Jan Geuns

Steroid hormones and plant growth and development

Abstract The occurrence of steroid hormones in plants is briefly reviewed. Their effects on plant growth, development and flowering are also considered.

Steviol Glycosides: Chemical Diversity, Metabolism, and Function

Steviol glycosides are a group of highly sweet diterpene glycosides discovered in only a few plant species, most notably the Paraguayan shrub Stevia rebaudiana. During the past few decades, the nutritional and pharmacological benefits of these secondary metabolites have become increasingly apparent. While these properties are now widely recognized, many aspects related to their in vivo biochemistry and metabolism and their relationship to the overall plant physiology of S. rebaudiana are not yet understood. Furthermore, the large size of the steviol glycoside pool commonly found within S. rebaudiana leaves implies a significant metabolic investment and poses questions regarding the benefits S. rebaudiana might gain from their accumulation. The current review intends to thoroughly discuss the available knowledge on these issues.

Metabolism of Stevioside by Healthy Subjects

Stevioside (250-mg capsules) was given thrice daily for 3 days to 10 healthy subjects. Blood samples were collected and blood pressure measured after nocturnal fasting, before and at different time points during the third day of the administration of stevioside. No significant differences were found between the control and the stevioside condition for blood pressure and blood biochemical parameters. The 24-hr urinary volume and urinary excretion of electrolytes were not significantly different. Likewise, no significant difference was found for mean blood glucose and insulin between control and stevioside conditions. Thus, oral stevioside is not directly effective as a hypotensive or hypoglycemic agent in healthy subjects at the dose administered in this study. Stevioside, free steviol, and steviol metabolites were analyzed in blood, feces, and urine after 3 days of stevioside administration. No uptake was found of stevioside by the gastrointestinal tract or the amounts taken up were very low and below the detection limit of the UV detector. Stomach juice did not degrade stevioside. All the stevioside reaching the colon was degraded by micro-organisms into steviol, the only metabolite found in feces. In blood plasma, no stevioside, no free steviol or other free steviol metabolites were found. However, steviol glucuronide (SV glu) was found in maximum concentrations of 33 μg/ml (21.3 μg steviol equivalents/ml). In urine, no stevioside or free steviol were present, but SV glu was found in amounts of up to 318 mg/24-hr urine (205 mg steviol equivalents/24 hrs). No other steviol derivatives were detected. In feces, besides free steviol, no other steviol metabolites or conjugates were detected. Steviol was excreted as SV glu in urine.

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL‐4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL‐4‐dependent E‐cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL‐4‐induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1‐deficient macrophages display rather enhanced IL‐4‐induced polyamine production, suggesting that an Arg1‐independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS‐induced expression of several proinflammatory genes was increased significantly in polyamine‐depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL‐4‐induced expression of several AAM mediators, they inhibit the LPS‐mediated expression of proinflammatory genes in CAMs.

Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway

Abstract As shown from [1- 13 C]glucose incorporation, steviol, the diterpene aglycone moiety of stevioside, is synthesized in Stevia rebaudiana Bertoni via the mevalonate-independent methylerythritol phosphate pathway.

Evaluation of supplementary stevia (Stevia rebaudiana, bertoni) leaves and stevioside in broiler diets: effects on feed intake, nutrient metabolism, blood parameters and growth performance

