**2. Results**

#### *2.1. Mini Plant 1 Mutants Were Severely Dwarfed Due to Shorter and Fewer Cells*

Legumes are the third largest family of angiosperms, including many important crops, such as soybeans and peanuts [44]. To gain a better understanding of the molecular basis of plant height regulation in legumes, we screened the *Tnt1* retrotransposon insertion mutant collection of the model plant *M. truncatula* [45] to isolate mutants with significant changes in plant height. Two allelic mutants with similar severely dwarf phenotypes were identified and designated as *mini plant 1-1* (*mnp1-1*) and *mini plant 1-2* (*mnp1-2*), respectively, because all F1 progenies derived from a cross between

*mnp1-1* and *mnp1-2* were dwarf plants. Compared with the wild type, the mutants are severely dwarf, with increased branches and dark green leaves (Figure 1A–C and Figure 2C).

**Figure 1.** Phenotypic characterization of *mnp1-1* mutant. (**A**–**C**) Morphologies of wild type (WT) and *mnp1-1* mutant at different developmental stages. (**A**) Ten days after sowing. Scale bar = 0.75 cm. (**B**) Six weeks after sowing. Scale bar = 2 cm. (**C**) The reproductive stage of plants. Scale bar = 6.5 cm. (**D**) The length of the third internode beneath the shoot apex. Values are means ± *SD* (*n* = 20 internodes). Two-sample *t*-test, **\*\*\*** *p* < 0.001. (**E**) The length of epidermal cells of the third internode beneath the shoot apex. Values are means ± *SD* (*n* = 20 cells from three biological replicates). Two-sample *t*-test, **\*\*\*** *p* < 0.001. (**F**) Scanning electron microscope images and cell outlines of a representative third internode beneath the shoot apex. Scale bar = 50 um. (**G**) Number of epidermal cells in the third internode beneath the shoot apex. The cell number was calculated from the ratio of the average internode length (**D**) to the average cell length (**E**). Error bars represent the standard deviation of the cell number of 20 independent internodes. Two-sample *t*-test, **\*\*\*** *p* < 0.001. (**H**) Expression analysis of cell division marker gene *MtCYCB1;1.* Values are means ± *SD*. Two-sample *t*-test, **\*\*** *p* < 0.01. (**I**) Expression analysis of cell division marker gene *MtKNOLLE.* Values are means ± *SD*. Two-sample *t*-test, **\*\*\*** *p* < 0.001.

During the growth and development from seedlings to adult plants, the height gap between the wild type and *mnp1-1* mutant was becoming bigger (Figure 1A–C). By measuring the length of the third internode beneath the shoot apex, we confirmed that the *mnp1-1* mutants have reduced internode length compared with the wild type (Figure 1D). Then, scanning electron microscopy (SEM) analysis was used to determine the reasons for the shorter internode of *mnp1-1* mutants. The epidermal cells of the *mnp1-1*

internode were considerably shorter than those of the wild type (Figure 1E,F). In addition, the number of internode cells was also greatly reduced in *mnp1-1* mutants (Figure 1G), indicating that cell division was significantly suppressed. This speculation would be in agreemen<sup>t</sup> with the quantitative analysis of the reduced cell cycle activity of the *mnp1-1* internode. The expression of the G2/M phase cell cycle marker *MtCYCB1;1* and the cytokinesis marker *MtKNOLLE* [46] were both dramatically lower in *mnp1-1* than that of wild type (Figure 1H,I). Therefore, both decreased length and number of internode cells contributed to the shortened stem of *mnp1-1*. In addition to the decrease of stem length, the petiole of *mnp1-1* was shortened as well (Figure S1). In conclusion, these results demonstrated that *MNP1* plays an important role in the length determination of stem and petiole in *M. truncatula*.

