The ribosome-associated N-terminal acetyltransferase B coordinates global proteostasis and autophagy in plants by creating Ac/N-degrons

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
The N-terminal acetyltransferase B (NatB) acetylates ~20% of the eukaryotic proteome. However, the role of NatB-mediated N-terminal acetylation (NTA) for the regulation of the proteome fate remains unclear in eukaryotes. In this study, we demonstrate that CRISPR-Cas9-mediated deletion of NatB activity in plants results in significantly lowered global protein turnover due to decreased ubiquitin-proteasome system (UPS) activity and protein translation. Quantitative proteomics uncovers that NatB substrates are significantly enriched in the fraction of stabilized proteins in natb mutants. We provide direct evidence that the absent NTA of KIN11, a subunit of the autophagy-controlling energy sensor SnRK1, protects it from UPS-mediated destruction. The resulting accumulation of KIN11 is responsible for the increased resistance of natb mutants to energy limitation induced by prolonged darkness. Our findings establish NatB as a central regulator of UPS–autophagy interplay and highlight its role in maintaining proteome stability and enabling dynamic stress responses in plants.

Introduction
N-terminal acetylation (NTA) is one of the most prevalent protein modifications in eukaryotes, catalyzed by a set of evolutionarily conserved N-terminal acetyltransferases (NATs)1,2. The substrate specificity of NATs is mainly determined by the first few amino acids of their N-termini3. Among the eight identified NATs (NatA-NatH), NatA and NatB are two major NATs, associating with ribosomes to cotranslationally acetylate ~60% of the proteins in eukaryotes1,4. NatA acetylates proteins that are subject to removal of the initiator methionine (iMet) by methionine aminopeptidases (MetAPs) and display small amino acids (Ala, Ser, Thr, Val, Gly, and Cys) at their N-terminus. NatB acetylates proteins starting with an iMet followed by a polar or acidic amino acid (Asp, Asn, Glu, Gln) with nearly 100% efficiency5,6. Reduced activity of NatA and NatB results in severe developmental defects in different organisms5–9. Unlike in yeast, where NatA deletion is tolerable, loss of NatA is lethal in all so far analyzed higher eukaryotes8,10–13. However, deletion of NatB, which is responsible for the NTA of ~20% of the proteomes in humans and plants, is lethal in Drosophila melanogaster14, whereas two plant NatB T-DNA insertion mutants are viable but exhibit increased sensitivity to reductive stress15. This raises the question of whether the physiological role of NatB is evolutionarily conserved in multicellular eukaryotes of the green and red lineage.

NTA influences various aspects of protein behaviors, including protein subcellular localization, protein-protein interactions, protein complex formation, and protein aggregation1. Notably, NTA of certain proteins has been reported to create specific signals for degradation via a specific branch of the ubiquitin proteasome system (UPS), termed the Ac/N-degron pathway, in which the Ac/N-degron is recognized by a specific E3 ubiquitin ligase termed N-recognin16. Recently, NTA has been revealed to play a critical role in proteome stability and protein quality control across different species17. At a global level, depletion of NatA accelerates protein turnover of non-Nt-acetylated proteins via the newly defined GASTC/N-degron pathway and destabilizes the proteome in human, plants, and yeast18–24. NatB is not considered to be a main factor in yeast proteostasis, since no global effect on protein stability was observed in NatB-deficient yeast cells, albeit the translation rate was decreased by 50%25. In Drosophila, NatB-mediated NTA in specific organs shields key target proteins from proteasomal degradation by the ubiquitin E3 ligase UBR1, thereby controlling male germline stem cell differentiation and reproduction26. However, the UBR1 protein is not directly conserved in plants; instead, the degradation of proteins bearing type 1 and type 2 N-degrons is undertaken by separate plant-specific N-recognins, PRT6 and PRT1, respectively27,28. Therefore, the role of NatB in global proteome stability and protein quality control via the UPS in higher eukaryotes remains elusive.

Besides the UPS, autophagy is another key cellular quality control mechanism29. Autophagy is activated under nutrient starvation or other stress conditions to degrade protein complexes, protein aggregates, and damaged organelles, which enables recycling of nutrients for dynamic stress responses and clearance of unwanted proteins30. However, the connection between NTA and autophagy in eukaryotes is rarely investigated. In yeast, NatB is reported to be a positive regulator of autophagy, promoting autophagosome formation and autophagosome-vacuole fusion through the acetylation of Act1 and Vps31. In contrast, in human cell lines, NatB exhibits an inhibitory effect on autophagy by regulating the LKB1-AMPK-mTOR axis32. These contrasting findings imply that the impact of NTA on autophagy regulation is a consequence of the evolution of NatB substrates in different organisms.

Since the UPS and autophagy are the two major pathways for regulating cellular protein accumulation, the interplay of UPS and autophagy is crucial for cellular homeostasis and the fine regulation of the organismal development and stress responses33. Previous studies have shown that certain autophagy-related (ATG) proteins can be degraded via UPS, while proteasomal proteins themselves can be degraded via autophagy34–39. However, how NTA contributes to the regulation of UPS-autophagy interplay remains unclear in plants. In this study, we demonstrate that NatB regulates global protein turnover and destabilizes the plant proteome at least partially by the generation of Ac/N-degrons. Consequently, NatB substrates are substantially enriched in the fraction of accumulated proteins in NatB-deficient plants. We uncover that NatB-mediated NTA destabilizes KINASE 11 (KIN11), a subunit of the eukaryotic energy sensor SNF1-related protein kinase 1 (SnRK1) to attenuate autophagy in plants. This KIN11-mediated induction of autophagy is essential for the remarkable tolerance of NatB-deficient mutants to energy-limitation stress and also compensates for the significantly lowered UPS activity in NatB-deficient plants. In summary, we elucidate the molecular mechanisms by which NatB regulates protein stability by the UPS and autophagy to balance plant growth and nutrient stress responses in plants. These findings extend our knowledge on the physiological function and the regulatory mechanism of NTA in plants. In contrast to animals, NatB is non-essential in plants due to an unknown compensatory mechanism ensuring partial NTA of many NatB substrates in NatB-deficient mutants.

Results
Knockout of the NAA20 does not result in plant lethality nor abolish NTA of NatB substrates
In a previous study, we reported delayed plant development in T-DNA insertion mutants of the NatB catalytic subunit gene NAA20 and the auxiliary subunit gene NAA25, both of which retained detectable levels of NatB complex transcripts and NAA25 protein9. To determine whether the complete loss of NatB in plants is lethal, as observed for NatB knockout mutants in animals14, we generated NAA20 null mutants using CRISPR/Cas9. Two independent mutations within the N-terminal coding region of NAA20 introduced frameshifts and premature stop codons, abolishing translation of the catalytic subunit and thereby fully inactivating NatB (Fig. 1a, Supplementary Fig. 1a). Both CRISPR/Cas9 knockout lines, designated naa20-cr1 and naa20-cr2, exhibited developmental retardation indistinguishable from the naa20-1 T-DNA mutant, including reduced rosette leaf size and fresh weight (30–40% growth delay) (Fig. 1b–d). These results indicate that complete loss of NAA20 is detrimental but not lethal in plants. Genetic complementation of naa20-cr1 with a NAA20–GFP fusion restored normal development (Fig. 1e, Supplementary Fig. 1b, c).

The N-acetylome was analyzed to evaluate the impact of NAA20 deletion on the NTA status of NatB substrates. N-terminomics identified 758 quantified non-redundant N-terminal peptides, of which 274 began at residue two due to iMet cleavage. Ninety-one N-termini retained the iMet, and 72 of these exhibited D/E/N/Q at position two, consistent with NatB substrate specificity (Supplementary Data 1).

In the wild type, 66 of 73 iMet-retaining proteins were fully N-acetylated. In contrast, both naa20-cr1 and naa20-1 mutants showed a pronounced reduction in NTA specifically for iMet-retaining peptides, while iMet-cleaved peptides (primarily acetylated by NatA) remained unaffected (Fig. 1f, g, Supplementary Fig. 1d, e). Notably, 33 iMet-retaining proteins displayed a more than 20% reduction in their Nt-acetylation in naa20 mutants compared with wild type (Supplementary Data 2). All of these proteins displayed the canonical NatB substrate sequence (ME, MD, MN or MQ). Twenty-five of the NatB substrates were found in both naa20 mutants; in those cases, the level of NTA was similarly decreased in both mutants.

Unexpectedly, not all NatB substrates in the mutants were unacetylated; many retained partial or full acetylation relative to wild type (Fig. 1f, g), suggesting the presence of a compensatory mechanism. Similar phenomena have been observed in vertebrates40, potentially involving unidentified NATs or alternative known NATs acting on iMet-initiated peptides (e.g., NatC/E/F). To test the latter hypothesis, we overexpressed the catalytic subunits of NatC (NAA30), NatE (NAA50), and NatF (both long and short NAA60 isoforms). However, none of them restored NTA levels or rescued the developmental defects in the naa20-cr1 mutant (Fig. 1h, i, Supplementary Fig. 2).

NatB regulates protein turnover and destabilizes the plant proteome
Recently, the role of NatA in stabilizing the proteome has been revealed in various species17. NatB is responsible for the NTA of more approximately 20% of the plant proteome. To investigate its role in protein turnover and proteome stability, we measured the chymotrypsin-like protease activity of the proteasome in soluble protein extracts of the wild type and the natb mutants by monitoring the release of fluorescent AMC from the model substrate Z-Leu-Leu-Leu-AMC. We found that the proteasome activity was significantly reduced in the natb mutants compared with wild type (Fig. 2a). Consistent with this finding, both overall and K48-linked polyubiquitination levels showed reduced levels in the soluble protein fraction of natb mutants (naa20-cr1 and naa25-1) compared with wild type when proteasome activity was inhibited by MG132 (Fig. 2b, c, Supplementary Fig. 3a, b). These findings imply a lower in vivo degradation rate by the UPS in natb mutants. Quantification of protein translation rates by feeding of isotope-labeled amino acids or azidohomoalanines showed that translation rates were also reduced in the natb mutants (Fig. 2d, Supplementary Fig. 3c, d).

To determine whether the lowered protein turnover in naa20-cr1 specifically leads to the accumulation of NatB substrates, we performed a quantitative proteomic analysis. A total of 3788 proteins were identified, with NatA and NatB targeting 61% and 19% of them, respectively (Fig. 2e, Supplementary Data 3). In the wild type, only 16% of stabilized proteins are NatB substrates, indicating that NatB substrates are not enriched in the stabilized protein fraction (as 19% of all identified proteins are NatB substrates). In the natb mutant, 30% of stabilized proteins are NatB substrates, representing a substantial enrichment of NatB substrates in this fraction when NatB is absent. The percentage of stable NatB proteins increased from 16% in the wild type to 30% in the natb mutant (Fig. 2e). This finding demonstrates that NAA20 is critical for controlling the stability of its substrate in plants. A gene ontology (GO) analysis using agriGO v2.041 revealed that the proteins with increased abundance in the naa20-cr1 mutant were mainly enriched in translation and amino acid metabolic process pathways (Fig. 2f), which may be explained by an unsuccessful attempt to compensate for the impaired translation rate in naa20-cr1 (Fig. 2d).

Proteins with reduced abundance in the naa20-cr1 mutant were mainly enriched in photosynthesis and proteins synthesis pathways (Fig. 2f), reinforcing the proposed role of NAA20 in determining plant protein homeostasis and plant growth.

NatB may not directly regulate the plant proteome by modulating the stability of individual NatB substrate
To further investigate the mechanism by which NatB regulates plant proteome stability, we performed an ubiquitin capture (Ubicapture) approach to enrich ubiquitinated proteins. The Ubicapture pulldown was followed by mass spectrometry to identify and classify differentially polyubiquitinated proteins in the wild type and naa20-cr1. The Ubi capture results confirmed dramatically reduced polyubiquitination levels in naa20-cr1 compared with wild type (Fig. 3a). Consistent with this, the vast majority (310 out of 314) of significantly changed polyubiquitinated proteins identified by mass spectrometry were more abundant in the wild type than in naa20-cr1 (Supplementary Data 4), suggesting that NatB destabilizes the plant proteome. Classification of NAT substrates for less ubiquitinated proteins in naa20-cr1 showed that NatB substrates were not significantly enriched, with only 16% of the proteins identified as NatB substrates, while NatA substrates represented 68% of the less ubiquitinated proteins in naa20-cr1 (Fig. 3b). GO analysis using agriGO v2.041 revealed that a large number of less polyubiquitinated proteins in the naa20-cr1 mutants are involved in protein hydrolysis, translation, and response to abiotic stimuli (Fig. 3c). Among these, 86% of the identified proteins involved in the translation process are NatA substrates, whereas only 4% of them are NatB substrates (Fig. 3d). However, 42% of the identified proteins involved in the ubiquitin-dependent protein catabolic process are NatB substrates (Fig. 3e). These findings suggest that NatB may not directly regulate the plant proteome by modulating the stability of all NatB substrates. Alternatively, NatB-mediated substrates may play a role upstream of the protein homeostasis cascade.

