ERF9 of Poncirus trifoliata (L.) Raf. undergoes feedback regulation by ethylene and modulates cold tolerance via regulating a glutathione S-transferase U17 gene

Abstract

Plant ethylene-responsive factors (ERFs) play essential roles in cold stress response, but the molecular mechanisms underlying this process remain poorly understood. In this study, we characterized PtrERF9 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-hardy plant. PtrERF9 was up-regulated by cold in an ethylene-dependent manner. Overexpression of PtrERF9 conferred prominently enhanced freezing tolerance, which was drastically impaired when PtrERF9 was knocked down by virus-induced gene silencing. Global transcriptome profiling indicated that silencing of PtrERF9 resulted in substantial transcriptional reprogramming of stress-responsive genes involved in different biological processes. PtrERF9 was further verified to directly and specifically bind with the promoters of glutathione S-transferase U17 (PtrGSTU17) and ACC synthase1 (PtrACS1). Consistently, PtrERF9-overexpressing plants had higher levels of PtrGSTU17 transcript and GST activity, but accumulated less ROS, whereas the silenced plants showed the opposite changes. Meanwhile, knockdown of PtrERF9 decreased PtrACS1 expression, ACS activity and ACC content. However, overexpression of PtrERF9 in lemon, a cold-sensitive species, caused negligible alterations of ethylene biosynthesis, which was attributed to perturbed interaction between PtrERF9, along with lemon homologue ClERF9, and the promoter of lemon ACS1 gene (ClACS1) due to mutation of the cis-acting element. Taken together, these results indicate that PtrERF9 acts downstream of ethylene signalling and functions positively in cold tolerance via modulation of ROS homeostasis by regulating PtrGSTU17. In addition, PtrERF9 regulates ethylene biosynthesis by activating PtrACS1 gene, forming a feedback regulation loop to reinforce the transcriptional regulation of its target genes, which may contribute to the elite cold tolerance of Poncirus trifoliata.

Keywords: ACC synthase; glutathione S-transferase; ERF; ROS homeostasis; Trifoliata orange; cold tolerance; ethylene biosynthesis.

Figure 1

The expression of ERF genes analysis under cold treatment in Poncirus trifoliata. (a) Expression profiles of 44 ERF genes in trifoliate orange under cold stress. (b) Relative expression of PtrERF9 was quantitated by qPCR in response to low temperature. Error bars represent ± SE (n = 3). (c, d) GUS staining (c) and relative GUS intensity (d) analysis in sweet orange callus transformation with empty vector (EV) and pPtrERF9: GUS in response to cold treatment. Error bars represent ± SE (n = 3). Asterisks indicate significant difference between the vectors under before and after the cold treatment (***P < 0.001).

Figure 2

Ethylene enhanced trifoliate orange cold tolerance and PtrERF9 expression was increased association with ethylene production under cold stress. (a) Cold induced ethylene production in trifoliate orange. (b) Phenotype of ACC‐, water‐ or AVG‐pretreated wild‐type plants in response to freezing treatment. Scale bars = 1 cm. (c, d) Electrolyte leakage (c) and MDA levels (d) in ACC‐, water‐, AVG‐pretreated wild‐type plants after cold treatment. Error bars indicate ± SE (n = 3). Asterisks indicate significant differences between different groups (**P < 0.01, ***P < 0.001). (e, f) The expression of PtrERF9 under ACC treatment (e), cold and cold with AVG treatment (f) in trifoliate orange. Error bars indicate ± SE (n = 3).

Figure 3

PtrERF9 is localized in the nucleus. (a) The fusion construct (35S: PtrERF9‐YFP) or an empty vector (35S: YFP) was co‐transformed with a nucleus marker gene VirD2NLS fused to mCherry in tobacco (Nicotiana benthamiana) leaves. Confocal microscopic images of the epidermal cells were taken under bright filed and yellow (for YFP), red (for mCherry) fluorescence signals. (b) DAPI staining, shown in blue, was used to stain the nucleus. The overlapped images are shown on the right. Scale bars = 25 µm.

