Changes in Metabolite Profiling and Expression Levels of Key Genes Involved in the Terpenoid Biosynthesis Pathway in Garden Sage (Salvia officinalis) under the Effect of Hydrazine Hydrate

Mutagenesis is a highly efficient tool for establishing genetic variation and is widely used for genetic enhancement in various plants. The key benefit of mutation breeding is the prospect of enhancing one or several characteristics of a variety without altering the genetic background. In this study, we exposed the seeds of Salvia officinalis to four concentrations of hydrazine hydrate (HZ), i.e., (0%, 0.1%, 0.2%, and 0.3%) for 6 h. The contents of terpenoid compounds in the S. officinalis plantlets driven from the HZ-treated seeds were determined by GC-MS, which resulted in the identification of a total of 340 phytochemical compounds; 163 (87.48%), 145 (84.49%), 65 (97.45%), and 62 (98.32%), from the four concentrations of HZ (0%, 0.1%, 0.2%, and 0.3%), respectively. Furthermore, we used the qRT-PCR system to disclose the “transcriptional control” for twelve TPS genes related to terpenoid and terpene biosynthesis, namely, SoGPS, SoMYRS, SoNEOD, SoCINS, SoSABS, SoLINS, SoFPPS, SoHUMS, SoTPS6, SoSQUS, SoGGPS, and SoGA2. Altogether, results are likely to ensure some positive relationship between the concentrations of the chemical mutagen HZ used for treating the seeds, the type and amount of the produced terpenes, and the expression of their corresponding genes.


Introduction
Plant improvement has been the cornerstone of the ever-growing human population's food security for many years. Despite the availability of large germplasm collections, crop development still depends on effective genetic diversity evaluation [1][2][3]. Furthermore, genetic diversity is essential for crop improvement and climate adaptation, especially in plants with low genetic diversity that are more susceptible to stresses [4][5][6][7][8][9]. Genetic variation is greatly influenced by the degree of DNA damage and the cell's capacity to repair it [10]. According to estimates made by McCulloch and Kunkel (2008) [11], each

Plant Material and Growth Conditions
Seeds of S. officinalis were pre-soaked in distilled water for 12 h, then soaked in four different concentrations (0%, 0.1%, 0.2%, and 0.3%) of HZ solution for 6 h by placing seeds in 10 mm × 100 mm petri plates (about 60 seeds per plate in a single layer), as described by [34][35][36]. The seeds from each treatment were then washed with distilled water three times to remove the traces of the HZ solution. The S. officinalis seeds were then surface sterilized with 75% (v/v) ethanol for 1:30 min and then in 2.5% (v/v) sodium hypochlorite solution for 12 min, thoroughly washed three times with sterilized distilled water, and sown in solid Murashige and Skoog (MS) [37] with pH 5.8 medium containing 30 g L −1 sucrose and 2.5 g L −1 phytagel. The seeds were incubated in the dark for three days then grown at 23 • C under a photoperiod of 8 h dark and 16 h light (110 µmol m −2 s −1 ) in a controlled growth chamber until the plantlets were grown for 6 weeks. After sowing, the percentage of surviving seedlings (survival rate, SR) was investigated.

RNA Extraction and cDNA Library Preparation
The total RNAs from the three biological plantlets replicates from each S. officinalis line (three plantlets from each treatment) were extracted using the plant TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. RNA purity was analyzed using a Nano-Photometer ® spectrophotometer (IMPLEN, CA, USA), and the quality was examined on 1.4% agarose gels as described previously [23,24,31,38,39]. For quantitative RT-PCR, the first strand of complementary DNA (cDNA) was synthesized from 1 µg total RNA, which was previously treated with DNase I (Takara), using an M-Malva Reverse Transcriptase (RNase H) kit, according to the manufacturer's protocol. The second strand of cDNA was synthesized in the presence of DNA Polymerase I and Rnase-H, as described previously [23,24,31,38,39].

Metabolite Extraction from S. officinalis Plantlet after Treatment with Different Concentrationsof HZ
For extraction and analysis of terpenoid compounds from the non-treated (control) and treated S. officinalis plantlets under different concentrations from hydrazine hydrate, we followed the procedures described by Ali et al., 2017Ali et al., , 2018Ali et al., , 2021Ali et al., , 2022Ali et al., , 2022Ali et al., , 2022. In short, the plantlets from each S. officinalis line (three plantlets from each treatment) were collected. Then all collected samples from non-treated (control) and treated S. officinalis plantlets were homogenized in liquid nitrogen and the powder was directly soaked in n-hexane in 60 mL bottles. Then, the bottles were incubated with shaking at 36 • C and 210 rpm for 73 h. Afterward, the solvent was purified using a centrifuge at 5050 rpm for 9 min at 5 • C to remove plant debris. The extract was concentrated and transferred to fresh 1.5 mL crimp neck vial amber glass screw-top vials. The terpenoid content in the extract solution was determined by gas chromatography-mass spectrometer (GC-MS: Shimadzu model GCMS-QP2010 Ultra (Tokyo, Japan) system). Three libraries: NIST Library (2014 edition), Volatile Organic Compounds (VOC) Analysis S/W software, and Wiley GC/MS Library (10th Edition), were used to identify the terpenoid constituents by parallel comparison of terpenoid recorded mass spectra with the data that stored in these previous Libraries [23,24,31,38,39]. All of the experiments were performed simultaneously three times under the same conditions for each isolation technique, with a total GC running time of 80 min.