Next, we examined the protein abundance of six NatB substrates (V-ATPase subunit, RPN12a, MAT3, PBA1, HSP101, and NBR1) in the wild type and naa20-cr1 by immunological detection. Among these, V-ATPase subunit and RPN12a have been experimentally confirmed to have reduced NTA levels in the naa20 mutants (Supplementary Data 2), whereas the other four candidates are predicted NatB substrates based on their sequences. The results suggested that only NBR1, a selective autophagy cargo receptor, showed higher protein abundance in naa20-cr1 compared with wild type (Fig. 3f), implying that NatB may not directly affect the protein stability of all individual NatB substrates.

NatB-mediated NTA destabilizes NBR1 in a UPS-dependent manner
NBR1 was not quantified by the proteomics approach (Fig. 2e). To further investigate how NatB regulates the protein abundance of NBR1, we excluded the possibility of elevated NBR1 transcripts in mutants by RT-qPCR (Supplementary Fig. 4a). In vitro acetylation assays demonstrated that the endogenous NatB complex, immunoprecipitated from a NAA20-GFP/naa20-cr1 line using GFP beads, catalyzes the acetylation of the NBR1 N-terminus. The N-terminally acetylated NBR1 (Ac-NBR1) and the mutagenized NBR1E2P N-terminus served as negative controls and were not accepted as substrates by NatB (Supplementary Fig. 4b, c). Next, we applied cycloheximide (CHX) chase experiments and found that NBR1 was significantly slower degraded in naa20-cr1 than in the wild type, implying that NatB-mediated NTA promotes degradation of soluble NBR1 (Fig. 3g). To further support this hypothesis, we examined the degradation rate of the NBR1E2P variant, which cannot be acetylated in vivo, and found that the degradation rate of NBR1E2P was indeed significantly reduced compared with the wild-type NBR1 (Fig. 3h). Previous studies have shown that NBR1 is a selective cargo receptor for autophagy and is degraded by autophagy in the vacuole. However, we found that treatment with MG132, an inhibitor of the proteasome, also caused its protein accumulation, and after combining Concanamycin A (ConA) to block autophagy, the amount of soluble NBR1 accumulated further (Supplementary Fig. 4d). These findings imply that the UPS regulates the amount of soluble NBR1 in an NTA-dependent manner, making it available to serve as a membrane-associated autophagy cargo receptor. To further support this conclusion, we analyzed NBR1 degradation dynamics in naa20-cr1, when autophagy, the UPS or both are suppressed by MG132 and/or ConA treatments. When protein synthesis was inhibited by Cycloheximide (CHX), both MG132 and ConA alone inhibited NBR1 degradation, with MG132 exhibiting a more pronounced effect in plants grown under optimal growth conditions. When MG132 and ConA were applied together, NBR1 degradation was completely inhibited, indicating that NBR1 is degraded via both the UPS and autophagy pathways in vivo (Supplementary Fig. 4e). In line with this hypothesis, we found that NBR1 was also degraded in vitro in a membrane-free system. The degradation rate of NBR1 in this membrane-free system was slower in the naa20-cr1 and could be inhibited in the wild type by the addition of MG132 (Supplementary Fig. 4f). Taken together, these results demonstrate that, besides autophagic degradation, NBR1 undergoes UPS-mediated degradation by creating Ac/N-degron.

N-terminal acetylation can influence not only protein stability but also subcellular localization and protein–protein interactions. Next, we investigated the localization of NBR1 using stable NBR1-mCherry-GFP/WT and NBR1-mCherry-GFP/naa20-cr1 transgenic lines. Microscopy results revealed that in wild-type root tip cells, NBR1 shows a cytosolic and a punctate localization pattern, which is consistent with previous studies42. In naa20-cr1 mutant, the localization pattern of NBR1 remains unchanged, indicating that the absence of NTA does not affect NBR1 localization (Supplementary Fig. 4g). To investigate whether loss of N-terminal acetylation alter the interaction between NBR1 and ATG8, we performed Co-IP assay using stable NBR1-HA/WT and NBR1-HA/naa20-cr1 transgenic plants. The results showed that NBR1 still interacts with ATG8 in naa20-cr1, suggesting that the absence of NTA does not affect the interaction between NBR1 and ATG8 (Supplementary Fig. 4h).

NatB-deficient plants display higher autophagic flux
Previous reports have demonstrated that a lowered proteasome abundance could be caused by enhanced autophagy43. To investigate whether the lower protein turnover via the UPS in the NatB mutant might be partially a consequence of a higher autophagy level in NatB-deficient mutants, we examined the K63-type polyubiquitination signals in the wild type and naa20-cr1 plants after inhibition of autophagy by ConA. In contrast to the K48-linked polyubiquitination (Fig. 2c), the K63-linked polyubiquitination was significantly increased in naa20-cr1 compared with wild type, indicating a higher autophagic flux in the naa20-cr1 mutant (Fig. 4a, b). This conclusion was further supported by the detection of autophagy-related proteins ATG8 and ATG3, which are essential for autophagosome formation, in natb mutants and the wild type. The abundance of these ATG proteins was significantly increased in natb mutants (Fig. 4c–e). This increase also applied to ATG8-PE, a post-translationally lipidated proteoform of ATG8 covalently conjugated to phosphatidylethanolamine, which is essential for autophagosome membrane expansion (Fig. 4c), and the ATG5-ATG12 conjugated complex, which is essential for autophagosome formation by acting as an E3-like enzyme that promotes ATG8 lipidation (Fig. 4e). Neither ATG8–PE nor ATG5-ATG12 conjugated complex was detected in the atg5-1 mutant (Fig. 4c, d). Real-time PCR analysis revealed no significant increase in the expression levels of these genes in the mutant, suggesting that the increase of ATG proteins may be associated with changes in post-translational modifications (Fig. 4f).

Next, we applied the autophagy marker GFP-ATG8a to analyze the autophagic flux in the wild type and the naa20-cr1 mutant. Non-invasive confocal laser scanning microscopy of GFP-ATG8a/naa20-cr1 and GFP-ATG8a/wild type demonstrated that after selective inhibition of autophagic flux by ConA application, darkness-treated naa20-cr1 mutants accumulated significantly more autophagic bodies in their vacuole than the wild type (Fig. 4g, h). Moreover, to further substantiate the increased autophagic flux in the naa20-cr1 mutant, a GFP-cleavage assay was performed. Darkness treatment caused a substantially stronger induction of GFP release from GFP-ATG8a in the naa20-cr1 when compared with the wild type as indicated by the higher GFP to GFP-ATG8a ratio in naa20-cr1 at all times of darkness treatment (4, 8, and 12 h, Fig. 4i). Taken together, these data demonstrate a higher autophagic flux in the absence of NatB.

NatB represses autophagy in plants
The connection between N-terminal protein acetylation and autophagy remains unaddressed in plants so far. To verify whether the constitutively elevated autophagic flux could cause more efficient physiological adaptation to stress conditions in naa20 mutants, we subjected the wild type, two naa20 mutants, and the autophagy-deficient mutant atg5-1 to elongated darkness stress. Both naa20 mutants exhibited significantly higher survival rates compared with the wild type upon darkness treatment. As expected, the autophagy mutant atg5-1 was susceptible to prolonged darkness stress (Fig. 5a, b). Consistent with this, the natb mutants exhibit delayed senescence after growth in short-day condition for 10 weeks and transfer to long-day condition for 2 weeks (Supplementary Fig. 5a). Furthermore, the naa20-cr1 showed higher resistance and more fresh weight accumulation upon nitrogen and sulfur starvation conditions. The autophagy-deficient mutant performed worse than the wild type under both nutrient deficiencies. However, under control conditions, naa20-cr1 and atg5-1 displayed similarly decreased growth compared to the wild type (Fig. 5c, d). These results suggest that, unlike in yeast, where NatB is critical for autophagy induction31, NatB functions as a negative regulator of autophagy in plants.

To further investigate the role of the induced autophagic flux in natb mutants, we crossed naa20-cr1 with two atg mutants, atg5-1 and atg7-1, respectively. We found that blocking autophagy did not rescue the growth defects of naa20-cr1 under optimal growth conditions (Supplementary Fig. 5b, c). Nevertheless, both naa20-cr1 atg5-1 and naa20-cr1 atg7-1 double mutants lost the resistance to prolonged darkness (Fig. 5e, f). Inactivation of autophagy in the naa20-cr1 atg double mutants restored wild-type levels of polyubiquitination, which is in agreement with the hypothesis that induction of autophagy is partially responsible for the disturbed protein turnover in naa20-cr1 (Fig. 5g, h). In support of a substantial crosstalk between autophagy and the UPS, the autophagy-deficient single mutants exhibited higher proteasome activity and polyubiquitination levels compared with the wild type (Fig. 5g, h, Supplementary Fig. 5d), demonstrating that the UPS is generally activated after the blockage of autophagy. To verify that crossing of atg5-1 into naa20-cr1 indeed inhibited autophagy in the double mutant, we crossed the GFP-ATG8a marker into this line and demonstrated the absence of autophagosome formation in this mutant (Supplementary Figs. 5e, f). Taken together, these results demonstrate that NAA20 regulates resistance to carbon starvation through autophagy and strongly suggest that constitutively induced autophagy may contribute to the downregulation of UPS in NatB-deficient plants.

NatB-mediated NTA regulates protein stability of KIN11, a component of the energy sensor SnRK1
To understand how the darkness stress-resistant phenotype is induced in natb mutants, we crossed naa20-cr1 and nbr1-2 and found that the naa20-cr1 nbr1-2 double mutant is still resistant to darkness (Supplementary Fig. 6a, b), implying that the accumulation of NBR1 in naa20-cr1 does not contribute to the darkness resistance phenotype of naa20-cr1. We then examined the N-terminal sequences of autophagy-related proteins, as well as those related to the upstream regulatory network of autophagy induction. We found that the two catalytic subunits of the plant energy sensor SnRK1, KIN10 and KIN11, are predicted to be NatB substrates. Thus, we detected the activation status of SnRK1 and found that pKIN10 and pKIN11, two proteoforms that are more phosphorylated at the kinase activation loop, accumulated in naa20-cr1, demonstrating that SnRK1 activity is substantially enhanced in the naa20-cr1 mutant (Fig. 6a, Supplementary Fig. 6c). Consistent with the activation of SnRK1, we found that the transcriptional levels of energy-related genes downstream of SnRK1 (DIN1, DIN6 and DIN10) were induced in natb mutants. Remarkably, the transcription factor bZIP63, which is directly phosphorylated by SnRK1 and controls transcription of these DIN genes, is not induced, suggesting that DIN gene activation is caused by post-translational modification of bZIP63. At the same time, endogenous activity of the sensor kinase TARGET OF RAPAMYCIN (TOR), as determined by the ratio of phosphorylated-RPS6 to RPS6, was suppressed in natb mutants, which may explain the decreased translation and lowered growth of these mutants (Supplementary Fig. 6d, e). Based on these findings, we hypothesized that the absence of NTA on KIN10 and KIN11 directly causes the accumulation of pKIN10 and pKIN11, thereby enhancing SnRK1 activity in natb mutants. In vitro acetylation experiments provided evidence that NatB indeed acetylates the major proteoforms of KIN10 and KIN11 (Fig. 6b). In contrast, the low-abundant proteoform of KIN10 (KIN10L), which starts with an MF, is not acetylated by NatB in vitro. Destruction of the canonical NatB substrate recognition signal in the mutagenized major protoeforms of KIN10 D2P or KIN11 D2P impaired acetylation of the major SnRK1 isoforms by NatB (Fig. 6b). Since we found that several NatB substrates accumulated in natB mutants (Fig. 2e), we tested the abundance of KIN10 and KIN11 in natb and the wild type by immunological detection. Only KIN11 but not KIN10 significantly accumulated in naa20-cr1 (Fig. 6c, d). In this context, it is remarkable that KIN10 and KIN11 exhibit 89.2% similarity, and that only three protein domains show greater variability between the proteins (Supplementary Fig. 6f). The domain with the highest difference between both proteins is the N-terminus, suggesting that the distinct N-terminal sequences of KIN11 (MDHSSNRFGN10-) and KIN10 (MDGSGTGSRS10-) contribute substantially to the observed differences in accumulation.

