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Autophagy is a process found ubiquitously in eukaryotic cells that uses specific molecular machinery incorporating the products of AUTOPHAGY (ATG) genes for the formation of double-membrane vacuoles, named autophagosomes (Li & Vierstra, 2012; Yoshimoto, 2012). Autophagosome biogenesis starts with the nucleation of lipid membranes which enlarge and surround the cell material to be discarded. After enclosure, autophagosomes are targeted to lytic vacuoles or lysosomes. The outer membrane of the autophagosome then fuses to the vacuolar membrane to deliver its cargo into the lumen. The damaged proteins and unwanted organelles are then digested by proteases, lipases and other hydrolases. Present in all eukaryotic cells at a basal level, autophagy is induced in response to stress and during ageing (Liu et al., 2009; Breeze et al., 2011). Although it was considered for a long time as a nonselective mechanism, the recent discovery of protein motifs and of protein interactions between the membrane-bound ATG8 proteins and some protein adaptors provides evidence that autophagic vesicles can be selective for their cargos (Noda et al., 2010).
In plants, as in animals and yeast, ATG genes appear to be essential for cell longevity, especially under nutrient-limiting conditions (Hanaoka et al., 2002). Autophagy mutants indeed display early senescence symptoms and hypersensitivity to carbon (C) and nitrogen (N) nutrient shortages (Liu & Bassham, 2012). Because plants are static organisms, their ability to recycle and remobilize nutrients, and especially N, is crucial for adaptation to a fluctuating environment. Processes allowing the recycling of organic N at the whole-plant level are therefore essential for plant survival and fitness (Masclaux-Daubresse & Chardon, 2011). In a recent report, we provided evidence that the autophagy machinery is an important factor for N remobilization efficiency and for grain-filling in Arabidopsis (Guiboileau et al., 2012). Autophagy mutants are senescing earlier than the wild type, especially when grown under low nitrate nutrition. Their capacity to remobilize nitrogen efficiently and their fitness are also greatly affected. Using salicylic acid-deficient autophagy mutants (atg5.sid2) that presented a green phenotype similar to that of the wild type and similar defects in autophagy machinery to those of atg mutants, we showed that the double mutants remained inefficient for N remobilization. The N remobilization capacity of atg mutants therefore seems to be independent of their senescence phenotype.
It is well known that the progressive degradation of chloroplast proteins occurring during leaf senescence and in response to N starvation is essential for N remobilization (Feller & Fischer, 1994). The ways in which proteins, especially chloroplast proteins, are degraded during leaf senescence are not fully understood (Martinez et al., 2008). There is evidence that the degradation of chloroplastic proteins such as Rubisco and chloroplastic glutamine synthetase 2 (GS2) can occur both inside and outside the chloroplast. When the chloroplast environment is oxidizing, the oxidation of a critical cysteine residue of Rubisco results in the fragmentation of its large subunit and in the binding of the oxidized peptides to the chloroplast envelope (García-Ferris & Moreno, 1993, 1994). Similarly, the chloroplastic glutamine synthetase has been shown to be rapidly degraded under conditions that cause photo-oxidative stress in leaves, and this has been linked to the oxidative carbonylation of histidine residues on the protein (Palatnik et al., 1999; Nakano et al., 2006). In addition to its localization in the chloroplasts, Rubisco was also detected in cytoplasmic vesicular compartments named ‘Rubisco-containing bodies’ (RCBs) or ‘Rubisco vesicular bodies’ (RVBs) depending on the authors (Chiba et al., 2003; Prins et al., 2008). It was shown that RCBs contain stroma proteins but not thylakoid proteins. Recently, several lines of evidence that RCB trafficking from the cytosol to the vacuole is autophagy dependent were provided (Ishida et al., 2008; Wada et al., 2009; Izumi et al., 2010). Tagging chloroplast stroma proteins with GFP, Wada et al. (2009) observed the appearance of many small vesicles in the vacuolar lumen which exhibited Brownian motion in the darkened leaves in which autophagic body degradation was blocked using the V-ATPase inhibitor concanamycin A. The accumulation of these vesicles, identified as RCBs, was impaired in autophagy mutants.
The over-expression of the cystatin protease inhibitor in tobacco (Nicotiana tabacum) leaves leads to the over-accumulation of RCBs and Rubisco in transgenic plants (Prins et al., 2008). This provides evidence that cysteine proteases can also be involved in the regulation of chloroplast protein degradation (Prins et al., 2008). Several cysteine proteases are induced during leaf ageing (Roberts et al., 2012), and it was shown that some protease inhibitors are down-regulated during leaf ageing and while N remobilization occurs. It is then likely that both proteases and protease inhibitors control the fine-tuning of protein degradation for N remobilization during leaf ageing (Etienne et al., 2007; Desclos et al., 2008). The cysteine proteases induced in response to leaf senescence are mainly described as vacuolar enzymes. Therefore, autophagy might play a role in the delivery of chloroplast components to the vacuole for degradation and then contribute to RCB proteolysis.
