Overexpression of Terpenoid Biosynthesis Genes Modifies Root Growth and Nodulation in Soybean (Glycine max)

Root nodule formation in many leguminous plants is known to be affected by endogen ous and exogenous factors that affect formation, development, and longevity of nodules in roots. Therefore, it is important to understand the role of the genes which are involved in the regulation of the nodulation signaling pathway. This study aimed to investigate the effect of terpenoids and terpene biosynthesis genes on root nodule formation in Glycine max. The study aimed to clarify not only the impact of over-expressing five terpene synthesis genes isolated from G. max and Salvia guaranitica on soybean nodulation signaling pathway, but also on the strigolactones pathway. The obtained results revealed that the over expression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes enhanced the root nodule numbers, fresh weight of nodules, root, and root length. Moreover, the terpene content in the transgenic G. max hairy roots was estimated. The results explored that the monoterpenes, sesquiterpenes and diterpenes were significantly increased in transgenic soybean hairy roots in comparison with the control. Our results indicate the potential effects of terpenoids and terpene synthesis genes on soybean root growth and nodulation. The study provides novel insights for understanding the epistatic relationship between terpenoids, root development, and nodulation in soybean.


Introduction
Soybean (Glycine max) is considered one of the oldest polyploidy (pa leopolyploid) plants and one of the most domesticated food crops in the world; it is expected to contribute to sustainable agriculture through its ability for symbiotic nitrogen fixation [1]. The symbiotic interaction between soybean roots and B. japonicum bacteria, leads to the formation of unique structures known as root nodules. Hosted inside the root nodule, rhizobia can transform the molecular nitrogen gas (N 2 ) from atmosphere into ammonia (NH 3 ), which will be readily available to the plant, and for this exchange of benefits deal, rhizobia are amended with plant carbohydrates [1,2]. Various factors regulate root nodule formation such as certain plant hormones, some metabolic enzymes, and definite transcription factors from the approach of the nodulation signal all the way to nodule initiation, development, and maturation [3,4]. Furthermore, several genes related to secondary metabolism (e.g., Phenylpropanoids, terpenoid and isoflavonoids biosyntheses) were identified by microarray analysis from Lotus japonicu nodule with higher frequency in nodule parenchyma (NP) and nodule vascular bundle (NC), compared with un-nodulated root [5]. Previously, we found that, the knockdown of MtHMGR1 gene form Medicago plant (Medicago truncatula) led to a decrease in nodules formation, which is considered a key gene in the Mevalonate (MVA) pathway that can interact with the DMI2 gene for induced symbiotic interaction and nodule development. Moreover, the use of RNA interference (RNAi) tool for silencing both genes GmMAX1a and GmMAX4a led to a dramatic decrease in nodule numbers in soybean plants. Recently, we found that the overexpression of SoTPS6, SoNEOD, SoLINS, SoSABS, SoGPS, and SoCINS genes from Salvia officinalis in soybean hairy roots, produces a drastic increase in root growth and nodulation [6].
Over the years, Agrobacterium rhizogenes-mediated transformation of soybean (G. max) hairy roots has become a powerful agent to investigate the responsibility of genes that are concerned in root biological roles such as plant-microbe communication, nutrient uptake, and hormone transport [6,28]. We have successfully used this system to clarify the role of various genes such as: GmMAX1a, GmMAX4a and GmIMaTs, that are involved in soybean nodulation [27,29]. Here, we characterized five genes from G. max and S. guaranitica that are involved in terpenoid and terpene biosynthesis, and determined theirbiological role in the interaction with rhizobia and promotion of nodulation in G. max. The inclusion methodologies that were employed to reach this goal are the following: (i) overexpression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes in the domesticated soybean hairy roots; (ii) investigating nodulation and root phenotypes at 10 and 20 days after B. japonicum inoculation; (iii) profiling terpenoid in transgenic G. max hairy roots by GC-mass; and (iv) monitoring the transcription of genes implicated in nodulation signaling and strigolactones biosynthesis by qPCR. In the context that, an important question has been raised: what are the key roles of terpenoid genes in root development and nodulation? This question was difficult to answer before conducting the present work because there was a lack of information at the genetic level regarding the terpenoid biosynthetic pathway and the roles of these genes in root development and nodulation. Interestingly, we may be able to answer the question through our findings, which support the significance of the previous terpenoid genes in rhizobial infection by elucidating the associations between the overexpression of terpenoid, nodulation-signaling, and strigolactone-biosynthesizing genes.

