Cloning and expression analysis of CaPIP1-1 gene in pepper (Capsicum annuum L.).

Gene 563 (2015) 87–93

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Research paper

Cloning and expression analysis of CaPIP1-1 gene in pepper (Capsicum annuum L.) Yan-Xu Yin a,1, Shu-Bin Wang b,1, Huai-Xia Zhang a, Huai-Juan Xiao a, Jing-Hao Jin a, Jiao-Jiao Ji a, Hua Jing a, Ru-Gang Chen a, Mohamed Hamed Arisha a,c, Zhen-Hui Gong a,⁎ a b c

College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, P. R. China Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, P. R. China Horticulture Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt

a r t i c l e

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Article history: Received 29 October 2014 Received in revised form 16 January 2015 Accepted 5 March 2015 Available online 11 March 2015 Keywords: Capsicum annuum L. CaPIP1-1 Gene expression Abiotic stresses Biotic stress

a b s t r a c t Plant aquaporins are responsible for water transmembrane transport, which play an important role on abiotic and biotic stresses. A novel plasma membrane intrinsic protein of CaPIP1-1 was isolated from the pepper P70 according to transcriptome databases of Phytophthora capsici inoculation and chilling stress library. CaPIP1-1, which is 1155 bp in length with an open reading frame of 861 bp, encoded 286 amino acids. Three introns, exhibited CT/AC splice junctions, were observed in CaPIP1-1. The numbers and location of introns in CaPIP1-1 were the same as observed in tomato and potato. CaPIP1-1 was abundantly expressed in pepper fruit. Increased transcription levels of CaPIP1-1 were found in the different stresses, including chilling stress, salt stress, mannitol stress, salicylic acid, ABA treatment and Phytophthora capsici infection. The expression of CaPIP1-1 was downregulated by 50 μM HgCl2 and 100 μM fluridone. The pepper plants silenced CaPIP1-1 in cv. Qiemen showed growth inhibition and decreased tolerance to salt and mannitol stresses using detached leaf method. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Water is indispensable to life, and sufficient water supply is also essential for plants to survive. For horticultural crops, water supply has become an important restriction factor of high yield. About 70% of water was consumed on agriculture every year, according to statistics of UNFAO (Reuscher et al., 2013). In recent years drought has become more and more serious in Chinese agriculture, especially in the northwest area, in which drought even ruins the crop up. Water supply has been studied extensively during the last few years. Water transport in different tissues mainly relies on plasma membrane intrinsic proteins (PIPs) (Zhou et al., 2007). Chaumont found that plant aquaporins (AQPs) play an important role in water transmembrane transport and improving efficiency of water use (Steudle, 2000; Chaumont et al., 2001). Further research revealed that the AQPs are not only responsible for water transport, but also transport some solutes across cell membranes (Soto et al., 2012). The AQP1 in animal cells can transport CO2 in Xenopus oocyte, which may be controversial. Moreover, Abbreviations:AQPs, aquaporins; ESTs, expressed sequence tags; hpi, hour post inoculation; MDA, malondialdehyde; PIPs, plasma membrane intrinsic proteins; qRT-PCR, quantitative real-time PCR; RACE-PCR, rapid amplification of cDNA ends PCR; RWC, relative water content; SA, salicylic acid; SE, standard error; SSH, suppression subtractive hybridization; VIGS, virus induced gene silencing. ⁎ Corresponding author. E-mail address: [email protected] (Z.-H. Gong). 1 These authors contributed equally to this study.

http://dx.doi.org/10.1016/j.gene.2015.03.012 0378-1119/© 2015 Elsevier B.V. All rights reserved.

