Site-directed mutagenesis of soybean PEAPOD genes using the CRISPR/Cas9 system alters tissue developmental transition

Abstract

In general, plant organ size is determined using cell number and expansion. In our previous study, we generated soybean (Glycine max) mutants of the PEAPOD (PPD) genes GmPPD1 and GmPPD2 using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 system. Some of these mutants exhibited extremely abnormal phenotypes, such as twisted pods and limited seeds. These phenotypes were attributed to the frameshift mutation in both GmPPD loci. In this study, the physiological and molecular biological properties of mutant plants with two knocked-out GmPPD loci (ppd-KO) were characterized. The ppd-KO mutant exhibited a delayed growth phase from the time of development of the unifoliolate leaves to that of first trifoliolate leaves and a stay-green phenotype, which were not observed in the other mutants of soybean or ppd mutants of other plant species. Gene expression analysis revealed considerably decreased expression of SPIRAL1-LIKE 5 (GmSP1L5), mainly causing the twisted pod phenotype observed in the ppd-KO mutant. The relationship between PPD and SP1L5 has not been previously reported, and in this study, we showed that that loss of PPD functioning affects SP1L5 expression in soybean. In this study, we revealed that the decrease in PPD function contributed to organ enlargement and that complete knockout of PPD has a negative effect on soybean organogenesis.

Introduction

Enlargement of plant organs can lead to an increase in plant biomass. In general, plant organ size is determined using cell number and expansion. Spontaneous or artificial mutations can result in increasing size of plant organs. Mutations in PEAPOD (PPD), a transcription factor for genes involved in cell proliferation, are closely associated with organ enlargement. The locus of PPD was identified for the first time in a mutant line of Arabidopsis (Arabidopsis thaliana); this mutant had increased leaf lamina size and dome-shaped leaves (White 2006). Because PPD overexpression promotes the initial arrest of mitotic cell proliferation during leaf and silique development and reduces leaf lamina size, the increased lamina growth observed in the ppd mutant was attributed to the increased duration of mitotic cell proliferation during leaf development (White 2006).

Loss-of-function mutations in PPD have been also observed in mutants of other plant species. The Medicago truncatula big seeds1-1 (mtbs1-1) mutant exhibits larger seeds and leaf tissues than its wild-type counterpart (Ge et al. 2016). In a Vigna mungo multiple organ gigantism (mog) mutant, although the total seed weight was not considerably different from that of the wild-type plant, the weight of 100 seeds was approximately 1.7 times higher than that of the wild-type plant (Naito et al. 2017). Map-based cloning of both mutants revealed that the genetic causes were mutations at the PPD locus (Ge et al. 2016; Naito et al. 2017). The orthologs of PPD (GmPPD1 and GmPPD2) were knocked down in transgenic soybean (Glycine max) plants using RNA interference (RNAi) or artificial microRNA (amiRNA) to elucidate the molecular mechanism of the PPD mutations found in the mtbs1-1 and mog mutants (Ge et al. 2016; Naito et al. 2017). Knock down of GmPPD1 and GmPPD2 resulted in enlarged seed and leaf tissue phenotype (Ge et al. 2016; Naito et al. 2017). Gene expression analysis of PPD knockdown-plants, using amiRNA technology, revealed that GROWTH-REGULATING FACTOR 5 (GRF5), GRF-INTERACTING FACTOR 1 (GIF1), and CYCLIN D3 (CYCD3;3) were noticeably upregulated in these plants compared with their expression in control plants (Ge et al. 2016). These genes are known to be closely associated with organ development in Arabidopsis (Baekelandt et al. 2018; Gonzalez et al. 2010, 2015; Kim et al. 2003). These functions of GRF, GIF, and CYCD3;3 are in line with the finding of Ge et al. (2016) that PPD knockdown in transgenic soybean plants elevates the expression of GRF5, GIF1, and CYCD3;3 and enlarges seed and leaf tissues.

