Response to Rogers Letter

The original aim of our work was to develop a set of fluorescent reporters that would allow storage and lytic vacuoles to be separately highlighted within Arabidopsis cells. We were consequently surprised that our reporters consistently identified only a single type of vacuole. This inevitably led us into a systematic testing of the multiple vacuole hypothesis in Arabidopsis using our novel panel of reporters. Judging from the number of material requests received within a month of our article appearing online, these reporters have been broadly welcomed by the community. In our article, we were very careful in limiting discussion to the behavior of our reporters. We stated that, if Arabidopsis cells did contain multiple vacuoles, our reporters were unable to distinguish them (Hunter et al., 2007). Furthermore, we did not stretch the interpretation of our findings to other species because the work carried out in our laboratory was limited to Arabidopsis. Regarding the TIPs, we state that YFP fusions to their complete genomic sequences resulted in seed expression of α-TIP-YFP but not of γ- or δ-TIP-YFP. No fluorescence was detected in seeds for γ- or δ-TIP-YFP. We make it clear that only mature seeds were studied, and not developing seeds. We do not extend such claims to the endogenous TIPs (indeed, we did not look for them), but we show the transcriptomic data to underline that their transcriptional patterns mirror the fluorescence patterns observed for our reporters. Others have attempted and failed to detect γ-TIP in Arabidopsis seeds (Höfte et al., 1992; Otegui et al., 2006). We saw no evidence of the TIP-YFP being detrimental to the plants. The western blot in our supplemental figure S5B shows only that dry seeds have detectable α-, but not γ- or δ-TIP-YFP. We accept that even the “genomic” constructs are overexpressed. However, overexpression is a necessary evil in our field and was also used in the Oufattole et al. (2005) article cited by Rogers. Our 35S-driven constructs produced identical patterns of localization regardless of YFP being fused N- or C-terminally. We believe this rules out YFP-induced missorting. Finally, our results with the soluble red fluorescent protein luminal markers, which corroborate our findings with the TIP-YFP reporters, are not criticized. Although we by no means reject the multivacuole hypothesis tout court (as clearly stated in our “Discussion”), the simplest interpretation of our results remains that our reporter proteins do not confirm such a hypothesis in the Arabidopsis tissues that we studied.