Inhibitory Effects of Copper Ions on Stylar RNase Activity in the Japanese Pear ‘Kosui’

Japanese pear [Pyrus pyrifolia (Burm. F.) Nakai] has a S-RNase-based self-incompatibility system and inactivation of S-RNase causes self-incompatibility breakdown in this system. Copper ions (Cu++) strongly inhibited the RNase activity of crude stylar proteins, but did not overcome self-incompatibility in the Japanese pear ‘Kosui’ (S4S5). To elucidate the cause of this phenomenon, the present study aimed to separate stylar RNases and clarify their responses to Cu++ in ‘Kosui’. Using an anion exchange chromatograph equipped with a Mono-Q column, one strong non-S-RNase was separated, and at least six non-S-RNases together with 2 S-RNases were isolated, by cation exchange chromatography with the Mono-S column. S-RNases were identified by dot blot analysis using S-RNase antiserum, and hereafter, the seven non-S-RNases isolated are designated as NS1 to NS7 in eluting order. NS1 and NS2 were abundant RNases with strong activities, and their activities were reduced to 6.6 and 4.5% of the control by 1 mM CuSO4, respectively. NS3, NS4, NS5, NS6, and NS7 were weak RNases, and NS7 showed intermediate inhibition (31%) with Cu++, whereas inhibition of other RNases was weak, from 44 to 74% of the control. Meanwhile, the degree of inhibition was quite low in S4- and S5-RNase (86 to 89%). Thus, RNase inhibition of crude stylar proteins by Cu++ is due to strong repression of NS1 and NS2 activities. These results indicate that active S-RNase can cause a self-incompatibility reaction despite large decreases in non-S-RNase activities in the pear style.