In-Silico Differntial Gene Expression and Phylogenetic Analysis
Phylogenetic tree was created via MEGA6 using the Neighbor-Joining method with 1000 bootstraps. G. max and S. guaranitica terpenoid biosynthetic pathway genes and the deduced amino acid sequences were searched using RNA-Seq Data Analysis and Phytozome database (phytozome.jgi.doe.gov) accessed on 12 April 2021 and identified soybean proteins with high sequence similarity (≥90% normalized identity) to annotated terpenoid biosynthesis genes of G. max and S. guaranitica through BlastP. Moreover, to investigate the putative accumulated transcript of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS across nine different tissues, we used public RNA-Seq meta-analyses from multifarious studies were presented from the Atlas of soybean (http://bar.utoronto.ca/eplant_soybean/, accessed on 25 March 2021). Additionally, GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS predicted subcellular localization was inferred from its Arabiposis homologous genes as retrieved from the Arabidopsis Information Resource (https://phytozome.jgi.doe.gov/pz/ portal.html#!info?Alias=Org_Athaliana, accessed on 25 March 2021). Ultimately, the image that showed the subcellular localization was built using Cell Electronic Fluorescent Pictograph Browsers (Cell eFP: http://bar.utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi, accessed on 25 March 2021).

Cloning of Full-Length Terpenoid Synthase cDNAs
The GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS full-length cDNAs were amplified with short and long gene-specific primers designed based on our transcriptome sequencing of S. guaranitica leaves as well as the soybean database (https://phytozome. jgi.doe.gov/pz/portal.html, accessed on 21 March 2021) (Table S1). Polymerase Chain Reaction (PCR) reaction was performed using short primers, KOD-Plus DNA polymerase enzyme (Toyobo, Japan) and leaf cDNA as a template with the following program (94 • C: 3 min, 98 • C: 10 s, 57 or 60 • C: 30 s, 68 • C: 1.5 min, 68 • C: 10 min, and 35 cycles), for the first PCR. For the second PCR we used the first PCR products as at templates, long primers and the same previous compounds and program. Afterwards, the PCR products were purified, cloned into the pDONR221 Gateway entry vector, and then subcloned into the pB2GW7 Gateway destination vector as described earlier [6,[8][9][10]. Following this, the destination vector was used to introduce our previous genes into the G. max hairy roots via Agrobacterium rhizogenes-K599 by electroporation. Sanger sequencing was used to verify the success of the cloning steps. The soybean hairy root transformation and rhizobial inoculation were performed as described earlier [6]. Briefly, soybean seeds were sterilized and germinated in sterilized vermiculite under controlled conditions. Hereafter, the bacterial suspension of the recombinant A. rhizogenes was prepared accordingly and was injected into the hypocotyl proximal of vigorous soybean seedlings with flattened cotyledons. Then, the transformed seedlings were grown under suitable and controlled conditions. On the tenth day from root transformation, rhizobial inoculation by B. japonicum USDA-110 was properly performed in ten to twelve replicates. Finally, transformed plants with properly established hairy roots and nodules were harvested for photographing or further differential gene expression analyses by qRT-PCR.

Differential Gene Expression by qRT-PCR
According to the manufacturer's methods and instructions, total RNA was extracted from different biological replicates using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was treated with DnaseI (Takara, Beijing, China), and its integrity was checked using 1.2% agarose-formaldehyde gel and with ethidium bromide staining. However, its purity and concentration were analyzed by NanoDrop™ 2000/2000c Spectrophotometers (Wilmington, MA, USA). For either cloning or qRT-PCR, cDNA synthesis was performed with a reverse transcription kit (M-MLV, Beijing, China) using 10 µg of RNA [6,[8][9][10]. To elucidate the differential gene expression of terpenoid biosynthesis, Strigolactone biosynthesis and early nodulation signaling genes (Tables S2 and S3) [6,[8][9][10]. The primers were designed via the IDTdna tool (https://eu.idtdna.com/scitools/Applications/RealTimePCR/, accessed on 12 June 2021), listed in (Tables S2 and S3). To calculate the cycle threshold (CT) of the target genes, GmActin was used as a reference gene to normalize the gene expression levels. Finally, the delta delta Ct method was used to calculate relative gene expression levels [30].

