Although the differentiation and variations between the Opisthopappus species were performed using diverse molecular markers [28, 30, 32-34], the availability of data on the molecular mechanisms of evolution and adaptation remained generally limited. To reveal the molecular mechanisms of the two Opisthopappus species associated with their surroundings, comparative transcriptome analysis was performed, which provided some clues for an improved elucidation of major differences under natural selection.
Adaptive characteristics of TFs
Transcription factors (TFs) are significant key elements of various physiological and biochemical pathways in higher plants. The activation and repression of TFs can primarily regulate the expression of genes related to growth, stress responses of plants, and other developmental processes [53, 40]. The activities of TFs often depend on the developmental stage, exogenous stimuli, and/or the presence of co-regulatory proteins. Various TFs are employed for the regulation of the expression of different genes. Thus, TF-based gene expression regulation allows plants to respond to changes in their environments [54, 40].
Based on transcriptome data, we initially obtained 406 TFs (Table 1). The bHLH, FAR1, NAC, MYB, and WRKY family members were highest among the obtained factors. These factors were found to be significantly expressed for lipid, secondary, amino acid, and nucleotide metabolism, which constituted the main biological processes. The responses of plants to various environments are pertinent for successful growth and reproduction . The proactive responses to environmental/developmental cues were explicitly depicted in O. taihangensis, as many TFs were involved in the amino acid metabolism pathways (Figure 1).
In particular, for our study, O. longilobus had a higher expression level in the terpenoid metabolic pathway than did O. taihangensis (Figure 5). Terpenoids are one of the most common compounds in the secondary metabolites of plants, which have numerous physiological and ecological functions, such as attracting pollinated insects, regulating growth and development, resisting environmental stress, and participating in the defense of pests . Meanwhile terpenoids are important components of the fragrances of flowers. For the two species of Opisthopappus under study, these secondary metabolites are primarily emitted by their leaves and flowers.
According to the Flora Reipublicae Popularis Sinicae (FRPS), the surfaces of O. longilobus leaves are smooth and glabrous, while O. taihangensis is sparsely pubescent on both leaf surfaces, which include glandular trichomes and non-glandular trichomes . The terpenoids produced in O. taihangensis can be released through glandular trichomes give rise to the unique aroma for communication and defense . For O. longilobus, owing to the lack of glandular trichomes on the surfaces of its leaves, terpenoid biosynthesis genes are more highly expressed to compensate for this deficit through the use of alternate emission strategies.
Basic helix-loop-helix (bHLH) transcription factors, which are one of the largest families and a large superfamily in plants, play relevant roles in a variety of developmental and evolutionarily conserved processes, including cell-fate specification, tissue differentiation, and secondary metabolites [56, 57, 24]. For example, the bHLH factor VvMYC1 can regulate anthocyanin and/or proanthocyanidin (PA) synthesis of the flavonoid pathway . In the terpenoid biosynthesis pathway of Opisthopappus, the involved TFs are all bHLH family members (Figure 5). Several bHLH TFs have been found in Medicago truncatula , Chenopodium quinoa , Panax notoginseng , and Panax ginseng . Meanwhile, many bHLH genes respond to various forms of stress such as drought, salt, and cold stresses [59, 53, 63]. Other TFs (e.g., NAC, MYB, and WRKY) have been investigated for their capacities to improve tolerance and resistance in many plants [64-68], while participating in abiotic and biotic stress responses [69 , 70,17]. Both bHLH and MYB can modulate secondary metabolism biosynthesis in plants that are subject to environmental stress. This suggested that TFs (particularly bHLH family members) might play critical roles in the responses of the two Opisthopappus species to heterogeneous environments.
