Pear PbrPAO14 modulates pollen tube growth in vitro via altering tip-localized H₂O₂ content

Polyamine oxidase (PAO)-derived hydrogen peroxide (H 2 O 2 ) is known to participate in various plant growth and developmental processes. However, its role in pollen tube growth (PTG) of fruit trees remain elusive. In this study, we first identified 63 PAO members across four pear genomes, including 15 in Pyrus bretschneideri , and 16 each in P. betulifolia, P. pyrifolia , and P. communis . Phylogenetic analysis of 258 PAOs from 38 plant species classified them into seven distinct subclasses. The 63 pear PAOs were distributed across Subclasses I, III, IVa, IVb, and IVc, with members within each subclass exhibiting relatively conserved gene structures. Secondly, using P. bretschneideri as a model, tissue-specific expression analysis revealed that PbrPAO14 were specifically and highly expressed in pollen and pollen tube (PT), was selected for further functional characterization. Thirdly, transient suppression of the peroxisome-localized PbrPAO14 expression via antisense oligodeoxynucleotides significantly decreased PAO activity (by average 40 %), reduced apical H 2 O 2 levels (by average 16 %) to increase PT length (by average 16 %). Furthermore, exogenous spermine and spermidine treatment markedly up-regulated PbrPAO14 expression, while its expression suppression led to significant spermine accumulation in PT. Recombinant His-PbrPAO14 protein preferentially used spermine as a substrate, converting spermine to spermidine and putrescine via back conversion (BC) pathway, and generating H 2 O 2 . Our results suggested that pear PbrPAO14 mainly regulates PTG by producing H 2 O 2 via the BC pathway. This study advances our understanding of a PAO-mediated reactive oxygen species (ROS) source at the PT apex in the regulation of PTG, which significantly broadens the perspective on ROS dynamics in polarized cell growth.