Abstract:

Objective: This study aims to functionally characterize the PdHyPRPs genes in the nationally approved elite variety of Populus × euramericana ‘Bofeng 3’, and generate novel salt-tolerant poplars by CRISPR/Cas9-mediated editing of PdHyPRPs. Method: With the tomato SlHyPRP1 amino acid sequence as a query, PdHyPRPs family genes were screened from the Populus deltoides reference genome. The gene structures and their protein types were analyzed through bioinformatics methods such as phylogenetic tree construction and homologous protein sequence alignment. Quantitative real-time PCR was employed to analyze the expression patterns of PdHyPRPs genes in leaves, stems, and roots of P. × euramericana ‘Bofeng 3’, and their responses to drought and salt stress. The homologous cloning method was used to obtain the coding sequences (CDS) of PdHyPRP1 and PdHyPRP2. CRISPR/Cas9 and homologous recombination techniques were used to construct gene editing and RNAi vectors targeting PdHyPRP1/2, which were then introduced into P. × euramericana ‘Bofeng 3’ by Agrobacterium-mediated transformation, to obtain PdHyPRP1/2-edited, PdHyPRP1/2-RNAi, and PdHyPRP1/2 overexpressed lines. Under salt stress, the contents of superoxide anion (O₂^–), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) in leaves and roots of each line were measured to evaluate salt tolerance and verify gene function. Result: A total of 15 PdHyPRP genes were identified in P. deltoides, with 360 to 1317 bp CDS. Each gene contained a single exon, encoding 119?438 amino acids. The encoded proteins all possessed an N-terminal signal peptide, a proline-rich repeat region, and a C-terminal 8-cysteine motif (8CM), belonging to the typical 8CM protein family and clustering into six evolutionary branches. Expression analysis revealed that, except for PdHyPRP13, 14 genes were expressed in leaves, stems, and roots of P. × euramericana ‘Bofeng 3’ and were induced by salt and drought stress, but with tissue specificity. PdHyPRP4, PdHyPRP5, PdHyPRP6, PdHyPRP7, and PdHyPRP8 showed high and relatively stable expression across all three tissues, while PdHyPRP1, PdHyPRP2, PdHyPRP9, PdHyPRP11, and PdHyPRP12 were mainly highly expressed in stems and roots. Under NaCl stress, the expression patterns of PdHyPRP1 and PdHyPRP2 exhibited similar expression patterns across three tissues, showing upregulation under low concentration stress and an initial upregulation followed by downregulation over time under high concentration. However, under PEG stress, their expression patterns varied among leaves, stems, and roots. PdHyPRP1 and PdHyPRP2 were successfully cloned from P. × euramericana ‘Bofeng 3’, with 840 bp and 597 bp CDS, respectively. Dual-gene editing and silencing vectors were constructed, and two edited-lines and two RNAi-lines simultaneously targeting PdHyPRP1 and PdHyPRP2 were obtained. Salt tolerance evaluation demonstrated that compared to the wild type, both edited- and RNAi-lines displayed milder leaf damage and significantly reduced accumulation of O₂^–, H₂O₂, and MDA in leaves and roots (0.60–0.85 times and 0.60–0.86 times of wild-type, respectively), indicating an improvement in salt tolerance. Conclusion: A total of 14 PdHyPRPs are expressed in leaves, stems, and roots of P. × euramericana ‘Bofeng 3’ with tissue specificity and respond to drought and salt stress. PdHyPRP1 and PdHyPRP2 play negative regulatory roles in salt stress response of P. × euramericana ‘Bofeng 3’, and their mutation or silencing can enhance salt tolerance by reducing reactive oxygen species accumulation and membrane lipid peroxidation. The PdHyPRP1/2 editing vector has been successfully constructed, and PdHyPRP1/2-edited lines of P. × euramericana ‘Bofeng 3’ with improved salt tolerance are obtained, providing new germplasm and target genes for stress resistance breeding in poplar.

Key words: poplar, gene editing, HyPRP gene, salt tolerance, Populus × euramericana ‘Bofeng 3’