The plant N-degron pathways of ubiquitin-mediated proteolysis; a review.

The amino-terminal residue of a protein (or amino-terminus of a peptide following protease 16 cleavage) can be an important determinant of its stability, through the Ubiquitin-Proteasome- 17 System associated N-degron pathways. Plants contain a unique combination of N-degron 18 pathways (previously called the N-end rule pathways) E3 ligases, PROTEOLYSIS (PRT)6 and 19 PRT1, recognising non-overlapping sets of amino-terminal residues, and others remain to be 20 identified. Although only very few substrates of PRT1 or PRT6 have been identified, substrates 21 of the oxygen and nitric oxide sensing branch of the PRT6 N-degron pathway include key 22 nuclear-located transcription factors (ETHYLENE RESPONSE FACTOR VIIs and LITTLE 23 ZIPPER 2) and the histone-modifying Polycomb Repressive Complex 2 component 24 VERNALISATION 2. In response to reduced oxygen or nitric oxide levels (and other 25 mechanisms that reduce pathway activity) these stabilised substrates regulate diverse 26 aspects of growth and development, including response to flooding, salinity, vernalisation 27 (cold-induced flowering) and shoot apical meristem function. The N-degron pathways show 28 great promise for use in the improvement of crop performance and for biotechnological 29 applications. Upstream proteases, components of the different pathways and associated 30 substrates still remain to be identified and characterised to fully appreciate how regulation of 31 protein stability through the amino-terminal residue impacts plant biology. and prt1 22 mutants, indicated a short half-life for the carboxyl-terminus (Ct-) proteoform of BB that was 23 dependant on Tyr61 and PRT1 . In addition, PRT1 was shown in vitro to bind to a peptide 24 mimicking Try61-BB. These data indicate that the PRT1 N-degron pathway is one mechanism 25 controlling the stability of BB Ct-proteoform produced following DA1-mediated cleavage. As 26 DA1 cleaves other proteins associated with cell proliferation, PRT1 may also be involved in 27 controlling their specificity, and it will be interesting to determine whether there is a role for 28 PRT1 in cell proliferation. of publicly available datasets that hypoxia-induced transcriptomes are in Agrobacterium tumefaciens . The high metabolic demand of A. tumefaciens crown-gall formation was shown to produce a steep hypoxia gradient within the gall, leading to up-regulation of hypoxia- responsive genes Crown gall symptoms were greatly reduced in the erfVII mutant but increased in mutants with constitutively stabilised ERFVIIs ( pco1 pco2 , ate1 ate2 and prt6 These data Indicate that tumour-forming pathogens utilise the endogenous plant oxygen sensing mechanism to enhance infection success, and also raise the interesting possibility that other Nt-Cys substrates may also be stabilised in response to pathogen attack and influence the host response.

living organisms, suggesting that a mechanism for degradation based on identification of the 1 Nt-residue evolved early for regulating protein fate. N-degrons are defined operationally as 2 components of substrate proteins that include an N-terminal 'destabilising' residue, an N-3 recognin (E3 ligase specific for N-degrons) available N-terminus, and a Lys residue for 4 ubiquitin addition (Gibbs et al. 2016). In eukaryotes N-degron pathways operate in conjunction

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The Arg/N-degron pathway has a hierarchical structure that enzymatically funnels 17 substrates to the N-recognin by sequential alteration of tertiary to secondary destabilising Nt-18 residues, that are then arginylated to provide an N-terminus with a primary destabilising 19 residue ( Figure 1A). Eukaryotic tertiary destabilising residues Gln and Asn can be converted      Nt-Leu requires additional yet-to-be discovered N-recognin components. The terminology for 2 N-degron pathways used in mammalian/yeast systems is not suitable in plants because of the 3 separation of functions of N-recognins into more than 2 distinct proteins. Therefore, it is 4 proposed the plant N-degron pathways are named by the associated N-recognin (e.g. PRT6 5 or PRT1 N-degron pathway) or by the destabilising residue where there is no known N-6 recognin (e.g. Leu/N-degron pathway), and branches of the pathways by associated enzymes

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Tertiary and secondary destabilising residues are converted to primary destabilising residues 12 through the action of Nt-residue-specific enzyme pathways ( Figure 1A). Conversion of the N-13 terminus from secondary to the primary destabilising residue Arg in arabidopsis is controlled 14 by ATE activity (Yoshida et al. 2002). ATE1 was shown in protoplasts from mesophyll cells to   Nt-Asn (NTAN1) and Nt-Gln (NTAQ1)-amidohydrolases respectively, and these were shown    (Gibbs et al. 2014b). It was considered that oxidation of cysteine to Cys-sulphinic (-SO2 -) or -7 sulphonic (-SO3 -) acid provided a bioisostere of Asp thereby acting as a substrate for ATE 8 (Kwon et al. 2002). A breakthrough in the field was the discovery that in plants oxidation of Nt-

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Cys requires the activity of PCO enzymes (Weits et al. 2014). These are monomeric, non-10 heme iron-dependent dioxygenases that use molecular oxygen to catalyse the formation of 11 Cys-sulphinic acid from Nt-Cys, that has been shown to then provide an efficient substrate for

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A key pre-requisite for entry into the majority of N-degron pathways (but not necessarily Met-34 or formyl-Met pathways, Figure 1A) is the action of intracellular peptidases to cleave their 35 targets, producing proteoforms with exposed N-degrons. In mammalian systems caspase and   that only a small proportion of these will be physiological PRT6 N-degron pathway substrates.     (Table 1). In particular it has become clear that the pathways play key roles in plant-36 environment interactions by controlling the half-lives of known regulator proteins, or through 37 yet to be identified substrates. Physiological roles were first identified by analysis of 1 phenotypes of mutants obtained through either forward or reverse genetic screens. The first 2 genetically-identified component of plant N-degron pathways shown to regulate a 3 physiological process was ATE1, as a determinant of leaf senescence (from the mutant 4 delayed-leaf-senescence (dls)1) (Yoshida et al. 2002). DLS1 (that encodes ATE1) was shown 5 to influence the progression of leaf senescence in an age-dependant way and in response to 6 dark, indicating that substrate(s) that require Nt-Arg addition are required for this process.

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Subsequently it was also shown that a double mutant ate1 ate2, that removes all ATE activity,

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B. Enhanced survival of arabidopsis seedlings following extended skotomorphogenic 5 development in hypoxia, compared to normoxia, when subsequently exposed to normoxia in 6 the light, is controlled by ERFVII transcription factors (images taken from (Abbas et al. 2015)).

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All seedlings except wild-type (WT) grown under hypoxia in the dark are dead.