could be used to clone a tagged gene. One such example, gene 2BC9, is predicted to code for a mitochondrial matrix protein. The tagging procedure will be optimized for cloning genes induced by nitrogen starvation, the cue for gametogenesis.
Agrobacterium tumefaciens‐mediated barley transformation
Authors: Tingay Sonia , McElroy David , Kalla Roger , Fieg Sarah , Wang Mingbo , Thornton Sarah , Brettell Richard
Published: March 2003

Genetically transformed barley was produced by eco‐cultivating immature embryo explants with Agrobacterium tumefaciens carrying a binary vector coding for chimaeric bacterial genes, bar and gus, and selecting for bialaphos‐resistant cultures from which plants were regenerated. Integration of both genes was confirmed by gel blot hybridization analysis of DNA from the transformed plants and their progenies. From 1282 embryos, plants were recovered for 54 independently transformed lines, giving a transformation efficiency of 4.2%. Transgene numbers in the different lines ranged from single copy insertion to at least ten copies. Sixteen out of 18 plants grown to maturity were fully fertile. Both marker genes, bar and gus, were expressed and co‐segregated in the T1 progeny plants. In the majority of cases, the genes showed Mendelian segregation predicted for transgene insertion at a single locus. In one family with multiple transgene insertions, molecular analysis of T1 and T2 plants suggested that the T‐DNA had inserted at two unlinked loci.
Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium‐mediated plant transformation
Authors: Schlaman Helmi R.M. , Hooykaas Paul J.J.
Published: March 2003

The usefulness of the E. coli codA gene encoding cytosine deaminase as a conditional toxic gene was explored during various stages of plant development and in different Agrobacterium‐mediated transformation protocols. To this end, several independent tobacco lines transgenic for codA were isolated and these were tested for their sensitivity to 5‐fluorocytosine (5‐FC) at different developmental stages. On media supplemented with 5‐FC, seedling proliferation was inhibited. Leaves failed completely to regenerate sprouts on 5‐FC‐containing medium. However, 40% of the shoots regenerated on non‐selective medium still formed roots on rooting medium with 5‐FC. In all these assays, control plants were unaffected by up to 1 mg m1−1 5‐FC. Transformation of a codA and nptll‐harbouring T‐DNA to tobacco leaf discs did not result in any regenerant using a combined 5‐FC and kanamycin selection, indicating that codA does not behave as a cell‐autonomous marker here. Nevertheless, transformation of the same T‐DNA to tobacco protoplasts resulted in some enrichment of codA−nptll+ calluses using the proper combination of 5‐FC and kanamycin for selection. Mixing of codA‐containing and codA‐lacking tobacco protoplasts revealed that the codA gene may behave as a cell autonomous marker under certain, appropriately chosen conditions, which seems to be in paradox with the total absence of escapes in tissue explant transformation. In all these experiments, 250 µg ml−1 5‐FC was found to be the most optimal for selection. Our results suggest that codA can be successfully used as a negative selectable marker in Agrobacterium‐mediated gene targeting protocols of tobacco whereby selection at the shoot regeneration level is the most effective.
Post‐transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats
Authors: Stam Maike , De Bruin Rob , Kenter Susan , Van Der Hoorn Renier A.L. , Van Blokland Rik , Mol Joseph N.M. , Kooter Jan M.
Published: March 2003

To induce post‐transcriptional silencing of flower pigmentation genes by homologous sense transgenes in transgenic petunias, it is not necessary for the transgenes to be highly transcribed. Even promoterless transgenes can induce silencing. Here it is shown that in these cases silencing is mediated by multimeric transgene/T‐DNA loci in which the T‐DNAs are arranged as inverted repeats (IRs). With the transgene constructs used, monomeric T‐DNA loci are unable to confer silencing even though they modulate IR‐induced silencing. IRsfs with the silencing sequences proximal to the centre (IRc) induce a more severe silencing than IRs with these sequences distal to the centre (IRn). Somatic reversion of silencing, as observed in a side branch of one of the chalcone synthase (Chs) transformants, was associated with a deletion of the IR locus from L1 cells, the meristematic cell layer that expresses the endogenous Chs genes in the flower corolla. Taken together, these data indicate that the post‐transcriptional silencing mechanism can be activated by inverted transgene repeats. It is also shown that a silent IR UidA—ChsA locus silences the expression of a monomeric 35S promoter‐driven UidA—ChsA transgene only in corollas where the endogenous Chs genes are highly transcribed. These results are consistent with a model in which an IR, by virtue of its palindromic sequence organization, is able to promote the production of aberrant RNAs from the endogenous homologs as a result of ectopic pairing.
Identification of the peroxisomal targeting signal for cottonseed catalase
Authors: Mullen Robert T. , Lee Michael S. , Trelease Richard N.
Published: March 2003

Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined. The most common peroxisome targeting signal (PTS) is a C‐terminal tripeptide composed of a conserved SKL motif (type 1 PTS). The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY‐2 suspension cells serving as an in vivo import system. To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA) epitope‐tagged at its N‐terminus. Bombardment with HA‐Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY‐2 cells. The C‐terminal tripeptide of Ccat, PSI, is necessary for import. Evidence for this were mislocalizations to the cytosol of PSI‐truncated Ccat and AGV‐substituted (for PSI) Ccat. PSI‐COOH, however, was not sufficient to re‐route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI‐COOH was sufficient. Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT‐KPSI) decreased import efficiency. However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g. CAT‐NVKPSI). Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non‐conserved residues (e.g. ‐AGVNVRPSI for ‐SRLNVRPSI). These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI‐COOH) whose residues are functionally dependent on a strict context of adjacent C‐terminal amino acid residues.
Expression of heterologous phytochromes A, B or C in transgenic tobacco plants alters vegetative development and flowering time
Authors: Halliday Karen J. , Thomas Brian , Whitelam Garry C.
Published: March 2003

