a, Relative mRNA level of the genotypes used for immunodetection of GFP in Fig. 2d under aerobic and hypoxic conditions (2% O 2 ), measured by RT–qPCR. Two biological replicates from two independent lines were used in the case of the constructs with wild-type ZPR2 (35S:ZPR2-GFP) and (MAC)ZPR2-GFP lines. Four biological replicates were instead used in the case of wild-type ZPR2-GFP in the prt6 background. The effect of hypoxia treatment versus aerobic conditions was evaluated by two-way ANOVA (P value = 0.342, n = 4 pools of 3 plants). The results of the RT–qPCR analysis exclude regulation by hypoxia at the transcriptional level and—combined with the immunoblot analysis—support the existence of a control checkpoint at the post-transcriptional level. b, Relative luciferase activity of a chimeric protein that consists of the whole ZPR2 coding sequence fused to the N terminus of a firefly luciferase (ZPR2-PpLUC). This construct was transfected together with a second one, which bears a Renilla luciferase gene driven by the same 35S CaMV promoter, into Arabidopsis mesophyll protoplasts. Renilla luciferase activity was used as a normalization control. One-way ANOVA followed by Tukey post hoc test; n = 6, 4 and 5 protoplast pools for wild-type ZPR2 (MC-ZPR2), (MAC)ZPR2 (here MAC-ZPR2) and wild-type ZPR2 in prt6, respectively. c, Relative GUS activity of a gZPR2-GUS construct expressed in Arabidopsis protoplasts. A 35S:PpLuc reporter was co-transformed to equalize for transfection efficiency. The addition of an Ala residue before the Cys2 led to enhanced stability of both reporter constructs, and the expression of the wild-type ZPR2 protein fusions in the prt6 mutant background also showed enhanced protein abundance. One-way ANOVA followed by Tukey post hoc test; n = 8, 5 and 6 protoplast pools for wild-type ZPR2, (MAC)ZPR2 and wild-type ZPR2 in prt6, respectively. d, Quantification of the relative staining intensity of GUS-stained plants that express a pZPR2:ZPR2-GUS construct at 2%, 21% and 80% O 2 . An example of GUS staining at each oxygen concentration is shown in Fig. 3b. Images of GUS-stained plants were converted to inverted greyscale images, and the staining intensity was measured using ImageJ. Wild-type plants that were de-stained in ethanol were used to correct for the background signal. Average relative staining intensity was calculated by comparing the corrected staining intensity at each O 2 concentration by the corrected staining intensity at 21% O 2 . These results show that ZPR2 stability in the SAM depends on oxygen availability. One-way ANOVA followed by Holm–Sidak post hoc test; n = 10, 7 and 8 plants for 2%, 21% and 80% O 2 , respectively. Source Data