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Article

Divergent Roles of CNGC2 and CNGC4 in the Regulation of Disease Resistance, Plant Growth and Heat Tolerance in Arabidopsis

by
Shan Lu
1,
Tianquan Zhu
1,
Lilin Luo
1,
Nana Ouyang
1,
Jian Hua
2,* and
Baohong Zou
1,*
1
The State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
2
Plant Biology Section, School of Integrated Plant Science, Cornell University, Ithaca, NY 14853, USA
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(9), 2176; https://doi.org/10.3390/agronomy12092176
Submission received: 13 June 2022 / Revised: 2 September 2022 / Accepted: 3 September 2022 / Published: 13 September 2022
(This article belongs to the Special Issue Molecular and Genetic Mechanisms of Plant Disease Resistance)
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Abstract

:
Arabidopsis cyclic nucleotide-gated channels (CNGC) 2 and 4 are shown to negatively regulate disease resistance and heat tolerance and to positively regulate plant growth. Whether or not their functions in these processes are interdependent is largely unknown. Here, using the mutation of phytoalexin deficient 4 (PAD4) to inhibit the enhanced defense response and programmed cell death (PCD), we assessed the contribution of the altered defense response to the heat tolerance and plant growth in the cngc2 and cngc4 single and double mutants. The pad4 mutation reverted the enhanced disease resistance of the cngc2 and cngc4 mutants at the normal temperature (22 °C) but not at the elevated temperature (28 °C). The pad4 mutation slightly alleviated the dwarfism of the cngc2 and cngc4 mutants at 22 °C but not at 28 °C, indicating a small contribution from the defense response to plant growth regulation. The pad4 mutation also reduced the enhanced heat tolerance in the cngc mutants, suggesting an involvement of PCD in heat tolerance. In addition, a higher heat tolerance was correlated with more opened stomata under heat treatment among the wild type and mutants of the PAD4, CNGC2 and CNGC4 genes. In sum, this study suggests that the regulation of heat tolerance and plant growth by CNGC2 and CNGC4 is almost independent of their regulation of disease resistance. It also reveals a PAD4-dependent role of CNGC2 and CNGC4 in stomatal aperture regulation and heat tolerance.

1. Introduction

As nonselective and ligand-gated cation channels, cyclic nucleotide-gated channels (CNGCs) transport Ca2+, K+, Na+ and some other cations and mediate signaling in response to diverse environmental stimuli [1]. Arabidopsis thaliana has 20 CNGC family members [2], many of which have been shown to regulate growth and development, and abiotic and biotic stress tolerance [3].
The first reported and well-studied function of CNGCs in Arabidopsis is the regulation of disease resistance. The cngc2/dnd1 (defense, no death 1) and cngc4/dnd2 mutants were isolated by their loss of the HR (hypersensitive response), a form of PCD (programmed cell death), to the avirulent pathogen Pseudomonas syringae expressing avrRpt2, but the mutants exhibited an enhanced resistance to the pathogen [4,5,6]. Both the cngc2 and cngc4 mutants have constitutive defense responses with elevated levels of SA (salicylic acid), high expression of PR (pathogenesis-related) genes and enhanced broad-spectrum resistance to virulent bacterial, fungal and viral pathogens [4,6]. The CNGC2 and CNGC4 proteins interact with each other to form a heteromeric complex to mediate PAMP (pathogen-associated molecular patterns)-triggered Ca2+ influx from the apoplast to cytosol [7,8]. Introduction of a salicylate hydroxylase NahG gene that abolishes SA accumulation reverts the enhanced disease resistance and increased PR gene expression but not the loss of HR in the cngc2 and cngc4 mutants [5,6]. PAD4 (phytoalexin deficient 4), a key regulator of SA biosynthesis, SA signaling and PCD, is required for enhanced resistance in cngc2 and cngc4 but not for SA accumulation of these mutants [9,10,11]. In contrast to the negative role of CNGC2 and CNGC4 in disease resistance, CNGC11, CNGC12, CNGC19 and CNGC20 are shown to be positive regulators of pathogen defense responses in Arabidopsis [12,13,14].
CNGC2 and CNGC4 also play important roles in temperature stress in Arabidopsis, and their roles are developmental-stage-dependent. CNGC2 and CNGC4 proteins are thought to be thermosensors by controlling heat-induced cytoplasmic Ca2+ influx, and they repress HSR (heat stress response) and acquired heat tolerance in seedlings [15]. The loss of CNGC2 leads to enhanced basal heat tolerance at the seedling stage but reduced heat tolerance at the reproductive stage, and the heat tolerance in seedlings and flowers is correlated with accumulation of heat-response-related proteins such as HSPs (heat-shock proteins), APXs (ascorbate peroxidases) and MBF1c (multiprotein bridging factor 1c) [16]. In addition, CNGC2 and CNGC4 are shown to be positive regulators of freezing tolerance and chilling growth at the seedling stage, and their mutants displayed shorter hypocotyls and greater reduction in fresh weight in chilling conditions and had lower survival rates after freezing treatment [17].
Stomatal pores in leaf epidermis play a crucial role in heat tolerance by influencing leaf temperature and water loss [18,19]. Stomatal opening increases transpiration and, therefore, facilitates leaf cooling at elevated temperature, while stomatal closure protects leaves from water loss under extreme high temperature [20]. Heat stress is often accompanied by the generation of cellular ROS (reactive oxygen species) that promotes stomatal closure and reduces water loss [18,21]. A few genes were reported to regulate heat tolerance associated with H2O2 accumulation in guard cells under heat treatment [22,23,24]. These studies suggest that stomatal aperture control is associated with heat tolerance, but whether more opened or more closed stomata leads to an enhanced heat tolerance is likely dependent on the environment.
Here, we investigated whether or not the roles of CNGC2 and CNGC4 in growth promotion and heat tolerance repression are dependent on their role in inhibiting disease resistance and/or promoting PCD by using the pad4 mutation. We reveal that PAD4 was required for the enhanced disease resistance of the cngc mutants at 22 °C, and partially required for the dwarfism at 22 °C and heat tolerance of the cngc mutants, but was not required for the enhanced disease resistance and dwarfism of the cngc mutants at 28 °C.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

