Phenylalanine ammonia-lyase2.1 contributes to the soybean response towards Phytophthora sojae infection

Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by Phytophthora sojae is a destructive disease worldwide. Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes related to plant responses to biotic and abiotic stresses. However, the molecular mechanism of PAL in soybean in response to P. sojae is largely unclear. Here, we characterize a novel member of the soybean PAL gene family, GmPAL2.1, which is significantly induced by P. sojae. Overexpression and RNA interference analysis demonstrates that GmPAL2.1 enhances resistance to P. sojae in transgenic soybean plants. In addition, the PAL activity in GmPAL2.1-OX transgenic soybean is significantly higher than that of non-transgenic plants after infection with P. sojae, while that in GmPAL2.1-RNAi soybean plants is lower. Further analyses show that the daidzein, genistein and salicylic acid (SA) levels and the relative content of glyceollins are markedly increased in GmPAL2.1-OX transgenic soybean. Taken together, these results suggest the important role of GmPAL2.1 functioning as a positive regulator in the soybean response to P. sojae infection, possibly by enhancing the content of glyceollins, daidzein, genistein and SA.

PAL is present in all higher plants studied and has also been found in some fungi 24,25 and cyanobacteria 26 . However, it has not yet been detected in Eubacteria, Archaea, and animals 27,28 . In plants, PAL is encoded by a multi-gene family. Four PAL isoforms have been detected in Arabidopsis 29,30 , two in Rubus 31 , five in Populus 32 , seven in cucumber 33 , and twelve in watermelon 34 . Other research has shown that the co-expression of different tobacco PAL proteins in Escherichia coli can produce functional heterotetrameric enzymes 35 . In soybean, PAL is encoded by a small gene family ranging from 2 to 3 members and can be divided into different subgroups 36 .
However, homo-or heterotetramers of the PAL protein and the different PAL genes are thought to be involved in plant development and in the response to different stress stimuli 37 . For instance, RiPAL1 is associated with early fruit ripening and the biosynthesis of flavonoids, whereas RiPAL2 correlates with late stages of flower and fruit development and the accumulation of anthocyanins in Rubus 31 . PtPAL1 is expressed in non-lignified tissues of shoots and roots, whereas PtPAL2 is expressed in the heavily lignified structural cells of shoots and in non-lignified cells of root tips in aspen 38 . Alternatively, several studies indicate that the gene expression of PAL is stimulated during developmental programming and by a variety of environmental stresses, including pathogenic attacks, wounding, UV irradiation, low temperatures, and low levels of nitrogen, phosphate, or ions 3,[39][40][41][42] . In French bean, an expression study of PvPAL showed that PvPAL is induced with tissue wounding and activated by fungi attack, suggesting that PAL may play a role in body injury and fungal responses 43 . In Arabidopsis, PAL1 and PAL2 double mutants are more sensitive to ultraviolet-B light and show a significant reduction in lignin accumulation than wild type plants 44 . In rice, a recent report shows that OsPAL4 and possibly OsPAL6 are key contributors to a broad spectrum of disease resistance 45 . Although PAL is extensively studied in various plants, the systematical research on PAL2 in soybean disease-resistance has not been reported.
In a previous study, a cDNA library enriched for mRNAs encoding ESTs that were increased in abundance during infection with P. sojae was constructed by suppression subtractive hybridization from leaf tissues of the highly resistant soybean cultivar 'Suinong 10' , and an EST homologous to a phenylalanine ammonia-lyase from Lotus japonicus was identified to be upregulated by microarray and real-time PCR 46 . In the present work, the full-length EST, designated GmPAL2.1 (GenBank accession no. NM_001250027, NCBI protein no. NP_001236956), was isolated using RT-PCR from 'Suinong 10' soybean. The expression patterns of GmPAL2.1 induced under abiotic and biotic stresses were examined. To gain insight into the function of GmPAL2.1 in soybean, the GmPAL2.1 gene was overexpressed in soybean plants under the control of the35S promoter, and RNA interference (RNAi) technology was applied to suppress the expression of GmPAL2.1 to generate knockdown soybean plants. Furthermore, the contents of SA and three kinds of isoflavone-daidzein, glycitein, and genistein-were analyzed. The relative content of glyceollins and the PAL activity in the transgenic plants were also investigated. Taking our findings together, we report insights into the function of a PAL gene in soybean, GmPAL2.1, in the defense response against P. sojae.

