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. 2021 Feb 20;27(3):515–522. doi: 10.1007/s12298-021-00949-9

Functional analysis of four Class III peroxidases from Chinese pear fruit: a critical role in lignin polymerization

Xi Zhu 3,#, Lan Jiang 1,#, Yongping Cai 4, Yunpeng Cao 2,4,5,
PMCID: PMC7981345  PMID: 33854280

Abstract

Pear fruit could be used as good medicine to relieve coughs, promote salivation, nourish lungs, and reduce the risk of many diseases for its phytochemical action. Lignin is a major secondary metabolite in Chinese pear fruit. Class III peroxidase (Class III PRX) is an important enzyme in the biosynthesis of lignin in plants. However, we poorly understand the role of PRXs in lignin biosynthesis in Chinese pear fruit. In our study, we cloned five PRXs from Chinese pear (Pyrus bretschneideri), namely PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75, which contained 978 bp encoded 326 amino acids (AA), 2607 bp encoded 869 AA, 972 bp encoded 324 AA, 687 bp encoded 229 AA, and 1020 bp encoded 340 AA, respectively. Enzyme activity analysis showed that four recombinant PbPRX proteins had catalytic activities for pyrogallol, guaiacol, ferulic acid, coniferyl alcohol, and sinapyl alcohol. Subcellular localization experiments showed that these genes were located in the cell wall or cell membrane. Enzyme activity and kinetics of PbPRX2 revealed its role in polymerization of lignin in Chinese pear fruit. The present study suggested that PbPRXs played critical roles in lignin biosynthesis in Chinese pear fruit.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-021-00949-9.

Keywords: Chinese pear, PRX, Enzymatic, Subcellular localization

Introduction

Lignin, a complex aromatic heteropolymer, participates in water transport, mechanical support, control of fruit taste, and response to abiotic and biotic stresses (Cesarino 2019; Pomar et al. 2002). Lignin is derived mainly from sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol (Ralph et al. 2019; Vanholme et al. 2010). The synthesis of lignin involves two steps, including monolignols synthesis and lignin polymerization (Ralph et al. 2019; Vanholme et al. 2010). Among them, Class III peroxidases (Class III PRXs) are unique to plants and play important roles in the polymerization of lignin (Fagerstedt et al. 2010; Pomar et al. 2002). The functions of PRX in the lignin biosynthesis pathway have been studied in many model plants, such as poplar (Populus trichocarpa), tobacco (Nicotiana tabacum), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) (Barceló and Aznar-Asensio 1999; Blee et al. 2003; Ehlting et al. 2005). These studies mainly focused on the localization of PRX protein in xylem, enzyme activity analysis, enzyme kinetic analysis, etc. (Barceló and Aznar-Asensio 1999; Blee et al. 2003; Ehlting et al. 2005). For example, Barceló and Aznar-Asensio found that a PRX protein was localized in the cell wall of Zinnia and have coniferyl alcohol oxidase activity (Barceló and Aznar-Asensio 1999). During the in vitro rooting of cherries, Dalet and Cornu found that the activity of PRX protein was positively correlated with the lignification of explants (Dalet and Cornu 1989). Sasaki et al. (2006) found that a PRX protein (CWPO-C) in poplar is located in the cell wall and can be combined with the substrate sinapyl alcohol to play an important role in the lignification process (Sasaki et al. 2006). Ren et al. (2014) identified the PRX family of poplar and analyzed the enzyme kinetics of 10 PRX proteins (Ren et al. 2014). Among them, 6 of the 10 PRX proteins had catalytic activity, and most of them had the highest catalytic activity for coniferol (Ren et al. 2014). Sinapyl alcohol, ferulic acid, and coniferyl alcohol are natural compounds that exist in plant cells (Cai et al. 2010; Ralph et al. 2019; Ren et al. 2014). Ferulic acid can increase the strength and rigidity of the cell wall by cross-linking hemicellulose, pentosan and arabinoxylan, and ultimately make the plant itself less susceptible to enzymatic hydrolysis when infected by foreign pathogens. Sinapyl alcohol and coniferyl alcohol are lignin monomers that involved in the formation of lignin polymers, so these two substances play a decisive role in the final biosynthesis of lignin.

