The role of PROTON1 in controlling the plasticity of ERD in
response to nitrogen
The significant association for plasticity of ERD in response to N
availability was detected based on the SNPs of 58 fully sequenced
accessions, but was not found when using the 250K SNPs due to the
absence of the identified SNPs in this dataset. To provide additional
support for this association, we sequenced the missing region in the
250K SNP data that contains the biallelic SNP for the 90 accessions that
were included in the initial phenotyping panel (Table S5). We then
looked at the associations of the sequenced SNP to the plasticity in ERD
in response to nitrogen. From all analyzed accessions, 42 carried the
minor alleles (A and T, for positions 6904858 and 6904935,
respectively), and 106 accessions carried the major alleles (C and G,
respectively). On average, the accessions with minor alleles showed 25
% lower FC in ERD in comparison to the accessions with major alleles
(0.55 and 0.41, respectively) (Figure 3d). The difference between the
ERD FC of the two haplogroups was significant (p = 0.0016, Mann Whitney
U test), giving further support to the association of PROTON1 to
the ERD plasticity in response to N.
Further investigations of growth using the proton1 T-DNA mutant
showed that its smaller FC in ERD was due to a significantly larger
early rosette under limiting N, and to a smaller rosette diameter under
optimal N in comparison to the WT (Figure 3c). This observation suggests
that the reduced plasticity, thus more stable growth, between limiting
and optimal N comes at a cost of decreasing the maximum attainable size.
Therefore, our findings point that PROTON1 mediates the trade-off
between stable and maximal growth under different N conditions.
Gene expression analysis of rosette leaves of plants grown under
limiting and optimal N revealed that proton1 is a strong
knockdown mutant, showing more than 1000 times reduced expression ofPROTON1 in comparison to WT (Figure 4a). Furthermore, the
expression of PROTON1 was increased in WT under limiting N
(Figure 4b). Additionally, we observed that the proton1 flowered
earlier than the WT under limiting N, but that its FT was not different
from that of the WT under optimal N (Figure 4c and d). To investigate if
PROTON1 controlled plasticity of ERD in response to N availability is
due to plasticity of N uptake, transport or assimilation, we evaluated
expression of 18 genes involved in these processes in WT andproton1 mutant grown under either limiting or optimal N. From the
18 analyzed genes, seven showed a significant expression difference
between WT and the mutant line in both optimal and limiting conditions
including the ammonium transporters AMT1;1 , AMT1;4 ,AMT2;1 ; NIA2 , involved in N assimilation; NLP7transcription factor; and NRT1;5 and NRT3;1 , which are
nitrate transporters (Figure 4e and 4f). Furthermore, the levels of N
assimilation NIA1 gene and NLP1 transcription factor under
limiting N, and the ammonium transporter AMT1;2 and NLP6transcription factor were also significantly different betweenproton1 and the WT under optimal N (Figure 4f). All of the
transcripts that showed significantly perturbed expression had higher
expression in the mutant line in comparison to the WT, indicating that
PROTON1 is involved in processes that lead to repression of expression
of genes involved in the aforementioned N-related processes. In
addition, it also suggests that the increased plasticity is associated
with increased expression of genes involved in N responses. The
mechanism by which PROTON1 modulates the expression of these
genes remains unknown.
To further confirm the role of PROTON1 in controlling the
plasticity of ERD to N availability, we searched the published genome
sequences of Arabidopsis accessions for polymorphisms in PROTON 1(At1g19880 ) gene. We identified that Xan-5 has a missense
substitution that results in a disruption in the start codon ofPROTON1 . The presence of this SNP in Xan-5 was further confirmed
by sequencing. When we grew Xan-5, Col-0 and proton1 under
optimal and limiting N, we observed reduction in the plasticity of ERD
in Xan-5 as well, thus further supporting the proposed role ofPROTON1 (Figure S3).