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.