Results
Isolation and sequence analysis of GmPAL2.1. The full-length cDNA sequence of the GmPAL2.1 gene (GenBank accession no. NM_001250027) was isolated from the total RNA of 'Suinong 10' using RT-PCR. Sequence analysis suggests that the full length of GmPAL2.1 is 2284 bp and contains an open reading frame (ORF) encoding a polypeptide of 717 amino acids ( Supplementary Fig. 1 Fig. 3). The prediction of the three-dimensional (3D) structure of GmPAL2.1 based on the data from Phyre (http://www.sbg.bio.jc.ac.uk/phyre/) shows that this protein is a HAL/PAL-like family member that belongs to the L-aspartase-like superfamily ( Supplementary  Fig. 2B).

Transcript abundance of GmPAL2.1 under various stresses. To evaluate the expression pattern of
GmPAL2.1, quantitative RT-PCR was used to examine the transcript abundance of GmPAL2.1 in 'Suinong 10' plants (resistant cultivar) and 'Dongnong 50' plants (susceptible cultivar). In 'Suinong 10' plants, quantitative real-time PCR shows that GmPAL2.1 is induced by treatment with SA, MeJA, ABA and GA (Fig. 1). Under UV radiation, a low-temperature treatment (4 °C) and dark treatments, the transcripts of GmPAL2.1 mRNA increase and reach a maximum level at 6, 6 and 12 h, respectively (Fig. 1).
The tissue-specific transcript abundance of GmPAL2.1 in 'Suinong 10' and 'Dongnong 50' shows that GmPAL2.1 is constitutively and highly expressed in the leaves followed by the cotyledons, stems, and roots ( Fig. 2A,B). The transcript levels of GmPAL2.1 were also determined after treatment with P. sojae. A significant upregulation of GmPAL2.1 expression is detected in the leaves at 6 h after the treatments and reaches a maximum level at 36 h, followed by a rapid decline in 'Suinong 10' (Fig. 1). However, it is slightly down-regulated at 36 h in 'Dongnong 50' plants, revealing differential expression for GmPAL2.1 in resistant and susceptible cultivars (  Fig. 3, confocal laser scanning microscopy reveals that GFP fluorescence is dispersed throughout the entire cell that was bombarded with the control plasmid 35S. GFP and the fusion GmPAL2.1-GFP protein are observed in the cell membrane and cytoplasm, similar to GmPRP 49 , indicating that GmPAL2.1 is present in both the cell membrane and cytoplasm. It should be noted that PAL was mainly located in cytoplasm and chloroplast, mitochondria, glyoxysome, peroxisoame, and other membrane organelles 50 . However, the enzymes encoded by different PAL genes could differ in subcellular location, and membrane associated PAL might' channel' cinnamic acid through interactions with membrane protein cinnamate 4-hydroxylase (C4H) for the second step in phenylpropanoid biosynthesis 51, 52 . Resistance to P. sojae in transgenic soybean plants. To

Analyses of PAL activity. To determine whether there are changes in the PAL activity in
GmPAL2.1-transgenic soybean leaves during P. sojae infection, the PAL activity was analyzed after 36 h of incubation with P. sojae. As shown in Fig. 6, the PAL activity in GmPAL2.1-OX transgenic soybean is significantly higher than that of non-transgenic plant leaves after infection with P. sojae. In all the GmPAL2.1-RNAi soybean lines, the PAL activity is distinctly compromised by P. sojae infection compared with that in non-transgenic plants. These results indicate that the expression of GmPAL2.1 affects PAL activity in soybean leaves after infection with P. sojae.

Isoflavone and glyceollin levels in transgenic soybean seeds.
