Pinellia ternata is one of most important medicinal herbs in China, which makes it vital for genomic studies. With the transformation of cultivation areas and scarcity of wild PT resources, to ensure clinical efficacy, there is a growing need for the genetic characterization of wild and cultivated PT. Using transcriptomic evidence, we provided an in-depth view of the differences between wild and cultivated PT, which may explain the better clinical efficacy of wild PT. Among the DEGs screened in this experiment, compared with cultivated PT, wild PT had significantly more upregulated DEGs, which were involved in the synthesis and metabolic pathways of the active ingredients widely known to exist in PT (including amino acids, nucleosides, etc.).
Carbohydrate metabolism
Pinellia ternata deposits carbohydrates, particularly starch and polysaccharides. 15 key target DEGs involved in carbohydrate metabolism and pathways were found to be downreguated in PT from cultivated fields. These genes were fructokinase (ScrK), GDP-L-galactose phosphorylase (GGP), malate dehydrogenase (MDH), fructose-bisphosphate aldolase (ALDO), aconitate hydratase (ACO), L-ascorbate peroxidase (APX), beta-glucosidase (bglX), SPS, trehalose 6-phosphate synthase (TPS), beta-fructofuranosidase (Inv), mannose-1-phosphate guanylyltransferase (GMPP), catalase (CAT), βGlu, FFase and succinic semialdehyde reductase (GLYR). Five of these DEGs were found to be upregulated: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), glucose-1-phosphate adenylyltransferase (glgC), 1,4-alpha-glucan branching enzyme (glgB) and inositol 3-alpha-galactosyltransferase (GOLS).
Ascorbic acid (AsA) plays important roles as a major antioxidant and free-radical scavenger in plants. The L-galactose pathway is the main pathway for AsA biosynthesis in higher plants (LinsterC and ClarkeS 2008). As a key enzyme, GGP catalyzes GDP-L-galactose to L-galactose-1-phosphate, which is beneficial to the formation of AsA and thus responds to abiotic stress. In addition to GPX (downregulated) in cultivated PT), APX also plays a vital role in plant antioxidation and abiotic stress, including abiotic stress responses induced by light and temperature (Yoshimura et al. 2000; Kim et al. 2007). CAT has a high affinity for hydrogen peroxide in plant cells. It regulates the plant stress response mainly by removing hydrogen peroxide generated during mitochondrial electron transfer and fatty acid oxidation (Jiang et al. 2002; Xing et al. 2007).
We also found that the DEGs involved in the metabolism of glutathione (GSH) and related to the plant stress response were also upregulated in wild PT, including GPX, GST, glutathione reductase (GR), and aminopeptidase N (pepN).
GSH is one of the main sulfur reducers in living organisms and can be resistant to stress, such as cold damage and drought (Grill 1978; Schupp and Rennenberg, 1988). It combines the electrophilic groups of certain harmful substances with the thiol group of reduced glutathione to form more soluble and nontoxic derivatives of these substances (Coleman 1997). Increased GST activity protects plants from stresses, for example, from organic pollutants and heavy metals (Kirchhoff and Pflugmacher et al. 2000; Pflugmacher et al. 2007). GR deoxidizes oxidized glutathione disulfide to reduced glutathione, which helps to eliminate reactive oxygen species (ROS) and participates in the plant ascorbic acid GSH cycle. Therefore, wild PT may suffer more environmental stress, making its AsA biosynthesis and metabolic activity significantly stronger than those of cultivated PT.
bglX can hydrolyze β-glucoside and cellulose to glucose together with other cellulases; SPS and SPP recombine degraded sucrose for transport, storage and metabolism. The catalytic effect of SPS is irreversible. Huber (1983) pointed out that SPS activity is negatively correlated with starch accumulation but directly proportional to sucrose formation. Inv hydrolyzes sucrose into fructose and glucose, and fructose needs to be phosphorylated by ScrK or hexokinase before further metabolism. ScrK plays a major role in the metabolism and distribution of library tissues (David 2007), and it can be used as a hexose sensor and signal molecule to regulate the metabolism and growth of plants (Rolland et al. 2006). Trehalose is the most stable sugar of natural disaccharides catalyzed by TPS and TPP. Studies have shown that trehalose could enhance the resistance of plant cells to various abiotic stresses (Elbein et al. 2003). TPP has little effect on trehalose accumulation (Blázquez et al. 1998); thus, TPS may have a decisive effect on trehalose synthesis. It can be concluded that starch and sucrose metabolism in PT from wild fields was more active than in PT from cultivated fields.
