New strategy to enhance soybean pod shattering resistance with quadruple <scp> <i>GmMYB26</i> </scp> mutations

Reducing harvest loss in soybean (Glycine max (L.) Merr.) is critical for tackling the growing issue of food insufficiency and for conserving natural resources used in fertilizer production. Although global soybean production has doubled in the last two decades, reaching 396.95 million metric tons by 2023/2024 (United States Department of Agriculture, 2024), an annual increase of 2.4% is required to feed the expected global population by 2050 (Ray et al., 2013). Since no additional fertilizers are required, reducing harvest loss may be a sustainable alternative to approaches focusing on increasing yields. Pod shattering-resistant soybean could ameliorate this issue, as pod shattering causes significant harvest loss during mechanization, especially in arid and/or hot climates (Parker et al., 2021; Lyu et al., 2023). During domestication, soybean acquired pod shattering resistance by thickening the fiber cap over the dehiscence zone, regulated by GmSHAT1-5 and by reducing the pod torsion, regulated by GmPdh1 (Dong et al., 2014; Funatsuki et al., 2014). However, cultivars with pod shattering-resistant alleles at GmSHAT1-5 and GmPdh1 still experience 19.8% harvest loss, even in humid countries (Yamada et al., 2017). Although GmSh1, which represses GmSHAT1-5 expression, and GmNST1A, a paralog of GmSHAT1-5, also appear to be involved in pod shattering, no other effective gene loci have been identified, even in large-scale genome-wide association studies (Hu et al., 2019; Zhang & Singh, 2020; Li et al., 2024). However, recent studies revealed that the cultigens of the genera Vigna and Phaseolus acquired pod shattering resistance differently from soybean (Takahashi et al., 2020; Parker et al., 2021). These legumes harbor nonfunctional mutations in the MYB26 homolog, which in Arabidopsis functions as an upstream transcription factor in secondary cell wall biosynthesis (Zhao & Dixon, 2011). Truncated versions of a single locus of the MYB26 homolog appear to confer superior pod shattering resistance by reducing lignin in the pod sclerenchyma, which generates pod torsion in azuki bean (Vigna angularis Ohwi et H. Ohashi) and cowpea (Vigna unguiculata (L.) Walp.) (Takahashi et al., 2020). Although the resistant allele of GmPdh1 also reduces pod torsion, it does not reduce lignin abundance (Funatsuki et al., 2014). Thus, the mechanisms of pod shattering resistance differ between soybean and other legumes, highlighting a new strategy for their improvement. Here, we adopted a reverse genetic approach to obtain GmMYB26 mutants. Since there are four GmMYB26 homologs in the soybean genome, we developed a quadruple mutant that showed significantly enhanced pod shattering resistance, thereby reproducing the pod shattering resistance mechanism of the genera Vigna and Phaseolus. Based on these results, we propose a new strategy to develop pod shattering-resistant soybean by pyramiding the GmMYB26-resistant alleles with the existing resistant alleles of the genes. Four homologs of Arabidopsis MYB26 exist in the soybean genome (Wm82.a6.v1), whereas two exist in that of azuki bean (Vangularis_v1.a1). GmMYB26-13g and GmMYB26-15g, from soybean, were homologs of azuki bean VaMYB26a, while GmMYB26-7g and GmMYB26-8g in soybean were homologs of azuki bean VaMYB26b (Fig. 1a; Supporting Information Table S1). We developed a quadruple mutant by crossing each single nonsense mutant (see also the detailed Materials and Methods in Methods S1), as four GmMYB26 genes without significant defects showed the highest expression in the pods (Fig. 1b; Table S2). First, we selected each single nonsense mutant for four GmMYB26 genes from a population mutagenized by ethyl methanesulfonate to ‘Enrei’ with pod shattering-resistant alleles at GmSHAT1-5 and GmSh1 genes and susceptible (or nonresistant) alleles at the GmPdh1 gene (Tsuda et al., 2015; Fig. S1; Tables S2, S3). The nonsense mutations were located in the first exons of GmMYB26-7g, GmMYB26-13g, and GmMYB26-15g and in the third exon of GmMYB26-8g. We then created double mutant 7g/8g by crossing GmMYB26-7g and GmMYB26-8g before creating double mutant 13g/15g by crossing GmMYB26-7g and GmMYB26-8g. Finally, we obtained a quadruple mutant after crossing the 7g/8g and 13g/15g mutants, selfing, and selecting the homozygous mutant alleles. In the following experiments, we used the quadruple mutant, 7g/8g mutant, 13g/15g