Discussion
Plants cope with changing environments by adjusting their phenotype
through phenotypic plasticity. The four adaptive phenotypes studied
here, namely early and final rosette size, flowering time and seed
yield, are known to respond to varying N availability (de Jong et al.,
2019; Ikram et al., 2012; Masclaux-Daubresse & Chardon, 2011; Meyer et
al., 2019; North et al., 2009). However, the genetic basis of their
phenotypic plasticity in response to N availability is unknown. In this
study, we quantified plasticity of the four phenotypes in response to N
by using both CV and FC and used GWA analysis to identify candidate
genes. None of the significantly associated loci contained genes known
to be involved in N response, indicating that the plasticity to N
availability has independent genetic basis in comparison to the focal
N-related traits. This was further supported with our observation that
the loci associated to the mean trait value of the accessions in
different N condition were different from the loci associated with the
trait plasticities to N. By using T-DNA mutant lines for the candidate
genes, we verified that a regulator of chromosome condensation (RCC1)
family protein, named here as PROTON1, influenced the plasticity of ERD
in response to N availability. The T-DNA mutant line for PROTON1(SALK_117261) showed significantly larger size under limiting N and was
significantly smaller under optimal N (Figure 3c), resulting in reduced
plasticity of ERD across the conditions (Figure 3b). This suggests that
RCC1-mediated stability in growth is attained by improved performance in
the limiting conditions, but with reduced performance in optimal
conditions.
RCC1-family proteins were first identified to function in regulating
cell cycle, but since then different members of this family are known to
be involved in diverse functions (Hadjebi et al., 2008). Arabidopsis
contains 24 RCC1-family genes, but only four of them have been
characterized so far. PROTON1 is the most highly expressed in roots and
young leaves (BAR eFP Browser, Arabidopsis.org), and we found that it
was induced by limiting N in young leaves (Figure 4b). Co-expression
analysis of PROTON1 showed that it is co-expressed with eight
genes with function in the spliceosome machinery (Figure S5) that
mediates alternative splicing plants. The four genes in primary
co-expression network of PROTON1 include two RNA-binding family
protein members. One of these is SR34, a known splicing factors in
Arabidopsis (Stankovic et al., 2016). The role of alternative splicing
controlling PROTON1-mediated plasticity of ERD and FT in response to N
availability remains to be investigated. Alternative splicing regulates
gene expression and protein diversity by producing multiple mRNA
isoforms from a single gene. In Arabidopsis , alternative splicing
is known to regulate transitioning to flowering, but less is known how
the alternative splicing is regulated by environmental cues. An
intriguing question for future studies would be to investigate if
variability in spliceosome, similar to the HSP90 system(Queitsch,
Sangster, & Lindquist, 2002; Salathia & Queitsch, 2007; Sangster &
Queitsch, 2005; Sangster et al., 2008; Zabinsky, Mason, Queitsch, &
Jarosz, 2019), plays a central role in regulating plasticity in plants
and animals.
In addition to the genetic basis of plasticity, we used correlation
analysis to investigate whether plasticity of specific primary
metabolites was associated with the plasticity of any of the four
phenotypes. We identified that plasticities of the fumarate and
glycerate levels showed significant negative correlation with the
plasticity of FRD. The mean levels of fumarate and glycerate were
previously found to negatively correlate with the biomass of Arabidopsis
plants grown under low N conditions (Sulpice et al., 2013). In our
experiments, the mean levels of fumarate showed negative correlation
with the mean ERD only under optimal N conditions (Figure S4c). This
suggests, similar to the four phenotypes, that the mean metabolites
levels associated with N responses are different from the plasticity of
metabolite levels in response to N.
To rapidly respond to changes in surroundings, plasticity in an
important trait for survival of homozygous organisms, such as
Arabidopsis. We found that plasticities of ERD and FT and the plasticity
of FRD correlated in across the studied accessions (Figure 2c). In
addition, proton1 mutant line was associated with both altered
ERD and FT plasticity in response to N availability. Further, inproton1, the ERD and FT plasticities were due to faster
development and earlier FT in limiting N conditions (Figure 4c, d).
These findings indicate that these traits respond simultaneously to the
changing N. Furthermore, we found that the different environments had
different impact on the focal traits. This enhances our understanding of
the complexity of possible constraints and consequences of selection
when acting on plasticity in response to climate change. Altogether,
these results highlight the importance to investigate plasticity of
different phenotypes in multiple environments alone and in combination,
when understanding the past, present and future relationships between
the plants and environment.