Search
Submit Manuscript
Advanced Search
MAP  >  Medicinal Plant Biology
2024 Volume 3
Article Contents
Abstract
Introduction
Materials and methods
Datasets and identification of PIP genes
Sequence alignment, phylogenetic analysis, and identification of conserved motifs
Synteny analysis, definition of orthogroups, and calculation of the evolutionary rate
Plant materials
Gene expression analysis
Protein quantification
Subcellular localization analysis
Results
Characterization of 14 PIP genes in tigernut
Gene localization and synteny analyses reveal complex evolution patterns of CePIP genes
Comparative genomics analyses reveals lineage-specific evolution of PIP genes in Cyperaceae
Tissue-specific expression analysis of CePIP genes identifies three dominant members
Expression analysis reveals that CePIP transcripts in leaves are subjected to development regulation
qRT-PCR analysis reveals that the transcription of CePIP1;1, -2;1, and -2;8 in mature leaves is subjected to diurnal regulation
Expression profiles of CePIP genes during tuber development
Protein abundances of CePIP1;1, -2;1, and -2;8 in tubers are positively correlated with the moisture content during tuber development
Subcellular localization analysis reveals that CePIP1;1, -2;1, and -2;8 function in the cell membrane
Discussion
The tigernut genome encodes 14 PIP genes with three introns and gene expansion was mainly contributed by WGD and transposed/tandem duplications
CePIP1;1, -2;1, and -2;8 are three dominant members that are constitutively expressed in most tissues
Transcription of CePIP1;1, -2;1, and -2;8 in leaves is subjected to development and diurnal regulation
mRNA and protein abundances of CePIP1;1, -2;1, and -2;8 are positively correlated with the moisture content during tuber development
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
About this article
Next Previous
REVIEW   Open Access    

Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions

  • 1.

    Hunan Key Laboratory of Traditional Chinese Veterinary Medicine, Hunan Agricultural University, Changsha 410128, Hunan, China

  • 2.

    Traditional Chinese Medicine Breeding Center of Yuelushan Laboratory, Changsha 410128, Hunan, China

  • 3.

    College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, Hunan, China

  • 4.

    Hunan Institute of Agricultural Environment and Ecology, Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China

  • 5.

    Micolta Bioresource Inc., Changsha 410016, Hunan, China

  • 6.

    National and Local Union Engineering Research Center of Veterinary Herbal Medicine Resource and Initiative, Hunan Agricultural University, Changsha 410128, Hunan, China

More Information
  • Macleaya cordata, a medicinal plant in the Papaveraceae family, is rich in bioactive benzylisoquinoline alkaloids. Recent research has elucidated the mechanisms by which these active components promote livestock and poultry growth and exhibit anti-inflammatory effects on the intestines. These findings have led to the development of two raw medicinal materials and two veterinary drug formulations, widely used in China's livestock industry. Advances in multi-omics technologies, such as whole-genome sequencing and transcriptomics, have clarified the chemical composition of alkaloids and the biosynthesis of sanguinarine, enabling its de novo synthesis in yeast. Efforts in plant breeding have focused on cultivar selection and germplasm innovation, establishing DUS (Distinctness, Uniformity, and Stability) testing guidelines. Genetic engineering techniques have re-edited the sanguinarine pathway and induced hairy roots and cell suspension cultures in M. cordata. These advancements reduce production costs, ensure product stability, and promote sustainable production. This paper reviews the species origin, current research status, and prospects of M. cordata, offering guidance for further research on this valuable resource.

  • Introduction

    Aquaporins (AQPs) constitute a large family of transmembrane channel proteins that function as regulators of intracellular and intercellular water flow[1,2]. Since their first discovery in the 1990s, AQPs have been found not only in three domains of life, i.e., bacteria, eukaryotes, and archaea, but also in viruses[3,4]. Each AQP monomer is composed of an internal repeat of three transmembrane helices (i.e., TM1–TM6) as well as two half helixes that are formed by loop B (LB) and LE through dipping into the membrane[5]. The dual Asn-Pro-Ala (NPA) motifs that are located at the N-terminus of two half helixes act as a size barrier of the pore via creating an electrostatic repulsion of protons, whereas the so-called aromatic/arginine (ar/R) selectivity filter (i.e., H2, H5, LE1, and LE2) determines the substrate specificity by rendering the pore constriction site diverse in both size and hydrophobicity[5−9]. Based on sequence similarity, AQPs in higher plants could be divided into five subfamilies, i.e., plasma membrane intrinsic protein (PIP), tonoplast intrinsic protein (TIP), NOD26-like intrinsic protein (NIP), X intrinsic protein (XIP), and small basic intrinsic protein (SIP)[10−17]. Among them, PIPs, which are typically localized in the cell membrane, are most conserved and play a central role in controlling plant water status[12,18−22]. Among two phylogenetic groups present in the PIP subfamily, PIP1 possesses a relatively longer N-terminus and PIP2 features an extended C-terminus with one or more conserved S residues for phosphorylation modification[5,15,17].

    Tigernut (Cyperus esculentus L.), which belongs to the Cyperaceae family within Poales, is a novel and promising herbaceous C4 oil crop with wide adaptability, large biomass, and short life period[23−27]. Tigernut is a unique species accumulating up to 35% oil in the underground tubers[28−30], which are developed from stolons and the process includes three main stages, i.e., initiation, swelling, and maturation[31−33]. Water is essential for tuber development and tuber moisture content maintains a relatively high level of approximately 85% until maturation when a significant drop to about 45% is observed[28,32]. Thereby, uncovering the mechanism of tuber water balance is of particular interest. Despite crucial roles of PIPs in the cell water balance, to date, their characterization in tigernut is still in the infancy[21]. The recently available genome and transcriptome datasets[31,33,34] provide an opportunity to address this issue.

    In this study, a global characterization of PIP genes was conducted in tigernut, including gene localizations, gene structures, sequence characteristics, and evolutionary patterns. Moreover, the correlation of CePIP mRNA/protein abundance with water content during tuber development as well as subcellular localizations were also investigated, which facilitated further elucidating the water balance mechanism in this special species.

    Materials and methods

    Datasets and identification of PIP genes

    PIP genes reported in Arabidopsis (Arabidopsis thaliana)[10] and rice (Oryza sativa)[11] were respectively obtained from TAIR11 (www.arabidopsis.org) and RGAP7 (http://rice.uga.edu), and detailed information is shown in Supplemental Table S1. Their protein sequences were used as queries for tBLASTn[35] (E-value, 1e–10) search of the full-length tigernut transcriptome and genome sequences that were accessed from CNGBdb (https://db.cngb.org/search/assembly/CNA0051961)[31,34]. RNA sequencing (RNA-seq) reads that are available in NCBI (www.ncbi.nlm.nih.gov/sra) were also adopted for gene structure revision as described before[13], and presence of the conserved MIP (major intrinsic protein, Pfam accession number PF00230) domain in candidates was confirmed using MOTIF Search (www.genome.jp/tools/motif). To uncover the origin and evolution of CePIP genes, a similar approach was also employed to identify homologs from representative plant species, i.e., Carex cristatella (v1, Cyperaceae)[36], Rhynchospora breviuscula (v1, Cyperaceae)[37], and Juncus effusus (v1, Juncaceae)[37], whose genome sequences were accessed from NCBI (www.ncbi.nlm.nih.gov). Gene structure of candidates were displayed using GSDS 2.0 (http://gsds.gao-lab.org), whereas physiochemical parameters of deduced proteins were calculated using ProtParam (http://web.expasy.org/protparam). Subcellular localization prediction was conducted using WoLF PSORT (www.genscript.com/wolf-psort.html).

    Sequence alignment, phylogenetic analysis, and identification of conserved motifs

    Nucleotide and protein multiple sequence alignments were respectively conducted using ClustalW and MUSCLE implemented in MEGA6[38] with default parameters, and phylogenetic tree construction was carried out using MEGA6 with the maximum likelihood method and bootstrap of 1,000 replicates. Systematic names of PIP genes were assigned with two italic letters denoting the source organism and a progressive number based on sequence similarity. Conserved motifs were identified using MEME Suite 5.5.3 (https://meme-suite.org/tools/meme) with optimized parameters as follows: Any number of repetitions, maximum number of 15 motifs, and a width of 6 and 250 residues for each motif. TMs and conserved residues were identified using homology modeling and sequence alignment with the structure resolved spinach (Spinacia oleracea) SoPIP2;1[5].

    Synteny analysis, definition of orthogroups, and calculation of the evolutionary rate

    Synteny analysis was conducted using TBtools-II[39] as described previously[40], where the parameters were set as E-value of 1e-10 and BLAST hits of 5. Duplication modes were identified using the DupGen_finder pipeline[41], and Ks (synonymous substitution rate) and Ka (nonsynonymous substitution rate) of duplicate pairs were calculated using codeml in the PAML package[42]. Orthologs between different species were identified using InParanoid[43] and information from synteny analysis, and orthogroups (OGs) were assigned only when they were present in at least two species examined.

    Plant materials

    Plant materials used for gene cloning, qRT-PCR analysis, and 4D-parallel reaction monitoring (4D-PRM)-based protein quantification were derived from a tigernut variety Reyan3[31], and plants were grown in a greenhouse as described previously[25]. For expression profiling during leaf development, three representative stages, i.e., young, mature, and senescing, were selected and the chlorophyll content was checked using SPAD-502Plus (Konica Minolta, Shanghai, China) as previously described[44]. Young and senescing leaves are yellow in appearance, and their chlorophyll contents are just half of that of mature leaves that are dark green. For diurnal fluctuation regulation, mature leaves were sampled every 4 h from the onset of light at 8 a.m. For gene regulation during tuber development, fresh tubers at 1, 5, 10, 15, 20, 25, and 35 d after tuber initiation (DAI) were collected as described previously[32]. All samples with three biological replicates were quickly frozen with liquid nitrogen and stored at −80 °C for further use. For subcellular localization analysis, tobacco (Nicotiana benthamiana) plants were grown as previously described[20].

    Gene expression analysis

    Tissue-specific expression profiles of CePIP genes were investigated using Illumina RNA-seq samples (150 bp paired-end reads) with three biological replicates for young leaf, mature leaf, sheath of mature leaf, shoot apex, root, rhizome, and three stages of developmental tuber (40, 85, and 120 d after sowing (DAS)), which are under the NCBI accession number of PRJNA703731. Raw sequence reads in the FASTQ format were obtained using fastq-dump, and quality control was performed using fastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc). Read mapping was performed using HISAT2 (v2.2.1, https://daehwankimlab.github.io/hisat2), and relative gene expression level was presented as FPKM (fragments per kilobase of exon per million fragments mapped)[45].

    For qRT-PCR analysis, total RNA extraction and synthesis of the first-strand cDNA were conducted as previously described[24]. Primers used in this study are shown in Supplemental Table S2, where CeUCE2 and CeTIP41[25,33] were employed as two reference genes. PCR reaction in triplicate for each biological sample was carried out using the SYBR-green Mix (Takara) on a Real-time Thermal Cycler Type 5100 (Thermal Fisher Scientific Oy). Relative gene abundance was estimated with the 2−ΔΔCᴛ method and statistical analysis was performed using SPSS Statistics 20 as described previously[13].

    Protein quantification

    Raw proteomic data for tigernut roots, leaves, freshly harvested, dried, rehydrated for 48 h, and sprouted tubers were downloaded from ProteomeXchange/PRIDE (www.proteomexchange.org, PXD021894, PXD031123, and PXD035931), which were further analyzed using Maxquant (v1.6.15.0, www.maxquant.org). Three dominant members, i.e., CePIP1;1, -2;1, and -2;8, were selected for 4D-PRM quantification analysis, and related unique peptides are shown in Supplemental Table S3. Protein extraction, trypsin digestion, and LC-MS/MS analysis were conducted as described previously[46].

    Subcellular localization analysis

    For subcellular localization analysis, the coding region (CDS) of CePIP1;1, -2;1, and -2;8 were cloned into pNC-Cam1304-SubN via Nimble Cloning as described before[30]. Then, recombinant plasmids were introduced into Agrobacterium tumefaciens GV3101 with the helper plasmid pSoup-P19 and infiltration of 4-week-old tobacco leaves were performed as previously described[20]. For subcellular localization analysis, the plasma membrane marker HbPIP2;3-RFP[22] was co-transformed as a positive control. Fluorescence observation was conducted using confocal laser scanning microscopy imaging (Zeiss LMS880, Germany): The wavelength of laser-1 was set as 730 nm for RFP observation, where the fluorescence was excited at 561 nm; the wavelength of laser-2 was set as 750 nm for EGFP observation, where the fluorescence was excited at 488 nm; and the wavelength of laser-3 was set as 470 nm for chlorophyll autofluorescence observation, where the fluorescence was excited at 633 nm.

