Excessive accumulation of auxin inhibits protocorm development during germination of Paphiopedilum spicerianum

Excessive auxin accumulation inhibits protocorm development during germination of Paphiopedilum spicerianum, delaying shoot meristem formation by downregulating boundary genes (CUC1, CUC2, CLV3) and promoting fungal colonization, essential for seedling establishment. Paphiopedilum, possess high horticultural and conservational value. Asymbiotic germination is a common propagation method, but high rates of protocorm developmental arrest hinder seedling establishment. Our study found that the key difference between normally developing protocorm (NDP) and arrested developmental protocorm (ADP) is their capability for continuous cell differentiation. In ADP, cells divide without differentiating, with indole-3-acetic acid (IAA) levels being 20 times higher than that in NDP. This suggests that auxin level plays a role in protocorm cell fate determination. Exogenous application of NAA demonstrated that elevated auxin level can delay the formation of the shoot apical meristem (SAM) inside the protocorm. Gene expression analysis revealed that elevated auxin can inhibit or even halt the SAM formation through down-regulation of SAM-related genes such as CLV3, CUC1 and CUC2. High auxin levels also led to reduced cell wall rigidity by up-regulation of cell wall expanding protein (EXPB15), thereby creating ideal conditions for fungi entry. Inoculation with a compatible orchid mycorrhizal fungus (OMF) resulted in successful cell differentiation of ADP and eventually triggered the conversion of ADP to NDP. Since the protocorm is a distinct structure that facilitates the establishment of symbiotic associations with compatible OMF, we propose that the excessive auxin accumulation inside Paphiopedilum protocorm can pause the further development of protocorm and soften the cell wall. This strategy likely serves to enhance the attraction and colonization by OMFs in the native habitat of Paphiopedilum, facilitating essential symbiotic relationships necessary for their survival and growth.