The transcription factors GmSTF1 and GmSTF2 promote photosynthesis and lignin biosynthesis in soybean

Abstract

Photosynthesis, as the primary biochemical reaction for carbon fixation, provides the carbon skeletons for diverse secondary metabolites in plants. Here, we report that the transcription factors GmSTF1 and GmSTF2 regulate both photosynthesis and lignin biosynthesis in soybean (Glycine max). GmSTF1 and GmSTF2 directly bound to a TGACG motif in the promoter of the photosynthetic gene GmLHCA4 and a Z-box element in the promoter of the lignin biosynthetic gene GmCAD1, activating their transcription. Loss-of-function mutants (gmstfs-dm) displayed light-green leaves with reduced chlorophyll levels, photosynthetic rates, soluble sugar contents, and lignin contents. Conversely, transgenic soybean plants overexpressing these genes (GmSTF1-YFP and GmSTF2-YFP) showed enhanced chlorophyll accumulation, photosynthetic efficiency, soluble sugar production, and lignin deposition. Our findings identify GmSTF1 and GmSTF2 as critical regulators of carbon assimilation and lignin production in soybean.

Dear Editor,

Plants harness solar energy through photosynthesis, converting carbon dioxide and water into carbohydrates and oxygen and storing the energy in chemical form [1]. These carbohydrates serve as precursors for the biosynthesis of secondary metabolites via various pathways, ultimately producing a wide array of organic compounds [2,3].

As the primary pigment in photosynthesis, chlorophyll captures light energy to drive photosynthetic process [4,5], and its cellular concentration directly dictates photosynthetic capacity [5,6]. This complex process involves over 50 intermediate reactions within chloroplasts, where light energy harvested by photosystems II (PSII) and I (PSI) is converted into chemical energy. Through the Calvin-Benson cycle, atmospheric CO₂ is fixed into organic carbon skeletons [1,7]. These fixed carbons are subsequently channeled through the shikimate pathway to generate phenylpropanoids [8,9].

Lignin, constituting approximately 30% of the biosphere's organic carbon, is derived from the phenylpropanoid pathway. It is crucial for the structural integrity of the cell wall and the stiffness and strength of the stem. In addition, lignin waterproofs the cell wall, enabling the transport of water and solutes through the vascular system, and helps protect plants against insects and pathogens [10,11]. At least 13 genes encode cytosolic enzymes involved in lignin biosynthesis in plants [10,12]. Various developmental and environmental cues affect the transcription of distinct lignin biosynthetic genes and thus lignin biosynthesis, but the regulatory mechanisms remain to be fully elucidated.

In this study we reported that the transcription factors GmSTF1 and GmSTF2 promote the photosynthesis and lignin biosynthesis in soybean. GmSTF1 and GmSTF2 directly bind to the promoters of the photosynthetic gene GmLHCA4 and the lignin biosynthetic gene GmCAD1 and activate their transcription. Our findings thus shed light on the molecular framework underlying the regulation of photosynthesis and lignin biosynthesis in soybean.

Our previous phenotypic analysis revealed that the gmstfs-dm mutants consistently displayed light-green leaves compared to wild-type Williams 82 (W82) in both the greenhouse and field (Fig. 1A, Fig. S1A), suggesting that GmSTF1 and GmSTF2 promote chlorophyll biosynthesis and accumulation in soybean. We measured chlorophyll contents in the leaves of gmstfs-dm plants, as well as transgenic soybean plants overexpressing these genes (GmSTF1-YFP and GmSTF2-YFP). Chlorophyll content was significantly lower in gmstfs-dm than in wild-type W82 (Fig. 1B), whereas GmSTF1-YFP and GmSTF2-YFP plants accumulated more chlorophyll than wild-type Tianlong 1 (TL1) (Fig. 1C).

Fig. 1.