    Results

    Characterization of 14 PIP genes in tigernut

    As shown in Table 1, a total of 14 PIP genes were identified from eight tigernut scaffolds (Scfs). The CDS length varies from 831 to 882 bp, putatively encoding 276–293 amino acids (AA) with a molecular weight (MW) of 29.16–31.59 kilodalton (kDa). The theoretical isoelectric point (pI) varies from 7.04 to 9.46, implying that they are all alkaline. The grand average of hydropathicity (GRAVY) is between 0.344 and 0.577, and the aliphatic index (II) ranges from 94.57 to 106.90, which are consistent with the hydrophobic characteristic of AQPs[47]. As expected, like SoPIP2;1, all CePIPs include six TMs, two typical NPA motifs, the invariable ar/R filter F-H-T-R, five conserved Froger's positions Q/M-S-A-F-W, and two highly conserved residues corresponding to H193 and L197 in SoPIP2;1 that were proven to be involved in gating[5,48], though the H→F variation was found in CePIP2;9, -2;10, and -2;11 (Supplemental Fig. S1). Moreover, two S residues, corresponding to S115 and S274 in SoPIP2;1[5], respectively, were also found in the majority of CePIPs (Supplemental Fig. S1), implying their posttranslational regulation by phosphorylation.

    Table 1.  Fourteen PIP genes identified in C. esculentus.
    Gene name Locus Position Intron no. AA MW (kDa) pI GRAVY AI TM MIP
    CePIP1;1 CESC_15147 Scf9:2757378..2759502(–) 3 288 30.76 8.82 0.384 95.28 6 47..276
    CePIP1;2 CESC_04128 Scf4:3806361..3807726(–) 3 291 31.11 8.81 0.344 95.95 6 46..274
    CePIP1;3 CESC_15950 Scf54:5022493..5023820(+) 3 289 31.06 8.80 0.363 94.57 6 49..278
    CePIP2;1 CESC_15350 Scf9:879960..884243(+) 3 288 30.34 8.60 0.529 103.02 6 33..269
    CePIP2;2 CESC_00011 Scf30:4234620..4236549(+) 3 293 31.59 9.27 0.394 101.57 6 35..268
    CePIP2;3 CESC_00010 Scf30:4239406..4241658(+) 3 291 30.88 9.44 0.432 98.97 6 31..266
    CePIP2;4 CESC_05080 Scf46:307799..309544(+) 3 285 30.44 7.04 0.453 100.32 6 28..265
    CePIP2;5 CESC_05079 Scf46:312254..314388(+) 3 286 30.49 7.04 0.512 101.68 6 31..268
    CePIP2;6 CESC_05078 Scf46:316024..317780(+) 3 288 30.65 7.68 0.475 103.06 6 31..268
    CePIP2;7 CESC_05077 Scf46:320439..322184(+) 3 284 30.12 8.55 0.500 100.00 6 29..266
    CePIP2;8 CESC_14470 Scf2:4446409..4448999(+) 3 284 30.37 8.30 0.490 106.90 6 33..263
    CePIP2;9 CESC_02223 Scf1:2543928..2545778(–) 3 283 30.09 9.46 0.533 106.47 6 31..262
    CePIP2;10 CESC_10007 Scf27:1686032..1688010(–) 3 276 29.16 9.23 0.560 106.05 6 26..256
    CePIP2;11 CESC_10009 Scf27:1694196..1696175(–) 3 284 29.71 9.10 0.577 105.49 6 33..263
    AA: amino acid; AI: aliphatic index; GRAVY: grand average of hydropathicity; kDa: kilodalton; MIP: major intrinsic protein; MW: molecular weight; pI: isoelectric point; PIP: plasma membrane intrinsic protein; Scf: scaffold; TM: transmembrane helix.
     | Show Table
    DownLoad: CSV

    To uncover the evolutionary relationships, an unrooted phylogenetic tree was constructed using the full-length protein sequences of CePIPs together with 11 OsPIPs and 13 AtPIPs. As shown in Fig. 1a, these proteins were clustered into two main groups, corresponding to PIP1 and PIP2 as previously defined[10,49], and each appears to have evolved into several subgroups. Compared with PIP1s, PIP2s possess a relatively shorter N-terminal but an extended C-terminal with one conserved S residue (Supplemental Fig. S1). Interestingly, a high number of gene repeats were detected, most of which seem to be species-specific, i.e., AtPIP1;1/-1;2/-1;3/-1;4/-1;5, AtPIP2;1/-2;2/-2;3/-2;4/-2;5/-2;6, AtPIP2;7/-2;8, OsPIP1;1/-1;2/-1;3, OsPIP2;1/-2;4/-2;5, OsPIP2;2/-2;3, CePIP1;1/-1;2, CePIP2;2/-2;3, CePIP2;4/-2;5/-2;6/-2;7, and CePIP2;9/-2;10/-2;11, reflecting the occurrence of more than one lineage-specific whole-genome duplications (WGDs) after their divergence[50,51]. In Arabidopsis that experienced three WGDs (i.e. γ, β, and α) after the split with the monocot clade[52], AtPIP1;5 in the PIP1 group first gave rise to AtPIP1;1 via the γ WGD shared by all core eudicots[50], which latter resulted in AtPIP1;3, -1;4, and -1;2 via β and α WGDs; AtPIP2;1 in the PIP2 group first gave rise to AtPIP2;6 via the γ WGD, and they latter generated AtPIP2;2, and -2;5 via the α WGD (Supplemental Table S1). In rice, which also experienced three WGDs (i.e. τ, σ, and ρ) after the split with the eudicot clade[51], OsPIP1;2 and -2;3 generated OsPIP1;1 and -2;2 via the Poaceae-specific ρ WGD, respectively. Additionally, tandem, proximal, transposed and dispersed duplications also played a role on the gene expansion in these two species (Supplemental Table S1).

    Figure 1.  Structural and phylogenetic analysis of PIPs in C. esculentus, O. sativa, and A. thaliana. (a) Shown is an unrooted phylogenetic tree resulting from full-length PIPs with MEGA6 (maximum likelihood method and bootstrap of 1,000 replicates), where the distance scale denotes the number of amino acid substitutions per site. (b) Shown are the exon-intron structures. (c) Shown is the distribution of conserved motifs among PIPs, where different motifs are represented by different color blocks as indicated and the same color block in different proteins indicates a certain motif. (At: A. thaliana; Ce: C. esculentus; PIP: plasma membrane intrinsic protein; Os: O. sativa).
    DownLoad: Full-Size Img PowerPoint

    Analysis of gene structures revealed that all CePIP and AtPIP genes possess three introns and four exons in the CDS, in contrast to the frequent loss of certain introns in rice, including OsPIP1;2, -1;3, -2;1, -2;3, -2;4, -2;5, -2;6, -2;7, and -2;8 (Fig. 1b). The positions of three introns are highly conserved, which are located in sequences encoding LB (three residues before the first NPA), LD (one residue before the conserved L involved in gating), and LE (18 residues after the second NPA), respectively (Supplemental Fig. S1). The intron length of CePIP genes is highly variable, i.e., 109–993 bp, 115–1745 bp, and 95–866 bp for three introns, respectively. By contrast, the exon length is relatively less variable: Exons 2 and 3 are invariable with 296 bp and 141 bp, respectively, whereas Exons 1 and 4 are of 277–343 bp and 93–132 bp, determining the length of N- and C-terminus of PIP1 and PIP2, respectively (Fig. 1b). Correspondingly, their protein structures were shown to be highly conserved, and six (i.e., Motifs 1–6) out of 15 motifs identified are broadly present. Among them, Motif 3, -2, -6, -1, and -4 constitute the conserved MIP domain. In contrast to a single Motif 5 present in most PIP2s, all PIP1s possess two sequential copies of Motif 5, where the first one is located at the extended N-terminal. In CePIP2;3 and OsPIP2;7, Motif 5 is replaced by Motif 13; in CePIP2;2, it is replaced by two copies of Motif 15; and no significant motif was detected in this region of CePIP2;10. PIP1s and PIP2s usually feature Motif 9 and -7 at the C-terminal, respectively, though it is replaced by Motif 12 in CePIP2;6 and OsPIP2;8. PIP2s usually feature Motif 8 at the N-terminal, though it is replaced by Motif 14 in CePIP2;2 and -2;3 or replaced by Motif 11 in CePIP2;10 and -2;11 (Fig. 1c).

    Gene localization and synteny analyses reveal complex evolution patterns of CePIP genes

    As shown in Fig. 2a, gene localization of CePIPs revealed three gene clusters, i.e., CePIP2;2/-2;3 on Scf30, CePIP2;4/-2;5/-2;6/-2;7 on Scf46, and CePIP2;10/-2;11 on Scf27, which were defined as tandem repeats for their high sequence similarities and neighboring locations. The nucleotide identities of these duplicate pairs vary from 70.5% to 91.2%, and the Ks values range from 0.0971 to 1.2778 (Table 2), implying different time of their birth. According to intra-species synteny analysis, two duplicate pairs, i.e., CePIP1;1/-1;2 and CePIP2;2/-2;4, were shown to be located within syntenic blocks (Fig. 2b) and thus were defined as WGD repeats. Among them, CePIP1;1/-1;2 possess a comparable Ks value to CePIP2;2/-2;3, CePIP1;1/-1;3, and CePIP2;4/-2;8 (1.2522 vs 1.2287–1.2778), whereas CePIP2;2/-2;4 harbor a relatively higher Ks value of 1.5474 (Table 2), implying early origin or fast evolution of the latter. While CePIP1;1/-1;3 and CePIP2;1/-2;8 were characterized as transposed repeats, CePIP2;1/-2;2, CePIP2;9/-2;10, and CePIP2;8/-2;10 were characterized as dispersed repeats (Fig. 2a). The Ks values of three dispersed repeats vary from 0.8591 to 3.0117 (Table 2), implying distinct times of origin.

    Figure 2.  Duplication events of CePIP genes and synteny analysis within and between C. esculentus, O. sativa, and A. thaliana. (a) Duplication events detected in tigernut. Serial numbers are indicated at the top of each scaffold, and the scale is in Mb. Duplicate pairs identified in this study are connected using lines in different colors, i.e., tandem (shown in green), transposed (shown in purple), dispersed (shown in gold), and WGD (shown in red). (b) Synteny analysis within and between C. esculentus, O. sativa, and A. thaliana. (c) Synteny analysis within and between C. esculentus, C. cristatella, R. breviuscula, and J. effusus. Shown are PIP-encoding chromosomes/scaffolds and only syntenic blocks that contain PIP genes are marked, i.e., red and purple for intra- and inter-species, respectively. (At: A. thaliana; Cc: C. cristatella; Ce: C. esculentus; Je: J. effusus; Mb: megabase; PIP: plasma membrane intrinsic protein; Os: O. sativa; Rb: R. breviuscula; Scf: scaffold; WGD: whole-genome duplication).
    DownLoad: Full-Size Img PowerPoint
    Table 2.  Sequence identity and evolutionary rate of homologous PIP gene pairs identified in C. esculentus. Ks and Ka were calculated using PAML.
    Duplicate 1 Duplicate 2 Identity (%) Ka Ks Ka/Ks
    CePIP1;1 CePIP1;3 78.70 0.0750 1.2287 0.0610
    CePIP1;2 CePIP1;1 77.20 0.0894 1.2522 0.0714
    CePIP2;1 CePIP2;4 74.90 0.0965 1.7009 0.0567
    CePIP2;3 CePIP2;2 70.50 0.1819 1.2778 0.1424
    CePIP2;4 CePIP2;2 66.50 0.2094 1.5474 0.1353
    CePIP2;5 CePIP2;4 87.30 0.0225 0.4948 0.0455
    CePIP2;6 CePIP2;5 84.90 0.0545 0.5820 0.0937
    CePIP2;7 CePIP2;6 78.70 0.0894 1.0269 0.0871
    CePIP2;8 CePIP2;4 72.90 0.1401 1.2641 0.1109
    CePIP2;9 CePIP2;10 76.40 0.1290 0.8591 0.1502
    CePIP2;10 CePIP2;8 64.90 0.2432 3.0117 0.0807
    CePIP2;11 CePIP2;10 91.20 0.0562 0.0971 0.5783
    Ce: C. esculentus; Ka: nonsynonymous substitution rate; Ks: synonymous substitution rate; PIP: plasma membrane intrinsic protein.
     | Show Table
    DownLoad: CSV

    According to inter-species syntenic analysis, six out of 14 CePIP genes were shown to have syntelogs in rice, including 1:1, 1:2, and 2:2 (i.e. CePIP1;1 vs OsPIP1;3, CePIP1;3 vs OsPIP1;2/-1;1, CePIP2;1 vs OsPIP2;4, CePIP2;2/-2;4 vs OsPIP2;3/-2;2, and CePIP2;8 vs OsPIP2;6), in striking contrast to a single one found in Arabidopsis (i.e. CePIP1;2 vs AtPIP1;2). Correspondingly, only OsPIP1;2 in rice was shown to have syntelogs in Arabidopsis, i.e., AtPIP1;3 and -1;4 (Fig. 2b). These results are consistent with their taxonomic relationships that tigernut and rice are closely related[50,51], and also imply lineage-specific evolution after their divergence.

