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

Abstract

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.

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

“…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.…”

“…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 ].…”

“…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, 2017, 2018, 2021, 2022, 2022, 2022 [ 23 , 24 , 31 , 38 , 39 ]. In short, the plantlets from each S. officinalis line (three plantlets from each treatment) were collected.…”

“…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 of80 min.…”

“…The values are means ± SE of the three replicates normalized using SoActin as a reference gene. The relative expression levels were calculated by comparing the cycle thresholds (CTs) of the target genes with that of the reference gene SoActin using the 2 −ΔΔCt method [ 23 , 24 , 31 , 38 , 39 ]. The sizes of amplification products were 150–161 bp.…”