Objective To reveal LaSPL2 and LaSPL3 function during somatic embryogenesis, based on their molecular characteristics and expression patterns.

Method The full-length cDNA sequences of LaSPL2 and LaSPL3 were obtained by homologous cloning and RACE techniques. The amino acid sequences for known SPL family proteins were aligned using the ClustalX 2.1 software. The full-length protein sequence alignment was used to construct a phylogenetic tree according to the neighbor-joining method of the MEGAX program. Subcellular localization was performed by transient expression system of tobacco. The expression patterns of LaSPL2 and LaSPL3 during somatic embryogenesis were detected by qRT-PCR.

Result In this study, two SPL homologous genes, LaSPL2 and LaSPL3, were isolated from Japanese larch, which encoded 532 amino acids and 191 amino acids respectively. Further analyses revealed that LaSPL2 and LaSPL3 mRNA were targeted by miR156. The deduced amino acid sequences of the LaSPL2 and LaSPL3 harbored a highly conserved SBP domain. A transient expression analysis of the LaSPLs-GFP fusion proteins in tobacco leaf epidermal cells revealed that the LaSPL2 and LaSPL3 proteins were localized in the nucleus. Moreover, the expression of LaSPL2 and LaSPL3 was down regulated by ABA during the early stage of somatic embryogenesis. With the further development of somatic embryos, the expression levels of LaSPL2 and LaSPL3 peaked at 10 and 14 days, respectively. Subsequently, with the maturation of somatic embryos, their transcription levels decreased gradually to the lowest level at 42 days.

Conclusion The effects of ABA removal on the expression of LaSPL2 and LaSPL3 indicate that ABA might be the main factor in their down-regulation during the early stage of somatic embryogenesis. Furthermore, the expression of LaSPL2 and LaSPL3 will reach the peak at the early stage of somatic embryogenesis, implying it might encode an important regulator of early embryonic pattern formation. The sequence analysis and expression patterns of LaSPL2 and LaSPL3 indicated that they may be regulated by miR156 during somatic embryogenesis and play an important role in the maturation of somatic embryos.