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First published online September 12, 2008; 10.1104/pp.108.126714 Plant Physiology 149:220-234 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
The ATG Autophagic Conjugation System in Maize: ATG Transcripts and Abundance of the ATG8-Lipid Adduct Are Regulated by Development and Nutrient Availability1,[W],[OA]Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706–1574
Plants employ sophisticated mechanisms to recycle intracellular constituents needed for growth, development, and survival under nutrient-limiting conditions. Autophagy is one important route in which cytoplasm and organelles are sequestered in bulk into vesicles and subsequently delivered to the vacuole for breakdown by resident hydrolases. The formation and trafficking of autophagic vesicles are directed in part by associated conjugation cascades that couple the AUTOPHAGY-RELATED8 (ATG8) and ATG12 proteins to their respective targets, phosphatidylethanolamine and the ATG5 protein. To help understand the importance of autophagy to nutrient remobilization in cereals, we describe here the ATG8/12 conjugation cascades in maize (Zea mays) and examine their dynamics during development, leaf senescence, and nitrogen and fixed-carbon starvation. From searches of the maize genomic sequence using Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) counterparts as queries, we identified orthologous loci encoding all components necessary for ATG8/12 conjugation, including a five-member gene family expressing ATG8. Alternative splicing was evident for almost all Atg transcripts, which could have important regulatory consequences. In addition to free ATG8, its membrane-associated, lipidated form was detected in many maize tissues, suggesting that its conjugation cascade is active throughout the plant at most, if not all, developmental stages. Levels of Atg transcripts and/or the ATG8-phosphatidylethanolamine adduct increase during leaf senescence and nitrogen and fixed-carbon limitations, indicating that autophagy plays a key role in nutrient remobilization. The description of the maize ATG system now provides a battery of molecular and biochemical tools to study autophagy in this crop under field conditions.
The recycling of intracellular constituents is critical to all organisms for optimal growth and development and survival under nutrient-limiting conditions. This recycling is especially important throughout a plant's life cycle, especially as plants scavenge available nutrients from storage tissues and older senescing leaves for the synthesis of reproductive organs and new leaves and during whole plant senescence as fixed carbon (C) and nitrogen (N) are mobilized to reproductive or storage organs (Vierstra, 1996  ; Hopkins et al., 2007; Lim et al., 2007). The process is initiated in source tissues by dramatic changes in gene expression, in which genes involved in basic metabolism, including photosynthesis and protein biosynthesis, are down-regulated while those involved in programmed cell death and the stress response and/or encoding various hydrolytic enzymes are up-regulated (Hopkins et al., 2007; Lim et al., 2007). Nutrient mobilization in plants, as in yeast and animals, involves a battery of catabolic processes, including a sophisticated recycling system called autophagy or "self-eating." Here, cytoplasm and organelles are sequestered in bulk into special autophagic vesicles, which then deliver their contents to the vacuole (or lysosome) for breakdown by resident hydrolases (Thompson and Vierstra, 2005; Bassham, 2007). The release products can be either consumed by the host cell or transported to other tissues and organs for reuse.