Following starch and sucrose metabolism, genes become involved in glycolysis and the TCA cycle. ALDO catalyzes the conversion of fructose 1,6-diphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate during glycolysis. This reaction is reversible. GOLS and PK are upregulated genes in cultivated PT that generate 1,3-bisphosphoglycerate and pyruvate during first and second substrate level phosphorylation, respectively. PK is one of the most critical rate-limiting enzymes in glycolysis. MDH catalyzes the dehydrogenation reaction of malic acid to oxaloacetate, which is accompanied by the reduction of NAD to NADH that may improve the TCA cycle (Chang et al. 2013). ACO is an important iron-thioproteinase in plant cells. In the TCA cycle, ACO catalyzes the production of isocitrate from citric acid in the cell via the intermediate product acis. This indicated that the TCA cycle was stronger, while glycolysis was weaker in wild PT than in cultivated PT.
Amino acid metabolism
Amino acids are the basic units that make up proteins and the precursors for many metabolites that have multiple functions in plant growth and stress responses. Thirteen DEGs were involved in amino acid metabolism, including pyrroline-5-carboxylate reductase (P5CR), acetyl-CoA acyltransferase 1 (ACAA1), GDA, peroxidase (POX), amino-acid N-acetyltransferase (argAB), 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase (ADI1), GS, hydroxypyruvate reductase 1 (HPR1), glycine dehydrogenase (GDC), polyamine oxidase (PAO), primary amine oxidase (AOC), 3-methylcrotonyl-CoA carboxylase alpha subunit (MCCA), and L-pipecolate oxidase (PIPOX). Among these DEGs, 12 were upregulated and 1 was downregulated in PT from wild fields.
GS and GAD are two vital enzymes in the amino acid metabolism pathway. GS reduces the toxicity of excessive ammonium ions to plants by catalyzing the synthesis of glutamine. Glutamine is the main storage and transport form of ammonia. NH4+ participates in the process of glutamate formation through the glutamine synthetase/glutamic acid synthase cycle. As an osmotic regulator, proline is formed through the glutamic acid pathway and ornithine pathway and could improve the salt tolerance of plants. Nancy’s (Roosens et al. 1999) study proved that GS could promote the synthesis of proline. Glutamate-semialdehyde (GSA) synthesizes proline through the glutamate pathway under the catalysis of P5CR (Rocha et al. 2012). Many studies have shown that the overexpression of the P5CR gene can improve plant resistance to drought. For example, under salt stress, P5CR-overexpressing sweet potatoes have increased proline contents and enhanced salt tolerance via the regulation of osmotic pressure (Gut et al. 2009). GAD is a pyridoxal phosphate-dependent enzyme that catalyzes the decarboxylation of glutamic acid to γ-aminobutyric acid. GAD activity is mainly regulated by the combination of pH and calcium ion/calmodulin (CAM) (Gut et al. 2009). When exposed to environmental stimulation (hypoxia, low temperature, acid stimulation, high salt penetration, etc.), H+ and Ca2+ in the plant will increase significantly and promote GAD activity.
MCCA and carbamoyl phosphate synthetase belong to the same enzyme family that could convert the leucine intermediate metabolite 3-methylcrotonyl-CoA to 3-methylpentadienoyl-CoA (Wurtele and Nikolau 2000). In Arabidopsis thaliana, the gene numbered AT1G03090 encodes MCCA, a subunit of methylcrotonyl-CoA carboxylase, which is biotinylated to participate in the degradation of leucine and further affects plant development (Ding et al. 2012). In plants containing purine alkaloids, such as cocoa and coffee, MCCA is essential in the purine alkaloid synthesis pathway by participating in the synthesis of the precursors of these substances (Zrenner et al. 2006; Ashihara et al. 2008).
During serine metabolism, hydroxypyruvate is reduced to glyceric acid by HPR and returned to the chloroplast, and glycerol is catalyzed to the final product 3-phosphoglycerate by glycerol 3 kinase (Bauwe et al. 2010).
Polyamines (PAs) in plants mainly include putrescine (Put), sub-spermidine (Spd), and spermine (Spm). PAO can oxidatively degrade polyamines and reduce excess free PAs. Its diverse degradation products are also vital for plant growth and development and stress adaptation (An et al. 2008). In summary, amino acid metabolism in PT from wild fields was more active than in PT from cultivated fields.