Quantitative GC-MS of Terpenoids
Fresh in vitro hairy roots from either GmFDPS-OE, GmGGPPS-OE, SgGPS-OE, SgFPPS-OE, and SgLINS-OE or GUS lines were promptly frozen using liquid nitrogen. Then, powder samples were soaked in 10 mL of n-hexane and incubated with shaking at 37 • C and 200 rpm for 72 h as described earlier [6]. Afterwards, the supernatant was concentrated to 1.5 mL, and transferred to fresh crimp 1.5-mL vial amber glass. The vials were then placed on the GC-MS auto-sampler. Following that, the quantification of terpenoids was done via GCMS-QP2010 Ultra (Shimadzu, Tokyo, Japan) with HP-5 fused silica capillary column (30 m × 0.25 mm ID, 0.25 µm film thicknesses), Helium gas at flow rate 1.0 mL/min and 1-µL aliquot injection volume. We used n-Hexadecane (CAS Number: 544-76-3; https://www.sigmaaldrich.com/EG/en/product/mm/820633, accessed on 25 March 2021) as an internal standard. Finally, the type and relative % concentration for each component was determined by comparison of their mass spectra with the mass spectra data were that stored in the various Libraries, as previously described by [6,[8][9][10].

Statistical Analyses
Soybean hairy root measurements were analyzed by the Student's t-test to estimate the effects of gene overexpression and time on the root length (cm), fresh root weight (gram), fresh nodule weight (gram) and nodule numbers, and compared to the control roots (GUS-overexpressing hairy roots). Each column represents the mean ± SD of the parameter, and statistical significance was based on the Student's t-test (* p < 0.05; ** p < 0.01; n.s., not significant) with GUS-overexpressing hairy roots as control.

Identification of Terpenoid Biosynthesis Genes from Soybean and Sage Plants
With a focus on the putative biosynthetic genes in the soybean genome, we managed a BLASTP search against the soybean genome using functionally characterized of S. guaranitica terpenoid biosynthesis proteins as queries. This approach identified several proteins closely related to SgGPS, SgFPPS, and SgLINS genes. These sequences were submitted to phylogenetic analysis ( Figure S1). The putative expression patterns of terpenoid biosynthesis genes of soybean were uncovered by transcript analysis across nine tissues using the Phytozome database (phytozome.jgi.doe.gov/). Interestingly, we observed the highest expression levels of these genes in root hairs, roots, and nodules ( Figure S2). In plants, there are two pathways responsible for terpene biosynthesis: the plastidial (MEP, methylerythritol 4-phosphate: MD:M00096) pathway; and the cytosolic (MVP, mevalonate: MD:M00095) pathway that produces different terpenoids (e.g., monoterpenes, sesquiterpenes, diterpenes, triterpenes, carotenoids, and sterols) [31][32][33]. Moreover, interconnection exists between SLs, nodulation signaling molecules and terpene since all of these compounds are derived from terpenoids/isoprenoids. For that reason, we closelyinvestigated the prospective subcellular localization for these genes, relying upon Arabidopsis protein localization to identify the probable synthesis sites using the Cell eFP browsers (http://bar.utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi). From this analysis, the previous genes are localized mainly to the cytosol, mitochondrion, nucleus, and plastid ( Figure S3).

Overexpression of Terpenoid Genes Changed Soybean Root Growth
To evaluate the effect of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes on soybean root phenotypes after25days without inoculation (non-nodulating), these genes were cloned from G. max and S. guaranitica and over-expressed in soybean hairy root as a transgenic expression system. The stable constitutive overexpression of those genes in hairy roots were carried out by the infection of G. max green seedling cotyledons using A. rhizogenes carrying pB2GW7-GmFDPS, pB2GW7-GmGGPPS, pB2GW7-SgGPS, pB2GW7-SgFPPS and pB2GW7-SgLINS under the control of 35S promoter. Transgenic hairy roots were successfully generated, which have higher root length and fresh weight than the GUS control ( Figure 1A,B). These results are in line with Ali et al. [6] and Samudin and Kuswantoro [34] who found the overexpression of terpenoid genes in soybean roots led to higher fresh root weight and length compared to control in cases without inoculation by B. japonicum, which confirmed the decisive role of terpenoids genes in soybean root development [6]. In previous reports, various TPSs family genes were highly coordinated in root and cell-specific processes, such as: marneral; β-amyrin and thalianol synthesis as a triterpene; rhizathalene synthase (AtTPS08) as diterpene; (Z)-γ-bisabolene synthases as a sesquiterpene; and 1,8-cineole synthase as a monoterpene [35][36][37][38][39][40][41][42]. These previous genes were co-expressed primarily in the root epidermis cells, the stele of the root elongation, differentiation/maturation zones, epidermis and cortex of older roots and other different root tissues for producing a "superhairy" different root phenotype [35][36][37][38][39][40][41][42]. These previous reports and results demonstrated the role of TPSs family genes in root growth and development [6].