Uehlein et al. found that transmembrane transport CO2 in tobacco is possible (Uehlein et al., 2003). Now more and more evidences show that AQPs also play a critical role in plant regeneration, cell elongation, opening stomata, fruit ripening and seed germination (Forrest and Bhave, 2007). Besides, AQPs have a positive effect on abiotic and biotic stresses of plants. Sreedharan et al. had reported that content of malondialdehyde (MDA) was lower compared with controls, but relative water content (RWC), proline content and photosynthetic efficiency were higher in transgenic MusaPIP1;2 banana under abiotic stress. Furthermore, the transgenic lines also showed better recovery ability after different abiotic stresses (Sreedharan et al., 2013). Results of Siefritz et al. showed that inhibition of NtAQP1 expression can decrease root hydraulic conductivity and result in lower capability of plant tolerance to water stress. All these results demonstrated the important role of AQPs in the water supply by the symplast pathway in plants (Siefritz et al., 2002). Yu et al. found that OsPIP1-1 and OsPIP2-1 might play a crucial role in recovery of water balance after chilling stress (Yu et al., 2006). In recent years, many researchers have revealed the positive effect of PIPs, Aharon et al. found that PIP1b could enhance the vitality of transgenic tobacco under favorable growth condition, while it had some negative influence on plants under salt and drought stresses (Aharon et al., 2003). The activity of PIP1s subfamily was less than that of PIP2s in Xenopus oocyte (Jang et al., 2004), which might result from different gene structures, or the regulation of phosphorylation. In our previous study, we obtained some stress-related expressed

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sequence tags (ESTs) from the suppression subtractive hybridization (SSH) cDNA library (Guo et al., 2013) and the transcriptome after Phytophthora capsici infection, combined with the database of gene expression under chilling stress (Hwang et al., 2005). Then we selected stress-related EST and cloned the full-length of CaPIP1-1 (GenBank accession no. JX402929) by rapid amplification of cDNA ends PCR (RACE-PCR). So far there were few reports on the expression characterization under stress and function of PIPs in pepper. In this study, we analyzed expression patterns of CaPIP1-1 gene under different stresses by quantitative real-time PCR (qRT-PCR). Additionally, we also reported the function of CaPIP1-1 under osmotic stresses with virus induced gene silencing (VIGS) method. This study will certainly contribute to explore the roles of PIPs genes in abiotic and biotic stresses.

and Kpn I. Agrobacterium tumefaciens strain GV3101 harboring pTRV1 was respectively mixed with pTRV2 (as the negative control), TRV2CaPDS (as the positive control) or TRV2-CaPIP1-1 at 1:1 ratio, then, the mixtures were inoculated into the fully expanded cotyledons of the cv. Qiemen. After injection, all the seedlings were kept at 18 °C and 60% relative humidity for 2 days, and then cultured in a growth chamber according to the protocol. The gene-silenced leaf discs were used for 0.1 M salt or 0.3 M mannitol stress. The special primers (CaPIP1-1F4 and CaPIP1-1R4, Supplemental Table 1) near the 5′-end were used to determine CaPIP1-1 expression levels in silenced pepper. The PCR product was sequenced in Sangon Biotech Company (Shanghai, China).

2. Materials and methods

Statistical analysis was performed using Statistical Analysis System software (SAS 8.2, North Carolina State University, USA), and the mean separation was analyzed using the Duncan's multiple range test, taking p b 0.05 as a significant difference. Values were expressed as the mean ± standard error (SE). All experiments were performed and analyzed separately with three biological replicates.

2.1. Plant materials and seedling treatment Seeds of Capsicum annuum L. cv. P70 and Qiemen were used in this study. The seedlings were grown in a growth chamber at 25 °C with a 16 h light/8 h dark photoperiod cycle. Different stress [chilling stress, salt stress (0.15 M NaCl), mannitol stress (0.3 M), ABA treatment (100 μM), 5 mM salicylic acid (SA), 50 μM HgCl2 and 100 μM fluridone] treatments were performed as previously described (Yin et al., 2014a). Leaves were harvested at 0, 1, 3, 6, 12 and 24 h, quickly frozen with liquid nitrogen and stored at −80 °C. Pepper plants were used for P. capsici infection (HX-9 strain) at the six-true-leaf stage with root-drenching method, and as described by Wang et al., the control plants were inoculated treated with sterile distilled water. All the infection plants were cultured according to the protocols (Wang et al., 2013b). The P. capsici-infected pepper leaves and roots were collected at 0, 6, 12, 24, 48 and 72 h after inoculation and stored at −80 °C for RNA isolation. 2.2. Cloning the CaPIP1-1 gene in pepper The EST of transcriptome databases of P. capsici inoculation was used as the query probe to search against the pepper EST database at GenBank. According to the nucleotide sequences (GenBank accession no. GO345801, GD065213, BM064594 and CK901684), primers of CaPIP1-1F and CaPIP1-1R (Supplemental Table 1) were designed to identify if they come from the same gene. After that, the special gene primers were designed to clone the 5′ and 3′ DNA sequence using Smart RACE cDNA amplification kit. The primers of CaPIPF3 and CaPIPR3 were used to clone the full cDNA of the pepper CaPIP1-1 gene with pfu polymerase.

