Increases in the Number of SNARE Genes Parallels the · Genome Analysis Increases in the Number of SNARE Genes Parallels the Rise of Multicellularity among the Green Plants1[W][OA]

Genome Analysis

Increases in the Number of SNARE Genes Parallels theRise of Multicellularity among the Green Plants1[W][OA]

Anton Sanderfoot*

Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108

The green plant lineage is the second major multicellular expansion among the eukaryotes, arising from unicellular ancestorsto produce the incredible diversity of morphologies and habitats observed today. In the unicellular ancestors, secretion ofmaterial through the endomembrane system was the major mechanism for interacting and shaping the external environment.In a multicellular organism, the external environment can be made of other cells, some of which may have vastly differentdevelopmental fates, or be part of different tissues or organs. In this context, a given cell must find ways to organize itssecretory pathway at a level beyond that of the unicellular ancestor. Recently, sequence information from many green plantshave become available, allowing an examination of the genomes for the machinery involved in the secretory pathway. In thiswork, the SNARE proteins of several green plants have been identified. While little increase in gene number was seen in theSNAREs of the early secretory system, many new SNARE genes and gene families have appeared in the multicellular greenplants with respect to the unicellular plants, suggesting that this increase in the number of SNARE genes may have somerelation to the rise of multicellularity in green plants.

The flowering plants are the most recent of a longlineage of green plants that first set to land nearly 450million years ago in the Ordovician period (for review,see Sanderson et al., 2004). In turn, the early landplants arose from a group of aquatic green algaerelated to Chara, which themselves are related tofamiliar unicellular green algae like Chlamydomonas.Thus, the multicellular plants arose from unicellularancestors completely independent from the animallineage, and represent a chance to examine a distinctway to assemble a multicellular organism. Multicellu-larity requires both the ability to perform as a cell andas part of a larger entity. One major requirement forboth of these abilities is the proper management of theendomembrane system, since within a multicellularorganism, the neighbors of a given cell can have a dif-ferent developmental fate, be part of a different tissue,or even a different organ. Basically, different neighborsmay require different aspects of cell-to-cell signaling(developmental signals, cell wall components, etc.),and thus it becomes important to control the secretoryaspects of the cell. Thanks to the large-scale sequenc-ing and annotation of many green plant genomes, we

can now examine some of the molecular basis of howone major player in the endomembrane system hasevolved during the rise of multicellular plants, that ofthe SNARE family of proteins.

Proteins of the SNARE family function as molecularmachines, self-assembling into a cluster of four coiled-coil helices that drive membrane fusion (for review, seeHong, 2005). The helices, termed Qa, Qb, Qc, and R areeach contributed by individual SNARE proteins, exceptin the SNAP25 family, which carries two SNARE helices(Qb and Qc) in a single protein. As a vesicle forms atthe donor membrane, a single type of SNARE is incor-porated into the vesicle membrane. Termed a vesicle(v)-SNARE, this protein has some level of specificity inits ability to interact with a cognate set of target(t)-SNAREs that contribute the other helices. The classicexample is the mammal synaptic complex where thev-SNARE of the synaptic vesicle, R-synaptobrevin/VAMP-2, interacts with the presynaptic t-SNARE com-plex of Qa-Syntaxin and Qb 1 Qc-SNAP25 (Hong,2005). Similarly, the v-SNARE of the endoplasmicreticulum (ER)-derived COPII vesicle, Qc-Bet1p, inter-acts with the cis-Golgi t-SNARE complex of Qa-Sed5p,Qb-Bos1p, and R-Sec22p (Hong, 2005). This theory hasbeen well supported by both in vivo and in vitrosystems, and likely represents the typical situationthroughout the endomembrane system of eukaryotes.

Full genome sequence assemblies have been pro-duced for three angiosperms, two dicots and onemonocot: Arabidopsis (Arabidopsis thaliana; eudicot:eurosids: malvids: Brassicales), Populus trichocarpa(eudicot: eurosid: fabids: Malpighiales), and rice (Oryzasativa; monocot: commelinids: Poales). In addition, fourchlorophyte algae have also been sequenced: Chlamy-domonas reinhardtii (Chlorophyta: Chlorophyceae), Vol-vox carteri (Chlorophyta: Chlorophyceae), Ostreococcus

1 This work was supported by the Department of Plant Biology atthe University of Minnesota, Twin Cities, and by a Grant-in-Aidaward from the graduate school of the University of Minnesota.

* E-mail [email protected]; fax 612–625–1738.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Anton Sanderfoot ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.106.092973

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tauri, and Ostreococcus lucimarinus (Chlorophyta: Prasi-nophyceae). Recently, these extremes of the green plantlineage were connected with the release of the genomesequence of the moss Physcomitrella patens (Bryophyta).For each of these genome sequences, all genes of theSNARE family were identified and classified withrespect to the major clades identified in previous anal-yses of the Arabidopsis SNAREs (Sanderfoot et al.,2000; Pratelli et al., 2004; Uemura et al., 2004), the riceSNAREs (Sutter et al., 2006), and a recent algorithm-based analysis of SNAREs for several sequenced eu-karyotes (Yoshizawa et al., 2006). In addition to thoseclades, some additional SNAREs were identified fromnew information in other organisms, and by compari-son among the green plant sequences.

Overall, the number of SNARE-encoding genes in-creases among the land plants compared to the unicel-lular plants. In addition to an increase in the netnumber of genes, new gene families and subfamiliesappear among the SNARE genes with products that areassociated with secretion and with the endosomal sys-tem. The increase in gene number for the endosomalSNAREs might be associated with the development ofthe typical large central vacuole morphology of theplant cell. Meanwhile, the large increase in genesencoding secretory SNAREs seems to occur in multi-cellular plants in comparison to their presumed unicel-lular ancestors. Others have hypothesized that thesekinds of increases in gene number may be associ-ated with the rise of multicellularity (e.g. Dacks andDoolittle, 2002). For example, once the cell above isdifferent from the cell beside, it may become importantto be able to deliver different material (cell-cell signals,extracellular matrix components, etc.) to the top of thecell than to the side. One mechanism for this polariza-tion of secretion can be provided by duplication andspecialization of the secretory SNAREs. Already, someevidence support the specialization of secretory SNAREsin distinct processes (Lukowitz et al., 1996; Collinset al., 2003; Sutter et al., 2006). Collecting and identi-fying the potentially specialized SNAREs of the greenplants with particular achievements in multicellularitymay lead to more efficient studies of this process.

