Hypericum perforatum
, also known as “natural fluoxetine,” is a commonly used herbal remedy for treating depression. It is unclear whether melatonin in plants regulated by the endogenous circadian clock system is like in vertebrates. In this work, we found that the melatonin signal and melatonin biosynthesis gene, serotonin
N
-acetyltransferase
HpSNAT1
, oscillates in a 24-hour cycle in
H. perforatum
. First, we constructed a yeast complementary DNA library of
H. perforatum
and found a clock protein HpLHY that can directly bind to the
HpSNAT1
promoter. Second, it was confirmed that HpLHY inhibits the expression of
HpSNAT1
by targeting the Evening Element. Last, it indicated that
HpLHY
-overexpressing plants had reduced levels of melatonin in 12-hour light/12-hour dark cycle photoperiod, while loss-of-function mutants exhibited high levels, but this rhythm seems to disappear as well. The results revealed the regulatory role of LHY in melatonin biosynthesis, which may make an important contribution to the field of melatonin synthesis regulation.