Golgi Apparatus-Localized Synaptotagmin 2 Is Required for Unconventional Secretion in Arabidopsis

Background Most secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYGR) can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYGR in plant cells remain unknown. Synaptotagmins (SYTs) are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear. Methodology/Principal Findings We found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYGR caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYGR, which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYGR-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYGR-GFP was truncated at carboxyl terminus of HYGR shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYGR-GFP,resulting in HYGR-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYGR-GFP trafficking and secretion. Conclusion/Significance These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYGR-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells.


Introduction
The secretory pathway traditionally contains a number of biochemically distinct inter-related membrane organelles that continuously communicate with each other and exchange materials through membrane trafficking. The classical secretory proteins are often extended at their N-terminus by a 'leader' or 'signal' sequence of 13-30 hydrophobic amino acids. This directs the nascent protein to co-translate and vectorially transfer across the membrane of the endoplasmic reticulum (ER), and is often cleaved before completion of the transmembrane transport of the protein [1,2]. Secretory proteins are then transported to the Golgi apparatus and trans-Golgi network where they undergo further glycosylation, and sorting and being packaged into vesicles, respectively. Finally the secretory vesicles are delivered to and fuse with the plasma membrane, resulting in releasing their contents into the extracellular space [3].
However, numerous secretory proteins with normal extracellular functions have been shown to be devoid of functional signal sequences and do not appear substrates for the ER membrane translocation machinery. In addition, the secretion of these proteins is not affected by the presence of brefeldin A, a drug that blocks ER/Golgi-dependent secretory transport [4][5][6]. These observations suggest that alternative secretory mechanisms that are independent of ER/Golgi secretory pathway exist in eukaryotic cells. Secretion of proteins without an N-terminal signal sequence is currently known as the unconventional/nonclassical secretory pathway or leaderless secretion. Up to date, several unconventional secretory pathways have been reported for a few biomedically important factors, including proangiogenic mediators such as fibroblast growth factors 2 and inflammatory cytokines such as interleukin 1a and 1b in mammalian cells [5,7]. Plant secretome revealed that more than half of the total identified proteins were leaderless secretory proteins, which is distinctly higher than in human and yeast secretomes, implying that this unconventional secretory mechanism is common to all eukaryotes and it is more largely used than in other eukaryotes [8]. Furthermore, plants exposed to biotic and abiotic stresses usually significantly contained more leaderless secretory proteins in the extracelluar space than non-stressed plants, suggesting that environmental component might be involved in release of leaderless secretory proteins into the extracelluar space [8]. However, until now, only one leaderless secretory protein, mannitol dehydrogenase (MTD) in celery, has been shown to bypass the ER-Golgi-plasma membrane exocytic pathway for its delivery to the extracellular space by molecular biology and biochemistry approaches [6].
Synaptotagmins (SYTs) constitute a family of membranetrafficking proteins that are characterized by an N-terminal transmembrane region, a linker of variable size, and two Cterminal C2 domains in tandem [9]. SYTs are reported to play a vital role in neurotransmitter release and insulin exocytosis in mammalian cells [10][11][12][13].The synaptotagmin family in Arabidopsis has five members. SYT1, the only one characterized so far, is ubiquitously expressed and predominantly localized to the plasma membrane [14]. Disruption of SYT1 function in Arabidopsis leads to abiotic stresses hypersensitivity due to a reduced integrity of the plasma membrane [14,15]. However, the subcellular localization and the functions of other SYTs remain unknown.
Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells by inhibiting protein synthesis [16,17]. It has been reported that hygromycin B acts by interfering with translocation and causes mistranslation [18]. An Escherichia coli gene has been identified that confers resistance in transgenic plants against hygromycin B. The resistance gene codes for hygromycin B phosphotransferase (HYG R , E.C. 2.7.1.119) that adds phosphate to position 7 of the destomic acid ring of hygromycin B, which results in complete loss of biological activity both in vitro and in vivo [19]. Although HYG R has been mainly used as a positive selective marker for transgenic cells [20], few studies have examined the subcellular localization and trafficking of HYG R and the putative elements that regulate the tolerance of HYG R -expressing cells to hygromycin B.
Here, we provided several lines of evidence about localization of Arabidopsis synaptotagmin SYT2. More importantly, we found that HYG R is present both in the cytoplasm and the extracelluar space in HYG R -GFP-transgenic plants. The loss of SYT2 caused inhibition of HYG R -GFP trafficking. Based on the fact that HYG R -GFP lacks a signal sequence and its secretion is not sensitive to brefeldin A treatment, we propose that HYG R -GFP is not secreted via the conventional secretory pathway and SYT2 plays an important role in regulating the unconventional protein trafficking from the cytosol to the extracelluar matrix in plant cells.

