Abstract
Homeodomain-Leucine Zipper (HD-Zip) proteins are important ubiquitous and diverse molecular chaperones in plants. We characterized a gene CaATHB-12 derived from HD-Zip I subfamily which was intensively induced by exogenous abscisic acid (ABA), salt, and mannitol applications in a pepper cultivar. Efficient gene silencing lines were created from pepper, and stable heterologous overexpression lines were created from Arabidopsis to achieve a comprehensive exploration of gene function. The functional study of CaATHB-12 in pepper increased plant sensitivity to ABA stress, while the over-expressing CaATHB-12 in Arabidopsis lines revealed that tolerance to ABA, salt, and mannitol stresses was decreased. Furthermore, CaATHB-12 plays a fundamental role in elevating the tolerance to these stresses through the increased expression of other stress related genes, increasing the activities of antioxidant enzymes and scavenging the reactive oxygen species. The studied functions of the CaATHB-12 gene may provide some insights in exquisite molecular detail by pursuing signal transduction mechanisms that converge on gene expression patterns.
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Introduction
Plants produce substantial amount reactive oxygen species (ROS) to carry out key cellular functions in normal conditions as well as in response to stresses caused by biotic and abiotic factors (Foyer 2020; Khan and Khan 2017). Environmental stresses affect the plant growth, development and reduce the yield, nutrition, and quality of the crops. To cope up with these environmental stresses, plants have developed sophisticated defense mechanisms, including antioxidant enzyme systems (glutathione peroxidase, GPX; superoxide dismutase, SOD; catalase, CAT; peroxidase, POD; ascorbate peroxidase, APX) and bioactive substances (phenolic compounds, flavonoids, carotenoids, tocopherols) (Gill and Tuteja 2010; Nafees et al. 2011). The homeodomain-leucine zipper (HD-Zip) proteins with a highly conserved and unique sequence which plays an extremely key role in the growth and development of plants (Ariel et al. 2007; Ré et al. 2014). The homeobox gene acts as a major regulator of all aspects of plant development, while some HD-Zip proteins participate in fruit development and also responds to different abiotic stimuli (Ariel et al. 2007). The HD-Zip proteins (I–IV) are pressure-sensitive HD-Zip I proteins and are studied in recent years (Ariel et al. 2007; Henriksson et al. 2005; Hjellstrom et al. 2003). The ATHB6, ATHB7, and ATHB12 showed an up-regulation to externally applied ABA and water-deficit treatments, suggesting their important roles in regulating crops responses to drought stress (Olsson et al. 2004; Soderman et al. 1996). Similar findings were observed in the model plant Arabidopsis, where overexpression of ATHB-7 facilitated photosynthesis, increased chlorophyll content and leaf growth (Hjellstrom et al. 2003; Soderman et al. 1996). Studies on ATHB-12 gene in response to stress in the Arabidopsis (Olsson et al. 2004; Ré et al. 2014), and many of the studies focused on salt stress and water deficit induction (Henriksson et al. 2005). In the Craterostigma plantagineum, CpHB4–CpHB7 genes are reported to show response to drought stress. The expression CpHB6 and CpHB7 were induced by ABA treatment and drought stress, while CpHB4 have showed down-regulation to ABA treatment (Deng et al. 2002; Frank et al. 1998). In rice, the HD-Zip I gene Oshox22 mediated drought and salt stresses following the ABA-mediated signal transduction pathway (Zhang et al. 2012). The sunflower HD-Zip I protein Hahb-4 was reported for the improved drought tolerance in Arabidopsis (Manavella et al. 2006). Recently, MtHB1, a Medicago truncatula HD-Zip I protein has shown an induced expression in response to salinity stress (Ariel et al. 2010). Although many HD-Zip I genes have been studied, no systematic study of HD-Zip I proteins has been conducted in pepper.
Abscisic acid (ABA), as a stress signal, enhances the plants tolerance to several environmental stresses (Yu et al. 2006), including extreme temperatures (Verslues and Zhu 2005), drought (Bartels and Sunkar 2005), and salinity (Ahuja et al. 2010). Exogenous application of ABA can regulate the accumulation of secondary metabolites in fruits (Satoru et al. 2009; Zhu et al. 2012). It has also been shown that ABA treatment increased carotenoid and chlorophyll concentrations in tomato leaves and fruit (Barickman et al. 2014). Previous studies have shown that the stress tolerance of plants against adverse environmental conditions is affected by ABA, which is the key regulator in response to environmental stresses (Taylor et al. 1988). It can greatly improve the resistance of higher plants to adversities (Bartels and Sunkar 2005). It has been reported that ABA treatment can lead to a sharp increase in POD activity, while POD can neutralize part of H2O2 and reduce the damage of H2O2 in plants (Bueno et al. 1998). Under drought stress, ABA treatment can reduce the active oxygen species in maize and further improve its antioxidant capacity (Jiang and Zhang 2002). ABA treatment can also affect the accumulation of carotenoids in tomato (Barickman et al. 2014; Zhu et al. 2012). Furthermore, due of its function as a signaling molecule, ABA can stimulate the change of fruit color. Exogenous ABA treatment of grapes resulted in quicker carotenoid production in the peel (Coombe and Hale 1973). Recently, Tian et al. (2016) reported that exogenously ABA (150 mgL−1) treated fruits of pepper, resulted in a significant increase the Capsaicin synthesis. A 150 mg L−1 of ABA treatment was used by Xiao (2014) to treat pepper leaves, resulting in a rapid decrease in chlorophyll and yellowing of leaves. It has also been found that treating tomato fruits with ABA (100 mg L−1) during ripening stimulates an increase in lycopene content, which is mainly affected by the negative effect of ABA on the GA3 content (Yu et al. 2016).
In higher plants, carotenoids are composed of the skeleton C40, which is cleaved to form ABA (Zhang et al. 2009). ABA regulates the expression of some chlorophyll degradation-related genes and accelerates the decomposition of chlorophyll (Li et al. 2015). Similar investigations have been conducted in apples and tomatoes (Sun et al. 2012; Yu et al. 2016). Simultaneously,, the endogenous ABA treatment was also up-regulated the carotenoid synthesis pathway genes in grapevine (Enoki et al. 2017). There are currently very few research findings on whether ABA can prevent the formation of carotenoids in pepper fruits or not.
Our previous studies have shown that CaATHB-12 gene could regulates carotenoid content under cold stress, and potential associated with oxygen scavenging mechanism (Zhang et al. 2020). Here, our aim to explored the function of CaATHB-12 using overexpression (OE) and virus-induced gene silencing (VIGS) in both Arabidopsis and pepper. Our results provide further insights into the function of CaATHB-12 in plant ABA, salt, and osmotic stresses response.
