Cuong Nguyen Huu, Barbara Keller,  Elena Conti,  Christian Kappel, and Michael Lenhard

PNAS September 15, 2020 117 (37) 23148-23157

Significance

Heterostyly is an adaptation to promote outbreeding in plants. In heterostylous primroses, plants form flowers either with long styles and low anthers or with short styles and high anthers. This difference is due to a chromosomal segment containing five predicted genes, yet their roles and the evolution of this segment remain unclear. Here we identify the gene responsible for raising the anthers in short-styled flowers. This gene arose by duplication from a classical floral-organ identity gene and gained a novel function. Surprisingly, the responsible chromosomal segment appears to have evolved by stepwise gene duplications rather than duplication of an entire chromosomal block. These findings thus provide detailed insight into the evolution of complex polymorphisms involving different individual traits.

Abstract

Heterostyly represents a fascinating adaptation to promote outbreeding in plants that evolved multiple times independently. While L-morph individuals form flowers with long styles, short anthers, and small pollen grains, S-morph individuals have flowers with short styles, long anthers, and large pollen grains. The difference between the morphs is controlled by an S-locus “supergene” consisting of several distinct genes that determine different traits of the syndrome and are held together, because recombination between them is suppressed. In Primula, the S locus is a roughly 300-kb hemizygous region containing five predicted genes. However, with one exception, their roles remain unclear, as does the evolutionary buildup of the S locus. Here we demonstrate that the MADS-box GLOBOSA2 (GLO2) gene at the S locus determines anther position. In Primula forbesii S-morph plants, GLO2 promotes growth by cell expansion in the fused tube of petals and stamen filaments beneath the anther insertion point; by contrast, neither pollen size nor male incompatibility is affected by GLO2 activity. The paralogue GLO1, from which GLO2 arose by duplication, has maintained the ancestral B-class function in specifying petal and stamen identity, indicating that GLO2 underwent neofunctionalization, likely at the level of the encoded protein. Genetic mapping and phylogenetic analysis indicate that the duplications giving rise to the style-length-determining gene CYP734A50 and to GLO2 occurred sequentially, with the CYP734A50 duplication likely the first. Together these results provide the most detailed insight into the assembly of a plant supergene yet and have important implications for the evolution of heterostyly.

See https://www.pnas.org/content/117/37/23148

Figure 1:

VIGS of GLO2 results in short-homostylous flowers. (A) Short-homostylous phenotypes in VIGS-GLO2–treated S (short-styled)-morph plants. Images were taken in dissected flowers (Top) and from the top of flowers (Bottom). Arrow indicates the position of anthers. (Scale bars, 1 mm.) (B) Expression of GLO2 in untreated S- and L (long-styled)-morph plants and in VIGS-GLO2–treated S- and L-morph plants. Values represent the mean ± SD from three biological replicates. Asterisk indicates significant difference from S-morph flowers by Student’s t test with ***P < 0.001. (C) Expression of GLO1 in untreated S- and L-morph plants and in VIGS-GLO2–treated S- and L-morph plants. Values represent the mean ± SD from three biological replicates. Student’s t test showed no significant difference (ns) between samples. (D–F) Anther height (D), length of corolla tube (E), and diameter of corolla mouth (F) as measured in untreated S- and L-morph and in VIGS-GLO2–treated S-morph plants. The lines in the boxes indicate the median, the boxes show the interquartile range and the whiskers indicate the largest and smallest values within 1.5x interquartile ranges above the 75th or below the 25th percentile, respectively, from n = 10 flowers. Asterisks indicate significant difference from S-morph flowers by Student’s t test with *P < 0.05 and ***P < 0.001. (G and H) Length of corolla cells above (G) and below (H) the anther in untreated S- and L-morph plants and in VIGS-GLO2–treated S- and L-morph plants. Ten corolla tubes from each phenotype were measured and values are the mean ± SD. Asterisks indicate significant difference from S-morph flowers by Student’s t test with ***P < 0.001.