JXB Advance Access originally published online on September 12, 2005
Journal of Experimental Botany 2005 56(421):2839-2849; doi:10.1093/jxb/eri276
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants
1Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel
2Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
3Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel
4Institute of Cell Biology, Comenius University, Bratislava 81107, Slovakia
To whom correspondence should be addressed. Fax: +972 8 9344112. E-mail: bmzevi{at}wicc.weizmann.ac.il; Fax: +972 8 9344181. E-mail: gad.galili{at}weizmann.ac.il
Received 21 February 2005; Accepted 2 August 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Arabidopsis plants possess a family of nine AtAtg8 gene homologues of the yeast autophagy-associated Apg8/Aut7 gene. To gain insight into how these genes function in plants, first, the expression patterns of five AtAtg8 homologues were analysed in young Arabidopsis plants grown under favourable growth conditions or following exposure to prolonged darkness or sugar starvation. Promoters, plus the entire coding regions (exons and introns) of the AtAtg8 genes, were fused to the ß-glucuronidase reporter gene and transformed into Arabidopsis plants. In all plants, grown under favourable growth conditions, ß-glucuronidase staining was much more significant in roots than in shoots. Different genes showed distinct spatial and temporal expression patterns in roots. In some transgenic plants, ß-glucuronidase staining in leaves was induced by prolonged darkness or sugar starvation. Next, Arabidopsis plants were transformed with chimeric gene-encoding Atg8f protein fused to N-terminal green fluorescent protein and C-terminal haemagglutinin epitope tags. Analysis of these plants showed that, under favourable growth conditions, the Atg8f protein is efficiently processed and is localized to autophagosome-resembling structures, both in the cytosol and in the central vacuole, in a similar manner to its processing and localization under starvation stresses. Moreover, treatment with a cocktail of proteasome inhibitors did not prevent the turnover of this protein, implying that its turnover takes place in the vacuoles, as occurs in yeasts. The results suggest that, in plants, the cellular processes involving the Atg8 genes function efficiently in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.
Key words: Apg8, Arabidopsis, autophagy, germination, starvation, stress
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Vesicular trafficking to vacuoles consists of a number of processes that serve multiple functions (Bryant and Stevens, 1998
). Among these, the process of starvation-induced autophagy was recently the focus of extensive research, particularly in yeast (Huang and Klionsky, 2002; Shintani et al., 2002; Wang and Klionsky, 2003). Genetic and molecular studies in yeast have identified novel proteins that control autophagy, many of which also participate in direct cytoplasm-to-vacuole transport of proteins (Harding et al., 1996; Baba et al., 1997). One of these proteins, which participate both in autophagy and cytoplasm-to-vacuole transport, is the Atg8 protein (formerly known as Apg8/Aut7) (Klionsky et al., 2003). In autophagy, this protein is known to conjugate to the autophagosomal membrane through phosphatidylethanolamine lipidation and to take part in autophagosome formation and expansion (Kirisako et al., 1999, 2000).
Unlike the single Atg8 gene in yeast, higher eukaryotes possess families with multiple members of Atg8 genes; for example, eight genes in human and nine in the model plant Arabidopsis thaliana (Doelling et al., 2002). The occurrence of multiple-gene families in higher eukaryotes suggests that the different Atg8 proteins may serve different functions, in addition to autophagy. Indeed, one of the mammalian Atg8 proteins (termed GATE-16) was found to promote intra-Golgi trafficking (Legesse-Miller et al., 2000; Elazar et al., 2003) and post-mitotic Golgi reassembly (Muller et al., 2002). Two other mammalian Atg8 proteins, GABA-RAP and MAP-LC3, have different functions; the former interacts with the 
-amino butyric acid (GABA) receptor in nerve cells (Wang et al., 1999), while the latter interacts with microtubule-associated proteins (Mann and Hammarback, 1994). MAP-LC3 is the only mammalian homologue that was implicated in autophagy.
Despite extensive studies on Atg8 proteins in animals and yeasts, little is known about their functions in plants. Using green fluorescent protein (GFP) fusion constructs, it had been shown recently that, under starvation stresses, Atg8 proteins are processed by the Atg4 protease in a similar manner in plant as in animal cells and in yeasts (Yoshimoto et al., 2004). Yet, in plants, the potential functions of members of this gene family under favourable growth conditions, and a comparison of their functions under stress conditions, has not been deeply addressed. This may be obtained by analysing the spatial and temporal expression patterns of their genes, as well as the processing and intracellular localization of their encoded proteins under stress and non-stress conditions. In the present report, ß-glucuronidase (GUS) fusion constructs of five representatives out of the nine Arabidopsis AtAtg8 genes were expressed, as well as a GFP fusion construct of the Atg8f gene in transgenic Arabidopsis plants, in order to address these issues. The results suggest that, in plants, the cellular processes using the AtAtg8 proteins have significant functions in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Construction of chimeric genes
The promoters plus the entire coding regions (including exons and introns) of five AtAtg8 genes were extracted from Arabidopsis genomic DNA by polymerase chain reaction (PCR) using primers whose sequences are given in Table 1. The 3'-end primers were designed so that the endogenous AtAtg8 stop codons were abolished and replaced by a SmaI restriction enzyme site and the 5'-end primers included a SalI or XbaI restriction enzyme site for further manipulation. The PCR fragments were then inserted into the pPZP111 Ti plasmid (Hajdukiewicz et al., 1994
) using SmaI and SalI/XbaI enzymes. The GUS coding region was amplified out of the Ti plasmid pBI101 (Novagen), using the appropriate 5'- and 3'-end primers that added SmaI and KpnI restriction enzymes sites into the 5' and 3' ends, respectively. pPZP111-AtAtg8 constructs were cut with SmaI and KpnI and the GUS fragment was inserted downstream to the AtAtg8 coding region to generate the AtAtg8-GUS chimera.