mutant, wild-type (WT) ‘Enrei’, and ‘Enrei-no-sora’ (NIL-pdh1) – a near-isogenic line of ‘Enrei’ with the resistant allele of GmPdh1 replaced. Detailed information can be found in Methods S1. The percentage of unshattered pods was recorded over time across two consecutive treatments: air-drying at 35–45% relative humidity and silica gel treatment at 10–12% relative humidity. We used pods grown from three independent cultivations: two from glasshouses and one from field cultivation (Table S4; Fig. S2). For the first glasshouse products, the percentages of unshattered pods were 0%, 30%, 67%, and 100% in the WT, 7g/8g mutant, 13g/15g mutant, and quadruple mutant plants, respectively, under the air-drying treatment (Fig. S3; Table S5). Under subsequent silica gel treatment, the quadruple mutant retained 83% unshattered pods, whereas those of the others were completely shattered. For the second glasshouse and field products, NIL-pdh1 was added to compare the effects of GmPdh1 and GmMYB26. For the second glasshouse products, the quadruple mutant and NIL-pdh1 showed significantly higher pod shattering resistance than the WT, while the two double mutants showed no significant difference from the WT under the air-drying treatment (Fig. S3; Table S5). Under subsequent silica gel treatment, the quadruple mutant retained 38% of its original number, whereas those of NIL-pdh1 were completely shattered. For the field products, the quadruple mutant, 7g/8g mutant, and NIL-pdh1 showed significantly higher pod shattering resistance than the WT, while the 13g/15g mutant showed no significant difference from the WT due to large variances under the air-drying treatment (Fig. 1d; Table S5). Under subsequent silica gel treatment, the quadruple mutant and NIL-pdh1 retained 88% and 44% unshattered pods, respectively. These results suggest that the GmMYB26 quadruple mutations provide superior pod shattering resistance compared to the resistant GmPdh1 allele. Although the resistant GmPdh1 allele showed high pod shattering resistance at 30% relative humidity or with high temperature treatments (Funatsuki et al., 2014), the resistance was not sufficient at lower humidity conditions (Fig. 1d). The GmMYB26 double mutations may provide moderate pod shattering resistance, although this varies greatly according to the environment. We resolved the physical properties of the underlying pod shattering resistance using GmMYB26 compared with GmPdh1. For the first glasshouse product, the pod torsion rate (the degree of pod torsion per pod length after desiccation) of the quadruple mutant was significantly lower than that of the others (Fig. S4). For the second glasshouse and field products, the pod torsion rates of the quadruple mutant, 13g/15g mutant, and NIL-pdh1 were significantly lower than those of the WT (Figs 1e, S4). Lignin layer thickness was investigated by microscopy of mature pod sections, showing that the quadruple mutant was 59% thinner for the first glasshouse products and 48% thinner for the field products than the WT (Figs S5, S6). The two double mutants were not significantly different from the WT in the first glasshouse products, whereas the 7g/8g mutant was significantly different from the WT in the field products (Figs S5, S6). For our first experiment to determine lignin content, we used the thioglycolic acid method, which allows analysis from a single pod. For glasshouse products, lignin content was 12% lower in the 13g/15g mutant and 60% lower in the quadruple mutant than in the WT, although there was no significant difference in the 7g/8g mutant (Fig. S7). For the field products, the quadruple mutant showed significantly lower lignin content than the others, including NIL-pdh1 (Fig. 1f). These results suggested that both GmMYB26 and GmPdh1 confer pod shattering resistance by reducing pod torsion, albeit the mechanisms differ, with GmMYB26 being involved in pod lignin biosynthesis while GmPdh1 was not. Double mutants showed partially reduced pod torsion and lignin content depending on the environment, similar to pod shattering resistance behavior. To elucidate the effect of GmMYB26, we compared the transcriptomic profiles of young pods of the WT and quadruple mutants. The quadruple mutant had 61 upregulated and 32 downregulated differentially expressed genes. Among the 32 downregulated genes, there were homologs for six laccases, three peroxidases, one cellulose synthase, and one trichome birefringence-like gene (Fig. 2a; Table S6). Because these homologs are