Expression of the SPL-like gene LaSPL9 in Japanese larch (Larix leptolepis) is regulated by miR156 during somatic embryogenesis

Abstract

The Larix leptolepis SPL-like gene LaSPL9 was identified. Additionally, premiR156b and LaSPL9 expression patterns during somatic embryogenesis and the effects of gibberellin and salicylic acid on their transcription revealed the post-transcriptional regulatory functions of the miR156–LaSPL9 module. A full-length cDNA sequence of a SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) gene was isolated from Japanese larch (Larix leptolepis) and designated as LaSPL9. The cDNA comprised 2735 bp, including a 1779-bp open reading frame encoding a protein (592 amino acids) with conserved SBP domains. The LaSPL9 promoter region included several gibberellin (GA)- and salicylic acid (SA)-inducible elements. Further analyses revealed that LaSPL9 mRNA is targeted by miR156. An examination of the LaSPL9-GFP fusion protein in tobacco leaf epidermal cells indicated that LaSPL9 is a nuclear protein. Additionally, LaSPL9 expression was induced by GA and the miR156b precursor (premiR156b) transcript accumulation was inhibited by SA. Moreover, abscisic acid (ABA) was identified as the main factor down-regulating LaSPL9 expression during the early stage of somatic embryogenesis (SE), which can induce premiR156b expression. Furthermore, LaSPL9 transcription peaked at 10 days, implying it might encode an important regulator of early embryonic pattern formation. The LaSPL9 transcript level subsequently decreased gradually and was lowest at 42 days, which is consistent with the gradual increase in miR156 expression during SE. The miR156–LaSPL9 module might regulate SE in L. leptolepis by responding to ABA. Overall, our results provide evidence that miR156–LaSPL9 is important for the crosstalk between GA and SA pathways during SE.