|
The chimeric GFP-Atg8-HA gene was constructed as follows. The full-length coding sequence of the AtAtg8f, lacking its initiator ATG and stop codons, was first amplified with primers shown in Table 2. The PCR product was then subcloned into another cassette of a pZP111 Ti plasmid to generate a chimeric gene containing a single open reading frame, which included fused GFP-Atg8-HA from its 5'- to its 3'- end, connected to an upstream 35S promoter and downstream OCS terminator.
|
Transformation of Arabidopsis plants and selection of transformed plants
Agrobacterium tumefaciens strain EHA105 was transformed with each of the pPZP111-AtAtg8-GUS and pZP111-GFP-Atg8-HA plasmids and then used to transform Arabidopsis thaliana plants (ecotype WS) as previously described (Clough and Bent, 1998
). Kanamycin-resistant plants were selected and selfed to obtain T3 plants homozygous for the transgenes.
Plant material and growth conditions
Wild-type and transgenic seeds of Arabidopsis (ecotype WS) were surface-sterilized in 50% bleach and selected on solid Nitsch medium (Nitsch, 1970) containing 50 µg ml–1 of kanamycin and incubated for 2 d at 4 °C. The plates were then incubated in a growth chamber under a regime of 16 h light/8 h dark cycle at 24 °C. All media were purchased from Duchefa.
Exposing plants to dark or starvation
For the dark treatment, 2-week-old seedlings were exposed to darkness for 10 d. For the starvation treatment 7-d-old seedlings were transferred to Gamborg B5 liquid medium supplemented with 2% sucrose for 3 d, then washed with deionized water and transfered to a Gamborg B5 medium lacking sucrose for an additional 3 d.
GUS staining
Different plant organs were collected and submerged for 4 h in the following staining solution: 50 mM Na3PO4 pH 7.0, 5 mM ferricyanide, 5 mM ferrocyanide, 0.1% dimethyl sulphoxide (DMSO), 0.1% triton X-100, and 1 mg ml–1 final concentration of 5-bromo-4-chloro-3-indolyl glucuronide (Duchefa) dissolved in dimethyl formamide. Plants were washed once in water and destained twice in 95% ethanol and observed in the light microscope.
Concanamycin A treatment
Roots of 10-d-old Arabidopsis plants transformed with GFP-AtAtg8f-HA construct grown on solid Nitsch medium with kanamycin were cut and incubated in liquid Nitsch medium containing 1 µM concanamycin A (Sigma) for 24 h at 24 °C under agitation at 80 rpm. After the treatment, the roots were washed in water and observed in a confocal microscope. Concanamycin A was prepared as a 100 µM stock solution in absolute DMSO. For the control, roots were incubated in liquid Nitsch medium containing an equal amount of DMSO as in the treatment.
Proteasome inhibitors treatment
The treatment was carried out as described previously (Valverde et al., 2004). Briefly, 10-d-old plants grown on solid Nitsch medium with kanamycin were covered with deionized water containing a cocktail of proteasome inhibitors and incubated for 6 h at 24 °C. Proteasome inhibitors [N-acetyl-Leu-Leu-Norleu-al, Z-Leu-Leu-Norvalinal, Z-Leu-Leu-Leu-al, Z-Ile-Glu(O-t-butyl)-Ala-Leucinal; Sigma] were diluted in absolute DMSO and each used at a final concentration of 10 µM. For a control, the plants were covered with water containing an equal amount of DMSO as in the treatments. After the treatments, the plants were harvested, immediately frozen in liquid nitrogen, and stored at –70 °C before protein extraction.
Protein and immunoblot analyses
Protein extraction, SDS-PAGE, and western blot analyses were performed as previously described (Stepansky and Galili, 2003). Monoclonal anti-haemagglutinin (HA) and anti-GFP antibodies were purchased from Sigma.
Microscopy analysis
For light and fluorescence imaging, an Olympus SZX12 microscope with a x0.3 DF PLFL lens, equipped with a GFP filter and an Olympus U-CMAD3 digital camera, was used. Images were processed with Olympus DP Controller software and Adobe Photoshop 7.0 ME software.
