Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), INRAE USC1328, University of Orleans, 21 rue de Loigny la Bataille, F-28000 Chartres, France.
Bioactifs et Cosmétiques, CNRS GDR3711, CEDEX 2, 45067 Orléans, France.
Int J Mol Sci. 2020 Jul 2;21(13):4730. doi: 10.3390/ijms21134730.
Mature fruits (i.e., achenes) of milk thistle ( (L.) Gaertn., Asteraceae) accumulate high amounts of silymarin (SILM), a complex mixture of bioactive flavonolignans deriving from taxifolin. Their biological activities in relation with human health promotion and disease prevention are well described. However, the conditions of their biosynthesis in planta are still obscure. To fill this gap, fruit development stages were first precisely defined to study the accumulation kinetics of SILM constituents during fruit ripening. The accumulation profiles of the SILM components during fruit maturation were determined using the LC-MS analysis of these defined developmental phases. The kinetics of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and peroxidase (POX) activities suggest in situ biosynthesis of SILM from l-Phenylalanine during fruit maturation rather than a transport of precursors to the achene. In particular, in contrast to laccase activity, POX activity was associated with the accumulation of silymarin, thus indicating a possible preferential involvement of peroxidase(s) in the oxidative coupling step leading to flavonolignans. Reference genes have been identified, selected and validated to allow accurate gene expression profiling of candidate biosynthetic genes (, , , , and ) related to SILM accumulation. Gene expression profiles were correlated with SILM accumulation kinetic and preferential location in pericarp during fruit maturation, reaching maximum biosynthesis when desiccation occurs, thus reinforcing the hypothesis of an in situ biosynthesis. This observation led us to consider the involvement of abscisic acid (ABA), a key phytohormone in the control of fruit ripening process. ABA accumulation timing and location during milk thistle fruit ripening appeared in line with a potential regulation of the SLIM accumulation. A possible transcriptional regulation of SILM biosynthesis by ABA was supported by the presence of ABA-responsive cis-acting elements in the promoter regions of the SILM biosynthetic genes studied. These results pave the way for a better understanding of the biosynthetic regulation of SILM during the maturation of fruit and offer important insights to better control the production of these medicinally important compounds.
成熟的果实（即瘦果）含有高水平的水飞蓟素（SILM），这是一种源自栎精的生物活性类黄酮木脂素的复杂混合物。其与人类健康促进和疾病预防相关的生物学活性已有详细描述。然而，其在植物体内生物合成的条件仍不清楚。为了填补这一空白，首先精确定义了果实发育阶段，以研究在果实成熟过程中 SILM 成分的积累动力学。通过对这些定义的发育阶段进行 LC-MS 分析，确定了 SILM 成分在果实成熟过程中的积累谱。苯丙氨酸解氨酶（PAL）、查尔酮合酶（CHS）和过氧化物酶（POX）活性的动力学表明，SILM 是在果实成熟过程中由 l-苯丙氨酸原位合成的，而不是前体物质向瘦果的运输。特别是，与漆酶活性相反，POX 活性与水飞蓟素的积累相关，这表明过氧化物酶（s）可能参与了导致类黄酮木脂素的氧化偶联步骤。已经鉴定、选择和验证了参考基因，以允许对与 SILM 积累相关的候选生物合成基因（、、、、和）进行准确的基因表达谱分析。基因表达谱与 SILM 积累动力学和果皮中的优先位置相关，在果实干燥时达到最大生物合成，从而强化了原位生物合成的假说。这一观察结果使我们考虑了脱落酸（ABA）的参与，ABA 是控制果实成熟过程的关键植物激素。ABA 在水飞蓟果实成熟过程中的积累时间和位置与 SILM 积累的潜在调节一致。通过研究的 SILM 生物合成基因启动子区域中存在 ABA 反应顺式作用元件，支持了 ABA 对 SILM 生物合成的转录调节。这些结果为更好地理解果实成熟过程中 SILM 的生物合成调节铺平了道路，并为更好地控制这些具有药用重要性的化合物的生产提供了重要的见解。
