A Gene Regulatory Network Controlled by BpERF2 and BpMYB102 in Birch under Drought Conditions

Abstract

Gene expression profiles are powerful tools for investigating mechanisms of plant stress tolerance. Betula platyphylla (birch) is a widely distributed tree, but its drought-tolerance mechanism has been little studied. Using RNA-Seq, we identified 2917 birch genes involved in its response to drought stress. These drought-responsive genes include the late embryogenesis abundant (LEA) family, heat shock protein (HSP) family, water shortage-related and ROS-scavenging proteins, and many transcription factors (TFs). Among the drought-induced TFs, the ethylene responsive factor (ERF) and myeloblastosis oncogene (MYB) families were the most abundant. BpERF2 and BpMYB102, which were strongly induced by drought and had high transcription levels, were selected to study their regulatory networks. BpERF2 and BpMYB102 both played roles in enhancing drought tolerance in birch. Chromatin immunoprecipitation combined with qRT-PCR indicated that BpERF2 regulated genes such as those in the LEA and HSP families, while BpMYB102 regulated genes such as Pathogenesis-related Protein 1 (PRP1) and 4-Coumarate:Coenzyme A Ligase 10 (4CL10). Multiple genes were regulated by both BpERF2 and BpMYB102. We further characterized the function of some of these genes, and the genes that encode Root Primordium Defective 1 (RPD1), PRP1, 4CL10, LEA1, SOD5, and HSPs were found to be involved in drought tolerance. Therefore, our results suggest that BpERF2 and BpMYB102 serve as transcription factors that regulate a series of drought-tolerance genes in B. platyphylla to improve drought tolerance.

Keywords: Betula platyphylla; RNA-Seq; drought stress; expression regulatory network; transcription factor; transient transformation.

Figure 1

Drought treatment of birch plants. (a) The growth phenotype of birch under drought or normal (control) growth conditions. Watering of birch plants grown in soil was stopped for 120 h, and the well-watered birch plants served as the controls; (b) measurements of soil moisture; (c) leaf water content study; (d) measurements of total chlorophyll; (e) MDA content analysis; (f) electrolyte leakage assay; (g) NBT, DAB, and Evans blue staining for birch leaves under normal or drought stress conditions. FW: fresh weight. Asterisks indicate a significant difference between treatment and control plants (p < 0.05).

Figure 2

Quantitative RT-PCR confirmation of the differentially expressed genes identified using RNA-Seq. (a) The families of differentially expressed transcription factors in birch under drought stress conditions. (b–h) Comparison of the expression level of differentially expressed genes in the function involved in: transcription factors (b), late embryogenesis abundant proteins (c), heat shock proteins (d), ROS-scavenging-related proteins (e), water deprivation-related proteins (f), stress-related proteins (g), and others (h) between qRT-PCR and RNA-Seq.

Figure 2

Quantitative RT-PCR confirmation of the differentially expressed genes identified using RNA-Seq. (a) The families of differentially expressed transcription factors in birch under drought stress conditions. (b–h) Comparison of the expression level of differentially expressed genes in the function involved in: transcription factors (b), late embryogenesis abundant proteins (c), heat shock proteins (d), ROS-scavenging-related proteins (e), water deprivation-related proteins (f), stress-related proteins (g), and others (h) between qRT-PCR and RNA-Seq.

Figure 3

Analysis of drought stress tolerance of BpERF2 and BpMYB102 in birch. Three kinds of transgenic plants were compared: plants transiently transformed with 35S:BpERF2, 35S:BpMYB102, and empty p1307-Flag (as the control). (a) The study of the expression of BpERF2 and BpMYB102 in transiently transformed plants using qRT-PCR. (b) NBT, DAB, and Evans blue staining of leaves from the three kinds of plants. (c–e) Comparison of ROS content (c), MDA content (d), and electrolyte leakage (e) among the three kinds of plants under normal or drought stress conditions. 1/2 MS: the plants grown on 1/2 MS medium as the control; PEG6000: the plants grown on 1/2 MS medium supplying with 20% PEG6000, which is used as drought stress. Asterisks indicates a significant difference between treatment and control plants (p < 0.05).