To provide direct evidence for the specific stabilization of the non-acetylated KIN11 protein in natb mutants, we firstly determined the in vivo NTA levels of KIN11-HA in wild-type and naa20-cr1 background by immunoprecipitation followed by mass spectrometry and found that the loss of NatB resulted in a two-fold lower KIN11 acetylation in naa20-cr1 when compared to wild type (Supplementary Fig. 6g, h). We then excluded the possibility of elevated KIN11 transcripts in the natB mutants as the reason for KIN11 accumulation (Supplementary Fig. 6i). CHX time-chase experiment revealed that the degradation of KIN11 could be inhibited by the proteasome inhibitor MG132 (Supplementary Fig. 6j) and that the degradation rate of KIN11 was significantly lower in the naa20-cr1 mutant than in the wild type (Fig. 6e), strongly suggesting that NTA of KIN11 leads to its degradation in a UPS-dependent manner. To further support this hypothesis, we examined the degradation rate of the KIN11 D2P variant, which cannot be acetylated in vivo, and found that the degradation rate of KIN11 D2P-GFP was significantly decreased when compared with the regular KIN11-GFP fusion protein starting with MD (Fig. 6f). Moreover, immunoprecipitation of KIN11-GFP followed by immunological detection of polyubiquitination revealed substantially higher accumulation of polyubiquitylated KIN11-GFP in the wild-type than in the naa20-cr1 (Fig. 6g). Furthermore, the non-acetylated KIN11 D2P-GFP was less polyubiquitinated than the acetylated KIN11-GFP in the wild type, demonstrating that the NTA status of the KIN11-GFP determines its degradation rate by the UPS (Fig. 6g). In summary, our results uncover that decreased NTA of KIN11 impairs its degradation by the UPS, which leads to the accumulation of the activated KIN11 protein. In agreement with our hypothesis that KIN10 is not destabilized in natb mutants due to its significantly different N-terminus when compared to KIN11, we found that KIN10 is not destabilized in naa20-cr1 (Supplementary Fig. 6k).

The activation of KIN11 contributes to the dark resistance of the naa20-cr1 mutant
Previous studies have shown that despite the substantial redundancy between KIN10 and KIN11, both isoforms also have specialized functions44,45. Motivated by these reports, we investigated the darkness resistance of kin10 and kin11 mutants and found that only kin11, but not kin10, was more sensitive to darkness treatment (Supplementary Fig. 7a, b).

To investigate whether the elevated KIN11 protein level is responsible for the increased darkness resistance of naa20-cr1, we crossed naa20-cr1 with kin10 and kin11. Remarkably, the darkness resistance of the naa20-cr1 kin11 double mutant was attenuated compared with that of naa20-cr1. In contrast, inactivation of KIN10 in the naa20 cr-1 kin10 double mutant did not affect the darkness resistance of the NatB-deficient mutant (Fig. 7a, b). Correspondingly, two independent KIN11-overexpressing lines exhibited higher survival rates than the wild type upon darkness treatment, suggesting a role for KIN11 in regulating carbon starvation response in plants (Supplementary Fig. 7c, d). Detection of the phosphorylated SnRK1 catalytic units showed that the phosphorylated KIN11 was absent in the naa20-cr1 kin11 double mutant, while the kin11 and naa20-cr1 kin11 mutants still showed comparable levels of phosphorylated KIN10 that were higher than those of the wild type. These findings demonstrate that the specific activation of KIN11 is critical for the darkness resistance of naa20-cr1 (Fig. 7c). However, the knockout of KIN11 fails to suppress the elevated ATG proteins in naa20-cr1 kin11 double mutant, implying that activation of either KIN10 or KIN11 in the natb mutants is sufficient for autophagy induction under non-stressed conditions (Supplementary Fig. 7e, f). Taken together, we found that NatB regulates the interplay between UPS and autophagy in plants by fine-tuning the SnRK1 activity as well as the abundance of autophagy-associated proteins, which in turn coordinates plant growth and development and nutrient stress accordingly.

In summary, we found that the absence of NTA on many NatB substrates causes their lowered degradation by the UPS (Figs. 2c, 3g, 3h, 6e–g), which causes them to accumulate (Figs. 2e, 3f, 6c). A decreased global protein turnover and lowered UPS accompany this (Figs. 2a–d, 3a). However, we could not solely explain the decreased polyubiquitination levels in natb mutants by the lowered polyubiquitination of NatB substrates (Fig. 3b). Instead, we found that decreased NTA of the catalytic subunits of the energy sensor SnRK1 (KIN10 and KIN11) caused their activation (Fig. 6a), which resulted in a constitutive induction of autophagy in NatB-deficient mutants (Figs. 4, 7a), most likely contributing to compensatory general downregulation of the UPS (Figs. 2a-c, 3a, 3b). Finally, we uncovered the molecular mechanism of the darkness stress resistance of NatB-deficient mutants, which is caused by the specific accumulation of activated KIN11 protein (Figs. 5a, 5b, 5e, 5f, 7a, 7b). Our findings demonstrate that NatB controls the stability of diverse NatB substrates by the UPS and attenuates autophagy levels in plants by regulating KIN11 abundance via the generation of an Ac/N-degron (Figs. 3g, 3h, 6e–g, Supplementary Fig. 4). Consequently, NatB is a critical regulator of the UPS-autophagy interplay, which explains its resistance to diverse nutrient limitation stresses (Fig. 7d).

Methods
Plant materials and growth conditions
Arabidopsis thaliana ecotype Col-0 was used as the wild type. The T-DNA insertion mutants naa20-1 (SALK_027687) and naa25-1 (GK-819A05) were previously published in ref. 9. The T-DNA insertion mutants kin11 (WiscDsLox320B03) and kin10 (GABI_579E09) were previously published in ref. 60. The T-DNA insertion mutants nbr1-2 (GABI_246H08), agt5-1 (SAIL_129_B07), atg7-1 (N39995) were obtained from the Nottingham Arabidopsis Stock Centre (www.arabidopsis.info). The pUb:GFP-ATG8a lines in both wild type and agt5-1 backgrounds were previously published in ref. 61. The pUb:GFP-ATG8a lines in naa20-cr1 and naa20-cr1 agt5-1 backgrounds were generated by genetic crossing naa20-cr1 with the respective pUb:GFP-ATG8a lines. The naa20-cr1 agt5-1, naa20-cr1 agt7-1, naa20-cr1 nbr1-2, naa20-cr1 kin11 and naa20-cr1 kin10 double mutants were generated by genetic crossing the naa20-cr1 with respective single mutants.

Seeds were sown in the pot filled with well-watered turf mixture (Ökohum, Herbertingen) supplemented with 10% [v/v] vermiculite and 2% [v/v] quartz sand. Then the seeds were vernalized in the dark at 4°C for 2–3 days and transferred to short-day conditions (8.5h light per day, light intensity: 100 μmol m−2 s−1; day temperature: 22°C; night temperature: 18°C; relative humidity: 50%) in plant growth chambers (ThermoTec). For seeds production or crossing, eight-week-old plants were transferred to long-day growth chambers (16-hour light per day; other conditions were the same as in short-day conditions).

Constructs for genetic transformation and selection of transgenic plants
To generate NAA20 CRISPR/Cas9 construct, single-guide RNA (sgRNA) targeting the first exon of NAA20 was designed (Supplementary Data 5) and cloned according to the protocol62. Selection of NAA20 CRISPR/Cas9 seedlings was performed by applying Hygromycin B (25 mg/L) on solid ½ MS medium plates. The surviving plants were sequenced using specific primers to identify mutations (Supplementary Data 5). The CRISPR/Cas9 construct was eliminated from naa20-cr mutants by self-pollination and segregation.

To generate the NAA20-GFP, NBR1-HA, NBR1-mCherry-GFP, KIN11-GFP, KIN11D2P-GFP, KIN11-HA tagged constructs and NBR1E2P, NAA30, NAA50, NAA60S, NAA60L overexpression constructs, the original and variant cDNA sequences of the respective genes were amplified using the PCRBIO HiFi Polymerase (PCR BIOSYSTEMS) with specific primers (Supplementary Data 5). The resulting PCR fragments were cloned into the GreenGate cloning system under the control of the Ubiquitin promoter. Final vectors were sequenced to confirm sequence identity (EUROFINs) and then stably transformed into respective plants by A. tumefaciens–mediated floral dipping. Selection of one-week-old soil-grown transgenic plants was performed by spraying glufosinate-ammonium solution (0.2 g/liter) three times within 6 days. The surviving plants were genotyped by PCR using specific primers or sequenced to confirm the variants and backgrounds (Supplementary Data 5).

RNA extraction, RT-qPCR and RT-PCR
Total RNA was isolated from 100 mg of leaf materials of soil-grown plants using the Universal RNA Kit (Roboclon). Subsequently, first-strand cDNA synthesis was carried out with the FastGene Scriptase II cDNA Synthesis Kit (NIPPON Genetics) following the manufacturer’s instructions to generate templates for both RT-qPCR and RT-PCR analyses. RT-qPCR was performed using the qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems) following the manufacturer’s instructions. RT-PCR was performed using the FastGene Taq 2× ReadyMix (NIPPON Genetics). The specific primer pairs used are listed in Supplementary Data 5. The expression levels were normalized to the expression of Actin7 (At5g09810) or PP2A (AT1G69960).

Protein extraction and immunoblot detection
The soluble proteins were extracted from 100 mg of leaf materials from six-week-old soil-grown plants under short day condition with 300 μl of extraction buffer (50 mM HEPES pH 7.4, 10 mM KCl, 1 mM EDTA, 11 mM EGTA, and 10% [v/v] glycerol) supplemented with 10 mM DTT, 0.5 mM PMSF and 1 x complete protease inhibitor cocktail (Roche). For the detection of RPS6 and autophagy-related proteins, NuPage sample buffer (150 mM Tris-HCl pH 8.5, 2% LDS, 1 mM EDTA, 10% [v/v] glycerol, 0.22 mM Coomassie Blue G250, 0.166 mM Phenol Red and 50 mM DTT) was used to extract total protein. Subsequently, proteins were separated by SDS-PAGE or Tricine-SDS-PAGE (for ATG8-PE separation) and blotted onto PVDF membranes. Antibodies against NAA25 (1:5,000)9, NAA50 (1:5,000)57, GFP (ChromoTek, Pabg1-100, 1:2,000 and Santa-Cruz Biotechnology, sc-9996 HRP, 1:2,000), K48-Ubi (Cell signaling, 8081, 1:5,000), K63-Ubi (Cell signaling, 5621, 1:1,000), Neutravidin-HRP (Invitrogen, 31001, 1:10,000), mono- and poly-ubiquitination (Enzo life Sciences, ENZ-ABS840HRP, 1:10,000), RPN12a (Agrisera, AS19 4268, 1:2,000), V-ATPase (Agrisera, AS07 213, 1:2,000), HSP101 (Agrisera, AS07 253, 1:2,000), SAM1-4 (Agrisera, AS16 3148, 1:2,000), PBA1 (Agrisera, AS19 4260, 1:2,000), ATG3 (Agrisera, AS19 4275, 1:3,000), ATG5 (Agrisera, AS15 3060, 1:2,000), ATG8 (Agrisera, AS14 2769, 1:2,000), RPS6 (Cell signalling, 2317, 1:2,000), p-RPS6 (1:5,000)63, KIN10 (Agrisera, AS21 4581, 1:2,000), KIN11 (Agrisera, AS10 920, 1:1,000), Phospho-AMPKα (Thr172) Antibody (Cell signaling, 2531S, 1:2,000) or the secondary anti-rabbit IgG-HRP (Agrisera, AS09 602, 1:25,000) were diluted in 1× Tris-buffered saline (50 mM Tris, pH 7.6, 150 mM NaCl, and 0.05% [v/v] Tween 20) supplemented with 0.5% [w/v] BSA. Antibodies against NBR1 (Agrisera, AS14 2805, 1:5,000) was diluted in 1× TBST supplemented with 8% [w/v] nonfat milk. The resulting signals were quantified with the Fiji image processing application. All the uncropped blots are provided in the Source Data file.