    Comparative genomics analyses reveals lineage-specific evolution of PIP genes in Cyperaceae

    As described above, phylogenetic and syntenic analyses showed that the last common ancestor of tigernut and rice is more likely to possess only two PIP1s and three PIP2s. However, it is not clear whether the gene expansion observed in tigernut is species-specific or Cyperaceae-specific. To address this issue, recently available genomes were used to identify PIP subfamily genes from C. cristatella, R. breviuscula, and J. effuses, resulting in 15, 13, and nine members, respectively. Interestingly, in contrast to a high number of tandem repeats found in Cyperaceae species, only one pair of tandem repeats (i.e., JePIP2;3 and -2;4) were identified in J. effusus, a close outgroup species to Cyperaceae in the Juncaceae family[36,37]. According to homologous analysis, a total of 12 orthogroups were identified, where JePIP genes belong to PIP1A (JePIP1;1), PIP1B (JePIP1;2), PIP1C (JePIP1;3), PIP2A (JePIP2;1), PIP2B (JePIP2;2), PIP2F (JePIP2;3 and -2;4), PIP2G (JePIP2;5), and PIP2H (JePIP2;6) (Table 3). Further intra-species syntenic analysis revealed that JePIP1;1/-1;2 and JePIP2;2/-2;3 are located within syntenic blocks, which is consistent with CePIP1;1/-1;2, CePIP2;2/-2;4, CcPIP1;1/-1;2, CcPIP2;3/-2;4, RbPIP1;1/-1;2, and RbPIP2;2/-2;5 (Fig. 2c), implying that PIP1A/PIP1B and PIP2B/PIP2D were derived from WGDs occurred sometime before Cyperaceae-Juncaceae divergence. After the split with Juncaceae, tandem duplications frequently occurred in Cyperaceae, where PIP2B/PIP2C and PIP2D/PIP2E/PIP2F retain in most Cyperaceae plants examined in this study. By contrast, species-specific expansion was also observed, i.e., CePIP2;4/-2;5, CePIP2;10/-2;11, CcPIP1;2/-1;3, CcPIP2;4/-2;5, CcPIP2;8/-2;9, CcPIP2;10/-2;11, RbPIP2;3/-2;4, and RbPIP2;9/-2;10 (Table 3 & Fig. 2c).

    Table 3.  Twelve proposed orthogroups based on comparison of representative plant species.
    Orthogroup C. esculentus C. cristatella R. breviuscula J. effusus O. sativa A. thaliana
    PIP1A CePIP1;1 CcPIP1;1 RbPIP1;1 JePIP1;1 OsPIP1;3 AtPIP1;1, AtPIP1;2,
    AtPIP1;3, AtPIP1;4,
    AtPIP1;5
    PIP1B CePIP1;2 CcPIP1;2, CcPIP1;3 RbPIP1;2 JePIP1;2 − −
    PIP1C CePIP1;3 CcPIP1;4 RbPIP1;3 JePIP1;3 OsPIP1;1, OsPIP1;2 −
    PIP2A CePIP2;1 CcPIP2;1 RbPIP2;1 JePIP2;1 OsPIP2;1, OsPIP2;4,
    OsPIP2;5    		 AtPIP2;1, AtPIP2;2,
    AtPIP2;3, AtPIP2;4,
    AtPIP2;5, AtPIP2;6
    PIP2B CePIP2;2 CcPIP2;2 RbPIP2;2 JePIP2;2 OsPIP2;2, OsPIP2;3 −
    PIP2C CePIP2;3 CcPIP2;3 RbPIP2;3, RbPIP2;4 − − −
    PIP2D CePIP2;4, CePIP2;5 CcPIP2;4, CcPIP2;5 RbPIP2;5 − − −
    PIP2E CePIP2;5 CcPIP2;5 RbPIP2;6 − − −
    PIP2F CePIP2;6 CcPIP2;6 − − − −
    PIP2G CePIP2;7 CcPIP2;7 RbPIP2;7 JePIP2;3, JePIP2;4 − −
    PIP2H CePIP2;8 CcPIP2;8, CcPIP2;9 RbPIP2;8 JePIP2;5 OsPIP2;6 AtPIP2;7, AtPIP2;8
    PIP2I CePIP2;9, CePIP2;10,
    CePIP2;11    		 CcPIP2;10, CcPIP2;11 RbPIP2;9, RbPIP2;10 JePIP2;6 OsPIP2;7, OsPIP2;8 −
    At: A. thaliana; Cc: C. cristatella; Ce: C. esculentus; Je: J. effuses; Os: O. sativa; Rb: R. breviuscula; PIP: plasma membrane intrinsic protein.
     | Show Table
    DownLoad: CSV

    Tissue-specific expression analysis of CePIP genes identifies three dominant members

    Tissue-specific expression profiles of CePIP genes were investigated using transcriptome data available for young leaf, mature leaf, sheath, root, rhizome, shoot apex, and tuber. As shown in Fig. 3a, CePIP genes were mostly expressed in roots, followed by sheaths, moderately in tubers, young leaves, rhizomes, and mature leaves, and lowly in shoot apexes. In most tissues, CePIP1;1, -2;1, and -2;8 represent three dominant members that contributed more than 90% of total transcripts. By contrast, in rhizome, these three members occupied about 80% of total transcripts, which together with CePIP1;3 and -2;4 contributed up to 96%; in root, CePIP1;1, -1;3, -2;4, and -2;7 occupied about 84% of total transcripts, which together with CePIP2;1 and -2;8 contributed up to 94%. According to their expression patterns, CePIP genes could be divided into five main clusters: Cluster I includes CePIP1;1, -2;1, and -2;8 that were constitutively and highly expressed in all tissues examined; Cluster II includes CePIP2;2, -2;9, and -2;10 that were lowly expressed in all tested tissues; Cluster III includes CePIP1;2 and -2;11 that were preferentially expressed in young leaf and sheath; Cluster IV includes CePIP1;3 and -2;4 that were predominantly expressed in root and rhizome; and Cluster V includes remains that were typically expressed in root (Fig. 3a). Collectively, these results imply expression divergence of most duplicate pairs and three members (i.e. CePIP1;1, -2;1, and -2;8) have evolved to be constitutively co-expressed in most tissues.

    Figure 3.  Expression profiles of CePIP genes in various tissues, different stages of leaf development, and mature leaves of diurnal fluctuation. (a) Tissue-specific expression profiles of 14 CePIP genes. The heatmap was generated using the R package implemented with a row-based standardization. Color scale represents FPKM normalized log2 transformed counts, where blue indicates low expression and red indicates high expression. (b) Expression profiles of CePIP1;1, -2;1, and -2;8 at different stages of leaf development. (c) Expression profiles of CePIP1;1, -2;1, and -2;8 in mature leaves of diurnal fluctuation. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (p< 0.01). (Ce: C. esculentus; FPKM: Fragments per kilobase of exon per million fragments mapped; PIP: plasma membrane intrinsic protein)
    DownLoad: Full-Size Img PowerPoint

    Expression analysis reveals that CePIP transcripts in leaves are subjected to development regulation

    As shown in Fig. 3a, compared with young leaves, transcriptome profiling showed that CePIP1;2, -2;3, -2;7, -2;8, and -2;11 were significantly down-regulated in mature leaves, whereas CePIP1;3 and -2;1 were up-regulated. To confirm the results, three dominant members, i.e., CePIP1;1, -2;1, and -2;8, were selected for qRT-PCR analysis, which includes three representative stages, i.e., young, mature, and senescing leaves. As shown in Fig. 3b, in contrast to CePIP2;1 that exhibited a bell-like expression pattern peaking in mature leaves, transcripts of both CePIP1;1 and -2;8 gradually decreased during leaf development. These results were largely consistent with transcriptome profiling, and the only difference is that CePIP1;1 was significantly down-regulated in mature leaves relative to young leaves. However, this may be due to different experiment conditions used, i.e., greenhouse vs natural conditions.

    qRT-PCR analysis reveals that the transcription of CePIP1;1, -2;1, and -2;8 in mature leaves is subjected to diurnal regulation

    Diurnal fluctuation expression patterns of CePIP1;1, -2;1, and -2;8 were also investigated in mature leaves and results are shown in Fig. 3c. Generally, transcripts of all three genes in the day (8, 12, 16, and 20 h) were higher than that in the night (24 and 4 h). During the day, both CePIP1;1 and -2;8 exhibited an unimodal expression pattern that peaked at 12 h, whereas CePIP2;1 possessed two peaks (8 and 16 h) and their difference was not significant. Nevertheless, transcripts of all three genes at 20 h (onset of night) were significantly lower than those at 8 h (onset of day) as well as 12 h. In the night, except for CePIP2;1, no significant difference was observed between the two stages for both CePIP1;1 and -2;8. Moreover, their transcripts were comparable to those at 20 h (Fig. 3c).

    Expression profiles of CePIP genes during tuber development

    To reveal the expression patterns of CePIP genes during tuber development, three representative stages, i.e., 40 DAS (early swelling stage), 85 DAS (late swelling stage), and 120 DAS (mature stage), were first profiled using transcriptome data. As shown in Fig. 4a, except for rare expression of CePIP1;2, -2;2, -2;9, and -2;10, most genes exhibited a bell-like expression pattern peaking at 85 DAS, in contrast to a gradual decrease of CePIP2;3 and -2;8. Notably, except for CePIP2;4, other genes were expressed considerably lower at 120 DAS than that at 40 DAS. For qRT-PCR confirmation of CePIP1;1, -2;1, and -2;8, seven stages were examined, i.e., 1, 5, 10, 15, 20, 25, and 35 DAI, which represent initiation, five stages of swelling, and maturation as described before[32]. As shown in Fig. 4b, two peaks were observed for all three genes, though their patterns were different. As for CePIP1;1, compared with the initiation stage (1 DAI), significant up-regulation was observed at the early swelling stage (5 DAI), followed by a gradual decrease except for the appearance of the second peak at 20 DAI, which is something different from transcriptome profiling. As for CePIP2;1, a sudden drop of transcripts first appeared at 5 DAI, then gradually increased until 20 DAI, which was followed by a gradual decrease at two late stages. The pattern of CePIP2;8 is similar to -1;1, two peaks appeared at 5 and 20 DAI and the second peak was significantly lower than the first. The difference is that the second peak of CePIP2;8 was significantly lower than the initiation stage. By contrast, the second peak (20 DAI) of CePIP2;1 was significantly higher than that of the first one (1 DAI). Nevertheless, the expression patterns of both CePIP2;1 and -2;8 are highly consistent with transcriptome profiling.

    Figure 4.  Transcript and protein abundances of CePIP genes during tuber development. (a) Transcriptome-based expression profiling of 14 CePIP genes during tuber development. The heatmap was generated using the R package implemented with a row-based standardization. Color scale represents FPKM normalized log2 transformed counts, where blue indicates low expression and red indicates high expression. (b) qRT-PCR-based expression profiling of CePIP1;1, -2;1, and -2;8 in seven representative stages of tuber development. (c) Relative protein abundance of CePIP1;1, -2;1, and -2;8 in three representative stages of tuber development. Bars indicate SD (N = 3) and uppercase letters indicate difference significance tested following Duncan's one-way multiple-range post hoc ANOVA (p < 0.01). (Ce: C. esculentus; DAI: days after tuber initiation; DAS: days after sowing; FPKM: Fragments per kilobase of exon per million fragments mapped; PIP: plasma membrane intrinsic protein).
    DownLoad: Full-Size Img PowerPoint

    Protein abundances of CePIP1;1, -2;1, and -2;8 in tubers are positively correlated with the moisture content during tuber development

    Since protein abundance is not always in agreement with the transcript level, protein profiles of three dominant members (i.e. CePIP1;1, -2;1, and -2;8) during tuber development were further investigated. For this purpose, we first took advantage of available proteomic data to identify CePIP proteins, i.e., leaves, roots, and four stages of tubers (freshly harvested, dried, rehydrated for 48 h, and sprouted). As shown in Supplemental Fig. S2, all three proteins were identified in both leaves and roots, whereas CePIP1;1 and -2;8 were also identified in at least one of four tested stages of tubers. Notably, all three proteins were considerably more abundant in roots, implying their key roles in root water balance.

