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