2.5. Statistical analysis

3. Results 3.1. Cloning, sequence character and structural analysis of the CaPIP1-1 gene in pepper 3.1.1. Cloning of CaPIP1-1 gene The cDNA of cultivar “P70” was used as template in the cloning of CaPIP1-1 gene. Firstly we predicted a 1100 bp fragment from the comparative analysis of transcriptome databases of P. capsici inoculation and chilling stress cDNA library. Then a 611 bp 5′-end fragment was cloned by 5′ RACE and a 590 bp 3′-end cDNA sequence was amplified by 3′ RACE. Finally, a full-length cDNA, designated CaPIP1-1 and consisted of 1155 bp, was assembled using ContigExpress software and verified by sequencing the fragment with primers designed according to the assembled sequence (Supplemental Table 1). It is predicted that CaPIP1-1 encoded 286 amino acids (Supplemental Fig. 1). Because of the high homology to Arabidopsis PIP1 family, we named this gene CaPIP1-1 (Fig. 1). 3.1.2. Analysis of phylogenetic and transmembrane structure of CaPIP1-1 The amino acid sequence alignment of PIPs in different plants, including tomato, potato, tobacco, Arabidopsis and petunia, is shown in

2.3. RNA isolation and quantitative real-time PCR analysis Reverse transcription was performed using the Primescript™ first strand cDNA Synthesis Kit (TaKaRa, Dalian, China). qRT-PCR was performed as described by Guo et al. (2012). qRT-PCR cycling conditions were as follows: 95 °C for 1 min and 45 cycles of 95 °C for 15 s, 52 °C for 20 s and 72 °C for 30 s. Fluorescence data were collected during the 52 °C step. The ubiquitin-conjugating protein (CaUbi3, accession no. AY486137.1), actin mRNA (CaActin2, accession no. AY572427) and translation elongation factor 1α (CaTEF1A, accession no. AF242732) were used as pepper reference genes (Wan et al., 2011; Wang et al., 2012). 2.4. VIGS assay of CaPIP1-1 in pepper plants The TRV-based VIGS system was used for gene silencing as described previously (Wang et al., 2013a). To generate the CaPIP1-1/TRV2 construct, a 309 bp fragment near the 3′-end of the CaPIP1-1 gene was PCR amplified from pepper. The resulting product was cloned into TRV2 vector using the double digested method with enzymes of Xba I

Fig. 1. Phylogenetic comparison of CaPIP1-1 (CaPIP) with PIP proteins from Arabidopsis. Phylogenetic tree generated using MEGA5.0 program. The rooted gene tree (majorityrule consensus from 1000 bootstrap replicates) resulted from heuristic searching in MEGA5.0. The amino acid sequences of 13 PIPs are from the Arabidopsis genome database. GenBank accession no. is in parentheses after each gene name. The asterisk indicates the CaPIP1-1 (CaPIP).

