John Kobza

Metabolism of 2-carboxyarabinitol 1-phosphate and regulation of ribulose-1,5-bisphosphate carboxylase activity

Metabolism of 2′-carboxy-D-arabinitol 1-phosphate (CA1P) is an important component in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity and whole leaf photosynthetic CO2 assimilation in many species, and functions as one mechanism for regulating Rubisco activity when photosynthesis is light-limited. Species differ in their capacity to accumulate CA1P, ranging from those which can synthesize levels of this compound approaching or in excess of the Rubisco catalytic site concentration, to those which apparently lack the capacity for CA1P synthesis. CA1P is structurally related to the six carbon transition state intermediate of the carboxylation reaction and binds tightly to the carbamylated catalytic site of Rubisco, making that site unavailable for catalysis. Under steady-state, the concentration of CA1P in the leaf is highest at low photon flux density (PFD) or in the dark. Degradation of CA1P and recovery of Rubisco activity requires light and is stimulated by increasing PFD. The initial degradation reaction is catalyzed by an enzyme located in the chloroplast stroma, CA1P phosphatase, which yields carboxyarabinitol (CA) and inorganic phosphate as its products. The pathway of CA metabolism in the plant remains to be determined. Synthesis of CA1P occurs in the dark, and in Phaseolus vulgaris this process has been shown to be stimulated by low PFD. The pathway of CA1P synthesis and its relationship to the degradative pathway remains unknown at the present time. The discovery of the existence of this previously unknown carbon pathway in photosynthesis indicates that we still have much to learn concerning the regulation of Rubisco activity and photosynthesis.

Isolation of photosynthetically active protoplasts and intact chloroplasts from Phaseolus vulgaris

A method is described for the isolation of protoplasts and intact chloroplasts from leaves of Phaseolus vulgaris L. The protocol includes digestion of the cell walls with a mixture of Onozuka RS cellulase and Pectolyase Y-23. After a 3.5-h digestion and isolation period, a typical yield of protoplasts was 0.1–0.2 mg chlorophyll per g fresh weight of leaf tissue. At saturating light levels, protoplasts demonstrated photosynthetic rates of 100–125 μmol O2 evolved (mg chlorophyll)−1 h−1. Chloroplasts were isolated by gentle lysis of the protoplasts and were purified by centrifugation through Percoll. These chloroplasts were >90% intact and were capable of CO2-dependent O2 evolution at rates of 100–120 μmol (mg chlorophyll)−1 h−1. The chloroplasts required ethylenediamine tetraacetic acid during purification and in the assay medium for maximum rates of photosynthesis. The response of photosynthesis to inorganic phosphate (Pi) by chloroplasts was biphasic with two observed optima at approximately 25 and 200 μM Pi.