HC1 Promotes Hilum Development, Oil Accumulation, and Nodulation in Soybean

The soybean (Glycine max) hilum is the mark remaining after the funicle connecting the seed to the pod is broken, which is the channel of nutrients transported in developing seeds (Li et al. 2024). To date, five loci (I, T, W1, R and O) have been found to synergistically influence the deposition of flavonoids in the hilum (Yang et al. 2010). The flavonoid pathway regulates soybean seed colour while influencing quality traits. For example, black-seeded soybeans contain higher folate (Agyenim-Boateng et al. 2022). Our phenotypic analysis revealed significant quality differences between cultivated soybean with darker versus lighter hilum colours (Figure S1). Nevertheless, the relationship between flavonoids and soybean quality remains largely unknown. Therefore, uncovering the functions of genes regulating hilum colour, an important domestication-related trait, will provide an important reference for precise prediction of soybean quality traits. To examine the regulation of hilum colour, we constructed an F2 population by crossing Qihuang 34 (black hilum) with an unknown edamame variety (brown hilum) (Figure 1a). F2:3 segregated with a 3:1 ratio, black hilum to brown hilum (χ2 = 0.0286 < χ20.05 = 3.84), indicating that a single dominant gene controls hilum colour in this population. We used bulked-segregant analysis to map the associated locus to a 1020-kb interval on chromosome 6, and designated it Hilum Colour 1 (HC1). By genotyping a large segregating population, we identified eight recombinants in the vicinity of HC1. A progeny test of the informative recombinants delimited HC1 to a 34-kb region. According to Williams 82 (Wm82) reference genome (Wm82.a2.v1), there is only one open reading frame (ORF), Glyma.06G202300, in this 34-kb interval (Figure 1b). Sequencing revealed a single-base deletion in exon 3 of Glyma.06G202300 in the brown-hilum lines, causing a frameshift with premature termination and deletion of the functional domain (Figure 1c; Figure S2). Glyma.06G202300 is an orthologue of Arabidopsis TT7 gene, encoding flavonoid 3′-hydroxylase (F3′H). F3′H can accept apigenin or kaempferol as a substrate, converting them to luteolin and quercetin, respectively (Figure S3). HC1 is constitutively expressed in soybean, with particularly high expression levels in early‑ to mid‑developing seeds (Figure S4a). HC1 is localised in the endoplasmic reticulum (ER) (Figure S4b), implying a potential role in flavonoid biosynthesis in the ER. To validate the candidate gene, we knocked out HC1 in the Wm82 background (Figure S5a). The hilum colour of seeds from knockout lines (hc1-KO) was lighter than that of seeds of Wm82 (Figure 1d). Flavonoid metabolite profiling showed that HC1 knockout mainly suppressed the downstream production of dihydroquercetin and quercetin, such as (−)-epicatechin and procyanidin B2 (Figure S6; Table S2). Additionally, two independent HC1-overexpression lines, OE1 and OE2, were generated by introducing the fragment of the HC1 coding sequence from Wm82 into Tianlong No. 1 (TL01), resulting in significantly higher HC1 transcript levels (Figure S5b). Seeds from TL01 have a light-brown hilum whereas seeds from the overexpression plants had a grey hilum (Figure 1d; Figure S5c). Interestingly, we observed that black-hilum lines in the F3 population had a larger hilum than brown-hilum lines (Figure S7a,b). Consistently, seeds from RHLHC1 had a larger hilum than those from RHLhc1 (Figure S7c,d). Importantly, the knockout of HC1 results in a significantly smaller hilum (Figure 1e; Figure S7e). However, the similar hilum size in TL01 and OE lines might be attributed to other genetic factors (Figure S7f). Thus, HC1 regulates hilum development, including colour and size. Then, we analysed nucleotide polymorphisms in the genomic region of HC1 and identified 2 major variations which assigned the population as three haplotypes: hap_1, hap_2, and hap_3 (Figure 1f; Table S3). Since hap_2 and hap_3 encode equally truncated and nonfunctional proteins (Figure S8), they could be regarded as the same allele (hc1). Notably, hap_2 and hap_3 were absent in wild soybeans (Glycine soja) but detected in 51% of landraces and 72% of cultivars (Figure 1g). The overall frequency of hap_2 and hap_3 was 30% in southern China but was 91% in northern China (Figure 1h). A reduction in nucleotide diversity was observed from wild to landrace and then to cultivar (Figure S9a). Particularly in northern China, the π value surrounding the HC1 region decreased markedly in the cultivated population compared to the wild population (Figure S9b). Moreover, the Tajima's D-value for cultivars in northern China was negative, whereas the other subpopulation did not have this nature (Figure S9c). These results show that HC1 may have undergone selection during domestication only in northern China. 