The number of the PRX family have been identified in Chinese pear (Cao et al. 2016b), while the function of PRX was still unkonown, especially their role in the polymerization of lignin. In plants, PRXs can use H2O2 as an oxidant to produce lignin monomer phenoxy radicals, and finally form lignin polymers (Lewis and Yamamoto 1990). In A. thaliana, eight AtPRXs (At5g05340, At3g28200, At2g43480, At1g30870, At4g37520, At4g37530, At5g42180, and At2g37130) are involved in the synthesis and polymerization of lignin (Ehlting et al. 2005). In our previous research, the expression patterns of PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75 significantly correlated with the change in lignin content, indicating that these five genes play important roles in lignin polymerization during fruit development of Chinese pear (Cao et al. 2016b). In this study, we isolated and cloned 5 PRX genes, including PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75. The substrates of pyrogallol, guaiacol, ferulic acid, coniferyl alcohol, and sinapyl alcohol were used to detect the activity of PbPRX proteins. This study will help us understand the role of PbPRXs in the lignin polymerization of Chinese pear, and lay foundation for selecting suitable candidate genes to improve the quality of Chinese pear fruit at the molecular level in the future.

Materials and method

Cloning of PbPRX genes

The RNAprep pure Plant Kit (Tiangen, Beijing, China) was used to extract total RNA from Chinese pear fruits. The First Strand cDNA synthesis Kit (Takara, Dalian, China) was used to synthesize the first-strand complementary DNA (cDNA). The Primer (version 5.0) was used to design the specific primers of target genes. Using specific primers (Table S1), we isolated and cloned 5 PbPRX genes from the Chinese pear fruit cDNA library.

Multiple sequence alignment

The ExPASy ProtParam (Gasteiger et al. 2005) was used to predict the theoretical molecular weight and isoelectric point of PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75 genes. In recent years, researchers have analyzed the crystal structure of PRX proteins (Østergaard et al. 2000; Schuller et al. 1996) for comprehensive and systematic study of their functions and structures. The MAFFT software (Katoh et al. 2005) was used for multiple sequence alignments of these five proteins and other related proteins, and the ESpript online tool (Gouet et al. 2005) was used for visualization.

Purification of PbPRX proteins

The open reading frames (ORFs) of PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75 were subcloned into pMAL-c2X vector, respectively, and transformed into E. coli BL21 (GENERAL BIOL, Hefei, China) competent cells. Strains with completely correct sequencing were cultured at 37 °C until the OD600 reached 0.6, IPTG was added to it to make the final concentration reach 1.0 mmol/L, and then the protein expression was induced overnight at 28 °C. The bacterial pellet were collected by centrifugation at 4 °C in a cryocentrifuge tobe (5000 rpm/min, 10 min), the supernatant was removed, and a suitable buffer solution was added to suspend the bacterial pellet. After the suspended cells were broken by ultrasonication, the supernatant was centrifuged and diluted with buffer solution, and finally the expressed protein was purified by affinity chromatography, as described by Han et al. (2017).

Analyses of enzymatic activity and enzyme kinetics of PbPRXs

The catalytic activity of PbPRX proteins for pyrogallol and guaiacol was determined by the method of Kvaratskhelia et al. (1997). The catalytic activity of PbPRX proteins for coniferyl alcohol and sinapyl alcohol was determined by the method of (Barceló and Aznar-Asensio 1999). The catalytic activity of PbPRX proteins for ferulic acid was determined by the method of Sanchez et al. (1996). All the above reactions were carried out at room temperature, and the protein concentration was measured using A280. The kinetic analyses of PbPRXs proteins was performed according to the method of Ren et al. (2014).

