A DIRIGENT Gene GmDIR26 Regulates Pod Dehiscence in Soybean

Pod dehiscence brings much loss for modern agricultural production, and multiple pod dehiscence components have been identified in many plant species. However, the pod dehiscence regulation factors in soybean are limited. In this study, we investigate the function of GmDIR26, a close homologues gene of pod dehiscence genes GmPdh1, PvPdh1, and CaPdh1, in the regulation of pod dehiscence in soybean. The secondary and tertiary structure analysis reveals that GmDIR26 protein has a similar structure with GmPdh1, PvPdh1, and CaPdh1 proteins. Synteny analysis of soybean and chickpea genomes shows that the genomic region surrounding GmDIR26 and CaPdh1 might be evolved from the same ancestor, and these two genes might have similar function. GmDIR26 shows an increased expression pattern during pod development and reaches a peak at beginning seed stage. Meanwhile, GmDIR26 exhibits high expression levels in dorsal suture and pod wall, but low expression pattern in ventral suture. In addition, GmDIR26 shows higher expression levels in pod dehiscence genotype than that in pod indehiscence accessions. Overexpression of GmDIR26 in soybean increases pod dehiscence in transgenic plants, of which the lignin layer in inner sclerenchyma pods is thicker and looser. The expression levels of several pod dehiscence genes are altered. Our study provides important information for further modification of pod dehiscence resistance soybean and characterization of soybean pod dehiscence regulation network.


Introduction
Pod dehiscence is an essential process in wild soybean (Glycine soja) for seed dispersal.The pod of wild soybean opens at the dorsal suture or ventral suture section when it matures and then spreads its seeds to the environment.However, pod dehiscence brings much loss and decreases soybean yield in modern agricultural production [1].The ancient wild soybean accessions are transited to modern cultivated soybean (Glycine max) during evolution, and loss of seed dispersal is an important agronomic trait during soybean domestication [2].To further improve the pod dehis-cence resistance in soybean, it is necessary to investigate the pod dehiscence functional genes.With the development of soybean reference genomes, many soybean functional genes have been identified [3,4].In the past decades, the molecular factors involved in soybean pod dehiscence have been analyzed, and several functional genes participating in soybean pod dehiscence regulatory network have been identified [5][6][7][8][9][10][11][12][13][14].
GmSHAT1-5 is the first identified gene involved in soybean pod dehiscence regulation during domestication [5,12,15].GmSHAT1-5 activates soybean pod secondary wall biosynthesis and promotes the thickening of fibre cap cells of secondary walls in soybean pod.GmSHAT1-5 expression pattern is associated with the content of sclerenchymatous cells and shows higher expression levels in fibre cap cells in pod indehiscence lines than that in pod dehiscence accessions.A 20 bp deletion in the promoter region approximately 4.0 kb upstream of GmSHAT1-5 initiation codon, which destroys the integrity of a GARP protein binding site of "AGAT," results in the high expression of GmSHAT1-5 in the pod indehiscence accession and is responsible for the domestication of soybean pod indehiscence [5].Moreover, GmNST1A, which shares 92.8% amino acid similarity to its paralog gene GmSHAT1-5, is associated with pod dehiscence in soybean [12].In addition, GmRNF1a interacts with a MADS-box gene GmAGL1, which is involved in pod dehiscence regulation, to accelerate pod dehiscence in transgenic Arabidopsis.Further investigation reveals that GmRNF1a is artificially selected during soybean domestication [16,17].L1, encoding a hydroxymethylglutaryl-coenzyme A lyaselike domain protein, is responsible for black pods.L1 plants show more dehisced pods than l1 because dark pigmentation increases photothermal efficiency [18].In common bean, PvMYB26 is located closely to the major QTL of pod dehiscence and is the best candidate gene for pod dehiscence because of its specific differential expression pattern between pod dehiscence and indehiscence genotypes [19].VrMYB26a is located in a hard selective sweep in mung bean genome and shows reduced polymorphism in the promoter region of cultivars [20].
GmPdh1, a DIRIGENT (DIR) gene family member, is another important gene involved in soybean shatter resistance domestication.GmPdh1 is mainly expressed in the lignin-rich inner sclerenchyma of soybean pod walls and exhibits the highest level at the lignin deposition initiation stage.The alteration of "A" to "T" in the coding sequence of GmPdh1 results in the change of a lysine amino acid codon to a stop codon, which is responsible for the transition from pod dehiscence to indehiscence in soybean.Knockout of GmPdh1 using CRISPR/Cas9 improves pod dehiscence resistance in soybean [6,21].GmDIR27, a close paralog of GmPdh1, exhibits increased expression pattern during pod development before full pod stage.Overexpression of GmDIR27 increases pod dehiscence in soybean, and the expression of soybean SEEDSTICK, INDEHISCENT, and ALCATRAZ homologous genes is altered in GmDIR27 transgenic plants [22].In addition, the orthologs of GmPdh1 have been identified to be involved in the regulation of pod dehiscence in other legumes.For example, PvPdh1 has been identified to be involved in lignin biosynthesis and associated with pod dehiscence in common bean (Phaseolus vulgaris) [13,23].CaPdh1, the homologous gene of GmPdh1 and characterized using a RIL (recombinant inbred line) population, is significantly associated with pod dehiscence in chickpea (Cicer arietinum) [24].VrPdh1 is considered to be a domesticated gene from shatter to shatter resistance in mung bean (Vigna radiata) [25].
Although several soybean pod dehiscence-related genes have been identified in recent years, the genes are still limited for further modification of soybean plants.The molecular mechanism of pod dehiscence regulation network still needs further investigation.In this study, we analyzed the function of GmDIR26, which shared close homology to the identified pod dehiscence genes GmDIR27 and GmPdh1, in the regulation of pod dehiscence.Our study provides important information for further characterization of soybean pod dehiscence regulation molecular networks.

