**3. Discussion**

Although the genes encoding CPS have been identified in many species [48–52], the pea LS is the only CPS protein characterized from legumes in general before the present study, and *mnp1* appears to be the first dwarf mutant related to GA biosynthesis in *M. truncatula*. We found that the *mnp1* dwarf phenotype is caused by the decrease of the cell elongation and cell division in the stem. This result is consistent with the previously reported function of GA in promoting cell elongation and division. Cell elongation is regulated by cell wall-loosening protein expansin (EXP) and xyloglucan endo-transglycosylases (XET) which play a role in cell wall reconstruction. Some *XET* and *EXP* genes have been shown to be specifically upregulated by GA, which is believed to cause cell elongation in *Arabidopsis* and rice [56–59]. GA also promotes plant growth via upregulating the transcription levels of cell division-related genes including cell cycle genes *CYCA1;1* and *CDC2Os-3* in deepwater rice [60]. However, the underlying mechanism by which GA regulates the expression of these genes remains to be studied. The identification of *mnp1* provides a very good model to further study this mechanism in *M. truncatula*.

Focusing on the phylogenetic analysis of MNP1 and its homologous proteins, we found that CPS proteins belonging to legumes were grouped into two clades (clade I and II), and each clade was identified in all selected legumes, suggesting that a lineage-specific duplication of CPS genes may have occurred in legumes during the evolution process. There are just single copies from *Arabidopsis* and the tomato outgroup of the legume CPS proteins, while the CPS proteins of grasses gather together and are significantly separated from those of eudicots (Figure 3A). Consistent with this result, the conserved DXDD motif of CPS shows some degree of sequence divergence between monocots and eudicots (Figure 3B). In *M. truncatula*, MNP2/Medtr7g011770 appears to be a very close paralogue of MNP1/Medtr7g011663, because MNP1 and MNP2 are tightly clustered on chromosome 7 and shared high sequence identity. MNP1 and MNP2 belong to the legume CPS clade I, while in the legume CPS clade II, two members were found in the *M. truncatula* genome, namely MNP3/Medtr7g094970 and MNP4/Medtr5g030050. Since a highly conserved DXDD motif existed in all these four CPS proteins of

*M. truncatula* (Figure S6), it will be interesting to explore the possibilities of functional redundancy and diversification between MNP1 and the rest members.

GA homeostasis is important for the regulation of many developmental processes and has been found to be maintained by feedback regulation of GA metabolism genes in a variety of plant species [61]. *GA20ox* and *GA3ox* are the main participants in the negative feedback regulation of GA. The expression of these two kinds of genes was upregulated in the GA biosynthesis deletion mutant [20]. In our study, we found that the expression of *MtGA20ox7* was significantly upregulated, up to thousands of times in *mnp1-1* compared with the wild type. Therefore, it seems that MtGA20ox7 may be a key member in the regulation of GA homeostasis in *M. truncatula.*

The *Arabidopsis cps1* mutants are male sterility caused by defective pollen development [55]. In *M. truncatula*, the expression of *MNP1* gene was also detected in stamens (Figure S7), suggesting that *MNP1* may play a potentially important role in stamen development. However, *mnp1* is fully fertile in *M. truncatula*, with its flower organ, pods and seeds being relatively smaller when compared with the wild type (Figure S8A–E). Pollen viability, tested by Alexander's staining, indicated no significant difference between the wild type and *mnp1-1* as well (Figure S8C). It is a common phenomenon that GA deficiency leads to dwarfing and male sterility in various species, such as *Arabidopsis*, maize and tomato, but this scenario does not appear to be the case in legumes. Fertile pollens can be produced in all the reported dwarf mutants with GA deficiency in peas [43,51], and *mnp1* is similar to pea *ls* mutants, unlike *cps* mutants of other species. Given that a legume species usually contain multiple *CPS* genes, it can be argued that the mechanism of GA biosynthesis for plant height and pollen development in legumes may be conserved and distinct from that of other species. In terms of the dwarfed but fertile phenotypes of *mnp1* and *ls* mutants, the identification of *MNP1*/*LS* and other key genes involved in GA metabolism would be of grea<sup>t</sup> potential utility in legume breeding.

#### **4. Materials and Methods**

#### *4.1. Plant Materials and Growth Conditions*

*M. truncatula* ecotype R108 and *A. thaliana* ecotype Col-0 were used for this study. The *mnp1-1* (NF0500), *mnp1-2* (NF13564) and *mnp1-3* (NF10616) mutants (all in ecotype R108 background) were isolated from the *Tnt1* retrotransposon-tagged mutant collection of *M. truncatula* as previously reported [45]. Among them, the *mnp1-3* mutants were screened from the *Tnt1* population by a reverse genetic approach. Seeds of *Arabidopsis cps1* mutant (SALK\_109115) were purchased from the *Arabidopsis* Biological Resource Center (ABRC). The *p35S::MNP1-GFP* transgenic plants were generated in *cps1* background.

