The Plant Journal

Callos, Joseph D.; Medford, June I.

doi: 10.1046/j.1365-313X.1994.6010001.xpmid: N/A

Harrison, Maria J.; Dixon, Richard. A.

doi: 10.1046/j.1365-313X.1994.6010009.xpmid: N/A

A combination of Northern blot analysis and in situ hybridization was used to measure and localize changes in the levels of transcripts encoding five enzymes of phenylpropanoid/flavonoid/isoflavonoid metabolism in Medicago truncatula roots following colonization with the mycorrhizal fungus Glomus versiforme. In colonized roots, elevated levels of phenylalanine ammonia‐lyase (PAL) and chalcone synthase (CHS) transcripts were detected specifically in the cortical cells containing arbuscules. This localization was discrete, and elevated levels of transcripts were not observed in adjacent, non‐colonized cells. In contrast, isoflavone reductase (IFR) transcripts, which were detected at relatively high levels in the cortical cells of non‐colonized roots, were almost undetectable in the colonized cortical cells containing arbuscules and were also lowered in adjacent cells. The expression of chalcone isomerase (CHI) and isoliquiritigenin 2′‐O‐methyltransferase (ChalOMT) was unaffected by colonization, and the tissue‐specific patterns of gene expression were the same as in non‐colonized roots. It is concluded that the establishment of the mycorrhizal interaction results in cell type‐specific differential expression of genes of phenylpropanoid/flavonoid/isoflavonoid biosynthesis, which may be causally related to arbuscule development.

Franken, Philipp; Schrell, Susanne; Peterson, Peter A.; Saedler, Heinz; Wienand, Udo

doi: 10.1046/j.1365-313X.1994.6010021.xpmid: 7920701

Two maize genes, Zm 1 and Zm 38, related to the regulatory anthocyanin gene C1 were analyzed molecularly and used for fusion constructs in transient domain swapping experiments with the C1 wild‐type gene. It was shown that both genes (Zm 1 and Zm 38) influence the expression of the A1 locus, a target gene for C1. Zm 1 activates the A1 promoter, however it does not turn on the whole anthocyanin pathway. The Zm 38 gene product shows functions similar to C1‐I, a dominant inhibitor of the C1 wild‐type gene. Concerning the trans‐inhibition by C1‐I two effects seem to be involved, competition for binding and formation of heterodimers. Further analysis of C1 function was carried out by a fine structure analysis of C1 mutants induced by the insertion and excision of transposable elements. These experiments indicate that for the activating domain of the protein, the formation of an alpha helix seems to be more important than a high negative charge.

Warner, Simon A.J.; Gill, Aideen; Draper, John

doi: 10.1046/j.1365-313X.1994.6010031.xpmid: 7920702

Previous reports have described the induction, by either wounding or attempted pathogen invasion, of an Asparagus officinalis intracellular pathogenesis‐related (AoPR1) promoter—GUS gene fusion in transgenic tobacco. Here we describe the unexpected developmental expression pattern of the AoPR1—GUS gene which correlates well, temporally and spatially, with the developmental expression observed for GUS fusions with promoters derived from genes coding for enzymes in the ‘core phenylpropanoid pathway’. Analysis of endogenous AoPR1 gene expression in asparagus and both AoPR1—GUS and AoPR1—luciferase gene fusions in transgenic tobacco suggests that the AoPR1 promoter directs similar cell‐specific transcription patterns in both asparagus and transgenic tobacco. The AoPR1 promoter contains sequence motifs similar to those implicated as important in the regulation of phenylpropanoid pathway genes and another ‘intracellular’ PR gene. Treatment with salicylic acid enhances AoPR1 promoter gene activity both in tobacco and in asparagus.

Bouvier, Florence; Hugueney, Philippe; D'Harlingue, Alain; Kuntz, Marcel; Camara, Bilal

doi: 10.1046/j.1365-313X.1994.6010045.xpmid: 7920703

The late steps of carotenoid biosynthesis in plants involve the formation of xanthophylls. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xanthophyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6‐epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionality, the name capsanthin‐capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a full‐length cDNA clone encoding a capsanthin capsorubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capsanthin‐capsorubin synthase gene is specifically expressed during chromoplast development in fruits accumulating ketocarotenoids, but not in mutants impaired in this biosynthetic step.

Houba‐Hérin, Nicole; Domin, Monique; Pédron, Jacques

doi: 10.1046/j.1365-313X.1994.6010055.xpmid: 7920704

Nicotiana plumbaginifolia haploid protoplasts were co‐transformed with two plasmids, one with a NPT‐II/Ds element and one with a gene encoding an amino‐terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

Sauer, Norbert; Stolz, Jürgen

doi: 10.1046/j.1365-313X.1994.6010067.xpmid: 7920705

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy‐dependent, uncoupler‐sensitive loading into phloem cells via a sucrose‐H+ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy‐dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy‐dependent manner. The KM‐values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m‐chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH‐group inhibitors. The VMAX but not the KM‐values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1‐His6 cDNA encoding a histidine tag at the SUC1 C‐terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.

Debel, Karsten; Knack, Gaby; Kloppstech, Klaus

doi: 10.1046/j.1365-313X.1994.6010079.xpmid: N/A

The expression of the 23 kDa plastid heat‐shock protein (HSP) of Chenopodium rubrum has been studied at various light intensities at a temperature of 38°C where the 23 kDa protein accumulates to its highest levels. It was observed that the level of mRNA which is induced at this heat‐shock temperature is independent of the light intensity between 0 and 1000 W m−2. Labelling in vivo of all investigated HSP is also not dependent on the light fluxes applied. In clear contrast the accumulation of the mature chloroplast HSP 23 is light dependent: while almost no protein is detectable in the dark the level of the accumulated protein reaches a maximum at a light intensity of 300 W m−2. The accumulated levels of HSP 23 correlate well with resistance against photoinhibition; photoinhibitory effects are observed at a light intensity of 300 W m−2 or above as measured by the decline of PS II activity.

Kawaoka, Akiyoshi; Kawamoto, Tomohiro; Sekine, Masami; Yoshida, Kazuya; Takano, Mitsuo; Shinmyo, Atsuhiko

doi: 10.1046/j.1365-313X.1994.6010087.xpmid: 7920706

The mechanisms that control the wound‐induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′‐deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β‐glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP‐1, that bound specifically to the Box 1 sequence was identified. The putative TFHP‐1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP‐1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP‐1 suppressed the expression of prxC2 (−529)/GUS.

Bonfiglioli, Roderick G.; McFadden, Geoffrey I.; Symons, Robert H.

doi: 10.1046/j.1365-313X.1994.6010099.xpmid: N/A

Viroids, small single‐stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin‐labelled RNA probes were used with subsequent detection by gold‐labelled monoclonal anti‐biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.