Abstract
In plants, neither the contribution of the plasmotype in controlling circadian clock plasticity and overall plant robustness, nor what may be the fitness consequences of clock plasticity on genetic make-up has been fully elucidated. Here, we investigated the cytonuclear genetics underlying thermal plasticity of clock rhythmicity and fitness traits in reciprocal doubled haploid population and a diversity panel of wild barley (Hordeum vulgare ssp. spontaneum ). We identified a positive correlation between the thermal plasticity of clock and vegetative growth with the robustness of reproductive output. Moreover, we identified significant linkage disequilibrium and epistatic interactions between previously identified drivers of clock (DOC) loci and the chloroplastic RpoC1 genes, indicating adaptive value for specific cytonuclear gene combinations. Finally, heterologous over-expression of two barley RpoC1 alleles in Arabidopsisshowed significantly differential plasticity under elevated temperatures. Our results unravel previously unknown cytonuclear interactions as well as specific alleles within the chloroplastic genome that control clock thermal plasticity while also having pleiotropic effects on plant fitness in the field. The evolutionary and functional relationship between nuclear and chloroplastic DOCs suggest that adaptation to warming environments involve cytonuclear changes to confer local adaptation.
Introduction
Plants are composed of cells in which three different organelle genomes co-evolved to cope with a dynamic environment: the nuclear genome (nucleotype), and the chloroplast and mitochondrial genomes (plasmotype). Environmental constraints promote the selection of causal mutations in all of those genomes. At the same time, epistatic relationships between nucleotypic and plasmotypic loci, and co-evolution of adaptive gene complexes are able to promote adaptation to dynamic environment and further to shape genetic make-up owing to preference of specific gene complexes (Groen et al., 2022). In recent years, several studies have shown that phenotypic effects are related to the genetic diversity of the plasmotype and its interactions with the nucleotype (Joseph et al. , 2013; Tang et al. , 2014; Rouxet al. , 2016). An elegant use of the haploid-inducer line available in Arabidopsis (GFP-tailswap ) (Ravi et al., 2014), allowed the generation of a set of reciprocal and isogenic cybrids from several accessions that were phenotyped for metabolism and photosynthesis under different light conditions (Flood et al.,2020). Genetic analysis revealed that the nucleotype, plasmotype and their interaction accounted for 91.9%, 2.9% and 5.2% of genetic variation, respectively, thus highlighting the importance of interactions between nucleotype and plasmotype.
In crop plants and their wild relatives, few reports exist on the contribution of cytonuclear interactions (CNI) to a plant’s phenotype and even less on its effects on its phenotypic plasticity. Examples, where the contribution of plasmotype to yield and grain quality has been demonstrated, exist in grasses (Frei et al. 2003; Sanetomo & Gebhardt, 2015). In cucumber, (Gordon & Staub, 2011) used reciprocal backcrosses between chilling-sensitive and chilling-tolerant lines to show that tolerance to reduced temperature is maternally inherited. Likely, these traits are the result of a local adaption of the original wild alleles, since for example in bread wheat (Trictium aestivum ), cytoplasmic influence on fruit quality is affected by genotype-by-environment interactions (Ekiz et al., 1998). Nevertheless, many of these examinations of alloplasmic lines, which contained cytoplasm from distantly related wild relatives showed that effects on agronomic traits (rather than protein quality) are not frequent (Frei et al., 2010). In maize, although cytoplasmic effects were not significant between the direct and reciprocal populations, the interactions among the plasmotype and the nucleotype quantitative trait loci (QTL) were detected for both days to tassel and days to pollen shed (Tang et al., 2014), further enforcing the increased explained variation between Arabidopsis cybrids when CNI are included (Flood et al., 2020).
Circadian clock rhythms in plants are interwined with chloroplastic activities including photosynthetic parameters such as NPQ and ΦPSII, whose values correlate with plant productivity (Kromdijk et al.,2016). This insight led to the development of several high-throughput methods that measure the rhythmicity of the leaf chlorophyll fluorescence as an approximation to the period, phase and amplitude of the core clock (Gould et al. , 2009; Tindall et al. , 2015; Dakhiya et al. , 2017). The ability to measure hundreds of plants allowed for a comparison between species (Rees et al., 2019), and to quantify the impact of temperature and soil composition on period and amplitude (Dakhiya et al. , 2017). Using the SensyPAM platform, which allows to infer photosynthetic rhythms based on repeated measurements of chlorophyll fluorescence (Bdolach et al., 2019), we recently analyzed wild, landrace, and cultivar panels of barley as well as interspecific populations. We showed that some of the nuclear loci that control the photosynthetic rhythms were under selection during domestication. This could explain how modern crops lost the thermal plasticity of photosynthetic rhythms while maintaining a robust core clock (Prusty et al., 2021). Furthermore, pleiotropic effects of these drivers of clocks (DOCs ) loci on grain yield under stress indicate the adaptive value of clock plasticity. Nevertheless, this study did not consider the possible role of plasmotype diversity in modulating the effect of DOCs loci on circadian clock and fitness outputs, nor it examined the possibility that these effects on the clock plasticity may have been under selection also within the wild. Notably, previous studies identified the correlation of molecular evolution (i.e. dN/dS ratio) between genes encoding the plastid-encoded RNA polymerase (PEP) protein complex and nuclear genes (sig1-6) (Zhang et al.,2015), which are ruled by the core clock genes (Belbin et al.,2017). However, whether such selective forces acted on loci that regulate the output rather than the core rhythm of the clock remains unknown.
Here, we followed up on the photosynthetic rhythm analysis of a reciprocal bi-parental doubled haploid (DH) population segregating for both nucleotype and plasmotype (either “Ashkelon” or “Mount Hermon”) (Bdolach et al., 2019). Photosynthetic rhythms measurements previously showed a significant difference of 2.2 h in the period between the carriers of the different plasmotypes (Bdolach et al., 2019). Whole chloroplast genome sequencing of the two chloroplast identified several non-synonymous candidate polymorphism that could underlie these changes, including a N571K in the rpoC1. In the current study, we wished to, 1) extend the analysis of the plasmotype effects by including fitness traits and test if there is pleiotropy for QTL identified for clock output rhythmicity and life history traits, and 2) extend the breadth of diversity tested by examining the larger Barley1K collection with a new SNP genotyping array and identify possible DOC loci and, 3) to look into cytonuclear interactions (CNI) and their possible consequences on genetic make-up by analyzing B1K collection and a derived reciprocal F2 population, and finally, 4) test the functional consequence of variation in the RpoC1 gene (Bdolach et al., 2019) on photosynthetic rhythm plasticity by heterologous expression of two barley alleles in model plant Arabidopsis .
MATERIALS AND METHODS