Coexpression of PalbHLH1 and PalMYB90 Genes From Populus alba Enhances Pathogen Resistance in Poplar by Increasing the Flavonoid Content

Abstract

Secondary metabolites of the flavonoid pathway participate in plant defense, and bHLH and MYB transcription factors regulate the synthesis of these metabolites. Here, we define the regulatory mechanisms in response to pathogens. Two transcription factors from Populus alba var. pyramidalis, PalbHLH1 and PalMYB90, were overexpressed together in poplar, and transcriptome analysis revealed differences in response to pathogen infection. The transgenic plants showed elevated levels of several key flavonoid pathway components: total phenols, proanthocyanidins (PAs), and anthocyanins and intermediates quercetin and kaempferol. Furthermore, PalbHLH1 and PalMYB90 overexpression in poplar enhanced antioxidase activities and H₂O₂ release and also increased resistance to Botrytis cinerea and Dothiorella gregaria infection. Gene expression profile analysis showed most genes involved in the flavonoid biosynthesis pathway or antioxidant response to be upregulated in MYB90/bHLH1-OE poplar, but significant differential expression occurred in response to pathogen infection. Specifically, expression of PalF3H (flavanone 3-hydroxylase), PalDFR (dihydroflavonol 4-seductase), PalANS (anthocyanin synthase), and PalANR (anthocyanin reductase), which function in initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, was significantly upregulated in D. gregaria-infected MYB90/bHLH1-OE poplar. Our results highlight that PalbHLH1 and PalMYB90 function as transcriptional activators of flavonoid pathway secondary-metabolite synthesis genes, with differential mechanisms in response to bacterial or fungal infection.

Keywords: Botrytis cinerea; Dothiorella gregaria Sacc; PalbHLH1 and PalMYB90; Populus alba var. pyramidalis; flavonoid.

Figure 1

Phylogenetic analyses and multiple alignments of PalbHLH1 and PalMYB90. (A) Phylogenetic tree of MYB90 and selected R2R3-Myb proteins from other plant species obtained using the Maximum Likelihood (ML) method in MEGA v.5.1 software. (B) Phylogenetic tree of bHLH1 with other bHLH sequences. The scale bar represents 0.05 substitutions per site. (C, D) The bHLH domains of bHLH1 and R2R3-Myb of MYB90 are both labeled with black lines. Identical amino acids are shaded in red, and similar amino acids are shaded in blue and green.

Figure 2

Characterization of transgenic poplar plants. (A) Construction of the MYB90/bHLH1-OE plasmid. (B) Growth of transgenic (MYB90/bHLH1-OE) and wild-type (WT) poplar plants. (C) Color of anthocyanin extraction. (D) Accumulation of total anthocyanins in the leaves of MYB90/bHLH1-OE and WT plants. (E) Accumulation of total phenolics in the leaves of MYB90/bHLH1-OE and WT plants. (F, G) Contents of quercetin and kaempferol in the leaves of MYB90/bHLH1-OE and WT poplar plants, respectively. The leaves of 2-month-old poplar grown in pots were used in these experiments. Error bars indicate ± SE. Asterisks (*) indicate significant differences, P < 0.05. Asterisks (**) indicate significant differences, P ≤ 0.01.

Figure 3

Quantification of insoluble and soluble tannin (PA) and reactive oxygen species in wild-type (WT) and transgenic lines (MYB90/bHLH1-OE 2 and MYB90/bHLH1-OE 5). (A) PA accumulation detected by DMACA staining. (B) Quantification of soluble PA. (C) Quantification of insoluble PA. (D) Changes in H₂O₂ content and (E, F) activities of two antioxidases, POD and SOD, in MYB90/bHLH1-OE poplar leaves. Values represent the means of three replications. Error bars indicate ± SE. Asterisks indicate significant differences: *P < 0.05. Asterisks (**) indicate significant differences, P ≤ 0.01.

Figure 4

Resistance of transgenic poplar to pathogen infection. Disease symptoms of three leaves of wild-type (WT) and MYB90/bHLH1-OE plants after B. cinerea infection for 4 days (A) or D. gregaria infection for 7 days (B). (C) Ratios of the lesion areas of infected leaves from WT and MYB90/bHLH1-OE plants (OE-2 and OE-5 lines). Values represent the means of three replications. Error bars indicate ± SE. Asterisks indicate significant differences: **P ≤ 0.01.

Figure 5

Expression patterns of flavonoid pathway genes in transgenic and control plants infected by two pathogens. Among them, pink blocks are gene expression patterns based on transcriptome data analysis, while blue blocks are qRT-PCR validation results for these genes. Error bars indicate ± SE.

Figure 6

Quantitative real-time PCR analysis of the transcript levels in WT and both independent MYB90/bHLH1-OE lines before and after being infected by two types of pathogens. The CYC063 expression was set as the internal control. In the figure, light gray bar represents WT, dark gray and black bars represent MYB90/bHLH1-OE2 and MYB90/bHLH1-OE5 lines, respectively. Each measurement was carried out in triplicate, and error bars represent the SE of the mean of fold changes for three biological replicates. Asterisks indicate significant differences: *P < 0.05, **P ≤.01.

Figure 7

Relationships of anthocyanin content and responses to pathogen infection in P. alba var. pyramidalis and P. euphratica leaves. (A) Disease symptoms induced by D. gregaria on the leaves of two sister poplars, P. alba var. pyramidalis and P. euphratica. (B) Colors of anthocyanin extracted from P. alba var. pyramidalis and P. euphratica leaves. (C) Total anthocyanin contents in P. alba var. pyramidalis and P. euphratica leaves. (D) Relative lesion areas of the third leaves of P. alba var. pyramidalis and P. euphratica plants after D. gregaria infection for 7 days. (E) Relative expression levels of bHLH1, DFR, and F3H in P. alba var. pyramidalis and P. euphratica leaves. Error bars indicate the standard error (SE) of the average anthocyanin content, **P < 0.01.