Comprehensive Assessment of Houttuynia cordata Thunb., an Important Medicinal Plant and Vegetable

: Houttuynia cordata Thunb., called Yuxingcao in Chinese, is an important medicinal plant and vegetable consumed in the southern regions of China. This review aims to summarize studies on the phyto-physiological chemistry, cytology, molecular biology, and genomics of H. cordata . Studies on the physiology and biochemistry of H. cordata have grown over the past few decades. Phenotypic and agronomic traits, tissue culture, elemental analysis, photosynthetic studies, bioactive compound identiﬁcation, and antioxidant research have been reported. Molecular biological studies, such as those of molecular markers, microRNAs, DNA variations, protein variations, and transcriptomes have also advanced. Recent studies have focused on the rDNA and chloroplast genome of this plant. This review could serve as a basis to perform the genetic breeding, genomic advance, and cultivation of this valuable diversiﬁed plant resource for medicinal applications and vegetable production.


Introduction
Houttuynia cordata Thunb., also called Yuxingcao in China (Figure 1), is the sole species in the genus Houttuynia of the family Saururaceae, which consists of the genera of Saururus (S. chinensis and S. cernus), Gymnotheca (G. involucrate and G. chinensis), Anemopsis (A. californica), and Houttuynia (H. cordata); however, the species of A. californica and S. cernus are not found in China [1][2][3][4]. H. cordata is a perennial, polyploid (diploid or tetraploid) flowering herb native to China, Japan, Korea, northeast India, and Southeast Asia [5,6]). The predominant chromosome of H. cordata is X = 9 (2n = 18~108). It is distributed in the central, southeastern, and southwestern regions of China. Some scholars speculated that it originated in the southwest of China. It was then transplanted and formed three groups [4,7,8]. This plant grows well in moist to soggy soil, on shady hillsides, waysides, and ridges of fields with an altitude of 300~2600 m, produces a unique fishy smell, and blooms white flowers in early summer [9][10][11]. Interestingly, this herb propagates through the formation and separation of underground stems and parthenogenesis rather than sexual reproduction [12].
As a common aromatic, medicinal, and vegetable plant, H. cordata is an important herb plant resource to the food, pharmaceutical, and aromatic industries. It has great value in the fields of ethnopharmacology, fermented products, and natural products chemistry [13][14][15]. It has been traditionally used as a therapeutic plant in folk medicine during the last century, and its phytochemical ingredients (such as essential oils, alkaloids, organic acids, fatty acids, sterols, amino acids, and microelements) are used in some pharmaceutical formulas. It has been applied to treat various diseases because of its pharmacological functions, As a common aromatic, medicinal, and vegetable plant, H. cordata is an important herb plant resource to the food, pharmaceutical, and aromatic industries. It has great value in the fields of ethnopharmacology, fermented products, and natural products chemistry [13][14][15]. It has been traditionally used as a therapeutic plant in folk medicine during the last century, and its phytochemical ingredients (such as essential oils, alkaloids, organic acids, fatty acids, sterols, amino acids, and microelements) are used in some pharmaceutical formulas. It has been applied to treat various diseases because of its pharmacological functions, such as diuretic, anti-microbial, anti-viral, anti-cancer/anti-tumor, anti-inflammatory, anti-oxidative, anti-diabetic, anti-allergic, and anti-mutagenic effects [16][17][18][19][20][21].
However, limited information has been obtained on the germplasm resources, from phenotypic diversity to heritable variation in H. cordata. Thus, this review aims to summarize studies on the phyto-physiological chemistry, cytology, molecular biology, and genomics of H. cordata. Hopefully, this review could provide some usefultheoretical and practical information to assist breeding research of H. cordata. Further studies are warranted to obtain further information, including the genomics, of H. Cordata. However, limited information has been obtained on the germplasm resources, from phenotypic diversity to heritable variation in H. cordata. Thus, this review aims to summarize studies on the phyto-physiological chemistry, cytology, molecular biology, and genomics of H. cordata. Hopefully, this review could provide some usefultheoretical and practical information to assist breeding research of H. cordata. Further studies are warranted to obtain further information, including the genomics, of H. Cordata.

