Our major research focus over the past two decades has been on self-incompatibility, a self/non-self recognition mechanism that operates during sexual reproduction in flowering plants.In addition, we have studied the role of a pollen-specific receptor-kinase in pollen development.Currently, we are continuing to investigate the biochemical and molecular bases of the type of self-incompatibility that has been found in three families of flowering plants, including the Solanaceae (nightshade) family.We are using Petunia inflata (a wild species of garden petunia) in the Solanaceae family as a model.
Self-incompatibility is an intraspecific reproductive barrier that prevents flowering plants from self-fertilizing and promotes out-crossing.It is estimated that more than half of the flowering plant species possesses self-incompatibility.To date, however, only a small number of families have been studied at the molecular level.Among them, three families, the Solanaceae, Rosaceae and Scrophulariaceae, employ the same self-incompatibility mechanism (Kao and Tsukamoto 2004).Here, the outcome of pollination is controlled by a highly polymorphic locus named the S-locus.If the S-haplotype of pollen matches one of the two S-haplotypes carried by the pistil, the pollen is recognized by the pistil as self-pollen and the growth of self-pollen tubes in the style is inhibited.If the S-haplotype of pollen is different from the two S-haplotypes carried by the pistil, the pollen is recognized as non-self pollen and the growth of the pollen tubes in the style is not inhibited.The non-self-pollen tube grows down through the style to the ovary to effect fertilization.
We are interested in addressing two fundamental questions about self-incompatibility.First, how does a pistil distinguish between self and non-self pollen?Second, how does the recognition of self-pollen lead to growth arrest of self-pollen tubes?We first identified a gene located at the S locus, named the S-RNase gene, and used gain-of-function and loss-of-function approaches to show that the S-RNase gene controls pistil function in recognition and rejection of self-pollen (Lee et al. 1994).We subsequently showed that the RNase activity of S-RNases is essential for rejection of self-pollen (Huang et al. 1994), and that the recognition function of S-RNases lies in the protein backbone but not in the carbohydrate moiety (Karunanandaa et al. 1994).Since the S-RNase gene does not control pollen function in self-incompatibility, it is imperative that the gene that controls pollen specificity in self-incompatibility interaction also be identified.To achieve this end, we identified pollen-expressed genes that are linked to the S-locus (McCubbin et al. 2000a), constructed a BAC library of S2-haplotype (McCubbin et al. 2000b), and then isolated BAC clones that contained DNA fragments in the S-locus region (Wang et al. 2003).From sequence analysis of a 328-kb S-locus region (constructed from three BAC clones), we identified a pollen-expressed gene, named PiSLF (Petunia inflataS-locus F-box), that is located 161 kb downstream of the S2-RNase gene (Wang et al. 2004).We subsequently obtained in vivo evidence that PiSLF indeed encodes the pollen determinant in self-incompatibility (Sijacic et al. 2004).
PiSLF contains an F-box domain, and most F-box proteins that have been characterized so far are involved in ubiquitin-mediated protein degradation.Specifically, F-box proteins are a component of a type of E3 ubiquitin ligase called SCF complex, which, in conjunction with E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme, mediates the transfer of a polyubiquitin chain to protein substrates.Each F-box protein interacts with specific proteins, and the ubiquitinated proteins are recognized by the 26S proteasome and degraded.We propose that PiSLF functions as a conventional F-box protein, and a PiSLF mediates specific degradation of its non-self S-RNases inside a pollen tube.Consequently, only self S-RNase is able to exert its cytotoxic effect to degrade pollen tube RNA, resulting in growth inhibition of self-pollen tubes in the pistil.For example, when an S1 pollen tube is growing down through a pistil of S-1S2 genotype, both S1-RNase and S2-RNase produced in the pistil are taken up by the S1 pollen tube; however, PiSLF1 produced in the S1 pollen tube would mediate degradation of S2-RNase (a non-self S-RNase with respect to PiSLF1), but allow S1-RNase to degrade RNA inside the S1 pollen tube.We have used a variety of molecular, biochemical, and genetic approaches to examine this hypothesis, and some of the key results obtained to date are summarized below (Hua and Kao 2006):(1) PiSLF is a component of a novel E3 ubiquitin, which also contains a Cullin-1 (named PiCUL1-G) and a RING-finger protein (named PiSBP1); (2) S-RNases are ubiquitinated and degraded in pollen tube extracts; (3) a PiSLF interacts with its non-self S-RNases more strongly than with its self-S-RNase; (4) an S-RNase interacts with its non-self PiSLFs more strongly than with its self PiSLF.These results are consistent with our hypothesis, as preferential interaction between a PiSLF and its non-self S-RNases would allow PiSLF to specifically mediate the ubiquitination and degradation of non-self S-RNases.
The questions we are currently addressing include the following.(1) Since there are a large number of F-box proteins in plants, we are interested in determining whether PiSLF is unique in its function in self-incompatibility, and if so, what features of PiSLF confer on it this unique function.We have studied several F-box proteins that share a number of properties with PiSLF, and found that none of them function in self-incompatibility.Sequence comparison between these PiSLF-like proteins and PiSLF has revealed regions that are unique to PiSLF.Chimeric proteins between PiSLF and PiSLF-like proteins will be generated for the study of the role of these regions in vitro and in vivo (i.e., in transgenic plants).(2) We are interested in determining the biochemical basis for the binding affinity differences between self-interactions and non-self interactions of PiSLF and S-RNase.Sequence comparison of different allelic variants of PiSLF has identified regions that may be involved in general interactions with both self and non-self S-RNases, and regions that may be involved in specific interactions with self-S-RNase.We will again use chimeric proteins to examine the role of these regions in vitro and in vivo.(3) We are interested in reconstituting the complex that contains PiSLF, and examining whether it preferentially mediates ubiquitination of non-self S-RNases.
Selective protein degradation via the ubiquitin-mediated pathway has rapidly emerged as an important regulatory mechanism for a variety of cellular and developmental processes in diverse organisms.In plants, this mechanism has been implicated in regulating floral organ identity, circadian rhythm, and auxin and jasmonate responses.PiSLF is among the very few F-box proteins whose substrates have been identified.Moreover, because there are literally hundreds of S-RNases and their corresponding PiSLFs in species that possess S-RNase-mediated self-incompatibility, this self-incompatibility system will be useful for biochemical characterization of the interactions between an F-box protein and its substrates.The information gained from our research will be valuable to understanding not only this self-incompatibility system, but also many cellular and developmental processes in a variety of organisms in which regulation of protein degradation has been implicated.