Introduction

Plants have evolved multiple defense signaling pathways to respond to environment stress and pathogen attack1. The phenylpropanoid pathway is one of the important secondary metabolism pathways and produces a variety of secondary metabolites2,3,4,5,6. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway7, in which L-phenylalanine undergoes deamination to yield trans-cinnamate and ammonia8. Formation of lignin, suberin, phytoalexins, stilbenes, coumarins and other flavonoids all depends on PAL activity3, 9. It has been reported that the biosynthesis of glyceollins occurs via the phenylpropanoid pathway in soybean7. Glyceollins are soybean-derived phytoalexins that accumulate in the seeds in response to environmental stimulus10. In general, glyceollins protect plant tissues from environmental challenges, possibly by reducing the oxidative damage induced by stress factors. Thus, these compounds have significant cellular anti-oxidative activities10,11,12. Moreover, glyceollins also inhibit several pathogen species and the growth of Phytophthora megasperma var. sojae 13,14,15. Research has revealed that glyceollins are a major factor in the restriction of Phytophthora sojae during compatible and incompatible interactions of soybean with the pathogen16. The accumulation of glyceollins is also related to deploying the defense responses to the cell wall glucan elicitor from the pathogen P. sojae in soybean17, 18. There are several enzymes involved in the glyceollin biosynthetic pathways19. PAL acts as a key-control enzyme, which catalyzes the first step in the synthesis of glyceollins20,21,22,23.

PAL is present in all higher plants studied and has also been found in some fungi24, 25 and cyanobacteria26. However, it has not yet been detected in Eubacteria, Archaea, and animals27, 28. In plants, PAL is encoded by a multi-gene family. Four PAL isoforms have been detected in Arabidopsis29, 30, two in Rubus31, five in Populus32, seven in cucumber33, and twelve in watermelon34. Other research has shown that the co-expression of different tobacco PAL proteins in Escherichia coli can produce functional heterotetrameric enzymes35. In soybean, PAL is encoded by a small gene family ranging from 2 to 3 members and can be divided into different subgroups36.

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 stimuli37. 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 Rubus31. 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 aspen38. 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 ions3, 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 responses43. 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 plants44. In rice, a recent report shows that OsPAL4 and possibly OsPAL6 are key contributors to a broad spectrum of disease resistance45. 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 PCR46. 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), with a predicted molecular mass of 78.116 kDa and a theoretic isoelectric point (pI) of 5.83. Phylogenetic tree and alignment analyses revealed that GmPAL2.1 has 71–87% identity for its overall amino acid sequence to Lotus japonicus LjPAL (BAF36972.1), Astragalus membranaceus AmPAL (EF567076.1), Medicago truncatula MtPAL (XM_003590423.1), Trifolium pratense TpPAL (AB236800.1), Manihot esculenta MePAL (AAK62030.1), Daucus carota DcPAL (BAG31931.1), Fragaria vesca FvPAL (XM_004304392.1), Morus alba MaPAL (HM064433.1), Robinia pseudoacacia RpPAL (ACF94716.1), Pisum sativum PsPAL (D10001.1), Oryza sativa OsPAL (NM_001054017.1) and Arabidopsis thaliana AtPAL (NM_111869.3) (Supplementary Fig. 2A,C). In addition, there are eight GmPAL members identified in soybean genomes47, 48. GmPAL2.1 shares 97.07%, 88.56%, 88.15%, 87.64%, 83.15%, 83.01% and 73.41% identity with GmPAL2.3 (XM_003542493), GmPAL1.1 (XM_003554334), GmPAL1.2 (XM_003521349), GmPAL1.3 (XM_003521348), GmPAL2.2 (XM_003556190), GmPAL2.4 (XM_006589357) and GmPAL3.1 (XM_003518532) at the amino acid level, respectively (Supplementary 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).

Figure 1
figure 1

The relative transcript levels of GmPAL2.1 at various time points post-treatment with SA, MeJA, ABA, GA, UV radiation, low temperature (4 °C), darkness and P. sojae ‘Suinong 10’ soybean plants. Fourteen-day-old plants were used for the treatments and analyses. The amplification of the soybean Actin (GmActin4) gene was used as an internal control to normalize all the data. The relative transcript levels of GmPAL2.1 were quantified compared with mock plants at the same time points. 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.

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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. 2C).

Figure 2
figure 2

Expression patterns of GmPAL2.1 in ‘Dongnong 50′ soybean plants. (A) The transcript abundance of GmPAL2.1 in various tissues of ‘Suinong 10’ soybean under normal condition. (B) The transcript abundance of GmPAL2.1 in various tissues of ‘Dongnong 50’ soybean under normal conditions. (C) The transcript levels of GmPAL2.1 in ‘Dongnong 50’ soybean under P. sojae treatment. The roots, stems, leaves and cotyledons were prepared from 14-day-old seedlings. The relative transcript levels of GmPAL2.1 were quantified compared with mock plants at the same time points. The amplification of the soybean Actin (GmActin4) gene was used as an internal control to normalize the data. For each sample, three biological replicates were analyzed with their respective three technical replicates. Bars indicate the standard error of the mean.

