Introduction

Plants are sessile organisms that are unable to escape from unfavorable environmental conditions, such as cold, heat, drought and high salinity stresses. These environmental abiotic stresses can detrimentally affect plant growth and development and ultimately decrease crop productivity. Plants protect themselves from the detrimental effects of abiotic stresses by increasing the expression of a large number of stress-responsive genes. According to their functions during stress, the products of these genes can be classified into two groups (Hirayama and Shinozaki 2010). One group contains proteins involved in stress-inducible gene expression and further regulation of signal transduction, such as protein kinases and transcription factors. The other group includes proteins functioning in direct abiotic stress tolerance, such as osmotin, chaperones, late embryogenesis-abundant (LEA) proteins and detoxification enzymes.

LEA proteins are highly hydrophilic proteins that play a key role in the response to abiotic stresses in plants (Amara et al. 2012). Plant dehydrins (DHNs) are the group II (D-11 family) of the LEA proteins that are accumulated in the later stages of embryogenesis when the water content in seeds declines (Brini et al. 2007). The DHNs genes are expressed at high levels in response to low temperature, salinity or drought stresses (Szabala et al. 2014). They are believed to stabilize metabolism during cellular dehydration (Kosova et al. 2007), probably via stabilization of membranes by chaperone activity (Kovacs et al. 2008), hydrophobic interactions (Campbell and Close 1997; Koag et al. 2003) or antioxidant activity preventing excessive reactive oxygen species (ROS) formation (Hara et al. 2005; Sun and Lin 2010).

DHNs are classified according to their highly conserved lysine-rich domain (EKKGIMDKIKEKLPG), also known as the K-segment, which are present at between 1 and 11 copies near the C-terminus of all DHNs characterized to date (Close 1997; Svenson et al. 2002). The K-segment takes on an amphipathic α-helical structure and is believed to play a role in protein-lipid interactions (Close 1997). Many DHNs include a tract of phoshorylatable serine residues named the S-segment, and the consensus sequence (V/T) DEYGNP, known as the Y-segment, is sometimes present near the N-terminus. Besides these three conserved segments, a second less conserved DHN domain (the \(\Phi\)-segment) that is rich in polar amino acids may be present. Based on the number and order of these highly conserved segments, DHNs are categorized into five subgroups: YnSKn, SKn, Kn, YnKn and KnS (Allagulova et al. 2003; Rorat 2006).

In recent years, several studies have reported a positive correlation between the accumulation of DHN transcripts and/or proteins and tolerance to low temperature, drought and salinity stresses. Borovskii et al. (2002) reported that cold, freezing, drought or exogenous ABA treatment resulted in accumulation of thermostable DHNs in plant mitochondria. Moreover, cryotolerant species such as wheat and rye accumulate more DHNs than cryosensitive species such as maize. The acidic SK3 dehydrin DHN24 from Solanum sogarandinum is constitutively expressed, and its expression is associated with cold acclimation (Rorat et al. 2006). In addition, the peach Y2K9-type DHN gene ppdhn1 is induced by cold, drought or ABA, and the gene product exhibits cryoprotective and antifreeze activities (Artlip et al. 1997; Wisniewski et al. 2006). In blueberries (Vaccinium spp.), three DHN cold-responsive genes have been identified, and accumulation of their transcripts was positively correlated with cold hardiness in several different genotypes (Dhanaraj et al. 2005). Hara et al. (2003) isolated WCor410 that encodes a wheat dehydrin and introduced it into strawberry, which resulted in transgenic plants with enhanced tolerance against freezing and chilling stress.

Despite numerous studies on DHNs in various plants, pepper proteins have not been investigated extensively. In our previously studies, we characterized an EST (GenBank no: JZ198814) homologous to DHNs by differential screening of a previously reported cold-associated pepper seedling cDNA library (Guo et al. 2013). So in this study, based on the sequence of this EST sequence, the full-length ORF was obtained using the rapid amplification of cDNA ends (RACE) method, and the designated gene CaDHN1 showed tissue-specific expression in different organs in pepper plants. Moreover, the possible role of CaDHN1 in the response against cold, salt, osmotic and heavy metal stresses and plant hormone (abscisic acid and salicylic acid) signaling was investigated using RT-qPCR. Furthermore, CaDHN1 loss-of-function plants were engineered using a virus-induced gene silencing (VIGS) system (Liu et al. 2002; Chung et al. 2004; Wang et al. 2013). Together, the results suggested that CaDHN1 is a potentially important player in the regulation of plant defense responses.

