SNARE molecules play important roles in cell-plate vesicle fusion during plant cytokinesis 
. Based on the sequence information from available genome assemblies, VAMP721
are classified into R-SNAREs of the VAMP72 group 
. Recent studies implicate that VAMP721 and VAMP722 are essential for plant growth in other aspects in addition to their roles in plant immune responses 
. In the present study, our results suggest that VAMP721 and VAMP722 are essential R-SNARE molecules required for cell plate formation. This conclusion is based on four key findings: (1) the homozygous vamp721vamp722
double mutant exhibited a strong cytokinesis-defective phenotype, lethal dwarf seedlings, characterized by the frequent appearance of bi-nucleate cells, cell wall stubs, or gaps. However, we did not detect any seedling-level cytokinetic defects in single vamp721
mutants or in heterozygous double mutants (Figure S6
), indicating that VAMP721 and VAMP722 have redundant functions in cytokinesis; (2) vamp721vamp722
mutations retarded cell plate expansion; (3) Confocal images revealed that both GFP-tagged VAMP721 and VAMP722 were localized to both the growing cell plates with strong signals and postcytokinetic walls with decreased intensity during cytokinesis. These labeling patterns are different from those of exocyst components that started to substantially accumulate at cell plate insertion sites after fusion with the mother wall 
; and (4) The resumed plant growth of complemented double mutant was probably due to the reestablishment of proper cytokinesis. Yet, we can not explain the precise mechanism leading to the incomplete cell wall phenotype in the vamp721vamp722
mutant. The most probable explanation is that VAMP721 and VAMP722 mediate homotypic membrane fusion during the entire process of cytokinesis. In this scenario, vesicle fusion is severely impaired when the key components of R-SNARE are absent 
. Alternatively, VAMP721 and VAMP722 may mediate heterotypic fusion of later-arriving vesicles with the nascent cell plate, which attributes to a VAMP721- and VAMP722-independent mechanism. However, such a two-step formation of cell plate during cytokinesis has not been documented. In any case, the fact that VAMP721 and VAMP722 contribute to the cell plate maturation is beyond doubt. These results suggest that VAMP721 and VAMP722 are the newly identified R-SNARE components for cell-plate membrane fusion.
Charting the subcellular localization facilitates the functional analysis of the interested genes. However, few studies deal with the subcellular localization and trafficking of VAMP721 and VAMP722 in detail except the early data that these two proteins are localized to plasma membrane and unknown organelles in Arabidopsis
protoplasts by transient assays 
. In our present study, we confirmed the PM localization of VAMP721 and VAMP722 in Arabidopsis
root cells. Importantly, we demonstrated that both VAMP721 and VAMP722 were localized to the cell plate during cytokinesis and cytosolic TGN/early endosomal compartments overlapped with VHA-a1-labled TGN domains. BFA treatment induces the accumulation of endocytosed proteins, FM4-64 and early endosome markers such as VHA-a1 and RabA2/A3 in large aggregates of membranes referred to as BFA compartments in Arabidopsis 
. Our results showed that when FM4-64 was added to root cells pretreated with BFA, FM4-64 accumulated into the BFA compartments in a region enriched with VHA-a1 proteins and surrounded by the trans-Golgi marker N-ST-YFP. Furthermore, we found that endosomes labeled with GFP-VAMP721 or GFP-VAMP722 were induced to accumulate at the core of BFA compartments that were enriched with FM4-64 after BFA treatment, indicating that GFP-VAMP721 and GFP-VAMP722-labeled endosomes are sensitive to BFA treatment, similar to the response of the VHA-a1 compartment.
The late enodosome markers were very sensitive to wortmannin treatment and the inhibitory effect of wortmannin can be monitored by the Rab GTPase RabF2b, as it is the most accepted PVC marker 
. In contrast to the response of GFP-RabF2b, wortmannin treatment did not induce vacuolation of endosomes labeled with GFP-VAMP721 and GFP-VAMP722, indicating that GFP-VAMP721- and GFP-VAMP722-labeled endosomes were not the targets of wortmannin. The co-labeling experiments using plants expressing mCherry-tagged VAMP721 or VAMP722 and the trans-Golgi marker N-ST-YFP or the PVC marker GFP-RabF2b showed that both the VAMP721 and VAMP722 compartments were distinct from the marker-labeled Golgi stacks and the PVC, although they were often close to these organelles. Deducing from the effects of the trafficking inhibitors on transgenes-labeled endosomes and the colocalization analysis, we can conclude that the organelles labeled with GFP-VAMP721 and GFP-VAMP722 do not behave as typical Golgi apparatus and PVC, providing clues for early endosomal compartments.
