ALBERT

Notes: In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein–starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as α-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473–477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, α-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated α-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both α-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in vitro studies in which the binding of purified β-amylase (EC 3.2.1.2) to starch granules isolated from turions following various pretreatments was monitored. The enzyme did bind to starch granules prepared from dark-stored turions (in which starch degradation had not been initiated), but not to those isolated from illuminated (starch degrading) turions.