Figure 4.

Analysis of PAO and RCCR activities (A), protein abundance (B), and gene expression (C) during dark-induced senescence of Arabidopsis leaves. A, For the determination of their activities, PAO and RCCR, respectively, were isolated from the tissues of interest and assessed in RFF-containing assays that were supplemented with purified His₆-AtRCCR (determination of PAO activity, white circles) and a standard preparation of PAO (determination of RCCR, black circles), respectively. Values are data from several independent experiments showing the great variability of PAO activity. B, Representative immunoblots of PAO and RCCR extracts used for measuring enzyme activities as shown in A. Gel loadings are based on equal amounts of fresh weight. Blots were labeled and developed as described in “Materials and Methods” using polyclonal antibodies against PAO and RCCR, respectively. C, RNA abundance of Pao (gray) and Rccr (black) was quantified by real-time RT-PCR and normalized to levels of mRNA encoding the actin 2 protein. Values are means of three replicates. Error bars indicate sd. n.d., Not determined.