Characterization of the candidate gene using T-DNA lines
Based on the linkage disequilibrium, we considered genes that were located ± 10 Kb and ± 20 Kb of the significantly associated SNPs for ERD and FT, respectively, as candidates. This resulted in eleven candidates around the SNPs associated with the plasticity of FT to N, and six candidate genes for the SNPs associated with the plasticity of ERD to N. To investigate the candidates, we examined 14 and 8 T-DNA mutant lines in Colombia (Col-0) background for the eleven and six candidate genes controlling the plasticity of FT and ERD, respectively (Table S4). After confirming the homozygosity of the T-DNA insertion, the lines were grown under the three N conditions as used for the initial screening, and the plasticity was compared to the Col-0 wild type (WT) in two independent experiments.
We did not detect significantly altered FC nor CV in FT in response to N in any of the tested mutant lines in comparison to Col-0 (Figure S2). We reasoned that the extremely low FT plasticity of Col-0 accession in response to N availability decreases the power for detecting significant differences. Indeed, the CV of FT ranged from 0.01 to 0.05 and the FC of FT ranged from 0.93 to 1.07, reflecting the low plasticity of this trait (Figure S2). For the ERD plasticity, homozygous-T-DNA mutant lines for five of the candidate genes were identified and further characterized. The homozygous SALK_117261 line, with T-DNA insertion inAt1g19880 gene, exhibited approximately 80 % reduced FC in ERD as compared to Col-0 in two independent experiments (Figure 3b, c). Analysis of homology demonstrated that At1g19880 encodes for an uncharacterized regulator of chromosome condensation (RCC1) family protein, which we further named as PLASTICITY OF ROSETTE SIZE TO NITROGEN 1 (PROTON1 ). The RCC1 family proteins include one or more RCC1- like domains (RLDs), and are known to have diverse function (Hadjebi, Casas-Terradellas, Garcia-Gonzalo, & Rosa, 2008). In Arabidopsis, there are 24 RCC1 family proteins, from which so far only UV RESISTANCE LOCUS 8 (UVR8) (Christie et al., 2012; Wu et al., 2012), RCC1/UVR8/GEF3-like 3 (RUG3) (Kuhn et al., 2011), TOLERANT TO CHILLING AND FREEZING 1 (TCF1) (Ji et al., 2015) and SENSITIVE TO ABA 1 (SAB1) (Ji et al., 2019) have been characterized.