#### *2.2. Molecular Cloning of MNP1 Gene*

Analysis of the F2 generation resulting from a cross between *mnp1-2* and wild type showed a segregation ratio of 3:1 between wild-type-like and dwarf phenotypes (36:13, χ2 = 0.0068 < χ20.05 = 3.84) (Figure S2A), indicating that the *mnp1-2* phenotype was controlled by a single recessive gene. To clone the target gene corresponding for the mutant phenotype, *mnp1-1* and *mnp1-2* were backcrossed with the wild type, respectively, and mutant plants were isolated from both F2 populations, followed by whole-genome resequencing at 20× coverage. Then, the resequencing data were analyzed using the bioinformatics tool Identification of Transposon Insertion Sites (ITIS) as previously described (Table S1) [47]. ITIS identified nine and seventy-one *Tnt1* insertions in the genomes of the *mnp1-1* and *mnp1-2* mutants, respectively. There were two *Tnt1* insertion sites on chromosome 7 that appeared to be nearby from the genomic sequence data of *mnp1-1* and *mnp1-2*; one was inserted into an intergenic region, and another was inserted into a genic region corresponding to the *Medtr7g011663* gene (annotated in A17 genome v4.0) (Figure 2A; Table S1). Then, PCR-based genotyping and sequencing analysis confirmed that the *mnp1-1* and *mnp1-2* mutants harbored *Tnt1* insertions in the fourth exon and the seventh exon of the candidate gene/*Medtr7g011663*, respectively (Figure 2B–D and Figure S2B). To determine whether the mutation of *Medtr7g011663* is responsible for the *mnp1* mutants' phenotype, an additional mutant line with a predicted *Tnt1* insertion in *Medtr7g011663* locus was identified via BLAST searching of the public mutant database [45], and thus was designated as *mnp1-3.* The *mnp1-3* plants displayed a severely dwarfed phenotype similar to *mnp1* alleles when growing in the greenhouse (Figure 2C). PCR-based sequencing confirmed that there is indeed a *Tnt1* insertion in the sixth exon of *Medtr7g011663* in *mnp1-3* (Figure 2D and Figure S2B). Thus, we considered *Medtr7g011663* as the putative *MNP1* gene.

**Figure 2.** Molecular cloning of the *MNP1* gene. (**A**) Adjacent *Tnt1* insertion sites were found on chromosome 7 of *mnp1-1* and *mnp1-2*. The *x*-axis represents chromosome 7. Rhombus and squares represent *Tnt1* insertions in *mnp1-1* and *mnp1-2*, respectively. The rhombus and square on a black line show nearby *Tnt1* insertions in an intergenic region. The rhombus and square on a red line show nearby *Tnt1* insertions in *Medtr7g011663* and the right image is an enlarged view in the same region. (**B**) Schematic illustration of *MNP1* gene structure and *Tnt1* insertion sites in *mnp1* alleles. The *mnp1-3* (blue color) mutants were screened from the *Tnt1* population using a reverse genetics approach. Filled black boxes represent exons and lines between them denote introns. Arrows indicate *Tnt1* orientation. (**C**) The phenotype of *mnp1* alleles. Scale bar = 6.5 cm. (**D**) Genotyping of *mnp1* alleles. The primers (*MNP1*-GT-F/R) were designed for detecting *MNP1* genomic fragments, and the primer pair TntF2/R2 were *Tnt1*-specific primers.

