Cloning and functional study of GmRPI2, which is the critical gene of photosynthesis in soybean

Abstract

Light provides energy for photosynthesis and is also an important environmental signal that regulates plant growth and development. Ribose-5-phosphate isomerase plays a crucial role in photosynthesis. However, ribose-5-phosphate isomerase has yet to be studied in soybean photosynthesis. To understand the biological function of GmRPI2, in this study, GmRPI2 was cloned, plant overexpression vectors and gene editing vectors were successfully constructed, and transformed into recipient soybean JN74 using the Agrobacterium-mediated method. Using qRT-PCR, we analyzed that GmRPI2 gene expression was highest in leaves, second highest in roots, and lowest in stems. Promoter analysis revealed the presence of multiple cis-acting elements related to light response in the promoter region of GmRPI2. Compared with the control soybean plants, the net photosynthetic rate and transpiration rate of the overexpression lines were higher than those of the control and gene editing lines, while the intercellular CO₂ concentration was significantly lower than that of the control and gene editing lines; the total chlorophyll, chlorophyll a, chlorophyll b contents and soluble sugar contents of the overexpression plants were significantly higher than those of the recipient and editing plants, indicating that the GmRPI2 gene can increase The GmRPI2 gene can increase the photosynthetic capacity of soybean plants, providing a theoretical basis and genetic resources for improving soybean yield by regulating photosynthetic efficiency.

Introduction

Soybean (Glycine max (Linn.) Merr.) is one of the five major cash crops and an important source of vegetable protein and oil worldwide (Li et al. 2014, Zhao et al. 2018). Crop yield is a global challenge, and there is a need to increase food production from the available land area to meet the demand of future populations (Hubbart et al. 2007, Takai et al. 2013, Taylaran et al. 2011). Photosynthesis is one of the critical factors affecting crop yield, and studies have shown that 90–95% of dry crop matter comes from photosynthesis (Rodriguez et al. 2014, Zhiponova et al. 2020). Bioengineering of plant photosynthesis and the development of existing sources of genetic variation for photosynthetic traits are potent ways to increase the genetic yield potential of plants (Long et al. 2015, Pan et al. 2018).

Photosynthesis is the physiological basis of biomass production, and regulating photosynthesis can improve growth and yield (Yamori et al. 2016). Improving the photosynthetic efficiency of crops is considered a very effective way to increase crop productivity (Makino 2011, Ort et al. 2015). The leaf is the main organ of photosynthesis, which absorbs light energy from sunlight through an antenna system and transmits it to reaction centers. The absorbed light energy is used to synthesize carbohydrates through carbon dioxide and water, which is the essential life process of plants (Liu et al. 2016). Chlorophyll (Chl) is the most crucial pigment in plants and is essential for photosynthesis (Fromme et al. 2003). Another reason for the change in photosynthetic rate could be the direct effect of carbohydrate accumulation in the leaves (Morita et al. 2016). Overexpression of purple acid phosphatase (AtPAP2) in potatoes increased photosynthesis, accelerated plant growth and tuber starch content, and introduced an early flowering phenotype (Zhang et al. 2014). Soluble sugars, sucrose, and starch are the main products of plant carbon metabolism, and their contents and changes are closely related to photosynthesis (Wilcox 2001).

Ribose-5-phosphate isomerase (RPI) is a naturally widespread and highly conserved protease that plays a critical enzymatic role in the pentose phosphate oxidation cycle (Hamada et al. 2003); it is also an essential player in cellular carbohydrate metabolism, participating in the prokaryotic and eukaryotic phosphate pentose pathway (PPP) and the Calvin cycle in plants for CO₂ fixation (Chen et al. 2020, Gontero et al. 1988, Zhang et al. 2003). Genes encoding ribulose-5-phosphate isomerases have been cloned from a variety of organisms. In Arabidopsis, four RPIs exist, each with a different catalytic function. Previous studies have shown that RPI1 is transcribed in roots, leaves, and seedlings and to a lesser extent in inflorescences and angiosperms, whereas RPI2 is actively expressed in leaves, floral tissues, and angiosperms but not in roots or seedlings (Howles et al. 2006) RPI1 deletion results in swelling of root hair-forming cells, reduced cellulose levels and altered levels of specific monosaccharides in non-cellulosic polysaccharides. RPI2 deletion disrupts chloroplast structure, reduces photosynthetic capacity, and affects chlorophyll content (Xiong et al. 2009); Vasilios et al. found in Arabidopsis that deletion of RPI3 affects embryo development and affects plant growth (Andriotis and Smith 2019).

