Relationship between aluminum stress and caffeine biosynthesis in suspension cells of Coffea arabica L.

Toxicity by aluminum is a growth-limiting factor in plants cultivated in acidic soils. This metal also promotes signal transduction pathways leading to the biosynthesis of defense compounds, including secondary metabolites. In this study, we observed that Coffea arabica L. cells that were kept in the dark did not produce detectable levels of caffeine. However, irradiation with light and supplementation of the culture medium with theobromine were the best conditions for cell maintenance to investigate the role of aluminum in caffeine biosynthesis. The addition of theobromine to the cells did not cause any changes to cell growth and was useful for the bioconversion of theobromine to caffeine. During a short-term AlCl3-treatment (500µM) of C. arabica cells kept under light irradiation, increases in the caffeine levels in samples that were recovered from both the cells and culture media were evident. This augmentation coincided with increases in the enzyme activity of caffeine synthase (CS) and the transcript level of the gene encoding this enzyme (CS). Together, these results suggest that actions by Al and theobromine on the same pathway lead to the induction of caffeine biosynthesis.

Effect of salicylic acid on the attenuation of aluminum toxicity in Coffea arabica L. suspension cells: A possible protein phosphorylation signaling pathway.

The protective effect of salicylic acid (SA) on aluminum (Al) toxicity was studied in suspension cells of Coffea arabica L. The results showed that SA does not produce any effect on cell growth and that the growth inhibition produced by aluminum is restored during simultaneous treatment of the cells with Al and SA. In addition, the cells exposed to both compounds, Al and SA, showed evident morphological signals of recovery from the toxic state produced in the presence of Al. The cells treated with SA showed a lower accumulation of Al, which was linked to restoration from Al toxicity because the concentration of Al(3+) outside the cells, measured as the Al(3+)-morin complex, was not modified by the presence of SA. Additionally, the inhibition of phospholipase C by Al treatment was restored during the exposure of the cells to SA and Al. The involvement of protein phosphorylation in the protective effect of SA on Al-toxicity was suggested because staurosporine, a protein kinase inhibitor, reverted the stimulatory effect of the combination of Al and SA on protein kinase activity. These results suggest that SA attenuates aluminum toxicity by affecting a signaling pathway linked to protein phosphorylation.

Determination of phospholipase C activity in vitro.

Measurement of phospholipase C (PLC) activity in vitro is a valuable biochemistry technique easily applicable in samples from different organisms. It quantifies the enzymatic activity of a key protein involved in critical developmental functions in organisms such as plants, animals, and bacteria. A protocol is described which assays the formation of two main products of the PLC hydrolysis reaction on radioactively labeled phospholipid substrates.

Aluminum induces changes in oxidative burst scavenging enzymes in Coffea arabica L. suspension cells with differential Al tolerance.

The accumulation of reactive oxygen species (ROS) and concomitant oxidative stress have been considered deleterious consequences of aluminum toxicity. However, several lines of evidence suggest that ROS can function as important signaling molecules in the plant defense system for protection from abiotic stress and the acquisition of tolerance. The role of ROS-scavenging enzymes was assayed in two different coffee cell suspension lines. We treated L2 (Al-sensitive) and LAMt (Al-tolerant) Coffea arabica suspension cells with 100 µM AlCl(3) and observed significant differences in catalase activity between the two cell lines. However, we did not observe any differences in superoxide dismutase or glutathione reductase activity in either cell line following Al treatment. ROS production was diminished in the LAMt cell line. Taken together, these results indicate that aluminum treatment may impair the oxidative stress response in L2 cells but not in LAMt cells. We suggest a possible role for Al-induced oxidative bursts in the signaling pathways that lead to Al resistance and protection from Al toxicity.

Phospholipidic signaling and vanillin production in response to salicylic acid and methyl jasmonate in Capsicum chinense J. cells.