In yeast (Saccharomyces cerevisiae) and animal cells, autophagy (also referred to as macroautophagy) begins with the formation of a sequestering membrane called the phagophore (or isolation membrane), which engulfs portions of the cytoplasm to form the double membrane-bound autophagosome vesicle (Ohsumi, 2001
Despite its relevance to the C/N economy of plants, we are only now beginning to appreciate autophagy at the molecular level. Key to this understanding was the discovery of an elaborate recycling system in yeast involving a suite of autophagy-related (ATG) proteins (Ohsumi, 2001
The conjugation cascades center on two Ub-fold proteins, ATG8 and ATG12, which become covalently attached to their respective targets, the lipid phosphatidylethanolamine (PE) and the ATG5 protein, by a process mechanistically similar to ubiquitination (Ohsumi, 2001
The biochemical roles of ATG8-PE and ATG12-ATG5 conjugates have recently been revealed. Based on cell-free studies, the ATG8-PE adduct appears to promote the expansion of autophagosomal membrane and hemifusion of this membrane with the vacuolar membrane (Nakatogawa et al., 2007
Over the past few years, obvious orthologs of various yeast ATG proteins have been detected in both higher and lower plants, indicating that a similar autophagic system likely exists throughout the plant kingdom. The Arabidopsis (Arabidopsis thaliana) genome, for example, encodes proteins related to subunits of the yeast ATG1 kinase and ATG6/Beclin1 phosphatidylinositol 3-kinase complexes and all components of the ATG8/12 conjugation cascades, including small gene families encoding the two tags (Doelling et al., 2002
While our understanding of the Arabidopsis ATG system is progressing, little is currently known about this system or autophagy in other plant species. Given the potential significance of autophagic recycling to many agriculturally relevant processes, including seed germination, N and C mobilization from leaves into seeds, fruits, and roots, survival under suboptimal growth conditions, pathogen protection, and senescence (Liu et al., 2005
Identification of Rice and Maize Atg Genes
Previous genome searches of Arabidopsis identified a suite of proteins structurally and functionally related to many ATG components present in yeast (Thompson and Vierstra, 2005
As shown in Figure 1A and Table II, this survey identified five maize loci encoding the ATG8 protein tag, two encoding the ATG4 processing protease, and one gene each encoding the ATG12 protein tag, the common ATG7 E1 activating enzyme, the ATG3 and ATG10 E2 conjugating enzymes specific for ATG8 and ATG12, respectively, and the ATG5 target of ATG12. For almost all loci, strong amino acid sequence conservation was evident when compared with their rice, Arabidopsis, and yeast orthologs (Table II). For example, the five maize Atg8 genes (Zm Atg8a to Zm Atg8e), which are 100% to 95%/100% to 84% similar/identical to each other, are 94%/86% to 85% and 92% to 90%/75% to 73% similar/identical to Arabidopsis ATG8a and yeast ATG8, respectively (Fig. 2 ; Table II). The Zm Atg8b and Zm Atg8c loci are on different chromosomes but encode identical proteins, suggesting that this pair arose by a recent gene duplication. Furthermore, key amino acids necessary for the function of each component were detected, indicating that the maize conjugation cascades are mechanistically identical to those present in yeast and Arabidopsis. These residues include the active-site Cys residues in ATG3, ATG7, and ATG10 that form the thiolester adducts with ATG8 and/or ATG12, the Lys acceptor site in ATG5, the positionally conserved Glys in ATG8 and ATG12 that directly participate in covalent binding, and the Cys essential for the proteolytic activity of ATG4 (Fig. 1A).
Our maize gene models were further validated by sequence analysis of a collection of transcripts for each Zm Atg gene (Table II). With the exception of Zm Atg5, for which we were unable to generate a cDNA by reverse transcription (RT)-PCR, presumably due to the high GC content of its 5' end, cDNAs encompassing the full coding region for each locus were obtained. These cDNA sequences not only confirmed that each Zm Atg gene is transcriptionally active but also identified a number of splice variants for many loci (Fig. 1, B and C; Table III ; Supplemental Table S1). The number of Zm Atg genes with splice variants (11 of 12 genes) was higher than for Arabidopsis (seven of 17) or rice (11 of 14; Table III). The average number of splice variants per gene was also higher in maize, suggesting that alternative pre-mRNA splicing is more extensive in this species.
Some of the splice variants substantially changed the corresponding reading frames, indicating that the resulting mRNAs would direct the synthesis of altered proteins if translated. For example, several splice variants of Zm Atg4b could affect the activity of the encoded protease by removing a stretch of amino acids near its active-site Cys (Fig. 1B). Additionally, several splice variants of Zm Atg10 eliminate the exonic region encoding the active-site Cys that binds ATG12 during its conjugation cascade (Fig. 1C), thus inactivating the variant ATG10 protein. Whether any of these variant proteins actually accumulate and adversely affect the ATG8 or ATG12 conjugate cascades is not yet known.
