The Plant Journal

Elmayan, Taline; Vaucheret, Hervé

doi: 10.1046/j.1365-313X.1996.9060787.xpmid: N/A

The bacterial UidA gene cloned between the 35S promoter with a double enhancer and the terminator sequences of the pea rbcS 9C gene was introduced into tobacco plants. All 11 transformants carrying the transgene at a single locus showed silencing at each generation, but the timing of silencing occurred at different rates in the different transformants. Two plants showed high levels of UidA mRNA accumulation and GUS activity in young seedlings and a rapid decline of these levels during the first month of growth irrespective of the allelic state of the T‐DNA. The other plants showed high levels of UidA mRNA accumulation and GUS activity in young seedlings and a slow decline of these levels during the first 4 months of growth in homozygous plants, whereas these levels decline only after 1 year of growth in hemizygous plants. Haploids showed the same kinetics of silencing as the homozygous plants from which they derived. Silencing correlated with a strong decrease of the steady‐state level of UidA mRNA, while the transcription of the transgene in the nucleus was not affected. Since haploids and hemizygous plants derived from transformants carrying a single copy of the T‐DNA show silencing, and since all the plants express the transgene at a very high level before the triggering of silencing, the results suggest that post‐transcriptional silencing occurs through a dose effect and not through DNA‐DNA interaction.

Lahjouji, Karim; Carrasco, Antoine; Bouyssou, Huguette; Cazaux, Louis; Marigo, Gérard; Canut, Hervé

doi: 10.1046/j.1365-313X.1996.9060799.xpmid: N/A

The effect of N‐(4‐azido‐salicylyl) aspartic acid (AzSA), a photolysable analogue of malate, was tested on the malate transport activity of tonoplast vesicles isolated from Catharanthus roseus cell suspension cultures. AzSA inhibited malate uptake in a competitive manner with a Kti of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Photolysis of 125I‐labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after analysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate‐binding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also protected the 37 kDa polypeptide from labelling. Citrate and, to a lesser extent, quinate afforded protection from labelling whilst other organic acids or aspartic acid (100 millimolar) did not. These photoprotection results are in good agreement with the data concerning the specificity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both in tonoplast vesicles and in isolated vacuoles. These results suggest the involvement of the 37 kDa polypeptide in vacuolar malate transport.

Kriete, G.; Niehaus, K.; Perlick, A.M.; Pühler, A.; Broer, I.

doi: 10.1046/j.1365-313X.1996.9060809.xpmid: 8696362

A system for the inducible destruction of plant tissues based on the deacetylation of the non‐toxic compound N‐acetyl‐l‐phosphinothricin (N‐ac‐Pt) has been developed. The argE gene product of Escherichia coli, representing a N‐acetyl‐l‐ornithine deacetylase was identified to remove the acetyl‐group from N‐ac‐Pt giving the cytotoxic compound l‐phosphinothricin (Pt, glufosinate). Transgenic Nicotiana tabacum plants constitutively expressing the argE gene were constructed. No effect of the bacterial N‐acetyl‐l‐ornithine deacetylase on plant growth and reproduction could be traced. However, application of N‐ac‐Pt on leaves of the transgenic plants led to the formation of necrotic areas due to the release of Pt. Additionally, due to the uptake of the N‐ac‐Pt by roots, transgenic shoots grown on medium containing N‐ac‐Pt bleached within 6–7 days and finally died. Untransformed controls showed no reaction to high amounts of N‐ac‐Pt applied, either under sterile or under unsterile conditions.