To obtain confocal and differential interference contrast images, an Olympus IX-70 microscope with a UPLAPO x40 0.85 NA lens, equipped with laser-scanning Fluoview 500 confocal unit, 488 nm argon laser, and a GFP filter (505–525 nm), was used. Images were processed with Fluoview software (Olympus, Japan) and Adobe Photoshop 7.0 ME software.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Multiple alignment analysis reveals distinct subclasses of the Arabidopsis Atg8 proteins
By contrast to the single autophagy-related Atg8 gene in yeast, Arabidopsis plants possess nine AtAtg8 homologues (Doelling et al., 2002
; Hanaoka et al., 2002), suggesting multiple functions of gene members. Moreover, the nine Arabidopsis AtAtg8 proteins, although relatively highly conserved, exhibit distinct differences in the sequence of amino acids (data not shown). As shown in the evolutionary tree (Fig. 1), the nine Arabidopsis AtAtg8 isoforms (AtAtg8a–AtAtg8i) can be divided into three distinct subgroups according to their amino acid sequence similarities. The first subgroup includes four proteins (AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8f), the second three proteins (AtAtg8b, AtAtg8e, and AtAtg8g), and the third two proteins (AtAtg8h and AtAtg8i).
|
Different AtAtg8-GUS constructs exhibit distinct GUS staining patterns in different root tissues of young seedlings
To elucidate the expression patterns of the various Arabidopsis AtAtg8 genes in young, non-senescing tissues, five representatives were selected out of the nine AtAtg8 members (AtAtg8a, AtAtg8c, and AtAtg8f from subgroup 1, AtAtg8e from subgroup 2, and AtAtg8h from subgroup 3). The promoters (1.6–3 kb regions upstream of the initiator ATG codon in the various genes) plus the entire coding regions of these genes (including exons and introns) were fused in-frame with the reporter GUS gene, and the various chimeric genes were introduced into Arabidopsis plants. Independently transformed lines expressing a given AtAtg8 isoform showed comparable GUS staining patterns and therefore photographs derived from different lines of each genotype are presented.
Transgenic Arabidopsis plants expressing the five different AtAtg8-GUS constructs were grown under favourable growth conditions and young seedlings were stained for GUS. In these young seedlings, GUS staining generally appeared preferentially in the roots, as depicted by the line expressing the AtAtg8h-GUS construct (Fig. 2A). Yet, a lower level of GUS staining also appeared in the shoots (Fig. 2A, right leaf); this staining could be enhanced upon longer incubation of the GUS staining (data not shown). GUS fusions of some of the different genes yielded different spatial GUS staining patterns, particularly along the distal region of the roots. As shown in Fig. 2B, the AtAtg8e-GUS construct generated significant GUS staining in the vascular tissue of the maturation zone as well as in the root cap, but only trace staining in the root meristem and root elongation zone. The AtAtg8h-GUS and AtAtg8f-GUS constructs generated similar staining patterns to AtAtg8e-GUS, but GUS was more abundant in the maturation zone. The AtAtg8c-GUS construct produced significant GUS staining in the elongation and maturation zones, but only trace staining in the root cap or in the apical meristem (Fig. 2B). The AtAtg8a-GUS construct generated GUS staining along the entire root, but exhibited a higher intensity adjacent to the elongation zone, the apical meristem, and the root cap. However, the GUS staining in the distal root regions of the AtAtg8a-GUS-expressing plants appeared more intense and with a different pattern compared with the staining in plants expressing the other AtAtg8-GUS genes. Cross-sectioning of the highly stained root region in plants expressing AtAtg8a-GUS revealed that staining was confined to the outer cell layers (Fig. 2B, right; see arrow).
|
Response of the five AtAtg8 genes to prolonged darkness and sucrose starvation
The yeast Atg8 gene is known to be involved in starvation-induced autophagy (Kirisako et al., 1999
; Ichimura et al., 2000) and a similar function has been indirectly attributed to this gene family in plants (Doelling et al., 2002; Hanaoka et al., 2002; Yoshimoto et al., 2004). Since GUS staining suggested that the AtAtg8 genes are weakly expressed in shoots (Fig. 2A), a test was done to find out if the expression of any of the five selected AtAtg8-GUS genes was stimulated by prolonged (10 d) exposure to darkness, which inhibits photosynthesis, causing sucrose starvation. GUS staining in two out of the five constructs (AtAtg8a-GUS and AtAtg8h-GUS) was extensively stimulated by this treatment (Fig. 3A; compare top and bottom panels for each construct), while the other three constructs showed a significantly lower response (data not shown).
|
Also tested was the response of the five AtAtg8-GUS constructs to direct sugar starvation. Arabidopsis seedlings were transferred to complete liquid medium for 3 d, after which they were transferred to a new medium, either complete or lacking sucrose, for an additional 3 d. As shown in Fig. 3B, GUS staining of AtAtg8a and AtAtg8h was also extensively induced upon exposure to sugar starvation (left leaf in each construct), compared with the control (right leaf in each construct), while the other constructs showed a significantly lower response (data not shown). These experiments cannot show, however, if the stimulated GUS staining after dark treatment or sugar starvation is due to increased synthesis or to decreased turnover of either the mRNA or proteins encoded by the GUS constructs.