In our previous study, we performed site-directed mutagenesis of two PPD genes using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system in soybean (Kanazashi et al. 2018). We revealed that site-directed mutagenesis of GmPPD1 and GmPPD2 resulted in various changes in the morphological characteristics of soybeans (Kanazashi et al. 2018). We divided the double mutant plants into two main types based on their morphological characteristics. One type had rippled trifoliolate leaves that were thicker and deep green in color, had longer petioles, bigger pods, and bigger seeds than wild-type plants (Kanazashi et al. 2018). The other type had dome-shaped trifoliate leaves and extremely twisted pods with remarkably low seed yields (Kanazashi et al. 2018). The second phenotype was due to both GmPPD loci being knocked out with frameshift mutations (Kanazashi et al. 2018), but the molecular mechanisms underlying the phenotype without tissue enlargement remain unknown. Understanding the molecular biology and physiology of these phenotypic differences will provide novel insights into PPD function in soybean.

In this study, we selected plants from the mutant soybean line generated by Kanazashi et al. (2018) with both GmPPD loci knocked-out and analyzed their physiological characteristics. Transcriptome analysis of these plants was also performed. Our study provides novel insights into the physiological and molecular biological characteristics of the ppd mutant plants in soybean.

Materials and methods

Plant materials

Soybean ppd mutant lines were generated using the CRISPR/Cas9 system as previously described (Kanazashi et al. 2018). The gene IDs Glyma.10G244400 and Glyma.20G150000 corresponded to the GmPPD1 and GmPPD2 loci, respectively. We selected mutant plants that had dome-shaped trifoliate leaves and prominently twisted pods with markedly low seed yields as the plant material. Because these mutants produced very few seeds, it was difficult to maintain these mutants through subsequent generations. We found that the later generation mutant plants of the mutant line generated by Kanazashi et al. (2018) could be segregated based on two different phenotypes (bigger pods, and bigger seeds than wild-type plants, or dome-shaped trifoliate leaves and extremely twisted pods with extremely low seed yields). Therefore, the progenies of this mutant were used as the plant materials. When mutant alleles in both GmPPD loci were homozygous of frameshift mutations, the mutant plants exhibited the phenotype of prominently twisted pods with considerably reduced seed yield. Therefore, selection for homozygosity of frameshift mutant alleles at both GmPPD loci was performed for every generation. The Japanese soybean variety Kariyutaka was used as the control plant. The mutant lines and control plants were grown in a closed greenhouse at 26°C.

Genotyping of the mutants at the GmPPD loci

The genotypes of the mutants at the GmPPD loci were confirmed via sequencing of each generation of the mutant plants. Genomic DNA was extracted from leaf pieces (approximately 5 mm×5 mm) or mature seeds according to the method described by Sugano et al. (2020). PCR fragments were amplified using specific primers (Supplementary Table S1) and sequenced either directly or after cloning into the pGEM-T-Easy vector (Promega, Madison, WI, USA) using the Big Dye Terminator Cycle method with the ABI3100 or ABI3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). DNA sequencing was performed by the Instrumental Analysis Division of the Graduate School of Agriculture, Hokkaido University.

Evaluation of the morphological and physiological characteristics of the mutants

The growth period was divided into three stages. The first growth phase was from sprouting to the development of unifoliolate leaves. The secondary growth phase was from the development of the unifoliolate leaves to that of the first trifoliolate leaves. The third growth phase was from the development of the first trifoliolate leaves to the reproductive R3 stage (1 cm-sized pod). The size of the unifoliolate leaves of the mutant lines was measured, and leaf area was determined as leaf size multiplied by the length and width of the unifoliolate leaf. SPAD values of the first trifoliolate leaves were measured using a chlorophyll analyzer every 2 days from 14 to 36 days after flowering (DAF). The cell size of the unifoliolate leaves was determined. Briefly, manicure was applied to the surface of unifoliolate leaves and allowed to dry. Then, the epidermal cells in the leaves were peeled off along with the manicure and observed under the BX50 optical microscope (Olympus, Tokyo, Japan). The cell size of the epidermal cells in the microscopic images was measured using ImageJ software (https://imagej.nih.gov/ij/index.html).