*Arabidopsis* GA-deficient mutant *cps1* cannot germinate in the soil. For this reason, *Arabidopsis* plants need to be grown on solid 1/2 MS medium for approximately 2 weeks and then be transplanted into the soil. All plants (*M. truncatula* and *A. thaliana*) were grown under the following greenhouse conditions: 16 h day/8 h night cycle, 150 uE/m<sup>2</sup>/s light intensity, 22 ◦C day/18 ◦C night temperature and 70% humidity.

#### *4.2. Statistical Analysis of Cell Length and Number*

For the measurement of the internode length, twenty individual plants of both the wild type and *mnp1-1* genotypes were grown simultaneously in the same greenhouse, and the third internode beneath the shoot apex of each plant (2-month-old) was collected and considered as an independent biological sample. Thus, a total of twenty internodes were processed to calculate the average length. Then, three internodes of each genotype were randomly selected from the above twenty samples, and were submerged in fixative solution (5% formaldehyde, 5% acetic acid and 50% ethanol) for over 12 h at room temperature. Subsequently, the samples were dehydrated in a graded ethanol series (50%, 70%, 90%, 95%, 100%), critical-point dried in liquid CO2 and sputter-coated with gold. The three dried internodes for each genotype were individually examined using scanning electron microscopy

(SEM) by an EVO LS10 (Zeiss, Oberkochen, Germany) at an accelerating voltage of 5 kV. Therefore, three SEM images of the third internode were obtained for each genotype (wild type and *mnp1-1*).

For the measurement of epidermal cell length of the internode, 20 cells were randomly selected from the SEM images (6–7 cells per image) for both the wild type and *mnp1-1* genotypes, and the lengths were measured by ImageJ.

The cell number was calculated from the ratio of the average internode length (that was evaluated from a total of 20 internodes) to the average cell length (that was evaluated from 20 cells of three biological replicates).

All above experiments were repeated twice independently with similar results.

#### *4.3. Molecular Cloning of MNP1*

The molecular cloning of the *MNP1* gene referred to the method reported previously [62]. We screened the *Tnt1* retrotransposon insertion mutant collection of *M. truncatula* (ecotype R108) and isolated two *mnp1* alleles (*mnp1-1*, NF0500 and *mnp1-2*, NF13564) with severely dwarfed phenotypes. Then, these two *mnp1* alleles were backcrossed with the wild type to purify the genetic background for reducing incoherent *Tnt1* insertions, and *mnp1-1* and *mnp1-2* F2 segregation populations were generated, respectively. Equal amounts of leaf material were harvested from 12 independent mutant individuals of each population to make two mixed samples. The genomic DNA of the mixed samples was extracted using the Plant Genomic DNA Kit (Tiangen, Beijing, China). Whole-genome resequencing was carried out at 20× coverage. Then, the data of whole-genome resequencing were analyzed by a novel bioinformatics tool, Identification of Transposon Insertion Sites (ITIS) to identify all *Tnt1* insertion sites in the genome [47]. The common *Tnt1* insertion sites in *mnp1-1* and *mnp1-2* genomes were found in *Medtr7g011663* locus (annotated in A17 genome v4.0). Subsequently, PCR experiments using *mnp1-1* and *mnp1-2* genomic DNA as templates were performed to verify the insertion of *Tnt1* in *Medtr7g011663*. An additional allele *mnp1-3* (NF10616) was screened from the *Tnt1* population by a reverse genetics approach, which also displayed a mini-plant phenotype. Genomic PCR analysis confirmed that the *mnp1-3* mutant does carry a *Tnt1* insertion in *Medtr7g011663.* Thus, *Medtr7g011663* was regarded as the putative *MNP1* gene. The analysis data of ITIS and the primers used for PCR are shown in Tables S1 and S3, respectively.

#### *4.4. Phylogenetic Analysis and Sequences Alignment*

The sequences of MNP1 homologs were identified through BLAST from Phytozome (https: //phytozome.jgi.doe.gov/pz/portal.html), URGI (https://urgi.versailles.inra.fr/Species/Pisum) and maizeGDB (https://maizegdb.org/) in the protein databases of *Medicago truncatula, Glycine max, Solanum lycopersicum, Arabidopsis thaliana*, *Oryza sativa*, *Pisum sativum* and *Zea mays*. Multiple amino acid sequences were aligned by ClustalX2 (v2.1) at default parameters and beautified by DNAMAN V6. The phylogenetic tree was performed by the maximum likelihood method with IQTREE v1.6.10 as previously reported [63]. The JTT + F + G4 model was selected as suggested by the IQTREE model test tool (BIC criterion) with 1000 ultrafast bootstrap replicates and 5000 iterations.

