Overexpression of RgPAL family genes involved in phenolic biosynthesis promotes the replanting disease development in Rehmannia glutinosa
Introduction
Rehmannia glutinosa, a species of the Orobanchaceae family, is a perennial herbaceous plant. Its tuberous roots contain a number of pharmacologically active compounds that are used in traditional Chinese medicine (Wen et al., 2002). However, R. glutinosa is also affected by the autotoxic effect (so-called replanting disease), which is defined as the direct/indirect harm of replanted plants via the production of allelochemicals that escape into the environment (Weston et al., 2012; Li et al., 2017a; Wu et al., 2018). As such the continuous monoculture of R. glutinosa leads to severe negative influences on both plant health and rhizosphere soils, resulting in a severe decline in both the biomass and quality of belowground tuber roots (Li et al., 2012; Zhang et al., 2016, 2018, 2020; Li et al., 2017a). Root exudates are perceived as chemical signals of communication between roots and microorganisms (Wu et al., 2018; Ding et al., 2019; Zhang et al., 2020). A growing body of evidence suggests that autotoxic harm to plants mediated by root exudates plays a crucial role in the replanting disease development (Ghimire et al., 2019; Li et al., 2020a; Zhang et al., 2018, 2020). Some endogenous phenolics that are important root exudates are involved in allelopathy and are prone to directly or indirectly causing autotoxicity, resulting in the replanting disease in some plant species (Ghimire et al., 2019; Zhang et al., 2020). Several phenolic compounds (e.g., 4-hydroxybenzoic acid, p-coumaric acid, ferulic acid and caffeic acid) have been identified as allelopathic autotoxic agents in R. glutinosa and other plant species (Loffredo et al., 2005; Li et al., 2012; Wu et al., 2015). Many of these compounds are derivatives of cinnamic acid and are synthesized via the phenolics/phenylpropanoid pathway (Ferrer et al., 2008; Huang et al., 2010).
Phenylalanine ammonia-lyase is the first enzyme of the phenolics/phenylpropanoid pathway, catalysing the conversion of l-phenylalanine to trans-cinnamic acid, a reaction considered to represent a key point at which carbon flux into this pathway is controlled (Jiang et al., 2013; Shi et al., 2013; Christopoulos and Tsantili, 2015; Nag and Kumaria, 2018). Following the reaction steps of the phenylpropanoid pathway, trans-cinnamic acid serves as the raw material that is eventually converted to other phenolic acids or their derivatives by the catalysis of a series of enzymes, such as 4-coumarate-CoA ligase (4CL), cinnamate 4-hydroxylase (C4H), p-coumarate-3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), hydroxycinnamoyl-CoA quinate hydroxycinnamoyl (CQT), cinnamyl alcohol dehydrogenase (CAD), and phenylcoumaran benzylic ether reductase (PCBER) (Ferrer et al., 2008; Rawal et al., 2013; Barros and Dixon, 2019). Due to the importance of PAL for phenolics/phenylpropanoid biosynthesis, PAL genes are generally well studied and are commonly found composing a small multigene family in many plant species (Reichert et al., 2009; Huang et al., 2010; Hui et al., 2010; Rawal et al., 2013; Fang et al., 2020). To date, several PAL family genes have been identified and functionally characterized in various species, including Arabidopsis thaliana (Huang et al., 2010), Populus trichocarpa (Shi et al., 2013), Scutellaria baicalensis (Hui et al., 2010), rice (Fang et al., 2013, 2020) and Juglans regia (Yan et al., 2019). A number of studies have established correlations between PAL and phenolic biosynthesis (Xiong et al., 2010; Fang et al., 2013; Jong et al., 2015). However, to date, only one RgPAL1 gene from R. glutinosa has been identified (Lee et al., 2003); information about the characterization of PAL family genes and their function in the phenolics/phenylpropanoid pathway is still limited. The molecular isolation and functional characterization of PAL family genes of R. glutinosa are therefore important steps for elucidating phenolic biosynthesis pathways.