99% of the black-hilum cultivars carry hap_1, and 86% of the yellow-hilum cultivars carry hap_3 (Figure S10a,b). It is conceivable that the hc1 allele accumulates in northern cultivars either due to the breeding preference for light-coloured hilum or being subject to two maturity genes located close to HC1, QNE1 (Glyma.06G204300) and E1 (Glyma.06G207800). In the F4 generation, a co-segregation pattern was observed between hilum colour and seed oil content, wherein the black-hilum lines demonstrated a markedly elevated seed oil content relative to brown-hilum lines (Figure S11a). Consistently, seeds from NILHC1 exhibited higher oil content than those from NILhc1 (Figure S11b). To determine whether HC1 regulates oil content, the oil contents of the seeds of transgenic lines and their background genotypes were quantified. Compared with the wild type (WT), seeds of hc1-KO had a 0.77% lower oil content (Figure 1i). Under nutrient-deficient soil conditions, seeds from the WT maintained a significantly higher oil content than those from hc1-KO (Figure S11c,d), demonstrating that HC1 can enhance oil accumulation stably. Compared with those of TL01, seeds from HC1-OE lines had a 0.69%–1.07% greater oil content (Figure 1j). Concurrently, the experimental materials with higher oil contents had lower protein contents (Figure S11e), which conforms to the commonly observed negative correlation between protein and oil contents. However, HC1 did not have an impact on yield (Figure S12a–d). Similar FA components were present in the transgenic plants and their background genotypes, indicating that HC1 regulates oil content without any dependency on specific FA metabolism pathways (Figure S11f). Flavonoids are well-established signalling molecules in symbiotic nodulation. In field experiments, WT individuals produced significantly more nodules than hc1-KO individuals at the R6 stage (Figure 1k,l), suggesting that HC1 may have an influential role in increasing nodule number. Meanwhile, leaves of WT plants had higher chlorophyll content and photosynthetic efficiency than those of hc1-KO (Figure 1m,n). The nodule number of hc1-KO and WT was also compared after inoculation with Bradyrhizobium diazoefficiens USDA110 and Sinorhizobium fredii CCBAU 05684/CCBAU 83666 in pot experiments. The nodule number for hc1-KO was significantly lower than that for WT only when inoculated with CCBAU 83666 (Figure S13a–c). In a word, HC1 can facilitate the colonisation of certain strains resulting in more nodules. The accumulation of oil in soybean is caused by various factors. HC1 can enhance antioxidant activity in the hilum (Toda et al. 2012), which is crucial for maintaining intracellular homeostasis. A larger size and stabler hilum may enhance the transport of photosynthates and thus increase seed oil content. Enhanced nodulation and photosynthesis may increase nutritional components flux to the developing embryos at the rapid-seed-growth stage, potentially leading to higher oil content. Furthermore, RNA sequencing identified 58 differential expression genes between WT and hc1-KO (Table S4), including lipoxygenase-encoding Glyma.07G034800, which implies the potential pathway mediated by HC1. Future studies are necessary to investigate how HC1-mediated flavonoid metabolism promotes oil accumulation during seed development. In summary, we successfully cloned the hilum colour gene HC1 through map-based cloning. Moreover, our findings demonstrated a previously uncharacterized function by which HC1 has a pleiotropic effect for boosting hilum development, oil content, and nodulation symbiosis in soybean, which offers evidence for the extension of black-hilum accessions. L.S., J.H., and J.L. designed the research project; J.L., L.W., and Q.H. performed experiments; J.L., R.M., W.H., and W.F. wrote the manuscript. R.X., D.Z. and X.W. provided the population and managed material. All authors read and approved its content. The authors declare no conflicts of interest. The data that supports the findings of this study is available in the Supporting Information of this article. Figure S1–S13. Table S1–S7. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.