To test whether the GmPAL2.1 expression level can cause change in the isoflavone and glyceollins content in soybean, the contents of three kinds of isoflavones (daidzein, genistein and glycitein) and the relative content of the glyceollins were measured in the seeds of transgenic soybean plants and non-transgenic soybean plants. The results show that the daidzein and genistein levels in the GmPAL2.1-overexpressing soybean plants are significantly higher than those of non-transgenic plants, and those levels are significantly compromised in GmPAL2.1-RNAi soybean plants (Fig. 7A,C). However, the levels of glycitein show little change compared to those of the control (Fig. 7B). As shown in Fig. 7D, the relative content of glyceollins in transgenic soybean seeds also varies markedly compared with that of the control. These results suggest that GmPAL2.1 may play a role in the defense resistance to P. sojae by participating in synthesis of isoflavones and glyceollins.

Discussion
In this study, we isolated and functionally characterized the PAL gene (GmPAL2.1), which acts as a positive regulator in resistance to P. sojae in soybean (Glycine max) plants. PAL, an entry-point enzyme in the phenylpropanoid biosynthesis pathway, was first isolated from barley (Hordeum vulgare L.) 53 . Since then, there have been many reports concerning the biochemical characterization and structures of PAL proteins in organisms, such as the PAL from Petroselinum crispum, Arabidopsis thaliana, Streptomyces maritimus, Rhodobacter sphaeroides, Cyanobacteria, Rhodotorula glutinis, and Musa paradisiaca 26,[54][55][56][57][58][59] . In soybean, the gene encoding PAL was cloned by Frank and Vodkin in 1991 36 . However, there is little knowledge about the biological function of PAL in soybean. Here, we report for the first time that GmPAL2.1 transgenic soybean plants inoculated with P. sojae display significantly altered responses to pathogen infection.
There are many studies that show PAL genes are involved in the response of plants to infection by pathogens 9, 60, 61 . In Arabidopsis thaliana, a pal1/pal2/pal3/pal4 quadruple knockout mutant showed increased susceptibility to the virulent bacterial pathogen Pseudomonas syringae 40 . In transgenic tobacco, it has been reported that a partial suppression of the PAL gene gives rise to increased fungal susceptibility 55 . In the present study, we determined that the overexpression of GmPAL2.1 transgenic soybean improves resistance to P. sojae and GmPAL2.1-RNAi soybean plants exhibits increased susceptibility. Moreover, PAL has been proposed to play important roles in biotic and abiotic stress responses in plants 3,41,43,62 . In this work, the transcript abundance of GmPAL2.1 following various stress treatments was analyzed. The results show that inoculation with P. sojae as a biotic stress and UV-B radiation, cold and dark treatments as abiotic stresses significantly increase the accumulation of GmPAL2.1 mRNA in soybean plants (Fig. 1). This study also found that the transcript levels of GmPAL2.1 are also remarkably increased by SA stress (Fig. 1), and further evidence showed that GmPAL2.1-transgenic soybean positively regulated the expression of the GmNPR1, GmPR1 and GmPR5 genes as well as SA accumulation (Fig. 8). NPR1 has been identified to be involved in SA-mediated PR gene expression and resistance 63 . PR1, PR2 and PR5 are considered to be the effector genes for systemic acquired resistance (SAR), which was mediated by SA 64,65 . It has  The non-transgenic soybean plants were used as controls. The experiment was performed on three biological replicates with their respective three technical replicates and statistically analyzed using Student's t-test (*P < 0.05, **P < 0.01). Bars indicate the standard error of the mean.
been found recently that soybean sprouts germinated under red light improve resistance to Pseudomonas putida 229 through the regulation of the de novo synthesis of SA and up-regulation of PR genes 66 . Therefore, we speculate that GmPAL2.1 might play an important role in soybean plant resistance to P. sojae, depending mainly on the SA signaling pathway.