Materials and Methods
2.1.Plant Materials and Growth Conditions.Williams 82 (W82) and wild soybean PI 468916 were used in this study [3].For gene expression analysis of GmDIR26 in development pods, the different growth stage pods were collected from Williams 82 grown in the field in Qingdao, China, including beginning bloom (R1), full bloom (R2), beginning pod (R3), full pod (R4), beginning seed (R5), and full seed (R6) [26].To analyze the expression of GmDIR26 in Williams 82 and PI 468916, R5 pods were sampled from plants grown in the field.To investigate the expression of GmDIR26 in different sections of soybean pods, the dorsal suture, ventral suture, and pod wall of R3, R4, and R5 stage pods were collected from Williams 82 grown in the field.To analyze the expression of GmDIR26 in transgenic plants, R5 stage pods of GmDIR26 transgenic lines and Williams 82 grown in pots in the green house were sampled for analysis.The growth conditions were set as follows: 25 °C 16 h light/ 25 °C 8 h dark, and the humidity was maintained at 30%.

Phylogenetic Analysis of DIR Orthologs.
To identify the relationship of GmDIR26 with other DIR genes, the amino acid sequence of GmDIR26 protein was used as a blast query against TAIR10 (https://www.arabidopsis.org)and Phytozome 13 (https://phytozome-next.jgi.doe.gov/) to search for its homologous genes.The amino acid sequences of DIR orthologs from Arabidopsis, common bean (Phaseolus vulgaris), chickpea (Cicer arietinum), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum) were aligned using MUSCLE software (multiple protein sequence alignment) and used to construct a phylogenetic tree using FastTree with default parameters [27].The iTOL (https:// itol.embl.de/itol.cgi)software was used for optimization of the phylogenetic tree [28].For the sequence alignment of GmDIR26 protein and its homologous genes, the amino acid sequences of these proteins were aligned using MEGA X and then presented in DNAMAN (version 10) [29].

Synteny Analysis of GmDIR26
Gene Regions.To identify the synteny regions of soybean and chickpea, the genome sequences of soybean and chickpea were obtained from NCBI database (National Center for Biotechnology Information), and then, the genome information was submitted to MCscanX software to identify the synteny regions of soybean and chickpea genomes with default parameters [30].The synteny regions of soybean and chickpea were 2 International Journal of Genomics connected using gray lines, and the connection between GmDIR26 and CaPdh1 was highlighted using a red line.