Many RPIs have been identified in previous studies, but the function of ribose-5-phosphate isomerase has yet to be identified in soybean. In this study, we cloned and identified the GmRPI2 gene and analyzed the expression pattern of GmRPI2 in roots, stems, and leaves using real-time fluorescence quantitative PCR. Overexpression of GmRPI2 in soybean increased photosynthetic efficiency and photosynthetic product accumulation, whereas knockdown of GmRPI2 produced the opposite result. These results suggest that GmRPI2 provides a theoretical basis and genetic resource for high-light efficiency breeding.

Materials and Methods

Materials

The experimental materials were selected from the natural population materials constructed in the previous stage, and JN74 showed high adaptability, early maturity, and good resistance to inversion during the growth cycle after many years of multi-location field cultivation.

Strains and plasmids: E. coli strain DH5α, Agrobacterium strain EHA105, cloning vectors pMD-18T, and pCAMBIA3301 were provided by the Plant Biotechnology Center of Jilin Agricultural University.

Seeds of the recipient soybean JN74, two overexpression lines OE1 and OE2, and two gene editing lines KO1 and KO2 were sown and cultured in the experimental field for subsequent experiments. Field management conditions were 4 m row length, 20 cm monopoly spacing, and a test field with excellent and consistent soil conditions was selected with one protected row on the side.

Experimental methods

Sequence analysis and vector construction

Total RNA was extracted from young soybean leaves using RNAiso-Pluskits (TaKaRa). for reverse transcription PCR, one ug of total RNA was treated with RNase-free DNase I, and first-strand cDNA was synthesized using SuperScriptTM III reverse transcriptase (Invitrogen). The cDNA was used as a PCR amplification template with primer sequences as in Supplemental Table 1. pCAMBIA3301-GmRPI2 overexpression vector was subsequently constructed using the Seamless Cloning Kit (Solarbio), with CaMV35S as the promoter. The gRNA target sequence was designed by the online software CRISPR-P, and ATU6-26 was the promoter to construct the gene editing vector pCBSG015-GmRPI2.

The cDNA sequence of the GmRPI2 (GenBank accession number: XM_003555894.5) gene was obtained from NCBI (https://www.ncbi.nlm.nih.gov/), and the homologous sequence of GmRPI2 gene was retrieved from the Uniprot database (UniProt). The MEGA11 software was used for Sequence alignment. Online predictive analysis of cis-acting elements of cloned sequences using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), a database of plant cis-acting elements.

Genetic transformation and identification

In this experiment, the recombinant vector was introduced into the recipient JN74 regarding an optimized Agrobacterium infesting soybean cotyledon node method to improve transformation efficiency (Li et al. 2017). The genomic DNA of T₀ generation overexpression plants, gene editing plants, and recipient plant leaves were extracted for PCR to identify T₀ generation positive plants. The obtained positive plants were subjected to additive generation in the greenhouse and field to obtain T₂ generation positive plants, and the T₂ generation gene editing plants were tested for targeting.

Southern blot analysis

The genomic DNA of the leaves of transformed plants positive for T₂ generation PCR was extracted in bulk by the CTAB method, and the Bar gene PCR product was used to prepare the probe. The GmRPI2 gene expression vector plasmid was used as a positive control, and the recipient control check was used as a negative control to detect the integration of the exogenous marker gene Bar in soybean lines.

RNA extraction and qRT-PCR analysis

Total RNA from different tissues (root, stem, and leaf) of T₂ generation PCR positive transgenic lines was extracted with RNAiso-Pluskits (TaKaRa), and the RNA was reverse-transcribed to cDNA using a fluorescent quantitative reverse transcription kit (TakaraBio, RR036A) as a template for the qRT-PCR reaction. The primers required for fluorescent quantitative PCR were designed (Supplemental Table 1). qRT-PCR amplification was performed using Prime-ScriptTM RT Master Mix (Fermentas) reagents. The Mx3000P fluorescent qRT-PCR instrument (Agilent Technologies, USA) was used for analysis. The relative expression of the target genes in the roots and leaves of the transformed plants was detected and calculated using the 2^–ΔΔCt method, and the measurements were repeated three times, and the average value was taken (Chi et al. 2016, Imoto et al. 2022).

