The proteins of the translational apparatus possess a remarkable resiliency

In a survey of protein half-lives in barley leaves, Nelson et al. (Reference Nelson, Alexova, Jacoby and Millar2014) found those proteins associated with the translational apparatus (no specific Category) to be particularly long-lived, suggesting that these proteins might somehow be inherently resilient or that they are a class of the proteome to which partiality is shown by the protective and/or repair mechanisms of the cell. Indeed, the ribosomal proteins (r-proteins; Category 1) from diverse species, once assembled into ribosomes, are particularly long-lived, at least in the cytoplasm (Boisvert et al., Reference Boisvert, Ahmad, Gierlinski, Charriere, Lamont, Scott, Barton and Lamond2012; Christiano et al., Reference Christiano, Nagaraj, Frohlich and Walther2014; Li et al., Reference Li, Nelson, Trosch, Castleden, Huang and Millar2017a).

The r-proteins of the large- and small-ribosomal subunits are intimately involved in translation. Some insight into the long-lived nature of some of the r-proteins has been linked to their high positive net charge at physiological pH, with some species’ large subunit r-proteins all possessing a net charge at physiological pH greater than 7 (Fedyukina et al., Reference Fedyukina, Jennaro and Cavagnero2014). This has relevance for protein and ribosomal stability because Lawrence et al. (Reference Lawrence, Phillips and Liu2007) demonstrated a surprising capacity of protein variants, engineered to impart to them either a positive or a negative ‘supercharge’, to re-gain structure and function following stressful events (in this case supra-optimal temperatures). They demonstrated that supercharging prevents protein aggregation upon denaturation and yet does not dramatically inhibit subsequent refolding into an active protein. Inherent r-protein positive supercharging is not strictly due to functional demands on proteins that must associate with negatively charged r-, t- and mRNA. Intriguingly, Fedyukina et al. (Reference Fedyukina, Jennaro and Cavagnero2014) have recorded a surprising plasticity in the net charge of r-proteins of the large subunit with those from halophiles considerably less positively charged than those from non-halophilic organisms. Upon further investigation, it was evident that r-proteins of the large subunit are generally segregated into positively and negatively charged protein moieties, a characteristic that becomes more pronounced in the halophile. The halophile negatively charged amino acids were found to be placed in solvent exposed positions, potentially better competing with salt for water for protein hydration while the positively charged portions of the protein tended to be buried within the protein-ribosomal RNA core (Fedyukina et al., Reference Fedyukina, Jennaro and Cavagnero2014). In keeping with Job's rule, such durability is a useful attribute if the proteins comprising the translational apparatus must remain in an active form throughout quiescence and are of such vital importance to the longevity of seeds following imbibition.

In some organisms, including plants, ribosomal protein (r-protein) families have numerous paralogous members (Barakat et al., Reference Barakat, Szick-Miranda, Chang, Guyot, Blanc, Cooke, Delseny and Bailey-Serres2001; Carroll et al., Reference Carroll, Heazlewood, Ito and Millar2008; Carroll, Reference Carroll2013; Hummel et al., Reference Hummel, Dobrenel, Cordewener, Davanture, Meyer, Smeekens, Bailey-Serres, America and Hanson2015). One hypothesis concerning this r-protein diversity is that the r-protein paralogues can impart to the translational machinery selectivity regarding which mRNAs are preferentially translated (Hummel et al., Reference Hummel, Dobrenel, Cordewener, Davanture, Meyer, Smeekens, Bailey-Serres, America and Hanson2015), constituting a so-called ‘ribosome filter’ (Mauro and Edelman, Reference Mauro and Edelman2002). Indeed, such are the subtleties of r-protein paralogue alterations to the ribosome, influencing the preference of the ribosome for translating specific mRNAs, that, in yeast, paralogues of the LARGE SUBUNIT RIBOSOMAL PROTEIN1 (RPL1; a or b), although identical in amino acid sequence, somehow dramatically bias the mRNA species translated by the ribosomes containing one or the other RPL1 paralogue (Segev and Gerst, Reference Segev and Gerst2018). Despite this redundancy, many genes encoding r-proteins belonging to cytoplasmic, mitochondrial (Tzafrir et al., Reference Tzafrir, Pena-Muralla, Dickerman, Berg, Rogers, Hutchens, Sweeney, McElver, Aux, Patton and Meinke2004; Zhang et al., Reference Zhang, Luo, Day, Talbot, Ivanova, Ashton, Chaudhury, Macknight, Hrmova and Koltunow2015; Robles and Quesada, Reference Robles and Quesada2017), as well as plastidal (Tsugeki et al., Reference Tsugeki, Kochieva and Fedoroff1996; Gong et al., Reference Gong, Jiang, Xu, Zhang, Teng, Lin and Dong2013) ribosomes are known to be lethal when mutated (Lloyd and Meinke, Reference Lloyd and Meinke2012). As our sophistication regarding translation during germination increases, the identities of r-protein paralogues, and their post-translational modifications (Carroll, Reference Carroll2013; Sanchez-de-Jimenez et al., Reference Sanchez-de-Jimenez, Aguilar and Dinkova1997), that are crucial to selective translation at various stages of germination, may be revealed to be absolutely required for the recovery from quiescence and the completion of seed germination (Galland et al., Reference Galland, Huguet, Arc, Cueff, Job and Rajjou2014; Basbouss-Serhal et al., Reference Basbouss-Serhal, Soubigou-Taconnat, Bailly and Leymarie2015; Galland and Rajjou, Reference Galland and Rajjou2015; Bai et al., Reference Bai, Peviani, van der Horst, Gamm, Snel, Bentsink and Hanson2017). In this vein, it should be noted that some of the first transcripts to be translated following imbibition, are those in the stored transcriptome encoding ribosomal proteins (Beltran-Pena et al., Reference Beltran-Pena, Ortiz-Lopez and Sanchez de Jimenez1995; Tatematsu et al., Reference Tatematsu, Kamiya and Nambara2008; Weitbrecht et al., Reference Weitbrecht, Muller and Leubner-Metzger2011) prioritizing the replacement of critical translational capacity with what might be the last functional translation of which the rehydrated, aged ribosome is capable! This is entirely consistent with the tenets of Job's rule.