    To further uncover their profiles during tuber development, 4D-PRM-based protein quantification was conducted in three representative stages of tuber development, i.e., 1, 25, and 35 DAI. As expected, all three proteins were identified and quantified. In contrast to gradual decrease of CePIP2;8, both CePIP1;1 and -2;1 exhibited a bell-like pattern that peaked at 25 DAI, though no significant difference was observed between 1 and 25 DAI (Fig. 4c). The trends are largely in accordance with their transcription patterns, though the reverse trend was observed for CePIP2;1 at two early stages (Fig. 4b & Fig. 4c).

    Subcellular localization analysis reveals that CePIP1;1, -2;1, and -2;8 function in the cell membrane

    As predicted by WoLF PSORT, CePIP1;1, -2;1, and -2;8 may function in the cell membrane. To confirm the result, subcellular localization vectors named pNC-Cam1304-CePIP1;1, pNC-Cam1304-CePIP2;1, and pNC-Cam1304-CePIP2;8 were further constructed. When transiently overexpressed in tobacco leaves, green fluorescence signals of all three constructs were confined to cell membranes, highly coinciding with red fluorescence signals of the plasma membrane marker HbPIP2;3-RFP (Fig. 5).

    Figure 5.  (a) Schematic diagram of overexpressing constructs, (b) subcellular localization analysis of CePIP1;1, -2;1, and -2;8 in N. benthamiana leaves. (35S: cauliflower mosaic virus 35S RNA promoter; Ce: C. esculentus; EGFP: enhanced green fluorescent protein; kb: kilobase; NOS: terminator of the nopaline synthase gene; RFP: red fluorescent protein; PIP: plasma membrane intrinsic protein).
    DownLoad: Full-Size Img PowerPoint

    Discussion

    Water balance is particularly important for cell metabolism and enlargement, plant growth and development, and stress responses[2,19]. As the name suggests, AQPs raised considerable interest for their high permeability to water, and plasma membrane-localized PIPs were proven to play key roles in transmembrane water transport between cells[1,18]. The first PIP was discovered in human erythrocytes, which was named CHIP28 or AQP1, and its homolog in plants was first characterized in Arabidopsis, which is known as RD28, PIP2c, or AtPIP2;3[3,7,53]. Thus far, genome-wide identification of PIP genes have been reported in a high number of plant species, including two model plants Arabidopsis and rice[10,11,13−17,54−56]. By contrast, little information is available on Cyperaceae, the third largest family within the monocot clade that possesses more than 5,600 species[57].

    The tigernut genome encodes 14 PIP genes with three introns and gene expansion was mainly contributed by WGD and transposed/tandem duplications

    Given the crucial roles of water balance for tuber development and crop production, in this study, tigernut, a representative Cyperaceae plant producing high amounts of oil in underground tubers[28,30,32], was employed to study PIP genes. A number of 14 PIP genes representing two phylogenetic groups (i.e., PIP1 and PIP2) or 12 orthogroups (i.e., PIP1A, PIP1B, PIP1C, PIP2A, PIP2B, PIP2C, PIP2D, PIP2E, PIP2F, PIP2G, PIP2H, and PIP2I) were identified from the tigernut genome. Though the family amounts are comparative or less than 13–21 present in Arabidopsis, cassava (Manihot esculenta), rubber tree (Hevea brasiliensis), poplar (Populus trichocarpa), C. cristatella, R. breviuscula, banana (Musa acuminata), maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and switchgrass (Panicum virgatum), they are relatively more than four to 12 found in eelgrass (Zostera marina), Brachypodium distachyon, foxtail millet (Setaria italic), J. effuses, Aquilegia coerulea, papaya (Carica papaya), castor been (Ricinus communis), and physic nut (Jatropha curcas) (Supplemental Table S4). Among them, A. coerulea represents a basal eudicot that didn't experience the γ WGD shared by all core eudicots[50], whereas eelgrass is an early diverged aquatic monocot that didn't experience the τ WGD shared by all core monocots[56]. Interestingly, though both species possess two PIP1s and two PIP2s, they were shown to exhibit complex orthologous relationships of 1:1, 2:2, 1:0, and 0:1 (Supplemental Table S5). Whereas AcPIP1;1/AcPIP1;2/ZmPIP1;1/ZmPIP1;2 and ZmPIP2;1/AcPIP2;1 belong to PIP1A and PIP2A identified in this study, AcPIP2;2 and ZmPIP2;2 belong to PIP2H and PIP2I, respectively (Supplemental Table S5), implying that the last common ancestor of monocots and eudicots possesses only one PIP1 and two PIP2s followed by clade-specific expansion. A good example is the generation of AtPIP1;1 and -2;6 from AtPIP1;5 and -2;1 via the γ WGD, respectively[17].

    In tigernut, extensive expansion of the PIP subfamily was contributed by WGD (2), transposed (2), tandem (5), and dispersed duplications (3). It's worth noting that, two transposed repeats (i.e., CePIP1;1/-1;3 and CePIP2;1/-2;8) are shared by rice, implying their early origin that may be generated sometime after the split with the eudicot clade but before Cyperaceae-Poaceae divergence. By contrast, two WGD repeats (i.e., CePIP1;1/-1;2 and CePIP2;2/-2;4) are shared by C. cristatella, R. breviuscula, and J. effusus but not rice and Arabidopsis, implying that they may be derived from WGDs that occurred sometime after Cyperaceae-Poaceae split but before Cyperaceae-Juncaceae divergence. The possible WGD is the one that was described in C. littledalei[58], though the exact time still needs to be studied. Interestingly, compared with Arabidopsis (1) and rice (2), tandem/proximal duplications played a more important role in the expansion of PIP genes in tigernut (5) as well as other Cyperaceae species tested (5–6), which were shown to be Cyperaceae-specific or even species-specific. These tandem repeats may play a role in the adaptive evolution of Cyperaceae species as described in a high number of plant species[14,41]. According to comparative genomics analyses, tandem duplicates experienced stronger selective pressure than genes formed by other modes (WGD, transposed duplication, and dispersed duplication) and evolved toward biased functional roles involved in plant self-defense[41].

    As observed in most species such as Arabidopsis[10,14−17], PIP genes in all Cyperaceae and Juncaceae species examined in this study, i.e., tigernut, C. cristatella, R. breviuscula, and J. effuses, feature three introns with conserved positions. By contrast, zero to three introns was not only found in rice but also in other Poaceae species such as maize, sorghum, foxtail millet, switchgrass, B. distachyon, and barley[54,55], implying lineage/species-specific evolution.

    CePIP1;1, -2;1, and -2;8 are three dominant members that are constitutively expressed in most tissues

    Despite the extensive expansion of PIP genes (PIP2) in tigernut even after the split with R. breviuscula, CePIP1;1, -2;1, and -2;8 were shown to represent three dominant members in most tissues examined in this study, i.e., young leaf, mature leaf, sheath, rhizome, shoot apex, and tuber, though the situation in root is more complex. CePIP1;1 was characterized as a transposed repeat of CePIP1;3, which represents the most expressed member in root. Moreover, its recent WGD repeat CePIP1;2 was shown to be lowly expressed in most tested tissues, implying their divergence. The ortholog of CePIP1;1 in rice is OsPIP1;3 (RWC-3), which was shown to be preferentially expressed in roots, stems, and leaves, in contrast to constitutive expression of OsPIP1;1 (OsPIP1a) and -1;2[59−61], two recent WGD repeats. Injecting the cRNA of OsPIP1;3 into Xenopus oocytes could increase the osmotic water permeability by 2–3 times[60], though the activity is considerably lower than PIP2s such as OsPIP2;2 and -2;2[61−63]. Moreover, OsPIP1;3 was shown to play a role in drought avoidance in upland rice and its overexpression in lowland rice could increase root osmotic hydraulic conductivity, leaf water potential, and relative cumulative transpiration at the end of 10 h PEG treatment[64]. CePIP2;8 was characterized as a transposed repeat of CePIP2;1. Since their orthologs are present in both rice and Arabidopsis (Supplemental Table S3), the duplication event is more likely to occur sometime before monocot-eudicot split. Interestingly, their orthologs in rice, i.e., OsPIP2;1 (OsPIP2a) and -2;6, respectively, are also constitutively expressed[61], implying a conserved evolution with similar functions. When heterologously expressed in yeast, OsPIP2;1 was shown to exhibit high water transport activity[62,64−66]. Moreover, root hydraulic conductivity was decreased by approximately four folds in OsPIP2;1 RNAi knock-down rice plants[64]. The water transport activity of OsPIP2;6 has not been tested, however, it was proven to be an H2O2 transporter that is involved in resistance to rice blast[61]. More work especially transgenic tests may improve our knowledge of the function of these key CePIP genes.

    Transcription of CePIP1;1, -2;1, and -2;8 in leaves is subjected to development and diurnal regulation

    Leaf is a photosynthetic organ that regulates water loss through transpiration. In tigernut, PIP transcripts in leaves were mainly contributed by CePIP1;1, -2;1, and -2;8, implying their key roles. During leaf development, in contrast to gradual decrease of CePIP1;1 and -2;8 transcripts in three stages (i.e. young, mature, and senescing) examined in this study, CePIP2;1 peaked in mature leaves. Their high abundance in young leaves is by cell elongation and enlargement at this stage, whereas upregulation of CePIP2;1 in mature leaves may inform its possible role in photosynthesis[67]. Thus far, a high number of CO2 permeable PIPs have been identified, e.g., AtPIP2;1, HvPIP2;1, HvPIP2;2, HvPIP2;3, HvPIP2;5, and SiPIP2;7[68−70]. Moreover, in mature leaves, CePIP1;1, -2;1, and -2;8 were shown to exhibit an apparent diurnal fluctuation expression pattern that was expressed more in the day and usually peaked at noon, which reflects transpiration and the fact that PIP genes are usually induced by light[11,71−73]. In rice, OsPIP2;4 and -2;5 also showed a clear diurnal fluctuation in roots that peaked at 3 h after the onset of light and dropped to a minimum 3 h after the onset of darkness[11]. Notably, further studies showed that temporal and dramatic induction of OsPIP2;5 around 2 h after light initiation was triggered by transpirational demand but not circadian rhythm[74].

    mRNA and protein abundances of CePIP1;1, -2;1, and -2;8 are positively correlated with the moisture content during tuber development

    As an oil-bearing tuber crop, the main economic goal of tigernut cultivation is to harvest underground tubers, whose development is highly dependent on water available[32,75]. According to previous studies, the moisture content of immature tigernut tubers maintains more than 80.0%, followed by a seed-like dehydration process with a drop of water content to less than 50% during maturation[28,32]. Thereby, the water balance in developmental tubers must be tightly regulated. Like leaves, the majority of PIP transcripts in tubers were shown to be contributed by CePIP1;1, -2;1, and -2;8, which was further confirmed at the protein level. In accordance with the trend of water content during tuber development, mRNA, and protein abundances of CePIP1;1, -2;1, and -2;8 in initiation and swelling tubers were considerably higher than that at the mature stage. High abundances of CePIP1;1, -2;1, and -2;8 at the initiation stage reflects rapid cell division and elongation, whereas upregulation of CePIP1;1 and -2;1 at the swelling stage is in accordance with cell enlargement and active physiological metabolism such as rapid oil accumulation[28,30]. At the mature stage, downregulation of PIP transcripts and protein abundances resulted in a significant drop in the moisture content, which is accompanied by the significant accumulation of late embryogenesis-abundant proteins[23,32]. The situation is highly distinct from other tuber plants such as potato (Solanum tuberosum), which may contribute to the difference in desiccation resistance between two species[32,76]. It's worth noting that, in one study, CePIP2;1 was not detected in any of the four tested stages, i.e., freshly harvested, dried, rehydrated for 48 h, and sprouted tubers[23]. By contrast, it was quantified in all three stages of tuber development examined in this study, i.e., 1, 25, and 35 DAI (corresponding to freshly harvested tubers), which represent initiation, swelling, and maturation. One possible reason is that the protein abundance of CePIP2;1 in mature tubers is not high enough to be quantified by nanoLC-MS/MS, which is relatively less sensitive than 4D-PRM used in this study[30,46]. In fact, nanoLC-MS/MS-based proteomic analysis of 30 samples representing six tissues/stages only resulted in 2,257 distinct protein groups[23].

