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. 2024 Jun 11;22(10):2859–2872. doi: 10.1111/pbi.14409

Soybean hypocotyl elongation is regulated by a MYB33‐SWEET11/21‐GA2ox8c module involving long‐distance sucrose transport

Tong Su 1, † , Huan Liu 1, † , Yichun Wu 1, † , Jianhao Wang 2, † , Fanglei He 1,3, † , Haiyang Li 1, Shichen Li 1, Lingshuang Wang 1, Lanxin Li 1, Jie Cao 1, Qiulian Lu 1, Xiaohui Zhao 1, Hongtao Xiang 4,5, Chun Lin 3, Sijia Lu 1,✉, Baohui Liu 1,✉, Fanjiang Kong 1,✉, Chao Fang 1,✉
PMCID: PMC11536460  PMID: 38861663

Summary

The length of hypocotyl affects the height of soybean and lodging resistance, thus determining the final grain yield. However, research on soybean hypocotyl length is scarce, and the regulatory mechanisms are not fully understood. Here, we identified a module controlling the transport of sucrose, where sucrose acts as a messenger moved from cotyledon to hypocotyl, regulating hypocotyl elongation. This module comprises four key genes, namely MYB33, SWEET11, SWEET21 and GA2ox8c in soybean. In cotyledon, MYB33 is responsive to sucrose and promotes the expression of SWEET11 and SWEET21, thereby facilitating sucrose transport from the cotyledon to the hypocotyl. Subsequently, sucrose transported from the cotyledon up‐regulates the expression of GA2ox8c in the hypocotyl, which ultimately affects the length of the hypocotyl. During the domestication and improvement of soybean, an allele of MYB33 with enhanced abilities to promote SWEET11 and SWEET21 has gradually become enriched in landraces and cultivated varieties, SWEET11 and SWEET21 exhibit high conservation and have undergone a strong purified selection and GA2ox8c is under a strong artificial selection. Our findings identify a new molecular pathway in controlling soybean hypocotyl elongation and provide new insights into the molecular mechanism of sugar transport in soybean.

Keywords: SWEET11/21, MYB33, sucrose, hypocotyl, soybean

Introduction

Soybean (Glycine max [L.] Merr.) is a major crop that provides over a quarter of the world's protein for human and animal consumption (Graham and Vance, 2003; South et al., 2019). At current average annual yield increases, soybean production will reach only 55% of the increase in supply necessary by 2050 (De Souza et al., 2022; Wang et al., 2020). In seedlings, the hypocotyl is responsible for transporting nutrients such as sugar, supporting physiological activities and promoting growth by connecting roots, stems and cotyledons via long‐distance transport (Jiang et al., 2014; Sliwinska et al., 2009; Wang et al., 2018). Hypocotyl length is also an important component of plant height, which affects lodging and yield of soybean (Fang et al., 2023).

Hypocotyl elongation is regulated by diverse endogenous and exogenous stimuli, such as light, temperature, hormones and the availability of photosynthate (Zhang et al., 2021). Genetic and biochemical evidence in Arabidopsis thaliana (Arabidopsis) suggests that multiple signalling pathways are integrated to regulate hypocotyl elongation through PIF proteins (phytochrome‐interacting factors) (Leivar and Monte, 2014; Lucas and Prat, 2014; Nozue et al., 2007; Zhang et al., 2021). For example, in signalling of the growth hormone gibberellin (GA), DELLA proteins interact with PIFs to inhibit their expression (Zhao et al., 2023). Meanwhile, over‐expression of GA2‐oxidase (GA2ox) genes inhibits high‐temperature‐induced hypocotyl elongation by reducing bioactive GA levels (Li et al., 2019). Sucrose, as the primary supplier and signalling molecule, also affects Arabidopsis hypocotyl elongation (Zhang et al., 2021). In the dark, sucrose promotes hypocotyl elongation by up‐regulation PIFs and GA synthases (Liu et al., 2011; Zhang et al., 2010). In the light, sucrose attenuates the dephosphorylation of BRASSINAZOLE‐RESISTANT 1 (BZR1), the master transcription factor of the brassinosteroid (BR) signalling pathway to inhibit hypocotyl elongation (Liu et al., 2011; Zhang et al., 2010, 2021). By contrast, studies on soybean hypocotyl elongation are scant, and known regulators include phytochromes and CCA1‐like genes (Shan et al., 2021; Zhang et al., 2023; Zhao et al., 2022). Regulatory mechanisms underlying soybean hypocotyl elongation therefore remain poorly explored.

Cotyledons serve as the major storage and photosynthetic organ to provide essential carbohydrates for all stages of seedling development and morphogenesis (Long et al., 2022; Saito et al., 1990; Xu et al., 2021). In Arabidopsis, sucrose moves towards the developing seedling hypocotyl from cotyledons via SUT (SUCROSE TRANSPORTER) and SWEET (SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER) proteins (Aoki et al., 2003; Chen et al., 2010a; Kelly et al., 2021; Sonnewald, 2011). In apoplasmic phloem loading, SUTs are responsible for importing sucrose into the CC–SE (companion cell–sieve element) complex, while SWEETs, which contain an MtN3_slv transmembrane domain, diffuse sucrose down concentration gradients (Ayre, 2011; Braun et al., 2006; Chen et al., 2012, 2015; Dhungana and Braun, 2021; Guan et al., 2008; Haritatos et al., 2000; Yuan and Wang, 2013). Seventeen SWEETs have been identified in the Arabidopsis genome, with AtSWEET11 and AtSWEET12 localizing to the plasma membrane of phloem parenchyma cells, to mediate sucrose efflux from phloem parenchyma cells to the apoplastic space (Chen et al., 2012; Gupta et al., 2021). The Arabidopsis sweet11/sweet12 double mutant over‐accumulates sucrose in leaves and grows slowly at high light levels due to defective phloem loading (Chen et al., 2012). Similarly, in crops such as maize, rice and potato, mutation of major phloem‐expressed SWEETs severely impairs plant growth and reduces yield (Abelenda et al., 2019; Antony et al., 2010; Bezrutczyk et al., 2018; Wu et al., 2018).

Previous studies have mainly focused on the regulation of soybean hypocotyl elongation by light and hormones. However, relatively few studies have addressed the regulation of hypocotyl elongation by sucrose. The paleopolyploid soybean genome is predicted to encode a large SWEET family of 52 members, but few SWEET‐related functional studies have been published. These studies mainly focus on sugar transport to sink organs or arbuscular mycorrhizal symbiosis (Miao et al., 2019; Wang et al., 2019, 2020; Zhang et al., 2020; Zheng et al., 2023), but none of the SWEETs expressed in source organs have been functionally characterized. Here, we found that the two closely related soybean paralogues SWEET11 and SWEET21 function as major sucrose transporters in the phloem of source organs, are transcriptionally regulated by MYB33, which responds to sucrose in cotyledons, and identified a GA oxidase that responds to sucrose in the hypocotyl to regulate hypocotyl elongation. We propose a novel module for soybean hypocotyl elongation in seedlings and reveal selection signatures for its components MYB33, SWEET11, SWEET21 and GA2ox8c throughout soybean domestication.