The r-proteins are only one of seven different categories listed as crucial for translation (Grosjean et al., Reference Grosjean, Breton, Sirand-Pugnet, Tardy, Thiaucourt, Citti, Barre, Yoshizawa, Fourmy, de Crecy-Lagard and Blanchard2014). In studies of aged seeds, certain members of Categories 3 and 7, (translation factors; initiation and elongation factors, chaperonins) have been documented to decline in abundance as ageing progresses (Min et al., Reference Min, Lee, Cheon, Han, Ko, Kang, Kim, Agrawal, Rakwal, Gupta and Kim2017; Wang et al., Reference Wang, Ma, Song, Shu and Gu2012). The distinction to be made here is that the studies indicating that Category 3 and 7 proteins declined in abundance were conducted using hydrated seeds exposed to high temperatures (accelerated ageing conditions) which may or may not reflect the dynamics of protein destabilization under natural ageing conditions (Schwember and Bradford, Reference Schwember and Bradford2010).

Protection: Proteins involved in translation may be protected from oxidation in the quiescent seed

Oxidation is a stress assailing the components of cells of the seed to which lipids, nucleic acids and proteins are all susceptible (El-Maarouf-Bouteau et al., Reference El-Maarouf-Bouteau, Meimoun, Job, Job and Bailly2013). The reactive oxygen species (ROS) are also potent signalling molecules. Hence the hydrated cell must permit sufficient ROS generation to fulfil relevant signalling while dampening ROS quantities below a threshold where cellular constituents are damaged by them (Bailly et al., Reference Bailly, El-Maarouf-Bouteau and Corbineau2008) and the hydrated cell employs a plethora of mechanisms to establish and maintain redox balance (Apel and Hirt, Reference Apel and Hirt2004). The sulfur-containing amino acids (AAs; cysteine and methionine) are prone to oxidation (Levine et al., Reference Levine, Moskovitz and Stadtman2000). Both of these oxidations, proceeding no further than cysteine sulfenic acid or methionine sulfoxide, respectively, are repairable by enzymatic reduction (Tarrago et al., Reference Tarrago, Laugier and Rey2009; Meyer et al., Reference Meyer, Belin, Delorme-Hinoux, Reichheld and Riondet2012; Akter et al., Reference Akter, Huang, Waszczak, Jacques, Gevaert, Van Breusegem and Messens2015; Waszczak et al., Reference Waszczak, Akter, Jacques, Huang, Messens and Van Breusegem2015) and even cysteine sulfinic acid is reversible in some cellular compartments (Rey et al., Reference Rey, Becuwe, Barrault, Rumeau, Havaux, Biteau and Toledano2007). Other AAs (proline, histidine, lysine, arginine, tyrosine and tryptophan) are also subject to oxidation but, to date, no reductive repair mechanism for these has been identified (Rinalducci et al., Reference Rinalducci, Murgiano and Zolla2008; Sweetlove and Moller, Reference Sweetlove and Moller2009). Some proteins in cell-free extracts from desiccated seeds appear to be preferentially oxidized; this damage occurring to these specific proteins at frequencies far surpasses their proportionate abundance in the cell (Oracz et al., Reference Oracz, El-Maarouf Bouteau, Farrant, Cooper, Belghazi, Job, Job, Corbineau and Bailly2007; El-Maarouf-Bouteau et al., Reference El-Maarouf-Bouteau, Meimoun, Job, Job and Bailly2013). This oxidation is presumed to have occurred during the sojourn of the seed in the dehydrated state (Gao et al., Reference Gao, Rampitsch, Chitnis, Humphreys, Jordan and Ayele2013) although it is impossible to emphatically state that the oxidation does not take place immediately upon addition of aqueous buffer used to extract the proteins from the desiccated seed, an ubiquitous caveat dogging most assays of dehydrated tissues. Nevertheless, in support of Job's rule, of those proteins identified as preferentially oxidized from desiccated seed, only one is involved in translation (eukaryotic ELONGATION FACTOR2 [eEF2]; Category 7; Job et al., Reference Job, Rajjou, Lovigny, Belghazi and Job2005; Oracz et al., Reference Oracz, El-Maarouf Bouteau, Farrant, Cooper, Belghazi, Job, Job, Corbineau and Bailly2007) and its oxidation in dry seeds is associated with dormancy alleviation in Helianthus annuus L. (sunflower), stimulating the completion of germination upon subsequent hydration, constituting a time-dependent signal eliciting a specific event (dormancy alleviation), rather than random protein damage.

