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Plant Cell. Dec 2003; 15(12): 2885–2899.
PMCID: PMC282822

The VTI Family of SNARE Proteins Is Necessary for Plant Viability and Mediates Different Protein Transport PathwaysW in Box

Abstract

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

INTRODUCTION

The plant endomembrane system plays a variety of roles throughout plant development. It is composed of a series of membrane-bound organelles that include the endoplasmic reticulum, the Golgi apparatus, the trans-Golgi network (TGN), the prevacuolar compartment (PVC), the vacuole, and endosomes. Vesicle transport is the major means by which proteins travel to and from these organelles. The correct targeting of proteins to the vacuole or other organelles of the endomembrane system is dependent on the action of SNAREs (soluble NSF attachment protein receptors). The majority of SNAREs are type-II membrane proteins with coiled-coil domains that are important for interacting with other SNAREs. SNAREs are divided into two groups—vesicle-SNAREs (v-SNAREs) and target-SNAREs (t-SNAREs), which are located on the target membrane. Generally, three t-SNAREs form a cis-SNARE complex, which is recognized by a v-SNARE that aligns its coiled-coil region to form a four-helix bundle. The formation of this complex drives the fusion of the target and vesicle membranes, allowing the delivery of cargo protein to the target compartment. Once delivery is complete, the complex is dissociated. Dissociation of the four-helix bundle requires soluble NSF and α-SNAP proteins. After the SNARE complex is dissociated in an energy-requiring process, the v-SNARE is recycled back to its original compartment.

Although the basic machinery of vesicular transport is conserved among all eukaryotic cells, the plant endomembrane system also has unique components. In mammals and yeast, each cell has only one type of vacuole, whereas in plants, different types of vacuoles—lytic and protein storage—frequently coexist in one cell (Vitale and Raikhel, 1999). It is possible that this complex vacuolar system requires more sophisticated transport machinery in plants than in yeast. Based on genome analysis, there are 21 SNAREs in Saccharomyces cerevisiae. In Arabidopsis, there are 55 SNAREs, and these can be subgrouped into several families (Sanderfoot et al., 2000). These multigene families could reflect a more complex endomembrane system in plants.

The VTI1-type v-SNARE has multiple functions. In yeast, although encoded by a single gene, it can form SNARE complexes with five syntaxins (Abeliovich et al., 1999; Fischer von Mollard and Stevens, 1999). Accordingly, it has been shown to function in multiple pathways to the vacuole: the CPY pathway via the PVC, the ALP pathway via the Golgi apparatus, and the Cvt pathway, which transports proteins such as API from the cytoplasm to the vacuole. In mouse, MmVTI1a is localized to the Golgi and involved in intra-Golgi trafficking. MmVTI1b is found on the Golgi, the TGN, and possibly the endosome (Xu et al., 1998).

In Arabidopsis, the VTI1 family is composed of three closely related members: VTI11, VTI12, and VTI13. VTI11 and VTI12 are highly expressed in all organs. VTI13 does not produce any mRNA that is detectable by reverse transcriptase–mediated (RT) PCR, although Basic Local Alignment Search Tool (BLAST) searches show that there is one VTI13 EST clone in the database, compared with ~12 each for VTI11 and VTI12 (M. Surpin and N. Raikhel, unpublished data). VTI11 and VTI12 are 60% identical. They are ~25% identical to yeast Vti1p and are closer to mammalian VTI1a than to mammalian VTI1b (Sanderfoot et al., 2000). Previously, we described the characterization of VTI11 (referred to as AtVTI1a by Zheng et al. [1999]). VTI11 is localized on the TGN with the NTPP cargo receptor ELP, and it colocalizes with the SYP2 and SYP5 groups of syntaxins on the PVC (Zheng et al., 1999; Bassham et al., 2000; Sanderfoot et al., 2001b). Recently, a defect in the VTI11 gene was identified as the zig mutation in a screen for mutants impaired in the shoot gravitropic response (Kato et al., 2002). In the zig mutant, amyloplasts do not sediment to the bottom of endodermal cells, and abnormal vesicular structures are found in several tissues. Endodermis-specific expression of wild-type VTI11 in the zig mutant complements the shoot gravitropism defect. These results suggest that vacuole formation is important for the shoot gravitropic response (Morita et al., 2002).

There is evidence that VTI11 and VTI12 function in different vesicle transport pathways. First, VTI11 and VTI12 complement different yeast vti1 alleles that block different vacuolar protein transport pathways (Zheng et al., 1999). VTI11 expressed in yeast vti temperature-sensitive and null mutants suppresses the CPY mistargeting and growth-defect phenotypes, whereas VTI12 restores API transport via the Cvt pathway as well as the transport of vacuolar ALP. Arabidopsis VTI12 also rescues the yeast vti1p null mutant, but the cells do not grow as quickly as those complemented with Arabidopsis VTI11 (Zheng et al., 1999). Second, in plant cells, VTI12 forms a SNARE complex with the SYP4- and SYP6-type SNAREs (Bassham et al., 2000; Sanderfoot et al., 2001b). These results indicate that VTI12 plays a role in vesicle trafficking different from that of VTI11.

We took a genetic approach to determine the respective functions of VTI1 family members. Here, we describe the Arabidopsis mutant vti12, further characterize the zig mutant, and present the results of a genetic cross between these two lines. We show that VTI11 and VTI12 mediate different protein transport pathways, and although they do not fully complement each other at a functional level, they can substitute for each other in their respective SNARE complexes. We also show that a double-mutant cross between zig and vti12 is embryo lethal, indicating that at least some VTI1 family protein must be present to ensure viability.

