Objectives This study aimed to elucidate the role of phenylalanine ammonia-lyase (PAL) activity in regulating root endodermal differentiation and cadmium (Cd) accumulation in rice, thereby providing a theoretical basis for breeding rice cultivars with low Cd accumulation.

Methods Hydroponic experiments were conducted using the low-Cd-accumulating japonica rice cultivar Nipponbare and the high-Cd-accumulating indica rice cultivar IR32307. First, the effects of the phenylalanine ammonia-lyase inhibitor (AIP; 5 and 10 μmol/L) on root PAL activity were evaluated under both Cd-stress and non-stress conditions to determine the optimal AIP concentration for subsequent experiments. Four treatments were then established: control without AIP or Cd stress (CK), 10 μmol/L Cd stress treatment (Cd), 10 μmol/L AIP treatment (AIP), and combined Cd+AIP treatment (Cd+AIP). After one week of treatment, Cd content in shoots and roots was determined using inductively coupled plasma mass spectrometry (ICP-MS). Endodermal barrier development was examined using confocal laser scanning microscopy, Cd transport pathways were analyzed using short-distance transport tracing techniques, and the expression levels of endodermal differentiation-related genes were quantified by real-time quantitative PCR (RT-qPCR).

Results Cd stress significantly increased PAL activity in rice roots, with PAL activity in Nipponbare and IR32307 increasing by 107.49% and 21.45%, respectively, compared with the CK. Cd-induced PAL activation promoted lignin and suberin deposition and upregulated the expression of OsPAL, OsCASP1, OsCYP450, and OsKCS20, thereby enhancing endodermal barrier development. In contrast, inhibition of PAL activity by AIP (Cd+AIP treatment) reduced root lignin and suberin contents by an average of 60.40% and 37.42%, respectively, relative to the Cd treatment. Relative to CK, AIP treatment delayed the development of the casparian strip and suberin lamellae, as evidenced by significant increases in root the ratio of the distance from the Casparian strip developmental site to the root apex to the total root length (D_TIP-CSs, 16.62% in Nipponbare; 16.41% in IR32307) and the ratio of the distance from the suberin lamellae developmental site to the root apex to the total root length (D_TIP-SL, 14.10% in Nipponbare; 8.95% in IR32307), resulting in weakened endodermal barrier function. Concurrently, AIP treatment increased Cd accumulation in the shoots and roots of Nipponbare by 27.37% and 4.36%, respectively, with IR32307 exhibiting a similar trend.

Conclusions PAL-mediated lignin and suberin biosynthesis is a key regulatory process governing root endodermal barrier development. Nipponbare exhibited a stronger capacity for PAL activity induction and earlier endodermal differentiation compared to IR32307; these physiological and structural characteristics serve as the key basis for its restriction of Cd uptake and subsequent translocation to the shoots.