Characterization of Arabidopsis SYT2 Protein
The synaptotagmin 2 gene (SYT2, At1g20080) is one of five putative SYTs in Arabidopsis. It comprises 12 exons and 11 introns, based on information available in the Arabidopsis Information Resource database (TAIR; http://www.arabidopsis.org/) ( Figure 1A). Homology analysis using amino acid sequence data showed that SYT1 is the closest relative of SYT2 in Arabidopsis, with about 66% amino acid identity between them [15]. Compared to SYT1, all amino acid residues thought to play crucial roles in coordinating calcium (Ca) ions are conserved in the C2A domain of SYT2 ( Figure 1B). Unlike SYT1, however, only four putative amino acids of the SYT2 C2B domain are involved in Ca binding (lacking the fourth putative residue) ( Figure 1C). According to SYT1 expression profiles based on microarray expression data obtained from Geneinvestigator (http://www. genevestigator.ethz.ch), SYT2 is highly expressed in pollen grains, whereas expression in other organs, such as roots or leaves, is detectable but low ( Figure S1). A secretory signal peptide was predicted in the SYT2 amino acid sequence but neither a chloroplast transit peptide nor a mitochondrial targeting peptide was identified using the TargetP 1.1 server (http://www.cbs.dtu. dk/services/TargetP/).

SYT2 does not Colocalize with BFA Compartment
To investigate the subcellular localization of SYT2, we fused the gene encoding green fluorescent protein (GFP) to the SYT2 gene (C-terminal end of the encoded protein) under the control of the 35S promoter of the cauliflower mosaic virus (CaMV35S) and used these constructs to transiently or stably transform Nicotiana tabacum and Arabidopsis. The resulting fusion protein (SYT2-GFP) was primarily detected in mobile punctate structures in leaf cells of transiently transformed N. tabacum and Arabidopsis (Figure 2A and 2B). Plant lines stably expressing SYT2-GFP were also analyzed by laser scanning confocal microscope (LSCM) to localize the fusion protein. The fluorescence signals appeared as punctate structures with a dim cytosolic background in root hairs, root meristem cells and elongation cells ( Figure 2C to 2F).
To investigate whether the SYT2-positive structures were of endosomal origin, transgenic SYT2-GFP Arabidopsis seedlings were incubated with FM4-64, a fluorescent marker internalized by a clathrin-dependent process and sequentially labels early endosomal, late endosomal, and vacuolar compartments [21][22][23][24]. As shown in Figure 3, the internalized FM4-64 dye rarely co-localized with SYT2-GFP-containing compartments in root cells even after 2 h of incubation, during which time FM4-64 was detected in vacuolar membranes ( Figure 3A to 3I). Co-localization studies were also performed using seedlings expressing VHA-a1-GFP, ARA6-GFP, and ARA7-GFP, all of which have been reported to reside on endosomes and regulate endosomal fusion [21,23,25]. Co-localization of FM4-64 with large amounts of VHA-a1-GFP and ARA6-GFP ( Figure 3J to 3O) and lesser amounts of ARA7-GFP ( Figure 3P to 3R), was detected after 30 min, demonstrating that SYT2-GFP is targeted to a compartment independent of endosomal membranes.
The endosomes in Arabidopsis root tips are the main target of the fungal toxin brefeldin A (BFA). This drug inhibits certain ADP ribosylation factor/guanine nucleotide exchange factors (ARF-GEFs) and causes the endocytic tracer FM4-64 to rapidly aggregate throughout vesicle agglomerations known as BFA compartments [26][27][28]. To investigate whether SYT2-GFP was associated with BFA-sensitive endosomes, the transgenic plants were treated with 25 mM BFA. After BFA treatment, the punctate SYT2-GFP structures were almost intact and did not accumulate in BFA compartments ( Figure S2A to S2F), while VHA-a1-GFP (early endosome marker) and ARA6-GFP (late endosome marker) perfectly overlapped with and was located at the periphery of the BFA compartments, respectively ( Figure S2G to S2L) [23,29].
To further demonstrate that SYT2-GFP-containing structures are excluded from the late endosomes, we analyzed the effect of wortmannin, which inhibits the biosynthesis of phosphatidylinositol 3-and 4-phosphates as well as phospholipids in plant cells [30]. Exogenous application of wortmannin causes the late endosomes to dilate or form ring-shaped structures, but has no effect on the Golgi apparatus and early endosomes [29,31]. The morphology of SYT2-GFP structures was not altered and the two markers were almost separated ( Figure S3A to S3C). As previously reported for ARA6-GFP and ARA7-GFP, both of which localize  on the late endosomes [29], wortmannin caused the formation of ring-shaped structures ( Figure S3D to S3I). Furthermore, SYT2 protein did not colocalize with ARA7-GFP by immunofluorescent labelingusing anti-SYT2 and anti-GFP antibodies ( Figure S3J to S3L). Taken together, these data demonstrate that SYT2-GFP does not localize on the late endosomes.