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Supplemental Fig. 2. CaPIP1-1 protein shared high homology to PIPs in other Solanaceae plants, including NtAQP1 (similarity 93%, accession no. AAB81601 or CAA04750), PhPIP (95%, accession no. AF452010) and StPIP (95%, accession no. ABJ97677) except for AtPIP (58%, accession no. At3g61430). Furthermore, the protein structure of CaPIP1-1 was predicted by online software of TMHMM Server2.0. Transmembrane domains (TM1–TM6) were predicted as shown in Supplemental Fig. 2. 3.1.3. Analysis of the CaPIP1-1 structure Analysis revealed that three introns existed in the CaPIP1-1 and PIP1s of tomato and potato. But these introns were different in size (Table 1). The size of CaPIP1-1 introns was similar to that of StPIP1-3. Interestingly, all introns exhibited CT/AC or GT/AG splice junction's model, but only one splice junction's model was shown in the same gene. 3.2. Expression patterns of CaPIP1-1 in different tissues The expression of CaPIP1-1 in different tissues had significant differences (pb 0.05) (Fig. 2). CaPIP1-1 transcripts were detectable in all tested tissues, including roots, stems, leaves, flowers, fruits and seeds. The highest expression level of CaPIP1-1 was detected in the fruits, which was 26-fold (Fig. 2A) or 54-fold (Fig. 2B) than that in the seeds. The CaPIP1-1 expression level in the roots was lower than in the leaves and flowers, but there were non-significant differences. CaPIP1-1 transcripts in the stems were slightly higher than in the seeds. The smaller differences observed estimation using CaUbi3 than CaActin2 reference genes. 3.3. Expression analysis of CaPIP1-1 in response to abiotic stresses 3.3.1. Expression analysis of CaPIP1-1 under HgCl2 stress The expression of CaPIP1-1 gene in roots and leaves was suppressed at different levels after treating roots with HgCl2 (50 μM). Meanwhile the inhibition of CaPIP1-1 expression in roots is higher than that in leaves (Fig. 3). CaPIP1-1 transcription levels were not nearly detected in roots at 1 h after HgCl2 treatment, while the gene expression in leaves reduced to 60% of control (Fig. 3A). After treatment for 6 h, CaPIP1-1 expression in leaves nearly came back to the same level as control, while in roots it is still lower (Fig. 3A). During the HgCl2 treatment period, the expression level of CaPIP1-1 in leaves was lower than control (0 h), and in roots it was stable but also lower than control. The inhibition of CaPIP1-1 expression in roots was relatively larger, and the recovery of CaPIP1-1 expression was not significant. Relative amounts recalculated to that of CaUbi3 were higher than that of CaTEF1A. 3.3.2. Effect of chilling, ABA and fluridone stresses on CaPIP1-1 gene expression Under the stresses of chilling, ABA and fluridone, the expression of CaPIP1-1 was regulated by endogenous rhythm. Its expression was increased firstly, then reduced and tended to be stable at the last in

Fig. 2. Expression of CaPIP1-1 in different tissues of pepper plants. Relative transcription of CaPIP1-1 in different tissues of pepper was analyzed using real-time quantitative RT-PCR. Relative amounts were recalculated to that of CaUbi3 (A) or CaActin2 (B). The results represent the mean ± SE from three independent biological experiments. Bars with different lower case letters indicate significant differences using Duncan's multiple range test (pb 0.05).

the photoperiod stage, with control treatment (with water) as shown in Fig. 4. Compared with the control, CaPIP1-1 expression was upregulated with chilling and ABA stresses but downregulated with fluridone stress. There was an increase under the stresses of both ABA and chilling followed by ABA. However, there were significant differences between the expression patterns regulated by ABA and chilling followed by ABA. Up-regulation mediated by ABA firstly significantly increased, then decreased, and finally increased (Fig. 4A). However the upregulation mediated by chilling followed by ABA was on the contrary. Expression of CaPIP1-1 recalculated to that of CaActin2 showed upregulation and then down-regulation (Fig. 4B). 3.3.3. Effect of mannitol, salt and SA stresses on gene expression of CaPIP1-1 Under mannitol, salt and SA stresses, CaPIP1-1 gene expression was up-regulated in the case of using water treatment as control. The gene expression pattern was firstly up-regulated and then down-regulated (Fig. 5). With mannitol treatment, the transcription level of CaPIP1-1 reached a maximum of 4-fold at 3 h higher than that of control. Then the CaPIP1-1 transcript started downregulation, which was 5.2 fold higher than that of the control at 24 h (Fig. 5A). Expression patterns of CaPIP1-1 under salt stress were similar to that under ABA treatment, the expression of CaPIP1-1 firstly significantly increased, then sharply

Table 1 Comparison of intron lengths and numbers of PIP1s in Solanaceae. Genes and accession no.

