Soybean sucrose transporter GmSUT4 regulated plant growth and development through sugar metabolism
Research Article
Functional characterization of a soybean GmSUT4 gene reveals its involvement in plant growth and development regulation through sugar metabolism
https://doi.org/10.21203/rs.3.rs-2254720/v1
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Soybean sucrose transporter GmSUT4 regulated plant growth and development through sugar metabolism
Sucrose is the main important source of carbon metabolism in plants, and the production (synthesis source), transportation and storage (sink) of sucrose are important for normal plant growth and development (Julius, et al. 2017; Ljung, et al. 2015; Yoon, et al. 2021). It is loaded into the phloem either symplastically by plasmodesmata complexes or apoplastically by plasma membrane sucrose transporters or carriers (SUTs or SUCs). SUTs are also thought to be important in regulating flowering time, nectar production, seed filling, fruiting, plant growth, and total yield (Gu, et al. 2020; Hackel, et al. 2006; Sivitz, et al. 2008; Stadler, et al. 1999).
Since the discovery of first sucrose transporter in spinach, several other sucrose transporters have been studied in various plants (Sun and Ward 2012; Wang, et al. 2016; Weise, et al. 2008). Sucrose transporters belong to the Major Facilitator Super Family (MFS), which is one of the largest known transporter families (Reddy, et al. 2012). Based on sequence homology analysis, SUT genes are divided into five subgroups by phylogenetic tree analysis, namely SUT1-5 (Reinders, et al. 2012). Among them, SUT1 is indigenous to dicotyledons, SUT3 and SUT5 are native to monocotyledons, while SUT2 and SUT4 are found in both monocotyledons and dicotyledons (Kuhn and Grof 2010). The physiological function of the SUT4 family members of sucrose transporters is very diverse and still unclear. Mutants or transgenic plants with reduced or increased expression of the SUT4 genes showed different effects in different plant species. For example, down-regulation of the StSUT4 gene resulted in early flowering, fewer leaves at flowering time, shorter internodes, and higher tuber production, which has been attributed to increased sucrose efflux (Chincinska, et al. 2008). Rice OsSUT2 mutants exhibited a significantly lower ability to export sucrose from the source (leaves) compared to the wild type. As a result, sucrose, glucose, and fructose accumulate in leaves, leading to growth retardation and reduced root and grain development (Atkins, et al. 2011). In Arabidopsis, overexpression of AtSUC4 seedlings showed a 30% reduction in sucrose content (Schneider, et al. 2012). SUT4 mutant seeds germinated significantly more frequently than wild-type seeds in sucrose supplementary media indicating that AtSUT4 mutants exhibit a sucrose-insensitive phenotype in seed-germination inhibition assays (Li, et al. 2012). Moreover, overexpression of MdSUT4.1 in apples significantly decreased sucrose accumulation and total sugar content in fruits, which likely mediates efflux of sucrose from the vacuole in apples (Peng, et al. 2020). Therefore, it is doubtful to draw a general conclusion about the function of SUT4 family members from different plant species.
Sugar-mediated signaling and regulation of sugar transporter proteins play an important role in the precise transition of growth and development and its regulatory mechanism. The activity of these sugar transporter has been found to be modulate by sugar signals. For example, the sucrose signaling genes SnRK2 and AREB2, which respond to ABA regulate sucrose transport from mature leaves at the bolting stage of B. napus (Lee, et al. 2020). A positive correlation between SnRK1-mediated sugar signaling and the expression of many SUTs has also been found (Luo, et al. 2020). In addition, there are studies showing that SUCs can act as secondary regulators of the SnRK2–ABF pathway, which is primarily regulated by sucrose signaling in plants (Jia, et al. 2015). There is growing evidence that sugar transport and signaling coordination are a cornerstone of plant development. However, sucrose signaling and other metabolic pathways involved in sugar remobilization and transport are not well understood. In this study we aimed to improve our understanding of the SUTs mechanisms by which they mediate sucrose transport in soybean plants to support the growth and development process. In this context, the isolation and functional characterization of GmSUT4 was performed, as this transporter protein has a function in sucrose transport in soybean. In this study, GmSUT4 was analyzed by determining its subcellular localization and transport ability using a heterologous yeast system. Furthermore, the EMS induced GmSUT4 soybean mutant and Agrobacterium mediated GmSUT4 overexpression Arabidopsis were generated and used to investigate its possible physiological and molecular role in soybean growth and development.