In contrast, hydration of the seeds prior to protein extraction results in the oxidation of many proteins directly involved in translation (Job et al., Reference Job, Rajjou, Lovigny, Belghazi and Job2005) so these translation-associated proteins, present in the desiccated cells of the seed, are not impervious to oxidation but seem, rather, to be specifically protected from it while dehydrated. The means by which the proteins that are prone to oxidation are rendered susceptible to it remains relatively unknown, as does the means by which proteins involved in translation are seemingly protected from oxidation while desiccated. One intriguing example of the former is the use, in animal mitochondria, of AA substitution through alternative codon translation, resulting in methionine (or N-formylmethionine) residues for both codons AUG and AUA (usually isoleucine). In this instance, solvent-exposed Met residues are thought to act as oxidation decoys, sacrificing their integrity to protect other mitochondrial constituents from ROS while, due to their peripheral placement in the protein they act like a ROS sponge, minimally influencing the ROS sponge protein's structural integrity and hence, function (Bender et al., Reference Bender, Hajieva and Moosmann2008). A repair pathway capable of reducing methionine sulfoxide also exists in the cell, which can render this damage temporary (Achilli et al., Reference Achilli, Ciana and Minetti2015). The seed storage proteins (one class of seed stored proteins) which are degraded to supply energy, nitrogen and carbon for the establishing seedling, are prone to all manner of damage during quiescence. Oxidative alterations have been suggested to be evidence of a role for the abundant storage proteins (Nguyen et al., Reference Nguyen, Cueff, Hegedus, Rajjou and Bentsink2015) in seeds to soak up ROS, reducing the titre of the damaging ROS during quiescence. An alternative opinion is that the storage proteins, destined for degradation and present in a compartment bereft of some repair mechanisms, are simply unworthy of repair (Dinkins et al., Reference Dinkins, Majee, Nayak, Martin, Xu, Belcastro, Houtz, Beach and Downie2008), slated as they are for destruction. There is even a role for phytic acid (frequently associated with seed storage proteins as inclusion bodies) as an anti-oxidant contributing to the seed longevity of maize (Zea mays) seeds (Doria et al., Reference Doria, Galleschi, Calucci, Pinzino, Pilu, Cassani and Nielsen2009).