Results

Cloning and sequence analysis of CaDHN1

A pepper cDNA clone (GenBank accession no. JZ198814) was selected from the differential screening of the previously characterized cold-associated pepper seeding cDNA library (Guo et al. 2013). Based on the sequence of the fragment, 3′- and 5′-RACE primers were designed and RACE was performed, which generated two fragments of 862 bp and 318 bp, respectively. Following alignment and assembly of these three sequences, the full-length cDNA was deduced, amplified by PCR, and confirmed by sequencing. The 1104 bp full-length cDNA contains a 660 bp ORF and encodes a 219 amino acid polypeptide (Fig. 1) with a calculated isoelectric point of 5.41 and a theoretical molecular mass of 24.7 kDa. This gene was designated as CaDHN1 and submitted to GenBank with accession No. JX402924.

Fig. 1
figure 1

Multiple sequence alignment of CaDHN1 with the amino acid sequences of Solanum tuberosum C17 cold stress dehydrin inducible protein, (GenBank accession no. AAB53203.1), Solanum sogarandinum 25 kDa dehydrin protein (GenBank accession no. AAp44575.1), Solanum peruvianum dehydrin protein (GenBank accession no. ADQ73992.1), Nicotiana tabacum dehydrin protein (GenBank accession no. BAD13499.1), Solanum commersonii dehydrin 2 protein (GenBank accession no. AAK66763.1). S and K motifs are underlined

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An NCBI blast search indicated that the deduced CaDHN1 amino acid sequence showed moderate homology with other dehydrin proteins from Solanum tuberosum (C17, cold stress dehydrin inducible protein, GenBank accession no. AAB53203.1, 84.9 % identity), Solanum sogarandinum (25 kDa dehydrin protein, GenBank accession No. AAp44575.1, 83.1 % identity), Solanum peruvianum (dehydrin protein, GenBank accession no. ADQ73992.1, 75.8 % identity), Nicotiana tabacum (dehydrin protein, GenBank accession no. BAD13499.1, 78.7 % identity), and Solanum commersonii (dehydrin 2 protein, GenBank accession no. AAK66763.1, 65.3 % identity).

Following comparison CaDHN1 with other members of the DHN family, CaDHN1 was shown to contain characteristic DHN motifs. There is one S-segment near the N-terminus and three K-segments near the C-terminus (Fig. 1). CaDHN1 belongs to the SKn-type dehydrin subfamily, and the K-segment closely matches that of the reported consensus sequence (EKKGIMDKIKEKLPG) with only a few conservative substitutions (Campbell and Close 1997).

In order to evaluate the molecular evolutionary relationships between CaDHN1 and other DHN proteins, a phylogenetic tree was generated using Mega5.1 (Fig. 2). Based on our phylogenetic results, the DHNs could be divided into five groups, corresponding to SKn-, YK-, YnSKn-, Kn-, and KS-type proteins. CaDHN1 falls into the SKn-type DHN protein family.

Fig. 2
figure 2

Phylogenetic tree of CaDHN1 and homologous DHN proteins from other species. The tree was constructed with MEGA 5.1 program using neighbor-joining method. Genbank accession numbers are as follows: CaDHN1 [JX402924], COR47 (At1g20440) [AY114699], ERD10 (At1g20450) [AF360351], At1g54410 [NM_104319], ERD14(At1g76180) [AF339722], At2g21490 [BT000900], XERO2(At3g50970) [NM_114957], XERO1(At3g50980) [NM_114958], At4g38410 [NM_120003], At4g39130 [NM_120073], RAB18(At5g66400) [AY093779], HvDhn8 [AF181458], HvDhn13 [AY681974], ShDHN [AHB20199.1], ScDHN [AK224734], SlDHN [M97211], SpDHN [ADQ73992], NtDHN [BAD13499], WCOR410 [XM_006647600], PvSR3 [U54703], BjDHN2 [DQ441470]

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Expression of CaDHN1 in response to abiotic stress and plant hormones

In order to investigate the expression pattern of CaDHN1 in different tissues of pepper cv P70 plants, RT-qPCR was performed on RNA from pepper roots, stems, leaves, flowers, immature fruits and seeds (Fig. 3). Clear differences were apparent, and expression of CaDHN1 was highest in fruits, followed by flowers and stems. However, CaDHN1 was expressed at very low levels in roots.

