Identification of pear BAHD superfamily genes and screening of stone cell differentiation related members

Abstract:【Objective】Kuerlexiangli pear (Pyrus sinkiangensis Yü) is a native species of Xinjiang Au- tonomous Region，northwest China. The fruit is known for its unique aroma, juicy flesh and crisp tex- ture. The formation of rough-skin has been affecting external and internal quality of Kuerlexiangli pear fruits, and becoming more common in recent years. With regular fruits, the stone cells are unperceiv- able. The rough-skin, also called“hard-end”, is a physiological disorder, in which mild symptoms can be observed only at the calyx end of pear fruits. But in severe cases, most of the fruit surface can be cov- ered with rough-skin. The increased stone cell content is one of the main factors that cause the forma- tion of rough-skinned fruits at later stage of fruit development. However, the molecular mechanisms of differentiation of stone cells and formation of rough-skin on pear fruits are still not fully understood. The BAHD superfamily genes participate in many biological processes through the modification of me- tabolites during plant metabolism, including lignin biosynthesis. The lignin is one the main components of stone cells which commonly present in fruits of all Pyrus species. Therefore, the aim of this study is to systemically investigate BAHD superfamily genes on pear genome, and thoroughly analyze their ex- pression profiles at key stages of stone cell differentiation during pear fruit development and in rough- skinned phenotype, so as to obtain the potential BAHD members that are related to the stone cell differ- entiation in pear fruits. 【Methods】The pfam structural model of transferase family proteins (PF02458) and HCT genes from model plants were used as references to identify the BAHD family genes in the pear genome. Based on conserved domains of this superfamily (HXXXD and DFGWG), the BAHD members were reconfirmed. Then the transcriptome analysis were performed on the fruit samples that were collected at three developmental stages (20, 50 and 80 days after flowering, DAF), representing initial, late and stationary stage of stone cell differentiation, respectively, and on rough-skinned and nor- mal fruits at mature stage to identify the members of BAHD superfamily genes that may participate in the process of the stone cell differentiation in Kuerlexiangli pear fruits. 【Results】A total of 92 transfer- ase family genes were identified on the genome of Pyrus bretschneideri‘Dangshansu’, among which the 81 genes contained the typical conserved motifs of BAHD superfamily. These genes were unevenly distributed on the 15 chromosomes of P. bretschneideri. The duplication analysis showed that among the pear BAHD superfamily genes, 14 of which were located in tandem on same chromosomes, and 15 of which had collinearity on the pear genome. The transcriptome analysis showed that 19 BAHD super- family genes were expressed in Kuerlexiangli pear fruits with higher abundance, and they were also dif- ferentially expressed at key stages of stone cell differentiation in Kuerlexiangli pear fruits. Among of them, four lignin synthetic genes (LOC103945380, LOC103964288, LOC103955861 andLOC103945376) were up- regulated at critical period of stone cell differentiation, and two of them (LOC103955861 and LOC103945376) were also up-regulated in rough-skinned fruits at mature stage; one gene (LOC103944254) related to the coumarin biosynthesis was up-regulated at the initial stage of stone cell differentiation and in rough-skinned fruits; one gene (LOC103954416) encoded the enzyme that participated in the synthesis of long chain fatty acids of cuticular wax, which was up-regulated in samples from 20 DAF and rough- skinned fruits; six genes (LOC103949999, LOC103944336,LOC103949890, LOC103951983, LOC103951985 and LOC103938506) encoded suberin synthetic en- zymes, all of them were up-regulated at least one critical stage of stone cell differentiation during fruit development, and two of them (LOC103951983 and LOC103951985) were up- regulated in rough- skinned fruits; two genes (LOC103955585 and LOC103963251) encoded the enzymes that related to the synthesis of green leaf volatile (GLV), and both of them were up-regulated during the fruit develop- ment and in rough- skinned phenotype; other four genes (LOC103961715, LOC103963031,LOC103958630 and LOC103945860) encoded the modifying enzymes of brassinolide and flavonoids.【Conclusion】The differentially expressed members of BAHD superfamily genes with higher abun- dance in Kuerlexiangli pear fruits not only participated in the process of synthesizing lignin, coumarin, cuticular wax and suberin to promote the differentiation of stone cells and the formation of rough-skin, but also encoded the modifying enzymes of flavonoids and volatile compounds. Lignin, coumarin, cutic- ular wax and suberin are the main components of secondary cell walls and epicarp of pear fruits. The flavonoids and volatiles are crucial for the regulation or maintaining the proper amount of reactive oxy- gen species (ROS) in pear fruits that are necessary for the oxidative linkage of cell wall monomers dur- ing the secondary thickening of cell walls of parenchyma cells to form the stone cells and rough-skin. In sum, the results of this study illustrate that some BAHD superfamily candidates participate in the formation of stone cells in pear fruits, which to some extend enlightens the fact that the formation of rough- skin is induced by abiotic stresses.