Regulation of the maizerab17 gene promoter in transgenic heterologous systems

The maizerab17 gene is expressed in different plant parts in response to ABA and osmotic stress (J. Vilardellet al., Plant Mol Biol 14 (1990) 423–432). Here we demonstrate that 5 upstream sequences of therab17 gene confer the appropriate patterns of expression on the chloramphenicol acetyl transferase (CAT) reporter gene in transgenic tobacco plants, as well as in protoplasts derived from cultured rice cells. Specifically, a CAT construct containing a large 5 upstream fragment ofrab17 (–1330/+29) results in high levels of CAT activity in embryos, leaves and roots of transgenic plants subjected to water stress or ABA treatment. Transient expression assays in rice protoplasts transfected with CAT genes fused torab17 promoter deletions indicate that a 300 bp DNA fragment (–351/–102) is sufficient to confer ABA responsiveness upon the reporter gene. Furthermore, a 100 bp sequence (–219/–102) is capable of conferring ABA responsiveness upon a minimal promoter derived from the 35S CaMV promoter. Gel retardation experiments indicate that maize nuclear proteins bind to this fragment. This region of 100 bp contains a sequence (ACGTGGC) which has been identified as an abscisic acid response element in studies of other ABA-responsive plant genes.     

Nature of light requirement for the flowering of Chenopodium rubrum L. (Ecotype 60° 47′N)

It has been shown that induction of flowering in Chenopodium rubrum L. (ecotype 60° 47 N) seedlings in BCJ light conditions is intensity dependent (Cumming, 1969, Canad. J. Bot. 47, 1247–1250) and that, this intensity dependence is not based on photosynthesis (Sawhney and Cumming, 1971, Can. J. Bot. 49, 2133–2137). Since BCJ light emits a high proportion of energy in the far-red, and the High Energy Reaction (HER) has its action maxima in the far-red and blue regions of the spectrum, we tested the involvement of HER in the light induction of flowering in C. rubrum. Our results show that optimum intensities of blue light are effective in inducing flowering in C. rubrum. Red light exposure does not lead to flower induction. We suggest the HER may be involved in the flower induction of C. rubrum in light. However, when high energy in blue and/or farred is provided in presence of energy between 500–700 nm wavebands, there is no flowering in C. rubrum. We suggest that flower inducing activity of HER may be counteracted by flower inhibitory action of red wavebands.This paper constitutes a part of a Ph. D. thesis submitted to the University of Western Ontario, London, Ontario     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects.The 1.9 kb 5-upstream fragment (–1559 to +342) of the CatA gene was fused with the Escherichia coli -glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between –1564 to –699. Abscisic acid (ABA) at a final concentration of 10^–6 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at –266 to –254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light.The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.author for corresondenceThe nucleotide sequence data reported will appear in the DDBJ EMBL and GenBank Nucleotide Sequence Databases under the accession number D29966.     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5 ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5 region of the MVK gene to the -glucuronidase (GUS) reporter gene, indicated that the MVK 5-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 5-flanking region are crucial for high-level MVK gene expression.     

Constitutive and light-induced DNAseI hypersensitive sites in the rbcS genes of pea (Pisum sativum)

The chromatin structure of pea (Pisum sativum) rbcS genes in inactive (root), potentially active (dark-grown leaf), and active states (light-grown leaf) was analysed using (a) pancreatic DNAseI to detect general DNAseI sensitivity and DNAseI-hypersensitive sites, and (b) methyl-sensitive restriction endonucleases to probe for cytosine methylation within the promoter region. We showed that within the same organ individual members of the pea rbcS multigene family are differentially sensitive to DNAseI suggesting differential protection in nuclei. During light activation general DNAseI sensitivity increases in some genes, especially their 5 upstream regulatory sequences. DNAseI-hypersensitive sites are constitutively present in 5 upstream regulatory sequences around positions –335, –465, –650, and –945 (5 constitutive domain) and in the coding region around position +340, +450, +530, +640, and +810 (3 constitutive domain). One additional hypersensitive site appears after light induction (inducible site). This region is centred around position –190 and flanked by light-responsive elements (LREs). In spite of changes in the chromatin structure of rbcS genes during their transition from an inactive to an active state, their cytosine methylation at Alu I, Fnu 4HI, Hae III, Sau 3AI and Sau 96I sites in the promoter region remains uniform.     

Dissection of a pollen-specific promoter from maize by transient transformation assays

We have previously reported the isolation and characterization of a gene (Zm 13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm 13 5 flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5 regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the -glucuronidase (GUS) gene under the control of various sized fragments of the Zm13 5 flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from –100 to –54, while other sequences which amplify that expression reside between –260 and –100. The replacement of the normal terminator with a portion of the Zm13 3 region containing the putative polyadenylation signal and site also increased GUS expression. While the –260 to –100 region contains sequences similar to other protein-binding domains reported for plants, the –100 to –54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

Primary structure of the Synechococcus PCC 7942 PAPS reductase gene

The structural gene encoding a thioredoxin-dependent 5-phosphoadenylyl sulphate (PAPS) reductase (EC 1.8.4.-) from cyanobacterium Synechococcus PCC 7942 (Anacystis nidulans) was detected by heterologous hybridization with the cysH gene from Escherichia coli K12. The cyanobacterial gene (further called par gene) comprised 696 nt which are 57.8% homologous to the enterobacterial gene. The putative open reading frame encoded a polypeptide consisting of 232 amino acid residues (deduced molecular weight 26635) which showed significant homologies to the polypeptide from E. coli (50.8%) and to the polypeptide from Saccharomyces cerevisiae (30.3%). A single cysteine located at the C-terminus of the polypeptide of E. coli (Cys₂₃₉) was conserved in Synechococcus. Conservation of this cysteinyl residue seems indispensable for catalysis. Complementation of a cysH-deficient mutant of E. coli by the cyanobacterial gene indicated that the cloned DNA is the structural gene of the PAPS reductase.Abbreviations IPTG isopropyl-1-thio--D-galactoside - PAPS 3-phosphoadenosine-5-phosposulphate     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