FM4-64 has been demonstrated as a useful tool to chart the endocytic pathway in plant cells 
. Our double labeling experiments showed that FM4-64 rapidly and significantly accumulated in the VAMP721 and VAMP722 compartments before it reached the PVC labeled with GFP-RabF2b within 6 min, similar to the labeling pattern of the VHA-a1-GFP compartment. Our results are consistent with recent findings that VHA-a1 and Rab-A2/A3 compartments are the early sites of FM4-64 labeling within 5–6 min, whereas the PVC compartment identified by BP80 or the Rab-F2 subclass apparently separates from the FM4-64 signal within the same time and even after 10 min or more after dye application in Arabidopsis
root tip cells 
. These results suggest that the VAMP721 and VAMP722 compartments also represent early sites of FM4-64 accumulation. The colocalization analyses showed that the VAMP721 and VAMP722 compartments colocalized with VHA-a1-labeled TGN membrane domain, indicating that VAMP721 and VAMP722 define the TGN/early endosomal compartments that either give rise to or are derived from compartments that carry VHA-a1 proteins. Thus, the TGN/early endosomal compartment localization of VAMP721 and VAMP722 might imply the putative trafficking pathways mediated by these two proteins either trafficking from Golgi to TGN or internalization from PM to TGN.
Our results also confirmed the inhibitory effect of ConcA on post-TGN trafficking and cell plate formation in dividing cells 
. Using this inhibitor, we found that GFP-KNOLLE-labeled vesicles were induced to form aggregates, resulting in an incomplete cell wall, similar to previous results 
. We also observed that ConcA treatment induced cellular accumulation of GFP-VAMP721- and GFP-VAMP722-labeled organelles and impaired the maturation of cell plate in dividing cells. Our results indicate that the vesicles and endosomes labeled with GFP-VAMP721 and GFP-VAMP722 are indeed transported from the TGN to the division plane during cytokinesis, similar to the trafficking pathway of the cell plate marker GFP-KNOLLE. Moreover, the evidence presented here highlights the specialized TGN function in mitotic cells, which has been strongly suggested by other studies in Arabidopsis
. For example, KNOLLE positive vesicles move to the cell plate through the TGN during cytokinesis. After cell plate formation, KNOLLE is retrieved to PVC, possibly via TGN, and finally to the lytic vacuole for degradation 
. Additionally, Chow et al. 
revealed that the small GTPases RAB-A2/A3 proteins, which define a new TGN/early endosomal membrane domain, colocalized with KNOLLE throughout mitosis and contributed substantially to the cell plate. Recently, it was shown that the MPK6 localized to the secretory TGN vesicles is involved in cell division plane control 
. Thus, our findings suggest that the VAMP721- and VAMP722-labeled vesicles and endosomal compartments sorted from TGN/early endosomal membrane domains are required for cell plate construction.
It has long been accepted that the newly synthesized material from Golgi apparatus-originated secretory vesicles mainly contributes to the cell plate formation. Inhibition of ER-Golgi trafficking with BFA treatment suppressed the transport of newly synthesized KNOLLE from Golgi to the cell plate via TGN and resulted in binucleate cells and cell wall stubs in gnl1
. RAB-A2/A3 compartment lay on the secretory pathway from Golgi to plasma membrane and dominant-inhibitory mutants of RAB-A2a
prolonged the retention at Golgi or plasma membrane, thus impairing cytokinesis by titrating their interactors 
. Golgi-derived membrane and proteins, however, are not the only source for cell plate construction. In BY-2 cells and Arabidopsis
seedlings, the endocytic tracers FM4-64 or the fluid phase markers Alexa 633 and Lucifer Yellow clearly labeled the forming cell plate within minutes after addition 
. Moreover, several PM marker proteins and parental cell wall-derived pectins were found to internalize and target into cytokinetic cell plate, in parallel with an increasing rate of endocytosis when the cell plate was forming 
, supporting the role of the endocytic pathway in cell plate building. However, the relative contribution between secretory and endocytic trafficking to cell plate formation remained to be further determined. Our results showed that in vamp721vamp722
mutant seedlings, the PM marker proteins were abnormally aggregated in the cytoplasm almost without plasma membrane localization, while the tonoplast marker proteins appeared normal localization, demonstrating that VAMP721 and VAMP722 are required for PM proteins trafficking and vesicle fusion at the plasma membrane. We also found that vamp721vamp722
mutations retarded cell plate expansion, probably due to the impaired membrane fusion at the division plane. Given the link between secretion of PM proteins and membrane targeting during cytokinesis, our findings suggest that VAMP721 and VAMP722 are essential for vesicle delivery, in particular for vesicle fusion, at the cell-division plane to complete cell plate expansion during plant cytokinesis. Based on our results together with recent publications, a hypothetical model for vesicle trafficking during plant cytokinesis, in which VAMP721- and VAMP722-labeled TGN/early endosomal compartments converge the secretory and endocytic pathways, is presented in .
Hypothetical model of vesicle trafficking during cell plate formation in root tip cells of Arabidopsis.