Overexpression of Terpene Synthase Genes Changed the Terpene Profiles in Transgenic Soybean Hairy Roots
To explore the consequence of overexpression of terpenoid biosynthesis genes (e.g., GUS as a control, GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS) in transgenic G. max hairy roots. GC-Mass was performed to analyze the qualitative and quantitative changes in the terpene profiles in transgenic G. max hairy roots. The analysis confirmed that various terpene profiles were significantly increased in transgenic hairy roots overexpressing terpene synthetic genes compared with GUS as reported in Table 1 and Figure 2A.
Moreover, the six hexane extracts from the different overexpression of terpenoid biosynthesis genes (e.g., GUS as a control, GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS), have unique, common, and major phytochemical compounds (Table 1 and Figure 2A). For example, the extracts of GmFDPS essential oils (B) had 11 unique compounds, two common compounds shared with extracts from SgGPS essential oils, one common compound shared with extracts from SgFPPS, one common compound shared with extracts from GmGGPPS and SgLINS, one common compound shared with extracts from GmGGPPS and SgFPPS, one common compound shared with extracts from SgGPS and SgLINS, one common compound shared with extracts from GmGGPPS, SgGPS, SgFPPS and SgLINS essential oils (Table 1 and Figure 2B). Furthermore, the GmGGPPS essential oils (C) contained seven unique compounds, one common compound shared with extracts from SgGPS, five common compounds shared with extracts from SgFPPS, one common compound shared with extracts from SgLINS, nine common compounds shared with extracts from SgGPS and SgFPPS essential oils. Moreover, the extracts from SgGPS essential oils (D) had 17 unique compounds, nine common compounds shared with extracts from SgFPPS essential oils. (B) In vivo hairy roots' fresh weight (gram) and root length (cm). Root phenotypes were examined for at least 10 independent lines (n = 10). Each column represents the mean ± SD of the parameter and statistical significance was based on the Student'st-test (* p < 0.05; ** p < 0.01) with GUS-overexpressing hairy roots as control.

Overexpression of Terpene Synthase Genes Changed the Terpene Profiles in Transgenic Soybean Hairy Roots
To explore the consequence of overexpression of terpenoid biosynthesis genes (e.g., GUS as a control, GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS) in transgenic G. max hairy roots. GC-Mass was performed to analyze the qualitative and quantitative changes in the terpene profiles in transgenic G. max hairy roots. The analysis confirmed that various terpene profiles were significantly increased in transgenic hairy roots overexpressing terpene synthetic genes compared with GUS as reported in Table 1  In roots overexpressing GmFDPS, sesquiterpenes represented the main compounds (11.87%), followed by monoterpenes (10.5%) and one diterpene compound (9.62%). In In vivo hairy roots' fresh weight (gram) and root length (cm). Root phenotypes were examined for at least 10 independent lines (n = 10). Each column represents the mean ± SD of the parameter and statistical significance was based on the Student's t-test (* p < 0.05; ** p < 0.01) with GUS-overexpressing hairy roots as control. Moreover, the extracts from SgFPPS essential oils (E) and the SgLINS essential oils (F) had 19 and five unique compounds, respectively ( Figure 2B). On the other hand, we found two common compounds named ((8) Annulene and Isomenthol) shared with all six extracts.