Protein Structure Analysis of GmDIR26
Protein.The amino acid sequence of GmDIR26 protein was used to analyze its secondary and tertiary structures.For the secondary structure, GmDIR26 was analyzed using PSIPRED software with default parameters [31], and GmDIR26 protein tertiary structure was predicted using AlphaFold 2 software with default parameters [32,33].

Plasmid Construction and Soybean Transformation.
To construct the GmDIR26 overexpression plasmid, the coding sequences of GmDIR26 was amplified from the pod dehiscence genotype PI 468916 using specific primers.The PCR products and pPTN1171 plasmid were disgusted using Xho I and Xba I, and then, the GmDIR26 fragment and linearity pPTN1171 were ligated using T 4 ligase as described [34,35].
The constructed plasmid was transformed into Agrobacterium LBA4404 and then transferred into soybean Williams 82 using Agrobacterium-mediated transformation method [22].The GmDIR26 transgenic plants were identified using PCR and sequencing of GmDIR26 PCR fragment.And then, the transgenic GmDIR26 plants were further confirmed using phosphinothricin-N-acetyltransferase/bar rapid test kit (Artron) according to the manufacturer's instructions.
The primers are listed in Supplementary Table S1.
2.6.Pod Dehiscence Phenotyping.For pod dehiscence analysis, two GmDIR26 transgenic soybean lines, Williams 82 and PI 468916, were used and grown in the pots under natural conditions in 2021 in Qingdao, China.The full maturity pods (R8 stage) were sampled as described [22].The collected soybean pods were transferred into an oven, the temperature of which was set at 37 °C, to investigate the dehisced pods.After 60 days, the dehisced pods were analyzed, and the percentage of GmDIR26 transgenic plants and Williams 82 was calculated.For the cross section analysis, soybeanmatured pods were used.Cross sections of pod walls were stained with 10% toluidine blue and observed using a microscope (Olympus CX23, China).

RNA Isolation and Gene Expression Analysis.
To analyze the expression of target genes, total RNA of soybean GmDIR26 transgenic plants and Williams 82 samples were isolated using a RNeasy mini kit (Qiagen) according to the manufacturer's instruction.Then, 1.0 μg total RNA for each soybean sample was used for the synthesis of cDNA with SuperScript II reverse transcriptase (Promega) as described by the manufacturer's instruction.The quantitative realtime PCR (qRT-PCR) was performed using ABI QuantStu-dio®5 (ABI, USA) machine as described [36].The amplification program was set as follows: 95 °C for 5 s and 60 °C for 30 s, 40 cycles.The expression level of the analyzed soybean genes was normalized to a soybean GmCons4 gene [26].Each soybean sample was analyzed using three biological replicates.The primers used for each gene are listed in Supplementary Table S1.

Evolutionary Relationship Analysis of GmDIR26.
To analyze the evolutionary relationship between GmDIR26 and DIR proteins from other plant species, a phylogenetic tree was constructed using DIR proteins from the model plant Arabidopsis; legume crops including soybean, common bean, chickpea, lima bean, and cowpea; and monocotyledons, including rice, corn, and wheat (Figure 1).GmDIR26 was classified into the same subgroup with GmPdh1, PvPdh1, CaPdh1, and GmDIR27, which were involved in pod dehiscence regulation [6,22,23], indicating that GmDIR26 might participate in the regulation of pod dehiscence (Figure 1 and Supplementary Figure S1).To identify the similarity of GmDIR26 and its homologous genes, the sequences of GmDIR26 protein and GmPdh1, PvPdh1, CaPdh1, and GmDIR27 were aligned (Figure 1).We found that all these proteins contained the conserved DIR domain.GmDIR26 protein sequence showed 62.3%, 66.7%, 61.6%, and 58.5% similarities to GmPdh1, GmDIR26, CaPdh1, and PvPdh1, respectively (Figure 1).In addition, the whole genome sequence of soybean was compared with other legumes, and the results revealed that the genomic region surrounding GmDIR26 showed synteny with that surrounding CaPdh1, indicating that GmDIR26 and CaPdh1 might be evolved from the same ancestor and have similar functions (Figure 2).