Protein degradation rate analysis
Leaf materials from six-week-old soil-grown plants or seedlings from ½ MS plates under short day condition were used to determine the protein degradation rate. For in vitro cell-free protein degradation assay, the soluble proteins were extracted from leaf materials by adding two volumes of extraction buffer (25 mM Tris–HCl pH 7.5, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM dithiothreitol, and 10 mM ATP). Supernatants were cleared by centrifugation at 20,000 g for 20 min at 4°C, and incubated at 22°C for indicated time. For CHX time chase assay, protein synthesis was inhibited by floating leaf discs or seedlings from ½ MS plates on ½ Hoagland medium (2.5 mM Ca(NO3)2, 0.5 mM MgSO4, 2.5 mM KNO3, 0.5 mM KH2PO4, 4 μM Fe-EDTA, 25 μM H3BO3, 2.25 μM MnCl2, 1.9 μM ZnCl2, 0.15 μM CuCl2, 50 nM (NH4)6Mo7O24, and 0.6% (w/v) microagar pH 5.8) supplemented with 100 μM CHX (Sigma Aldrich) for indicated time under the light at 22°C while shaking at 60 rpm/min. MG132 (50 μM) or ConA (1 μM) were applied to inhibit UPS and autophagy-mediated degradation. The collected samples were subjected to SDS-PAGE and immunoblot analysis using specific antibodies.

Co-immunoprecipitation assay
HA magnetic beads and GFP agarose beads based immunoprecipitation was performed following the manufacturer’s instructions. Proteins were extracted from transgenic plants expressing the respective HA or GFP fusion proteins with extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM PMSF and 1× complete protease inhibitor cocktail and 0.1% Nonidet P-40). To confirm the presence of the NatB complex in the IP products of NAA20-GFP/naa20-cr1 plants, IP products were detected with GFP and NAA25 antibodies. To detect polyubiquitinated KIN11-GFP and KIN11D2P-GFP in KIN11-GFP/WT, KIN11-GFP/naa20-cr1 and KIN11D2P-GFP/WT plants, 50 μM of MG132 was applied to leaf discs and incubated for 5h to accumulate polyubiquitination. The respective IP products were detected with GFP and ubiquitin antibodies. To investigate the interaction between NBR1 and ATG8, stable NBR1-HA/WT and NBR1-HA/naa20-cr1 transgenic plants were used.

Determination of in vivo N-terminal acetylation of KIN11 by mass spectrometry
SDS-PAGE gels containing HA-immunoprecipitated products from KIN11-HA/WT and KIN11-HA/naa20-cr1 were manually excised after coomassie staining. The in-gel Trypsin digestion was performed following by Nanoflow LC-MS2 analysis using the Vanquish Neo coupled to an Orbitrap Eclipse (Thermo Fisher Scientific). An in-house packed analytical column (75 μm x 200 mm, 1.9 μm Reprosil Pur-AQ 120 C18 material was used. The fragment ion mass tolerance was set to 0.02 Da, and the parent ion mass tolerance to 10 ppm. The following variable modifications were allowed: Oxidation (O), Met-loss (M), Acetyl (N-terminus), Deamidation (N, Q), whereas Carbamidomethylation (C) was set as a fixed modification. Peptide quantification was done using a precursor ion quantifier node with the Summed Intensity method set for protein abundance calculation.

DTNB-based N-terminal acetylation assay
The in vitro NTA assay was performed as previously described in ref. 64. The purified NatB complex using GFP agarose-beads from NAA20-GFP/naa20-cr1 plants was used. Samples without NatB complex or with heat-inactivated NatB complex were used for negative controls. The absorbance of negative controls without NatB complex was averaged and subtracted from the absorbance of the samples. The product concentration was calculated using the Lambert Beer equation and the extinction coefficient of TNB2- (13700/M cm). The following synthetic peptides (GenScript, >95% purity) were used: NBR1: [H]MESTANALVVRVSYGGVLRR[OH]; NBR1E2P: [H]MPSTANALVVRVSYGGVLRR[OH]; Ac-NBR1: ac-MESTANALVVRVSYGGVLRR[OH]; KIN11: [H]MDHSSNRFGNNGVESILPNY[OH]; KIN11D2P: [H]MPHSSNRFGNNGVESILPNY[OH]; ac-KIN11: ac-MDHSSNRFGNNGVESILPNY[OH]; KIN10S: [H]MDGSGTGSRSGVESILPNY[OH]; KIN10D2P: [H]MPGSGTGSRSGVESILPNY[OH]; ac-KIN10S: ac-MDGSGTGSRSGVESILPNY[OH] and KIN10L: [H]MFRRVDEFNLVSSTIDHRI[OH]. The lysine residue in NBR1 peptides was replaced by arginine residues to minimize any potential interference by Nϵ-terminal acetylation.

Determination of free N-termini
Free N-termini groups in foliar proteins from six-week-old soil-grown plants were labeled with 0.5 mM NBD-Cl (4-Chloro-7-nitrobenzofurazan, Sigma-Aldrich) as previously described in ref. 21. Prior to labeling, free amino acids and other low-molecular-weight compounds were removed using a PD SpinTrap G-25 column (Cytiva).

Determination of proteasome activity
Proteasome activity was determined in leaves of six-week-old soil-grown plants with the proteasome substrate I (Z-Leu-Leu-Leu-AMC, Sigma-Aldrich) as previously described in ref. 22.

Long-term carbon, nitrogen and sulfur starvation assays
For the darkness treatment and survival rate analysis, plants were germinated on soil and grown under short-day conditions for two weeks. After that, every fourth plant was picked to a pot filled with soil. After one week of recovery growth, plants were moved to a chamber set to night conditions (same conditions as in the short-day chamber, but no light) for 8–14 days. Then the plants were moved back to the short-day conditions for 7–14 days for recovery. The survival rate was calculated by counting the plants with clearly visible intact green meristems and new leaves.

For nitrogen and sulfur starvation assays, Arabidopsis seeds were surface sterilized and seeded on solid ½ Hoagland medium plates (full, -N and -S). Plates were then kept at 4°C in dark for two days before being moved to LD conditions. After two weeks of growth, the fresh weight of every tenth seedling was weighed. Four replicates were calculated for each phenotype or condition.

GFP-ATG8a cleavage assay
The GFP-AtATG8a cleavage assay was performed as described previously65. Briefly, five-day-old seedlings grown on ½ MS medium plates containing 1% sucrose under long day conditions were exposed to continuous light for 1 day before being subjected to liquid ½ MS-C medium and kept in dark for 0, 4, 8, and 12 h. Then, the seedlings were harvested to isolate total proteins using NuPAGE buffer for immunoblotting analysis with anti-GFP antibody (Santa-Cruz Biotechnology, sc-9996 HRP).

Quantification of global translation
Protein translation rates in leaves of six-week-old soil-grown plants were assessed by monitoring the incorporation of 35S radiolabeled cysteine and methionine using the EasyTag™ EXPRESS35S Protein Labeling Mix (7 mCi, PerkinElmer, Waltham). In vivo protein labeling and quantification of incorporated radioactivity were performed as previously described in ref. 21.

Labeling and detection of newly synthesized proteins via L-azidohomoalanine incorporation was performed as previously described21. In brief, leaf discs from six-week-old soil-grown wild type and natb mutant were incubated for 3 h in ½ Hoagland medium supplemented with 50 μM azidohomoalanine. The incorporated azidohomoalanine was conjugated to Biotin Azide (Invitrogen) using the Click-iT™ Protein Reaction Buffer Kit (Invitrogen), and labeled proteins were detected with Neutravidin-HRP (1:100,000 dilution, Invitrogen, 31001). DMSO-treated wild-type leaf discs served as negative controls to distinguish endogenous biotinylated proteins.

Enrichment of polyubiquitinated proteins with UbiQapture-Q matrix and quantification of ubiquitome
Enrichment of all type- and K48-polyubiquitinated proteins was performed using UbiQapture-Q matrix kit (Enzo life Sciences) as previously described in ref. 21. Proteasome and deubiquitination activity were inhibited in the leaves of wild type and natb plants by floating leaf discs on ½ Hoagland medium supplemented with 50 μM of MG132 (Santa-Cruz Biotechnology) and 20 μM deubiquitinase inhibitor PR-619 (Sigma Aldrich) for 6 h. To quantify the K63-polyubiquitinated proteins, 1 μM ConA (Santa-Cruz Biotechnology) instead of 50 μM MG132 was applied to the ½ Hoagland medium to inhibit autophagy pathway.

Ubiquitome were analyzed as previously described in ref. 21. LFQ values were calculated by MaxQuant and used for quantitative data analysis. Heatmap was plotted by https://www.bioinformatics.com.cn (last accessed on 1st April 2025), an online platform for data analysis and visualization.

LC-MS/MS measurement of whole proteomes
Proteins were extracted from dried leaves of 6-week-old soil grown plants using lysis buffer containing 75 mM HEPES pH 7.5, 150 mM KCl, 1.5 mM EGTA pH 8.0, 1.5 mM MgCl2, 10% (v/v) glycerol, 0.075% NP-40 supplemented with 1 mM DTT, PhosSTOP phosphatase inhibitors, and Protease Inhibitor Mix. Proteins were precipitated in acetone and resolved in 8 M urea following a standard in-solution digest protocol as above. Peptides were separated on an EASY-nLC 1200 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) using a 110 min gradient from 5–60% acetonitrile with 0.1% formic acid and then directly sprayed in an Orbitrap HF-X Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The mass spectrometer was operated in a data-independent acquisition mode. The MS1 scans were acquired in a mass range of m/z 350–1000, with an Orbitrap resolution of 120,000 and a normalised AGC target of 3e6. MS2 scans were performed at a resolution of 15,000 and a normalised AGC target of 1e6. 100 windows with an isolation width of 5.6 Th (data points per peak=6) were acquired per cycle. MS raw files were searched against the UniProt FASTA protein database of Arabidopsis thaliana (release April 2023). Proteins quantified in at least 3 out of 4 replicates in either WT or naa20-cr1 mutant were filtered out, log2-transformed and imputated. Proteins with a fold change (FC) ≥ 1.5 and a Student’s t-test p-value < 0.05 were considered as significantly different. Heatmap was plotted by https://www.bioinformatics.com.cn (last accessed on 1st April 2025), an online platform for data analysis and visualization.

Quantification of the N-terminal acetylome
The N-acetylome was analyzed using the SILProNAQ method on protein extracted from leaves of six-week-old soil-grown plants66. In brief, tissues were frozen, ground, and extracted in buffer (50 mM HEPES pH 7.2, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 2 mM PMSF, 150 mM NaCl, and protease inhibitors cocktail). Free N-termini were labeled with d3-acetyl groups, proteins digested with trypsin, and peptides fractionated by SCX chromatography. Enriched N-terminal peptides were analyzed by LC-MS/MS using an LTQ-Orbitrap Velos, and data were processed with Mascot Distiller and the EnCOUNTer tool67.

Seedling treatment and confocal microscopic analysis
Seeds of GFP-ATG8a in WT, naa20-cr1, atg5-1, naa20-cr1 atg5-1 backgrounds were surface sterilized and seeded on ½ MS medium plates (with 1% sucrose). Plates were then kept at 4°C in dark for two days before being moved to LD conditions for one week. Subsequently, plates were transferred to continuous light for one day and then immersed in liquid ½ MS medium (without 1% sucrose) with 1 μM ConA for 6 h in the dark. Seedlings treated with 1 μM DMSO under light were used as control. Roots cells in the elongation zone were imaged using the Leica Stellaris 8, HC PL APO CS2, 20x water-immersed objective and software LASX 4.5.0.25531 (Leica, Wetzlar). The microscope was set to Pinhole 1AU, Zoom 1.5, as well as detection with bidirectional scan with phase x30, speed 200 Hz and line average 4, line accumulation 1. The laser intensity was between 5 and 15%. Excitation at 489 nm, GFP fluorescence was recorder with HyD S1 detector at EM = 498–540 nm. Autophagic puncta labeled by GFP-AtATG8a in confocal images were identified using the ImageJ. In brief, images were background-subtracted with 25 pixels of rolling ball radius. Each image was subsequently thresholded using the MaxEntropy method and was converted to an 8-bit grayscale image. Threshold values were adjusted according to the puncta signals in original confocal images. The number of puncta in thresholded images was counted by the Analyze Particles function in ImageJ. For all puncta quantification, puncta with the size between 0.10 and 5.00 μm2 were counted.