As we found that autophagy mutants have a low N-remobilization efficiency at the whole-plant level (Guiboileau et al., 2012), in this study we investigated the physiology of N management and remobilization in rosettes of autophagy mutants (the atg5 and atg9 mutants) and an RNAi line in which ATG18a expression is suppressed (RNAi18) (Supporting Information Fig. S1). From the results obtained, with respect to enzyme activities and metabolite contents in plants grown under nitrate-sufficient or nitrate-limiting conditions, we can provide a picture of the physiological symptoms presented by the autophagy mutants and compare them to the senescence symptoms observed in Arabidopsis leaves by Diaz et al. (2008). The protein profiles described in this report strongly suggest that protein degradation is incomplete and aberrant in autophagy mutants, and the identification of some proteins accumulating in the rosettes of atg mutants leads us to propose potential protein substrates for autophagy.
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In plants, especially in Arabidopsis, most of the studies and reports dealing with autophagy have focused on the dissection of the molecular machinery involved in the regulation of the autophagy pathway, in the formation of autophagosome structures and more recently in the specificity of the autophagy process (for reviews, see Bassham et al., 2006; Li & Vierstra, 2012; Yoshimoto, 2012). These studies have provided a large set of atg mutants that, with very few exceptions, show similar phenotypes, such as early leaf senescence, hypersensitivity to N and C limitation, defects in plant immunity and hypersensitivity to a large panel of stresses such as drought, salt stress and pathogen attack, especially by necrotrophs. Because of the hypersensitivity of atg mutants to nitrate limitation, it was assumed that autophagy is essential for N recycling (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005), and we have recently documented the defect of N remobilization in autophagy mutants (Guiboileau et al., 2012). Although there are several lines of evidence that autophagy defects have strong impacts on the physiology of plants and on their response to the environment, no physiological data have been available until now. The combination of cytological approaches and mutant studies has provided evidence that the autophagy process could be important for the degradation of stroma proteins, especially Rubisco (Ishida et al., 2008; Wada et al., 2009). The aim of the work presented here was to gain insights into the physiological disorders affecting atg mutants.
Because the principal autophagy mutant phenotype, as observed in all studies performed by us and other groups, is early and severe leaf senescence, especially under nitrate limitation conditions, we decided to monitor some physiological and metabolic markers before and after the appearance of any leaf-yellowing phenotype, and three time-points were selected at which to perform the analysis.
At the time of the first harvest used in this study, 30 DAS, there was no visible yellowing phenotype in any of the mutants or in the wild type. As several autophagy genes were already significantly induced at this date in wild-type rosettes, by contrast with the SAG12 (senescence associated gene 12) senescence marker whose expression was very low (Fig. S6), we speculated that it may be possible to detect at 30 DAS precocious autophagy defects, unrelated to typical leaf senescence. Indeed, we observed that biomass and the C : N ratio were slightly lower in atg mutants and RNAi18 than in the wild type at 30 DAS under low-nitrate conditions. No significant difference in sugar and N compound concentrations was, however, detected at 30 DAS between mutants and the wild type. In contrast, immunoblotting allowed us to reveal differences in the abundance of certain specific proteins between the wild type and atg5 under low- and high-nitrate conditions. Taken together, these results provide evidence that physiological disorders are present in atg mutants even before the appearance of any visible senescence phenotype.
The differences between the wild type and atg mutants became more significant later, at 60 and 75 DAS. The C : N defect detected in atg mutants and RNAi18 was clearly linked to the accumulation of N compounds such as ammonium, amino acids and proteins, and to lower concentrations of sugars. Ammonium and amino acid over-accumulations in atg mutants and RNAi18 were not related to changes in GS activity, which was maintained at the same level in mutants and in the wild type throughout plant development. Surprisingly, we observed that both protein concentrations and protease activities are higher at 60 DAS in atg mutants relative to the wild type. This suggests that protein accumulation in mutants is not a result of protease deficiency. Immunoblots revealed that protein accumulation in atg mutants is selective. Relative accumulation of RPS6, RPL13, GDH and CAT was observed in atg5 from 30 DAS onwards; changes in GS1, GS2 and LSU contents and incomplete degradation products of GS2, LSU and IDH were clearly observed at 60 DAS in atg mutants and RNAi18.
All these results provide evidence that mutants accumulate physiological and metabolic disorders, which are on the one hand side effects of their early leaf senescence symptoms and on the other hand direct effects of autophagic activity defects. Indeed, comparison of the present data with those of our previous studies shows that, while the autophagy mutants display some modifications that are similar to the typical leaf senescence phenotypes as described in several plant species by Masclaux et al. (2000), Diaz et al. (2005, 2008) and Roberts et al. (2012), they also present modifications that are not related to senescence symptoms. The decreases in FW and sugar concentrations and the increases in GS1 protein content and protease activities observed in atg mutants and RNAi18, under low nitrate nutrition especially, are well-known markers of leaf senescence. However, while leaf senescence is characterized by decreases in total N, soluble proteins, total RNA and total amino acids, we observed exactly the opposite in atg mutants and RNAi18 irrespective of nitrate conditions. The autophagy defect is clearly characterized by the accumulation in leaves of various N compounds such as proteins, amino acids and ammonium. All other symptoms may be indirectly attributable to their early leaf senescence phenotype.
