Early phenylpropanoid biosynthetic steps in Cannabis sativa: link between genes and metabolites

Abstract

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

Figure 1

Phenylpropanoid pathway in Cannabis sativa. PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumaric acid: CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FS flavonol synthase, F3′H flavonol 3′ hydroxylase; COMT, caffeic acid O-methyltransferase; CCR, cinnamoyl-CoA reductase; CAD, cinnamyl alcohol dehydrogenase.

Figure 2

Gene structure and phylogenetic analysis of hemp PAL. (a) Representation of CsPAL genomic sequence (KC970302) and cDNA (KC970300). Black and white boxes indicates CsPAL coding sequence (white box indicates region not covered by PAL genomic clone), grey box indicates the intronic region, solid line represents the fragment used as probe for Southern-blot; (b) The PAL proteins identified from other species were aligned using Clustal X, and the PAL phylogeny was constructed using the neighbor-joining method with the MEGA 5.1 program. The branch lengths are indicated above the branch lines. The clades indicate monophyletic groups of dicots, monocots and fungi. CsPAL is highlighted by a red diamond. * Indicates similarities of C. sativa ESTs (EC 5006722/EC 55372 and EC JK497725) to M. alba and C. roseus PAL, respectively. Accession numbers for protein sequences used to build the PAL tree are reported in Table S2.

Figure 3

Gene structure and phylogenetic analysis of hemp 4CL. (a) Representation of Cs4CL genomic sequence (KC970303) and cDNA (KC970301). Black and white boxes indicates Cs4CL coding sequence (white box indicates region not covered by 4CL genomic clone), grey box indicates the intronic region, solid line represents the fragment used as probe for Southern-blot; (b) The 4CL proteins identified from other species were aligned using Clustal X, and the 4CL phylogeny was constructed using the neighbor-joining method with the MEGA 5.1 program. The branch lengths are indicated above the branch lines. The clades indicate monophyletic groups of dicots, monocots/mosses and fungi. Cs4CL is highlighted by a red diamond. Accession numbers for protein sequences used to build the 4CL tree are reported in Table S2.

Figure 3

Gene structure and phylogenetic analysis of hemp 4CL. (a) Representation of Cs4CL genomic sequence (KC970303) and cDNA (KC970301). Black and white boxes indicates Cs4CL coding sequence (white box indicates region not covered by 4CL genomic clone), grey box indicates the intronic region, solid line represents the fragment used as probe for Southern-blot; (b) The 4CL proteins identified from other species were aligned using Clustal X, and the 4CL phylogeny was constructed using the neighbor-joining method with the MEGA 5.1 program. The branch lengths are indicated above the branch lines. The clades indicate monophyletic groups of dicots, monocots/mosses and fungi. Cs4CL is highlighted by a red diamond. Accession numbers for protein sequences used to build the 4CL tree are reported in Table S2.

Figure 4

Expression analysis by qRT-PCR of PAL, 4CL, C4H, CHS in different hemp tissues: young, mature leaves, stems and roots. (a) Transcript abundances of PAL and 4CL and (b) transcript abundances of C4H and CHS relative to β-tubuline as reference gene were plotted as fold differences compared to stems, with stem expression assigned a value of 1. Values are expressed as the means ± SD of three biological replicates and two technical replicates.

Figure 5

Specific activities of PAL and 4CL in different hemp tissues: young, mature leaves, stems and roots. (a) PAL and TAL activities were measured toward phenylalanine and tyrosine substrates, respectively. (b) 4CL activities were measured toward p-coumaric, cinnamic, caffeic, ferulic, sinapic acids substrates. Specific activities are expressed as the mean values ± SD of three biological replicates.

Figure 6

Aromatic region of ¹H-NMR spectra from different hemp tissues: (a) roots, (b) stems, (c) young leaves, (d) mature leaves. 1: trigonellin, 2: formiate, 3: apigenin-7-O-glucoside, 4: cytidine, 5: unknown, 6: luteolin-7-O-glucoside, 7: fumarate, 8: unknown, 9: unknown.