Navarro, María Teresa; Prieto, Rafael; Fernández, Emilio; Galván, Aurora

doi: 10.1046/j.1365-313X.1996.9060819.xpmid: N/A

The effect of constitutive expression of nitrate reductase (NR) on the regulation pattern of nitrite and nitrate transporters (NT) has been analysed in Chlamydomonas reinhardtii. Strain Tx11‐8, bearing the chimeric Nia1 gene for NR under the control of the cabll‐1 gene promoter, showed a constitutive NR which was inactive in ammonium‐grown cells. In contrast to the wild‐type, incubation of Tx11‐8 in ammonium media containing nitrate at micromolar concentrations resulted in reactivation of inactive NR and excretion of nitrite. In addition, negligible amounts of NT gene transcripts were induced by nitrate in this strain. However, NT transcripts from Tx11‐8 cells were induced in amounts similar to those in the wild‐type, by preventing constitutive NR expression from the cabll‐1 gene promoter in the dark. Strains lacking high‐affinity NT and nitrite reductase activity and having the chimeric Nia1 gene have been constructed by genetic crosses. All these strains were sensitive to chlorate in ammonium‐containing media and excreted nitrite when nitrate was present at millimolar concentrations. Spontaneous chlorate‐resistant mutants isolated from these strains lacked constitutive NR activity though they constitutively expressed NR mRNA. The data strongly suggest that nitrate plays an important post‐transcriptional regulatory role and that NR is involved in the regulation of the nitrate/ nitrite transport activities in C. reinhardtii.

Heins, Lisa; Schmitz, Udo Klaus

doi: 10.1046/j.1365-313X.1996.9060829.xpmid: 8696363

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.

Weber, Hwans; Buchner, Peter; Borisjuk, Ljudmilla; Wobus, Ulrich

doi: 10.1046/j.1365-313X.1996.9060841.xpmid: 8696364

The roles of sucrose‐phosphate synthase (Sps) and sucrose synthase (Sus) in developing embryos of Vicia faba have been characterized. In the cotyledons the expression of both Sps and Sus is initiated in cells differentiating into storage tissue. This stage is characterized by a switch in the carbohydrate state from a high to a low hexoses to sucrose ratio. The carbohydrate state was found earlier to be controlled by seed coat‐associated invertase. During cotyledon development the Sps‐enzyme undergoes a cycle of deactivation and reactivation: the activated state is associated with the prestorage phase, desiccation and germination and the deactivated state with the storage phase. Sus activity is associated with the storage phase. Sps and Sus are differentially influenced by free sugars. Feeding hexoses to storage phase cotyledons increases levels of Sps‐mRNA but not Sus‐mRNA, Sps activity and Sps activation state and impairs storage functions evidenced by an increased sucrose to starch ratio and a downregulation of storage protein legumin B‐mRNA. Sus enzyme activity is inhibited by free hexoses in vitro. It is proposed that the changing carbohydrate state during cotyledon development controls the ratio of Sps to Sus. Sps may have some significance for the initiation of the storage process possibly decreasing hexoses and/ or increasing sucrose. The relevance of the changing carbohydrate state with respect to development and storage processes is discussed.

Bennett, Mark; Gallagher, Matthew; Fagg, Jean; Bestwick, Charles; Paul, Teresa; Beale, Michael; Mansfield, John

doi: 10.1046/j.1365-313X.1996.9060851.xpmid: N/A

The expression of resistance to Bremia lactucae determined by the resistance genes Dm5/8 and Dm7 in lettuce was examined; incompatibility involved the hypersensitive reaction (HR) which occurred only within penetrated cells at early and late stages of fungal development, respectively. Autofluorescence observed under UV and blue light excitation in cells undergoing the HR was associated with the accumulation of ester‐linked syringaldehyde and caffeic acid on plant cell walls. Two phases of phenolic deposition were identified. The first was highly localized around penetration points and occurred during incompatible and compatible interactions. The second and major phase was only activated after the occurrence of irreversible membrane damage in the penetrated cell and was reduced by inhibitors of mRNA synthesis. Fungal structures, primary and secondary vesicles, intercellular hyphae and haustoria also became autofluorescent during incompatible interactions. Changes in the fluorescence due to preformed phenolics located in the plant cell vacuole were found just before plasma membrane damage became irreversible during the HR. In addition to localized deposition of phenolics, increases in the concentrations of the major free phenolic esters identified as dicaffeoyl tartaric and chlorogenic acids also occurred during incompatible interactions. The results suggest that membrane damage in penetrated cells occurs at different rates in resistance controlled by Dm5/8 and Dm7 and indicate an important role for irreversible membrane damage in lettuce as a key signalling event leading to widespread activation of defence responses in surrounding cells.