Seeds of soybean cultivar Guixia1 were obtained from Guangxi Academy of Agricultural Sciences (Nanning, China). The yeast mutant strain SUSY7/ura3 and expression vector PDR196 were provided by Professor Qiusheng Yang, College of Horticulture, Henan Agricultural University (Henan, China). Soybean seeds of EMS-induced GmSUT4 mutagenesis and wide type Williams 82 were provided by Professor Qingxin Song, Nanjing Agricultural University (Nanjing, China).
Soybean cultivar ‘Guixia1’ was used for the experiment was germinated in vermiculite. 80 seeding with the same growth rate (at true three leaf stage) were selected and divided into 2 groups. The one group plants used for GmSUT4 expression analysis was grown in potting soil, the samples (root, leaf and stem of 14dyas seeding; source leaf at maturity; 15, 25, 35 and 45days seed after flowering) were harvested and stored at -80℃ for further experimentation. The second group seedlings used for gene dependence analysis of exogenous sucrose were exposed to 1% sucrose in 1/4 Hoagland nutrient solution, while the seedlings grown in only 1/4 Hoagland solution were designated as control. Subsequently, samples were collected at 0, 1, 2, 4, 8, 12, 24 and 48 hrs, for qRT-PCR analysis.
Molecular cloning and analysis of GmSUT4 gene
The coding sequence of the GmSUT4 gene was downloaded from SoyBase (https://www.soybase.org) and primers used to clone the open reading frame (ORF) of GmSUT4 were designed using Primer Premier 5.0 (Supplemental Table S1). Total RNA was extracted from soybean leaves by guanidine isothiocyanate method. RNA concentration and quality were checked by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). First-strand cDNA was synthesized from total RNA using a reverse transcription kit (Vazyme Biotech Co.,Ltd). PCR amplification reactions were carried out under the following conditions: 95 ℃ for 5 min followed by 34 cycles of 95 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ for 1 min with final amplification at 72℃ for 5 min. PCR products were recovered by FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co.,Ltd) and cloned using pEASY®-Blunt Cloning Kit (TransGen Biotech). After screening the positive clones, the strains were sequenced from BGI Genomics Co., Ltd.
The composition of the conserved domain and motif of GmSUT4 was analyzed using the CD-search function of NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and MEME tool (http://meme-suite.org/tools/meme) respectively. The ProParam tool and Proscale in ExPASy (https://web.expasy.org/) were used to predict the protein composition and hydrophilicity. Multiple sequence alignment of GmSUT4 with other soybean SUT proteins was performed with DNAMAN8.0 and phylogenetic analysis was constructed using MEGA7.0 software.
Subcellular localization of GmSUT4
The PCR amplified products of the GmSUT4 gene without the terminator region were ligated and cloned into the vector pBI121-GFP at restriction sites X-bal and Kpn I to generate pBI121-GmSUT4-GFP. The resulting recombinant plasmid was designated GmSUT4-GFP and transferred into DH5α. The positive clones were screened out by the colony PCR identification method and confirmed by sequencing. The purified plasmid construct was introduced into Agrobacterium tumefaciens EHA105 and the recombinant strains were grown on LB liquid medium containing 25 mg L− 1 Rif+, 50 mg L− 1 Kan+ and 25 mg L− 1 Str+, and cultured overnight at 28 ℃ and 220 rpm. When the cell reached a final concentration corresponding to an absorbance of 0.8-1.0, they were washed with sterile water (containing 10 mM MgCl2 and 100 µM acetylsynthon) and injected into the lower epidermis of leaves of 3-week old tobacco seedling. The injected leaves were incubated in the dark at 24 ℃ for 48 hours and were photographed under laser confocal microscope.
Functional analysis of GmSUT4 in yeast
To verify the sucrose transport activity of the SUT4 gene, the recombinant plasmid PDR196-GmSUT4 was constructed and transferred into the yeast strain SUSY7/ura3 which is involved in cells sucrose metabolism. Positive transformants were selected and grown in SD/-ura liquid medium (containing 6.7 g L− 1 yeast nitrogen base without amino acids, 1.29 g L− 1 SD/-ura base, 2% glucose, and a pH of 5.8) at 30 ℃ with continuous shaking at 220 rpm until an OD600 value of 1.2 was reached. The supernatant was removed by centrifugation at 4000 rpm for 5 min, and the pellet was resuspended in sterile water to an optical density of 1.0 at OD600. The yeast cell suspension was then diluted 10, 102, 103, and 104 fold, and 2 mL of each dilution reaction was added to modified SD/-ura agar medium (SD/-ura liquid medium, 20 g L− 1 agar) supplemented with 2% sucrose or 2% glucose. Yeast growth conditions were observed and recorded after 3 days of incubation at 30°C in the dark.
