Raffinose in chloroplasts is synthesized in the cytosol and transported across the chloroplast envelope

Abstract

In chloroplasts, several water-soluble carbohydrates have been suggested to act as stress protectants. The trisaccharide raffinose (α-1,6-galactosyl sucrose) is such a carbohydrate but has received little attention. We here demonstrate by compartmentation analysis of leaf mesophyll protoplasts that raffinose is clearly (to about 20) present in chloroplasts of cold-treated common bugle (Ajuga reptans L.), spinach (Spinacia oleracea L.) and Arabidopsis [Arabidopsis thaliana (L.) Heynh.] plants. The two dedicated enzymes needed for raffinose synthesis, galactinol synthase and raffinose synthase, were found to be extra-chloroplastic (probably cytosolic) in location, suggesting that the chloroplast envelope contains a raffinose transporter. Uptake experiments with isolated Ajuga and Arabidopsis chloroplasts clearly demonstrated that raffinose is indeed transported across the chloroplast envelope by a raffinose transporter, probably actively. Raffinose uptake into Ajuga chloroplasts was a saturable process with apparent Km and vmax values of 27.8 mM and 3.3 μmol mg-1 Chl min-1, respectively.