To study the function of CNGC2 and CNGC4 and their relationship with PAD4, Arabidopsis thaliana mutants cngc2-1 (CS6523, dnd1), cngc2-2 (SALK_019922), cngc4-1 (CS6524, dnd2), cngc4-2 (GK-106G06) and pad4 (pad4-1) [25] were obtained from ABRC (Arabidopsis Biological Resource Center). For generating combined mutants, cngc2-2 was crossed with cngc4-2, cngc2-2 was crossed with pad4, cngc4-2 was crossed with pad4, and then cngc2-2 pad4 was crossed with cngc4-2 pad4. The homozygous of cngc2-2 cngc4-2, cngc2-2 pad4 and cngc4-2 pad4 double mutants and cngc2-2 cngc4-2 pad4 triple mutants were isolated from the F2 population. All primers used for mutant identification are summarized in Supplementary Table S1.
The Arabidopsis seeds were sown in soil and grown in growth chambers (16 h light/12 h dark, 100 µmol/s/m2, 65% humidity, 22 °C) after being stratified at 4 °C for 3 days. For pathogen growth assay, plants were grown in chambers with short day condition of 12 h light/12 h dark. For growth phenotypic analysis at 28 °C, plants were moved to 28 °C for 18 days after germination and grown at 22 °C for one week.

2.2. Pathogen Growth Assay

The resistance was analyzed through pathogen growth assays. The bacteria strain Pst DC3000 (Pseudomonas syringae pv. tomato DC3000) grown on King’s B medium plates for 2 days were collected and diluted with 10 mM MgCl2 and 0.02% (v/v) Silwet L-77. The bacteria were resuspended to an OD600 of 0.05 and dip-inoculated into two-week-old seedlings grown at 22 °C or 12-day-old seedlings grown at 28 °C. Pathogen growth assays were performed as previously described [26]. The amounts of bacteria in plants were analyzed at 0 (keeping covered for 1 h after dipping), 4 dpi at 22 °C and 0, 3 dpi at 28 °C.

2.3. Histochemical Staining

Accumulation of H2O2 and cell death were detected by DAB (3, 3′-diaminobenzidine) and trypan blue staining, respectively, following the methods described previously [27]. Two-week-old seedlings grown at 22 °C or 12-day-old seedlings grown at 28 °C were dip-inoculated with Pst DC3000, and the staining was performed at 4 dpi at 22 °C and 3 dpi at 28 °C, respectively.

2.4. Gene Expression Assay

Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Three-week-old seedlings grown at 22 °C or 28 °C were inoculated with Pst DC3000 and seedling tissues were collected at 24 h after dipping for RNA isolation. Total RNAs were extracted from seedling tissues using TRIpure Reagent (RP1001, Bioteke, Beijing, China) following the manufacture’s protocols. cDNA was synthesized from 1 μg total RNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme, Nanjing, China). The cDNAs were used as templates for expression analysis by qRT-PCR using AceQ® qPCR SYBR® Green Master Mix (Q111-02, Vazyme, Nanjing, China) with AtActin2 (AT3G18780) as internal control. The qRT-PCR was run on the Bio-Rad CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) with three biological replicates. All primers used for qRT-PCR are summarized in Supplementary Table S1.

2.5. Heat Tolerance Assay

For heat treatment of Arabidopsis seedlings, seeds were sown on soil neatly, stratified at 4 °C for 3 days, and then grown at 22 °C for 9 days. Plants were then transferred to 35 °C for heat treatment, and the survival rate were calculated at 10 days and 20 days after treatment.

