The ETHYLENE RESPONSE FACTOR6-GRETCHEN HAGEN3.5 module regulates rooting and heat tolerance in Dimocarpus longan

Abstract

Heat stress can seriously affect plant growth and development. Ethylene response factors (ERFs) play important roles in plant development and physiological responses. Here, we identified DlERF6, an ERF family transcription factor that promotes heat tolerance in Dimocarpus longan. DlERF6 was strongly induced by heat stress and IAA treatment in longan roots. Overexpression of DlERF6 generated abundant, fast-growing hairy roots and enhanced longan heat stress tolerance by promoting IAA biosynthesis and reactive oxygen species (ROS) scavenging. Additional assays indicated that DlERF6 directly binds to the DlGH3.5 promoter and represses its expression. Overexpressing DlGH3.5 reduced hairy root number, root length, and heat tolerance, concomitant with a reduction in IAA content and ROS scavenging. Collectively, these results reveal the molecular mechanism through which the DlERF6-DlGH3.5 module regulates root growth and heat stress tolerance, providing a gene network that can be used for the genetic improvement of longan.

Figure 1.

Phenotypic and molecular characterization of DlERF6 transgenic roots. A) The phenotype of DlERF6 transgenic roots. Bars = 5 cm. Images were digitally extracted for comparison. B) PCR analysis for GUS in independent transgenic hairy roots. C) Longitudinal section of root tip region of hairy roots of wild-type (WT) and DlERF6 transgenic lines. Roots were collected, in paraffin section, and observed with 0.5% toluidine blue staining. D) GUS staining of the wild-type (WT) and DlERF6 transgenic roots. Images were digitally extracted for comparison. E) RT-qPCR analysis of DlERF6 in wild-type (WT) and transgenic roots. Overexpression (OE) 1, 2, different DlERF6-overexpressing roots. RNAi (erf1, erf2), different DlERF6-RNAi roots. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a”, “b” and “c” . Images were digitally extracted for comparison. F) The statistical results of the hairy roots length of Wild-Type (WT) and DlERF6 OE, RNAi transgenic lines. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” G) The statistical results of the hairy roots numbers of wild-type (WT) and DlERF6 OE, RNAi transgenic lines. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 10), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.”

Figure 2.

DlERF6 overexpression plants are more tolerant to heat stress. A) Phenotype of plants wild-type (WT), DlERF6 overexpressing (OE), and RNAi in the roots under heat treatment. Scale bar = 5 cm. Images were digitally extracted for comparison. B) Nitroblue tetrazolium (NBT) was used to detect the accumulation of superoxide and hydrogen peroxide (H₂O₂) in wild-type (WT) and DlERF6 transgenic roots. The arrow indicates the staining site. Images were digitally extracted for comparison. Images were digitally extracted for comparison. C) The H₂O₂ content of wild-type (WT), DlERF6 transgenic roots under normal and heat stress treatment. FW: Fresh weight. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” D–G) The SOD, POD, CAT, and GST activities of wild-type (WT) and DlERF6 transgenic roots under normal and heat treatment. FW: Fresh weight. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.”

Figure 3.

Expression of auxin metabolism genes in DlERF6 overexpression roots in response to heat stress. A) The IAA (Indole-3-Acetic Acid) content of wild-type (WT), DlERF6 transgenic roots under normal and heat stress. FW: Fresh weight. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” B) Quantification of IAA-amino acid (IAA-aa) conjugates in wild-type (WT), DlERF6 overexpression roots under heat stress (n = 1). FW: Fresh weight. C) Diagram of the auxin biosynthesis and conjugate pathways in DlERF6 overexpression (OE) roots in response to heat stress. The downward arrows, upward arrows, and N(ns) indicate the downregulated genes, upregulated genes, and non-regulated genes respectively in DlERF6 overexpression (OE) roots. The black frame represents different auxin components. D) Heatmap showing the relative expression level of genes involved in auxin metabolism in DlERF6 overexpression (OE) roots in response to heat stress.