Overexpressing Terpenoid and Terpene Biosynthesis Genes under the Effect of Different Concentrations of HZ
To unveil the effects of different concentrations of HZ on various terpenoid and terpene genes expression, we used the qRT-PCR system to measure the level of expression. The results showed that the expression patterns of our candidate genes at different concentrations of HZ (e.g., 0.1%, 0.2%, and 0.3%) were detected, and their expression profiles were compared with the control (0.0% HZ) (Figure 3). For example, the expression levels of SoFPPS and SoHUMS were the highest under the effect of HZ at 0.0% concentration. Moreover, the highest expression levels for SoMYRS, SoSABS, SoSQUS, and SoGGPS were observed at a concentration of 0.1% of HZ. On the other hand, the expression of SoGPS, SoCINS, and SoLINS were the highest in HZ at a concentration of 0.2%. Furthermore, the highest expression levels for SoNEOD, SoTPS6, and SoGA2 were detected in a concentration of 0.3% HZ (Figure 3). Therefore, different concentrations of HZ may have an impact on the level of gene expression for genes involved in terpenoid and terpene biosynthesis, according to differences in expression patterns for these genes.

Validation of the Relationship between the Type and Amount of Terpenoid and Gene Expression under Different Concentrations of HZ
We used the qRT-PCR system to reveal the "transcriptional control", which represents the natural link between the "number of mRNA copies" and "the number of copies of enzyme", which follow the end-product quantity, in order to ascertain the relationship between the type and amount of terpenoid that was produced under the effect of different concentrations of HZ and gene expression. Therefore, the expression patterns of our twelve candidate genes with various expression levels were detected, and their quantity expression profiles were compared with our GC-MS analysis data. Therefore, according to the gene's expression levels and the findings of our GC-MS analysis, we found that the SoGPS gene showed the highest expression levels at 0.2% concentration, followed by 0.3%, 0.1%, and 0.0% of HZ, and these qRT-PCR results are in line with our GC-MS analysis data, which indicate that the main group of monoterpene compounds was observed at 0.2% concentration, followed by 0.3%, 0.0%, and 0.1% of HZ. Moreover, the highest expression levels for the SoFPPS2 gene were observed at a concentration of 0.0% of HZ, followed by 0.2%, 0.1%, and 0.3%. In this context, the most abundant sesquiterpene compounds group was detected at a concentration of 0.0% of HZ, followed by 0.1%, 0.2%, and 0.3%.
Additionally, we discovered a favorable link between the gene expression levels of 1, 8-cineole synthase at various concentrations of HZ. For instance, the higher of the 1, 8-cineole synthase gene product and expression level presented at 0.2% concentration, followed by 0.3%, 0.1%, and 0.0%of HZ. In addition, we found an engagement between the (+)-germacrene-D product and germacrene-D-synthase (SoTPS-6):the highest product and gene expression were discovered at a concentration of 0.3% of HZ, followed by 0.2%, 0.1%, and 0.0%. Likewise, there is a linkage detected between the (E)-β-caryophyllene, gamma-caryophyllene, humulene, caryophyllene oxide, and 1,2-humulene epoxide as a product and the expression of humulene synthase (SoHUM) gene at different concentrations of HZ. Our findings concur with those of other researchers and our earlier research [23,24,31,[38][39][40][41][42][43][44][45][46][47], which discovered and discussed a connection between gene expression and the end product, which gives the impression that the production of the terpene compounds under study can be controlled by the gene transcription process.
On the other hand, we discovered that some genes (e.g., SoMYRS, SoSABS, SoSQUS, SoNEOD, SoLINS, and SoGA2) fluctuate in their gene expression level, and also, some terpene compounds (e.g., camphene, geranylisobutyrate, cajeputol, Cis-β-terpineo, thujan-3-one, thujone, l-2-camphanol, ledene, elemene, labda-8(20),14-dien-13-ol, (13R)-, and labda-8(20),14-dien-13-ol, (13R)-) were not detected or were detected in quantities that are not commensurate with the expression levels of their genes under the influence of some concentrations of the HZ. There are numerous explanations for this. First, the expression of some terpene synthase genes may be controlled by the cell's circadian rhythm. Secondly, the fact that there is a positive correlation between transcript levels and terpene emission which suggests that changes in transcript level are an important determinant of terpene production, even though changes in transcript levels might not directly affect protein levels or enzyme activities due to potential posttranscriptional, post-translational, or enzyme-regulatory mechanisms. Third, the reason why some terpene compounds do not appear may be due to the different rates of protein synthesis, protein modifications, protein degradation, and protein proteolytic turnover. Additionally, by converting some compounds to other compounds through oxidation or glycosylation of monoterpene olefins and sequestration [23,24,48,49]