In situ monitoring of chlorophyll fluorescence to assess the synergistic effect of low temperature and high irradiance stresses inSpirulina cultures grown outdoors in photobioreactors

A chlorophyll fluorescence technique was applied to anin situ study on the effects of low temperature and high light stresses onSpirulina cultures grown outdoors in controlled tubular photobioreactors at high (1.1 g L^–1) and low (0.44 g L^–1) biomass concentrations. Diurnal changes in PSII photochemistry (F _v/F _m) after 15 min of darkness, or in the light (dF/F _m), and non-photochemical (qN) quenching were measured using a portable, pulse-amplitude-modulated fluorometer. The depression of theF _v/F _m ratio ofSpirulina cultures grown outdoors at 25°C (i.e. 10°C below optimum for growth) and 0.44 g L^–1, reached 30% at the middle of the day. At the same time of the day thedF/F _m ratio showed a reduction of up to 52%. The depression of bothF _v/F _m anddF/F _m was lower in the cultures grown at 1.1 g L^–1. Photoinhibition reduced the daily productivity of the culture grown at 0.44 g L^–1 and 25°C by 33% with respect to that grown at 35°C. Changes in the growth yields of the cultures grown under different temperatures and growth rates correlate well with analogous changes in photon yield (dF/F _m). Simple measurements of photochemical yield (F _v/F _m) can be used to test the physiological status ofSpirulina cultures. The results indicate that the saturating pulse fluorescence technique, when usedin situ, is a powerful tool for assessment of the photosynthetic characteristics of outdoor cultures ofSpirulina.     

Role of red light in hair-whorl formation induced by blue light in Acetabularia mediterranea

After a pre-treatment with red light, hair formation at the growing tip of the siphonaceous green alga Acetabularia mediterranea Lamour. (= A. acetabulum (L.) Silva) can be induced by a pulse of blue light. Red light is needed again after the inductive blue-light pulse if the new whorl of hairs is to develop within the next 24 h. In order to investigate the role of this red light, the duration of the red irradiation was varied and combined with periods of darkness. The response of hair-whorl formation was dependent on the total amount of red light, regardless of whether the red irradiation followed the blue pulse immediately or was separated from it by a period of darkness. Furthermore, periods of exposure to the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1-1dimethylurea had a similar effect to darkness. Both observations indicate that this red irradiation acts as a light source for photosynthesis. Whether or not the red light had an additional effect via phytochrome was tested in another type of experiment. The dependence of hair-whorl formation on red-light irradiance in the presence of simultaneous far-red irradiation was determined for the pre-irradiation period as well as for the irradiation period after the blue pulse. In both experiments, far-red light caused a small promotion of hair-whorl formation when low irradiances of red light were used. However, these differences were attributable to a low level of photosynthetic activity (which in fact was measurable) caused by red light reflected in the growth chamber. Furthermore, lowering the proportion of active phytochrome by far-red light would be expected to suppress hair-whorl formation. The influence of far-red light was also tested in a strain of Acetabularia mediterranea that developed hair whorls in about 20% of cells even when kept in complete darkness after the blue-light pulse. Far-red irradiation had no effect. These results strongly indicate that phytochrome is not involved in hair-whorl formation. Rather it is concluded that the effects of red light are caused by photosynthesis.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy ABA (7OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2′ promoter wound-responsive elements

A 318 bp mannopine synthase 2 (mas2) promoter element from the T-DNA of Agrobacterium tumefaciens can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants. Wound inducibility is further enhanced by sucrose in the medium. Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from –318 to –213. DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors. Regions extending from –103 to +66 and from –213 to –138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated (–209) CaMV 35S promoter, respectively. DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2 TATA box. In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves. Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves. We propose a model to describe the involvement of nuclear factors with mas2 promoter elements in wound-inducible gene expression.     

Regulation of electrical coupling between Arabidopsis root hairs

Voltage clamp was used to measure the voltage dependence of cell-to-cell coupling via plasmodesmata between higher-plant cells (root hairs of Arabidopsis thaliana (L.) Heynh.). In addition, ionophoresis was used to introduce a variety of ions [Ca²⁺, inositol-trisphosphate, Li⁺, K⁺, Mg²⁺, ethylene glycol-bis(-aminoethyl ether)-N,N,N, N-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), H⁺, and OH^–] to examine whether they regulate cell-to-cell coupling. Electrical coupling showed high variability in this single cell type at the same developmental stage; the coupling ratio ranged from near 0% to about 90% with a mean value of 32%. It was voltage independent for intracellular voltage gradients (transplasmodesmatal) of -163 to 212 mV. While Ca²⁺ closes the plasmodesmatal connections (at concentrations higher than those causing cessation of cytoplasmic streaming), inositol-trisphosphate and lithium are without effect. Apparently, inositol-trisphosphate may not cause increased cytosolic Ca²⁺ in root hairs. Alkalinization by OH ionophoresis caused a modest decline in cell-to-cell coupling, as did acidification by H⁺ ionophoresis (to an extent causing the cell to become flacid). Increases in cytosolic K⁺, Mg²⁺, and the calcium chelator BAPTA by ionophoresis had no effect on cell-to-cell coupling. The regulation (and lack thereof) reported here for plant plasmodesmata is quite similar to that of gap junctions.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid