The Plant Journal

Unterlinner, Bernhard; Lenz, Rainer; Kutchan, Toni M.

doi: 10.1046/j.1365-313X.1999.00470.xpmid: 10417697

The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum . Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH‐dependent reduction of codeinone to codeine. In this study, we have isolated and characterized four cDNAs encoding codeinone reductase isoforms and have functionally expressed them in Escherichia coli . Heterologously expressed codeinone reductase‐calmodulin‐binding peptide fusion protein was purified from E. coli using calmodulin affinity column chromatography in a yield of 10 mg enzyme l ‐1 . These four isoforms demonstrated very similar physical properties and substrate specificity. As least six alleles appear to be present in the poppy genome. A comparison of the translations of the nucleotide sequences indicate that the codeinone reductase isoforms are 53% identical to 6′‐deoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthesis. By sequence comparison, both codeinone reductase and 6′‐deoxy‐ chalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH‐dependent oxidoreductases, and thereby possibly arise from primary metabolism.

De Santis‐Maciossek, Giovanna; Kofer, Waltraud; Bock, Armin; Schoch, Siegrid; Maier, Rainer M.; Wanner, Gerhard; Rüdiger, Wolfhart; Koop, Hans‐Ulrich; Herrmann, Reinhold G.

doi: 10.1046/j.1365-313X.1999.00473.xpmid: 10417698

Plieth, Christoph; Hansen, Ulf‐Peter; Knight, Heather; Knight, Marc R.

doi: 10.1046/j.1365-313X.1999.00471.xpmid: 10417699

Cold elicits an immediate rise in the cytosolic free calcium concentration ([Ca 2+ ] c ) of plant cells. We have studied the concerted action of the three underlying mechanisms, namely sensing, sensitisation and desensitisation, which become important when plants in the field are subjected to changes in temperature. We applied different regimes of temperature changes with well‐defined cooling rates to intact roots of Arabidopsis thaliana expressing the calcium‐indicator, aequorin. Our results indicate that temperature sensing is mainly dependent on the cooling rate, dT/dt, whereas the absolute temperature T is of less importance. Arabidopsis roots were found to be sensitive to cooling rates of less than dT/dt = 0.01°C/s. However, at cooling rates below 0.003°C/s (i.e. cooling 10°C in 1 h) there is no detectable [Ca 2+ ] c response at all. At low temperature, the sensitivity of the plant cold‐detection system is increased. This in turn produces greater cooling‐induced [Ca 2+ ] c elevations. Prolonged or repeated cold treatment attenuates the [Ca 2+ ] c responses to subsequent episodes of cooling.

Cerdán, Pablo D.; Yanovsky, Marcelo J.; Reymundo, F. Carolina; Nagatani, Akira; Staneloni, Roberto J.; Whitelam, Garry C.; Casal, Jorge J.

doi: 10.1046/j.1365-313X.1999.00475.xpmid: 10417700

Phytochrome A (phyA) and phytochrome B (phyB) share the control of many processes but little is known about mutual signaling regulation. Here, we report on the interactions between phyA and phyB in the control of the activity of an Lhcb1*2 gene fused to a reporter, hypocotyl growth and cotyledon unfolding in etiolated Arabidopsis thaliana. The very‐low fluence responses (VLFR) induced by pulsed far‐red light and the high‐irradiance responses (HIR) observed under continuous far‐red light were absent in the phyA and phyA phyB mutants, normal in the phyB mutant, and reduced in the fhy1 mutant that is defective in phyA signaling. VLFR were also impaired in Columbia compared to Landsberg erecta. The low‐fluence responses (LFR) induced by red‐light pulses and reversed by subsequent far‐red light pulses were small in the wild type, absent in phyB and phyA phyB mutants but strong in the phyA and fhy1 mutants. This indicates a negative effect of phyA and FHY1 on phyB‐mediated responses. However, a pre‐treatment with continuous far‐red light enhanced the LFR induced by a subsequent red‐light pulse. This enhancement was absent in phyA , phyB , or phyA phyB and partial in fhy1 . The levels of phyB were not affected by the phyA or fhy1 mutations or by far‐red light pre‐treatments. We conclude that phyA acting in the VLFR mode (i.e. under light pulses) is antagonistic to phyB signaling whereas phyA acting in the HIR mode (i.e. under continuous far‐red light) operates synergistically with phyB signaling, and that both types of interaction require FHY1.