RNA-seq analysis

For transcriptome analysis, total RNA was extracted from the 1 cm-sized pods of ppd-KO and control plants using the LiCl precipitation method (Adachi et al. 2021). The analysis was performed without any biological replicates. Sequencing samples were prepared and paired-end sequencing using NovaSeq 6000 (Illumina, CA, USA) was performed by Rhelixa Inc. (Tokyo, Japan). The sequences were mapped with a Glycine max reference (Glycine max Wm82.a2.v1) from Phytozome (https://phytozome-next.jgi.doe.gov/info/Gmax_Wm82_a2_v1 (Accessed Feb 20, 2020)) using Bowtie 2.3.5.1 (Langmead and Salzberg 2012). Splice junctions were mapped using TopHat 2.1.1 (Kim et al. 2013). Differentially expressed genes were identified using Cufflinks 2.2.1 (Trapnell et al. 2012). A false discovery rate of Q<0.05 was considered as the threshold for differently expressed genes. The descriptions of differently expressed genes were explored via PhytoMine (https://phytozome-next.jgi.doe.gov/phytomine/bag.do?subtab=upload (Accessed Feb 20, 2020)).

Gene expression analysis via qRT-PCR

qRT-PCR was performed in a 20 µl reaction volume containing 9.2 µl of diluted cDNA solution, 0.8 µl of each primer (1 µM), and 10 µl of TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, Shiga, Japan). PCR was performed using the CFX96 Real-Time System (Bio-Rad, California, USA). The amplification conditions were as follows: 40 cycles at 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. The specificity of the amplification was verified via melting curve analysis. The expression of each gene was normalized to that of Bic-C2 (Glyma.03G064800) using a specific primer set for each gene (Supplementary Table S1).

Statistical analysis

Tests of significance among means of data were performed using the Student’s t-test. A p-value of <0.05 was considered statistically significant.

Results

Molecular characterization of mutants

Sequences of both GmPPD loci in the mutant plants were determined. Two mutant alleles were detected at each GmPPD locus in the mutant plants that segregated the phenotypes in the later generations (Table 1). At the GmPPD1 locus, 7- and 39-nucleotide deletions were detected (Table 1). In contrast, 2- and 45-nucleotide deletions were confirmed at the GmPPD2 locus (Table 1). The mutant plants that exhibited dome-shaped trifoliate leaves and appreciably twisted pods with markedly low seed yields harbored homozygous mutant alleles with 7- and 2-nucleotide deletions at the GmPPD1 and GmPPD2, respectively. All of these mutant alleles carried frameshift mutations (Supplementary Figure S1). This mutant line was designated as ppd-KO. On the contrary, the mutant that harbored a homozygous frameshift mutant allele (7-nucleotide deletion) at the GmPPD1 locus and a homozygous in-frame mutant allele (45-nucleotide deletion) at the GmPPD2 locus was designated as ppd1 (Table 1, Supplementary Figure S1). The ppd2 mutant plant harbored a homozygous in-frame mutant allele (39-nucleotide deletion) at the GmPPD1 locus and a homozygous frameshift mutant allele (2-nucleotide insertion) at the GmPPD2 locus (Table 1, Supplementary Figure S1). In our study, site-directed mutagenesis of the GmPPD genes did not yield mutants in which either GmPPD locus was wild-type, and all plants harbored mutant alleles at the both GmPPD loci (Kanazashi et al. 2018). The ppd1 and ppd2 mutants possessing partial PPD functions were used as plant materials in this study; the in-frame mutation might have altered the original PPD function.

Table 1. Alleles of the mutants at the GmPPD loci.

Mutant lines	GmPPD1 locus	GmPPD2 locus	Morphological evaluation1
ppd1	7-nucleotide deletion	45-nucleotide deletion	Moderate phenotype alteration
ppd2	39-nucleotide deletion	2-nucleotide insertion	Moderate phenotype alteration
ppd-KO	7-nucleotide deletion	2-nucleotide insertion	Severe phenotype alteration

¹Morphological evaluation was based on the characteristics described by Kanazashi et al. (2018).

Morphological characteristics of the mutants

The size of the mature seeds of the ppd2 mutant was similar to that of control plants (Figure 1A). The seed weight of the ppd1 mutant was significantly (p<0.05) higher than that of the ppd2 mutant and control plants (Figure 1A). Further, the seed weight of the ppd1 mutant was approximately 21% higher than that of control plants. In contrast, the number of seeds in the ppd1 mutant was reduced to approximately 42% of that in control plants (Figure 1B). However, no differences in total seed weight were observed between the ppd1 and ppd2 mutants and control plants (Figure 1C). The ppd-KO mutant plants rarely produced seeds (Figure 1A).

