The interplay of malic acid and hormone signaling in adventitious root formation in MdWOX11-overexpressed transgenic apple microshoots

Adventitious root (AR) formation is a bottleneck for the mass propagation of apples. Malic acid (MA), an organic acid naturally found in various fruits and plants, serves as a key regulator in the rooting process. However, the role of MA in AR formation has not been investigated in apples. Thus, this study investigates the effects of MA treatments on the AR morphology and the underlying physiological and molecular mechanisms in MdWOX11-OE transgenic and GL3 (wild type, WT) microshoots. Preliminary experiments revealed that control microshoots exhibited higher AR numbers compared to MA-treated microshoots, with a medium level of MA treatment markedly improving AR length. MdWOX11-OE microshoots outperformed WT microshoots in both AR quantity and length, especially at the medium MA level, indicating a greater response to MA. Morphological observations over time showed a significant increase in AR primordia in MdWOX11-OE microshoots by day 8, correlated with increased mitotic activity in cambial cells. Endogenous hormone analysis showed higher indole-3-acetic acid (IAA) levels and lower methyl jasmonate (JA-me) in MdWOX11-OE microshoots, suggesting hormonal influences on AR formation. A total of 4104 differentially expressed genes (DEGs) were identified through RNA sequencing, with significant enrichment of plant hormone signaling and stress-responsive pathways. Key auxin- and cytokinin-related DEGs revealed distinct expression patterns, illuminating the regulatory networks involved in AR formation. Moreover, DEGs related to the brassinosteroid (BR), gibberellin (GA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) signaling pathways were systematically analyzed, indicating complex regulatory networks. The identification of DEGs associated with starch and sucrose metabolism underscores the metabolic adjustments following adventitious rooting. These results suggest important insights into the molecular mechanisms regulating AR formation in response to MA treatment, highlighting the interplay between hormone signaling and genetic factors in improving adventitious rooting in apples.