Protection: LEA protein association with proteins involved in translation

The LATE EMBRYOGENESIS ABUNDANT proteins (LEA proteins) are thought to protect intracellular membranes and proteins of seeds while drying and during their sojourn in the desiccated state (Hundertmark et al., Reference Hundertmark, Buitink, Leprince and Hincha2011). LEA proteins have been suggested to act as molecular spacers in the increasingly crowded intracellular milieu during dehydration (Goyal et al., Reference Goyal, Walton and Tunnacliffe2005) and some have been shown to possess remarkable anti-aggregation properties (Boucher et al., Reference Boucher, Buitink, Lin, Boudet, Hoekstra, Hundertmark, Renard and Leprince2010). Some of the LEA proteins have been demonstrated to exert a protective function for membranes of particular composition (Thalhammer et al., Reference Thalhammer, Hundertmark, Popova, Seckler and Hincha2010; Tolleter et al., Reference Tolleter, Hincha and Macherel2010; Eriksson et al., Reference Eriksson, Kutzer, Procek, Grobner and Harryson2011; Thalhammer et al., Reference Thalhammer, Bryant, Sulpice and Hincha2014; Eriksson et al., Reference Eriksson, Eremina, Barth, Danielsson and Harryson2016) and some to stabilize sugar glasses (Wolkers et al., Reference Wolkers, McCready, Brandt, Lindsey and Hoekstra2001; Shimizu et al., Reference Shimizu, Kanamori, Furuki, Kikawada, Okuda, Takahashi, Mihara and Sakurai2010). Certainly the cosmopolitan distribution of usually two or more members of the LEA proteins within compartments of the plant cell (Candat et al., Reference Candat, Paszkiewicz, Neveu, Gautier, Logan, Avelange-Macherel and Macherel2014) augurs well for a general, shielding mechanism, redundantly backed up (Chakrabortee et al., Reference Chakrabortee, Tripathi, Watson, Schierle, Kurniawan, Kaminski, Wise and Tunnacliffe2012). Recently, this view is being altered due to the recovery of a diversity of specific phenotypes upon mutation of a single LEA (Manfre et al., Reference Manfre, Lanni and Marcotte2006; Chen et al., Reference Chen, Nayak, Majee, Lowenson, Schafermeyer, Eliopoulos, Lloyd, Dinkins, Perry, Forsthoefel, Clarke, Vernon, Zhou, Rejtar and Downie2010; Olvera-Carrillo et al., Reference Olvera-Carrillo, Campos, Reyes, Garciarrubio and Covarrubias2010; Salleh et al., Reference Salleh, Evans, Goodall, Machin, Mowla, Mur, Runions, Theodoulou, Foyer and Rogers2012), scarcely possible if LEAs all acted as general shield molecules with multiple members present in cellular compartments. While demonstrations of certain of the LEA proteins safeguarding, to some degree, the function of commercial enzyme preparations abound (e.g. Hara et al., Reference Hara, Fujinaga and Kuboi2004; Reyes et al., Reference Reyes, Rodrigo, Colmenero-Flores, Gil, Garay-Arroyo, Campos, Salamini, Bartels and Covarrubias2005; Kovacs et al., Reference Kovacs, Kalmar, Torok and Tompa2008; Liu et al., Reference Liu, Zheng, Zhang, Wang and Li2010; Zhang et al., Reference Zhang, Lu, Jiang, Wang, Lv, Shen and Ming2014), efforts to identify particular endogenous proteins that are bound by explicit, conspecific LEA proteins have also been successful (Xie et al., Reference Xie, Zhang, Qu, Miao, Zhang, Shen, Wang and Dong2012; Rivera-Najera et al., Reference Rivera-Najera, Saab-Rincon, Battaglia, Amero, Pulido, Garcia-Hernandez, Solorzano, Reyes and Covarrubias2014; Zhang et al., Reference Zhang, Lu, Jiang, Wang, Lv, Shen and Ming2014; Hernandez-Sanchez et al., Reference Hernandez-Sanchez, Maruri-Lopez, Graether and Jimenez-Bremont2017). Consistent with the tenets of Job's rule, preferred client proteins bound by a SEED MATURATION PROTEIN (SMP) family Arabidopsis LEA protein, and by its soybean orthologue, belonged to Categories 1, 4 and 7 of translation (Kushwaha et al., Reference Kushwaha, Lloyd, Schafermeyer, Kumar and Downie2012) providing evidence that at least some of the LEA proteins can bind (and presumably protect) proteins of the translational apparatus (Kushwaha et al., Reference Kushwaha, Downie and Payne2013). Loss of function of the Arabidopsis SMP LEA, while not lethal to the seeds, does remove the capacity of smp1 seeds to enter thermodormancy in response to thermal insult applied during germination (Chen et al., Reference Chen, Nayak, Majee, Lowenson, Schafermeyer, Eliopoulos, Lloyd, Dinkins, Perry, Forsthoefel, Clarke, Vernon, Zhou, Rejtar and Downie2010), seemingly negating the seed hydration memory of the stress (Fig. 1).