The root cells of stable transgenic NBR1-mCherry-GFP/WT and NBR1-mCherry-GFP/naa20-cr1 seedlings from ½ MS plates were used to detect the subcellular localization of NBR1 using the Leica Stellaris 8, HC PL APO CS2, 40x water-immersed objective and software LASX 4.5.0.25531 (Leica, Wetzlar).

Quantification and statistical analysis
Data are presented as mean ± SEM. The number of replicates and experimental details are provided in the respective figure legends. Statistical analyses were performed using GraphPad Prism 9. For comparisons between two groups, an unpaired two-tailed Student’s t-test was applied. Statistically significant differences are indicated with by asterisks (*p < 0.05, **p < 0.01). For analyses involving more than two groups, one-way ANOVA followed by a Tukey´s test was used for comparisons.

Accession numbers
Sequence data from this article can be found in The Arabidopsis Information Resource (www.arabidopsis.org) under the following accession numbers: AT1G03150 (NAA20), AT5G58450 (NAA25), AT4G24690 (NBR1), AT5G61500 (ATG3), AT5G17290 (ATG5), AT5G45900 (ATG7), AT4G21980 (ATG8a), AT2G45170 (ATG8e), AT2G38130 (NAA30), AT5G11340 (NAA50), AT5G16800 (NAA60), AT1G64520 (RPN12a), AT4G11150 (V-ATPase), AT2G36880 (MAT3), AT1G74310 (HSP101), AT4G31300 (PBA1), AT5G09810 (ACT7), AT1G69960 (PP2A), AT3G01090 (KIN10) and AT3G29160 (KIN11), AT4G35770 (DIN1), AT3G47340 (DIN6), AT5G20250 (DIN10), AT5G28770 (bZIP63).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The mass spectrometry proteomics data associated to N-terminomics of naa20 mutant lines have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD066411. The mass spectrometry proteomics data associated to proteomic changes of naa20-cr1 mutant have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD066676. The mass spectrometry proteomics data associated to determination of polyubiquitinated proteins enriched with UbiQaputre-Q matrix in the wild-type and naa20-cr1 mutant have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD054814. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This very interesting paper uses a range of different approaches to explore the role(s) of N-terminal acetylation (NTA) catalysed by the NatB complex on the plant proteome. Originally, NTA was thought to stabilize proteins but there is a growing body of evidence that NTA, in certain contexts, acts as a signal for degradation.\nUsing a CRISPR-generated knockout line (naa20-cr1), the authors demonstrated a modest decrease in acetylation of NatB substrates, but some substrates retained NTA status, indicating an unexplained compensation mechanism, which was considered not to involve other known N-acetyl transferases, since they could not rescue the naa20-cr1 mutant. Polyubiquitination levels were lower in the mutant, suggesting that NatB destabilizes the plant proteome, but the authors did not find strong evidence that NatB regulates the proteome by modulating the stability of all NatB substates. Indeed, of six NatB substrates tested, only NBR1 (a selective autophagy receptor) was stabilized in naa20-cr1.\nImportantly, the authors demonstrated that loss of NatB function in the naa20-cr1 line results in decreased ubiquitin proteasome activity and decreased protein translation. They then explored whether lowered proteasome activity could be caused by enhanced autophagy and provided evidence that NatB-deficient plants display higher autophagic flux, indicating that NatB represses autophagy. This is contrary to the situation in yeast, where NatB is important for autophagy induction. They discovered that NatB-mediated NTA specifically destabilises KIN11, a subunit of the energy sensor kinase, SnRK1. Intriguingly, the closely related subunit, KIN10 was not regulated in the same way. Taken together, the data provide evidence for NatB as a central regulator of UPS-autophagy interplay. Once thought of a discrete processes, it has become increasingly evident that the autophagy and the UPS are linked but the underlying mechanisms have largely remained obscure. Therefore, this study represents an important advance in proteostasis.\nIssues to address:\nThe fact that other NATs don’t rescue naa20-cr1 does not necessarily mean that they don’t contribute to the residual NTA activity in the mutant. Perhaps they provide the residual activity but are not active towards the substrates that lack acetylation in naa20-cr1? Potentially this could be tested using double mutants although the combinations could be deleterious. At least this possibility could be discussed.\nAlthough NatB does not appear to control stability of RPN12a and V-ATPase (Fig. 3F), they do have reduced NTA according to Table S2. Perhaps this could be mentioned?\nAGI codes are provided in Table S3; please could the annotations be added?\nLine 202: please clarify whether the six NatB substrates are predicted substrates or whether they have been validated previously.\nThe manuscript contains several quantified western blots. Whilst several use 3-4 technical replicates, it is not clear whether these experiments have been repeated. Other experiments seem to have little replication: e.g. in Fig. 4A the K63 blot presented only has one lane per sample; similarly, in Fig. 5G and H, only two replicates are presented, and the loading control looks quite uneven. Have these experiments been repeated? Overall, it was not clear to me how many independent quantification experiments have been performed.\nAltered translation is presented at the beginning of the manuscript but is not touched upon in the Discussion. It would be helpful to revisit this. Also, it is mentioned that the potential effect of NatB-regulation of KIN11 activity on TOR kinase remains to be investigated but it would be interesting and relatively straightforward to do a preliminary test with phosphorylation of ribosomal protein S6 (RPS6) as a readout (western with anti-P-RPS6 antibody).\nPlease check the labelling on Fig. S6F\nMethods: please provide dilutions for all antibodies.", "reviewer_2": "This study by Gong et al is focussed on assessing the role of NATB mediated N-terminal acetylation (NTA) on protein stability and overall proteostasis (via the UPS and autophagy) in Arabidopsis. NTA is a widespread modification that has received increased attention in recent years as more and more functions have been ascribed, many by the authors of this study. The work here shows hat NATB influences global protein turnover through a combination of the creation of so-called Ac/N-degrons in certain target proteins (e.g. NBR1 and KIN11, where the data are convincing) and through enhancing autohpagy. This is in keeping with some prior reports in yeast and animals connecting NTA to protein destabilisation, but contrasts with findings for NATB substrates in drosophila, and indeed with the effects of NATA-mediated NTA on protein fate in plants. As such the study adds significantly to our knowledge of the intriguing and varied effects of NTA on protein stability, and will be of broad interest to the community. It also provides interesting new insights into the cross talk between UPS and autophagy pathways, and how NTA-mediated control of Kin11 stability, part of the SnRK1 complex, may orchestrate this.\nThe paper is well written and overall the data/figures are presented in an accessible manner. I have a few comments and queries, listed here in order of appearance in the manuscript.\nLine 42: consider changing the word “stunning”.\nLine 64: I think the word vital is incorrect here, as this suggests that they are needed, when in fact Arabidopsis is still able to live without them.\nFor completeness, in the introduction when introducing plant-specific NATB studies, the authors should also cite another of their papers that has focussed on assessing roles for NATB in plants (with respect to reductive stress sensitivity) – Huber et al 2022, Frontiers in Plant Science.\nLine 82: Here it might be interesting to comment on the fact that the UBR1 protein is not directly conserved in plants (Arabidopsis), where instead the degradation of proteins bearing type 1 and type2 N-degrons is undertaken by separate, plant specific N-recognins (PRT6 and PRT1, respectively). This might also contribute to kingdom specific differences in the effect of NATB on substrate fate.\nLine143: The meaning of “>20% decrease” could be made clearer here. Do you mean that 22 proteins had a more than 20% reduction, or that they had less than 20% levels of acetylation. It was hard to directly match this statement to any of the figures by eye (e.g., 1F and 1G), so it might be better to spell it out more clearly. Related to this, obviously 22 proteins is a very small amount to base proteome-wide conclusions on, so some extra details of what percentage of the total numer of potential NATB substrates this covers (I.e., 20% of the whole proteome) would be useful.\nLine 157: Please provide some further in-text details of what was actually measured in terms of proteasome stability? Did you measure single or combined protease activities etc. It would be useful to be clearer about this in the main text.\nLine 159: Could the authors comment on why the reduced levels of polyubiquitylated proteins are only apparent upon proteasome inhibition (Fig 2B), when the relative proteasome activity (Fig 2A) is significantly reduced even under non-treated conditions? Also, is this result not paradoxical? If natb mutants have reduced proteasome activity, would one not expect to see enhanced polyubiquitylated proteins compared to WT, due to reduced capacity to degrade them? I realise that later in the paper the authors also examine autophagy, and show elevated K63-linked substrates in natb mutants, but it would be good to add some extra information here.\nLine 171: should that say “more than twice the percentage of stabilsied NATA substrates”?\nLine172: It is interesting that “translation” associated GO terms are up, given that basal translation rates are reduced in the natb. Is this likely linked to an unsuccessful attempt to compensate? Please add some more thoughts/clarification.\nFigure 4A: It would be important to repeat this K63 blot across multiple biological replicates, or (ideally) to show it in different natb mutants alleles(CRISPR and T-DNA), as was done for the K48/proteasome assays in Fig. 3.\nFigures 4B-D: In the text it says the ATG proteins are significantly enhanced in the mutant – were statistics performed on multiple repeats? It might be better to perform a dilution (e.g., 1x, 0.5x and 0.25x) in both the mutants vs WT to see if any differences are clearer for ATG8, ATG3 and ATG5. By eye think the proposed increase looks to only be subtle, so alternatively I would consider rewording. Did you also look at expression levels by qPCR to see if their increase linked is transcriptional or post-translational changes?\nLine 355: This refers to a figure 7E, which is non-existent.\nConclusion: This paper is quite complex with lots of molecular details, and sometimes seemingly contradictory findings. As such, I appreciated the conclusion paragraph, which helped to reiterate and synthesise all the key findings.", "reviewer_3": "This manuscript reports that loss of NATB activity in naa20 mutant reduces proteasomal function, resulting in slower degradation of a subset of proteins in Arabidopsis. The authors further show that autophagy activity is elevated in the naa20 mutant and that NBR1 accumulates due to reduced degradation, although the authors conclude that NBR1 accumulation is not the cause of enhanced autophagy. They also report increased phosphorylation of KIN10 and KIN11 in the naa20 mutant, along with KIN11 protein accumulation, which leads to elevated autophagy and enhanced tolerance to carbon starvation (dark stress). Although the topic is of some interest, the overall novelty is limited, that prevents the manuscript from being suitable for publication in Nature Communications.\nConcerns\n1. Limited novelty due to a similar regulatory pathway well established in mammals. In mammalian cells, NAA20 was previously shown to regulate N-terminal acetylation of LKB1, which is the upstream kinase of AMPK (SnRK1 in plants), and thereby inhibiting activation of the LKB1–AMPK pathway (Experimental & Molecular Medicine, 2020, 1831–1844). Thus, NATB functions as a negative regulator of AMPK signaling. In this study, the authors report a similar mechanism in plants, proposing that NAA20 regulates KIN11 stability via N-terminal acetylation and thereby negatively regulates SnRK1 signaling. Because this regulatory logic has already been established in mammals, the conceptual novelty of this work is limited. Moreover, in plants, NATB may regulate SnRK1 signaling through SnAK2, the upstream kinase of SnRK1. The authors should experimentally test whether NATB affects SnAK2 activity.\n2. In the naa20 mutant, only ~30% of the accumulated proteins are predicted NATB substrates, whereas ~60% are predicted NATA substrates (Fig. 2). Likewise, among proteins with reduced ubiquitination in naa20, ~60% are NATA substrates (Fig. 3B). How do the authors explain that depletion of NATB more substantially affect NATA substrates? Given that most affected proteins are not NATB substrates, performing GO enrichment analysis using the entire set of proteins (Fig. 3C–E) may not be appropriate. I recommend performing GO analysis specifically on the predicted NATB substrates.\n3. The authors conclude that NBR1 accumulates in naa20 due to impaired proteasomal degradation, and that increased autophagy does not counteract this accumulation. However, this cannot be firmly concluded without directly testing NBR1 turnover when proteasome activity is inhibited (e.g., MG132 treatment) or when autophagy is blocked (e.g., ConA treatment). The authors should analyze NBR1 degradation dynamics in naa20 under both/ and either MG132 and ConA treatments.