Results

Identification of SWEET11 and SWEET21 in soybean

In Arabidopsis, sucrose inhibits hypocotyl elongation under continuous light conditions (Liu et al., 2011; Zhang et al., 2010). Here, we confirmed a similar phenomenon in soybean, with soybean hypocotyls gradually shortening in response to increases in exogenous sucrose supplementation (Figure S1). To explore the potential SWEETs involved in this process, we constructed a phylogenetic tree containing annotated Arabidopsis and soybean SWEETs. Soybean SWEETs group into four clades, and six genes belonging to Clade III share a high similarity with AtSWEET11 and AtSWEET12 known to transport sucrose (Figure S2). Next, we examined the tissue‐specific expression of these six Clade III soybean SWEETs by RT–qPCR. SWEET11 (Glyma.05G202700) and SWEET21 (Glyma.08G010000) are predominantly expressed in cotyledons and other source organs such as the unifoliolate leaf (the first true leaf to emerge) and trifoliolate leaves (Figure S3a,b). The other four SWEET paralogues are highly expressed in non‐photosynthetic organs including roots and symbiotic root nodules, with only SWEET4 (Glyma.04G198400) and SWEET17 (Glyma.06G167000) showing slight but detectable expression in cotyledons (Figure S3c–f). Based on these observations, we hypothesize that SWEET11 and SWEET21 are the key SWEETs involved in sucrose export from soybean source organs.

Towards confirming the function(s) of SWEET11 and SWEET21, we developed knockout mutants in the reference cultivar Williams 82 (Wm82) background using CRISPR–Cas9 gene editing with two target sites per gene and obtained two double‐mutant lines: sweet11/21–1 and sweet11/21–2 (Figure S4a,b). Both have longer hypocotyls compared with Wm82 under long‐day and short‐day photoperiods (Figure 1a,b; Figure S4c,d). Additionally, besides elongation, the hypocotyl of sweet11/21–1 becomes notably slender, with both fresh weight and dry weight significantly smaller than that of Wm82 (Figure S5). As expected, total sucrose and sucrose specifically derived from 14C‐labelled photosynthates in the hypocotyls of sweet11/21–1 mutants were both statistically significantly lower than Wm82, whereas cotyledon samples had the opposite results (Figure 1c,d), suggesting that loss of SWEET11 and SWEET21 disturbs sucrose transport from cotyledons to hypocotyls. To further confirm that the hypocotyl elongation observed in sweet11/21–1 is due to a reduction in sucrose content within the hypocotyl, we subjected sweet11/21–1 and Wm82 to treatments with either 2% sucrose or water to detect the recovery of hypocotyl length (Figure 1e). After a 6‐day treatment, the untreated sweet11/21–1 exhibited significantly longer hypocotyls compared to Wm82; however, the disparity in hypocotyl length between sweet11/21–1 and Wm82 was relatively minor following sucrose treatment (Figure 1f). These results suggest that sucrose can partially restore the phenotype of sweet11/21–1, underscoring the crucial role of sucrose in causing the elongated hypocotyl phenotype in sweet11/21–1. These results imply that SWEET11 and SWEET21 facilitate long‐distance sucrose transport and thus affect hypocotyl elongation.

Figure 1.

Figure 1

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SWEET11 and SWEET21 facilitate sucrose transport to regulate hypocotyl elongation in soybean. (a) Wm82, sweet11/21–1 and sweet11/21–2 seedlings at 6 DAE grown under long‐day photoperiod. Scale bar = 1 cm. (b) Hypocotyl length at 6 DAE for Wm82, sweet11/21–1 and sweet11/21–2 under long‐day photoperiod. (c) Sucrose contents of 6‐DAE Wm82 and sweet11/21–1 cotyledons and hypocotyls. FW, fresh weight. (d) 14C‐labelled sucrose contents of 6‐DAE Wm82 and sweet11/21–1 cotyledons and hypocotyls. (e) Exogenous sucrose rescued the hypocotyl elongation of sweet11/21–1 under long‐day conditions. (f) Hypocotyl length of sweet11/21–1 and Wm82 treated with or without sucrose. Samples were collected at DAE 6. Scale bar = 1 cm. (g) SWEET11 and SWEET12 can complement a sucrose transporter‐deficient yeast strain. Cultures were grown for 3 days on plates before photography. AtSWEET11 was used as a positive control. (h–m) In situ hybridization of SWEET11 transcripts in 6‐DAE cotyledons. (h) Cross‐section of cotyledon. (i) Longitudinal section of cotyledon. (j) Negative control for in situ hybridization using a SWEET11 sense probe in cotyledons. (k–m) In situ hybridization of SWEET21 transcripts in 6‐DAE cotyledons. (k) Cross‐section of cotyledon. (l) Longitudinal section of cotyledon. (m) Negative control for in situ hybridization using a SWEET21 sense probe in cotyledons. For h–m, red arrows indicate target signals. X, xylem; P, phloem; SE, sieve element; CC, companion cell. Scale bars = 50 μm. (n, o) Subcellular location of SWEET11–GFP (n) and SWEET21–GFP (o) in transiently transgenic Arabidopsis protoplasts by confocal laser microscopy. OsRac3–mCherry was used to mark the plasma membrane. Scale bars = 10 μm. The data represent the means ± SE of n = 3 or 8. The value of each measurement/plant is represented by a dot; Student's t tests were used to determine statistical significance (*P < 0.05; **P < 0.01).

SWEET11 and SWEET21 participate in sucrose transport

To confirm whether SWEET11 and SWEET21 can transport sucrose, we sought to determine whether they could complement the hexose‐transport‐ and SUC2‐deficient Saccharomyces cerevisiae strain CSY4000, with AtSWEET11 serving as a positive control. All strains grew similarly well on maltose (Figure 1g). However, except for the empty vector control, strains expressing SWEET11, SWEET21 and AtSWEET11 all grew well on 2% (w/v) sucrose (Figure 1g). This experiment suggests that soybean SWEET11 and SWEET21 have sucrose transport functionality, at least in yeast.

Towards understanding SWEET11 and SWEET21 function in planta, we profiled their spatiotemporal expression patterns in hypocotyls and cotyledons by RT–qPCR during the development of the hypocotyl from 1 DAE (day after emergence) through to 8 DAE when the unifoliolate leaf is fully expanded (Figure S6). SWEET11 and SWEET21 share similar trends in cotyledons, both peaking at 6 DAE under long‐day and short‐day photoperiods, while their expression in hypocotyls is generally much lower than in cotyledons (Figure S6). Considering that this experiment was carried out in both short‐day and long‐day photoperiod conditions and expression patterns show similar trends, subsequent experiments were carried out under long‐day photoperiod. To elucidate the influence of sucrose on the expression of SWEET11 and SWEET21, we treated hypocotyls with low sucrose content to discern any alterations in the expression levels of SWEET11 and SWEET21. Upon exogenous supplementation with 2% sucrose, both SWEET11 and SWEET21 were up‐regulated, suggesting that sucrose is capable of inducing the expression of SWEET11 and SWEET21 (Figure S7).