Post-translational processing of the Arabidopsis AtAtg8 proteins
The operation of Atg8 proteins in stress-associated autophagy in yeast and mammals generally requires a processing step that removes a C-terminal portion from these proteins and enables their lipidation into membranes and the formation of autophagosomes (Ichimura et al., 2004). The fate of the Atg8 proteins following the delivery and subsequent degradation of the autophagosomes in the vacuoles is still unknown. These proteins may either penetrate together with the autophagosomes into the vacuoles, and be degraded inside the organelle (Klionsky, 2005), or stick to the outer vacuolar membrane, and then be removed from the outside membrane by an unknown processing event that may use Atg4 (Kirisako et al., 2000). To find out if the AtAtg8 proteins follow similar processing events in young, non-senescing plants grown under favourable growth conditions, the coding sequence of AtAtg8f was fused to GFP at its N-terminus and to an HA epitope tag at its C-terminus. This chimeric construct was fused to the 35S promoter and transformed into Arabidopsis plants. Independently transformed lines expressing GFP-AtAtg8f-HA generally showed very similar polypeptide patterns and GFP fluorescence and, therefore, will be presented in the following examples derived from different independently transformed lines. The processing of the GFP-AtAtg8f-HA polypeptide was studied by germinating the seeds under favourable growth conditions for increasing time periods and analysing proteins in western blots with either anti-HA or anti-GFP antibodies. As shown in Fig. 4A, panel a, the anti-HA antibodies recognized the full-length GFP-AtAtg8f-HA, which was not detected in mature seeds. This full-length protein was first detected 1–2 d after the initiation of germination, and its intensity was progressively reduced from 3 to 7 d after the initiation of germination. The GFP antibodies recognized only a faint band of the full-length GFP-AtAtg8f-HA, as well as two additional smaller polypeptide bands not detected by the anti-HA antibodies (Fig. 4A, panel b, compare with Fig. 4A, panel a). The upper band of these two smaller polypeptides corresponded in size to the expected C-terminal-processed AtAtg8, implying that the AtAtg8 protein was processed in its C-terminus, similar to the processing of its yeast and mammalian counterparts. The size of the lower band corresponded approximately to the size of the GFP domain alone, implying further processing of the AtAtg8 protein. As antibodies against the AtAtg8 proteins were not available, it was not possible to distinguish whether the appearance of this smaller band, corresponding to GFP alone, represents a cleavage of the AtAtg8 domain from the GFP domain or that it represents a complete degradation of the Atg8 domain. Yet, it is believed that the appearance of the smaller band signifies the degradation of the Atg8 protein, leaving the relatively stable GFP protein quite intact (see Discussion). Notably, only the full-length GFP-AtAtg8-HA band and that corresponding to the C-terminal-processed GFP-AtAtg8-HA protein were clearly detected 1–2 d after the initiation of germination (Fig. 4A, panel b), while the lower band corresponding in size to the GFP domain alone was detected only 3–4 d after the initiation of germination (Fig. 4A, panel b). This implies that processing of the C-terminal region precedes the second processing step of the Atg8 polypeptide during seed germination. In addition, the intensity of the C-terminal-processed GFP-AtAtg8-HA band was progressively reduced, while that of the GFP band was progressively increased with the progression of seed germination (Fig. 4A, panel b), implying that the C-terminal-processed form, but not the full-length GFP-AtAtg8-HA is converted into the GFP domain alone. The band corresponding to the GFP protein was also detected in mature seeds (Fig. 4A, panel b), implying that Atg8 proteins are also synthesized and processed during seed development.
|
Germination in the dark enhances the processing of the AtAtg8 proteins
Tests were also carried out to see if germination in the dark would affect the timing of appearance of the various protein bands associated with GFP-AtAtg8-HA. Similar to germination in the light, upon germination in the dark, the full-length GFP-AtAtg8-HA (detected by the anti-HA antibodies), as well as its C-terminal-processed intermediate (detected by the anti-GFP antibodies), first appeared at around 2 d after the initiation of germination and then gradually reduced as the germination progressed (Fig. 4B, panels a, b, compare with Fig. 4A, panels a, b). By contrast, upon germination in the dark, the GFP domain started accumulating alone 1–2 d after the initiation of germination, significantly before its appearance in seeds germinated in the light (Fig. 4B, panel b, compare with Fig. 4A, panel b).
The AtAtg8 proteins are also processed in a comparable manner both in roots and shoots under favourable growth conditions
Since the GFP-AtAtg8-HA construct was driven by the constitutive 35S promoter, it was possible to use the GFP-AtAtg8-HA-expressing transgenic plants to test whether the machinery needed for the two processing events of the Atg8 proteins is active in both roots and shoots. To address this, 10-d-old seedlings, grown under favourable growth conditions, were separated into roots and shoots, and proteins were reacted with the anti-GFP antibodies. As shown in Fig. 5, the C-terminal-processed form of GFP-AtAtg8-HA, as well as the GFP domain alone, was detected in both roots and shoots, implying that the GFP-AtAtg8-HA was processed in a similar manner both in roots and shoots.
