The introduction and domestication of ornamental plant species is mainly affected by climate, especially in seasonally frozen regions at mid and high latitudes. The long cold winters in Northeastern China severely restrict the range of ornamental plants that can be introduced. Many fine ornamental plants have failed to domesticate due to climate inadaptability. Hosta ventricosa is widely used for planting under forest in Jilin Province because of its excellent low temperature tolerance. To date, studies on low temperature resistance in Hosta species have been limited to the exogenous application of antifreeze proteins (AFPs) to protect H. capitata from cold stress , and the majority of the research on low temperature resistance has focused on rice , cabbage  and other annual crops or model plants. Therefore the investigation of low temperature resistance in H. ventricosa may not only identify the mechanism of such resistance in this species, but also form the basis for the development of new varieties of Hosta that can tolerate low temperatures. Transcriptomic and metabolomic technologies are often used to identify regulatory mechanisms, to explore the growth, development, and physiological and pathological response mechanisms of organisms, and by combining transcriptomics and metabolomics through the KEGG metabolic pathway to identify genes and metabolites involved in the same biological process. Significant changes in genes and metabolites are a very effective method.
4.1 The transmission of low temperature signals in H. ventricosa
The cold response and cold defense mechanisms of plants are very complex, involving multiple synergistic signal transduction and metabolic pathways, such as ROS protection, Ca2+ signal transduction, and changes in cell membrane structure and components . In the present study, a total of 12 059 DEGs were obtained, of which 3188 DEGs were annotated to 131 KEGG pathways, and these differential genes were mainly concentrated in plant–pathogen interactions and signal transduction (MAPK signaling pathways). The DEGs in the four pathways of plant hormone signal transduction and phenylpropanoid biosynthesis accounted for 20.36% of the DEGs that had been annotated on KEGG. Plants perceive cold stimuli through changes in the plasma membrane. Low temperature affects the composition and structure of the cell membrane, triggering the process whereby Ca2+ enters the cell via ion channels on the cell membrane, and activating the cell’s defense or immune response through Ca2+ signalling.
In this study, it was found that at least three channel proteins, namely COLD, CNGCs and CRLK, interact with Ca2+ to transmit cold signals across the root cell membrane in H. ventricosa (Fig. 5). COLD1 is a cold-sensing signal sensor found in rice. It can recognize changes in cell membranes to sense cold signals, and activate Ca2+ channels through the action of the G protein α subunit. Rice (Oryza sativa) that lacks COLD1 is more sensitive to cold. Furthermore, overexpression significantly improves the cold tolerance of rice . There is only one gene annotated as COLD1 in H. ventricosa, and its expression is significantly up-regulated under cold stress. It is speculated that in this species COLD1 may have a similar function to OsCOLD1. Plays an important role in sensing low temperature during the low temperature stress stage. CNGCs are another type of non-selective cation channel located on the cell membrane, and have been shown to be related to a variety of external stimuli and to influence plant immune activities. OsCNGC6 and OsCNGC16 in rice can be significantly induced at low temperatures . There is also significant differential expression of different types of CNGCs in the root system of H. ventricosa. This includes the up-regulated expression of CNGC4, CNGC5, CNGC20 and CNGC2, and the down-regulated expression of CNGC15C. These CNGCs may participate in the cold mediation process. However, a study of CNGCs in mustard found that they are also responsible for the regulation of salt, heavy metals, germs, and growth processes , so the role of CNGCs in the regulatory mechanism of H. ventricosa may be more complex. CRLK1 has two calmodulin-binding sites with different affinities, which can sense cold and hydrogen peroxide. In A. thaliana, CRLK1 knockout mutant plants exhibit significant low temperature sensitivity . In H. ventricosa, two genes have been identified, namely CRLK1 and CRLK2, both of which are up-regulated. This finding is consistent with the results reported for OsCRLK1 in rice .
Although there are multiple low-temperature-related ion channels or sensors on the cell membrane of Hosta, they all interact with Ca2+ to transmit low-temperature signals. Many studies have indicated that the MAPK signaling pathway is one of the most important signaling mechanisms in plants. This pathway can be activated by a variety of signals, such as hydrogen peroxide, abscisic acid (ABA), ethylene, low temperature and salt, and it then triggers transcription factors through gradual phosphorylation. Changes in the activity of proteins such as phospholipase, or phosphorylation of specific targets in response to environmental stimuli [26, 27]. Studies have shown that MAPK is involved in the conduction of signals related to external stimuli such as non-essential heavy metals , drought , low temperature  and pathogenic injury . A large number of DEGs have been identified in the MAPK pathway in H. ventricosa. Ca2+ signaling positively regulates the MAPK-mediated plant response to cold stress through CPKs. In H. ventricosa, CPK17 is up-regulated and expressed, triggering MEKK1-MKK3-MPK4. In the cascade of 6, MKK3 and MPK4/6 are up-regulated, but the expression of MEKK1 remains unchanged. There are also reports that MEKK1 can be phosphorylated by CRLK1 .