Analysis of photosynthetic characteristics parameters

The net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO₂ concentration of soybean plants at the seedling, flowering, and pod-filling stages were measured with a Li-6400 (Li-Cor Inc., Lincoln, NE) portable photosynthesis meter starting at 9:00 a.m. on a sunny day according to Michelmore et al. (1991). Five plants were randomly selected for each strain, and the measurement site was the third leaf from top to bottom, and the measurement was repeated three times, and the average value was taken.

Measurement of photosynthetic pigment content

Soybean leaves of consistent growth at the three reproductive stages were selected and immersed in 80% aqueous acetone solution at room temperature to 24 h in the dark (Li et al. 2014), and the absorbance values of the extracts at 663, 645, and 470 nm were measured using an Infinite M200PRD ELISA (TECAN, Switzerland) and replicated three times. The following equation (Porra et al. 1989, Schoefs et al. 2001) was used to calculate.

C_a (mg L^–1) = 12.21A₆₆₅ – 2.82_A649

C_b (mg L^–1) = 20.13A₆₄₉ – 5.03_A665

Car (mg L^–1) = (1000A470 – 3.27C_a – 104C_b)/229

Where Chla (mg g^–1): chlorophyll a; Chlb (mg g^–1): chlorophyll b; Car (mg g^–1): carotenoid.

Determination of carbon metabolism-related indicators

The third leaf from top to bottom of soybean with consistent growth in three reproductive stages was selected and snap frozen in liquid nitrogen at –80°C. Soluble sugar, sucrose, and starch contents were measured (Du et al. 2020).

Data analysis

All data in the tables and graphs in this paper are reported as mean ± standard error. The materials were compared using a two-way analysis of variance (ANOVA) in GraphPad Prism 8 or IBM SPSS Statistics 27, depending on the type of analysis. Where * indicates a significant difference (P < 0.05); ** indicates a highly significant difference (P < 0.01).

Results

Cloning of GmRPI2 gene and vector construction

In this study, the gene GmRPI2 was cloned from soybean JN74, which has a CDS of 996 bp (Fig. 1A) and encodes a polypeptide containing 245 amino acid residues with a predicted molecular weight of 28.49 kD. By comparing the homologous sequence analysis of this gene (Fig. 2), GmRPI2 contains the same structural domain as in rice, maize, and Arabidopsis. Double digestion of the overexpression vector plasmid using BglII and BstEII enzymes resulted in a large fragment of the base vector pCAMBIA3301 and a 996 bp target fragment (Fig. 1B). The sequence comparison of SG1 (small guide RNA 1) and SG2 (small guide RNA 2) of the gene editing vector pCBSG015-GmRPI2 was also performed (Fig. 3), and the sequencing results showed no base mutation, indicating that the overexpression vector pCAMBIA3301-GmRPI2 and the gene editing vector pCBSG015-GmRPI2 were successfully constructed.

Fig. 1.

A: Extraction of total RNA from soybean leaves; B: cloning of target genes; C: erification results of double digestion. M: DL2000 DNA marker; 1–4: products of PCR.

Fig. 2.

Evolutionary tree and sequence comparison of GmRPI2 gene.

Fig. 3.

Comparison of sequencing results of pCBSG015-GmRPI2. A: The comparison diagram of SG1 sequence; B: The comparison diagram of SG2 sequence.

Since the upstream promoter regulates the expression level of the gene, the promoter element analysis was performed for 2000 bp upstream of their GmRPI2 gene, and the analysis results showed (Table 1) that the upstream promoter of GmRPI2 gene contains several elements that interact with reactive oxygen species, abscisic acid, and light response.

Table 1. Cis-elements in the promoter region (~2 kb) of GmRPI 2

Site name	Strand	Numbers of bases	Sequence	Function
TATA-box	+	4	TATATA	core promoter element around –30 of transcription start
CAAT-box	–	7	CAAAT	common cis-acting element in promoter and enhancer regions
AE-box	–	1	AGAAACTT	part of a module for light response
GT1-motif	+	1	GGTTAAT	light responsive element
A-box	–	2	CCGTCC	cis-acting regulatory element
GATA-motif	–	2	GATAGGG	part of a light responsive element
G-Box	+	2	CACGAC	cis-acting regulatory element involved in light responsiveness
TC-rich repeats	+	1	ATTCTCTAAC	cis-acting element involved in defense and stress responsiveness
TCCC-motif	+	1	TCTCCCT	part of a light responsive element

Genetic transformation and identification of GmRPI2 gene

The constructed expression vector was genetically transformed to obtain soybean-transformed plants (Supplemental Fig. 1). PCR assays were performed on the T₂ generation positive plants obtained by their addition generation, and some of the results are shown below (Supplemental Fig. 2); target mutation assays were also performed on the T₂ generation gene editing plants, and the mutation types were mainly base deletion, substitution and insertion (Fig. 4).

