Naoac

The extraction of vacuolar and cell wall-bound proteins from cucumber and Nicotiana benthamiana leaves essentially followed the protocol described in Link et al. [58 (link)]. Bound proteins were eluted from the resuspended cell wall fraction with 500 mM NaCl (Sangon, Shanghai, China) for 1 h at 4 °C, using an overhead shaker, followed by centrifugation at 10,000× g at 4 °C. Soluble and salt-eluted proteins (cell wall-bound fraction) were washed and concentrated by centrifugal filter (Millipore, Darmstadt, Germany) with 50 mM NaOAc (Sangon, Shanghai, China) buffer pH 5. Protein concentrations were determined by Bradford assay (Sangon, Shanghai, China). For total soluble carbohydrates extraction from cucumber seedlings, method as described by Wei et al. [59 (link)].

GmCWI4 recombinant protein was kindly gifted by Su et al. [40 (link)]. CSVI1 or GmCWI4 protein was incubated with 110–500 mM sucrose (Sangon, Shanghai, China) in 50 mM NaOAc (Sangon, Shanghai, China) buffer, pH 5.0 at 37 °C for different time intervals. After incubation, the reaction was stopped by heating at 95 °C for 5 min. Released fructose and glucose were determined using HPAEC-PAD as described in Wei et al. [60 (link)]. In parallel, glucose and fructose were also determined using a coupled spectrophotometric enzyme assay as described in Link et al. [58 (link)]. All enzyme measurements were performed under conditions in which activities were proportional to enzyme amounts and incubation times.