RESULTS AND DISCUSSION

Examining the sequences available from many eu-karyotes has indicated that most eukaryotes have acommon set of SNARE proteins, a core set that likelyrepresents the genes from the last common ancestor ofthe extant eukaryotes (Table I; Dacks and Doolittle,2002, 2004; Yoshizawa et al., 2006). Often, SNAREs thatare single genes in unicellular organisms are present assmall gene families, or even seem to have divergedinto novel gene families that have gained specializedfunctions in several organisms. In particular, an in-crease in the net number of SNARE-encoding genes isseen in at least two places, among the green plants andamong the animals (Fig. 1). This observation that the

increase in SNAREs may be associated with the rise ofmulticellularity in animals and land plants has beenmade before (Dacks and Doolittle, 2002), though theincrease in the sequence information from the pre-sumed unicellular ancestors of these groups has pro-duced more support for this hypothesis.

Overall, the green plants examined in this workhave a remarkably consistent and homologous set ofSNARE proteins that generally recapitulate their pre-sumed evolutionary relationships (see below). In con-trast, based upon the genome sequences of twounicellular thermoacidophilic red algae and EST evi-dence from the multicellular red algae Porphyra ye-zoensis, the SNARE proteins of the red algae are nomore similar to those of the green plants than eitherare to the heterokont diatoms or oomycetes (see belowfor examples). Therefore, even though red algae areoften considered to be a part of a larger plant kingdomalong with the green and glaucophyte algae (definedby those organisms that are descendants of the pri-mary plastid symbiosis; see Cavalier-Smith, 1998; Adlet al., 2005), this work will focus on the more clearlymonophyletic green lineage (i.e. green plants).

The SNAREs of the Early Secretory Pathway HaveChanged Little in Land Plants

Green plants have proteins similar to the yeast(Saccharomyces cerevisiae) or mammalian SNAREs thatoperate between the ER and Golgi apparatus (Fig. 2;Supplemental Figs. S1, S2, and S6; Table I). In yeastcells, the SNARE complex that mediates retrogradetraffic back to the ER seems to consist of Qa-Ufe1p 1Qb-Sec20 1 Qc-Use1p 1 R-Sec22p (for review, seeHong, 2005). As in mammals, plants have proteins thatare similar to each of these SNAREs: Qa-SYP81, Qb-SEC20, Qc-USE1, and R-SEC22. These proteins arefound in all angiosperms examined, and also in thechlorophyte algae, indicating that this complex wasinherited vertically through the green plant lineage. Insome angiosperms, multiple copies of some of thesegenes have been identified in completely sequencedgenomes, though any specialized functions for theadditional copies have not been examined. Due to amisannotation in an early version of the Arabidopsisgenome, the gene encoding Qc-USE11 (At1g54110)was fused to an adjacent gene that encodes a cationexchanger (CAX10; At1g54115). Though this genemodel was later split, the CAX10 name stayed withthe USE1-encoding gene, and has subsequently beenpassed through annotation of several other plant ge-nomes (and missed in the Yoshizawa et al., 2006analysis). Nonetheless, this gene does encode a proteinwith a structure similar to other SNARE proteins,groups with the USE1-like genes of animals and fungiin phylogenetic analysis (Supplemental Fig. S1) and isfound in all green plants (Table I).

Similarly, proteins similar to those of the yeast oranimal Golgi SNARE complex (Hong, 2005) are alsofound in green plants: Qa-SYP3, Qb-MEMB1, Qc-BET1,

The SNARE Proteins of Green Plants

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and R-SEC22. Investigations into a few of these geneshas indicated that the Arabidopsis proteins functionsimilar to the yeast and animal homologs (Chatre et al.,2005). Again, these proteins are present in all greenplants (Fig. 2; Supplemental Figs. S2, S3, S5, and S6).In Arabidopsis, two of these genes (SYP31 and SYP32)may have specialized functions: Instead of the cis-Golgi,SYP31 is found on the peripheral ER and may par-ticipate in a specialized role in cytokinesis (Rancouret al., 2002).

Later in the Golgi stacks, it has been shown thatsome of the SNAREs in the early Golgi complex arereplaced by other SNAREs (Parlati et al., 2002; Volchuket al., 2004). In mammals, Qb-Membrin is replaced byQb-GS28 and Qc-mBet1 is replaced by a related pro-

tein (Qc-GS15; Volchuk et al., 2004). Plants also havehomologs of the Qb-GS28/Gos1p and a BET1-likeprotein called Qc-SFT1 (Table I; Fig. 2; SupplementalFigs. S2, S3, and S5). An annotation error led to theassociation of the Arabidopsis SFT1-family genes withthe KOG (clusters of orthologous groups for eukary-otic complete genomes; Tatusov et al., 2003) for themitochondrial termination factor (KOG1267). This ledto spread of this name for the SFT1-like genes through-out subsequent autoannotations of genomes. As withUSE1, the plant SFT1-like genes have a clear SNARE-like structure and group within the brevin-like Qc-SNAREs (Supplemental Fig. S5), suggesting that it isreasonable to identify these as a green plant-specificclade of SNARE.

Table I. Major groups of green plant SNAREs (with outgroups)

As, Angiosperms; Gs, gymnosperms; Bp, bryophytes (mosses); Ch, Chlorophyceae; Pr, Prasinophyceae; Rp, rhodophytes; Hk, heterokonts; Am,amoebozoa; Fn, fungi; An, animals; *, at least one SNARE of this type is present. When one has been assigned, a KOG number is given. Group namesin bold represent green-plant-specific groups or subgroups.