Fig. 4.

The results of the T₂ gene editing plant target. –: Base deletion; S: base replacement; +: base insertion.

Southern detection of GmRPI2 overexpression gene strains

Southern blot hybridization assay was performed on the T₂ overexpression transformed plants that were positive by PCR (Fig. 5), and hybridization signals appeared in lanes 1, 2, 3, 4, and 5, but at different positions. The test results indicated that the GmRPI2 gene was integrated into the genome of T₂ generation transformed plants mainly in single-copy and multi-copy manner and at different integration sites.

Fig. 5.

Southern blot detection results of T₂ generation transgenic GmRPI2 plants. M: Southern DNA marker; P: positive control; N: water; CK: untransformed plants; 1–4: Transforming plants.

qRT-PCR assay of T₂ generation transformed plants

The results of relative expression analysis using the β-Lactin (GenBank accession number: NM_001252731.2) gene as an internal reference gene (Fig. 6) showed that the relative expression of the GmRPI2 gene in both roots and leaves was significantly increased in the seedling, flowering and pod-filling stages of the overexpressing transgenic lines compared with the control lines; the relative expression of GmRPI2 gene in both roots and leaves was significantly decreased in the gene editing vector transformed lines.

Fig. 6.

Relative expression of GmRPI2 gene at different periods in each transgenic strain (A: seedling stage; B: flowering stage; C: pod-filling stage). * indicates significant difference (P < 0.05); ** indicates highly significant difference (P < 0.01).

Measurement of photosynthetic pigment content of soybean by GmRPI2 gene

As shown in Table 2, the leaf chlorophyll content of the soybean strains transformed with the GmRPI2 gene was significantly regulated during the three reproductive periods of seedling, flowering, and pod-filling stages. The chlorophyll content of OE1 and OE2 leaves at the seedling stage increased by ->54.06% and ->43.08%, respectively, compared with the control strain; the chlorophyll content of KO1 and KO2 leaves decreased by 66.02% and 64.43%, respectively, compared with the control strain. The chlorophyll content of OE1 and OE2 leaves at the flowering stage increased by ->55.69% and ->59.46%, respectively, compared with the control strain; the chlorophyll content of KO1 and KO2 leaves decreased by 77.18% and 75.98%, respectively, compared with the control strain. The chlorophyll content of OE1 and OE2 leaves at the pod-filling stage increased by ->58.45% and ->48.59%, respectively, compared with the control strain; the chlorophyll content of KO1 and KO2 leaves decreased by 76.53% and 76.29%, respectively, compared with the control strain.

Table 2. Photosynthetic pigment content of each transgenic strain at different periods

Growth stage	Materials	Chlorophyll (ug/ml)			Chlorophyll (a/b)	Caroniods (ug/ml)
		Chlorophyll a	Chlorophyll b	Chlorophyll (a + b)
V3	WT	2.06 ± 0.03c	1.18 ± 0.09b	3.24 ± 0.12b	1.76 ± 0.11	0.66 ± 0.03b
	OE	3.03 ± 0.04b	1.98 ± 0.01a	4.94 ± 0.06a	1.54 ± 0.02	0.92 ± 0.01ab
	KO	3.12 ± 0.01a	1.9 ± 0.01a	5.02 ± 0.01a	1.64 ± 0.00	0.91 ± 0.01ab
R2	WT	1.35 ± 0.03e	0.82 ± 0.23c	2.16 ± 0.2c	1.71 ± 0.51	0.82 ± 0.23ab
	OE	1.33 ± 0.04e	0.96 ± 0.01c	2.29 ± 0.06c	1.39 ± 0.02	0.96 ± 0.01a
	KO	3.49 ± 0.12b	1.34 ± 0.04c	4.82 ± 0.08b	2.62 ± 0.16a	0.91 ± 0.01c
R6	WT	4.06 ± 0.07a	2.09 ± 0.01b	6.15 ± 0.06a	1.99 ± 0.01b	1.13 ± 0.01b
	OE	4.15 ± 0.06a	1.93 ± 0.08a	6.08 ± 0.15a	2.15 ± 0.06b	1.23 ± 0.03a
	KO	1.93 ± 0.01c	1.02 ± 0.02e	2.94 ± 0.03c	1.9 ± 0.04c	0.82 ± 0.01e

Different letters in the same column indicate significant differences (P < 0.05).