Phenotypic Diversity and Agronomic Traits
Previous studies have described and classified morphologies of the leaves, stems, flowers, and rhizomes of H. cordata. The geo-herbalism of H. cordata is largely determined by its genetic factors, which have been used to guide the cultivation, breeding, and utilization of wild H. cordata populations [22][23][24]. Guan (2010) analyzed the genetic variation in morphological characteristics of 20 H. cordata germplasm populations, and the populations were divided into 3 groups through cluster grouping with 15 measurable traits, showing a rich variation among these populations [25]. Shun et al. (2014) analyzed and showed that environmental factors exert different effects on the formation and accumulation of active physiochemical compounds in H. cordata [26]. Li et al. (2018a) analyzed the phenotypic diversity of leaf traits (9 phenotypic traits and 6 relative traits) in cultivated H. cordata populations from Hunan province in China, and showed that the diversity of leaf phenotypic traits is mainly determined by genetic factors, providing a theoretical basis for the breeding of excellent germplasms [27].

Elemental Analysis
H. cordata is a vegetable rich in proteins, minerals, vitamins, and amino acids, giving H. cordata its high nutritional and economic value [28,29]. Many studies analyzed the elemental composition of H. cordata. Qin and Yang (2015) measured the contents of 5 trace elements (calcium: Ca, magnesium: Mg, iron: Fe, zinc: Zn, and copper: Cu) through atomic absorption spectrophotometry, and the results showed that the order of element content in the same organ is Mg, Ca, Fe, Zn, and Cu [30]. Mo et al. (2015) compared the contents of 10 macro-elements and micro-elements in the leaves, aerial stems, and underground stems of wild H. cordata from different regions, and found that this plant is rich in sulphur (S), phosphorus (P), potassium (K), Ca and Mg [31]. Li et al. (2018b) detected the contents of 14 heavy metals (manganese: Mn, Zn, Cu, cobalt: Co, titanium: Ti, stannum: Sn, antimony: Sb, barium: Ba, chromium: Cr, nickel: Ni, arsenic: As, plumbum: Pb, mercury: Hg and cadmium: Cd) in H. cordata via the inductively coupled ICP-MS (inductively coupled plasma mass spectrometry) method, and the results showed that the linear relationship between the concentration of each element ranges from 0.15 to 695 g·L −1 [32]. Fotev et al. (2018) concluded that the micro-elements of H. cordata accumulated Mn, Fe, and Cu in the leaves, whereas Fe, Co, Cu, and Zn are detected mainly in the roots [11].

Photosynthetic Transpiration
The photosynthetic transpiration of plants is directly related to their growth potential and the economic benefits for farmers. Therefore, photosynthesis studies of H. cordata are important. Huang et al. (2006) measured the photosynthetic and transpiration characteristics of H. cordata, and found that photo flux quantum (PFD), relative humidity (RH), stomatal conductance (Gs), and leaf temperature (Tl) significantly correlate with the netphotosynthetic rate (Pn) and transpiration rate (Tr). PFD and Ca (air CO 2 concentration) are the major Pn-affecting factors, and PFD and Ta (air temperature) are the main Tr-affecting factors [33]. Xu et al. (2011b) explored the optimal potassium concentration for the proper growth and physiological response of H. cordata and found that 1.28 mM potassium is the optimum concentration to obtain the optimal values of dry weight, shoot height, root length and number, H 2 O 2 content, superoxide dismutase activity, and net photosynthetic rate [34]. Lv et al. (2012) tested the effect of different concentrations of NaCl on the photosynthesis of H. cordata at different habitats, and concluded that H. cordata from Huaihua County, Hunan Province showedimproved photosynthesis and adapt to salt stress [35].