Introns, donor and acceptor splice sites Number 1

Number 2

Number 3

SlPIP1-1a (AB845604) SlPIP1-2a (AB845605) StPIP1-1b (PGSC0003DMG400004980) StPIP1-2b (PGSC0003DMG400005780) StPIP1-3b (PGSC0003DMG400012337) StPIP1-4b (PGSC0003DMG400020742) StPIP1-5b (PGSC0003DMG400000045) CaPIP1-1 (JX402929)

GT/AG (1383 bp) GT/AG (947 bp) GT/AG (486 bp) CT/AC (618 bp) CT/AC (118 bp) CT/AC (159 bp) GT/AG (939 bp) CT/AC (109 bp)



a b

Named by Reuscher et al. (2013). Named by Venkatesh et al. (2013).

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Fig. 3. Negative effects of HgCl2 on CaPIP1-1 expression in pepper. Relative expression of CaPIP1-1 in the leaves and roots of pepper plants at 0, 1, 3, 6, 12 and 24 h after treatment of 50 μM HgCl2 was detected. Relative amounts were recalculated to that of CaUbi3 (A) or CaTEF1A (B). Bars with different lower case letters in each group indicate significant differences using Duncan's multiple range test at pb 0.05. Three biological experiments were performed, which produced similar results.

decreased, and ultimately increased again followed by a decreased trend (Figs. 4A, 5A). While compared with the control, CaPIP1-1 transcription level was up-regulated (Fig. 5). After SA treatment, the

Fig. 5. Expression of CaPIP1-1 in pepper under salt, mannitol and SA stresses. Relative expression of CaPIP1-1 was calculated in pepper plants when exposed to 0.15 M NaCl, 0.3 M mannitol and 5 mM SA at 0, 1, 3, 6, 12 and 24 h. Relative amounts were recalculated to that of CaUbi3 (A) or CaTEF1A (B). Bars with different lower case letters in each group indicate significant differences, as determined using Duncan's multiple range test at pb 0.05. Three biological experiments were performed, which produced similar results. Water (untreated) indicates the control.

CaPIP1-1 transcript firstly increased, then decreased, and finally increased again. 3.4. Effect of CaPIP1-1 gene expression in response to biotic stress Pepper cv. P70 was inoculated with the virulent P. capsici HX-9 stain. Compared with the mock control, the CaPIP1-1 transcript in the leaves was upregulated and reached 2-fold at 1 h, and peaked (4-fold) at 3 h, which was significantly higher than that of the control (p b 0.05). After inoculation for 72 h, the CaPIP1-1 expression of leaves increased again, this was coincident to expression profile of transcriptome sequencing (Fig. 6A). The CaPIP1-1 gene expression in roots was significantly higher from 6 h to 48 h after P. capsici inoculation (Fig. 6B). 3.5. Phenotype of CaPIP1-1 gene silenced plants When the positive control (inoculated with TRV2:CaPDS) showed large bleaching symptom, young leaves of CaPIP1-1-silenced plants and negative control (inoculated with pTRV2) were collected to detect the silencing efficiency. Compared to negative control, CaPIP1-1 silencing rate reached nearly 80% (Fig. 7A). Meanwhile, the growth of plants with high CaPIP1-1 silencing rate was inhibited obviously (Fig. 7C). Leaf discs (0.5 cm in diameter) were obtained from the young leaves with above 50% gene silencing rate to perform osmosis stresses. The CaPIP1-1-silenced leaves became brown, and the leaf disc blades appeared as necrotic or chlorotic phenotypes (Fig. 7B).

