Investigation of PtSGT1 and PtSGT4 Function in Cellulose Biosynthesis in Populus tomentosa Using CRISPR/Cas9 Technology.

Yinxuan Xue,Siyan Li,Sai Huang,Bin Guo,Xin-Min An,Shanwen Li,Deyu Miao

doi:10.3390/ijms222413200

Abstract

Cellulose synthesis is a complex process in plant cells that is important for wood processing, pulping, and papermaking. Cellulose synthesis begins with the glycosylation of sitosterol by sitosterol glycosyltransferase (SGT) to produce sitosterol-glucoside (SG), which acts as the guiding primer for cellulose production. However, the biological functions of SGTs in Populus tomentosa (P. tomentosa) remain largely unknown. Two full-length PtSGT genes (PtSGT1 and PtSGT4) were previously isolated from P. tomentosa and characterized. In the present study, CRISPR/Cas9 gene-editing technology was used to construct PtSGT1-sgRNA and PtSGT4-sgRNA expression vectors, which were genetically transformed into P. tomentosa using the Agrobacterium-mediated method to obtain transgenic lines. Nucleic acid and amino acid sequencing analysis revealed both base insertions and deletions, in addition to reading frame shifts and early termination of translation in the transgenic lines. Sugar metabolism analysis indicated that sucrose and fructose were significantly downregulated in stems and leaves of mutant PtSGT1-1 and PtSGT4-1. Glucose levels did not change significantly in roots and stems of PtSGT1-1 mutants; however, glucose was significantly upregulated in stems and downregulated in leaves of the PtSGT4-1 mutants. Dissection of the plants revealed disordered and loosely arranged xylem cells in the PtSGT4-1 mutant, which were larger and thinner than those of the wild-type. This work will enhance our understanding of cellulose synthesis in the cell walls of woody plants.

Highlights

Cell walls are important components of plant cells responsible for their characteristic structures
Following amplification with primers PtSGT1F, PtSGT1R, PtSGT4F, and PtSGT4R, a 1.9-kb fragment was obtained by 1.5% agarose gel electrophoresis using P. tomentosa cDNA as the template (Figure 1)
PtSGT1 was thought to be located in the chloroplast, while PtSGT4 may be in the cytoplasm

Summary

Introduction

Cell walls are important components of plant cells responsible for their characteristic structures. Sterol biosynthesis occurs through the mevalonate and non-mevalonate pathways [1], which have highly diverse skeletal and glycosylated structural forms. Sterols and their derivatives are involved in the structure of cell membranes [2], and act as precursors for hormone and vitamin biosynthesis [3]. Other important cellular functions of sterols include anti-stress and antiheat shock actions, signal transduction, and protection of bacteria from the host immune response [4]. Some sterols, such as sitosterol, are ubiquitous in plants [5]

Methods

Results

Conclusion