Genome-wide analysis of the SPL family in Zanthoxylum armatum and ZaSPL21 promotes flowering and improves salt tolerance in transgenic Nicotiana benthamiana.

Z. armatum is an economically valued crop known for its rich aroma and medicinal properties. This study identified 45 members of the SQUAMOSA-PROMOTER BINDING PROTEIN LIKE (SPL) gene family in the genome of Z. armatum. Phylogenetic and collinearity analyzes demonstrated a close relationship between ZaSPLs and ZbSPLs from B subgenomes of Zanthoxylum bungeanum. Our miRNA sequencing revealed a high degree of conservation of miR156a within Z. armatum, with the za-miR156a sequence identical to miR156-5p in Arabidopsis thaliana and Citrus sinensis. Of the 45 genes identified by ZaSPLs, 21 were targeted by za-miR156a, transient co-expression experiments in N. benthamiana demonstrated the targeting relationship between za-miR156 and ZaSPL21. Furthermore, RNA-seq and qRT-PCR analysis revealed that ZaSPL genes exhibited elevated expression levels in juvenile tissues of Z. armatum. The expression of nine representative ZaSPL genes were upregulated under polyethylene glycol (PEG) and abscisic acid (ABA). Overexpression of ZaSPL21 delayed the germination of transgenic tobacco and facilitated the flowering process in transgenic N. benthamiana. Significant up-regulation in the expression levels of flowering-related genes such as NbFT1, NbPIP2;1, NbTCP1, NbCOL1, NbGI2, NbGAI1, NbCKX2, and NbARR4 was observed in transgenic plants, suggesting that ZaSPL21 may stimulate plant flowering by regulation of these genes. Furthermore, ZaSPL21 also increased the germination speed of transgenic tobacco seeds during drought and salt stress conditions, and improved the salt tolerance of transgenic seedlings. In conclusion, our study contributes to understanding the functional analysis of the SPL gene family in Z. armatum and emphasizes the crucial role of ZaSPL21 in improving tolerance to salt and promoting flowering. The results offer potential strategies for the further utilization of these genes to improve the salt tolerance of Z. armatum.