involved in lignin, cellulose, or hemicellulose biosynthesis, we performed detergent fiber analysis of mature pods as our second trial to determine the lignin, cellulose, and hemicellulose contents. The quadruple mutant had 43% less lignin, 14% less cellulose, and 30% less hemicellulose than the WT (Fig. 2b). In Arabidopsis, laccases and peroxidases play a role in lignin assembly by catalyzing the oxidative polymerization of two major monolignols: coniferyl alcohol and sinapyl alcohol (Zhao et al., 2013). Thus, when laccase and peroxidase expression is downregulated, monolignols accumulate excessively in cells. Liquid chromatography-mass spectrometry of young pods showed that the quadruple mutant had 76% more coniferyl alcohol than the WT, whereas there was no significant difference in sinapyl alcohol (Fig. 2c; Methods S1; Table S7 for detailed monolignol analysis). Such quantitative changes in secondary metabolites are due to the downregulated genes in the GmMYB26 quadruple mutant that may be involved in pod shattering resistance. To verify the effects of GmMYB26 on other organs, we observed plants, particularly their stem characteristics. The plant architecture of the WT and quadruple mutants showed no major differences until flowering in both glasshouse and field products (Figs S8, S9). However, drooping petioles were observed in quadruple mutants at the R6 stage (Fig. S8c,d). We compared the lignin thickness and content of the first node of the main stem at the flowering stage and found no significant difference in any combination (Figs 2d, S10). Meanwhile, detergent fiber analysis of the 1st–10th nodes of the main stem revealed that the quadruple mutant had 12% more lignin and 14% less cellulose than the WT, with no difference in hemicellulose (Fig. 2b). These results suggest that GmMYB26 is involved in stem lignin and cellulose biosynthesis. Here, we demonstrated that MYB26 homologs are appropriate targets for enhancing pod shattering resistance in soybean and possibly other legume crops. Our reverse genetic approach successfully demonstrated that nonsense mutations significantly reduced pod wall fiber abundance, thus improving pod shattering resistance. In particular, the GmMYB26 quadruple mutant conferred superior pod shattering resistance compared with the widely used resistant allele of GmPdh1. As GmMYB26 genes may have redundant functions in pod sclerenchymal formation, the degree of pod shattering resistance showed some correlation with the number or combination of mutated loci. We also validated for the first time that MYB26 homologs are indispensable for pod sclerenchymal development in legumes, including lignin, cellulose, and hemicellulose biosynthesis. Lignin is assembled via the oxidative polymerization of two major monolignols, coniferyl alcohol and sinapyl alcohol (Zhao et al., 2013), after the AtMYB26 gene activates laccase and peroxidase, respectively, via other transcription factors to catalyze monolignol polymerization (Zhao & Dixon, 2011). GmMYB26 quadruple mutations suppress the expression of laccase and peroxidase homologs and the overaccumulation of monolignol in young pods. Thus, GmMYB26 initiates monolignol polymerization by activating laccase and peroxidase expression such as AtMYB26. Additionally, the GmMYB26 quadruple mutant showed reduced cellulose and hemicellulose levels in mature pods. These results agreed well with our previous reports on cowpeas, showing that a recessive locus containing the VuMYB26a gene is responsible for pod shattering resistance and reduced pod wall fibers, including lignin, cellulose, and hemicellulose (Suanum et al., 2016; Takahashi et al., 2020). However, we cannot conclude whether pod shattering resistance was caused by reduced lignin alone or by reduced pod wall fibers as a whole. While lignin, cellulose, and hemicellulose were reduced in the pods of the quadruple mutants, in stems, only cellulose was reduced, but lignin and hemicellulose were increased (Fig. 2b). This could be due to tissue-specific regulation of GmMYB26 genes, leading to differential regulation of downstream players in pod shattering, such as laccase, cellulose, and peroxidase (Fig. 2a; Yong et al., 2024). For example, two laccase homologs, Glyma.11G137500 and Glyma.12G060900, which were downregulated in the GmMYB26 quadruple mutant, were expressed in the pods but not in the stems (Table S8). The retention of lignin and hemicellulose in the stem could explain why the quadruple mutant was able to remain