Green Plants

As Gs Bp Ch Pr Rp Hk Am Fn An

Qa-SNAREsQa-SYP8 KOG3894 * * * * * * * * * *Qa-SYP3 KOG0812 * * * * * * * * * *Qa-SYP4 KOG0809 * * * * * * * * * *Qa-SYP2 KOG0811 * * * * * * * * *Qa-SYP1 KOG0810 * * * * * * * * * *

SYP13 – * * *SYP12 – * * *SYP11/KNOLLE – * *PEN1/ROR2 – *SYP124 – *

Qb 1 Qc-SNAREsSNAP33 KOG3065 * * * * * * *

Qb-SNAREsQb-SEC20 – * * * * * * * * * *Qb-MEMB1 KOG3251 * * * * * * * * * *Qb-GOS1 KOG3208 * * * * * * * * * *Qb-VTI1 KOG1666 * * * * * * * * * *Qb-NPSN1 – * * * * * * *

Qc-SNAREsQc-USE1 – * * * * * * * *Qc-BET1 KOG3385 * * * * * * * * * *

plant-SFT1 – * * * * *Qc-SYP6 KOG3202 * * * * * * * * * *Qc-SYP5 – * * * * * * * * *Qb-SYP7 – * * * * * * *

R-SNAREsR-SEC22 KOG0862 * * * * * * * * * *R-YKT6 KOG0861 * * * * * * * * * *R-VAMP71 KOG0859 * * * * * * * * *

VAMP711 – * *VAMP714 – * *

R-VAMP72 KOG0859 * * * * *VAMP721 – * *VAMP727 – * *VAMP724 – *

R-brevins KOG0860 * * * * *R-tomosyn KOG1983 * * * * * *

Sanderfoot

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Additional Endosomal SNARE Genes May Be Associatedwith Development of the Large Central Vacuole

At the far end of the Golgi complex are thosecomplexes of the trans-Golgi network (TGN) that func-tion in anterograde traffic from earlier Golgi stacks orin retrograde traffic from the endosomes. Plants againhave the SNAREs similar to the mammal and fungalproteins that are posited to be involved in these steps(Fig. 2; Supplemental Figs. S2–S4, and S6). In greenplants, Qa-SYP4 is similar to mammalian syntaxin 16or fungal Tlg2p, and at least in Arabidopsis, has beenshown to localize to the TGN like the mammalianequivalents (Bassham et al., 2000). Some plants havemultiple copies of Qa-SYP4, and in Arabidopsis, SYP41and SYP42 have been shown to localize to distinctdomains of the same TGN (Bassham et al., 2000), indi-cating some functional distinction. Qa-SYP41 hasbeen shown to interact with Qb-VTI12 and Qc-SYP61(Bassham et al., 2000), similar to homologous proteinsfrom yeast and mammals (Mallard et al., 2002), and toalso interact with R-YKT6 in Arabidopsis (Chen et al.,2005). It was also shown that Qa-SYP41 can distinguishbetween two members of the Qb-VTI1 gene family, indi-cating that some specialization has occurred amongthe multiple copies of these SNAREs in angiosperms(Bassham et al., 2000; Sanderfoot et al., 2001).

Defining the SNARE complexes that function in thelate endosomes and vacuole/lysosomes has been dif-ficult and may be very lineage specific. For example,

yeast has two Qa-SNAREs that are functionally dis-tinct: Pep12p that functions on the late endosome/prevacuolar compartment and Vam3p that functionson the vacuole (for review, see Burri and Lithgow,2004). Meanwhile, most other fungi have only a singlePep12p-like Qa-SNARE. Mammals also have two re-lated Qa-SNAREs, syntaxin 7 and syntaxin 13, thatmay also have a functional distinction between thelysosome and endosomes (Mullock et al., 2000; Sunet al., 2003); though, again, some animals have onlyone Qa-SNARE that presumably does both. Similarly,while chlorophyte algae have a single Qa-SYP2, an-giosperms often have multiple genes that appear tohave functional differences. For example, in Arabi-dopsis, SYP21 appears to function on the late endo-somes, while SGR3/SYP22 functions on the vacuole(Rojo et al., 2003; Yano et al., 2003). Regardless, theseQa-SNAREs each make complexes with Qb-VTI11 andQc-SYP5 (homologous to mammalian syntaxin 8; Sup-plemental Fig. S4), and like Qa-SYP41, can distinguishbetween Qb-VTI11 and -VTI12 (Sanderfoot et al., 2001).The R-SNARE partner has yet to be conclusivelydemonstrated, but preliminary indications are thatR-YKT61 can interact with each of these SNARE com-plexes (A. Sanderfoot, unpublished data). Moreover,members of the R-VAMP71 clade have been shown tolocalize to the vacuolar membrane (Carter et al., 2004;Uemura et al., 2004), suggesting that these may also beinvolved with vacuolar/late endosomal trafficking

Figure 1. SNARE genes in several eukaryotic genomes. SNAREs are divided into three basic modules based upon the site ofaction: ER/Golgi, TGN/endosomal (including vacuole), and secretory/PM. Among the unicellular eukaryotes, this division isbased upon homology to SNAREs with known functions and does not exclude multiple roles for some proteins (especially inCyanidioschyzon where the Qb, Qc, and R roles in secretion must be played by proteins from other modules). SNARE genes areindicated by individual boxes with the type of SNARE labeled with a color (Qa, orange; Qb, purple; Qb 1 Qc, violet; Qc, red; R,blue). Each box indicates a single genomic locus and does not include alternatively spliced isoforms of SNARE genes that arecommon in some lineages (especially vertebrates). Lineages are specified to the left with arrows indicating relationships amongthe major groups (Adl et al., 2005): Green, Green plants; embryo., embryophytes (land plants); chloro., chlorophytes (greenalgae); red, red algae; hetero., heterokonts (brown algae, diatoms, oomycetes); amoeba, amoebozoa (slime molds); fungi;animals. Species abbreviations: Arath, Arabidopsis; Poptr, P. trichocarpa; Orysa, O. sativa; Phypa, P. patens; Chlre, C.reinhardtii; Volca, V. carteri; Ostta, O. tauri; Ostlu, O. lucimarinus; Cyame, Cyanidioschyzon merolae; Thaps, Thalassiosirapseudonana; Phatr, Phaeodactylum tricornutum; Physo, Phytophthora sojae; Phyra, Phytophthora ramorum; Dicdi, Dictyo-stelium discoideum; Sacce, S. cerevisiae; Schpo, Schizosaccharomyces pombe; Caeel, Caenorhabditis elegans; Drome,Drosophila melanogaster; Homsa, Homo sapiens.