The phospholipidic signal transduction system involves generation of second messengers by hydrolysis or changes in phosphorylation state. Several studies have shown that the signaling pathway forms part of plant response to phytoregulators such as salicylic acid (SA) and methyl jasmonate (MJ), which have been widely used to stimulate secondary metabolite production in cell cultures. An evaluation was made of the effect of SA and MJ on phospholipidic signaling and capsaicinoid production in Capsicum chinense Jacq. suspension cells. Treatment with SA inhibited phospholipase C (PLC) (EC: 3.1.4.3) and phospholipase D (PLD) (EC: 3.1.4.4) activities in vitro, but increased lipid kinase activities in vitro at different SA concentrations. Treatment with MJ produced increases in PLC and PLD activities, while lipid kinase activities were variable and dose-dependent. The production of vanillin, a precursor of capsaicinoids, increased at specific SA or MJ doses. Preincubation with neomycin, a phospholipase inhibitor, before SA or MJ treatment inhibits increase in vanillin production which suggests that phospholipidic second messengers may participate in the observed increase in vanillin production.

The location of aluminium in protoplasts and suspension cells taken from Coffea arabica L. with different tolerance of Al.

Biotechnological advances in coffee research (in vitro manipulation, multiplication, generation and development of transgenic coffee plants with specific traits like high yield and good quality) have contributed to description of the metabolic pathways involved in the response mechanisms to environmental factors like abiotic stress. Coffea arabica L. plants grow in acidic soils, and therefore aluminium (Al) toxicity is a major negative impact on crop productivity. To understand Al toxicity mechanisms in cells via the Al absorption kinetic, we isolated protoplasts from two C. arabica L. suspension cell lines: Al-sensitive (L2) and Al-tolerant (LAMt). Protoplasts of LAMt line exhibited lower Al absorption levels than protoplasts of the L2 line. Use of two fluorescent tracers (morin and calcofluor white) indicated that Al interacts with internal cell structures, such as the plasma membrane and nucleus, with differences in both cell lines. Al-tolerance in the LAMt is probably associated with the cell wall as well as intracellular structures. These data will help to better understand Al toxicity in C. arabica, and Al toxicity mechanisms in plant cells.

Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells.

In acid soils, aluminium (Al) toxicity and phosphate (Pi) deficiency are the most significant constraints on plant growth. Al inhibits cell growth and disrupts signal transduction processes, thus interfering with metabolism of phospholipase C (PLC), an enzyme involved in second messenger production in the cell. Using a Coffea arabica suspension cell model, we demonstrate that cell growth inhibition by Al toxicity is mitigated at a high Pi concentration. Aluminium-induced cell growth inhibition may be due to culture medium Pi deficiency, since Pi forms complexes with Al, reducing Pi availability to cells. Phosphate does not mitigate inhibition of PLC activity by Al toxicity. Other enzymes of the phosphoinositide signal transduction pathway were also evaluated. Aluminium disrupts production of second messengers such as inositol 1,4,5-trisphosphate (IP(3)) and phosphatidic acid (PA) by blocking PLC activity; however, phospholipase D (PLD) and diacylglycerol kinase (DGK) activities are stimulated by Al, a response probably aimed at counteracting Al effects on PA formation. Phosphate deprivation also induces PLC and DGK activity. These results suggest that Al-induced cell growth inhibition is not linked to PLC activity inhibition.

Aluminium-induced phospholipid signal transduction pathway in Coffea arabica suspension cells and its amelioration by silicic acid.

Coffee (Coffea arabica L.) is of economic importance worldwide. Its growth in organic-rich acidic soils is influenced by aluminium such that coffee yield may be impaired. Herein we have used the Al-sensitive C. arabica suspension cell line L2 to analyse the effect of two different Al species on the phosphoinositide signal transduction pathway. Our results have shown that the association of Al with coffee cells was affected by the pH and the form of Al in media. More Al was associated with cells at pH 4.3 than 5.8, whereas when Al was present as hydroxyaluminosilicates (HAS) the association was halved at pH 4.3 and unchanged at pH 5.8. Two signal transduction elements were also evaluated; phospholipase C (PLC) activity and phosphatidic acid (PA) formation. PLC was inhibited ( approximately 50%) when cells were incubated for 2 h in the presence of either AlCl(3) or Al in the form of HAS. PA formation was tested as a short-term response to Al. By way of contrast to what was found for PLC, incubation of cells for 15 min in the presence of AlCl(3) decreased the formation of PA whereas the same concentration of Al as HAS produced no effect upon its formation. These results suggest that Al is capable to exert its effects upon signal transduction as Al((aq))(3+) acting upon a mechanism linked to the phosphoinositide signal transduction pathway.