#### *2.3. MNP1 Encodes a Putative CPS Protein in M. truncatula*

To figure out the type of protein encoded by *MNP1*, phylogenetic analysis of MNP1 and its homologous proteins from *M. truncatula* and related legume plants (pea and soybean), dicotyledonous model plants (*Arabidopsis* and tomato) and grasses (rice and maize) was performed. MNP1 protein was closely grouped with numerous homologs from legumes, and each selected legume species has at least two homologous copies. When compared to the reported homologous proteins, MNP1 showed the most homology to the pea LS and significant homology to the GIB-1 in tomatoes, CPS1/GA1 in *Arabidopsis*, OsCPS1 in rice and An1 in maize (Figure 3A), all of which are in the CPS family belonging to type-B cyclase and take part in the first step of GA biosynthesis [48–52]. The loss-of-function mutants of *ls*, *gib-1*, *cps1*/*ga1*, *Oscps1* and *an1* all show dwarfed phenotypes. In addition, the alignment of multiple amino acid sequences shows that MNP1 exhibits a high degree of amino acid sequence identities with these CPS proteins (Figure S3). Furthermore, there is an aspartate-rich motif DXDD near the N-terminal region of MNP1 (Figure 3B), which is conserved among type-B cyclase and important for the catalysis of the type-B cyclization reactions [52,53]. Taken together, we believe that MNP1 would be a conserved CPS protein involved in the GA biosynthesis pathway in *M. truncatula*.

**Figure 3.** Phylogenetic analysis and sequences alignment of MNP1 and its closely related homologs. (**A**) Phylogenetic analysis of MNP1 and its homologs. Proteins from the species *Medicago truncatula* (Medtr), *Pisum sativum* (Psat), *Glycine max* (Gm), *Solanum lycopersicum* (Solyc), *Arabidopsis thaliana* (At), *Oryza sativa* (Os) and *Zea mays* (Zm). Bootstrap values are indicated upon the branches. Red rhombus indicates MNP1 protein and red circles indicate the reported CPS proteins. (**B**) The sequences alignment of MNP1 and the reported CPS proteins. The amino acid color indicates the homology of sequences between these species: black = 100%, pink ≥ 75% and blue ≥ 50%. The DXDD motifs in the sequences are indicated by the black line.

#### *2.4. Subcellular Localization of MNP1*

The CPS1/GA1 has been reported to be localized on plastids in *Arabidopsis* with a chloroplast transit peptide (cTP) at its *N*-terminus [50]. Then, we carried out cTP prediction using the ChloroP program (http://www.cbs.dtu.dk/services/ChloroP) and found that the MNP1 is also highly predicted to have a cTP at its *N*-terminus, with a score of 0.591 (strong).

Based on the ChloroP prediction results, the sequence encoding the N-terminal truncation of 1–100 amino acids of MNP1 (TPMNP1) was used to generate *p35S::TPMNP1-GFP* constructs, which was

then transiently expressed in epidermal cells of tobacco (*Nicotiana benthamia*). The green fluorescence signal of the fusion protein was observed in a chloroplast (Figure 4). This result was further confirmed by the subcellular localization analysis of the GFP fusion protein with full-length MNP1 (Figure S4). Thus, these data sugges<sup>t</sup> that MNP1 may play the same role as *Arabidopsis* CPS1 in chloroplasts and participate in GA biosynthesis.

**Figure 4.** Subcellular localization of MNP1. According to ChloroP prediction, there is a chloroplast transit peptide (cTP) at the *N*-terminus of MNP1 protein, so a sequence encoding 100 amino acids containing cTP was used to generate *p35S::TPMNP1-GFP* constructs. Then, the constructs were transformed into tobacco (*Nicotiana benthamia*) leaf epidermal cells by *Agrobacterium*-mediated transformation. *p35S::GFP* was used as a positive control. Images were taken 36 h after transformation with dual GFP (green) and chlorophyll (red) channels. Scale bar = 40 um.

