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Alfalfa (Medicago sativa L.) is a nutritious forage plant in dryland regions with strong drought tolerance and broad adaptability. To understand the architectural characters and physiological functions of the root systems in response to different drought stress and rehydration, two experiments were conducted in a glasshouse. Experiment 1 used three alfalfa cultivars and four soil moisture treatments in a split-plot design with four replicates to investigate the response of root systems of different alfalfa cultivars to drought stress and subsequent rehydration. Experiment 2 used one alfalfa cultivar and four soil moisture treatments in a randomized complete block (RCB) design with three replicates to measure reactive oxidant species and antioxidant defense systems of alfalfa root. The soil moisture treatments included (1) 75–80% of the water-holding capacity (CK); (2) 60–65% of the water-holding capacity and subsequent rehydration to 75–80% of the water-holding capacity; (3) 45–50% of the water-holding capacity and subsequent rehydration to 60–65% and 75–80% of the water-holding capacity; and (4) 30–35% of the water-holding capacity and subsequent rehydration to 45–50%, 60–65% and 75–80% of the water-holding capacity. Our results demonstrated that plants had higher root biomasses (mean = 0.77 g), fractal dimensions (1.47), fractal abundances (3.84), and branching ratios (0.39) under severe water deficit and rehydration than under less stressful soil moisture conditions. Plant root systems tended to exhibit herringbone branching (Topological Index = 0.63) when soil moisture was at 60–65% water-holding capacity with the soil being then rehydrated, whereas they tended to have a smaller lateral root length, root surface area, root volume, and branching ratio and larger root diameter under more favorable soil moisture conditions for less stressful soil moisture treatments. Severe water stress and subsequent considerably improved superoxide (O2−) generation rate, the content of hydrogen peroxide (H2O2) and Malondialdehyde (MDA), and non-enzyme antioxidant glutathione (GSH), and reduced the content of ascorbate (AsA) and activity of catalase (CAT), ascorbate peroxidase (APX), and glutathione peroxidase (GPX). It is concluded that Medicago sativa L. cv Zhaodong (ZD) and cv Aohan (AH) exhibited higher drought resistances than cv Golden empress (GE) using topological and fractal analyses. This could be a key contributor to altering optimum soil moisture conditions and screening alfalfa cultivars based on root system architecture and antioxidant defense systems. The molecular functions warrant further investigation which could help explain the observed root phenotypes after drought conditions and rehydration.

Fractal and Topological Analyses and Antioxidant Defense Systems of Alfalfa (Medicago sativa L.) Root System under Drought and Rehydration Regimes