Fig. 3
figure 3

Tissue-specific expression of CaDHN1 in different tissues of pepper plants. Bars with different lower case letters in each group were significantly different, as determined using Duncan’s multiple range tests (p < 0.05)

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The transcription expression of CaDHN1 was further investigated by abiotic (cold, salt, osmotic, and heavy metal) stresses and abscisic acid (ABA), salicylic acid (SA) treatments in seedlings. When seedlings of pepper exposed to cold stress, the expression of CaDHN1 was not significantly affected in the initial stages (0–6 h) 6 °C treatments, but was upregulated threefold 12 h after treatment (Fig. 4a). The strongest response to cold treatment was highest at 24 h, at which point expression levels were elevated 15-fold. Similar to the results for cold stress, CaDHN1 expression was also significantly enhanced in pepper leaves following salt and osmotic stress (Fig. 4b, c). By salt stress, the transcript of CaDHN1 was dramatically increased with a 15-fold increase at 3 h and reached a peak of 25-fold at 6 h, then subsequently decreased to a 7-fold elevation by 24 h. By osmotic stress, expression of CaDHN1 was strongly and rapidly upregulated with a sixfold elevation at 1 h after a 0.3 M mannitol treatment. Upregulation continued and reached tenfold by 3 h, before diminishing slightly. By 24 h, expression of CaDHN1 was again increased by 13-fold. However, in this study, expression of CaDHN1 was not significantly affected by heavy metal stress (Fig. 4d).

Fig. 4
figure 4

RT-qPCR analysis of CaDHN1 expression in the leaves of pepper plants following abiotic stresses and plant hormone treatments. The results are the mean ± standard error (SE), replicated thrice. Different lower case letters indicates significant difference when compared with the control at a p value <0.05

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To investigate the possible involvement of CaDHN1 in defense-associated signaling, expression was analyzed in pepper leaves treated with 0.57 mM ABA or 5 mM SA using RT-qPCR. CaDHN1 expression was not significantly changed between control plants and following exogenous ABA treatment plants (Fig. 4e). Following treatment with 5 mM SA, however, the transcription levels of CaDHN1 were strongly increased by 25-fold at 1 h, before expression levels fell sharply and were maintained at low levels between 3 and 12 h (Fig. 4f). By 24 h, expression increased again and peaked at 28-fold.

Suppression of CaDHN1 diminishes tolerance to abiotic stresses

To examine further the role of CaDHN1 in the response to cold, salt and osmotic stress, virus-induced gene silencing (VIGS) was performed on pepper cv P70 using the tobacco rattle virus (TRV)-induced gene silencing technique (Wang et al. 2013; Chen et al. 2014). A fragment from the 3′ end of the CaDHN1 ORF was cloned into the pTRV2 vector to generate the pTRV2:CaDHN1 construct (empty pTRV2:00 was used as a negative control). The pTRV2:CaPDS construct silences the phytoene desaturase (PDS) gene and induces a bleaching phenotype (about 70 percent plants have these phenotype), and was used as a positive control to determine the success of gene silencing (Fig. 5a).

Fig. 5
figure 5

VIGS of CaDHN1 in pepper. a Schematic representation of the pTRV1, pTRV2, pTRV2:CaPDS and pTRV2:CaDHN1 constructs. b Phenotypes of gene-silenced pepper plants. (1), control (pTRV2:00); (2), CaPDS-silenced (pTRV2:CaPDS); (3), CaDHN1-silenced (pTRV2:CaDHN1). c Phenotypes of gene-silenced pepper plants after 24 h of 6 °C cold treatments. (1), control (pTRV2-00); (2), CaDHN1-silenced (pTRV2:CaDHN1). d RT-qPCR analysis of CaDHN1 expression in gene-silenced (pTRV2:CaDHN1) and control (pTRV2:00) plants 35 days after inoculation. e Effects of low temperature stress on MDA, chlorophyll (f) and EC1/EC2 (g) in CaDHN1-silenced pepper seedlings. Asterisk indicates significant differences compared with the control at a p value <0.05

