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.

Protoplasts: a friendly tool to study aluminum toxicity and coffee cell viability.

OBJECTIVE: Aluminum toxicity is a major limiting factor with regard to crop production and quality in most acidic soils around the world. We propose the use of C. arabica L. protoplasts to evaluate the toxic effects of aluminum, the nuclear localization of aluminum and propensity of aluminum to cause DNA damage. RESULTS: After protoplasts were exposed to aluminum (Al) for varying periods of time (0, 5, 10, 20 and 30 min), we detected a reduction in protoplast viability. Additionally, we observed a rapid decline in the ability of protoplasts to synthesize DNA following exposure to Al for 30 min. Furthermore, DNA damage was observed after 10 min of treatment with Al. CONCLUSIONS: Protoplasts can be used to evaluate the effects of Al upon entry into the cell, which affects the structure of the nucleus. These results indicate that protoplasts provide a useful model for the study Al toxicity at the cellular level.

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.

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.

Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway.

Aluminum (Al(3+)) has been recognized as a main toxic factor in crop production in acid lands. Phosphatidic acid (PA) is emerging as an important lipid signaling molecule and has been implicated in various stress-signaling pathways in plants. In this paper, we focus on how PA generation is affected by Al(3+) using Coffea arabica suspension cells. We pre-labeled cells with [(32)P]orthophosphate ((32)Pi) and assayed for (32)P-PA formation in response to Al(3+). Treating cells for 15 min with either AlCl(3) or Al(NO(3))(3) inhibited the formation of PA. In order to test how Al(3+) affected PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. The Al(3+) inhibited mas-7 induction of PA response, both before and after Al(3+) incubation. The PA involved in signaling is generated by two distinct phospholipid signaling pathways, via phospholipase D (PLD; EC: 3.1.4.4) or via Phospholipase C (PLC; EC: 3.1.4.3), and diacylglycerol kinase (DGK; EC 2.7.1.107). By labeling with (32)Pi for short periods of time, we found that PA formation was inhibited almost 30% when the cells were incubated with AlCl(3) suggesting the involvement of the PLC/DGK pathway. Incubation of cells with PLC inhibitor, U73122, affected PA formation, like AlCl(3) did. PLD in vivo activation by mas-7 was reduced by Al(3+). These results suggest that PA formation was prevented through the inhibition of the PLC activity, and it provides the first evidence for the role of Al toxicity on PA production.

Exposure to toxic concentrations of aluminum activates a MAPK-like protein in cell suspension cultures of Coffea arabica.

Addition of a toxic concentration of aluminum (Al) to cell suspension cultures of Coffea arabica L. induced the rapid and transient activation of a protein kinase that phosphorylates myelin basic protein (MBP), as revealed by in-gel kinase assays. This enzyme with an apparent molecular mass of 58 kDa was activated shortly after cells were exposed to 50 microM AlCl(3), a concentration previously shown to produce toxicity in plant cells in vitro. The activity of this kinase dropped to basal levels after 20 min of Al addition; this activity is specific for MBP as it could not be detected when casein or histone H1 were used as substrates. Analysis of the same cell extracts with antibodies that specifically recognize bis-phosphorylated (active) mitogen-activated protein kinases (MAP kinases), revealed the presence of a phosphoprotein with an apparent molecular mass of 58 kDa, which showed the same response to Al as the protein kinase revealed by the in-gel kinase assays. Furthermore, immunoprecipitation with an antibody directed against mammalian MAP kinases depleted both the enzymatic activity and the phosphoprotein from the cell extracts, suggesting that the 58 kDa kinase and the 58 kDa phosphoprotein from C. arabica cells are the same protein, and that it can be actually a member of the MAP kinase family of protein kinases. Since its activity is enhanced dramatically after addition of AlCl(3) to the medium, we can speculate that Al toxicity in plants could be perceived through the MAP kinase signal transduction pathway.

Interaction of spermine with a signal transduction pathway involving phospholipase C, during the growth of Catharanthus roseus transformed roots.

In Cantharanthus roseus transformed roots, the application of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50), inhibited the root growth in a dose-dependent manner with a DL(50) of about 300 micro m. Spermidine and spermine (Spm) levels and SAMDC and phospholipase C (PLC; EC 3.1.4.3) activities were reduced in the presence of the inhibitor. The inhibition was reversed by the addition of Spm. Radioactivity from [(14)C]Spm was detected in an immunoprecipitated fraction with an antibody anti-PLC-delta. To our knowledge, this is the first direct evidence that demonstrates an interaction of Spm with the signal transduction cascade phosphoinositide-Ca(2+).