|
The AtAtg8 proteins associate with autophagosome-resembling structures and are transported into vacuoles under favourable growth conditions
Although the function of the Atg8 proteins is generally associated with stress, their abundant expression, particularly in roots of young, non-senescing plants grown under favourable growth conditions, prompted tests to find out if the Atg8 proteins are also associated with structures resembling autophagosomes under favourable growth conditions. This was analysed by in planta GFP fluorescence. Individual plants of a given line showed variable GFP fluorescence, which varied from no fluorescence to intense fluorescence. Moreover, despite the fact that the GFP-AtAtg8-HA was produced at comparable levels in roots and shoots (Fig. 5), GFP fluorescence was mostly detected in roots (Fig. 6A). Also, in some cases, GFP fluorescence was observed only in some lateral roots, but not in others of the same plant, while in others, as demonstrated in Fig. 6A and B, it was detected in the entire root. The same kind of variable fluorescence was also detected upon exposure to starvation stresses (data not shown), indicating that it was not related to stress conditions. The reason for this variable GFP fluorescence pattern is not clear, but comparing the GFP fluorescence data with those obtained from the western blots suggests that the variable fluorescence may be related to the GFP itself and not to its attached Atg8 polypeptides. It is possible that the apparent second processing step of the AtAtg8 may, in some cases, trim portions from the N-terminus of the GFP, which is essential for its activity (Li et al., 1997
). Indeed, in some experiments, the GFP-related band migrated slightly more rapidly than the intact GFP (data not shown). Therefore, in the following, only higher magnifications derived from root sections that showed GFP fluorescence will be considered.
|
Higher magnification analyses of root tip cells showed that GFP fluorescence is mostly associated with autophagosome-resembling spots, which are most abundant in the cytoplasm surrounding the central vacuoles (Fig. 6C, E). Such structures were not observed in roots of control plants expressing the GFP polypeptide alone (Fig. 6I, J), implying that they were specific to the AtAtg8 polypeptide. Similar appearance of GFP fluorescence in autophagosome-resembling spots, located mostly in the cytosol surrounding the central vacuoles, was also observed in larger epidermal cells above the root tips, which contain large central vacuoles occupying most of the cell volume (Fig. 6G). Although not clearly seen in Fig. 6G, some GFP-fluorescing spots were also detected inside the central vacuoles of these large epidermal cells. In some experiments involving plants expressing GFP-AtAtg8f-HA, GFP fluorescence appeared more dispersed in the cytosol and apparently localized to much smaller spots than those shown in Fig. 6E (data not shown). Whether this reflects biological variation or technical variation associated with the preparation of the plants for analysis in the confocal microscope is not known.
Since in yeast the Atg8 proteins deliver autophagosome into the vacuoles (Lang et al., 1998), it was of interest to verify further whether the very weak GFP fluorescence inside the central vacuoles was due to the acidic pH of the vacuoles that may interfere with GFP fluorescence (Tamura et al., 2003). To address these issues, root cells of the GFP-AtAtg8-HA-expressing plants were incubated for 24 h with concanamycin A, which inhibits the activities of vacuolar ATPases and hence slows down the acidification of the vacuole sap (Drose et al., 1993). As shown in Fig. 7C, concanamycin A treatment caused a general enhancement of GFP fluorescence in epidermal cells, above the root tips (compare the fluorescence intensities in Fig. 7C and A showing cells of untreated plants relative to the laser intensity provided in the legend). Moreover, following the concanamycin A treatment, a large number of fluorescing spots was also present inside the vacuoles (Fig. 7C). A similar concanamycin A treatment did not cause any significant internalization of a control GFP polypeptide into the vacuole (Fig. 7E, G), implying that the AtAtg8-containing structures are specifically and efficiently internalized into the central vacuoles in root cells of plants grown under favourable growth conditions. Notably, the control GFP polypeptide, but not GFP-AtAtg8-HA, was also localized to the nucleus (Fig. 7G, see arrow). In a few concanamycin A-treated root sections of plants expressing the control GFP polypeptide, a few cells appeared in which a low amount of GFP was also present in the vacuole (Fig. 7G, see asterisk). Yet, no GFP was detected in the vacuoles in other cells near them in the same section (Fig. 7G). In addition, the intensity of GFP in the vacuoles of these cells was significantly lower then that in root cells expressing the GFP-AtAtg8 polypeptide. The reason for this is not yet clear. Concanamycin A had no effect on the steady-state levels of the processing products of the GFP-AtAtg8-HA polypeptides, as determined in western blots with anti-GFP and anti-HA antibodies (data not shown), implying that the concanamycin A effect was largely due to increased vacuolar pH rather then the stability of the GFP-AtAtg8-HA polypeptides.
