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

  • ️Sun Jan 01 2017

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

Chuanzhong Zhang et al. Sci Rep. 2017.

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

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.

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.

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.

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.

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.

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.

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.

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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References

    1. Jones JDG, Dangl JL. The plant immune system. Nature. 2006;444:323–329. doi: 10.1038/nature05286. - DOI - PubMed
    1. Pellegrini L, Rohfritsch O, Fritig B, Legrand M. Phenylalanine ammonia-lyase in tobacco. Molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor. Plant Physiol. 1994;106:877–886. doi: 10.1104/pp.106.3.877. - DOI - PMC - PubMed
    1. Dixon RA, Paiva NL. Stress-Induced Phenylpropanoid Metabolism. Plant Cell. 1995;7:1085–1097. doi: 10.1105/tpc.7.7.1085. - DOI - PMC - PubMed
    1. Liu R, Xu S, Li J, Yl, Lin Z. Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis. Plant Cell Rep. 2006;25:705–710. doi: 10.1007/s00299-005-0072-7. - DOI - PubMed
    1. Mahesh V, et al. Isolation and genetic mapping of a Coffea canephora phenylalanine ammonia-lyase gene (CcPAL1) and its involvement in the accumulation of caffeoyl quinic acids. Plant Cell Rep. 2006;25:986–992. doi: 10.1007/s00299-006-0152-3. - DOI - PubMed

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