Materials and Methods
Plant Materials and Growth Conditions
The gene silencing lines were created from Capsicum annuum and the overexpressed lines were created from Arabidopsis thaliana to explore the CaATHB-12 gene function from both positive and negative aspects. Two plant materials were used. Capsicum annuum cv. R24 was obtained from the pepper research group, College of Horticulture, Northwest A&F University, P.R. China. Seedlings of pepper were maintained in a growth chamber under 16 h light at 25 °C and 8 h dark cycles at 20 °C as following in (Ul Haq et al. (2019). Fruit was harvested at 25, 35, and 50 days post full blooming of flowers. Fruit samples were stored in frozen form in liquid nitrogen for later chemical analysis and gene expression analysis through quantitative real-time PCR (RT-qPCR). Arabidopsis thaliana ecotypes Columbia-0 was procured from College of Horticulture, Northwest A&F University, P.R. China, and maintained temperatures at 22 °C to light and 18 °C at night and a 65% of relative humidity (Wang et al. 2017).
Sequence and Phylogenetic Analysis of CaATHB-12 in Pepper
The CaATHB-12 sequence analysis was performed using the NCBI BLASTp program (Available online: http://blast.ncbi.nlm.nih.gov/Blast.cgi). The protein sequences were analyzed for finding the HD-Zip domain with other plant species by using the CLUSTALW following the methods of Guo et al. (2016). The alignment of CaATHB-12 proteins with other plant species were done through the DNAMAN (Version 5.0) software and the phylogenetic tree was generated by the MEGA 6.0 program with the default parameters as described by Benson (Benson et al. 2000). Other physico-chemical properties such as the molecular weight (MW) and isoelectric point (PI) of the CaATHB-12 were determined by EXPASY program (Available online: https://www.expasy.org/) according to the method of He et al. (2018).
Expression of CaATHB-12 Gene in Different Tissues of Pepper Using RNA‑Seq Data
The RNA-seq database (Version 1.5) of CM334 was used for analyzing the tissue-specific expression (http://pepperhub.hzau.edu.cn/index.php, Kim et al. 2014). Data regarding RPKM (reads per kilo base per million mapped reads) of the CaATHB-12 gene for different organs including leaves, stems, roots, as well as for the placenta and pericarp at 6 days post anthesis, 16 days post anthesis, and 25 days post anthesis was recorded and normalized at log2 while a the heatmap was constructed by program ImageGP (Available online: http://www.ehbio.com/ImageGP/) as described by Huo et al. (2019).
VIGS Assay of CaATHB-12 in Pepper Fruits
The tobacco rattle virus (TRV) based silencing method was used to knockdown the CaATHB-12 gene in pepper fruits (cv. ‘R24’). Tobacco rattle virus RNA1 (TRV1) and RNA2 (TRV2) sequences were used as vectors in the pepper plant (Macfarlane 1999). A 307 bp portion of the CaATHB-12 ORF was sequenced confirmed from pepper cDNA using the specific primer pair (Supplementary Table S1) with the restriction enzymes sites XbaI and KpnI. Special primers of CaATHB-12 and CaPDS were designed and then the target genes were injected into the TRV vector to generate TRV2:CaATHB-12 and TRV2:CaPDS (phytoene desaturase gene, the positive control) (Tian et al. 2014). The TRV:00 was acted as a negative control. The TRV1, TRV2 and TRV2:CaATHB-12 vectors were injected into the target fruits by using the Agrobacterium tumefaciens strain GV3101. The suspensions with Agrobacterium and TRV1, TRV2, and TRV2:CaATHB-12 (OD600 = 1.0) were injected into the green mature stage of pepper fruits (25 days after full bloom). The fruits were packed in sterilized filter papers in a clean and sterilized container and were placed in a growth chamber in dark condition for 48 h maintain the temperature at 18 °C with 35% of relative humidity. After 2 days in the dark, the treated pepper fruits were shifted to 16 h/23°C on light day and 8 h/20 °C a dark day, following the methods of Tian et al. (2014). The control and silenced fruits were used for further analysis after 15 days of treatment as described by Tian et al. (2014). To reduce experimental error, all the experiments were independently repeated three times.
CaATHB-12 Transgenic Arabidopsis Lines
The full-length ORF of the CaATHB-12 was cloned from pepper cDNA with specific primer pairs having restriction enzymes site XbaI and KpnI (Supplementary Table S1). The transgenic plants were grown on Murashige and Skoog (MS) medium supplemented with 50 mmol/L kanamycin and their screening was done through PCR. For further experiments, the third generation (T3) Arabidopsis seeds were used following the method of Zhang et al. (2020).
Stress Treatments and Sample Collection
To investigate the response of CaATHB-12 to abiotic stresses, leaf discs of 0.5 cm in diameter were collected from the CaATHB-12-overexpressed Arabidopsis leaves and floated in different concentrations of ABA (0, 50, 100, 150, 200 and 250 mg L−1). The excised leaf discs of the CaATHB-12 overexpressed plants exposed to ABA stresses were put in a control environment at 26 °C with continuous fluorescent light for 3 days, and the method of Xiao et al. (2014) was followed to conduct the treatments with a little modification.
The selected TRV2:CaATHB-12 and TRV2:00 detached fruits were sprayed with 150 mg L−1 ABA following the methods of Zhang (2016) and Tian et al. (2016) with a little modification. Fruit samples collection was conducted at 0, 6, 12, 24 and 48-hours post treatment. Three independent biological replicates were conducted for each treatment experiments.
Additionally, the 3-week-old CaATHB-12 overexpressed (OE1 and OE2) and WT lines of A. thaliana were selected to further analyze their ABA, salt, and mannitol stress tolerance. For ABA stress, the seedlings (raised under normal growing conditions) were sprayed with 150 mg L−1 ABA and leaf samples were collected at 48 h. And then, the MDA, superoxide anion free radical, antioxidant enzymes activities, chlorophyll and carotenoid contents were measured. For salt stress, seedlings were watered 150 mM NaCl every two days for 7 days. For mannitol stress, seedlings were watered 200 mM mannitol every two days for 7 days. Arabidopsis seedlings incubated under normal conditions were calculated as the following the control. Three separate seedlings samples were collected randomly, immediately kept in liquid nitrogen and stored at − 80 °C. Three independent biological replicates were used in experiments.
Fruit Color Measurement
Color measurement of the fruit samples were conducted according to the method of Michael et al. (1997), and the colorimetric system (CR-400, KONICA MINOLTA, Japan) was used to record the L, a, b, and C values of the fruits (Hunter 1987). The above-mentioned parameters represent respectively the “luminance”, “degree of red/green”, “degree of yellow/blue”, and “Chroma (saturation or vividness of color); Chroma= (a2 + b2)1/2”. After measurement of each fruit, the instrument was recalibrated and for reading the fruits were directly put on the diam aperture. These experiments were conducted in three biological replicates.
RNA Extraction and Quantitative Real-Time PCR (RT-qPCR) Analysis
The total RNA extraction, synthesis of cDNA and RT-qPCR was done according to the methods of (Guo et al. 2014; Khan et al. 2018). The ubiquitin binding gene (CaUBI3) of pepper (Wan et al. 2011), and AtActin2 gene of Arabidopsis were correspondingly used as reference genes. NCBI Primer BLAST was used to design all the primer pairs (Supplementary Table S1) for RT-qPCR. Relative gene expression levels were determined following the 2−△△CT method (Schmittgen and Livak 2008).
