Journal of Cell Science SHORT REPORT Degradation of S-RNase in compatible pollen tubes of Solanum chacoense inferred by immunogold labeling Nicolas Boivin, David Morse and Mario Cappadocia* ABSTRACT The flowering plant Solanum chacoense uses an S-RNase-based self-incompatibility system in order to reject pollen that shares the same genes at the S-locus (S-haplotype) with the style (an incompatible reaction). Two different models have been advanced to explain how compatible pollen tubes are protected from the cytotoxic effects of the S-RNase, sequestration of the S-RNase in a vacuolar compartment or degradation of the S-RNase in the cytoplasm. Here, we examine the subcellular distribution of an S 11 -RNase 18 and 24 h post pollination (hpp) in compatible and incompatible crosses by immunogold labeling and transmission electron microscopy. We find that the S-RNase is present in the cytoplasm of both compatible and incompatible crosses by 18 hpp, but that almost all the cytoplasmic S-RNase is degraded by 24 hpp in compatible crosses. These results provide compelling evidence that S-RNases are degraded in compatible but not in incompatible pollen tubes. KEY WORDS: Gametophytic self-incompatibility, Pollination, S-RNase INTRODUCTION Self-incompatibility is the most important and widespread genetic mechanism used by flowering plants to promote outcrossing and thus avoid the deleterious effects of inbreeding. Self- incompatibility operates to block the formation of zygotes after self-pollination or crosses between genetically related individuals (de Nettancourt, 1977). The phenomenon involves interactions between gene products expressed in the pollen, and those expressed in specialized cells of the pistil. Present in ,60% of all angiosperm species, self-incompatibility is assumed to have played a key role in the successful evolution of the angiosperm, as assessed by current phylogeny studies (Allen and Hiscock, 2008). In the vast majority of species characterized by self- incompatibility, the interactions between pollen and pistil are under the control of elements of a single, highly polymorphic locus, the S-locus (de Nettancourt, 2001), that encodes for both the male and female determinants to self-incompatibility. In Solanaceae, Rosaceae and Plantaginaceae, the breeding behavior of the pollen is determined by the S-allele constitution of each individual pollen grain. Pollen rejection occurs when the S-haplotype of the pollen grain matches any of the S-alleles expressed in the pistillar tissues. In these families the pistillar gene product that mediates self-incompatibility is an extremely polymorphic ribonuclease termed an S-RNase (McClure et al., 1989). S-RNases are synthesized by the specialized cells of the transmitting tissue, secreted into the surrounding extracellular matrix (ECM) where the pollen tubes grow, and taken up from the ECM by the pollen tubes. Rejection of incompatible pollen thus occurs in the style. Electron microscopy studies have shown differences after compatible and incompatible crosses in the pistil ultrastructure (Herrero and Dickinson, 1979), as well as in pollen tubes during either intraspecific (de Nettancourt et al., 1973a; Herrero and Dickinson, 1980; Herrero and Dickinson, 1981) or interspecific crosses (de Nettancourt et al., 1974; de Nettancourt et al., 1973b), whereas immunolocalization studies have resolved the longstanding debate concerning whether S-RNases entered into the pollen tubes in a haplotype-specific manner or whether they were able to enter both compatible and incompatible pollen tubes. Using S-RNase- specific antibodies, Grey et al. (Gray et al., 1991) were the first to show that S-RNases enter Nicotiana alata pollen tubes in an S- haplotype-independent manner. However, these studies were performed on pollen tubes grown in vitro, and an in vivo confirmation was not provided until almost ten years later (Luu et al., 2000). These authors used immunogold electron microscopy to demonstrate accumulation of S-RNases in both compatible and incompatible pollen tubes of Solanum chacoense, although in that study no distinction was made between different subcellular compartments and only a single time post-pollination was used. The in vivo results were later extended to N. alata using confocal microscopy (Goldraij et al., 2006). Interestingly, observations made in this latter species indicated that S-RNases remain sequestered in a vacuole space after their entry inside compatible pollen tubes (see McClure et al., 2011), and were only released into the cytoplasm during incompatible crosses. These observations led to development of the sequestration model for S-RNase-based gametophytic self-incompatibility, in contrast to the degradation model derived from results using Petunia hybrida, which proposes degradation of presumably cytoplasmic S-RNases in compatible pollen tubes (see Meng et al., 2011). Intriguingly, previous studies on the self-incompatibility reaction of S. chacoense have furnished support for both sequestration and degradation models. On the one hand, measurements of the amount of RNA and protein in pollen tubes as a function of time during compatible and incompatible crosses appeared to support the sequestration model (Liu et al., 2012). This comes from studies using transgenic pollen expressing GFP, where compatible and incompatible crosses only developed a marked difference in the amounts of both GFP mRNA and protein between 18 and 24 h post pollination (hpp), a result suggestive of a sudden release of S-RNase into the cytoplasm at this crucial time. Furthermore, when the amount of Institut de Recherche en Biologie Ve ´ge ´ tale (IRBV), De ´ partement de Sciences Biologiques, Universite ´ de Montre ´ al, 4101 Sherbrooke est, Montre ´al, QC H1X 2B2, Canada. *Author for correspondence (mario.cappadocia@umontreal.ca) Received 15 April 2014; Accepted 11 July 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4123–4127 doi:10.1242/jcs.154823 4123