Initially, there was no clear full-length cDNA model for Zm Atg7, even though our BLAST analyses detected ESTs corresponding to the 3' end of the transcript. Fortunately, we were able to generate a putative full-length Atg7 cDNA by RT-PCR using a forward primer designed from a partial 5' genomic sequence and a reverse primer based on the partial 3' EST. This longer Zm Atg7 cDNA matched (1) our partial maize gene models, (2) a full-length gene model generated from a more recently released BAC sequence of maize, and (3) a predicted full-length rice Atg7 transcript. Surprisingly, when aligned with the Arabidopsis ATG7 coding region, we found that the rice and maize transcripts encoded substantially longer polypeptides (1,021 and 1,042 residues for maize and rice ATG7, respectively, versus 697 residues for Arabidopsis ATG7).
Dot plot amino acid sequence comparisons of the maize and rice polypeptides with Arabidopsis ATG7 revealed that the increased length was created solely by a sizable duplication of the C-terminal region of the maize and rice proteins (Fig. 3A
; data not shown). This arrangement has not yet been described in other plants, fungi, or animals, implying that it was generated by a cereal-specific tandem duplication. The region involved in the duplication is essential for the E1 activating activity of ATG7, as it contains both the nucleotide-binding pocket and the conserved Cys that binds ATG8/12 via thiolester bond (Doelling et al., 2002 To test whether the extended maize and rice ATG7 proteins retained their ability to interact with ATG8/12, we examined by yeast-two hybrid (Y2H) analysis the interaction of Zm ATG7 with Arabidopsis and maize ATG8 and ATG12. As shown in Figure 3B, Zm ATG7, like its Arabidopsis ortholog, could bind both tags, including the At ATG8a and Zm ATG8a isoforms, Zm ATG12, and both isoforms of At ATG12. The interactions of the tags with At ATG7 were not abolished by the C558A variant, in which the active-site Cys was substituted for an Ala (Fig. 3B), indicating that the binding was likely to be noncovalent and did not require formation of the thiolester adduct. Taken together, we conclude that the maize (and likely rice) ATG7 protein retained its ability to interact with its ATG8 and ATG12 substrates despite the C-terminal duplication.
The modification of ATG8 with PE is an important molecular and cytological marker for autophagic compartments (Ohsumi, 2001
To study the lipidation of maize ATG8, we exploited the assay developed by Yoshimoto et al. (2004) that enriches for the ATG8-PE adduct by the centrifugal isolation of a crude membrane fraction, solubilizes the membranes with Triton X-100, and then separates lipidated ATG8 from the free form by SDS-PAGE in the presence of 6 M urea. Lipidation of ATG8 can then be confirmed by treatment of the solubilized membranes with phospholipase D (PLD), which converts the faster migrating ATG8-PE adduct into the slower migrating free form (Tanida et al., 2004; Fujioka et al., 2008). When the maize samples were subjected to SDS-PAGE in the presence of urea, we detected a faster migrating species in addition to the slower migrating free ATG8 protein (Fig. 4C; data not shown). This faster migrating species could even be detected in crude extracts prior to membrane isolation and became highly enriched in the solubilized membranes, as expected for a membrane-anchored protein. The abundance of this faster migrating form was substantially reduced upon incubation of the solubilized membranes with PLD, indicating that this species is a lipidated form of Zm ATG8 (Fig. 4C). While not proven here, this species likely represents the PE adduct (Fujioka et al., 2008).
