Genome-wide identification and expression analysis of PIN gene family during adventitious rooting of Juglans regia L. microcuttings

Walnut tree (Juglans regia L.) is one of the most widely spread crops worldwide, highly valued for its fruits. The propagation of J.regia cultivars is mostly done through grafting based on seed propagation or through in vitro microcuttings. However, even when used optimized in vitro protocols several walnut cultivars exhibit a recalcitrant behavior upon the stimulus of adventitious roots development (AR). The auxin indole-3-butyric acid (IBA) is the phytohormone most used in AR protocols. Genes involved in IBA metabolism, including auxin influx/efflux transporters, have been widely characterized in several plant species due to their involvement in AR formation (Velada et al., 2020).

PIN-Formed Auxin Efflux Carrier genes encode one of the most important group of polar auxin transporters in plants, being involved in many developmental processes (Cardoso et al., 2022). To further investigate the involvement of PIN in AR efficiency in walnut genotypes, an in silico characterization of this gene family was performed. PIN sequences retrieved from Olea europaea were used as queries in walnut tree genome databases ( https://plants.ensembl.org/index.html), and the resulting sequences were used as secondary queries.

In total, fourteen JrPIN genes were identified, exhibiting a well-conserved exon-intron structure, with most members comprising six exons. To classify the identified JrPIN sequences, a phylogenetic tree was constructed using PIN sequences of nine Magnoliopsida species. Cluster analysis showed a distribution of the JrPIN members across 5 PIN subfamilies, with some members belonging to the canonical proteins, characterized by larger sequences comprising a central hydrophilic intercellular loop (HL) (named JrPIN1a to JrPIN1f, JrPIN2a to JrPIN2b and JrPIN3a to JrPIN3b), and 4 members grouped as non-canonical proteins (JrPIN5 and JrPIN8a to JrPIN8c), characterized by shorter sequence lacking the HL. Details related with protein features and exon-intron sequence analysis will be presented.