Plant BioDynamics Laboratory

Chara

The Green algae Chara as model system for intra and inter cellular transport processes.

Program

• Improvement of laboratory cultures of (ecortical) Chara species.

In The Netherlands the ecorticate species Chara australis and Chara coralline are not indigeneous, while the ecorticate Chara braunii is very rare. Therefore, building up and cherishing an international network, to secure the availability of clean (not contaminated) and healthy Chara material is of the utmost importance. In the past we have encountered problems with maintaining cultures, due to a sudden collapse of cultures. We have now overcome this problem and we can maintain cultures for several years. Special attention will be paid to other propagation methods, both asexually and sexually.

• Measuring intra- and intercellular auxin transport.

The natural auxin IAA (Indole-3-acetic acid) has been detected in Chara species, but its developmental role is unknown. When we started the program, the occurrence of PAT was still unknown as well. However, adapting our donor-receiver PAT assay (see above) to thallus of Chara coralline, we could demonstrate PAT in internodal cells. The velocity of this PAT in these giant (3-5 cm long) internodal cells was relatively high (approx. 4-5 cm/hr), which excluded transport by simple diffusion. In addition it was not sensitive to a specific inhibitor of cytoplasmatic streaming. Further characterization of the dynamics of PAT in Chara is in progress.

• Visualization of distribution of IAA in internode cells.

A challenging part of the program is the question: what kind of mechanism is at the root of PAT in internode cells of Chara? In order to address this question, we decided to use a fluorescent active derivative of IAA as probe to visualize distribution and transport of auxin in internodal cells, thereby using advanced microscopic techniques, such as CSLM (confocal scanning laser microscopy). Since a few attempts by other groups to synthesize fluorescent auxin did not give convincing results, we decided to try it ourselves in collaboration with the Leiden Institute of Chemistry (LIC).

• Isolation and sequencing of a potential Chara PM H⁺-ATPase (CHA1).

Bio-informatics and functional analysis of CHA1 revealed a new amino acid pattern which could be essential for structural stabilization of ATP hydrolysis and proton transport. This suggests a regulation mechanism that is different from the known plant and yeast PM H⁺-ATPases which energize the PM by generating an electrochemical proton gradient. This might explain the observed different effect of exogenous auxin on trans PM H⁺-fluxes in Chara internode cells (see above). A follow-up of this study, which may shed light on the evolution of PM H⁺-ATPases from green algae to higher plants, is in progress.

• Investigation of trans plasma membrane ion-fluxes, in particular K⁺ and H⁺, in Chara internodal cells, using the non-invasive ion-selective vibrating scanning electrode technique, in collaboration with Prof. B. de Boer (VU). Interestingly, it was found that physiological concentrations of exogenous auxin (IAA) had, in contrast to higher plants, no effect on H⁺ fluxes, which suggests the presence of different plasma membrane (PM) protonpumps (H⁺-ATPases) in Chara.