Proline (Pro) metabolism in plants is involved in various cellular processes mediated during abiotic stress. However, the Pro-regulatory mechanisms are unclear. We used a suppressor mutation technique to isolate novel genes involved in the regulation of Pro metabolism in Arabidopsis. Using atrzf1 as a parental plant for T-DNA tagging mutagenesis, we identified a suppressor mutant, termed praline content alterative 21 (pca21), that displayed reduced Pro contents compared with the atrzf1 under osmotic stress conditions. Genomic Thermal Asymmetric Interlaced (TAIL)-PCR revealed pca21 harbored an inserted T-DNA in the region of At2g36620 that encodes Ribosomal Protein L24A. In general, the pca21 mutant partially suppressed the insensitivity of atrzf1 to osmotic stress and abscisic acid during seed germination and early seedling stage. Additionally, the pca21 mutant had increased MDA content and lower expression of several Pro biosynthesis-related genes than the atrzf1 mutant during drought condition. These results suggest that pca21 acts as partial suppressor of atrzf1 in the osmotic stress response through the Pro-mediated pathway. (C) 2017 Elsevier Inc. All rights reserved.