Summary
In plants, the contribution of the plasmotype (mitochondria and chloroplast) in controlling of the circadian clock plasticity and possible consequences on cytonuclear genetic make-up has not been fully elucidated. Here, we investigated the cytonuclear genetics underlying thermal plasticity of clock rhythmicity and fitness traits in reciprocal hybrid (RH) and B1K diversity panels of wild barley (Hordeum vulgare ssp. spontaneum ). Phenotypic analysis of the RH panel, showed higher abundance of plasmotype effects on chlorophyll fluorescence and its rhythmicity than plant phenology and growth. Performing a genome wide association study in the B1K panel found overlap with previously reported drivers of clock (DOC ) loci yet due to intra-chromosomal linkage disequilibrium these loci encompass shorter intervals. Moreover, by incorporating long-range chloroplastic sequencing we identified significant inter-chromosomal linkage disequilibrium and epistatic interactions between previouslyDOC3.2 and 5.1 loci and the chloroplastic RpoC1genes, indicating adaptive value for specific cytonuclear gene combinations. Finally, heterologous over-expression of two barleyRpoC1 alleles in Arabidopsis showed significantly differential plasticity under elevated temperatures. Our results unravel previously unknown cytonuclear interactions as well as alleles within the chloroplastic genome that control clock thermal plasticity.
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 (G x E) (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 non-photochemical quenching (NPQ) and photosystem II (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 (Dakhiyaet 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 and showed a clear loss of thermal plasticity of photosynthetic rhythms under domestication. Notably, to corroborate more on the nature of the rhythms obtained by chlorophyll fluorescence (chlF) we also performed time-lapse transcription analysis. Core clock genes were compared to photosynthetic genes by RNAseq between wild vs cultivated genotypes (Prusty et al., 2021), or by qPCR for pooled genotypes based on a single QTL affecting period plasticity (Bdolach et al., 2019). In these experiments, unlike the match seen between the SensyPAM phenotype and photosynthetic genes (e.g.PHOTOSYSTEM I SUBUNIT F (PsaF ), LIGHT-HARVESTING Chl-BINDING (Lhcb5 : CP26 )) under OT and HT conditions, none of the core genes (e.g., TIMING OF CAB EXPRESSION 1(TOC1 ), CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1 )) showed a significant difference in their amplitude or period. These observations for the core clock genes support the existence of temperature-compensation mechanisms (Gould et al. , 2006b; Sorek & Levy, 2012; Ford et al., 2016) and indicate another layer of regulation between core clock and its outputs. Nevertheless, with SensyPAM analysis of 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 in the wild.
Here, we follow up on the photosynthetic rhythm analysis of a reciprocal wild barley bi-parental doubled haploid (DH) population segregating for both nucleotype and plasmotype (either “Ashkelon” (B1K-09-07) or “Mount Hermon” (B1K-50-04)) (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 , which is part of the plastid-encoded RNA polymerase (PEP) protein complex. This complex is composed of four subunit α, β, β’ and β” encoded by RpoA , RpoB ,RpoC1 and RpoC2 respectively (Hajdukiewicz et al.,1997) and it requires sigma factor for promoter recognition and initiation of transcription. There are six sigma factors encoded by theArabidopsis genome, and all these factors are regulated by the circadian clock therefore communicating circadian timing information from the nucleus to chloroplasts (Noordally et al., 2013). 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) (Zhanget al., 2015), which are ruled by the core clock genes (Belbinet al., 2017). However, whether such selective forces acted on loci that regulate the core clock or its output rhythms remains unknown.
In the current study, we wished to, 1) extend the breadth of plasmotype diversity tested by examining reciprocal hybrids between 11 wild B1K that represent the different genetic clades (Hubner et al., 2009) and, 2) genetically characterize the B1K collection with a new SNP genotyping array and identify possible DOC loci and, 3) to look into CNI and their possible consequences on genetic make-up by analyzing B1K collection and a derived reciprocal F2 population that segregates for one of theDOC loci, 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