Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper

Abstract

Plant basic helix-loop-helix (bHLH) transcription factors (TFs) play central roles in various abiotic stresses. However, its role in plant cold resistance is largely unknown. Previously, we characterised CaNACOSS in pepper, which positively regulates tolerance to cold, salt and drought stresses tolerance. Here, we identified CabHLH035, a CaNAC035-interacting protein in pepper. To explore its functions in cold stress tolerance, we silenced the gene in pepper via virus-induced gene silencing (VIGS) and overexpressed the gene in Arabidopsis. The results showed that CabHLHOSS expression was induced by cold treatment, and silencing of CabHLHOSS decreased cold stress tolerance. Conversely, overexpression of CabHLHOSS in Arabidopsis increased cold stress tolerance. To investigate homologs genes of C-repeat binding factor (CBF) pathway proteins and reactive oxygen species (ROS) marker gene expression blocking by CabHLHOSS, we performed yeast one-hybrid (Y1H), dual luciferase and electrophoretic mobility shift assay experiments. The results showed that CabHLH035 bound to the region upstream of the CaCBFlA and CaAPX promoters. Additionally, CaCBFlA bound to the CaDHN4 promoter. Taken together, our results showed that CabHLHOSS plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops. The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

Headnote

Received 16 July 2022; Received in revised form 5 October 2022; Accepted 26 December 2022

Available online 16 March 2023

ABSTRACT

Plant basic helix-loop-helix (bHLH) transcription factors (TFs) play central roles in various abiotic stresses. However, its role in plant cold resistance is largely unknown. Previously, we characterised CaNACOSS in pepper, which positively regulates tolerance to cold, salt and drought stresses tolerance. Here, we identified CabHLH035, a CaNAC035-interacting protein in pepper. To explore its functions in cold stress tolerance, we silenced the gene in pepper via virus-induced gene silencing (VIGS) and overexpressed the gene in Arabidopsis. The results showed that CabHLHOSS expression was induced by cold treatment, and silencing of CabHLHOSS decreased cold stress tolerance. Conversely, overexpression of CabHLHOSS in Arabidopsis increased cold stress tolerance. To investigate homologs genes of C-repeat binding factor (CBF) pathway proteins and reactive oxygen species (ROS) marker gene expression blocking by CabHLHOSS, we performed yeast one-hybrid (Y1H), dual luciferase and electrophoretic mobility shift assay experiments. The results showed that CabHLH035 bound to the region upstream of the CaCBFlA and CaAPX promoters. Additionally, CaCBFlA bound to the CaDHN4 promoter. Taken together, our results showed that CabHLHOSS plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops. The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

Keywords: Pepper; CabHLHOSS; Silencing; Overexpression; VIGS; Cold tolerance

1. Introduction

Plant growth and development are vulnerable to harmful or stressful environments (Zhu 2016). Adverse environmental conditions, such as high temperatures, drought, high salinity, cold, and nutrient deficiency are serious constraints for pepper growth and development, decreasing yield and quality, even crop death in severe cases (Xu et al., 2019). In particular, cold stress can have an adverse effect on plant growth and production by destroying destruction of the cell membrane and inhibiting important enzymes activities. However, the reaction mechanisms and regulatory patterns of cold stress in pepper are not well understood. C-repeat binding factor (CBF) transcription factors (TFs) are believed to play a central role in the response to cold stress via direct binding to the promoters of cold responsive (COR) and dehydration (DHN) genes (Tang et al., 2022; Lin et al., 2023). For example, MdMYB88 and MdMYB124 TFs promote cold resistance through a CBF-dependent regulatory pathway in apple (Xie et al., 2018), and apple В-box protein BBX37 promotes cold resistance through the CBF pathway (An et al., 2021).

Plant basic helix-loop-helix (bHLH) TFs play an important role in adjusting and controling plant growth, development, and various stress responses (Wu et al., 2022; Song et al., 2023). These proteins, are named for their bHLH structure, they are the second largest family of TFs, and they are found in diverse plants including Arabidopsis (Bailey et al., 2003), tomato (Sun et al., 2015), apple (Wang et al., 2022a). Cloning and characterisation has been performed for BRII EMS SUPPRESSOR 1/BRASSINAZOLE RESISTANT 1 (BES1/BZR1) transcription factor genes in maize (Li et al., 2006) and apple (Mao et al., 2017), and bHLHs were found to play critical roles in signalling pathways (Guo et al., 2008). The bHLH TF domain spans ~60 amino acids, and is divided into two parts; a basic amino acid region, and a HLH structure. According to evolutionary relationships and gene structure, bHLH TFs in animals are split into six groups (A-F).

