4.3 Validation of the poC1G1713Tcausality in Arabidopsis and implication for understanding PEP complex
role in clock plasticity
Our heterologous expression of the B1K-50-04 and B1K-09-07 RpoC1alleles in the Arabidopsis chloroplast support the role of N571K
substitution in manifesting heat responses and changes in the
photosynthetic rhythmicity (Figure 6 ). Zooming in on this
significant and hitherto unknown relationship between PEP variation and
clock thermal plasticity will require a more thorough analysis of more
advanced and isogenic lines. In the PEP complex, one major functional
group is comprised of PAPs involved in DNA/RNA metabolism and gene
expression regulation, while the second group is related to redox
regulation and reactive oxygen species protection (Steiner et al.2011). Moreover, the PEP is somehow coordinated with the nuclear
encoding RNA polymerase (Pfannschmidt et al., 2015). Therefore,
presumably non-synonymous variations (such as those between RpoC2alleles that we identified but not yet tested; Table S7 ) could
be as effective as non-synonymous ones (between RpoC1 alleles) in
the functionality and variation we observed. It would be therefore
required to look at different layers (transcriptome, proteome) between
nearly isogenic and not necessarily knockout mutant lines to achieve
relevant causal variation. Recent developments in plastid gene editing,
also in cereals, may assist in generating and analyzing both types of
mutations in barley and learn how they might modulate physiology and
development of the plant under normal and high temperatures. Recent
experiments suggest that most recent developments of TALLEN-based allele
editing tested in Arabidopsis (Nakazato et al., 2021)
could also be applied in barley (Fridman and Arimura, Personal
communication) to allow such multi-layer analysis of isogenic mutants.
Conclusions
This study indicates that the relationship between plasticity of the
clock under warming with robustness of plants could have a significant
effect on shaping the cytonuclear diversity. However, one way of testing
the possible cause behind these LD and CNI will require cloning of the
underlying genes in the DOC and figuring out how they may interact with
chloroplastic genes such as rpoC1 , and to learn what makes one allele
different than other and what cellular mechanisms may be involved.
ACKNOWLEDGEMENTS
We thank Dr Stephan Greiner (Max-Planck-Institut für Molekulare
Pflanzenphysiologie, Golm, Germany) for sharing barley chloroplasts
sequence data. The authors are grateful to Royi Levav Oded Anner and
Daniel Shamir (SensyTIV, Amiel, Israel) for their assistance in
maintaining the SensyPAM as a system for measuring circadian rhythms. We
also wish to thanks the technical assistance of laboratory member Avital
Beery and Orit Amir-Segev.
LIST OF AUTHOR CONTRIBUTIONS
E.B, L.D.T. and E.F. designed the experiments, collected, analyzed and
interpreted data, with A.B., and wrote the manuscript. M.R.P and A.F.D
collected and designed the B1K SNP platform, and E.Y. performed the QxE
GWAS. E.B., L.D.T., M.R.P., S.B, E.Y., were involved in the data
analyses, their interpretation and in writing the manuscript.