Identification of functional single nucleotide polymorphism of Populus trichocarpa PtrEPSP‐TF and determination of its transcriptional effect

Abstract

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that Populus trichocarpa PtrEPSP‐TF, an isoform of 5‐enolpyruvylshikimate 3‐phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in Populus. In this study, we report the identification of single nucleotide polymorphism (SNP) of PtrEPSP‐TF that defines its functionality. Populus natural variants carrying this SNP were shown to have reduced lignin content. Here, we demonstrated that the SNP‐induced substitution of 142nd amino acid (PtrEPSP‐TFD142E) dramatically impairs the DNA‐binding and transcriptional activity of PtrEPSP‐TF. When introduced to a monocot species rice (Oryza sativa) in which an EPSP synthase isoform with the DNA‐binding helix‐turn‐helix (HTH) motif is absent, the PtrEPSP‐TF, but not PtrEPSP‐TFD142E, activated genes in the phenylpropanoid pathway. More importantly, heterologous expression of PtrEPSP‐TF uncovered five new transcriptional regulators of phenylpropanoid biosynthesis in rice. Collectively, this study identifies the key amino acid required for PtrEPSP‐TF functionality and provides a strategy to uncover new transcriptional regulators in phenylpropanoid biosynthesis.

Accelerating wood domestication in forest trees through genome editing: Advances and prospects

“…However, the contribution of individual SNPs to the overall phenotype is often limited, and highly depends on the genotype and the environment [90]. Regardless of their small effects, a GWAS does identify genes that play a causal role in establishing the trait [91][92][93][94]. Through the use of multiplex genome editing in elite germplasm, a large number of novel allelic variants at multiple QTLs can be generated, as exemplified in a model maize variety for yield genes [95].…”

Lignin engineering in forest trees: From gene discovery to field trials

“…Another strategy for steering metabolic flux in the lignin biosynthesis pathway is via transcriptional regulation. 5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) is a key enzyme in the shikimate biosynthetic pathway and has recently been identified as a transcriptional regulator of the phenylpropanoid pathway in poplar ( Xie et al., 2018 , 2020 ; Yao et al., 2021 ). The transcriptional regulatory functionality of EPSPS is defined by a single-nucleotide polymorphism (SNP), and natural poplar variants carrying this SNP have reduced lignin content (the amount of lignin reduction and the biomass yield were not reported) ( Xie et al., 2018 , 2020 ).…”

“…5-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) is a key enzyme in the shikimate biosynthetic pathway and has recently been identified as a transcriptional regulator of the phenylpropanoid pathway in poplar ( Xie et al., 2018 , 2020 ; Yao et al., 2021 ). The transcriptional regulatory functionality of EPSPS is defined by a single-nucleotide polymorphism (SNP), and natural poplar variants carrying this SNP have reduced lignin content (the amount of lignin reduction and the biomass yield were not reported) ( Xie et al., 2018 , 2020 ). Another example in which an allelic form of a transcription factor results in a reduced lignin content is the expression of a phosphorylation-null mutant of LIGNIN BIOSYNTHESIS-ASSOCIATED TRANSCRIPTION FACTOR 1 (LTF1) in poplar ( Gui et al., 2019 , 2020 ).…”

“…Genome-wide association studies (GWAS) are of particular interest because GWAS can be used to identify genes/alleles that are causal for a specific trait in a population ( Fahrenkrog et al., 2017 ). For instance, association genetics in different poplar species has revealed associations between lignin amount or composition and genes encoding β-TUBULIN 15, CCoAOMT1, an FAD-binding domain-containing protein, ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) domain 11, the KANADI transcription factor, and EPSPS ( Guerra et al., 2013 ; Porth et al., 2013 ; Muchero et al., 2015 ; Xie et al., 2018 , 2020 ; Yao et al., 2021 ). The roles of CCoAOMT1 and EPSPS in lignification have been validated by reverse genetics ( Meyermans et al., 2000 ; Zhong et al., 2000 ; Xie et al., 2020 ).…”

“…For instance, association genetics in different poplar species has revealed associations between lignin amount or composition and genes encoding β-TUBULIN 15, CCoAOMT1, an FAD-binding domain-containing protein, ADP-ribosylation factor (ARF) GTPase-activating protein (GAP) domain 11, the KANADI transcription factor, and EPSPS ( Guerra et al., 2013 ; Porth et al., 2013 ; Muchero et al., 2015 ; Xie et al., 2018 , 2020 ; Yao et al., 2021 ). The roles of CCoAOMT1 and EPSPS in lignification have been validated by reverse genetics ( Meyermans et al., 2000 ; Zhong et al., 2000 ; Xie et al., 2020 ). Further reverse genetics support is needed to validate the roles of β-TUBULIN 15, the FAD-binding domain-containing protein, ARF-GAP domain 11, and KANADI in lignin biosynthesis.…”