|
Since the AtAtg8 proteins were efficiently processed and transported to vacuoles under favourable growth conditions, it was of interest to determine whether the proteasome machinery is involved in the processing of the Atg8 protein, particularly the removal of the Atg8 protein, leaving the ‘GFP alone’ band shown in Figs 4 and 5. To address this, 10-d-old seedlings of the GFP-AtAtg8-HA-expressing plants, grown under favourable conditions, were treated with a cocktail of proteasome inhibitors for 6 h, and proteins were reacted in a western blot with the anti-GFP antibodies. This cocktail affected neither the pattern nor the actual levels of the various GFP-AtAtg8-HA-associated polypeptides (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Expression and intracellular localization studies suggest that the cellular process involving Atg8 operates in plants both under favourable growth conditions and under starvation stresses
While yeast cells possess a single Atg8 protein, higher eukaryotes (animals and plants) contain large families of Atg8 proteins, suggesting multiple functions. Indeed, recent studies in mammals suggest that the Atg8 proteins function not only in autophagy but also in other cellular processes such as vesicular transport via the Golgi (Sagiv et al., 2000
) and post-mitotic Golgi reassembly (Muller et al., 2002). In the present report it was of interest to study whether in young, non-senescing tissues of Arabidopsis plants, the cellular process using Atg8 operates both under starvation stresses and under favourable growth conditions. For this, young transgenic plants expressing GUS fusions to the promoters plus the entire coding regions (introns and exons) of five Arabidopsis AtAtg8 genes, as well as transgenic plants expressing a GFP-AtAtg8-HA fusion protein, were used. Analysis of the plants used in this study, as discussed later in more detail, shows that, in young plants, the cellular processes involving Atg8 operates both under favourable growth conditions and under starvation stresses. Due to the nature of the GUS constructs that contained promoter as well as exons and introns of the AtAtg8 genes, it is not possible to determine whether the observed changes in GUS staining are due to the promoter regions alone or to additional sequences derived from the coding regions of the various AtAtg8 genes.
Differential GUS staining patterns in young roots and plausible function of distinct AtAtg8 genes in this organ under favourable growth conditions
Under favourable growth conditions, all five Arabidopsis AtAtg8 homologues were abundantly expressed in young roots. However, certain members of this family generated differential GUS staining patterns in various root regions, suggesting differential non-redundant functions that may be associated with root development and physiology. Based on the GUS staining patterns, the AtAtg8 genes function predominantly in root caps and in the region of maturation, root parts that are strongly associated with protein degradation. Root caps function in penetration into the soil, a process associated with extensive cell death due to mechanical friction and the formation of new cells. Extensive degradation of macromolecules in this region may enable metabolite remobilization from old to newly formed cells. Root tissues in the region of maturation are strongly associated with the degradation process associated with the mobilization of metabolites (particularly nitrogenous compounds) to the above-ground tissues. Hence, it is feasible that plant Atg8 proteins are involved in macromolecule degradation in root tissues grown under favourable growth conditions, in a similar manner to their function in macromolecule degradation during starvation-induced autophagy (Kirisako et al., 1999).
The GUS staining patterns also suggest that some AtAtg8 genes operate in root meristems and elongation zones, which are largely engaged in cell division and differentiation, respectively. As such, these regions are characterized by extensive biosynthesis, rather than catabolic activities. Hence, it is likely that in these tissues the cellular machinery using Atg8 proteins is involved in cellular remodelling associated with root growth and development.
The AtAtg8 genes are weakly expressed in shoots under favourable growth conditions, but the expression of some of them is induced by starvation stresses
By contrast to roots, GUS staining was very weak in young shoots of transgenic plants grown under favourable growth conditions (Fig. 2A), indicating that under favourable growth conditions the cellular process using Atg8 is not as strongly needed as in young roots. In shoots of plants grown under favourable conditions, the process of autophagy is apparently utilized profoundly only in mature leaves entering senescence to enable efficient macromolecule degradation for metabolite mobilization into the floral organs (Hanaoka et al., 2002). Nevertheless, extensive stimulation of GUS staining was generated in leaves by two AtAtg8-GUS constructs in response to prolonged darkness or sugar starvation. This implies that the process involving Atg8 may be particularly needed in leaves under starvation stresses, supporting previous reports on the induction of autophagy in plant cells exposed to starvation (Aubert et al., 1996). The present results do not exclude the possibility that, in mature plants entering senescence, the expression of additional AtAtg8 genes is stimulated by sugar starvation.
AtAtg8 proteins are processed at their C-termini and are internalized into vacuoles in young plants under favourable growth conditions
The function of Atg8 proteins is not only dependent on their synthesis, but also on their C-terminal processing that enables them to attach to lipids (Ichimura et al., 2004). Indeed, it has been shown here that the AtAtg8f protein is efficiently processed at its C-terminus in young plants grown under favourable growth conditions, and also follows a second yet unclear processing event. Moreover, it has also been shown that in young plants grown under favourable conditions, the AtAtg8f protein is efficiently associated with autophagosome-resembling structures that are internalized into the vacuoles. The present results therefore imply that, in young plants, the machineries responsible for these processes operate with comparable efficiency under favourable growth conditions and starvation stresses, and support the possible functions of the Atg8 proteins in multiple degradation processes associated with cellular remodelling during plant growth and development. Notably, under favourable growth conditions, the processing machinery of the AtAtg8 proteins was also active in shoots in which the various AtAtg8 genes are not abundantly expressed, indicating that this machinery may not only regulate processes involving Atg8.
