Figure 2.

Mapping, expression pattern and functional analysis of ClAGA2. A, GWAS of the Raf content trait in 135 watermelon accessions. The significantly associated SNPs, marked with a black arrow, are located in the promoter of ClAGA2. B, RNA-Seq expression profiles of ClAGA family genes at different developmental stages in CF from 10 to 34 DAP, cultivated mesocarp (CM) from 10 to 34 DAP, nonsweet wild accession fruit from 10 to 50 DAP, stem (S) from 10 to 34 DAP, carpopodium (C) from 10 to 34 DAP, leaf from 10 to 34 DAP, and in root. Average values ± sd for n = 2 independent fruit replicates are shown, three technical replicates for each fruit were performed. C, Bright field (left) and RFP emission merged channels (right). The red areas indicate the xylem zone labeled by Texas red dextran in vascular bundles of watermelon fruit. D, In situ hybridization of ClAGA2 expression in the watermelon fruit (left). Vascular bundles are labeled, with the IP and EP marked with black arrows. Vascular parenchyma cells are outlined by the dotted line; FP, fruit parenchymal cells. The sense probe was used for in situ hybridization in fruit as a negative control (right). E and F, Relative alpha-galactosidase activity in different watermelon tissues (E) and in vitro expressed ClAGA2 (F). Average values ± sd for n = 3 independent replicates of watermelon tissues or ClAGA2 enzyme. G, Kinetics of ClAGA2 enzyme activity with 50 mM Raf or Sta as substrates. Error bars, sd for n = 3 independent in vitro expressed ClAGA2 enzyme. H and J, Fruit maturity (H), sugar content (I), and alpha-galactosidase activity (J) after injection of alpha-galactosidase inhibitor DGJ. Three independent fruits at 18 DAP for each treatment are shown. Bar = 10 cm. Average values ± sd for n = 3 independent biological replicates are shown. *t-test significant at P < 0.05, **t-test significant at P < 0.01.