Table_3_In Silico Genome-Wide Analysis of the Pear (Pyrus bretschneideri) KNOX Family and the Functional Characterization of PbKNOX1, an Arabidopsis BREVIPEDICELLUS Orthologue Gene, Involved in Cell Wall and Lignin Biosynthesis.xlsx

Stone cells are a characteristic trait of pear fruit, but the contents and sizes of stone cells negatively correlate with fruit texture and flavor. Secondary cell wall thickening and lignification have been established as key steps of stone cell development. KNOTTED-LIKE HOMEOBOX (KNOX) proteins play important roles in plant cell growth and development, including cell wall formation and lignification. Although the characteristics and biological functions of KNOX proteins have been investigated in other plants, this gene family has not been functionally characterized in pear. Eighteen PbKNOX genes were identified in the present study, and all of the identified family members contained the KNOX I and/or KNOX II domains. Based on the phylogenetic tree and chromosomal localization, the 18 PbKNOX genes were divided into five subfamilies [SHOOT MERISTEMLESS (STM)-like, BREVIPEDICELLUS (BP)-like, KNOTTED ARABIDOPSIS THALIANA 2/6 (KNAT2/6)-like, KNAT7-like, and KNAT3-5-like] and were distributed among 10 chromosomes. In addition, we identified 9, 11, and 11 KNOX genes in the genomes of grape, mei, and strawberry, respectively, and the greatest number of collinear KNOX gene pairs formed between pears and peaches. Analyses of the spatiotemporal expression patterns showed that the tissue specificity of PbKNOX gene expression was not very significant and that the level of the PbKNOX1 transcript showed an opposite trend to the levels of stone cells and lignin accumulation. Furthermore, PbKNOX1 has high sequence identity and similarity with Arabidopsis BP. Compared with wild-type Arabidopsis, plants overexpressing PbKNOX1 not only showed an approximately 19% decrease in the secondary cell wall thickness of vessel cells but also exhibited an approximately 13% reduction in the lignin content of inflorescence stems. Moreover, the expression of several genes involved in lignin biosynthesis was downregulated in transgenic lines. Based on our results, PbKNOX1/BP participates in cell wall-thickening and lignin biosynthesis and represses the transcription of key structural genes involved in lignin synthesis, providing genetic evidence for the roles of KNOX in cell wall thickening and lignin biosynthesis in pear.

石细胞是梨果的特征性状，但石细胞的含量与体积与果实质地和风味呈负相关。次生细胞壁增厚与木质化已被证实为石细胞发育的关键步骤。KNOX蛋白（KNOTTED-LIKE HOMEOBOX）在植物细胞生长发育（包括细胞壁形成与木质化）中发挥重要作用。尽管KNOX蛋白的特征与生物学功能已在其他植物中得到研究，但该基因家族在梨中的功能特征尚未被阐明。本研究共鉴定出18个梨PbKNOX基因，所有家族成员均包含KNOX I和/或KNOX II结构域。基于系统发育树与染色体定位分析，这18个PbKNOX基因可划分为5个亚家族：类SHOOT MERISTEMLESS（STM）、类BREVIPEDICELLUS（BP）、类KNOTTED ARABIDOPSIS THALIANA 2/6（KNAT2/6）、类KNAT7以及类KNAT3-5，且这些基因分布于10条染色体上。此外，本研究分别在葡萄、梅花和草莓的基因组中鉴定出9、11和11个KNOX基因，其中梨与桃之间形成的共线性KNOX基因对数量最多。时空表达模式分析显示，PbKNOX基因的表达组织特异性并不显著，其中PbKNOX1的转录本水平与石细胞含量及木质素积累量呈相反的变化趋势。进一步研究发现，PbKNOX1与拟南芥BP的序列一致性和相似性均较高。与野生型拟南芥相比，过表达PbKNOX1的植株不仅表现出导管细胞次生细胞壁厚度约19%的降低，同时花序茎的木质素含量也下降了约13%。此外，转基因株系中多个参与木质素生物合成的关键基因的表达量均被下调。综上，PbKNOX1/BP参与细胞壁增厚与木质素生物合成过程，并抑制木质素合成关键结构基因的转录，本研究为KNOX家族在梨的细胞壁增厚与木质素生物合成中的作用提供了遗传证据。