Identification of the PHT phosphate transporter family in strawberry and functional analysis of FaPHT2.1

【Objective】Inorganic phosphorus is the primary form of phosphorus nutrition that plants absorb and utilize, and the phosphate transporter (PHT) family is central to the plant's uptake and transport of inorganic phosphorus. This study aimed to perform bioinformatics analysis on the physicochemical properties and molecular evolutionary relationships of the diploid strawberry (Fragaria vesca) phosphate transporter (PHT) family, and explore the function of the phosphate transporter PHT2.1 in plant growth, development, and strawberry fruit ripening.【Methods】The coding sequences (CDSs) of 22 PHT genes from Arabidopsis thaliana, obtained from NCBI (https://www.ncbi.nlm.nih.gov/), were used as query sequences for BLAST analysis against the whole genome CDSs of the woodland strawberry (F. vesca Genome v4.0) in the GDR (https://www.rosaceae.org/). All seqences were then submitted to Protparam analysis (https://www.expasy.org/resources/protparam) to predict their physicochemical properties. The Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) was used to predict the subcellular localization of the strawberry PHT proteins. The amino acid sequences of all identified PHT genes in F. versa and the query sequences of Arabidopsis were aligned using MEGA11 with the ClustalW program and default options. The phylogenetic tree for PHT proteins was constructed usingMEGA11 with the Neighbor- Joining (NJ) algorithm and the following parameters: complete deletion, Poisson model, and 1000 replicates of the bootstrap method. Chromosomal localization data was retrieved from the GDR JBrowse (F. vesca Whole Genome v4.0.a2). Genes were mapped to the chromosomes using MG2C (http://mg2c.iask.in/mg2c_v2.1/). The online Gene Structure Display Server (GSDS 2.0, https://gsds.gao-lab.org/) was utilized to analyze the exon-intron structure of the FvPHT genes based on the CDS and the corresponding genomic sequence, which were obtained from NCBI (https:// www.ncbi.nlm.nih.gov/). The sequence of FaPHT2.1 was constructed into the pSupper1300-GFP vector driven by the 35S promoter. LeCDJ1 was also constructed to the p1300-mCherry as a chloroplast marker. These recombinant vectors were transformed into Agrobacterium tumefaciens GV3101 (carrying P19), and further transiently transformed into epidermal cells of Nicotiana benthamiana to obtain subcellular localization images. A phosphate-uptake deficient yeast mutant YP101 was used to validate the phosphate transport activity of FaPHT2.1 by growing on culture media with different phosphate concentrations (10 μM, 500 μmol · L- 1 , 5 mmol · L- 1 , 10 mmol · L- 1 , 20 mmol · L- 1 ). Transgenic Arabidopsis FaPHT2.1/atpht2.1, Arabidopsis mutant atpht2.1 and transiently transformed strawberry fruits FaPHT2.1 OE, FaPHT2.1 RNAi were utilized to characterize the biological functions of FaPHT2.1. 【Results】16 PHT family members were found in woodland strawberries, which belonged to 5 subfamilies, and they were named as FvPHT1.1, FvPHT1.2, FvPHT1.3, FvPHT1.4, FvPHT1.5, FvPHT1.6, FvPHT1.7, FvPHT1.8, FvPHT2.1, FvPHT3.1, FvPHT3.2, FvPHT4.1, FvPHT4.2, FvPHT5.1 and FvPHT5.2. Bioinformatics analysis indicated that strawberry PHT family proteins were predominantly stable hydrophobic proteins. The pI (theoretical isoelectric point) was distributed between 5.73 and 9.43, predominantly falling within the range of 8.00 to 9.43. The number of amino acids in PHTs protein family ranged from 317 to 720, with relative molecular weights between 34.99 and 80.44 kDa. PHT1s were predicted to be localized on the cytoplasmic membrane, PHT2 on the chloroplast, PHT3s on the mitochondria, PHT4s on the plastid or Golgi apparatus, and PHT5s on the vacuole. The 16 FvPHT genes were distributed on six strawberry chromosomes. There were four FvPHT genes on chromosomes LG2 and LG7, respectively. LG4 and LG5 each had three, and LG3 and LG6 each had one, which were the FvPHT4.1 and FvPHT3.1. RT-qPCR using four strawberry organs (roots, stems, leaves, and flowers) and fruits from seven different developmental stages (small green stages, large green stages, degreening stages, white fruit stages, initial red stages, partial red stages, and full red stages) indicated that the FaPHT2.1 expression was highest in green fruit, followed by leaves, flowers and stems, almost absent in roots, and the expression level of the FaPHT2.1 in fruit continuously decreased as strawberries ripened. The subcellular localization observation showed that FaPHT2.1 was located in chloroplasts. The growth recovery experiment in a phosphate-uptake deficient mutant yeast showed that both the recombinant strains YP101- pRS426:FaPHT2.1 and two truncated strains (FaPHT2.141- 569 and FaPHT2.171- 569) were able to partially restore the growth defects exhibited by the strains YP101 transformed with empty plasmid pRS426, and the restoration effect gradually diminished with decreasing Pi concentration. The physiological analysis of transgenic Arabidopsis revealed that the mutant atpht2.1 exhibited significantly lower phosphorus content, chlorophyll content, and fresh weight compared to the wild-type (WT) and FaPHT2.1/atpht2.1 transgenic lines. The results from transiently transformed strawberries showed that the expression of the FaPHT2.1 influenced fruit quality. The FaPHT2.1 overexpression (OE) fruits had a higher total soluble sugar content, soluble solids content and a lower fruit firmness than the control fruits, whereas the RNA interference (RNAi) fruits were opposite to the OE fruits. 【Conclusion】Our research identified strawberry PHT1, PHT2, PHT3, PHT4, and PHT5 family pro-teins, predicted their physicochemical properties and subcellular localization, and demonstrated the physiological functions of strawberry PHT2.1 in regulating plant growth and fruit ripening, laying a theoretical foundation for future molecular breeding and fruit quality improvement.