Nicotinamide; antioxidative and DNA hypomethylation effects in plant cells.

The effects of nicotinamide (NIC) and its natural plant metabolites nicotinic acid (NIA) and trigonelline (TRIG) were studied with respect to defense in plant cell cultures. NIC and NIA could protect against oxidative stress damage caused by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which generates free radicals. Damage was analyzed as DNA strand breaks in cell cultures of Pisum sativum (garden pea), Daucus carota (carrot), Populus tremula L. × P. tremuloides (hybrid aspen) and Catharanthus roseus (Madagascar periwinkle), monitored by single cell gel electrophoresis (comet assay), and assays of cell leakage in C. roseus. The activities of aconitase and fumarase enzymes, which have key roles in energy metabolism, were analyzed in P. sativum cultures after treatment with NIC or NIA. Aconitase activity was increased by NIA, and fumarase activity was increased by both compounds. These compounds were shown to promote glutathione metabolism in P. sativum cultures, and NIC was shown to have a global DNA hypomethylating effect. Neither TRIG nor poly(ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide offered any protection against DNA damage or cell leakage, nor did they promote aconitase or fumarase activities, or glutathione metabolism. By this broad approach addressing multiple biochemical factors and different plant species, we demonstrate that NIC and NIA protect plant cells from oxidative stress, and that NIC clearly exerts an epigenetic effect; decreased DNA methylation. This indicates that these compounds have important roles in the regulation of metabolism in plant cells, especially in connection to stress.

UV-B exposure of indoor-grown Picea abies seedlings causes an epigenetic effect and selective emission of terpenes.

Terpenoids are involved in various defensive functions in plants, especially conifers. Epigenetic mechanisms, for example DNA methylation, can influence plant defence systems. The purpose of the present study was to investigate the influence of UV-B exposure on the release of terpenoids from spruce seedlings and on needle DNA methylation. Ten-week-old seedlings grown indoors were exposed to UV-B radiation during 4 h, and the volatile compounds emitted from the seedlings were analysed. Analysis of the volatiles 1, 3, and 22 d after this UV-B exposure showed that bornyl acetate, borneol, myrcene, and limonene contents increased during the first 3 days, while at day 22 the level of emission had returned to the control level. UV-B exposure decreased the level of DNA methylation in needles of young seedlings, reflected in methylation changes in CCGG sequences. Exposure of young seedlings to UV-B radiation might be a way to potentiate the general defensive capacity, improving their ability to survive in outdoor conditions. UV-B-induced defence is discussed in the light of epigenetic mechanisms.

Increased metal tolerance in Salix by nicotinamide and nicotinic acid.

We have earlier shown that nicotinamide (NIC) and nicotinic acid (NiA) can induce defence-related metabolism in plant cells; e.g. increase the level of glutathione. Here we investigated if NIC and NiA could increase the metal tolerance in metal sensitive clones of Salix viminalis and whether this would be mediated via increased glutathione level. Salix clones, sensitive or tolerant to zinc (Zn), copper (Cu) and cadmium (Cd) were grown in the presence of heavy metals (Cd, Cu or Zn) or NIC and NiA as well as in combination. In addition, the influence of N-acetyl-cystein (NAC) and l-2-oxothiazolidine 4-carboxylate (OTC), stimulators of reduced glutathione (GSH) biosynthesis, and the glutathione biosynthesis inhibitor buthionine sulfoximine (BSO) was analysed. Tolerance was measured as effects on root and shoot dry weight, and the glutathione and metal concentrations in the tissues were analysed. Results showed that NIC and NiA decreased the toxic effects of Cd, Cu and Zn on growth significantly in sensitive clones, but also to some extent in tolerant clones. However, the glutathione level and metal concentration did not change by NIC or NiA addition. Treatment with NAC, OTC or BSO did not per se influence the sensitivity to Cd, although the glutathione level increased in the presence of NAC and OTC and decreased in response to BSO. The results suggest that NIC and NiA increased the defence against heavy metals but not via glutathione formation per se.

Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.

Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. x P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis.

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.