Adaptability of PSGs
To describe genome-wide levels of coding sequence evolution and estimate the effects of natural selection on lineage divergence, we calculated the dN/dS ratios for O. longilobus and O. taihangensis orthologs. This ratio has frequently been employed as an indicator of frequency and selection modes under which protein-coding genes evolved . In this study, the average dN/dS ratios across the pairs of the Opisthopappus genus were much lower than 0.9, which suggested that purifying selection had a general influence on the evolution of most protein-coding regions (ORF) of the two species, as has been observed in other plants [71, 16]. Actually, the genes that were most under the influence of purifying selection primarily contained structural or “housekeeping” genes, for example tyrosyl-tRNA synthetase. As these genes are involved in processes that are crucial for organisms, purifying selection serves to eliminate deleterious, nonsynonymous mutations [72, 73, 16]. Among the 38986 orthologs shared between lineages, 1203 gene pairs significantly exhibited dN/dS ratios >1.5 (P < 0.05) (Figure 2A, 2B). These genes were involved in several biological functions (e.g., metabolic processes) and may constitute candidates that are under the effect of positive selection; thus, potentially associated with species divergence.
Adaptive divergence at molecular level may be reflected by an increased rate of non-synonymous changes within genes involved in adaptation . Analyses of the PSGs identified from O. taihangensis and O. longilobus yielded significantly different landscapes of biological processes, cellular component and molecular function (Figure 3). O. taihangensis and O. longilobus would experience divergent adaptation during their evolution, and that of genes related to adaptation was under rapid evolution and/or have signs of positive selection. These findings implicated that Opisthopappus were experienced the ongoing accelerated evolution under different environment. Thus, more than 1000 genes under positive selection between the two Opisthopappus species might have played a role in shaping the divergence of this genus, given that O. longilobus and O. taihangensis are exposed to sub-humid warm temperate, and temperate continental monsoon climates, respectively.
Subsequently, KEGG enrichment and MapMan analyses suggested that significant GO terms, mainly related to metabolic regulation (Figure 3B), were important among the Opisthopappus species (Figure 4). Secondary metabolism was also significantly represented for O. longilobus and O. taihangensis in our PSG analysis (Figure 5).
Beta-amylase, a member of family 14 of glycosyl hydrolases , can hydrolyzes the α-1,4-glucosidic linkages in starch, removing successive maltose units from the non-reducing ends of the chains . Starch is the main form of carbon storage in higher plants and it accumulates in different organs, which can be consumed by the cell in which they are produced, transported to nonphotosynthetic sink tissues, or stored for later use. According to studies using transgenic and mutant plants, beta-amylase seems to be important for normal degradation of the transient starch accumulated in chloroplasts . Meanwhile, beta-amylases possibly facilitating the rapid starch degradation under heat and drought . In this study, we found that the PSG expression levels involved in beta-amylase for O. longilobus was significantly higher than those for O. taihangensis (Figure 4A). Starch degradation as a major response in conditions prohibiting photosynthesis or prolonged drought , therefore, O. longilobus seems better adapted to weak light or drought conditions than O. taihangensis.
Beta-amylase can convert starch into maltose and glucose as the predominant form in which sugars are translocated in plants presented in Figure 4C. Consistent with this notion, O. longilobus exhibited higher levels of cellular glucose and fructose than did O. taihangensis. Collectively, these data signified that the higher expression levels of beta-amylase genes in O. longilobus might accelerate its cell growth processes (in contrast to O. taihangensis), which have important roles in the evolution of the two species. Ultimately, glycan degradation resulted in the accumulation of sucrose, which is critical for the protection of plants against xenobiotic and oxidative stresses , and which might facilitate the enhanced survival of O. longilobus for relatively more drought habitats over than O. taihangensis.
The electron transport chain (ETC, respiratory chain) is a series of protein complexes that transfer electrons from electron donors to electron acceptors via redox reactions (both reduction and oxidation occurring simultaneously) and couples this electron transfer with the transfer of protons (H+ ions) across a membrane. The electron transport chain is built up of peptides, enzymes, and other molecules. In this study, the expression levels of relative PSGs involved in complex I (NADH ubiquinone oxireductase) and complex IV (cytochrome c oxidase, EC 18.104.22.168) for O. longilobus was higher than those for O. taihangensis (Figure 4B). The ETC sustains the major mitochondrial function of ATP generation, in relation to the metabolic dynamics of tricarboxylic acids (TCAs), acetyl-CoA, ADP, oxidized (NAD+) or reduced (NADH) β-nicotinamide adenine dinucleotide, oxidized (FAD) or reduced (FADH2) flavin adenine dinucleotide . Thus, we concluded that the changes in ETC can affect nicotinate and nicotinamide metabolism, glycan degradation metabolism and transmembrane transport activity, which was supported by the KEGG and MapMan results (Figures 3 and 4). The various expression levels of relative PSGs between these two species were also regarded as different adaption signals to habitats.