Effects of Bioactive Compounds onAntioxidantCapacitiesof H. cordata
Some studies investigated the nutritional quality and health benefits, and other studies analyzed the bioactive substances and antioxidant capacity of different parts of H. cordata. Li et al. (2007) tested the effects of Pb, Zn, and their interactions on the chlorophyll content and antioxidant enzyme systems of H. cordata and found that treatment with Pb or Zn alone exerts more beneficial effects on chlorophyll content and antioxidant enzyme systems than their combination. It was also shown that H. cordata might have a high tolerance to Pb [36]. Cai et al. (2013) analyzed the total phenol and flavonoid contents and antioxidant activity of leaves from 16 H. cordata cultivars, showing that it isdivided into two types (I and II type) based on comprehensive agronomic characteristics. The total phenol and flavonoid contents and antioxidant activities of all materials were different due to their genetic backgrounds, in particular, the chromosome number (more than 80) of the material which had the highest total phenol and flavonoid contents and antioxidant activity [37]. Wang et al. (2015) tested the effects of LED (lightemitting diode) light spectra on the active oxygen metabolism and expression of antioxidant is ozymes in H. cordata seedlings and found that the antioxidant system of H. cordata seedlings is more sensitive to short light wavelength than to long light wavelength [38]. Liu et al. (2016) analyzed the bioactive compounds and antioxidant capacities in H. cordata, and the results indicated that (1) the levels of carotenoids, chlorophyll, anthocyanins, vitamin C, pro-anthocyanidins, total phenolics, flavonoids, and antioxidantcontents can be arranged in the order ofleaves > stems > rhizomes; (2) asignificant positive correlation exists between antioxidant capacities and seven bioactive compounds (p < 0.05); and (3) the leaves contain more bioactive compounds and anti-oxidant capacity than the stems and rhizomes. Furthermore, a comparison with 8 types of common vegetables revealed that H. cordata has great potential for development because this plant, especially its leaves, contains more bioactive substances and higher antioxidant capacity than other vegetables [39].
Some studies also focused on the anti-tumor/anti-cancer effects of H. cordata [54,55]. H. cordata extract inhibits cell growth and induces apoptotic cell death in human primary colorectal cancer cells through a mitochondria-dependent signaling pathway [56]. The extract of H. cordata modulates G 0 /G 1 phase arrest and Fas/CD95-dependent apoptotic cell death in human lung cancer A549 cells [57]. Meanwhile, extract of H. cordata induces A375 programmed cell death in human melanoma cells by p38 phosphorylation associated with HMGB1 reduction and by activating the caspase-dependent pathway [58]. Kim et al. (2017) demonstrated that H. cordata promotes the activation of HIF-1A-FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells, indicating that this plant is a promising candidate for anti-tumor drug development [59]. Lou et al. (2019) showed that H. cordata and its bioactive compound 2-undecanone can significantly suppress benzo(a)pyrene-induced lung tumorigenesis byactivating the Nrf2-HO-1/NQO-1 signaling pathway significantly [60].

Physiochemical Compounds
Many researchers authenticated the ingredients and function of the physiochemical compounds, such as polyphenols, and analyzed the relationship between the contents of flavonoids and their biological characteristics in H. cordata by using high-performance liquid chromatography (HPLC) [61,62]. Wu et al. (2002aWu et al. ( ,2002b analyzed the peroxidase and esterase isozymes variations of the germplasm resources of H. cordata from Sichuan Province in China and found 6 types of peroxidase isozyme bands (each band type was composed of from 4 to 6 bands, with the number in the north being higher than that in southern areas) and 10 types of esterase isozyme bands (each band type was composed of from 4 to 8 bands which had no geographic differences) [63,64]. Wu et al. (2009c) analyzed the content variation of flavonoids, including hyperin, quercitrin, and quercetin in H. cordata from 22 provinces or geographic origins in China and found thatthe levels of the three major flavonoids vary remarkably from different provinces, the contents of quercetin is not correlated with the geographic region, and concentrations from the highest to the lowest were in leaves, stems, and rhizomes [65]. Shun et al. (2014) concluded that appropriate shade treatment not only avoids the inhibitory effect of strong light on its growth, but also significantly increases the contents of active components [26]. Li et al. (2015a) examined the contents of major nutrient components, including protein, soluble sugar, fat, volatile oils, and total flavonoids, and the composition and contents of medicinal substances under field conditions and natural light at full intensity, 40% intensity, and 20% intensity in H. cordata from sprouting to harvesting. This study showed that sufficient light is necessary when growing H. cordata as medicinal and functional food, but appropriate shading or intercropping is necessary when the crop is grown as food [66]. Se (2015) detected the pharmacological volatiles emanating from3 different parts of H. cordata (leaves, aerial stems, and underground stems) from South Korea through fast gas chromatography-surface acoustic wave sensor (GC/SAM) and identified 16 compounds from the leaves (71.0%) and aerial stems (50.1%), including the monoterpenes β-myrcene, cis-ocimene, and decanal in underground stems (74.6%), and2-undecanone (1.3%) and lauraldehyde (3.5%) in leaves [67]. Zhang et al. (2018) determined the four main volatile components (4-terpenol, α-terpilenol, bornyl acetate, and methylnonylketone) of 47 H. cordata germplasm resources. They screened out the high content of the volatile components and provided a theoretical basis for aspecial type of breeding [68]. Chen et al. (2004) studied the relationship between the essential oil constituents and the chromosome number of H. cordata and found a tendency for the number of essential oil spots to increase with the chromosome number [69].

