Identification of plum PsNAC gene family and its expression patterns in development of fruit hollowness and browning

[Objective] The NAC transcription factor family is one of the plant-specific transcription factor families and plays a pivotal role in plant growth and development and responses to biotic and abiotic stresses. Huangguan plum is a newly developed high-quality plum variety bred by our team specifically suited for cultivation in Fujian Province, China. Its fruit has the advantages of excellent taste, pleasant flavor, and rich nutrient profile. However, in our previous long-term observations, Huangguan plum has been found to be prone to fruit hollowness and browning (HB), characterized by rough and crystalline fruit pulp surfaces undergoing lignification and browning. We found that lignin biosynthesis and accumulation is one of the predominant biochemical responses to HB. The NAC transcription factor is recognized as the primary regulator in the transcriptional control of plant secondary wall synthesis. This study aims to characterize the PsNAC gene family members in plum and investigate their association with fruit HB in Huangguan plum. [Methods] The molecular weight, theoretical isoelectric point, and other physicochemical properties were predicted by the online tool ExPASy. The subcellular localization of PsNACs was predicted by the online software WoLF PSORT. The MEGA 11 software was used to construct a phylogenetic tree. The online tool Simple MEME Wrapper was used to analyze the motifs. The conserved motifs and gene structure maps were drawn by Tbtools. To analyze the cis-acting elements in the promoter region of PsNACs, the upstream 2000 bp promoter sequences were extracted from the genomic sequences and submitted to the Plant TBDB website for the identification of cis-elements in the promoter region. The analysis results were organized and displayed using Simple BioSequence Viewer. The expression of ten PsNACs in Huangguan fruit with and without HB was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). [Results] There are 115 PsNAC members identified in Prunus salicina Lindl., with protein sequences ranging from 182 to 861 amino acids, molecular weights from 20.98 to 95.97 ku, theoretical isoelectric points from 4.43 to 9.55, and the instability index from 27.84 (PsNAC60) to 61.36 (PsNAC087). The grand average of hydropathy values of PsNAC gene family members were negative, indicating that these proteins are hydrophilic in nature. Transmembrane structure analysis revealed that 94% of PsNAC gene family members do not possess a transmembrane domain. The subcellular localization prediction results showed that 91 PsNAC gene family members were located in the nucleus, and the rest were distributed in structures such as the cytoplasm, Golgi apparatus, peroxisome, cytoskeleton, mitochondria, chloroplasts, plasma membrane, and vacuole. Chromosomal localization analysis revealed uneven distribution across the plum's eight chromosomes, with chromosomes 2 and 3 harboring the highest number counts (17.4%), followed by chromosome 5 (15.2%), and the fewest on chromosomes 6 and 7 (10 PsNACs each). Phylogenetic tree analysis between Arabidopsis thaliana and P. salicina Lindl. classified PsNAC genes into 17 subfamilies, with 6 and 10 members clustered in OsNAC003, and OsNAC7, respectively, which are associated with plant secondary wall biosynthesis. The number of coding sequence segments in PsNACs ranged from 1 to 8, with most containing 3 to 6 segments. Analysis of gene annotation files identified a total of 10 conserved motifs among PsNACs, with varying positions and frequencies. Motif 1, motif 2, motif 3, and motif 6 were found in the majority of PsNACs, typically located towards the N-terminus of the sequences. PsNAC members within the same subfamily exhibited similar motif distributions and gene structure characteristics including the CDS and UTR regions, suggesting potential functional similarity. Analysis of the 2000 bp upstream sequences from the transcription start site of PsNACs identified a total of 3069 cis-elements. The most significant core elements included phytohormone-responsive elements, MYB binding sites, low temperature responsiveness, drought-inducible elements, and light-responsive elements. Intraspecific synteny analysis revealed that the PsNAC gene family contained 13 pairs of duplicated genes within the plum genome. The relative expression levels of PsNAC26, PsNAC57, PsNAC77, and PsNAC95 were highest at the fruit expansion period and gradually decreased as fruit development progressed. Conversely, PsNAC54 and PsNAC74 exhibited their lowest expression levels at the fruit expansion period, and increased gradually with fruit development and ripening. The expression levels of PsNAC26, PsNAC57, PsNAC77, PsNAC95, PsNAC54, and PsNAC74 were higher in Huangguan fruit showing hollowness and browning compared to those without. [Conclusion] This study represents the first comprehensive analysis of the PsNAC gene family in plum, identifying and characterizing 115 members of the PsNAC gene family. We explored their physical and chemical properties, gene structures, chromosome locations, phylogenetic relationships, and subcellular localization characteristics. Furthermore, using qRT-PCR technology, we investigated the gene expression patterns of PsNAC gene family members in Huangguan fruit exhibiting HB and non-HB across various developmental stages. The findings of this study will serve as a crucial foundation for further exploration into the biological functions of PsNAC gene family and the molecular mechanism by which PsNAC gene family members regulate fruit HB in Huangguan plum.