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Abstract PremiseA multi‐omic approach was used to explore proteins and networks hypothetically important for establishing filament dimorphisms in heterostylousTurnera subulata(Sm.) as an exploratory method to identify genes for future empirical research. MethodsMass spectrometry (MS) was used to identify differentially expressed proteins and differentially phosphorylated peptides in the developing filaments between the L‐ and S‐morphs. RNAseq was used to generate a co‐expression network of the developing filaments, MS data were mapped to the co‐expression network to identify hypothetical relationships between theS‐gene responsible for filament dimorphisms and differentially expressed proteins. ResultsMapping all MS identified proteins to a co‐expression network of the S‐morph's developing filaments identified several clusters containing SPH1 and other differentially expressed or phosphorylated proteins. Co‐expression analysis clustered CDKG2, a protein that induces endoreduplication, and SPH1—suggesting a shared biological function. MS analysis suggests that the protein is present and phosphorylated only in the S‐morph, and thus active only in the S‐morph. A series of CDKG2 regulators, including ATM1, and cell cycle regulators also correlated with the presence of reciprocal herkogamy, supporting our interest in the protein. ConclusionsThis work has built a foundation for future empirical work, specifically supporting the role of CDKG2 and ATM1 in promoting filament elongation in response to SPH1 perception. 

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen. 

Heterostyly employs distinct hermaphroditic floral morphs to enforce outbreeding. Morphs differ structurally in stigma/anther positioning, promoting cross-pollination, and physiologically blocking self-fertilization. Heterostyly is controlled by a self-incompatibility (S)-locus of a small number of linked S-genes specific to short-styled morph genomes. Turnera possesses three S-genes, namely TsBAHD (controlling pistil characters), TsYUC6, and TsSPH1 (controlling stamen characters). Here, we compare pistil and stamen transcriptomes of floral morphs of T. subulata to investigate hypothesized S-gene function(s) and whether hormonal differences might contribute to physiological incompatibility. We then use network analyses to identify genetic networks underpinning heterostyly. We found a depletion of brassinosteroid-regulated genes in short styled (S)-morph pistils, consistent with hypothesized brassinosteroid-inactivating activity of TsBAHD. In S-morph anthers, auxin-regulated genes were enriched, consistent with hypothesized auxin biosynthesis activity of TsYUC6. Evidence was found for auxin elevation and brassinosteroid reduction in both pistils and stamens of S- relative to long styled (L)-morph flowers, consistent with reciprocal hormonal differences contributing to physiological incompatibility. Additional hormone pathways were also affected, however, suggesting S-gene activities intersect with a signaling hub. Interestingly, distinct S-genes controlling pistil length, from three species with independently evolved heterostyly, potentially intersect with phytochrome interacting factor (PIF) network hubs which mediate red/far-red light signaling. We propose that modification of the activities of PIF hubs by the S-locus could be a common theme in the evolution of heterostyly. 

Summary Distyly is an intriguing floral adaptation that increases pollen transfer precision and restricts inbreeding. It has been a model system in evolutionary biology since Darwin. Although theS‐locus determines the long‐ and short‐styled morphs, the genes were unknown inTurnera. We have now identified these genes.We used deletion mapping to identify, and then sequence,BACclones and genome scaffolds to constructS/shaplotypes. We investigated candidate gene expression, hemizygosity, and used mutants, to explore gene function.Thes‐haplotype possessed 21 genes collinear with a region of chromosome 7 of grape. TheS‐haplotype possessed three additional genes and two inversions.TsSPH1was expressed in filaments and anthers,TsYUC6in anthers andTsBAHDin pistils. Long‐homostyle mutants did not possessTsBAHDand a short‐homostyle mutant did not expressTsSPH1.Three hemizygous genes appear to determine S‐morph characteristics inT. subulata. Hemizygosity is common to all distylous species investigated, yet the genes differ. The pistil candidate gene,TsBAHD, differs from that ofPrimula, but both may inactivate brassinosteroids causing short styles.TsYUC6is involved in auxin synthesis and likely determines pollen characteristics.TsSPH1is likely involved in filament elongation. We propose an incompatibility mechanism involvingTsYUC6andTsBAHD.