e., initial activity, and comparing this activity to the activity of the fully carbamylated enzyme, i.e. total activity (Perchorowicz et al. 1981). To measure Rubisco activation, ARS-1620 price the standard assay described above (Fig. 1a) or a modified version (Fig. 1b) can be used as a stand-alone assay for either purified Rubisco or Rubisco in leaf extracts (Fig. 1b). The modified version still uses dPGM-ST and enolase to convert 3-PGA to PEP, but couples PEP formation to NADH oxidation via pyruvate kinase and lactate dehydrogenase. The

pyruvate kinase-lactate dehydrogenase link requires ADP, a potent inhibitor of RCA, but not Rubisco. Thus, these linking enzymes, while suitable for measuring Rubisco activity per se, cannot be used for measuring the effects of RCA on Rubisco activity in a continuous C59 nmr assay. The main advantage of the modified assay for

measuring Rubisco is that the two linking enzymes are commercially available and inexpensive. To demonstrate the usefulness of the assay for measuring Rubisco activation, the effect of irradiance on the activation state of Rubisco was determined in wild-type and transgenic Arabidopsis using the modified assay (Fig. 1b). As shown in Table 1, the results demonstrated that the assay was capable of measuring light-dependent changes in Rubisco activation that occur in wild-type plants. The measurements also confirmed that (1) deactivation of Rubisco in response to low light was minimal in the rwt43 transformant, a transgenic Arabidopsis that expresses only the ADP-insensitive β-isoform of RCA (PD173074 manufacturer Carmo-Silva and Salvucci 2013) and (2) the rates

of Rubisco activity most in crude leaf extracts of wild-type and transgenic plants were similar to those determined with a 14C-based Rubisco assay (Salvucci et al. 2006). In a separate set of experiments, the non-radioactive assay was used to detect the decrease in Rubisco activation state that occurred in camelina plants subjected to heat stress (Supplemental Table S2). These results confirmed previous findings obtained using the 14C assay (Carmo-Silva and Salvucci 2012). Table 1 Effect of irradiance on the activation state of Rubisco in wild type Arabidopsis and the transgenic line, rwt43 Arabidopsis line Irradiance (μE m−2 s−1) Rubisco activity Activation (%) Initial Total (μmol min−1 mg−1 prot) Wild type 1200 0.40 ± 0.03 0.46 ± 0.08 86 ± 3a   75 0.35 ± 0.03 0.59 ± 0.02 60 ± 4b   25 0.13 ± 0.01 0.59 ± 0.03 23 ± 2c rwt43 1200 0.42 ± 0.08 0.45 ± 0.07 91 ± 4a   75 0.45 ± 0.04 0.53 ± 0.04 85 ± 4a   25 0.47 ± 0.03 0.55 ± 0.04 87 ± 3a Leaf discs were exposed to the indicated irradiance for 120 min prior to sampling. Letters indicate activation states that are statistically different at the P = <0.