Photosynthesis by Isolated Protoplasts, Protoplast Extracts, and Chloroplasts of Wheat

Abstract

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material) and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O2 evolution of about 100-150 .mu.mol of O2/mg of chlorophyll per h at 20.degree. C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 mM N-2-hydroxyethylpiperazine-N''-2-ethanesulfonic acid KOH (pH 7.6), and 10 mM NaHCO3 with protoplast, plus a requirement of 1-10 mM ethylenediaminetetraacetate (EDTA) and 0.2-0.5 mM Pi with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 .mu.mol of O2/mg of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset. Photosynthesis by chloroplasts from 6 day old wheat plants under optimum levels of Pi was similar to that with the addition of 5 mM PPi plus 0.2 mM ADP. Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2-6 min, photosynthesis was strongly inhibited by 5 mM PPi and this inhibiting was relieved by including ATP or ADP (0.2-0.6 mM). Chloroplasts from 9 day old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP. Chloroplasts isolated from protoplasts and assayed with 0.3 mM Pi added before illumination have an induction time from less than 1 min up to 16 min depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO3 and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20-30 min storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.