The involvement of the LEA protein SMP1 in binding (presumably protecting) proteins involved in translation may be used as a paradigm to suggest that certain LEA proteins may physically shelter oxidation-sensitive AAs of their client proteins from this modification in the quiescent seed. Certainly the region of the client proteins to which both Arabidopsis and soybean orthologues of SMP1 bound was consistently the most, or among the most, hydrophyllic of the client proteins (Kushwaha et al., Reference Kushwaha, Downie and Payne2013), suggesting that these regions are solvent exposed and therefore would be the most susceptible to oxidative damage. Such physical protection from oxidation has been demonstrated for the subunit proteins comprising the 12S cruciferin storage proteins where the β-subunits, thought to be buried within the α-subunits, are 6-fold less oxidized in the desiccated seed (Job et al., Reference Job, Rajjou, Lovigny, Belghazi and Job2005). Following imbibition, at least some LEA proteins, when hydrated, would lose any structural features helping to induce a fit with their client proteins, leaving their client proteins more susceptible to oxidation, which was the case for proteins involved in translation upon seed hydration (Job et al., Reference Job, Rajjou, Lovigny, Belghazi and Job2005).

Using a Tandem Affinity Purification approach repeated four times (twice in the light and twice in the dark) Shaw (Reference Shaw2016) recovered and identified a group of client proteins binding to a TAP-LEA5 protein (At4g02480, which is LEA38 of the LEA3 family; Hundertmark and Hincha, Reference Hundertmark and Hincha2008). Of the five client proteins recovered two or more times using this approach, two were involved in translation and these were recovered most consistently from the screen. The Arabidopsis DEAD-Box RNA Helicase 22 (RH22) (Category 3) was recovered each time the assay was performed. RH22 is required for proper maturation and assembly of plastidial ribosomal RNA crucial for translation in the organelle (Chi et al., Reference Chi, He, Mao, Li, Ma, Ji, Zou and Zhang2012). The next most consistently recovered client protein (three out of four times) was PUMILIO24, an RNA binding protein of the PUF family (Tam et al., Reference Tam, Barrette-Ng, Simon, Tam, Ang and Muench2010), also involved in ribosomal RNA maturation (Category 3) and assembly in the nucleolus (Shanmugam et al., Reference Shanmugam, Abbasi, Kim, Kim, Park, Park, Oh, Park, Muench, Choi, Park and Choi2017; Maekawa et al., Reference Maekawa, Ishida and Yanagisawa2018). Although these efforts to identify legitimate, conspecific LEA protein client proteins have indicated, for two different families of LEAs, that proteins involved in translation are preferred targets, many LEA proteins are known to bind membranes rather than proteins (Thalhammer et al., Reference Thalhammer, Hundertmark, Popova, Seckler and Hincha2010; Tolleter et al., Reference Tolleter, Hincha and Macherel2010; Eriksson et al., Reference Eriksson, Kutzer, Procek, Grobner and Harryson2011). Additionally, for those LEA proteins that do bind proteins, it is still unclear whether proteins involved in translation are an ubiquitous set of client proteins or if the two LEA proteins investigated thus far coincidentally had proteins involved in translation as their targets. Regardless, what is clear is that some proteins of the translational apparatus are preferred client proteins bound (potentially protected) by some members of the LEA family, entirely consistent with Job's rule.

Protection: Difficulty assigning protection of protein associated with translation to altered soluble carbohydrate profiles

The alteration of the sugar profile during dehydration of tissues is a well-documented occurrence in pollen, seeds and resurrection plants (Griffiths et al., Reference Griffiths, Gaff and Neale2014). One of the attributes of increasing sugar concentration concomitant with water loss is the transition to an amorphous state in the cytoplasm, and presumably, the organelles in which the cellular constituents are embedded. The increase in viscosity is sufficient to greatly impede diffusion rates while dampening metabolism to such an extent that it is difficult to measure. Carbohydrate-mediated protection is thought to be a general phenomenon (ElSayed et al., Reference ElSayed, Rafudeen and Golldack2014) encompassing both membranes and proteins that, although not preferentially focused on proteins (let alone proteins involved in translation), by protecting proteins promiscuously, protects those of the translational apparatus as well (Buera et al., Reference Buera, Schebor and Elizalde2004).