Bougri, Oleg; Grimm, Bernhard

doi: 10.1046/j.1365-313X.1996.9060867.xpmid: 8696365

In plants tetrapyrrole synthesis is initially light regulated on the level of 5‐aminolevulinate (ALA) synthesis. ALA is formed from glutamate in three enzymatic steps. Glutamyl tRNA reductase (GluTR) catalyses the NADPH‐dependent reduction of glutamyl tRNA to glutamate 1‐semialdehyde. GluTR is encoded by a low‐copy gene family consisting of three to four genes. Three different cDNA clones are presented. Full‐length clones BHA1 and 87 differ in the length of the 3′ untranslated region and code for a 58.5 kDa protein. The sequence of the partial clone, BHA13, contains at least 87 base mismatches in the coding region for the mature GluTR resulting in 11 amino acid substitutions. Synthesis of a recombinant mature and a truncated GluTR lacking the first 19 amino terminal amino acids in Escherichia coli lead only in the latter case to complementation of an E. coli hemA mutant. Steady‐state level of BHA1‐ and BHA13‐specific mRNA encoding GluTR were analysed by Northern blot hybridization using cDNA‐specific oligo nucleotides and quantitative reverse transcriptase‐polymerase chain reaction. Accumulation of the two RNA species is light induced in greening barley and controlled during cellular development. In contrast to BHA13, BHA1 transcripts are present in roots and are elevated after cytokinin treatment of dark‐grown seedlings. Furthermore, BHA1 mRNA shows oscillation under circadian growth conditions. GluTR transcript levels correlate with the capacity for ALA synthesis indicating that the rate‐limiting substrate flux through the ALA synthesizing pathway can be at least partially attributed to GluTR expression. Consequences of the initial control of the chlorophyll metabolic pathway on the level of ALA formation are discussed.

Xu, Wei; Campbell, Paul; Vargheese, Ajay K.; Braam, Janet

doi: 10.1046/j.1365-313X.1996.9060879.xpmid: 8696366

Enzymes that modify cell wall components most likely play critical roles in altering size, shape, and physical properties of plant cells. Regulation of such modifying activity is expected to be important during morphogenesis and in eliciting developmental and physiological alterations that arise in response to environmental conditions. Previous work has shown that the Arabidopsis TCH4 gene encodes a xyloglucan endotransglycosylase (XET) which acts on the major hemicellulose of the plant cell wall. The expression of TCH4 is dramatically upregulated in response to several environmental stimuli (including touch, wind, darkness, heat shock, and cold shock) as well as the growth‐enhancing hormones, auxin and brassinosteroids. This paper reports the presence of an extensive XET,related (XTR) gene family in Arabidopsis. In addition to TCH4, this family includes two previously identified genes, EXT and Meri‐5, and at least five additional genes. The cDNAs of the XTR family share between 46 and 79% sequence identity and the predicted XTR proteins share from 37 to 84% identity. All eight proteins include potential N‐terminal signal sequences and most have a conserved motif (DEIDFEFLG) that is also found in Bacillusβ‐glucanase and may be important for enzyme activity. The members of the XTR gene family are differentially sensitive to environmental and hormonal stimuli. Magnitude and kinetics of regulation are distinct for the different genes. Differential regulation of expression of this complex gene family suggests a recruitment of related, yet distinct, cell wall‐modifying enzymes that may control the properties of cell walls and tissues during development and in response to environmental cues.

Schnorr, Kirk Matthew; Gaillard, Catherine; Biget, Elisabeth; Nygaard, Per; Laloue, Michel

doi: 10.1046/j.1365-313X.1996.9060891.xpmid: 8696367

Adenine phosphoribosyltransferase (APRTase) is an important enzyme for its ability to convert adenine, a by‐product of many biochemical reactions, into AMP. By functional complementation of an Escherichia coli mutant, cDNAs encoding two APRTases have been cloned from Arabidopsis thaliana. One of the cDNAs (ATapt1)has been previously identified while the second (ATapt2) is of a previously unknown type. Kinetic analysis of the two enzymes purified from E. coli expressing the two cDNAs indicates that ATapt2 has a higher affinity for cytokinin than the ATapt1. RNase protection studies indicate that the ATapt2, is not expressed in leaves. Analysis of the gene structure indicates that ATapt2 has identical intron positions to ATapt1, but neither the intron sequence nor intron size are conserved between the two genes. The implications of a second, differentially expressed APRTase with affinity for both adenine and cytokinin are discussed.