PtrABF of Poncirus trifoliata functions in dehydration tolerance by reducing stomatal density and maintaining reactive oxygen species homeostasis

Abstract

Abscisic acid-responsive element (ABRE)-binding factors (ABFs) play important roles in abiotic stress responses; however, the underlying mechanisms are poorly understood. In this study, it is reported that overexpression of Poncirus trifoliata PtrABF significantly enhanced dehydration tolerance. The transgenic lines displayed smaller stomatal apertures, reduced stomatal density/index, and lower expression levels of genes associated with stomatal development. PtrABF was found to interact with PtrICE1, a homologue of ICE1 (Inducer of CBF Expression 1) that has been shown to be critical for stomatal development. Microarray analysis revealed that a total of 70 genes were differentially expressed in the transgenic line, 42 induced and 28 repressed. At least two units of ABREs and coupling elements were present in the promoters of most of the induced genes, among which peroxidase and arginine decarboxylase were verified as bona fide targets of PtrABF. Transgenic plants exhibited higher antioxidant enzyme activities and free polyamine levels, but lower levels of reactive oxygen species (ROS) and malondialdehyde. Polyamines were revealed to be associated with ROS scavenging in the transgenic plants due to a modulation of antioxidant enzymes triggered by signalling mediated by H2O2 derived from polyamine oxidase (PAO)-mediated catabolism. Taken together, the results indicate that PtrABF functions positively in dehydration tolerance by limiting water loss through its influence on stomatal movement or formation and maintaining ROS homeostasis via modulation of antioxidant enzymes and polyamines through transcriptional regulation of relevant target genes.

Keywords: ABRE; Poncirus trifoliata; ROS; antioxidant enzyme; arginine decarboxylase; polyamine; polyamine oxidase; stomatal development..

Fig. 1.

Overexpression of PtrABF in trifoliate orange confers enhanced dehydration tolerance. (a) Phenotypes of leaves detached from 60-day-old transgenic lines (#8 and #10) and the wild type (WT) before and after 90min of dehydration treatment under an ambient environment. (b) Rates of water loss from the detached leaves of the WT and transgenic lines during the dehydration treatment, measured every 15min. Error bars indicate the SD (n=3). (c, d) Electrolyte leakage (c) and cell death (d) in the WT and transgenic lines measured after dehydration stress. Error bars indicate the SD (n=3). Asterisks indicate a significant difference between the WT and transgenic lines (***P<0.001). (This figure is available in colour at JXB online.)

Fig. 2.

Comparison of stomatal parameters between the wild-type (WT) and transgenic lines. (a) Stomatal aperture size of WT and transgenic lines, measured in fully expanded leaves before and after 90min of dehydration. Error bars indicate the SD (n≥30). (b) Scanning electron microscopy (SEM) images showing representative stomata of the WT and transgenic lines before (upper panel) and after (bottom panel) 90min of dehydration. Scale bars=5 μm. (c) Stomatal density in fully expanded leaves collected from WT and transgenic plants grown under normal conditions. Error bars indicate the SD (n=22). (d) Representative SEM photographs showing the difference in the number of stomata between the WT and transgenic lines. Scale bars=5 μm. (e–g) Stomatal index (e), stomatal area (f), and leaf areas (f) of the WT and transgenic lines. Asterisks indicate significant differences between the transgenic lines and the WT at the same time point (**P<0.01; ***P<0.001).

Fig. 3.

Expression profiles of genes associated with stomatal development, using fully expanded leaves sampled at the same stage as experimental tissues. (a–c) Expression patterns of SPCH (a), FAMA (b), and MUTE (c) in the transgenic lines (#8 and #10) and wild type (WT) based on qPCR analysis. Error bars indicate the SD (n=4). Asterisks indicate significant differences between the transgenic lines and the WT (**P<0.01, ***P<0.001).

Fig. 4.

Analysis of the interaction between PtrABF and PtrICE1 by yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC). (a) Yeast two-hybrid analysis of the interaction between PtrABF and PtrICE1. Growth of the yeast cells on SD/-Leu/-Trp/-Ura or SD/-Leu/-Trp/-His with or without added 3-AT. The blue colour shows the examination of X-gal activity of the corresponding yeast cells. (b) BiFC assay of the interaction between PtrABF and PtrICE1 using tobacco leaf epidermis. Images taken under bright field and fluorescence are shown. Positive and negative controls are used to verify the reliability of the approach.

Fig. 5.

Differentially expressed genes (DEGs) in the PtrABF-overexpressing line (#10). (a) Expression patterns of the DEGs in #10 relative to the wild type (WT). (b) A scatterplot of the expression profiles of the complete gene set in the PtrABF-overexpressing line relative to the WT. The red and green dots indicate the probe sets with a signal ratio >2 or <0.05, respectively, between #10 and the WT. (c) Expression patterns of 10 DEGs in the WT and #10 before and after dehydration treatment. Transcript levels of the genes in the sampled leaves were examined by qPCR. Error bars indicate the SD (n=4). (d) GO analysis of the DEGs based on cellular component, biological process, and molecular function.