Hairy roots were generated according to the previously described method with slight modifications (Fan, et al. 2020; Kereszt, et al. 2007). Soybean seeds were surface sterilized with a mixture of 12 N HCl ( 4 mL ) and 5.25% NaClO (50 mL) in an airtight dryer for 14 hours (Di, et al. 1996), cleaned three times with distilled water and then germinated in MS medium for 3 days. The explants of the cotyledon nodes running parallel to the hypocotyl axis were gently scratched 3–4 times with a scalpel, and the wounded explants were then transferred to the Agrobacterium co-cultivation medium (CCM) for 30 mints at 28°C with constant shaking at 120 rpm. For Agrobacterium CCM medium, expression of GmSUT4 was mediated by Agrobacterium rhizogenes K599 with exogenous plasmid Pcambia1301-GmSUT4. After infection, 9 explants were placed evenly on each agar CCM coated with filter paper and co-cultured at 24°C in the dark for 3 days. After co-culture, explants were washed six times with distilled water containing 300 mg L− 1 cephalosporin. Then explants were transferred to rooting medium (RM) and kept in an incubator at 24°C for 12 days on 16/8 h light/dark cycle.
GmSUT4 overexpression vector construction and transgenic Arabidopsis plants acquisition
Gateway BP clonase II (Invitrogen, USA) recombination method was used for amplification of the full-length coding GmSUT4 region, purification and construction of vector. The recombinant Pcambia1301-GmSUT4 plasmid was transformed into E.coli DH5α. The positive clones were picked out by the colony PCR identification method and confirmed by sequencing. The purified recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101, which was then used to transfect Arabidopsis plants by the floral dip method (Clough 2005). The T1 generation seedlings of the transgenic plants were tested on 1/2 MS media supplemented with 30 mg L–1 hygromycin and further verified by PCR and qRT-PCR.
The expression of GmSUT4 gene and other genes in this study were determined by quantitative real-time reverse transcription PCR (qRT-PCR) and three biological replicates with three technical replicates for each sample were conducted. All primers of gene and the internal reference gene actin are listed in Supplementary Table S2. First-strand cDNA was used as template for qRT-PCR and the reaction was performed with TransStart Top Green qPCR SuperMix (TransGen Biotech) using the Bio-Rad CFX96 (Bio-Rad Laboratories) machine. Changes in gene expression levels were calculated using the 2−ΔΔCt calculation method (Zhang et al. 2020).
All experiments described above were repeated at least three times independently, and the data were expressed as mean ± SEM. Statistical analyses and drawing were performed using Excel 2010 soft-ware (Microsoft Corp., Albuquerque, NM, USA) and GraphPad 8.2 software. The sig-nificance level was defined as *(P < 0.05) and **(P < 0.01) based on the analysis results of One-way ANOVA or Student’s T test.
Molecular cloning and sequence analysis of GmSUT4
To investigate the function of the soybean GmSUT4 gene, we cloned the GmSUT4 gene (accession number: XM_006578191.3) by PCR amplification. The total nucleotide sequence length of GmSUT4 gene was 1765 bp, with 1542 bp open reading frame (ORF) (Fig. S1a). The encoded protein had a length of 513 amino acid residues and contained 12 transmembrane domains (Fig. S2a). The molecular weight of the GmSUT4 protein was 57869.35 Da, and the theoretical isoelectric point was 9.43 (Fig. S2b). Conserved domain analysis of the protein revealed the presence of GPH and MFS functional domains in the GmSUT4 protein (Fig. S2c). The amino acid sequences of GmSUT4 shared several highly conserved regions with other soybean SUT amino acid sequences (Fig.S3), but the similarity index ranged from 39.90–50.18% (Table S3). The result of Phylogenetic analysis showed that GmSUT4 was closely related to LjSUT4 from Lotus japonicus and AiSUT4 from Arachis hypogaea and only distantly related to other SUT4 proteins (Fig.S4).
GmSUT4 encodes a plasma membrane protein and has sucrose transport activity
In this study, the GmSUT4-GFP vector of the GmSUT4 gene was constructed and transformed into tobacco leaf subepidermal tissue using Agrobacterium-mediated transformation. After 48hours of co-cultivation, the tissues were observed at 480 nm using a fluorescence microscope. GFP signals were clearly visible in the cell membranes of tobacco cells (Fig. 1a), but not in other parts of the cells, indicating that GmSUT4 is localized in the cell membranes of tobacco cells. This experimental result is consistent with the location predicted by PSORT (https://www.psort.org).