2.6. Stomatal Aperture Assay

Stomatal apertures were measured from the leaves before (20-day-old plants grown at 22 °C) or after heat treatment of 40 °C for 6 h. The leaf epidermal stomata observation was performed using clear nail polish for epidermal impressions [28]. Dried nail polish was peeled from leaf epidermal and observed under an Olympus BX53 microscope. Stomatal apertures were measured using ImageJ software and calculated as the ratio of the width/length of stomata. One hundred stomatal apertures were measured for each sample from about four independent leaves.

2.7. Statistical Analysis

All the experiments were performed in three biological repeats and the obtained results are shown as average values ± SD (standard deviations) from three biological repeats. Significant differences among samples were determined based on Duncan’s multiple range test and one-way ANOVA at the level of p value of 0.05 or 0.01.

3. Results

3.1. Enhanced Disease Resistance of the cngc2 and cngc4 Mutants Is Dependent on PAD4 at Normal Temperature

Early studies of CNGC2 and CNGC4 were mostly done on their missense mutants cngc2-1/dnd1 and cngc4-1/dnd2. We verified their function using the T-DNA insertion mutants cngc2-2 and cngc4-2 that are both knockout mutants [7,16]. A cngc2-2 cngc4-2 double mutant was also generated to fully assess the interaction of these two genes. The cngc single and double mutants were identified by sequencing and PCR analysis (Supplementary Figure S1). Disease resistance was assayed by virulent bacterial pathogen Pst DC3000 for these mutants. At 4 days after inoculation, pathogen growth in cngc2-1, cngc2-2, cngc4-1, cngc4-2 and cngc2-2 cngc4-2 mutants was reduced (10% decrease) compared to that in the wild-type Col-0 (Figure 1a). There was no significant difference of pathogen growth among the cngc2-2, cngc4-2 and cngc2-2 cngc4-2 mutants (Figure 1a).
The cngc2-1 and cngc4-1 mutants were shown to be more resistant to virulent pathogen Psm E4326 (Pseudomonas syringae pv. Maculicola E4326) in a PAD4-dependent manner [10]. We determined whether or not the function of CNGC2 and CNGC4 in regulating disease resistance to the virulent bacterial pathogen Pst DC3000 is also dependent on PAD4. We generated the cngc2-2 pad4 and cngc4-2 pad4 double mutants, as well as the cngc2-2 cngc4-2 pad4 triple mutants, through crossing. The cngc pad4 mutants were isolated by sequencing and PCR analysis (Supplementary Figure S1). The enhanced resistance in the cngc2 and cngc4 single and double mutants was suppressed by the pad4 mutation. The three cngc pad4 mutants all had a higher pathogen growth compared to the cngc mutants (17% increase), to the same level as the pad4 mutant (Figure 1a).
Cell death and ROS accumulation are correlated with disease resistance [29], but cngc2-1 and cngc4-1 had enhanced resistance without cell death [7]. We detected ROS accumulation and cell death in the leaves of the cngc mutants after infection. Lower H2O2 accumulation and cell death was observed in the cngc2 and cngc4 mutants compared with the wild type, and all the cngc pad4 mutants had the same level of H2O2 accumulation and cell death as the pad4 mutant, which had more H2O2 accumulation and cell death than the wild type (Figure 1b,c). These observations were further supported by quantifying the average optical density of DAB staining. The optical density values of DAB staining were significantly lower in the cngc2 and cngc4 mutants and higher in the cngc pad4 mutants compared to the wild type after infection (Supplementary Figure S2a). These results indicate that CNGC2 and CNGC4 negatively regulate disease resistance to the virulent pathogen Pst DC3000 at normal growth temperature and this function is PAD4-dependent. Thus, CNGC2 and CNGC4 are positive regulators of cell death and ROS accumulation after pathogen infection, and PAD4 appears to function downstream of CNGC in this regulation.

3.2. Enhanced Disease Resistance of the cngc2 and cngc4 Mutants Is Not Dependent on PAD4 at Moderately Elevated Temperature

