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Phytophthora root rot, caused by Phytophthora sojae is a destructive disease of soybean ( Glycine max ) worldwide. We previously confirmed that the bHLH transcription factor GmPIB1 ( P. sojae -inducible bHLH transcription factor) reduces accumulation of reactive oxygen species (ROS) in cells by inhibiting expression of the peroxidase-related gene GmSPOD thus improving the resistance of hairy roots to P. sojae . To identify proteins interacting with GmPIB1 and assess their participation in the defense response to P. sojae , we obtained transgenic soybean hairy roots overexpressing GmPIB1 by Agrobacterium rhizogenes mediated transformation and examined GmPIB1 protein-protein interactions using immunoprecipitation combined with mass spectrometry. We identified 392 proteins likely interacting with GmPIB1 and selected 20 candidate genes, and only 26S proteasome regulatory subunit GmPSMD (Genbank accession no. XP_014631720) interacted with GmPIB1 in luciferase complementation and pull-down experiments and yeast two-hybrid assays. Overexpression of GmPSMD ( GmPSMD- OE) in soybean hairy roots remarkably improved resistance to P. sojae and RNA interference of GmPSMD ( GmPSMD -RNAi) increased susceptibility. In addition, accumulation of total ROS and hydrogen peroxide (H 2 O 2 ) in GmPSMD- OE transgenic soybean hairy roots were remarkably lower than those of the control after P. sojae infection. Moreover, in GmPSMD -RNAi transgenic soybean hairy roots, H 2 O 2 and the accumulation of total ROS exceeded those of the control. There was no obvious difference in superoxide anion (O 2 - ) content between control and transgenic hairy roots. Antioxidant enzymes include peroxidase (POD), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) are responsible for ROS scavenging in soybean. The activities of these antioxidant enzymes were remarkably higher in GmPSMD -OE transgenic soybean hairy roots than those in control, but were reduced in GmPSMD -RNAi transgenic soybean hairy roots. Moreover, the activity of 26S proteasome in GmPSMD -OE and GmPIB1 -OE transgenic soybean hairy roots was significantly higher than that in control and was significantly lower in PSMD -RNAi soybean hairy roots after P. sojae infection. These data suggest that GmPSMD might reduce the production of ROS by improving the activity of antioxidant enzymes such as POD, SOD, GPX, CAT, and GmPSMD plays a significant role in the response of soybean to P. sojae . Our study reveals a valuable mechanism for regulation of the pathogen response by the 26S proteasome in soybean., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Wang, Liu, Pang, Zhang, Zhao, Ning, Song, Liu, He, Wei, Wu, Liu, Xu and Zhang.)

Phytophthora root and stem rot, a destructive disease of soybean [ Glycine max (L.) Merr.], is caused by the oomycete Phytophthora sojae . However, how the disease resistance mechanisms of soybean respond to P. sojae infection remains unclear. Previously, we showed that GmWRKY31, which interacts with a sucrose non-fermenting-1(SNF1)-related protein kinase (SnRK), enhances resistance to P. sojae in soybean. Here, we report that the membrane-localized SnRK GmSnRK1.1 is involved in the soybean host response to P. sojae . The overexpression of GmSnRK1.1 ( GmSnRK1.1 -OE) increased soybean resistance to P. sojae , and the RNA interference (RNAi)-mediated silencing of GmSnRK1.1 ( GmSnRK1.1 -R) reduced resistance to P. sojae . Moreover, the activities and transcript levels of the antioxidant enzymes superoxide dismutase and peroxidase were markedly higher in the GmSnRK1.1 -OE transgenic soybean plants than in the wild type (WT), but were reduced in the GmSnRK1.1 -R plants. Several isoflavonoid phytoalexins related genes GmPAL , GmIFR , Gm4CL and GmCHS were significantly higher in "Suinong 10" and GmSnRK1.1 -OE lines than these in "Dongnong 50," and were significantly lower in GmSnRK1.1 -R lines. In addition, the accumulation of salicylic acid (SA) and the expression level of the SA biosynthesis-related gene were significantly higher in the GmSnRK1.1 -OE plants than in the WT and GmSnRK1.1 -R plants, moreover, SA biosynthesis inhibitor treated GmSnRK1.1 -R lines plants displayed clearly increased pathogen biomass compared with H 2 O-treated plants after 24 h post-inoculation. These results showed that GmSnRK1.1 positively regulates soybean resistance to P. sojae , potentially functioning via effects on the expression of SA-related genes and increased accumulation of SA.

Although the effect of elevated CO 2 (eCO 2 ) on soybean yield has been well documented, few studies have addressed seed quality, particularly at the fresh edible (R6) and mature stages (R8). Under the current global scenario of increasing CO 2 levels, this potentially threatens the nutritional content and quality of food crops. Using four soybean cultivars, we assessed the effects of eCO 2 on the concentrations of crude protein, crude oil, and isoflavones and analyzed the changes in free amino acids, fatty acids, and mineral elements in seeds. At R6, eCO 2 had no influence on soybean seed protein and oil concentrations. At R8, eCO 2 significantly decreased seed protein concentration but increased seed oil concentration; it also significantly decreased total free amino acid concentration. However, at the same stage, the proportion of oleic acid (18:1) among fatty acids increased in response to eCO 2 in the cultivars of Zhongke-maodou 2 (ZK-2) and Zhongke-maodou 3 (ZK-3), and a similar trend was found for linoleic acid (18:2) in Zhongke-maodou 1 (ZK-1) and Hei-maodou (HD). Total isoflavone concentrations increased significantly at both the R6 and R8 stages in response to eCO 2 . Compared with ambient CO 2 , the concentrations of K, Ca, Mg, P, and S increased significantly under eCO 2 at R6, while the Fe concentration decreased significantly. The response of Zn and Mn concentrations to eCO 2 varied among cultivars. At R8 and under eCO 2 , Mg, S, and Ca concentrations increased significantly, while Zn and Fe concentrations decreased significantly. These findings suggest that eCO 2 is likely to benefit from the accumulation of seed fat and isoflavone but not from that of protein. In this study, the response of seed mineral nutrients to eCO 2 varied between cultivars.