Importantly, many PAL family genes mediate various plant biotic and abiotic stresses, such as fungal infection, tissue wounding, low temperature, salinity, and allelopathic phenolics, by being involved in the phenolics/phenylpropanoid biosynthesis pathway (Fang et al., 2013; Christopoulos and Tsantili, 2015; Nag and Kumaria, 2018; Gho et al., 2019; Yan et al., 2019). In particular, previous studies have shown that OsPAL functions in rice as a positive regulator of allelopathic potential by being involved in phenolic biosynthesis (He et al., 2005; Fang et al., 2013, 2020; Li et al., 2020b). However, it is still unclear whether RgPAL family genes regulate the allelopathic autotoxic formation of R. glutinosa exposed to continuous monoculture stress. A thorough understanding of RgPAL function in terms of allelopathic potential is therefore important for elucidating the mechanisms underlying the development of the replanting disease.
In this study, four sequences from the R. glutinosa transcriptome were investigated, and putative RgPAL family genes whose complete open reading frame (ORF) sequences have been isolated and characterized were identified. Their basic function in phenolic biosynthesis was determined by their overexpression in R. glutinosa. To explore the regulatory roles of RgPAL involvement in the replanting disease formation, we assessed the amount of allelopathic phenolics released from the roots of RgPAL-overexpressing transgenic seedlings in vitro and determined the physio-biochemical and molecular characteristics of the transgenic plants under continuous monoculture stress, laying a foundation for the clarification of the molecular mechanism responsible for the disease development.
Section snippets
Isolation of RgPAL cDNAs
The R. glutinosa cultivar “Wen 85-5” was cultured at the Wen Agricultural Institute, Jiaozuo City, Henan Province, China. To clone gene sequences, R. glutinosa roots were sampled at the root elongation stage (60 days after seedling emergence). Total RNA from R. glutinosa was isolated using TRIzol™ reagent (Invitrogen, Carlsbad, USA) according to the manufacturers’ instructions. A Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA) instrument was used to determine the RNA concentration. A
Isolation and sequence analysis of the RgPAL genes
Based on the R. glutinosa transcriptomic data, four putative sequences were annotated and exhibited to possess high homology to the species’ RgPAL1 and other plant species’ PALs; these sequences were named RgPAL2 to RgPAL5. The complete cDNA sequences of 2,182-2,321 bp in length were isolated via PCR and verified by sequencing. The RgPAL genes were inputted into the NCBI GenBank database (Assession number MT780187-MT780187). The RgPAL proteins ranged in length from 702 to 711 amino acids, with
Characterization of RgPAL family genes in R. glutinosa
PAL is the first enzyme that catalyses the deamination of l-phenylalanine into trans-cinnamic acid in the phenolics/phenylpropanoid pathway (Tsai et al., 2006; Barros et al., 2019). PAL family genes have been functionally characterized in many plant species (Reichert et al., 2009; Rawal et al., 2013; Nag and Kumaria, 2018). Our study identified four novel full-length RgPAL (i.e., RgPAL2-RgPAL5) cDNAs from R. glutinosa. Sequence analysis revealed that the four RgPALs have retained the
Author contribution statement
YYH: designed the research, performed data curation and wrote the manuscript. WCJ and YH: performed the experiments. LRF and ZZY: revised the manuscript. CCY and LJT: performed in silico modelling. All authors have read and agreed to the published version of the manuscript.
Funding
This research was supported by grants from the National Natural Science Foundation of China (No. 81973417), the Key Research and Development Special Project of Henan Province (No. 182102310606), the Science Foundation of Henan University of Technology (No. 2017RCJH05), the Program for Innovative Research Team (in Science and Technology) of the University of Henan Province (No. 19IRTSTHN008) and the National Key Research and Development Program of China (2017YFC1700705).
CRediT authorship contribution statement
Yan Hui Yang: Conceptualization, Funding acquisition, Project administration, Writing - original draft. Chao Jie Wang: Data curation, Formal analysis, Investigation, Methodology. Rui Fang Li: Funding acquisition, Resources, Supervision, Writing - review & editing. Zhong Yi Zhang: Funding acquisition, Resources, Supervision, Writing - review & editing. Heng Yang: Visualization, Validation. Chen Yang Chu: Formal analysis. Jia Tian Li: Formal analysis.
Declaration of Competing Interest
The author(s) declare no competing interests.
Funding Sources
This research was supported by grants from the National Natural Science Foundation of China (No. 81973417), the Key Research and Development Special Project of Henan Province (No. 182102310606), the Science Foundation of Henan University of Technology (No. 2017RCJH05), the Program for Innovative Research Team (in Science and Technology) of the University of Henan Province (No. 19IRTSTHN008) and the National Key Research and Development
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