There are some cryptic correlations between the abundance of sugars in stressed cells and desiccation tolerance. One of the relationships that has been gaining attention is the importance of the ratio between the raffinose family oligosaccharides (RFO) and sucrose (first identified by Chen and Burris, Reference Chen and Burris1990) in the acquisition of desiccation tolerance (Pereira Lima et al., Reference Pereira Lima, Buitink, Lalanne, Rossi, Pelletier, da Silva and Leprince2017) and/or enhancement of seed longevity (Li et al., Reference Li, Zhang, Wang, Liu, Dirk, Goodman, Downie, Wang, Wang and Zhao2017b). In vitro studies suggest that the mixture of the sugars may not benefit protein longevity or desiccation tolerance, potentially diverting research efforts to examine how the sugar mixture may influence membrane stabilization and/or the propensity of the mixture to remain amorphous (Davidson and Sun, Reference Davidson and Sun2001). However, it is still possible that a favourable RFO/sucrose ratio may protect proteins in vivo due to the complexity of sugar metabolism in the cell being beyond the capacity of in vitro studies to unveil. For instance, trehalose abundance in stressed yeast cells has been linked to the propensity of these cells to reduce protein aggregation generally. Up-regulation of trehalose production has been demonstrated to result in increased heat shock protein (HSP104; a Category 3 chaparonin) abundance, and increased autophagic clearing of protein aggregates (Chaudhary et al., Reference Chaudhary, Kardani, Singh, Banerjee and Roy2014). The beneficial influence in preventing protein aggregation was demonstrated to be, in part, the dual action of trehalose and HSP104 when they were present concurrently and in a strict stoichiometry (Saleh et al., Reference Saleh, Gune, Chaudhary, Turakhiya and Roy2014) that was not entirely dependent on the up-regulation of autophagy by trehalose accumulation. This level of synergy would not be evident with in vitro studies. However, none of these features of RFO/sucrose ratios or trehalose-mediated protection (chaparonin-induction, synergistic action, or autophagy stimulation) has been categorically identified as specifically targeted to proteins of the translational apparatus.

Repair: Protein repair pathways

Certain AAs in a polypeptide are prone to damage through oxidation, isomerization and spontaneous conversion. For some of these forms of damage, cellular repair mechanisms have been identified in most forms of life, testifying to both the ubiquity of the agents of damage (e.g. oxidation; Gracy et al., Reference Gracy, Talent, Kong and Conrad1999) as well as to the importance of rectifying damage, when possible, to maintain a functional proteome (Clarke, Reference Clarke2003). Following the discovery, using radio-labelling and two-dimensional gel electrophoresis (2DGE), that seed proteins involved in translation decreased in abundance 1 day after imbibition in the proteome of aged – relative to unaged – seeds (Rajjou et al., Reference Rajjou, Lovigny, Groot, Belghazi, Job and Job2008), it was noted (again using 2DGE, membrane transfer, and on-blot methylation) that there were certain proteins acting as preferred substrates of the protein repair enzyme PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) (Dinkins et al., Reference Dinkins, Majee, Nayak, Martin, Xu, Belcastro, Houtz, Beach and Downie2008).

Repair: PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT)

Isoaspartate (IsoAsp) formation is a common form of damage incurred under physiological conditions (Geiger and Clarke, Reference Geiger and Clarke1987) and its non-enzymatic production (or at least the production of its immediate succinimidyl precursor) is heightened under stressful conditions (Mudgett and Clarke, Reference Mudgett and Clarke1994) and in desiccated, aged tissues (Mudgett et al., Reference Mudgett, Lowenson and Clarke1997). There are numerous studies demonstrating increased seed longevity due to up-regulation of PIMT or a reduction in this seed attribute upon pimt dysfunction (Oge et al., Reference Oge, Bourdais, Bove, Collet, Godin, Granier, Boutin, Job, Jullien and Grappin2008; Verma et al., Reference Verma, Kaur, Petla, Rao, Saxena and Majee2013; Wei et al., Reference Wei, Xu, Diao, Zhu, Xie, Cai, Wu, Wang, Zhang and Xie2015). In an endeavour to identify those proteins in seeds that were particularly prone to isoAsp formation and/or preferentially repaired by the PIMT enzyme, many proteins involved in translation were identified as preferred PIMT targets (or particularly susceptible to isoAsp formation; Chen et al., Reference Chen, Nayak, Majee, Lowenson, Schafermeyer, Eliopoulos, Lloyd, Dinkins, Perry, Forsthoefel, Clarke, Vernon, Zhou, Rejtar and Downie2010). These included proteins responsible for ribosomal maturation (AT3G51270; AT5G62190; Category 3); a component of the mitochondrial inner membrane translocon (AT5G51150; Category 8); rRNA modification (AT5G55920; Category 2); and an r-protein (AT3G22230; Category 1). Moreover, this was in stark contrast with PIMT target proteins identified in organisms incapable of surviving desiccation (Reissner et al., Reference Reissner, Paranandi, Luc, Doyle, Mamula, Lowenson and Aswad2006; Zhu et al., Reference Zhu, Doyle, Mamula and Aswad2006; Dai et al., Reference Dai, Ni, Patananan, Clarke, Karger and Zhou2013). In these desiccation-sensitive organisms, with a large number of PIMT target proteins identified, there is only a single report of a protein (eIF4E-binding protein2; Category 7) intimately associated with the eukaryotic translational complex, being a target of PIMT (Bidinosti et al., Reference Bidinosti, Martineau, Frank and Sonenberg2010).

