Brassica rapa expansin-like B1 gene (BrEXLB1) regulate growth and development in transgenic Arabidopsis and elicits response to abiotic stresses
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The functional role of expansin-like A and B family members remains unclear in plants. In this study, we investigate the functional role of Brassica rapa expansin-like B1 (BrEXLB1) by overexpressing BrEXLB1 sense, antisense and BrEXLB1 specific promoter: GUS in Arabidopsis. The overexpressors of sense BrEXLB1, antisense BrEXLB1 showed variable and unstable pleiotropic effects on leaf growth. Interestingly, overexpressors of sense BrEXLB1 enhances plant height than antisense overexpressors and wild types. GUS reporter-aided analysis of BrEXLB1 promoter showed their activity prominently in seeds, roots and carpels. This is further confirmed by relative quantification of BrEXLB1 among various tissue samples of B. rapa by semi-quantitative Reverse transcription-polymerase chain reaction assay (RT-PCR). We found that BrEXLB1 promoter has several cis-acting elements including light-responsive, phytohormone-responsive, environmental/stress-responsive and tissue-specific elements in promoter sequences. Hence, we analysed the promoter activity of BrEXLB1 by GUS reporter-aided approach, in which stress, phytohormones and other factors regulated changes in GUS transcripts were measured using quantitative RT-PCR. This study suggests that promoter activity is inducible to exogenous application of phytohormones such as indole-3-acetic acid, jasmonic acid, and other factors such as white light and drought stress. These results suggest that BrEXLB1 under its specific promoters may participate in regulation of leaf, plant growth and responds to hormone availability, light quality, dark periods, developmental stages, and drought conditions.
KeywordsBrassica rapa Expansin-like B1 gene Plant growth and development GUS Promoter analysis Transgenic Arabidopsis
Brassica rapa expansin-like B1 gene
Indole acetic acid
Quantitative reverse-transcription PCR
Expansins are cell wall proteins, present in all plants, play crucial role in cell wall remodeling (Marowa et al. 2016). Plant expansins are necessary for growth and development as it helps the plants to uptake water, cell stress relaxation and or cell enlargement through its cell wall loosening activity (Cosgrove 2001; Sasidharan et al. 2011). It is a non-enzymatic proteins that extend cell walls by weakening non-covalent linkages between cellulose microfibrils and xyloglucans (Santiago et al. 2018; Wang et al. 2013). Based on the phylogenetic relationships, plant expansins are classified into four families: α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB) proteins (Kende et al. 2004). Members of EXPA and EXPB families have been clearly shown to be involved in most plant growth and developmental processes, whereas EXLA and EXLB proteins are only known from their homologous conserved gene sequences and their functions yet to be discovered (Cosgrove 2015).
Following initial investigations of expansins in cucumber hypocotyls (McQueen-Mason and Cosgrove 1994; McQueen-Mason et al. 1992), numerous studies have shown that expansins are involved in all cell wall modification processes, from abscission (Cho and Cosgrove 2000), seed number increase (Bae et al. 2014), root formation (Greenwood et al. 2006), fruit ripening (Brummell et al. 1999), plant architecture definition (Dal Santo et al. 2011) to plant–microbe interactions (Wieczorek et al. 2006). Although the exact mechanism underlying expansin activity is not fully understood (Cosgrove 2015), several studies have successfully correlated the involvement of expansins with leaf growth and development using sense and antisense transgenic plants. Examples in Arabidopsis include overexpression of NtEXPA5 increasing leaf and stem sizes (Kuluev et al. 2013), sense and antisense overexpression of AtEXPA10 effectively altering leaf and petiole sizes (Cho and Cosgrove 2000), and overexpression of AtEXPA10 and PnEXPA1 producing larger leaves and longer stems (Kuluev et al. 2012). Overexpression of NtEXPA1 increases tobacco leaf size (Kuluev et al. 2014), and PttEXPA1 overexpression increases stem internode elongation and leaf expansion in aspen (Gray-Mitsumune et al. 2008). Furthermore, CsEXP1 overexpression has been found to reduce leaf sizes and internodes in tomato plants (Wieczorek et al. 2006). Overexpression of OsEXP4 sense and antisense constructs significantly increases and decreases, respectively, the number of leaves in rice plants (Choi et al. 2003). Because EXLA and EXLB gene sequences are conserved across many plant species and are highly homologous to those of EXPA and EXPB members (Sampedro and Cosgrove 2005), EXLA and EXLB genes may be directly and/or indirectly involved in plant growth and developmental processes. Concurrence with this hypothesis, Arabidopsis EXLA2 gene has been recently reported to involve in plant development and defense (Abuqamar et al. 2013).