Overexpression of Terpenoid Biosynthesis Genes after Soybean Hairy Root Nodulation
Five genes from G. max and S. guaranitica were cloned and overexpressed in soybean roots, then soybean roots were inoculated with B. japonicum (USDA110), to explore the effects of these genes on soybean root phenotypes and nodulation after 10 and 20 days Figure 3A-H. The following root and nodule characteristics were investigated: root length (cm), fresh root weight (gram), fresh nodule weight (gram) and nodule number after 10 and 20 days, as indicated in Figure 4A. GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS expression were validated in roots and nodules after 10 and 20days by qRT-PCR, with substantial overexpression compared with GUS-containing plants ( Figure 4B-E). Our results reveal that overexpression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS led to a significant increase in root length and fresh root weight after 10 days. Moreover, the overexpression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS led to a significant increase in root length after 20days, while only the overexpression of GmFDPS and SgGPS led to a significant increase in fresh root weight after 10days ( Figure 4A). Furthermore, when compared to GUS lines, overexpression of the GmGGPPS, SgGPS, and SgFPPS resulted in higher nodule counts and dramatically increased nodules fresh weight for a given amount of root after 10 and 20 days ( Figure 4A). On the contrary, the overexpression of SoLINS after 10 days from inoculation led to formation of a few numbers from ultra-fine unmature nodules with meager fresh weight compared to the GUS. Our results are in agreement with [6,43] who found the nodule grow and formation at soybean and japans cultivars peanut need for a longer period, which means that there is a suitable and standard diameter of the 1st-order lateral roots for nodule formation that related with each growth

Overexpression of Terpenoid Biosynthesis Genes after Soybean Hairy Root Nodulation
Five genes from G. max and S. guaranitica were cloned and overexpressed in soybean roots, then soybean roots were inoculated with B. japonicum (USDA110), to explore the effects of these genes on soybean root phenotypes and nodulation after 10 and 20 days Figure 3A-H. The following root and nodule characteristics were investigated: root length (cm), fresh root weight (gram), fresh nodule weight (gram) and nodule number after 10 and 20days, as indicated in Figure 4A. GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS expression were validated in roots and nodules after 10 and 20days by

Relative Expression Analysis of Nodulation and Strigolactone Biosynthesis Genes in Transgenic Soybean Hairy Roots at 10 DAI by B. japonicum
The creation of root nodules is mediated by successful communication between the legume root and the rhizobia, which is established by transmitting chemical signals from both sides to recognize one other and initiate the infection thread [44]. Therefore, the successful production of these chemical signals is crucial for nodulation. These signals are biosynthesized by specific genes; for example, the early nodulation signaling genes such as GmNINa, GmNINb, GmNRF5, GmDMI2a, GmDMI2b, GmNSP2a, GmNSP2b, GmNSP1a, GmNSP1b, GmDMI3a and GmDMI3b and SL biosynthetic genes such as GmMAX3, Gm-MAX1a, GmMAX1b, GmMAX2, GmMAX4a, and GmMAX4b. These previous genes are well-known to control the biosynthesis of these chemical signals. At 10-DAI, the chosen seventeen early nodulation signaling, and SL biosynthetic genes were expressed differently in the transgenic hairy roots ( Figure 5). For example, the expression levels of GmNSP2a, GmNSP1a, GmMAX1a, and GmMAX2, were highest in hairy roots overexpressing SgGPS. GmMAX1b and GmMAX4a transcription levels were markedly increased in hairy roots overexpressing GmFDPS, while the highest expression levels for GmNINa, GmNINb, Gm-MAX3, and GmMAX4b were observed in hairy roots overexpressing SgFPPS. Moreover, GmNRF5, GmDMI2a, GmNSP2b, GmDMI3a, and GmDMI3b were at the highest expression levels in hairy roots overexpressing SgLINS ( Figure 5). Additionally, the impact of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes overexpression on the expression of nodulation signaling genes and SLs biosynthesis in nodules at 10 DAI by B. japonicum, were investigated to determine whether it plays a role during rhizobial infection, early phases of nodule formation, and nodule growth.
The results show that the previously mentioned list of selected genes was differently expressed in nodules at 10 DAI ( Figure 6). Intriguingly, the expression of GmNSP2b and GmMAX4a were highest in nodules overexpressing GmFDPS. On the other hand, GmNINb and GmDMI2a transcription levels were markedly increased in nodules by overexpressing GmGGPPS. Moreover, the highest expression levels for GmNRF5, GmNSP2a, GmNSP1a, GmNSP1b, GmDMI3a, GmDMI3b, and GmMAX3 were observed in nodules overexpressing SgGPS. Additionally, GmMAX4b expression was highest in nodules overexpressing SgFPPS ( Figure 6). The effect of the GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS transgene on these genes' expressions in the transgenic hairy roots and nodules after 10 days of B. Japonicum infection, concludes the involvement of our genes in nodule formation. This finding suggests that tepene synthese genes perform important functions during nodulation signaling and early nodule development by controlling the transcription of the main genes responsible for nodulation.