Effect of GmRPI2 gene on photosynthetic properties of soybean

The net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO₂ concentration data showed (Fig. 7) that the net photosynthetic rate and transpiration rate of soybean plants transgenic to the GmRPI2 gene were significantly higher than those of the control and gene editing strains as fertility progressed, and were significantly higher at the pod-filling stage; the net photosynthetic rate and transpiration rate of the overexpression strains (OE1, OE2) was 40.65%, 42.71%, 33.5%, and 31.24% of those of the control strain, respectively, at the pod-filling stage. The gene-edited strains (KO1, KO2) were 23.39%, 26.93%, 35.95%, and 45.29% lower, respectively, compared to the control strains. The overall change in stomatal conductance was insignificant. The increase in stomatal conductance of the overexpression strain was higher than that of the control and gene editing strains but did not reach a significant level. For the intercellular CO₂ concentration, the overexpression strain was lower than the control and gene editing strains throughout the reproductive period. This may be due to this plant’s enhanced photosynthetic capacity and higher photosynthetic assimilation rate. It showed that the soybean phytoplankton transgenic to the GmRPI2 gene accelerated the utilization of CO₂ and intercellular CO₂ diffusion, which promoted soybean photosynthesis.

Fig. 7.

Photosynthetic characteristics analysis of the GmRPI2 gene in each transgenic strain at different periods (A: net photosynthetic rate; B: transpiration rate; C: stomatal conductance; D: intercellular CO₂ concentration). * indicates a significant difference (P < 0.05); ** indicates a highly significant difference (P < 0.01).

Effect of GmRPI2 gene on soluble sugar content, sucrose, and starch in soybean

Significant differences in leaf-soluble sugar content existed at different periods as the fertility period increased (Fig. 8). Compared with the control strain JN74, the leaf soluble sugar content of overexpression strains (OE1 and OE2) was increased at seedling, flowering, and pod-filling stages; OE1 and OE2 significantly increased leaf soluble sugar and sucrose content by 17.5%, 19.14%, 25.87%, and 25.52%, respectively, at seedling stage compared with WT; at flowering stage leaf soluble sugar and sucrose were significantly increased by 30.5%, 31.66%, and 32.52%, respectively. at pod-filling stage, leaf soluble sugars and sucrose were significantly increased by 48.99%, 49.34%, 42.01%, and 42.33%, respectively. Among them, the soluble sugar content of leaves of KO1 and KO2 was significantly reduced by 11.16%, 12.65%, 18.18%, 18.16%, 34.43%, and 33.69% at seedling, flowering, and pod-filling stages, respectively, compared with the control strain; the sucrose content was significantly reduced by 14.18%, 14.11%, 19.27%, 19.18%, 26.98% and 26.28%, respectively, throughout the growth period compared with the control strain.

Fig. 8.

Analysis of related metabolites of the GmRPI2 gene in different transgenic strains at different periods (A: soluble sugar content; B: sucrose content; C starch content. * indicates a significant difference (P < 0.05); ** indicates a highly significant difference (P < 0.01).

As for the starch content, the starch content of leaves of the gene-editing lines (KO1, KO2) was significantly reduced compared to the control lines, with a decrease of 15.06% and 14.04% at the seedling stage, respectively, while the starch content of leaves overexpressed with the GmRPI2 gene (OE1, OE2) was significantly increased compared with the control lines, with 19.92% and 20.07%, respectively, indicating that the GmRPI2 gene overexpression strain was beneficial in promoting the accumulation of starch content (Fig. 8C).