In plants, bHLH TFs participate in multiple ways including signal transduction, biosynthesis and catabolism. For example, in rice, OSBI and OSB2 participate in the regulation of anthocyanin biosynthesis (Gou et al., 2011), and in Arabidopsis, the IND gene is involved in signal transduction related to gibberellin (Arnaud et al., 2010). The bHLH TFs also perform roles in various plant processes including growth, development and stresses responses. For example, the OsbHLH107 gene is an important regulator of grain size, and might be useful for grain yield improvement, like its homolog in maize (Yang et al., 2018). Under the effect of blue light, the AtMYC2 gene in Arabidopsis plays a positive regulatory role in lateral root formation (Yadav et al., 2015). These proteins also participate in modulating stress responses to low/high temperature (Chinnusamy et al., 2013), and salt (Zhou et al., 2009). For example, in apple, MdbHLH130 enhances drought stress resistance in transgenic tobacco by modulating homeostasis (Zhao et al., 2020). MfbHLH38, a Myrothamnus ßabellifolia bHLH TF, improves tolerance to drought and salt stresses in Arabidopsis (Qiu et al., 2020). Similarly, the TF TabHLH49 plays a positive role in regulating the expression of the dehydrin WZY2 gene and improves drought stress tolerance in wheat (Liu et al., 2020). CsbHLH041 is a positive regulators of salt and abscisic acid (ABA) tolerance in transgenic Arabidopsis and cucumber seedlings (Li et al., 2020). In pear, PbrbHLH195 is involved in the production of reactive oxygen species (ROS) in response to cold stress (Dong et al., 2021).

One hundred and twenty-two genes of the CabHLH family have been identified in pepper (Zhang et al., 2020a, 2020b). However, functional analyses of only a few bHLH genes have been conducted in this important crop species. In the present study, based on genespecific expression patterns, CabHLH035 (LOC107866727) was upregulated in response to cold treatment. We demonstrated that CabHLH035 plays a significant role in the growth and development of this plant. The present work provides valuable information for further analysis of the physiological and biochemical characteristics of bHLH TFs in peppers and other plant species.

2. Materials and methods

2.1. Plant materials, growth conditions and cold treatment

The experimental materials, Arabidopsis ecotype Columbia-0, T3 homozygous transgenic lines and pepper (Capsicum annuum L.) cold-tolerant cultivar P70, were grown in the Laboratory of Vegetable Plant Biotechnology and Germplasm Innovation, Northwest A & F University, Yangling, China. Pepper was germinated on moist fabric at 28 °C in the dark for 5 d. Arabidopsis was vernalized at 4 °C in the dark for 1 d and thereafter germinated at 24 °C on Murashige and Skoog (MS) medium. Pepper and Arabidopsis were grown in a climate-controlled growth chamber under a 22 °C/18 °C 16 h light/8 h dark photocycle with 70% relative humidity. For cold stress, pepper plants at the 6-8 leaf stage were subjected to low temperature (4 °C) for 3 days as described by Chen et al. (2019).

2.2. Transient expression in pepper

In order to transiently express the gene in pepper, the coding sequences (CDS) of CabHLH035 and CaCBFlA were separately amplified and cloned into a 35:GFP plasmid vector. Recombinant plasmids were confirmed by DNA sequencing and transformed into A. tumefaciens strain GV3101 as previously described (Ma et al., 2021). Transient expression in pepper plants was verified by PCR, and mRNA levels of transiently expressed genes in pepper were examined by both PCR and quantitative Real-time PCR (qRT-PCR). Gene-specific primers used to determine the relative expression levels are listed in Table SI.

2.3. Virus-induced gene silencing (VIGS) of CabHLH035

Forknockdown of CabHLH035, a 300 bp open reading frame (ORF) was inserted into the tobacco rattle virus-based 2 (TRV2) vector. The recombinant vector pTRV2-CabHLH035 and pTRV2 (negative control), pTRV2:CaPDS (positive control) and pTRVl vectors were transformed into A. tumefaciens strain GV3101. After co-infiltration into cotyledons of 2-week-old pepper plants, plants separately harbouring pTRV2:CabHLH035, pTRV2:CaPDS and pTRV2:00 were grown in darkness for two days, then transferred to normal growth conditions as previously described (Ma et al., 2019). After 28 days, the silencing efficiency of CabHLH035 was assessed by qPCR.

2.4. Generation of Arabidopsis CabHLH035-overexpressing lines

For the transformation of Arabidopsis, the CDS of CabHLH035 was amplified from pepper cDNA using gene-specific primers. Amplified products were then cloned into the 35S:GFP vector to obtain 35S:CabHLH035:GFP. The recombination plasmids 35S:CabHLH035:GFP and 35S:GFP were separately transformed into the A. tumefaciens strain GV3101 and CabHLH035 transgenic Arabidopsis plants were generated via the floral dipping method (Clough and Bent, 1998). Homozygous transgenic plants were selected in the T2 generation and confirmed in the T3 generation through kanamycin screening and PCR analysis.