It has been reported that PAL is one of the branch point enzymes between primary and secondary metabolism 67 . The gateway from primary metabolism into phenylpropanoid metabolism is the deamination of L-phenylalanine  Three technical replicates were averaged and statistically analyzed using Student's t-test (**P < 0.01). Bars indicate the standard error of the mean. The data are the means ± SD from three independent experiments. Asterisks indicate significant differences as determined by Student's t-test (P < 0.05).
by PAL to form trans-cinnamic acid 68 . The enzymatic activity of PAL determines the flux through the phenylpropanoid pathway and the rate of phenylpropanoid production, which has important functions in the plant defense against abiotic and biotic stresses [69][70][71] . Therefore, PAL activity has been suggested to play important roles in the plant defense against pathogens, typically as a physiological marker for measuring the resistance of plants, such as pea 72 , tomato 73 , cucumber 74 , and lupine 75,76 . The determination of PAL activity also has great significance. A spectrophotometric assay was previously developed for testing the activity of PALs in plants, which accords to the formation of trans-cinnamic acid determined at 290 nm 77 . Some other similar spectrophotometric assays 78 were also developed. In addition, studies have reported that the high performance liquid chromatography (HPLC) technique is a rapid and sensitive method to analyze PAL activity 79,80 . In our study, the PAL activity in the leaf extract was determined by spectrophotometry following the method described by Song and Wang 81 . PAL activity in GmPAL2.1-OX soybean plants is significantly higher than that in non-transgenic plants after P. sojae infection and is markedly lower in GmPAL2.1-RNAi soybean plants (Fig. 6). Isoflavonoids belong to an important group of secondary metabolites derived from the phenylpropanoid pathway and play important roles in plant defense 82,83 . To investigate whether the isoflavone content changes in the transgenic soybean lines, six T 2 transgenic soybean seeds (OX-1, OX-2, OX-29, RNAi-24, RNAi-27 and RNAi-32) and non-transgenic soybean seeds were used to analyze the content of daidzein, glycitein and genistein. The results show that the daidzein content and the genistein content are positively and significantly correlated with the expression levels in the transgenic soybean seeds (Fig. 7A,C). But interestingly, it's been found that the combination of transcription factor activation of multiple phenylpropanoid pathway genes and cosuppression of a single gene, F3H, the enzyme catalyzes the conversion of flavanones to dihydroflavonols, provided an effective metabolic engineering strategy for producing high levels of isoflavones in soybean seed 23 . IFR is an enzyme involved in the synthesis of glyceollins from daidzein and the daidzein content greatly reduced in GmIFR-overexpression soybean seeds 84 . The reason is maybe PAL produces trans-cinnamic acid, which serves as a precursor for the synthesis of all phenylpropanoids, including isoflavones 3 . These data suggest that the expression levels of GmPAL2.1 might have an effect on PAL activity and the accumulation of isoflavones in response to P. sojae infection.
Several reports have shown that phytoalexins constitute a chemically heterogeneous group of low-molecular-weight antimicrobial compounds that are synthesized de novo and accumulate in plants in response to stress [85][86][87][88] . Glyceollins represent another group of phytoalexins whose biosynthesis is increased in soybean in response to various stress signals, such as fungal infection 87,88 . Another study has suggested that the fungi Aspergillus flavus, Aspergillus niger, Aspergillus oryzae, and Aspergillus flavus are all capable of the inductive synthesis of glyceollins in soybean 89 . As isoflavonoid-type phytoalexins, glyceollins have exhibited antifungal activity 90 . Glyceollins have a significant antimicrobial effect against Phytophthora capsici and Sclerotinia sclerotiorum 12,14 and exhibit resistance to Phytophthora megasperma var. sojae in soybean [91][92][93] . Previous research suggests that the biosynthesis of glyceollin is via the isoflavonoid branch of the phenylpropanoid pathway 94 . More specifically, the glyceollin biosynthetic pathway includes the enzymes involved in phenylpropanoid metabolism, flavonoid/isoflavonoid synthesis and those dedicated to the biosynthesis of pterocarpan phytoalexins 95 . Thus, PAL involves the synthesis of glyceollins, which are a mixture of structurally related pterocarpans 7,96 . In this work, we detected the relative content of glyceollins in transgenic soybean seeds and nontransgenic soybean seeds. The relative content of glyceollins in GmPAL2.1-OX soybean plants is significantly higher than that in non-transgenic plants, while that in GmPAL2.1-RNAi soybean plants is lower (Fig. 7D). Therefore, we suggest that GmPAL2.1 may play an important role in the biosynthesis of glyceollins to improve resistance to P. sojae in soybean.