SYT2 is Localized on the Golgi Apparatus
The punctate structures labeled by SYT2-GFP were insensitive to BFA and wortmannin treatment and did not become labeled with FM4-64, reminiscent of the Golgi apparatus. In addition to its punctate appearance, the Golgi apparatus did not become colocalized with the endocytic tracer FM4-64 or with BFA compartments in Arabidopsis root-tip cells [32][33][34]. To determine if SYT2-containing structures were associated with Golgi apparatus, we performed an immunofluorescent study on wild-type and SYT2-GFP-overexpressing plants. Antibodies were generated against the cytoplasmic region (300 aa-535 aa) of SYT2 (anti-SYT2). Affinity purified anti-SYT2 antibodies was analyzed by western blotting of wild type and SYT2-GFP transgenic Arabidopsis seedlings, recognizing proteins of 61 KD and 87 kD corresponding to both native SYT2 and recombinant SYT2-GFP respectively ( Figure 4A). In order to determine whether the generated antibodies were specific for SYT2 and did not recognize other SYT family members, including the closely related SYT1, a mutant in SYT2 from the SALK collection (SALK_135307, syt2-1) with the T-DNA located in the 9th exon of At1g20080 was isolated and further analyzed. No mRNA transcripts and proteins were detected in the homozygous syt2-1 line, despite the fact that SYT1 was expressed at wild-type levels in the mutant ( Figure 4A and 4B). In view of the lower SYT2 transcripts in vegetative tissues analyzed by microarray ananlysis ( Figure S1), it is suggested that SYT2 protein production is probably regulated at the level of translation.
As shown in Figure 4D, SYT2 became immunolocalized into punctate structures were similar to those observed in plants expressing SYT2-GFP. Furthermore, most of the co-localization between SYT2 and SYT2-GFP occurred in transgenic plants overexpressing SYT2-GFP, as confirmed using anti-SYT2 and anti-GFP antibodies ( Figure 4E). SYT2 localization was further analyzed by double-immunofluorescent labeling with anti-SYT2 and anti-GFP antibodies in plants expressing ST-YFP, a well described Golgi marker [23,32]. SYT2 likewise co-localized with ST-YFP ( Figure 4F). Immuno-labeling of ultra-thin sections of Arabidopsis root cells using anti-SYT2 antibodies showed that gold particles mainly deposited on the Golgi apparatus ( Figure 4G to 4L).

Co-expression of SYT2 and HYG R Leads to Hypersensitivity to Hygromycin B
Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells [16]. The hygromycin B phosphotransferase (HYG R ) phosphorylates and inactivates hygromycin B, and has been widely used as a selectable marker in the generation of transgenic plants. To investigate the subcellular localization of SYT2 in plant cells as described above, the binary vector (pCambia1301-SYT2/HYG R ), which was generated by pCam-bia1301 from T-DNA containing a SYT2-GFP expression cassette and a hygromycin B-selectable marker, was introduced into Arabidopsis seedlings by Agrobacterium-mediated transformation. However, we noticed that the positively transgenic plants (named SYT2/HYG R ) grew weakly when selected on 20 mg/mL hygromycin B-containing medium. These plants had low viability or showed slow growth and the apparent loss of apical dominance (inhibition of the primary inflorescence) following the development of two symmetrical axillary buds after their transfer to soil ( Figure   S4A to S4C). T2 and T3 seedlings exhibited wild-type growth in the absence of hygromycin B. To observe whether SYT2 was associated with uptake of hygromycin B, wild-type and syt2-1 plants were incubated on K Murashige and Skoog (MS) medium containing different concentrations of hygromycin B. As shown in Figure S5, the phenotype of syt2-1 is not obviously different from that of wild type under hygromycin B treatment, indicating that SYT2 is not probably related with the uptake of hygromycin B in Arabidopsis. Therefore, it is presumed that SYT2 contributes to the detoxification of HYG R in HYG R -containing plants. To investigate the effect of SYT2 on hygromycin B tolerance, wild-type and syt2-1 plants were transformed with a 35S-HYG R construct and the transgenic plants were named as HYG R and syt2-1/HYG R , respectively.
We analyzed the expression of the HYG R gene in these transgenic lines using semi-quantitative RT-PCR ( Figure 5A). The expression level of HYG R gene was similar in the selected lines. We further investigated the growth of these lines on K MS medium agar plates with different concentrations of hygromycin B. The growth of the primary roots and hypocotyls of SYT2/HYG R seedlings was greatly inhibited even on the medium containing as low as 5 mg/mL hygromycin B ( Figure 5B). The roots of SYT2/ HYG R seedlings were 30.1%, 9.3% and 7.1% of that of HYG R seedlings in the presence of 5, 10 and 20 mg/mL of hygromycin B, respectively ( Figure 5C). Microscopic observation also revealed that the root hairs and roots of SYT2/HYG R seedlings were greatly shortened ( Figure S4D to S4L), suggesting that a reduced function of HYG R caused by the over-expression of SYT2.