Analyses of subcellular localization of PbPRXs

The ORFs of PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75 were subcloned into pCambia1304 vector by using GenRec Assembly Master Mix Kit (GENERAL BIOL, Hefei, China), respectively. The constructed pPbPRX-GFP vectors were electroporated into Agrobacterium tumefaciens EHA105 (GENERAL BIOL, Hefei, China) by using a Gene Pulser Xcell (BIO-RAD, USA). As described by Cao et al. (2016a, b), the suspensions were infiltrated into the tobacco leaves. The laser scanning microscopy (CarlZeiss LSM710, Germany) was used to observe the expressed PbPRX-GFPs.

Results

Characteristics of PbPRX genes

Five PbPRX (PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75) genes were cloned from the Chinese pear fruit. Their full-length of cDNAs are 978 bp, 2607 bp, 972 bp, 687 bp, and 1020 bp, respectively, encoding 326 amino acids (AA), 869 AA, 324 AA, 229 AA, and 340 AA (Table 1). The ExPASy ProtParam was used to predict their molecular weights and isoelectric points. The molecular weights of PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75 were 35.27 kilodaltons (kDa), 98.12 kDa, 34.41 kDa, 25.11 kDa, and 37.26 kDa, respectively, and the isoelectric points were 8.82, 6.33, 5.88, 8.93, and 5.36, respectively (Table 1).

Table 1.

The basic information of fivePbPRXs genes

Gene name Accession number Molecular weight (kD) of proteins cDNA length (bp) ORF length (bp) Size (Amino acids) pI
PbPRX2 Pbr035186.1 35.27 978 978 326 8.82
PbPRX22 Pbr013845.1 98.12 2607 2607 869 6.33
PbPRX34 Pbr020590.1 34.41 972 972 324 5.88
PbPRX64 Pbr039193.1 25.11 687 687 229 8.93
PbPRX75 Pbr007872.1 37.26 1020 1020 340 5.36

Determination of enzyme activity of PbPRX proteins

After induction and fragmentation, the fusion proteins of pMAL-PbPRX2, pMAL-PbPRX22, pMAL-PbPRX34, pMAL-PbPRX64, and pMAL-PbPRX75 were purified. Among them, pMAL-PbPRX22 was mainly obtained in the precipitate, so we only studied the four proteins PbPRX2, PbPRX34, PbRX64, and PbPRX75 in further experiments. These four purified proteins had certain catalytic activities for pyrogallol, guaiacol, ferulic acid, coniferyl alcohol, and sinapyl alcohol. Among them, PbPRX2 showed higher activity for both sinapyl alcohol and coniferyl alcohol, compared with the other three PbPRX proteins (Table 2). These data suggested that PbPRX2 might play a decisive role in the lignin biosynthesis of Chinese pear fruit.

Table 2.

Specific activities of the Pyrus bretschneideri PbPRX proteins for five substrates

Specific activity (μmol/min per mg)
Pyrogallol Guaiacol Ferulic acid Coniferyl alcohol Sinapyl alcohol
PbPRX2 1108.25 ± 21.33 426.76 ± 23.69 1311.23 ± 28.27 5003.13 ± 9.65 2004.21 ± 10.54
PbPRX34 1321.62 ± 19.24 428.13 ± 9.32 2142.27 ± 32.14 3002.93 ± 110.32 111.81 ± 5.32
PbPRX64 634.21 ± 12.33 233.18 ± 9.87 634.23 ± 19.42 3303.21 ± 9.32 80.32 ± 1.22
PbPRX75 719.11 ± 19.29 215.24 ± 4.97 691.75 ± 23.65 2331.31 ± 132.10 47.31 ± 4.52

Enzyme kinetic analyses of PbPRXs

Using pyrogallol, ferulic acid, and coniferyl alcohol as substrates, the kinetic constants of the four purified PbPRX proteins (PbPRX2, PbPRX34, PbRX64, and PbPRX75) were tested. Compared with pyrogallol, these four PbPRX proteins have better kcat/Km (higher catalytic efficiency), bette kcat (higher conversion number), and bette 1/Km (higher affinity) for ferulic acid and coniferyl alcohol (Table 3).