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Subcellular localization of the GmPAL2.1 protein

To determine the subcellular localization of the GmPAL2.1 protein, Arabidopsis protoplasts were examined to analyze the expression of GmPAL2.1-GFP fusion protein by the control of the 35S promoter. As shown in 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 GmPRP49, 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 organelles50. 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 biosynthesis51, 52.

Figure 3
figure 3

Subcellular localization analysis of the GmPAL2.1-GFP protein in Arabidopsis protoplasts. Subcellular localization was investigated in Arabidopsis protoplasts using a confocal microscope. The images of bright-field (B,F and J), the GFP fluorescence (green) only (A,E and I), the chlorophyll autofluorescence (red) only (C,G and K) and combined ones (D,H and L) are shown. All scale bars indicate 10 µm.

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Resistance to P. sojae in transgenic soybean plants

To investigate whether the relative expression of GmPAL2.1 in soybean has an effect on Phytophthora root rot resistance, six T2 transgenic soybean plants (namely, OX-1, OX-2, OX-29, RNAi-24, RNAi-27 and RNAi-32), of which the T1 transgenic soybean plants was confirmed through PCR and Southern hybridization (Supplementary Fig. 4), were selected. Real-time PCR was used to assay the pathogen response (Fig. 4B). As shown in Fig. 4A, six transgenic soybean plants showed that GmPAL2.1 enhances resistance to P. sojae after root infection. As shown in Fig. 5A, after 96 h of incubation with P. sojae, the cotyledons of the GmPAL2.1-RNAi soybean plants exhibit clear and large lesions, and the lesion area of the GmPAL2.1-OX soybean lines is obviously milder than that of non-transgenic and GmPAL2.1-RNAi soybean lines. The lesion area of the six transgenic lines were different than that of non-transgenic soybean plants at 96 h after inoculation (Fig. 5B). Moreover, the relative biomass of P. sojae in infected cotyledons after 24 h, 48 h and 96 h of incubation with zoospore suspensions of P. sojae was also analyzed by qPCR. The results indicate that the biomass of P. sojae, based on the transcript level of the P. sojae TEF1 gene, is significantly lower (P < 0.01) in the GmPAL2.1-OX soybean plants than that in non-transgenic and GmPAL2.1-RNAi soybean lines during the course of the infection (Fig. 5C). These results indicate that the expression of GmPAL2.1 in soybean plants plays an important role in resistance to P. sojae.

Figure 4
figure 4

GmPAL2.1 enhances resistance to P. sojae in transgenic soybean roots. (A) Disease symptoms on the roots of the transgenic lines and non-transgenic lines treated with P. sojae at 7days. (B) qRT-PCR was used to determine the relative abundance of GmPAL2.1 in three GmPAL2.1-overexpressing soybean plants (a) and three GmPAL2.1-RNAi soybean plants (b) Non-transgenic soybean plants were used as controls. For each sample, three biological replicates were analyzed 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.

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Figure 5
figure 5

GmPAL2.1 enhances resistance to P. sojae in transgenic soybean cotyledons. (A) Disease symptoms on the living cotyledons of transgenic lines and non-transgenic lines treated with P. sojae inoculum at 48 h and 96 h. (B) The relative lesion area of transgenic soybean cotyledon infection with P. sojae after 96 h. The average lesion area of each independent transgenic line (n = 3) was calculated, and their relative lesion areas are shown in columns after a comparison with the average lesion area on non-transgenic soybean. (C) Quantitative real-time PCR analysis of the P. sojae relative biomass in three GmPAL2.1-overexpressing soybean plants (a) and three GmPAL2.1-RNAi soybean plants (b) based on the transcript level of the P. sojae TEF1 gene. 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.

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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.

Figure 6
figure 6

PAL activity in non-transgenic and transgenic soybean leaves treated with P. sojae inoculum at 36 h. 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.

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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.

Figure 7
figure 7

The content of the isoflavone components and the relative content of glyceollins in seeds of transgenic and non-transgenic soybeans. (A) The daidzein levels in seeds of transgenic and non-transgenic soybeans. (B) The glycitein levels in seeds of transgenic and non-transgenic soybeans. (C) The genistein levels in seeds of transgenic and non-transgenic soybeans. (D) The relative content of glyceollins in the seeds of transgenic and non-transgenic soybeans. Non-transgenic soybean plants were used as controls. The experiment was performed using 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.

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The expression of SA marker genes and SA accumulation in transgenic soybeans

To test whether the GmPAL2.1 protein could regulate SA marker genes and SA accumulation, the content of SA and the expression of GmNPR1, GmPR1, GmPR2 and GmPR5 genes were analyzed. As shown in Fig. 8A, the SA accumulation in GmPAL2.1-OX transgenic soybean leaves is significantly higher than that of non-transgenic plants and GmPAL2.1-RNAi soybean plants. The transcripts of GmNPR1, GmPR1 and GmPR5 changed significantly in all the transgenic lines compared with the control (Fig. 8B,C,D). Although there is difference in the content of SA between non-transgenic plants and GmPAL2.1-RNAi soybean plants, it does not reach a significant level.