To investigate the function of GmSUT4 as a sucrose-uptake carrier, we constructed the PDR196-GmSUT4 vector and transferred it into SUSY7/ura3. Yeast overexpressing the GmSUT4 gene have grew normally on the medium SD-ura medium contained 2% sucrose, even at dilution ratio of 102 compared with the control (Fig. 1b). On the other hand, the transgenic and control yeast grew normally at all dilutions on the SD medium containing 2% glucose (Fig. 1b). Expression of GmSUT4 in SUSY7/ura3 was able to recover a normal sucrose transport capacity for yeast, suggesting that GmSUT4 encodes a functional transporter in sucrose transport.
Tissue-specific and exogenous sucrose sensitivity analysis of GmSUT4
Root, stem, leaf, flower and seed samples of soybean were used to analyze the tissue-specific expression of GmSUT4 by qRT-PCR. The GmSUT4 gene was found to be differentially expressed in different tissues of soybean plants. The highest transcript abundance was found in roots, followed by seed 35 DAF, and mature leaf (Fig. 2a) and the lowest expression abundance was found in stem, seed 15 DAF and flower. Additional experiments of the sucrose-dependent response analysis of sucrose transporter GmSUT4 in roots reveled GmSUT4 expression level was upregulated by 1% exogenous sucrose and reached a maximum at 24 h (Fig. 2b).
Overexpression GmSUT4 promotes sucrose uptake in soybean hairy roots
To determine the effects of exogenous sucrose on plant growth, transgenic soybean hairy roots overexpressing GmSUT4 and control roots were exposed to sucrose concentrations of 0.1% (MS0),1% (MS1) and 3% (MS3) in MS medium. The transgenic roots grew well on MS1 and MS3 medium compared to control roots under normal conditions. However, no significant difference was observed in the growth rate of transgenic and control roots grown on MS0 medium (Fig. 3a). Furthermore, the metabolic conversion of exogenous sucrose to soluble sugar and endogenous sucrose was investigated. The results showed that the content of soluble sugar and sucrose in both transgenic and control roots gradually increased with increasing sucrose concentration. Compared with the control roots, the GmSUT4 overexpressing roots grown on MS1 and MS3 medium showed an increase in soluble sugar content of 55.31% and 43.95% respectively, and sucrose content of 28.08.0% and 9.85% respectively (Fig. 3b, c). Similar content of sucrose (1.22 mg/g and1.25 mg/g) and soluble sugar (4.40 mg/g and 4.49 mg/g) were observed in transgenic and control roots grown on MS0 medium Fig. 3b, c). These results suggest that the expression of the sucrose transporter GmSUT4 causes rapid uptake of sucrose in overexpressing hairy roots.
Overexpression of GmSUT4 regulates plant growth in Arabidopsis
To further elucidate the functional role of GmSUT4 in sugar transport, homozygous T3 generation of Arabidopsis lines overexpressing GmSUT4 gene was generated. The transgenic lines were verified by PCR and qRT-PCR. The band of the required length was detected in 4 independent transgenic lines by PCR amplification whereas no band was detected in wild-type plants (Fig.S5a). Real-time fluorescence quantification results showed that transcription of GmSUT4 was up-regulated in transgenic Arabidopsis thaliana (Fig. S5b).
Phenotypic observation showed delayed leaf expansion in the transgenic plants compared with the wild type (Fig. 4a). Compared with the fully open rosette leaves of the wild type, the leaves of the transgenic plants were contracted in the middle and formed a central bundle, resulting in small and numerous rosette leaves (Fig. 4a, b). Transgenic line 1–4 had 25, 19, 23 and 21 rosette leaves respectively, with an average of 22, which was much more than the number in WT (average 13.5 rosette leaves) (Fig. 4d). The emergence of primary rosette branches (RI) was also delayed in the transgenic plants compared with the wild type (Fig. 4a). The number of rosette branches (average 2.5 RI branches) was higher in the transgenic plants than in the wild-type (average 1.5 RI branches) (Fig. 4c, d). However, the number of cauline branches (CI) of the transgenic lines was lower than that of wild type (Fig. 4d). Analysis of the sugar content of rosette leaves at day 30 (at the time of axillary bud formation) showed higher accumulation of sucrose, soluble sugar, and starch in the transgenic plants than in the wild-type plants (Fig. 4e). This increase in photosynthetic metabolites content in transgenic plants is accompanied by up-regulation of gene expression involved in sugar metabolism (Fig. 5a, b).
