Gene inactivation of SmHQT simultaneously improved the browning tolerance and the accumulation of anthocyanins and flavonols of eggplant fruit

The biosynthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) are important enzymes in these pathways. However, the spatiotemporal expression patterns of SmHQT and its relationship to the CGA biosynthesis in eggplant remain largely unclear. In this study, 89 BAHDs were identified from the eggplant genome using PF02458 (transferase) as Hidden Markov Model (HMM) to screen the BAHDs and divided into 7 subfamilies according to the phylogenetic tree. Six eggplant acyltransferases clustered in a subgroup of Clade Ib, clustered with StHQT. We examined the expression patterns of 6 BAHDs in clade Ib in 14 eggplant tissues, and screened out SmHQT for further study. RNAi suppression of SmHQT resulted in over a 90% reduction in CGA, but no change in antioxidant activity suggests that flux was rerouted into other phenylpropanoids. LCMS results showed that flavonols were significantly increased in young leaves, mature leaves, flesh and peel, and anthocyanins were significantly increased in leaves and peel. The genotypes of knockout were chimeric, suggesting that our multiplex gene knockout CRISPR/Cas9 system worked in later stages of plant regeneration. Collectively, those results showed that CGA in eggplant is synthesized through SmHQT, and SmHQT suppression redirected phenylpropanoid flux and altered phenotype.

绿原酸（CGA）在植物体内可通过多条不同途径完成生物合成，而羟基肉桂酰辅酶A:奎宁酸羟基肉桂酰转移酶（HQT）是这些途径中的关键酶。然而，茄子中SmHQT的时空表达模式及其与CGA生物合成的关联仍未完全阐明。
本研究以隐马尔可夫模型（HMM）PF02458（转移酶结构域）为检索工具，从茄子基因组中鉴定得到89个BAHD家族酰基转移酶，并基于系统发育树将其划分为7个亚家族。其中6个茄子酰基转移酶聚类于进化枝（Clade）Ib的一个亚组中，并与StHQT聚为一类。
我们对进化枝Ib亚家族中的6个BAHD家族基因在茄子14种组织中的表达模式进行了检测，并筛选出SmHQT用于后续研究。对SmHQT进行RNA干扰（RNAi）抑制后，CGA含量下降超过90%，但植株抗氧化活性未发生变化，这表明代谢流被重定向至其他苯丙烷类代谢途径。液相色谱-质谱联用（LC-MS）结果显示，幼叶、成熟叶、果肉和果皮中的黄酮醇含量显著升高，而叶片和果皮中的花青素含量显著增加。
基因敲除株系为嵌合体型，这表明本研究构建的多基因敲除成簇规律间隔短回文重复序列相关蛋白9（CRISPR/Cas9）系统可在植物再生后期发挥功能。综上，上述结果证实茄子中的CGA通过SmHQT催化合成，且SmHQT的表达抑制可重定向苯丙烷类代谢流并改变植株表型。