Methods
Plant materials and stress treatments. The soybean cultivar 'Suinong 10' , which is resistant to dominant physiological race 1 of P. sojae in Heilongjiang, China 97 , was used in this study. The seeds were grown in a glasshouse maintained at 22 °C and 70% relative humidity under a photoperiod of 16/8 h light/dark. Fourteen days after planting, seedlings at the first-node stage (V1) 98 were used for various treatments.
'Dongnong 50' soybean, which is susceptible to P. sojae dominant physiological race 1 and was provided by the Key Laboratory of Soybean Biology in the Chinese Ministry of Education, Harbin, was used for P. sojae treatment and gene transformation experiments.
Isolation of the GmPAL2.1 gene. A suppression subtractive hybridization library coupled with cDNA microarrays was queried using a soybean expressed sequence tag (EST) encoding an EST homologous to a phenylalanine ammonia-lyase from Lotus japonicus, previously shown to be upregulated in the highly resistant soybean 'Suinong 10' infected with P. sojae 46 . Here, the full-length cDNA (termed GmPAL2.1, GenBank accession no. NM_001250027, NCBI protein no. NP_001236956) of the EST was amplified using RT-PCR with the cDNA of 'Suinong 10' using the primer pairs GmPAL2.1F and GmPAL2.1R (see Supplementary Table 1 for primer sequences). The primers for GmPAL2.1 were used for PCR under the following condition: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 8 min. The amplification product was gel purified and cloned into the PMD18-T vector (TaKaRa, Dalian, China), then transformed into E. coli DH5α cells (Shanghai Biotech Inc, Shanghai, China) and sequenced (GENEWIZ, Beijing, China). Sequence alignments were performed using DNAMAN software (http://www.lynnon.com/). A phylogenetic analysis of GmPAL2.1 and various heterologous PAL proteins was performed using MEGA4 software 101 . The three-dimensional (3D) structure of GmPAL was predicted using the online program Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2).
Quantitative RT-PCR. For expression analysis of GmPAL2.1 under abiotic and biotic stresses, the total RNA was isolated from 'Suinong 10' soybean leaves using TRlzol reagent ((Invitrogen, Shanghai, China). The first-strand cDNAs were synthesized using 1 µg of RNA with the Moloney murine leukemia virus reverse transcriptase kit (Takara, Dalian, China) according to the manufacturer's protocol. The qRT-PCR analysis was performed using a real-time RT-PCR kit (Takara, Japan) with a CFX96 Touch TM Real-Time PCR Detection System (BioBad, USA). DNA accumulation was measured using SYBR Green as the reference dye. The soybean housekeeping gene GmActin4 (GenBank accession no. AF049106) was used as the internal control. Each qRT-PCR was run in three technical replicates.

Subcellular localization.
To investigate the subcellular localization of GmPAL2.1, the full-length GmPAL2.1 was cloned in frame into the 5′-terminus of the GFP coding sequence in the 35 S:GFP vector using the primer pairs GmPAL2.1-GF and GmPAL2.1-GR (Supplementary Table 1), generating the fusion construct 35 S:GmPAL2.1-GFP. Arabidopsis protoplasts were acquired using the method described by Lin 102 . Arabidopsis protoplast transformation was performed as described by Yoo et al. 103 with minor modifications. After incubation of the transfected Arabidopsis protoplasts cells for 16 h at 25 °C, the GFP signal was imaged using a TCS SP2 confocal spectral microscope imaging system (Leica, Germany).  Table 1) were designed to amplify 310 bp fragments of GmPAL2.1. The fragments were cloned into the pjawoh18 vector. The plant expression vector was introduced into Agrobacterium tumefaciens LBA4404 and EHA105 using the freezing and thawing method as described by Holsters et al. 104 . 'Dongnong 50' soybean was used for the gene transformation experiments using the Agrobacterium-mediated transformation method described by Paz et al. 105 . To confirm transgene insertion in the soybean plants, genomic DNA was extracted from the transformants, and PCR analysis was conducted. Transgenic soybean plants (T 1 ) were identified by PCR amplification and Southern blot hybrid-ization using a DIG High Prime DNA Labeling and Detection Starter kit II (Roche, Germany). Transgenic soybean plants (T 2 ) were also identified by quantitative RT-PCR (see Supplementary Table 1 for primer sequences).

