Background and aims Utilizing drought-tolerant genotypes with appropriate adaptive characteristics is a crucial mitigation strategy to improve wheat productivity in dry conditions. Understanding rhizosphere processes (e.g., enzyme traits) involved in nutrient acquisition and adaptation to drought stress across different genotypes is critical for the development of drought-resistant genotypes. Methods We grew three wheat genotypes with varying drought tolerance -Baran (rainfed drought-tolerant), Sirvan (drought-tolerant), and Marvdasht (non-drought-tolerant)- in rhizoboxes under drought stress. Through in-situ zymography and ex-situ enzyme kinetic analysis, we examined the localization and dynamic behaviors of key enzymes, acid phosphatase (ACP) and β-glucosidase (GLU), in the rhizosphere and their relationship with root traits. Results Baran displayed a more extensive root system with abundant lateral roots compared to other genotypes. Its rhizosphere exhibited a higher hotspot of GLU and ACP than Sirvan (1.5- and 1.2-fold higher, respectively) and Marvdasht (2- and 2.7-fold higher, respectively). Baran also demonstrated a broader enzyme activity expansion in the rhizosphere, showcasing its superior nutrient exploration capability. Drought-tolerant genotypes displayed elevated GLU and ACP activity in the rhizoplane, indicating enhanced root exudation. Notably, Vmax values of GLU were approximately 2-fold lower in drought-tolerant genotypes than in Marvdasht, revealing an energy conservation strategy in dry conditions. Additionally, drought-tolerant genotypes exhibited a higher affinity of GLU and ACP to substrates, enabling efficient nutrient extraction from soil organic matter despite lower enzyme activity. Conclusion Our findings demonstrate that drought-tolerant genotypes can better withstand water stress by having a broader rhizosphere extent and an effective enzyme system, both of which are primarily facilitated by lateral root growth.

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