酚酸和类黄酮积累对古侧柏扦插愈伤组织木质化的影响分子机制

Stem cuttings are widely used to preserve endangered ancient tree even if they exhibit excellent longevity and stress resistance. However, a serious decrease in adventitious root (AR) formation in stem cuttings of old trees. Phenolics and flavonoids accumulation is a major factor that causes the callus lignification instead of rooting. To date, the molecular mechanisms regulating the effects of aging and lignification on AR formation have not been characterized. Then, we compared the proteomic changes during adventitious root (AR) formation of cuttings from 5-, 100-, and 700-year-old Platycladus orientalis cuttings using iTRAQ-based workflows. Based on the metabolites pathways, proteins in phenylalanine metabolism, flavonoid, and phenylpropanoid biosynthesis pathways were affected in 100-, and 700-year-old donors. Consistently, the contents of secondary metabolites such as flavonoids, phenolics and lignin were implicated in callus lignification in 100-, and 700-year-old donors. On this basis, tissue sections showed that the callus of old tree cuttings was heavily lignified, and secondary metabolites including lignin, phenolics, and flavonoids content were detected and significantly higher than 5-year-old cuttings. The relationship among DEPs of accumulation of phenolics and flavonoids, tree age, and rooting factors were comprehensively investigated. Therefore, the effects of phenolics and flavonoids accumulation on the callus lignification of P. orientalis cuttings and their molecular physiology mechanisms were elucidated. Our findings provide new insights regarding calli instead of roots, which may be relevant for enhancing the rooting of ancient trees.

古树树势日益衰弱，扦插繁殖对保存古树优良遗传资源意义重大，然而随着树龄的增加扦插生根率严重减少。酚酸和类黄酮在古树中积累并造成愈伤组织木质化抑制不定根形成，但其机制尚缺乏研究。本项目已经利用iTRAQ技术测定了5年、100年和700年古侧柏扦插过程中3个不定根形成的阶段，发现次生代谢相关的苯丙氨酸代谢，类黄酮和苯丙素生物合成途径在100和700年古侧柏的插穗不定根形成过程中显著上调。在此基础上组织切片发现古树插穗愈伤组织木质化严重，并测定次生代谢产物发现木质素、酚酸、类黄酮含量显著高于5年生插穗。从而阐明酚酸和类黄酮积累对古侧柏扦插愈伤组织木质化的影响及其分子生理机制，最终为提高古树扦插生根率奠定基础。

古树树势日益衰弱，扦插繁殖对保存古树优良遗传资源具有重要意义，然而随着树龄的增加扦插生根率严重减少。酚酸和类黄酮在古树中积累并造成愈伤组织木质化，从而影响古树扦插不定根形成，但其机制尚缺乏研究。本项目利用5年、100年和700年古侧柏扦插过程中3个不定根形成的阶段的材料进行蛋白质组测序和代谢组分析，蛋白质组测序发现次生代谢相关的苯丙氨酸代谢，类黄酮和苯丙素生物合成途径在100和700年古侧柏的插穗不定根形成过程中显著上调。在此基础上组织切片发现古树插穗愈伤组织木质化严重，并测定次生代谢产物发现木质素、酚酸、类黄酮含量显著高于5年生插穗。代谢组测序共检测到7244种代谢产物，对代谢物表达量分析，5年、100年、700年的的不同生根阶段的代谢物变化明显不同，表明在第二阶段是愈伤组织形成时期，其响应机制方面有显著不同。通过分析也能够发现同一生根阶段中5年、100年、700年的部分代谢物的表达量也发生了明显变化。对黄酮类合成进行关联分析，表明代谢物质绿原酸、花青素、二氢杨梅素、圣草酚、木犀草素、柚皮素查耳酮、柚皮素、山奈酚、儿茶素、杨梅素、高圣草酚等以及与查耳酮合酶。研究在愈伤组织木质化中的表达机制及其调控网络，综合探讨酚酸和类黄酮积累与代谢相关差异蛋白、代谢物、生根因子三者间的关系。从而阐明酚酸和类黄酮积累对古侧柏扦插愈伤组织木质化的影响及其分子生理机制，最终为提高古树扦插生根率奠定基础。