2.5. Cold stress tolerance assay

To analyse tolerance to cold stress, 3-week-old T3 transgenic and wild-type (WT) Arabidopsis lines were tested, the CabHLH035 transgenic and WT plants were treated at 4 °C for three days. Similarly, to assess the cold tolerance of pepper plants, CabHLH035-silenced and control plants were exposed to low temperature of 4 °C for three days. Full details of the cold stress conditions are given in our previous study (Zhang et al., 2019).

2.6. Diaminobenzidine (DAB) and nitro-blue tétrazolium (NBT) staining

In order to measure the accumulation of H2O2 and superoxide anion (O2 ), we performed DAB and NBT staining following the previously described procedure of Dai et al. (2018). In brief, the harvested leaves were treated with 1 mg^mL 1 DAB or NBT for 24 h in the dark, and then discoloured by boiling in 75% ethanol.

2.7. Physiological analysis and histochemical staining

Relative electrolyte leakage (REL) was determined following the methods as described by Dionisio-Sese and Tobita (1998). The MDA content was measured using the thiobarbituric acid methods according to Buege and Aust (1978). Total chlorophyll content was determined as previously described by Arkus et al. (2015). The H2O2 and O2' levels were assayed following the protocol of Alexieva et al. (2001). The Fv/Fm ratio was calculated by using an imaging pulse amplitude-modulated (PAM) chlorophyll fluorimeter (FC800, PSI,Germany). Antioxidant enzyme, superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) activities were estimated following the protocol of Guo et al. (2012).

2.8. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays

The full-length CaNAC035 sequence was obtained using gene-specific primers and inserted into pGBKT7 to generate pGBKT7-CaNAC035, as bait plasmid. The full-length CabHLH035 gene was acquired using gene-specific primers and inserted into pGADT7 to generate pGADT7-CabHLH035 as prey plasmid. To further authenticate the interaction region between CaNAC035 and CabHLH035, different deletion fragments of CaNAC035 were cloned using gene-specific primers, and each PGR product was cloned into the pGBKT7 vector. Similarly, the pGADT7-CabHLH035 and each pGBKT7-CaNAC035 mutant plasmid were introduced into Y2HGold yeast cells, and strains were grown on SD (-Trp/-Leu), SD (-Trp/-Leu/His/-Ade) and SD (-Trp/-Leu/-His/-Ade + X-a-gal) solid media for 3-5 days.

For the BiFC assay, the ORF of CaNAC035 was amplified using gene-specific primers, and the PGR product was harvested and inserted into pSPYNE-35S/pUC-SPYNE. The ORF of CabHLH035 was cloned using the gene-specific primers, then inserted into pSPYCE-35S/pUC-SPYCE, and the recombination plasmids were co-transformed into A. tumefaciens GV3101 and infiltrated into the tobacco leaves. The fluorescence signal was measured and images were collected using an automatic positive fluorescence microscope (Olympus, Tokyo, Japan).

2.9. Co-immunoprecipitation (Со-IP) assay

The recombinant vectors CaNAC035-MYC and CabHLH035-HA were transformed into Nicotiana benthamiana leaves and total proteins were extracted as described by Cai et al. (2021). Total proteins using HA antibody were at 4 °C for 12 h. The eluted sample was separated by SDS-PAGE method and immunoblotted using HA and MYC antibodies.

2.10. Yeast-one-hybrid assay

Promoter sequences (1 500 bp upstream of ATG) were acquired from the pepper genome platform (PGP). The CDS of CabHLH035 was inserted into the pGADT7, and promoter sequence fragments of CaCBFlA, CaDHN4 and CaAPX were introduced into the pAbAi vector. Recombinant vectors were co-transformed into Y1H yeast strains and the Y1H experiment was performed following the Clontech method. The bait yeast strain was coated on the SD/Leu/medium with or without 200 ng-mL 1 aureobasidin A (AbA), and incubated at 30 °C for 5-7 days.

2.11. LUC/REN ratio assays

Full-length CaCBFlA, CaAPX and CaDHN4 promoters were cloned into the pGreenII0800-LUC vector, and the CDS of CabHLH035 and CaCBFlA were cloned into pGreenll 62-SK to construct an effector vector. Recombinant vectors were transformed into the A. tumefaciens GV3101 strain then injected into the leaves of tobacco. Transient express were measured following the luciferase (LUC) and Renifla (REN) luciferase ratio using the Dual-Luciferase® Reporter Assay System (Promega, WI, USA).

2.12. Electrophoretic mobility shift assay (EMSA)

The ORFs of CabHLH035 and CaCBFlA were amplified and inserted into the pMAL-c5x (MBP) vector using the Gateway method. The resulting constructs were transformed into Escherichia coli BL21 (DE3), and the recombinant proteins were purified using a Ni-NTA Kit (Qiagen) based on the manufacturer's instructions. EMSA was performed according to the LightShift Chemiluminescent EMSA Kit and the procedure employed by Xie et al. (2018).