GmSTF1 and GmSTF2 promote the chlorophyll biosynthesis and photosynthesis in soybean. A Leaf phenotypes of W82 and gmstfs-dm soybean grown in the greenhouse. B–K Analysis of photosynthesis-related parameters in W82, gmstfs-dm, TL1, GmSTF1-YFP and GmSTF2-YFP soybean. Chlorophyll content (B–C), Photosynthetic rate (D-E), Conductance to H₂O (F-G), Transpiration rate (H–I), Intercellular CO₂ concentration (J-K). L-M Soluble sugar content in W82, gmstfs-dm, TL1, GmSTF1-YFP, GmSTF2-YFP. N–O The expression of GmLHCA4 in W82, gmstfs-dm, TL1, GmSTF1-YFP, GmSTF2-YFP. In the panels of (B–O), asterisks indicate significant differences (∗∗∗P < 0.001, ∗∗P < 0.01, and ∗P < 0.05) determined by two-tailed Student's t-test. P Schematic representation of the GmLHCA4 promoter. The numbers represent the locations of the GmLHCA4 promoter regions relative to the translation start site (referred to as position +1). Q Yeast one-hybrid assays showing that GmSTF1 and GmSTF2 activate the GmLHCA4pro:LacZ reporter. R EMSA showing that GmSTF1 and GmSTF2 bind to GmLHCA4 promoter sub-fragments in vitro. ‘‘-’’ indicates the absence of corresponding probes or proteins. “+” indicates the existence of corresponding probes or proteins. Competitor indicates the non-biotin-labeled GmLHCA4pro probe. FP, free probe. The arrowheads indicate protein–DNA complexes. S Promoter structures of GmLHCA4 showing the positions of fragments used for chromatin immunoprecipitation (ChIP)–qPCR. T ChIP–qPCR assays showing that GmSTF1 and GmSTF2 associate with the GmLHCA4 promoters in vivo. Thirty-day-old TL1 and GmSTF1-YFP and GmSTF2-YFP soybean leaves were used for the ChIP assays. Chromatin fragments were immunoprecipitated using GFP-Trap antibodies. The GmActin gene was used as the negative control. Error bars represent SE (n = 3). Asterisks indicate significant differences (∗∗∗P < 0.001) determined by two-way ANOVA with Tukey hypothesis test.

Since chlorophyll is crucial for photosynthesis [6], we measured photosynthetic rates in gmstfs-dm, GmSTF1-YFP, and GmSTF2-YFP plants. The photosynthetic rate, stomatal conductance of H₂O, and transpiration rate were significantly lower in gmstfs-dm than in W82 plants but were higher in both GmSTF1-YFP and GmSTF2-YFP plants than in TL1 (Fig. 1D–I). The intercellular CO₂ concentrations were comparable in gmstfs-dm and W82 and significantly higher in both GmSTF1-YFP and GmSTF2-YFP plants than in TL1 (Fig. 1J–K). Consequently, the soluble sugar content was significantly reduced in gmstfs-dm but elevated in both GmSTF1-YFP and GmSTF2-YFP plants (Fig. 1L-M). These data suggest that GmSTF1 and GmSTF2 promote chlorophyll accumulation, photosynthetic efficiency, and soluble sugar production.

To investigate the molecular basis of the GmSTF1- and GmSTF2-mediated promotion of photosynthesis, we analyzed RNA-seq data, which indicated that GmSTF1 and GmSTF2 regulate the expression of 40 genes involved in chlorophyll biosynthesis and 65 genes involved in photosynthesis (Fig. S2). RT-qPCR confirmed that GmSTF1 and GmSTF2 positively regulate the expression of six photosynthetic genes: GmLHCA4, GmSVR1, GmGATB, GmHEMC, GmCHLG, and GmFD11 (Fig. 1N-O, Fig. S3). GmLHCA4 encodes a subunit of the light-harvesting complex of PSI [13,14]. GmLHCA4 was downregulated in gmstfs-dm and upregulated in GmSTF1-YFP and GmSTF2-YFP leaves and stems (Fig. S4A–B). The expression of GmLHCA4 in gmstfs-dm decreased at both the V3 and V4 stages, whereas it increased in GmSTF1-YFP and GmSTF2-YFP at the V1–V3 stages but not at V4 (Fig. S4C–D).