Because a moderately elevated temperature (28 °C) could inhibit the defense response [30], we investigated resistance to the virulent pathogen Pst DC3000 in the cngc2 and cngc4 mutants at 28 °C. At 3 days after inoculation by dipping, pathogen growth in cngc2-2, cngc4-2 and cngc2-2 cngc4-2 was lower than that in the wild type (Figure 2a). The reduction in pathogen growth in the mutants compared to the wild type was smaller at 28 °C compared to at 22 °C (Figure 1a and Figure 2a). There was no significant difference of pathogen growth among the cngc2, cngc4 and cngc2 cngc4 mutants at 28 °C (Figure 2a). Surprisingly, unlike at 22 °C, the pad4 mutation did not alter the disease resistant phenotype of cngc2, cngc4 or cngc2 cngc4 mutants, and all three cngc pad4 mutants had the same pathogen growth level as the cngc mutants at 28 °C (Figure 2a).
ROS accumulation and cell death were assayed in the leaves after 3 days of infection at 28 °C. Similar to at 22 °C, the cngc mutants had lower H2O2 accumulation and cell death compared to the wild type or pad4 at 28 °C (Figure 2b,c). Unlike at 22 °C, the three cngc pad4 mutants also had lower H2O2 accumulation and cell death, to the same level as the cngc mutants, while the pad4 mutant had higher accumulation and cell death than the wild type (Figure 2b,c). The optical density values of DAB staining were significantly lower in all the cngc and cngc pad4 mutants compared to the wild type after infection (Supplementary Figure S2b). These results suggest that CNGC2 and CNGC4 appear to negatively regulate disease resistance through a PAD4-independent process at moderately elevated temperatures.

3.3. Expression of SA-Associated Genes at Normal and Moderately Elevated Temperatures after Infection

It has been reported that both the cngc2 and cngc4 mutants display enhanced disease resistance to Psm E4326 associated with constitutively elevated levels of SA and high expression of PR genes [4,6]. So, we further analyzed expression of SA-associated genes at both 22 °C and 28 °C after infection. We detected the expression of genes associated with SA biosynthesis (ICS1 and CPB60g) [31], SA-associated defense regulators EDS1 and PAD4, and also the PR1 in the seedlings of Col-0, pad4, cngc2-2 cngc4-2 and cngc2-2 cngc4-2 pad4 grown at 22 °C or 28 °C at 1 dpi.
At 22 °C, the expression of ICS1, EDS1, PAD4 and CPB60g was all significantly lower in the CNGC-related mutants than in the wild type and higher than in pad4, and there was no difference between the cngc2-2 cngc4-2 and cngc2-2 cngc4-2 pad4 mutants (Figure 3). At 28 °C, EDS1 expression was higher in cngc2-2 cngc4-2 compared to the wild type, and the pad4 mutation decreased the expression in cngc2-2 cngc4-2 but was still higher than the wild type (Figure 3). CPB60g expression was lower in all the mutants compared to the wild type at 28 °C (Figure 3). PR1 expression was much higher in cngc2-2 cngc4-2 compared with the wild type and the pad4 mutation decreased its expression but was still higher than the wild type at 22 °C, while the cngc2-2 cngc4-2 pad4 mutant still had similar high expression level as cngc2-2 cngc4-2 at 28 °C (Figure 3). Therefore, the pad4 mutation reverted the enhanced disease resistance of the cngc mutants at 22 °C but not at 28 °C and was correlated with the expression level of PR1 gene.

3.4. The Growth Defect of the cngc Mutants Is Partially Dependent on PAD4 at Normal Temperature

It was hypothesized that the dwarf phenotype of the cngc2 and cngc4 mutants resulted from the high accumulation of SA and high expression of PR genes expression [4,6]. We tested this by analyzing the cngc pad4 mutants where the enhanced disease resistance of the cngc mutants was abolished by pad4 at 22 °C. Analysis of seedlings growth at 22 °C for 3 weeks revealed that the cngc single and double mutants had a 90% decrease in growth compared to the wild type, while the cngc pad4 mutants had 80% growth reduction (Figure 4a). The rosette weights were around 14 mg for all three cngc mutants, 25 mg for all the cngc pad4 mutants, 132 mg for the wild type and 123 mg for pad4 (Figure 4b). The rosette areas were around 2 cm2 for all three cngc mutants, 4 cm2 for all the cngc pad4 mutants, 22 cm2 for the wild type and 20 cm2 for pad4 (Figure 4c). There was no significant growth difference among the cngc2, cngc4 and cngc2 cngc4 mutants, and there was also no significant growth difference among the cngc2 pad4, cngc4 pad4 and cngc2 cngc4 pad4 mutants (Figure 4b,c). In sum, the dwarfism of the cngc single and double mutants is partially dependent on PAD4 at 22 °C.

3.5. The Growth Defect of the cngc Mutants Is Not Dependent on PAD4 at Moderately Elevated Temperatures

We then analyzed the growth phenotypes of the cngc and cngc pad4 mutants at 28 °C. After 18 days grown at 28 °C, the three cngc mutants and three cngc pad4 mutants all had reduced growth (90–96% decrease) compared to the wild type and pad4 (Figure 5a). The rosette weights were around 5 mg for all three cngc mutants and three cngc pad mutants, 51 mg for the wild type and 50 mg for pad4 (Figure 5b). The rosette areas were around 1 cm2 for all cngc and cngc pad mutants and 26 cm2 for the wild type and pad4 (Figure 5c). There was no significant growth difference among cngc mutants and their combination mutants with pad4 (Figure 5b,c). These results indicated that the dwarfism of the cngc single and double mutants was not dependent on PAD4 at moderately elevated temperatures.

