The genes related to lignin biosynthesis pathway regulate juice sac granulation in Guanxi pomelo

Abstract:【Objective】Guanxi pomelo is one of the representative subtropical fruits, which produced in Pinghe county, Fujian province of China. However, the juice sac granulation is a physiological disease in the ripening period of the pomelo, which can seriously affect the taste and nutritional value of the fruits. In this study, the pomelo juice sacs in 4 different growth and development stages were used as experimental materials. In order to reveal the molecular regulation mechanism of lignin biosynthesis during juice sac granulation, the differentially expressed key genes related to lignin biosynthesis were screened through transcriptome data, and the expression characteristics of the genes during fruit juice sac granulation were studied.【Methods】Guanxi pomelo fruits on 135, 165, 195 and 215 days after anthesis in 2018 were collected as experimental materials. The juice sac granulation rates of the pomelo indifferent periods were calculated, and the juice sac slices were observed used microscope for cell wall. The genes related to the lignin biosynthesis pathway screened from transcriptome data were analyzed, the accuracy of transcriptome data was verified by qRT-PCR, and the expressions of the genes related to the lignin biosynthesis pathway were also analyzed. Then the enzyme activities related to the key genes of the lignin biosynthesis and the lignin content were determined.【Results】There was no juice sac granulation in the fruit development stage of Guanxi pomelo before 195 days after anthesis, but the juice sac granulation rate gradually increased and the lignin content of the juice sac also showed a significant increase trend on 195 days after anthesis. These results showed that the fruit juice sac granulated with the accumulation of lignin content. The microscopic observation results showed that the cell walls of the inner side cells in the fruit juice sacs on 195 days after anthesis were dyed red by safranin O staining compared with those prior to 195 days after anthesis, when the juice cells were lignified and the lignin content increased slightly. The cell walls in the inner cells and the epidermic cells of the fruit juice sacs were red and the color was deeper on 215 days after anthesis than those on 195 days after anthesis. These results confirmed that the lignification of the juice cells aggravated significantly from 195 to 215 days after anthesis. Thirteen differentially expressed lignin biosynthesis pathway genes were selected from the transcriptome of the juice sac at 4 developmental stages. The 13 genes showed an upward trend with the process of fruit development and maturation, including CrPAL1, CrPAL3, CrC4H1, CrC4H2, Cr4CL, CrHCT1, CrCCR3, CrCAD3, CrCOMT4, CrPOD2, CrPOD6, CrPOD7 and CrPOD8, especially from 195 to 215 days after anthesis. Furthermore, the expressions of these genes on 215 days after anthesis were up-regulated by 2 to 9 times compared with those on 195 days after anthesis. qRT-PCR results confirmed that 9 of the 13 genes had similar expression pattern in the fruit juice sac with the transcriptome sequencing dada, including CrPAL1, CrPAL3, CrC4H1, CrC4H2, Cr4CL, CrCCR3, CrCAD3, CrPOD2 and CrPOD7. However, qRT-PCR results of the other 4 genes, including CrHCT1, CrCOMT4, CrPOD6 and CrPOD8, were slightly inconsistent with the transcriptome sequencing data at 4 developmental stages of pomelo fruit, but their relative expression levels were rapidly upregulated from 195 to 215 days after anthesis with significantly difference, which were consistent with the data. The enzyme activities of PAL, C4H, 4CL, CAD and POD related to lignin biosynthesis pathway were determined. The results showed that the activities of these 5 enzymes in the fruit juice sac were consistent with the expressions of the genes related to lignin biosynthesis pathway and the content of lignin during 4 different growth and development stages, indicating that these enzymes played an important role in lignin synthesis.【Conclusion】The juice sac granulation rate increased with the accumulation of lignin content, especially at the mature stage in Guanxi pomelo fruit. Nine genes in the pomelo juice sac including CrPAL1, CrPAL3, CrC4H1, CrC4H2, Cr4CL, CrCCR3, CrCAD3, CrPOD2 and CrPOD7 were the key regulatory genes of the lignin biosynthesis pathway. The significantly up-regulated expressions of these genes regulated the activity of important enzymes in lignin biosynthesis pathway, such as PAL, C4H, 4CL, CAD and POD, would promote the accumulation of lignin content in pomelo juice sacs and cause juice sac granulation of Guanxi pomelo. In the present study, the relationship between the expressions of the genes of juice sac lignin biosynthesis pathway and the juice sac granulation during the fruit development and maturation of Guanxi pomelo was systematically investigated at molecular and physiological levels, which would provide a theoretical basis for the variety improvement and molecular breeding of Guanxi pomelo in the future.