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. 2024 Jun 10;24(1):520.
doi: 10.1186/s12870-024-05178-2.

Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures

Shahla Amani  1 , Mehdi Mohebodini  2 , Shahram Khademvatan  3 , Morad Jafari  4 , Vinod Kumar  5
Affiliations

Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures

Shahla Amani et al. BMC Plant Biol. .

Abstract

Background: One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah.

Methods: The elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control.

Results: Elicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3'-hydroxylase (F3'H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds.

Conclusions: This study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.

Keywords: Piriformospora indica; Rhizobium (Agrobacterium) rhizogenes; Fungal elicitor; Hairy roots; Methyl jasmonate; Secondary metabolites.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The in vitro culture of F. carica cv. Siah followed by induction, establishment and elicitation of hairy roots: in vitro F. carica cv. Siah plantlet (a); induced hairy root on the leaf of shoot explant (b); hairy root lines (c); highest growth hairy root line (L10) on solid medium culture (d); L10 hairy root line transferred to liquid culture (e); the growth of L10 hairy root line on liquid culture (f and g); L10 hairy root line before elicitation (h); L10 hairy root line after MeJA elicitation (i); L10 hairy root line after fungal elicitation (j)
Fig. 2
Fig. 2
The growth curve of the superior hairy root line (L10) of F. carica cv. Siah. Lag phase: delayed growth phase, exponential (log) growth phase: logarithmic growth phase, stationary phase: plateau phase
Fig. 3
Fig. 3
Gel electrophoresis of extracted DNA, polymerase chain reaction products amplified by rol and virD2 primers, and extracted RNA from hairy roots. a: DNA extracted from four hairy root lines; b1: PCR amplicons of four hairy root lines using rolA-B (1794 bp) primer and b2: confirmation of the absence of bacterial contamination in these four lines by PCR using virD2 (338 bp); c: PCR amplicons of three hairy root lines using rolB (780 bp); d: PCR amplicons of L10 hairy root line by rolC-D (1105 bp) and virD2; e-g: RNA extracted from non-elicited and/or elicited L10 hairy roots. M: DNA marker (50 bp or 1 kb) Ladder, GoldBio), 1–4: hairy root lines, C+: A7 Agrobacterium rhizogenes plasmid as positive control, C_: normal root as negative control, C0: PCR reaction without DNA as blank control, HR: L10 hairy root line (Additional edges and unrelated wells of gel have been cropped, from images, but the original image of all blots is available in the supplementary file.)
Fig. 4
Fig. 4
Gel electrophoresis of RTq-PCR amplicons using specific primers for CHS, PAL, UFGT, F3’H, MYB3, bHLH, and ACT1 (as reference gene) genes. M: DNA marker (50 bp or 1 kb) Ladder (Additional edges and unrelated wells of gel have been cropped, from images, but the original image of all blots is available in the supplementary file.)
Fig. 5
Fig. 5
RT-qPCR analysis of different genes from the phenolic/flavonoid biosynthetic pathway, a: PAL; b: CHS; c: F3’H; d: UFGT; e: MYB3 gene expression in F. carica cv. Siah hairy root cultures after elicitation treatments. Values represent the means of three replicates ± SD. Values followed by the same letters in each column are not significantly different according to Duncan’s test (P ≤ 0.05)
Fig. 6
Fig. 6
RT-qPCR analysis of bHLH in F. carica cv. Siah hairy root cultures after MeJA elicitation treatments. Values represent the means of three replicates ± SD. Values followed by the same letters in each column are not significantly different according to Duncan’s test (P ≤ 0.05)
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