HYG R -GFP is Exported via an Unconventional Secretory Pathway
To obtain the clues as to why co-expression of SYT2-GFP and HYG R caused hypersensitivity to hygromycin B in Arabidopsis, we first analyzed the subcellular localization of a translational fusion between HYG R and GFP under the control of the constitutive promoter (CaMV35S) in stable transgenic lines. The fluorescent signals from HYG R -GFP were present on the cell surface of root cells and interestingly HYG R -GFP was preferentially expressed in leaf-tip zones ( Figure 6A to 6C). In the plasmolyzed cells, HYG R -GFP was found in the cytoplasm as well as in the cell walls ( Figure 6D and 6E). As it has been mentioned that the HYG R protein could be secreted in plants [35], we first investigated the secretory property of the HYG R -GFP protein by protein gel blot using mesophyll protoplasts of HYG R -GFP plants. Unexpectedly, in protoplast lysates, the expected band of full length HYG R -GFP (about 65 kD) was not detected when anti-HYG R antibody was applied, but a band with a molecular weight between 34-43 kD (about the molecular weight of HYG R protein) was detected both in the medium and in the protoplast lysates ( Figure 6F), suggesting that HYG R protein was secreted into extracellular space. When anti-GFP antibody was applied, the HYG R-GFP protein appeared as a single band of approximately 30 kD, slightly bigger than GFP, in both the medium and the protoplast lysates of HYG R-GFPexpressing plants. Using tubulin as an intracellular marker, it was found that contamination of the medium with intracellular proteins was below the level of detection. These results suggest that HYG R -GFP had been efficiently truncated at carboxyl terminus of HYG R shortly after it was synthesized in HYG R-GFPexpressing plants. To assess whether HYG R -GFP was secreted via the conventional secretory pathway, protoplasts were treated with BFA. Although the Golgi apparatus in Arabidopsis root tissues appears to be BFA-resistant [32][33][34], it turns out that BFA indeed exerts a marked effect on the Golgi apparatus in non-root tissues of Arabidopsis. Confocal microscopy revealed that the classic reabsorption of Golgi membranes back into the ER in BFA-treated Arabidopsis leaves [34,36]. Furthermore, the secretion of acid phosphatase inhibited by BFA treatment was also reported in mesophyll protoplasts of tobacco, indicating that BFA inhibits the conventional secretory pathway in leaf cells [37]. In order to analyze whether BFA affected the secretion of HYG R -GFP by immunoblot analysis, the total protein of protoplast lysates and the medium was harvested after 5-h BFA treatment, respectively. As shown in Figure 6G, HYG R was detected in similar amounts in the absence and presence of BFA in the protoplast lysates or in the medium, respectively. The effectiveness of BFA on the conventional ER/Golgi pathway was verified by measuring the activity of acid phosphatase (AcPase) at hourly intervals in the medium and protoplast lysates ( Figure 6H). As expected, an obvious inhibition of AcPase secretion upon BFA treatment was observed at each individual measurement time as previously reported [37]. The facts that BFA inhibited AcPase secretion but did not inhibit secretion of HYG R -GFP suggested that HYG R -GFP secretion indeed follows an alternative secretory pathway.

SYT2 is Required for the Unconventional Secretion of HYG R
To further examine whether SYT2 was involved in the unconventional secretory process of HYG R -GFP in Arabidopsis, the HYG R -GFP was introduced into syt2-1 plants by Agrobacteriummediated transformation and the resultant transgenic plants (syt2-1/HYG R -GFP) had similar phenotype to syt2-1/HYG R plants under hygromycin B treatments ( Figure S6). As shown in Figure 7A to 7C, expression of HYG R -GFP resulted in an increase in GFP fluorescence owing to intracellular accumulation of HYG R -GFP. HYG R -GFP accumulated in whole leaf cells besides in leaf-tip zones ( Figure 7A). When we examined the tissues at higher resolution by LSCM, the fluorescence signals were found on punctate structures in cytoplasm ( Figure 7B and 7C). After being plasmolyzed, syt2-1/HYG R -GFP plants showed that fluorescence signals of HYG R -GFP were primarily localized on intracellular punctate structures and vacuoles ( Figure 7D to 7F). To characterize the fluorescent proteins in syt2-1/HYG R -GFP plants, total proteins extracted from protoplasts and medium were analyzed by protein gel blot using anti-GFP antibody. As shown in Figure 7G, the total protein in the medium contained no detectable tubulin, indicating that the medium was not obviously contaminated by protoplast proteins. HYG R-GFP in the medium extracts of syt2-1/HYG R -GFP protoplasts similarly exhibited a single band that co-migrated with the GFP-fusion protein in the extracts from HYG R -GFP protoplasts and medium. However, the protoplast extracts had three forms of GFP fusion protein in syt2-1/HYG R -GFP plants. The greatest band migrated with an apparent molecular weight of about 55 kD which was lower than the expected full-length of HYG R-GFP. Apart from this upper GFP fusion protein, two less intense bands with the molecular weight of about 43 kD and 30 kD were recognized below it, implying that HYG R -GFP had undergone partial truncation at its amino terminus with different extents in the syt2-1/HYG R -GFP plants. Immunogold-labeled ultrathin sections for electron microscopy showed the gold particles situated on the cell wall both in concentrated and dispersed manner in the root cells of HYG R-GFPexpressing plants ( Figure 7H to 7K). Little or no gold particles were detected on the cell wall in the root cells of the syt2-1 plants expressing HYG R-GFP. However, several gold particles well deposited close to, or in the vacuoles in these cells ( Figure 7L to 7O). No obvious signals were found in the vacuoles of the HYG R-GFP transgenic plants ( Figure 7P) or in the whole cells of non-transformed plants ( Figure 7Q).
It was of interest to note that syt2-1/HYG R seedlings also exhibited the inhibition of root elongation under higher hygromycin B treatments (.5 mg/ml) ( Figure 5). To confirm that the sensitivity of syt2-1/HYG R to hygromycin B in root tip growth is caused by the deficiency of SYT2, the binary construct containing SYT2-GFP and HYG R was introduced into syt2-1 mutants by Agrobacterium-mediated transformation. T3 progeny were subjected to hygromycin B and it was found that the SYT2/HYG R plants restored the syt2-1 phenotype to the HYG R transgenic plants with respect to the root elongation and root morphology ( Figure S7). These results confirmed that the deficiency of SYT2 in syt2-1 resulted in the increased sensitivity to higher concentrations of hygromycin B during Arabidopsis seedling growth.

