4.2 Validation of the rpoC1G1713Tcausality 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 PEP-associated proteins (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 RpoC2 alleles that we identified but not yet tested;
Table S7 ) could be as effective as non-synonymous ones (betweenRpoC1 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 optimal and high
temperatures. Recent experiments suggest that most recent developments
of TALLEN-based allele editing tested in Arabidopsis (Nakazatoet al., 2021) could also be applied in barley (Fridman and
Arimura, Personal communication) to allow such multi-layer analysis of
isogenic mutants.
Conclusions
Naturally, occurring evolution and adaptation of wild populations
reflect on the genetic make up their genomes, including the relatively
neglected organelles. By combining an efficient clock phenomics tool and
new genotyping platforms, we were able to show the importance of
considering chloroplast diversity for gaining better understanding of
plants behavior under warming temperatures and determine the genetic
network underlying heat-conditioned effects on the circadian clock
output. We also identified linkage disequilibrium between some of these
DOC and the plasmotype loci therefore indicating selection on genes
controlling clock output. Furthermore, heterologous complementation of
barley RpoC1 alleles in the model plant Arabidopsisindicate the significant role of the PEP complex in regulating
rhythmicity of photosynthesis under changing environments and may
suggest its adaptive role in a yet to be defined heat sensing mechanism.
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, and wrote the manuscript. M.R.P and A.F.D collected
and designed the B1K SNP platform, and 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.