CBL is a Ca2+ signal sensor. It interacts with CIPK to relay cold-triggered Ca2+ signals into phosphorylation . In H. ventricosa, CBL1 significantly up-regulates the expression, but its associated CIPKs expression is not inconsistent. In rice and Arabidopsis, CIPK1 and CIPK7 are induced by low temperatures. Research on the effect of low temperature on cassava (Manihot esculenta) also showed that CIPK10 is induced by cold stress in the root system. These findings are similar to the CBL-CIPK effect in H. ventricosa [ [33–35]. In addition, studies have shown that both MAPKs and CBL-CIPK can interact with ICE1 to bind to CBF or DREB and trigger plant cold defense responses . Arabidopsis ICE1 has been confirmed to be a cold-mediating gene, and overexpressed ICE1 can interact with CBF3/DREB1A to enhance the freezing resistance in this genus . H. ventricosa SCRM is an ICE1-like transcription factor. In the present study, SCRM had the same effect as ICE1, and mediated H. ventricosa DREB1A under conditions of cold stress. However, transcriptome analysis has shown that there are many members of the DREB family, including DREB2A, DREB1B, DREB2B, DREB1D, DREB1E and DREB1F, among others. Although these genes are similar to DREB1A and are all down-regulated, it is unclear whether they are affected by other functions. In addition, there have been many reports that CAMTA3 is a calmodulin transcription factor, and the Arabidopsis knockout mutants show higher sensitivity to low temperatures. CBF1 can also recognize CAMTA3 , but this study did not CAMTA3 is found in H. ventricosa, which may require more experiments to verify.
In addition, hydrogen peroxide, ethylene, abscisic acid and jasmonic acid are involved in the signaling and defense responses of plants to low temperatures. Combined with the determination of plant hormones, more important information may be obtained.
4.2 Involvement of the phenylpropanoid and flavonoid metabolic pathways in the cold defense response of H. ventricosa
The transcriptomics and metabolomics of H. ventricosa show that a large number of differentially expressed genes and metabolites are enriched in the metabolic pathways of phenylpropanoid and flavonoid biosynthesis. Many studies have shown that the phenylpropanoid biosynthetic pathway is affected by drought, heavy metals, temperature and other abiotic stresses. Its activation leads to the accumulation of a range of phenolic compounds, which include metabolites such as phenolic acid and flavonoids. During these biological processes, multiple genes are involved in the synthesis of metabolites . In the leaves of maize (Zea mays), the levels of phenolic compounds and anthocyanins increase as the temperature decreases, and there is an increase in the expression of genes that encode enzymes of the phenylpropanoid pathway, which in turn results in increased activity of cinnamate-4-hydroxylase (C4H) and chalcone synthase (CHS) . When Fagopyrum tataricum is subjected to low temperature treatment, most of the transcripts in the phenylpropanoid biosynthetic pathway are up-regulated, and the levels of anthocyanin and proanthocyanidin increase significantly . In cold-acclimated tobacco (Nicotiana tabacum) it has been demonstrated that a number of DEGs and differential metabolites that have a role in signal transduction, carbohydrate metabolism and phenylpropanoid biosynthesis are involved in the low-temperature defense process .
Metabolome analysis of H. ventricosa identified 32 differentially expressed phenolic compounds, including cinnamic acid, the first intermediate in the pathway of phenylpropanoid biosynthesis. In that metabolic pathway, PAL catalyzes the conversion of phenylalanine to trans-cinnamic acid (t-CA), which is the precursor for many active metabolites. However, cinnamic acid has two isomers in plants, namely cis-cinnamic acid (c-CA) and trans-cinnamic acid (t-CA). Only t-CA enters the phenylpropanoid pathway. c-CA may be an ultraviolet-light-mediated t-CA isomerization product, which has a role in regulation of the dynamic balance of auxin . The UPLC-MS/MS technology that was used in the present study cannot distinguish between isomers, so it is assumed that the cinnamic acid involved in the phenylpropanoid biosynthetic pathway is t-CA.