Upon the progression of seed germination, the C-terminal-processed intermediate of GFP-AtAtg8-HA appeared alone significantly before the GFP domain, implying that, during germination, the AtAtg4 protease-dependent machinery responsible for the C-terminal processing of the AtAtg8 proteins (Yoshimoto et al., 2004) is synthesized much earlier than the as yet unknown machinery required for the second processing event of the AtAtg8 proteins. The physiological and mechanistic consequences of this observation await future studies.
Interestingly, germination in the dark, which apparently causes sugar starvation, stimulated the biogenesis of the machinery regulating the second processing step of the AtAtg8f protein (resulting in the appearance of the ‘GFP alone’ band; Figs 4, 5), supporting a stronger need for the machinery controlling this second processing event during seed germination under starvation stresses (Moriyasu and Ohsumi, 1996; Takatsuka et al., 2004). The exact nature of this second processing event is still unknown. However, since the GFP-AtAtg8f polypeptide was efficiently internalized into vacuoles, it is believed that this processing event signifies the degradation of the AtAtg8 protein in the vacuole. This is also supported by the fact that proteasome inhibitors did not affect this second processing event.
Atg8, a multifunctional protein family
The presence of multiple AtAtg8 isoforms and their differential expression patterns in distinct tissues and under both favourable growth conditions and starvation stresses imply that the proteins serve multiple non-redundant functions in plants at both their young and mature stages. Performance of such multiple functions could operate through the capacity of the different Atg8 isoforms to interact with a variety of macromolecules, such as proteins and lipids (Ichimura et al., 2000; Sagiv et al., 2000). The Atg proteins of yeast and mammals adopt a ubiquitin-like structure (Paz et al., 2000) and, based on the high homology of plant, yeast, and mammal Atg8 proteins, it is probable that the plant Atg8 proteins also adopt a similar structure. Since a unique characteristic of ubiquitin is its ability specifically to interact, via conjugation, with a wide range of target proteins and mark them for degradation by the 26 proteasomes (Hershko and Ciechanover, 1998), it is possible that plant Atg8 also adapted similar structural characteristics to enable their functions.
|
Acknowledgements |
|---|
We thank Vladimir Kiss for his invaluable help in the confocal microscopy. This work was supported by The United States–Israel Binational Science Foundation (BSF) Grant No. 1999-355, and by the European Union Research Training Network Contract No. HPRN-CT-2002-00262 to GG, and by the Israeli Science Foundation (ISF) Grant no. 1042/03 and WIS Minerva Foundation to ZE. GG is an incumbent of the Bronfman Chair in Plant Sciences.
|
Footnotes |
|---|
* These authors contributed equally to this work.
Abbreviations: DMSO, dimethyl sulphoxide; GFP, green fluorescent protein; GUS, ß-glucuronidase; HA, haemagglutinin; PCR, polymerase chain reaction.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aubert S, Gout E, Bligny R, Marty-Mazars D, Barrieu F, Alabouvette J, Marty F, Douce R. 1996. Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates. Journal of Cell Biology 133, 1251–1263.
Baba M, Osumi M, Scott SV, Klionsky DJ, Ohsumi Y. 1997. Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. Journal of Cell Biology 139, 1687–1695.
Bryant NJ, Stevens TH. 1998. Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole. Microbiology and Molecular Biology Reviews 62, 230–247.
Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743.[CrossRef][Web of Science][Medline]
Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD. 2002. The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana. Journal of Biological Chemistry 277, 33105–33114.
Drose S, Bindseil KU, Bowman EJ, Siebers A, Zeeck A, Altendorf K. 1993. Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32, 3902–3906.[CrossRef][Medline]
Elazar Z, Scherz-Shouval R, Shorer H. 2003. Involvement of LMA1 and GATE-16 family members in intracellular membrane dynamics. Biochimica et Biophysica Acta –Molecular Cell Research 1641, 145–156.[CrossRef]
Hajdukiewicz P, Svab Z, Maliga P. 1994. The small, versatile Ppzp family of Agrobacterium binary vectors for plant transformation. Plant Molecular Biology 25, 989–994.[CrossRef][Web of Science][Medline]
Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y. 2002. Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiology 129, 1181–1193.
Harding TM, Hefner-Gravink A, Thumm M, Klionsky DJ. 1996. Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein targeting pathway. Journal of Biological Chemistry 271, 17621–17624.
Hershko A, Ciechanover A. 1998. The ubiquitin system. Annual Review of Biochemistry 67, 425–479.[CrossRef][Web of Science][Medline]
Huang WP, Klionsky DJ. 2002. Autophagy in yeast: a review of the molecular machinery. Cell Structure and Function 27, 409–420.[CrossRef][Web of Science][Medline]
Ichimura Y, Kirisako T, Takao T, et al. 2000. A ubiquitin-like system mediates protein lipidation. Nature 408, 488–492.[CrossRef][Web of Science][Medline]
Ichimura Y, Mamura Y, Emoto K, Umeda M, Noda T, Ohsumi Y. 2004. In vivo and in vitro reconstitution of atg8 conjugation essential for autophagy. Journal of Biological Chemistry 279, 40584–40592.
Kirisako T, Baba M, Ishihara N, Miyazawa K, Ohsumi M, Yoshimori T, Noda T, Ohsumi Y. 1999. Formation process of autophagosome is traced with Apg8/Aut7p in yeast. Journal of Cell Biology 147, 435–446.
Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y. 2000. The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. Journal of Cell Biology 151, 263–276.
Klionsky DJ, Cregg JM, Dunn WA, et al. 2003. A unified nomenclature for yeast autophagy-related genes. Developmental Cell 5, 539–545.[CrossRef][Web of Science][Medline]
Klionsky DJ. 2005. The molecular machinery of autophagy: unanswered questions. Journal of Cell Science 118, 7–18.
Lang T, Schaeffeler E, Bernreuther D, Bredschneider M, Wolf DH, Thumm M. 1998. Aut2p and Aut7p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole. EMBO Journal 17, 3597–3607.[CrossRef][Web of Science][Medline]
Legesse-Miller A, Sagiv Y, Glozman R, Elazar Z. 2000. Aut7p, a soluble autophagic factor, participates in multiple membrane trafficking processes. Journal of Biological Chemistry 275, 32966–32973.
Li X, Zhang G, Ngo N, Zhao X, Kain SR, Huang CC. 1997. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. Journal of Biological Chemistry 272, 28545–28549.
Mann SS, Hammarback JA. 1994. Molecular characterization of light chain 3. A microtubule binding subunit of MAP1A and MAP1B. Journal of Biological Chemistry 269, 11492–11497.
Moriyasu Y, Ohsumi Y. 1996. Autophagy in tobacco suspension-cultured cells in response to sucrose starvation. Plant Physiology 111, 1233–1241.[Abstract]
Muller JMM, Shorter J, Newman R, Deinhardt K, Sagiv Y, Elazar Z, Warren G, Shima DT. 2002. Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion. Journal of Cell Biology 157, 1161–1173.
Nitsch JP. 1970. Experimental androgenesis in Nicotiana. Phytomorphology 19, 389–404.[Web of Science]
Paz Y, Elazar Z, Fass D. 2000. Structure of GATE-16, membrane transport modulator and mammalian ortholog of autophagocytosis factor aut7p. Journal of Biological Chemistry 275, 25445–25450.
Sagiv Y, Legesse-Miller A, Porat A, Elazar Z. 2000. GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28. EMBO Journal 19, 1494–1504.[CrossRef][Web of Science][Medline]
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425.[Abstract]
Shintani T, Huang WP, Stromhaug PE, Klionsky DJ. 2002. Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway. Developmental Cell 3, 825–837.[CrossRef][Web of Science][Medline]
Stepansky A, Galili G. 2003. Synthesis of the Arabidopsis bifunctional lysine-ketoglutarate reductase/saccharopine dehydrogenase enzyme of lysine catabolism is concertedly regulated by metabolic and stress-associated signals. Plant Physiology 133, 1407–1415.
Takatsuka C, Inoue Y, Matsuoka K, Moriyasu Y. 2004. 3-Methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions. Plant and Cell Physiology 45, 265–274.
Tamura K, Shimada T, Ono E, Tanaka Y, Nagatani A, Higashi SI, Watanabe M, Nishimura M, Hara-Nishimura I. 2003. Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. The Plant Journal 35, 545–555.[CrossRef][Web of Science][Medline]
Valverde F, Mouradov A, Soppe W, Ravenscroft D, Samach A, Coupland G. 2004. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303, 1003–1006.
Wang CW, Klionsky DJ. 2003. The molecular mechanism of autophagy. Molecular Medicine 9, 65–76.[Web of Science][Medline]
Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW. 1999. GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton. Nature 397, 69–72.[CrossRef][Medline]
Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y. 2004. Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. The Plant Cell 16, 2967–2983.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Chung, A. Suttangkakul, and R. D. Vierstra The ATG Autophagic Conjugation System in Maize: ATG Transcripts and Abundance of the ATG8-Lipid Adduct Are Regulated by Development and Nutrient Availability Plant Physiology, January 1, 2009; 149(1): 220 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Slavikova, S. Ufaz, T. Avin-Wittenberg, H. Levanony, and G. Galili An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses J. Exp. Bot., October 3, 2008; (2008) ern244v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Phillips, A. Suttangkakul, and R. D. Vierstra The ATG12-Conjugating Enzyme ATG10 Is Essential for Autophagic Vesicle Formation in Arabidopsis thaliana Genetics, March 1, 2008; 178(3): 1339 - 1353. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fujioka, N. N. Noda, K. Fujii, K. Yoshimoto, Y. Ohsumi, and F. Inagaki In Vitro Reconstitution of Plant Atg8 and Atg12 Conjugation Systems Essential for Autophagy J. Biol. Chem., January 25, 2008; 283(4): 1921 - 1928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Muntz Protein dynamics and proteolysis in plant vacuoles J. Exp. Bot., July 1, 2007; 58(10): 2391 - 2407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Inoue, T. Suzuki, M. Hattori, K. Yoshimoto, Y. Ohsumi, and Y. Moriyasu AtATG Genes, Homologs of Yeast Autophagy Genes, are Involved in Constitutive Autophagy in Arabidopsis Root Tip Cells Plant Cell Physiol., December 1, 2006; 47(12): 1641 - 1652. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||