    Taken together, our results imply a key role of CePIP1;1, -2;1, and -2;8 in tuber water balance, however, the mechanism underlying needs to be further studied, e.g., posttranslational modifications, protein interaction patterns, and transcriptional regulators.

    Conclusions

    To our knowledge, this is the first genome-wide characterization of PIP genes in tigernut, a representative Cyperaceae plant with oil-bearing tubers. Fourteen CePIP genes representing two phylogenetic groups or 12 orthogroups are relatively more than that present in two model plants rice and Arabidopsis, and gene expansion was mainly contributed by WGD and transposed/tandem duplications, some of which are lineage or even species-specific. Among these genes, CePIP1;1, -2;1, and -2;8 have evolved to be three dominant members that are constitutively expressed in most tissues, including leaf and tuber. Transcription of these three dominant members in leaves are subjected to development and diurnal regulation, whereas in tubers, their mRNA and protein abundances are positively correlated with the moisture content during tuber development. Moreover, their plasma membrane-localization was confirmed by subcellular localization analysis, implying that they may function in the cell membrane. These findings shall not only provide valuable information for further uncovering the mechanism of tuber water balance but also lay a solid foundation for genetic improvement by regulating these key PIP members in tigernut.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design, supervision: Zou Z; analysis and interpretation of results: Zou Z, Zheng Y, Xiao Y, Liu H, Huang J, Zhao Y; draft manuscript preparation: Zou Z, Zhao Y. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All the relevant data is available within the published article.

    Acknowledgments

    This work was supported by the Hainan Province Science and Technology Special Fund (ZDYF2024XDNY171 and ZDYF2024XDNY156), China; the National Natural Science Foundation of China (32460342, 31971688 and 31700580), China; the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2022-66), China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

  • The authors declare that they have no conflict of interest.

  • [1]

    Liu X, Liu Y, Huang P, Ma Y, Qing Z, et al. 2017. The genome of medicinal plant Macleaya cordata provides new insights into benzylisoquinoline alkaloids metabolism. Molecular Plant 10(7):975−89

    doi: 10.1016/j.molp.2017.05.007

    CrossRef   Google Scholar

    [2]

    Huang P, Zhang Y, Xiao K, Jiang F, Wang H, et al. 2018. The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids. Microbiome 6:211

    doi: 10.1186/s40168-018-0590-5

    CrossRef   Google Scholar

    [3]

    Lin L, Liu YC, Huang JL, Liu XB, Qing ZX, et al. 2018. Medicinal plants of the genus Macleaya (Macleaya cordata, Macleaya microcarpa): a review of their phytochemistry, pharmacology, and toxicology. Phytotherapy Research: PTR 32:19−48

    doi: 10.1002/ptr.5952

    CrossRef   Google Scholar

    [4]

    Tani C, Takao S. 1967. Studies on the alkaloids of fumariaceous plants. IX. alkaloids of Bocconia frutescens. Yakugaku Zasshi: Journal of the Pharmaceutical Society of Japan 87:699−701

    doi: 10.1248/yakushi1947.87.6_699

    CrossRef   Google Scholar

    [5]

    Takao N, Yasumoto Y, Iwasa K. 1973. Studies on the alkaloids of papaveraceous plants. XV. on the alkaloids of Bocconia cordata. (2). Yakugaku Zasshi: Journal of the Pharmaceutical Society of Japan 93(2):242−44

    doi: 10.1248/yakushi1947.93.2_242

    CrossRef   Google Scholar

    [6]

    Qing ZX, Yang P, Yu K, Yang XY, Liu JH, et al. 2017. Mass spectrometry-guided isolation of two new dihydrobenzophenanthridine alkaloids from Macleaya cordata. Natural Product Research 31:1633−39

    doi: 10.1080/14786419.2017.1285300

    CrossRef   Google Scholar

    [7]

    Qing ZX, Xu YQ, Yang P, Yu K, Cheng P, et al. 2016. Mass spectrometry-guided isolation of two new benzoquinoline alkaloids from Macleaya cordata. Natural Product Research 30(9):1030−35

    doi: 10.1080/14786419.2015.1101695

    CrossRef   Google Scholar

    [8]

    Zou HL, Li HY, Liu BL, Zhou GX. 2015. A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata. Journal of Asian Natural Products Research 17(8):856−60

    doi: 10.1080/10286020.2015.1016000

    CrossRef   Google Scholar

    [9]

    Yu K, Peng Y, Qing Z, Yang P, Zuo Z, et al. 2017. Two pairs of new dihydrobenzophenanthridine alkaloid isolated from the root of Macleaya cordata. Phytochemistry Letters 22:9−12

    doi: 10.1016/j.phytol.2017.08.002

    CrossRef   Google Scholar

    [10]

    Qing Z, Yan F, Huang P, Zeng J. 2021. Establishing the metabolic network of isoquinoline alkaloids from the Macleaya genus. Phytochemistry 185:112696

    doi: 10.1016/j.phytochem.2021.112696

    CrossRef   Google Scholar

    [11]

    Qing ZX, Cheng P, Liu XB, Liu YS, Zeng JG, et al. 2014. Structural speculation and identification of alkaloids in Macleaya cordata fruits by high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry combined with a screening procedure. Rapid Communications in Mass Spectrometry: RCM 28(9):1033−44

    doi: 10.1002/rcm.6874

    CrossRef   Google Scholar

    [12]

    Qing ZX, Liu XB, Wu HM, Cheng P, Liu YS, et al. 2015. An improved separation method for classification of Macleaya cordata from different geographical origins. Analytical Methods 7(5):1866−71

    doi: 10.1039/C4AY02600D

    CrossRef   Google Scholar

    [13]

    Qing ZX, Cheng P, Liu XB, Liu YS, Zeng JG. 2015. Systematic identification of alkaloids in Macleaya microcarpa fruits by liquid chromatography tandem mass spectrometry combined with the isoquinoline alkaloids biosynthetic pathway. Journal of Pharmaceutical and Biomedical Analysis 103:26−34

    doi: 10.1016/j.jpba.2014.11.002

    CrossRef   Google Scholar

    [14]

    Qing ZX, Yang P, Tang Q, Cheng P, Liu XB, et al. 2017. Isoquinoline alkaloids and their antiviral, antibacterial, and antifungal activities and structure-activity relationship. Current Organic Chemistry 21(18):1920−34

    doi: 10.2174/1385272821666170207114214

    CrossRef   Google Scholar

    [15]

    Qing ZX, Huang JL, Yang XY, Liu JH, Cao HL, et al. 2018. Anticancer and reversing multidrug resistance activities of natural isoquinoline alkaloids and their structure-activity relationship. Current Medicinal Chemistry 25(38):5088−114

    doi: 10.2174/0929867324666170920125135

    CrossRef   Google Scholar

    [16]

    Liu ZH, Wang WM, Zhang Z, Sun L, Wu SC. 2022. Natural antibacterial and antivirulence alkaloids from Macleaya cordata against methicillin-resistant Staphylococcus aureus. Frontiers in Pharmacology 13:813172

    doi: 10.3389/fphar.2022.813172

    CrossRef   Google Scholar

    [17]

    Cheng P, Zeng J. 2012. Progresses in synthesis of benzophenanthridine alkaloids and their derivatives. Chinese Journal of Organic Chemistry 32(9):1605−19

    doi: 10.6023/cjoc201204008

    CrossRef   Google Scholar

    [18]

    Harayama T, Akamatsu H, Okamura K, Miyagoe T, Akiyama T, et al. 2001. Synthesis of trisphaeridine and norchelerythrine through palladium-catalyzed aryl–aryl coupling reaction. Journal of the Chemical Society, Perkin Transactions 1(5):523−28

    doi: 10.1039/b008683p

    CrossRef   Google Scholar

    [19]

    Vicario JL, Badı́a D, Domı́nguez E, Carrillo L. 2000. Stereocontrolled synthesis of 2-aryl tetralones. Application in the synthesis of B/C hexahydrobenzo[c]phenanthridine alkaloids. Tetrahedron: Asymmetry 11(5):1227−37

    doi: 10.1016/s0957-4166(00)00042-2

    CrossRef   Google Scholar

    [20]

    Ishikawa T, Shimooka K, Narioka T, Noguchi S, Saito T, et al. 2000. Anomalous substituent effects in the bischler-napieralski reaction of 2-aryl aromatic formamides. The Journal of Organic Chemistry 65(26):9143−51

    doi: 10.1021/jo0012849

    CrossRef   Google Scholar

    [21]

    Watanabe T, Ohashi Y, Yoshino R, Komano N, Eguchi M, et al. 2003. Total synthesis of 12-methoxydihydrochelerythrine and anti-tumour activity of its quaternary base: toward an efficient synthetic route for 12-alkoxybenzo[c]phenanthridine bases via naphthoquinone monooxime from 2-benzofuranyl-1-tetralone derivative. Organic & Biomolecular Chemistry 1(17):3024−32

    doi: 10.1039/b304216m

    CrossRef   Google Scholar

    [22]

    Ishihara Y, Azuma S, Choshi T, Kohno K, Ono K, et al. 2011. Total synthesis of benzo[c]phenanthridine alkaloids based on a microwave-assisted electrocyclic reaction of the aza 6π-electron system and structural revision of broussonpapyrine. Tetrahedron 67(6):1320−33

    doi: 10.1016/j.tet.2010.11.066

    CrossRef   Google Scholar

    [23]

    Le TN, Gang SG, Cho WJ. 2004. A versatile total synthesis of benzo[c]phenanthridine and protoberberine alkaloids using lithiated toluamide-benzonitrile cycloaddition. The Journal of Organic Chemistry 69(8):2768−72

    doi: 10.1021/jo035836+

    CrossRef   Google Scholar

    [24]

    Lynch MA, Duval O, Sukhanova A, Devy J, MacKay SP, et al. 2001. Synthesis, biological activity and comparative analysis of DNA binding affinities and human DNA topoisomerase I inhibitory activities of novel 12-alkoxy-benzo[c]phenanthridinium salts. Bioorganic & Medicinal Chemistry Letters 11(19):2643−46

    doi: 10.1016/s0960-894x(01)00520-0

    CrossRef   Google Scholar

    [25]

    Sai CM, Li DH, Li SG, Han T, Guo YZ, et al. 2016. Racemic alkaloids from Macleaya cordata: structural elucidation, chiral resolution, and cytotoxic, antibacterial activities. RSC Advances 6(47):41173−80

    doi: 10.1039/C6RA05423D

    CrossRef   Google Scholar

    [26]

    Deng AJ, Qin HL. 2010. Cytotoxic dihydrobenzophenanthridine alkaloids from the roots of Macleaya microcarpa. Phytochemistry 71(7):816−22

    doi: 10.1016/j.phytochem.2010.02.007

    CrossRef   Google Scholar

    [27]

    Wang L, Wang X, Wang W, Liu W, Liu Y, et al. 2021. Visible-light-promoted biomimetic reductive functionalization of quaternary benzophenanthridine alkaloids. Journal of Natural Products 84(8):2390−97

    doi: 10.1021/acs.jnatprod.1c00512

    CrossRef   Google Scholar

    [28]

    Wang X, Wang L, Zhang J, Liu Y, Xie H, et al. 2023. Photoredox catalysed reductive aminomethylation of quaternary benzophenanthridine alkaloids. Natural Product Research 37(21):3551−55

    doi: 10.1080/14786419.2022.2092732

    CrossRef   Google Scholar

    [29]

    Qing Z, Cao H, Cheng P, Wang W, Zeng J, et al. 2018. Visible light photoredox catalyzed semisynthesis of the analogues of maclekarpine E: a series of 6-vinyl substituted dihydrobenzophenanthridine alkaloids. Organic Chemistry Frontiers 5(3):353−57

    doi: 10.1039/C7QO00617A

    CrossRef   Google Scholar

    [30]

    Cheng P, Wang W, Wang X, Wang L, Liu W, et al. 2022. Biomimetic synthesis of 6-substituted dihydrobenzophenanthridine alkaloids based on visible-light promoted radical addition reaction. Natural Product Research 36(1):341−47

    doi: 10.1080/14786419.2020.1784171

    CrossRef   Google Scholar

    [31]

    Liu W, Zheng X, Zeng J, Cheng P. 2017. Visible light promoted C-H functionalization reactions of tertiary amines. Chinese Journal of Organic Chemistry 37(1):1−19

    doi: 10.6023/cjoc201607040

    CrossRef   Google Scholar

    [32]

    Zeng J, Liu Y, Liu W, Liu X, Liu F, et al. 2013. Integration of transcriptome, proteome and metabolism data reveals the alkaloids biosynthesis in Macleaya cordata and Macleaya microcarpa. PLoS One 8:e53409

    doi: 10.1371/journal.pone.0053409

    CrossRef   Google Scholar

    [33]