similar to the some of the roles indicated for VAMP7 inmammals (Ward et al., 2000; Pryor et al., 2004). Thisrole may have been elaborated in the land plantsbecause a second clade of VAMP71-type SNAREs (theVAMP714-like subgroup) was found in angiosperms(Table I; Fig. 2), though in the absence of experimentalevidence, the role of this angiosperm-specific subcladeis unclear. Ultimately, this very complicated and es-sential (Rojo et al., 2001) region of the plant cell willrequire further study to clearly identify the roles forthe different complexes in traffic to and from the en-dosomes and the vacuole.

Is the Expansion of Secretory SNAREs Associated

with Multicellularity?

The land plants have greatly expanded the moresimple complement of secretory SNAREs present intheir unicellular ancestors (Fig. 1; Supplemental Figs. S1and S6). Among green plants, it is likely that SYP1(and related proteins) represents the Qa-SNAREs ofthe plasma membrane (PM), which work along witha Qb 1 Qc-SNARE similar to Arabidopsis SNAP33 (oralternately with the Qb-NPSN1 and Qc-SYP7). Lack-ing brevins, green plants are widely believed to use aspecial branch of the VAMP7-type R-SNAREs, theVAMP72-clade, which appears to be specific to greenplants (Table I; Supplemental Fig. S6). Many membersof the VAMP72-type of SNARE have been localized tothe PM in Arabidopsis (Uemura et al., 2004), though

that these proteins act as the R-SNARE for a secretorycomplex has yet to be shown conclusively. Since boththe Qa-SYP1 and the VAMP7 clades have greatly in-creased among the transition from chlorophytes to landplants (Fig. 2), these groups deserve more investigation.

The SYP1 Lineage and Specialization and Differentiation

Among the vertebrates, many distinct types of Qa-SNAREs (syntaxins) reside on the PM (syntaxins 1, 2,3, 4, 11, and 19) that have diverse and specializedfunctions in delivery of vesicles to various domains ofthe PM (Hong, 2005). Other animals typically have asyntaxin 1-like protein and then at least one otherSNARE distantly related to this syntaxin 1 to 4 clade(Bock et al., 2001). Fungi tend to have a single (or aparalogous pair) of PM-type syntaxins (e.g. Burri andLithgow, 2004), as do most other unicellular eukary-otes that have been examined (Fig. 3). Others haveargued that this diversity of vertebrate syntaxins re-flects their complex multicellular lifestyle in contrastto their simpler unicellular ancestors (Bock et al., 2001;Dacks and Doolittle, 2002).

Based on phylogenetic analysis, the single-copy chlo-rophyte SYP1 lies at the base of the embryophyte SYP1clade (Fig. 3). Among the embryophytes, the SYP1 cladesplits into two large, well-supported groups: SYP12 andSYP13 (Fig. 3). A common feature of the genes of theSYP13 group is that all have multiple introns, almost allof which occur at equivalent positions with the open

Figure 2. Clustering of SNARE gene families among eukaryotic groups. SNARE-encoding genes from several fully sequencedeukaryotes are indicated by boxes as described in Figure 1. Hatched boxes in the row for pine (Pinta, Pinus taeda) indicate thatthese genes are derived from searches of the EST database only and likely represent an underestimate of the number of SNAREgenes in this organism. Each vertical stack of boxes indicates a particular related group of SNARE proteins (see SupplementalFigs. S1–S6) with the names given to the plant groups across the top and to the vertebrate groups across the bottom. See Figure1 for abbreviations and notations. See Supplemental Table S1 for a list of individual genes for each organism.

Sanderfoot




reading frames (Supplemental Fig. S7). Little work hasbeen done on this type of syntaxin in the land plants,aside from the finding that the protein is found on thePM when overexpressed in Arabidopsis protoplasts(Uemura et al., 2004) and that it plays some role inRhizobium symbiogenesis in legumes (Catalano et al.,2007). The second group of PM syntaxins, the SYP12group, includes members from all the embryophytesexamined (Fig. 3). The SYP12 group is distinct from theSYP13 in that all the genes are encoded by a single exon,or have only one intron, typically in an equivalentposition within the ORF (Supplemental Fig. S7). Likethe SYP13 group, members of the SYP12 group havebeen shown to be found on the PM and be involved inaspects of secretion (Lauber et al., 1997; Geelen et al.,2002). The SYP12 group is itself split into three reason-ably well-supported subgroups (Fig. 3).