Overexpression of GmSUT4 in Arabidopsis increase seed yield and seed germination rate
After investigating the effect of GmSUT4 on Arabidopsis vegetative growth, we also evaluated seed development of the transgenic lines. The number of pods per plant was significantly higher (32.83%) in transgenic Arabidopsis than in the wild type (Fig. 6a, b). Consequently, the seed yield of the transgenic plants per plant was increased by 36.52% (p = 0.0421) compared to the wild-type plants. However, the 100-seed weight, seed size and number of seeds per silique were found without significant difference in transgenic plants compared with the wild type (Fig. 6b).
Previous studies have shown that the Arabidopsis sucrose transporter SUT4 interacts with Cyb5 to mediate sucrose sensing or signaling in the process of seed germination (Li, et al. 2012). Seeds from transgenic and WT plants were germinated on medium supplemented with different concentration of sucrose. The results showed decreased germination rate with increasing sucrose concentration, but the inhibition of germination decreased with time. The germination rate of the seeds of the transgenic plant was higher than that of WT (Fig. 7).
EMS-induced soybean GmSUT4 mutation decreased biomass, organs size and yield To further confirm the effects of GmSUT4 on soybean plants, we used and uncharacterized mutant allele in a collection of M4 EMS-mutagenized NJAU0264 and NJAU1191 lines described previously (Zhang, et al. 2022). NJAU0264 (named GmSUT4-M1) carries a EMS-induced G-to-A mutation in GmSUT4 at position of 204 amino acid in motif1 within the coding region (Fig. S6a). NJAU1191 (GmSUT4-M2) carries a EMS-induced C-to-T mutation in GmSUT4 at 446 amino acids that introduces a premature stop codon in place of Asparagine (Fig. S6b).Individual plants carrying the M1 and M2 alleles were identified from the original mutant material by PCR genotyping, and the nature of the mutation was confirmed by sequencing of pooled-DNA.
Phenotypic observation of mutant and wild-type plants (WT) in the greenhouse showed that non-synonymous mutations of GmSUT4 in soybean significantly inhibited plant growth (Fig. 8a). The small leaf and low height phenotype of GmSUT4 mutants was evident at the development stage VC (the single leaf is fully extended) (Fig. 8b, c). The defective phenotype gradually becomes evident with the development of the leaf and seriously affects the growth of plants in the R stages at SD conditions (Fig. 8d, e). The contents of primary metabolites in the early R stage showed a decrease in sucrose content by 57.63% and 43.91% and total soluble sugar content by 60.19% and 51.18% in leaves of GmSUT4-M1 and GmSUT4-M2 respectively, which were significantly lower than those of wild-type plants. Similarly, the starch content of the mutant plants was also decreased compared to the wild type (Fig. 8f). Consequently, the R3 stage mutants accumulated significantly lower shoot and leaf dry biomass compared with the wild-type plants at R3 stage (Fig. 8g). Moreover, yield-related traits such as number of branches, number of pods, number of seed per pod and number of seed per plant as well as total yield were examined. Growth of GmSUT4 mutants was poor compared to wild-type plants, with fewer branches, pods, and seeds, but similar plant height and nodes at the R7 stage (Fig. 8h, i). Accordingly, the total yield was significantly lower (p < 0.05) in mutants than wild type plants (Fig. 8i).
Mutations in the GmSUT4 gene decreased expression of genes related to sucrose metabolic pathways
qRT-PCR analysis revealed the differences in the expression levels of six soybean genes related to sugar distribution metabolism in the mutants (GmSUT4-M1, GmSUT4-M2) and wide type, including known genes involved in sugar transport (GmSUC2, GmSWEET6, GmSWEET11), sucrose metabolism (GmSPP2, GmCInv1), and sucrose signaling (GmSnRK1). The results showed that the expression of GmSPP2, GmCInv1, and GmSnRK1 was significantly down-regulated in the mutants compared with wide type williams82 (Fig. 9a), but the expression of three sucrose transport genes was up-regulated in GmSUT4-M1 and GmSUT4-M2 (Fig. 9b).