    Zuo Z, Zheng Y, Liang Z, Liu Y, Tang Q, et al. 2017. Tissue-specific metabolite profiling of benzylisoquinoline alkaloids in the root of Macleaya cordata by combining laser microdissection with ultra-high-performance liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry: RCM 31(5):397−410

    doi: 10.1002/rcm.7804

    CrossRef   Google Scholar

    [34]

    Xu Z, Xia L, Sun M, Huang P, Zeng J. 2022. Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme. World Journal of Microbiology and Biotechnology 38:77

    doi: 10.1007/s11274-022-03265-w

    CrossRef   Google Scholar

    [35]

    Sun M, Zhong X, Zhou L, Xu Z, Huang P, et al. 2022. Plant–microbe hybrid synthesis provides new insights for the efficient use of Macleaya cordata. World Journal of Microbiology and Biotechnology 38(6):110

    doi: 10.1007/s11274-022-03295-4

    CrossRef   Google Scholar

    [36]

    Liu T, Gou Y, Zhang B, Gao R, Dong C, et al. 2022. Construction of ajmalicine and sanguinarine de novo biosynthetic pathways using stable integration sites in yeast. Biotechnology and Bioengineering 119(5):1314−26

    doi: 10.1002/bit.28040

    CrossRef   Google Scholar

    [37]

    Gou Y, Li D, Zhao M, Li M, Zhang J, et al. 2024. Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast. Nature Communications 15:5238

    doi: 10.1038/s41467-024-49554-w

    CrossRef   Google Scholar

    [38]

    Huang P, Xu M, Xia L, Qing Z, Tang Q, et al. 2017. Establishment of an efficient Agrobacterium-mediated genetic transformation method in Macleaya cordata. Scientia Horticulturae 226:302−6

    doi: 10.1016/j.scienta.2017.09.004

    CrossRef   Google Scholar

    [39]

    Huang P, Liu W, Xu M, Jiang R, Xia L, et al. 2018. Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata. Scientific Reports 8:17988

    doi: 10.1038/s41598-018-36211-8

    CrossRef   Google Scholar

    [40]

    Sun M, Liu X, Liu W, Xu Z, Wang Y, et al. 2022. The effects of protopine 6-hydroxylase (P6H) overexpression on benzylisoquinoline alkaloids in Macleaya cordata. Plant Cell, Tissue and Organ Culture 148:429−37

    doi: 10.1007/s11240-021-02183-3

    CrossRef   Google Scholar

    [41]

    Huang P, Xia L, Liu W, Jiang R, Liu X, et al. 2018. Hairy root induction and benzylisoquinoline alkaloid production in Macleaya cordata. Scientific Reports 8:11986

    doi: 10.1038/s41598-018-30560-0

    CrossRef   Google Scholar

    [42]

    Zheng Q, Xu Z, Sun M, Liang H, Wang Y, et al. 2021. Hairy root induction and benzylisoquinoline alkaloid production in Macleaya microcarpa. Plant Cell, Tissue and Organ Culture 147:189−96

    doi: 10.1007/s11240-021-02109-z

    CrossRef   Google Scholar

    [43]

    Sun M, Zhong X, Zhou L, Liu W, Song R, et al. 2024. CRISPR/Cas9 revolutionizes Macleaya cordata breeding: a leap in sanguinarine biosynthesis. Horticulture Research 11:uhae024

    doi: 10.1093/hr/uhae024

    CrossRef   Google Scholar

    [44]

    European Commission. 2014. European Union Register of Feed Additives Pursuant to Regulation (EC) No 1831/2003. Brussels, Belgium: European Commission
    [45]

    Liu Y, Han K, Liu H, Jia G, Comer L, et al. 2024. Macleaya cordata isoquinoline alkaloids attenuate Escherichia coli lipopolysaccharide-induced intestinal epithelium injury in broiler chickens by co-regulating the TLR4/MyD88/NF-κB and Nrf2 signaling pathways. Frontiers in Immunology 14:1335359

    doi: 10.3389/fimmu.2023.1335359

    CrossRef   Google Scholar

    [46]

    Drsata J, Ulrichová J, Walterová D. 1996. Sanguinarine and chelerythrine as inhibitors of aromatic amino acid decarboxylase. Journal of Enzyme Inhibition 10(4):231−37

    doi: 10.3109/14756369609036530

    CrossRef   Google Scholar

    [47]

    He X, Zhang S, Yue L, Lin Q. 2015. Sanguinarine instead of tryptophan on growth and metabolism of pig under conditions of lower-protein diets. The FASEB Journal 29:742.10

    doi: 10.1096/fasebj.29.1_supplement.742.10

    CrossRef   Google Scholar

    [48]

    Song H, Wang X, Zhang M, Zou Z, Yang S, et al. 2024. Dual effects of feed-additive-derived chelerythrine in combating mobile colistin resistance. Engineering 32:163−73

    doi: 10.1016/j.eng.2023.06.012

    CrossRef   Google Scholar

    [49]

    Li Y, Xu F, Tong X, Chen R, Shen C, et al. 2020. Effects of Macleaya cordata extract on small intestinal morphology and gastrointestinal microbiota diversity of weaned pigs. Livestock Science 237:104040

    doi: 10.1016/j.livsci.2020.104040

    CrossRef   Google Scholar

    [50]

    Chen J, Kang B, Yao K, Fu C, Zhao Y. 2019. Effects of dietary Macleaya cordata extract on growth performance, immune responses, antioxidant capacity, and intestinal development in weaned piglets. Journal of Applied Animal Research 47:349−56

    doi: 10.1080/09712119.2019.1636800

    CrossRef   Google Scholar

    [51]

    Chen J, Kang B, Zhao Y, Yao K, Fu C. 2018. Effects of natural dietary supplementation with Macleaya cordata extract containing sanguinarine on growth performance and gut health of early-weaned piglets. Journal of Animal Physiology and Animal Nutrition 102(6):1666−74

    doi: 10.1111/jpn.12976

    CrossRef   Google Scholar

    [52]

    Wang F, Yin Y, Yang M, Chen J, Fu C, et al. 2021. Effects of combined supplementation of Macleaya cordata extract and Benzoic acid on the growth performance, immune responses, antioxidant capacity, intestinal morphology, and microbial composition in weaned piglets. Frontiers in Veterinary Science 8:708597

    doi: 10.3389/fvets.2021.708597

    CrossRef   Google Scholar

    [53]

    Matulka RA, Dohms J. 2018. Effects of the consumption of Macleaya cordata extract preparation by sows. Open Journal of Animal Sciences 8(3):294−302

    doi: 10.4236/ojas.2018.83022

    CrossRef   Google Scholar

    [54]

    Li Y, Fan M, Qiu Q, Wang Y, Shen X, et al. 2022. Nano-selenium and Macleaya cordata extracts improved immune function and reduced oxidative damage of sows and IUGR piglets after heat stress of sows in late gestation. Biological Trace Element Research 200(12):5081−90

    doi: 10.1007/s12011-022-03103-y

    CrossRef   Google Scholar

    [55]

    Liu ZY, Wang XL, Ou SQ, Hou DX, He JH. 2020. Sanguinarine modulate gut microbiome and intestinal morphology to enhance growth performance in broilers. PLoS ONE 15:e0234920

    doi: 10.1371/journal.pone.0234920

    CrossRef   Google Scholar

    [56]

    Wang M, Zhang J, Huang X, Liu Y, Zeng J. 2022. Effects of dietary Macleaya cordata extract on growth performance, biochemical indices, and intestinal microbiota of yellow-feathered broilers subjected to chronic heat stress. Animals 12(17):2197

    doi: 10.3390/ani12172197

    CrossRef   Google Scholar

    [57]

    Song B, He J, Pan X, Kong L, Xiao C, et al. 2023. Dietary Macleaya cordata extract supplementation improves the growth performance and gut health of broiler chickens with necrotic enteritis. Journal of Animal Science and Biotechnology 14:113

    doi: 10.1186/s40104-023-00916-2

    CrossRef   Google Scholar

    [58]

    Aljumaah MR, Suliman GM, Abdullatif AA, Abudabos AM. 2020. Effects of phytobiotic feed additives on growth traits, blood biochemistry, and meat characteristics of broiler chickens exposed to Salmonella typhimurium. Poultry Science 99(11):5744−51

    doi: 10.1016/j.psj.2020.07.033

    CrossRef   Google Scholar

    [59]

    Shi Y, Liu Y, Xie K, Dai J, Wang Y, et al. 2023. Dietary sanguinarine ameliorates growth impairment and intestinal dysfunction in Ctenopharyngodon idellus fed a high cottonseed meal diet. Aquaculture 576:739864

    doi: 10.1016/j.aquaculture.2023.739864

    CrossRef   Google Scholar

    [60]

    Liu YL, Zhong L, Chen T, Shi Y, Hu Y, et al. 2020. Dietary sanguinarine supplementation on the growth performance, immunity and intestinal health of grass carp (Ctenopharyngodon idellus) fed cottonseed and rapeseed meal diets. Aquaculture 528:735521

    doi: 10.1016/j.aquaculture.2020.735521

    CrossRef   Google Scholar

    [61]

    Shi Y, Liu Y, Xie K, Zhang J, Wang Y, et al. 2023. Sanguinarine improves intestinal health in grass carp fed high-fat diets: involvement of antioxidant, physical and immune barrier, and intestinal microbiota. Antioxidants 12(7):1366

    doi: 10.3390/antiox12071366

    CrossRef   Google Scholar

    [62]

    Chen K, Liu Y, Cheng Y, Yan Q, Zhou C, et al. 2020. Supplementation of Lactobacillus plantarum or Macleaya cordata extract alleviates oxidative damage induced by weaning in the lower gut of young goats. Animals 10(4):548

    doi: 10.3390/ani10040548

    CrossRef   Google Scholar

    [63]

    Yang C, Cheng Y, Li X, Li H, Yan Q, et al. 2021. Effects of dietary Macleaya cordata extract inclusion on transcriptomes and inflammatory response in the lower gut of early weaned goats. Animal Feed Science and Technology 272:114792

    doi: 10.1016/j.anifeedsci.2020.114792

    CrossRef   Google Scholar

    [64]

    Ling H, Xiao H, Zhang Z, He Y, Zhang P. 2023. Effects of Macleaya cordata extract on performance, nutrient apparent digestibilities, milk composition, and plasma metabolites of dairy goats. Animals 13(4):566

    doi: 10.3390/ani13040566

    CrossRef   Google Scholar

    [65]

    Boulware R, Southard G, Yankell S. 1985. Sanguinaria extract, a new agent for the control of volatile sulfur-compounds in the oral cavity. Journal of the Society of Cosmetic Chemists 36(4):297−302

    Google Scholar

  • Cite this article

    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019
    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019
    shu

Figures(6)

Download PDF

Article Metrics

Article views(2004) PDF downloads(410)

Other Articles By Authors

REVIEW   Open Access    

Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions

  • 1.

    Hunan Key Laboratory of Traditional Chinese Veterinary Medicine, Hunan Agricultural University, Changsha 410128, Hunan, China

  • 2.

    Traditional Chinese Medicine Breeding Center of Yuelushan Laboratory, Changsha 410128, Hunan, China

  • 3.

    College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, Hunan, China

  • 4.

    Hunan Institute of Agricultural Environment and Ecology, Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China

  • 5.

    Micolta Bioresource Inc., Changsha 410016, Hunan, China

  • 6.

    National and Local Union Engineering Research Center of Veterinary Herbal Medicine Resource and Initiative, Hunan Agricultural University, Changsha 410128, Hunan, China

  • Received: 20 May 2024
  • Revised: 19 July 2024
  • Accepted: 26 July 2024
  • Published online: 09 October 2024
Medicinal Plant Biology  3,  Article number: e020  (2024)  |  Cite this article

Abstract: Macleaya cordata, a medicinal plant in the Papaveraceae family, is rich in bioactive benzylisoquinoline alkaloids. Recent research has elucidated the mechanisms by which these active components promote livestock and poultry growth and exhibit anti-inflammatory effects on the intestines. These findings have led to the development of two raw medicinal materials and two veterinary drug formulations, widely used in China's livestock industry. Advances in multi-omics technologies, such as whole-genome sequencing and transcriptomics, have clarified the chemical composition of alkaloids and the biosynthesis of sanguinarine, enabling its de novo synthesis in yeast. Efforts in plant breeding have focused on cultivar selection and germplasm innovation, establishing DUS (Distinctness, Uniformity, and Stability) testing guidelines. Genetic engineering techniques have re-edited the sanguinarine pathway and induced hairy roots and cell suspension cultures in M. cordata. These advancements reduce production costs, ensure product stability, and promote sustainable production. This paper reviews the species origin, current research status, and prospects of M. cordata, offering guidance for further research on this valuable resource.

