Huaqin Kuang

The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR-Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR-Cas9 as a mutant screening tool. Here, we report a pooled CRISPR-Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR-Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1-1/1-2/1-3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.

Beany flavor induced by three lipoxygenases (LOXs, including LOX1, LOX2, and LOX3) restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) strategy targeting three GmLox genes (GmLox1, GmLox2, and GmLox3) was applied and 60 T0 positive transgenic plants were generated, carrying combinations of sgRNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1gmlox2gmlox3 triple mutants and GmLox-40 was a gmlox1gmlox2 double mutant. Sequencing of T1 mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T2 generation of lipoxygenase-free mutant lines (GmLox-28 and GmLox-60). These transgene- and lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.

Soybean (Glycine max) is a major source of plant-based protein for people worldwide, providing a healthy, affordable, and environmentally friendly alternative to animal-based protein (Montgomery, 2003Montgomery K.S. Soy protein.J. Perinat. Educ. 2003; 12: 42-45https://doi.org/10.1624/105812403X106946Crossref Google Scholar). It has been estimated that there are currently over 12 000 food products that contain soy protein (Montgomery, 2003Montgomery K.S. Soy protein.J. Perinat. Educ. 2003; 12: 42-45https://doi.org/10.1624/105812403X106946Crossref Google Scholar). Different food applications of soy protein require distinct properties that affect its function and sensory features. For instance, the sensory quality of soymilk is significantly affected by protein emulsibility, whereas tofu quality is determined by protein-gelling ability (Kinsella, 1979Kinsella J.E. Functional properties of soy proteins.JAOCS J. Am. Oil Chem. Soc. 1979; 56: 242-258https://doi.org/10.1007/BF02671468Crossref Scopus (1128) Google Scholar). Improving the functional properties of soybeans has long been a major aim in the soy food industry, yet it can be difficult to balance processing methods to achieve desired properties while retaining nutritional value and controlling cost (Kinsella, 1979Kinsella J.E. Functional properties of soy proteins.JAOCS J. Am. Oil Chem. Soc. 1979; 56: 242-258https://doi.org/10.1007/BF02671468Crossref Scopus (1128) Google Scholar; Rubio et al., 2020Rubio N.R. Xiang N. Kaplan D.L. Plant-based and cell-based approaches to meat production.Nat. Commun. 2020; 11: 6276https://doi.org/10.1038/s41467-020-20061-yCrossref PubMed Scopus (180) Google Scholar). The functional properties of soy protein are affected by storage protein composition, particularly β-conglycinin (7S) with high emulsibility and glycinin (11S) with high gelling abilities (Kinsella, 1979Kinsella J.E. Functional properties of soy proteins.JAOCS J. Am. Oil Chem. Soc. 1979; 56: 242-258https://doi.org/10.1007/BF02671468Crossref Scopus (1128) Google Scholar). Mutations in 7S or 11S subunits can affect the functional properties of soy protein (Poysa et al., 2006Poysa V. Woodrow L. Yu K. Effect of soy protein subunit composition on tofu quality.Food Res. Int. 2006; 39: 309-317https://doi.org/10.1016/j.foodres.2005.08.003Crossref Scopus (135) Google Scholar). However, breeding an elite variety with pyramided 7S/11S mutations would be time consuming and, with uncertain effects on seed-quality traits, is unlikely to meet the numerous and rapidly changing demands of the food industry (Montgomery, 2003Montgomery K.S. Soy protein.J. Perinat. Educ. 2003; 12: 42-45https://doi.org/10.1624/105812403X106946Crossref Google Scholar). The development of CRISPR-Cas9 technology enables rapid and precise editing of multiple genes in crops, including soybean (Cai et al., 2018Cai Y. Chen L. Liu X. Guo C. Sun S. Wu C. Jiang B. Han T. Hou W. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.Plant Biotechnol. J. 2018; 16: 176-185https://doi.org/10.1111/pbi.12758Crossref PubMed Scopus (215) Google Scholar; Li et al., 2020Li J. Li H. Chen J. Yan L. Xia L. Toward precision genome editing in crop plants.Mol. Plant. 2020; 13: 811-813https://doi.org/10.1016/j.molp.2020.04.008Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In this study, we sought to combine two soybean varieties created using genome editing, one mutant lacking 7S subunits (ultra-low 7S:11S ratio) and another lacking 11S subunits (ultra-high 7S:11S ratio). We reasoned that the seeds of the two varieties could be mixed in different proportions to allow the precise custom formulation of functional properties of soy protein for food processing. The 7S globulin is a heterotrimer composed of three subunits (α, α′, and β) encoded by 7 genes, and the 11S globulin is a heterohexamer of 5 subunits (group I: A1aB1b/A1bB2 and A2B1a, group II: A3A4 and A5A4B3, and group III: Gy7) encoded by 5 genes. To facilitate efficient multiplex editing of more than 5 target loci, we developed a soybean-optimized multiplex CRISPR vector, designated pGES401, based on the single-transcript-unit system (Tang et al., 2019Tang X. Ren Q. Yang L. Bao Y. Zhong Z. He Y. Liu S. Qi C. Liu B. Wang Y. et al.Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing.Plant Biotechnol. J. 2019; 17: 1431-1445https://doi.org/10.1111/pbi.13068Crossref PubMed Scopus (100) Google Scholar). We then constructed two vectors, pGES401-7S to target the seven 7S subunit genes (Figure 1A) and pGES401-11S to target the five 11S subunit genes (Figure 1B; Supplemental Figure 1). The vectors were individually transformed into an elite food-grade cultivar, QiHuang34 (QH34), by Agrobacterium-mediated transformation (Bai et al., 2020Bai M. Yuan J. Kuang H. Gong P. Li S. Zhang Z. Liu B. Sun J. Yang M. Yang L. et al.Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean.Plant Biotechnol. J. 2020; 18: 721-731Crossref PubMed Scopus (91) Google Scholar). In the T0 transformants, the genotypes of target loci were evaluated by Hi-TOM sequencing (Liu et al., 2019Liu Q. Wang C. Jiao X. Zhang H. Song L. Li Y. Gao C. Wang K. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems.Sci. China Life Sci. 2019; 62: 1-7https://doi.org/10.1007/s11427-018-9402-9Crossref PubMed Scopus (167) Google Scholar). Simultaneous frameshift editing of all target genes was identified in 3 of the 8 pGES401-7s-null events and 5 of the 13 pGES401-11s-null events (Figures 1C and 1D). Transgene-free septuple 7S and quintuple 11s mutant lines were identified among T2 progeny, designated as 7s-null, and 11s-null, respectively (Figure 1E). qRT–PCR and SDS–PAGE showed that the expression of the targeted 7S or 11S subunits was largely abolished in the mutant seeds, and genes encoding other storage proteins accumulated due to a compensatory effect (Figures 1F and 1G). We next examined the seed yield and quality traits of the T3 mutant lines and found that seed yield per plant and oil and protein content in the 7s-null or 11s-null lines were similar to wild type (WT) (Figure 1H; Supplemental Figure 2). Therefore, seed traits other than the 7S/11S composition were apparently not affected by genome editing. We then tested whether the protein functional properties were affected in the mutants and whether custom formulation is feasible by proportional mixing 7s-null and 11s-null mutant seeds. We first monitored the emulsion stability of the soybean protein isolates of WT, 7s-null, 11s-null, or proportionally mixed samples using a LUMiSizer, in which the slopes of the curves of the integral light-transmission intensity versus time are negatively associated with the stabilities of the emulsions (Figure 1I). Compared with the QH34 WT samples, the emulsions prepared from 11s-null mutants exhibited greatly enhanced stabilities, while 7s-null emulsions had the lowest stabilities (Figure 1I). Consistent with our hypothesis, the 2:1, 1:1, or 1:2 mixtures of 7s-null:11s-null seeds have intermediate or higher emulsion stabilities (Figure 1I). Consistently, the variation in emulsions stabilities was visually distinguishable after standing for 30 days at room temperature (Figure 1J). Analysis using a laser particle-size analyzer revealed that the variations in emulsion stability were associated with differences in their particle-size distribution (Figure 1K). The gelling ability of soy protein gels was assessed using a texture analyzer to monitor the textural characteristics (Supplemental Figure 3). The gelling hardness, springiness, gumminess, and chewiness were significantly increased in the 7s-null compared with WT protein gels, while they were largely decreased in 11s-null protein gels. The 1:2, 1:1, or 2:1 seed mixtures exhibited gelling abilities that were intermediate to the 7s-null and 11s-null lines (Figure 1L). In summary, we demonstrated a solution to custom formulize the functional properties of soy protein by using a combination of two multiplex genome-edited varieties, without apparent effects on other seed quality traits. Such a solution could be useful in producing traditional and novel soy foods (e.g., plant-based meat and milk analogs), which require distinct functional properties (Rubio et al., 2020Rubio N.R. Xiang N. Kaplan D.L. Plant-based and cell-based approaches to meat production.Nat. Commun. 2020; 11: 6276https://doi.org/10.1038/s41467-020-20061-yCrossref PubMed Scopus (180) Google Scholar). In addition, the individual 7s-null and 11s-null varieties might also be valuable for purification of 11S or 7S protein isolates as emulsifiers or food supplements (Figure 1M). This work was supported by the ChuYing scholar program of Fujian province to Y.G.

Abstract Legumes can utilize atmospheric nitrogen via symbiotic nitrogen fixation, but this process is inhibited by high soil inorganic nitrogen. So far, how high nitrogen inhibits N 2 fixation in mature nodules is still poorly understood. Here we construct a co-expression network in soybean nodule and find that a dynamic and reversible transcriptional network underlies the high N inhibition of N 2 fixation. Intriguingly, several NAC transcription factors (TFs), designated as Soybean Nitrogen Associated NAPs (SNAPs), are amongst the most connected hub TFs. The nodules of snap1/2/3/4 quadruple mutants show less sensitivity to the high nitrogen inhibition of nitrogenase activity and acceleration of senescence. Integrative analysis shows that these SNAP TFs largely influence the high nitrogen transcriptional response through direct regulation of a subnetwork of senescence-associated genes and transcriptional regulators. We propose that the SNAP-mediated transcriptional network may trigger nodule senescence in response to high nitrogen.