Protein Structure Analysis of GmDIR26 and Its Homologous
Genes.In legumes, GmPdh1, PvPdh1, CaPdh1, and GmDIR27 displayed important roles in the regulation of pod dehiscence, and GmDIR26 showed close relationships with these genes (Figure 1) [6,22,23].To identify the similarity of GmDIR26 with these proteins, we analyzed their secondary and tertiary structures (Figure 3 and Supplementary Figure S1).These proteins showed some similarities in tertiary structures and contained 0-2 alpha helices, 9-11 beta turns, and 9-10 random coils in the conserved DIR domain, respectively (Supplementary Figure S1).However, these proteins also exhibited some differences in tertiary structures.GmPdh1 contained three alpha helices and 11 beta turns, GmDIR27 had one alpha helix and 11 beta turns, PvPdh1 had three alpha helices and 10 beta turns, CaPdh1 contained two alpha helices and 10 beta turns, and GmDIR26 contained four alpha helices and 13 beta turns, respectively (Figure 3).

Expression of GmDIR26 during Pod Development.
The expression of GmDIR27 and GmPdh1, the close homologous genes of GmDIR26, displayed different levels during pod development [6,22], and we analyzed the expression patterns of GmDIR26 during different pod development stages, including R1 to R6 (Figure 4).The expression of GmDIR26 showed low levels from R1 to R3 stages and increased from R3 to R4 stages.GmDIR26 expression reached a peak at R5 stage and decreased at R6 stage (Figure 4 Figure 1: Phylogenetic relationship analysis and sequence alignment of GmDIR26 and its homologous genes: (a) phylogenetic tree of GmDIR26 and DIR proteins from several plant species (the phylogenetic tree is constructed using amino acid sequences of these proteins); (b) amino acid sequence alignment of GmDIR26 and pod dehiscence regulation genes GmDIR27, GmPdh1, CaPdh1, and PvPdh1.The red line indicates the conserved DIR domain in these proteins.

International Journal of Genomics
GmDIR26 in different sections of pods, including the dorsal suture, ventral suture, and pod wall, which were sampled from R3, R4, and R5 stage pods.GmDIR26 showed low expression levels in dorsal suture, ventral suture, and pod wall at R3 and R4 stages, respectively (Figure 4(b)).However, GmDIR26 exhibited high expression levels in dorsal suture and pod wall at R5 stage, but low expression pattern in ventral suture at R5 stage, indicating that GmDIR26 might be involved in pod development in dorsal suture and pod wall at R5 stage (Figure 4(b)).To compare the expression pattern of GmDIR26 in pod dehiscence and indehiscence genotypes, we selected PI 468916 and Williams 82 for analysis, which showed 98.7% and 5.6% dehisced pods, respectively (Figures 5(a) and 5(b)).GmDIR26 showed higher expression levels in PI 468916, indicating that GmDIR26 might be involved in pod dehiscence regulation (Figure 5(c)).

Cicer arietinum
Glycine max To identify the function of GmDIR26 in the regulation of pod dehiscence, we constructed a GmDIR26 overexpression plasmid under the control of the cauliflower mosaic virus 35S promoter and transformed it into the pod indehiscence variety Williams 82 (Figure 6(a)).The transgenic plants were firstly identified with PCR (Figure 6(b)) and then confirmed with bar gene antibody using phosphinothricin-N-acetyltransferase/bar rapid test kit (Figure 6(c)).Then, the expression levels of GmDIR26 were analyzed in Williams 82 and transgenic plants.GmDIR26 showed higher expression levels in two transgenic plants than that in the control plant Williams 82 (Figure 6(d)).To investigate the pod dehiscence of Williams 82 and transgenic plants, the matured pods were sampled from soybean plants and transferred into 37 °C oven to be analyzed [22].After 60 days, the two lines of GmDIR26 transgenic plants exhibited 37% and 33% dehisced pods, respectively, while the pod dehiscence in the control plants was 6.25%, indicating that GmDIR26 accelerates pod dehiscence in soybean (Figure 7).The anatomical characteristics of GmDIR26 transgenic pods were analyzed, and the lignin layer in inner sclerenchyma of GmDIR26 transgenic pods was thicker and looser, while Williams 82 is thinner and compact (Figure 8), indicating that GmDIR26 has similar function to GmPdh1 [21].