Fig. 6.

Overexpression of PtrABF alters the expression of PtrPOD and PtrADC. (a, b) Expression patterns of PtrPOD (a) and PtrADC (b) in the wild type (WT) and transgenic lines before and after dehydration treatment. Transcript levels of the tested genes were determined by qPCR. Error bars indicate the SD (n=4). Asterisks indicate significant differences between the transgenic lines and the WT at the same time point (**P<0.01; ***P<0.001).

Fig. 7.

Interaction between PtrABF and the promoters of PtrPOD and PtrADC. (a) Schematic diagrams of the promoter of POD (pPOD) and ADC (pADC). The filled and open circles are ABREs and coupling elements (CEs), respectively. The short lines with pPOD’ or pADC’ show the partial promoter fragments used for the analysis. (b) The effector and reporter vectors used for yeast one-hybrid assay. (c) Growth of yeast cells of the positive control (P; p53-AbAi+pGAD-p53), negative control (N; pPOD’-AbAi+pGADT7 in the upper panels, pADC’-AbAi+pGADT7 in the bottom panels), and the effector–reporter co-transformant (Pp) on SD/-Leu medium without (left) or with (right) addition of 300mM AbA. (d) Schematic diagrams of the effector and reporter constructs used for transient expression assay. PtrABF driven by the CaMV 35S promoter was used as the effector. In the reporter construct, the POD or ADC promoter fragments (pPOD’, pADC’) were fused to the upstream region of the LUC gene. The REN gene under the control of the CaMV 35S promoter was used as a control for activity normalization. (e) Transient expression assay of transcriptional activation of POD and ADC promoters by PtrABF based on the relative LUC activities in tobacco protoplasts co-transformed with the effector and the reporter. LUC/REN ratio of the control (in the absence of the effector, –PtrABF) was taken as 1, while co-transformation of PtrABF and a promoter fragment (m-pADC’) without an ABRE element was used to examine specificity. Asterisks indicate that the values are significantly different from each other (**P<0.01).

Fig. 8.

The transgenic lines exhibited higher antioxidant enzyme activity and polyamine levels, but lower levels of ROS and MDA. (a–c) Activities of POD (a), SOD (b), and CAT (c) in the wild type (WT) and transgenic lines (#8, #10) before and after dehydration treatment. Error bars indicate the SD (n=3). The insets in b and c indicate transcript levels of SOD and CAT genes before and after dehydration treatment. (d) Free polyamine contents in the WT and transgenic lines under normal growth conditions. Error bars indicate the SD (n=3). (e) Accumulation of H₂O₂ (left panels) and O₂ ^– (right panels) in the WT and transgenic lines after dehydration treatment, as revealed by histochemical staining with DAB and NBT, respectively. (f–h) Quantitative measurement of H₂O₂ (f), O₂ ^– (g), and MDA (h) in the WT and transgenic lines after dehydration. Asterisks indicate significant differences between the transgenic lines and the WT at the same time point (*P<0.05, **P<0.01, ***P<0.001).

Fig. 9.

d-Arginine (d-arg) and guazatine treatments altered ROS accumulation, activity, and transcripts of antioxidant enzymes. (a–d) Treatment with d-arg increased ROS accumulation in the transgenic lines (#8, #10) after dehydration stress. The transgenic lines were pre-treated with water (0mM) or 1, 5, and 10mM d-arg for 3 d prior to 90min of dehydration. Accumulation of H₂O₂ (a, b) and O₂ ^– (c, d) was quantitatively measured (a, c) or histochemically detected using DAB (b, for H₂O₂) and NBT (d, for O₂ ^–). (e, f) Guazatine treatment reduced the accumulation of H₂O₂ in the wild type (WT) and transgenic lines after dehydration. The transgenic lines and the WT were pre-treated with water (0mM) or 5mM and 10mM guazatine for 3 d before they were exposed to 90min of dehydration. Accumulation of H₂O₂ was quantitatively measured (e) or histochemically detected using DAB staining (f). (g, h) Expression levels (g) and activities (h) of the antioxidant enzymes SOD, POD, and CAT in transgenic lines and the WT pre-treated with water (0mM) or 5mM and 10mM guazatine. Asterisks indicate significant differences between inhibitor treatment and water treatment of the same line (*P<0.05, **P<0.01, ***P<0.001).

Fig. 10.

A proposed mode of action of PtrABF based on the current study. In this model, PtrABF mediates dehydration tolerance via two major mechanisms: (1) mitigation of water loss by stimulating stomatal closure and decreasing stomatal density through interacting with PtrICE1; and (2) transcriptional regulation of target genes encoding enzymes associated with antioxidant (POD) or polyamine synthesis (ADC), by interacting with ABRE cis-acting elements. Higher levels of polyamines trigger catabolism mediated by PAO. This generates low levels of H₂O₂ at the initial stage of stress, which serves as a signalling molecule that activates stress-associated genes, including those encoding antioxidant enzymes. As a result, antioxidant capacity is enhanced, which in turn expedites detoxification of excess ROS and leads to a reduction in the oxidative stress and the alleviation of cell death.