RESULTS

Characterization of vti12

A reverse-genetics approach was taken to address the function of VTI12. To identify a T-DNA insertion mutant of VTI12, pools of Arabidopsis Col-gl seeds (see Methods) mutagenized with T-DNA were screened using gene-specific primers and T-DNA border primers as described by Sanderfoot et al. (2001a). One mutant plant with a T-DNA inserted at ~76 nucleotides upstream of the transcription start site (Figure 1A) was identified. This allele was named vti12 for its mutation in VTI12. Total RNAs prepared from wild-type plants, heterozygous plants, and homozygous vti12 plants were used as templates for RT-PCR. As shown in Figure 1B, RT-PCR of homozygous vti12 plants detected no mRNA for VTI12. RT-PCR of an unrelated gene (NPSN12) using the same RNA samples was used as a control. Wild-type plants and plants with homozygous insertions were characterized further by protein gel blot analysis. As shown in Figure 1C, the VTI12 antibody recognized a protein in wild-type plants at ~28 to 30 kD that was missing from the homozygous vti12 plants. By contrast, when VTI11 antibodies were used for protein gel blot analysis, there was no significant difference between proteins from wild-type and vti12 plants. Under normal growth conditions, vti12 plants had the same morphology and growth characteristics as wild-type plants.

Figure 1.
vti12 Is a Null Mutant for VTI12.

VTI11 and VTI12 Functional Redundancy

Previously, we showed that VTI1 family proteins, although closely related, form SNARE complexes with different sets of syntaxins (Bassham et al., 2000; Sanderfoot et al., 2001b). Furthermore, although vti12 appeared the same as the wild type under normal growth conditions, plants that are mutated at the zig locus have a distinctive visible phenotype (Kato et al., 2002; Morita et al., 2002). Thus, VTI11 and VTI12 have functional differences as well. In addition, the double homozygous mutant vti11 vti12 was embryo lethal (see below), suggesting that (1) some VTI family protein was essential for viability and (2) both expressed VTI proteins were able to substitute functionally for each other. To evaluate the second possibility, we analyzed the VTI11- and VTI12-containing SNARE complexes in wild-type, vti12, and zig-1 plants. As shown in Figure 2, when VTI11 antibodies were used for immunoprecipitation, SYP2 and SYP5 families of syntaxins were coprecipitated at similar levels in both wild-type and vti12 plants. However, there were significantly more SYP4- and SYP6-type SNAREs coprecipitated with VTI11 from vti12 than from wild-type plants (Figure 2). Thus, we concluded that VTI11 forms a SNARE complex with SYP4- and SYP6-type SNAREs and possibly performs the function of VTI12 in the vti12 mutant.

Figure 2.
Immunoprecipitation of VTI12 from Wild-Type and zig Plants and of VTI11 from Wild-Type and vti12 Plants.

When we performed coimmunoprecipitations on zig-1 extracts from both roots and shoots, we detected VTI12 in a complex with SYP5- and SYP2-type SNAREs (Figure 2). The experimental procedure was the same as that used with vti12 and was performed using purified anti-VTI12 antibodies (see Methods). Equal amounts of VTI12 were immunoprecipitated from wild-type and zig-1 plants. SYP6 was coprecipitated from both wild-type and zig-1 plants at similar levels (Figure 2). We observed no significant differences in the amounts of coprecipitated SYP4-type SNAREs (data not shown). However, more SYP2- and SYP5-type SNAREs were immunoprecipitated from zig-1 than from wild-type plants, likely as a result of their interaction with VTI12 in zig-1.

We examined whether VTI12 can substitute functionally for VTI11 in plants. First, we doubled the copy number of VTI12 in the zig-1 mutant by introducing a genomic fragment containing VTI12 with expression driven by its own promoter. There was no effect on the zig-1 phenotype in these transgenic lines (data not shown). We then overexpressed VTI12 in zig-1 plants using the 35S promoter. We analyzed a number of independent lines of zig-1 transgenic plants carrying the 35S-VTI12 cDNA construct. Although we observed some slight variation among these lines, the majority exhibited the phenotype shown in Figure 3. Plants homozygous for the transgenic construct had normally shaped leaves and stems, although the stems were slightly smaller and thinner than those of the wild type (Figures 3A and 3D). Plants heterozygous for the transgenic construct had an intermediate phenotype for stem morphology but were wild type with respect to leaf shape (Figures 3B and 3E, left). The zygosity of the transgene also affected the gravitropic response. zig-1 plants that were homozygous for the 35S-VTI12 construct had a normal gravitropic response. Plants that were either heterozygous or did not have any copies of the transgene showed reduced or no gravitropic response, respectively. In addition, we examined plants for relative amounts of VTI12. The amount of VTI12 correlated with the zygosity of the transgene. zig-1 plants that were homozygous for the 35S-VTI12 construct contained excessive amounts of VTI12 (Figure 3F), and plants with one or no copies contained intermediate or normal levels of VTI12, respectively (Figure 3F). These results suggested that an excess of VTI12 was able to form a SNARE complex with SYP2 and SYP5 in the absence of VTI11 (Figure 2), and the complex was functional to the extent that it was able to support embryonic development (see below). However, normal amounts or a fewfold difference in the quantity of VTI12 was not enough to complement the leaf and stem morphogenesis defects in zig-1.

Figure 3.
Excess VTI12 Can Complement Defects in the Atvti11/zig-1 Mutant.

Based on the data described above, we propose that both VTI11 and VTI12 can substitute for each other in their respective SNARE complexes at the molecular level. We also believe that these complexes are, to some degree, functional.

VTI12 Functions in the Plant Autophagy Pathway

The vti12 plants were checked for defects in NTPP vacuolar protein transport. It has been shown that Arabidopsis aleurain has a typical NTPP signal and possibly is transported through a pathway common for NTPP proteins (Ahmed et al., 2000). In total protein extracts, no difference in the total amount of mature aleurain was found between wild-type and vti12 plants (data not shown), nor was there any obvious abnormal accumulation of higher molecular variants of aleurain in the vti12 plants. We concluded that the VTI12 v-SNARE does not act in any of the previously characterized NTPP cargo transport pathways.