Figure 1. Seed traits of the mutants and control plants. (A) Seed weight of control plants and the ppd1, ppd2, and ppd-KO mutants. (B) Total seed number of control plants and the ppd1, ppd2, and ppd-KO mutants. (C) Total seed weight per plant of control plants and the ppd1, ppd2, and ppd-KO mutants. * indicates significant differences between control plants and mutants at the 5% level. All data shown are the mean±SE of 5–9 plants. ND denotes not determined.

The vertical and horizontal widths of the unifoliolate leaves of the mutants and control plants were measured, and the multiplied values were considered as leaf area. The mutants exhibited different unifoliolate leaf sizes. The ppd1 mutant had the largest unifoliolate leave sizes (Figure 2A). The leaf size of the ppd1 mutant increased by approximately 50% compared with that of control plants. Further, the size of the unifoliolate leaves of the ppd2 mutant was larger than that of control plants (Figure 2A). The size increased by approximately 28% compared with that of control plants (Figure 2A). The unifoliolate leaves of the ppd-KO mutant were wrinkled and domed-shaped; therefore, vertical and horizontal widths could not be accurately measured (Figure 2A). Epidermal cells were isolated from unifoliolate leaves, and cell size was measured using ImageJ. The size of the epidermal cells in control plants was approximately 1,110 µm². However, no differences were observed in the size of the epidermal cells in the mutants (Figure 2B). Furthermore, the shape of the epidermal cells was similar in control plants and the ppd-KO mutant (Figure 2C, D).

Figure 2. Sizes and epidermal cells of the unifoliolate leaves of control plants and the ppd mutants. (A) Sizes of the unifoliolate leaves of control and mutant plants. The unifoliolate leaves of the ppd-KO mutant were wrinkled and domed-shaped; therefore, the vertical and horizontal widths were not measured. * and ** indicate significant differences between control plants and the mutants at the 5% and 1% levels, respectively. All data shown are the mean±SE of 6–7 plants. ND denotes not determined. (B) Size of the epidermal cells in control plants and the mutants. The size of the epidermal cells is expressed in a boxplot diagram. Every 10 cells were measured at five areas per one unifoliolate leaf. All data are collected from 6–7 plants. (C) Epidermal cells in control plants. (D) Epidermal cells in the ppd-KO mutant. Scale bars: 200 µm.

Physiological characteristics of the mutants

The growth durations of control plants and the ppd-KO mutant were considerably different. The ppd-KO mutant did not develop pods by the time the control plants did (Figure 3A, B). The first growth phase was not different between the mutants and control plants (Figure 3C). The first growth phase lasted approximately 6 days (Figure 3C). However, the second growth phase of the ppd-KO mutant was approximately 14 days longer than the other mutants and control plants (Figure 3C). This resulted in delayed flowering time in the ppd-KO mutant compared with that in the other mutants and control plants. Nevertheless, there was no difference in the third growth phase among the mutants, and the phase lasted approximately 18 days (Figure 3C). The ppd-KO mutant exhibited leaf senescence characteristics different from those of the other mutants and control plants. The control plants showed a decrease in SPAD values 22 DAF (Figure 3D). However, the ppd1 and ppd2 mutants showed the same leaf senescence pattern (data not shown). The SPAD value of the ppd-KO mutant was slightly lower than that of control plants; however, the SPAD value remained unchanged after 36 DAF. Further, the ppd-KO mutant exhibited a stay-green phenotype (Figure 3D).

Figure 3. External morphological and physiological characteristics of control plants and the mutants. External morphological character of control plants (A) and the ppd-KO mutant (B) 63 days after flowering. (C) Growing periods of the 1 cm-sized pods from sprouting to development of control plants and the mutants. ** indicates significant differences between control plants and the mutants at the 1% level. All data shown are the mean±SE of 3–10 plants. (D) SPAD value of the first trifoliolate leaves of control plants and the ppd-KO mutant from 14 days after flowering. All data were collected as four area of one trifoliolate region of three plants each. All data shown are mean±SE. * and ** indicate significant differences between control plants and the mutants at the 5% and 1% levels, respectively.

