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Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr (serine/threonine) protein kinases that play very important roles in plant responses to biotic and abiotic stressors. However, the MAPK gene family in the important crop walnut (Juglans regia L.) has been less well studied compared with other species. We discovered 25 JrMAPK members in the Juglans genome in this study. The JrMAPK gene family was separated into four subfamilies based on phylogenetic analysis, and members of the same subgroup had similar motifs and exons/introns. A variety of cis-acting elements, mainly related to the light response, growth and development, stress response, and hormone responses, were detected in the JrMAPK gene promoters. Collinearity analysis showed that purification selection was the main driving force in JrMAPK gene evolution, and segmental and tandem duplications played key roles in the expansion of the JrMAPK gene family. The RNA-Seq (RNA Sequencing) results indicated that many of the JrMAPK genes were expressed in response to different levels of Colletotrichum gloeosporioides infection. JrMAPK1, JrMAPK3, JrMAPK4, JrMAPK5, JrMAPK6, JrMAPK7, JrMAPK9, JrMAPK11, JrMAPK12, JrMAPK13, JrMAPK17, JrMAPK19, JrMAPK20, and JrMAPK21 were upregulated at the transcriptional level in response to the drought stress treatment. The results of this study will help in further investigations of the evolutionary history and biological functions of the MAPK gene family in walnut.

AT-hook motif nuclear localization (AHL) proteins play essential roles in various plant biological processes. Yet, a comprehensive understanding of AHL transcription factors in walnut (Juglans regia L.) is missing. In this study, 37 AHL gene family members were first identified in the walnut genome. Based on the evolutionary analysis, JrAHL genes were grouped into two clades, and their expansion may occur due to segmental duplication. The stress-responsive nature and driving of developmental activities of JrAHL genes were revealed by cis-acting elements and transcriptomic data, respectively. Tissue-specific expression analysis showed that JrAHLs had a profound transcription in flower and shoot tip, JrAHL2 in particular. Subcellular localization showed that JrAHL2 is anchored to the nucleus. Overexpression of JrAHL2 in Arabidopsis adversely affected hypocotyl elongation and delayed flowering. Our study, for the first time, presented a detailed analysis of JrAHL genes in walnut and provided theoretical knowledge for future genetic breeding programs.