Alleva K , Marquez M , Villarreal N , Mut P , Bustamante C , Bellati J , Martínez G , Civello M , Amodeo G .

	Fig. 1. Phylogenetic analysis of full-length deduced amino acid sequences of plant aquaporins, including our clones, FaPIP2;1 and FaPIP1;1. Deduced amino acid sequences from full-length plant aquaporins encoding genes from Arabidopsis thaliana, Vitis vinifera, and other plant PIP2s with high identity to FaPIP2;1 were compared using Clustal W. Phylogenetic analyses were conducted using MEGA version 4 (Tamura et al., 2007).
	Fig. 2. Alignment of predicted amino acid sequence of Fragaria×ananassa aquaporin (FaPIP2;1) with other aquaporins (ClustalX). The predicted amino acid sequence of FaPIP2;1 was compared with aquaporins from different sources (aquaporins with the higher identity with FaPIP2;1 or very well studied in the literature). Transmembrane domains are shown with a dashed line below the alignment; triangles indicate a potential diacidic motif (putative ER signal); circles indicate the NPA selectivity filter; a star indicates His199, and inverted triangles putative phosphorylated Ser residues.
	Fig. 3. Functional expression of FaPIP2;1 in Xenopus oocytes. Calculated mean water permeabilities (Pf±SEM) of oocytes under hypo-osmotic conditions for NI (non-injected, negative control) or expressing 25 ng of FaPIP1;1, FaPIP2;1, and AtPIP2;3 (used as positive control) are shown. The number of measured oocytes for each condition is 5 to 8. Pf of oocytes expressing both FaPIP2;1 and AtPIP2;3 present significant differences from negative control (p <0.05).
	Fig. 4. Co-expression of FaPIP2;1 and FaPIP1;1. (A) Co-expression of 3 ng of cRNA of FaPIP2;1 with 12 ng of cRNA of FaPIP1;1 is shown (white bar). As a control, 3 ng of FaPIP2;1 (black bar) and 12 ng of FaPIP1;1 (light grey bar) were injected separately. The co-expression increased significantly the water permeability six times compared with the expression of FaPIP2;1 alone (p <0.05). Data are expressed as mean Pf±SEM, n=5 or 6 oocytes. NI are non-injected oocytes (grey bar). (B) Increasing cRNA mass of FaPIP2;1 injected alone in oocytes (from 6 ng to 18 ng) shows a increasing Pf. This relationship is not observed in FaPIP2;1-FaPIP1;1 co-expressing oocytes. Pf remains high but constant no matter the cRNA mass ratio injected. Data are shown as mean Pf ±SEM, n=6–8 oocytes.
	Fig. 5. Pf inhibitory response after cytosolic acidification. (A) FaPIP2;1 expressing oocytes were exposed to different external (pHe) or internal (pHi) pH conditions. Negative controls (NI) are non-injected oocytes. Pf for oocytes expressing FaPIP2;1 exposed to internal acidification is statistically different from its control (i.e. pHi=7.5), while treatment with pHe=6.0 does not result in a significant inhibition (p <0.05) when compared with its control (pHe=7.5). (B) FaPIP2;1-FaPIP1;1 was co-expressed in Xenopus oocytes and exposed to different external (pHe) or internal (pHi) pH conditions. Negative controls (NI) are non-injected oocytes. Data are shown as mean Pf±SEM, n=8–10 oocytes.
	Fig. 6. FaPIP1 and FaPIP2 expression pattern during strawberry fruit ripening. (A, C). Northern blot showing the accumulation of FaPIP mRNA in different ripening stages of strawberry fruits: large green (LG), white (W), 25% red (25%R), 50% red (50%R), and 100% red (100%R) for two different cultivars, a firm (Camarosa) and a softer one (Toyonoka). (B, D) Quantification of FaPIP1 and FaPIP2 expression relative to Toyonoka LG stage is shown for Camarosa and Toyonoka cultivars (Gel Pro Analizer v 3.0 was used). Data are shown as mean ±SEM, n=2.
	Fig. 7. Hypothetical and schematic representation of the role of aquaporins in fruit ripening. Section of a strawberry fruit showing fibrovascular strands (vascular bundles), achenes, the interior of the receptacle (pith), and main parenchyma; all marked tissues are possible targets of PIP expression due to their participation in water and solute balance in strawberry fruit.

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