Table 3.

Kinetic constants of the Pyrus bretschneideri PbPRXs proteins for three substrates

Ferulic acid Pyrogallol Coniferyl alcohol
1/Km Kcat Kcat/Km 1/Km Kcat Kcat/Km 1/Km Kcat Kcat/Km
(mM−1) (S−1) (mM−1S−1) (mM−1) (S−1) (mM−1S−1) (mM−1) (S−1) (mM−1S−1)
PbPRX2 2.11 12,543.24 26,466.24 0.13 698.32 90.78 4.3 997.35 4288.61
PbPRX34 3.12 12,501.29 39,004.02 0.21 832.22 174.77 2.23 9975.19 22,244.67
PbPRX64 2.22 5229.13 11,608.67 0.32 312.25 99.92 3.16 4543.56 14,357.65
PbPRX75 4.45 2176.45 9685.21 3.56 74.32 264.58 4.09 8114.43 33,188.02

Multiple sequence alignment analyses of PbPRX proteins

In this study, the amino acid sequences of the two PRX proteins, viz. 1SCH (Schuller et al. 1996) and 1PA2 (Østergaard et al. 2000) were used as templates to analyze the secondary structure of PbPRX2, PbPRX34, PbRX64, and PbPRX75 proteins. As shown in Fig. 1, the typical conserved domains of peroxidase existed in these four proteins of Chinese pear. The most conserved regions in these PbPRX proteins mainly include residues used to maintain the folding and catalytic activity of peroxidase. For example, the eight Cys residues involved in the four disulfides are highly conserved in all PRX tested.

Fig. 1.

Fig. 1

Structural alignment of PbPRX2, PbPRX34, PbRX64 and PbPRX75 with two PRXs from the research collaboratory for structural bioinformatics PDB

Subcellular localization of PbPRX proteins

To determine the subcellular localization of PbPRX2, PbPRX34, PbRX64, and PbPRX75, we constructed pPbPRX-GFP expression vectors and then transformed them into tobacco, respectively. The green fluorescence signals from the expressed fusion PbPRX2-GFP, PbPRX34-GFP, PbPRX64-GFP, and PbPRX75-GFP proteins were specifically distributed on the cell membrane or cell wall, as shown in Fig. 2. However, the green fluorescence from the expressed GFP alone was distributed on the whole cell, which means that it was a constitutive expression pattern.

Fig. 2.

Fig. 2

Subcellular localization of PbPRX from Chinese pear (Pyrus bretschneideri)

Discussion

Lignin is a kind of biological macromolecule with complex and extremely stable structure in plants (Hatakeyama and Hatakeyama 2009; Martínez et al. 2008; Ralph et al. 2019). Lignin is mainly composed of three structural units, i.e., hydroxyphenyl propane, guaiacyl propane, and syringyl propane (Ralph et al. 2019; Vanholme et al. 2010). S-lignin (syringyl lignin) consists of syringyl structural units, while H-lignin (hydroxyphenyl lignin) is composed of p hydroxyphenylpropane units, and G-lignin (guaiacyl lignin) consists of guaiacyl units (Cai et al. 2010; Ralph et al. 2019). The stone cells of pear fruit, which have adversely affected pear quality and flavor, are mainly composed of lignin (Cai et al. 2010; Rogers and Campbell 2004). Therefore, lignin content is one of the important factors affecting the fruit quality of Chinese pear (Jin et al. 2013; Yan et al. 2014). The core lignification gene families in Chinese pear, including C4H, CSE, COMT, 4CL, CCR, HCT, CCoAOMT, C3H, PAL, F5H, and CAD, have been studied in previously published articles (Cao et al. 2016a, 2019; Cheng et al. 2017; Ding et al. 2020; Li et al. 2021). For the polymerization of lignin, although Cao et al. (2016a, b) also identified the number of the PbPRX family in Chinese pear, but the function of PRXs still remains underexplored.