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At 35 days after the induction of TRV-mediated gene silencing, pepper plants infiltrated with pTRV2:CaDHN1 showed no visible phenotypic differences from negative control plants (Fig. 5b), but bleaching was evident on the leaves of CaPDS-silenced plants. To verify that the CaDHN1 transcript was effectively downregulated by VIGS, RT-qPCR was carried out (Fig. 5c). CaDHN1 expression was dramatically reduced by 70 % in the new leaves of silenced pepper plants grown at 22 °C under non-stress conditions, suggesting that VIGS was successful and effective for CaDHN1 gene silencing.

Under 24 h 6 °C cold stress, the pTRV2:CaDHN1 plants aggravated the visible symptoms of leaf damage in seedlings. There was more seriously wilting appeared in pepper seedlings in pTRV2:CaDHN1 plants than that of pTRV2:00 plants (Fig. 5c). In order to confirm the influence of silencing of CaDHN1 in the cold stress defense response, malondialdehyde (MDA), chlorophyll and electrical conductivity were measured in control and silenced pepper plants. After 24 h of 6 °C cold treatments, the MDA and electrical conductivity measurements were both significantly increased, while chlorophyll levels were markedly decreased in CaDHN1-silenced plants (Fig. 5e–g).

To determine whether the silencing of CaDHN1 led to reduced tolerance to salt and osmotic stresses, leaf discs from empty vector control (pTRV2:00) and CaDHN1-silenced (pTRV2:CaDHN1) plants were exposed to 300 mM NaCl and 300 mM mannitol solution, respectively, with continuous lighting for 3 days (Fig. 6). CaDHN1-silenced leaf discs were more yellow than those of control plants, and some were blackened after 3 days of salt treatment, and the same was true of mannitol-treated plants. Chlorophyll levels were also significantly reduced in CaDHN1-silenced plants compared with controls.

Fig. 6
figure 6

Reduced tolerance of CaDHN1-silenced pepper plants to salt and mannitol stress. a Phenotypes; b chlorophyll content in leaf discs of 3 days after NaCl and c mannitol treatments in gene-silenced and control plants. The results are the mean ± standard error (SE), replicated thrice. Asterisk indicates significant differences compared with the control at a p value <0.05

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To elucidate the mechanism of the reduced tolerance to cold stress in CaDHN1-silenced plants, the expression patterns of cold stress- and antioxidant system-related genes (KIN, a cold-induced gene, Mn-SOD, POD) were monitored in control (pTRV2:00) and CaDHN1-silenced (pTRV2:CaDHN1) plants by RT-qPCR (Fig. 7). The result indicated that cold stress induced Mn-SOD, POD and KIN1 expression in both control and CaDHN1-silenced plants (Fig. 7). However, after cold treatment, expression of these genes in CaDHN1-silenced plants was noticeably lower than in control plants.

Fig. 7
figure 7

RT-qPCR was used to assess the transcript levels of cold stress- and antioxidant system-relative genes (KIN1, POD and Mn-SOD) in control and CaDHN1-silenced plants subjected to cold stress. Results are the mean ± standard error (SE), replicated thrice. Asterisk indicates significant differences when compared with the TRV2:00 leaves at a p value <0.05

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Discussion

In this study, we isolated a dehydrin gene from Capsicum annuum leaves, designated CaDHN1. Amino acid alignment of this gene has the DHN-specific and conserved K- and S-domain and belongs to the SKn-type dehydrin subfamily. Tissue distribution expression indicated that CaDHN1 was expressed in all tested tissues and high level in fruits (Fig. 3). The relatively high expression in fruits may be due to the fruits have the developing seeds which contain high levels of LEA family transcripts and proteins, and DHNs belong to the LEA group II gene family (Chung et al. 2003).