To localize SWEET11 and SWEET21 expression in the context of sucrose transport in cotyledons, in situ hybridization was performed. Both transcripts gave strong signals with anti‐sense probes in cotyledon phloem companion cells (Figure 1h–m). In transiently transgenic Arabidopsis protoplasts expressing SWEET11–GFP and SWEET21–GFP, both fusion proteins localized to the plasma membrane and GFP fluorescence overlapped with that of the rice plasma membrane marker Rac3–mCherry (Figure 1n,o) (Chen et al., 2010b). These findings suggest that SWEET11 and SWEET21 may function at the plasma membrane of phloem companion cells in soybean cotyledons.

MYB33 positively regulates SWEET11 and SWEET21

Loss of SWEET11 and SWEET21 impairs long‐distance sucrose transport from cotyledons to hypocotyls, resulting in sucrose over‐accumulating in cotyledons and becoming deficient in hypocotyls, and actually forms a higher sucrose content environment in cotyledon and a lower sucrose content environment in hypocotyl. Unlike common transcriptomic analyses, these particular mutants enabled identification of differentially expressed genes that respond to sucrose levels both upstream or downstream of SWEET11 and SWEET21 by RNA‐seq.

Transcriptomic analysis identified 801 differentially expressed genes (DEGs) in cotyledons between Wm82 and sweet11/21–1 (fold change ≥1.5, P < 0.05) (Figure S8; Figure S9a; Table S4). To identify candidate upstream regulators of SWEET11 and SWEET21, we analysed promoter elements using PlantRegMap (http://plantregmap.gao‐lab.org/binding_site_prediction.php) and found 371 proteins predicted to bind the SWEET11 promoter and 365 predicted to bind the SWEET21 promoter (Table S6; Table S7). After integrating the DEG list and candidate promoter‐binding proteins, we extracted seven candidate genes that might be upstream of SWEET11 and SWEET21 (Table S8). Among them, MYB33 (Glyma.13G187500) was selected for detailed analysis on account of the transcription factor being highly expressed in cotyledons and responsive to sucrose (Figure S10a–c), and MYB transcription factors are known to regulate cotton SWEETs (Sun et al., 2019).

To confirm that MYB33 is in fact a regulator of SWEET11 and SWEET21, we performed dual‐luciferase (LUC) transactivation reporter assays in Nicotiana tabacum. Relative LUC:REN activity from SWEET11 and SWEET21 promoters fused to LUC was enhanced over effector controls when co‐infiltrated with MYB33, suggesting MYB33 activates the SWEET11 and SWEET21 promoters (Figure 2a,b). Chromatin immunoprecipitation (ChIP)–qPCR assay demonstrated MYB33–FLAG directly associates with the SWEET11 and SWEET21 promoters (Figure 2c‐f). We assayed SWEET11 and SWEET21 expression by RT–qPCR in two ethyl methanesulphonate‐induced (EMS) mutants of MYB33 (designated myb33‐1 and myb33‐2) isolated in this study. Both SWEETs were statistically significantly down‐regulated, but not abolished, in myb33‐1 and myb33‐2 mutants, indicating regulation by other genes (Figure 2g, h; Figure S10d). In addition, myb33‐1 and myb33‐2 hypocotyls were statistically significantly longer than Wm82 (Figure 2i, j) and total sucrose and sucrose specifically derived from 14C‐labelled photosynthates in the hypocotyls and cotyledons of myb33‐1 were both disturbed (Figure 2k, l), consistent with sweet11/21–1 mutant phenotypes. These results imply a regulatory circuit acting in soybean cotyledons in which MYB33 is indirectly regulated by sucrose and is a direct positive regulator of SWEET11 and SWEET21, which in turn regulates long‐distance transport of sucrose to the hypocotyl.

Figure 2.

Figure 2

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MYB33 positively regulates SWEET11 and SWEET21. (a) Schematic diagram of the constructs used for the transient reporter assay. A GFP effector was used as a negative control. Effectors and reporters were infiltrated into N. benthamiana leaves. UBI, ubiquitin; Pro35S, CaMV 35S promoter; LUC, firefly luciferase; REN, Renilla luciferase. (b) Relative LUC:REN luminescence for SWEET11 and SWEET21 reporters in N. benthamiana leaves. The combinations of numbers in (b) derived from (a). (c) Schematic diagram of the SWEET11 gene and regions tested for MYB33–FLAG enrichment by ChIP–qPCR in (d). (d) ChIP–qPCR assay reveals MYB33–FLAG is enriched in the SWEET11 promoter in transgenic hairy roots. (e) Schematic diagram of the SWEET21 gene and regions tested for MYB33–FLAG enrichment by ChIP–qPCR in (f). (f) ChIP–qPCR assay reveals MYB33–FLAG is enriched in the SWEET21 promoter in transgenic hairy roots. BS, binding site. (g, h) SWEET11 (g) and SWEET21 (h) are down‐regulated in myb33‐1 and myb33‐2 6‐DAE cotyledons relative to wild‐type Wm82. Relative expression was normalized to TUB, and data are the mean ± SD of three biological replicates. (i) Phenotypes of Wm82, myb33‐1 and myb33‐2 seedlings at 6 DAE under long‐day photoperiod. Scale bar = 1 cm. (j) Hypocotyl length at 6 DAE in Wm82, myb33‐1 and myb33‐2. (k) Sucrose contents in Wm82 and myb33‐1 in cotyledons and hypocotyls at 6 DAE under long‐day photoperiod. FW, fresh weight. (l) 14C‐labelled sucrose contents in cotyledons and hypocotyls of 6‐DAE Wm82 and myb33‐1 grown under long‐day photoperiod. The data represent the means ± SE of n = 3 or 8. The value of each plant/measurement is represented by a dot. Student's t tests were used to determine statistical significance (*P < 0.05; **P < 0.01).

Sucrose regulates hypocotyl elongation through GA2ox8c

Gibberellins have long been reported to regulate hypocotyl length (Liu et al., 2011; Zhang et al., 2010). Among the differentially expressed genes in hypocotyl transcriptomes, GA2ox8c (encoding a gibberellin 2‐oxidase that inactivates bioactive GAs by 2β‐hydroxylation) is a homologue of GA2ox8a and GA2ox8b with established roles in regulating internode length (Figure 3a; Figures S8 and S9b; Table S5) (Li et al., 2019; Wang et al., 2021). GA2ox8c is predominantly and highly expressed in hypocotyls and epicotyls (Figure 3b) and was up‐regulated in hypocotyls upon sucrose supplementation (Figure 3c). Given these characteristics, we assigned GA2ox8c as a candidate for regulating soybean hypocotyl elongation in response to sucrose.

Figure 3.

