Abstract
Summary The N‐end rule pathway of targeted protein degradation is an important regulator of diverse processes in plants but detailed knowledge regarding its influence on the proteome is lacking.To investigate the impact of the Arg/N‐end rule pathway on the proteome of etiolated seedlings, we used terminal amine isotopic labelling of substrates with tandem mass tags (TMT‐TAILS) for relative quantification of N‐terminal peptides in prt6, an Arabidopsis thaliana N‐end rule mutant lacking the E3 ligase PROTEOLYSIS6 (PRT6). TMT‐TAILS identified over 4000 unique N‐terminal peptides representing c. 2000 protein groups. Forty‐five protein groups exhibited significantly increased N‐terminal peptide abundance in prt6 seedlings, including cruciferins, major seed storage proteins, which were regulated by Group VII Ethylene Response Factor (ERFVII) transcription factors, known substrates of PRT6. Mobilisation of endosperm α‐cruciferin was delayed in prt6 seedlings. N‐termini of several proteases were downregulated in prt6, including RD21A. RD21A transcript, protein and activity levels were downregulated in a largely ERFVII‐dependent manner. By contrast, cathepsin B3 protein and activity were upregulated by ERFVIIs independent of transcript.We propose that the PRT6 branch of the pathway regulates protease activities in a complex manner and optimises storage reserve mobilisation in the transition from seed to seedling via control of ERFVII action.
Highlights
The transitions from dormant seed to photosynthetically active plant are key steps in the life cycle of plants (Holdsworth et al, 2008; Wu, 2014; de Wit et al, 2016)
Etiolated seedlings were selected for analysis because PRT6 is active at this developmental stage, as demonstrated by stabilisation of the artificial Arg/N-end rule substrate, R-GUS, in the prt6 mutant background (Fig. S2)
Labelling of proteins with TMTsixplexTM reagents was used in combination with TAILS to identify and quantify Nt peptides in seedlings of Col-0 and the null mutant, prt6-5 (Graciet et al, 2009)
Summary
The transitions from dormant seed to photosynthetically active plant are key steps in the life cycle of plants (Holdsworth et al, 2008; Wu, 2014; de Wit et al, 2016). Dependent on the light environment following germination, a seedling may undergo skotomorphogenesis (hypocotyl elongation in the dark) or photomorphogenesis (opening of the apical hook and development of the photosynthetic apparatus). In both cases, mobilisation of seed storage reserves fuels growth until plants become fully photoautotrophic (Penfield et al, 2006b; Theodoulou & Eastmond, 2012). Lipids in the form of triacylglycerol (TAG) and specialised seed storage proteins (SSPs), but the relative proportions differ considerably between species (Baud et al, 2008) In oilseed plants, such as Arabidopsis, TAG is the most abundant storage reserve but the endosperm and embryo of Arabidopsis seeds contain numerous protein storage vacuoles (PSVs). Tissue-specific analysis of abscisic acid (ABA) signalling has shown that mobilisation of embryo and endosperm lipid reserves is under distinct hormonal control (Penfield et al, 2004, 2006a)
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