Temperature stress is one of the most important abiotic factors limiting the growth, development and geographical distribution of plants (Nguyen et al. 2009), and the expression of many DHNs is altered in response to low or high temperature stress. In this study, expression of CaDHN1 was upregulated under cold stress (6 °C treatments) (Fig. 4a) and reached a peak of 5-fold at 48 h. This expression pattern was reminiscent of the findings of Qiu et al. (2014) who found that the expression of SiDhn2, a DHN protein-encoding gene from Saussurea involucrata, was increased under low temperature stress conditions. After low temperature treatment for 6 h, expression of SiDhn2 was enhanced 3-fold compared to controls. In another study, five DHN/LEA/RAB (dehydrin, late embryogenesis-abundant, responsive to ABA)-related genes (COR47, DHNX, LTI30, LTI45 and RAB18) from Arabidopsis thaliana exhibited different expression levels in response to chilling stress, and LTI30, LTI45 and COR47 primarily responded to low temperature stress (Welin et al. 1994). However, expression of RABl8 was strongly induced both in water-stressed and ABA-treated plants, but was only slightly responsive to cold stress. Together, these results suggest that different DHN genes exhibit different expression patters under cold stress conditions.

Many DHNs are upregulated in response to salt and osmotic stress, and transgenic plants overexpressing exogenous DHNs are highly tolerant to this abiotic stress (Brini et al. 2007; Santos and Mazzafera, 2012). In this study, expression of CaDHN1 was strongly and rapidly upregulated by salt and osmotic stresses (Fig. 4b, c). The result is in accordance with previous studies on the DHNs PpDHNA and PpDHNB from Physcomitrella patens, which were strongly upregulated by NaCl and mannitol treatment (Ruibal et al. 2012). Choi and Close (2000) found that the barley dehydrin DHN3 was induced by drought, ABA and salt treatment, whereas expression of DHN12 was embryo-specific and not induced by dehydration, cold or salt stress, or ABA treatment. These results suggested a possible role for CaDHN1 in response to salt and osmotic stress in pepper.

Some Dehydrin gene transcript could be induced by heavy metals. For example, the Brassica juncea dehydrin genes BjDHN2 and BjDHN3, which were both upregulated by heavy metals, and transgenic tobacco plants overexpressing BjDHN2/BjDHN3 exhibited enhanced tolerance of heavy metal stress (Xu et al. 2008). Similarly, Zhang et al. (2006) isolated the heavy metal-inducible SK2-type dehydrin gene PvSR3, and found that PvSR3 was strongly induced by heavy metals. However, in this study, expression of CaDHN1 was not significantly affected by heavy metal stress (Fig. 4d). These results suggest that different DHNs may have different functions, and pepper CaDHN1 may not be involved in response to heavy metal stress.

The plant hormone ABA plays a key role in a broad array of developmental processes and adaptive stress responses to environmental stimuli (Cutler et al. 2010; Fujita et al. 2011). SA acts as an important endogenous signaling molecule, and is involved in establishing the local and systemic disease response to pathogen attack, as well as in the induction of defense-related genes in response to abiotic stress (Chini et al. 2004; Li et al. 2004). Numerous dehydration-responsive genes including DHNs have been identified, and many are induced by exogenous ABA and SA application (Yamaguchi-Shinozaki and Shinozaki 2006; Ochoa-Alfaro et al. 2012). However, in this study, CaDHN1 expression was slightly lower than that of control plants following exogenous ABA treatment, but rapidly and strongly increased by SA treatment (Fig. 4e, f). This was consistent with a previous study in which PvSR3, encoding an acidic DHN from bean (Phaseolus vulgaris L), was not responsive to ABA, but strongly induced by SA (Zhang et al. 2006). These results indicated that CaDHN1 may operate through ABA-independent but SA-dependent signaling pathways.

It is well documented that the primary site of stress injury in plants is the cell membrane, and electrolyte leakage is a typical symptom of stress-associated injury following membrane damage causing expansive lysis or phase transitions (Thomashow 1999; Xu et al. 2008). Additionally, dehydration has been suggested to cause decreased membrane fluidity and lipid peroxidation, which can be estimated from the MDA content. MDA and electrical conductivity measurements are established indicators of the extent of cell membrane injury and electrolyte leakage, which are known to correlate well with the severity of the visual damage index (Li et al. 2011). Similarly, chlorophyll levels can be used to estimate the degree of leaf senescence. In this study, cold stress significantly increased MDA levels and electrical conductivity, and reduced the chlorophyll levels in CaDHN1-silenced plants. Addition, leaf discs were more yellow and chlorophyll levels were also significantly reduced in CaDHN1-silenced plants than those of control after 3 days of salt and osmotic treatment. This suggests loss of CaDHN1 reduced the tolerance of pepper seedlings to cold, salt and osmotic treatment and accelerated leaf senescence, indicating a potential role for CaDHN1 in resistance to these abiotic stresses.

Moreover, in plant cells, cold stresses induce the production of the reactive oxygen species (ROS). Cell membrane lipid peroxidation is mainly induced by ROS and/or free radicals. To protect cell membrane during oxidative stresses, plants possess very efficient enzymative antioxidant defense systems, such as SOD, POD (Choudhury et al. 2013; Liu et al. 2015). Meanwhile, earlier studies found KIN1, a cold-induced gene, has similar functions to fish antifreeze proteins (AFPs) genes (Kurkela and Franck 1990). In this study, we found that knock-down of CaDHN1 suppressed the expression of Mn-SOD, POD and KIN1genes transcripts (Fig. 7). These changes maybe lead to more ROS accumulation in the gene knock-down lines than the wild-type under stress conditions and this result indicated that CaDHN1 may act as a positive regulator of cold stress-responsive gene expression, consistent with the results of the leaf chilling assays described above.

Experimental section

Plant materials and growth conditions

Pepper cv P70 was provided by the pepper breeding group in Northwest A&F University, China. Seeds were treated with warm water (55 °C) for 20 min to promote germination, then placed on moist gauze in an incubator at 28 °C, 60 % relative humidity in darkness and rinsed twice per day. When seeds were at least 80 % germinated, they were sown in pots containing compost. Pepper seedlings were grown in a growth chamber with temperature conditions at a 25/20 °C day/night temperature cycle, 75 % relative humidity, and a 16 h light and 8 h dark photoperiod cycle.

Bacteria Strains, Vectors and Restriction Enzymes

pTRV2 (Liu et al. 2002; Chung et al. 2004) and Vector pMD19-T (TaKaR, Dalian, China) were used to engineer constructs, and Agrobacterium tumefaciens strain GV3101 was used for subsequent plant transformation. Pfu DNA polymerase (Promega Corporation, Madison, WI, USA) was used for PCR, and products were subsequently sequenced. Xba I and BamH I restriction enzymes and T4 DNA ligase (TaKaR, Dalian, China) were used for vector construction.

Cloning of CaDHN1 Gene and Sequence Analysis

The DHN-homologous EST R031 (GenBank accession no.: JZ198814) characterized from the differential screening of a cold-related pepper seedling cDNA library was previously reported (Guo et al. 2013). The full-length CaDHN1 ORF was obtained using the rapid amplification of cDNA ends (RACE) method. First-strand cDNA synthesis was performed using the Smart RACE cDNA amplification kit (Clontech, Mountain View, CA, USA). Gene-specific primer GSP1 (5′-ACTTCCTACAATATTCACGAC-3′) was used for 3′-RACE and GSP2 (5′-GGAATGAATGTTTATTTGTTG-3′) was used for 5′-RACE. Universal primers for 5′ and 3′ RACE were provided in the kit. The full-length cDNA sequence of CaDHN1 was obtained by PCR amplification using forward (5′-GTCAAAACTTTTACTTTAGTGATCAT-3′) and reverse (5′-GAAATCAAAATCACTGCATTTTAC-3′) primers. PCR products were cloned into the pMD19-T vector (TaKaR, Dalian, China) and sequenced (Shanghai GeneCore Biotechnologies Co. China). The sequence analysis was performed as described by Chen et al. (2014).

CaDHN1 Gene Expression Patterns Analysis

Tissue-specific Expression of CaDHN1 Gene

To evaluate the expression levels of CaDHN1 in different tissues under normal conditions, roots, stems, leaves, flowers, fruits and seeds were collected from pepper cv P70 plants, frozen in liquid nitrogen and stored at −80 °C until needed for gene expression analysis.

