Integrated application of organic acids and earthworms improves rhizosphere microbiome, reduces health risks, and modulates proteomic and transcriptomic responses in rice (Oryza sativa L.) under aluminum stress.

Abstract

Soil contamination with toxic heavy metals such as aluminum (Al) is becoming a serious global problem due to the rapid development of the social economy. Organic acid and earthworms (Eisenia fetida) are efficient, environmentally friendly, and biodegradable and they enhance the solubility, absorption, and stability of metals. Therefore, the present study was conducted to investigate the individual and combined effects of organic chelating agents such as ascorbic acid and malic acid (5.0 µM L⁻¹ each) and earthworms (Eisenia fetida, 10 individuals per pot) on plant growth and biomass, photosynthetic pigments, gas exchange attributes, oxidative stress and response of antioxidant compounds (enzymatic and nonenzymatic), ASA-GSH cycle, cellular fractionation, and their specific gene expression, sugars, nutritional status of the plant, Al accumulation from the different parts of the plants, Al uptake, rhizosphere microbiome, health risk, and proteomic responses, in rice (Oryza sativa L. cv. IR-64) grown in soil spiked with Al [100 mg kg⁻¹ ]. Results from the present study revealed that the Al toxicity induced a substantial decrease in shoot length, root length, number of leaves, leaf area, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight, chlorophyll-a, chlorophyll-b, total chlorophyll, carotenoid content, net photosynthesis, stomatal conductance, transpiration rate, soluble sugar, reducing sugar, non-reducing sugar contents, calcium (Ca^2 +)^, magnesium (Mg²⁺), iron ( Fe²⁺), and phosphorus (P) contents in the plants. However, Al stress also induced oxidative stress in the plants by increasing malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), which also led to an increase in various enzymatic and non-enzymatic antioxidants and also the gene expression and sugar content. Furthermore, a significant (P < 0.05) increase in proline metabolism, the AsA-GSH cycle, and the pigmentation of cellular components was observed. Addition of organic acid and E. fetida into the soil significantly alleviated Al toxicity effects on O. sativa by improving photosynthetic capacity and ultimately plant growth. Increased activities of antioxidant enzymes in organic acid and E. fetida-treated plants seem to play a role in capturing stress-induced reactive oxygen species as was evident from lower levels of MDA and H₂O₂. Moreover, the application of organic acids and E. fetida enhanced both the abundance and diversity of the rhizosphere microbiome, with bacterial population levels and Shannon diversity indices significantly increasing. A marked reduction in daily Al intake and associated health risks was also observed under these treatments, and proteomic responses showed downregulation of HSP70, MT2A, and PRP under Al stress. Research findings, therefore, suggested that individual and combined application of organic acid and E. fetida can ameliorate Al toxicity in O. sativa seedlings and resulted in improved plant growth and composition under metal stress.