Regulation of Ribulose-, 5-Bisphosphate Carboxylase Activity in Response to Diurnal Changes in Irradiancel

The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (Rubisco) activity and metabolite pool sizes in response to natural diumal changes in photon flux density (PFD) was examined in three species (Phaseolus vulgaris, Beta vulgaris, and Spinacia oleracea) known to differ in the mechanisms used for this regulation. Diumal regulation of Rubisco activity in P. vulgaris was primarily the result of metabolism of the naturally occurring tight-binding inhibitor of Rubisco, 2-carboxyarabinitol 1-phosphate (CAIP). In B. vulgaris, the regulation of Rubisco activity was the result of both changes in activation state and CAIP metabolism. In S. oleracea, Rubisco activity was regulated by a combination of changes in activation state and the binding/ release of another tight binding inhibitor, probably RuBP. Despite these different mechanisms for the light regulation of Rubisco activity, the relationship between the in vivo activity of Rubisco and the PFD was the same for all three species. Rates of CAIP metabolism were thus sufficient to allow this mechanism to participate in the diumal regulation of Rubisco activity as PFD changed at its normal rate. Furthermore, under natural conditions this regulatory mechanism was found to be important in controlling Rubisco activity over approximately the same range of PFD as did changes in activation state of the enzyme. Finally, this regulation of Rubisco activity resulted in relatively similar and saturating RuBP pool sizes for photosynthesis at all but the lowest PFD values in all three species. The light-dependent regulation of Rubisco' activity involves a number of components, including carbamylation/ decarbamylation of a specific lysine residue (13), synthesis/ breakdown of a tight-binding inhibitor of catalysis, CAIP (2, 7, 10, 21, 26), and a protein factor, Rubisco activase, involved in the release of noncatalytically bound RuBP from the enzyme (17). Regulation of Rubisco activity by these mechanisms is important in matching the leaf s capacity for RuBP regeneration (light harvesting, electron transport, photophosphorylation) with its capacity for RuBP carboxylation and starch and sucrose synthesis (28). Different species ofC3 plants do not necessarily use these regulatory mechanisms in the same proportions, however, (10, 23). In a previous study, we examined the light regulation of Rubisco activity and photo' This research was supported by the National Science Foundation under grant DCB 8796314 to J. R. S. 2Abbreviations: Rubisco, RuBP carboxylase (EC 4.1.1.39); CAIP, 2-carboxyarabinitol 1-phosphate; kcat, catalytic constant; PFD, photon flux density; RuBP, ribulose 1,5-bisphosphate. synthesis under steady-state light conditions in three species which differed in the mechanisms used for Rubisco regulation. In this study, we have examined how Rubisco activity is regulated in these same three species under a natural diurnal irradiance pattern, the mechanisms ofthis regulation, and the consequences of this regulation on the pool size of RuBP. MATERIALS AND METHODS Plant Growth and Termination of Metabolism Plants were grown from seed in 4 L pots in a mixture of compost:sand:perlite (2:1:1, v:v:v). Phaseolus vulgaris L. var Linden, Beta vulgaris L. var SSBN1, and Spinacia oleracea were moved to a common greenhouse for the diurnal experiment. Throughout the day, leaf samples were obtained using a hand-held freeze-clamp with copper heads (designed in our laboratory) cooled to the temperature of liquid N2. This ensured rapid termination of the leafs metabolism. The leaf samples were also bisected into two equal halves by the freeze clamp. Samples were stored in liquid N2 until further processing could occur. At each sampling point during the d 3 samples per species were generated and the mean is reported here. The PFD was monitored using a model LI-185B quantum-radiometer-photometer (LiCor, Inc.). Metabolite and Rubisco Analyses RuBP assays were carried out with HCIO4 acid extracts of one-halfofthe leafdisc, as described by Seemann and Sharkey (24). CA1P content in the acid extract was determined by the inhibition ofpurified and activated spinach Rubisco produced by the metabolite extract in comparison to a standard curve of activity produced in the absence of any inhibitor (21). The second halfofthe leafsample was extracted for enzyme analysis in 4 mL of a C02-free, ice-cold buffer containing 100 mM Bicine (pH 7.8), 5 mM MgCl2, 0.1 mm EDTA, 5 mM DTT, and 1.5% (w/v) polyvinylpolypyrrolidone. The homogenate was clarified by a 10 s spin (total time) in an Eppendorf model 5414 microfuge and duplicate aliquots were immediately assayed for Rubisco activity. This activity is referred to as the 'initial activity'. The initial activity is dependent upon the state of activation of the enzyme and the concentration of tight-binding inhibitors in the leaf (23) and represents the substrate saturated in vivo activity as affected by these regulatory mechanisms. Another aliquot of the leaf homogenate was made 10 mm and 20 mm with HC03and Mg2+, respectively (10% dilution), and allowed to incubate on ice for 10 min. We have previously demonstrated that this procedure