Expression Profiling of Flavonoid Biosynthesis Genes and Secondary Metabolites Accumulation in Populus under Drought Stress

Abstract

Flavonoids are key secondary metabolites that are biologically active and perform diverse functions in plants such as stress defense against abiotic and biotic stress. In addition to its importance, no comprehensive information has been available about the secondary metabolic response of Populus tree, especially the genes that encode key enzymes involved in flavonoid biosynthesis under drought stress. In this study, the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the expression of flavonoid biosynthesis genes (PtPAL, Pt4-CL, PtCHS, PtFLS-1, PtF3H, PtDFR, and PtANS) gradually increased in the leaves of hybrid poplar (P. tremula × P. alba), corresponding to the drought stress duration. In addition, the activity and capacity of antioxidants have also increased, which is positively correlated with the increment of phenolic, flavonoid, anthocyanin, and carotenoid compounds under drought stress. As the drought stress prolonged, the level of reactive oxygen species such as hydrogen peroxide (H₂O₂) and singlet oxygen (O₂^-) too increased. The concentration of phytohormone salicylic acid (SA) also increased significantly in the stressed poplar leaves. Our research concluded that drought stress significantly induced the expression of flavonoid biosynthesis genes in hybrid poplar plants and enhanced the accumulation of phenolic and flavonoid compounds with resilient antioxidant activity.

Keywords: Populus; drought stress; flavonoid biosynthesis pathway; gene expression; secondary metabolites.

Figure 1

Illustration of the biosynthetic pathways leading to the major groups of phenolic compounds in Populus. [22].

Figure 2

Morphological changes of basal leaves in poplar 717 under different relative soil moisture contents (RSMC). (A) D0 plant, (B) D5 plant, (C) D10 plant, (D) D15 plant, (E) D0 basal leaf, (F) D5 basal leaf, (G) D10 basal leaf, (H) D15 basal leaf.

Figure 3

Photosynthetic attributes of poplar leaves under drought stress. (A) Net photosynthetic rate (Pn), (B) Intercellular CO₂ (Ci), (C) Stomatal conductance (gs), (D) Transpiration rate (Tr), (E) Chlorophyll a, (F) Chlorophyll b. The values are presented as means ± standard error (SE) (n = 3). The different small letters indicate a significant difference (p < 0.05) among different treatments.

Figure 4

Relative expression of genes involved in the flavonoid biosynthesis pathway under drought stress in poplar 717 leaves. (A) PAL, phenylalanine ammonia-lyase, (B) 4-CL, 4-coumarate CoA ligase, (C) CHS, chalcone synthase, (D) FLS-1, flavonol synthase, (E) F3H, flavanone 3-hydroxylase, (F) DFR, dihydroflavonol-4-reductase, (G) ANS, anthocyanidin synthase. The gene expression values are presented as means ± standard error (SE) (n = 3). The different small letters indicate a significant difference (p < 0.05) among different treatments.

Figure 5

Accumulation of secondary metabolites in Populus leaves under various drought stress treatments. (A) Total phenolic contents (TPC), (B) Total flavonoid contents (TFC), (C) Total carotenoid contents (TCC), (D) Proanthocyanidin contents (PAC), (E) Total anthocyanin contents (TAC). The data are presented as fold change relative to the control (samples at 0 days of treatment). The values are presented as means ± standard error (SE) (n = 3). The different small letters indicate a significant difference (p < 0.05) among different treatments.

Figure 6

Antioxidant, reactive oxygen species, and salicylic acid response to drought stress in Populus leaves under drought stress. (A) Antioxidant capacity, (B) Antioxidant activity, (C) O₂^– production rate, (D) H₂O₂ production, (E) Salicylic acid. The values are presented as means ± standard error (SE) (n = 3). The different small letters indicate a significant difference (p < 0.05) among different treatments.

Figure 7

Correlational matrix of secondary metabolic compounds in poplar 717 leaves under drought stress. The colored gradient legends represent coefficients of correlation r-values from +1.0 (dark green) to −1.0 (dark red). Significant effects are indicated in boldface as follows: *** p ≤ 0.001, ** p ≤ 0.01, and * p ≤ 0.05. All coefficients were computed by the Pearson correlation for possible pairs of variables in the matrix.