Figure 3

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Sucrose regulates hypocotyl elongation through GA2ox8c. (a) Phylogenetic relationships among soybean GA2ox8c proteins. Arabidopsis GA2ox8 was used as an outgroup. Sequences were obtained from Phytozome v13, and the dendrogram was constructed using the minimum‐evolution method in MEGA11. (b) Tissue‐specific expression pattern of GA2ox8c. Cot, cotyledon; Uni, unifoliolate leaf; Tri, trifoliolate leaf; SAM, shoot apical meristem; Hyp, hypocotyl; Epi, epicotyl. (c) GA2ox8c is up‐regulated in hypocotyls upon sucrose supplementation. – suc represents soybean seedlings treated with water; + suc represents soybean seedlings treated with 2% w/v sucrose. Expression was normalized to TUB, and data are the mean ± SD of three biological replicates. Hypocotyls were harvested at 6 DAE. (d) Phenotypes of Wm82, ga2ox8c‐1 and ga2ox8c‐2 seedlings under long‐day conditions at 6 DAE. Scale bar = 1 cm. (e) Hypocotyl lengths of Wm82, ga2ox8c‐1 and ga2ox8c‐2 mutants under long‐day conditions at 6 DAE. (f) LC–MS/MS determination of GA4 contents in Wm82, ga2ox8c‐1 and ga2ox8c‐2 hypocotyls grown under long‐day conditions at 6 DAE. (g) LC–MS/MS determination of GA4 contents in Wm82 and sweet11/21–1 hypocotyls grown under long‐day conditions at 6 DAE. (h) Hypocotyl lengths of Wm82 and ga2ox8c‐1 with or without 2% sucrose supplementation. The figures in the histograms indicate the shortening degrees of Wm82 and ga2ox8c‐1 treated with sucrose. The data represent the means ± SE of n = 3 or 8. The value of each plant/measurement is represented by a dot. Student's t tests were used to determine statistical significance (**P < 0.01).

Towards further characterizing GA2ox8c involvement in this process, we obtained two new EMS‐induced mutants in GA2ox8c (assigned ga2ox8c‐1 and ga2ox8c‐2), the hypocotyl lengths of which were longer than Wm82, thus confirming a role for GA2ox8c in hypocotyl elongation (Figure 3d,e; Figure S11a). LC–MS/MS quantification of the bioactive GA molecule GA4 revealed its over‐accumulation in ga2ox8c‐1 and ga2ox8c‐2 mutants (Figure 3f). These results suggest a role for GA2ox8c in regulating hypocotyl length, whereby the enzyme reduces the abundance of GA4 to consequently down‐regulate hypocotyl elongation. Towards linking these observations to SWEETs facilitating sucrose export to hypocotyls, the sweet11/21–1 mutant has attenuated GA2ox8c expression and over‐accumulated GA4 in hypocotyls (Figure S11b; Figure 3g). We then supplied Wm82 and ga2ox8c‐1 seedlings with 2% sucrose and saw that shortening of hypocotyls was weaker in ga2ox8c‐1 than in Wm82, indicating that loss of GA2ox8c reduces the sensitivity to sucrose in hypocotyls (Figure 3h).

In soybean cotyledons, MYB33 responds to sucrose and promotes the expression of SWEET11 and SWEET21 to form a valve between cotyledons and hypocotyls, thus facilitating the long‐distance transport of sucrose from cotyledons to hypocotyls. Upon movement to the hypocotyls, sucrose indirectly activates the expression of GA2ox8c to ultimately reduce hypocotyl length. Mutations in MYB33, SWEET11 and SWEET21 partially block the export of sucrose from cotyledons to hypocotyls, which down‐regulates the expression of GA2ox8c and consequently leads to hypocotyl elongation. Loss of GA2ox8c function increases GA4 levels and thus promotes hypocotyl elongation (Figure 4).

Figure 4.

Figure 4

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Model of MYB33–SWEET11/21–GA2ox8c‐mediated control of soybean hypocotyl length. MYB33 is indirectly induced by sucrose and up‐regulates the expression of SWEET11 and SWEET21 by binding directly to their promoters. SWEET11 and SWEET21 form a valve between the cotyledons and hypocotyls to facilitate long‐distance sucrose transport from cotyledons to hypocotyls. Sucrose in the hypocotyl indirectly activates GA2ox8c, reducing the length of hypocotyl by catalysing GA4 breakdown. Impairment of MYB33, SWEET11 and SWEET21 partially reduces sucrose transport from cotyledons to hypocotyls, leading to hypocotyl elongation. Loss of GA2ox8c over‐accumulates bioactive GA4 and ultimately results in hypocotyl elongation.

Selection on MYB33, SWEET11, SWEET21 and GA2ox8c

To gain insight into the selection history for MYB33, SWEET11, SWEET21 and GA2ox8c, we conducted a haplotype analysis using a genetically diverse panel of 559 sequenced soybean accessions (Lu et al., 2020), comprising 121 wild soybeans, 207 landraces and 231 improved cultivars.

For MYB33, 11 polymorphisms in the coding sequence were identified and assigned into 12 haplotypes (Figure S12a). A T→A transversion at nucleotide 450 leads to a substitution from histidine at residue 150 to glutamine and is carried by haplotypes 2–4. An alignment of MYB33 proteins showed that MYB33 A is the primitive haplotype (Figure S12b). A transient transactivation assay revealed MYB33A (haplotype 4) carries stronger activation capacity on the SWEET11 and SWEET21 promoters compared with MYB33T (haplotype 12) (Figure 5a–c). The proportion of accessions with MYB33 A gradually increases from wild soybeans (10%) to landraces (23%) and to cultivars (30%) (Figure 5d). These results indicate that the stronger activator of SWEET11/21, MYB33H4/A, has been subjected to weak but apparent levels of artificial selection during soybean domestication and improvement.

Figure 5.

Figure 5

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Selection on MYB33, SWEET11, SWEET21 and GA2ox8c. (a) Schematic diagram of constructs used for transient reporter assays of MYB33‐haplotype effector activity. GFP was used as a negative control. Effectors and reporters were infiltrated into N. benthamiana leaves. UBI, ubiquitin; Pro35S, CaMV 35S promoter; LUC, firefly luciferase; REN, Renilla luciferase. (b, c) Relative LUC:REN luminescence for SWEET11 (b) and SWEET21 (c) promoters co‐infiltrated with different MYB33 effector alleles in N. benthamiana leaves. Lowercase letters above the histograms denote statistically significant differences in relative LUC:REN activity based on one‐way ANOVA (P < 0.05). Means ± SE of three independent biological replicates is plotted, and each replicate is represented by a dot. (d–f, h) Proportions of MYB33 (d), SWEET11 (e), SWEET21 (f) and GA2ox8c (h) alleles and their co‐occurrence within each of the three germplasm groups. (g) Ka/Ks ratios in different species. DnaSP6 software was used to calculate Ka/Ks ratios. (i) Fst and Pi in wild soybeans, landraces and improved cultivars across the 2‐Mb genomic region surrounding GA2ox8c. The red arrow represents the location of GA2ox8c, and the grey dotted line indicates the threshold. Data are combined from 559 sequenced accessions (121 wild soybeans, 207 landraces and 231 improved cultivars).