A preangiosperm subgroup includes all of theSYP12-like proteins from moss and SYP12-like pro-teins found encoded in the EST collections of gymno-sperms (Fig. 3). The angiosperm SYP12 subgroup isrepresented in two further subgroups, the PEN1/ROR2 and the SYP124-like subgroups. In addition toa role in general secretion (Geelen et al., 2002), the

PEN1/ROR2 subgroup may have taken on a special-ized role in defense to fungal pathogens (Collins et al.,2003; Nuhse et al., 2003; Assaad et al., 2004). Arabi-dopsis mutants that lack both members of the PEN1/ROR2 subgroup (i.e. syp121/pen1, syp122 double mu-tants) are severely dwarfed and show necrosis (Assaadet al., 2004), suggesting that the function of the sub-group cannot be completely replaced by the SYP124subgroup. Nonetheless, any specialized role for theSYP124 subgroup syntaxins among the angiospermshas yet to be shown. The third subgroup of SYP12syntaxins is represented by the SYP11 group. A SYP11-like protein is encoded among the gymnosperm ESTs(Fig. 3), suggesting that this group may be common toall the seed plants, though no SYP11-like protein isfound in the moss genome. Among the angiosperms,this class of syntaxin is best represented by theKNOLLE-like proteins. In Arabidopsis, KNOLLE pro-tein is produced only during cell division, is found onthe cell plate, and is essential for completion of cyto-kinesis (Lukowitz et al., 1996; Lauber et al., 1997).Interestingly, the cell-plate-mediated form of cytoki-nesis is found throughout all the land plants and iseven found in many green algae (Lopez-Bautista et al.,

Figure 3. Qa-SNAREs of the PM have greatlyexpanded in the land plants. Full-length proteinsequences of the Qa-PM orthologs from variousorganisms were aligned by ClustalW, distanceswere estimated with a neighbor-joining algorithmand visualized in a phylogram. The tree wasrooted using the Qa-PM SNAREs of heterokonts.Bootstrap support is indicated to the left ofbranches. The clades and subclades of land plantsyntaxins are also indicated as described in thetext (also see Supplemental Fig. S7). See Figure1 for species abbreviations.





2003), suggesting that other PM syntaxins must medi-ate this process in plants that lack the SYP11 group,and that KNOLLE was a later invention of the seedplants. A further subgroup of PM syntaxins (SYP112)is found only among the eudicots Arabidopsis andpoplar (Fig. 3), and no obvious relative to SYP112 hasbeen found in the EST databases of other eudicots.Because this clade is only found in two completegenomes and has no known role (Muller et al., 2003), itis unclear what role this SNARE may play.

The presence of three major groups (and severalsubgroups) of PM syntaxins suggests a complexity andspecialization of roles that occurs at a point in evolu-tionary time coincident with the rise of land plants andthe associated multicellularity (see Fig. 5). The hypoth-esis that the expansion of the plant PM syntaxins maybe linked to multicellularity has been made before (e.g.Dacks and Doolittle, 2002), but the new information inthis work now suggests this point clearly lies closer tothe radiation of the land plants. One could hypothesizethat the SYP13 group represents the basal PM syntaxininherited vertically from the chlorophyte ancestors,and would be involved in general housekeeping roles(e.g. constitutive secretion). The SYP12 group wouldarise in the mosses (or earlier) to support more spe-cialized roles of secretion (e.g. defense related, orperhaps cell-plate formation). Finally, a specializedsyntaxin (SYP11/KNOLLE) would evolve to exclu-sively operate the essential process of cytokinesis in theseed plants (or earlier). Later specializations (PEN1/ROR2 and SYP124) would arise to further differentiatesome additional functions. Intensive research intomembers of these clades in particular model systemsmay support this hypothesis, as could additional se-quence information from other plants that lie at evo-lutionarily significant points.

The VAMP7 Lineage in the Green Plants

Within the animal and fungi lineage, the brevinsserve the functions associated with secretion (Hong,2005), and some other lineages (e.g. Dictyostelium) havesimilar brevins that may act similarly (SupplementalTable S1). On the other hand, the only potential brevinsin the green plant lineage are found in Chlamydomonas,where a paralogous pair of genes (VAMP like orVMPL) with unknown function are found. Instead,plants only have the longin types of R-SNARE (Fig. 2;Supplemental Fig. S6). Longin proteins have a con-served N-terminal domain that contains a profilin-related fold that is also found in other non-SNAREproteins (Rossi et al., 2004). In particular, plants have alarge group of longin SNAREs similar to the mamma-lian VAMP7 (Table I). In mammals, VAMP7 (also calledTI-VAMP) is involved in intraendosomal trafficking aswell as specialized aspects of exocytosis (Pryor et al.,2004). Green plants seem to have two major groups ofVAMP7-like proteins (Table I; Supplemental Fig. S6).The VAMP71 group is most similar to the mammalian

VAMP7 and likely plays a similar role to the mamma-lian VAMP7 in the endosomal system (see above). Onthe other hand, the second group (VAMP72) appears tobe specific to the green plant lineage (Table I; Fig. 4)and likely represents the R-SNARE component forsecretion among the green plants.

As in the VAMP71 group (see above), there has beena divergence among the VAMP72 group in the seedplants, where VAMP724 and VAMP727 subgroups canbe identified (Table I; Fig. 4). Some results have indi-cated that the VAMP727 of Arabidopsis is localized tothe early endosomes while other VAMP72 gene familymembers from angiosperms have been found on thePM at steady state (Marmagne et al., 2004; Uemuraet al., 2004). The VAMP727-like proteins of the seedplants have a common 20-residue insertion in theN-terminal domain that distinguishes these proteinsfrom the other VAMP72 proteins (Supplemental Fig.S8). The split to make the VAMP724 subgroup seemsto have happened in the seed plants and perhaps inthe angiosperms, suggesting a more recent specializa-tion of function. Importantly, together with the PMsyntaxins, this parallel enlargement of the VAMP72group seems to have occurred during the time of therise of complex multicellularity among the green plantlineage (Fig. 5), and suggests that specializationsamong the secretory machinery may have played anessential role in the rise of multicellularity.

The Strange Case of Qb 1 Qc-SNAREs (SNAP25 Like)

SNAP25, a protein with two tandem SNARE do-mains (an N-terminal Qb and a C-terminal Qc domain)was one of the original proteins to be defined as aSNARE, and is also a member of the prototypicalSNARE complex of the mammalian synapse (for re-view, see Hong, 2005). In SNAP25, a stretch of Cysresidues following the N-terminal Qb-SNARE domainare palmitoylated vivo and serve to increase the mem-brane interactions of these proteins (Gonzalo et al.,1999). A third protein, SNAP29, is also found inmammalian cells, and is thought to be involved insimilar fusion events among endosomal membranes(Steegmaier et al., 1998). A fourth type of Qb 1 Qc-SNARE, SNAP47, has recently been identified inmammals (Holt et al., 2006). Fungi also have a Qb 1Qc-SNARE, Sec9p, which operates in the secretorySNARE complex similar to SNAP25 in their cells (forreview, see Burri and Lithgow, 2004). Though thefungal proteins are highly divergent from their animalequivalents, it is generally thought that they are eachderived from a common ophistokont ancestor.