Secretory Tissues
Few studies focused on the roots, aerial stems, leaves, rhizomes, and other organs (such as secretory tissues), which greatly differ in anatomical structure among different populations of H. cordata [28,70]. Lin et al. (2013) showed that many oil cells in the leaves, stems, and rhizomes of H. cordata are the storage sites of volatile oil [71]. Some research also showed the structural, componential, and functional characteristics of its secretory tissues in the floral and vegetative parts of H. cordata [72].

Embryology
Although some researchers have studied the embryology of Saururaceae [73][74][75], studies on the sexual reproduction of H. cordata are not comprehensive. In particular, previous studies were insufficient for helping cross-breeding attempts [76]. Nuclear extrusion and abnormal meiosis possibly lead to the high pollen abortion rate (99.6%) of H. cordata [25]. Subsequently, Guan et al. (2012) observed the cytomixis and meiotic abnormalities during microsporogenesis in two populations of H. cordata with different ploidy levels (2n = 38~96), and found the origin of the intraspecific polyploidy, and suggested that it might lead to a large variation in chromosome numbers in this species [77]. Lin et al. (2011) worked on the histochemistry of anther development in H. cordata, and results showed that (1) some starches are distributed in the epidermis and endothelium; (2) plasmolysis in the tapetal cells occurs at the period of microspore mother cells; and (3) lipid accumulation in pollen grains is insufficient during anther development [78]. Li et al. (2010) reported that H. cordata pollen grains are abortive for deficiency of lipids in mature pollen grains [79]. Li et al. (2014) determined the pollen viability by I 2 -KI staining and examining the developmental course of male and female gametophytes using paraffin sections, and the results showed that (1) H. cordata pollen viability is extremely low (3.18%); and (2) the male gametophyte is normal in the early development stage and disintegrates in the dyad period of the tapetum cell. Then, the cytoplasm of free microspores gradually disappears and shows an empty flat shape, and the microspore shape changes from sub-orbicular to irregular. Finally, microspore abortion occurs before pollen sac cracking, which might lead to male sterility, and the seed might come from apomixes [80]. Li et al. (2017) reported that they are closely correlated with inflorescence size, anther color, and length at the meiosis stage, suggesting that the uninuclear pollen development rather than the abnormal division of pollen mother cell meiosis is the main reason behind the low pollen counts in H. cordata [81].

Intraspecific Polyploidy
Hsu (1967) studied the chromosomes of many vascular plants, including H. cordata from Taiwan, and suggested that the number of chromosomes of H. cordata is 24 [82]. Many researchers have studied the phylogenetic development of the Saururaceae plant, but their conclusions were contradictory (Table 1). The position of the centromere is expressed by the ratio of its long arm to its short arm. M: the ratio value is 1.0; m:the ratio value isfrom 1.0 to 1.7; sm: the ratio value is from 1.7 to 3.0; st: the ratio value isfrom 3.0 to 7.0; t: the ratio value is from 7.0 to ∞.

microRNA
Recent studies have suggested that miRNAs play regulatory roles at the intracellular and intercellular levels, and even in the inter-species manner. He et al. (2019) identified 163 conserved miRNAs and 30 novel miRNAs through high-throughput sequencing of H. cordata and found that they are enriched in the endocrine and other factor-regulated Ca reabsorption pathways, the insulin signaling pathway, melanogenesis, and aldosteroneregulated sodium reabsorption pathways, via bioinformatics analysis on the targets of H. cordata miRNAs to study the cross-kingdom functions of active compounds in H. cordata [97].

