Research articleEvaluation of buffers toxicity in tobacco cells: Homopiperazine-1,4-bis (2-ethanesulfonic acid) is a suitable buffer for plant cells studies at low pH

- β-alanine was clearly cytotoxic and dimethylgluturate and biphthalate were found to be cytostatic.
- MES buffer is still useful, for not presenting cytotoxicity, but does not present pKa suitable for studies at low pH.
- Homopipes did not cause cytotoxicity and provided buffering power at experimental conditions combined with suitable pKa.
- Homopipes can be a useful buffer in low pH experiments, especially for aluminium toxicity experiments.

Low pH is an important environmental stressor of plant root cells. Understanding the mechanisms of stress and tolerance to acidity is critical; however, there is no widely accepted pH buffer for studies of plant cells at low pH. Such a buffer might also benefit studies of Al toxicity, in which buffering at low pH is also important. The challenge is to find a buffer with minimal cellular effects. We examined the cytotoxicity and possible metabolic disturbances of four buffers that have adequate pKa values and potential use for studies in the pH range of 4.0-5.0. These were homopipes (homopiperazine-1,4-bis (2-ethanesulfonic acid); pKa1 4.4), 3,3-dimethylglutaric acid (pKa1 3.73), β-alanine (pKa1 3.70) and potassium biphthalate (pKa1 2.95; pKa2 5.41). First, tobacco BY-2 cells were grown in a rich medium containing 10 mM of each buffer or MES (2-(N-morpholino) ethanesulfonic acid) as a control, with the pH initially adjusted to 5.7. β-alanine was clearly toxic and dimethylgluturate and biphthalate were found to be cytostatic, in which no culture growth occurred but cell viability was either unaffected or decreased only after 5 days. Only homopipes allowed normal culture growth and cell viability. Homopipes (10 mM) was then tested in cell cultures with an initial pH of 4.3 ± 0.17 in minimal medium to examine whether its undissociated species (H2A) displayed any cellular effects and no cytotoxic effects were observed. It is possible to conclude that among tested buffers, homopipes is the most suitable for studies at low pH, and may be especially useful for aluminum toxicity experiments.