GmSUT4 mediated sucrose metabolism and transport
SUT, a member of the MFS family, is ubiquitous in plants. Currently, there are many reports of this family gene in various plant species, including monocotyledonous plants such as rise Oryza sativa (Aoki, et al. 2003), maize (Zea mays L) (Bezrutczyk, et al. 2018), sorghum (Sorghum bicolor (L.))(Babst, et al. 2021), sugarcane (Saccharum officinarum) (Reinders, et al. 2006), and dicotyledons such as Arabidopsis(Meyer, et al. 2004; Wippel and Sauer 2012; Xu, et al. 2020), cotton (Gossypium sp.)(Yadav, et al. 2022), potato (Solanum tuberosum L.) (Wang, et al. 2020). However, very little information is available on the functional expression of the SUT4 gene in soybean in different tissues or under different culture conditions which is important to understand the expression and regulation of this gene in sugar uptake and transport. In this study, the coding sequence of the GmSUT4 gene was cloned by PCR amplification (Fig.S1). GmSUT4 shared several highly conserved regions with other soybean SUT genes based on amino acid sequence analysis (Fig. 1a), but the overall similarity index of the gene sequences is not high (Table S2). The GmSUT4 gene is thought to have the same function as other soybean SUT genes, but it may have other functions. Phylogenetic analysis based on the amino acid sequence of GmSUT4 and homologs from other species revealed that the GmSUT4 gene has the closest homology relationship to two SUT4 proteins in lotus japonicas and peanut, suggesting that they serve similar functions in these species (Fig. 1b). Studies have shown that sucrose transporter genes belonging to the SUT4 subfamily have dynamic targeting to the plasma membrane and vacuole as well as the chloroplast. The sugar transporter OsSUT2 was identified as a plasma membrane protein during functional characterization of yeast, but GFP fusion proved that fluorescence was present in vacuole (Eom, et al. 2011). The sugar transporter AtSUT4 is located in the plasma membrane (Weise, et al. 2000), vacuole (Schneider, et al. 2012)and chloroplast(Rolland, et al. 2003). The expression of GmSUT4 in SUSY7/ura3 yeast can recover the sucrose-uptake ability which requires at least the localization of the GmSUT4 protein to the plasma membrane (Fig. 4), Moreover, the cytoplasmic membrane localization of GmSUT4 in tobacco leaves was confirmed by the GmSUT4-GFP signals (Fig. 2a). Previously, SUTs were reported to be down-regulated by high sucrose concentrations (Ransom-Hodgkins, et al. 2003) but up-regulated by sucrose starvation (Li, et al. 2003). qRT-PCR analysis showed that GmSUT4 gene expression was up-regulated in soybean roots under 1% exogenous sucrose treatment (Fig. 3b). This indicates that sucrose transporter activity was increased under 1% exogenous sucrose treatment. Sucrose transport in phloem vascular tissues was affected by the application of exogenous sucrose. The response appears to be specific to the sucrose transporter, as other transporter activities were not decreased by exogenous sucrose(Yoon, et al. 2021). This dynamic change in the transcriptional level of the GmSUT4 gene in response to exogenous sucrose suggests that GmSUT4 may play an important role in regulating phloem loading.
The individual role of GmSUT4 was further investigated in transgenic Arabidopsis overexpressing GmSUT4. Seed germination rate was decreased with the increase of sucrose concentration, but the difference disappeared on the fifth and sixth day after germination (Fig. 7). Sucrose may function as a sugar signal to regulate plant developmental processes (Chiou and Bush 1998; Huijser, et al. 2000; Rolland, et al. 2006), and seed germination is inhibited by high sugar content via sucrose signaling (Jang, et al. 1997; Rolland, et al. 2006). Sucrose suppresses the interaction between SUT4 and Cyb5-2/A, a member of Cyb5 (Li, et al. 2012). It should be noted that this germination inhibitory effect is specific to the low-affinity SUT4 transporter, which belongs to the AtSUT4 subfamily. In our study, the germination rate of overexpressed transgenic seeds on medium supplemented with sucrose (0.1%, 1%, 3%) was consistently higher than that of wild-type seeds (Fig. 7 ). The increased activity of GmSUT4 in Arabidopsis has an opposite effect on seed germination than AtSUT4, suggesting a different role of GmSUT4 in plants. It can be speculated that the soybean sucrose transporter GmSUT4 reduced seeds sensivity to sucrose and thus improves the seed germination rate, however, the underlying mechanism still remains unclear. A recent report suggests that overexpression of exogenous sucrose transporters in Arabidopsis may have a regulatory function on AtSUTs and further reduce sucrose hypersensitivity to promote seed germination on high-sucrose media (Cai, et al. 2020), which may partly explain the GmSUT4-mediated molecular mechanism of the sucrose metabolism and transport during seed development.
