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).