In yeast one-hybrid assays, GmSTF1 and GmSTF2 activated the GmLHCA4pro:LacZ reporter (Fig. 1P-Q). To map the binding site(s) of GmSTF1 and GmSTF2 within the GmLHCA4 promoter, we divided the promoter fragment into three portions (A, B, and C). GmSTF1 and GmSTF2 activated GmLHCA4proB:LacZ, but not GmLHCA4proA:LacZ or GmLHCA4proC:LacZ (Fig. 1P-Q), suggesting that the GmSTF1 and GmSTF2 binding sites are likely located within the B region and do not require the C region (Fig. 1P). The GmSTF1- and GmSTF2-mediated activation of the reporter in yeast cells was completely abolished upon mutation of the TGACG motif, but not the ACE cis-element (Fig. 1Q), suggesting that the TGACG motif in the GmLHCA4 promoter is required for GmSTF1 and GmSTF2 binding. To support these results, we performed an in vitro Electrophoretic Mobility Shift Assay (EMSA). Recombinant GST-GmSTF1 and GST-GmSTF2, but not GST alone, bound to sub-fragments of the GmLHCA4 promoter containing an intact TGACG motif (Fig. 1R). As the amount of DNA probes containing the non-biotin-labeled GmLHCA4 promoter (competitor) increased, the binding affinity of GST-GmSTF1 and GST-GmSTF2 to the biotin-labeled probes decreased (Fig. 1R). ChIP-qPCR assays verified the binding of GmSTF1 or GmSTF2 to the GmLHCA4 promoter in vivo: GmSTF1 or GmSTF2 was specifically enriched on P3 fragments of the GmLHCA4 promoter containing an intact TGACG motif (Fig. 1T). These results suggest that GmSTF1 and GmSTF2 directly associate with the GmLHCA4 promoter to activate its transcription, thereby promoting photosynthesis.

Given that photosynthetic products such as soluble sugars provide the carbon sources needed for lignin biosynthesis in plants (Fig. 2A) [2,8], we examined whether GmSTF1 and GmSTF2 regulate this process. Lignin content was significantly reduced in gmstfs-dm plants compared to W82, whereas GmSTF1-YFP and GmSTF2-YFP plants accumulated more lignin than TL1 under both greenhouse and field conditions (Fig. 2B–C, Fig. S1B). Following phloroglucinol-HCl lignin staining, gmstfs-dm showed lighter staining in xylem compared to W82, whereas GmSTF1-YFP and GmSTF2-YFP produced robust xylem vessels with more intense red coloration than TL1 (Fig. 2D). Therefore, GmSTF1 and GmSTF2 promote lignin biosynthesis and accumulation in soybean.

Fig. 2.

GmSTF1 and GmSTF2 promote lignin biosynthesis by directly activating GmCAD1 transcription. A Schematic representation of key enzymes and steps involved in lignin biosynthesis pathways. B–C Lignin content in W82, gmstfs-dm, TL1, GmSTF1-YFP, GmSTF2-YFP soybean grown in the greenhouse. D Cross-sections of stems at the 4th internode of W82, gmstfs-dm, TL1, GmSTF1-YFP and GmSTF2-YFP at the V5 stage stained with phloroglucinol-HCl, respectively. Scale bar = 100 μm. E-F The transcript levels of GmCAD1 in W82, gmstfs-dm, TL1, GmSTF1-YFP, GmSTF2-YFP. Error bars represent SE (n = 3). Asterisks indicate significant differences (∗∗∗P < 0.001) determined by two-way ANOVA with Tukey hypothesis test. G Schematic representation of various constructs used in the transient transfection assay in Nicotiana benthamiana leaves. H Transient transcriptional activation assay showing that GmSTF1 and GmSTF2 activated the GmCAD1pro:LUC.I Promoter structures of GmCAD1pro showing the positions of fragments used for ChIP–qPCR. J ChIP–qPCR assays showing that GmSTF1 and GmSTF2 associate with the GmCAD1 promoter in vivo. The GmActin gene was used as the negative control. Error bars represent SE (n = 3). Asterisks indicate significant differences (∗∗∗P < 0.001) determined by two-way ANOVA with Tukey hypothesis test. K EMSA showing that GmSTF1 and GmSTF2 bind to GmCAD1 promoter sub-fragments in vitro. ‘‘-’’ indicates the absence of corresponding probes or proteins. “+” indicates the existence of corresponding probes or proteins. Competitor indicates the non-biotin-labeled GmCAD1pro probe. FP, free probe. The arrowheads indicate protein–DNA complexes. L A proposed working model showing how GmSTF1 and GmSTF2 enhance photosynthesis and lignin biosynthesis in soybean. They directly activate the key photosynthetic gene GmLHCA4, thereby enhancing photosynthesis and soluble sugar production. Concurrently, they promote lignin biosynthesis by upregulating the biosynthetic gene GmCAD1 in soybean.