Figure 4

Determination of the genes regulated by BpERF2. (a–g) Investigation of the genes regulated by BpERF2 using qRT-PCR. Transcription factors (a), late embryogenesis abundant proteins (b), heat shock proteins (c), ROS-scavenging-related proteins (d), water deprivation-related proteins (e), stress-related proteins (f), and others (g). The transcripts of the genes in control plants (transiently transformed with empty p1307-Flag) were used to normalize their expression in the plants that transiently overexpressed BpERF2 according to qRT-PCR. (h) Investigation of whether BpERF2 directly regulates its target genes using ChIP. BpERF2 was fused with a Flagtag, transiently transformed into the plants, and used for ChIP. ChIP+: sonicated chromatin immunoprecipitated with anti-Flag antibody; ChIP−: sonicated chromatin immunoprecipitated without any antibody. Asterisks indicates a significant difference between treatment and control plants (p < 0.05 and |log2FC| ≥ 1).

Figure 4

Determination of the genes regulated by BpERF2. (a–g) Investigation of the genes regulated by BpERF2 using qRT-PCR. Transcription factors (a), late embryogenesis abundant proteins (b), heat shock proteins (c), ROS-scavenging-related proteins (d), water deprivation-related proteins (e), stress-related proteins (f), and others (g). The transcripts of the genes in control plants (transiently transformed with empty p1307-Flag) were used to normalize their expression in the plants that transiently overexpressed BpERF2 according to qRT-PCR. (h) Investigation of whether BpERF2 directly regulates its target genes using ChIP. BpERF2 was fused with a Flagtag, transiently transformed into the plants, and used for ChIP. ChIP+: sonicated chromatin immunoprecipitated with anti-Flag antibody; ChIP−: sonicated chromatin immunoprecipitated without any antibody. Asterisks indicates a significant difference between treatment and control plants (p < 0.05 and |log2FC| ≥ 1).

Figure 5

Determination of the genes regulated by BpMYB102. (a–g) Investigation of the genes regulated by BpMYB102 using qRT-PCR. Transcription factors (a), late embryogenesis abundant proteins (b), heat shock proteins (c), ROS-scavenging-related proteins (d), water deprivation-related proteins (e), stress-related proteins (f), and others (g). The transcripts of the genes in control plants (transiently transformed with empty p1307-Flag) were used to normalize their expression in the plants that transiently overexpressed BpMYB102 according to qRT-PCR. (h) Investigation using ChIP of whether BpMYB102 directly regulates its target genes. BpMYB102 was fused with a Flag tag, transiently transformed into the plants, and used for ChIP. ChIP+: sonicated chromatin immunoprecipitated with anti-Flag antibody; ChIP−: sonicated chromatin immunoprecipitated without any antibody. Asterisks indicates a significant difference between treatment and control plants (p < 0.05 and |log2FC| ≥1).

Figure 6

Analysis of the drought stress tolerance of several BpERF2 or BpMYB102 target genes. The plants were transiently transformed with several target genes of BpERF2 and BpMYB102 for overexpression, and empty p1307-myc was transiently transformed into plants as the control. (a) Analysis of the expression of the transgenes in transiently transformed plants using qRT-PCR. (b) NBT, DAB, and Evans blue staining analysis. (c–e) Comparison of ROS content (c), MDA content (d), and electrolyte leakage (e) among the studied plants under normal or drought stress conditions. Asterisks indicate a significant difference between treatment and control plants (p < 0.05).

Figure 7

The regulatory network of BpERF2 and BpMYB102 in response to drought stress. Drought stress induces a series of transcription factors, including BpERF2 and BpMYB102, and the induced BpERF2 and BpMYB102 positively or negatively regulate a series of genes to improve drought tolerance. The solid blue lines indicate that BpERF2 or BpMYB102 directly regulates or induces the target gene expression; the solid green lines indicate that BpERF2 or BpMYB102 directly regulates or inhibits the target gene expression; the dotted lines indicate indirect regulation. The red hexagons highlight genes that were confirmed to confer drought tolerance to transgenic plants; the green hexagon indicates a gene that is related to drought sensitivity; the yellow hexagon indicates a gene that is not involved in drought tolerance; the hexagon without color shows genes that were not studied for their function in drought stress here.