Association Analysis for prediction of candidate genes
It is not feasible to determine the consequences of the missense variants on gene expression, protein structure and function of an elevated number of putative candidate genes. Alternatively, candidate gene lists can be bounded conducting association analysis such as the successfully applied to identify genes associated with plant architecture in Brassica napus [48], water stress responses in wheat [49], or chilling stress tolerance in rice [50]. Here, we performed an association analysis by combining the genomic and transcriptomic data described above. The candidate gene lists obtained from the BSA-Seq and the parental comparison analysis (Dataset SD6-8) was compared with the root and leaf DEGs exclusive of the bic treatment in A1(c+) vs T6(c-) comparison (Dataset SD3), resulting in 18 matching genes (Figure 5A&B). The expression pattern of these genes confirms that generally they are more expressed in the roots of A1(c+) but also in the leaves of the sensitive T6(c-) deme (Figure 5C&D, Dataset SD9). This suggests that roots of the tolerant A1(c+)are reacting quickly to the adverse alkaline condition avoiding secondary effects in the leaves. Contrastingly, in the sensitive T6(c-) leaves are promptly stressed, especially due to the inhibition of Fe translocation (Figure 1C), and leaf gene expression adjusts to these alterations in order to alleviate further injury.
The SNP changes present in these 18 genes were explored and several missense variants were identified in both demes. The high number of SNPs present in AHH and PCH1 of A1(c+), and in INV-E of T6(c-) stands out (Dataset SD9, Figure S4). Some of these variants could be responsible for the observed gene expression differences or be linked to structural changes that affect the regulation of the gene. In order to evaluate the consequences of the no-expression of these genes under carbonate stress, the knock-out mutants of the 18 genes (T-DNA lines, Dataset SD1), together with the wild type of Col-0 and A1(c+) and T6(c-), were cultivated on the same carbonated soil (LP) and under the same control (pH 5.9) and bicarbonate hydroponic conditions (10 mM NaHCO3, pH 8.3) used in our previous studies.
Seeds of m03 (Sulphotransferase12) did not germinate in any condition and therefore sot12 could not been tested. Instead, gdh2(m08) germinated normally under carbonated soil and we observed an enhanced fitness of this mutant in this condition (Figure S3A&B). Both mutants csld2 (m15) and fbn6 (m16) coding, respectively, for Cellulose Synthase Like D2 and Fibrillin6 died only when treated with bicarbonate stress under hydroponic conditions (Figure S3A&B), suggesting hypersensitivity to this stress. CSLD2, is required for normal root hair development [51]. However, the inhibition of root hair development alone may not cause plant death. The failure to survive is more likely related to the putative role of CSLD2 in collaboration with CSLD3 and CSLD5 in hemicellulose biosynthesis [52] affecting early plant development. FBN6 is also required for normal plant development and fbn6 mutants have stunted root growth. This has been related to alteration in sulphate reduction, enhanced glutathione, and cadmium tolerance [53]. We have previously shown that bicarbonate tolerance in A. thaliana was associated with the upregulation of leaf genes related to sulphate acquisition [38]. So far FBN6 has mainly been studied in relation to chloroplasts, while the role in root development is still unknown. Clearly, further characterization of the role of FBN6 in plant development under bicarbonate is required.
On the other hand, dao1 (m01), aah (m07), and jaz10(m10) grew significantly better than the WT under bic stress (Figure S3A) but only dao1 exhibited higher fitness when it was cultivated on carbonated soil (Figure S3B). DAO1, coding for a 2-oxoglutarate and Fe (II)-dependent dioxygenase, is a main player in irreversible auxin degradation [54] and a higher production of adventitious roots has been shown in the dao1-1 loss of function mutants [55]. Downregulation of DAO1 could enhance root development and Fe acquisition, being beneficial in high pH habitats. Allantoate Amido Hydrolase (AHH) catalyzes the degradation of allantoin yielding CO2 and four molecules of NH4[56]. High levels of allantoin and downregulation of allantoin degradation has been found to enhance salt stress tolerance in A. thaliana [57]. Our findings regarding the bicarbonate tolerance of the ahh mutant (Figure S3A) and the lower root expression of AHH in A1(c+) indicate (Figure 5C&D) a role for allantoin also in bicarbonate tolerance. InĀ A. thaliana , JAZ proteins act as repressors of JA signaling and plants overexpressing AtJAZ10are insensitive to JA [58, 59]. In our case, jaz10 exhibited a better response to bicarbonate stress than the WT and JAZ10 was less expressed in the tolerant A1(c+) deme, suggesting that a downregulation of this gene could trigger JA signals important for tolerant responses.
Trichome Birefringence Like 19 -tbl19 (m11) did not grow or produced more siliques than Col-0 but were not affected by the carbonate treatment, exhibiting higher tolerance (Figure S3A&B). In both demesTBL19 was downregulated in roots and upregulated in leaves (Figure 5). Under bicarbonate exposure, the knock-out mutant had high root biomass and the highest fitness as expressed in number of siliques (Figure S3). TBL19, also known as AtXYBAT1 [63], has O-acetyltransferase activity and is probably involved in the acetylation of cellohexaose, a hemicellulose in A. thaliana cell walls. Root apoplastic iron in A. thaliana is mainly stored in the operationally defined HC1 fraction of hemicellulose and this bound-Fe can be mobilized by coumarin-type phenolic exudation [60]. Under bicarbonate exposure, the tolerant A1(c+) has higher exudation of these phenolics than the sensitive T6(c-)[5]. The decrease of acetylation may enhance Fe binding to cell wall hemicellulose and its accessibility to the coumarin-type phenolics, so enhancing the root to shoot Fe translocation specifically in A1(c+).
Alkaline/Neutral Invertase (INV-E) encodes a chloroplast-targeted protein, although it is expressed in root tissues in A. thaliana(TAIR-BAR). INV-E catalyzes the irreversible cleavage of sucrose into glucose and fructose and some At INV have been linked to the control of root cell elongation mediated by sugars [61].inv-e (m17) mutant was clearly affected by the bicarbonate content both in hydroponics and in soil (Figure S3A&B). The SNP changes detected in A1(c+) and T6(c-) were evaluated and we found that T6(c-) INV-E gene contains a large number of variants that could affect the expression of the gene (Figure S4, Dataset SD9), supporting the hypothesis that a proper regulation of INV-E could play an important role in bicarbonate tolerance.
In conclusion, differential bicarbonate tolerance in A. thalianais associated with enhanced root to shoot translocation of iron, proper distribution of C/N fixation, and better root development. Association analysis of genomics and transcriptomics with natural demes well- or mal-adapted to soil carbonate allowed the selection of 18 candidate genes for this differential tolerance. Evaluation of genomic variation and the corresponding knock-out mutants suggested implications of AHH, CSLD2, DAO1, GDH2, FBN6, INV-E, JAZ10 and TBL19. We propose these genes as candidates for being further explored through complementary analysis under bicarbonate stress conditions.