Lipid metabolism
In cultivated PT, there were 2 upregulated and 8 downregulated DEGs associated with lipid metabolism: allene oxide cyclase (AOC), acyl-coenzyme A thioesterase 1/2/4 (ACOT), 2-acylglycerol O-acyltransferase 2 (MGAT2), lysophospholipid acyltransferase (LPLATs), secretory phospholipase A2 (PLA2), phosphoethanolamine N-methyltransferase (PEAMT), glycerophosphoryl diester phosphodiesterase (GDPD), enoyl-[acyl-carrier protein] reductase I (FabI), very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (HACD) and long-chain acyl-CoA synthetase (ACSLS).
PLA2 catalyzes the hydrolysis of the acyl bond at the 2-position of glycerol phosphate to generate free fatty acids and lysates, which play an important role in cell signal transduction, biosynthesis of bioactive lipids, and the release of linolenic acid from phospholipids. GDPD catalyzes the hydrolysis of glycerol phosphodiesters into an alcohol molecule and glycerol triphosphate. Its products are involved in many biochemical pathways. Alpha-linolenic acid is the substrate of a synthetic pathway of jasmonic acid. AOC synthesizes 12-oxo-plant dienoic acid (OPDA) in this pathway. OPDA then undergoes reduction and oxidation to synthesize jasmonate. Jasmonic acid can induce tuber formation during plant growth and development (McConn 1996). Triacylglycerol TAG is a storage lipid and an important carbon source to support seedling development after seed germination. DGAT catalyzes the last step of the TAG synthesis pathway and is the only rate-limiting enzyme in this pathway (Settlage et al. 1998). MGAT2 is a membrane-bound acyltransferase that belongs to the diacylglycerol acyltransferase (DGAT) gene family. ACOT hydrolyzes CoA into free fatty acids (FFA) and coenzyme A (CoASH) and participates in essential physiological processes by maintaining appropriate levels of intracellular lactoyl CoA, FFA and CoASH (HuntilM and Alexson 2002). As a methyltransferase, PEAMT is a rate-limiting enzyme that catalyzes the methylation of phosphoethanolamine to choline. Recent studies have shown that PEAMT not only plays a role in the process of plant growth and development (Fulgencio et al. 2012; Mouet al. 2002) but also participates in the synthesis of the osmolyte betaine and stress-related second messenger phosphatidic acid to aid in the plant response to salt stress (Kosová et al. 2013; Huang 2000). ACSLs participate in the first reaction step of long-chain fatty acid degradation in various organisms (Fulda et al. 2002). It can be concluded that lipid metabolism in wild PT was stronger than in cultivated PT.
Biosynthesis of other secondary metabolites
As an abundant and widely studied effective substance in PT, nucleoside alkaloids are considered to be more suitable for quality control than succinic acid (Lua et al. 2011) which makes an interesting study topic in future research on the differences between wild and cultivated PT an interesting target of study in future PT research. We found increased expression of 2 DEGs related to nucleoside alkaloids in wild accessions: 5'-NT and adenosine deaminase (ADA). Various nucleotides in plants are catalyzed to nucleosides through 5'-NT. ADA, a thiolase, catalyzes the conversion of adenine nucleoside to hypoxanthine nucleoside and then generates hypoxanthine through the action of nucleoside phosphorylase, which plays a vital role in purine nucleoside metabolism. This may indicate that nucleoside alkaloid biosynthesis was stronger in wild PT than in cultivated PT.
Vitamin E is a kind of fat-soluble vitamin necessary for the human body. It is an important effector substance on the cell membrane of plants that can remove singlet oxygen and inactivate reactive oxygen radicals under oxidative stress (Valentin et al. 2005). The tocopherol O-methyltransferase (γ-TMT) and tocopherol cyclase (TC) genes were upregulated in this study, and are responsible for the synthesis of vitamin E. γ-TMT is the last step in the biosynthetic pathway of vitamin E that catalyzes the methylation of γ and δ-tocopherol to α and β-tocopherol. TC catalyzes different intermediates in tocopherol synthesis to form aromatic rings. Kumar R’s (2005) study found that overexpression of the TC gene in rapeseed could increase the total tocopherol levels and change the proportion of each component of tocopherol. It can be concluded that the biosynthesis of other SMs in wild PT was stronger than that in cultivated PT.