Among the green plants, the SNAP33-type proteinsare another example of a Qb 1 Qc-SNARE and havebeen shown to play similar roles to that of the mam-malian SNAP25. SNAP33-like proteins have beenshown to be essential in such roles as general secretion(Kargul et al., 2001), pathogen defense (Collins et al.,2003), and for cell plate formation during cytokinesis

Sanderfoot




(Heese et al., 2001). Searches in the sequences of othergreen plants have identified proteins similar toSNAP33 in gymnosperms and mosses (Fig. 2), indi-cating that these proteins have been in green plantssince the adaptation to land. Compared to SNAP25,the land plant SNAP33 proteins have an N-terminalextension (that does not bear any strong resemblanceto the N-terminal extensions of SNAP29, SNAP47, orSec9p), and lack the palmitoylation site of theSNAP25-like proteins. Outside of the land plants, thechlorophyte algae Chlamydomonas and Volvox alsoencode SNAP25-like proteins (SNAP34). Oddlyenough, these proteins have an N-terminal extensionsimilar to that of the land plant SNAP33, yet theseproteins also have a Cys-rich sequence after the Qb-

SNARE domain similar to the sequence that is pal-mitoylated in SNAP25 (A. Sanderfoot, unpublisheddata). Whether this domain is palmitoylated in thesealgae has not been tested. The prasinophyte greenalgae Ostreococcus, two thermoacidophilic red algae(Cyanidioschyzon and Galdieria), and all the heterokontsexamined do not have any SNAP25-like SNAREs(Table I; Fig. 2).

Since the divergence between the animal and fungiand green plant lineages span the many SNAP25-lacking unicellular protist groups (Table I; Dacks andDoolittle, 2001), the presence of this gene in these dis-parate branches is difficult to reconcile by strict par-simony. Furthermore, that Chlamydomonas and Volvoxcould have a Qb 1 Qc-SNARE that is palmitoylated

Figure 4. R-SNAREs of the VAMP7 group havegreatly expanded in the land plants. Full-lengthprotein sequences of the R-VAMP7 orthologsfrom various organisms were aligned byClustalW, distances were estimated with a neigh-bor-joining algorithm and visualized in a phylo-gram (top). The tree was rooted using the VAMP7SNAREs from heterokonts. Bootstrap support isindicated to the left of branches. The ubiquitousVAMP71 and the green-plant-specific VAMP72clades are indicated at right, along with thesubclades of the land plants (see text and Sup-plemental Fig. S8). See Figure 1 for species ab-breviations.





at a remarkably similar sequence to that of the verte-brate SNAP25 proteins is strange, especially consider-ing that this trait is not shared with organisms withintheir separate lineages. Did the green plants inventSNAP33 distinct from the animals and fungi, or havemany other lineages lost the ancestral gene retainedin the plants, animals, and fungi. Finally, consideringthe essential role of SNAP25-like proteins in the se-cretory process of those cells that possess them, whatSNAREs have replaced SNAP25 in those eukaryotesthat lack this class of SNARE?

Additional SNAREs First Identified in Plants: Qb-NPSN1and Qc-SYP7

Through searches in the genomic sequence of themodel plant Arabidopsis, new types of Qb- and Qc-SNAREs that were not found in animal or fungalgenomes were identified (Sanderfoot et al., 2000). Asmore sequence information later became available

from nonanimal/fungal unicellular eukaryotes, ho-mologs of SYP7 and NPSN have turned up in alveo-lates, chromists/heterokonts, and amoebae, thoughnever in an animal or fungi (Table I; Fig. 2; Supple-mental Figs. S3 and S4). Since these proteins do notexist in the heavily studied animal or fungal systems,they did not receive as much attention as other types ofSNAREs, but recent work has begun to indicate thatthese proteins may be worthy of more study.

Outside of green plants, these types of Qb- and Qc-SNAREs are found in most eukaryotes that lack theQb 1 Qc-SNAP25-type SNARE that seems to be in-volved in secretion (see above). This leads to the hy-pothesis that these SNAREs have replaced (or werereplaced by) SNAP25-like SNAREs in secretion/exo-cytosis in these important groups of eukaryotes. Evi-dence for a role in secretion comes from experimentsin the angiosperm Arabidopsis where it has beenshown that Qb-NSPN11 and Qc-SYP71 interact withthe cell-plate-specific Qa-KNOLLE, and have a role in

Figure 5. Hypothetical expansion ofsecretory SNARE gene families in thegreen plants. Relevant taxa of greenplants are indicated with boxes repre-senting the hypothesized state of theQa-SYP1 and R-VAMP7 gene familiesat the point in green plant evolutionindicated by the arrow. New genefamilies are indicated by indentednames with arrows indicating the hy-pothetical descent of that gene from arelated gene family (see text and Sup-plemental Figs. S7 and S8 for moredetails).