Lejay, Laurence; Tillard, Pascal; Lepetit, Marc; Olive, Francesc Domingo; Filleur, Sophie; Daniel‐Vedele, Françoise; Gojon, Alain

doi: 10.1046/j.1365-313X.1999.00480.xpmid: 10417701

Root NO 3 – uptake and expression of two root NO 3 – transporter genes ( Nrt2;1 and Nrt1) were investigated in response to changes in the N‐ or C‐status of hydroponically grown Arabidopsis thaliana plants. Expression of Nrt2;1 is up‐regulated by NO 3 – starvation in wild‐type plants and by N‐limitation in a nitrate reductase (NR) deficient mutant transferred to NO 3 – as sole N source. These observations show that expression of Nrt2;1 is under feedback repression by N‐metabolites resulting from NO 3 – reduction. Expression of Nrt1 is not subject to such a repression. However, Nrt1 is over‐expressed in the NR mutant even under N‐sufficient conditions (growth on NH 4 NO 3 medium), suggesting that expression of this gene is affected by the presence of active NR, but not by N‐status of the plant. Root 15 NO 3 – influx is markedly increased in the NR mutant as compared to the wild‐type. Nevertheless, both genotypes have similar net 15 NO 3 – uptake rates due to a much larger 14 NO 3 – efflux in the mutant than in the wild‐type. Expressions of Nrt2;1 and Nrt1 are diurnally regulated in photosynthetically active A. thaliana plants. Both increase during the light period and decrease in the first hours of the dark period. Sucrose supply prevents the inhibition of Nrt2;1 and Nrt1 expressions in the dark. In all conditions investigated, Nrt2;1 expression is strongly correlated with root 15 NO 3 – influx at 0.2 m m external concentration. In contrast, changes in the Nrt1 mRNA level are not always associated with similar changes in the activities of high‐ or low‐affinity NO 3 – transport systems.

Savage, Linda J.; Ohlrogge, John B.

doi: 10.1046/j.1365-313X.1999.00478.xpmid: 10417702

We have examined whether chloroplast acetyl‐CoA carboxylase is a phosphoprotein. Pea ( Pisum sativum ) chloroplasts were incubated in the presence of [γ‐ 33 P]‐ATP and radiolabeled proteins were examined after immunoprecipitation with antibodies against all four known subunits of heteromeric chloroplast acetyl‐CoA carboxylase. The β‐subunit of the carboxyltransferase was found to be labeled by 33 P. Phosphoamino acid analysis of the immunoprecipitated β‐subunit of the carboxyltransferase indicates that it is phosphorylated on serine residues. Incorporation of 33 P into carboxyltransferase β‐subunit decreased in chloroplasts transferred to dark conditions after labeling in the light. Dephosphorylation of pea chloroplast extracts by an alkaline phosphatase‐agarose conjugate reduced in vitro acetyl‐CoA carboxylase activity by 67%. Furthermore, while acetyl‐CoA carboxylase activity and its carboxyltransferase half‐reaction were reduced in dephosphorylated extracts, the biotin carboxylase half‐reaction was not inhibited. The evidence presented here points to the carboxyltransferase β‐subunit of chloroplast acetyl‐CoA carboxylase as a candidate for regulation by protein phosphorylation/dephosphorylation.