Figure 8
figure 8

The content of SA and expression analysis of GmNPR1, GmPR1 and GmPR5 in transgenic and non-transgenic soybeans. The content of SA in leaves of transgenic and non-transgenic soybeans. (B) The relative transcript abundance of GmNPR1 (NM_001251745) in transgenic and non-transgenic soybeans. (C) The relative transcript abundance of GmPR1 (AF136636) in transgenic and non-transgenic soybeans. (D) The relative transcript abundance of GmPR5 (M21297) in transgenic and non-transgenic soybeans. The amplification of the soybean Actin (GmActin4) gene was used as an internal control to normalize all the data. 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).

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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 199136. 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 pathogens9, 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 susceptibility55. 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 plants3, 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 resistance63. PR1, PR2 and PR5 are considered to be the effector genes for systemic acquired resistance (SAR), which was mediated by SA64, 65. It has 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 genes66. 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 metabolism67. The gateway from primary metabolism into phenylpropanoid metabolism is the deamination of L-phenylalanine by PAL to form trans-cinnamic acid68. 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 stresses69,70,71. 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 pea72, tomato73, cucumber74, and lupine75, 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 nm77. Some other similar spectrophotometric assays78 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 activity79, 80. In our study, the PAL activity in the leaf extract was determined by spectrophotometry following the method described by Song and Wang81. 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 defense82, 83. To investigate whether the isoflavone content changes in the transgenic soybean lines, six T2 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 seed23. IFR is an enzyme involved in the synthesis of glyceollins from daidzein and the daidzein content greatly reduced in GmIFR-overexpression soybean seeds84. The reason is maybe PAL produces trans-cinnamic acid, which serves as a precursor for the synthesis of all phenylpropanoids, including isoflavones3. 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 stress85,86,87,88. Glyceollins represent another group of phytoalexins whose biosynthesis is increased in soybean in response to various stress signals, such as fungal infection87, 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 soybean89. As isoflavonoid-type phytoalexins, glyceollins have exhibited antifungal activity90. Glyceollins have a significant antimicrobial effect against Phytophthora capsici and Sclerotinia sclerotiorum 12, 14 and exhibit resistance to Phytophthora megasperma var. sojae in soybean91,92,93. Previous research suggests that the biosynthesis of glyceollin is via the isoflavonoid branch of the phenylpropanoid pathway94. More specifically, the glyceollin biosynthetic pathway includes the enzymes involved in phenylpropanoid metabolism, flavonoid/isoflavonoid synthesis and those dedicated to the biosynthesis of pterocarpan phytoalexins95. Thus, PAL involves the synthesis of glyceollins, which are a mixture of structurally related pterocarpans7, 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, China97, 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.

For abiotic treatments, soybean leaves were each subjected to seven different stresses including UV radiation, low temperature (4 °C), dark treatment, ABA, SA, GA and MeJA. For UV treatment, the seedlings were exposed for 0, 3, 6, 9, 12, 24 or 36 h to an ultraviolet lamp. For low-temperature (4 °C) and dark treatments, soybean seedlings were incubated separately in a cold chamber and a darkroom for 0, 6, 12, 24, 36, 48 or 72 h. Untreated leaves of soybean were used as controls. ABA (50 µM), SA (0.2 mM), GA (250 mg L−1) and MeJA (100 µM) were dissolved in 0.01% Tween 20 and sprayed onto young leaves for 0, 1, 3, 6, 9, 12 or 24 h. The control leaves were sprayed with an equivalent volume of 0.01% (v/v) Tween 20. For P. sojae treatment, the soybean plants were infected with P. sojae race 1 using the procedure of Ward et al.99 and Morris et al.100 with minor modifications. Zoospores were developed following the method described by Ward et al.99, and the concentration was estimated using a hemocytometer to approximately 1 × 105 spores mL−1. The unifoliolate leaves were harvested at 0, 1, 3, 6, 9, 12, 20, 24, 30, 36, 48 and 72 h after inoculation.

‘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 software101. 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 TouchTM 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 Lin102. 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).

Vector construction and transformation of soybean

The full length GmPAL2.1 coding region was amplified with two specific primers, GmPAL2.1-F and GmPAL2.1-R (Supplementary Table 1), for transformation assays. The following PCR cycling parameters were used: 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min 30 s and a final cycle at 72 °C for 8 min. To overexpress the GmPAL2.1 gene, the open reading frame of GmPAL2.1 was cloned in frame into the pCAMBIA3301 vector. To suppress the gene expression of GmPAL2.1, GmPAL2.1-attB-F and GmPAL2.1-attB-R (Supplementary 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 (T1) 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 (T2) 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 Thorne107 with minor modifications. The roots and living cotyledons of three T2 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 T2 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 Wang81, 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 method109 with bovine serum albumin as the protein standard.

Isoflavone and glyceollin analysis

Approximately 0.1 g sample of seeds from T2 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 T2 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.