#### *4.5. Exogenous GA3 Application Method*

Bioactive GA3 (Genview, Lot: 5209010140) was dissolved in ethanol (0.1 M) and diluted with water before being applied [64]. About 600 mL of 70 uM bioactive GA3 working solution was sprayed to a total of twelve *mnp1-1* mutant plant one time. The first spray was applied at 10-day-old seedlings after sowing, and the later sprays performed once a week for two months in total. An equivalent group (*n* = 12) of *mnp1-1* mutant plants was treated similarly with a solution without GA3 at each same time. All *mnp1-1* mutants with the treatments (+GA3 and −GA3) were grown simultaneously in the same greenhouse. Experiments were repeated twice independently with similar results.

#### *4.6. RNA Extraction, RT-PCR and Quantitative RT-PCR (qRT-PCR)*

*Medicago* stem tissues and *Arabidopsis* rosette leaves for RNA extraction were harvested from 7-week-old and 6-week-old plants, respectively. Total RNA was isolated using TransZol (TransGen, Beijing, China) according to the manufacturer's protocol and then was reverse transcribed into cDNA by HiScript ® II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The resulting cDNAs were used as templates for RT-PCR and qRT-PCR. *AtACTIN* (*Actin2*/*At3g18780*) was used as an internal control for *Arabidopsis* RT-PCR. qRT-PCR was performed using 2 × T5 Fast qPCR mix (SYBR Green I) (TsingKe, Beijing, China) on the Roche Light Cycler 480II real-time PCR machine (95 ◦C, 1 min; 95 ◦C, 10 s, 60 ◦C, 10 s, 72 ◦C, 15 s, 40 cycles). *MtACTIN* (*Medtr3g095530*) was used as an internal control for *Medicago* qPCR. Three independent biological replicates were used for RNA extraction and subsequent cDNA synthesis. All samples were selected randomly under the same greenhouse conditions. Three technical replicates for each biological replicate were used in qRT-PCR analysis. The genes involved in this study and the primers used for qPCR are listed in Tables S2 and S3, respectively.

### *4.7. Plasmid Construction*

Coding sequences of target genes were isolated by RT-PCR from wild type root tissue of seedlings (3 weeks old). For subcellular localization experiments, the coding sequences of the *N*-terminus of MNP1 (100-amino acid, TPMNP1) and the full-length coding sequence of MNP1 were inserted into the *pCAMBIA3301MP* vector between NcoI and AvrII site via the ClonExpress II One Step Cloning Kit (Vazyme) to generate *p35S::TPMNP1-GFP* and *p35S::MNP1-GFP* constructs, respectively. The *p35S:: MNP1-GFP* constructs were also used for plant transformation. The primers used for plasmid construction are listed in Table S3.

### *4.8. Subcellular Localization*

The constructs, *p35S::TPMNP1-GFP* and *p35S::GFP*, were introduced into *Agrobacterium tumefaciens* EHA105 strain, and then they were transiently expressed in tobacco (*Nicotiana benthamia*) leaves by *Agrobacterium*-mediated transformation [65]. The *p35S::GFP* constructs were served as a positive control. The TPMNP1-GFP fusion protein was examined using a confocal laser scanning microscope (FV1000; Olympus, Japan). This experiment was repeated three times independently with similar results.

### *4.9. Plant Transformation*

The *p35S::MNP1-GFP* constructs were introduced into *Agrobacterium tumefaciens* EHA105 strain, which was subsequently used to transform *cps1* heterozygotes by *Agrobacterium*-mediated transformation using the floral dip method [66]. Through resistance screening with 20 mg/<sup>L</sup> Basta (BBI Life Sciences, Lot: C707BA0017) and the subsequent PCR genotyping, the *p35S::MNP1-GFP* transgenic lines in the *cps1* homozygous background were isolated. The primers used for PCR are shown in Table S3.

### *4.10. Alexander's Staining*

Mature pollens were stained with Alexander's staining solution as previously described [67]. Mature anthers of WT and *mnp1-1* from the same developmental stage were immersed directly in a drop of staining solution, covered with a coverslip respectively, and then kept them in an oven at 50 ◦C for 1 h. Next, a microscopic examination was conducted via a fluorescence microscope (Olympus BX63) using the bright field channel. The fertile pollen would be stained red to deep red, while aborted pollen would be green. Experiments were repeated more than three times independently.

*Int. J. Mol. Sci.* **2020**, *21*, 4968

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/14/ 4968/s1.

**Author Contributions:** S.G., X.Z. and J.C. designed the research. Q.B. and S.Z. supported constructive comments on the research. S.G. and X.Z. performed most of the experiments. W.Z. and Y.F. assisted the experiments. S.G., X.Z., L.H. and B.Z. analyzed the data. S.G. wrote the original manuscript. J.C. and L.H. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, gran<sup>t</sup> number 31700244, U1702234 and 31471171. L.H. and J.C. were also supported by the STS of CAS (KFJ-STS-ZDTP-076).

**Acknowledgments:** We are grateful to Zhijia Gu and Yanxia Jia (Kunming Institute of Botany, Chinese Academy of Sciences) for their technical help in SEM and laser confocal experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.
