Functional Characterisation of the Poplar Atypical Aspartic Protease Gene PtAP66 in Wood Secondary Cell Wall Deposition

Secondary cell wall (SCW) deposition is an important process during wood formation. Although aspartic proteases (APs) have been reported to have regulatory roles in herbaceous plants, the involvement of atypical APs in SCW deposition in trees has not been reported. In this study, we characterised the Populus trichocarpa atypical AP gene PtAP66, which is involved in wood SCW deposition. Transcriptome data from the AspWood resource showed that in the secondary xylem of P. trichocarpa, PtAP66 transcripts increased from the vascular cambium to the xylem cell expansion region and maintained high levels in the SCW formation region. Fluorescent signals from transgenic Arabidopsis plant roots and transiently transformed P. trichocarpa leaf protoplasts strongly suggested that the PtAP66-fused fluorescent protein (PtAP66-GFP or PtAP66-YFP) localised in the plasma membrane. Compared with the wild-type plants, the Cas9/gRNA-induced PtAP66 mutants exhibited reduced SCW thickness of secondary xylem fibres, as suggested by the scanning electron microscopy (SEM) data. In addition, wood composition assays revealed that the cellulose content in the mutants decreased by 4.90–5.57%. Transcription analysis further showed that a loss of PtAP66 downregulated the expression of several SCW synthesis-related genes, including cellulose and hemicellulose synthesis enzyme-encoding genes. Altogether, these findings indicate that atypical PtAP66 plays an important role in SCW deposition during wood formation.