Figure 4

Overexpression of PtrERF9 improved cold tolerance of transgenic plants. (a) Phenotypes of wild‐type (WT) and PtrERF9 overexpression transgenic tobacco lines (#2 and #4) in response to cold treatment. Scale bars = 1 cm. (b) Survival rate of transgenic tobacco plants after the growth recovery for 3 d at ambient environment. (c, d) Electrolyte leakage (c), MDA content (d) of transgenic tobacco plants before and after the growth recovery for 3 d at 25 °C. (e, f) Chlorophyll fluorescence (e) and F _v/F _m ratios (f) of transgenic tobacco plants before and after cold treatment and the growth recovery for 3 d at 25 °C. (g) Phenotypes of WT and PtrERF9 overexpression transgenic lemon plants (#15 and #18) before and after cold treatment. Scale bars = 1 cm. (h‐k) Electrolyte leakage (h), MDA content (i), chlorophyll fluorescence (j) and F _v/F _m (k) of WT and transgenic lemon before and after the cold treatment. Error bars represent ± SE (n = 3). Asterisks indicate significant difference between WT and the transgenic lines in response to cold treatment (***P < 0.001).

Figure 5

Silencing of PtrERF9 reduced the cold tolerance in trifoliate orange. (a) Phenotypes of TRV plants and TRV‐PtrERF9 plants before and after cold treatment. Scale bars = 5 cm. (b–e) Electrolyte leakage (b), MDA content (c), chlorophyll fluorescence (d) and F _v/F _m (e) of TRV control and TRV‐PtrERF9 plants before and after the cold treatment. Error bars indicate ± SE (n = 3). Asterisks indicate significant difference between the VIGS line and the TRV control plants under same conditions (***P < 0.001).

Figure 6

Silencing of PtrERF9 leads to transcriptional reprogramming of a larger number of genes in trifoliate orange. (a) Scatterplots of gene expression patterns in the VIGS plants compared with TRV control under normal condition. Blue and red circles represent down‐regulated and up‐regulated genes, respectively. (b) GO analysis of enrichment of down‐regulated genes in terms of biological process. (c) The top 20 enriched KEGG pathways among the down‐regulated DEGs.

Figure 7

PtrERF9 binds to PtrGSTU17 promoter and positively regulates its expression. (a) Schematic diagram of the PtrGSTU17 promoter. The black rectangle indicates the position of the partial promoter fragment (P1) within GCC‐box elements. (b) The prey and bait vectors were used for yeast one‐hybrid assay (Y1H). mP1 is mutated form P1 fragment as the GCCGCC was changed to TCCTCC. (c) Growth of yeast cells co‐transformed with baits (P1 or mP1) and prey combinations (pGADT7‐PtrERF9), positive control (p53‐AbAi + pGAD‐p53), negative control (bait + pGADT7) on SD/‐Ura/‐Leu medium without (left) or with (right) AbA. (d) EMSA assay of specific binding of PtrERF9 to the GCC‐box of the PtrGSTU17 promoter. The purified His‐PtrERF9 protein was incubated with the biotin‐labelled probe containing the wild‐type or mutated GCC‐box element, with or without unlabelled probe was used as a competitor. Open and closed arrows indicate bound and free probes, respectively. +, presence; −, absence. (e) Schematic diagram of two PtrGSTU17 promoter fragments (F1, F2), shown as grey bars, used for ChIP‐qPCR analysis. GCC‐box element is marked by black bar. (f) ChIP‐qPCR assays revealed the enrichment of PtrERF9 in the promoter of PtrGSTU17 by using specific primers as indicated by arrows below the grey bars in (e). (g) Schematic diagrams of the effector and reporter constructs used for dual‐LUC transient expression assay. P_35S and T_35S, the promoter and terminator of CaMV 35S, respectively. MCS, multiple cloning sites. LUC, firefly luciferase. REN, Renilla luciferase. (h) Dual‐LUC transient expression assays of the promoter activity were measured by the LUC/REN ratios in tobacco (Nicotiana benthamiana) protoplasts. LUC/REN ratio of the negative control (SK‐PtrERF9 + pGreen0800 empty vector) was considered as 1 for normalization. (i) Representative bioluminescence image of the capability of PtrERF9 to regulate the PtrGSTU17 promoter activity in tobacco leaves. Error bars indicate ± SE (n = 3). Asterisks indicate that the value is significantly different from that of the control (***P < 0.001).