2.13. Statistical analysis

All the statistical analyses of experimental data were performed using SPSS 17.0 software (IBM, Chicago, IL, USA). One-way analysis of Tukey's HSD was applied at P < 0.05.

3. Results

3.1. CaNACOSS interacts with СаЬНШОЗБ

In our previous study, we identified CabHLH79 that binds directly to the CaNAC035 promoter to regulate the expression of CaNAC035 in a dynamic mode, and CabHLH79 was found to enhance cold tolerance (Wang et al., 2022b). In the present study, to confirm the interaction between CaNAC035 and CabHLH035, pGBKT7-CaNAC035 and pGADT7-CabHLH035 were cotransformed into Y2H gold yeast cells. As shown in Fig. 1, A, yeast cells expressing both CaNAC035 and CabHLH035 grew normally and turned blue on SD/-Trp/-Leu/-His/-Ade + X-a-Gal solid medium. However, when pGBKT7-CaNAC035+pGADT7 or pGBKT7+ pGADT7-CabHLH035 were co-transformed into Y2H, cells did not grow or turn blue on the above solid medium, indicating that CaNAC035 and CabHLH035 interacted in yeast cells. To further explore the region of CabHLH035 that interacts with CaNAC035, the full-length sequence of CaNAC035 was divided into different fragments (Fig. 1, C). Recombination plasmids harbouring each CaNAC035 mutant and the pGADT7-CabHLH035 plasmid were co-transformed into yeast cells, and CabHLHOSS was found to interact with the D-domain region of CaNAC035 (Fig. 1, D).

To confirm that CabHLH035 interacts with CaNAC035, bimolecular fluorescence complementation (BiFC) was assayed. Tobacco was used as injection material for the BiFC assay, and recombinant CabHLH035-nYFP and CaNAC035-cCFP plasmids were co-transformed into tobacco cells. Fluorescence signals showed that when CaNAC035 and CabHLH035 were cotransformed, green fluorescence was observed in the cell nucleus (Fig. 1, B). In addition, Со-IP results also indicated that CabHLHOSS interacts with CaNACOSS in vitro (Fig. 1, E). These results indicated that CabHLHOSS interacts with CaNACOSS in the cell nucleus.

3.2. Silencing the CabHLHOSS gene reduces cold tolerance in pepper

To explore whether CabHLHOSS responds to cold stress in pepper plants, qRT-PCR was used to measure CabHLHOSS mRNA expression levels. We found that after cold stress treatment, CabHLHOSS was up-regulated at 6 h, but expression decreased rapidly thereafter (Fig. 2, A). To explore the molecular function of CabHLHOSS under cold stress, pepper plants were injected with pTRV2: CabHLHOSS and pTRV2:CaPDS after 30 days. As shown in Fig. SI, plants injected with the CaPDS gene exhibited photo-bleaching characteristics in leaves. The silencing efficiency examined by qRT-PCR was almost 85%, and CabHLHOSS-silenced and control (pTRV2:00) plants were used for further experiments. Leaves of CabHLHOSS-silenced and control plants were treated at 4 °C for three days. No significant phenotype change was observed under normal conditions between CabHLH035-silenced and control plants. However, after cold stress for 3 days, CabHLH035-silenced leaves showed obvious shrinking symptoms compared with control plants (Fig. 2, B). Similarly, no obvious difference in Fv/ Fm was observed between CabHLH035-silenced and control plants. Cold stress caused severe leaf wilting, and a significant decrease in the Fv/Fm ratio of plants. The Fv/Fm ratio of CabHLH035-silenced plants was significantly lower than that of control plants (Fig. 2, C). Next, we investigated REL, and MDA and chlorophyll levels in CabHLH035-silenced and control plants. Under normal conditions, CabHLH035-silenced and control plants showed no differences in REL, MDA or chlorophyll content. By contrast, after CabHLH035-silenced and control pepper plants were treated at 4 °C for three days, CabHLH035-silenced pepper plants showed higher REL (22.3%) and MDA (26.5%) levels, but lower chlorophyll (13.2%) content than control pepper plants (Fig. 2, D-F). Our results demonstrated that knockdown of CabHLH035 made plants more sensitive to cold stress.

3.3. Overexpression of CabHLH035 in Arabidopsis increases cold tolerance

To further confirm the function of CabHLH035 in cold tolerance, we overexpressed the CabHLH035 gene in Arabidopsis. We selected three overexpressing (OE) transgenic lines of Arabidopsis for the experiment, and the relative expression of CabHLH035 in all three lines (#1, #2 and #3) was significant higher than in wildtype (WT) plants (Fig. S2). The 3-week-old WT and transgenic Arabidopsis lines were treated at 4 °C for three days. As shown in Fig. 3, A, no growth differences were observed between transgenic Arabidopsis and WT plants without stress. However, after treatment at 4 °C for three days, the leaves of CabHLH035 transgenic lines showed a little wilting, but the leaves of WT lines showed severe wilting, and some had even died. Accordingly, the Fv/Fm ratios of CabHLH035 transgenic lines were obviously higher (5%-7%) than those of WT plants (Fig. 3, B and C). Following exposure to cold treatment, CabHLH035 transgenic lines exhibited markedly increased cold tolerance than control plants, accompanied by obviously decreased REL and MDA levels and higher chlorophyll content (Fig. 3, D-F). Thus, overexpression of CabHLH035 promoted cold stress tolerance in Arabidopsis.

