Transcription factors TgbHLH95 and TgbZIP44 cotarget terpene biosynthesis geneTgGPPSinTorreya grandisnuts

Abstract

Terpenes are volatile compounds responsible for aroma and the postharvest quality of commercially important xiangfei (Torreya grandis) nuts, and there is interest in understanding the regulation of their biosynthesis. Here, a transcriptomics analysis of xiangfei nuts after harvest identified 156 genes associated with the terpenoid metabolic pathway. A geranyl diphosphate (GPP) synthase (TgGPPS) involved in production of the monoterpene precursor GPP was targeted for functional characterization, and its transcript levels positively correlated with terpene levels. Furthermore, transient overexpression of TgGPPS in tobacco (Nicotiana tabacum) leaves or tomato (Solanum lycopersicum) fruit led to monoterpene accumulation. Analysis of differentially expressed transcription factors identified one basic helix-loop-helix protein (TgbHLH95) and one basic leucine zipper protein (TgbZIP44) as potential TgGPPS regulators. TgbHLH95 showed significant transactivation of the TgGPPS promoter, and its transient overexpression in tobacco leaves led to monoterpene accumulation, whereas TgbZIP44 directly bound to an ACGT-containing element in the TgGPPS promoter, as determined by yeast one-hybrid test and electrophoretic mobility shift assay. Bimolecular fluorescence complementation, firefly luciferase complementation imaging, co-immunoprecipitation and GST pull-down assays confirmed a direct protein-protein interaction between TgbHLH95 and TgbZIP44 in vivo and in vitro, and in combination these proteins induced the TgGPPS promoter up to 4.7-fold in transactivation assays. These results indicate that a TgbHLH95/TgbZIP44 complex activates the TgGPPS promoter and up-regulates terpene biosynthesis in xiangfei nuts after harvest, thereby contributing to its aroma.

Gas Chromatography-Mass Spectrometry Metabolites and Transcriptome Profiling Reveal Molecular Mechanisms and Differences in Terpene Biosynthesis in Two Torrya grandis Cultivars during Postharvest Ripening

“…The results revealed that aroma compounds were mainly classified into seven major groups, including terpenes, alcohols, aldehydes, esters, oxygenated terpenes, alkanes, and ketones, with terpenes being the most abundant, accounting for 85.19–93.90% in ‘Xi Fei’ ( Figure 2 A) and 67.75–93.24% in ‘Xiangya Fei’ ( Figure 2 B), suggesting that terpenes are the main aroma-presenting substances during the postharvest ripening process of T. grandis . We previously explored changes in the aroma composition of ‘Xi Fei’ at 20 °C and a 90% RH environment during postharvest ripening, revealing that terpenes accounted for 56–87%, and that the terpene content increased significantly on days 4 and 8 [ 36 ]. Our current results showed that the total terpene content in both cvs.…”

“…The screened nuts were mixed, divided into three groups each of 5 kg, and then placed at 25 ± 2 °C and in a 90% RH ± 2% RH environment for postharvest ripening treatment [ 35 ]. Generally, the postharvest ripening process of Torreya grandis nuts usually takes a period of 10–15 days under appropriate temperature and humidity conditions [ 36 ]. Therefore, the nuts were then sampled on days 0, 5, 10, 15, and 20.…”

Genome and Transcriptome Analysis of the Torreya grandis WRKY Gene Family during Seed Development

“…Collection of the seeds occurred monthly from May to September. Subsequently, the seeds were carefully de-scaled using a manual peeling method based on established protocols for T. grandis seed kernels [ 26 , 27 , 28 ], ensuring clean and intact seeds, then rapidly frozen in liquid nitrogen and preserved at −80 °C for subsequent analyses.…”

Genome-wide identification of GA2ox genes family and analysis of PbrGA2ox1-mediated enhanced chlorophyll accumulation by promoting chloroplast development in pear

“…To induce transient overexpression in pear leaves, the full-length PbrGA2ox1 gene without the stop codon was amplified using the primer pair PbrGA2ox1 -EGFP-F/R (Table S5 in Additional File 2 ) and framed into the p1300-EGFP vector digested at XbaI and BamHI sites to generate overexpression fusion constructs PbrGA2ox1 _OE with upstream CaMV 35S promoter, termed as 35S: PbrGA2ox1 in our previous work [ 30 ]. Then, the PbrGA2ox1 _OE fusion vector was transformed into the Agrobacterium tumefaciens GV3101-pSoup cells via thermal stimulation .…”

“…Previously, RNA-seq helped to reveal the fundamental determinants that contribute to interstock-induced dwarfism in sweet persimmon [ 26 ], and altitude-dependent potato tuber coloring [ 27 ]. Moreover, RNA-seq has also been widely utilized to derive novel insights into the functions of several genes, such as the SlWRKY35 -activated carotenoid biosynthesis in tomatoes [ 28 ], MdMYB94 -induced ester biosynthesis in apples [ 29 ], and TgbHLH95- and TgbZIP44 -targeted terpene biosynthesis in ‘xiang fei’ ( Torreya grandis ) nuts [ 30 ]. Thus, RNA-seq can help efficiently explore the potential mechanisms underlying phenotypic alterations arising from gain-of-function or loss-of-function of genes.…”