We showed previously (Zheng et al., 1999) that the VTI12 (AtVTI1b) cDNA complements the cytoplasm-to-vacuole (Cvt) pathway in yeast vti1p mutants. In yeast, the Cvt pathway shares many components with the autophagy pathway (Reggiori and Klionsky, 2002). Thus, we determined whether vti12 mutants show a similar phenotype under the same conditions as seen in known Arabidopsis autophagy mutants. Seeds were germinated on Gamborg's B-5 medium with 2% sucrose. At 2 weeks, seedlings were transferred to plates containing no nitrogen/no carbon (No N/C) medium for varying amounts of time. The plants were transferred back to Gamborg's B-5 medium, and the number of plants recovered (scored by new green leaves) was counted. Figure 4A shows the recovery curve of wild-type, vti12, and zig-1 plants that were on No N/C medium for 32 days. vti12 plants clearly recovered from starvation more slowly than either wild-type or zig-1 plants, and by this time point, a small proportion of the vti12 plants did not recover at all.

Figure 4.
The vti12 Mutant Has a Defect in the Autophagy Pathway.

Once recovered, vti12 plants were smaller than identically treated wild-type plants (Figure 4B). Seedlings were grown on Gamborg's B-5 medium for 2 weeks, transferred to No N/C medium for 30 days, and then transferred back to Gamborg's B-5 medium. At 13 days after transfer, wild-type plants were significantly larger and more robust than vti12 plants. Furthermore, vti12 plants on No N/C medium developed their primary inflorescence (bolted) earlier than wild-type plants under the same conditions (see supplemental data online).

Previously characterized Arabidopsis autophagy mutants have shown accelerated leaf senescence (Doelling et al., 2002; Hanaoka et al., 2002). We performed detached leaf assays to determine whether vti12 mutants also have this phenotype. First and second true leaves were excised from 2-week-old seedlings and were either placed on filter paper soaked in Mes buffer or floated on water. In either condition, the leaves were placed in the dark and retrieved at various time points. Figure 4C shows that vti12 leaves became chlorotic ~1 day before either wild-type or zig-1 leaves. We also examined the induction of the SEN1 and YSL4 genes, which are markers for leaf senescence in Arabidopsis, by semiquantitative RT-PCR (Hanaoka et al., 2002) (Figure 4D). In wild-type plants, there was initially a low level of SEN1 transcript and then the level of transcript increased between 24 and 48 h. By contrast, the leaves of the vti12 mutant accumulated a large amount of SEN1 transcript between 12 and 24 h, and this high level of accumulation was maintained through the 48-h time point. In wild-type plants, YSL4 showed a large increase in the accumulation of transcript between 24 and 48 h. In vti12, YSL4 mRNA accumulation appeared to be increased already at time 0. It increased significantly at ~12 h and decreased subsequently at 24 and 48 h. Together, these results indicated that vti12 plants not only entered senescence prematurely after leaf detachment but also may have existed already in a senescent state before leaf excision.

Electron Microscopic Examination of Wild-Type and vti12 Plants Grown under Starvation Conditions

The results from the starvation and detached-leaf assays suggested a role for VTI12 in the autophagy pathway. We microscopically examined wild-type and mutant cells under conditions that are known to induce autophagy. Plants were grown on rich medium for 2 weeks and then transferred to plates containing No N/C medium. Ultrathin sections were made from the leaves of wild-type and vti12 plants at various times, and using electron microscopy, we examined cells from the two lines. We also made corresponding thick sections to orient ourselves to the gross morphology of the leaf sections (data not shown). We noted that similar effects of starvation occurred in both palisade and spongy parenchyma cells. Figure 5 shows electron micrographs of wild-type and vti12 cells at different time points. Cells from time 0 for both wild-type and vti12 plants (Figures 5A and 5B) showed similar morphologies. The vacuole did not take up the entire cellular space, and there was a perimeter of cytoplasm that contained a number of organelles. After 6 days on No N/C medium, the vacuole from wild-type plants was expanded and appeared to have engulfed the organelles, whereas the cytoplasmic perimeter disappeared in wild-type cells (Figure 5C). In vti12 cells, however, the cytoplasmic perimeter persisted and the vacuole did not expand to the same extent as in wild-type cells (Figure 5D). After 12 days on No N/C medium, the organelles from wild-type plants appeared to have been fully engulfed and were in the process of being digested, whereas in vti12 plants, the organelles still appeared to be intact and remained outside the vacuole (Figures 5E and 5F, respectively). By 19 days on No N/C medium (Figures 5G and 5H), the wild-type cells had vacuolar inclusions that contained remnants of organelles with some intact, but disorganized, membrane structure. By contrast, vti12 plants had vacuolar inclusions that were bubble-like in appearance (Figure 5H) and appeared to have been composed of many small, empty vesicles. We observed a number of these unique multivesicular structures in vti12 cells but not in any wild-type cells. Clearly, the progression of the autophagic process was impeded in vti12 cells. Besides being slower, there appeared to be a defect in autophagosome formation and/or fusion.

Figure 5.
Electron Microscopic Examination of Autophagy in Wild-Type and vti12 Cells.

Double-Mutant Studies with vti12 and zig-1

We took a genetic approach to better understand the individual and possibly overlapping functions of the VTI family of proteins. Toward this end, we created a double-mutant cross, but we were unable to detect any homozygous double mutants in the course of our genotyping experiments. We examined siliques from F1 plants to test whether the double homozygous mutant combination was embryo lethal. Whereas seeds from wild-type plants rarely aborted (Figure 6A), self-pollinated vti12/vti12 ZIG-1/zig-1 and VTI12/vti12 zig-1/zig-1 siliques frequently had nonviable progeny (Figures 6Ai to 6Aviii). As shown in Figures 6Aiii to 6Av, there were many shriveled seeds and vacant spaces in these siliques. Upon close examination, we detected the traces of ovaries and funiculi in some vacancies. Thus, we categorized the nonviable progeny into three classes: shriveled seed (Figure 6Avii), aborted seed (Figure 6Aviii), and unnatural interval (Figure 6Av, arrowheads). We calculated the percentage of nonviable progeny from vti12, zig-1, vti12/vti12 ZIG-1/zig-1, and VTI12/vti12 zig-1/zig-1. The results are summarized in Table 1. In both single mutants, a percentage of progeny did not develop into normal seeds (vti12, 13%; zig-1, 32%). In the vti12/vti12 ZIG-1/zig-1 and VTI12/vti12 zig-1/zig-1 plants, 31 and 58% of the self-fertilized progeny were dead, respectively, whereas 25% of the self-progeny of vti12/vti12 ZIG-1/zig-1 and VTI12/vti12 zig-1/zig-1 would have been expected to be lethal. The actual lethality scores in the progeny of vti12/vti12 ZIG-1/zig-1 or VTI12/vti12 zig-1/zig-1 were higher than 25%, suggesting that the single mutations and the double homozygous mutation affect viability independently. In accordance with this hypothesis, the theoretical lethality (expected lethality) was calculated as shown in Table 1. Actual lethality scores (lethality of embryo) in vti12/vti12 ZIG-1/zig-1 and VTI12/vti12 zig-1/zig-1 were very similar to the theoretical values, in agreement with our statistical expectations.

