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.