3.4. Altered expression of genes related to the CBF pathway

To further explore whether CabHLH035-regulated cold stress is related to the CBF pathway, we measured cold-induced relative expression levels of CBF/DREB1 genes and their dependent cold response-related genes, including ICE, CBF1A, CBF1B, COR47-Like, and KINI. The qRT-PCR results showed that expression of CaCBFlA was significantly upregulated in CabHLH035-OE lines and CabHLH035-To plants compared with control plants (Fig. 4, A, C). However, the relative expression of CaCBFlA in CabHLH035 VIGS plants were significantly lower than that in control plants (Fig. 4, B). These results suggest that CabHLH035 acts as a positive regulator of cold signalling by upregulating CBF1A expression.

3.5. CaCBFlA is a direct target of CabHLH035

We then explored whether CabHLH035 directly regulates the expression of CBF1A. We analysed the characteristics of CaCBFlA promoter sequences, and found that the CaCBFlA promoter contained a G-box domain (Fig. 5, A). The Y1H results revealed that yeast strains expressing the full-length gene grew well on SD/Leu and SD/Leu/AbA solid medium. However, when the G-box sequences were mutated, yeast cells did not grow on SD/Leu or SD/Leu/AbA solid medium (Fig. 5, C). These results confirmed the direct binding of CabHLH035 to the promoter sequence of CaCBFlA.

To further confirm that CabHLH035 binds to the CaCBFlA promoter and positively regulates its expression, LUC/REN ratios were calculated. We next carried out a dual LUC reporter assay to investigate the in vivo regulation of CaCBFlA promoter by CabHLH035. CabHLH035 expressed under the control of CaMV35S promoter was used as an effector, while CaCBFlA promoter was used to drive LUC in the reporter vectors (Fig. 5, B). Dual LUC assay results showed that CabHLH035 could upregulated CaCBFlA expression (Fig. 5, D). To further authenticate the specific binding of CabHLH035 to the G-box of CaCBFlA promoter, EMSA was performed. A DNA-protein complex was formed, but the complex was decreased when the competitor was added, and the complex disappeared when the G-box was mutated (Fig. 5, E). Next, we analysed the expression of CBF1A in CabHLH035-silenced, CabHLH035-OE and CabHLH035-To plants. The results showed that after cold stress, CaCBFlA mRNA levels in CabHLH035silenced plants were downregulated compared with pTRV2:00 plants, but CBF1 mRNA levels were significantly increased in CabHLH035-OE and CabHLH035-To plants compared with the control plants (Fig. 5, F-H). The results confirmed that CabHLH035 binds to the CaCBFlA promoter and positively regulates its expression.

3.6. Altered expression of DHN genes

DHNs are a known targets of CBFs. To identify potential target genes of CaCBFlA, we measured expression levels of DHNs in CaCBFlA-To and CaCBFlA-silenced plants. We found that expressions of DHN genes was modified, including CaDHNl, CaDHN2, CaDHN3, CaDHN4, CaDHN5, CaDHN6, and CaDHN7. The CaDHN4 gene showed the most significant increase in expression in CaCBFlA-To pepper plants, and an obvious decrease in expression in CaCBFlA-silenced pepper plants (Fig. S3). These results indicate that CaCBFlA plays an important role in cold tolerance by moderating the expression of CaDHN4.

3.7. CaDHN4 is a direct targets of CaCBFlA

To identify potential target genes of CaCBFlA, we performed Y1H experiments. Based on the Y1H results, we performed sequence analysis of CaDHN4 promoters, and found that CaDHN4 contain dehydration-responsive element/C-repeat element A/ GCCGAC (DRE/CRT) features. To test whether the DRE/CRT elements of the CaDHN4 promoter could directly bind to CaCBFlA, we performed Y1H experiments. The results showed that yeast cells expressing the full-length gene grew well on SD/Leu and SD/ Leu/AbA solid medium. However, when the DRE/CRT elements were mutated, there was no growth of yeast cells on SD/Leu and SD/Leu/AbA solid medium (Fig. 6, A and B). These results revealed direct binding of CaDHN4 to the CaCBFlA promoter.