Fig. 4. Expression of CaPIP1-1 in pepper leaves under chilling, ABA and fluridone stresses. Relative transcription levels of CaPIP1-1 were detected in pepper leaves treated with chilling, 100 μM ABA, chilling after ABA and 100 μM fluridone stress (inhibitor of ABA biosynthesis) using qRT-PCR technology. Pepper seedlings, incubated under standard culture conditions (25 °C under fluorescent lighting, 14 h light/10 h dark cycle, 200 μmol m−2 s−1, 70% relative humidity), were used as controls. Relative amounts were recalculated to that of CaUbi3 (A) or CaActin2 (B). Bars with different lower case letters in each group indicate significant differences, as determined using Duncan's multiple range test at pb 0.05. Three biological experiments were performed, which produced similar results.

4. Discussion Compared to Arabidopsis, there were few reports on AQPs in pepper (Johanson et al., 2001; Kim et al., 2014; Qin et al., 2014; Yin et al., 2014a). Most of the AQPs researches in plants were focusing on gene cloning and expression analysis (Yin et al., 2014b). In our early study we isolated the CaPIP1-1 gene by the comparative analysis of the transcriptome database, SSH cDNA library (Guo et al., 2013) and the EST

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Fig. 6. Expression of CaPIP1-1 gene in response to Phytophthora capsici infection. Pepper plants (P70) were infiltrated with zoospores of P. capsici (1 × 105 zoospores/mL). The transcription levels of CaPIP1-1 in the leaves (A or C) and roots (B or D) were analyzed for mRNA levels by quantitative RT-PCR at different time points (0, 1, 3, 6, 12, 24, 48 and 72 h) after root inoculation of P. capsici. Relative amounts were recalculated to that of CaUbi3 (A and B) or CaTEF1A (C and D). Bars with asterisks in each group indicate significant differences, as determined using Duncan's multiple range test at pb 0.05.

database under chilling stress (Hwang et al., 2005). The CaPIP1-1 protein encoded 286 amino acids. Our results showed that the CaPIP1-1 gene belongs to the PIP1s family, which suggested that subcellular localization of CaPIP1-1 mainly located on the plasma membrane or vacuolar membranes (Uehlein et al., 2008). Other research also showed that PIPs mainly located on the endoplasmic reticulum membrane and mitochondria (Soto et al., 2012). Bioinformatic analysis showed that no signal peptide sequence existed, and further research should be done on the exact location of CaPIP1-1.

NtAQP1 is an aquaporin which its mechanism is comparatively clear (Biela et al., 1999; Flexas et al., 2006; Otto and Kaldenhoff, 2000; Porcel et al., 2005; Sade et al., 2014; Siefritz et al., 2001, 2004; Uehlein et al., 2003). QRT-PCR analysis revealed that expression of the CaPIP1-1 gene was different from NtAQP1. Northern hybridization analysis showed that expression of NtAQP1 was detected in all tissues including roots, stems and leaves, especially higher expression in the young tissues in tobacco. In this study, CaPIP1-1 transcription levels could be found in all tissues, especially in the young fruits where these were abundant

Fig. 7. Phenotypes and CaPIP1-1 expression level of gene-silenced pepper plants. (A) Expression of CaPIP1-1 in gene-silenced pepper (TRV2: CaPIP1-1) cv. Qiemen and control plants (TRV2:00) was tested at 45 d after inoculation. Relative amounts were recalculated to that of CaUbi3 or CaTEF1A or CaActin2. (B) Phenotypes of leaf discs (0.5 cm in diameter) of the gene-silenced plants in response to 0.1 M salt stress and 0.3 M mannitol stress. (C) Phenotypes of gene-silenced plants were imaged at 45 d after infection, and 30 plants were used for each infection. This experiment was independently repeated three times, and similar results were obtained.