Measurement of the Contributed Parameters in Pepper Silenced Fruits and Transgenic Arabidopsis
The lipid peroxidation in cell plasma membranes of pepper fruits and Arabidopsis lines were assessed by measuring the antioxidant system and antioxidant substances. The malonaldehyde (MDA) content was measured using 0.5% 2-thiobarbituric acid (TBA) containing 5% (w/v) trichloroacetic acid reaction following the method of Buege and Aust (1978). The chlorophyll and carotenoid contents were quantified and calculated by the method described by Porra et al. (1989). Anthocyanins content was measured according to the method of Christie et al. (1994). The determination of total phenols and flavonoids were measured according to the method of Rodov et al. (2010) with slight modification, we used (OD280/g) and (OD325/g) to indicate the relative amounts of total phenols and flavonoids, respectively, while the catalase (CAT) activity was determined using the method of Aebi (1984), superoxide dismutase (SOD) and peroxidase (POD) activities were measured following the method of Stewart and Bewley (1980), glutathione peroxidase (GPX) activity was measured according to Flohé and Günzler (1984), ascorbate peroxidase (APX) activity was determined the protocol of Nakano and Asada (1987), superoxide anion free radical (O2−·) accumulation was determined as described previously by Zweier (1988), hydrogen peroxide (H2O2) content was measured by the method of Brennan and Frenkel (1977).
Statistical Analysis
Statistical analysis was executed through Statistical Analysis System software (IBM SPSS Statistics 19.0, USA) for analysis of variance (ANOVA). A least significant difference (P ≤ 0.05) test was used to identify significant differences among the treatments. All experiments were performed and analyzed with three independently biological replicates.
Results
Expression of CaATHB-12 at Different Developmental Stages and Analysis of Color Parameters of Pepper Fruit
To illuminate the function of CaATHB-12, with tissue-specific analysis of the vegetative (roots, stems, and leaves) and reproductive parts (three different developmental stages of pericarp and placenta) were conducted using the Pepper Hub from the pepper CM334 (http://pepperhub.hzau.edu.cn/index.php, Kim et al. 2014). As revealed in Supplementary Fig. S1, at 6 days post-anthesis (DPA) of the pericarp (PC) displayed the highest expression of CaATHB-12 gene, followed by stems, PC-16 DPA, roots, and PL-6 DPA, while leaves had the lowest expression at PL-25 DPA. However, there were no obvious changes in the expression profile of carotenoid-biosynthetic genes. The transcriptomic results indicated that the expression of CaATHB-12 was higher in the pericarp development process. Furthermore, the transcript levels of CaATHB-12 under normal condition in three continuous developmental stages of ‘R24’ pepper fruit (Fig. 1A) was investigated by RT-qPCR. Results obtained showed that “L” (represented “luminance”), “b” and “C” values initially decreased, followed by an increase at the final stage of fruit ripening, while the “a” value increased in the whole period of fruit ripening (Fig. 1B). CaATHB-12 transcripts were detectable in all stages, with Stage 3 (50 days after full bloom) and Stage 2 (35 days after full bloom) having the highest expression level, and the least expression at stage 1 (25 after full bloom) (Fig. 1C). These dynamic changes in the above-mentioned parameters proposed that fruit color change follows the rule: green, bottle green, light-colored, and red.
Virus-Induced Gene Silencing (VIGS) of CaATHB-12 in Pepper Detached Fruit
TRV2 vector carrying the CaATHB-12 gene was vaccinated into the Capsicum annuum cv. R24 detached fruits. 15 days post-inoculation, different colors were noted in the CaATHB-12-silenced fruits as compared to the control (Fig. 2A). The color of the CaATHB-12-slienced fruits changed from green to yellow, while that of the control fruits were from green to red color. Simultaneously, the TRV2:CaPDS (positive control) detached fruits were green to orange-yellow color. Furthermore, silencing efficiency measured through RT-qPCR affirmed that CaATHB-12 transcript level in the silenced fruits were 86% lower as compared to the control (Fig. 2B). Similarly, the carotenoids content in the silenced fruit was also significantly lower than the control fruit (Fig. 2C).
Effect of ABA Stress on CaATHB-12 Silenced Pepper Fruit
Further investigated the function of CaATHB-12 under ABA treatment, the CaATHB-12-silenced and control fruits were treated with ABA solution (150 mg L−1). To study the silencing effect of CaATHB-12 in pepper fruits, the ROS, MDA contents, chlorophyll contents, and ROS scavenging antioxidants enzymes were measured at different time points (0, 6, 12, 24 and 48 h) post treatment in pepper fruits. The H2O2 content of the empty vector (control) fruits were significantly higher (4 folds) at 48 h than the CaATHB-12-silenced fruits (Fig. 3A). Correspondingly, the malondialdehyde (MDA) and O2−· levels followed similar trend of increment after ABA treatment in both the control and silenced fruits (Fig. 3B, C).
The antioxidant enzyme system of the pepper plants was stimulated in response to stress in order to mitigate the ROS associated damage. The activities of CAT, POD, SOD and APX gradually increased at each time point (Fig. 4). However, the antioxidant enzyme activities of the above-mentioned enzymes were significantly higher in the CaATHB-12-silenced fruits than the control. The POD activity increased at all time points and reached their peaks in both TRV2:00 and TRV2:CaATHB-12 at 48 h, which was about 4-fold and 5.8-fold respectively (Fig. 4A). Both the SOD and POD activities levels followed the same trends of enhancement after ABA treatment in both control and silenced pepper fruits, but their respective peaks were higher than control fruits (Fig. 4B, C). Though the GPX activity significantly increased up to 24 h post stress in the CaATHB-12-silenced fruit and then decreased at 48 h (Fig. 4D).
Thus, we measured the total carotenoid, anthocyanin, flavonoid and total phenolic contents from both CaATHB-12-silencedand control pepper fruits at different time points. In control fruits, a slight increase in total carotenoid contents was noted until 48 h post-treatment, whereas in the CaATHB-12-silenced fruits, the ABA treatment caused a significant and dynamic increase in the total carotenoid contents (Fig. 5A). Similarly, the measured anthocyanin contents at 48 h were also almost significantly higher (7 folds) in the silenced fruits as compared to the control fruit (4.5 folds) (Fig. 5B). Flavonoid and total phenolic contents followed the similar trend of increment after ABA stress in both silenced and control pepper fruits. In addition, after ABA stress the flavonoid at 24 h and total phenolic contents at 12 h were significantly higher in the silenced fruits compared to control, and then a bit decrease was recorded in the silenced fruits, but they were still significantly higher than control fruits (Fig. 5C, D).
Additionally, exogenous application of ABA (150 mg L−1) induced the transcript level of CaATHB-12 in the both silenced and control fruits, but the expression level of the silenced fruits was lesser than that in the control fruits at 0 and 6 h, while its expression abruptly increased at 12 h, reached to maximum at 48 h, and was significantly higher than the control (Fig. 6A). The carotenoid synthesis related genes (CaPSY, CaZEP, CaBCH, CaLCYB) were also differentially induced, and their expression levels were significantly higher in the CaATHB-12-silenced fruits than that in the control at 48 h, expect CaLCYB gene (Fig. 6B, E). Furthermore, the transcript levels of the defense-related genes (CaPOD, CaSOD and CaMYB44) were significantly higher at 24 and 48 h in the CaATHB-12-silenced fruits as compared to the control except CaSOD which reached to peak at 12 h (Fig. 6F, H). These results partially revealed that CaATHB-12 played a negative role in the plant defense response against ABA osmosis stress.