By contrast, variation in the coding sequence of SWEET11 is sparse, with only one non‐synonymous mutation retained by the population of 559 sampled accessions (Figure S13a). This mutation is in the non‐conserved domain and was carried by only two of the 559 sequenced varieties (Figure S13b; Figure 5e). This sparseness was also the case for SWEET21 (Figure S13c): the sole non‐synonymous mutation of SWEET21 is also in the non‐conserved domain and is carried by one accession (Figure S13d; Figure 5f). These results suggest SWEET11 and SWEET21 are likely undergoing strong purifying selection. To confirm this possibility, we calculated the Ka/Ks ratios for SWEET11 and SWEET21 and their homologues in soybean, rice, maize, Arabidopsis, Medicago truncatula and common bean. Ka/Ks ratios ranged from 0.0631 to 0.5488, with the Ka/Ks ratios for SWEET11 and SWEET21 being the lowest, indicating these genes are subject to strong purification (Figure 5g). A potential reason is that SWEET11 and SWEET21 are so important for proper plant development that soybean cannot tolerate such mutations. To verify and support this hypothesis, we quantified sucrose contents of sweet11/21–1 in trifoliolate leaves and saw an almost doubling compared with that of Wm82 (Figure S14a). Plant height and internode length for sweet11/21–1 were also statistically significantly increased, while the 100‐seed weight, total grain per plant and grain weight per plant were all statistically significantly reduced (Figure S14b–h). These developmental and yield defects underscore the essential roles of SWEET11 and SWEET21 in soybean growth and development and are consistent with evidence that SWEET11 and SWEET21 are under strong purifying selection.

Finally, we found six SNPs in GA2ox8c that form seven haplotypes (Figure S15a). Among them is a stop–loss mutation resulting from a T→G mutation at nucleotide 1045 (GA2ox8c G ). To explore the functional relevance of GA2ox8c G and GA2ox8c T , we introduced GA2ox8c G and GA2ox8c T into soybean hairy roots and measured their lengths. Roots expressing GA2ox8c T were shorter than those expressing GA2ox8c G , and both transgenic lines had roots statistically significantly shorter than controls (Figure S15b,c), indicating GA2ox8c T confers stronger function than GA2ox8c G . The allele frequency of GA2ox8c T (stop–loss allele) shows a sharp increase from wild soybean (21%) to landraces (72%) and is present in 95% of sampled cultivars (Figure 5h). Fst and Pi values around the GA2ox8c locus did not however exceed the standard threshold (0.56) to consider it as a classic domestication gene (Figure 5i). These data suggest that although GA2ox8c T is likely not a domestication gene, GA2ox8c T remains under strong artificial selection during domestication and subsequent improvement.

Discussion

In this study, we have identified and proposed a molecular model for the regulation of hypocotyl length in soybean: sucrose produced by photosynthesis in the cotyledons can indirectly induce MYB33, which subsequently binds to and activates the expression of SWEET11 and SWEET21, which serve as valves for long‐distance sucrose transport from cotyledons to hypocotyl sink tissues. In the hypocotyl, sucrose indirectly activates GA2ox8c to shorten hypocotyl length through greater breakdown of the bioactive GA4 molecule. We also explored the selection patterns of MYB33, SWEET11, SWEET21 and GA2ox8c in a genetically diverse panel of sequenced accessions.

Genetic compensation is an adaptive mechanism that permits organisms to adapt to changes in their environment to maintain viability and fitness (El‐Brolosy and Stainier, 2017; Li et al., 2021). The soybean paleopolyploid genome underwent a whole‐genome duplication, which generated 52 predicted SWEET homologous genes (Patil et al., 2015; Schmutz et al., 2010). When mutations occur in SWEET11 and SWEET21, the expression levels of homologues SWEET4 and SWEET17 increase according to cotyledon transcriptomes, implying genetic compensation (Table S4) (Bouche and Bouchez, 2001). That is, when SWEET11 and SWEET21 lose (or change) their function in cotyledons, the expression of SWEET4 and SWEET17 increases to compensate for the impaired sugar transport capacity. Therefore, sweet11/21–1 is still capable of maintaining basic physiological activities, despite the morphological and developmental phenotypes of sweet11/21–1 seedlings changed. SWEET4, SWEET17, SWEET5 and SWEET16 are predominantly expressed in roots and root nodules, indicating potential roles in sugar transport to and within nodules (Figure S3c–f). SWEET10 and SWEET15, which are responsible for sucrose transport in seed development, along with SWEET6 which is required for arbuscular mycorrhizal symbiosis, indicate that the additional functions of soybean SWEETs warrant further investigation (Miao et al., 2019; Wang et al., 2019, 2020; Zhang et al., 2020; Zheng et al., 2023).

Sugar not only provides carbon source and energy for plant growth, but also interacts with various plant signalling pathways as signalling molecules to regulate plant growth. It has been reported that trehalose‐6‐phosphate (T6P) acts as a sugar signalling molecule to coordinate thermoresponsive hypocotyl growth with endogenous sugar utilization (Hwang et al., 2019). Additionally, the sucrose non‐fermenting 1‐related kinase (SnRK1)‐mediated metabolic enzyme activity and growth‐related transcripts enhance hypocotyl availability to sucrose (Simon et al., 2018). In this study, sucrose, in addition to its basic function as an energy source, also acts as a signal for regulating hypocotyl development. The reduction of sucrose signalling molecules in the mutations of SWEET11 and SWEET21 led to longer hypocotyls. Notably, the mutation of SWEET11 and SWEET21 also blocks the transport of sucrose serving as a carbon nutrient and energy source for anabolism. Thus, the hypocotyl of sweet11/21–1 becomes significantly thinner, with both fresh weight and dry weight significantly smaller. This case simultaneously demonstrates the dual functions of sucrose as both a signalling molecule and a source of nutrition or energy.

Typical RNA‐seq analyses tend to focus on identifying candidate genes based on the differential expression of genes between treatment and control. Here, the low sucrose content in sweet11/21–1 hypocotyls guided the identification of GA2ox8c as a candidate gene, on account of it being specifically up‐regulated in hypocotyls in response to sucrose (Figure 3b,c). Unique to this particular situation, the DEG list contains sucrose‐responsive transcripts—some of which might function upstream of SWEET11 and SWEET21. We identified a total of seven candidate upstream‐regulatory genes for SWEET11 and SWEET21 upon integration of the predicted binding proteins and transcriptome data. Among these, six genes were up‐regulated and one gene was down‐regulated in sweet11/21–1 (Table S8). We therefore selected MYB33 for further characterization, but we do not exclude the possibility that the other six genes also contribute to the regulation of SWEET11 and SWEET21 in these conditions. Arabidopsis homologues of the other six candidates are involved in widespread physiological processes including seed dormancy, floral organ and leaf development (Bou‐Torrent et al., 2012; Fu et al., 2023; Preciado et al., 2021; Vaistij et al., 2013). Therefore, we speculate that these candidate genes are likely to regulate SWEET11 and SWEET21 in sucrose‐related processes and even other developmental processes in plants and are worthy of further study. Here, we found that MYB33 activates SWEET11 and SWEET21 by directly binding to their promoters, and their expression is also sucrose‐responsive (Figure 2c–f; Figures S7 and S10c). The sucrose over‐accumulation in sweet11/21–1 cotyledons led to MYB33 up‐regulation that was accompanied by up‐regulated incomplete transcripts of SWEET11 and SWEET21, suggesting that MYB33, SWEET11 and SWEET21 contribute to the maintenance of dynamic sucrose balancing in cotyledons.