3.6. Enhanced Heat Tolerance of the cngc Mutants Is Partially Dependent on PAD4

We further determined whether or not the heat tolerance function of CNGC2 and CNGC4 is dependent on PAD4. After 10 days of heat treatment at 35 °C, the survival rates were more than 85% for the cngc2-2, cngc4-2 and cngc2-2 cngc4-2 mutants, much higher (70% increase) than the 50% survival rate for the wild type (Figure 6a,b). The cngc pad4 mutants had the same survival rates as the cngc mutants, much higher than the 50% survival rate for pad4 or the wild type. After 20 days of heat treatment, almost no seedlings of the wild type or the pad4 mutant survived, while 43-50% of the cngc2-2, cngc4-2 and cngc2-2 cngc4-2 plants survived and 18–27% of the cngc pad4 mutants survived (Figure 6a,b). No significant difference was found for the survival rates among the cngc2, cngc4 and cngc2 cngc4 mutants or among the cngc2 pad4, cngc4 pad4 and cngc2 cngc4 pad4 mutants (Figure 6b). These results indicate that PAD4 partially mediates the enhanced heat tolerance in the CNGC2 and CNGC4 mutants, especially under longer heat treatment.

3.7. The Stomatal Apertures of the cngc Mutants Are Partially Suppressed by the PAD4 Mutation under Heat

We further investigated whether or not CNGC2 and CNGC4 affect stomatal movement in response to heat. Stomatal apertures (ratio of width and length of the pore) were measured in cngc, cngc pad4, pad4 and the wild type before and after heat treatment. At 22 °C, all the genotypes had similar apertures, ranging from 0.68 to 0.69 (Figure 7a,b). The wild type and the pad4 mutant had more closed stomata at 6 h of heat treatment at 40 °C compared to no heat treatment, and their apertures decreased to around 0.60 (Figure 7a,b). In contrast, cngc2-2, cngc4-2, and cngc2-2 cngc4-2 mutants had more opened stomata at 6 h of heat treatment compared to no heat treatment, and their apertures increased to around 0.78 (Figure 7a,b). The cngc single and double mutants had a 30% increase in stomatal aperture compared to the wild type, while the cngc pad4 mutants had a 16% stomatal aperture increase after heat treatment (Figure 7a,b). No significant difference was observed among the cngc2, cngc4 and cngc2 cngc4 mutants or among the cngc2 pad4, cngc4 pad4 and cngc2 cngc4 pad4 mutants (Figure 7b). Therefore, the stomatal aperture was correlated with the survival rate under heat among cngc, cngc pad4 and the wild type, with a larger aperture associated with a higher survival rate.

