Primary Structure of a Novel Stylar RNase Unassociated with Self-Incompatibility in a Tobacco Plant, Nicotiana Alata

Abstract

Self-incompatiblity is a system for segregating self pollen from non-self pollen in the female organ of flowering plants (de Nettancourt, 1977). This system is not caused by malfunction of reproductive organs since self-incompatibility does not appear when the pistil accepts pollen with different S-alleles. Nicotiana alata has gametophytic self-incompatiblity in which pollen bearing the same S-allele as one of those in the pistil is rejected by arresting pollen tube growth. This rejection takes place in the style where S-allele specific glycoproteins (S-glycoproteins) responsible possibly for both segregation of self and non-self pollens and arrest of pollen tube growth are synthesized prior to anthesis (Anderson et al.,1986). Recently, we have found that S-glycoproteins associated with self-incompatibility are RNases in the RNase T2 family (McClure et al.,1989), based on predictions made by amino acid sequence comparisons and chemical modification experiments (Kawata et al.,1990). Subsequently, a number of studies have been reported for the primary structures of S-glycoproteins mainly from Solanaceae. These studies have revealed that all the proteins contain two conserved amino acid sequences, including two histidine residues corresponding to those existing in the active site of RNase T2. According to very recent investigations (Huang et al.,1994; Lee et al.,1994; Murfett et al.,1994), RNase activity is indispensable for the function of S-glycoprotein in petunia and tobacco. However, the presence of this enzyme activity in the style is not sufficient for the appearance of self-incompatibility. In fact, we detected the same RNase activity in the style (with the stigma) of either a self-incompatible variant (Nijisseiki) of Japanese pear (Pyrus pyrifolia) or its self-compatible mutant (Osa-Nijisseiki). Arabidopsis thaliana, a substantially self-compatible plant also synthesizes enzymes of the RNase T2 type in the style (Taylor and Green, 1991). Moreover, two or more RNases have been detected in the style of heterozygous petunia (Lee et al.,1992) and tobacco (McClure et al.,1989), implying that the style of self-incompatible plants synthesize RNase that is not associated with self-incompatibility. Our investigation was undertaken to characterize the RNase in the style of N.alata in order to seek a structure motif(s) specific for S-RNase or non-S-RNase. We then separated three stylar RNases, assigned individual RNases to S-RNase and non-S-RNase by analyzing their appearance in the style during flower development and elucidated the amino acid sequences of these RNases. This paper summarizes the result of some of these experiments and analyses of the structural information for RNase MS1, a novel non-S-RNase.