Figure 6.
The Genetic Cross of vti12 and zig (vti11) Mutants.
Table 1.
Summary of the Lethality of vti1 Mutant Progeny

We observed that ~2 of every 15 plants had a zig-1 phenotype and that ~1 of every 20 plants had a novel phenotype best described as an enhanced zig phenotype. These plants were smaller than zig-1 plants, had extremely wrinkled leaves, and exhibited a severe developmental delay (Figure 6Bi). The life span of these plants was correspondingly and significantly longer. Mature plants also exhibited varying degrees of fasciation at the shoot apical meristem (Figure 6Biii) and an altered phyllotaxy (data not shown). Despite their rather severe phenotype, however, the plants that reached adulthood were fertile and yielded viable progeny. Genotyping of a large number of F2 progeny showed that all of the plants with this enhanced zig phenotype were VTI12/vti12 zig-1/zig-1. Based on the principle of independent assortment, we would have expected 2 of every 15 plants to have an enhanced zig phenotype. A ratio of 1:20 suggests that as a group these plants were not entirely viable. Plants with the inverse genotype, vti12/vti12 ZIG/zig-1, had the same phenotype as their vti12 parents (i.e., they had no apparent abnormal phenotype under normal growth conditions).

Histological Studies of Mutant Lines

VTI11 was shown recently to encode ZIG, which, when mutated, causes a shoot gravitropism defect (Kato et al., 2002). The endodermal cells of inflorescence stems contain starch-filled amyloplasts that sediment to the bottom of the cell and act as statoliths (Fukaki et al., 1998). The endodermal cell layer of the zig mutant does not completely sediment starch-containing amyloplasts, as in wild-type plants (Morita et al., 2002). We extended this study to plants with the enhanced zig phenotype by making longitudinal thin sections from wild-type, vti12, zig-1, and VTI12/vti12 zig-1/zig-1 inflorescence stems (Figure 7A). As expected, amyloplasts sedimented almost exclusively to the bottom of cells in the endodermal layer in wild-type and vti12 plants. Amyloplasts could be found in approximately equal numbers at the tops and bottoms of endodermal cells from zig-1 inflorescence stems. In the VTI12/vti12 zig-1/zig-1 line, the cortex and endodermal cell layers were highly disorganized and the individual cell types were not readily distinguished. Cells that did contained amyloplasts featured them on the tops, bottoms, and occasionally the sides of these cells.

Figure 7.
Histological Analysis of the Wild Type, vti12, zig-1 (vti11), and VTI12/vti12 zig-1/zig-1.

In wild-type and vti12 plants, a single epidermal layer encloses approximately three to seven cortex cell layers and one endodermal cell layer (Figures 7Ai, 7Aii, 7Bi, and 7Bii). The cell shapes and sizes in all of these layers were highly uniform and regular. These layers were easily distinguishable in zig-1, although the cell shapes and sizes were slightly irregular. In the VTI12/vti12 zig-1/zig-1 line, the epidermal, cortex, and endodermal cell layers were highly disorganized and often indistinguishable. We made thick cross-sections in all four lines, and the results are shown in Figure 7B. Both the wild-type and vti12 lines showed similar patterns of tissue organization (Figures 7Bi and 7Bii), although in the vti12 section there appeared to be some regions where there were two layers of endoderm cells. The zig-1 cross-section had recognizable vascular bundles, and the xylem cells appeared to be of normal size, shape, and number. However, the phloem cells were spread out over a larger area than in wild-type or vti12 cross-sections (Figure 7Biii). In the enhanced zig phenotype cross-section (Figure 7Biv), the epidermal layer appeared to be intact and composed of regularly sized cells, but the cortex and endodermal layers showed irregularly shaped cells and there did not appear to be any distinction between the cell types. The vascular bundles were not as prominent as in the wild-type and vti12 sections. The xylem cells were small with almost no sclerotic cells, and the phloem appeared to form an almost continuous ring around the cross-section, with correspondingly fewer interfascicular cells. The inner portion of the stele had cells that were smaller and had a more regular shape than in the wild-type, vti12, and zig-1 lines. There also were no gaps between the stele cells in the enhanced zig line.

It appears, then, that the absence of VTI11 caused tissue identity defects. The cross-sections all were obtained from tissue approximately halfway down the floral inflorescence stem at ~1 week after bolting. It may be possible, however, that even though we waited until comparable times after bolting, the ages of the tissue were not strictly equivalent, because both the zig-1 and VTI12/vti12 zig-1/zig-1 lines have significantly expanded life spans compared with the wild-type and vti12 lines. Nonetheless, it is clear that VTI11 plays some role in tissue identity and organization.