In our previous comparative genomic analysis, we identified three EXLB genes in the Chinese cabbage (Brassica rapa subsp. pekinensis) genome (Krishnamurthy et al. 2015). In this study, we cloned and characterized the physiological effect of the BrEXLB1 gene in transgenic Arabidopsis expressing BrEXLB1 sense and antisense constructs driven by the CaMV35S promoter. We also isolated the BrEXLB1 promoter sequence and characterized its activity under various environmental stimuli, phytohormones, and stress conditions. The promoter activity was effectively measured using reporter gene expression levels in transgenic Arabidopsis via quantitative reverse-transcription PCR (qRT-PCR). This study will help to understand the likely function of BrEXLB1 in plant growth and development.
Materials and methods
Full-length coding, amino acid, and upstream (promoter region: − 1600 bp to + 1 bp) sequences of EXLB genes of B. rapa (BrEXLB1) and Arabidopsis thaliana (AtEXLB1) were retrieved from Brassica (http://brassicadb.org/brad/index.php; B. rapa genome version 1.5) and Phytozome v10.3 (http://phytozome.jgi.doe.gov/ pz/portal.html#) databases, respectively. To identify putative cis-acting elements of BrEXLB, the promoter region (− 1500 bp to + 1 bp) was searched against known motifs of plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database. All tools and programs mentioned in this study were used with default settings unless otherwise specified.
Construction of expression cassette for BrEXLB1-sense, antisense and BrEXLB1 promoter::GUS
The BrEXLB1 coding sequence was amplified by polymerase chain reaction (PCR) with gene-specific forward (5′-AATATGAAGACATTTAACGTCTTG-3′) and reverse (5′-GGAATCAAGTAAGTAGAATGTTGG-3′) primers. The EcoR I digested PCR amplicons (0.773 kb) were inserted in the transgene orientation between the cauliflower mosaic virus 35S promoter (CaMV35Sp) and the nopaline synthase terminator of a pCAMBIA1390 vector to generate binary vectors pCAMBIA1390::35S-Pro + BrEXLB1sense (pLSI09) and pCAMBIA1390::35S-Pro + BrEXLB1antisense (pLSI12). The sense and the antisense orientation of BrEXLB1 was confirmed by Sanger sequencing. Similarly, the BrEXLB1 promoter region was PCR amplified using forward and reverse primers 5′-GAAACGAACACGGCTATTATACG-3′ and 5′-CATATTGTTATATGTAACTATTGTA-3′, respectively. The amplified promoter sequence (1.5 kb) was ligated into a pGEM-T Easy vector followed by transformation into Escherichia coli DH-5α cells for amplification, and then introduced into a pCAMBIA1391Z vector at EcoR I site to generate the binary vector pCAMBIA1391Z + BrEXLB1promoter::GUS (pLSI13). All primer pairs were obtained from GenoTech (Daejeon, Korea).
