Transcriptional changes in response to bicarbonate stress
Microarray analyses in roots were performed to characterize, at the gene
expression level, the mechanisms underlying the differential response to
bicarbonate of the two naturally selected accessions. Gene responses
were assessed by using samples after 48 h of treatment exposure, when
the plants still did not exhibit any foliar symptoms of the stress
treatments. We performed pairwise comparation to understand the
differences between A1(c+)-tolerant and
T6(c-) -sensitive lines in response to different
treatments (pH 8.3 vs pH 5.9 and bic vs control pH 5.9)
(Dataset SD3). A total of 4595 differentially expressed genes (DEGs)
were identified, considering accession and treatment (Figure 2). The
bicarbonate treatment caused a higher number of DEGs than the high pH
treatment, indicating that bicarbonate stress involves more complex
processes than simply the specific responses to alkaline pH (Figure 2A).
For that, we further focused our analysis on the pathways found in the
plants exposed to the bic treatment.
Previously, RNA-Seq of leaves from A1(c+) and
T6(c-) individuals revealed that bicarbonate exposure
quickly upregulated Fe-deficiency related genes in the sensitive
T6(c-) but not in the tolerant A1(c+)[38]. In leaves, the highest number of DEGs was observed in
T6(c-) but in roots the tolerant A1(c+)exhibited a huge response when exposed to bic (Figure 2B). These
differences in DEG number may denote contrasted deme strategies toward
bicarbonate stress. Gene Ontology (GO) terms indicated that in roots of
A1(c+) the main pathways activated after 48 h of
exposure were related to activation of biological processes and
metabolism located in the extracellular region (Figure 2C, Dataset SD4).
Also, enzymatic regulatory activity, transport and transcription factors
were altered. In T6(c-) only up and down modifications
in catalytic activity were found. The analysis of the KEEG pathways
revealed that the sensitive line activated only two principal pathways
involved in catalytic activity while in A1(c+) more than
20 pathways were up or down regulated (Figure 2D, Dataset SD4). This
analysis indicates that the modifications are being produced in the
sugar, lipid and protein metabolism of the tolerant line.
In rice, better tolerance to saline-alkaline conditions was due to
superior Fe acquisition and higher root to shoot Fe translocation
[39]. This was attributed to enhanced root development and higher
expression of genes involved in Fe acquisition and transport in the
tolerant variety in comparison to the sensitive one. Contrastingly,
among the DEGs in our tolerant A. thaliana deme, no differential
enhancement of main Fe-acquisition related genes was observed. Moreover,
root expression of bHLH101 was more than 4 times higher in the
sensitive T6(c-) than in the tolerant
A1(c+) (Dataset S5). bHLH101 is a transcription factor
that controls plant Fe homeostasis in a FIT independent way [40]. It
is required for proper growth under Fe deficiency conditions. These
results further confirm that Fe deficiency is earlier perceived by the
sensitive T6(c-).
Focusing on A1(c+) vs T6(c-)comparison under the bic treatment and excluding the genes also up or
down regulated in control conditions, 208 DEGs were identified in roots,
2503 DEGs in leaves, and 26 DEGs in both tissues (Figure 2D, Dataset
SD3). Two contrasted root strategies imply the possibility of
differences in signal perceiving and transduction. To further explore
these differential mechanisms, we performed protein-protein interaction
network functional enrichment analysis with the STRING database on the
208 genes (Figure 3, Dataset SD4). These genes can be classified in 5
main keywords according to FLAME: catalytic, binding and ubiquitin
activity; phosphorus metabolic process; and oxidoreductase activity. We
found two main cores of genes with high network connectivity: one lead
by genes involved in nucleus binding (TOPII, CDC20.2, MAD2,
AUR1, FZR3, CYCB1;3) and microtubule movement regulation (AT5G60930,
ATK5, AT3G20150, AT4G15830) that are indispensable for normal plant
development and fertility but have not previously been associated with
abiotic stress resistance; and another controlled by the NIA1 and NIR1
genes. NIA1 (or GNR1) encodes the cytosolic minor isoform of nitrate
reductase and Nitrite Reductase 1 (NIR1) is involved in the second step
of nitrate assimilation. Contrastingly, NRT2 .3 was less expressed
in A1(c+) than in the sensitive T6(c-).
NRT2.3 is a high affinity nitrate transporter with still poorly defined
functions but linked to the signaling of several phytohormones [41].
The occurrence of inorganic carbon and nitrogen in karst and carbonated
soils affects the carbon/nitrogen metabolism of plant species. Under
bicarbonate stress, growth reduction is enhanced -due to the inhibition
of the photosynthesis and nitrogen metabolism- but water use efficiency
is promoted in tolerant plants [42]. This balance is vital for
plants to adapt to alkaline environments and the regulation of the
‘nitrogen hub genes’ found here might be important for C fixation and
bicarbonate tolerance.
Phenotypic variation for soil carbonate tolerance traits among demes
The natural habitat of A1(c+) and T6(c-)differs mainly in soil pH (A1(c+) : 7.4;
T6(c-) : 6.5) and soil carbonate content
(A1(c+) : 12%; T6(c- ) : 0.8%)
[5]. Natural carbonated soil from a location closer to
A1(c+) deme (pH = 7.9; CaCO3 = 33%) was
excavated for studying the tolerance capability of each deme and their
progeny. F1 progeny of both crosses (A1xT6 & T6xA1) exhibited similar
growth and fitness as the tolerant parental A1(c+)(Figure 4A&C), while F2 progeny showed segregation (Figure 4B&C). In
addition to symptoms of iron deficiency and reduced growth,
T6(c-) grown on alkaline soil suffers from delayed
flowering and infertility that substantially hamper its fitness (Figure
S2, Dataset SD5). Only the 35% of F2 progeny plants were able to flower
and reproduce (Figure 4B). In consequence the fitness of both crosses
did not strictly fulfill the Mendelian phenotype ratio (3:1) (Figure
4B&C), suggesting that the ‘soil carbonate tolerance phenotype’ in our
demes is partial-dominant or a polygenic trait. Moreover, considering
only the plants that were able to flower, seed production was higher in
the A1xT6 than in the T6xA1 offprings (Figure 4C) pointing to a
potential parental effect.