Relative Expression Analysis of Nodulation and Strigolactone Biosynthesis Genes in Transgenic Soybean Hairy Roots at 20 DAI by B. japonicum
Investigating the impact of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes overexpression on the expression of nodulation signaling and SLs biosynthesis genes in soybean hairy roots and nodules at 20 DAI may better understand the long-term influence on root and nodule development. Therefore, the expression analysis of the selected genes involved in nodulation signaling and SLs biosynthesis were analyzed by qRT-PCR.The results showed that the nodulation signaling, and SLs biosynthesis genes were upregulated in hairy roots and nodules at 20 DAI (Figures 7 and 8). For example, the expression of GmDMI3a was highest in hairy roots overexpressing GmFDPS, while the highest level of GmNSP2b transcription was observed in hairy roots overexpressing GmGGPPS. Moreover, the expression levels of GmNINb, GmNSP2a, GmMAX3, GmMAX1b and GmMAX2 were highest in hairy roots overexpressing SgGPS. Furthermore, GmNINa expression was highest in hairy roots overexpressing SgFPPS (Figure 7). In addition, expression of Gm-NRF5, GmDMI2a, GmNSP2a, GmMAX1a, GmMAX1b, GmMAX2, and GmMAX4a were the highest in nodules overexpressing GmFDPS. The highest expression levels for GmMAX3, GmMAX4b, GmNINb, GmNSP1a, GmDMI2b, GmDMI3a, and GmDMI3b were observed in nodules overexpressing GmGGPPS. Besides, GmNINa expression was highest in nodules overexpressing SgGPS, while GmNSP2b transcription was highest in nodules overexpressing SgFPPS (Figure 8). Consequently, these data indicate that the terpenoid biosynthesis genes expression orchestrates nodulation signaling and SL biosynthesis genes in hairy roots and nodules at 20 DAI. The results show that the previously mentioned list of selected genes was differently expressed in nodules at 10 DAI ( Figure 6). Intriguingly, the expression of GmNSP2b and GmMAX4a were highest in nodules overexpressing GmFDPS. On the other hand, GmN-
Phylogenetic analysis showed that the close homolog to GmFDPS, GmGGPPS, Sg-GPS, SgFPPS, and SgLINS from G. max and S. guaranitica were Glyma.02G059000.1, Glyma. 19G144800.1, Glyma.05G100400.4, and Glyma.15G121400.1, respectively ( Figure S1). To identify GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS biological functions, their expression patterns in nine different tissues based on their increased resemblance to genes from G. max were identified and predicted. The results show that their constitutively expressed for all of these genes are mostly expressed in (root_hairs, root and nodules) ( Figure S2). Forexample, the expression levels of Glyma.02G059000.1, Glyma.05G100400.4, and Glyma.15G121400.1 genes were highest in all the nine tissues, especially in nodules, root, and root_hairs. Therefore, their expression patterns are similar to other orthologous putative terpenoid genes Glyma.07G073800.2, Glyma.03G014300.1 and Glyma.07G074600.2 from cultivated soybean ( Figure S2) [6]. Moreover, putative subcellular localization studies based on Arabidopsis protein localization for recognized synthesis sites from the Cell eFP database revealed that the GmFDPS, GmG-GPPS, SgGPS, SgFPPS, and SgLINS genes are presents mainly in the cytosol, mitochondrion, nucleus, and plastid ( Figure S3). These in silico results align withearlier studies that exhibited all organelles such as cytosol, mitochondrion, nucleus, and plastid can be considered as main loci for terpenoids synthesis and activity [6,[8][9][10]. The putative expression patterns and subcellular localization of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS underscore the possible roles of terpenoids in yielding terpene found at infection sites and during infection to attract rhizobia and establish nodulation [6]. Therefore, cloning the full-length cDNA of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS and examining their roles in soybean root and nodule development through overexpressing in hairy root systemsis crucial to proving this hypothesis ( Figures 1A and 3A-H). The results demonstrated that this gene plays a significant role in promoting root and nodule growth parameters compared with the GUS control in transgenic G. max hairy roots ( Figure 3A-H). Terpenoids and their derivatives have been shown to operate in the root nodules of G. max to enhance legume nodulation.
Ahmad et al. [2] reported that the overexpression of GmMAX2 in the G. max hairy roots system enhances the expression of early nodulation genes such as DMI2α, DMI3α, NSP2β, NSP1α, NFR5α, and NFR1α, but compromised in GmMAX2 knockdown compared with the control. In addition, hormones such as strigolactones and brassinosteroids are likely to autoregulate nodulationand maintain meristematic activity during nodule development [24,25,27,55,74,75]. In context to that, Ali et al. [6] found that the overexpression of SoCINS, SoNEOD, SoSABS, SoLINS, SoGPS and SoTPS6 from S. officinalis in the G. max hairy roots system enhances the expression of the most nodulation signaling and SL synthesis genes compared with the control. Generally speaking, our findings support that the GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS from G. max and S. guaranitica enhances the transcription of nodulation signaling and SL biosynthesis genes. On the other hand, root growth and nodulation showed different phenotypic plasticity when grow under the same conditions, and the reason behind this phenotypic plasticity may be due to the control of nodulation signaling and SL synthesis genes whose expression is modulated by the overexpression of GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS genes [76]. Therefore, these data highlighting GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS roles in legume root growth and nodulation are valuable if we harness its benefits to increase legume nodulation, growth, and productivity. In context, the terpenoid genes that we have identified can be used in a gene-editing experiment to augment the value of nodule numbers, fresh weight of nodules, root, and root length [77][78][79][80]. Finally, terpenoid genes donated from wild plant species or landraces plants can be introduced into other cultivated elite lines, for the development ofroot development, and nodulation in soybean and other leguminous plants [77][78][79][80].