\n4. N-terminal acetylation can influence not only protein stability but also subcellular localization and protein–protein interactions. Does loss of N-terminal acetylation alter NBR1 localization or its interaction with ATG8?\n5. The confocal images in Fig. 4E include excessively large fields of view, making autophagosomes difficult to identify. Higher-magnification images are needed. Additionally, in the WT Dark + ConA (6 h) panel, two central columns of cells show no autophagosomes, which contradicts previously published studies. Moreover, the authors should perform a rigorous GFP-cleavage assay to accurately quantify autophagic flux during a time-course of carbon starvation treament.\n6. Both KIN10 and KIN11 are predicted NATB substrates, and both show increased phosphorylation in the naa20 mutant. However, only KIN11 protein accumulates. What accounts for this striking difference? How do variations in the N-terminal sequences of KIN10 and KIN11 lead to such divergent outcomes? Additionally, how does loss of N-terminal acetylation influence phosphorylation levels of these kinases?\n7. Are the expression levels of known KIN11 target genes increased in naa20? This would demonstrate functional activation of KIN11.\n8. Is KIN11 a direct NATB substrate? Is it possible to perform in vitro acetylation assays to directly test whether KIN11 is acetylated by NATB?\n9. Do KIN10 and KIN11 undergo ubiquitin-mediated degradation? If so, how does KIN11 N-terminal acetylation affect its ubiquitination and turnover?\n10. Multiple studies have shown that KIN10 is the major catalytic subunit of SnRK1 protein kinase complex, and kin10 mutants are hypersensitive to abiotic stresses. Why does kin10 not display stress-sensitive phenotypes in this study?\n11. What are the weak protein bands observed in the WT sample in Fig. S1B?\n12. Because proteasome activity is reduced in naa20, one would expect accumulation of ubiquitinated proteins under normal conditions. Why is this not observed?\n13. Does NBR1 accumulate in the proteomics dataset of the naa20 mutant? This should be reported explicitly.\n14. Figures 4A–D show levels of autophagy-related proteins, but the treatment conditions are not specified. These must be clearly stated in the figure legends or main text.\n15. Given the increased autophagy activity in naa20, do these plants exhibit delayed senescence, particularly under short-day conditions?\n16. The statement “only three point 7 (3.788) proteins were identified” should be a typographical error."}, "author_response": "Response: We agree with the reviewer that this is a possible scenario, which we have discussed in a new paragraph in the Discussion section (Lines 415-432).\nResponse: As suggested by reviewer 1, we have rephrased the corresponding statements in the improved manuscript version and highlighted this relevant information and its consequence for the regulation of protein stability by N-terminal acetylation of NatB substrates (Lines 207-209).\nResponse: We thank the reviewer for carefully reading our manuscript and for this helpful suggestion. The annotations have been added to Supplementary Table 3 and Supplementary Table 4.\nResponse: Among these six NatB substrates, V-ATPase and RPN12a have been experimentally confirmed in our study using a proteomics approach (Supplementary Table 2), whereas the other four are predicted NatB substrates based on their sequences. We have rephrased the corresponding sentences and added the information in Lines 205-209. The identity of NBR1 as a NatB substrate was later experimentally confirmed by in vitro enzymatic assays using immunoprecipitated plant NatB (Supplementary Fig. 4b, Lines 216-221).\nResponse: All experiments were repeated at least three times. In most cases, each independent repetition included multiple replicates from different individuals except for some IP experiments (e.g., Fig. 4a) or when too many genotypes needed to be investigated (e.g., Fig. 5g, h). We have specified the number of replicates throughout the figure legends and added the information in the “Source Data File”. For Fig. 4A, we have prepared a new graph showing the quantification of three independent repetitions with one replicate and added the calculated p-value. The immunoblots of repetition 2 and 3 are included in the Source Data file. The blot shown in the manuscript represents the blot that is closest to the calculated mean value for the difference between K63-polyubiquitination in naa20 mutants and the wild type.\nResponse: We appreciate the reviewers' comments and suggestions. As suggested, we detected TOR activity in the natb mutants by monitoring the ratio of p-RPS6 to RPS6 levels as a readout (New Supplementary Fig. 6e). The results revealed reduced p-RPS6 levels in the natb mutants compared to the wild type, suggesting a lower TOR activity, which might help explain the generally lowered translation rate in the natb mutants (Lines 332-335). These novel findings are discussed in a new paragraph of the Discussion section, highlighting the NatB depletion-induced changes in translation (Lines 492-495).\nResponse: We thank the reviewer for carefully reading the manuscript. The mistake in the description of the x-axis has been corrected (New Supplementary Fig. 6h).\nResponse: This information has been added to the improved version of the manuscript (Lines 603-616).\nResponse: We appreciate the reviewers' suggestion. The word “stunning” has been replaced by “increased” (Line 42).\nResponse: The word “vital” has been replaced by “viable” (Line 60).\nResponse: We appreciate the reviewers' suggestion. The reference has been added (Line 61).\nResponse: We appreciate the reviewers' suggestion and added the proposed information and references to Lines 77-80.\nResponse: We thank the reviewer for pointing this out. We meant that the NTA level of these proteins was more than 20% lower in naa20 mutants than in the wild type. We have rephrased the corresponding sentences to make this statement more straightforward (Lines 137-142). In fact, we have identified only the N-termini of 33 NatB substrates, whose NTA levels are more than 20% lower in naa20 mutants, out of the potential 8948 NatB substrates of the whole proteome. This discrepancy arises because we need to identify and quantify the acetylated and the non-acetylated N-terminal peptides for a given protein in one of the replicates for each genotype to determine the N-terminal acetylation level of the respective proteins. Indeed, the number of quantified protein N-termini is low, but the method used here is the current state-of-the-art.\nResponse: We apologize that his information was hidden in the material and method section. We have determined the chymotrypsin-like protease activity of the proteasome as a proxy for the overall proteasome abundance. In this assay, the extractable proteasome activity in the soluble protein fraction is determined by monitoring the release of fluorescent AMC after the proteasome cleaves the model substrate Z-Leu-Leu-Leu-AMC. We have rephrased the corresponding sentence and added the relevant information in the result text (Lines 154-157).\nResponse: This is because, in addition to the relative proteasome activity, the endogenous polyubiquitination rate is decreased in natb mutants. As a consequence, the steady-state polyubiquitin level of the proteome in the WT and natb is comparable. Please note that proteasome inhibition also results in an increase of poly-ubiquitinated proteins in natb mutants, but this increase is lower than in the wild type. Thus, natb mutants possess a lower endogenous polyubiquitination rate. One of our claims is that this lowered endogenous polyubiquitination rate is caused by reduced activity of N-recognins in the Ac/N-degron pathway, as these N-recognins do not recognize the non-acetylated NatB substrates in natb mutants.\nResponse: This assumption would be correct if the translation rate and the E3-ligase activity (defining the endogenous polyubiquitination rate) in natb mutants were not affected. We show in Fig. 2a that natb mutants exhibit lower extractable proteasome activity, and at the same time, a lower global translation rate (Fig. 2d). Since global degradation and global translation in the natb mutant are decreased, the steady-state proteome level (Fig. 2e) is almost unaffected, and the “global amount” of ubiquitinated proteins is not affected (Fig. 2c, compare WT DMSO with naa20 DMSO). Furthermore, the accumulation of ubiquitinated proteins also depends on the endogenous E3 ligase activity. We claim that lowered NTA of NatB substrates causes less ubiquitination of NatB substrates. Even if the proteasome activity is decreased, polyubiquitinated proteins should only accumulate in naa20, if these proteins are translated and ubiquitinated with the same rate. Both translation (Fig. 2d) and global endogenous polyubiquitination rate (Fig. 2c, now compare the difference between DMSO and MG132 in the wild type versus the difference between DMSO and MG132 in naa20) are decreased in naa20. As a consequence, we do not observe a significant accumulation of polyubiquitinated proteins in naa20.\nResponse: We apologize for failing to explain this comparison in the previous version of the manuscript. In the wild type, only 16 % of stabilized proteins were NatB substrates. In the natb mutant, 30 % of stabilized proteins are NatB substrates. This is almost twice the percentage of NatB proteins stabilized in the wild type, and strongly suggests that, in general, NatB substrates tend to be more stable in natb mutants. The improved version of the manuscripts provides more information, hopefully making this comparison more straightforward to understand (Lines 169-174).\nResponse: We greatly appreciate the reviewers’ helpful comments to improve the manuscript´s readability and have added this possibility to the results section (Lines 178-179).\nResponse: We fully agree with the reviewer about the importance of independent repetitions. The improved Figure 4A now shows the results from three independent replicates. A representative Western blot is shown in addition to allow the reader to evaluate this assay. The complete blots for the other replicates have been shown in the manuscript's Source Data file.\nResponse: We apologize for the potentially unclear statement in the previous manuscript version. Yes, the experiments have been independently repeated and always showed an increase of ATG-related proteins in the natb mutants. We have specified the number of replicates in the figure legends and added the p-values for the exemplary shown repetitions, which included four independent biological replicates, to the “Source Data File”. For ATG8, we quantified the lipidated ATG8 (ATG-PE), a post-translationally lipidated proteoform of ATG8 covalently conjugated to phosphatidylethanolamine. This ATG8 proteoform is essential for autophagosome membrane expansion. In case of ATG5, we quantified the amount of ATG5 bound in the ATG5-ATG12 conjugated complex amount, which is essential for autophagosome formation by acting as an E3-like enzyme that promotes ATG8 lipidation.\nResponse: As suggested, we performed qPCR on the autophagy-related genes tested here. We did not find a significant increase in the steady-state transcript levels of these genes in naa20cr1, suggesting that the rise in ATG proteins is caused by post-transcriptional regulation or post-translational modifications. The novel results are shown in Figure 4F and described in the results text (Lines 272-274).\nResponse: We thank the reviewer for carefully reading our manuscript and pointing out this mistake. Indeed, we wanted to refer to Supplementary Fig.7c and Supplementary Fig.7d. This mistake is corrected in the improved manuscript version.\nResponse: We thank the reviewer for appreciating our efforts to improve the readability of this complex manuscript.\nResponse: We thank the reviewer for the opportunity to reaffirm the novelty of our work. We agree with the reviewer that NatB-mediated regulation of energy-sensing kinases has been previously reported in the mammalian system via the LKB1–AMPK–mTOR axis [1]. However, we would like to emphasize that (1) Although LKB1 was identified as a substrate of NatB in vitro, Jung and coworkers failed to confirm the decrease in LKB1 acetylation level in NatB-depleted mammalian cells. Thus, a direct action of NatB on LKB1 is still questionable. Furthermore, the authors do not even propose a direct action of NatB on the catalytic AMPK subunits [1], as we did. As a consequence, their proposed mechanism for NatB-mediated AMPK regulation is fundamentally different from the regulation we propose for SnRK1 by NatB in plants. (2) Human AMPK is fundamentally different from plant SnRK1 in its regulation. In contrast to the AMP-activated Protein Kinase (AMPK), SnRK1 is not activated by AMP at all. Plant SnRK1 senses Trehalose-6-P levels and redox changes [2, 3], which is explained by the substantially different motif composition in the C-terminal located regulatory domain of the catalytically active α-subunits of SnRK1 when compared to AMPK. On top of these differences, the regulatory β subunit of AMPK is replaced by the βγ-subunit in the plant SnRK1 complex [4]. Thus, one would not expect a conserved mechanism for regulating AMPK/SnRK1 in eukaryotes between the red and green lineages. (3) In yeast, NatB is essential for autophagy; thus, natb yeast mutants are deficient in autophagy [5]. In stark contrast, NatB depletion in humans induces autophagy, potentially via the proposed LKB1-AMPK axis [1]. Given the fundamental differences in AMPK1 and autophagy regulation between fungi, animals, and plants, we do not believe that the study by Jung et al. undermines the novelty of our manuscript. In contrast to Jung et al. and Shen et al., we demonstrate that NatB inhibits autophagy in plants by providing direct evidence that the absence of NTA of KIN11, a subunit of the energy sensor SnRK1, protects it from UPS-mediated Ac/N-degron pathway. Our findings not only identify the role of NTA in regulating autophagy in plants for the first time but also establish NatB as a central regulator of UPS–autophagy interplay. In addition to this finding, we demonstrate, for the first time, that NatB controls general protein turnover in a eukaryote by imprinting a destabilizing mark on a subset of its substrates. We strongly believe that these findings are of significant interest to the broad readership of Nature Communications. We have added a new paragraph in the Discussion section to summarize our key findings (Lines 486-498).