A photosynthesis operon in the chloroplast genome drives speciation in evening primroses

Abstract

Genetic incompatibility between the cytoplasm and the nucleus is thought to be a major factor in species formation, but mechanistic understanding of this process is poor. In evening primroses (Oenothera spp.), a model plant for organelle genetics and population biology, hybrid offspring regularly display chloroplast-nuclear incompatibility. This usually manifests in bleached plants, more rarely in hybrid sterility or embryonic lethality. Hence, most of these incompatibilities affect photosynthetic capability, a trait that is under selection in changing environments. Here we show that light-dependent misregulation of the plastid psbB operon, which encodes core subunits of photosystem II and the cytochrome b6f complex, can lead to hybrid incompatibility, and this ultimately drives speciation. This misregulation causes an impaired light acclimation response in incompatible plants. Moreover, as a result of their different chloroplast genotypes, the parental lines differ in photosynthesis performance upon exposure to different light conditions. Significantly, the incompatible chloroplast genome is naturally found in xeric habitats with high light intensities, whereas the compatible one is limited to mesic habitats. Consequently, our data raise the possibility that the hybridization barrier evolved as a result of adaptation to specific climatic conditions.

Figure 1

Distribution of Oenothera AA-I, BB-III and AB-II/BA-III species, and compatibility/incompatibility relations upon hybridization. A, Native distribution of Oenothera A and B genome species (pure colors) and their hybridization zones (two-color pattern). Yellow and magenta gradients indicate occurrence of distinct species or subspecies. B, Genetic species concept of evening primroses, based on plastome/nuclear genome compatibility and incompatibility, exemplified for the A and B genome species. Species are defined by their combinations of nuclear and chloroplast genomes (boxed in magenta), and are genetically separated by PGIs that occur upon hybridization and vary in the severity of the hybrid phenotype (BB-I white, AB-I and BB-II yellow-green, and AA-III bleaching leaf phenotype). C, Association of AA-I and B genome (BB and AB) species of Oenothera to the xeric and mesic habitats of North and Central America. Distribution maps redrawn from Dietrich et al. (1997). Climate data are from SolarGis and North American Environmental Atlas.

Figure 2

Light-dependent phenotype and physiology of AB-I plants. A, Yellow-green (lutescent) leaf phenotype and growth retardation under HL conditions (right). Plants are pictured at the end of the early rosette stage ∼30 days after germination. All molecular genetic and physiological analyses presented in this work were performed at this developmental stage. Bar = 5 cm. B, Analysis of photosynthetic parameters and photooxidative damage. (Left) Quantification of the components of the photosynthetic electron transport chain by difference absorbance spectroscopy. Note that AB-I plants under HL condition are not able to perform a typical light acclimation response by strongly increasing the content of all redox-active components of the electron transport chain relative to LL conditions. Bars represent mean values ± sd (n = 6–8 plants grown alongside). Right: Severe photooxidative damage of AB-I plants under HL conditions, exemplified by measurement of the maximum quantum efficiency of PSII in the dark-adapted state (F_V/F_M). Bars represent mean values ± sd (n = 6–8 plants grown alongside). Different lower case letters indicate significant differences (P < 0.05) according to two-way ANOVA with interactions followed by Tukey’s post-hoc testing (Supplemental File S4). C, Blue-native PAGE independently confirming the reduction of the electron transport chain complexes in AB-I under HL. Protein extracts equivalent to 30 μg chlorophyll were loaded in each lane. This experiment was performed two times independently with similar results.

Figure 3

Molecular genetic analyses of the psbM–petN spacer region in compatible (AA-I, AA-II, and AB-II) and incompatible (AB-I) material under HL and LL conditions. A, Sequence context and indels (blue triangle) in the spacer that is specific to plastome I. Arrows indicate transcription start sites. B, C, RNA gel blot analyses of psbM (B) and petN (C) transcript accumulation under LL and HL conditions. These experiments were performed three times with similar results. D, Immunoblot analysis of PetN accumulation under HL. N.t. = N. tabacum wild type, ΔpetN = petN knockout in N. tabacum (Hager et al., 1999). 100% corresponds to 5 µg chlorophyll equivalent. Tobacco control lines were grown in tissue culture as described in “Methods.” The experiment was performed independently three times with similar results.

Figure 4

Regulation of the psbB operon in compatible (AA-I, AA-II, and AB-II) and incompatible (AB-I) plants under HL and LL conditions. A, Physical map of the region in the chloroplast genome containing the clpP and psbB operons. The 144-bp deletion in the intergenic spacer upstream of the psbB operon promoter is indicated. Transcription start sites and mRNA processing sites are indicated by arrows. Note that pbf1 (encoded on the opposite strand) is transcribed from its own promoter. B, RNA gel blot analysis of psbB transcript (representative of the whole psbB operon; also see Supplemental Figure S2). C, 5'-RACE with and without TAP treatment, a method to map transcription start sites and RNA processing sites of the psbB operon. For details, see “Methods.” D, Run-on transcription analysis of the psbB operon (psbB and petB), including appropriate controls (rrn16, rrn23), and pbf1. E, RNA gel blot analysis of pbf1 transcripts. F, Immunoblot analysis of Pbf1 accumulation. 100% corresponds to 3 µg chlorophyll equivalent. G, RNA gel blot analysis of petA transcript accumulation, serving as a control for a gene outside of the psbB operon. H, Ribosomal profiling and translation efficiency of the psbB operon of AA and AB genotypes under HL conditions. Note a tendency of increased translation in AA-I versus AA-II (left). In turn, upon presence of the B genome, translation of psbT and pbf1 is reduced in incompatible AB-I plants when compared to compatible AB-II (right). B–G, With the exception of (F) all experiments were performed independently three times with similar results. F, was performed two times with similar results. H, The experiment is based on three replicates from a pool of ∼20 individuals per genotype. Bars represent mean values ± sd.