Table II. Web sites of genome assemblies used in this work

Green Plants Genome Assembly Sites

Arabidopsis v6.0 www.arabidopsis.orgRice v4.0 http://www.tigr.org/tdb/e2k1/osa1/Poplar v1.0 http://genome.jgi-psf.org/Poptr1/Poptr1.home.htmlChlamydomonas v3.0 http://genome.jgi-psf.org/Chlre3/Chlre3.home.htmlVolvox v0.0 (not publicly released, available through JGI Chlamydomonas site)O. tauri v2.0 http://genome.jgi-psf.org/Ostta4/Ostta4.home.htmlO. lucimarinus v2.0 http://genome.jgi-psf.org/Ost9901_3/Ost9901_3.home.htmlP. patens v1.0 http://genome.jgi-psf.org/Phypa1/Phypa1.home.htmlRed algae

C. merolae v1.0 http://merolae.biol.s.u-tokyo.ac.jp/Galdieria sulfuraria v0.0 http://genomics.msu.edu/galdieria/about.html

HeterokontsT. pseudonana v1.0 http://genome.jgi-psf.org/thaps1/thaps1.home.htmlP. tricornutum v1.0 http://genome.jgi-psf.org/Phatr1/Phatr1.home.htmlP. sojae v1.1 http://genome.jgi-psf.org/Physo1_1/Physo1_1.home.htmlP. ramorum v1.1 http://genome.jgi-psf.org/Phyra1_1/Phyra1_1.home.html

Sanderfoot




the secretion-related process of cytokinesis (Zhenget al., 2003; L. Conner and A. Sanderfoot, unpublisheddata). Moreover, these proteins are found on variousorganelles in the late secretory system in nondividingcells (Zheng et al., 2003; Marmagne et al., 2004;Mongrand et al., 2004; Morel et al., 2006). Whenmembers of the Qc-SYP7 group were fused to afluorescent protein and overexpressed in protoplasts,these fusion proteins were reported to localize to theER (Uemura et al., 2004). This result is at odds with theresults of others when examining tobacco (Nicotianatabacum) cells (Marmagne et al., 2004; Mongrand et al.,2004; Morel et al., 2006). Whether this result is simplyan overexpression artifact or a reflection of a special-ized role for these proteins under certain conditionsremains to be seen. Still, it seems possible that theseproteins may be the major mediators of secretion inorganisms that lack SNAP25-like Qb 1 Qc-SNAREs,which includes many medically relevant organismslike the unicellular pathogens and parasites. Obvi-ously, this requires more work in both plants and inother nonanimal/fungal eukaryotes, where the me-chanical aspects of secretion have not been thoroughlyinvestigated. Meanwhile, as an additional set of secre-tory Qb- and Qc-SNAREs in the green plant lineage(along with the Qb 1 Qc-SNAP33 group), they pro-vide an additional flexibility for the SNARE complexesthat can mediate secretory vesicle trafficking, and thuscan be considered to be part of the enlarged comple-ment of secretory SNAREs among the multicellulargreen plants.

CONCLUSION

The SNARE family of proteins has greatly enlargedamong the land plants with respect to their unicellularancestors. This is apparent in Figures 1 and 2, as wellas in the ordered list of SNAREs for each organismgiven in Supplemental Table S1. The chlorophytealgae, perhaps representing the state of unicellulargreen plants, have the basic core of the eukaryoticSNARE proteins among their 30 to 35 SNARE-encodinggenes. The moss Physcomitrella, arguably represent-ing the state of the first land plants, increases thenumber of SNAREs to a total of 63 that representalmost all of the major groups and subgroups of theangiosperm SNAREs. Among the angiosperms, thenumbers of SNAREs vary from 62 to 76 with only asmall number of new groups with respect to themosses. Clearly, the doubling of SNARE-encodinggenes that occurs between the chlorophyte algae andthe mosses spans several morphological innovations.The observation that most of these new SNAREs arerelated to secretory function, and the importance of aspecialized secretory pathway in multicellular organ-isms, it seems reasonable to hypothesize that thisincrease in SNARE number is related to multicellular-ity. Others have made this suggestion in the past, bothin relation to the green plants and the animals (Dacks

and Doolittle, 2002, 2004), and the increased genomicinformation available today has supported and ex-tended this hypothesis. Other lineages, such as somegroups of red algae and the brown algal kelps, havealso undergone well-documented transitions to mul-ticellularity from unicellular ancestors. Unfortunately,there is no genome sequence currently available fromthese groups to test if a comparable expansion ofSNARE proteins is found among these multicellulareukaryotes. Additional sequence information may oneday be available from representatives of multicellularbrown and red algae, as well as some of the charo-phyte ancestors of plants. Such data may provide moreevidence on the link between SNAREs and multicel-lularity in the near future.

Even with eight green plants, we have only exam-ined the tips of plant diversity, and many evolution-arily significant green plants are not yet available to beexamined. In the next few years, three relatives ofArabidopsis (Arabidopsis lyrata, Capsella rubella, andThellungiella halophila), fabids like Medicago truncatula,and asterids like monkey flower (Mimulus) and tomato(Solanum lycopersicum) will become available for morethorough examination of the dicot SNARE gene fam-ilies. There will also be several grass and nongrassmonocots available soon to better define the angio-sperm clades, as well as the lycophyte Selaginellamoellendorffii to aid in broader comparisons amongthe basal plants. Based upon the relatively smalldifferences between the already available angiospermsand moss, there may not be great differences in thefinal numbers of SNAREs revealed in these plants, buteach may help to narrow down the appearance ofparticular gene families and better define how eachhas helped to establish the unique aspects of the verysuccessful green plant lineage.

MATERIALS AND METHODS

Sequence Databases

The genome assemblies searched for this work are listed in Table II. In

addition to the green plant sequences, SNAREs were also examined in several

other nongreen plant algae for whom sequence assemblies were available (see

Supplemental Table S1). The Volvox assembly has not been publicly released, but

can be accessed by searches through the Joint Genome Institute (JGI) Chlamy-

domonas site. The plant gene indices (currently available through the Dana

Farber Computational Biology and Functional Genomics portal: http://compbio.

dfci.harvard.edu/tgi/) were also used to identify ESTs for several related or

taxonomically relevant plants. Though ESTs will definitely underestimate the

number of SNAREs in a given organism, the encoded protein sequences were

often useful for preventing long-branch issues (barley [Hordeum vulgare] as a

second representative of grasses), and for bridging gaps in the evolutionary

series (loblolly pine [Pinus taeda] for the gymnosperms). All other sequences

were acquired from GenBank accessions of the respective assemblies. The

tables and figures in this work refer to either the unique protein ID of the

organism-specific genome databases (see Table II), or to a GenBank accession.