\nResponse: The N-terminus of LKB1 is highly conserved in humans and animals. Its sequence suggests that LKB1 is a NatB substrate as it starts with ME [1]. In stark contrast, the only known upstream kinases of SnRK1 in plants, SnRK1 activating kinase 1 and 2 (SnAK1/2), exhibit an N-terminus (MF, see Response Fig. 1) that is not accepted by NatB in plants [6], humans [7], or yeast [8]. Whether NatB affects SnAK1/2 activity through a mechanism other than direct acetylation is an interesting question, but it is far beyond the scope of this study.\nResponse: We understand the reviewer's concerns regarding the number of stable NatA substrates in natb mutants. In this context, it is important to mention that NatA substrates are often stable and abundant housekeeping proteins (e.g., the cysteine synthesizing protein OAS-TL A [9]). For that reason, they are frequently overrepresented in quantitative proteomics studies. In our quantitative proteomics approach 61% of all identified proteins in the wild type are NatA substrates (Figure 2E). NatA acetylates only 40% of the proteome based on predictions of substrate N-termini encoded in the genome. The reviewer is correct that the number of affected NatA substrates in natb mutants is high. However, the percentage of stable NatA substrates is substantially lower in natb mutants (52%) than in the wild type (68%). This decrease of NatA substrates is caused by the higher number of stable NatB substrates in natb mutants. The number of non-NatB substrate proteins is still high because NatA substrates are, in general, stable proteins in the wild type. Moreover, the Ubi capture followed by mass spectrometry analysis clearly demonstrates that NatB destabilizes the plant proteome. In this approach, we found that out of 314 significantly changed polyubiquitinated proteins, 310 (99%) were more ubiquitinated in the wild type than in naa20-cr1 (Fig. 3a and Supplementary Table 4). These results suggest that NatB may not only regulate the plant proteome by modulating the stability of individual NatB substrates, but also indirectly by modulating the UPS. In line with this hypothesis, 42% of the identified proteins in the enriched GO term “ubiquitin-dependent protein catabolic process” are NatB substrates (Fig. 3e). This finding suggests that NatB substrates play a critical role in controlling protein homeostasis. To explain this apparent discrepancy, we have added a more comprehensive description and discussion of these findings in the results section (Lines 200-204, 456-461).\nResponse: We thank the reviewer for this suggestion. In fact, we thought along the same lines. We performed this GO analysis on the predicted NatB substrates right at the beginning, but were hampered by the technical difficulty that the number of NatB substrates (49) was too low to yield meaningful Gene Enrichment Ontology results. The algorithm needs a certain number of proteins to find pathways in which these proteins are enriched. Therefore, we performed GO analysis on all identified proteins because this approach allowed us to address how NatB affects the composition of the overall plant proteome.\nResponse: We thank the reviewers for this interesting suggestion for strengthening our claim. As suggested, we analyzed NBR1 degradation dynamics in naa20-cr1 under both/ and either MG132 and ConA treatments. When protein synthesis was inhibited by Cycloheximide (CHX) for 6 hours, the steady-state level of NBR1 dropped to 45% of the control level. Both MG132 (78%) and ConA (60%) alone partially inhibit NBR1 degradation, with MG132 exhibiting a more pronounced effect. When MG132 and ConA were applied together, NBR1 degradation was inhibited entirely (106%, Supplementary Fig. 4e). These findings indicate that NBR1 is degraded via both the UPS and autophagy pathways in vivo. Under the non-stressed conditions applied here, in which autophagy is not required for nutrient recycling, NBR1 turnover was slightly more dependent on the UPS. We report these findings in the result text (Lines 233-240) and Supplementary Fig. 4e.\nResponse: We agree with the reviewer that N-terminal acetylation can influence not only protein stability but also subcellular localization and protein–protein interactions. In the revised manuscript, we investigated NBR1 localization using stable transgenic NBR1-mCherry-GFP/WT and NBR1-mCherry-GFP/naa20-cr1 lines. In wild-type root tip cells, NBR1 exhibits a cytosolic, punctate localization pattern consistent with previous studies [10]. In the naa20-cr1 mutant, the localization pattern of NBR1 remains unchanged, indicating that the absence of NTA does not affect NBR1 localization (new Supplementary Fig. 4g). To investigate whether loss of N-terminal acetylation alters the interaction between NBR1 and ATG8, we performed a Co-IP assay using stable NBR1-HA/WT and NBR1-HA/naa20-cr1 transgenic plants. NBR1 still interacted with ATG8 in naa20-cr1, suggesting that the absence of NTA does not affect the interaction between NBR1 and ATG8 (new Supplementary Fig. 4h). This is consistent with the established concept that NBR1 interacts with ATG8 via its AIM (ATG8-interacting motif) domain located in the C-terminal region of NBR1. These novel results are included in Supplementary Fig. 4g and Supplementary Fig. 4h and reported in (Lines 245-255).\nResponse: We apologize for the excessively large fields of view images and the misleading image of the WT Dark+ConA in the previous manuscript version. We included higher-magnification images in the improved manuscript version, replacing the previous WT Dark+ConA image. The results presented in these higher-quality images are consistent with previous studies. Regarding the assessment of elevated autophagy, we have included six independent assays to quantify autophagy-induction in naa20: (1) Phenotypic analysis of nutrient-starved or elongated darkness-treated WT, naa20, and naa20atg double mutants (Figure 5); (2) Confocal laser scanning microscopy-based analyses of the GFP ATG8a marker in WT, naa20, and naa20atg5 backgrounds (Figure 4G); (3) Immunological detection of ATG3 protein levels in WT and natb mutants (Figure 4D); (4) Demonstration of enhanced ATG8 lipidation in natb mutants (Figure 4C); (5) Quantification of ATG5-ATG12 complex formation in WT and natb mutants (Figure 4E); (6) In addition, we showed increased K63-polyubiquitination levels in naa20-cr1 (Figure 4A). Therefore, we believe that the conclusion that autophagy is induced in natb mutants is supported by a sufficient number of independent, accepted assays outlined in the “Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)” and the review method paper ”Studying plant autophagy: challenges and recommended methodologies” [11, 12].\nResponse: We thank the reviewer for the opportunity to elaborate on this indeed striking difference. The team of authors was also surprised by this difference before we carefully evaluated the primary sequence of KIN10 and KIN11. Both proteins are almost identical, with only a few sequence differences, clustered in three domains. One of these domains is the N-terminus (See Supplementary Fig. 6f and below). The substantially different amino acid sequence (Residues 3 to 11) in this domain most likely evolved to enable the differential degradation of both isoforms by the Ac/N-degron pathway, in which specific E3-ubiquitin ligases (N-recognins) recognize specifically the N-terminus. N-recognins have been shown to distinguish between non-acetylated and acetylated N-termini and are highly selective for the amino acid sequences at the N-terminus (reviewed in [13]). Consequently, the substantial changes at the N-terminus of both KIN isoforms will cause recognition by different N-recognins, or only recognition of KIN11 but not KIN10 by a so far unidentified N-recognin. An example in which KIN11 is specifically degraded while KIN10 remains stable is the phosphate starvation response [14].\nResponse: How variations in the N-terminal KIN10/11 sequence can interfere with the selective binding to the N-recognin depends on structural features of the N-terminus and the N-recognin [15], which we cannot predict without knowing the responsible N-recognin. However, examples of specific binding motifs that depend on the addition of only 42 Da (acetyl group) at the N-terminus are widespread [16-19, reviewed in 20]. It is thus evident that significant changes in the surrounding amino acid sequence will interfere with the selective binding of a substrate to its specific N-recognin [reviewed in 13, 15].\nResponse: This is an interesting question that we will address in a separate study with a new PhD candidate, as it is outside the scope of this study. This comprehensive study aims to understand the function of NatB in global protein turnover and demonstrates that the NTA of several NatB substrates destabilizes them via the Ac/N-degron pathway. It furthermore connects the NTA-dependent differences in the stability of the two SnRK1 isoforms to autophagy induction and diverse stress responses.\nResponse: As suggested, we examined the expression levels of DIN1, DIN6, DIN10, and bZIP63, a transcription factor phosphorylated by SnRK1 that regulates transcription of the aforementioned DIN genes. The expression levels of DIN1, DIN6, and DIN10 were significantly elevated in the naa20 mutant, while bZIP63 expression remained unchanged. These findings suggest that increased SnRK1 activity in naa20 activates the expression of downstream energy-related genes. The absent transcriptional induction of bZIP63 suggests that bZIP63 activation occurs at the post-translational level (e.g., phosphorylation by stabilized KIN11) rather than the transcriptional level. We have included these results in the revised manuscript (Lines 327-332 and Supplementary Fig. 4d).\nResponse: We agree with the reviewer that it is crucial to show that KIN11 is a direct substrate of NatB. For that reason, we showed that immune-precipitated plant NatB acetylates the N-terminus of KIN11 in vitro(Fig. 6b). In addition, we quantified the N-terminal acetylation level of KIN11 by quantitative mass spectrometry of immune-precipitated KIN11-HA expressed in the wild type and naa20-cr1. We found that N-terminal acetylation of KIN11 is substantially reduced in naa20-cr1 when compared to wild type (old Figure S6E, F -> now Supplementary Fig. 6g, h). The transcript level of KIN11 was not affected in naa20-cr1 (old Figure S6G -> now Supplementary Fig. 6i).\nResponse: In the original manuscript, we showed that KIN11-GFP is polyubiquitinated in the wild type and that the degree of polyubiquitination is significantly lower in naa20-cr1(Fig. 6g). Furthermore, we inhibited N-terminal acetylation of KIN11-GFP expressed in the wild type by genetic engineering. The mutagenized KIN11D2P protein was substantially less poly-ubiquitinated in the wild type (Fig. 6g), strongly suggesting that the lowered degradation of KIN11 in naa20 (Fig. 6e) is caused by its lowered N-terminal acetylation status. In agreement with this claim, the non-acetylated KIN11D2P was substantially stabilized in the wild type (Fig. 6f). To provide additional evidence for UPS-mediated degradation of KIN11, we performed cycloheximide chase assays in the presence and absence of the proteasome inhibitor MG132. In the absence of MG132, KIN11 was quickly degraded. Inhibition of the proteasome by MG132 entirely inhibited the degradation of KIN11 in the wild type. We have included these results in the revised manuscript (Lines 357-358, Supplementary Fig. 6j).\nResponse: NTA of certain proteins has been reported to create specific signals for degradation via a particular branch of the ubiquitin proteasome system (UPS), termed the Ac/N-degron pathway, in which the Ac/N-degron is recognized by a specific E3 ubiquitin ligase termed N-recognin [16-19, reviewed in 20]. In our study, we demonstrated that KIN11 is a substrate of NatB both in vivo and in vitro. Our results indicate that N-terminally acetylated KIN11 is labelled for UPS-mediated degradation by an unidentified plant E3 ligase. This conclusion is further supported by showing that engineered KIN11 lacking N-terminal acetylation (KIN11D2P) exhibits a reduced degradation rate in the wild type, suggesting that N-terminal acetylation of KIN11 promotes its degradation.