Previous studies with Arabidopsis demonstrated that the genes encoding the ATG8/12 conjugation cascades are ubiquitously expressed, with senescence and nutrient limitations increasing transcript abundance (Contento et al., 2004
To gain further insights into the regulation of ATG8 conjugation during development, we examined the levels of the free ATG8 protein and its lipid adduct in selected maize tissues at various stages of development. In most cases, enrichment of the ATG8-PE adduct by membrane isolation was not needed to enable detection of the faster migrating lipidated form, indicating that some maize tissues accumulate high levels of this species. For example, both free ATG8 and the ATG8-PE adduct were easily detected during seed/seedling development in crude extracts prepared from both the endosperm and embryo at increasing times of maturity and in several parts of the seedling at 5 d after germination (DAG; Fig. 5B). In young embryos, almost all ATG8 was present in the free form, but as they developed, the ATG8-PE adduct appeared around 22 d after pollination (DAP). In mature embryos at 30 DAP, the level of free ATG8 was substantially reduced, while the level of ATG8-PE remained the same. The endosperm also accumulated higher levels of the ATG8-PE adduct as it matured, suggesting that autophagy may participate in programmed cell death during endosperm development. A high level of adduct was evident in the scutellum, but not in the plumule and radicle, during germination, which could reflect the autophagic mobilization of stored reserves in the scutellum to support new growth (Fig. 5B). Leaves harvested from soil-grown, well-fertilized seedlings also contained both the free and lipidated forms of ATG8, with a similar ratio evident in green leaves of various ages, as categorized by their positions on the plant (Fig. 5C). Strikingly, we found that both the total amount of ATG8 and the abundance of the ATG8-PE adduct in maize leaves were markedly affected by senescence. For example, when we compared the yellow and green sectors in an individual senescing leaf from a mature flowering plant, the yellow sectors had substantially more total ATG8 protein (Fig. 5D). Whereas the green sectors had a nearly equal ratio of lipidated ATG8 to free ATG8, similar to that found in the adjacent nonsenescing leaf positioned on the plant axis immediately above, the yellow sectors contained almost exclusively the ATG8-PE adduct, suggesting that high rates of autophagy were under way in these areas. This senescence-dependent effect could also be induced when the plants were grown hydroponically in a defined solution containing inorganic nutrients, a growth condition that accelerates the developmental senescence of leaves even though the plants appear well fertilized. As shown in Figure 5E for plants grown hydroponically for 10 DAG, the oldest leaf (L1) contained only traces of ATG8-PE, the next oldest leaf (L2) contained much higher levels, but almost all was lipidated, while younger leaves (L3) contained a mixture of the free and lipidated form, and the shoot apex contained almost exclusively the free form. Eventually, both forms of ATG8 disappeared along with most other proteins as the older leaves completed senescence (see L1 and L2 from 24-DAG seedlings in Fig. 5E).
The age-dependent accumulation of the ATG8-PE adduct in the hydroponically grown seedling leaves (Fig. 5E) suggested that the availability of macronutrients and/or micronutrients regulates Zm Atg gene expression and the lipidation of the ATG8 protein. One obvious candidate is N, since its availability is often limiting to plant growth and protein synthesis. To test this possibility, we germinated and grew maize seedlings hydroponically for 15 d on deionized water, Murashige and Skoog (MS) medium lacking N (MS-N), or MS-N supplemented with 0.5, 1, or 2 times the normal concentration of nitrate (see "Materials and Methods"). As expected, the growth of seedlings treated with only water or MS-N was significantly retarded at 15 DAG compared with N-fertilized plants (Fig. 6 ). Concomitant with this growth reduction was an increase in the oldest leaves (L1) of both the amount of total ATG8 and the proportion converted to the lipidated form. As increasing amounts of N were added to the MS-N, the amounts of the ATG8-PE adduct and total ATG8 dropped in the L1 leaf (Fig. 6). Doubling the strength of N (2x N) did not significantly affect the levels of total and lipidated ATG8 when compared with single strength medium (1x N).
To further investigate the effect of N limitation on ATG8 lipidation and Zm Atg gene expression, we collected leaf samples from 10-, 15-, and 20-DAG seedlings that were hydroponically grown with either MS or MS-N. At 10 DAG, no significant differences in seedling growth or in total and lipidated ATG8 levels were detected between leaf samples collected from MS- and MS-N-grown seedlings, suggesting that the plants had not yet experienced N deficiencies (data not shown). However, at 15 DAG, reductions in growth became evident for the seedlings fertilized with MS-N (Fig. 7A , left), indicating that they were now experiencing a N deficit. This deficiency became more obvious at 20 DAG, when the oldest leaves (L1) acquired the yellow/brown patches characteristic of strongly senescing leaves. In parallel with this N starvation was a substantial increase in the levels of the ATG8 protein. The 15- and 20-DAG plants grown on MS-N had higher levels of total and lipidated ATG8 than MS-grown seedlings, a difference that was especially apparent in the older (L1) leaves (Fig. 7B). In contrast to the effects on ATG8 levels in leaves, both the total amount of ATG8 and the ratio of lipidated ATG8 to the free form were unaffected by N limitation in the roots and shoot apex (Fig. 7C).