To further confirm that CaCBFlA binds to the CaDHN4 promoter to positively regulate gene expression, LUC/REN ratios were calculated. The dual LUC assay results showed that CaCBFlA could upregulate CaDHN4 (Fig. 6, C and D). EMSA also showed that CaCBFlA directly bound to the CaDHN4 promoter fragment (Fig. 6, E). We found that after cold stress, CaDHN4 expression levels in CaCBFlA-silenced plants were downregulated compared with pTRV2:00 plants, but CaDHN4 mRNA levels were obviously higher in CaCBFlA-ОЕ and CaCBFlA-To plants compared with control plants (Fig. 6, F and G). The results indicate that CaDHN4 is a direct target gene of CaCBFlA.

3.8. Silencing of CabHLH035 in pepper leads to excessive accumulation of ROS

We measured the accumulation of ROS in CabHLH035-silenced and control plants after cold stress, Diaminobenzidine (DAB) and nitro-blue tétrazolium (NBT) staining were performed. As shown in Fig. 7, A and B, the area stained by DAB and NBT in CabHLH035silenced plants was substantially greater than that in control plants. In addition, levels of H2O2 (42.5%) and O2 (52.5%) in CabHLH035-silenced plants were higher than that in control plants (Fig. 7, C and D). Cold stress leaded to the antioxidant enzyme system to reduce the ROS damage, and significantly promoted the product of SOD, POD, and APX activities. The results showed that under control conditions CabHLH035-silenced plants had obviously lower APX content than control plants whereas their POD and SOD contents did not differ. However, cold stress caused severe leaf wilting and a significant decrease of SOD, POD, and APX activities, and the SOD, POD, and APX activities of pTRV2:CabHLH035 plants was significantly lower than control plants (Fig. 7, E-G). These results showed that CabHLH035-silenced plants accumulated more ROS than control plants.

3.9. The overexpressing plants accumulate less ROS

To further investigate the cold-resistance CabHLH035, we performed DAB and NBT staining, and measured H2O2 and O2 levels. Under control conditions, there were no significant differences in DAB and NBT staining areas between CabHLH035 transgenic and WT plants. However, under cold-stress conditions, the DAB and NBT staining area of CabHLH035 transgenic plants was significantly smaller than that of WT plants (Fig. 8, A and B). There were no distinct differences in H2O2 or O2 contents in WT and transgenic plants under normal conditions. However, after cold stress transgenic plants had decreased levels of H2O2 (33%-36%) and O2 (46%-48%) compared to WT plants (Fig. 8, C and D). We also measured the SOD, POD, and APX activities. The results showed that under normal conditions CabHLH035 transgenic plants had obviously higher APX content than control plants whereas their POD and SOD contents did not differ. However, after cold stress the SOD, POD, and APX activities of CabHLH035 transgenic plants was significantly higher than WT plants, which was 1.09-, 1.33-, and 1.64-fold of that in WT, respectively (Fig. 8, E-G). Thus, overexpression of CabHLH035 promoted cold stress tolerance in Arabidopsis.

3.10. Altered expression of genes related to ROS scavenging

ROS-related genes play vital roles in ROS homeostasis. Therefore, expression levels of ROS marker genes (CAT, POD, SOD, APX, RbohA, RbohB and RbohD) were measured in CabHLH035-OE Arabidopsis, CabHLH035-To, and CabHLH035silenced plants. The results showed that APX was markedly upregulated in CabHLH035-OE and CabHLH035-To plants compared with control plants, but expression of APX was decreased in CabHLH035-silenced plants (Fig. S4). The above results imply that ROS marker genes may be key regulators that act downstream of the CabHLH035 gene, and overexpressing CabHLH035 may upregulate CaAPX in response to oxidative stress and other unfavourable conditions.

3.11. Identification of putative target genes of CabHLH035

The above results suggest that ROS marker genes may be key regulators that act downstream of the CabHLH035 gene, and overexpressing CabHLH035 may upregulate APX in response to oxidative stress and other unfavourable conditions. Hence, we suspected that CabHLH035 bind to CaAPX promoter. Y1H and LUC/REN assays were conducted to explore whether CabHLH035 could bind to the promoter sequences of CaAPX ROS marker genes. The promoter sequences were inserted into the pAbAi vector to generate effector plasmids, which were co-transformed with the AD-CabHLH035 exporter plasmid into the Y1H yeast strain (Fig. 9, A). Analysis of the growth characteristics showed that co-transformation of AD-CabHLH035 with pAbAi-CaAPX resulted in cells that grew well on SD/-Leu medium and SD/-Leu/ +AbA200 medium (Fig. 9, C), suggesting that CabHLH035 could bind to the promoter sequences of CaAPX.