Figure 8

PtrERF9 regulates PtrGSTU17 expression to control ROS levels in response to cold stress. (a–d) PtrGSTU17 expression (a, c) and GST activity (b, d) in lemon wild‐type (WT) and transgenic plants (a, b) and TRV control plants and trifoliate orange VIGS (TRV‐PtrERF9) plants (c, d) before and after cold stress. (e–h) Levels of H₂O₂ (e, g) and O₂ ^·− content (f, h) in transgenic lemon and WT (e, f) and TRV control and VIGS plants (g, h) before and after cold treatment. (i–l) In situ detection of H₂O₂ (i, k) and O₂ ^·− (j, l) in the WT and transgenic lemons (i, j) and TRV control and VIGS plants (k, l) after cold treatment, as revealed by histochemical staining with DAB and NBT, respectively. Error bars indicate ± SE (n = 3). Asterisks indicate significant differences between different groups under the same growth condition (*P < 0.05; ***P < 0.001).

Figure 9

PtrERF9 binds to PtrACS1 promoter and positively regulates its expression. (a) Schematic diagram of PtrACS1 promoter, black rectangle indicates the GCC‐box element in the partial promoter fragment (P2). (b) Prey and bait constructs were used for Y1H analysis, the mutated bait mP2 is a mutated form P2 by changing ‘GGCGGC’ into ‘GGTGGC’. (c) Growth of yeast cells co‐transformed with the prey and bait on selective medium with or without AbA. Positive control: pGADT7‐Rec‐p53 + p53‐AbAi; Negative control: pGADT7 + bait. (d) EMSA assay using purified His‐PtrERF9 fusion protein incubated with biotin‐labelled probe containing wild‐type or mutated probe, along with or without unlabelled competitor DNA. Open and closed arrows indicate bound and free probes, respectively. +, presence; −, absence. (e) Schematic diagrams of effector and reporter constructs used for dual‐LUC transient assay. (f) The various combinations of vectors were performed on Dual‐LUC transient expression assays in tobacco protoplasts. (g) Representative bioluminescence image of PtrERF9 activation on the PtrACS1 promoter in tobacco leaves. (h) Schematic diagrams of F3 and F4 regions, shown as grey bars, in the PtrACS1 promoter, in which only F3 contains a GCC‐box sequence (black bar). (i) Enrichment of PtrERF9 in the PtrACS1 promoter based on ChIP‐qPCR assays using an anti‐GFP antibody. The arrows below the grey bars in (h) show the specific primers used for ChIP‐qPCR. Error bars indicate ± SE (n = 3). Asterisks indicate that the value is significantly different from that of the control (***P < 0.001).

Figure 10

PtrERF9 regulates ethylene synthesis via controlling ACS expression in trifoliate orange for cold response. (a–c) ACS1 expression (a), ACS activity (b) and ACC content (c) in TRV control and PtrERF9‐silencing trifoliate orange. (d–f) ACS1 expression (d), ACS activity (e) and ACC content (f) in wild‐type and PtrERF9 overexpression transgenic lemon plants. Error bars indicate ± SE (n = 3). Asterisks indicate significant differences between different groups under the same growth condition (**P < 0.01; ***P < 0.001). (g) Schematic diagram of ClACS1 promoter, black rectangle indicated the GGTGGC motif in the partial promoter fragment (P3). (h) Prey and bait constructs were used for yeast one‐hybrid analysis. pGADT7‐PtrERF9 and pGADT7‐ClERF9 were used as prey. P3 fragment was used as bait. (i) Growth of yeast cells co‐transformed with prey (PtrERF9, ClERF9) and bait (P3) on selective medium added with or without AbA. positive control (p53‐AbAi + pGAD‐p53), negative control (bait + pGADT7).

Figure 11

A proposed model for the regulatory function of PtrERF9 under cold stress. Cold stress induces ethylene production and activates the expression of PtrERF9. PtrERF9 binds to the GCC‐box in the promoter of PtrGSTU17, improves the plant ROS scavenging captivity. Additionally, PtrERF9 involves in the signalling pathway and acts as a feedback enhancer to elevate ethylene biosynthesis gene (PtrACS1) expression and ethylene level, and finally increases plant cold tolerance. The GCC‐box elements are shown by red circles.