4. Discussion

In this study, we examined the biological functions of Arabidopsis CNGC2 and CNGC4 genes using their knockout mutants and probed the inter-dependence of their roles in disease resistance, plant growth and heat tolerance by using the pad4 mutation. Our findings suggest that CNGC2 and CNGC4 regulate these three processes largely independently, although a minor contribution of the defense response was found for the growth regulation. We also revealed a differential dependence of PAD4 for resistance repressed by CNGC2/4 at normal temperature and moderately high temperature. In addition, a role in stomatal aperture control under heat was found for CNGC2 and CNGC4, and this function is associated with the heat tolerance and PAD4.
The relationships of CNGC2/4 and PAD4 are different in regulating disease resistance at 22 °C and 28 °C. PAD4 is an essential mediator of plant immunity [9,32,33] and mediates temperature sensitive defense responses [30,34]. Consistent with the previous study, we found CNGC2 and CNGC4 regulate disease resistance at 22 °C in a PAD4-dependent manner, but the pad4 mutation did not alter the enhanced disease resistance in the cngc mutants at 28 °C (Figure 2a), suggesting that the regulation of disease resistance of CNGC2 and CNGC4 at 28 °C might be independent of PAD4. In addition, previous studies proved that SA accumulation in the cngc2 and cngc4 mutants to be completely PAD4-independent at 22 °C [10]. Consistent with this study, we found that the expression of SA biosynthetic and associated defense regulators [31] was almost at the same level in the cngc and cngc pad4 mutants (Figure 3). Because SA accumulation and PAD4-mediated defense are repressed at 28 °C, it is unknown what defense mechanisms are enhanced in the cngc mutants at elevated temperature. In addition, the loss of CNGC2 and CNGC4 function leads to growth defects at both 22 °C and 28 °C and the pad4 mutation slightly alleviated the dwarf phenotype at 22 °C but not at 28 °C (Figure 4 and Figure 5), indicating that the effect of CNGC2 and CNGC4 in disease resistance has only a small contribution to their regulation of plant growth. The genetic interaction between CNGC2/4 with PAD4 is correlated with the disease resistance phenotype: pad4 suppressed the enhanced resistance of the cngc mutants at 22 °C but not at 28 °C (Figure 1 and Figure 2). Therefore, the regulation of disease resistance by CNGC2 and CNGC4 is tightly associated with or perhaps resulting from their regulation of ROS and cell death. CNGC2 has been implicated in regulating Ca2+ influx during pathogen infection [35] and cytosolic Ca2+ elevation could activate downstream cellular machinery to promote stomatal closure [36]. Because Ca2+ channels are shown to be involved in stomatal closure regulation [37,38,39], it will be interesting to investigate in the future whether or not CNGC2 and CNGC4 regulate stomatal defense to restrict pathogen entry at the first line of plant immunity at 22 °C and 28 °C.
The negative regulation of heat tolerance in seedlings by CNGC2 and CNGC4 were attributed to their repression of heat-induced cytoplasmic Ca2+ and HSR, as well as their regulation of ROS homeostasis [15,16]. Here we identified a role of CNGC2 and CNGC4 in stomatal movement control and a correlation of stomatal aperture and heat tolerance. CNGC2 and CNGC4 inhibit stomatal opening in response to heat, and PAD4 is partially required for this function. PAD4 is also required for the heat-tolerance repression function of CNGC2 and CNGC4. It is likely that a larger stomatal aperture in the cngc2 and cngc4 mutants leads to more leaf cooling and, therefore, to enhanced heat tolerance. The role of CNGC2 and CNGC4 in stomatal movement control could come from their regulation of the calcium signal, which subsequently influences ROS and PCD. The role in heat tolerance is likely independent of their role in disease-resistance regulation, as PAD4-mediated resistance to Pst DC3000 is inhibited by an elevated temperature of 28 °C and is expected to be suppressed under the high temperature of 35 °C. PAD4 has been shown to be critical for high light-stress responses and this role is related to PCD and ROS regulation [40,41]. Therefore, PAD4 may mediate the heat tolerance regulation of CNGC2 and CNGC4 via PCD or ROS, which is independent of its regulation of resistance to Pst DC3000. It will be interesting to analyze the Ca2+ signal, as well as ROS accumulation, in guard cells under heat treatment in the future.
This study also supports that CNGC2 and CNGC4 function in a protein complex. It has been demonstrated that some Arabidopsis CNGC proteins can interact with each other to form heteromeric channel complexes [3], and the CNGC2 and CNGC4 proteins work together to negatively regulate the disease resistance of Arabidopsis [7,8]. Here, we found identical effects from the loss of CNGC2 and CNGC4 function either individually or together in all phenotypes we characterized. These include enhanced resistance at 22 °C and 28 °C (Figure 1 and Figure 2), growth defects at 22 °C and 28 °C (Figure 4 and Figure 5), heat tolerance and the stomatal opening (Figure 6 and Figure 7). The pad4 mutation also had the same effect on the cngc2, cngc4 and cngc2 cngc4 mutants of all the detected phenotypes. These findings support that CNGC2 and CNGC4 work in a protein complex to regulate disease resistance, growth and heat tolerance.
In sum, this study reveals the largely independent roles of CNGC2 and CNGC4 in disease resistance, plant growth and heat tolerance and identifies a new role of these genes in stomatal aperture control that is associated with heat tolerance. Furthermore, it suggests an involvement of PCD medicated by PAD4 in heat tolerance and disease resistance regulation at elevated temperatures. These findings have expanded our knowledge of the function of CNGC2 and CNGC4 and will further enhance our understanding of the molecular mechanisms underlying the diverse roles of these channel proteins in plants.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy12092176/s1: Supplementary Table S1. Primers for genotyping; Supplementary Figure S1. Identification of the mutants by PCR or sequencing; Supplementary Figure S2. Average optical density of DAB staining.

Author Contributions

Conceptualization, J.H. and B.Z.; methodology, S.L.; investigation, S.L., T.Z., L.L. and N.O.; data curation, S.L., T.Z.; writing—original draft preparation, S.L., and J.H. and B.Z.; writing—review and editing, B.Z. and J.H.; visualization, B.Z.; supervision, J.H. and B.Z.; project Administration, B.Z.; funding acquisition, S.L. and B.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work is supported by the National Natural Science Foundation of China (31700223, 31971827), Open Funds of the State Key Laboratory of Plant Physiology and Biochemistry (SKLPPBKF2102), and Jiangsu Collaborative Innovation Center for Modern Crop Production and Cyrus Tang Innovation Center for Crop Seed Industry.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Genes from Arabidopsis in this study can be accessed on TAIR (www.arabidopsis.org) (accessed on 21 January 2022) under the following accession numbers: CNGC2 (AT5G15410), CNGC4 (AT5G54250) and PAD4 (AT3G52430).

Conflicts of Interest

The authors have no conflicts of interest to declare.