GmSUT4 regulates Arabidopsis growth in a sucrose-dependent manner
Overexpression of GmSUT4 strongly affects leaf development of transgenic Arabidopsis plants, leading to cluster growth in the middle of the leaves (Fig. 4a). Compared with the wild type, the leaves of the overexpressed lines grow slowly (Fig. 4a). Leaf growth and development are primarily controlled by cell proliferation and expansion (Lee, et al. 2022). Recent studies have reported that sugar availability is closely associated with cell proliferation of the apical meristem, and affects overall growth responses and developmental transitions in short-lived plants such as Arabidopsis (Zhang, et al. 2019). In the sucrose uptake experiment of K599-mediated hairy roots, expression of GmSUT4 promoted sucrose uptake and conversion by transgenic roots and accumulated more sugar content in the roots (Fig. 3a, b). SnRK1 is a sugar signaling node and appears to be repressed by high-energy signals such as trehalose-6-P (T6P) (Crepin and Rolland 2019), and SnRK1 delays key life cycle events such as growth, flowering and senescence under a given available sugar (Wingler and Henriques 2022). T6P is a sucrose signaling metabolite and acts as a negative feedback regulator of sucrose levels (Figueroa and Lunn 2016; Yadav, et al. 2014). There are studies showing that SUCs can act as secondary regulators of the SnRK2–ABF pathway, which is primarily regulated by sucrose signaling in plants (Jia, et al. 2015). We hypothesized that early leaf development is influenced by the interaction of SnRK1 and GmSUT4 mediated sucrose signaling and metabolism. Final leaf organ development is primarily under the control of genetic programs that determine the rate and duration of cell proliferation and expansion, which increases the number of rosette leaves. These results require further research to verify this possibility.
Moreover, ectopic expression of GmSUT4 caused bud growth development and resulted in more rosette leaves and branches (Fig. 4b, d). Given the established function of sucrose as a signal for plant bud branch availability at the apex of advantage (Barbier, et al. 2015; Barbier, et al. 2019; Fichtner, et al. 2017; Martin-Fontecha, et al. 2018). Our first consideration was whether the content in sugar supply was affected by the expression of GmSUT4 in Arabidopsis thaliana. Determination of sugar content showed that the overexpressed lines accumulated more sucrose and soluble sugars in the leaves compared to the wild type (Fig. 4e). There is increasing evidence that SnRK1 plays a key role in carbohydrate metabolism. Further analysis revealed that GmSUT4 increased the expression of SnRk1 and Cinv1, as well as the expression of SPP and sucrose efflux genes (Fig. 5a, b). A similar result was obtained by Hall and Ellis (2013): Genes involved in sugar transport (SUC2,SWEET12,SUS) and sugar signaling(SnRK2,2) are differentially expressed in the inflorescence stem. The observed up-regulation of SWEET11, SWEET12 and SWEET13 in source leaves of the transgenic plants (Fig. 5b) could increase the export of sucrose from the source leaves and thus the potential supply of sucrose to the sink organs, including axillary buds. Increasing sucrose supply to axillary buds via such mechanisms would not only provide more carbon and energy for growth, but could also trigger their release from dormancy and growth into new shoots via a signaling pathway (Mason, et al. 2014). However, it can be ruled out that GmSUT4-mediated sucrose signaling and signal transduction may collide with other hormones (such as auxin).
It has also been shown that increasing the activity of GmSUT4 in the Col wild-type background has a strong additive effect on yield (Fig. 6a, b). Seed yield is determined by two components: the number of seeds produced and seed size (or weight) (Kihira, et al. 2017; Song, et al. 2017). In overexpressed plants, no differences were observed in the number of seeds per pod or seed size. Therefore, we explained the increase in seed yield by: (i) a higher transcript level of GmSUT4 in transgenic Arabidopsis increased the final number of rosette leaves and was associated with an up-regulation of SWEETs and related genes (SnRK1,SPP,Cinv1); and (ii) higher sucrose content in the leaves of transgenic GmSUT4 plant increased the supply of sucrose to strongly promote the release of buds, which formed more branches and pods and eventually increased the total seed yield (36.52% for GmSUT4 transgenic lines).