Assessment of the Effects of Hydrazine Hydrate on the Terpene Genes Expression and Terpenoid Production
Hydrazine hydrate (N 2 H 4 × H 2 O) is an important inorganic compound, which is mainly used in agrochemicals as a foaming agent [50]. Hydrazine hydrate is considered as one of the alkylating agents (e.g., ethyl methane sulphonate (EMS), ethyleneimides, alkyl methane sulphonates, sulfur mustards, methyl methane sulfonate, epoxides, and alkyl nitrosoureas), which can be used as chemical mutagens [13,14]. Chemical mutagens have been widely used to alter plants' genetic makeup in various ways, such as: changing the chemistry of the base pairs; nucleotides, and confusing the DNA replication machinery; stripping DNA nucleotides from the essential modifications; in sertion or deletion of some extra base pairs through a round of DNA replication; and two DNA nucleotides cross-linking together, introducing a single base pair (SNPs) [51]. Moreover, the chemical mutagens are usually high in induction and more applicable in the in-vitro compared to physical and radiation approaches [52][53][54][55]. Cells generally attempt to fix these mutations via the cell cycle checkpoints by nucleotide excision repair (NER) and base excision repair (BER) for the eradication of damaged bases and the repair of nucleotides, respectively [52]. Furthermore, in some cases, the disruption of the DNA nucleotide mutation repair mechanism could create mutations in the genetic makeup, which can alter the gene expression and encoding protein and create genetic variability for increased crop productivity through crop improvements, such as that seen in Mung bean (Phaseolus aureus Roxb.) [51,56]; for example, EMS, which belongs to the alkylating agents, and is used as a chemical mutagen in plants. EMS has the ability to induce GC → AT transitions in the genomic DNA, which results in mutant proteins that perform alternate roles to those of the normal protein [51]. Additionally, it has been demonstrated that the application of alkylating agents such as EMS and HZ is a practical and efficient way to develop distinctive gene pools in plants [34][35][36]51,56]. Therefore, we can assume that the alkylating compounds are the ones that are used the most commonly use dto cause point mutations, a sort of genetic modification in which only one nucleotide base from an organism's DNA or RNA sequence is changed, added, or removed [57]. According to the type of point mutation, these changes can have a variety of effects on protein function, composition, and synthesis, ranging from positive effects (synonymous mutations) to negative effects (nonsynonymous mutations) [58,59]. In this context, these mutations can lead to different effects on the differential protein expression levels, such as removing or adding stop codon, which causes the translated protein to be abnormally extended or shortened, change in chemical and physical properties of the amino acids, protein may lose its function and may exhibit a new function or become activated [60,61]. For example, in Yokoyama et al.'s 2022, 2021 study on the stability of mutations in aromatic amino acid (AAAs) compounds from A. thaliana seeds, that were mutagenized using EMS [62,63], they isolated a total of 351 suppressor of tyra2 (sota) mutants that lacked one of two TyrA genes that were associated with Tyr biosynthesis-used as a common substrate for the shikimate pathway [62]. This kind of mutant showed an increase in AAAs compared with other amino acids at F1 and F2 of the plant population with dominant or semidominant characteristics, and was accompanied by an increase in net CO 2 assimilation and flux through the shikimate pathway [62][63][64][65]. These results provide genetic evidence that the induction of point mutations using chemical mutagens has the ability to enhance plant metabolic levels in a dominant fashion.

Conclusions
The application of chemical mutagens on crops is an easy and effective method for the improvement of various agronomic traits. Seeds from many different plants have been widely used in investigations as the initial materials to produce plant mutations via chemical mutagenesis. There have been fewer studies about the effect of chemical mutagens on medicinal plants because it is difficult to determine the biological and molecular genetic effects of a chemical mutagen on the seeds and plantlets of these plants. This investigation was carried out in order to comprehend the potential impacts of HZ on S. officinalis terpenoids and terpene synthesis genes. The goal of the study was to shed light on the effects of four HZ concentrations on the expression levels of the genes involved in terpene synthesis, as well as terpene and terpenoid biosynthesis. The acquired results showed that varied HZ concentrations considerably raised the percentage of most monoterpenes, sesquiterpenes, and diterpenes while increasing the expression levels of twelve terpene genes. The findings of our study open the door to additional research on the use of chemical mutagens to improve a variety of features in medicinal and aromatic plants without changing their genetic makeup. This study might be widely applicable toother salvia species or other genera that principally belong to the Lamiaceae family plants.