GmDIR26 Affects the Expression of Pod Dehiscence-Related Genes.
To investigate the effects of GmDIR26 on the expression of pod dehiscence genes, we analyzed several functional genes, including GmPdh1 [6], GmDIR27 [22], and GmAGL1, which accelerated pod dehiscence in plants [16], and Glyma.08G156000, the homologous gene of Arabidopsis pod dehiscence regulation gene INDEHISCENT [37], in the pods of GmDIR26 transgenic lines and Williams 82 plants (Figure 9).GmPdh1 and Glyma.08G156000slightly increased in GmDIR26 transgenic plants than that in Williams 82.However, the expression of GmAGL1 was lower in two GmDIR26 overexpression lines than that in Williams 82, indicating that GmAGL1 was suppressed in two GmDIR26 transgenic plants (Figure 9).GmDIR27 showed no significant change between GmDIR26 transgenic plants and Williams 82 (Supplementary Figure S2).These results suggested that GmDIR26 affected the expression of pod dehiscence-related genes in soybean.

Discussion
Soybean is an important legume crop and provides essential oil and protein for human food and animal feed.Pod dehiscence brings much loss for the production of soybean, and the investigation of pod dehiscence molecular regulation system will provide genetic resources for soybean modification to improve soybean yield.However, the molecular mechanism regulating pod dehiscence is limited in soybean.In this study, we characterized the function of GmDIR26 in pod dehiscence regulation and provide important information for further soybean modification.
The homologous genes have the same conserved domains and might have similar functions in plants.GmSHAT1-5 and its close homologous gene GmNST1A, encoding NAC transcription factors, are considered to participate in pod development regulation in soybean [5,12].GmPdh1, a DIR domain protein, has been identified to be involved in the regulation of pod dehiscence in the lignin-rich inner sclerenchyma of pod walls [6].GmDIR26 and GmDIR27, the homologous gene of GmPdh1, are important factors to regulate soybean pod dehiscence (Figure 1).Synteny analysis reveals that GmDIR26 and CaPdh1 might be evolved from the same ancestor (Figure 2), and they have similar function in the regulation of pod development in legumes.Moreover, DIR proteins are found to regulate the formation of lignan and lignin in plants, which are important components of soybean pod structure, indicating that the DIR proteins are important components in pod development [38][39][40][41].These results suggest that there might be some other DIR genes involved in pod dehiscence regulation in legumes, such as GmDIR19, which is the close homologous gene of GmDIR26 (Figure 1).