Conclusions
In summary, this study focuses on cloning GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS from G. max and S. guaranitica, over-expressing them in hairy root systems of the cultivated soybean (G. max), and assessing the root growth characters and nodulation as has been affected by the transgenic. Transgenic cultivated soybean overexpressing GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS presented meaningful changes in root development, nodulation, and the expression levels of nodulation signaling and SL biosynthesis genes. In silico tools and the putative expression analysis were employed to predict GmFDPS, GmGGPPS, SgGPS, SgFPPS,and SgLINS functions in root and nodule development. Our data affirm that the GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS promote root development and nodulation signaling by activating nodulation signaling and SL synthetic genes. As a result, this study provides a clearer vision of the function of the GmFDPS, GmGGPPS, SgGPS, SgFPPS, and SgLINS in root development and nodulation, in conjunction with nodulation signaling and SL biosynthetic genes that are critical for legume nodulation production.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells11172622/s1, Figure S1: Phylogenetic tree of terpenoid genes from S. guaranitica with selected terpenoid genes from G. max plants. The MEGA6 program was used for the alignment of terpenoid genes through neighbor joining method with bootstrap values based on 1000 replicates; Figure S2: Heat maps representation the putative transcript levels of terpenoid genes from G. max at nine tissues (pod, leaves, root_hairs, root, nodules, seed, sam, stem and flower) from phytozome database (phytozome.jgi.doe.gov/). Green/red color-coded heat maps represent relative transcript levels of different terpenoid and terpene synthases genes in G. max, that were determined by alignment of terpenoid genes protein sequences from S. guaranitica with Glycine max genomic sequences database. MeV: Multi Experiment Viewer software was used to depict transcript levels; Figure S3: Putative subcellular localisations of terpenoid genes based on Arabidopsis protein localization at different cell organs. Cell sub-cellular localisations profile images were built using Cell ElectronicFluorescent Pictograph Browsers (Cell eFP browsers. The blue arrow points the expression scale (the more intense red color, the more gene expression), http://bar.utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi; Table S1: List of Glycine max and S. guaranitica genes and primer pairs used for full-length terpene synthases cDNAs clones; Table S2: List of Glycine max and S. guaranitica genes and primer pairs used for qRT-PCR; Table S3: List of Glycine max genes involved in nodules biosynthesis and signaling pathway and primer pairs used for qRT-PCR.

Institutional Review Board Statement:
This study did not involve humans or animals.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.