Integrated physiological and multiomic analyses reveal the multilayered aluminum tolerance mechanisms of acidophilic alga Coccomyxa sp. A2

ABSTRACT Acid mine drainage (AMD) is a pervasive global environmental challenge, and algal-based bioremediation represents one of the most robust strategies for its treatment. However, the mechanisms by which algae survive in metal-rich AMD remain largely unclear. Here, we integrated physiological, transcriptomic, and proteomic analyses to explore the tolerance mechanisms of the acidophilic alga Coccomyxa sp. A2 to aluminum (Al), a metal prevalent in AMD. The results demonstrated that A2 exhibited substantially higher Al tolerance than the model alga Chlamydomonas reinhardtii under their respective optimal pH conditions. Upon Al exposure, A2 significantly increased the extracellular levels of three strong organic acids (oxalic, citric, and tartaric acids), thereby facilitating chelation of free Al 3+ present in the acidic environment. In the near-neutral cytosol, A2 accumulated a broader array of organic acids at higher concentrations to sequester Al despite severe extracellular H + stress. To restrict Al influx, A2 enhanced the synthesis of cellulose, hemicellulose, and proteins across the distinct layers of its cell wall, increasing cell wall dry weight by 66%. This remodeling was probably supported by starch degradation, enhanced gluconeogenesis, and Calvin cycle activation, which together supplied sufficient monosaccharide precursors for polysaccharide synthesis. Concurrently, the upregulation of glycosyltransferases facilitated rapid polysaccharide chain elongation. To scavenge ROS, the alga likely initiated a temporally coordinated, biphasic antioxidant response, in which non-enzymatic ascorbic acid pools compensated for lagging superoxide dismutase activity during the early acute phase of stress. Calcium, abscisic acid, and jasmonic acid coordinated these defense mechanisms, with calcium serving as the primary signaling hub. IMPORTANCE The conventional treatment method for acid mine drainage (AMD) involves neutralization with lime, which is costly and generates large amounts of sludge that is difficult to manage. The application of sulfate-reducing bacteria (SRB) presents a promising alternative by eliminating secondary pollution and enabling metal sulfide recovery. However, a key challenge of this approach is ensuring an affordable and sustainable organic carbon source for SRB since AMD is oligotrophic. A potential solution is the in-situ cultivation of acidophilic algae. Given the high metal concentrations in AMD, investigating the metal tolerance mechanisms of acidophilic algae is vital for producing sufficient biomass to support SRB. Meanwhile, about 40% of the world’s arable soils are acidic, and these soils typically contain high concentrations of aluminum (Al), which is a major factor limiting crop production. Therefore, research on the exceptional Al tolerance mechanisms of acidophilic algae can provide insights into improving crop metal resistance through genetic engineering techniques.