To further explore whether CabHLH035 binds to the CaAPX promoters and positively regulates their expression, LUC/REN ratios were calculated. Dual LUC assay showed that CabHLH035 could upregulate CaAPX (Fig. 9, B, D). Next, we measured expression levels of CaAPX in CabHLH035-silenced plants. EMSA revealed that CaAPX was a direct target gene of CabHLH035 (Fig. 9, E). We then measured expression levels of CaAPX in CabHLH035-silenced, CabHLH035-OE and CabHLH035-To plants. The results showed that after cold stress, CaAPX transcripts in CabHLH035-silenced plants were markedly decreased compared with pTRV2:00 plants, but APX transcript levels were significantly higher in CabHLH035-OE and CabHLH035-To plants than control plants (Fig. 9, F-H). The results indicate that CabHLH035 does indeed bind to CaAPX promoters to positively regulate their expression.

4. Discussion

Plants survive in unfavourable environments by producing different secondary metabolites (Wang et al., 2015). Cold stress is a major factor that can affect plant growth, development, and crop yield. In our previous studies, we found that CaNAC035 is involved in responses to cold, salt and drought stresses (Zhang et al., 2020a, 2020b), and in the present study we identified CabHLH035, a CaNAC035-interacting protein in pepper. bHLHs are known to play diverse roles in plant growth, development, and various stress responses (Chakraborty et al., 2009). A number of bHLH TFs have been found to participate in different physiological and biochemical processes including plant growth and development, responses to different stresses, and signal transduction (Duek and Fankhauser, 2003; Hernandez et al., 2004). Members of the bHLH family have been authenticated in Arabidopsis (Toledo-Ortiz et al., 2003), rice (Li et al., 2006), apple (Yang et al., 2017), and other species.

The influences of cold stress on plants include reduced photosynthetic reactivity capacity (Damian and Donald, 2001). The CabHLH035 transgenic lines showed higher photosynthetic capacity, however CabHLH035-silenced plants showed lower photosynthetic capacity, we speculated that CabHLH035 plays a crucial role in safeguarding photosynthetic output and mediating photosynthetic yield. Previous studies showed that the MDA content is correlated with increased membrane injury (Hu et al., 2012). Under cold stress, the accumulation of MDA in CabHLH035-OE plants indicated reduced lipid peroxidation and membrane damage.

Furthermore, stress-related marker genes are involved in regulating stress responses in transgenic plants (Zhang et al., 2014; Nguyen et al., 2018). CBFs are important regulatory activators of cold responsive genes (Liu et al., 2018). CBF genes can be induced by cold stress, and overexpression can enhance cold tolerance in Arabidopsis, rice, tobacco, tomato, and apple (Gilmour et al., 2000; Hsieh et al., 2002; Ito et al., 2006; Yang et al., 2011). The ICE1-CBF-COR transcriptional model is used to understand cold resistance (Chinnusamy et al., 2007). In this pathway, CBFs are induced by cold and they modulate mRNA expression levels of stress-responsive genes by directly binding their promoters (Thomashow, 1999). In Arabidopsis, AtICEl binds the E-box domain in the AtCBF3 promoter to regulate its expression (Chinnusamy et al., 2013). In tobacco, overexpression of NtbHLH123 enhances cold tolerance and regulates the expression of NtCBF genes (Zhao et al., 2018). In the present study, overexpression of CabHLH035 significantly increased the levels of transcripts of CBF1A. The enhanced tolerance to cold stress in CabHLH035 transgenic Arabidopsis plants may be related to the expression of cold stress-responsive genes. ROS are known to regulate various biological processes, such as plant cell status, pathogen susceptibility, and abiotic stress tolerance (Capper and Dolan, 2006; Baxter et al., 2014). ROS are generated by numerous enzymatic systems, especially class III peroxidases, quinone reductases and NADPH oxidases (Apel and Hirt, 2004; Nanda et al., 2010; Marino et al., 2012). Moreover, Rboh genes participate in regulating transcriptional levels. For example, Arabidopsis NAG TF, which possesses a transmembrane motif 1-like 4 (NTL4) domain, regulates the ROS homeostasis by binding to the promoters of AtrbohC and AtrbohE under drought stress (Lee et al., 2012), and AtRbohD regulates ethylene-responsive factor (AtERF6) under oxidative stress conditions (Wang et al., 2013).

TFs and cis-acting elements are important for adjusting plant regulatory mechanisms. Identifying direct downstream target genes is a useful way to study the functions of TFs such as CabHLH035. Based on the Y1H, LUC/REN and EMSA results, we found that CabHLH035 could directly bind to the promoters of CaAPX genes. We speculated that CabHLH035 confers cold resistance mainly by controlling the expression of these ROS marker genes. During cold stress, damage to photosystem II (PSII) and decreased metabolic rates compromise photosynthetic ability (Kong et al., 2014). Inhibition of photosynthesis can cause a dramatic increase in ROS, which is damaging to photosynthetic potential Herein, ROS levels were lower in CabHLH035-OE lines and higher in CabHLH035-silenced lines than that in control plants under cold stress. These results showed that, under cold stress conditions, CabHLH035 could maintain chloroplast genome stability, resulting in a rapid recovery of photosynthetic ability. Additionally, CabHLH035 decreased the accumulation of ROS. H2O2 and O2' are important types of ROS that cause oxidative damage when their concentrations exceed a certain threshold; after cold stress, CabHLH035-silenced pepper plants exhibited higher H2O2 and O2' levels (Fig. 7, C and D), implying increased susceptibility following knockdown of CabHLH035. Conversely, CabHLH035 overexpression in Arabidopsis lowered H2O2 and O2' levels (Fig. 8, C and D). These results indicate that CabHLH035 is involved in cold tolerance. Cold stress includes chilling and freezing stress. In this study, we only investigated chilling stress. In future studies we will explore freezing stress.

