Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products

Abstract

Background: The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana.

Results: This study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3'-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase, secondary wall-associated NAC domain protein1, MYB58 and MYB63 resulting in higher accumulation of lignin content compared to the control.

Conclusions: These results indicate that the additions of microbially based soil additives have a perceptible influence on phenylpropanoid pathway gene regulation and its production of secondary metabolites. These findings open an avenue of research to investigate the mode of action of microbially-based soil additives which may assist in the sustainable production of food, feed, fuel and fiber.

Figure 1

Profiles of flavonol glycoside detected in Arabidopsis thaliana treated once (TI) and multiple times (TII) with SoilBuilder™-AF (SB). Kaempferol-3,7-O-bis-alpha-L-rhamnoside (F1), kaempferol-3-O-alpha-L-rhamnopyranosyl (1-2)-beta-D-glucopyranoside-7-O-alpha-L-rhamnopyranoside (F2), Kaempferol with rhamnoside (F3), Kaempferol with rhamnoside (F4), Kaempferol with rhamnoside (F5), Kaempferol in hydrolyzed (F6), quercetin 3,7-dirhamnoside (F7), apigenin 7-(2",3"-diacetylglucoside) (F8) and pentamethoxydihydroxyflavone (F9). Bars indicate standard error of three biological replicates at each sampling time-point. Different letters in different bar differ significantly from the control according to Fit Least Squares (FLS) test, P ≤ 0.05. CONT (black bar) indicates the untreated plants, TI (shaded) and TII (white) treated with microbial products only once and multiple times, respectively.

Figure 2

Profiles of anthocyanidins glycoside detected in Arabidopsis thaliana treated once (TI) and multiple times (TII) with SB. Cyanidin –Rhamnoglucoside (A1), cyanidin 3-(6-malonylglucoside)-7,3’-di-(6-feruloylglucoside) (A2), cyanidin 3-(6"-caffeyl-2"-sinapylsambubioside)-5-(6-malonylglucoside) (A3) and cyanidin 3-(2G-glucosylrutinoside) (A4) and cyanidin 3-(2G-glucosylrutinoside) (A5). Bars indicate standard error of three biological replicates at each sampling time-point. For significant level identification, see Figure  1.

Figure 3

Relative transcript abundance of phenylalanine ammonia lyase (PAL) of flavonoid pathway (PAL1, PAL2, PAL3 and PAL4) genes known to be involved in flavonoid biosynthesis in Arabidopsis thaliana after being treated once (TI) and multiple times (TII) with SB. Primers used in these studies, products size for the amplified fragments, accession numbers are shown in Additional file 6. Transcript abundance of each gene was normalized by the level of an actin and EF-1α gene. Bars indicate standard error of three biological replicates at each sampling time-point. For significant level identification, see Figure  1.

Figure 4

Relative transcript abundance of flavonoids pathway structural genes (CHS, CHI, F3H, F3´H, FLS1, UF3GT, DFR and LDOX) known to be involved in flavonoid biosynthesis in Arabidopsis treated once (TI) and multiple times (TII) with SB. Primers used in these studies, products size for the amplified fragments, accession numbers are shown in Additional file 6. Transcript abundance of each gene was normalized by the level of an actin and EF-1α gene. Bars indicate standard error of three biological replicates at each sampling time-point. For significant level identification, see Figure  1.

Figure 5

Relative transcript abundance of transcription factors (PAP1, PAP2, MYB11, MYB12, MYB111, MYB113, MYB114, GL3, EGL3, TT8 and TTG1) known to direct flavonoids biosynthesis-related gene expression in Arabidopsis treated once (TI) and multiple times (TII) with SB. Primers used in these studies, products size for the amplified fragments, accession numbers are shown in Additional file 6. Transcript abundance of each gene was normalized by the level of an actin and EF-1α gene. Bars indicate standard error of three biological replicates at each sampling time-point. For significant level identification, see Figure  1.

Figure 6

Relative transcript abundance of structural genes (C4H, 4CL1, HCT, C3’H1, CCoAOMT1, CCR1, CCR2, COMT1, F5H1, and SAT) known to involved in lignin biosynthesis in Arabidopsis treated once (TI) and multiple times (TII) with SB. Primers used in these studies, products size for the amplified fragments, accession numbers are shown in Additional file 6. Transcript abundance of each gene was normalized by the level of an actin and EF-1α gene. Bars indicate standard error of three biological replicates at each sampling time-point. For significant level identification, see Figure  1.

Figure 7

Influence of microbial products SB treated once (TI) and multiple times (TII) on lignin content in Arabidopsis thaliana. Bars indicate standard error of three biological replicates at each sampling time-point. For significant level identification, see Figure  1.