3.6 Arabidopsis complementation with barley RpoC1 alleles confer differential clock output plasticity
Previously, the comparison between B1K-09-07 and B1K-50-04 chloroplast genomes identified a non-synonymous SNP at the RpoC1 gene (position: 24530; N571K). We then speculated that this gene could be responsible for the difference in photosynthetic rhythm parameters between the two subpopulations within ASHER (Bdolach et al.,2019). In the current study, some of the interactions we tested for the same nonsynonymous SNP in the wider B1K collection support an association to the clock plasticity (Figure 4 ). Therefore, we first tested rpoC1 mutant in the Arabidopsis background. We procured T-DNA insertion rpoC1 mutant (cs835205 ) lines that showed integration in intron region of RpoC1 gene (Figure6a ). We analyzed the chloroplastic rpoC1 mutant for clock phenotype and noticed that, rpoC1 mutant showed plasticity in the period compared to wild type (WT) plants during temperature shift in short day entrainment (Table S10 ). Under OT, clock period inrpoC1 mutant and WT varied between 20.71-27.44 hr and 20.8-29.08 hr, respectively, with coefficient of variance (CV) 9.28% (rpoC1 ) and 8.67% (WT). The mean period of the rpoC1mutant and WT plants under the two temperatures differed significantly with clock rhythm accelerated from period length of 24.28 hr under OT to 22.58 hr under HT in rpoC1 mutant line as compared to relatively robust rhythmicity of the WT, i.e. 22.82 hr and 23.40 hr under OT and HT, respectively (Figure 6b ; S5a ).
Next, we wished to compare the possible consequences of the barleyRpoC1 variation on the clock output in the heterologousArabidopsis system. We therefore over-expressed (OE) the chloroplastic RpoC1 gene alleles from B1K-09-07 and B1K-50-04 barley in Col-0 Arabidopsis background. To direct translocation to the chloroplast we fused the barley chloroplastic RpoC1 coding region with chloroplast transit peptide under the control ofCaMV35S promoter (Figure 6c ). The homozygous OE lines were analysed for clock phenotype under OT and HT condition (TableS11 ). The Arabidopsis OE lines for B1K-09-07RpoC1 showed, on average, a significant plasticity or acceleration of the period during temperature shift from 23.73 to 22.18 hr, similar to the rpoC1 mutant line (rpoC1 : OT-23.54 hr, HT-22.41 hr). This is compared to the B1K-50-04 allele OE lines that showed robustness in the period (OT-22.93 hr, HT-22.85 hr) during temperature shift similar to WT lines (OT-23.27 hr, HT-24.23 hr) (Figure6d ; S5b ). Overall, in short day entrainment B1K-50-04 allelic OE lines and WT showed robustness while B1K-09-07 allelic OE lines and rpoC1 mutant showed plasticity during temperature shift. Similar to mutant and WT lines, OE lines from both allele showed amplitude remain same and decelerated during temperature shift.
Discussion