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(Fig. 2). The relation between the high expression of CaPIP1-1 and maturation of fruits needs to be thoroughly studied. Expression of PIP1s family genes in leaves was different from that in the roots by analysis of 13 PIPs of Arabidopsis. For example, expression of AtPIP1-1 in the aboveground part was significantly higher than that in the roots (Jang et al., 2004). Although all these genes have high homology with each other (Fig. 1), the difference of gene expression was significant. The abovementioned results indicated that the regulation mechanism of endogenous aquaporins was more complex. Water transport analysis in Xenopus oocytes through the tobacco aquaporins under hypotonic condition indicated that NtAQP1 is not sensitive to HgCl2 (Biela et al., 1999). In this study, we found that expression of CaPIP1-1 in the roots and leaves of the pepper plants under HgCl2 stress was significantly inhibited more than that under normal culture condition (Fig. 3). While, the expression levels in the roots were lower than those in the leaves. Heavy metal inhibited water transport through aquaporins by regulating the activity of the sulfhydryl protein, which increased activation energy (Ea), blocked water transport and caused the membrane to close (Ionenko et al., 2010). Therefore, the minimum Ea value of water transmembrane transport is always considered as one of the standards of water transport through AQPs. Earlier studies showed that the low concentration of HgCl2 stress only affected water transport in the roots (Martinez-Ballesta et al., 2003). The short-term inhibition of the membrane proteins in the roots of pepper may come from the inhibition of protein phosphorylation and the long-term effect had an impact on the water transport and gene expression. But it was still not clear that the mechanism of inhibition effect on leaves may be due to the insensitiveness of CaPIP1-1 protein to HgCl2, or interaction among the AQPs. In the future, further study will be performed to reveal the mechanism of inhibition regulation. Experiment of treatment with fluridone and ABA showed that the regulation of CaPIP1-1 belonged to ABA dependent pathway, and the transcription level was up-regulated. Expression of AtPIP1-1 and AtPIP1-4 increased more than four times in the roots and aerial parts of Arabidopsis after treating with ABA (Jang et al., 2004), while the expression patterns in the roots and aerial parts were different. In this study, expression of CaPIP1-1 increased 2 to 4 times under exogenous ABA treatment. PIP1s family genes were down-regulated in the aboveground part of Arabidopsis after treating with cold stress for 24 h, while AtPIP1-4 was up-regulated (Jang et al., 2004). In this study, CaPIP1-1 was also up-regulated under chilling stress, but in the aboveground parts the increase rate was relatively lower. Mahdieh and Mostajeran revealed that ABA increased the sap flow rate of excised tobacco roots and also the osmotic root hydraulic conductance by upregulating endogenous NtAQP1, NtPIP1;1 and NtPIP2;1 (Mahdieh and Mostajeran, 2009). Some stresses, such as drought stress, participate in water stress mediated by ABA (Zhou et al., 2012). These physiologic reactions may be related with response to plant drought stress. We found that expression patterns under salt stress were similar to that under exogenous ABA treatment. Salt stress participates in signal transduction pathway mediated by ABA (Zhou et al., 2012). It suggested that the regulation of CaPIP1-1 expression may be involved in ABA signaling pathways. Mannitol regulated the quick up-regulation expression of plasma membrane aquaporin, which was similar to drought stress mediated by PEG and ABA signal transduction pathway (Zhou et al., 2012). However it was different from the regulation mediated by ABA in CaPIP1-1. The signal transduction pathway mediated by SA is always related with plant defense reaction (An et al., 2008; Wang et al., 2013c). Expression of CaPIP1-1 in pepper leaves increased after P. capsici infection. Our early studies showed that P. capsici started to grow on leaves within 12 h post inoculation (hpi), and enter the leaves after inoculation via stomata at 24 hpi without forming appressoria-like structures (Du et al., 2013). In the stage of spore germination expression of the CaPIP1-1 gene significantly increased. The inoculated spores, when germinating around the root wound, might release material as signal to