    HTML

    Introduction

    • Macleaya cordata (Willd.) R. Br., commonly known as boluohui in China, is a perennial, upright herbaceous plant belonging to the Papaveraceae family (Fig. 1)[1]. Modern pharmacological research has identified that plants of the Macleaya genus are rich in benzylisoquinoline alkaloids (BIAs), which exhibit significant anti-inflammatory and antibacterial properties, regulate intestinal microflora, and promote animal growth[2]. The European Food Safety Authority (EFSA) has approved M. cordata as a safe plant for the manufacture of feed additives. In China, four alkaloids found in M. cordata, including sanguinarine (SAN), chelerythrine (CHE), allocryptopine (ALL), and protopine (PRO) have been successfully developed into veterinary medicines (Fig. 1).

      Figure 1. 

      Morphological characteristics of M. cordata. The figure shows various parts of M. cordata, commonly known as boluohui. The main image displays a field of mature plants with characteristic upright growth. The top left inset highlights a single leaf with a broad, lobed structure. The top middle inset shows the flower of M. cordata, featuring clustered blooms with delicate petals. The top right inset illustrates the capsule, the fruiting body containing seeds. These images collectively depict the key morphological features of M. cordata, which is valued for its rich content of benzylisoquinoline alkaloids.

      This review aims to provide a comprehensive overview of the latest advancements in the study of M. cordata. It will cover the plant's resources, chemical composition, pharmacological activities, biosynthesis mechanisms, and breeding strategies. By synthesizing current knowledge, this review seeks to offer a broad perspective on future research and potential applications of M. cordata in various fields.

    Origin and distribution, germplasm, and characteristics of M. cordata

    • The Macleaya genus comprises two species: M. cordata, and M. microcarpa. M. cordata is primarily distributed in China and Japan, originating in the regions south of the Yangtze River and north of the Nanling Mountains in China. It extends south to Guangdong, west to Guizhou, and northwest to southern Gansu. M. cordata predominantly grows in hills, low mountain forests, shrubs, or grasses at altitudes between 150 and 830 m. It is notably found in grasslands or shrubs on slopes at elevations ranging from 450 to 1,600 m in provinces such as Jiangsu, Jiangxi, Shanxi, Gansu, and Shaanxi.

      Both M. cordata, and M. microcarpa are upright herbaceous plants with a woody base, producing a milky yellow sap. Their stems are hollow, with the upper parts branching out, and the leaves are broad ovate or nearly circular. They possess large conical inflorescences that can be both terminal and axillary, with a flowering and fruiting period from June to November. Plants of the Macleaya genus are rich in various chemical constituents, including alkaloids, flavonoids, terpenes, and phenylpropanoids, with alkaloids being the predominant components[3]. Benzylisoquinoline alkaloids, such as sanguinarine, are among the most active components in this genus. Sanguinarine is also abundant in other plants like poppy (Papaver somniferum) and bloodroot (Sanguinaria canadensis); however, due to regulatory restrictions and over-exploitation, these plants are no longer viable sources. Consequently, M. cordata has emerged as the primary commercial source of sanguinarine.

    Chemical resources and biological activities of M. cordata

    • Japanese researchers such as Tani & Takao[4] and Takao[5] isolated alkaloids like sanguinarine, chelerythrine, berberine, and coptisine from M. cordata, initiating the study of its chemical constituents. Subsequently, numerous researchers contributed to the isolation and structural identification of secondary metabolites in the Macleaya genus[6−9]. From 2009 to 2024, Qing et al.[10] developed a new method for the discovery and structural identification of alkaloids in the Macleaya genus using mass spectrometry, identifying over 200 alkaloids. These alkaloids belong to ten major categories: benzylisoquinoline alkaloids, tetrahydroprotoberberine alkaloids, N-methyl-tetrahydroprotoberberine alkaloids, protopine alkaloids, protoberberine alkaloids, 7,8-dihydroprotoberberine alkaloids, aporphine alkaloids, benzophenanthridine alkaloids, dihydrobenzophenanthridine alkaloids, and benzophenanthridine dimers (Fig. 2). Using mass spectrometry-guided techniques, over 20 new compounds were isolated from Macleaya species, established a mass spectrometry database and metabolic profile for alkaloids in Macleaya, and proposed new biosynthetic pathways for berberine and sanguinarine[10−13]. In addition to a large number of alkaloids, Macleaya species also contain small amounts of flavonoids and polyphenols[3]. The rich chemical composition of Macleaya endows it with various biological activities. Modern pharmacological studies have shown that Macleaya possesses antibacterial, anti-inflammatory, insecticidal, antitumor, antifibrotic, hepatoprotective, antiviral, antioxidant, immune-enhancing, gut microbiota-regulating, animal growth-promoting, wastewater-purifying, and soil erosion-preventing properties[13−16].

      Figure 2. 

      Types of compounds identified in Macleaya species.

    Total synthesis, semi-synthesis, and structural modification of alkaloids in M. cordata

      Total synthesis of major secondary metabolites in M. cordata

    • The primary active secondary metabolites in Macleaya cordata are protopine alkaloids and quaternary benzo[c]phenanthridine alkaloids. To date, there have been no reports in the literature regarding the total chemical synthesis of protopine alkaloids. The total chemical synthesis of quaternary benzo[c]phenanthridine alkaloids is generally achieved through multi-step reactions using conventional chemical reagents[17]. In most reported synthetic routes, the cyclization reactions to form rings B and C constitute the final steps in constructing the tetracyclic system. Key chemical reactions for constructing ring B include Heck coupling to form the C10a-C11 bond[18], Pictet-Spengler reaction (PS reaction) or Bischler-Napieralski reaction (BN reaction) to form the C6-C6a bond[19,20], amidation to form the N5-C6 bond[21], and electrocyclization to form the N5-C5a bond[22]. For ring C, key reactions include enamide-aldehyde cyclization to form the C11-C11a bond[23] and Friedel-Crafts reaction to form the C12-C12a bond[24]. These reactions have been widely applied in the synthesis of benzo[c]phenanthridine derivatives with anticancer activity (Fig. 3).

      Figure 3. 

      Total synthesis of quaternary benzo[c]phenanthridine alkaloids, the major active components of M. cordata. This figure illustrates the chemical structure of quaternary benzo[c]phenanthridine alkaloids, highlighting the tetracyclic framework composed of rings A, B, C, and D. These alkaloids are notable for their complex ring system and significant biological activities. The positions of the rings are labeled for clarity, facilitating understanding of the synthetic pathways and structural modifications discussed in the study.

      Semi-synthesis and structural modification of novel 6-substituted dihydrobenzo[c]phenanthridine alkaloids in M. cordata

    • In recent years, with the deepening research on the chemical constituents of M. cordata, a series of 6-substituted dihydrobenzo[c]phenanthridine alkaloids have been successively isolated and structurally confirmed (Fig. 4)[6,9,25,26]. Currently, studies on the biological activities of these alkaloids are still in their infancy, and no biosynthetic pathways for 6-substituted dihydrobenzo[c]phenanthridine alkaloids have been reported. Based on the structural types and chemical properties of the C-6 substituents, it is speculated that these alkaloids may be biosynthesized in plants through radical reactions or nucleophilic substitution pathways. As shown in Fig. 4, quaternary benzo[c]phenanthridine alkaloids or dihydrobenzo[c]phenanthridine can be converted to α-amino carbon radicals through single-electron reduction[27,28] or oxidation[29,30]. These radicals can then undergo various types of radical reactions to achieve functionalization at C-6 of the benzo[c]phenanthridine ring structure, resulting in the semi-synthesis of the aforementioned alkaloids[31]. Additionally, the N5=C6 double bond in quaternary benzo[c]phenanthridine alkaloids can accept nucleophilic reagents, leading to the formation of 6-substituted dihydrobenzo[c]phenanthridine alkaloids.

      Figure 4. 

      Novel 6-substituted dihydrobenzophenanthridine alkaloids in Macleaya and their semi-synthesis.

    Multi-omics analysis of the biosynthesis mechanism of sanguinarine in M. cordata

      Discovery of the alkaloid biosynthesis pathway in M. cordata

    • To date, nearly 300 secondary metabolites, including 204 isoquinoline alkaloids have been identified from M. cordata using untargeted LC-MS metabolomics technology[10]. This breakthrough surpasses the limitations of traditional methods for isolating and identifying secondary metabolites in plants, guiding the targeted analysis of alkaloid metabolism and metabolic profiling in M. cordata. Using untargeted LC-MS metabolomics technology, nearly 2,000 characteristic ions were discovered in M. cordata. Through stable isotope 13C6-labeled tyrosine tracing experiments, 179 13C6-labeled compounds were identified[1]. A database search for the non-labeled counterparts of these 13C6-labeled compounds quickly identified 40 alkaloids and four non-alkaloids, providing evidence for determining the biosynthetic pathways of sanguinarine and chelerythrine in M. cordata.

      Targeted analysis of the metabolic accumulation patterns of intermediates in the biosynthesis of sanguinarine and chelerythrine in different developmental stages and organs of M. cordata, combined with transcriptomics, proteomics, and metabolomics analysis revealed the biosynthetic tissues and gene expression patterns of alkaloid biosynthesis at different growth stages. This study elucidated the biosynthetic pathways of sanguinarine and chelerythrine and discovered the pattern of synthesis by 'root-pod compartmentalized synthesis', where precursor compounds like protopine are primarily synthesized in the roots and then transported to the pods for the synthesis of sanguinarine[1,32]. Laser microdissection with fluorescence detection was used to separate different tissue cells in the roots of M. cordata. Combined with LC-MS targeted analysis of trace alkaloids in each tissue, this approach accurately identified the storage cells of the alkaloids, clarifying the 'synthesis-storage-transport-resynthesis' biosynthetic mechanism of sanguinarine, chelerythrine, and their precursors[33] (Fig. 5).

      Figure 5. 

      Isotope tracing of the biosynthetic pathways of sanguinarine and chelerythrine in Macleaya.

      Integrated analysis of genomics, transcriptomics, metabolomics, and proteomics in M. cordata to elucidate the mechanisms of sanguinarine storage, synthesis, and transport

    • In 2013, Zeng completed an integration of transcriptomic, proteomic, and metabolomic data to elucidate the biosynthesis of alkaloids in M. cordata and M. macrocarpa[32]. This study investigated the potential mechanisms of alkaloid biosynthesis in Macleaya species by analyzing transcriptomic, proteomic, and metabolomic data from 10 different samples collected at various times, tissues, and organs. The assembled and clustered transcriptomic data yielded 69,367 unigenes for M. cordata and 78,255 unigenes for M. macrocarpa. Through a multi-level comparative analysis of key gene expressions controlling enzymes in the alkaloid metabolic pathway, the research identified homologous genes for all functional genes in the sanguinarine biosynthesis pathway. This annotation provided foundational data for cloning functional genes in the sanguinarine pathway of M. cordata.

      In 2017, researchers first mapped the complete genome of M. cordata, making it the first Papaveraceae plant to have its whole genome sequenced[1]. The genomic results indicated that the genome size of M. cordata is 540.5 Mb with a heterozygosity rate of 0.92%, predicting 22,328 protein-coding genes, of which 43.5% are transposable elements. Additionally, 1,355 non-coding RNA (ncRNA) genes were identified in the M. cordata genome, including 216 rRNA, 815 tRNA, 75 miRNA, and 249 snRNA genes. Comparative genomics revealed a significant expansion of the flavoprotein oxidase gene family closely associated with sanguinarine synthesis, providing crucial insights into the evolutionary origin of the sanguinarine biosynthetic pathway in Macleaya. Through co-expression analysis of genes and metabolites, researchers successfully identified 14 genes, including flavoprotein oxidases, methyltransferases, and cytochrome P450 enzymes, involved in the biosynthesis of sanguinarine.