The increase in maize ATG8 protein levels during N starvation was consistent with previous data in Arabidopsis showing that the expression of the ATG system is up-regulated by nutrient stress (Contento et al., 2004 ; Yoshimoto et al., 2004; Buchanan-Wollaston et al., 2005; Sláviková et al., 2005; Thompson et al., 2005; Rose et al., 2006; van der Graaff et al., 2006; Osuna et al., 2007; Peng et al., 2007; Phillips et al., 2008). To test whether the suite of Zm Atg genes was affected similarly, we analyzed the transcript levels for each during N starvation along with several senescence-associated transcripts. Here, total RNA was isolated from maize leaves of various ages (L1–L3) from 15-DAG seedlings fertilized with either MS or MS-N, and the levels of individual mRNAs were determined by semiquantitative RT-PCR. Transcripts encoding the Ub-conjugating enzyme (Ubc9) and the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were used for normalization and as an internal control, respectively (Czechowski et al., 2005). As can be seen in Figure 7D, the Gapdh/Ubc9 transcript ratio was not significantly affected by leaf age or N availability under these growth conditions. In contrast, the senescence-associated See1a transcript (Smart et al., 1995) was significantly up-regulated by the MS-N growth condition, confirming that premature senescence was induced in these N-limited leaves. Accelerated leaf senescence in N-limited leaves was also evident from RT-PCR analysis of the Lhcb5 transcript encoding one of the chlorophyll-binding proteins in the chloroplast (van der Graaff et al., 2006). The older senescing L1 leaves contained a very low level of Lhcb5 transcript. Unexpectedly, the second leaves (L2) of MS-N seedlings had higher levels of Lhcb5 transcript than MS-grown leaves. This is consistent with the RT-PCR data for sedoheptulose-bisphosphatase (Sbp) transcript, which encodes a Calvin cycle enzyme and shows a light-dependent expression pattern (Willingham et al., 1994). The Sbp transcript levels in N-limited L2 leaves were also higher than in L2 leaves fertilized with MS. The reason for this accumulation of these photosynthesis-related transcripts in N-limited middle leaves is not clear. When the Zm Atg genes were analyzed similarly, we found that the transcript abundance of almost all were up-regulated by the absence of N and further up-regulated as the leaves aged (Fig. 7D). In most cases, the highest transcript abundance was found in the L1 leaves from MS-N-fertilized plants. This trend was particularly obvious for Atg3, Atg7, Atg10, and four of the five isoforms of Atg8 (Atg8b, -c, -d, and -e). Interestingly, in addition to Atg7, those genes encoding components required for ATG8 conjugation were more strongly affected by the combined action of N availability and leaf age (Atg3, Atg4b, and Atg8b to Atg8e) than those encoding components for ATG12 conjugation (Atg5, Atg10, and Atg12; Fig. 7D). This difference suggests that formation of the ATG8-PE adduct is more limiting to autophagic recycling during senescence.
Fixed-C limitation has also been shown to increase the abundance of various ATG transcripts in Arabidopsis using either the withdrawal of Suc from the growth medium of cell cultures (Contento et al., 2004
Transcript analysis of Gapdh, See1a, Lhcb5, and Sbp demonstrated that this covering affected the photosynthetic machinery well before senescence became apparent. While the mRNA levels for Gapdh were unaffected and those for the See1a transcript were only modestly up-regulated in the darkened and presumably fixed-C-limited sectors relative to illuminated sectors, the levels of both Lhcb5 and Sbp dropped dramatically, consistent with their light requirement for transcription (Willingham et al., 1994
Despite the increases in Atg mRNA abundance by darkening the leaf, the levels of the free ATG8 and the ATG8-PE adduct were not markedly altered. The steady-state levels of the free and conjugated forms were similar in the darkened and illuminated sectors even after 4 d of treatment (Fig. 8B). This lack of effect on ATG8 levels during fixed-C limitation as opposed to the strong effect induced by N limitation (Fig. 7B) could indicate that autophagy is more sensitive to N starvation. However, a more plausible interpretation is that our experiments with N starvation likely caused the entire plant to experience N deficits, while those testing fixed-C limitation subjected only part of the leaf to fixed-C deficits. Consequently, it is possible that the neighboring illuminated areas became a source of soluble sugars to the darkened areas, thus mitigating the drop in fixed-C in the absence of photosynthesis.