In this study, oat phytochrome A (phyA), Arabidopsis phytochrome B (phyB) or Arabidopsis phytochrome C (phyC) were expressed in both day‐neutral and photo‐period‐sensitive (short‐day) tobacco (Nicotiana tabacum cv. Hicks). Introgression of the Maryland Mammoth (MM) gene into cv Hicks was used to confer short‐day photo‐periodic sensitivity. Expression of oat phyA led to characteristic hypersensitivity of hypocotyls to red light (R) and far‐red light (FR) and an overall dwarfing of the mature plant. Expression of Arabidopsis phyB enhanced the sensitivity of hypocotyls to R and caused even more marked dwarfing of the mature plant. In contrast, the expression of Arabidopsis phyC had no detectable consequences for the photocontrol of hypocotyl elongation. However, phyC expression did lead to a R‐dependent increase in cotyledon expansion in de‐etiolating seedlings and to a significant increase in leaf area in mature plants. This provides the first experimental evidence that phyC is biologically active. The flowering time of cv Hicks plants grown under 8 h photoperiods was virtually unaffected by a 30 min white light (W) night break given 8 h into the dark period. In contrast, cv Hicks MM plants responded to a night break with a delay in flowering. Expression of phyA or phyB led to a night break‐dependent delay in flowering in cv Hicks plants. For cv Hicks MM plants, the expression of any of phyA, phyB or phyC caused a marked enhancement of the flower‐delaying effect of a night break. These observations indicate that transgenic phyA, phyB or phyC can interact with the endogenous mechanisms controlling flowering time in tobacco.
Ozone induction of ethylene emission in tomato plants: regulation by differential accumulation of transcripts for the biosynthetic enzymes
Authors: Tuomainen Jaana , Betz Christian , Kangasjärvi Jaakko , Ernst Dieter , Yin Zu‐Hua , Langebartels Christian , Sandermann Heinrich
Published: March 2003

Stress ethylene emission is positively correlated with ozone sensitivity in various plant species, indicating that ethylene may be involved in the control of ozone damage. This study shows that ozone exposure of tomato plants for 5 h at 85 nl l−1 and above leads to leaf injury within 24 h. 1‐aminocyclopropane‐1‐carboxylic acid (ACC) content and ACC synthase activity were accordingly elevated within 1–2 h. Pre‐treatment of leaves with inhibitors of ACC synthase and ACC oxidase significantly inhibited the evolution of ethylene and reduced ozone‐induced visible damage. Transcript levels for only one out of three S‐adenosyl‐l‐methionine (SAM) synthetase genes (SAM3), and one out of four ACC synthase genes (LE‐ACS2) were induced by ozone (maximum at 2 h). Treatment with protein kinase (K‐252a) and phosphatase inhibitors (calyculin A) revealed that ACC synthase activity was additionally regulated by protein phosphorylation/dephosphorylation. Transcripts of ACC oxidase (pTOM13 cDNA probe) displayed the fastest response of the parameters tested (maximum at 30 min), suggesting a regulatory role for ACC oxidase in ethylene formation of ozone‐exposed plants. The results demonstrate a highly selective ozone response by ethylene biosynthetic genes which resembles that of plant—pathogen interactions.
Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose‐6‐phosphate synthase
Authors: Blázquez Miguel A. , Santos Elisa , Flores Carmen‐lisset , Martínez‐Zapater José M. , Salinas Julio , Gancedo Carlos
Published: March 2003

An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose‐6‐phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose‐6‐phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose‐6‐phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single‐copy gene and is expressed constitutively at very low levels.
Ectopic expression of a novel MYB gene modifies the architecture of the Arabidopsis inflorescence
Authors: Kirik Victor , Kölle Kerstin , Wohlfarth Thomas , Miséra Simon , Bäumlein Helmut
Published: March 2003

The Arabidopsis thaliana mutants fus3, lec1 and abi3 have pleiotropic defects during late embryogenesis. Mutant embryos fail to enter the maturation programme and initiate a vegetative germination pathway instead. Screening for genes which are differentially expressed in the fus3 mutant of Arabidopsis resulted in the isolation of several members of the MYB family. MYB domain proteins in plants represent an extended gene family of transcription factors, suggesting their participation in a variety of plant specific cellular functions. Here, the authors describe one of these genes, designated AtMYB13, representing a novel member of the MYB gene family. The structure of the gene as well as its genomic organisation and localisation are reported. The expression of the gene is regulated by dehydration, exogenous abscisic acid, light and wounding. A chimeric AtMYB13 promoter/GUS gene is tissue‐specifically expressed in transgenic Arabidopsis plants. The GUS staining was predominantly detected in the shoot apex zone and at the basis of developing flowers. In addition, the AtMYB13 gene promoter is active at branching points of the inflorescence. Furthermore, ectopic expression of the AtMYB13 gene has a characteristic impact on the architecture of the inflorescence leading to peculiar hook structures at pedicel branching points. In addition, some transgenic plants exhibit a reversed order of first flowers and axillary buds. These data suggest a function of the AtMYB13 gene product in linking shoot morphogenic activity with environmental as well as intrinsic signals.