Nitrogen deficiency limits crop performance under elevated CO 2 (eCO 2 ), depending on the ability of plant N uptake. However, the dynamics and redistribution of N 2 fixation, and fertilizer and soil N use in legumes under eCO 2 have been little studied. Such an investigation is essential to improve the adaptability of legumes to climate change. We took advantage of genotype-specific responses of soybean to increased CO 2 to test which N-uptake phenotypes are most strongly related to enhanced yield. Eight soybean cultivars were grown in open-top chambers with either 390 ppm (aCO 2 ) or 550 ppm CO 2 (eCO 2 ). The plants were supplied with 100 mg N kg -1 soil as 15 N-labeled calcium nitrate, and harvested at the initial seed-filling (R5) and full-mature (R8) stages. Increased yield in response to eCO 2 correlated highly ( r = 0.95) with an increase in symbiotically fixed N during the R5 to R8 stage. In contrast, eCO 2 only led to small increases in the uptake of fertilizer-derived and soil-derived N during R5 to R8, and these increases did not correlate with enhanced yield. Elevated CO 2 also decreased the proportion of seed N redistributed from shoot to seeds, and this decrease strongly correlated with increased yield. Moreover, the total N uptake was associated with increases in fixed-N per nodule in response to eCO 2 , but not with changes in nodule biomass, nodule density, or root length.

Phytophthora root and stem rot caused by the oomycete pathogen Phytophthora sojae is a destructive disease of soybean worldwide. Plant dirigent proteins (DIR) are proposed to have roles in biosynthesis of either lignan or lignin-like molecules, and are important for defense responses, secondary metabolism, and pathogen resistance. In the present work, a novel DIR gene expressed sequence tag is identified as up-regulated in the highly resistant soybean cultivar 'Suinong 10' inoculated with P. sojae . The full length cDNA is isolated using rapid amplification of cDNA ends, and designated GmDIR22 (GenBank accession no. HQ_993047). The full length GmDIR22 is 789 bp and contains a 567 bp open reading frame encoding a polypeptide of 188 amino acids. The sequence analysis indicated that GmDIR22 contains a conserved dirigent domain at amino acid residues 43-187. The quantitative real-time reverse transcription PCR demonstrated that soybean GmDIR22 mRNA is expressed most highly in stems, followed by roots and leaves. The treatments with stresses demonstrated that GmDIR22 is significantly induced by P. sojae and gibberellic acid (GA 3 ), and also responds to salicylic acid, methyl jasmonic acid, and abscisic acid. The GmDIR22 is targeted to the cytomembrane when transiently expressed in Arabidopsis protoplasts. Moreover, The GmDIR22 recombinant protein purified from Escherichia coli could effectively direct E -coniferyl alcohol coupling into lignan (+)-pinoresinol. Accordingly, the overexpression of GmDIR22 in transgenic soybean increased total lignan accumulation. Moreover, the lignan extracts from GmDIR22 transgenic plants effectively inhibits P. sojae hyphal growth. Furthermore, the transgenic overexpression of GmDIR22 in the susceptible soybean cultivar 'Dongnong 50' enhances its resistance to P. sojae . Collectively, these data suggested that the primary role of GmDIR22 is probably involved in the regulation of lignan biosynthesis, and which contributes to resistance to P. sojae .

Phytophthora root and stem rot of soybean [ Glycine max (L.) Merr.] caused by the oomycete Phytophthora sojae , is a destructive disease worldwide. The molecular mechanism of the soybean response to P. sojae is largely unclear. We report a novel WRKY transcription factor (TF) in soybean, GmWRKY31, in the host response to P. sojae . Overexpression and RNA interference analysis demonstrated that GmWRKY31 enhanced resistance to P. sojae in transgenic soybean plants. GmWRKY31 was targeted to the nucleus, where it bound to the W-box and acted as an activator of gene transcription. Moreover, we determined that GmWRKY31 physically interacted with GmHDL56, which improved resistance to P. sojae in transgenic soybean roots. GmWRKY31 and GmHDL56 shared a common target GmNPR1 which was induced by P. sojae . Overexpression and RNA interference analysis demonstrated that GmNPR1 enhanced resistance to P. sojae in transgenic soybean plants. Several pathogenesis-related ( PR ) genes were constitutively activated, including GmPR1a , GmPR2 , GmPR3 , GmPR4 , GmPR5a , and GmPR10 , in soybean plants overexpressing GmNPR1 transcripts. By contrast, the induction of PR genes was compromised in transgenic GmNPR1 -RNAi lines. Taken together, these findings suggested that the interaction between GmWRKY31 and GmHDL56 enhances resistance to P. sojae by regulating defense-related gene expression in soybean.