Effect of CaATHB-12 Overexpressing on Transgenic Arabidopsis
To further explore the function of CaATHB-12, the CaATHB-12-overexpressed transgenic lines of Arabidopsis were generated. Under normal growth conditions, there are no discernible differences between CaATHB-12-overexpressed lines (CaATHB-12-OE) and wild type Arabidopsis plants (Fig. 7A). Furthermore, the yellowing symptoms in leaf discs of both transgenic and WT lines aggravated as the ABA concentration increases (Supplementary Fig. S2A). Though, as compared to the transgenic lines, the carotenoid content was significantly reduced in WT lines when treated with ABA, while the chlorophyll content of the transgenic lines displayed lower levels than that in WT, and the highest variation was noted with 150 mg L−1 ABA solution, where transgenic lines provided approximately 0.2 mg/g and WT lines provided 0.83 mg/g chlorophyll content, the former being 75% lower than the latter (Supplementary Fig. S2B-C). After treated with exogenous ABA (150 mg L−1) treatment at 48 h, no difference was detected in the withering symptom of both transgenic and WT lines (Fig. 7A). Interestingly, the carotenoid and total chlorophyll contents in OE lines were lower than that of the wild type (Fig. 7B-C). However, the ROS included H2O2 contents and O2−· levels in OE lines were higher than that of the wide type (Fig. 7D-E). Furthermore, the MDA content of the WT lines was slightly increased and was higher than transgenic seedlings (Fig. 7F). Furthermore, the CAT, POD, SOD, APX and GPX activities of the transgenic seedlings were significantly lower than the WT (Fig. 8), and a significant difference was found in the above-mentioned antioxidant enzymes activities of the transgenic lines and wild type Arabidopsis. Meanwhile, the transgenic Arabidopsis thaliana showed dehydration and wilting with weak growth under salt and mannitol treatment (Fig. 9A). However, the WT plants showed a slight yellowing phenotype with lower MDA, H2O2, O2−·content and higher CAT, POD activities than OE lines (Fig. 9B–F).
Discussion
As sessile organisms, plants are exposed to various environmental stresses during growth and development. If subjected to adversity environments such as drought, high salt and abscisic acid, the membrane integrity of the plants will be disrupted, resulting in adverse effects such as low photosynthetic rate and cell dysfunction, which will eventually lead to crop yield reduction (Wei et al. 2016), it will also lead to the formation of ROS, cell damage, metabolic disorders, and aging processes (Jaleel et al. 2009). The ROS in low concentrations are important signaling molecules, but the increased quantities of ROS result in the generation of oxidative secondary stress (Bailey-Serres and Mittler 2006). In higher plants, abiotic stressors (extremes heat/cold, salt) are due to the imbalance between pro-oxidants and antioxidants resulting in oxidative stress (Sreenivasulu et al. 2007). HD-Zip I transcription factors were involved in response to various environmental stresses such as low temperature, salt, and ABA in regulating fruit development (Jiang et al. 2017; Zhang et al. 2020), by regulating the expression of downstream related genes to promote plant oxidation stress response (Ariel et al. 2010; Harris et al. 2011). On the other hand, the HD-Zip transcription factors also help in the synthesis of color pigments such as chlorophyll and carotenoids (Lu et al. 2014; Manavella et al. 2008). In our previous studies, we identified and cloned the CaATHB-12 gene of the HD-Zip I subfamily which includes corresponding conserved HD and Zip motifs (Zhang et al. 2020), interact in vitro with the pseudo-palindromic sequence CAAT(A/T)ATTG (Ariel et al. 2010), and were involved in the tolerance to exogenous ABA application (Ribichich et al. 2013).
Previously, it was reported that the expression of photosynthesis-related gene was regulated by the HD-Zip transcription factor HAHB4 in sunflower, which further regulated the synthesis of carotenoids in transgenic Arabidopsis (Manavella et al. 2008). This gene interacted with MYB, bHLH and WD40 partners in the cytoplasm and participates in the regulation of related pigment accumulation (Jiang et al. 2017). The stability and integrity of the cell membrane is an important foundation for plant to grow (Rui et al. 2010). The production of MDA in plants has an important impact on the normal function of the cell membrane. The chlorophyll content is reduced by the influence of excess reactive oxygen, which approximately measures the degree of stress damage in the plant (Choudhury et al. 2017). Under normal growth conditions, ROS such as H2O2 and O2−· are in dynamic balance, but under abiotic stress, the balance is broken, and excess reactive oxygen will damage the cell structure and corresponding functional proteins in the cell. In this experiment, the control fruit accumulated more H2O2 and O2−· than CaATHB-12-silenced fruit, and the amount of H2O2 and O2−· of the OE plants were found significantly higher than that of WT. Our results revealed that the pepper CaATHB-12 gene plays a regulatory role in improving the scavenging capacity of ROS and lowering the oxidative stress.
Abscisic acid (ABA) being a “stress hormones” (Brandt et al. 2014), while the induction of exogenous ABA tolerance could be due to elevating the activities of the antioxidant enzymes system, which helped to reduce the ROS accumulation and protect the membrane structure from oxidative damage (Yu et al. 2019). Higher plants possess a sophisticated and complex system of antioxidant enzymes and non-enzymatic systems in response to various stresses (Ali et al. 2008), in which SOD acts as a large number of antioxidants (SOD, CAT, POD, APX, and GPX) can decompose O2−· into H2O2, which is further removed by POD and APX (Choudhury et al. 2017). On the other hand, non-enzymatic active oxygen scavenging systems mainly contain bioactive substances such as carotenoids, anthocyanins, polyphenols and flavonoids (Gill and Tuteja 2010). Relevant research reported that the ATHB-12 gene in Arabidopsis negatively regulates the elongation of plant stems and is induced by NaCl and ABA treatments (Olsson et al. 2004). Previously, we found that the expression of CaATHB-12 could be modulated significantly during cold stress (Zhang et al. 2020). In this study, the CaATHB-12 gene was heterogeneously expressed in Arabidopsis thaliana. After ABA (150 mg L−1) treatment, the activities of GPX, SOD, APX and POD in the transgenic lines were lower than that of WT, while the activity of CAT was contrary to the law. We speculate that overexpression of A. thaliana leads to a reduction in the activity of most antioxidant enzymes in plants, and the MDA content is higher than that of WT, indicated that the extent of cell membrane damage in transgenic plants caused by abiotic stress was higher than in WT plants. Although CAT is an important enzyme for removing H2O2 (Karpinski and Muhlenbock 2007; Lee et al. 2007), due to the reduction in the activity of other important antioxidant enzymes, it is not enough to remove excess O2−·, which in turn reduced the ability of the expression strain to clear ROS reduces the stress resistance of the plant.
The antioxidant capacity of plants is manifested through the synergistic effect of various antioxidants. Bioactive substances (such as total phenols, flavonoids, carotenoids, and anthocyanins) as antioxidants have a crucial role in improving plant resistance (Chang et al. 2019; Singh et al. 2017). Wang et al. (2020) reported that exogenous application of ABA (150 mg L−1) had a forceful inhibitory effect on the nitrogen accumulation of fruit, resulting in disorders of nitrogen metabolism, so affecting pigmentation in ‘Red Fuji’ apple fruit. At the same time participate in the construction of photosynthetic complex protein PSI and maintain the stability of thylakoid membrane (Gill et al. 2011; Niyogi et al. 2001). Havaux (2014) reported that carotenoids function as an oxidative stress signaling molecule. β-carotene is the main component of carotenoids, that can interact with hydrogen peroxide (H2O2), superoxide radicals (O2−·) accumulation, free radical reaction (Mahapatra et al. 2013), at sufficiently high concentrations, carotenoids are more effective in protecting lipids from peroxidative damage (Ahmad et al. 2010). Because of their unique structure, flavonoids can locate free radical molecules in cells and at the same time scavenge free radicals, making them important for plants under harsh environmental conditions (Løvdal et al. 2010). Studies have reported that the concentration of flavonoids will increase under conditions of cold damage, low temperature, and lack of hormones (Winkel-Shirley 2002). At the same time, it was found that flavonoids have good nitrogen tolerance under the condition of nitrogen deficiency (Peng et al. 2008). Interestingly, polyphenols are directly involved in the process of plant antioxidant stress response, and has the function of metal chelating agent (Ksouri et al. 2008). Studies have shown that the antioxidant activity of blueberries is closely related to the total phenolic content and anthocyanin content (Ehlenfeldt and Prior 2001), and the high content of total phenol and anthocyanin content improves the antioxidant activity of plants (Kalt et al. 2000). In our study, silencing the CaATHB-12 reduced the carotenoid content, and the expression levels of the related gene involved in carotenoid regulation were also reduced compared to the control fruit (Fig. 6). Previous studies also showed that RhHB1 (HD-Zip I) impacted the flower color of rose (Rosa hybrida) (Lu et al. 2014). Similarly, Jiang et al. (2017) showed that silencing of MdHB1 (HD-Zip I) caused the accumulation of anthocyanin in ‘Granny Smith’ flesh apple, whereas its overexpression reduced the flesh content of pigment in ‘Ballerina’ (red-fleshed apple). After exogenous ABA treatment, the carotenoid synthesis rate of the silenced fruits was significantly higher than that of the control fruits, while the contents of total phenol and flavonoids were higher than that of the control fruit (Fig. 5). On the other hand, after ABA treatment, the carotenoid content of the CaATHB-12-overexpressed lines was slightly lower than that of WT (Fig. 7). We speculated that the CaATHB-12 gene of pepper is involved in inducing the production of related bioactive substances and regulating the resistance of plants to abiotic stress.
Exogenous ABA also modulates the expression of gene networks that control other ameliorative and adaptive stress responses in plants (Lim et al. 2015). In previous studies, ABA responses have a wide and various range of downstream effects, and their network of the hormonal pathways is further complicated by interactions with ROS (Pinheiro and Chaves 2011). So, CaATHB-12 could become fundamental part of the ROS-mediated ABA signaling cascade in plants. On the other hand, loss-of-function ATHB12 mutants have illustrated that the gene activates clade a protein phosphatases 2 C (PP2C) genes to interact with proteins of the basal transcriptional machinery and repress AHG3 (Protein Phosphatase 2CA), ABI2 (ABA Insensitive 2), and ABI1 (ABA Insensitive 1) (Ma et al. 2009; Rubio et al. 2009), thus acting as negative regulators of ABA signaling networks, meanwhile the binding of some of these targets is ABA-dependent for ATHB12 (Valdés et al. 2012). In addition, ATHB20 and ATHB5 act as negative regulators of ABA sensitivity in germinating plants (Barrero et al. 2010; Johannesson et al. 2003) and ATHB6 has also been proposed as negative modulator of the ABA response (Himmelbach et al. 2002; Reyes et al. 2006). Moreover, over-expression of CpHB-7 isolated from Craterostigma plantagineum in Arabidopsis resulted in reduced sensitivity towards ABA treatment (Deng et al. 2006). Many stress-related genes in plants generally mediate the response of plants to stress. For example, AtMYB44 gene can regulate ABA signal-mediated plant response to NaCl and drought stress (Nguyen et al. 2019), AtDREB2A as an ABA signal response gene can be induced by low temperature expression (Nakashima et al. 2000), Mn-SOD and POD, as marker genes related to the antioxidant system, are involved in responding to various stresses (Guo et al. 2012). Rai et al. (2013) study revealed that under the control of stress-inducing factor (RD29A), the overexpression of AtDREB1A in tomatoes showed enhanced levels of antioxidant enzymes and antioxidant substances, and the ability to drought-induced oxidative stress greatly enhanced. Our research shows that after ABA treatment, the content of POD and SOD in the CaATHB-12-silenced fruits are higher than that of the control fruits. Altogether, our study indicating that the CaATHB-12 gene is involved in the regulation of ABA-mediated oxidative stress response, which is further induced by exogenous ABA, but the exact molecular regulatory mechanisms need further study. Therefore, reduced tolerance to ABA stresses of the CaATHB-12-overexpressed plants may be due to partially impeded expression of these genes. Furthermore, this research will closely focus on fundamental insights for future studies to precisely explore the role of the CaATHB-12 gene in regulatory pathways.
Conclusions
Taken together, we characterized a gene CaATHB-12 derived from HD-Zip I subfamily which was intensively induced by exogenous ABA, salt, and mannitol applications. Efficient gene silencing lines were created from pepper, and stable heterologous overexpression lines were created from Arabidopsis to achieve a comprehensive exploration of gene function. The functional study of CaATHB-12 in pepper increased plant sensitivity to ABA stress, while the over-expressing CaATHB-12 in Arabidopsis lines revealed that tolerance to ABA, salt, and mannitol stresses was decreased. Furthermore, CaATHB-12 plays a fundamental role in elevating the tolerance to these stresses through the increased expression of other stress related genes, increasing the activities of anti-oxidant enzymes and scavenging the ROS. The studied functions of the CaATHB-12 gene may provide some insights in exquisite molecular detail by pursuing signal transduction mechanisms that converge on gene expression patterns.
Data Availability
All data generated or analysed during this study are included in this manuscript and its supplementary information files.
References
Aebi H (1984) Catalase in vitro. Methods Enzymol 105(13):121–126
Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S (2010) Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Crit Rev Biotechnol 30(3):161–175
Ahuja I, Vos RCHd, Bones AM, Hall RD (2010) Plant molecular stress responses face climate change. Trends Plant Sci 15(12):0–674
Ali B, Hasan S, Hayat S, Hayat Q, Yadav S, Fariduddin Q, Ahmad A (2008) A role for brassinosteroids in the amelioration of aluminium stress through antioxidant system in mung bean (Vigna radiata L. Wilczek). Environ Exp Bot 62(2):153–159
Ariel FD, Manavella PA, Dezar CA, Chan RL (2007) The true story of the HD-Zip family. Trends Plant Sci 12(9):419–426
Ariel F, Diet A, Verdenaud M, Gruber V, Frugier F, Chan R, Crespi M (2010) Environmental regulation of lateral root emergence in Medicago truncatula requires the HD-Zip I transcription factor HB1. Plant Cell 22(7):2171–2183
Bailey-Serres J, Mittler R (2006) The roles of reactive oxygen species in plant cells. Plant Biol 141(2):311–311
Barickman TC, Kopsell DA, Sams CE (2014) Abscisic acid increases carotenoid and chlorophyll concentrations in leaves and fruit of two tomato genotypes. J Am Soc Hortic Sci Am Soc Hortic Sci 139(3):261–266
Barrero JM, Millar AA, Griffiths J, Czechowski T, Scheible WR, Udvardi M, Reid JB, Ross JJ, Jacobsen JV, Gubler F (2010) Gene expression profiling identifies two regulatory genes controlling dormancy and ABA sensitivity in Arabidopsis seeds. Plant J 61(4):611–622
Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24(1):23–58
Benson D, Karsch-Mizrachi I, Lipman D, Ostell J, Rapp B, Wheeler D (2000) GenBank Nucleic Acids Res 28(1):15–18
Brandt R, Cabedo M, Xie Y, Wenkel S (2014) Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment. J Integr Plant Biol 56(6):518–526
Brennan T, Frenkel C (1977) Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol 59(3):411–416
Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52(C):302–310
Bueno P, Piqueras A, Kurepa J, Savoure A, Verbruggen N, Van Montagu M, Inze D (1998) Expression of antioxidant enzymes in response to abscisic acid and high osmoticum in Tobacco BY-2 cell cultures. Plant Sci 138(1):27–34
Chang J, Wang M, Jian Y, Zhang F, Zhu J, Wang Q, Sun B (2019) Health-promoting phytochemicals and antioxidant capacity in different organs from six varieties of Chinese kale. Sci Rep 9(1):20344
Choudhury FK, Rivero RM, Blumwald E, Mittler R (2017) Reactive oxygen species, abiotic stress and stress combination. Plant J 90(5):856–867
Christie PJ, Alfenito MR, Walbot V (1994) Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194(4):541–549
Coombe BG, Hale C (1973) The hormone content of ripening grape berries and the effects of growth substance treatments. Plant Physiol 51(4):629–634
Deng X, Phillips J, Meijer AH, Salamini F, Bartels D (2002) Characterization of five novel dehydration-responsive homeodomain leucine zipper genes from the resurrection plant Craterostigma plantagineum. Plant Mol Biol 49(6):601–610
Deng X, Phillips J, Bräutigam A, Engström P, Johannesson H, Ouwerkerk PB, Ruberti I, Salinas J, Vera P, Iannacone R (2006) A homeodomain leucine zipper gene from Craterostigma plantagineum regulates abscisic acid responsive gene expression and physiological responses. Plant Mol Biol 61(3):469–489
Ehlenfeldt MK, Prior RL (2001) Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbush blueberry. J Agric Food Chem 49(5):2222–2227
Enoki S, Hattori T, Ishiai S, Tanaka S, Mikami M, Arita K, Nagasaka S, Suzuki S (2017) Vanillylacetone up-regulates anthocyanin accumulation and expression of anthocyanin biosynthetic genes by inducing endogenous abscisic acid in grapevine tissues. J Plant Physiol 21(9):22–27
Flohé L, Günzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105(1):114–121
Foyer CH (2020) How plant cells sense the outside world through hydrogen peroxide. Nature 578(7796):518–519
Frank W, Phillips J, Salamini F, Bartels D (1998) Two dehydration-inducible transcripts from the resurrection plant Craterostigma plantagineum encode interacting homeodomain-leucine zipper proteins. Plant J 15(3):413–421
Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48(12):909–930
Gill SS, Khan NA, Anjum NA, Tuteja N (2011) Amelioration of cadmium stress in crop plants by nutrients management: morphological, physiological and biochemical aspects. Plant Stress 5(1):1–23
Guo W, Chen R, Gong Z, Yin Y, Ahmed S, He Y (2012) Exogenous abscisic acid increases antioxidant enzymes and related gene expression in pepper (Capsicum annuum) leaves subjected to chilling stress. Genet Mol Res 11(4):4063–4080
Guo M, Yin YX, Ji JJ, Ma BP, Lu MH, Gong ZH (2014) Cloning and expression analysis of heat-shock transcription factor gene CaHsfA2 from pepper (Capsicum annuum L). Genet Mol Res Gmr 13(1):1865–1875
Guo M, Liu JH, Ma X, Zhai YF, Gong ZH, Lu MH (2016) Genome-wide analysis of the Hsp70 family genes in pepper (Capsicum annuum L.) and functional identification of CaHsp70-2 involvement in heat stress. Plant Sci 25(2):246–256
Harris JC, Hrmova M, Lopato S, Langridge P (2011) Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli. New Phytol 190(4):823–837
Havaux M (2014) Carotenoid oxidation products as stress signals in plants. Plant J 79(4):597–606
He YM, Luo DX, Khan A, Liu KK, Arisha MH, Zhang HX, Cheng GX, Ma X, Gong ZH (2018) CanTF, a novel transcription factor in Pepper, is involved in resistance to phytophthora capsici as well as abiotic stresses. Plant Mol Biology Report 36(5):776–789
Henriksson E, Olsson AS, Johannesson H, Johansson H, Hanson J, Engstrom P, Soderman E (2005) Homeodomain leucine zipper class I genes in Arabidopsis. Expression patterns and phylogenetic relationships. Plant Physiol 139(1):509–518
Himmelbach A, Hoffmann T, Leube M, Hohener B, Grill E (2002) Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. EMBO J 21(12):3029–3038
Hjellstrom M, Olsson A, Engström P, Söderman E (2003) Constitutive expression of the water deficit-inducible homeobox gene ATHB7 in transgenic Arabidopsis causes a suppression of stem elongation growth. Plant Cell Environ 26(7):1127–1136
Hunter RS (1987) The measurement of appearance. John Wiley, New York
Huo Y, Xiong W, Su K, Li Y, Yang Y, Fu C, Wu Z, Sun Z (2019) Genome-wide analysis of the TCP gene family in Switchgrass (Panicum virgatum L.). Int J Genom. 2019: 8514928
Jaleel CA, Riadh K, Gopi R, Manivannan P, Inès J, AlJuburi HJ, ChangXing Z, HongBo S, Panneerselvam R (2009) Antioxidant defense responses: physiological plasticity in higher plants under abiotic constraints. Acta Physiol Plant 31(3):427–436
Jiang MY, Zhang JH (2002) Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves. J Exp Bot 53(379):2401–2410
Jiang Y, Liu C, Yan D, Wen X, Liu Y, Wang H, Dai J, Zhang Y, Liu Y, Zhou B, Ren X (2017) MdHB1 down-regulation activates anthocyanin biosynthesis in the white-fleshed apple cultivar ‘Granny Smith’. J Exp Bot 68(5):1055–1069
Johannesson H, Wang Y, Hanson J, Engström P (2003) The Arabidopsis thaliana homeobox gene ATHB5 is a potential regulator of abscisic acid responsiveness in developing seedlings. Plant Mol Biol 51(5):719–729
Kalt W, McDonald J, Donner H (2000) Anthocyanins, phenolics, and antioxidant capacity of processed lowbush blueberry products. J Food Sci 65(3):390–393
Karpinski S, Muhlenbock P (2007) Genetic, molecular and physiological mechanisms controlling cell death, defenses, and antioxidant network in response to abiotic and biotic stresses in plants. Comp Biochem Physiol a-Molecular Integr Physiol 146(4):S60–S60
Khan MIR, Khan N (2017) Reactive oxygen species and antioxidant systems in plants: role and regulation under abiotic stress. Springer, Heidelberg
Khan A, Li RJ, Sun JT, Ma F, Gong ZH (2018) Genome-wide analysis of dirigent gene family in pepper (Capsicum annuum L.) and characterization of CaDIR7 in biotic and abiotic stresses. Sci Rep 8(1):5500
Kim S, Park M, Yeom S-I, Kim YM, Lee JM, Lee HA, Seo E, Choi J, Cheong K, Kim KT, Jung K, Lee GW, Oh SK, Bae C, Kim SB, Lee HY, Kim SY, Kim MS, Kang BC, Jo YD, Yang HB, Jeong HJ, Kang WH, Kwon JK, Shin C, Lim JY, Park JH, Huh JH, Kim JS, Kim BD, Cohen O, Paran I, Suh MC, Lee SB, Kim YK, Shin Y, Noh SJ, Park J, Seo YS, Kwon SY, Kim HA, Park JM, Kim HJ, Choi SB, Bosland PW, Reeves G, Jo SH, Lee BW, Cho HT, Choi HS, Lee MS, Yu Y, Do Choi Y, Park BS, van Deynze A, Ashrafi H, Hill T, Kim WT, Pai HS, Ahn HK, Yeam I, Giovannoni JJ, Rose JKC, Sørensen I, Lee SJ, Kim RW, Choi IY, Choi BS, Lim JS, Lee YH, Choi D (2014) Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet 46(3):270–278
Ksouri R, Megdiche W, Falleh H, Trabelsi N, Boulaaba M, Smaoui A, Abdelly C (2008) Influence of biological, environmental and technical factors on phenolic content and antioxidant activities of Tunisian halophytes. C R Biol 331(11):865–873
Lee SH, Ahsan N, Lee KW, Kim DH, Lee DG, Kwak SS, Kwon SY, Kim TH, Lee BH (2007) Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses. J Plant Physiol 164(12):1626–1638
Li D, Li L, Luo Z, Mou W, Mao L, Ying T (2015) Comparative transcriptome analysis reveals the influence of abscisic acid on the metabolism of pigments, ascorbic acid and folic acid during strawberry fruit ripening. PLoS ONE 10(6):e0130037
Lim CW, Baek W, Jung J, Kim JH, Lee SC (2015) Function of ABA in stomatal defense against biotic and drought stresses. Int J Mol Sci 16(7):15251–15270
Løvdal T, Olsen KM, Slimestad R, Verheul M, Lillo C (2010) Synergetic effects of nitrogen depletion, temperature, and light on the content of phenolic compounds and gene expression in leaves of tomato. Phytochemistry 71(5–6):605–613
Lu P, Zhang C, Liu J, Liu X, Jiang G, Jiang X, Khan MA, Wang L, Hong B, Gao J (2014) RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa Hybrida) petal senescence. Plant J 78(4):578–590
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324(5930):1064–1068
Macfarlane SA (1999) Molecular biology of the tobraviruses. J Gen Virol 80(11):2799–2807
Mahapatra S, Mohanta YK, Panda S (2013) Methods to study antioxidant properties with special reference to medicinal plants. Int J Pharm North Orissa Univ Baripada-757003 Odisha India 3(1):91–97
Manavella PA, Arce AL, Dezar CA, Bitton F, Renou JP, Crespi M, Chan RL (2006) Cross-talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb-4 transcription factor. Plant J 48(1):125–137
Manavella PA, Dezar CA, Ariel FD, Drincovich MF, Chan RL (2008) The sunflower HD-Zip transcription factor HAHB4 is up-regulated in darkness, reducing the transcription of photosynthesis-related genes. J Exp Bot 59(11):3143–3155
Michael A, and, Paul, Wilson (1997) Relationship between hunter color values and β-Carotene contents in white-fleshed African sweetpotatoes (Ipomoea batatas Lam). J Sci Food Agric 73(3):301–306
Nafees A, Khan, Naser A, Anjum, Narendra T, Sarvajeet S (2011) Amelioration of cadmium stress in crop plants by nutrient management: morphological, physiological and biochemical aspects. Plant Stress 5(1):1–23
Nakano Y, Asada K (1987) Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate-depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol 28(1):131–140
Nakashima K, Shinwari ZK, Sakuma Y, Seki M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K (2000) Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration-and high-salinity-responsive gene expression. Plant Mol Biol 42(4):657–665
Nguyen NH, Nguyen CTT, Jung C, Cheong JJ (2019) AtMYB44 suppresses transcription of the late embryogenesis abundant protein gene AtLEA4-5. Biochem Biophys Res Commun 511(4):931–934
Niyogi KK, Shih C, Chow WS, Pogson BJ, DellaPenna D, Björkman O (2001) Photoprotection in a zeaxanthin-and lutein-deficient double mutant of Arabidopsis. Photosynth Res 67(12):139–145
Olsson AS, Engstrom P, Soderman E (2004) The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol 55(5):663–677
Peng M, Hudson D, Schofield A, Tsao R, Yang R, Gu H, Bi YM, Rothstein SJ (2008) Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene. J Exp Bot 59(11):2933–2944
Pinheiro C, Chaves M (2011) Photosynthesis and drought: can we make metabolic connections from available data? J Exp Bot 62(3):869–882
Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys acta 975(3):0–394
Rai GK, Rai NP, Rathaur S, Kumar S, Singh M (2013) Expression of rd29A: AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants. Plant Physiol Biochem 6(9):90–100
Ré DA, Capella M, Bonaventure G, Chan RL (2014) Arabidopsis AtHB7 and AtHB12 evolved divergently to fine tune processes associated with growth and responses to water stress. BMC Plant Biol 14:150
Reyes D, Rodríguez D, González-García MP, Lorenzo O, Nicolás G, GarcíaMartínez JL, Nicolás C (2006) Overexpression of a protein phosphatase 2 C from beech seeds in Arabidopsis shows phenotypes related to abscisic acid responses and gibberellin biosynthesis. Plant Physiol 141(4):1414–1424
Ribichich KF, Arce AL, Chan RL (2013) Coping with drought and salinity stresses: role of transcription factors in crop improvement. Climate change and plant abiotic stress tolerance. Wiley, Hoboken, pp 641–684
Rodov V, Tietel Z, Vinokur Y, Horev B, Eshel D (2010) Ultraviolet light stimulates flavonol accumulation in peeled onions and controls microorganisms on their surface. J Agricultural Food Chem 58(16):9071–9076
