The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.     

Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum). II. Assessment of control coefficients of the triose phosphate/phosphate translocator

 Transgenic tobacco (Nicotiana tabacum L.) plants with decreased and increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were used to study the control the TPT exerts on the flux of starch and sucrose biosynthesis, as well as CO₂ assimilation, respiration and photosynthetic electron transport. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing a TPT gene from Flaveria trinervia (FtTPT) were used. In ambient CO₂, there was no or little effect of altered TPT transport activities on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the TPT exerted strong control on the rate of CO₂ assimilation (control coefficient for the wild type; C^JA _TPT=0.30) in saturating light. Similarly, the incorporation of ¹⁴C into starch in high CO₂ was increased in tobacco plants with decreased TPT activity, but was reduced in plants overexpressing the TPT from F. trinervia. Thus, the TPT exerted negative control on the rate of starch biosynthesis with a C^JStarch _TPT=−0.19 in the wild type estimated from a hyperbolic curve fitted to the data points. This was less than the positive control strength on the rate of sucrose biosynthesis (C^JSuc _TPT=0.35 in the wild type). Theoretically, the positive control exerted on sucrose biosynthesis should be numerically identical to the negative control on starch biosynthesis unless additional metabolic pathways are affected. The rate of dark respiration showed some correlation with the TPT activity in that it increased in FtTPT overexpressors, but decreased in αTPT plants with an apparent control coefficient of C^JRes _TPT=0.24. If the control on sucrose biosynthesis is referred to as “gain of carbon” (positive control) and the control on starch biosynthesis as well as dark respiration as a “loss of carbon” (negative control) for sucrose biosynthesis and subsequent export, the sum of the control coefficients on dark respiration and starch biosynthesis would be numerically similar to the control coefficient on the rate of sucrose biosynthesis. There was also some control on the rate of photosynthetic electron transport, but only at high light and in elevated CO₂ combined with low O₂. The control coefficient for the rate of photosynthetic electron transport was C^JETR _TPT=0.16 in the wild type. Control coefficients were also calculated for plants with elevated and lowered TPT activity. Furthermore, the extent to which starch degradation/glucose utilisation compensates for the lack of triose phosphate export was assessed. The TPT also exerted control on metabolite contents in air. Received: 26 March 1999 / Accepted: 21 August 1999     

Evidence that corticosterone is not an obligatory intermediate in aldosterone biosynthesis in the rat adrenal

CGS 16949A is a potent inhibitor of aromatase in vitro with an IC50 of 0.03 microM for the inhibition of LH-stimulated estrogen biosynthesis in hamster ovaries. In vivo, CGS 16949A leads to sequelae of estrogen deprivation (e.g. regression of DMBA-induced mammary tumors) without causing adrenal hypertrophy in adult rats. To complement these in vitro and in vivo findings, the effect of CGS 16949A on adrenal steroid biosynthesis in rats was investigated in vitro and in vivo. The surprising finding in vitro was that CGS 16949A inhibited aldosterone biosynthesis (IC50 = 1 microM) at concentrations 100 times lower than those for inhibition of corticosterone biosynthesis (IC50 = 100 microM). Moreover, deoxycorticosterone (DOC) concentrations were elevated at all concentrations of CGS 16949A which inhibited aldosterone synthesis. The classical biosynthetic pathway for aldosterone is DOC----corticosterone----18-OH-corticosterone----aldosterone. Thus inhibition of aldosterone biosynthesis, reflected in DOC accumulation, without affecting corticosterone concentrations, indicates that corticosterone is not an obligatory intermediate in the conversion of DOC to aldosterone in the rat. In vivo, CGS 16949A showed a suppression of plasma aldosterone in ACTH-stimulated male rats at doses which did not significantly affect plasma corticosterone. In conclusion, aldosterone measured both in vitro and in vivo must be derived primarily from a biosynthetic pathway in which corticosterone is not obligatory intermediate.     

The AAA‐type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana

The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant‐negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1) under the control of the trichome‐specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant‐negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant‐negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell‐cycle regulation.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Effect of Detachment on the Palatability of two Kelp Species

Many species of macroalgae survive after becoming dislodged from their primary substratum, but little is known about their capacity to express anti-herbivore defences after detachment. We examined the effect of detachment on the relative palatability of the two kelp species Lessonia nigrescens and Macrocystis integrifolia to mesograzers. Laboratory and field experiments were conducted on the northern-central coast of Chile to investigate whether (i) time after detachment and (ii) grazing on detached and attached algae could trigger internal defence mechanisms in the algae, which may have acted as deterrents to grazing. In order to examine palatability, feeding assays were run after each experiment using fresh algal pieces and artificial food. Time after detachment had a significant influence on palatability of L. nigrescens but not of M. integrifolia. During the first 12 days of detachment, detached L. nigrescens held in grazer-free laboratory tanks were not significantly more palatable than attached conspecifics from the field but thereafter detached individuals became more palatable. Floating individuals of M. integrifolia showed no effect of detachment, indicating that this alga maintains its defence after detachment. An experiment conducted in the field confirmed these results for M. integrifolia. An additional laboratory experiment confirmed that attachment status plays an important role on algal defence reaction for L. nigrescens when exposed to grazers. Detached and previously grazed individuals of this species were less palatable than grazer-free control algae, but grazing had no effect on palatability of attached algae. Our results indicate that kelps have varying capacities for development of anti-grazing responses once they become detached, possibly depending on their capacity to float and survive after detachment.