Additionally, the responses to sulfur between O. longilobus and O. taihangensis were significantly different, which suggested that the two species possessed different stress tolerances for S in response to heterogeneous environments.
Adaptive function of Terpenoids
Secondary metabolism produces large populations of specialized molecules that are required for plants to survive in their environments and are essential for communicating with other organisms in a mutualistic (e.g., to attract beneficial organisms such as pollinators), or antagonistic (e.g., to combat herbivores and pathogens) manner. Under baseline or non-stress conditions, it is expected that mutualistic metabolites, or those required for normal physiological processes, are expressed . This scenario, particularly in terms of terpenoid biosynthesis, was unambiguously observed in the present study as a difference between the two species TFs and PSGs (Figure 5).
The terpenoid pathway is intricately regulated by endogenous and environmental factors that enable spatially and temporally controlled metabolite production [79-81]. Terpenes are the major components of plant terpenoids, which comprise the largest and the most diverse class of natural products as a homologous series of molecules as isoprene polymers. They often play important roles in plant defenses by attracting the enemies or predators of herbivores and repelling herbivorous insect feeders .
Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the main intermediate compounds in the terpenoid pathway for terpene biosynthesis [83, 20]. Their precursor molecules (C5) are generated via the process of isoprenogenesis [84, 85]. In plants, isoprenogenesis occurs through two discrete biosynthetic pathways: the mevalonic acid (MVA) pathway in the cytosol, and the 2-C-methyl-D-erythritol 4-phosphate/1-deoxyD-xylulose 5-phosphate (MEP/DOXP) pathway within plastids. In the MEP pathways, the C5 units are catalyzed step by step via a series of enzymes to synthesize IPP. The 4-diphosphocytidyl-2C methyl-D-erythritol synthase (ISPD/CMS) and 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (ISPE/CMK) catalyze the synthesis of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDP-ME) and 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDP-ME2P), respectively. CDP-ME and CDP-ME2P are the intermediate compounds of IPP biosynthesis.
IPP or DMAPP may be linked through head to tail condensation reactions to generate terpenes of different classes (e.g., mono, di, and triterpenes), which are catalyzed by geranyl diphosphate synthase (GPPS) to synthesize geranyl pyrophosphate (GPP). The GPP results in farnesyl pyrophosphate (FPP) and geranylgeranyl diphosphate (GGPP) via farnesyl pyrophosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS) enzymes, respectively. GPP and GGPP are substrates for monoterpene and diterpene biosynthesis in the terpene pathway [86 -88].
Monoterpenes and diterpenes can impart the distinct flavors and aromas of plants [89, 20]. These terpenes are produced in plants as secondary bioactive metabolites, often for ecological adjustment and protection from microbial pathogens, fungi, pests, and predation . An overview of terpenoid metabolism of the two species revealed that the putative PSGs (trinity_dn129788_c0_g1 and trinity_dn155665_c0_g2) displayed different levels of expression for IPP biosynthesis, particularly in the synthesis of CDP-ME and CDP-ME2P (Figure 6).
Meanwhile, both the PSGs and TFs involved in the downstream steps of the MEP pathway exhibited a relatively higher expression in contrast to the two species under study, which displayed different expression levels for monoterpene and diterpene biosynthesis (Figure 6). These results indicated variable adaptability in the responses of the two species to different environments. During the evolutionary process, the genes involved in terpenoid biosynthesis pathway (in particular, monoterpene and diterpene biosynthesis), begun to diverge from their expression levels under different long-term environment stresses. Simultaneously, several bHLH TFs involved this metabolic process to further regulate the expression of these positive selection genes. Ultimately, the adaptive phenotypic characteristics occurred in O. longilobus and O. taihangensis, such as the presence of trichomes or not, and different fragrances.