SYT1 Expression is Up-regulated in Hygromycin B-treated syt2-1 Mutant
The detoxifying ability of HYG R in the syt2-1 mutant was reduced under higher concentrations of hygromycin B when compared with that in the wild-type plants, but was much higher than that in SYT2-overexpressing plants. We hypothesized that the other members of SYT family in Arabidopsis, especially the SYT1, which has the highest homology with SYT2, might contribute to the decreased resistance of syt2-1 to hygromycin B. To address this possibility, the expression level of SYT1 in hygromycin-treated syt2-1 plants was examined by semi-quantitative RT-PCR. Under normal condition, SYT1 is expressed at similar level in syt2-1 to that in wild-type plants. However, the expression of SYT1 was greatly enhanced in syt2-1 plants under hygromycin B treatment for 3 h and 15 h ( Figure 8A). To further examine whether the up-regulated expression level of SYT1 in syt2-1 has a role of enhancing the sensitivity to hygromycin B, we investigated the phenotype of syt1-2 (SYT1 knock-out mutant, Schapire et al., 2008) and SYT1-overexpresing plants both which contain HYG R gene. From Figure 8B and 8C, it is evident that coexpression of SYT1 and HYG R led to hypersensitivity to hygromycin B in Arabidopsis.

Discussion
Like most proteins involved in vesicular trafficking, the localization of synaptotagmins provides important information about the biological functions of these proteins [38]. Thus, we firstly investigated the subcellular distribution of SYT2 using different approaches. In all cases, SYT2 was detected in punctate structures, raising the possibility that it was targeted to the membrane trafficking pathway. This was further supported by the results obtained from immunolocalization studies using anti-SYT2 and anti-GFP antibodies. These studies showed that SYT2 was broadly distributed on the Golgi apparatus. This result is analogous to mammalian Syt 4, which localizes to the Golgi apparatus in undifferentiated neuroendocrine PC12 cells [39]. However, the localization of SYT2 is in contrast to the plasma membrane localization of SYT1 in Arabidopsis [14].
It has been long appreciated that the Golgi apparatus forms the heart of the secretory pathway and it is where secretory materials are posttranslationally modified before being sorted for delivery to their final destination, such as the plasma membrane or extracellular space [40,41]. Indeed, in plant cells, some Golgilocalized proteins have been shown to be involved in the transport of cargo from the Golgi apparatus to the cell surface [42,43]. Therefore, the localization of SYT2 on the Golgi apparatus in Arabidopsis suggests a role in the secretory pathway.
HYG R has been shown to be effective in selection with various plant species, including dicots, monocots and gymnosperms [44,45]. However, the data available at present appear insufficient to provide complete knowledge of mechanism of HYG R secretion. In the present experiment, HYG R -GFP was present in both intracellular and extracelluar space, suggesting that HYG R -GFP may be excreted from cytosol into the extracellular space. Furthermore, anti-GFP antibodies recognized a band with a molecular weight of about 30 kD in the protoplast lysates from HYG R -GFP plants, which was slightly greater than the full-length GFP, implying that HYG R -GFP was truncated at the carboxyl terminus of HYG R shortly after its synthesis and HYG R -GFP was secreted in its truncated form. Interestingly, co-expression of HYG R and SYT2 in Arabidopsis caused hypersensitivity to hygromycin B, suggesting that SYT2 may have a role in regulating the detoxification of HYG R for hygromycin B. To confirm whether SYT2 is involved in the trafficking of HYG R , we examined the existing form of HYG R -GFP in syt2-1 mutant. We found that the loss of SYT2 partially inhibited the truncation of HYG R -GFP at the carboxyl terminus of HYG R , which subsequently accumulated in intracellular punctate structures and vacuoles in several truncating forms, suggesting SYT2 has a vital role in regulating the trafficking of HYG R -GFP for its secretion in plant cells (Figure 9).