DNA Variation
DNA variation is the basis of the origin and diversity of species. Liu (2014) analyzed the DNA variation of 46 strains of H. cordata from the same GAP (good agricultural practices) origin with ISSR and RAPD molecular markers through cluster analysis. The results showed that 9 ISSR primers and 8 RAPD primers have amplification of 134 and 101 bands respectively, which helped to estimate the polymorphisms of the stripe numbers 115 and 72 respectively. Additionally, polymorphic rates were 85.8% and 71.3% and the average genetic similarity (GS) coefficients were 0.6786 and 0.6947, respectively. This result revealed that the gene possessed polymorphisms among H. cordata from the same GAP area [62].

Protein Variation
Some studies have suggested that the stronger frost resistance of the plants located at higher altitudes is associated with their massive accumulation of protein, fat, starch, and related substances [98]. Liu (2014) has shown a wide range of protein types, with molecular weights ranging from 6.5 KD to 97.2 KD from the 53 lines of H. cordata, but the protein types warrant further clarification [62].

Transcriptome
To date, genomic information about H. cordata is still limited. Kim

rDNA
Some researchers studied the intraspecific polymorphisms in the exon and group I intron of subunit rDNA (ribosomalDNA) of the obligate plant parasite Plasmodiophora brassicae [104]. Zhao (2008) analyzed the nuclear ribosomal DNA based on internal transcribed spacer (ITS) sequences of 23 H. cordata germplasm resources from Sichuan province and Chongqing city in China, including 11 cytotypes and 2 chemotypes, in total obtaining 147 ITS sequences, and the PCR product length varied from 670 bp to 684 bp and the length of ITS1 varied from 231 bp to 245 bp, whereas that of ITS2 was uniformly 275 bp for all accessions [105].

Chloroplast Genome
Although the chloroplast genome contains many noncoding regions, relatively few studies focused on interspecific phylogenetic and intraspecific phylogeographic characteristics [106]. Yu et al. (2019) reported that the complete chloroplast genome sequence of H. cordata is 161,090 bp in length, containing a large single copy (LSC) region of 88,180 bp and a small single copy (SSC) region of 19,204 bp, which were separated by a pair of 26,853 bp inverted repeat regions, and H. cordata is a sister of Piper cenocladum [107]. Zhu et al. (2020) carried out the de novo assembling of the complete chloroplast genome of H. cordata and showed a typical quadripartite cycle of 160,226 bp, including a pair of inverted repeats (IRa and IRb) is 26,853 bp. It is separated by an SSC region of 18,340 bp and an LSC region of 88,180 bp, including 112 unique genes, 79 protein coding genes, 29 tRNA genes, and 4 rRNA genes. In addition, 81, 13, and 17 two copy genes were located on the LSC, SSC, and IR region, respectively, and the chloroplast genome of H. cordata (48 repeat sequences, 86 SSR motifs) had a close relationship with the Aristolochia species (38~138 repeat sequences, from 95 to 156 SSR motifs), as revealed by mVISTA analysis [108].

Genetic Diversity
Intra-species genetic variability assessment was used in formulating genetic improvement and germplasm conservation strategy research. Wu et al. (2005b) tested the genetic diversity of 70 H. cordata accessions from Sichuan, Chongqing, Guizhou, and Jiangsu provinces in China by using RAMP, RAPD, and ISSR markers, and they arrived at the following conclusions: (1) a higher degree of genetic diversity exists among the germplasm resources of the genus Houttuynia at the molecular level (RAMP markers) [109], (2) the groups based on ISSR GS studies had been correlated with the same chromosome numbers which could have been classified together, while correlated with geographic distribution by RAPD GS [110]. Wei and Wu (2012) assessed the level and distribution of genetic diversity in 226 individuals from 15 populations of H. cordata in China by using ISSR markers and revealed low genetic variations within populations, but high genetic differentiations among populations [8]. Gupta and Bharalee (2020) also assessed the genetic diversity of H. cordata from four regions of northeast India, which contained 545 genotypes from 18 populations, and found that the genetic differentiation among 18 populations is high (Fst = 0.3894; p <0.001) with relatively restricted gene flow (Nm = 0.6564), which suggested that ex situ conservation could be an appropriate measure to adequately capture the total genetic diversity of H. cordata populations in northeast India [111].

