Torkel Berglund

Nicotinamide, a missing link in the early stress response in eukaryotic cells: A hypothesis with special reference to oxidative stress in plants

A hypothesis is presented suggesting that nicotinamide (NIC) is an initial signal substance in the response of eukaryotic cells to conditions which cause DNA‐strand breakage, especially in connection with oxidative stress. In the stressed cell, NIC is released as a result of the activity of poly(ADP‐ribose)polymerase (PADPRP). PADPRP is known to be activated by DNA‐strand breakage, caused by e.g. oxidative stress or mutagens. NIC and its metabolite trigonelline (N‐methylnicotinic acid) can induce defensive metabolism at the gene level. Connections between NIC and DNA‐methylation are also considered. This hypothesis is discussed in the light of own observations and literature reports.

In vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose by detergent extracts of membranes from cell suspension cultures of hybrid aspen

The aim of this work was to optimize the conditions for in vitro synthesis of (1→3)-β-D-glucan (callose) and cellulose, using detergent extracts of membranes from hybrid aspen (Populus tremula ×tremuloides) cells grown as suspension cultures. Callose was the only product synthesized when CHAPS extracts were used as a source of enzyme. The optimal reaction mixture for callose synthesis contained 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 8 mM Ca2+, and 20 mM cellobiose. The use of digitonin to extract the membrane-bound proteins was required for cellulose synthesis. Yields as high as 50% of the total in vitro products were obtained when cells were harvested in the stationary phase of the growth curve, callose being the other product. The optimal mixture for cellulose synthesis consisted of 100 mM Mops buffer pH 7.0, 1 mM UDP-glucose, 1 mM Ca2+, 8 mM Mg2+, and 20 mM cellobiose. The in vitroβ-glucans were identified by hydrolysis of radioactive products, using specific enzymes. 13C-Nuclear magnetic resonance spectroscopy and transmission electron microscopy were also used for callose characterization. The (1→3)-β-D-glucan systematically had a microfibrillar morphology, but the size and organization of the microfibrils were affected by the nature of the detergent used for enzyme extraction. The discussion of the results is included in a short review of the field that also compares the data obtained with those available in the literature. The results presented show that the hybrid aspen is a promising model for in vitro studies on callose and cellulose synthesis.

UV‐B‐ and oxidative stress‐induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP‐ribose)polymerase inhibitor in plant tissue

Nicotinamide and trigonelline contents increased in Catharanthus roseus tissue culture after exposure to 2,2′azobis(2‐amidinopropane) dihydrochloride (AAPH) or vanadylsulfate and in Pisum sativum leaves after exposure to UV‐B radiation. Vanadylsulfate increased phenylalanine ammonia‐lyase (PAL) activity and the content of reduced and oxidized glutathione in C. roseus tissue culture. The increases in PAL activity caused by 2 mM AAPH or 0.2 mM vanadylsulfate were prevented by 0.1 mM 3‐aminobenzamide (3‐AB), an inhibitor of poly(ADP‐ribose)polymerase. Present results support the hypothesis [Berglund T., FEBS Lett. (1994) 351, 145–149] that nicotinamide and/or its metabolites may function as signal transmittors in the response to oxidative stress in plants and that poly(ADP‐ribose)polymerase has a function in the induction of defensive metabolism.

Defensive and secondary metabolism in plant tissue cultures, with special reference to nicotinamide, glutathione and oxidative stress

The purpose of the presentation is to interconnect and illuminate certain parts of metabolism regarding stress signalling and defensive functions, including secondary metabolism in intact plants and plant tissue cultures. Increased cell/tissue levels of reactive oxygen species like H2O2, O2- and ·OH and the metabolism of glutathione, are linked to defensive/secondary metabolism and tissue differentiation. Special attention is paid to nicotinamide. A hypothetical role of nicotinamide and its metabolites as stress signals is also put forward especially in connection with hypomethylation of DNA. A role of DNA hypomethylation, as a link between various types of stressors and the induction of plant devensive metabolism, is discussed. We suggest that nicotinamide or nicotinamide based substances may be of value within biotechnology for the production of valuable substances as well as for plant protection.