Proteins can be secreted in plant cells via either the conventional or the unconventional secretory pathway. The unconventional secretory proteins not only lack of canonical signal sequence, but are also resistant to the export processes affected by BFA, an inhibitor of ER/Golgi-dependent protein secretion in both animals and plants [4,28,46]. Interestingly, no conventional signal peptide sequence was found in HYG R predicted by SignalP 3.0 Server (http://www.cbs.dtu.dk/services/SignalP/) or TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/). Therefore, it is possible that HYG R -GFP was synthesized on the free ribosomes in cytoplasm and exported by a signal peptide-independent secretory process (Figure 9). This unconventional secretion has been thoroughly demonstrated in mammalian and yeast cells. We further analyzed the secretion of HYG R -GFP protein in HYG R -GFP-expressing plants in response to BFA treatment by protein immunoblot. As expected, upon BFA treatment, HYG R -GFP secretion in the transgenic Arabidopsis was not perturbed. Thus HYG R -GFP secretion displays the features of leaderless or unconventional protein secretion, including the absence of a canonical signal peptide in the protein and the insensitive export in the presence of brefeldin A [4,46], Therefore, we can safely conclude that an unconventional secretion is involved in HYG Rtransgenic Arabidopsis plants.
It is unexpected that the detoxifying ability of HYG R in the lossof-function syt2-1 mutant was also destroyed, causing the plants to grow slowly and weakly under higher concentrations of hygromycin B, although these plants showed stronger resistance compared with SYT2/HYG R ones. The most probable explanation for this phenomenon is that the trafficking of HYG R -GFP in syt2-1 plants is inhibited and the protein is partly transported into vacuoles, as revealed by the localization of HYG R -GFP in this mutant, and may then be degradated. Co-expression of HYG R and SYT1, the latter, the most similar member to SYT2 in Arabidopsis SYT family, also led to hypersensitivity to hygromycin B. In any case, this result provided direct evidence that the contributor to the weakened resistance of syt2-1 to hygromycin B might be SYT1. We further found that the transcriptional expression of SYT1 in syt2-1 plants remained unchanged under normal conditions, but obviously enhanced under hygromycin B treatment. The SYT1 knock-out mutants also showed sensitivity to hygromycin B even at lower concentration (5 mg/mL), although they have much stronger tolerance than SYT1/HYG R plants. Considering the similar responses of SYT2 and SYT1 to hygromycin B, we conclude that SYT1 may contribute to the resistance of HYG R -harboring plants via a different secretory route (Figure 9).
Unconventional secretion can be classified into non-vesicular and vesicular mechanisms. Non-vesicular mechanisms are based on direct translocation of cytoplasmic proteins across the plasma membrane via a specific plasma membrane ATP-binding cassette transporter or some lipids, such as phosphatidylinositol 4,5 bisphosphate [PI(4,5)P 2 ] in the inner leaflet of the plasma membrane [47].Vesicular mechanisms of unconventional secretion involved multi-vesicular bodies and exosomes that need to fuse with plasma membranes to release cargo into the extracellular space [47,48]. In our study, SYT2 was not presented on the multivesicular bodies (PVC in plant cells), indicating that SYT2 protein may regulate the unconventional secretory pathway by a distinct manner from the multivesicular body-mediated secretion of exosome in mammalian cell. However, SYT2 is not the only Golgi-localized protein that regulates unconventional secretion. Golgi-localized protein GRASP (Golgi reassembly stacking protein) in Dictyostelium discoideum, is also required for Golgiindependent cell-surface transport of a non-signal-peptide-containing protein, acyl-CoA binding protein (AcbA), which triggers terminal differentiation of spore cells [49,50]. In Drosophila melanogaster, GRASP modulates Golgi-independent cell surface transport of a intergrin. In a D. melanogaster grasp mutant, the a integrin subunits are not properly deposited at the plasma membrane and instead retained intracellularly [51]. From sequence comparison of all the available genomes, it was revealed that plants lack a bona fide GRASP homolog [49]. Very recently, an Arabidopsis protein Exo70E2 was found to be present in some double membrane structures (named EXPO) and did not colocalize with the Golgi apparatus, the TGN or PVC. Exo70E2 served to release a leaderless protein (SAMS2) into the extracelluar space [52], indicating that there may be diverse proteins which localize on different organelles and modulate the release process of unconventional proteins in plant cells. Therefore, SYT2 is the first protein, to our knowledge, that resides on Golgi apparatus and regulates unconventional protein secretion in plants.