Numbers of guard cells on the unifoliolate leaves of the mutants

We determined the number of guard cells on the abaxial and adaxial sides of the unifoliolate leaves of the mutants and control plants. The number of guard cells was 22 cells/0.6 mm² on the adaxial side of the unifoliolate leaves of control plants (Figure 4A) and was considerably higher than that on the adaxial side of the unifoliolate leaves of the ppd1 and ppd-KO mutants (Figure 4A). The abaxial side of the unifoliolate leaves of the ppd-KO mutant, which had the lowest number of guard cells on the adaxial side, was compared with that of control plants. No difference was observed in the number of guard cells on the abaxial side of the unifoliolate leaves (Figure 4B).

Figure 4. Guard cell numbers in the unifoliolate leaves of control plants and the ppd-KO mutant. (A) The adaxial side of the unifoliolate leaves. (B) The abaxial side of the unifoliolate leaves. All data were collected from every five areas (0.6 mm²) of one the unifoliolate leaf in five plants. All data shown are mean±SE. ** indicates significant differences between control plants and the mutants at the 1% level.

Gene expression analysis of the ppd-KO mutant and control plants

To investigate why the ppd-KO mutant produced fewer seeds, the shape of the young pods and development of embryos were observed in the ppd-KO mutant and control plants 21 DAF. The ppd-KO mutant exhibited a prominently twisted pod phenotype (Figure 5A). However, developed embryos were found in these twisted pods and control plants (Figure 5B). Subsequently, transcriptome analysis was performed on 1 cm-sized pods of the ppd-KO mutant and control plants, which were collected 7 DAF. In total, 128 differentially expressed genes were identified between the ppd-KO mutant and control plants (Supplementary Table S2). The genes involved in cell division and organ enlargement were SPIRAL1-LIKE 5 (GmSP1L5; Glyma.08G017200), GmGIF1 (Glyma.03G249000; Glyma.19G246600; Glyma.20G226500; and Glyma.10G164100), and PACLOBUTRAZOL RESISTANCE 1 (GmPRE1; Glyma.18G258700). Therefore, qRT-PCR analysis was performed using 1 cm-sized pods of the mutants and control plants to analyze the expression of the abovementioned genes and GmCYCD3;2 (Glyma.17G167700), which is reported to be regulated by PPD. The expression of GIF1 family genes was noticeably higher in 1 cm-sized pod of the ppd-KO mutant than in that of control plants (Figure 5C). In addition, the expression of GmSP1L5 was considerably lower in 1 cm-sized pods of the ppd-KO mutant than in that of control plants (Figure 5C). These changes in gene expression level were also observed in the unifoliolate leaves of the ppd-KO mutant (Supplementary Figure S2). In contrast, there were no significant differences in these genes between the ppd1 or ppd2 mutants and the control plants (Figure 5C).

Figure 5. Morphological characteristics of the pods and gene expression analysis of control plants and the ppd-KO mutant. (A) Pods 21 days after flowering. Upside, Control plant; bottom side, ppd-KO mutant. (B) Embryos 21 days after flowering. Upside, Control plant; bottom side, ppd-KO mutant. (C) Gene expression analysis of 1 cm-sized pods of control plants and the ppd mutants. The expression of each gene was normalized to that of Bic-C2 (Glyma.03G064800). *** indicates significant differences between control plants and the ppd mutants at the 0.1% levels. All data shown are the mean±SE of 3–4 biological replicates.

Discussion

In this study, ppd1 and ppd2 mutants exhibited a phenotype of organ enlargement (Figures 1A, 2A) similar to that of GmPPD-knocked down plants (Ge et al. 2016; Naito et al. 2017). The ppd1 and ppd2 mutants lacked 15 and 13 amino acid residues at the GmPPD2 and GmPPD1 loci, respectively, owing to in-frame mutations (Supplementary Figure S1). These deleted regions were located away from the PPD and TIFY domains (Bai et al. 2011; Cuéllar Pérez et al. 2014), which play important roles as transcription factors in PPD. Our findings suggest that the ppd1 and ppd2 mutants partially retain the function of the PPD protein. In contrast, the ppd-KO mutant, which harbored frameshift mutations at both the GmPPD1 and GmPPD2 loci, exhibited drastic phenotype differences, such as wrinkled and dome-shaped leaves and twisted pods (Figures 3B, 5A). These observations indicate that partial retention of the PPD function would be essential for tissue enlargement in soybean.

