Extended Data Fig. 7: N-terminal tagging of proteins with a ZPR2 sequence confers degradability by the N-degron pathway.
From: An apical hypoxic niche sets the pace of shoot meristem activity
a, Relative mRNA level of the genotypes used for immunodetection of GFP in Fig. 2d under aerobic and hypoxic conditions (2% O2), 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% O2. 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 O2 concentration by the corrected staining intensity at 21% O2. 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% O2, respectively.