Plant materials, Agrobacterium mediated transformation and morphology of transgenic lines
Arabidopsis thaliana Columbia (Col-0) seeds were surface-sterilized by 70% ethanol for 15 min followed by 100% ethanol for 2 min and plated on Murashige and Skoog (MS) medium supplemented with vitamins, 1.5% sucrose, and 0.25% phytagel. Plates were stratified at 4 °C under dark for 2 days to induce synchronous germination and transferred to a growth chamber (16-h light/8-h dark photoperiod at 23 °C) for transformation and morphological character analyses. Transformation (A. tumefaciens strain GV3101) and transformant selection with hygromycin B were performed according to Hong et al. (2012). Leaf length, leaf width, and petiole length were measured on 15, 20, and 30 days after transferring the pots into the growth chamber. Measurements were taken from three independent biological replicates. Mean significant differences were compared by Duncan Multiple Range Test at P ≤ 0.05 using SAS package 9.1.3 service pack 4.
Histochemical localization of β-glucuronidase (GUS) activity
To evaluate the regulation of BrEXLB1 promoter under different developmental stages, seeds of transgenic Arabidopsis (pLSI13) lines were germinated as mentioned above. Samples were collected from the first day of germination, 7-, 14-, and 20-days old seedlings and reproductive organs of mature plants. Samples were directly used for the GUS assay. An assay was performed using a β-Glucuronidase Reporter Gene staining kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.
Phytohormone, stress treatments, and quantitative reverse-transcription real-time PCR (qRT-PCR)
Transgenic T1 Arabidopsis seedlings harboring pLSI13 cassette were surface sterilized as mentioned above and plated on MS medium supplemented with vitamins, 1.5% sucrose, 0.25% phytagel and 30 μg/ml hygromycin B. After 2 days stratification under dark at 4 °C and 5 days under growth chamber, transformants were selected based on the hypocotyl length (Harrison et al. 2006). Selected seedlings were divided for triplicate and independently grown hydroponically in MS solution for 3 days in Arabidopsis growth chamber. Then, three plates each (each having three seedlings) were moved to different light qualities namely white (photosynthetic photo flux density: 59 μmol m−2 s−1), blue (59 μmol m−2 s−1), red (10 μmol m−2 s−1), far-red (2 μmol m−2 s−1) and dark in our customized LED chamber. Concurrently, phytohormones, namely IAA (50 μM), GA (50 μM), JA (100 μM), SA (100 μM), and ABA (100 μM), as well as heavy metal (CdCl2; 200 μM) and polyethylene glycol (PEG6000; 4%) were added to the respective triplicate hydroponic cultures and kept in the growth chamber with gentle shaking (60 rpm). After incubation for 6 h, entire seedlings from all the treatments were harvested, frozen in liquid nitrogen, and stored at − 80 °C. Seedlings grown in growth chamber (80 μmol m−2 s−1) in MS solution were used as control. Temperature (25 ± 0.5 °C) and humidity (33 ± 2%) were maintained same in both the growth chamber and LED chamber.
Total RNA was extracted using Ambion Purelink® RNA Mini Kit (Thermo Fisher Scientific, MA, USA) from ground tissues samples (Qiagen TissueLyser II (Hilden, Germany). RNA (1 µg) was reverse transcribed into cDNA using Qiagen`s QuantiTect® Reverse Transcription Kit (Hilden, Germany) which included genomic DNA elimination step. cDNA products were diluted in a 1:9 ratio with nuclease-free water (Promega) and used as template for qRT-PCR. QRT-PCR was performed using the SYBR Premix Ex Taq kit® (TaKaRa, Japan) with GUS-specific primers (forward: 5′-GAATACGGCGTGGATACGTTAG-3′ and reverse 5′-GATCAAAGACGCGGTGATACA-3′) on StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) under the following cycling profile: 95 °C for 30 s, 95 °C for 5 s (40 cycles), 60 °C for 30 s, and melting curve analysis at 65 °C for 10 s with 61 cycles. The reaction mixture volume for qRT-PCR was 20 µL. Experimental repeat runs for three biological and three technical replicates were included in the analysis. The Arabidopsis Actin2 (AT3G18780) gene (forward primer: 5′-TCGGTGGTTCCATTCTTGCT-3′; reverse primer: 5′-GCTTTTTAAGCCTTT GATCTTGAGAG-3′) was used as an internal control to normalize the expression level of the target GUS gene. An analysis of the relative quantitation (RQ) values were directly inferred from the StepOnePlus Real-Time PCR System that mean significant differences were compared by Duncan Multiple Range Test at P ≤ 0.05 using SAS package 9.1.3 service pack 4.
Relative quantification of BrEXLB1 transcripts in different tissues of Brassica rapa
Brassica rapa ‘Chiifu-401-42’ seeds were germinated and grown according to Hong et al. (2010). Samples of apical meristems, cotyledons and hypocotyls were collected from 2-week-old B. rapa seedlings whereas leaves, roots, pollen, carpels, and siliques were harvested from mature B. rapa plants. All samples were frozen in liquid nitrogen and stored at − 80 °C throughout the sampling period and were ground using Qiagen TissueLyser II (Hilden, Germany). Total RNAs and cDNA were prepared as mentioned above and used as template for semi-quantitative (reaction volume 25 µL) PCR with BrEXLB1 primers under following cycling parameters: 95 °C for 5 min, followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, with a final extension step of 72 °C for 7 min. B. rapa β-actin (forward: 5′-TGGCATCACACTTTTCTACAA-3′; reverse: 5′-CAACGGAATCTCTCAGCTCC-3′) was used as an internal control to check the RNA quality (Hong et al. 2010).
Results and discussion
Implications on the structural characteristics of B. rapa EXLB genes and their promoter regions
Comparative sequence alignment among the promoter regions (− 1600 bp to + 1 bp of the upstream sequence) of BrEXLBs revealed that the BrEXLB1 promoter share only 48–50% similarity to BrEXLB2 and BrEXLB3 promoters, whereas 72.31% similarity was found between the latter two. The AtEXLB1 promoter showed 43–49% similarity to the BrEXLB promoters. Due to the limitation of PlantCARE database, the − 1500 bp to + 1 bp of the obtained promoter sequences were submitted to identify and compare cis-acting elements. In each sequence, 83–96 conserved cis-acting elements were identified corresponding to 55 unique elements that could be broadly classified into five types: (1) promoter-related, (2) light-responsive, (3) plant hormone-responsive, (4) environmental- and stress-responsive, and (5) tissue-specific (Table S2). Of the 55 cis-acting elements, 35–47% is contributed by promoter-related elements followed by light-responsive (20–33%) and phytohormone-responsive elements. BrEXLB promoters has several elements like phytohormone responsive (ABA and SA), fungal elicitor-responsive (Box-W1 (TTGACC), wound and pathogen responsive (W box: (TTGACC)), meristem expression-specific (CAT-box: GCCACT), endosperm-specific expression (Skn-1_motif: GTCAT), circadian control and element involved in negative regulation of phloem expression and responsible for restricting vascular expression (AC-II; [(C/T)T(T/C)(C/T)(A/C)(A/C)C(A/C)A(A/C) C(C/A) (C/A)C]. The comparative analysis with BrEXLB2, BrEXLB3 and its orthologous promoter AtEXLB1 shows some elements are specific to respective promoter sequences. For instance, BrEXLB1 has unique elements like Box-W1 and the W box (TTGACC), similarly, BrEXLB2 has MBS motif (CAACTG; involved in drought inducibility), BrEXLB3 has 5′-untranslated region Py-rich stretch element (TTTCTTCTCT) and AtEXLB1 has auxin, JA-, GA-responsive, and AT-rich sequence (TAAAAATACT; element for maximal elicitor-mediated activation).
Elements such as anaerobic induction-responsive (ARE motif: TGGTTT), stress-responsive (TC-rich repeats: GTTTTCTTAC) were commonly found in all BrEXLB promoters while CAT-box was found in BrEXLB1 and BrEXLB3 promoters. Similarly heat stress-responsive elements (HSE motif: AAAAAATTTC and AGAAAATTCG) were present in BrEXLB2 and BrEXLB3 promoters. Further comparison with AtEXLB1 revealed that AC-II as well as the Skn-1_motif: GTCAT are common among all the promoters, whereas circadian control elements were unique to BrEXLB promoters. As indicated in the present study and by the reports of Zhu et al. (2014), we expect that the presence of diverse cis elements may facilitate the differential expression of EXLB genes depending upon the environmental stumuli, presence of phytohormones or its exogenous application, developmental stages and stress conditions.
GUS reporter-aided analysis of BrEXLB1-specific promoter activity during plant development, application of phytohormones and stress in Arabidopsis
Similarly, the exogenous application of IAA, JA, and exposure to PEG6000 mediated drought stress and CdCl2 influence the activities of BrEXLB1 promoter. The quantitative RT-PCR analysis showed that application of IAA increased GUS transcripts by 2.8-fold and, JA and PEG exhibited 2.5-fold increase in pLSI13 lines. Intriguingly, cis-elements responsive to IAA, JA- and drought stress were not observed from the sequence analysis (Table S2). However, both IAA (auxin), JA-responsive elements were found in AtEXLB1 which is a homolog of BrEXLB1. It is reasonable to think that the technical limitation for sequence length with PlantCARE might reduce our chance in identification of auxin-, JA-responsive elements. Moreover, standardization of time periods for each treatment may provide most conceivable data. Further, the synergistic relationship between JA- and wound-responsive elements (W-box) may facilitate the induced activity of BrEXLB1 promoter against JA (Bari and Jones 2009). Up-regulation by PEG6000 and slightly down-regulation by CdCl2 suggests BrEXLB1 is sensitive to plant water balance, as both CdCl2 and PEG6000 decrease plant water potential and thereby inhibit plant growth and development (Lagerwerff et al. 1961; Perfus-Barbeoch et al. 2002). As evident from the results (Fig. 3), BrEXLB1 promoter mediated expression of GUS transcript is also regulated by exogenous application of GA, SA, ABA and CdCl2, although their exposure did not significantly affect the promoter activity. PEG induced activity of BrEXLB1 are encouraging as it may result in improved drought stress response as noted in tobacco (Chen et al. 2016). Nevertheless, our results show that BrEXLB1 promoter activity is up-regulated by white light, IAA, JA and PEG treatments and reduced under red, far-red and dark treatments indicating the possible involvement of BrEXLB1 gene in plant growth and development.
Impact of BrEXLB1 sense and antisense overexpressing transgenic Arabidopsis on plant growth and development
Moreover, the expression of GUS driven by promoter of BrEXLB1 in leaves and cotyledons of transgenic Arabidopsis was abundant while the expression of endogenous BrEXLB1 in B. rapa was undetectable. This difference in BrEXLB1 promoter activity suggests the likely interference of other factors and the different genetic constitution. Unlike in A. thaliana, three EXLB (EXLB1, 2 and 3) genes were present in B. rapa (Krishnamurthy et al.). Possibly, BrEXLB2 and BrEXLB3 would have direct or indirect effect in expression of BrEXLB1 in host plant, B. rapa. However, the effect of BrEXLB2 and BrEXLB3 is unlikely in transgenic Arabidopsis due to the absence of EXLB2 and EXLB3. In addition, other endogenous and exogenous factors including white light, red light, dark, IAA, JA and drought stress in B. rapa and A. thaliana would modulate the expression of BrEXLB1 differently as shown by transgenic promoter lines in the present study.
In conclusion, our findings suggest that BrEXLB1 may participate in germination, leaf development, and plant growth depending upon the phytohormone availability (esp. IAA, JA), interaction with other EXLB genes, transcriptional regulators, light types and intensity, dark periods and drought stress in a given physiological conditions. Moreover, BrEXLB1 may exhibit tissue preference as evidenced by GUS reporter-aided analysis of BrEXLB1-specific promoters in this study.
This work was supported by the Rural Development Administration (Korea) through the Rural Program for Agricultural Science and Technology Development (Project No. PJ01247202) and the Next Generation Bio-green 21 Program (Project No. PJ01334002).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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