Rubio S, Rodrigues A, Saez A, Dizon MB, Galle A, Kim TH, Santiago J, Flexas J, Schroeder JI, Rodriguez PL (2009) Triple loss of function of protein phosphatases type 2 C leads to partial constitutive response to endogenous abscisic acid. Plant Physiol 150(3):1345–1355
Rui H, Cao S, Shang H, Jin P, Wang K, Zheng Y (2010) Effects of heat treatment on internal browning and membrane fatty acid in loquat fruit in response to chilling stress. J Sci Food Agric 90(9):1557–1561
Satoru K, Siriwan M, Yusuke B, Takaya (2009) Effects of auxin and jasmonates on 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase gene expression during ripening of apple fruit. Postharvest Biol Technol 51(2):281–284
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3(6):1101–1108
Singh B, Singh JP, Kaur A, Singh N (2017) Phenolic composition and antioxidant potential of grain legume seeds: a review. Food Res Int 10(1):1–16
Soderman E, Mattsson J, Engstrom P (1996) The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. Plant J 10(2):375–381
Sreenivasulu N, Sopory S, Kishor PK (2007) Deciphering the regulatory mechanisms of abiotic stress tolerance in plants by genomic approaches. Gene 388(1–2):1–13
Stewart RRC, Bewley JD (1980) Lipid peroxidation associated with accelerated aging of soybean axes. Plant Physiol 65(2):245–248
Sun L, Yuan B, Zhang M, Wang L, Cui M, Wang Q, Leng P (2012) Fruit-specific RNAi-mediated suppression of SlNCED1 increases both lycopene and β-carotene contents in tomato fruit. J Exp Bot 63(8):3097–3108
Taylor I, Linforth R, Al-Naieb R, Bowman W, Marples B (1988) The wilty mutants flacca and sitiens are impaired in the oxidation of ABA-aldehyde to ABA. Plant. Cell Environ 1(1):739–745
Tian SL, Li L, Chai W-G, Shah SNM, Gong ZH (2014) Effects of silencing key genes in the capsanthin biosynthetic pathway on fruit color of detached pepper fruits. BMC Plant Biol 14(1):314
Tian SL, Li L, Tian YQ, Shah SNM, Gong ZH (2016) Effects of abscisic acid on capsanthin levels in Pepper fruit. J Am Soc Hort Sci 141(6):609–616
ul Ul Haq S, Khan A, Ali M, Gai W-X, Zhang HX, Yu QH, Yang SB, Wei AM, Gong ZH (2019) Knockdown of CaHSP60-6 confers enhanced sensitivity to heat stress in pepper (Capsicum annuum L). Planta 250(6):2127–2145
Valdés AE, Övernäs E, Johansson H, Rada-Iglesias A, Engström P (2012) The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities. Plant Mol Biol 80(4–5):405–418
Verslues PE, Zhu JK (2005) Before and beyond ABA: Upstream sensing and internal signals that determine ABA accumulation and response under abiotic stress. Biochem Soc Trans 33(Pt 2):375–379
Wan H, Yuan W, Ruan M, Ye Q, Wang R, Li Z, Zhou G, Yao Z, Zhao J, Liu S (2011) Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L). Biochem Biophys Res Commun 416(1–2):0–30
Wang H, Niu H, Zhai Y, Lu M (2017) Characterization of BiP genes from pepper (Capsicum annuum L.) and the role of CaBiP1 in response to endoplasmic reticulum and multiple abiotic stresses. Front Plant Sci 8(1):122
Wang F, Sha J, Chen Q, Xu X, Zhu Z, Ge S, Jiang Y (2020) Exogenous abscisic acid regulates distribution of 13C and 15N and anthocyanin synthesis in ‘Red Fuji’ apple fruit under high nitrogen supply. Front Plant Sci 10:1738
Wei T, Deng K, Gao Y, Liu Y, Yang M, Zhang L, Zheng X, Wang C, Song W, Chen C, Zhang Y (2016) Arabidopsis DREB1B in transgenic Salvia miltiorrhiza increased tolerance to drought stress without stunting growth. Plant Physiol Biochem 10(4):17–28
Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5(3):218–223
Xiao H, Juan (2014) Cloning and functional analysis of leaf senescence-related genes in capsicum annuum L, vol 3. Northwest A&F University, pp 0–107. (in Chinese)
Xiao HJ, Yin YX, Chai WG, Gong ZH (2014) Silencing of the CaCP gene delays salt-and osmotic-induced leaf senescence in Capsicum annuum L. Int J Mol Sci 15(5):8316–8334
Yu XC, Li MJ, Gao GF, Feng HZ, Geng XQ, Peng CC, Zhu SY, Wang XJ, Shen YY, Zhang DP (2006) Abscisic acid stimulates a calcium-dependent protein kinase in grape berry. Plant Physiol 140(2):558–579
Yu Y, Weng Q, Zhou B (2016) Effects of exogenous ABA on contents of lycopene and endogenous hormone in tomato pericarp. Biotechnol J Int 1(6):1–5
Yu W, Zhao R, Wang L, Zhang S, Li R, Sheng J, Shen L (2019) ABA signaling rather than ABA metabolism is involved in trehalose-induced drought tolerance in tomato plants. Planta 250(2):643–655
Zhang M, Leng P, Zhang G, Li X (2009) Cloning and functional analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits. J Plant Physiol 166(12):1241–1252
Zhang S, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, Schluepmann H, Liu CM, Ouwerkerk PB (2012) Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol 80(6):571–585
Zhang S, Xu (2016) Study on the effect of ABA signal in fruit development and maturation process in ‘Zunla-1’. Zhongkai University of Agriculture and Engineering (in Chinese) 65:0-195
Zhang RX, Zhu WC, Cheng GX, Yu YN, Li QH, ul Haq S, Said F, Gong H (2020) A novel gene, CaATHB-12, negatively regulates fruit carotenoid content under cold stress in Capsicum annuum. Food & Nutrition Research 64:3729
Zhu Y, Zheng P, Varanasi V, Shin S, Main D, Curry E, Mattheis JP (2012) Multiple plant hormones and cell wall metabolism regulate apple fruit maturation patterns and texture attributes. Tree Genet Genom 8(6):1389–1406
Zweier JL (1988) Measurement of superoxide-derived free radicals in the reperfused heart. Evidence for a free radical mechanism of reperfusion injury. J Biol Chem 263(3):1353–1357
Acknowledgements
This work was supported through funding from the National Natural Science Foundation of China (No. 31772309, No. 31860556 and No. U1603102) and China Agriculture Research System of MOF and MARA (CARS-24-G-01).
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RXZ and ZHG: Conceptualization, Methodology, Investigation, Data curation, Writing—original draft. RXZ, QHL, and GXC: Writing—review and editing. JJX: Conceptualization, Visualization, Investigation, Writing—review and editing. AK and SH: Methodology, Writing—review and editing. HLY and ZHG: Supervision, Visualization, Resources, Writing—review and editing. All authors have read and approved the final manuscript.
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Zhang, R., Li, Q., Xiao, J. et al. Characterization of a Homeodomain-Leucine Zipper Gene 12: Gene Silencing in Pepper and Arabidopsis-Based Overexpression During Abiotic Stress. J Plant Growth Regul 43, 1689–1706 (2024). https://doi.org/10.1007/s00344-023-11215-5
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DOI: https://doi.org/10.1007/s00344-023-11215-5