\nResponse: Indeed, KIN10 is suggested to be the major SnRK1 protein kinase in plants, because the KIN10 promoter is broadly active in all tissues, and the KIN11 promoter activity is highly active in generative organs [21]. Furthermore, immunoprecipitation assays using anti-SnRK1.1/SnRK1.2 antibodies revealed that complexes containing SnRK1.1 (KIN10) contribute approximately 90% of the total AMARA kinase activity. In comparison, complexes containing SnRK1.2 (KIN11) contribute only approximately 10% in crude extracts of A. thaliana cell cultures [22]. While using the same strategy and the same AMARA peptide, SnRK1 activity was assayed in the supernatant of seedling extracts after immunoprecipitation with antibodies to AKIN10 and AKIN11, and it was found that AKIN10 antibody removed 57.1% of SnRK1 activity, and AKIN11 antibody removed 38.3% of SnRK1 activity [23]. The latter finding suggests that KIN11 is more critical in Arabidopsis than previously thought. Moreover, based on the foundational research by Baena-González et al. and the latest transcriptomic analyses [24], KIN11 also exhibits very high expression levels in plants, if not comparable to those of KIN10 [25]. Consistent with this, both KIN10 and KIN11 single mutations display no phenotype under normal conditions, while double mutations are lethal, indicating functional redundancy between them [25]. Regarding the phenotype of the KIN10-depleted lines, depletion of KIN10 activity was reported to show no obvious phenotype after darkness, nitrogen starvation, drought, and submergence treatments [26]. In the initial study on SnRK1, Baena-González et al. demonstrated that soil-grown kin10-1 and kin10-2 lines did not show an obvious phenotype, whereas kin10 kin11 VIGS double-mutant (d-1, d-2) plants arrest growth and exhibit premature senescence [25]. In another study, the kin10kin11 sesqui mutant exhibited a dark-sensitive phenotype [27]. To the best of our knowledge, the kin10 mutant did not show sensitivity under dark conditions, and the kin11 single mutant has not been investigated previously. Ectopic overexpression of KIN10 confers resistance to drought stress [26], but a KIN11-overexpressing plant has not been tested in this study. Thus, we cannot conclude that this is a specific function of KIN10. In our study, we investigated the resistance to darkness in kin10 and kin11 single mutants and found that only kin11, but not kin10, was more sensitive to darkness treatment (Supplementary Fig. 7a, b). To investigate whether the elevated KIN11 protein level is responsible for the increased darkness resistance of naa20-cr1, we crossed naa20-cr1 with kin10 and kin11. Remarkably, the darkness resistance of the naa20-cr1 kin11 double mutant was attenuated compared with that of naa20-cr1. In contrast, inactivation of KIN10 in the naa20cr-1 kin10 double mutant did not affect the darkness resistance of naa20cr-1 (Fig. 7a, b). These results suggest that although the functions of KIN10 and KIN11 are largely redundant, the absence of KIN11 specifically impairs plants' resistance to darkness. Indeed, previous studies have also revealed functional differentiation between KIN10 and KIN11 in plants: (1) KIN10 is broadly expressed, while KIN11 expression is more pronounced in specific tissues and developmental stages [21]. (2) While gain-of-function transgenic plants overexpressing two different isoforms of SnRK1.1 flower late, SnRK1.2 overexpressors flower early. In addition, SnRK1.2 overexpressors have increased leaf size and rosette diameter during early development, which is the opposite of SnRK1.1 overexpressors. (3) SnRK1 isoforms KIN10 and KIN11 are differentially regulated in Arabidopsis plants under phosphate starvation. Upon phosphate limitation, KIN11 is destabilized. The KIN10 protein remained stable [14]. (4) KIN10, but not KIN11, can restore flowering and embryogenesis in the Arabidopsis trehalose 6-phosphate synthase1 (tps1) mutant [28]. (5) In maize, only ZmSnRKα2 (ZmKIN11), but not ZmSnRK1α1 nor ZmSnRK1α3, can interact with ZmWAK to regulate nutrient availability to defend against head smut disease [29].\nResponse: The weak protein signals observed in the WT samples are endogenous plant proteins that are recognized by the commercial GFP antibody (ChromoTek, Pabg1-100).We have attached the uncropped WB results below and uploaded them to the “Source Data File”.\nResponse: This assumption would be correct if the translation rate and the E3-ligase activity (defining the endogenous polyubiquitination rate) in natb mutants were not affected. We show in Fig. 2a that natb mutants exhibit lower extractable proteasome activity, but at the same time, they also exhibit a lower global translation rate (Fig. 2d). Since global degradation and global translation in the natb mutant are decreased, the steady-state proteome level (Fig. 2e) is almost unaffected, and the “global amount” of ubiquitinated proteins is not affected (Fig. 2c, compare WT DMSO with naa20 DMSO). Furthermore, the accumulation of ubiquitinated proteins also depends on the endogenous E3 ligase activity. We claim that lowered NTA of NatB substrates causes less ubiquitination of NatB substrates. Even if the proteasome activity is decreased, polyubiquitinated proteins should only accumulate in naa20, if these proteins are translated and ubiquitinated at the same rate. Both translation (Fig. 2d) and global endogenous polyubiquitination rate (Fig. 2c, now compare the difference between DMSO and MG132 in the wild type versus the difference between DMSO and MG132 in naa20) are decreased in naa20. As a consequence, we do not observe a significant accumulation of polyubiquitinated in naa20.\nResponse: NBR1 has not been identified in the quantitative proteomics approach, likely due to its low protein abundance under normal conditions. This information is added in (Line 214).\nResponse: We apologize for not clearly reporting the exact treatment conditions. In this case, we immunologically detected proteins in the total protein fraction extracted from leaves of 6-week-old wild type, naa20-cr1, and naa25-1 plants grown in soil under short-day conditions. We hope that the improved figure legend now contains all the necessary information to evaluate the obtained results.\nResponse: We thank the expert reviewer for this helpful suggestion. As expected by the reviewer, the natb mutants exhibit delayed senescence after 10 weeks of growth in short-day conditions, followed by a 2-week transfer to long-day conditions. The senescent phenotype is consistent with the increased autophagy activity in natb mutants. We have added these novel data to Supplementary Fig. 5a and report them in the result section (Lines 289-291).\nResponse: We thank the reviewer for carefully reading our manuscript and highlighting this error. We have corrected this statement (Line 168)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am satisfied that the authors have addressed my comments. They have also provided several pieces of additional data to address the comments of the other reviewers.", "reviewer_2": "I am satisfied with the revised version of the manuscript submitted to Nature Communications. The authors have adequately addressed all of my queries and comments.", "reviewer_3": "The authors have made an effort to address the previous concerns. However, several critical issues remain regarding the interpretation of the autophagy data and the genetic evidence supporting the KIN11 pathway. The following points should be addressed to improve the quality of the manuscript:\n1. The authors conclude that autophagy is upregulated in the naa20 mutant based on increased ATG8 lipidation, ATG5-ATG12 conjugation, and the presence of autophagic bodies. However, these markers can be misinterpreted if autophagic flux (the complete degradation process) is not properly distinguished from the mere accumulation of autophagosomes.\n(1) ATG8 Lipidation: Increased levels of ATG8-PE are often observed in mutants where autophagosome-vacuole fusion is disrupted (reduced flux), not just when autophagy is induced. Furthermore, the protein bands in Figure 4c are ambiguous; the lower faint band may not represent the lipidated form. The authors must include an atg5 or atg7 mutant (deficient in ATG8-PE formation) as a negative control to definitively identify the ATG8-PE band.\n(2) ATG5-ATG12 Conjugation: In plant systems, it is established that most endogenous ATG5 exists in the conjugated (ATG5-ATG12) form (see PMC3967041). In Figure 4e, the majority of ATG5 appears as a free protein. This contradicts established literature and requires a technical explanation or re-validation.\n(3) Confocal Imaging and Vacuolar Localization: The \"dots\" identified as autophagic bodies in Figure 4g appear to be localized in the cytoplasm rather than within the vacuole. If these structures are cytoplasmic, it suggests a block in trafficking/fusion, implying decreased rather than increased flux. The authors should redo confocal analysis using root elongation cells treated with Concanamycin A to stabilize autophagic bodies within the vacuole, and take images to demonstrate the vacuolar autophagic bodies following the procedures shown in some previous publications (e.g., Zhao et al., J Cell Biol, 2022; Stephani et al., eLife, 2020). Moreover, to provide definitive proof of autophagic flux, a standard GFP-cleavage immunoblotting assay should be performed.\n2. The authors claim that the kin11 single mutant is sensitive to dark treatment and posits that the dark-tolerant phenotype of naa20 is due to elevated KIN11 (but not KIN10) activity. To strengthen this causal link, the authors should provide evidence from gain-of-function lines. Have the authors tested the dark-tolerance phenotypes of KIN11-OE or KIN11(D2P)-OE (phosphomimetic) overexpression plants?"}, "author_response": "Response: We greatly appreciate the reviewer’s valuable evaluation and constructive suggestions, which have significantly improved the manuscript.\nResponse: We greatly appreciate the reviewer’s valuable evaluation and constructive suggestions, which have significantly improved the manuscript.\nResponse: We thank the expert reviewer for the critical interpretation of autophagic flux assessment and for providing detailed revision suggestions. As suggested, we added an atg5-1 mutant as a negative control to provide additional evidence for the identity of the ATG8–PE signal. Absence of the proposed ATG8-PE signal in atg5-1 mutant clearly demonstrates that this signal indeed corresponds to ATG8–PE, which accumulates in the natb mutants compared with the wild type (Fig. 4c, lines 263-264, also see Response Fig. 1). In this experiment, total proteins were extracted using NuPage sample buffer (150 mM Tris-HCl, pH 8.5, 2% LDS, 1 mM EDTA, 10% [v/v] glycerol, 0.22 mM Coomassie Blue G250, 0.166 mM Phenol Red, and 50 mM DTT) and subsequently separated by Tricine–SDS-PAGE (Schägger, Nature protocols, 2006), which enables us to distinguish the different ATG8 variants.\nResponse: We thank the reviewer for pointing out the potentially misleading interpretation of the Western blot. The reviewer is correct that most endogenous ATG5 exists in the conjugated ATG5–ATG12 form. This signal is indicated by a red arrow in the figure. The lower signal shown in the uncropped version of the previous manuscript was nonspecific (seeResponse Fig. 2A) and was therefore not labelled as free ATG5 in the resubmitted manuscript (seeResponse Fig. 2B -> old Figure 4E). To avoid misinterpretation, we will show only the specific signal for the ATG5-ATG12 complex in the revised version of the manuscript (revised Fig. 4e; included here as Response Fig. 2C). We did not detect any signal for free ATG5 with the ATG5 antibody (Agrisera, AS15 3060) used here.\nResponse: We appreciate the reviewer's careful assessment and detailed experimental instructions. We have repeated the confocal analysis following the procedures described in Yang et al., Mol. Plant, 2023, Zhao et al.,J Cell Biol, 2022and Stephani et al., eLife, 2020. The confocal images clearly showed that after Concanamycin treatment, autophagic bodies appeared in the vacuole of elongated root cells in the wild type and the naa20-cr1 plants (Fig. 4g, also seeResponse Fig. 3).\nResponse: As suggested, we have performed a standard GFP-cleavage assay to assess the impact of darkness stress on the autophagic flux in the wild type and the naa20-cr1 mutant. To this end, we suppressed autophagy as much as possible in both genotypes by growing seedlings on ½ MS medium plates containing 1% sucrose under long day conditions for five days, followed by 24 hours of continuous light (time point 0). Next, the wild type and naa20cr-1 mutant were subjected to darkness for up to 12 hours as described in Yang et al., Mol. Plant, 2023. Darkness treatment caused a substantially stronger induction of GFP release from GFP-ATG8a in the naa20-cr1 when compared with the wild type as indicated by the higher GFP to GFP-ATG8a ratio in naa20-cr1 at all times of darkness treatment (4, 8, and 12 hours) (seeResponse Fig. 4). These novel results are included in Fig. 4i and reported in Lines 271-276.\nResponse: As suggested by the referee, we have tested the darkness tolerance of two independent KIN11-overexpressing lines. As expected, overexpression of KIN11 in the wild type (KIN11-OE) resulted in higher survival rates of darkness-treated KIN11-OE plants when compared to wild type (seeResponse Fig. 5). This increased survival rate was comparable to that of the naa20-cr1 mutant upon darkness treatment. These novel results are included in Supplementary Fig. 7c, d, and are reported in Lines 379-382."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have made efforts to address my concerns by including new experimental data. I have no more questions and would like to suggest acceptance of this interesting story."}, "author_response": ""}]

Decision letter