regulate the expression of CaPIP1-1. Expression profile analysis of P. capsici infection might reveal total modulation and expression of PIPs. For the long period and low transformation rate of pepper (Kothari et al., 2010), VIGS could quickly identify the gene function (An et al., 2008; Wang et al., 2013a,c; Xiao et al., 2014; Zhang et al., 2013). VIGS analysis verified that effective silencing of CaPIP1-1 affected the growth of the plant which presents symptom of dwarfism. Detach leaves of CaPIP1-1-silenced plants with decreased stress resistance showed water spots earlier and the edge of leaves presented etiolation. The previously mentioned results suggested that CaPIP1-1 played an important role in water modulation, growth and improving pepper stress tolerance. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 31272163), Jiangsu Agriculture Science and Technology Innovation Fund [CX(12)1004], and the Shaanxi Provincial Science and Technology Coordinating Innovative Engineering Project (No. 2012KTCL02-09). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.03.012. References Aharon, R., Shahak, Y., Wininger, S., et al., 2003. Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress. Plant Cell 15, 439–447. An, S.H., Sohn, K.H., Choi, H.W., et al., 2008. Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance. Planta 228, 61–78. Biela, A., Grote, K., Otto, B., et al., 1999. The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury-insensitive and permeable for glycerol. Plant J. 18, 565–570. Chaumont, F., Barrieu, F., Wojcik, E., et al., 2001. Aquaporins constitute a large and highly divergent protein family in maize. Plant Physiol. 125, 1206–1215. Du, Y., Gong, Z.H., Liu, G.Z., et al., 2013. Scanning electron microscopic study of the infection process of Phytophthora capsici. Pak. J. Bot. 45, 1807–1811. Flexas, J., Ribas-Carbo, M., Hanson, D.T., et al., 2006. Tobacco aquaporin NtAQP1 is involved in mesophyll conductance to CO2 in vivo. Plant J. 48, 427–439. Forrest, K.L., Bhave, M., 2007. Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype. Funct. Integr. Genomics 7, 263–289. Guo, W.L., Chen, R.G., Gong, Z.H., et al., 2012. Exogenous abscisic acid increases antioxidant enzymes and related gene expression in pepper (Capsicum annuum) leaves subjected to chilling stress. Genet. Mol. Res. 11, 4063–4080. Guo, W.L., Chen, R.G., Gong, Z.H., et al., 2013. Subtractive hybridization analysis of genes regulated by application of exogenous abscisic acid in pepper plant (Capsicum annuum L.) leaves under chilling stress. PLoS ONE 8, e66667. http://dx.doi.org/10. 61371/journal.pone.0066667. Hwang, E.W., Kim, K.A., Park, S.C., et al., 2005. Expression profiles of hot pepper (Capsicum annuum) genes under cold stress conditions. J. Biosci. 30, 657–667. Ionenko, I.F., Anisimov, A.V., Dautova, N.R., 2010. Effect of temperature on water transport through aquaporins. Biol. Plant. 54, 488–494. Jang, J.Y., Kim, D.G., Kim, Y.O., et al., 2004. An expression analysis of a gene family encoding plasma membrane aquaporins in response to abiotic stresses in Arabidopsis thaliana. Plant Mol. Biol. 54, 713–725. Johanson, U., Karlsson, M., Johansson, I., et al., 2001. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol. 126, 1358–1369. Kim, S., Park, M., Yeom, S.I., et al., 2014. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat. Genet. 46, 270–278. Kothari, S.L., Joshi, A., Kachhwaha, S., et al., 2010. Chilli peppers — a review on tissue culture and transgenesis. Biotechnol. Adv. 28, 35–48. Mahdieh, M., Mostajeran, A., 2009. Abscisic acid regulates root hydraulic conductance via aquaporin expression modulation in Nicotiana tabacum. J. Plant Physiol. 166, 1993–2003. Martinez-Ballesta, M.C., Diaz, R., Martinez, V., et al., 2003. Different blocking effects of HgCl2 and NaCl on aquaporins of pepper plants. J. Plant Physiol. 160, 1487–1492. Otto, B., Kaldenhoff, R., 2000. Cell-specific expression of the mercury-insensitive plasmamembrane aquaporin NtAQP1 from Nicotiana tabacum. Planta 211, 167–172. Porcel, R., Gomez, M., Kaldenhoff, R., et al., 2005. Impairment of NtAQP1 gene expression in tobacco plants does not affect root colonisation pattern by arbuscular mycorrhizal fungi but decreases their symbiotic efficiency under drought. Mycorrhiza 15, 417–423.

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