The phenylpropionic acid in H. ventricosa is catalyzed by the up-regulated expression of PAL (PAL1, PAL2, PAL3) to form t-CA, t-CA
P-Coumaroyl-CoA is formed under the action of 4CL (4CL1, 4CL2) and cytochrome P450 (CYP73A100, CYP73A13), and p-Coumaroyl-CoA, as the core intermediate product of the phenylpropane metabolic pathway, can connect to the flavonoid metabolic pathway and form part of it Isoflavones, or other phenolic compounds formed by other means (Fig. 5). Four significantly different metabolites were produced by the metabolic pathways of phenylpropanoid and flavonoid biosynthesis in H. ventricosa, including the two isoflavones formononetin 7-O-glucoside (FG) and genistein 7-O-glucoside (genistein), and the two phenolic compounds scopolin and syringin. There was also an increase in content of the precursors of phenolic compounds.
FG is a type of isoflavone that is commonly found in legumes. Many in vitro experiments have shown that FG has significant antioxidant and anti-inflammatory effects on other organisms , but there has been very little research on plant resistance. Some studies have shown that the FG content of red clover (Trifolium pratense) increases sharply after 3 weeks of submergence stress . There is no direct evidence that FG is involved in plant cold stress, and FG can interact with plant hormones during plant growth. Leading to the decrease in plant hormones, it is speculated that the increase in FG content in H. ventricosa may be related to hormone cross-talk, which together affect the low temperature resistance of this species . Genistein is another type of isoflavone that is abundant in legumes, and it has a wide range of pharmacological properties. Many studies have reported that genistein can significantly improve the resistance of plants to biological stress . For example, it has been shown to increase the resistance of alfalfa (Medicago sativa) to Acyrthosiphon pisum , and of rice (O. sativa) to Magnaporthe oryzae j. Genistein can also improve the response of plants to adverse environmental conditions. One study reported that after soybean (Glycine max) was subjected to a low temperature (10°C) for 24 hours, the genistein content of the root system increased threefold compared with the untreated group . The same research is also reflected in the low temperature-Osmotic compound stress of soybeans. There are also some studies which suggest that genistein is linked to high temperature stress. The genistein content of soybean (G. max) increases significantly with rising temperature, but it has also been noted that the genistein content of different soybean genotypes does not always increase in response to high temperature stress . Although these studies did not identify the mechanism underlying the response of genistein to temperature change, they did show that genistein content is closely related to temperature change, which is consistent with the finding that in H. ventricosa under low temperature stress the genistein content increased significantly. The above-mentioned studies all used soybean as the experimental material. However, there are no reports on genistein content under abiotic stress in other crop plants, and further research is needed to clarify the mechanism underlying the response of genistein to changes in temperature.
Scopoletin is the precursor of scopolin, and both compounds are derivatives of coumarin. They can participate in the elimination of ROS and in defense against pathogens. The content of scopoletin and scopolin in H. ventricosa is significantly increased, which indicates that both compounds have a role in the low temperature defense process in this species. Low temperature stress can induce the accumulation of scopolin in Arabidopsis, and a decrease in scopolin accumulation is observed in Arabidopsis mutants that lack the genes related to scopolin production . Under a combination of drought and high temperature stress, the scopolin content of mandarin orange (Citrus reticulata) increases, enabling the plant to cope with these adverse environmental conditions. It has been suggested that scopolin reduces oxidative damage in citrus , grape (Vitis vinifera)  and tea tree (Camellia sinensis) .
Sinapyl alcohol is a constituent of lignin, which is a component of the cell wall that plays an important role in resisting abiotic stress . Cinnamyl alcohol dehydrogenase (CAD), which has been identified in sweet potato (Ipomoea batatas), catalyzes the last step of the lignin synthesis pathway and is involved in the synthesis of lignin monomers. Overexpression of IbCAD1 in cassava can cause increased accumulation of sinapyl alcohol in the root system, and at the same time increases the plant’s tolerance of ROS such as hydrogen peroxide. Lignin is mainly composed of guaiacyl (G), syringyl (S) and p-hydroxyphenyl (H) units, which are derived from coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol, respectively . The ratio between them can affect the adaptability of plants to environmental stress . Under low temperature conditions, the root system of Hosta violacea showed up-regulation of PAL, 4CL, F5H, CCOAOMT and almost all other genes related to the synthesis of sinapyl alcohol via the phenylpropanoid biosynthesis pathway, whereas CYP84A1, 7OMT and CAD8 were all down-regulated. These genes promote a significant increase in the content of sinapyl alcohol. At the same time, the content of coniferyl alcohol in H. ventricosa is significantly reduced, which indicates that this species controls the composition of lignin by adjusting the G/S ratio, thereby altering the morphological structure of the cell wall.