Systematic Analysis of Dof Gene Family in Prunus persica Unveils Candidate Regulators for Enhancing Cold Tolerance

Abstract

Late-spring frost events severely damage low-chill peach blossoms, causing significant yield losses. Although 5-aminolevulinic acid (ALA) enhances cold tolerance through the PpC3H37-PpWRKY18 module, the regulatory mechanism of ALA on PpC3H37 remains to be elucidated. Using yeast one-hybrid screening with the PpC3H37 promoter as bait, we identified PpDof9 as a key interacting transcription factor. A genome-wide analysis revealed 25 PpDof genes in peaches (Prunus persica). These genes exhibited variable physicochemical properties, with most proteins predicted as nuclear-localized. Subcellular localization experiments in tobacco revealed that PpDof9 was localized to the nucleus, consistent with predictions. A synteny analysis indicated nine segmental duplication pairs and tandem duplications on chromosomes 5 and 6, suggesting duplication events drove family expansion. A conserved motif analysis confirmed universal presence of the Dof domain (Motif 1). Promoter cis-element screening identified low-temperature responsive (LTR) elements in 12 PpDofs, including PpDof1, PpDof8, PpDof9, and PpDof25. The quantitative real-time PCR (qRT-PCR) results showed that PpDof1, PpDof8, PpDof9, PpDof15, PpDof16, and PpDof25 were significantly upregulated under low-temperature stress, and this upregulation was further enhanced by ALA pretreatment. Our findings demonstrate ALA-mediated modulation of specific PpDof TFs in cold response and provide candidates (PpDof1, PpDof9, PpDof8, PpDof25) for enhancing floral frost tolerance in peaches.

Keywords: 5-aminolevulinic acid (ALA); PpDofs; cold stress.

Figure 1

Yeast one-hybrid assay showing that PpDof9 interacted with the promoter of the PpC3H37 in yeast. SD-TL: -trp, -leu; SD-TLH: -trp, -leu, -his; 3-AT: 3-amino-1,2,4-triazole.

Figure 2

Phylogenetic trees of Dof genes in Arabidopsis thaliana, the strawberry, and the peach. Different colors represent different subfamilies.

Figure 3

Subcellular localization of PpDof9. GFP: green fluorescent protein. RFP (nuclear marker): red fluorescent protein. Scale bar: 20 μm.

Figure 4

PpDof chromosome localization (A) and a collinearity analysis (B).

Figure 5

The motifs of the PpDof gene family.

Figure 6

An analysis of cis-acting elements of PpDof gene family promoter.

Figure 7

The highly expressed PpDofs genes in response to low-temperature stress and regulated by ALA. The treatments of flowers were defined as follows. ① Control: Cultured in distilled water + maintained at ambient temperature (22 °C/15 °C). ② Low-temperature treatment (LT): Cultured in distilled water + subjected to −3 °C for 6 h. ③ ALA treatment (ALA + LT): Pretreated with 50 mg·L⁻¹ ALA + subjected to −3 °C for 6 h. The values are the means ± SE from three biological replicates. The same letters indicate no significant differences at p = 0.05 level.

Figure 8

The possible regulatory pathway of PpDof9 involvement in ALA-enhanced cold tolerance in peach flowers. Note: → represents a positive effect. The red arrow represents positive regulation and the blue arrow represents negative regulation.