GS Values of Cytotypes
Studies of the GS values of cytotypes provided the theoretical basis and the depth of research in another aspect of system diversity of H. cordata populations. Wu et al. (2002a) detected the genetic diversity of 92 H. cordata germplasm resources and found that the average GSs are from 0.52 to 0.572. Meanwhile, the interspecific GS is 0.517 among these 92 lines' populations. All materials had been divided into 11 types. In addition, the same number of chromosomes tended to cluster together, while the groups classified according to the RAMP GS coefficient were related to the geographic distribution [62]. Wu et al. (2003a) analyzed the genetic diversity of germplasm resources of H. cordata by ISSR marker and found that the chromosome number of 36 of H. cordata is the most similar in their cellular types. The average value of their GS factors is 0.618. As such, great genetic deference certainly exists at the cellular level in the germplasm resources of H. cordata, but the medicinal compositions are determined by its genetic factors [112]. Wu et al. (2005b) showed that the GS between the accessions within H. emeiensis and H. cordata are 0.660 and 0.575, respectively. Meanwhile, the GS value with the chromosome number of 36 is 0.559 [109]. Zhao (2008)

Gene Function Characterization
In recent years, cloning and studying the genetic functions of medicinal plants have become research hotspots. Thus, some studies focused on these aspects in H. cordata (Table 2). Furthermore, organic H. cordata harbors higher abundance and diversity of anti-bacterial resistance genes (ARGs) than that of non-organic origin, but the ARGs are still unknown [113].

Vegetativeand Sexual Production of H. cordata
The seed viability of H. cordata is lacking, and vegetative proliferation during winter is poor. Furthermore, H. cordata seeds are light-sensitive and small, with a low germination rate [120,121]. Therefore, tissue culture techniques have been used for the rapid propagation and conservation of elite clones of H. cordata throughout the years [122,123]. Moreover, studies about the embryo sac, embryo, and the low pollen fertility of H. cordata are rarely reported, and the mechanism of male sterility was not studied [124]. Wang (2013) suggested the following:(1) the stigma pollen is incompatible with self-pollination and cross-pollination in the populations, stigma papilla cells produce callose, or the fertility pollen grains germinate regularly but the pollen tube could not grow into the stigma, whereas the tapetum cells degenerate normally providing nutrition for microspore development; (2) the embryo sac development of H. cordata belongs to a monospore polygonum type; and (3) during embryonic development, starch gradually accumulates in nucellar cells, resulting in nucellar cells of mature seeds filled with starch grains. Therefore, stem propagation is the main method of reproduction in the practical production culture of H. cordata [125].

Transgenic Engineering
In recent years, molecular biology and genomic studies of H. cordata had been reported because of their research and economic value. Attempts to use H. cordata for transgenic engineering as a model plant have been carried out. Lai (2008) studied the Agrobacterium tumefaciens-mediated cecropin foreign gene transfer in H. cordata, which provided a theory for the anti-bacterial peptide expression in Chinese medicinal plants. Antimicrobial peptides are small molecules with broad-spectrum anti-microbial activity. These molecules have become research hotspots of disease resistance breeding in plant genetic engineering. They are expressed in many plants with enhanced disease resistance [126].

Conclusions
H. cordata is an important medicinal plant and vegetable. At present, genetic diversity, and GS intra-species and inter-species of H. cordata have become increasingly valuable, and comprehensive utilization of germplasm resources remains to be conducted. Data mining and utilization of genome information of H. cordata can be applied to fill this gap. These studies on pharmacology, cultivation, and breeding are equally important. In addition, the true value of H. cordata secondary metabolites should be considered [129,130]. Obviously, it holds great research value, not only in medicine but also in agricultural biodiversity. In-depth research, however, still needs to be carried out in the molecular, breeding, physiological, genetic, and genomics fields to understand H. cordata.

Conflicts of Interest:
The authors declare no conflict of interest.