Identification of the fructokinase gene family members in Hongyang kiwifruit and its expression analysis during postharvest softening

【Objective】Kiwifruit (Actinidia spp.) production in China maintains the world's leading position in both cultivation area and yield. However, postharvest softening of fruit notably reduces its quality. The initiation of fruit postharvest softening of kiwifruit primarily involves the hydrolysis of starch. The hydrolysis of starch into glucose and fructose necessitates phosphorylation for entry into the glycolytic pathway. Plant fructokinases (FRK) belong to the phosphofructokinase B family and possess characteristic substrate recognition regions of the FRK family. The FRK is a specific enzyme is involved in fructose phosphorylation, regulating fructose flux in plant cells and subsequently modulating respiratory metabolic pathways. Furthermore, the plant FRK may function similarly to hexokinases in sugar sensing, serving as a signaling molecule to regulate plant growth, development, starch accumulation, and environmental stress responses. However, little is known about the role of FRK in fruit postharvest softening and senescence. This study sought to identify the kiwifruit FRK gene family, investigate the differential expression patterns, and explore its association with kiwifruit softening.【Methods】Using Arabidopsis thaliana, Lycopersicon esculentum, and Manihot esculenta FRK nucleotide sequences as references, kiwifruit FRK family members were identified from the kiwifruit genome database Hong Yang v3, the HMMER online tool was used to verify the presence of the pfkB conserved domain in kiwifruit FRK family members, then the NCBI-CDD website was utilized to further validate the characteristic substrate recognition domain of AcFRK genes. We used the TBtools software to analyze the chromosomal localization of AcFRK genes; the subcellular location of the kiwifruit FRK proteins was predicated using WoLF PSORT online website; the protein molecular weight and isoelectric points were analyzed using EXPASY online website. A phylogenetic relationships of FRK proteins across kiwifruit, Arabidopsis, tomato, and cassava were established using MEGA (version 5.05) software; multiple sequence alignments were performed using Clustal X (version 1.81) and Gendoc 32; the conserved motifs of kiwifruit FRK proteins were analyzed using the MEME online website; the exon-intron gene structures were analyzed by TBtools; the FRK protein secondary structure was analyzed using SOPMA; the FRK protein tertiary model was constructed using SWISS-MODEL website; the promoter cis-acting elements of AcFRKs were predicted using PlantCARE online website; and the expression heatmap of AcFRKs during postharvest softening were generated using TBtools by normalizing FPKM values from transcriptomic data. The expression profiles of the AcFRK2 and AcFRK4 were verified with the realtime quantitative PCR technique after glucose, fructose and various hormones treatment.【Results】In the kiwifruit genome, a total of 6 FRK sequences were identified, randomly distributed on 5 chromosomes. The lengths of these 6 FRK proteins varied from 168 aa to 457 aa, with isoelectric points ranging from 4.88 to 6.03 and molecular weights spanning from 18.47 kDa to 50.01 kDa. Furthermore, the phylogenetic tree analysis revealed that kiwifruit FRK family could be divided into two groups: AcFRK1/AcFRK3/AcFRK4 (Group Ⅰ), AcFRK2/AcFRK5 (Group Ⅱ a), and AcFRK6 (Group Ⅱ b), each group comprising three FRK members. The multiple sequence alignments showed that AcFRK proteins possessed pfkB, substrate recognition, and ATP binding domains, members within the same group sharing highly similar conserved domains. The gene structure analysis showed that the AcFRK genes comprised 1 to 3 UTRs, 2 to 4 exons of GroupⅠ, and 6 to 9 exons of Group Ⅱ. Additionally, the amino acid sequence analysis unveiled 12 conserved motifs and demonstrated a high level of conservation among proteins within the same group, these findings were consistent with the multiple sequence alignments analysis. The protein structure analysis revealed a significant resemblance in the main structure among members of the same group, suggesting a strong conservation of functions within AcFRK proteins. The analysis of promoter cis-acting elements found a wide distribution of elements responsive to ethylene, abscisic acid, osmotic stress, and oxidative stress responses, suggesting that the AcFRK genes would be likely induced by the hormones ethylene or ABA, thereby participating in kiwifruit ripening and softening. The gene expression heatmap demonstrated an upward trend in the expression levels of the AcFRK2 and AcFRK4 as kiwifruit ripening progresses. The correlation analysis revealed a negative association between genes and fruit firmness as well as starch content, while showing a positive correlation with reducing sugar content, indicating that the AcFRK2 and AcFRK4 might positively regulate kiwifruit softening. Hexose treatment experiments demonstrated that the AcFRK2 and AcFRK4 exhibited upregulation in response to hexose, indicating their potential for efficient utilization of glucose and fructose. The hormone treatment experiments demonstrated that the AcFRK2 and AcFRK4 were upregulated by ethylene induction during the late storage period, with the AcFRK4 expression being further inhibited by AsA and NAA during room temperature storage, indicating that the AcFRK2 and AcFRK4 might be involved in the softening process through their response to different hormones.【Conclusion】Six members of the FRK gene family were identified, and two key genes, the AcFRK2 and AcFRK4, were selected, which would be involved in sugar metabolism, plant hormones, and kiwifruit softening. This would provide a theoretical basis for further exploring the biological functions of the FRK genes in kiwifruit postharvest softening.