Of the enzymes involved in translation that were preferentially repaired by PIMT in screens of the seed proteome, one of those involved in the processing of the ribosomal RNAs in the nucleolus (Category 3) was examined further (Galland and Rajjou, Reference Galland and Rajjou2015; Huang et al., Reference Huang, Shen, Huang, Wu, Yeh and Lu2016a; Lorkovic et al., Reference Lorkovic, Herrmann and Oelmuller1997; Nayak et al., Reference Nayak, Putnam, Addepalli, Lowenson, Chen, Jankowsky, Perry, Dinkins, Limbach, Clarke and Downie2013). An orthologue of this Arabidopsis DEAD-box RNA helicase (PLANT RNA HELICASE75, PRH75) had been shown to be the product of one of the few transcripts preferentially up-regulated in imbibed aged seeds of mung bean (Vigna radiata) relative to unaged seeds (Li et al., Reference Li, Chung and Chen2001). This up-regulation of transcript abundance may be a reaction to a decline in active protein abundances, similar to the findings in imbibed, aged Arabidopsis seeds (Rajjou et al., Reference Rajjou, Lovigny, Groot, Belghazi, Job and Job2008) where a DEAD-box RNA helicase, involved in mRNA export (Category 4; Kammel et al., Reference Kammel, Thomaier, Sorensen, Schubert, Langst, Grasser and Grasser2013) declines in aged seeds. Further studies in Arabidopsis determined that most prh75 mutants are embryo lethal and that weakly penetrant prh75 mutations, while viable, produced abnormally shaped (corkscrewed) seeds/embryos (or those with compromised completion of germination) at high frequency (Nayak et al., Reference Nayak, Putnam, Addepalli, Lowenson, Chen, Jankowsky, Perry, Dinkins, Limbach, Clarke and Downie2013; Huang et al., Reference Huang, Sie, Chen, Huang and Lu2016b), underlining how crucial the functionality of PRH75 is during plant development, a functionality safeguarded by PIMT (Nayak et al., Reference Nayak, Putnam, Addepalli, Lowenson, Chen, Jankowsky, Perry, Dinkins, Limbach, Clarke and Downie2013). Indeed, plants possess two different genes encoding PIMT (Xu et al., Reference Xu, Belcastro, Villa, Dinkins, Clarke and Downie2004) and dysfunction of one is sufficient to severely compromise seed longevity (Oge et al., Reference Oge, Bourdais, Bove, Collet, Godin, Granier, Boutin, Job, Jullien and Grappin2008; Verma et al., Reference Verma, Kaur, Petla, Rao, Saxena and Majee2013).

Repair: METHIONINE SULFOXIDE REDUCTASE

If the proteins of the translational apparatus are preferentially repaired (at least by PIMT and Met Sulfoxide Reductase; Caldwell et al., Reference Caldwell, Luk, Weissbach and Brot1978; Chen et al., Reference Chen, Nayak, Majee, Lowenson, Schafermeyer, Eliopoulos, Lloyd, Dinkins, Perry, Forsthoefel, Clarke, Vernon, Zhou, Rejtar and Downie2010; Nayak et al., Reference Nayak, Putnam, Addepalli, Lowenson, Chen, Jankowsky, Perry, Dinkins, Limbach, Clarke and Downie2013), this may be a contributing reason why they are among the more resilient proteins in the cytoplasm (Nelson et al., Reference Nelson, Alexova, Jacoby and Millar2014). At least isoAsp is relegated as problematic to its occurrence in a peptide chain. Oxidation of Met, on the other hand, can occur in a protein as well as to free methionine, or that bound as an aminoacyl-tRNA (Chousterman and Chapeville, Reference Chousterman, Chapeville and Moldave1981). In fact, oxidative stress has been shown to result in the preferential mis-acetylation of methionine to non-methionine tRNAs (Category 6) that, coupled with an oxidation-mediated reduction in the efficiency of AMINOACYL-tRNA SYNTHETASE editing capacity, decreases the fidelity of translation (Ling and Soll, Reference Ling and Soll2010). Such ‘controlled inaccuracy’ (Lee et al., Reference Lee, Kim, Kim, Yang, Hong, Kang, Oh, Kim, Han, Hwang, Kang, Kang, Kim, Kwon and Kim2014) is mediated through an oxidative environment leading to phosphorylation of key residues in the METHIONYL-tRNA SYNTHETASE (Category 5) that render it less discriminating in the tRNAs perceived as cognate (Lee et al., Reference Lee, Kim, Kim, Yang, Hong, Kang, Oh, Kim, Han, Hwang, Kang, Kang, Kim, Kwon and Kim2014). Due to the hypothesized role of solvent-exposed methionines acting as a ROS reductant (Bender et al., Reference Bender, Hajieva and Moosmann2008), it has been postulated that this mis-priming of tRNAs to increase MET incidence in the proteome, is a cryptic means by which cells protect themselves from oxidative stress (Netzer et al., Reference Netzer, Goodenbour, David, Dittmar, Jones, Schneider, Boone, Eves, Rosner, Gibbs, Embry, Dolan, Das, Hickman, Berglund, Bennink, Yewdell and Pan2009). Moreover, free, oxidized methionine can form diastereomers (Met-S-SO and Met-R-SO) for which metazoans and higher plants have not retained the enzyme capable of efficiently reducing Met-S-SO (Le et al., Reference Le, Lee, Marino, Zhang, Fomenko, Kaya, Hacioglu, Kwak, Koc, Kim and Gladyshev2009). Met-SO will not form S-adenosyl methionine (Achilli et al., Reference Achilli, Ciana and Minetti2015), the primary methyl donor in the cell, and the means through which PIMT functions to convert isoAsp to Asp.