A number of genetic studies with the ATG8/12 conjugation cascades in Arabidopsis have demonstrated the importance of autophagy to nutrient recycling and survival of plants under N/C-limiting growth conditions (see above). In an effort to determine the importance of autophagy to crop plants, we completed a molecular description of an orthologous ATG system in the cereals rice and maize. We then assayed in maize their expression patterns and the accumulation of the lipidated ATG8 adduct during senescence and in response to N or fixed-C limitation. All components required for ATG8/12 conjugation in yeast and Arabidopsis (Ohsumi, 2001 ; Thompson and Vierstra, 2005) were identified in both rice and maize, indicating that the pathway is highly conserved among plant species. As in Arabidopsis, four maize components are encoded by single genes (Atg3, Atg5, Atg7, and Atg10), whereas two are encoded by small gene families (two for Atg4 and five for Atg8). This increased diversity for the ATG8 protein tag and the ATG4 processing protease required for ATG8 maturation suggests both subfunctionalization of the ATG8 isoforms and distinct roles for the two ATG4 isoforms in processing the ATG8 precursor and/or recycling ATG8 modified with PE. As reported for the Arabidopsis ATG8 gene family (Sláviková et al., 2005; Thompson et al., 2005), individuals in the five-member maize Atg8 family display subtle differences in their tissue expression patterns and varying responses to N and fixed-C limitations, indicating that the individual ATG8 isoforms in maize have cell/tissue-specific and/or stress-specific roles.
Our description of the maize Atg genes is one of the earliest comprehensive genomic analyses on a specific metabolic pathway since the release of the draft genome sequence (http://maizesequence.org). Despite difficulties in determining the exact number of genes encoding each Zm ATG protein, given the incomplete annotation of the sequence and the large number of discontinuous segments still remaining, we are certain about the gene identifications described in Figure 1 and Table II for the following reasons. First, we focused on genes involved in the ATG8/12 conjugation cascades. They appear to be the most conserved members in the Atg system, which in turn simplified the identification of the rice and then the maize orthologs. For example, like its structural relative Ub, ATG8 is arguably one of the most conserved proteins in eukaryotes (Fig. 2; Supplemental Fig. S1; Doelling et al., 2002 Although we are confident in most of the rice and maize ATG gene models, several await full-length cDNAs and/or more refined chromosomal sequence data before final confirmation of their gene organizations. For example, we could not assign a BAC sequence to the Zm Atg8e transcript, although there are two candidate BACs (AC203867 and AC203875) predicted to encode very similar transcripts (Table II). In addition, the precise gene models for rice Atg3b and Atg10b await full-length cDNAs. cDNAs encoding partial polypeptides are available for both, indicating that these loci are transcribed. However, based on the predicted high expression of the paralogous Os Atg3a and Os Atg10a loci (based on the number of ESTs [Supplemental Table S1]), it is likely that the rice Atg3b and Atg10b loci are minor contributors to the pools of each enzyme. And lastly, the rice Atg8e gene is currently without any EST or cDNA data, thus leaving its gene model and transcriptional activity unsupported (Table I). While almost all of the rice and maize ATG proteins are structurally orthologous to their Arabidopsis and yeast counterparts, ATG7 is notably distinct. The rice and maize polypeptides contained an approximately 325-amino acid duplication of their C-terminal half that replicates the region encompassing the active-site Cys and the nucleotide-binding pocket necessary for ATG8/12 activation. While the nucleotide-binding pocket sequence is evident in both repeats, the more N-terminal repeat had its active-site Cys replaced with a Ser. Despite this duplication, maize ATG7 remains functional, as judged by the ability of the full-length protein to interact by Y2H analysis with ATG8 and ATG12 and by our detection of its ATG8-PE product in planta. At present, it is unclear how the catalytic regions of rice and maize ATG7s are organized and whether the more N-terminal predicted nucleotide-binding pockets can actually participate in ATG8/12 activation.