Searches and Annotations

The seed used for searches was the annotated list of Arabidopsis

(Arabidopsis thaliana) SNAREs kept by the author based upon prior work

(Sanderfoot et al., 2000; Pratelli et al., 2004; Sutter et al., 2006), as well as





information from other similar works (Bock et al., 2001; Hong, 2005; Yoshizawa

et al., 2006) and unpublished information. These proteins were used to

iteratively search through the genome databases using BLASTp, tBLASTn,

and related searches (including keyword searches of autoannotations when

possible) to assure that all possible SNARE-encoding genes were identified in

the databases. When possible, EST information was used to confirm exon

structure, otherwise the best possible model was chosen for each gene based

upon homology with a related genome sequence (either an internal paralog or

a homolog in a related organism). Each predicted SNARE protein was

iteratively compared (by BLASTp or PSI-BLAST) to the nonredundant protein

database at the National Center for Biotechnology Information (http://

www.ncbi.nlm.nih.gov), the predicted proteins of the individual genome

assemblies (including that of the source organism to identify gene family

members and help identify pseudogenes and poor gene predictions), the

Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/

cdd/cdd.shtml), and the KOGnitor (http://www.ncbi.nlm.nih.gov/COG/

grace/kognitor.html). Through such analysis, typically a single group of

SNAREs (e.g. a KOG) was identified as the most likely candidate based upon a

highly significant score (greater than 1e-20), and a significant distinction from

other groups. In cases where such a KOG (or other similar group) was not

identified, a temporary group was created to facilitate identification of groups

that lie outside the previously established groups. Finally, each protein was

subjected to phylogenetic analysis with members of well-known SNARE

groups (i.e. Sanderfoot et al., 2000; Bock et al., 2001; Pratelli et al., 2004) to

ensure that the assignment of the particular SNARE group based upon the

profile-based searches is consistent with the well-known phylogeny. As new

genome information was added, each subsequent set of predicted SNARE

proteins was added to the analysis until the groups indicated in this work

were apparent.

Because this work was based upon the information present in the genome

assemblies, it was common to identify poor gene predictions based upon the

automated gene prediction algorithms. Some of these predictions could be

fixed by additional cDNA/EST information, by changing to a different algo-

rithm model, or making adjustments to the model in attempts to create a gene

prediction with a typical SNARE structure (through addition or adjustments

to exons). In some cases, this was not possible, and the most likely predictions

appeared to be fragmentary genes, or pseudogenes. Gene fragments and

pseudogenes represent cases where the most similar sequence (both protein

and nucleotide) would be from a related gene in the same genome, but

the predicted gene would only represent a truncated protein or a protein

that would be missing functionally relevant domains found in SNARE

proteins. In this analysis, none of the predicted gene fragments had any

evidence of expression. Some pseudogenes are represented in EST databases,

but in many cases this expression was very limited. For example, Arabidopsis

VAMP723 (At2g33110) contains several mutations in the third exon (with

respect to other gene family members; this exon encodes the conserved central

core of the SNARE motif), and has evidence of only very low expression across

many tissues (as derived from AtGenExpress data; Schmid et al., 2005). Even if

such a protein were produced, it would be very unlikely to produce a

functional SNARE protein. Although such genes were included in Supple-

mental Table S1, they were generally not used in phylogenetic analysis since

pseudogenes and gene fragments can significantly affect alignment and

distance estimates.

Alignment and Phylogeny

The SNARE protein sequences, either full length or just the SNARE

domain were aligned by ClustalW (MEGA3.1 software; Kumar et al., 2004).

Alignments of only the SNARE domains were created by hand through

deletion of all residues except the traditionally defined heptad-repeat motif

common to all SNAREs. Trees were produced using a boot-strapped neighbor-

joining algorithm (Saitou and Nei, 1987; as implemented in MEGA3.1) using

standard settings. Other methods of phylogenetic analysis did not result in

any significant changes to topology identified by neighbor joining (data not

shown), nor did such methods help to resolve some poorly supported

branches. Among the green plant sequences, very little change in topology

occurred between analyses using full-length sequences and those using just

the SNARE domain. The latter were only used in cases where truncated

SNAREs (brevins, or the BET1/SFT1 groups) that lack the N-terminal exten-

sions were the object of study to prevent spurious alignments and in cases

where all-SNARE alignments were used to allow useful distance estimations.

Examination of the gene structure (intron position and number, etc.) for

several SNARE groups also helped support some weakly supported clusters.

Other information, such as the well-established evolutionary series or green

plants and algae (e.g. Sanderson et al., 2004) also helped to support some of

the phylogenetic analysis.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Phylogenetic tree of ER Qb- and Qc-SNAREs.

Supplemental Figure S2. Phylogenetic tree of Qa-SNAREs from five

major clades.

Supplemental Figure S3. Phylogenetic tree of Qb-SNAREs of the Golgi,

endosomes, and PM.

Supplemental Figure S4. Phylogenetic tree of Qc-SNAREs of the TGN,

endosomes, and PM.

Supplemental Figure S5. Phylogenetic tree of Brevin-like Qc-SNAREs.

Supplemental Figure S6. Phylogenetic tree of Longin-type R-SNAREs.

Supplemental Figure S7. Alignment of Qa-SYP1 SNAREs.

Supplemental Figure S8. Alignment of R-VAMP7 SNAREs.

Supplemental Table S1. List of predicted SNARE proteins from green

plant genomes.

ACKNOWLEDGMENTS

The author acknowledges the assistance of lab members Laura Conner

and Lingtian Kong in reading and commenting on the manuscript, as well as

the Chlamydomonas and Physcomitrella genome sequence projects for allowing

me to take part in the genome annotation process for those organisms. I also

appreciate the resources made available by the Department of Energy Joint

Genome Institute for genome annotation and analysis that were helpful in

preparation of this manuscript.

Received November 14, 2006; accepted March 6, 2007; published March 16,

2007.

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