SlSPL15: A Negative Regulator Targeted by SlmiR156a Participates in Regulating the Light-Induced Anthocyanin...

Anthocyanins are crucial compounds known for their antioxidant and health benefits. The Aft tomato (Solanum lycopersicum) line LA1996 can generate anthocyanins in fruits upon light exposure. Despite the identification of various regulatory genes, such as microRNAs and transcription factors involved in anthocyanin biosynthesis across different plant species, the function of the miR156/SPL module in Aft tomato fruit pigmentation is not well understood. In this research, 17 SlSPL family genes of Aft tomato were classified into six clades. SlSPL15 (Solyc10T002263.1) was grouped in Clade V, with AtSPL9, which is known to be involved in anthocyanin biosynthesis in Arabidopsis. Moreover, an inverse relationship between SlSPL15 and miR156a expression in mature green (MG) stage fruits was shown by quantitative RT-PCR analysis. Transient expression analysis of luciferase confirmed that SlSPL15 transcripts are degraded by SlmiR156a. Furthermore, overexpression of SlSPL15 in Aft tomato reduced the anthocyanin accumulation in MG fruits. In SlSPL15 overexpressed tomato plants, the transcription level of SlSPL15 was elevated compared to that in wild-type fruits, whereas the expression of SlPAL, SlCHS1, SlDFR, SlF3H, SlF3'5'H, and SlANS was reduced. Additionally, the expression of light-responsive regulatory genes SlHY5, SlAN2-like, and SlMYB12 in the anthocyanin biosynthetic pathway was also reduced in light-exposed fruits of 35S:SlSPL15 plants. Subcellular localization analysis verified that SlSPL15 is localized in the nucleus, while yeast two-hybrid assays demonstrated its interaction with SlAN2-like, a part of the MBW complex that participates in regulating anthocyanin biosynthesis in Aft tomato fruits. The findings expand our comprehension of the roles of SlSPL15, targeted by SlmiR156a, in regulating light-induced anthocyanin accumulation in tomatoes.