Although the ppd-KO mutant rarely produced seeds, immature embryos were formed in young pods 21 DAF (Figures 1A, 1B, 5B). These pods exhibited a twist phenotype (Figure 5A). These findings indicate that pod twisting may physically inhibit embryo development. Collectively, the findings suggest that complete loss of PPD function results in considerably impairment in soybean organogenesis. The ppd-KO mutant exhibited an extended growth phase from the development of the unifoliolate leaves to that of the first trifoliolate leave (Figure 3), which was about 2 weeks longer than that of control plant (Figure 3). The ppd-KO mutant also exhibited a stay-green phenotype that suppressed chlorophyll degradation during plant senescence (Figure 3D). This stay-green phenotype has not been previously reported in other PPD mutants. Studies have reported two types of spontaneous stay-green mutants in soybeans, and the genes responsible for this phenotype have been identified (Fang et al. 2014; Kohzuma et al. 2017; Nakano et al. 2014). However, these genes were not present among the differentially expressed genes identified via transcriptome analysis in the present study. Further, the ppd-KO mutant maintained this stay-green phenotype until the plant body died (data not shown). Collectively, loss of PPD function in soybean affects transition of developmental stage and photosynthetic pigment biosynthesis and metabolism. These findings may provide novel insights into the PPD function in higher plants.

In Arabidopsis, GIFs are transcriptional cofactors that regulate plant growth and development (Debernardi et al. 2014; Kim and Kende 2004). The gif1 mutant of Arabidopsis exhibited decreased leaf and petal sizes (Lee et al. 2009). G1F1 expression is controlled by the protein complex containing PPD (Liu et al. 2020). Knockdown of GmPPD via RNAi also increased the expression of GIF1 and the seed weight and leaf size of transgenic soybean plants (Ge et al. 2016). The expression of GIF1 family genes was significantly increased in 1 cm-sized pods of the ppd-KO mutant and the unifoliolate leaves of the ppd-KO mutant (Figure 5C, Supplementary Figure S2). However, unlike PPD-knocked down plants, the ppd-KO mutant did not exhibit the organ enlargement phenotype but exhibited a twisted pod phenotype and produced fewer seeds. It was not possible to determine whether increased expression of the GmGIF1 family genes is essential for the phenotype of the ppd-KO mutant, but the production of transgenic soybean plants overexpression the GmGIF1 gene may reveal the relationship between the expression level of GIF1 gene and the twisted pod phenotype. Gene expression analysis also revealed considerable downregulation of GmSP1L5 in the 1 cm-sized pods of the ppd-KO mutant (Figure 5C). In Arabidopsis, SPIRAL1 (SPR1) and SP1L act redundantly to maintain the cortical microtubule organization essential for anisotropic cell growth (Furutani et al. 2000; Nakajima et al. 2006). Multiple-mutant spr1 and sp1l plants exhibited right-handed tendril-like twinning growth (Nakajima et al. 2006). These findings support the hypothesis that considerable downregulation of SP1L5 results in twisted pods in the ppd-KO mutant. Of date, there are no reports on the relationship between PPD and SP1L5. We revealed that loss of PPD function affects the expression of SP1L5 in soybean.

In this study, we demonstrated that while the decrease in PPD function contributed to organ enlargement, complete knockout of PPD had a negative effect on soybean organogenesis. Loss of PPD function also delayed vegetative growth and inhibited chlorophyll metabolism in soybean plants. This information could be useful for improving crop yield and growing time.

Abbreviations

DAF

days after flowering

qRT-PCR

quantitative RT-PCR

RNA-seq

RNA sequencing

Conflict of interest

The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Author contribution

JS, YK, and TY designed the study. YK and TY produced the soybean mutants. JS analyzed the soybean mutants. JS and TY wrote the manuscript. All the authors contributed to the manuscript and approved the submitted version.

Funding

This work was supported by the Cabinet Office of the Government of Japan (Cross-Ministerial Strategic Innovation Promotion Program [SIP] awarded to TY).

Description of Supplementary Files

Supplementary Table S1. Primer sequences used for genotyping, sequencing, and gene expression analysis

Supplementary Table S2. Changes in gene expression identified in ppd-KO and control plants using RNA-seq

Supplementary Figure S1. Targeted region of the gRNA and nucleotide and predicted amino acid sequences of the targeted and flanking regions

Supplementary Figure S2. Gene expression analysis of 1 cm-sized pods of the control and ppd1 and ppd2 mutant plants