Abstract
Carbonate-rich soils limit plant performance and crop production. Previously, local adaptation to carbonated soils was detected in wildArabidopsis thaliana accessions, allowing the selection of two demes with contrasting phenotypes: A1 (carbonate tolerant, c+) and T6 (carbonate sensitive, c-). Here, A1(c+) and T6(c-) seedlings were grown hydroponically under control (pH 5.9) and bicarbonate conditions (10 mM NaHCO3, pH 8.3) to obtain ionomic profiles and conduct transcriptomic analysis. In parallel, A1(c+) and T6(c-) parental lines and their progeny were cultivated on carbonated soil to evaluate fitness and segregation patterns. To understand the genetic architecture beyond the contrasted phenotypes a bulk segregant analysis sequencing (BSA-Seq) was performed. Transcriptomics revealed 208 root and 2503 leaf differentially expressed genes (DEGs) in A1(c+)vs T6(c-) comparison under bicarbonate stress, mainly involved in iron, nitrogen and carbon metabolism, hormones, and glycosylates biosynthesis. Based on A1(c+) and T6(c-) genome contrasts and BSA-Seq analysis, 69 genes were associated with carbonate tolerance. Comparative analysis of genomics and transcriptomics discovered a final set of 18 genes involved in bicarbonate stress responses that may have relevant roles in soil carbonate tolerance.
Keywords: Arabidopsis, soil carbonate, bicarbonate stress, BSA-Seq; transcriptomics.
Introduction
Soil alkalinity is a highly stressful environmental factor that limits plant growth and crop yield [1-2]. High pH soils are present in 30% of the Earth surface specially located in areas with arid and semiarid climate. The pH values ​​of most calcareous soils are within the range of 7.5 to 8.5 [3] with bicarbonate concentrations between 5-35 mmol L-1. The main anions present in excess in alkaline soils are HCO3- and CO32- [4]. Soil carbonates act as pH buffers and play an important role in rhizosphere processes, hampering nutrient availability to plants. The low availability of nitrogen (N), phosphorus (P) and micronutrients such as iron (Fe), zinc (Zn), and manganese (Mn) produce nutrient deficiencies in many plant species cultivated on carbonated soils [5-7]. Moreover, the high pH surrounding plant roots can alter the membrane potential [8] and inhibit the uptake of essential ions, and thus further contribute to nutrient deficiency in sensitive plants.
The uptake of bicarbonate can cause important metabolic disturbances due to both alteration of cell pH homeostasis and dark fixation of inorganic carbon. Bicarbonate can be quickly incorporated into organic acids, mainly malate, causing inhibition of mineral nutrient transport from roots to shoot, reduction of root growth, and oxidative stress in sensitive calcifuges, but not in tolerant calcicole species [9,10]. Due to these multiple direct and indirect injuries, sensitive species exhibit a complex syndrome of stress symptoms, including morpho-anatomical changes in roots, disturbance of water relations, signs of iron and/or zinc deficiency, reduction of total photosynthetic pigments and photosynthetic activity; accumulation of osmo-protectants, soluble sugars, and organic acids; and activation of the biosynthesis of antioxidant enzymes [11,12,13].
A better understanding of plant alkaline tolerance mechanisms and cultivation of new varieties of alkali-tolerant crops is needed to improve carbonated soils and increase food production [14]. However, current knowledge of the bicarbonate stress response of plants is limited. Most studies have been conducted on roots of crop species such as Glycine max or Oryza sativa which are species with moderate bicarbonate tolerance [15, 16]. Nonetheless, after several decades of effort still a proper model is missing to understand the adaptive mechanism to carbonate stress and the tolerance mechanism at the molecular-genetic level. Early experiments revealed that organic acid accumulation in response to bicarbonate occurred both in sensitive and tolerant species and it was speculated that differential compartmentation of organic acids may play a role in the tolerance [10]. In fact, several anion transporters, and gene families such as ALMT, NRT/POT and SLAHs were related to bicarbonate response inGlycine max [16,17].
During recent years, combined omics are applied to successfully characterize plant tolerance responses to complex stress factors (e.g. [18]). Resequencing-based Bulk Segregation Analysis (BSA) utilizes a strategy of pooling individuals with extreme phenotypes to conduct rapidly linked marker screening [19]. Whole-genome studies are useful to detect signature of selection, QTLs, and SNP markers. However, some important changes affecting the regulation of key genes can remain hidden. The combination of genomic and transcriptomic studies allows a more accurate screening of the candidate genes involved in specific physiological processes. Here, we took advantage of the natural variation present in two A. thaliana populations with contrasting phenotypes of soil alkalinity tolerance [5] to highlight the loci involved in bicarbonate stress responses and adaptation to carbonated soils by combining BSA-Seq and RNA-Seq technologies.
Materials and Methods