RESULTS
pTAS2R20 was cotransfected into HEK-293T cells with the plasma membrane localization marker protein fused with GFP (mGFP) to determine whether pTAS2R20 was successfully localized at the cell membrane. Then, the cell membrane was visualized by using mGFP (green fluorescence, Fig. 2A). The immunocytochemical detection of pTAS2R20 (red fluorescence, Fig. 2B) showed that pTAS2R20 localized to the cell membrane. Superimposed red and green fluorescence appeared yellow, which indicated that the two markers colocalized at the cell membrane (Fig. 2C). Henceforth, pTAS2R20-expressing cells were stimulated with several common bitter compounds and bamboo-derived bitter chemicals. The results showed that all four pTAS2R20 variants were specifically responsive to quercitrin.
We quantified the contents of quercitrin in the leaves of B. fargesii and F. qinlingensis , consumed in the diet of Qinling pandas, and in the leaves of F. denudate and B. faberi , consumed in the diet of pandas from other areas. The quercitrin content of F. qinlingensis leaves was the highest among the four examined bamboo species, reaching 222.3 ng/mg, followed by that ofB. fargesii at 143.7 ng/mg. In contrast, the quercitrin contents quantified in F. denudata and B. faberi leaves were 98.2 ng/mg and 66.4 ng/mg, respectively, which are much lower than those of the two bamboo species sampled in the Qinling Mountains (P < 0.05).
To determine the optimum concentration of quercitrin required to activate pTAS2R20, we measured the highest potency of quercitrin, which activated pTAS2R20 at a low molar concentration, in a dose-response curve. When the concentration of quercitrin was increased, the activation of pTAS2R20 was demonstrated by a sigmoidal curve in which the concentration resulting in 50% of maximal effect (EC50) was 285 μM, and maximum activation occurred at a concentration of several thousand micromolar (Fig. 3C).
To characterize the effect of these two directionally selected nonsynonymous sites on the function of pTAS2R20 in the response to quercitrin, we activated each pTAS2R20 variant with the optimal activation concentration of quercitrin. Cell fluorescence images obtained at five time points before and after quercitrin treatment are shown in Fig. 4. The fluorescence intensity of the cells expressing pTAS2R20 significantly increased in comparison with that in the control groups (pcDNA, pTAS2R20, mG15), which showed no significant change. This confirmed that pTAS2R20 expressed in HEK-293T cells was responsive to quercitrin. Therefore, the experimental group harboring the pTAS2R20-AQ variant exhibited the strongest reaction, whereas the group with the pTAS2R20-VH variant showed the weakest reaction. Overall, the order of the reaction activities of the experimental groups was pTAS2R20-AQ > pTAS2R20-AH > pTAS2R20-VQ > pTAS2R20-VH. To further quantify the differences in the responses of different pTAS2R20 variants to quercitrin molecules, we quantified the fluorescence intensity of representative single cells in each group at 42 time points by using ImageJ (Fig. 5). The statistical results showed that the maximum value of fold of Ca2+ signal change in the VQ mutation group was 2.80, and those in the other groups were as follows: AH 3.34, VH 2.50, and pTAS2R20-AQ 4.10. These results consistently suggested that the two nonsynonymous sites, A52V and Q296H, greatly reduced the sensitivity of the receptor in the response to quercitrin in bamboo (Fig. 5).