    Metabolic engineering for the biosynthesis of sanguinarine

    • Recent advancements in genetic engineering have led to a series of modifications on the berberine bridge enzyme (McBBE), a rate-limiting enzyme in the biosynthesis of sanguinarine in M. cordata. These modifications included codon optimization and the design of N-terminal truncated mutants to enhance expression efficiency in heterologous hosts. By employing CRISPR-Cas9 gene editing technology, these optimized genes were successfully integrated into the genome of Saccharomyces cerevisiae. This integration resulted in a significant increase in the production of the key precursor, chelerythrine, achieving a yield 58 times higher than the original level[34]. This accomplishment not only underscores the tremendous potential of genetic engineering in enhancing the synthesis of secondary metabolites but also provides a crucial foundation for subsequent industrial production. Furthermore, the research team developed innovative methods for producing sanguinarine through a 'plant-microbe' co-fermentation system. This system involved the co-cultivation of engineered yeast strains with the non-medicinal parts of M. cordata — specifically, the leaves — in a specially designed co-fermentation reactor[35]. This novel approach effectively converted precursor substances from the leaves into sanguinarine, maximizing the utilization of the non-medicinal parts of M. cordata and reducing production costs. This co-fermentation system not only offers an efficient and sustainable new pathway for the production of valuable bioactive compounds like sanguinarine but also exemplifies the integration of plant biology and microbial biotechnology.

      In 2022, significant progress was made by Jiachang Lian's team, who successfully achieved the de novo biosynthesis of sanguinarine, reaching a yield of 16.5 mg/L[36]. After comprehensive metabolic engineering modifications, the current best-engineered yeast strain achieved a production titer of 448.64 mg/L for sanguinarine[37]. This achievement highlights the immense potential of modern biotechnology in promoting the effective utilization and industrial production of components from traditional medicinal plants. By leveraging advanced genetic engineering techniques, researchers have been able to enhance the efficiency of secondary metabolite production, opening up new possibilities for the pharmaceutical and biotechnology industries.

      The innovative approaches employed in this research, such as codon optimization, N-terminal truncation, and the use of CRISPR-Cas9 for gene editing, represent significant advancements in the field of synthetic biology. These methods not only improve the expression efficiency of key enzymes but also pave the way for the scalable production of complex bioactive compounds. The successful integration of plant and microbial systems in a co-fermentation setup demonstrates a promising strategy for sustainable and cost-effective production processes. This research not only advances our understanding of biosynthetic pathways but also sets the stage for future developments in the industrial application of traditional medicinal plant components

    Breeding research on M. cordata

      Molecular breeding of M. cordata

    • In the fields of plant science and biotechnology, artificial genetic transformation technology has become a key tool for molecular breeding. In 2016, a genetic transformation system for Macleaya cordata based on Agrobacterium tumefaciens was successfully established[38]. Utilizing this system, key genes involved in sanguinarine biosynthesis, such as berberine bridge enzyme MCBBE[39] and MCP6H[40], have been overexpressed, demonstrating the significant potential of genetic engineering in enhancing the production of plant secondary metabolites. Additionally, a hairy root culture system for M. cordata and M. microcarpa mediated by A. rhizogenes has also been successfully developed[41,42]. Recently, a CRISPR/Cas9 gene editing system for M. cordata has been established. By re-editing the sanguinarine biosynthesis pathway, researchers have achieved a significant increase in sanguinarine content in transgenic materials[43]. These advancements highlight the potential of molecular breeding techniques to enhance the yield of valuable secondary metabolites in M. cordata, providing a foundation for future research and industrial applications.

      Study on the domestication and DUS testing guidelines of M. cordata

    • Previously, wild M. cordata capsules were the primary source of raw materials for M. cordata-related products. However, the popularity of these products has led to a significant decline in wild resources year by year. In response, domestic research teams have developed a standardized cultivation technology system for M. cordata. This system encompasses various techniques, including seedling raising, field management, pest and disease control, harvesting, and primary processing, facilitating the shift from wild to cultivated varieties. Additionally, comprehensive research on the current state of resources and breeding has led to the establishment of 30 traits which form the M. cordata DUS testing guidelines. As a result of these efforts, the team has preliminarily identified a superior strain, 'Meibo 1', known for its high fruit yield and elevated haematoxylin content.

    The role of M. cordata in animal production and the mechanism and safety of sanguinarine as a feed antibiotic substitute

    • Sanguinarine from M. cordata as a bioactive compound was initially used in Europe as feed additives to enhance flavor and stimulate appetite in food animals[44]. Studies have shown that M. cordata extracts are safe, with no adverse effects observed in target animals even at ten times the recommended clinical dose. Pharmacodynamic and clinical data indicate that long-term addition of M. cordata extracts at recommended doses has anti-inflammatory and growth-promoting effects, and total protopine alkaloids are effective in treating Escherichia coli-induced diarrhea in poultry.

      Research on the mechanism of antibiotic substitution revealed that M. cordata extract enhances intestinal health by increasing the abundance of lactic acid bacteria, which inhibit pathogenic microorganisms through competitive exclusion[2]. Sanguinarine inhibits the phosphorylation and ubiquitination of I-κB proteins, reducing the dissociation of p50 and p60, thereby inhibiting the activation of the NF-κB signaling pathway. This results in decreased expression of pro-inflammatory cytokines and reduced inflammation levels in animals[45]. Additionally, sanguinarine was found to enhance protein synthesis in animals by inhibiting tryptophan decarboxylase activity, thereby increasing amino acid utilization[46,47]. Based on the growth-promoting effects of M. cordata extract as an antibiotic substitute, Jianguo Zeng's team has developed a comprehensive feed technology focused on 'intestinal health, anti-inflammatory properties, and growth promotion'. This approach avoids the risk of antibiotic resistance associated with traditional growth-promoting antibiotics while still enhancing animal growth performance. This technology supports the development of antibiotic-free feed solutions and the use of non-antibiotic inputs in animal husbandry. Furthermore, chelerythrine in M. cordata, when used as an animal feed additive, interacts with phospholipids on bacterial membranes, increasing membrane fluidity and impairing respiration by disrupting proton motive force and generating reactive oxygen species, leading to reduced intracellular ATP levels. This dual action of downregulating the mobile colistin resistance gene mcr-1 and associated genes offers a new strategy to circumvent colistin resistance. These findings provide strong technical support for the development of antibiotic-free feeding practices and the creation of alternative growth-promoting solutions in animal production[48].

      M. cordata extract is the first traditional Chinese veterinary medicine product approved for feed use in China. It is widely used to improve animal production performance, enhance animal health, and replace growth-promoting antibiotics in feed. Weaning stress is a major factor causing diarrhea and growth inhibition in piglets. Adding M. cordata extract to their diet can improve small intestine morphology, enhance intestinal barrier function, and regulate intestinal microbiota homeostasis, thereby reducing diarrhea and improving growth performance[49−52]. During pregnancy, progressive oxidative stress and parturition stress can lead to metabolic disorders and increased inflammation in sows. Supplementing their diet with M. cordata extract can reduce inflammation, enhance antioxidant capacity, shorten farrowing duration, increase feed intake and milk production during lactation, and ultimately increase the weaning weight of piglets[53,54]. Environmental stress and pathogenic infections are significant threats to poultry intestinal health. Adding M. cordata extract to poultry feed can improve intestinal mucosal morphology, enhance intestinal barrier function, regulate microbial populations, and improve broiler growth performance[55−58]. High-fat and high-protein feed can cause intestinal damage in fish. Supplementing their diet with M. cordata extract can enhance intestinal antioxidant capacity, alleviate intestinal barrier damage, improve microbiota homeostasis, and thus increase survival and growth rates[59−61]. Ruminants have a unique rumen physiology where the microbiota plays a crucial role in nutrient digestion and metabolism. Studies have shown that adding M. cordata extract to dairy cow diets can regulate rumen microbial populations and reduce methane emissions, alleviate intestinal inflammation in weaned lambs[62], promote growth in meat sheep[63], increase milk yield in dairy goats, reduce somatic cell counts in milk, and improves milk quality[64].

      In addition to its applications in animal production, M. cordata extracts have been developed into various products due to their significant insecticidal and antibacterial properties. In 2021, they were registered as a new botanical pesticide in China and the United States for controlling greenhouse plant diseases such as powdery mildew and leaf spot disease. In the bioenergy sector, the combination of M. cordata extracts with trace elements cobalt and nickel can effectively accelerate the fermentation process of agricultural waste, increasing biogas production. Furthermore, Macleaya alkaloids are used in toothpaste and mouthwash for their sustained antibacterial activity, which helps treat periodontal disease and prevent bad breath[65]. These findings highlight the diverse applications and benefits of M. cordata in improving animal health and productivity, controlling plant diseases, and enhancing bioenergy production (Fig. 6).

      Figure 6. 

      This illustration showcases the versatile applications of alkaloids derived from M. cordata. The molecular structures of key alkaloids—chelerythrine, sanguinarine, allocryptopine and protopine—are depicted in the center. These alkaloids are utilized in enhancing digestion and improving feed efficiency in ruminants, boosting immunity and promoting growth in pigs, promoting health and growth in fish, improving health in poultry, serving as natural pesticides for crops, reducing methane emissions from ruminants to lower greenhouse gases, acting as antibacterial ingredients in toothpaste and mouthwash, and exhibiting potential anti-cancer activity in cancer treatment. The alkaloids from M. cordata demonstrate significant potential across various fields, from agriculture to medicine.

    Future challenges and prospects

    • M. cordata, a plant rich in bioactive compounds, holds vast potential for applications in medical and agricultural fields. However, future research faces multiple challenges and opportunities. Firstly, although substantial research has been conducted on the primary alkaloids in M. cordata, their regulatory mechanisms remain unclear. Future studies need to integrate multi-omics data and systems biology approaches to elucidate the molecular mechanisms regulating alkaloid biosynthesis in M. cordata, providing a theoretical foundation for precise metabolic pathway regulation.

      Secondly, the application of molecular breeding and metabolic engineering in M. cordata is still in its infancy. Effectively increasing the yield of specific bioactive compounds through genetic engineering, particularly achieving efficient biosynthesis and accumulation of key alkaloids like sanguinarine, will be a focus of future research. Despite the achievement of de novo synthesis of sanguinarine in yeast, the scalability of this production method remains challenging. It is necessary to assess the economic feasibility of these methods to ensure they can meet industrial demands without compromising quality or sustainability. Additionally, the long-term stability and consistency of genetically engineered strains and their products need to be ensured to avoid potential issues in production and application.

      Lastly, as the medicinal value of M. cordata becomes better understood, ensuring its sustainable use and the conservation of wild resources, while developing more high-value products, will be crucial for future development. Large-scale cultivation and utilization of M. cordata may pose potential environmental impacts, including changes in soil health, biodiversity, and local ecosystems, which are not sufficiently studied and require further investigation and evaluation.

      In summary, while the research and application prospects of M. cordata are promising, they also present a series of scientific and technical challenges. By fostering interdisciplinary collaboration and enhancing the integration of basic and applied research, these challenges can be overcome, enabling the efficient utilization and industrial development of M. cordata resources. Addressing the limitations in current research, particularly in ecological impact and production methods, will provide a more balanced perspective and guide future research toward sustainable and scalable solutions.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Huang P, Zeng J; draft manuscript preparation: Huang P, Cheng P, Sun M, Liu X, Qing Z, Liu Y, Yang Z, Liu H, Li C, Zeng J. All the authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

      Acknowledgments

      • This work was supported by Hunan Provincial Natural Science Foundation of China (2023JJ40367 & 2023JJ30341).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  References (65)
  • About this article
    Cite this article
    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019
    Huang P, Cheng P, Sun M, Liu X, Qing Z, et al. 2024. Systemic review of Macleaya cordata: genetics, biosynthesis of active ingredients and functions. Medicinal Plant Biology 3: e020 doi: 10.48130/mpb-0024-0019
    shu

Catalog

  • HTML

  • Introduction
  • Origin and distribution, germplasm, and characteristics of M. cordata
  • Chemical resources and biological activities of M. cordata
  • Total synthesis, semi-synthesis, and structural modification of alkaloids in M. cordata

  • Total synthesis of major secondary metabolites in M. cordata

  • Semi-synthesis and structural modification of novel 6-substituted dihydrobenzo[c]phenanthridine alkaloids in M. cordata

  • Multi-omics analysis of the biosynthesis mechanism of sanguinarine in M. cordata

  • Discovery of the alkaloid biosynthesis pathway in M. cordata

  • Integrated analysis of genomics, transcriptomics, metabolomics, and proteomics in M. cordata to elucidate the mechanisms of sanguinarine storage, synthesis, and transport

  • Metabolic engineering for the biosynthesis of sanguinarine
  • Breeding research on M. cordata

  • Molecular breeding of M. cordata

  • Study on the domestication and DUS testing guidelines of M. cordata

  • The role of M. cordata in animal production and the mechanism and safety of sanguinarine as a feed antibiotic substitute
  • Future challenges and prospects
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
logo

Contents

Policies

Services

Partnerships

© 2025 Maximum Academic Press, except Open Access articles. MAP is a member of ALPSP, COPE, CrossRef, cOAlition S, OASPA and STM.

Privacy Policy  |  Site Map  |  Terms and Conditions

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return