Aspartic proteases gene family: Identification and expression profiles during stem vascular development in tobacco

Aspartic proteases (APs) constitute a large family in plants and are widely involved in diverse biological processes, like chloroplast metabolism, biotic and abiotic stress responses, and reproductive development. In this study, we focused on overall analysis of the APs genes in tobacco. Our analysis included the phylogeny and cis-elements in the cell wall-associated promoters of these genes. To characterize the expression patterns of APs genes in stem vascular development. The tissue expression analysis showed that NtAED3-like was preferentially expressed in the differentiating xylem and phloem cells of the vascular system. Based on histochemical staining analysis showed that the NtAED3-like gene was specifically expressed in stem vascular tissue, root vascular tissue, and petiole vascular tissue. The TdT-mediated dUTP nick-end labeling (TUNEL) assay illustrated a delayed progression of programmed cell death (PCD) within the xylem of the ko-ntaed3a-like mutant, relative to the wild type. The mutant ko-ntaed3a-like exhibited a phenotype of thinning stem circumference and changed in xylem structure and lignin content. In addition, the two-dimension heteronuclear single quantum coherent nuclear magnetic resonance (2D-HSQC) analysis of three milled wood lignins (MWLs) showed that the content of β-O-4 connection in ko-ntaed3a-like decreased slightly compared with wild type. In conclusion, this study provides our understanding of the regulation of vascular tissue development by the NtAED3-like gene in tobacco and provides a better basis for determining the molecular mechanism of the aspartic protease in secondary cell wall (SCW) development.