Plants Mutated in VTI11 Have Auxin Transport Defects

Endodermal expression of VTI11/ZIG is necessary for the gravitropic response (Morita et al., 2002). Other studies have shown that vesicular transport is necessary for some aspect of the shoot gravitropic response. Arabidopsis pin3 mutants, which contain defects in an auxin efflux carrier, have a shoot-agravitropism phenotype (Friml et al., 2002). Recent studies have shown that Arabidopsis seedlings treated with the drug brefeldin A, which inhibits vesicle transport, have mislocalized PIN proteins (Geldner et al., 2001; Friml et al., 2002). Therefore, we decided to determine whether the vesicle-transport defect in zig plants in turn causes a defect in the localization of auxin efflux carriers. First, we measured whole-plant auxin transport activity in vti12 and zig-1 seedling hypocotyls. The zig-1 mutant showed a significant reduction (~35%) in basipetal auxin transport through the vascular system from the shoot apical tip to the hypocotyl-root transition zone (Figure 7C). Thus, we examined the localization of PIN efflux carrier proteins in zig seedlings. The localization in vascular tissues of PIN1 and PIN3 did not appear to be altered dramatically in the zig mutants.

DISCUSSION

The purpose of this study was to determine the functions of the two expressed VTI1 family proteins, VTI11 and VTI12. Our results show that VTI12 functions in the plant autophagy pathway and that VTI11 plays a role in basipetal auxin transport and cell type differentiation and specificity in addition to NTPP-mediated vacuolar transport. Furthermore, we showed that both VTI1 family proteins substitute for each other at the molecular level and, to some extent, at the functional level. A double-mutant cross experiment showed that the double homozygous vti11 vti12 mutant was embryo lethal, thus demonstrating that some VTI1 family protein is required for plant viability.

Some VTI1 Protein Is Necessary for Plant Viability, and VTI11 and VTI12 Can Substitute for Each Other in Their Respective SNARE Complexes

The shoot gravitropism mutant zig has a lesion in the VTI11 gene and has a severe defect in the gravitropism of the inflorescence stem and hypocotyl (Kato et al., 2002). The vti12 mutant exhibited no visible phenotype under normal conditions. We created a double-mutant cross between the vti12 and zig mutants. The double homozygous mutant was embryo lethal. Furthermore, examination of progeny siliques from genotyped plants clearly demonstrated a reduction in embryo viability. The complete loss of VTI1 family protein appeared to affect the embryos quite early in development, whereas the loss of one or the other protein increased the number of deflated seeds. At this time, we do not know precisely at which stage the vti11 vti12 double mutant aborts. The vacuole biogenesis mutant vacuoless1 (vcl1) also is embryo lethal and terminates sometime near the torpedo stage (Rojo et al., 2001). The VCL1 gene encodes the Arabidopsis homolog of yeast Vps16p, a component of the class-C VPS complex. In yeast, the class-C VPS complex is required for Golgi-to-vacuole protein transport and has been shown to mediate the trans-SNARE pairing of the Vti1p-Vam7-Vam3 complex (Sato et al., 2000). In Arabidopsis, the VCL1 complex has been shown to contain SYP2-type syntaxins (Rojo et al., 2003), which interact with VTI11 (Bassham et al., 2000; Sanderfoot et al., 2001b). Therefore, we speculate that the Arabidopsis vti11 vti12 mutant perhaps no longer mediates vesicle fusion events involving the VCL1 complex and consequently may be embryo lethal at the same stage as the vcl1 mutant.

Our results from coimmunoprecipitation experiments using extracts from the vti12 and zig mutants suggested that both VTI family SNARE proteins were able to substitute for each other at the molecular level. We also complemented the zig-1 phenotype with a 35S-VTI12 construct. zig-1 plants that were homozygous for the transgenic construct showed a wild-type gravitropic response, whereas mutant plants that were heterozygous for the transgene showed an intermediate response. Thus, it appears that the mutant complexes are functional but probably are less efficient in their cargo delivery. Therefore, cargo is delivered, but perhaps not in a timely manner, and this affects the pathway defined by VTI11 much more than that defined by VTI12.

VTI1-type SNAREs are mobile proteins. Immunocytochemical studies have localized both VTI11 and VTI12 on the TGN and the PVC, although their distribution ratios between the TGN and the PVC may be different, as reflected by their density gradient fractionation pattern difference (Zheng et al., 1999; H. Zheng and N. Raikhel, unpublished results). VTI11 and VTI12 both may shuttle between the TGN and the PVC. When one is missing, the other has the chance to form a complex with its SNARE partner and fulfill its functions. Thus, although VTI1-type proteins may have critical functions in the plant, the loss of one or the other is tolerated.

VTI12 Is Involved in the Plant Autophagy Pathway

Thus arises the challenge of identifying the particular function of each VTI1 family protein. Reverse genetics is a powerful means by which to address questions regarding the functions of VTI12 and its SNARE complex. The vti12 mutant was viable, and the loss of active VTI12 protein caused no obvious defects in mutant plants grown under normal conditions. When expressed in yeast, VTI12 functions in vacuolar membrane protein transport and the Cvt pathway (Zheng et al., 1999). Because the yeast Cvt and autophagy pathways share many components, we examined whether Arabidopsis VTI12 was involved in the plant autophagy pathway. There have been electron microscopic descriptions of plant macroautophagy (Aubert et al., 1996), and recently, two groups have begun to address the molecular mechanisms of the plant autophagy pathway (Doelling et al., 2002; Hanaoka et al., 2002). Reports from both of these groups have demonstrated that the Arabidopsis genome contains orthologs for 12 of the 15 described yeast autophagy (or APG) genes. Mutations in two of these genes, APG7 and APG9, have strikingly similar phenotypes. When grown under normal conditions, both show accelerated senescence after bolting, and apg9 plants are reported to bolt earlier than wild-type plants. We did not detect any significant differences between wild-type and vti12 plants that were grown under nutrient-rich conditions. Both apg7 and apg9 are hypersensitive to nutrient-limiting conditions, showing accelerated chlorosis and premature leaf senescence compared with wild-type plants (Doelling et al., 2002; Hanaoka et al., 2002). Our own experiments clearly showed that vti12 plants grown under nutrient-limited conditions have a phenotype similar to that of the other known Arabidopsis autophagy mutants (Doelling et al., 2002; Hanaoka et al., 2002). The milder vti12 phenotype compared with apg7 and apg9 can be attributed to the fact that both APG7 and APG9 are single-copy genes, whereas there is both molecular and genetic evidence that VTI11 can compensate partially for VTI12 in the vti12 mutant. Vti1p has been shown to be involved in autophagosome docking and fusion in yeast (Fischer von Mollard and Stevens, 1999; Ishihara et al., 2001), and it is reasonable to speculate that VTI12 may play a similar role.