References

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Figure 1. Disease resistance phenotypes of the CNGC (cyclic nucleotide-gated channel) and PAD4 (phytoalexin deficient 4) mutations under normal temperature. (a) Growth of Pst DC3000 (Pseudomonas syringae pv. tomato DC3000) in the wild-type Col-0, pad4, cngc and cngc pad4 mutants at 0 and 4 d post-inoculation (dpi) at 22 °C. Values represent means ± SD (standard deviations) for three biological repeats. Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05). FW, fresh weight. (b) DAB (3, 3′-diaminobenzidine) staining (for H2O2 accumulation detection) of 4 dpi leaves of the wild-type Col-0, cngc and cngc pad4 mutants. Bars = 100 μm. (c) Trypan blue staining (for cell death detection) of inflorescences of 4 dpi leaves of the wild-type Col-0, cngc and cngc pad4 mutants. Bars = 100 μm.
Figure 1. Disease resistance phenotypes of the CNGC (cyclic nucleotide-gated channel) and PAD4 (phytoalexin deficient 4) mutations under normal temperature. (a) Growth of Pst DC3000 (Pseudomonas syringae pv. tomato DC3000) in the wild-type Col-0, pad4, cngc and cngc pad4 mutants at 0 and 4 d post-inoculation (dpi) at 22 °C. Values represent means ± SD (standard deviations) for three biological repeats. Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05). FW, fresh weight. (b) DAB (3, 3′-diaminobenzidine) staining (for H2O2 accumulation detection) of 4 dpi leaves of the wild-type Col-0, cngc and cngc pad4 mutants. Bars = 100 μm. (c) Trypan blue staining (for cell death detection) of inflorescences of 4 dpi leaves of the wild-type Col-0, cngc and cngc pad4 mutants. Bars = 100 μm.
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Figure 2. Disease resistance phenotypes of cngc and cngc pad4 mutants under moderately elevated temperatures. (a) Growth of Pst DC3000 in the wild-type Col-0, pad4, cngc and cngc pad4 mutants at 0 and 3 dpi at 28 °C. Values represent means ± SD for three biological repeats. Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05). FW, fresh weight. (b) DAB staining (for H2O2 accumulation detection) of 3 dpi leaves of the he wild-type Col-0, cngc and cngc pad4 mutants. Bar = 100 μm. (c) Trypan blue staining (for cell death detection) of inflorescences of 3 dpi leaves of the he wild-type Col-0, cngc and cngc pad4 mutants. Bar = 100 μm.
Figure 2. Disease resistance phenotypes of cngc and cngc pad4 mutants under moderately elevated temperatures. (a) Growth of Pst DC3000 in the wild-type Col-0, pad4, cngc and cngc pad4 mutants at 0 and 3 dpi at 28 °C. Values represent means ± SD for three biological repeats. Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05). FW, fresh weight. (b) DAB staining (for H2O2 accumulation detection) of 3 dpi leaves of the he wild-type Col-0, cngc and cngc pad4 mutants. Bar = 100 μm. (c) Trypan blue staining (for cell death detection) of inflorescences of 3 dpi leaves of the he wild-type Col-0, cngc and cngc pad4 mutants. Bar = 100 μm.
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Figure 3. Relative expression of SA-associated genes under normal or moderately elevated temperatures. Seedlings of Col-0, pad4, cngc2-2 cngc4-2 and cngc2-2 cngc4-2 pad4 grown at 22 °C or 28 °C were inoculated with Pst DC3000. Gene expression in these seedlings was measured by qRT-PCR (quantitative real-time PCR) at 1 dpi and represented as relative to the level of Col-0 in each temperature. Data are means ± SE (standard error, three biological repeats). Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
Figure 3. Relative expression of SA-associated genes under normal or moderately elevated temperatures. Seedlings of Col-0, pad4, cngc2-2 cngc4-2 and cngc2-2 cngc4-2 pad4 grown at 22 °C or 28 °C were inoculated with Pst DC3000. Gene expression in these seedlings was measured by qRT-PCR (quantitative real-time PCR) at 1 dpi and represented as relative to the level of Col-0 in each temperature. Data are means ± SE (standard error, three biological repeats). Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
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Figure 4. Growth phenotypes of cngc and cngc pad4 mutants under normal temperature. (a) Rosette leaves of the wild-type Col-0, pad4, cngc and cngc pad4 plants grown at 22 °C for 3 weeks. Bar = 2 cm. (b,c) Quantification of rosette weight (b) and area (c) of each genotype grown at 22 °C for 3 weeks. Data are means ± SD (n = 10). Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
Figure 4. Growth phenotypes of cngc and cngc pad4 mutants under normal temperature. (a) Rosette leaves of the wild-type Col-0, pad4, cngc and cngc pad4 plants grown at 22 °C for 3 weeks. Bar = 2 cm. (b,c) Quantification of rosette weight (b) and area (c) of each genotype grown at 22 °C for 3 weeks. Data are means ± SD (n = 10). Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
