Cryptochrome 1 mediates blue light induction of 4-coumarate:CoA ligase-3 gene expression and flavonoids accumulation in Arabidopsis
Jianzhong Lin^1§, Xiaoying Zhao^1§, Dongying Tang¹, Ming Guo¹, Derong Liu¹, Xuanming Liu^1*, Chentao Lin³, Bin Liu^2*

¹College of Life Sciences, Hunan University, Changsha 410082, China.
²Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
³Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA.
^§These authors contribute equally.
^* Corresponding author. E-mail: xml05@126.com (X.L.), liu-bin2011@caas.net.cn (B.L.)

Abstract
Cryptochrome 1 (CRY1) is a blue light receptor regulating photomorphogenesis and flavonoid biosynthesis in Arabidopsis. The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in the biosynthesis of plant secondary compounds at divergence point from general phenylpropanoid metabolism to several major branch pathways. The involvement of 4CL in the lignin biosynthesis have been extensively studied, whereas the roles of 4CL in the flavonoid biosynthesis are remain largely unknown. In this study, we analyzed the transcription levels of 4CL family genes, 4CL1, 4CL2, 4CL3 and 4CL5, and the flavonoids content in the Arabidopsis photo receptor mutants. Our results showed that the 4CL3 expression and flavonoids accumulation were mainly induced by CRY1-mediated blue light signal transduction pathway and synergistically by CRY2. Then the conditional nuclear localization of CRY1 was investigated, and the results showed that the transcription levels of 4CL3 and the flavonoids content in 35S::CRY1-GR increased several times in the presence of Dexamethasone, indicating that the 4CL3 expression and flavonoid biosynthesis were induced by nuclear CRY1 rather than cytoplasmic CRY1. Moreover, the 4CL3 expression profile was found to be highly correlated with the chalcone synthase gene (CHS), a known key enzyme in flavonoid biosynthesis, suggesting that 4CL3 is mainly involved in the flavonoid biosynthesis rather than lignin biosynthesis. Compared with that of wild type, the flavonoids content was increased 3-5 times in the 4CL3 over-expressing transgenic plants and reduced 20-40% in the 4cl3 mutant. Therefore we propose that the 4CL3 is a major enzyme involved in the flavonoid biogenesis pathway regulated by nuclear CRY1 in response to blue light.

Key words: Cryptochrome; 4CL3; flavonoid; phenylpropanoid; Arabidopsis