In our previous studies, we identified 18 proteins that may potentially interact with the CaNAC035 protein, and these potential interacting partners may be involved in various stress responses, and in photosynthesis. In our previous studies, silencing of CaNAC035 reduced cold, mannitol, and salt stress tolerance, while overexpressing CaNAC035 in Arabidopsis increased tolerance to cold, mannitol and salt stresses. Many studies have reported that NAC TFs play important roles in plants, including leaf senescence, secondary metabolism, and responses to various unfavourable environmental conditions (Puranik et al., 2012; Zhu et al., 2014). The present study provides a basis for further confirmation of the role of CabHLH035 in various hormonal signalling pathways under cold conditions in important crop plants.

According to the Y1H, LUC/REN and EMSA results, CabHLH035 binds to the upstream region of CaCBFlA and CaAPX promoters. Interestingly, CabHLH035 could bind to the CACGAG sequence of CaCBFlA. Moreover, based on the Y1H, dual luciferase and EMSA assays, we found that CaCBFlA could bind to the CaDHN4 promoter, which includes a DRE/CRT motif for cold responses, and it positively regulates CaDHN4 expression, resulting in improved cold tolerance.

Dehydrin (DHN) plays central role in regulating plant responses to various unfavourable conditions. Our previous studies identified DHNs with irregular structures that are associated with cold resistance (Hughes et al., 2013). DHNs may participate in regulating plant growth and development, and function in stress responses. In previous reports, we found that CaDHN4 knockdown decreased cold tolerance in plants, while CaDHN4 overexpression in Arabidopsis enhanced cold stress tolerance. Our current results demonstrated that CaDHN4 act as a positive regulator in the response to cold conditions (Zhang et al., 2019). This finding provides new insight into the roles of DHNs in adaptation to adverse environmental conditions. However, the underlying molecular mechanisms remain poorly understood and further studies are needed.

Low temperature stress may affect some unknown modifications of CabHLH035, which may affect its binding ability. It is also possible that unfavourable conditions may affect the DNA conformation, and thereby accessibility for CabHLH035. In the present study, we found that CabHLH035 not only binds to the promoter of CaCBFlA, but also interacts with CaNAC035. Thus, we propose a bHLH035-NAC035-CBF module, and identifies candidate genes for development of elite cold-tolerant pepper varieties. This knowledge will help us to explore the molecular mechanisms and transcription regulatory networks of bHLH TFs related to cold stress. In addition, our study demonstrates a new perspective for studying the role of CabHLH035 in cold stress responses. Under cold stress, CabHLH035 is upregulated, and CabHLH035 binds to the upstream region of CaCBFlA and CaAPX, thereby modulating their mRNA expression levels. In this way, CaCBFlA upregulates the expression of CaDHN4 genes. It is possible that CabHLH035 promotes cold tolerance in pepper via a CBF- and ROS-independent pathway.

5. Conclusions

In summary, the overexpression of CabHLH035 in Arabidopsis improved cold stress tolerance through the protection of plasma membrane integrity. Conversely, knockdown of CabHLH035 decreased cold stress tolerance. CabHLH035 can bind to the upstream region of CaCBFlA and CaAPX promoters, and CaDHN4 is a direct target gene of CaCBFlA. Further studies are needed to unveil the regulatory network of CabHLH035, and the molecular mechanism of CabHLH035 in response to various abiotic stresses in other important crops (Fig. 10). Our results provide new insight into networks related to cold stress in pepper, and may help to enhance cold tolerance in pepper and other crop plants through genetic engineering in future studies.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was funded by the Scientific & Technological Innovative Research Team of Shaanxi Province (Grant No. 2021TD-34), National Natural Science Foundation of China (Grant Nos. 32172582, 316721465), Agricultural Key Science and Technology Program of Shaanxi Province (Grant No. 2021NY-086) and the Natural Science Foundation of Shaanxi Province (Grant No. 2018JM3023).

Supplementary materials

Supplementary data to this article can be found online at https://doi.Org/10.1016/j.hpj.2023.03.007.

Footnote

1 These authors have contributed equally to this work.

* Corresponding author.

E-mail address: [email protected]

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

References

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