The zig-1 Mutant Is Affected in Auxin Transport

The zig-1 mutant has a distinctive visible phenotype, and we have some knowledge about VTI11 function at the molecular level. VTI11 has been shown, via subcellular fractionation and immunoelectron microscopy, to colocalize with the vacuolar sorting receptor ELP on the TGN and the PVC (Zheng et al., 1999) and to colocalize with NTPP proteins on the Golgi (Ahmed et al., 2000). Most likely, VTI11 functions as a v-SNARE that targets ELP and NTPP cargo-containing vesicles from the TGN to the PVC. We extended previous histological studies of the zig mutant to include the VTI12/vti12 zig-1/zig-1 line. It is well established that endodermal cells are the gravity-sensing cells in Arabidopsis (Masson et al., 2002). Starch-filled amyloplasts sediment to the bottom of the shoot endodermal cells in response to the gravity vector and act as statoliths. Expression of wild-type ZIG driven by the endoderm-specific SCR promoter in zig-1 plants complements the abnormal gravitropic response of the mutant and restores the normal sedimentation of amyloplasts (Morita et al., 2002). Histological analysis of longitudinal thick sections stained with toluidine blue show that amyloplasts do not sediment in the zig-1 line, as expected (Kato et al., 2002). The individual tissue layers (epidermis, cortex, endoderm, and stele) still are recognizable, if slightly less organized. In addition to the erratic distribution of amyloplasts, there also was both extreme disorganization of tissue layers and loss of recognizable cell identity in the VTI12/vti12 zig-1/zig-1 line. This loss of tissue identity suggests a specific role for the VTI11 protein, because we observed no obvious abnormal phenotype in the reciprocal genotype (i.e., vti12/vti12 ZIG/zig-1 plants).

Based on the gravitropic phenotype in zig-1 and recent reports that vesicle cycling is involved in the asymmetric distribution of auxin efflux carrier proteins (Friml et al., 2003), we hypothesized that VTI11 was involved in auxin efflux carrier cycling. Geldner et al. (2001) and Friml et al. (2002) have shown that PIN1 and PIN3 continuously cycle in membrane vesicles that travel along the actin cytoskeleton between the plasma membrane and the endosome. Functional PIN3 is required for gravitropism, and disruption of PIN3 cycling via auxin efflux or vesicle trafficking inhibitors or actin depolymerization agents results in a gravitropism defect. zig plants had a significant defect in auxin transport, whereas PIN1 localization in the hypocotyls and roots of the vti11 and vti12 mutants was not significantly different from that in the wild type in the vascular tissue. The 35% decrease in auxin transport observed in zig-1 may be attributable to the partial mislocalization of PIN1 in cells adjacent to the vascular tissue or to a deficiency in auxin transport in the improperly developed phloem cells. Evidence that PIN1 localization occurs in the cells adjacent to the vascular tissue in young seedlings was described recently (Noh et al., 2003). Given the fact that zig plants have tissue identity and organization phenotypes, perhaps basipetal transport is impeded because the vascular tissue is not developed correctly. Indeed, with respect to vascular patterning, the zig and enhanced-zig phenotypes described in this study are not entirely unlike those described for wild-type plants treated with the auxin transport inhibitor NPA (Zhong and Ye, 2001). A defect in basipetal auxin transport could, in turn, decrease the absolute amount of auxin moving through the system (Cambridge and Morris, 1996), but it may not have any effect on the auxin carrier proteins per se. Alternatively, it is possible that VTI11 plays a role in proper auxin efflux carrier localization, but because VTI12 can substitute for VTI11 in the zig mutant, the delivery of vesicle cargo is merely slowed and not impeded entirely. Further study will be necessary to resolve this issue. Therefore, VTI11 defines a cargo transport pathway that is a major player in the plant's morphology and development. Although the VTI12-SYP21/22-SYP51/52 SNARE complex rescues the mutant plants from lethality, it is clear from the distinctive zig phenotype that this complex is not capable of delivering pathway cargo in a completely efficient or timely matter.

The phenomenon that two functionally different SNARE proteins can substitute for each other in vivo has been observed in yeast (Liu and Barlowe, 2002), and noncognate SNAREs have been shown to form complexes in arbitrary combinations in vitro (Fasshauer et al., 1999). It is not difficult to imagine that the more closely related VTI11 and VTI12 could be exchanged in their SNARE complexes in vitro. In vivo, the possibility of substitution also exists because both VTI11 and VTI12 are localized on the TGN and the PVC membrane (Zheng et al., 1999). There are a number of reports of mutated genes in Arabidopsis causing no apparent abnormal phenotype. Normally, this lack of effect is attributed to gene redundancy. However, when we start to understand the machinery of the plant cell function in more detail, “redundant” genes may be found to have functional differences. The mechanism for structurally related but not fully redundant proteins to substitute for each other represents just one facet of the flexibility of plant cells.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana plants grown on plates were incubated under long-day conditions (16 h of light/8 h of dark) at 22°C. Seeds were surface-sterilized in either 50% bleach and 0.1% Triton X-100 solution or 70% ethanol and 0.05% Triton X-100 solution. Plates contained either 1× Murashige and Skoog (1962) Minimal Organics medium (Invitrogen, Carlsbad, CA) or, for the rich-growth phase of the autophagy experiments, Gamborg's B-5 medium with 2% sucrose. For the poor-medium phase of the autophagy experiments, seedlings germinated on Gamborg's B-5 medium with 2% sucrose were transferred to a no nitrogen/no carbon (No N/C) medium as described by Doelling et al. (2002). Plants grown on soil were cultivated in a growth facility that features long-day conditions and maintained at 22°C. Plants were watered with an automatic watering system every 3 days.