Given that stem lignin content is positively correlated with lodging resistance [10,12], we measured stem strength in gmstfs-dm, GmSTF1-YFP, and GmSTF2-YFP plants. Whereas gmstfs-dm plants exhibited weaker stem strength than W82, GmSTF1-YFP and GmSTF2-YFP plants showed stronger stems than TL1 under both greenhouse and field conditions (Fig. S5A–B). Moreover, gmstfs-dm plants were significantly taller than W82, whereas GmSTF1-YFP and GmSTF2-YFP plants were slightly shorter than TL1 when grown in the field (Fig. S5C). The yields per plant for gmstfs-dm, GmSTF1-YFP, and GmSTF2-YFP were comparable to those of the corresponding wild types (Fig. S5D). These results suggest that GmSTF1 and GmSTF2 simultaneously enhance stem strength and suppress stem elongation.

Considering that GmSTF1 and GmSTF2 promote lignin biosynthesis, we examined whether they regulate the expression of lignin biosynthetic genes. GmCAD1 (Cinnamoyl Alcohol Dehydrogenase 1), GmCCR1 (Cinnamoyl CoA Reductase 1), GmCCR2, and GmCCR3 were significantly downregulated in gmstfs-dm but upregulated in GmSTF1-YFP and GmSTF2-YFP compared to the wild type (Fig. 2E, Fig. S6). GmCAD1 catalyzes the reduction of cinnamaldehydes to the corresponding alcohols [10]. The expression of GmCAD1 was reduced in gmstfs-dm but higher in GmSTF1-YFP and GmSTF2-YFP leaves and stems compared to the wild type (Fig. S7A–B). GmCAD1 transcript levels were reduced in gmstfs-dm across all stages examined (V1–V4), whereas they were elevated in GmSTF1-YFP and GmSTF2-YFP plants, particularly from stages V2 to V4 (Fig. S7C–D).

Both GmSTF1 and GmSTF2 activated the GmCAD1pro:LUC reporter when transiently co-expressed in Nicotiana benthamiana (Fig. 2G–H). To test whether GmSTF1 and GmSTF2 bind to the GmCAD1 promoter, we performed ChIP-qPCR. These proteins were enriched on GmCAD1 promoter region P3 containing a Z-box motif (Fig. 2I–J). In addition, in EMSAs, purified recombinant GST-GmSTF1 and GST-GmSTF2, but not GST alone, bound to biotin-labeled GmCAD1 promoter fragments possessing a Z-box motif. As the amount of non-biotin-labeled DNA fragments (competitor) increased, the binding of GST-GmSTF1 or GST-GmSTF2 to the same biotin-labeled GmCAD1 promoter fragments decreased. GST-GmSTF1 and GST-GmSTF2 failed to bind to GmCAD1 promoter fragments in which the Z-box was mutated (Fig. 2K). Together, these data indicate that GmSTF1 and GmSTF2 can directly associate with the Z-box cis-element in the GmCAD1 promoter to activate transcription both in vivo and in vitro.

In summary, our data show that the transcription factors GmSTF1 and GmSTF2 both directly bind to the promoters of the photosynthetic gene GmLHCA4 and the lignin biosynthetic gene GmCAD1 to upregulate their expression (Fig. 2L). In addition to GmLHCA4 and GmCAD1, GmSTF1 and GmSTF2 regulate the expression of a suite of other genes involved in photosynthesis and lignin biosynthesis (Fig. S2–3, S6), suggesting that the metabolic network from photosynthetic carbon fixation to lignin deposition might be more complex than previously assumed. Although GmSTF1 and GmSTF2 function as key regulators promoting photosynthesis and lignin biosynthesis in soybean, their breeding potential remains to be validated through field trials, particularly regarding lodging resistance and yield performance of mutants and transgenic lines across varying planting densities.

1. Materials and methods

1.1. Plant materials and growth conditions

gmstfs-dm are in soybean (Glycine max (L.) Merr.) cultivar Williams 82 (W82) background. The GmSTF1-YFP and GmSTF2-YFP are in Tianlong 1 (TL1) background [15]. They were grown in long-day conditions (14 h light/8 h dark, light intensity: 524 μmol/m²/s) in the greenhouse that was maintained at 25 °C. Field experiments were conducted during the summer of 2022 and 2023 at the Transgenic Experimental Station of the Jiangsu Academy of Agricultural Science, Nanjing, Jiangsu (118°38′33.45″E, 32°28′39.59″N).