#### *2.5. Genes of GA Biosynthesis Pathway Are Significantly Up-Regulated in mnp1-1*

Based on the above evidence, we conclude that *MNP1* is the putative gene encoding a CPS protein that participates in GA biosynthesis in *M. truncatula*. Therefore, exogenous GA3 was used to investigate whether *mnp1* is a GA-sensitive mutant. As expected, the plant height of *mnp1-1* sprayed with GA3 was significantly higher than that of the control group without GA treatment (Figure 5A). Besides, the blade size and petiole length of *mnp1-1* mutants were also significantly restored after GA treatment (Figure S5). Thus, it could be stated that the lack of GA leads to the dwarf phenotype of *mnp1.* The GA biosynthesis pathway involves many genes besides *CPS* (Figure 5B). According to the reference [54], we tested the expression level of the putative genes (Table S2) in the GA biosynthesis pathway of *M. truncatula* stem tissue. The results showed that most GA biosynthesis genes were highly upregulated in the *mnp1-1* mutant, while a small proportion of the genes (*MtKS*, *MtKAO1*, *MtCYP714\_A1*, *MtCYP714\_C2*) showed a low level of upregulation, with no significant difference. Among these upregulated genes, *MtGA20ox7* was most significant and was thousands of times higher than that of the wild type (Figure 5C), which is coincident with the earlier statement that GA20ox has an important role in GA homeostasis regulation in plants [20]. The upregulation of the genes that lie downstream of the GA biosynthesis pathway in *mnp1-1* implied a negative feedback response to the low GA content, and *MtGA20ox7* may play a key role in GA feedback regulation in *mnp1-1*.

**Figure 5.** Expression analysis of GA biosynthesis genes in GA-sensitive mutant *mnp1-1*. (**A**) From left to right are *mnp1-1* without GA3 treatment and *mnp1-1* with 70 uM GA3 treatment. Scale bar = 4 cm. (**B**) GA biosynthesis pathway schematic diagram. The red, green and blue arrows represent the three stages of GA biosynthesis pathway. Gray ovals represent enzymes. (**C**) Relative expression levels of GA biosynthesis genes in the stem of WT and *mnp1-1*. The red, green and blue boxes represent the three stages of GA biosynthesis pathway as in (**B**). The significant difference was determined by unpaired two-sample *t*-test (**\*\*** *p* < 0.01, **\*\*\*** *p* < 0.001).

#### *2.6. MNP1 Could Partially Rescue the Phenotype of Arabidopsis cps1 Mutant*

The *cps1*/*ga1* mutant of *Arabidopsis* shows a severely dwarfed and sterile phenotype due to the loss of CPS function [55]. To examine the extent of the functional conservation between *M. truncatula* and *A. thaliana* CPS proteins, we obtained a homozygous *T-DNA* insertion mutant of *At4g02780* (SALK\_109115) from the *Arabidopsis* Biological Resource Center (ABRC), namely the *cps1* mutant. The *cps1* mutant showed extremely dwarf as expected, and was able to produce inflorescences, but no fertile seeds (Figure 6A). Next, we introduced *p35S::MNP1-GFP* constructs into *cps1* heterozygotes by the floral dip method. Through resistance screening and PCR genotyping, we isolated the *p35S::MNP1-GFP* transgenic plants in the *cps1* homozygous background, and found that the size of transgenic plants was partially restored (Figure 6B,C). RT-PCR analysis confirmed the expression of *MNP1* gene in the transgenic plants (Figure 6D). These results indicated that *MNP1* could partially recover the mini-plant phenotype of the *cps1* mutant, suggesting that CPS has functional conservation between *M. truncatula* and *A. thaliana*.

**Figure 6.** *MNP1* partially rescued the mini-plant phenotype of *Arabidopsis cps1* mutant. (**A**) The phenotype of *Arabidopsis* wild type (Col-0) and *cps1* mutant. The Col-0 and *cps1* mutant were 7 and 12 weeks old, respectively. Scale bar = 2 cm. (**B**) The *p35S::MNP1-GFP* transgenic plant of the *cps1* homozygous background partially restored the mini-plant phenotype of *cps1* mutant. The plants were 6 weeks old. Scale bar = 2 cm. (**C**) Genotyping of the transgenic plant. The homozygous *T-DNA* insertion in *CPS1*/*At4g02780* locus and *MNP1* coding sequence were detected in the transgenic plant. (**D**) RT-PCR amplification of *MNP1* from Col-0, the transgenic plant and *cps1* mutant. *AtACTIN* was used as an internal control.