Wild type in this study is the Columbia-0 (Col-0) ecotype. The vti12 mutation is in a Columbia background that also is homozygous for the glabrous (gl) mutation (see below). The Arabidopsis VTI11 mutant, zig/sgr4, is in a Columbia background and was described previously (Kato et al., 2002; Morita et al., 2002). The construct was transformed into Agrobacterium tumefaciens strain MP90 and then introduced into the Atvti11/zig-1 plants. T1 plants were selected based on resistance to kanamycin. The genotypes of the T3 siblings used for the experiment were confirmed by checking the segregation ratio of kanamycin resistance to kanamycin susceptibility in the T4 generation.

Isolation of the vti12 T-DNA Insertion Mutant

The isolation and identification of vti12 were performed essentially as described by Sanderfoot et al. (2001a). The following primers were used to screen T-DNA inserted into the VTI12 gene: 5′ end gene-specific primer, 5′-TATTTCCTGGACGAGTAATCTTGGTTCTGC-3′; 3′ end gene-specific primer, 5′-TCTGACGTGACAGTGGGTCTCCTGCCTGCG-3′; T-DNA left border, 5′-CTCATCTAAGCCCCCATTTGGACGTGAATG-3′; T-DNA left border nested, 5′-TTGCTTTCGCCTATAAATTACGACGGATCG-3′; T-DNA right border, 5′-TGGGAAAACCTGGCGTTACCCAACTTAAT-3′. The PCR conditions for amplification of the genomic DNA were standard conditions described in the manufacturer's recommendations (Invitrogen). For reverse transcriptase–mediated (RT) PCR, total RNA was extracted from wild-type Col-0 seedlings and heterozygous and homozygous seedlings as described by Zheng et al. (1999). Reverse transcription reactions were performed using the Superscript II reverse transcriptase system according to recommendations from the manufacturer (Invitrogen). The following gene-specific primers were used to generate first-strand cDNAs: for VTI12, 5′-GAGCCACGATTACCGATGT-3′; for NPSN12, 5′-AGTGTAATATGCACCAAACC-3′. The forward primers were as follows: for VTI12, 5′-GAAAATGTCACTCTGCATCG-3′; for NPSP12, 5′-GAGCCTGAAATAATCCGGCAGAT-3′.

Immunoprecipitation

Antibody characterization is described in the supplemental data online. Twenty grams of 21-day-old liquid-cultured Arabidopsis roots was homogenized on ice in extraction buffer (50 mM Hepes-KOH, pH 6.5, 10 mM potassium acetate, 100 mM NaCl, 5 mM EDTA, and 0.4 M sucrose) with Complete protease inhibitor tablets (Roche, Mannheim, Germany). To prepare total membranes, the homogenate was centrifuged at 1,000g for 15 min, and the supernatant was subjected to 100,000g ultracentrifugation for 3 h. The pellet was homogenized in TBS (Tris-balanced buffer; 0.14 M NaCl, 2.7 mM KCl, and 25 mM Tris, pH 8.0) with miniComplete tablets (Roche). The total membranes were solubilized by adding Triton X-100 to 1%. The solubilized membranes were further cleared by ultracentrifugation at 100,000g for 30 min. The supernatant was incubated with antibodies or preimmune columns for 2 h. After washes with TBST (TBS + 1% Triton X-100), the bound protein was eluted with 0.1 M glycine, pH 2.5. The eluted protein was precipitated by adding 10% trichloroacetic acid. After two acetone washes, the protein pellet was solubilized in 2× Laemmli buffer (125 mM Tris, pH 6.8, 20% glycerol, 2% SDS, 2% 2-mercaptoethanol). The protein was separated by SDS-PAGE, and different proteins were detected by protein gel blot analysis.

For immunoprecipitation of VTI12 protein from the wild type and the zig-1 mutant, total membranes were prepared from 10 g of liquid-cultured wild-type or zig-1 roots. After solubilization with 1% Triton X-100, the membrane preparation was cleared by passing it through a VTI11 antibody column to subtract any VTI11 in the solubilized membrane. VTI12 then was immunoprecipitated using purified anti-VTI12 antibodies as described above.

VTI12 Functional Substitution for VTI11

To create the zig-1 line containing the VTI12 transgenic construct, VTI12 cDNA was inserted into a binary vector derived from pBI121 that contains the 35S constitutive promoter of Cauliflower mosaic virus. The construct was transformed into A. tumefaciens strain MP90 and then introduced into the Atvti11/zig-1 plants. T1 plants were selected based on resistance to kanamycin. The genotypes of the T3 siblings used for the experiment were confirmed by checking the segregation ratio of kanamycin resistance to kanamycin susceptibility in the T4 generation. Gravitropic responses were measured as described previously by Fukaki et al. (1996). For immunoprecipitation analysis, membrane fractions were solubilized with 1% Triton X-100 and then used for SDS-PAGE and protein gel blot analysis.

Autophagy Experiments

All autophagy experiments were performed as described previously by Doelling et al. (2002) and Hanaoka et al. (2002). For the detached-leaf assay, first and second true leaves were excised from 2-week-old plants and either placed on filter paper saturated with 3 mM Mes, pH 5.7, or floated on water in the wells of 12-well tissue culture dishes. Detached leaves then were placed in the dark at room temperature for the times indicated in Results. Detached leaves were used for semiquantitative RT-PCR of senescence-related genes.

For the recovery experiments, seeds were germinated on Gamborg's B-5 (rich) medium (Invitrogen) with 2% sucrose and then transferred to No N/C medium when they were 2 weeks old. At the times indicated in Results, plants were transferred back to Gamborg's B-5 medium and scored as recovered upon the appearance of new green leaves.

For the SEN1 and YSL4 RT-PCR, total RNA was isolated from Col-0, vti12, and zig-1 leaves and stems using the RNEasy kit (Qiagen, Chatsworth, CA) with DNase I treatment. The primers for the SEN1 and YSL4 genes were the same as those described by Hanaoka et al. (2002). RT-PCR was performed using the Qiagen One-Step RT-PCR kit. The cycling conditions were identical to those described for semiquantitative RT-PCR by Hanaoka et al. (2002), except that 500 ng of starting material was used, there was an initial 30-min incubation at 50°C for the reverse transcription reaction, and a 15-min PCR activation step at 95°C and 26 cycles for both genes was used.