Hypocotyls remarkably elongate in darkness. Dark conditions effectively suppress photosynthetic sucrose production, yet it remains unclear whether the dark‐triggered longer hypocotyls are also induced by a lower sucrose concentration. To address this, we treated soybean seedlings with a 2% sucrose solution in darkness to compensate for the lack of sucrose supply resulting from the absence of photosynthesis. Our results showed that soybean seedlings treated with sucrose exhibited a significant reduction in hypocotyl length, suggesting low sucrose content is also a key factor triggering hypocotyl elongation in darkness, though not the only factor (Figure S16a,b). Furthermore, we found that light induces the expression of SWEET11 and SWEET21 (Figure S16c). Collectively, light or sucrose activates the expression of SWEET11 and SWEET21 to strengthen the sucrose transport valve and inhibit hypocotyl elongation in soybean.

Crops have undergone both natural selection by their wild ancestors and artificial selection as cultivated species have been domesticated and improved. Natural selection consists mainly of two types of selection: positive selection and negative (purifying) selection (Choudhuri, 2014). Positive selection fixes beneficial variants and promotes the emergence of new phenotypes, while purifying selection removes deleterious variants. Artificial selection occurs during domestication, and cultivar improvement and diversification generally enrich superior alleles. In this study, we analysed the haplotypes and selection histories and measures for MYB33, SWEET11, SWEET21 and GA2ox8c. SWEET11 and SWEET21 have undergone strong purifying selection and retained their primitive genotypes during artificial selection. Purifying selection acting on SWEET11 and SWEET21 is highly likely due to mutations on a pair of essential homeologues bringing a huge negative effect on yield, reducing the competitiveness of cultivated soybean, and undoubtedly reducing the competitiveness of wild soybean. MYB33 was subject to slight artificial selection during improvement and diversification. Its high expression in cotyledons and mature leaves suggests a role in facilitating sucrose transport throughout the entire growth and development of plants (Figure S10b). The stronger activator, MYB33 A , might bring a higher sucrose transport efficiency of the whole plants. GA2ox8c is a negative regulator of hypocotyl elongation, which is specifically expressed in hypocotyls and epicotyls (Figure 3b). The tissue expression of GA2ox8c led us to observe a longer epicotyl in ga2ox8c‐1, indicating that GA2ox8c not only inhibits epicotyl and hypocotyl elongation, but also acts as a crucial regulator affecting plant height in soybean seedlings (Figure S17). Although our study revealed sucrose activates the expression of GA2ox8c, we speculate that there are key intermediary factors between sucrose and GA2ox8c that respond to sucrose and subsequently activate GA2ox8c. Furthermore, GA2ox8c T is the enhanced allele that shortens hypocotyl length. The use of MYB33 A and GA2ox8c T could facilitate breeding programmes targeting hypocotyl length for lodging resistance and yield traits.

Materials and methods

Plant materials and growth conditions

The soybean reference cultivar Williams 82 (Wm82) was used as the wild type (WT) for both phenotypic and gene‐expression analyses. Plants were grown under short‐day (12 h light/12 h dark) or long‐day (16 h light/8 h dark) photoperiods in growth chambers (Conviron, Canada) with 60% relative humidity at a constant temperature of 25 °C with a light intensity of 500 μmol m−2 s−1. The sweet11/21–1 used for phenotyping were grown under natural long‐day conditions in the field (daylength >15 h) in 2022 at the Experimental Station of the Hebei Academy of Agricultural and Forestry Sciences in Shijiazhuang (37°27′ N, 113°30′ E), China. Plants were sown in the beginning of June, spaced 0.15 m apart in rows 2 m long, with 0.5 m between rows, and harvested in October. The days from emergence to pods attained mature colour, corresponding to the R8 stage (Fehr and Cavines, 1977).

Phylogenetic analysis and sequence alignment

Nomenclature for soybean SWEET naming is derived from Patil et al., (2015). The corresponding amino acid sequences of these genes for Wm82 were obtained from Phytozome 13 (https://phytozome‐next.jgi.doe.gov/) and aligned using CLUSTALW to create multiple alignments. Phylogenetic analysis was performed using MEGA11 with the minimum‐evolution method and with a bootstrap test comprising 1000 replicates (Tamura et al., 2021).

Plasmid construction and plant transformation

CRISPRdirect (http://crispr.dbcls.jp/) was used to design target sequences for SWEET11 and SWEET21 (Naito et al., 2015). Primers used are listed in Table S1. Target sequences were subcloned into different single‐guide RNA (sgRNA) expression cassettes and transferred into pYLCRISPR/Cas9‐DB vector, following the protocol by Ma et al., (2015). Constructs were introduced into Agrobacterium tumefaciens EHA101. For transformation, the cotyledon‐node method was used as previously described (Flores et al., 2008).

To identify the positive CRISPR/Cas9‐induced mutants, genomic DNA was extracted from the leaves of plants, and then, PCR analysis was performed by the mutant detection primers. After screening and examining, two positive lines (sweet11/21–1 and sweet11/21–2) were obtained from the progenies.

We obtained myb33‐1, myb33‐2, ga2ox8c‐1 and ga2ox8c‐2 from the Nanjing Agricultural University using iSoybean website. PCR analysis was performed to detect the mutations, and the homozygous lines were obtained in the progeny (Zhang et al., 2022a).

Generation of transgenic soybean hairy roots

The UBI pro :MYB33–FLAG–GFP and empty vectors were transformed into A. tumefaciens K599. Induction and transformation of soybean hairy roots were carried out as described previously (Jin et al., 2021). The PCR‐positive transgenic lines were used for further analysis.

RNA extraction and reverse transcription–quantitative polymerase chain reaction (RT–qPCR)

Total RNA was extracted from cotyledons, hypocotyls and unifoliolate leaves of Wm82 using an Ultrapure RNA Kit (CWBIO, China). cDNA was synthesized from 500 ng of RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). RT–qPCR was performed using a real‐time PCR kit (cat. no. RR430; Takara) on a Roche LightCycler 480 instrument (Roche Molecular Biochemicals, USA). Each 10 μL reaction contained 1 μL of 1:5 diluted cDNA with 0.2 μL of each primer (10 μM), 5 μL SYBR Green Master Mix and water to a final volume of 10 μL. Each sample was assayed in triplicate with three biological replicates. The expression levels of target genes were normalized using tubulin as the reference gene. RT–qPCR primers for SWEETs were derived from Patil et al., (2015), and primers used in this experiment are listed in Table S1.

Subcellular localization

The coding sequences of SWEET11 and SWEET21 from Wm82 were amplified and subsequently cloned in‐frame with GFP into a pCAMBIA1300 vector under the regulation of the cauliflower mosaic virus 35S promoter (Chen et al., 2010b). To investigate the subcellular localization of the SWEET11–GFP and SWEET21–GFP fusion proteins, transient expression was conducted in A. thaliana protoplasts following the protocol of Ma et al. (2008). The plasma membrane was labelled with pCAMBIA1300‐mCherry‐OsRac3 (Chen et al., 2010b). Localization was visualized using a FV1000 laser scanning confocal microscope (Olympus) using an excitation filter of 488 nm for GFP and 561 nm for mCherry. Primers used for vector construction are listed in Table S1.