While these damages impact translation generally, there is at least one occurrence of an oxidized methionine in an r-protein (Category 1) that is known to inhibit translation specifically but that can be repaired to regain functionality (Caldwell et al., Reference Caldwell, Luk, Weissbach and Brot1978). The reduction of both free methionine-sulfoxide and methionine-sulfoxide present in a polypeptide context is, obviously, an important facet of Job's rule. While METHIONYL-tRNA SYNTHETASE cannot be charged with MET-SO (Category 6) (Lemoine et al., Reference Lemoine, Waller and van Rapenbusch1968), Met can be oxidized once it has formed the aminoacyl tRNA^MET-SO (Category 5; Chousterman and Chapeville, Reference Chousterman, Chapeville and Moldave1981), so reduction to tRNA^MET is also an important aspect of Job's rule.

Repair: Chaperonins: PEPTIDYL-PROLYL CIS-TRANS ISOMERASE

In addition to being oxidized, proline can isomerize to/from trans-/cis-isomers, important for protein folding (Wedemeyer et al., Reference Wedemeyer, Welker and Scheraga2002). Peptidyl-prolyl cis-trans isomerases (PPIases) are a class of enzyme capable of isomerizing proline between cis- and trans-isomers at the N-terminal amide bond in proteins (Wedemeyer et al., Reference Wedemeyer, Welker and Scheraga2002; Camilloni et al., Reference Camilloni, Sahakyan, Holliday, Isern, Zhang, Eisenmesser and Vendruscolo2014). Proline isomerization has been documented to influence seed vigour through mutation of specific PPIases (Category 7; Bissoli et al., Reference Bissoli, Ninoles, Fresquet, Palombieri, Bueso, Rubio, Garcia-Sanchez, Fernandez, Mulet and Serrano2012). The ribosome inserts all prolines in the trans conformation (Feige et al., Reference Feige, Hendershot and Buchner2010), requiring PPIases to subsequently convert the trans proline to cis, if necessary, to fold the nascent polypeptide. Nevertheless, in yeast, concurrent elimination of 12 cyclophylin and FK506 binding proteins (PPIase enzymes) through mutagenesis was not lethal (Dolinski et al., Reference Dolinski, Muir, Cardenas and Heitman1997), leaving the authors to propose that each PPIase has a unique set of proteins on which it acts in yeast. Later studies recovered specific protein interactors with FK506 binding protein 12 (FKBP12) prolyl isomerase, a cyclophylin known to bind FK506 and cyclosporine, validating the preposition of the authors that PPIases will have specific client proteins with which they react (Dolinski and Heitman, Reference Dolinski and Heitman1999). However, to date, no protein associated with translation has been identified in any organism as specifically interacting with a PPIase in order to be refolded following stress.

In bacteria, the TRIGGER FACTOR chaperonin protein with PPIase activity, is physically associated with the 50S ribosomal-tunnel from which the amino-terminus of the protein being constructed emerges and is the first chaperonin the nascent polypeptide encounters (Kristensen and Gajhede, Reference Kristensen and Gajhede2003). In plants, the plastid is apparently the only compartment supporting translation that has retained a TRIGGER FACTOR chaperonin (Category 7; Ries et al., Reference Ries, Carius, Rohr, Gries, Keller, Lancaster and Willmund2017) which, due to its susceptibility to supraoptimal temperature stress, is thought to confer to plastidial translation some advantage during heat stress (Ries et al., Reference Ries, Carius, Rohr, Gries, Keller, Lancaster and Willmund2017). While none of the above mentioned isomerizations has been considered ‘damage’, or to occur preferentially in proteins involved in translation, in plants it does link PPIase activity tightly to translation, at least in the plastid.