PRXs play an important role in various physiological processes of plants, such as cell wall structure, response to biotic and abiotic stresses, and biosynthesis of secondary metabolites (Blee et al. 2003; Fagerstedt et al. 2010; Fernández-Fueyo et al. 2014; Hoffmann et al. 2020; Kidwai et al. 2020; Miller et al. 2007). PRXs usually exist in the form of the gene family in plants. For example, Wang et al. (2015) identified 119 ZmPRXs in maize (Zea mays), Li et al. (2020) cloned CsPRXs in tea (Camellia sinensis), and Yan et al. (2019) identified 374 TaPRXs in wheat (Triticum aestivum) (Li et al. 2020; Wang et al. 2015; Yan et al. 2019). PRXs are very important enzymes in plants that mainly catalyze the polymerization of lignin monomers in the last step of the lignin metabolism pathway (Davin et al. 2008). In our study, we cloned five PbPRXs (PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75), and found that four (PbPRX2, PbPRX34, PbPRX64, and PbPRX75) of these genes may be involved in the lignin biosynthesis in Chinese pear fruit. Sequence alignment analysis revealed that these five PbPRX proteins have typical conserved domains of peroxidase (Cosio and Dunand 2009; Passardi et al. 2004; Teixeira et al. 2004). Ren et al., (2014) found that PRX proteins are mainly located in the cell membrane or cell wall (Ren et al. 2014), which was also confirmed in our results (Fig. 2).

PRX proteins have catalytic activity for a variety of substrates, such as pyrogallol, guaiacol, ferulic acid, coniferyl alcohol, and sinapyl alcohol (Kokkinakis and Brooks 1979; Lai et al. 2006; Ren et al. 2014; Wariishi and Gold 1989). To study the catalytic activity of PbPRX2, PbPRX34, PbPRX64, and PbPRX75 for these substrates, these proteins were purified, and their enzyme activities and enzyme kinetic constants were analyzed. Enzyme activity analysis showed that PbPRX2 has high catalytic activity for both coniferyl alcohol and sinapyl alcohol. Previous studies have shown that coniferyl alcohol and sinapyl alcohol are precursors for the synthesis of lignin monomers (Amthor 2003; del Río et al. 2020; Jin et al. 2013; Vanholme et al. 2019; Yan et al. 2014), which suggested that PbPRX2 may play a vital role in the lignin biosynthesis of Chinese pear fruit. Indeed, Koutaniemi et al., (2005) found that one gene PaPRX5 encoded protein preferred coniferyl alcohol and contributed to the lignin biosynthesis in Norway spruce (Picea abies) (Koutaniemi et al. 2005).

Conclusion

In our study, we cloned PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75 genes from Chinese pear fruit. Subsequently, we performed sequence alignment, enzyme activity, enzyme kinetics, and subcellular localization analyses. Finally, we found that PbPRX2 played a key role in the polymerization of lignin in Chinese pear fruit. Our results provided candidate PbPRX genes for improving pear fruit quality at the molecular level in the future.

Supplementary Information

Below is the link to the electronic supplementary material.

Acknowledgements

We are grateful the supported by the Outstanding youth of the Education Department of Hunan Province (20B624), and the Talent Research Start-up Fund of Central South University of Forestry and Technology (2019YJ012).

Abbreviations

Class III PRX

Class III peroxidase

AA

Amino acids

ORFs

Open reading frames

cDNA

Complementary DNA

kDa

Kilodaltons

S-lignin

Syringyl lignin

H-lignin

Hydroxyphenyl lignin

G-lignin

Guaiacyl lignin

Funding

This work was supported by the Outstanding youth of the Education Department of Hunan Province (20B624), and the Talent Research Start-up Fund of Central South University of Forestry and Technology (2019YJ012).

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xi Zhu and Lan Jiang have contributed equally to this work.

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