Mario Cappadocia

S-RNase uptake by compatible pollen tubes in gametophytic self-incompatibility.

Many flowering plants avoid inbreeding through a genetic mechanism termed self-incompatibility. An extremely polymorphic S-locus controls the gametophytic self-incompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the S-alleles present in the diploid style. The only known product of the S-locus is an S-RNase expressed in the mature style. The pollen component to this cell–cell recognition system is unknown and current models propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.

Hypervariable Domains of Self-incompatibility RNases Mediate Allele-Specific Pollen Recognition

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

Production of an S RNase with Dual Specificity Suggests a Novel Hypothesis for the Generation of New S Alleles

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of selfincompatibility. The S11 and S13 RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S11 or S13 pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S11 RNase to match those in the S13 RNase was sufficient to completely replace the S11 phenotype with the S13 phenotype. We now show that an S11 RNase in which only three amino acid residues were modified to match those in the S13 RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S11 and S13 pollen). Thus, S12S14 plants expressing this hybrid S RNase rejected S11, S12, S13, and S14 pollen yet allowed S15 pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S13 allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dualspecific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.

Plastid ultrastructure defines the protein import pathway in dinoflagellates

Eukaryotic cells contain a variety of different compartments that are distinguished by their own particular function and characteristic set of proteins. Protein targeting mechanisms to organelles have an additional layer of complexity in algae, where plastids may be surrounded by three or four membranes instead of two as in higher plants. The mechanism of protein import into dinoflagellates plastids, however, has not been previously described despite the importance of plastid targeting in a group of algae responsible for roughly half the oceans net primary production. Here, we show how nuclear-encoded proteins enter the triple membrane-bound plastids of the dinoflagellate Gonyaulax. These proteins all contain an N-terminal leader sequence with two distinct hydrophobic regions flanking a region rich in hydroxylated amino acids (S/T). We demonstrate that plastid proteins transit through the Golgi in vivo, that the first hydrophobic region in the leader acts as a typical signal peptide in vitro, and that the S/T-rich region acts as a typical plastid transit sequence in transgenic plants. We also show that the second hydrophobic region acts as a stop transfer sequence so that plastid proteins in Golgi-derived vesicles are integral membrane proteins with a predominant cytoplasmic component. The dinoflagellate mechanism is thus different from that used by the phylogenetically related apicomplexans, and instead, is similar to that of the phylogenetically distant Euglena, whose plastids are also bound by three membranes. We conclude that the protein import mechanism is dictated by plastid ultrastructure rather than by the evolutionary history of the cell.

Study of microspore-culture responsiveness in oilseed rape (Brassica napus L.) by comparative mapping of a F2 population and two microspore-derived populations

RFLP segregation analyses were performed on a F2 population and two F1 microspore-derived populations from the same cross between a microspore culture-responsive parent (‘Topas’) and a non-responsive parent (‘Westar’). A total of 145 loci were detected with 87 cDNA clones. Eighty-two markers were common across all three populations. A total of 66 markers was assembled into 18 linkage groups and 16 markers remained unlinked. Segregation distortions were significant for 29% of the markers in the F2 population and 23% and 31% in microspore-derived populations M3 and M5, respectively. An equivalent number of markers showed biased segregation towards each parental allele in the F2 population while more markers showed a significant deviation from the expected Mendelian ratio towards the responsive parent in both microspore-derived populations. Different subsets of markers showed segregation distortions in the three populations indicating that the selective pressures leading to microsporederived plants are different from those acting during selfing of the F1. Linkage groups 1 and 18 were identified as putative chromosomal regions associated with microspore-culture responsiveness.

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.

Analysis of RFLP mapping inaccuracy in Brassica napus L.

Abstract We identified sources of mapping inaccuracy during the construction of RFLP linkage maps from one F2 population and two F1 microspore-derived populations from the same cross of oilseed Brassica napus. The genetic maps were compared using a total of 145 RFLP marker loci including 82 loci common to all three populations. In the process, we identified a series of mapping events that could lead to ambigous conclusions. Superimposed restriction fragments could be mistaken as a single dominant restriction fragment in a F2 population and, when analyzed as such, would yield inaccurate linkage information. Residual heterozygosity in parental lines resulted in complicated allelic assignment and yielded subsequent difficulties in linkage determination. Loose and spurious linkages occurred during mapping and were identified by comparing maps derived from different populations. LOD scores and χ2 test of independence were compared for their capacity to detect loose linkages or generate spurious ones. Extreme segregation distortions towards the same parental allele also contributed to an additional source of spurious linkage. Small but significant segregation distortions resulted in reduced estimates of the recombination fraction. The use of the same ‘probe× enzyme’ combinations in doubled haploid populations allowed the identification of the correct allele assignment as well as loose and spurious linkages. A translocation between two homoeologous linkage groups was observed. The consequences of such a chromosomal event as a source of error in mapping applications are discussed.

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 μmol m-2 s-1) on two induction media containing 2,4-D (4.5 or 22.5 μM), NAA (5.4 μM) and kinetin (0.5 μM). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 μM BA, or 5 μM kinetin and 2 μM TIBA or 9 μM BA and 4 μM TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.

Involvement of SLR1 genes in pollen adhesion to the stigmatic surface in Brassicaceae

Abstract The S-locus-related gene SLR1 is highly conserved and highly expressed in several species of the Brassicaceae family. Its function has not been determined, although several features would suggest a fundamental role in pollination. A second related gene (SLR2) is conserved and expressed in a subset of Brassica genotypes. We analysed the stigmatic expression of SLR1 and SLR2 genes among 11 different plants from various species or genera of the Brassicaceae and examined the extent of the pollen-stigma interaction during intraspecific, interspecific and intergeneric pollinations between them. Appropriate statistical tests on these variables (pollen adhesion, germination, penetration into the stigma, style and ovary, and SLR gene expression) showed that expression of SLR1 (but not SLR2) may be a factor in pollen-stigma adhesion. This hypothesis was supported by the observation of reduced pollen-stigma adhesion in transgenic B. napus plants modified for SLR1 expression.

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt.

SummaryIn this study, a novel approach was used to characterize the genetic architecture of plants produced by in vitro anther culture of two lines of self-incompatible Solanum chacoense Bitt. (2n=2x=24). We used cytological observations to determine the ploidy level of the regenerated plants and scanned genomic DNA of the anther donor plants to identify heterozygous sequences. Restriction fragment length polymorphism (RFLP) analyses permitted the visualization of DNA variations. Several heterozygous DNA markers were found within single anther donor plants. Completely homozygous lines could be easily identified. Somatically derived plants could be separated from diploid plants produced from 2n (unreduced) microspores. Our results demonstrate first division restitution (FDR) as the mechanism operating during the production of 2n microspores in one of our S. chacoense line. Potential applications of RFLP analyses for genetic mapping, identification of lethal alleles and quantitative trait loci (QTL) with haploid or homozygous diploid plants and determination of gene-centromere distance with diploid plants derived from 2n microspores will be discussed.