Plant Material and Growth Conditions
Seeds expressing ARA6-GFP and ARA7-GFP were kindly provided by Takashi Ueda and Thierry Gaude [29,53]. Construct of ST-YFP was kindly made available by Jingbo Jin [54]. Arabidopsis mutant syt2-1 (SALK_135307) was obtained from the Arabidopsis Biological Resource Center at Ohio State University. Other transgenic plants were generated based on the protocol in Text S1.
Arabidopsis seeds were pretreated in 70% ethanol for 5 min, surface-sterilized in 50% bleach for 1 min, and washed with sterile distillated water at least five times. Seeds were planted on 1% agar containing K MS salts with or without the indicated concentrations of hygromycin B, allowed to imbibe for 3 days at 4uC, and germinated in a vertical orientation. Seedlings were grown at 2263uC under a 16-h light/8-h dark regime. Experiments were performed using 3-to 4-day-old seedlings for microscopic observation, or 7-to 10-day-old seedlings for measurement of root and shoot lengths.

Antibody Preparation and Protein Gel Blot Analysis
For protein gel blot analysis, a polyclonal antibody was raised against a truncated form of SYT2. The C-terminal region of SYT2 (235 amino acid residues) and the full length of HYG R were expressed in E. coli respectively as recombinant proteins using the expression vector pET28b (Invitrogen, Carlsbad, CA). The recombinant proteins were expressed and purified according to the manufacturer's protocol, and the purified proteins were injected into a rabbit to raise antibody according to a published protocol [55]. The polyclonal antibody was purified according to Park et al. [56]. Monoclonal anti-GFP antibodies were purchased from Sigma-Aldrich (St. Louis, MO).
Total protein extracts were obtained by grinding 100 mg of wild-type, syt2-1, or SYT2-GFP-overexpressing plants in protein extraction buffer [20 mM Tris-HCl, pH 7.5, 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylene glycol tetraacetic acid (EGTA), 10 mM dithiothreitol (DTT), 0.05% sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The extracts were spun for 10 min at 4uC, and the resulting supernatant loaded on a SDS-PAGE gel with loading buffer. For HYG R protein hybridization, mesophyll protoplasts were prepared from the leaf tissues of 3-to 4-week-old Arabidopsis plants which stably expressed HYG R -GFP protein [57]. After being washed five times with W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, 2 mM 4-Morpholineethanesulfonic acid, pH 5.7), the protoplasts were incubated with 25 mM BFA for 5 hours. At the end of the incubation, the medium and the protoplasts were collected respectively. The protoplast proteins were extracted as described by Wu et al [58]. The medium proteins were precipitated by trichloroacetic acid method and resolved in the SDS-PAGE loading buffer. The samples were boiled for 10 min and loaded on polyacrylamide gel.
After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane for 2 h. The nitrocellulose membrane was then incubated in a 1:800 dilution of anti-SYT2, 1:500 anti-HYG R , 1:1000 anti-tubulin or 1:4000 anti-GFP antibodies in phosphate-buffered saline (PBS) buffer (pH 6.9). Horseradishperoxidase-conjugated secondary antibody (Sigma-Aldrich) was used at a 1:5000 dilution, and the results were interpreted using an enhanced chemiluminescence detection system, with visualization by enhanced chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China) according to the manufacturer's recommendations.

Fluorescent Dye and Treatments with BFA and Wortmannin
To visualize putative endosomes, seedlings were mounted in K MS liquid with 3 mM FM4-64 [Invitrogen; T13320; diluted from a 3 mM stock solution in dimethyl sulfoxide (DMSO)] on slides for a specified time. For BFA treatment, seedlings were incubated in K MS liquid containing 25 mM BFA diluted from a 50 mM stock solution in DMSO and then mounted on slides in the presence of BFA. For the wortmannin treatment, seedlings were incubated in K MS liquid containing 20 mM wortmannin diluted from a 20 mM stock solution in DMSO for 1 h before observation.

Immunofluorescent Labeling
Four-day-old seedlings were fixed in 4% paraformaldehyde in PEM buffer (50 mM PIPES, 5 mM EGTA, and 5 mM MgSO 4 , pH 6.9) for 1 h at room temperature, followed by washing with 0.1 M glycine in PEM buffer. Fixed cells were partially digested with 2% (w/v) driselase (Sigma-Aldrich) for 30 min at 37uC. The plasma membrane was permeabilized with 0.3% Triton X-100 and 10% DMSO in PBS for 1 h at room temperature. Seedlings were incubated in blocking solution for 1 h at room temperature and then incubated with primary antibodies of anti-SYT2 (1:50) or anti-GFP (1:200; Sigma-Aldrich) again for 1 h at room temperature. Primary antibodies were washed out with blocking solution three times for 5 min and the seedlings then incubated with fluorochrome-conjugated secondary antibodies in the dark at 37uC for 3 h. Secondary antibodies (purchased from Sigma-Aldrich) were used at the following concentrations: fluorescein isothiocyanate-conjugated anti-rat IgG, 1:100; fluorescein isothiocyanate-conjugated anti-rabbit IgG, 1:100; rhodamine (TRITC)conjugated anti-rabbit IgG, 1:100.

