Genome-wide identification of the TCP gene family in sweet cherry and expression profiling in Gisela 17 rootstock in response to abiotic stresses

【Objective】TCP transcription factors (TFs) are a class of plant-specific TFs that play a crucial regulatory role in processes such as seed germination, vegetative growth, flowering, fruit development, senescence, and stress response in plants. To date, although the TCP family has been extensively studied in model plants such as Arabidopsis thaliana and rice, there have been no systematic reports on this gene family in Prunus avium L., a globally important economic fruit crop. This study aims to conduct a comprehensive genome-wide identification and systematic expression analysis of the TCP family in sweet cherries, with a focus on exploring its stress (drought, salt and iron deficiency) resistance functions.【Methods】The latest genome sequences, protein sequences, and GFF3 annotation files of the sweet cherry cultivar Tieton v2.0 were retrieved from the Rosaceae Genome Database (https://www.ro-saceae.org/), a widely recognized repository for genomic resources of species in Rosaceae. A series of bioinformatics tools and pipelines were employed to characterize the TCP family members. Then, ExPASy ProtParam was used to analyze physicochemical properties (such as molecular weight, isoelectric point, instability index); MEGA 11 was utilized to construct a neighbor-joining phylogenetic tree with 1000 bootstrap replicates; Gene Structure Display Server (GSDS 2.0) was used to visualize gene structures (exons, introns, and UTRs); MEME Suite (v5.5.3) was conducted to identify conserved motifs. In addition, TBtools was used to map genes to chromosomes and analyze syntenic relationships; and PlantCARE was used to predict cis-acting elements in the 2000 bp upstream promoter regions. For expression analysis, in vitro rooted plantlets of Gisela 17, a commonly used rootstock with good adaptability, were subjected to three abiotic stress treatments: drought, salt, and iron deficiency. Total RNA was extracted from leaves using TRIzol reagent at 0, 12, 24, 48 and 72 hours after treatment, and the first strand cDNA was synthesized by reverse transcription using PrimeScript RT kit. Quantitative real-time PCR (qRT-PCR) was performed to analyze the gene expression levels.【Results】Candidate genes were further verified by domain confirmation via the SMART and NCBI CDD databases to exclude sequences with incomplete or truncated domains, resulting in the final identification of 19 TCP family members, designated as PaTCP1-PaTCP19 based on their chromosomal locations. These genes were unevenly distributed across 8 of the 16 sweet cherry chromosomes. The coding sequences of PaTCP genes ranged from 268 to 601 amino acids (aa), with corresponding molecular weights of 29.87-66.73 kDa. The isoelectric points (pI) varied from 6.14 to 9.51, including 10 alkaline proteins (pI＞7) and 9 acidic proteins (pI＜7). The instability index ranged from 48.14 to 78.73, indicating all PaTCP proteins are unstable (instability index＞40), while the aliphatic index (52.28-87.44) and negative hydrophobicity values confirmed their hydrophilic nature, consistent with their predicted nuclear (PaTCP1-17), mitochondrial (PaTCP18), and cytoplasmic (PaTCP19) localization. Motif analysis revealed 10 conserved motifs, among which motif 1 (corresponding to the core TCP domain) was present in all 19 PaTCP proteins, highlighting its high conservation. Gene structure analysis indicated 8 PaTCP genes contained a single exon, 4 had two or more exons, and 7 lacked untranslated regions (UTRs), with intron numbers ranging from 1 to 5. Phylogenetic analysis of 19 sweet cherry TCP proteins and 24 Arabidopsis TCP proteins classified PaTCP members into two major clades: ClassⅠ (PCF subfamily, 10 genes) and Class Ⅱ (9 genes total, including 7 in the CIN subfamily and 2 in the CYC/TB1 subfamily). Synteny analysis identified 6 pairs of homologous PaTCPs (PaTCP1- PaTCP9, PaTCP2- PaTCP15, PaTCP4- PaTCP5, PaTCP5- PaTCP14, PaTCP4- PaTCP14), all derived from segmental duplication (no tandem duplication events were detected), indicating large-fragment duplication as the primary mechanism driving the expansion of PaTCP family. The Ka/Ks ratios of all homologous gene pairs ranged from 0.13 to 0.29 (all＜1), suggesting strong purifying selection during evolution to maintain functional stability. The TCP genes in sweet cherries show significant selective conservation in evolution, which maintain a high degree of linearity with dicotyledonous model plants and closely related stone fruit trees. Among them, 32 pairs and 22 pairs of homologous genes were identified in Arabidopsis thaliana and tomato, respectively, while the collinear logarithms with peach, plum and apricot reached 38 to 40 pairs, confirming the closer evolutionary distance and more complete preservation of chromosomal segments within stone fruit trees. In contrast, the collinear relationship with the monocotyledonous plant rice is only 13 pairs, and the evolutionary distance between the two is relatively far. Promoter cis- acting element analysis identified 7 types of functional elements in the 2000 bp upstream regions of PaTCP genes, including abiotic stress- responsive elements (low temperature, drought, defense and stress) and hormone (gibberel-lin, salicylic acid, methyl jasmonate, auxin, abscisic acid)-responsive elements, indicating potential involvement of PaTCP genes in multiple stress and hormone signaling pathways. qRT- PCR results showed PaTCP6 was the only gene with no significant expression changes under all the three stresses at all time points, while most other PaTCP genes exhibited similar response patterns across different stresses. Specifically, PaTCP1, PaTCP3, PaTCP4, PaTCP7, PaTCP9, PaTCP10, PaTCP15, PaTCP17, PaTCP18, and PaTCP19 were significantly upregulated under drought, salt, and iron deficiency, with the most dramatic upregulation observed at 24 h and 48 h post-treatment (fold changes ranging from 2.3 to 12.7). In contrast, PaTCP2, PaTCP8, PaTCP11, PaTCP12, PaTCP13, and PaTCP16 were consistently downregulated under three stresses, while PaTCP14 showed no significant change under drought but exhibited significant upregulation under salt and iron deficiency stresses (fold changes of 3.1 and 4.5 at 48 h, respectively), indicating its specific role in responding to salt and ionic stresses.【Conclusion】 The research results indicate that the PaTCP genes play an "activation-inhibition" regulatory role under various abiotic stresses (such as drought, salt stress, and iron deficiency), and different members exhibit either conserved or specific response patterns. The stress- responsive PaTCP genes identified in this study provide valuable candidate resources for genetic improvement of stress resistance in sweet cherry via molecular breeding approaches.