Fu, Hongyong; Doelling, Jed H.; Rubin, David M.; Vierstra, Richard D.

doi: 10.1046/j.1365-313X.1999.00479.xpmid: 10417703

The 26S proteasome is a multi‐subunit ATP‐dependent protease responsible for degrading most short‐lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylin‐ drical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP‐dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, desig‐ nated RPT1–6 for regulatory particle triple‐A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub‐ units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3–6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non‐redundant functions.

Bouly, Jean‐Pierre; Gissot, Lionel; Lessard, Philippe; Kreis, Martin; Thomas, Martine

doi: 10.1046/j.1365-313X.1999.00476.xpmid: 10417704

AKINα1, a Ser/Thr kinase from Arabidopsis thaliana belongs to the highly conserved SNF1 family of protein kinases in eukaryotes. Recent data suggest that the plant SNF1‐related kinases (SnRK1 family) are key enzymes implicated in the regulation of carbohydrate and lipid metabolism. In Saccharomyces cerevisiae and mammals, the SNF1 and AMPKα protein kinases interact with two other families of proteins, namely SNF4/AMPKγ and SIP1/SIP2/GAL83/AMPKβ, to form active heterotrimeric complexes. In this paper, we describe the characterisation of three novel cDNAs. AKINβ1 and AKINβ2 encode proteins similar to SIP1, SIP2 and GAL83 and AKINγ codes for a protein showing similarity with SNF4. Using the two‐hybrid system, specific interactions have been shown between A. thaliana AKINβ1/β2, AKINγ and AKINγ as well as between the A. thaliana and S. cerevisiae subunits. Interestingly, AKINβ1, AKINβ2 and AKINγ mRNAs accumulate differentially in A. thaliana tissues and are modulated during development and under different growth conditions. These data suggest the presence in higher plants of a conserved heterotrimeric complex. Moreover, the differential transcription of different non‐catalytic subunits can constitute a first level of regulation of the SNF1‐like complex in plants.

Ninu, Luciano; Ahmad, Margaret; Miarelli, Carolina; Cashmore, Anthony R.; Giuliano, Giovanni

doi: 10.1046/j.1365-313X.1999.00466.xpmid: 10417705

Cryptochrome genes (CRY) are a novel class of plant genes encoding proteins that bear a strong resemblance to photolyases, a rare class of flavoproteins that absorb light in the blue (B) and UV‐A regions of the spectrum and utilise it for photorepair of UV‐damaged DNA. In Arabidopsis, both CRY1 and CRY2 are implicated in numerous blue light‐dependent responses, including inhibition of hypocotyl elongation, leaf and cotyledon expansion, pigment biosynthesis, stem growth and internode elongation, control of flowering time and phototropism. No information about the in vivo function of CRY genes is available in other plant species. The tomato CRY1 gene (TCRY1) encodes a protein of 679 amino acids, which shows 78% identity and 88% similarity to Arabidopsis CRY1. In order to verify the in vivo function of TCRY1, we constructed antisense tomato plants using the C‐terminal portion of the gene. Partial repression of both mRNA and protein levels was observed in one of the transformants. The progeny from this transformant showed an elongated hypocotyl under blue but not under red light. This character co‐segregated with the transgene and was dependent on transgene dosage. An additional, partially elongated phenotype was observed in adult plants grown in the greenhouse under dim light and short days with no artificial illumination. This phenotype was suppressed by artificial illumination of both short and long photoperiods. The synthesis of anthocyanins under blue light was reduced in antisense seedlings. In contrast, carotenoid and chlorophyll levels and second positive phototropic curvature were essentially unaltered.

Rupp, Hans‐Michael; Frank, Markus; Werner, Tomás; Strnad, Miroslav; Schmülling, Thomas

doi: 10.1046/j.1365-313X.1999.00472.xpmid: 10417706

This study investigates the consequences of endo‐ genously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Tran‐ scriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature‐dependent increased cytokinin production in transgenic plants. Heat‐treated plants accumulated higher levels of unbound and bound zeatin‐type cyto‐ kinins, the latter being preferentially N‐conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin‐overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin‐overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.