Fluorescence Microscopy
Fluorescence microscopy was performed using a TCS SP5 confocal laser-scanning microscope (Leica, Oberkochen, Germany). All LSCM images were obtained using the Leica Confocal software and a 636 water-immersion objective. GFP or GFP/ FM4-64 was excited at 488 nm and emission was detected between 500 and 530 nm for GFP and between 620 and 680 nm for FM4-64. To visualize GFP/RFP, GFP and RFP were excited at 488 nm and 543 nm, respectively, and emission detected at 500 and 530 nm for GFP and between 565 and 600 nm for RFP. Images were edited using the LAS AF Lite image browser (Leica) and Adobe Photoshop CS3 (Adobe Systems, San Jose, CA).

Immunoelectron Microscopy
For immunogold labeling of SYT2 and HYG R , roots of Arabidopsis were fixed with 4% paraformaldehyde and 1% glutaraldehyde for 4 h and then were embedded in LR White resin (Sigma) and polymerized by heat. Ultrathin sections were obtained and transferred to nickel grids that were then blocked with 5% BSA and incubated subsequently with the primary antibody (anti-SYT2, 1:200; anti-HYG R , 1:200) at 37uC for 1 h. After five washes with PBS for 20 min, the sections were treated with the secondary antibody (goat anti-rabbit IgG coupled to 10-nm gold particles, Sigma, 1:50) at 37uC for 1 h. Finally, the sections were stained with 2% uranyl acetate for 10 min and observed under JEM-1230 TEM (JEOL).

Acid Phosphatase Assay
The activity of the AcPase was measured according to Pfeiffer by measuring the release of p-nitrophenol (pNP) from pnitrophenyl phosphate (pNPP) [59]. Samples of 200 ml were incubated with 200 ml of reaction buffer containing 40 mM MES-Tris, pH 5.5, 5 mM pNPP, and 10 mM MgCl 2 , for 45 min at 30uC. The reaction was stopped by the addition of 5 ml of 40 mM NaOH, and the concentration of pNP was determined at 405 nm wavelength. All assays were performed as triplicate.

Supporting Information
Text S1 Material and Methods (Construction of Chimeric Genes and Transformation of Arabidopsis Plants, RT-PCR Analysis, Transient Expression in Tobacco and Arabidopsis). (DOCX) Figure S1 Expression profiles of Arabidopsis SYT2 based on microarray expression data from Genevestigator (https://www.genevestigator.com). (TIF) Figure S2 No co-localization between SYT2-GFP and BFA compartments. (A-F) Seedlings stably expressing SYT2-GFP were stained with FM4-64 for 10 min and treated with either DMSO (A-C) or 25 mM BFA for 60 min (D-F). FM4-64-labeled BFA compartments (red) mostly non-overlapped with SYT2-GFP punctuate structures (green). Arrows indicate BFA compartments. Bars = 10 mm. (G-L) Seedlings stably expressing VHA-a1-GFP or ARA6-GFP were stained with FM4-64 for 10 min before being treated with 25 mM BFA for 60 min. VHA-a1-GFP-labeled structures (green) aggregated and perfectly overlapped with BFA compartments (red) (G-I), and ARA6-GFP-positive structures (green) clustered at the periphery of BFA compartments (red) (J-L). Arrows indicate BFA compartments. Bars = 10 mm.  (D and E), syt2-1/HYG R (F), SYT2-GFP/HYG R (G) and wild-type (H) plants on hygromycin B-containing medium. Seedlings grew on K MS medium supplemented with 0 (D) or 5 mg/mL (E-H) hygromycin B for 3 days before images were taken. Bars = 500 mm. (I-L) Sensitivity of root tips of HYG R (I), syt2-1/HYG R (J), SYT2/HYG R (K) and syt2-1/ SYT2/HYG R (L) to hygromycin B. Seeds were germinated and grew on K MS medium with 10 mg/mL hygromycin B for 3 days before images were taken. Bars = 100 mm. (TIF) Figure S5 Responses of syt2-1 and wild-type seedlings to hygromycin B treatments. Seeds were germinated on K MS medium containing 0, 5 and 10 mg/mL hygromycin B and grown for 7 days before the pictures were taken. (TIF) Figure S6 Responses of HYG R -GFP and syt2-1/HYG R -GFP seedlings to hygromycin B treatments. Seeds were germinated on K MS medium containing 0, 10 and 20 mg/mL hygromycin B and grown for 10 days before the pictures were taken. syt2-1/HYG R -GFP1 and syt2-1/HYG R -GFP2 were different lines that HYG R -GFP is expressed in SYT2 knock-out plants.