Double-Mutant Studies

For the double-mutant studies, plants were grown at 23°C under constant white light as described by Fukaki et al. (1996). Arabidopsis ecotype Col-0 was used as the wild-type reference. To genotype the F2 progeny of the vti12 × zig-1 cross, we isolated genomic DNA samples using the quick DNA prep for PCR described by Weigel and Glazebrook (2002). The following primers were used for F2 genotyping of VTI12: 5′ VTI12, 5′-TTCCGAGAGCTGAAAAAGTGA-3′; 3′ VTI12, 5′-TCGTCTTCTAATCCAAGCAATG-3′; and T-DNA LB (to identify vti12), 5′-TTGCTTTCGCCTATAAATACGACGGATCG-3′. The following primers were used for F2 genotyping of zig-1: ZTAILF, 5′-CAAGACTTGCACGGTCAGAGACAG-3′; 3′ ZIG, 5′-GTTCATCCTCCTCGTCATG-3′; and MPO12.P7R, 5′-GAGCAGGTGCAAGAAGGTCC-3′. Seven-week-old plants were used for wild-type, zig-1/zig-1, and vti12/vti12, and 10- to 12-week-old plants were used for VTI12/vti12 zig-1/zig-1 and vti12/vti12 ZIG-1/zig-1. Siliques were collected from the main shoot, and the first four siliques were not used because embryo lethality was high even in wild-type plants. Clearing of siliques was performed as described by Aida et al. (1997). Siliques were fixed overnight in ethanol:acetic acid (9:1) solution at room temperature. After rehydration in a graded ethanol series (90, 70, 50, and 30%) for 20 min each, siliques were cleared with chloral hydrate:glycerol:water solution. Siliques were mounted with chloral hydrate:glycerol:water solution on a glass slide, covered with a cover slip, and observed with a stereomicroscope (SMZ-U; Nikon, Tokyo, Japan) and photographed (COOLPIX; Nikon). Then, the lethality of the seeds was reexamined closely and quantitated using a microscope with Nomarski optics (ECLIPSE E800; Nikon).

Histological and Electron Microscopic Analysis

We used the same method of fixation for both histological and electron microscopic analyses. Small pieces (~1 cm) were cut from the upright stems of Arabidopsis plants (wild-type and mutant lines) and fixed under vacuum in a buffer containing 4% formaldehyde, 0.5% glutaraldehyde, 0.1 M sucrose, and 0.05 M sodium phosphate. The sections were fixed for 6 h with a fixative change every hour. We used 0.2-mL tubes and maintained the growth orientation throughout the fixing process. After fixation, the specimens were rinsed in the same buffer and postfixed in OsO4 for 1 h. The dehydrated specimens were embedded in London Resin White (Electron Microscopy Sciences, Fort Washington, PA).

Thick (1-μm) and thin (80-nm) sections were made with an Ultracut UCT microtome (Leica, Solms, Austria) equipped with a diamond knife. For histological analysis, sections were stained with toluidine blue O. For electron microscopy, sections were stained with 2% uranyl acetate and lead citrate.

Optical images were taken on an MCID Elite workstation (Imaging Research, Saint Catherines, Ontario, Canada) equipped with a motorized Axiovert 100S microscope using a Planapo 40×/0.85NA objective (Carl Zeiss, Jena, Germany) and a CoolSnap fx monochrome camera (Roper Scientific, Trenton, NJ). Tiled-field mapping with automatic focus was used to automatically visit, capture, and align up to 16 fields of view per tissue section.

Auxin Transport Analysis

Auxin transport assays were conducted on intact light-grown seedlings as described previously (Noh et al., 2001) with the following exceptions: before assay, 10 seedlings were transferred to vertically discontinuous filter paper strips saturated in one-quarter-strength Murashige and Skoog (1962) medium and allowed to equilibrate for 2 h. Auxin solutions used to measure transport were made up in 0.25% agarose containing 2% DMSO and 25 mM Mes, pH 5.2. A 0.1-μL microdroplet containing 500 nM unlabeled indoleacetic acid and 500 nM 3H-indoleacetic acid (specific activity, 25 Ci/mmol; American Radiochemical, St. Louis, MO) was placed on the apical tips of seedlings using a modified microliter Hamilton syringe. Injections were performed using a Narishige micromanipulator (Narishige Scientific Instrument Lab, Tokyo, Japan) mounted on a Florod LFA microscope-guided x-y laser cutter (Florod Corp., Gardena, CA). Seedlings then were incubated in the dark for 5 h. After incubation, the hypocotyls and cotyledons were removed, and a 2-mm section centered on the root-shoot transition zone was harvested, along with a 1- to 2-mm basal section of each root. Data shown are means ± percent standard deviations for two independent experiments.

Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact Natasha Raikhel, ude.rcu.surtic@lehkiarn.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Klaus Palme for kindly providing PIN1 and PIN3 antibodies and Anton Sanderfoot, Diane Bassham, and members of the Raikhel laboratory for helpful discussions. Jocelyn Brimo prepared the figures and the manuscript. We are grateful for help from Francis Marty and Roland Douce in interpreting the electron micrographs. Optical imaging and sample preparation were performed at the Center for Plant Cell Biology Microscopy Core Facility. Electron microscopy was performed at the Central Facility of Advanced Microscopy and Microanalysis (University of California, Riverside). This study was funded by: the National Science Foundation (MCB 0296080) to N.R.; the United States-Israel Binational Science Foundation (1999355) to N.R.; a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan (14036222) to M.T.; a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (13440241) to M.T.; the Japan Society for the Promotion of Science (15570035) to M.T.M.; the U.S. Department of Agriculture National Research Initiative Grant 2002-35304-12290 to A.M.

Notes

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016121.

Footnotes

W in BoxOnline version contains Web-only data.

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