Pathogen response assays of transgenic soybean plants. To investigate whether the
GmPAL2.1-transformed plants have changes in resistance to pathogen infection, artificial inoculation procedures were performed according to the methods described by Dou et al. 106 and Morrison and Thorne 107 with minor modifications. The roots and living cotyledons of three T 2 GmPAL2.1-overexpressing soybean plants (OX-1, OX-2, and OX-29) and three GmPAL2.1-RNAi soybean plants (RNAi-24, RNAi-27, and RNAi-32) were treated with a P. sojae inoculum. The roots and living cotyledons were incubated in a mist chamber at 25 °C with 90% relative humidity under a 14 h photoperiod at a light intensity of 350 µmol photons m −1 s −1 for investigation. The cotyledons of non-transformed plants were used as controls. Disease symptoms on each cotyledon were observed and photographed after inoculation using a Nikon D7000 camera.
To further determine the responses of GmPAL2.1-transformed soybean plants to P. sojae ingress, the relative biomass of P. sojae in infected cotyledons of the selected T 2 transgenic plants at the first-node stage (V1) 98 were assessed after 24 h, 48 h and 96 h of incubation with zoospore suspensions of P. sojae. The assessment of the biomass of P. sojae was based on the transcript level of P. sojae TEF1 (GenBank accession no. EU079791) in reference to soybean EF1β according to the method of Chacón et al. 108 (see Supplementary Table 1 for the TEF1 and EF1β primer sequences). The pathogen response assays were performed on three biological replicates with their respective three technical replicates. PAL activity assay. Enzymes were extracted from four-week-old soybean seedlings leaves using 100 mM phosphate buffer (pH 6.0) containing 2 mM EDTA, 4 mM dithiothreitol, and 2% (w/w) polyvinylpyrrolidone. Fresh leaf samples were ground on ice for 5 min in 0.25 g · mL −1 of extraction buffer and then centrifuged for 25 min at 17,000 × g and 4 °C to obtain a solid-free extract. The PAL activity in the leaf extract was determined by the method of Song and Wang 81 , with slight modifications. Briefly, the protein extract (0.2 mL) was incubated at 30 °C for 60 min with 2 mL of 0.01 M borate buffer (pH 8.7) and 1 mL of 0.02 M L-phenylalanine (pre-dissolved in 0.01 M borate buffer, pH 8.7). This reaction was stopped by the addition of 1 mL of 6 M HCl. The reaction was then centrifuged for 10 min at 12,000× g to pellet the denatured protein. The absorbance was measured at 290 nm before and after incubation. One unit of activity (katal) was defined as the amount of PAL that produces 1 mole of cinnamic acid in 1 s and was expressed as nkat mg −1 of protein. A reaction without the substrate was our blank control. Triplicate assays were performed for each extract. The protein concentration was determined using the dye-binding Bradford method 109 with bovine serum albumin as the protein standard.
Isoflavone and glyceollin analysis. Approximately 0.1 g sample of seeds from T 2 transgenic soybean plants (lines OX-1, OX-2, OX-29, RNAi-24, RNAi-27 and RNAi-32) was used to analyze the content of daidzein, glycitein and genistein. The three kinds of isoflavones were extracted from the samples and separated using HPLC as described by Zeng et al. 110 .
Seeds of T 2 transgenic soybean plants (lines OX-1, OX-2, OX-29, RNAi-24, RNAi-27 and RNAi-32) were used for glyceollin extraction with 80% ethanol following the method described by Boue et al. 111 and isolated using HPLC as described by Zeng et al. 110 . Non-transformed seeds extracts were used as controls.
SA measurement. SA was extracted and measured from soybean plant leaves, as described previously by Aboul et al. 112 . Leaf tissues (0.5 g) were extracted in 1 mL of 90% methanol following homogenization in liquid nitrogen. 3-Hydroxybenzoic acid (Sigma) was used as an internal standard. The SA extracts were analyzed automatically using a fluorescence detector (excitation at 305 nm and emission at 405 nm) with reversed-phase high-performance liquid chromatography on a Waters 515 system (Waters, Milford, MA, USA) with a C18 column.