PpyNAC381 Transcriptionally Regulates Exocarp Suberization Of Russeted Sand Pear Fruit

Pyrus pyrifolia,also known as sand pear,originates from the Yangtze River basin and southern regions of China.It is one of the four pear species native to China,along with the Pyrus bretschneideri,Pyrus ussuriensis,and Pyrus sinkiangensis.Sand pear is known for its early fruiting,high yield,and strong adaptability to hot and humid climate conditions.However,sand pear fruit is susceptible to rust,which exhibited irregular and unevenly distributed brown patches in fruit skins and seriously affect its appearance and quality.Russeting resuls from the rupture of the epidermis and cuticle layers of the fruit skin and subsequent accumulation of suberized cells,and suberin is the main component of the russeted and semi-russeted sand pear skin.However,transcriptional regulation involved in russet formation of sand pear fruit is still limited and needs further study.This present study analyzed and compared the differences in fruit skin structure during sand pear fruit development among different cultivars,including rust-susceptible cultivars’Nijisseikki’ and ’Cuiguan’,and russet-free cultivars ’Cuiyu’ and ’Sucui NO.1’.Based on transcriptome sequencing data,we identified several differentially expressed PpyNAC transcription factors and analyzed their expression patterns during pear fruit development.Specifically,the study found that the expression of three NAC transcription factors,PpyNAC58,PpyNAC381,and PpyNAC382,and six suberin biosynthesis-related structural genes,PpyCYP86Al,PpyCYP86Bl,PpyHHT2/3,PpyFAR1,and PpyGPAT2,were upregulated in russet-susceptible cultivars and were correlated with the dynamic changes of russeting during fruit development.Promoter elements analysis revealed that all the selected six structural genes contained the cis-elements recognized by NAC transcription factors.Using a dual-luciferase reporter system,we found that PpyNAC58 and PpyNAC381 could transcriptionally activate the promoter of PpyGPAT2,reached 2.6-and 3.8-fold.Yeast one hybrid system revealed that PpyNAC381 could directly bind to the PpyGPAT2 promoter,thereby participating in suberin biosynthesis in pear fruit skin.The study further investigated the functions of PpyNAC381 and PpyGPAT2 by homologous transient over-expression.The results showed that overexpression of PpyNAC381 significantly increased the accumulation of fruit skin suberin monomers,such as fatty acids,α,ω-dicarboxylic acids,ω-hydroxy fatty acids,and alkanols,meanwhile overexpression of PpyGPAT2 significantly increased the accumulation of α,ω-dicarboxylic acids and co-hydroxy fatty acids.These findings suggest that PpyNAC381 and PpyGPAT2 involved in russet formation of sand pear fruit.In summary,this study identified a NAC transcription factor,PpyNAC381,which directly binds and activates the promoter of the suberin biosynthesis-related structural gene PpyGPAT2,promoting the accumulation of several suberin monomers and involved in russet formation of sand pear fruit.