An interesting aspect of the cereal Atg gene models is that most of the genes are associated with alternative splicing of pre-mRNA. Current estimates of genes with multiple splice variants are approximately 21% and 22% for Arabidopsis and rice, respectively (Wang and Brendel, 2006
As observed previously with Arabidopsis (Contento et al., 2004
What drives this substantial increase in the steady-state levels of the ATG8-PE adduct during seed development, leaf senescence, and N starvation is not yet known. In yeast, this lipidation is controlled by a set of nutrient-sensing systems (e.g. target-of-rapamycin, Snf1, and cAMP-dependent protein kinases) that suppress the activity of the ATG1 kinase complex, whose action ultimately impacts the ATG8/12 conjugation machinery (Klionsky, 2007
Whatever the mechanisms, the strong correlations between the steady-state abundance of the ATG8-PE adduct and leaf senescence and growth under N-limiting conditions suggest that the level of the adduct represents a sensitive indicator of both the amount of autophagic recycling and the nutrient status of the plant/organ/tissue. For specific tissues, it could also be a measure of their source/sink status. For example, in hydroponically grown plants, older leaves, which are expected to be important nutrient sources for the growing regions as they senesce, contained mostly the conjugated form of ATG8, while the shoot apex, composed of rapidly expanding young leaves that are strong nutrient sinks, contained mostly the free form. Taking this possibility one step further, our analysis of ATG8 lipidation also implies that autophagic recycling is regulated in a tissue/organ-dependent manner in maize and likely in other plants. In N-fertilized plants, the high ATG8-PE to free ATG8 ratio in roots relative to shoots and leaves would imply that roots are relatively more active in autophagy under N-rich growth conditions. When depleted of N, root autophagy appeared unaffected, as this ratio remained unchanged. Instead, a dramatic increase in the ATG8-PE adduct was observed in leaves, suggesting that autophagy in this tissue was specifically induced. This N starvation-induced increase further underscores the notion that senescing leaves are a major source of remobilized nutrients during nutrient starvation (Lim et al., 2007
Plant Materials and Growth Conditions All experiments were performed with the maize (Zea mays) inbred B73. Seeds were germinated in pots (125 cm3) with a 1:1:1 mixture of compost, peat moss, and vermiculite and were grown at 21°C in a growth chamber with a 16-h-light/8-h-dark photoperiod. For dark-induced fixed-C limitation, the basal part of the second leaf blade was wrapped with aluminum foil (75 mm in length) at 15 DAG. At various times, the wrapped part of the leaf blade was harvested along with a corresponding region from a light-exposed leaf blade of the same developmental age, which served as the control. For N limitations, seedlings were grown hydroponically in pots (450 cm3) with vermiculite. The pots were subirrigated with 10 mL of various liquid media every 5 d, starting at 1 DAG. The media included (1) MS medium (MS basal salts), (2) MS-N (MS micronutrient solution plus 3 mM CaCl2, 1.5 mM MgSO4, 1.25 mM KH2PO4, and 5 mM KCl [pH 5.7]), or (3) MS-N supplemented with 0.825 g L–1 NH4NO3 and 0.95 g L–1 KNO3 (MS-N + 0.5x N medium), 1.65 g L–1 NH4NO3 and 1.9 g L–1 KNO3 (MS-N + 1x N medium), or 3.3 g L–1 NH4NO3 and 3.8 g L–1 KNO3 (MS-N + 2x N medium). When needed, seedlings were grown to maturity in a greenhouse to allow for natural senescence and to obtain developing kernels following self-pollination. Extra lighting was provided to maintain a 14-h-light/10-h-dark photoperiod.