The higher concentration of total amino acids and soluble proteins in the rosettes of atg mutants relative to the wild type suggests that N resources are globally trapped in the leaves of mutants. This is in close accordance with the lower N remobilization efficiency we have reported for these mutants (Guiboileau et al., 2012). The over-accumulation of specific proteins and peptides in autophagy mutants suggests that mutants cannot properly degrade some of their proteins. The higher protease activities measured in mutants show that this is not attributable to defects in proteolysis. Regarding the pH responses of the endopeptidases and carboxypeptidases differentially active in mutants relative to the wild type, it seems that they are located in the vacuole and possibly also in the cytosol. Acidic endopeptidase and carboxypeptidase classes known to increase during leaf senescence are mainly located in the vacuole (Peoples & Dalling, 1988; Brouquisse et al., 2001; Roberts et al., 2012). It is, then, likely that the increase in peptidase activities in atg mutants is senescence-related.
The presence on western blot membranes of additional bands recognized by the LSU and GS2 proteins suggests that the LSU and GS2 proteins have undergone the first steps of their degradation process but that proteolysis of their peptides has not been completed. The release of peptides from the GS2 and Rubisco LSU proteins by reactive oxygen species (ROS) in chloroplasts is well documented in several plant species (see Feller et al., 2008 for a review). We know that autophagy mutants suffer from oxidative stress (Yoshimoto et al., 2009). The peptides observed on our western blots could have been released from GS2 and LSU by the effect of ROS within the chloroplast. Considering that the increased peptidase activities are mainly located in the vacuole and not in the same compartment as LSU, GS2 or IDH products, we can speculate that they cannot complete proteolysis unless the autophagy pathway causes the targeting of these substrates to the vacuole.
In addition to GS2 and LSU peptides, atg mutants also specifically accumulated several native proteins, such as ribosomal RPS6 and RPL13, IDH, GDH and CAT. Together with the higher total RNA concentrations (mostly represented by rRNA), the over-accumulation of RPS6 and RPS13 observed in atg mutants and RNAi18 suggests defects of ribophagic activity (MacIntosh & Bassham, 2011). However, the fact that the RPS14 content was not different between the wild type and atg mutants suggests that the role of autophagy in the degradation of the whole ribosome complex or in the degradation of each RP protein individually is more complex. Also, the fact that two mitochondrial proteins, IDH and GDH, accumulate in atg5 under low-nitrate conditions does not mean that mitophagy is globally affected in atg mutants. The mechanisms by which IDH and GDH accumulate in atg mutants can be different. The detection on IDH western blot membranes of peptides of lower mass than the native IDH protein suggests that, like that of GS2 and LSU, IDH protein degradation needs autophagy to be complete. The absence of additive bands on GDH western blot membranes and a recent transcriptome analysis performed in our laboratory (C. Masclaux-Daubresse, unpublished) suggest that, unlike IDH, GDH protein accumulates in atg mutants as a result of higher GDH gene expression.
Thus, the present study provides several lines of evidence of protein turnover defects in atg mutants. However, imperfect protein degradation cannot explain why atg mutants over-accumulate amino acids and ammonium. Ammonium and amino acid accumulation does not seem to be a result of defects in N assimilation, as GS activity was not different between the wild type and mutants and nitrate did not accumulate in mutants or in wild type (not shown). One explanation for amino acid and ammonium accumulation is the early and severe senescence symptoms observed on the leaves of atg mutants. Export of amino acids and ammonium requires efficient phloem loading, and vein tissue longevity is needed for that (Tegeder & Rentsch, 2010). One can suppose that phloem loading is affected when leaf senescence occurs too rapidly and too early. Consistent with this idea is the finding that ammonium and amino acid accumulation occurred at 60 DAS, when senescence symptoms were observed on leaves of mutants.
The accumulation of soluble proteins, total amino acids and ammonium in the rosettes of autophagy mutants can explain why they are less efficient at remobilizing N to their seeds (Guiboileau et al., 2012). Interestingly, we provide here some evidence that the nature of the proteins and peptides that accumulate in rosettes of atg mutants is specific and that autophagy is involved in the degradation of chloroplast protein cleavage fragments (Ono et al., 2013). Although it is probable that the increase of protease activities in leaves of atg mutants is related to their early senescence phenotype (Masclaux et al., 2000), we can also hypothesize that their respective genes are overexpressed in atg mutants as a response to the over-accumulation of unwanted proteins and peptides. The future characterization of the proteome and transcriptome of atg mutants and the study of the specific protease activities that increase in atg mutants are promising lines of enquiry that will enable identification of components of the machinery involved in the recycling of inorganic N for remobilization.