The total leaf N content significantly decreased in the low-Pi treatments and remained almost constant above 1.2 mM Pi treatments (Figure 4a). The Rubisco content showed a similar response to the total leaf N content. A low-Pi application slightly decreased the Rubisco content, but a higher Pi application did not change the Rubisco content (Figure 4b).
Rubisco activation maintained high in the low-Pi treatments, but decreased with increasing Pi application (Figure 4c). The carbamylation potential of Rubisco, which is lowered by a tight binging inhibitor, such as 2-carboxy-D-arabinitol 1-phosphate, was not different among different levels of Pi application (Figure 4d). Indeed, the Rubisco in the night-sampled leaves showed significantly lower activation and carbamylation potential compared with the Rubisco sampled under illumination (Figure 4c, d). Thus, one of the reasons for the decline in photosynthesis by excessive Pi application is a decline in in vivo Rubisco activation.
Because Rubisco activation is catalyzed by Rubisco activase (RCA) (Salvucci et al., 1985), we examined the RCA content of the leaves. Three different isoforms of RCA resulting from alternative splicing and limited proteolysis have been reported in rice plants (Portis 2003; Vargas-Suarez et al., 2004; Fukayama et al., 2012). According to Fukayama et al. (2012), an upper protein band corresponds to theα- form of RCA, which has redox-active cysteine residues; a middle protein band corresponds to the β -form, which lacks redox active Cys residues; and a lower protein band corresponds to the N-terminal processed β (β* )-form in the leaves (Figure 4e). The total amount of RCA, including the α- , β-, and β*- forms was similar among different Pi treatments (Figure 4f). However, a high-Pi application decreased the content of α - andβ -forms compared with the control-Pi conditions (Figure 4g). Contrary to the change in the α- and β -forms, theβ* -form content increased with increasing Pi application (Figure 4g). When the isoform ratios of α /β and β */βwere calculated, an increase in Pi application decreased theα /β ratio and increased the β */β ratio, compared to the low-Pi and control plants (Figure 4h, i). These results indicated that an increase in Pi application modified the RCA isoform composition of the leaves.