erect, although the reduced cellulose content might be responsible for the drooping petioles (Figs 2b, S8). Recently, GmSh1 was reported to be involved in soybean pod shattering resistance (Li et al., 2024). We then found that ‘Enrei’, which was used as the WT, has a susceptibility allele at GmPdh1 and a resistance allele at GmSh1; meanwhile, ‘Enrei-no-sora’, used as NIL-pdh1 in this study, has the opposite resistance and susceptibility allele pattern (Fig. S1). In other words, this study could not determine whether the effect of the GmMYB26 quadruple mutation was greater than the combined effect of the pod shattering-resistant alleles of GmPdh1 and GmSh1. Future studies evaluating the combined effects of the pod shattering-resistant alleles of GmMYB26, GmPdh1, and GmSh1 will provide valuable insights for developing pod shattering-resistant soybean varieties. By mimicking the domestication process of the genera Vigna and Phaseolus, we developed a novel pod shattering-resistant soybean that has not been identified among natural variations. Soybean is a paleotetraploid and possesses all four GmMYB26 genes, which may have redundant functions and are expressed in pods (Fig. 1b). This may explain why MYB26 homologs were not selected to improve pod shattering resistance during soybean domestication, unlike in diploid species of the genera Vigna and Phaseolus. Therefore, using a reverse genetic approach in soybeans can be an effective way of mimicking genetic variation in diploid species. This study demonstrated the applicability of domestication-related traits to other legume crops that have undergone independent domestication processes, reiterating the importance of conducting genetic studies across diverse crops. We thank Norihiko Tomooka for general advice, Masako Yamamoto and Kumi Kawamura for plant cultivation, and Mineko Aburano, Tomoko Kimura, Yuko Iwamoto, and Junko Kamiya for their assistance with genotyping. This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant nos. 19KT0016, 21KK0272, 24K01736) and the Genebank Project of the National Agriculture and Food Research Organization. None declared. YT conceptualized the study. RT, YT, AK, RN, KN and MI planned the study. RT, YT and AK generated the plant materials. RT, YT, AK and RN performed the experiments. RT, YT, AK, RN, KN and MI wrote the manuscript. The raw sequencing data of RNA-seq are deposited in DDBJ Sequence Read Archive under accession no. PRJDB20196. The accession number of each raw sequencing dataset is listed in Table S9. All the data needed to evaluate the conclusions in the paper are available in the main text or the Supporting Information. Fig. S1 Genetic variation of GmMYB26, GmPdh1, GmSh1, and GmSHAT1-5 genes. Fig. S2 Weather data during the cultivation period taken from the Agro-Meteorological Grid Square Data. Fig. S3 Pod shattering resistance for the first and second glasshouse products. Fig. S4 Pod torsion angle per pod length for the first and second glasshouse products. Fig. S5 Cross sections of mature pods, including replicates to determine the lignin layer thickness using the phloroglucinol–HCl reaction for the first glasshouse and field products. Fig. S6 Quantitative characterization of the pod lignin layer. Fig. S7 Pod lignin content determined by the thioglycolic acid method. Fig. S8 Plants of the first flowering stage and pod elongation stage of the wild-type and quadruple mutants cultivated in the first glasshouse. Fig. S9 Plants of V4 stage and the first flowering stage of the wild-type and quadruple mutant cultivated in the field. Fig. S10 Quantitative characterization of the stem lignin layer for products of the third glasshouse. Methods S1 Supporting Materials and Methods. Table S1 ID of the genes addressed in this study. Table S2 Partial genomic DNA sequences, genomic position, and predicted amino acids in four GmMYB26. Table S3 List of primer sequences and restriction enzymes to detect each GmMYB26 mutation. Table S4 Summary of experimental design. Table S5 Raw data of pod shatter resistance (%) for the first glasshouse, second glasshouse, and field production. Table S6 Differential expression genes between the wild-type and the quadruple mutant, and top hits of similarity search. Table S7 Inclusion list for SIM. Table S8 Expression atlas of differentially expression genes between the wild-types and the quadruple mutant. Table S9 SRR information. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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