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.