International Journal of Genomics
In soybean, GmSHAT1-5 and GmPdh1 have different haplotypes in different varieties, which have distinct functions, and different genotypes of soybean show different degrees of pod dehiscence [5,6].Mutation of GmPdh1 results in the change of a lysine amino acid codon to a stop codon, which leads the change of pod dehiscence to indehiscence [6].GmSHAT1-5 and Gmshat1-5 have some differences in promoter regions; as a result, GmSHAT1-5 exhibits high expression levels in pod indehiscence genotypes, and Gmshat1-5 shows low expression pattern in wild soybean, which exhibits pod dehiscence phenotype [5].GmDIR26 shows high expression levels in pod dehiscence wild soybean, and overexpression of GmDIR26 increases the percentage of pod dehiscence in soybean (Figure 7), indicating that the expression level of GmDIR26 is critical for soybean pod dehiscence, and different expression levels of GmDIR26 might have different degrees of pod dehiscence.A low expression of GmDIR26 genotype will be useful for soybean  The functional genes might be expressed when its function is needed, and the expression levels of genes in different development stages have a direct relationship with their functions.For example, the expression of soybean growth habit regulation gene Dt2 is mainly expressed at V2 stage (when the 1st trifoliate leaflet is fully expanded and before the 2ed trifoliate leaflet is unrolled), when it suppresses its downstream gene Dt1 [42].The pod dehiscence-related genes are expressed at different stages, and they might participate in different pod development stages in soybean.For example, GmSHAT1-5 shows low expression level at early pod development stage and reached a high level at approximately 18-day-old pods (approximately R5 stage) [5].The expression level of Pdh1 increases at early pod development stage and reaches a peak at 21-day-old pods (approximately R5 stage) [6].GmDIR27 shows increased expression pattern during R1 to R4 and reaches a peak at R4 stage [22].In addition, GmDIR26 exhibits low expression pattern during early pod development stages and reaches a high expression level at R5 stage (Figure 4).These results indicate that these genes might be involved in pod development at middle growth stages.
In plants, the pod dehiscence regulation system contains complex components to form an effective network, and many related genes are still unknown.The genes regulate the same agronomy trait that might have cooperative or antagonist effect on the phenotype [43].In soybean, the  International Journal of Genomics expressions of GmPdh1 and Glyma.08G156000 are slightly increased in GmDIR26 transgenic plants, indicating that GmDIR26 might have cooperative effect with these two genes in pod development regulation (Figure 9).However, GmAGL1 is decreased significantly in GmDIR26 overexpression lines, suggesting that they might be antagonist in pod dehiscence process (Figure 9).In addition, DIR proteins regulate the production of pinoresinol in plants, which is necessary of the synthesis of lignans and lignin [44,45]; thus, how GmDIR26 affects the expression of other pod dehiscence genes still needs further investigation.

Conclusions
In summary, we identified a DIR protein from soybean, which is the homologous gene of pod dehiscence genes GmPdh1 and GmDIR27.Synteny analysis reveals that GmDIR26 and CaPdh1 might be evolved from the same ancestor.The expression of GmDIR26 shows different expression levels in different development stages and different pod sections.Overexpression of GmDIR26 increased pod dehiscence by affecting the expression of several pod dehiscence genes.
(a)), indicating that GmDIR26 influenced pod development during seed beginning stage.Pod dehiscence indicates that soybean pods open at dorsal or ventral suture.To investigate the potential function of GmDIR26 in the pod, we analyzed the expression

Figure 2 :Figure 3 :Figure 4 :
Figure 2: Syntenic relationship analysis of chickpea (Cicer arietinum) and soybean (Glycine max) genomes.The gray lines indicate synteny blocks within chickpea and soybean genomes, and the red line indicates syntenic regions of GmDIR26 and CaPdh1.

Figure 6 :
Figure 6: Identification of GmDIR26 transgenic soybean plants: (a) the schematic diagram of 35S::GmDIR26 in the plasmid; (b) PCR of GmDIR26 in Williams 82 and transgenic lines using specific primers; (c) bar gene antibody analysis; (d) gene expression analysis of GmDIR26 in Williams 82 and GmDIR26 transgenic lines.R5 stage pods are used for analysis.The expression of GmDIR26 is normalized to the soybean GmCons4 gene.Significant differences relative to the control Williams 82 are indicated by asterisks, * * * P < 0 001.

Figure 7 :
Figure 7: Pod dehiscence analysis of GmDIR26 transgenic and Williams 82 plants: (a) pod dehiscence phenotype of GmDIR26 transgenic and Williams 82 plants (full maturity pods are used for analysis; bar = 4 cm); (b) the percentage of pod dehiscence in GmDIR26 transgenic and Williams 82 plants.Significant differences relative to the control plant Williams 82 are indicated by asterisks, * * * P < 0 001.

Figure 8 :Figure 9 :
Figure 8: Cross section of pod wall of Williams 82 and GmDIR26 transgenic soybean plants: (a) the red boxes indicate inner sclerenchyma in pod wall; (b) the arrows indicate the length of inner sclerenchyma in red boxes.