In situ hybridization assay

The experiment followed the protocol previously described by Deblock and Debrouwer (1993). To generate RNA probes for SWEET11 and SWEET21, SP6 and T7 RNA polymerase (catalogue no. 11175025910; Roche) were used for amplification with specific primers listed in Table S1. Anti‐sense and sense probes, labelled with digoxigenin, were generated from linear pGEM‐T Easy Vectors, in accordance with the manufacturer's instructions. Hybridization signals were detected and captured using a fluorescence microscope (Axio Imager A2; Zeiss).

Exogenous sucrose supplementation

Wm82 seeds were sown in vermiculite and grown in a growth chamber under either long‐day, short‐day, continuous light or continuous dark conditions. After being exposed to light, seedlings were treated with either 200 mL of water, 1% w/v or 2% w/v sucrose solutions, respectively. Seedlings were treated for a total of three times. The length of the hypocotyl was measured after 6 days of treatment.

14C‐labelling experiment

14C‐labelling of soybean was done according to Liu et al., (2022). Briefly, when the cotyledons unfolded, the hypocotyl was wrapped with aluminium foil to prevent photosynthesis, and the apex was removed (as it is the strongest sink). A sealable plastic bag was attached to cotyledons and supplied with 14CO2 generated from NaH14CO3 and excess 80% (v/v) lactic acid for 1 h under illumination. Excess 14CO2 was cleared by 3 M KOH. Treated plants were exposed to ambient air for another 4 h after removal of the bag. Tissue samples were snap‐frozen in liquid nitrogen and homogenized, and soluble sugars were extracted. Thin‐layer chromatography (TLC) containing acetic acid, chloroform and water (7:6:1) was used to separate the sugars on silica plates (Merck, Shanghai, China). Maximum separation was achieved after running the plates three times. Radiolabelled spots were visualized with X‐ray film (Kodak Biome MR film, Rochester, USA), and spots were removed and placed in scintillation fluid. The percentage of each scraped radiolabelled sugar was calculated as the proportion of total sugars in Ecoscint scintillation solution (National Diagnostics, Atlanta, GA, USA).

Heterologous expression in yeast

The coding sequences of SWEET11 and SWEET21 were cloned into the pDR196 vector (Zheng et al., 2023). Recombinant vectors and the empty vector control were introduced into Saccharomyces cerevisiae CSY4000, which was developed to characterize sucrose and hexose transporters (Rottmann et al., 2016). Transformed cells of the uptake‐deficient strain were grown on SD (synthetic deficient) medium supplemented with 2% (w/v) maltose and auxotrophic requirements. Yeast cell suspensions were subjected to serial dilutions (×10−1, ×10−2, ×10−3) and then dropped (2 μL) onto solid SD media containing either 2% maltose (w/v) or 2% (w/v) sucrose and auxotrophic requirements. After three days of growth at 30 °C, growth was documented by photography. Primers used in the experiment are listed in Table S1.

Determination of sucrose content

The cotyledons and hypocotyls of Wm82, sweet11/21–1 and myb33‐1 were collected at 6‐DAE plants grown under long‐day photoperiod. Sucrose contents were analysed using the Agilent 8890‐5977B GC–MSD platform and detected by Metware (http://www.metware.cn/). Agilent 7890B gas chromatograph coupled to a 7000D mass spectrometer with a DB‐5MS column (30 m length × 0.25 mm i.d. × 0.25 μm film thickness; J&W Scientific, USA) was employed for GC–MS analysis of sugars. Helium was used as carrier gas, at a flow rate of 1 mL/min. Injections were made in the split mode with a split ratio of 3:1, and the injection volume was 2 μL. The oven temperature was held at 150 °C for 1 min and then raised to 200 °C at 5 °C/min, raised to 300 °C at 16 °C/min, raised to 320 °C at 20 °C/min and held at the temperature for 5.5 min. All samples were analysed in selective ion monitoring mode. The ion source and transfer line temperatures were 230 °C and 280 °C, respectively (Sun et al., 2016). Metabolites with an absolute fold change of ≥1.5 or a fold change of ≤0.5 were considered to be significantly different. Hypocotyl data were further selected with a fold change of ≥1.2. Three biological replicates were quantified. Data analysis was carried out by Metware Biotechnology Co., Ltd., Wuhan, China.

Determination of GA content

Tissue samples were collected at 6‐DAE plants grown under long‐day photoperiod. Content of the endogenous active GA4 was determined by high‐performance liquid chromatography and mass spectrometry (HPLC–MS/MS). The quantitation was performed on Agilent 1290 HPLC system (Agilent) and QTRAP 6500 mass spectrometer (Sciex). The standards were purchased from Sigma. WATERS ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) was used, and the mobile phase was A: B = (methanol/0.1% formic acid): (water/0.1% formic acid). Three biological replicates were obtained for each sample. The method in this study was used as previously published (Qiu et al., 2021).

RNA‐seq

RNA samples were collected from 6‐DAE cotyledons and hypocotyls of Wm82 and sweet11/21–1 harvested grown under long‐day photoperiod. Three biological replicates were obtained for each genotype, and all samples were sequenced on an Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd. (Guangzhou, China). Reads were mapped to the soybean reference genome (Wm82.a2.v1) using HISAT (2.0.0) (Kim et al., 2015), and transcript abundance was calculated using FPKM (fragments per kilobase of exon per million fragments mapped). DEGs were identified based on an absolute fold change of ≥1.5 and FDR <0.05. GO analysis (http://geneontology.org/page/go‐enrichment‐analysis) was performed on the DEGs. P < 0.05 was considered statistically significantly enriched in pathways.

Transient expression assays

The full‐length coding sequence of MYB33 was cloned in‐frame into the UBI pro :FLAG–GFP vector (Zhang et al., 2022b). A 2425‐bp promoter sequence of SWEET11 and a 2592‐bp promoter sequence of SWEET21 were amplified from Wm82 gDNA and inserted into the pGreenII 0800‐LUC vector (Zhang et al., 2022b). The empty vector was used as a negative control. All constructs were then introduced into A. tumefaciens GV3101, and plant transformation and LUC detection methods were performed as previously described (Nie et al., 2023). The N. benthamiana used in this study were grown under long‐day photoperiods in growth chambers (Conviron, Canada) with 60% relative humidity at a constant temperature of 25 °C with a light intensity of 500 μmol m−2 s−1. Experiments were performed three times, and the primer sequences are listed in Table S1.

