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