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Figure 5. Growth phenotypes of cngc and cngc pad4 mutants under moderately elevated temperatures. (a) Rosette leaves of the wild-type Col-0, pad4, cngc and cngc pad4 plants grown at 28 °C for 18 days after germination at 22 °C for one week. Bar = 2 cm. (b,c) Quantification of rosette weight (b) and area (c) of each genotype grown at 28 °C for 18 days after germination at 22 °C for one week. Data are means ± SD (n = 10). Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
Figure 5. Growth phenotypes of cngc and cngc pad4 mutants under moderately elevated temperatures. (a) Rosette leaves of the wild-type Col-0, pad4, cngc and cngc pad4 plants grown at 28 °C for 18 days after germination at 22 °C for one week. Bar = 2 cm. (b,c) Quantification of rosette weight (b) and area (c) of each genotype grown at 28 °C for 18 days after germination at 22 °C for one week. Data are means ± SD (n = 10). Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
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Figure 6. Heat tolerance of cngc and cngc pad4 mutants. (a) Images of the wild-type Col-0, pad4, cngc2-2 and cngc2-2 pad4 plants after heat treatment. Plants were germinated and grown at 22 °C for 9 days and transferred to 35 °C for heat treatment. Images were taken at 10 days and 20 days after transfer. Bar = 1 cm. (b) Survival rates of heat-treated plants of the wild-type Col-0, pad4, cngc and cngc pad4 mutants at 35 °C for 10 days and 20 days. Values represent means ± SD for three biological repeats. Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
Figure 6. Heat tolerance of cngc and cngc pad4 mutants. (a) Images of the wild-type Col-0, pad4, cngc2-2 and cngc2-2 pad4 plants after heat treatment. Plants were germinated and grown at 22 °C for 9 days and transferred to 35 °C for heat treatment. Images were taken at 10 days and 20 days after transfer. Bar = 1 cm. (b) Survival rates of heat-treated plants of the wild-type Col-0, pad4, cngc and cngc pad4 mutants at 35 °C for 10 days and 20 days. Values represent means ± SD for three biological repeats. Letters indicate significant differences based on Duncan’s multiple range test among the means via ANOVA (p < 0.05).
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Figure 7. Stomatal apertures of cngc and cngc pad4 mutants under heat. (a) Microscopic observation of stomatal statements of the wild-type Col-0, pad4, cngc2-2 cngc4-2 and cngc2-2 cngc4-2 pad4 plants before or after heat treatment. Plants were germinated and grown at 22 °C for 20 days and transferred to 40 °C for 6 h. Bar = 25 μm. (b) Quantification of stomatal apertures of the wild-type Col-0, pad4, cngc and cngc pad4 mutants before (22 °C) or after heat treatment (40 °C for 6 h). Stomatal apertures were defined by the ratio between width and length. Data are means from 100 stomata for each genotype. Error bars show the SDs. Letters indicate significant differences based on Duncan’s multiple range test based on the ANOVA analysis (p < 0.01).
Figure 7. Stomatal apertures of cngc and cngc pad4 mutants under heat. (a) Microscopic observation of stomatal statements of the wild-type Col-0, pad4, cngc2-2 cngc4-2 and cngc2-2 cngc4-2 pad4 plants before or after heat treatment. Plants were germinated and grown at 22 °C for 20 days and transferred to 40 °C for 6 h. Bar = 25 μm. (b) Quantification of stomatal apertures of the wild-type Col-0, pad4, cngc and cngc pad4 mutants before (22 °C) or after heat treatment (40 °C for 6 h). Stomatal apertures were defined by the ratio between width and length. Data are means from 100 stomata for each genotype. Error bars show the SDs. Letters indicate significant differences based on Duncan’s multiple range test based on the ANOVA analysis (p < 0.01).
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Lu, S.; Zhu, T.; Luo, L.; Ouyang, N.; Hua, J.; Zou, B. Divergent Roles of CNGC2 and CNGC4 in the Regulation of Disease Resistance, Plant Growth and Heat Tolerance in Arabidopsis. Agronomy 2022, 12, 2176. https://doi.org/10.3390/agronomy12092176

AMA Style

Lu S, Zhu T, Luo L, Ouyang N, Hua J, Zou B. Divergent Roles of CNGC2 and CNGC4 in the Regulation of Disease Resistance, Plant Growth and Heat Tolerance in Arabidopsis. Agronomy. 2022; 12(9):2176. https://doi.org/10.3390/agronomy12092176

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Lu, Shan, Tianquan Zhu, Lilin Luo, Nana Ouyang, Jian Hua, and Baohong Zou. 2022. "Divergent Roles of CNGC2 and CNGC4 in the Regulation of Disease Resistance, Plant Growth and Heat Tolerance in Arabidopsis" Agronomy 12, no. 9: 2176. https://doi.org/10.3390/agronomy12092176

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