Maize Atg genes were identified by TBLASTN (Altschul et al., 1990
Sequence alignments between individual cDNAs and genomic sequences were manually inspected for consensus coding regions, intron splice sites, and potential splice variants. Sequence gaps were filled in by BLASTN of the resulting gene models to the maize HTGS BAC sequence database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Dot plots revealing local sequence similarities were generated by PLALIGN (Pearson, 2000
RNA was extracted from three biological replicates of various maize tissues using Trizol (Invitrogen). First-strand cDNA synthesis was achieved with SuperScript II (Invitrogen) reverse transcriptase and an oligo(dT)18 primer (Fermentas). The amount of RT product in each sample was standardized by a second round of PCR using primers against Zm Ubc9 (named because of its closest amino acid match to Arabidopsis UBC9; Czechowski et al., 2005
Y2H assays were conducted with full-length cDNAs as described previously with minor modifications (Gingerich et al., 2007
In most cases, maize tissue was harvested, immediately frozen to liquid N temperatures, and homogenized in SDS-PAGE sample buffer. The extracts were clarified by centrifugation at 16,000g and then heated before use. Protein amounts were determined by spotting aliquots of the extracts onto Whatman 3MM filter paper (Fisher Scientific) and comparing the Coomassie Brilliant Blue staining intensity of the spots with bovine serum albumin standards (Ghosh et al., 1988 To characterize the ATG8-PE adducts, various maize tissues were homogenized in TNPI buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, and 10 mM iodoacetamide). The extract was filtered through four layers of cheesecloth and clarified by centrifugation at 2,000g for 5 min, and the supernatant was centrifuged at 100,000g for 1 h. The resulting 100-k pellet was resuspended in TNPI buffer, solubilized by adding Triton X-100 at a final concentration of 0.5%, and clarified at 16,000g for 10 min to obtain the solubilized membrane fraction. This membrane fraction was incubated at 37°C for 1 h with Streptomyces chromofuscus PLD (Biomol; 250 units mL–1 final concentration) or an equal volume of its buffer. The reactions were stopped by the addition of SDS-PAGE sample buffer and then subjected to SDS-PAGE on 12% polyacrylamide gels with 6 M urea. Free ATG8 and the ATG8-PE adduct were detected by immunoblot analysis with anti-At ATG8 antibodies. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers FJ445019 and FJ444997 (Zm Atg3, splice variants 1 and 2), FJ444998 and FJ444999 (Zm Atg4a, splice variants 1 and 2), FJ445000 to FJ445004 (Zm Atg4b, splice variants 1, 3, 4, 5, and 6), FJ445005 (Zm Atg7), FJ445006 (Zm Atg8a, splice variant 1), FJ445007 (Zm Atg8b, splice variant 1), FJ445008 (Zm Atg8c, splice variant 1), FJ445009 and FJ445010 (Zm Atg8d, splice variants 1 and 4), FJ445011 (Zm Atg8e, splice variant 1), FJ445012 to FJ445014 (Zm Atg10, splice variants 1, 5, and 6), and FJ445015 and FJ445016 (Zm Atg12, splice variants 1 and 2), respectively.
The following materials are available in the online version of this article.
We acknowledge the Maize Genome Sequencing Consortium for making the sequence available prior to publication. We thank Joseph Walker and Dr. Allison Phillips for helpful advice and Ashvini Damodaran for assistance with the Y2H analyses. Received July 18, 2008; accepted September 9, 2008; published September 12, 2008.
1 This work was supported by grants from the National Research Initiative Competitive Grants Program (grant nos. 2005–35301–15768 and 2008–02545) of the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service to R.D.V. and by a Thailand Predoctoral Fellowship to A.S.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Richard D. Vierstra (vierstra{at}wisc.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.126714 * Corresponding author; e-mail vierstra{at}wisc.edu.
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ABSTRACT
RESULTS
-amino group of a conserved Lys in ATG5 is the attachment site. The ATG4 processing protease is also essential for ATG8 activity; it exposes the C-terminal Gly in the longer ATG8 precursor and recycles mature ATG8 by releasing it from the ATG8-PE adduct, presumably before fusion of the autophagosome with the vacuolar membrane. 
, respectively, and diploid yeast strains were then generated by mating. Protein-protein interactions were identified by testing for growth of the diploid strains on selection medium lacking His, Leu, and Trp and containing 5 mM 3-amino-1',2',4'-triazole. The At ATG7(C558A) mutant, in which the Cys-558 codon was replaced with that encoding Ala, was generated by the PCR-based QuickChange method (Stratagene) with appropriate mutagenic primers. 