GmSUT4 regulates soybean growth by changing the expression of related genes in sucrose metabolism pathway
The EMS-induced mutant appears visually smaller than wild type Williams 82 from the early stages of vegetative growth to the R stage (Fig. 8a-e). Leaf development is closely related to energy balance. Studies have reported that disruption of SUT4 gene function would result in increased sugar levels in source leaves (Chincinska, et al. 2008; Eom, et al. 2011; Leach, et al. 2017). Compared with the wild type, the ZmSUT2 mutant plants exhibited a twofold increase in sucrose, glucose, fructose, and starch levels in leaves. Similarly, inhibitory expression of StSUT4 reduced the leaf sugar levels the afternoon and increased leaf sugar levels in the evening. Interestingly, the sugar capacity was significantly reduced in the GmSUT4 mutants (Fig. 8f). In the relevant reports on sugar regulation in plant leaf development, the source leaf (Source) was found to be mainly through the T6P/SnRK (trehalose-6-phosphate/sucrose non-fermented protein kinase) pathway (Asim, et al. 2022). Overexpression of ZjSWEET2.2 increases carbon fixation of photosynthetic products by reducing carbohydrate content in mesophyll cells (Geng, et al. 2020). The results of quantitative fluorescence analysis showed that the expression of GmSnRK1 and GmCInv1 as well as GmSPP was significantly down-regulated in the GmSUT4-M1 and GmSUT4-M2 lines (Fig. 9a, b). It is suggested that the GmSUT4 gene may alter sugar and starch content by altering the expression of genes related to sugar synthesis, thereby regulating plant vegetative development. This result supports the hypothesis that smaller leaves and lower reduced accumulation of photosynthetic products are responsible for mutations in the GmSUT4 gene.
In addition, we observed a sharp decrease in in total yield of GmSUT4 mutants compared with wild type (Fig. 8h, i). Seed yield is determined by manipulation of the source-sink interaction. Sucrose transport from mature leaves at the bolting stage increases the expression of the SUT4 and SWEET11 genes, which are regulated by ABA-responsive sucrose signaling genes SnRK2 and AREB2, and is an significant determinant of seed yield in oilseed rape (Lee, et al. 2020). Expression of GmSnRK1 was significantly down-regulated, whereas sucrose transporter gene expression was up-regulated in the GmSUT4-M1 and GmSUT4-M2 lines (Fig. 9a, b). There is little evidence that SUT4 orthologs somehow mediate sucrose export from source leaves to sink organs in trees or herbaceous species (Julius, et al. 2017; Kuhn and Grof 2010; Payyavula, et al. 2011). The growth is severely reduced in null mutants of class III tonoplast SUT in rice and maize, while the sucrose export and long-distance translocation are normal (Julius, et al. 2017). Combined with the result that aboveground tissue biomass are significantly reduced in GmSUT4-M1 and GmSUT4-M1 mutant plants compared with wide type plants (Fig. 8g). This confirms our assumption that inactivation of GmSUT4 has an effect on source metabolism, which in conjunction with SnRK1 involved in sucrose metabolism, affects the growth and development ultimately leading to a decrease in yield.
In this study, the soybean sucrose transporter gene GmSUT4 was characterized. GmSUT4 gene belongs to SUT4 subfamily with highest homology to LjSUT4 and AiSUT4, and subcellular localized in the plasma membrane. The sucrose transport ability of GmSUT4 was verified through function recover of deficient yeast SUSY7/ura3 and soybean hairy roots transformation experiments. In addition, overexpression GmSUT4 in Arabidopsis showed that it plays key roles in positively regulating plant growth, development and thus to increase biomass and seed yield, while the GmSUT4 mutant soybean showed the opposite trend. Furthermore, the sugar signaling, sugar transport and distribution involved genes were characterized differentially expressed in transgenic lines compared with wild types. Taken together, the present research provides evidence that the GmSUT4 gene is involved in the regulation of plant growth and development through sugar metabolism, and lay a foundation for soybean seed yield and quality genetic improvement.
Conflict of interest
The authors declare no known competing financial interests.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
This research work was supported by the National Natural Science Foundation of China (Grant No. 31960368), and the China Agriculture Research System of MOF and MARA (CARS-16-E14).
PC, conceived and designed the research. XW, JN performed the experiments and analyzed the data. XW, wrote the manuscript and JY, MS revised the manuscript. DL, SC, CW, QW, HZ, JP, CN, MW participated in experiments and data collection. All authors read and approved the manuscript.
We are thankful to Professor Qiusheng Yang for providing mutant yeast SUSY7/ura3 and vector PDR196. We also thanks to Professor Qingxin Song for providing seeds of soybean cultivar Williams 82 and mutant line NJAU1191 and NJAU0264.
Functional characterization of a soybean GmSUT4 gene reveals its involvement in plant growth and development regulation through sugar metabolism
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