ChIP–qPCR assay

Samples were collected from PCR‐positive transgenic soybean hairy roots, and experiments were conducted using methods previously described (Dong et al., 2021). Solubilized chromatin was immunoprecipitated using anti‐Flag antibody (Sigma, F1804), and the co‐immunoprecipitated DNA was recovered and analysed by RT–qPCR in triplicate. The relative fold enrichment was calculated by normalizing the amount of the target DNA fragment against that of a genomic fragment of the reference gene ELONGATION FACTOR 1B (ELF1B) and then normalizing the value of the input DNA. The data, which were normalized with input transcripts, represent the means from three biological repeats. Primers used for amplification are listed in Table S1.

Calculation of Ka/Ks ratios

Non‐synonymous (Ka) to synonymous (Ks) substitution rates were used to estimate the selection mode for homologues of SWEET11 and SWEET21. A multiple sequence alignment of the corresponding DNA was converted into a codon alignment using MEGA11 software. DnaSP6 software was used to calculate Ks and Ka.

Statistical analyses

Statistical analysis was conducted using Excel's two‐tailed Student's t test or one‐way ANOVA with equal variance. Data were further analysed using GraphPad Prism 8 (ver. 8.0.2).

Funding

This work was supported by the National Key Research and Development Program (grant nos. 2021YFF1001203 to B. Liu, 2022YFD1201501 to F. Kong, 2022YFD1201400 to C. Fang and 2021YFF1001100 to S. Lu), the Major Program of Guangdong Basic and Applied Research (grant no. 2019B030302006 to F. Kong and B. Liu), the open competition program of the top‐ten critical priorities of Agricultural Science and Technology Innovations for the 14th Five‐Year Plan of Guangdong Province (grant no. 2022SDZG05 to F. Kong and B. Liu), the National Natural Science Foundation of China (grant nos. 32330074 to B. Liu, 32372139 to C. Fang, 32301825 to T. Su and U20A2027 to S. Lu) and the Technology Plan of Guangzhou, China (grant nos. 2023A04J1500 to C. Fang).

Conflicts of interest

The authors declare no competing interests.

Author contributions

S. Lu, B. Liu, F. Kong and C. Fang designed and supervised the experiments and managed the projects. T. Su, H. Liu, Y. Wu, J. Wang, F. He, H. Li, S. Li, L. Wang, L. Li, H. Xiang, X. Zhao, J. Cao and Q. Lu performed the experiment. T. Su, H. Liu, Y. Wu, J. Wang, F. He and C. Lin performed the data analysis. T. Su and C. Fang drafted the manuscript. S. Lu, B. Liu, F. Kong and C. Fang revised the manuscript.

Supporting information

Figure S1 Sucrose inhibits hypocotyl elongation in soybean.

Figure S2 Phylogenetic relationships of SWEET proteins in soybean and Arabidopsis.

Figure S3 Tissue‐specific expression of select soybean SWEET genes.

Figure S4 Analysis of SWEET11 SWEET21 double mutants.

Figure S5 The stem diameter of Wm82 and sweet11/21‐1.

Figure S6 Developmental time course of SWEET11 and SWEET21 expression.

Figure S7 Sucrose up‐regulates SWEET11 and SWEET21 expression.

Figure S8 Transcriptome analysis of sweet11/21‐1 and Wm82 cotyledons and hypocotyls.

Figure S9 Cluster analysis of DEGs in cotyledons and hypocotyls of sweet11/21‐1 versus Wm82.

Figure S10 MYB33 is induced by sucrose.

Figure S11 GA2ox8c is down‐regulated in sweet11/21‐1.

Figure S12 MYB33 haplotypes in genetically diverse soybean germplasm.

Figure S13 SWEET11 and SWEET21 haplotypes in genetically diverse soybean germplasm.

Figure S14 Agronomic performance of field‐grown sweet11/21‐1 mutants.

Figure S15 GA2ox8c haplotypes in genetically diverse soybean germplasm.

Figure S16 Dark‐triggered longer hypocotyls are induced by a lower sucrose concentration.

Figure S17 The epicotyl length of Wm82 and ga2ox8c‐1.

Table S1 List of primers used in this study.

Table S2 The accession numbers of SWEET genes.

Table S3 Differential metabolites in cotyledon and hypocotyl.

Table S4 Significantly differentially expressed genes in the cotyledons.

Table S5 Significantly differentially expressed genes in the hypocotyls.

Table S6 Binding site prediction of SWEET11.

Table S7 Binding site prediction of SWEET21.

Table S8 Upstream candidate genes of SWEET11 and SWEET21.

Table S9 Protein variation of EMS‐mutants.

Table S10 559‐accession.

PBI-22-2859-s001.zip (1.1MB, zip)

Acknowledgements

The authors are grateful to Dr. Xinliang Hou (South China Botanical Garden, Chinese Academy of Sciences) for providing the vector pDR196, to Dr. Xia Li and Dr. Jintao Cheng (Huazhong Agricultural University) for providing yeast strain and to Dr. Qingxin Song (Nanjing Agricultural University) for providing myb33‐1, myb33‐2, ga2ox8c‐1 and ga2ox8c‐2.

Contributor Information

Sijia Lu, Email: lusijia@gzhu.edu.cn.

Baohui Liu, Email: liubh@gzhu.edu.cn.

Fanjiang Kong, Email: kongfj@gzhu.edu.cn.

Chao Fang, Email: fangchao@gzhu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Sucrose inhibits hypocotyl elongation in soybean.

Figure S2 Phylogenetic relationships of SWEET proteins in soybean and Arabidopsis.

Figure S3 Tissue‐specific expression of select soybean SWEET genes.

Figure S4 Analysis of SWEET11 SWEET21 double mutants.

Figure S5 The stem diameter of Wm82 and sweet11/21‐1.

Figure S6 Developmental time course of SWEET11 and SWEET21 expression.

Figure S7 Sucrose up‐regulates SWEET11 and SWEET21 expression.

Figure S8 Transcriptome analysis of sweet11/21‐1 and Wm82 cotyledons and hypocotyls.

Figure S9 Cluster analysis of DEGs in cotyledons and hypocotyls of sweet11/21‐1 versus Wm82.

Figure S10 MYB33 is induced by sucrose.

Figure S11 GA2ox8c is down‐regulated in sweet11/21‐1.

Figure S12 MYB33 haplotypes in genetically diverse soybean germplasm.

Figure S13 SWEET11 and SWEET21 haplotypes in genetically diverse soybean germplasm.

Figure S14 Agronomic performance of field‐grown sweet11/21‐1 mutants.

Figure S15 GA2ox8c haplotypes in genetically diverse soybean germplasm.

Figure S16 Dark‐triggered longer hypocotyls are induced by a lower sucrose concentration.

Figure S17 The epicotyl length of Wm82 and ga2ox8c‐1.

Table S1 List of primers used in this study.

Table S2 The accession numbers of SWEET genes.

Table S3 Differential metabolites in cotyledon and hypocotyl.

Table S4 Significantly differentially expressed genes in the cotyledons.

Table S5 Significantly differentially expressed genes in the hypocotyls.

Table S6 Binding site prediction of SWEET11.

Table S7 Binding site prediction of SWEET21.

Table S8 Upstream candidate genes of SWEET11 and SWEET21.

Table S9 Protein variation of EMS‐mutants.

Table S10 559‐accession.

PBI-22-2859-s001.zip (1.1MB, zip)

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