Diagrammatic representation of the N-end rule pathway associated with PRT6 function. Components for which orthologous Arabidopsis genes have been identified are highlighted. Protein substrate (represented as a cylinder) is cleaved by a methionine aminopeptidase (MetAP) or a specific endopeptidase (Endo-P) at a unique amino acid sequence (dotted curve). Cleavage reveals destabilizing amino acid residues, which may be further modified by enzymatic (NTAN, amino-terminal amidohydrolase; ATE1/2, Arg-tRNA-protein transferase) or chemical (O₂, NO) action. 1°, 2°, and 3° indicate primary, secondary, and tertiary destabilizing amino-terminal residues, respectively. Generation of a primary destabilizing residue (1°) leads to targeting by E3 ubiquitin ligases PROTEOLYSIS6 (PRT6) or PRT1, depending on the nature of the destabilizing residue. C*, oxidized derivative of cysteine.

Influence of PRT6 and ATE1/2 on lipid degradation during establishment. (A) Establishment of prt6-4 and WT (Ler) seedlings after 7 days of growth on increasing concentrations of sucrose either in the presence (1/2MS) or absence (water) of 1/2 MS following 2 days of moist chilling. (B) Influence of exogenous sucrose on root length of prt6 and ate1 and ate2 alleles in relation to WT, measured at 7 days of imbibition on 1/2MS media following 2 days of moist chilling. (C and D) Representative confocal microscopy images of hypocotyl epidermal (C) and endosperm (D) cells (5 days of imbibition on 1/2MS, no added sucrose, following 2 days of moist chilling) stained with Nile Red to reveal oil bodies, which can be seen as red-staining spherical inclusions in prt6-1 and ate1 ate2, but not in Col-0. (Scale bar: 20 μm.) Brightfield images are presented in Fig. S5. (E) Eicosenoic acid (20:1) content of 5-day-old seedlings grown on 1/2MS media following 2 days of moist chilling. Data represent means ± SE of the mean.

ABA sensitivity of germination, after-ripening, and sucrose sensitivity of establishment. (A) After-ripening of WT (Ler) and prt6-4 seeds assayed on water agarose after 7 days. (B) Germination potential of seeds imbibed for 7 days on water-agarose media after increasing periods of cold, moist chilling. (C) Germination potential of seeds imbibed for 7 days on agarose media containing 1/2MS in the absence (black bars) and presence (white bars) of 100 μM GA₃. (D and E) Germination potential after 7 days of WT, mutant, and transgenic seeds assayed on 1/2MS media in the presence of exogenous ABA following 2 days of moist chilling. Seeds were stored for 2 months before assay. Respective WT and mutant seeds are: Col-0, prt1-1, prt6-1, prt6-2, ate1-2, ate2-1, abh1, ahg1, 2, 3 (3–5 and 36–39). Data points for ate2-1 are slightly obscured by those of prt1. Results obtained for ABI3OX, ABI5OX, and aip2 are presented in Fig. S2D. (F and G) Change in sensitivity of endosperm rupture (F) and testa rupture (G) to exogenous ABA, with time of dry storage of cold, chilled WT (Ler) and prt6-4 seeds, assayed on 1/2MS media and measured at 7 days following 2 days of moist chilling. (H) Sucrose sensitivity of establishment (greening of cotyledons) of WT, transgenic, and mutant seedlings grown on water-agarose supplemented with 1% (wt/vol) sucrose measured at 7 days, following 2 days of moist chilling. Dose–response curves are presented in Fig. S4. Data represent means ± SE of the mean.

Genetic interactions of PRT6 with components of ABA and GA biosynthesis and signaling. (A and B) Germination potential after 7 days of WT mutant and transgenic seeds on 1/2MS media in the presence of exogenous ABA, following 2 days of moist chilling. abi1-1 (Ler background) (26), aba1-1 (Ler) (25), rgl2-1 (Ler) (24), abi3-8 (Col-0), abi3-10 (Col-0) (28), abi5-1 (Ws) (27). (C) Germination potential, after 7 days, of freshly harvested seeds on water-agarose. (D) Seedling establishment, after 7 days, on water-agarose containing 1% (wt/vol) sucrose, following 2 days of moist chilling (dose–response curves are presented in Fig. S4.). (E) Light micrographs of hypocotyl epidermis of single- and double-mutant seeds showing retention of lipid bodies in prt6-4, but not in single- or double-mutant combinations with abi3-10 or abi5-1, on 1/2MS media after 5 days, following 2 days of moist chilling. (Scale bar: 20 μm.). Data represent means ± SE of the mean.