A perennial schrub, stevia, and its extracts are used as a natural sweetener and have been shown to possess antimicrobial properties. Stevia contains high levels of sweetening glycosides including stevioside which is thought to possess antimicrobial and antifungal properties. Little is known about the nutritional value of the schrub in livestock. This study determined the potential use of the shrub as a prebiotic animal feed supplement in light of the recent ban on the use of antibiotics in animal feed and the role of its constituent stevioside in the effects of the shrub. Male Cobb broiler chicks were fed a basal broiler diet without antibiotic but with performance enhancing enzyme mix (positive control), a basal diet without antibiotic and enzymes (negative control), or diets in which 2% of the negative control diet was replaced with either dried ground stevia leaves or 130 ppm pure stevioside during 2 week starter and 2 week grower periods. Body weight gains, feed conversion, abdominal fat deposition, plasma hormone and metabolites and caecal short chain fatty acids (SCFA) were measured in the broilers at 2 and 4 weeks of age. There was no significant effect of the treatments on feed intake during the starter period but birds fed diet supplemented with stevia leaves and stevioside consumed more feed (p < 0.05) than those fed the positive control diet during the grower period. Weight gain by birds fed the positive control and stevioside diets was higher (p < 0.05) than those fed other diets only during the starter period. Feed/gain ratio of birds fed the positive control and stevioside diets was superior (p < 0.05) to others. There was no effect of the treatments on nutrient retention and water content of the excreta. Dietary stevia leave and stevioside decreased total concentration of SCFA and changed their profile in the ceca. There was no effect of the treatments on pancreas weight. Dietary stevia reduced blood levels of glucose, triglycerides and triiodothyronine (T(3)) but had no effect on non-esterified fatty acids. In contrast, stevioside only decreased T(3). Both the stevia leaves and stevioside diets significantly increased abdominal fat content. It is concluded that dietary enzyme growth promoters are beneficial to the broilers only during the starter stage and that inclusion of stevia leaves or stevioside has no beneficial effect on the performance of broilers.

Polyamine transport systems in the LLC-PK1 renal epithelial established cell line

LLC-PK1 cells were brought to a quiescent state by treatment with DL-2-difluoromethylornithine (DFMO), a specific inhibitor of L-ornithine decarboxylase (ODC). The inhibition of ODC, which is the key enzyme for polyamine synthesis, strongly reduced the cellular content of putrescine and spermidine. The cells resumed DNA-synthesis followed by mitosis when exogenous putrescine was added. DFMO treatment strongly stimulated the putrescine uptake capability. A kinetic analysis of the initial uptake rates revealed a saturable Na+-dependent and a saturable Na+-independent pathway on top of non-saturable diffusion. The stimulation by DFMO was exclusively due to an effect on the Vmax values of the saturable pathways. The Na+-dependent transporter had a higher affinity for putrescine (apparent Km = 4.7 +/- 0.7 microM) than the Na+-independent transporter (apparent Km = 29.8 +/- 3.5 microM). As a consequence, although the latter transporter had a higher Vmax, the Na+-dependent transport was more important at a physiological putrescine concentration. Putrescine uptake by both transporters was inhibited with similar relative affinities by spermidine, spermine as well as by the antileukemic agent, methylglyoxal bis(guanylhydrazone), but not by amino acids. The activity of the Na+-dependent transporter was very much dependent on SH-group reagents, whereas the Na+-independent transporter was not affected. Both transporters were inhibited by metabolic inhibitors and by ionophores but the Na+-dependent transporter was affected to a greater extent. For both transporters there was a down-regulation in response to exogenous putrescine. This suggests that the polyamine transporters in LLC-PK1 are adaptively regulated and may contribute to the regulation of the cellular polyamine level and cellular proliferation.

Variations in sterol composition in etiolated mung bean seedlings

Abstract The sterol fractions of different morphological and physiological parts of etiolated Mung bean seedlings have been studied. Young growing tissues conta

UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides

A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.

Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni).

The effect of photoperiodism on steviol glycoside (SVgly) accumulation was investigated in Stevia rebaudiana. Topped plants were cultivated to develop new branches under a 16h or 8h photoperiod. During different ontogenetic phases, leaves, stems, lateral shoots, roots and reproductive organs were collected and analysed for nine SVglys. Long-day (LD) conditions prolonged vegetative growth, significantly increasing leaf biomass and total SVgly content. In both photoperiods, declines in SVglys were observed during reproductive development, occurring mainly in mature leaves under LDs or young leaves under SDs. When lateral shoots were included in plants under LDs, total leaf and SVgly yield per branch significantly increased, indicating a harvest during flowering is possible. The ratio of rebaudioside A (Reb A) to stevioside (ST) amounts was influenced by ontogeny and daylength, with larger ratios during vegetative growth under SDs. Linear correlations were observed between dry matter and total SVglys and between the major SVglys individually. Minor SVglys showed larger fluctuations, especially under SDs. Under LDs, the Reb A to ST ratio was inversely correlated with both leaf dry matter and total SVglys. The highly dynamic nature of the observed patterns suggests a complex regulation of SVgly metabolism on molecular and biochemical level.