1.2. Measurement of photosynthetic parameters

The LI-6400 portable photosynthesis system (LI-6400XT; LI-COR, USA) was used to measure the net photosynthetic rate, stomatal conductance, transpiration rate and intercellular carbon dioxide concentration according to the manufacturer's instructions.

1.3. Measurement of chlorophyll content

For chlorophyll content analysis, 0.1 g of fresh leaves were collected. 2 mL of 80% acetone was added, and then kept in the dark at room temperature overnight. The UV spectrophotometer was used to measure the absorbance of extraction at wavelengths of 645 nm, 663 nm, and 470 nm respectively.

1.4. Extraction and determination of soluble sugars

Soluble sugar contents were determined in leaves collected from 21-d-old greenhouse-grown plants, including the gmstfs-dm mutant, GmSTF1-YFP and GmSTF2-YFP overexpression lines, and two WT (W82 and TL1). Soluble sugar was determined as described by Du et al. [16].

1.5. Extraction and determination of lignin

Lignin content was measured in stems (the third main internode) harvested from 21-day-old greenhouse-grown plants. Measurements were performed according to Deng et al. [17].

1.6. Measurement of stem strength

The measurement of stem strength was previously described [18]. The W82, gmstfs-dm, TL1, GmSTF1-YFP and GmSTF2-YFP were grown in the greenhouse for 46 d or in the field harvested at the R6 stage. Stem strength was measured using a push-pull meter when the first internode was tilted to 40° from the vertical.

1.7. Yeast one-hybrid assay

Approximately 2.5 kb DNA promoter fragments from GmLHCA4 were cloned into the placZi-2μ vector. pB42AD-GmSTF1 or pB42AD-GmSTF2 were used as effectors. The two plasmids were transformed into the yeast strain EGY48. Positive transformants were screened on synthetic defined medium lacking Trp and Ura (SD/-Trp-Ura; Coolaber) and transferred to chromogenic medium containing raffinose, galactose, and X-gal for blue color development. The primers used for this assay are listed in Table S1.

1.8. Dual-luciferase reporter system

Nicotiana benthamiana plants were used for transient transactivation assay. Agrobacterium strain GV3101 cells carrying the 35S:GmSTF1-YFP, 35S:GmSTF2-YFP or GmCAD1pro:LUC constructs were transiently infiltrated into Nicotiana benthamiana leaves as indicated combinations. Firefly LUC and Renillia LUC (Ren) were detected using the Dual-LUC Reporter Assay System (Vazyme, DL101), according to the manufacturer's instructions.

1.9. Electrophoretic mobility shift assay (EMSA)

The EMSA was performed using a Light Shift Chemiluminescent EMSA Kit (Beyotime #GS009) according to the manufacturer's protocol. The binding activities of proteins were analyzed using an oligonucleotide labeled with biotin at the 5' end, using the EMSA Probe Biotin Labeling Kit (Beyotime #GS008). The specific probes used in vitro EMSA are listed in Table S1.

1.10. ChIP-qPCR assay

TL1, GmSTF1-YFP, and GmSTF2-YFP were collected and treated with formaldehyde to crosslink protein–DNA complexes. After washing three times, the excess liquid was removed. Samples were ground to a fine powder with a pestle and mortar in liquid nitrogen. After isolation and sonication of chromatin, samples were centrifuged at 16,000 g for 5 min, and the supernatants were collected. Anti-GFP antibodies were used for immunoprecipitation. 10% of each supernatant served as the input. The specific primers used in this experiment are listed in Table S1.

1.11. RNA extraction and RT-qPCR analysis

The FastPure® Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, RC112) is used for RNA extraction. cDNAs were synthesized using the HiScript® Ⅲ 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, R312) according to the manufacturer's instructions. Quantitative real-time PCR was performed using the LightCycler 480 Ⅱ detection system (Roche) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q511). The specific primers used in this experiment are listed in Table S1.

CRediT authorship contribution statement

Zhaoqing Song: Validation, Conceptualization. Fengyue Zhao: Investigation. Yeting Bian: Investigation. Jiaxuan Li: Investigation. Wubin Wang: Supervision, Conceptualization. Dongqing Xu: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

Gene sequences were referred to the Phytozome database (Glycine max W82.a4.v1) with following accession numbers: GmSTF1 (Glyma.18G117100), GmSTF2 (Glyma.08G302500), GmLHCA4 (Glyma.06G194900), GmCAD1(Glyma.14G221200), GmCCR1 (Glyma.19G006900), GmActin (Glyma.18G290800).