Discussion

Ribose-5-phosphate isomerase is a vital enzyme and an essential gene in plant photosynthesis. It has been previously reported that RPI is involved in various biological pathways that catalyze the conversion of ribose-5-phosphate to ribulose-5-phosphate (Tang et al. 2021). Expression of RPIs genes plays a vital role in response to abiotic stresses such as plant photosynthesis, drought, cold damage, and plant defense (Schreier et al. 2004, Xiong et al. 2009). In this study, we cloned the GmRPI2 gene and successfully constructed the plant overexpression vector pCAMBIA3301-GmRPI2 and the gene editing vector pCBSG015-GmRPI2. Based on this, we performed gene expression analysis of this gene in soybean under different tissues. The results showed that GmRPI2 transcripts were abundant in leaf tissues (Fig. 6), supporting its possible role in photosynthesis. In this study, the promoter element analysis was performed for 2000 bp upstream of GmRPI2, which contains a large number of light-responsive elements, among which GATA-motif and G-Box are mainly involved in light response, efficient and tissue-specific expression in plants (Chen et al. 2023, Kobayashi et al. 2012); by understanding the regulatory characteristics of the promoter and analyzing the promoter activity, we further suggest that this gene is related to photosynthesis, and we will further verify this conjecture through experiments.

Light provides energy for photosynthesis and is also an important environmental signal that regulates plant growth and development (Raines and Paul 2006). Recent studies have shown that genetic modifications of genes involved in the photosynthetic pathway can alter plants’ maximum photosynthetic efficiency and biomass (Heyneke and Fernie 2018). Increased SBPase activity in tomatoes resulted in higher photosynthetic efficiency, higher RuBP regeneration capacity, and quantum efficiency of photosystem II (Ding et al. 2016). Populus PtoPsbX1 will increase the accumulation of functional photosystem II complexes and improve photosynthetic efficiency and biomass accumulation (Xiao et al. 2020). In the present study, transgenic lines OE1 and OE2 showed improved chlorophyll a, chlorophyll b, chlorophyll a/b, chlorophyll a + b, and carotenoid contents (Table 2). This finding is consistent with the higher photosynthetic rate in overexpressed leaves due to the high transpiration rate and stomatal conductance (Fig. 7). It indicates that GmRPI2 increased photosynthetic efficiency in transgenic soybean.

Photosynthesis is a major contributor to crop yield (Eckhardt et al. 2004). Previous studies have shown that more than 50% of the carbon fixed by photosynthesis can be used for starch and sugar formation (Tang et al. 2018). In chloroplasts, starch is the primary energy storage compound in granular form and is used for the primary storage of excess carbohydrates produced during photosynthesis (Liu et al. 2016). Overexpression of GmFtsH25 leads to more basal granule-like vesicle accumulation in chloroplasts and increases photosynthetic efficiency and starch content, whereas knockdown of GmFtsH25 produces the opposite phenotype (Wang et al. 2023). The cotton GbRPI2 gene is involved in the Calvin cycle, and changes in enzyme activity directly affect the accumulation of related products (Babadzhanova et al. 2010, Hartwell et al. 1996). Our data show that overexpression of GmRPI2 significantly increases the photosynthetic rate and photosynthetic product accumulation in soybean plants, increasing soybean yield. In contrast, photosynthetic rate and photosynthetic product accumulation are suppressed in GmRPI2 gene editing mutants (Fig. 8). It indicates that the accumulation of carbohydrates may affect the photosynthetic rate of the transgenic strain.

Therefore, we determined that overexpression of the GmRPI2 gene increased chlorophyll content and net photosynthetic rate of soybean with more efficient photosynthesis; it also promoted the accumulation and translocation of sucrose in the leaves of soybean during the pre-reproductive period, resulting in a significant increase in sucrose content in the leaves. It promoted the accumulation of starch, which benefited the production of photosynthetic assimilation products in the leaves. Subsequently, we will conduct phenotypic, physiological, biochemical, and molecular experiments on transgenic soybean to further demonstrate the effect on photosynthesis and provide a basis for improving photosynthetic efficiency in soybean.

Author Contribution Statement

SYW was the executor of the experimental design and experimental study of this study; SYWcompleted the data analysis and wrote the first draft of the paper; WXY, LL, ZQ, and XYJ participated in the experimental design and the analysis of the experimental results; WPW was the conceptualizer and leader of the project and directed the experimental design, data analysis, and paper writing and revision. All authors read and agreed on the final text.

Acknowledgments

This research was supported by Jilin Province Major Science and Technology Innovation Project for Improved Seed of Main Grain Crops—Resource Identification, Functional Gene Discovery and Material Creation of High-yield and high-quality Special soybean. Project Number: 20210302002NC.

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