Stress treatments

Pepper plants at the sixth leaf expansion stage were treated with abiotic stress and plant hormone. The abiotic stress (cold, salt, osmotic and heavy metal) and plant hormone (ABA and SA) treatments were performed as described previously (Chen et al. 2014). After 0, 1, 3, 6, 12 and 24 h treatment, pepper leaves were harvested for examination of CaDHN1 expression pattern under various stress conditions. At each time point, three or four upper young leaves from four separate seedlings were collected to form one sample, wrapped with aluminum foil, immediately frozen in liquid nitrogen and stored at −80 °C.

Isolation of RNA and RT-qPCR analysis

RNA isolation, cDNA preparation and RT-qPCR were performed as described by Chen et al. (2014). The relative fold difference in mRNA levels was determined using the 2−ΔΔCT method. The CaUbi3 gene (GenBank accession no. AY486137.1) encoding the ubiquitin-conjugating protein was amplified from pepper plants as a reference gene for normalization of CaDHN1 cDNA samples (Wan et al. 2011). The cold stress- and antioxidant system-related genes-specific gene primers (Mn-SOD, POD, and KIN1) are presented in Table 1.

Table 1 Primer sequences used for RT-qPCR analysis
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Virus-induced gene silencing (VIGS) assay of CaDHN1 in pepper

The pTRV2: CaDHN1 construct was engineered to include a 338 bp fragment of CaDHN1 cloned from a pepper cDNA template using gene-specific forward (5′-GGAATGAATGTTTATTTGTTG-3′) and reverse (5′-CGCGGATCCGAAATCAAAATCACTGCATT-3′) primers containing a BamH I restriction site (underlined). The resulting PCR product was cloned into vector pMD19T (Takara, Dalian, China), the resultant construct was digested with XbaI and BamH I, and the CaDHN1 fragment was inserted into the XbaI–BamH I site of pTRV2 to form pTRV2:CaDHN1. Agrobacterium tumefaciens GV3101 containing either pTRV1 or pTRV2:CaDHN1 were injected into pepper and plants were grown as described by Wang et al. (2013). 50 plants were used for the silencing assay.

MDA, electrolyte leakage and chlorophyll level assay

The amount of lipid peroxidation in the chloroplast membranes was estimated by measuring MDA produced by the thiobarbituric acid reaction as described previously by Dhindsa et al. (1981). To assess membrane permeability, electrolyte leakage was measured according to the method described by Dionisio-Sese and Tobita (1998). Total chlorophyll was extracted from the treated samples and calculated by a spectrophotometric method as described by Arkus et al. (2005).

Statistical Analysis

RT-qPCR data analysis was carried out using Sigma Plot 10. The relative expression levels of DHN under various abiotic stress and hormonal treatments are mean ± SE of three biological replicate samples. Each replicate sample was a composite of leaves from three individual seedlings. For each treatment, the expression level at time point 0 was defined as 1.0. Statistical analysis were performed using the Statistical Analysis System software (SAS 8.2, North Carolina State University, Cary, NC, USA), and the means were compared using Duncan’s multiple range test, taking p < 0.05 as a significant difference. The values of MDA, electrolyte leakage and total chlorophyll are mean ± SE of three biological replicate samples. Each replicate sample was a composite of leaves from nine seedlings.

Conclusion

In summary, we isolated the pepper DHN gene CaDHN1 from the leaves of pepper cv P70. CaDHN1 transcription differed in different tissues, and CaDHN1 expression was strongly induced by high salt, osmotic and cold stress. Moreover, CaDHN1 expression was significantly upregulated by SA treatment but not by ABA. Virus-induced gene silencing (VIGS) revealed that mutant plants lacking CaDHN1 were more susceptible to low temperature, high salt and mannitol treatment. Overall, these results suggest that CaDHN1 plays an important role in resistance to abiotic stresses in pepper plants. Genetic transformation studies are needed to further explore the function of CaDHN1.

Author contribution statement

RGC, HJ, WLG, and ZHG conceived and designed the experiments; RGC, WLG, HJ and FM performed the experiments; SBW, BGP and WPD analyzed the data; ZHG contributed reagents/materials/analysis tools; RGC wrote the paper. All authors read and approved the final manuscript.