Figure 4.

DlERF6 directly binds to DlGH3.5. A) Schematic diagrams of effector DlERF6 and reporter DlGH3.5 vectors. B) The INTEGRATIVE GENOMICS VIEWER (IGV) image of DlERF6 binding to DlGH3.5 promoter by DAP-seq. C) RT-qPCR analysis of DlGH3.5 expression in wild-type (WT), DlERF6 transgenic roots. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” D, E) Dual-luciferase system for the detection of DlERF6 targeting DlGH3.5. Images were digitally extracted for comparison. LUC, firefly luciferase activity; REN, Renilaluciferase. Student's t-test was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” F) GUS staining assay and RT-qPCR showing that DlERF6 can inhibit proDlGH3.5 expression. Images were digitally extracted for comparison. Student's t-test was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a”, “b” and “c”. G) Yeast one-hybrid analysis of the interaction between DlERF6 and DlGH3.5 promoter.

Figure 5.

Determination of DlGH3.5 transgenic roots. A) The phenotype of wild-type (WT), DlGH3.5 transgenic roots. Bars = 5 cm. Images were digitally extracted for comparison. B) PCR analysis for GUS in independent wild-type (WT), DlGH3.5 transgenic hairy roots. C) Longitudinal section of root tip region of hairy roots of WT, DlGH3.5 transgenic lines. Roots were collected, in a paraffin section, and observed with 0.5% toluidine blue staining. D) GUS staining of the WT, DlGH3.5 transgenic roots. Images were digitally extracted for comparison. E) RT-qPCR analysis of DlGH3.5 in wild-type (WT), DlGH3.5 transgenic roots. OE 1,2, different DlGH3.5-overexpressing root lines. RNAi (gh3.5-1, gh3.5-2), different DlGH3.5-RNAi root lines. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c”. F) The statistical results of the hairy root numbers of wild-type (WT), DlGH3.5 transgenic lines. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 10), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” G) The statistical results of the hairy root length of WT, DlGH3.5 transgenic lines. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 10), and significant differences (P < 0.05) between groups are indicated by “a”, “b” and “c”.

Figure 6.

Overexpression of DlGH3.5 reduces longan resistance to heat stress. A) Phenotype of plants wild-type (WT), overexpressing (OE), and RNAi DlGH3.5 in the roots under heat treatment. Scale bar = 5 cm. Images were digitally extracted for comparison. B) Nitroblue tetrazolium (NBT) was used to detect the accumulation of superoxide and hydrogen peroxide (H₂O₂) in wild-type (WT), DlGH3.5 transgenic roots. Images were digitally extracted for comparison. C) The H₂O₂ content of wild-type (WT), DlGH3.5 transgenic roots under normal and heat treatment. FW: Fresh weight. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” D–F) The SOD, POD, and CAT activities of wild-type (WT), DlGH3.5 transgenic roots under normal and heat treatment. FW: Fresh weight. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” G) The IAA (Indole-3-Acetic Acid) content of wild-type (WT), DlGH3.5 transgenic roots under normal and heat treatment. FW: Fresh weight. One-way ANOVA was performed. Error bars represent the Sd of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by “a,” “b,” and “c.” H) Heatmaps of metabolites related to flavonoid and lignin synthesis in DlGH3.5 RNAi roots in response to heat stress.

Figure 7.

Proposed working model of DlERF6–DlGH3.5. DlERF6 directly inhibits the expression of DlGH3.5 by binding to a GCC-box motif in its promoter. DlERF6 positively regulates the Indole-3-Acetic Acid (IAA) accumulation and enhances the reactive oxygen species (ROS) scavenging ability. Arrows and bar ends indicate activation and repression effects, respectively. Dashed lines suggest potential regulatory. Solid lines indicate direct regulatory relationships.