Nicotinamide increases glutathione and anthocyanin in tissue culture of Catharanthus roseus

Summary Nicotinamide (NIC), at a concentration of 8.2 mM, increased the glutathione content to 300% of the control in an anthocyanin accumulating Catharanthus roseus (periwinkle) tissue culture when harvested 10 days after NIC addition. One day after NIC addition, the content of oxidized glutathione (GSSG) increased to 169% of the control, but the contents of total glutathione (GSH tot ) and reduced glutathione (GSH) were unchanged. Longer NIC exposure resulted in increasing contents of GSH tot and GSH, as well as GSSG and a concomitant increase in anthocyanin accumulation. The glutathione biosynthesis inhibitor, buthioninesulfoximine (BSO), decreased glutathione levels in the tissue and offset the NIC-induced increase in glutathione content. BSO also decreased the NIC-induced anthocyanin accumulation in C. roseus tissue culture.

Effects of High MnSO4 Levels on Cardenolide Accumulation by Digitalis lanata Tissue Cultures in Light and Darkness

Summary Elevation of the MnS0 4 concentration in culture medium from 0.1 mM to 10 mM influenced growth and cardenolide accumulation in tissue cultures of Digitalis lanata . The digitoxin content increased from 16.7 to 63.5 μg/g dry weight (d.w.) in strain S-2 in darkness. In strain S-l in light the digitoxin content decreased from 305 to 235 μg/g d.w., and the chlorophyll content decreased from 0.70 to 0.46 mg/g d.w. MnCl 2 also stimulated cardenolide accumulation in strain S-2. Addition of MnS0 4 to strain S-2 in the beginning of the culture period (days 0 and 6) resulted in higher cardenolide content than later addition (days 10 and 15). With gibberellic acid in the medium there was only a small increase in cardenolide accumulation in strain S-2 when the MnS0 4 concentration was elevated.

Effect of Nicotinamide on Gene Expression and Glutathione Levels in Tissue Cultures of Pisum sativum

Summary Nicotinamide (NIC; 8.2 mM) caused increased abundance of mRNA for chalcone synthase in shoot and root cultures of Pisum sativum . Increased gene expression was observed from 3.5 h after NIC addition in shoot cultures in light (PS-S) and root cultures in light (PS-R-L) and darkness (PS-R-D). Twelve h after NIC addition to the cultures, the amount of mRNA for chs2 was increased 10-fold in PS-R-L, 6-fold in PS-R-D and 2-fold in PS-S. The expression of the glutathione reductase (GR) gene and the photosynthetic cab gene was also studied. Increased GR expression was clearly seen 3.5 h after NIC addition in PSS and PS-R-L and after 12 h in PS-R-D cultures, while the amount of mRNA for cab initially (2–3.5 h) increased and later ( 6–12 h) decreased in PS-S. Glutathione content was determined in the cultures. After a decrease in glutathione content during the first 2–3.5 h following NIC addition, the level of glutathione increased. In light-grown cultures, an increase in the content of the oxidised (disulfide) form of glutathione (GSSG) was an earlier event than the rise in reduced glutathione (GSH) levels, after 12h for PSR- L and after 3.5-6h for PS-S. Five days after NIC addition, the GSSG contents in the root and shoot cultures in light were increased by up to 150 and 500%, respectively, whereas the amounts of GSH doubled in both cultures. In dark grown cultures, an early increase in the content of GSH, but not GSSG, was observed.

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.

Summary.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. × 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.

Gibberellic acid-induced changes in glutathione metabolism and anthocyanin content in plant tissue

Literature data point to a possible link between gibberellic acid (GA3) and glutathione metabolism in plant tissue, as both are connected to dormancy breakage. In order to study the influence of GA3 on glutathione metabolism, we treated an anthocyanin accumulating cell culture of periwinkle (Catharanthus roseus) and a shoot differentiated culture of pea (Pisum sativum) with GA3. Glutathione reductase (GR; E.C. 1.6.4.2) activity increased to 135% and 190% of the control in C. roseus and P. sativum, respectively. The level of oxidized glutathione (GSSG) decreased to 60% of the control in the C. roseus culture while no change in GSSG was observed in the P. sativum culture. No changes in the tissue concentration of total glutathione was observed in the cultures after GA3 treatment. Concomitant to the changes in GSSG and GR, an increase in anthocyanin accumulation was observed in the C. roseus culture in association with a strong increase in phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) activity in response to GA3. These data strongly suggest a link between GA3 and glutathione metabolism.

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.