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