The 1.5 kb JcAP1 promoter from the biofuel plant Jatropha curcas is predominantly active in the inflorescence buds of transgenic plants, in which the −1313/−1057 region is essential for maintaining the activity.
Arabidopsis thaliana APETALA1 (AP1) is a MADS-domain transcription factor gene that functions primarily in flower development. We isolated a homolog of AP1 from Jatropha curcas (designated JcAP1), which was shown to exhibit flower-specific expression in Jatropha. JcAP1 is first expressed in inflorescence buds and continues to be primarily expressed in the sepals. We isolated a 1.5 kb JcAP1 promoter and evaluated its activity in transgenic Arabidopsis and Jatropha using the β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis and Jatropha, the inflorescence buds exhibited notable GUS activity, whereas the sepals did not. Against expectations, the JcAP1 promoter was active in the anthers of Arabidopsis and Jatropha and was highly expressed in Jatropha seeds. An analysis of promoter deletions in transgenic Arabidopsis revealed that deletion of the −1313/−1057 region resulted in loss of JcAP1 promoter activity in the inflorescence buds and increased activity in the anthers. These results suggested that some regulatory sequences in the −1313/−1057 region are essential for maintaining promoter activity in inflorescence buds and can partly suppress activity in the anthers. Based on these findings, we hypothesized that other elements located upstream of the 1.5 kb JcAP1 promoter may be required for flower-specific activation. The JcAP1 promoter characterized in this study can be used to drive transgene expression in both the inflorescence buds and seeds of Jatropha.
As a potential oilseed plant for renewable biodiesel production, Jatropha curcas (hereafter referred to as Jatropha), a member of the Euphorbiaceae family, has been studied for decades (Heller 1996; Sujatha et al. 2008; Divakara et al. 2010; de Argollo Marques et al. 2013). Considering the low genetic diversity of Jatropha (Tatikonda et al. 2009; Cai et al. 2010), transgenic breeding is a highly promising approach for directionally modifying characteristics of Jatropha in a short time, without the limitation of germplasm resources. To accelerate the process of the transgenic breeding of Jatropha, many efforts have been made to establish a genetic transformation system (Li et al. 2008; Joshi et al. 2010; Kumar et al. 2010; Pan et al. 2010) to analyze gene expression patterns (Chen et al. 2011; Sato et al. 2011; Li et al. 2015) and functions (Tang et al. 2011; Qu et al. 2012; Wei et al. 2012; Li et al. 2014). However, few studies have evaluated promoters in Jatropha. The CURCIN promoter (CP1) and curcin-L promoter (CP2) isolated from Jatropha have been characterized in tobacco, in which CP1 is specifically active in the seeds (Qin et al. 2009a), and CP2 is a leaf-specific promoter induced by stresses (Qin et al. 2009b). The JcNAC1 promoter is active in the roots, stems and leaves of seedlings, and JcNAC1 promoter activity can be induced in guard cells by drought (Qin et al. 2014). The JcSDP1 promoter is only active in Jatropha seeds, especially in the endosperm (Kim et al. 2014). The JcMFT1 promoter is highly active in transgenic Arabidopsis seeds and can be induced by ABA in germinating seeds (Tao et al. 2014). The JcUEP promoter could serve as an alternative to the CaMV35S promoter for driving the constitutive overexpression of transgenes in Jatropha (Tao et al. 2015). It needs more efforts to characterize more promoters in Jatropha, which is one of the crucial regulation factors for efficient expression of transgenes in Jatropha.
In this study, we focused on isolating a reproductive tissue-specific promoter in Jatropha because the seed yield can be improved by modifying the flowering trait (Pan and Xu 2011; Chen et al. 2014; Pan et al. 2014). APETALA1 (AP1) is a MADS-domain transcription factor gene that specifies floral meristem identity and functions as an A-class gene involved in floral organ formation (Mandel et al. 1992; Mena et al. 1995; Berbel et al. 2001; Litt and Kramer 2010; Chi et al. 2011). In Arabidopsis, AP1 acts a hub that mediates the switch from floral initiation to flower formation (Kaufmann et al. 2010). Overexpression of Arabidopsis AP1 in tomato and citrus results in early flowering and a reduction of the generation time in transgenic plants (Pena et al. 2001; Ellul et al. 2004). As expected, the Arabidopsis AP1 promoter is active in floral primordia and young floral buds produced from primary and secondary inflorescences (Chou et al. 2001; Guan et al. 2002); thus, it has been used as a flower-specific promoter to analyze gene function in flowers or to modify flower traits. The AP1 promoter directs the expression of the floral regulatory gene SUPERMAN (SUP) in Arabidopsis and tobacco, and the flowers of transgenic plants of both species exhibit fewer floral organs, consistent with an effect of SUP on cell proliferation (Yun et al. 2002). Expression of the cytokinin synthesis gene IPT4 driven by the AP1 promoter in Arabidopsis results in an increased flower number (Li et al. 2010). In the horticultural plant torenia, AP1:MYB24-SRDX transgenic plants produce open flowers with wavy petals and normal leaves, whereas 35S:MYB24-SRDX transgenic plants exhibit unopened flower buds and glossy dark green leaves with curled margins (Sasaki et al. 2011). The use of flower-specific promoters has the advantage of defining modifications in flowers rather than other organs.
Here, we isolated the Jatropha curcas APETALA1 (JcAP1) promoter and characterized its activity in Arabidopsis and Jatropha. In Arabidopsis, the JcAP1 promoter was active only in inflorescence buds and anthers, and we found that the −1313/−1057 region was essential for promoter activity in inflorescence buds. However, in addition to the inflorescence buds, the JcAP1 promoter was also highly active in Jatropha seeds.
Materials and methods
Jatropha curcas plants cultivated in Xishuangbanna, Yunnan Province, China, were used as previously described (Pan and Xu 2011). The Arabidopsis thaliana Col-0 ecotype, employed for transformation was grown at 22 °C with a 16 h light/8 h dark photoperiod.
qRT-PCR analysis in Jatropha
A cDNA sequence (GenBank accession no. KM610239) of the APETALA1 (AP1) gene was identified from our Jatropha flower cDNA library (Chen et al. 2014). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to examine the expression levels of JcAP1 in various organs of adult Jatropha plants, including the roots, stems, leaves, inflorescence buds, female flowers, male flowers, pericarps and seeds at 42 days after pollination (DAP). Total RNA was isolated (Ding et al. 2008) and reverse transcribed using the PrimeScript® RT reagent kit with gDNA Eraser (TAKARA). qRT-PCR was performed with SYBR® Premix Ex Taq™ II (TAKARA) using the Roche 480 real-time PCR detection system (Roche Diagnostics). All of the gene expression data obtained via qRT-PCR were normalized to the expression of JcGAPDH. The primers used for qRT-PCR are listed in Table 1.
Cloning of the 5′ flanking region and determination of the transcription start site of JcAP1
The 5′ flanking region upstream of the translation start codon of JcAP1 was isolated from Jatropha genomic DNA through genome walking (Siebert et al. 1995). For nested PCR, the JcAP1 gene-specific primers GSP1 and GSP2 and the adaptor primers AP1 and AP2 were used. The JcAP1 promoter was amplified via PCR and cloned into the pGEM-T Easy vector for sequencing. The putative cis-acting elements of the JcAP1 promoter were analyzed using the PLACE database (Higo et al. 1999). To identify the transcription start site of JcAP1, rapid amplification of 5′-cDNA ends (5′-RACE) was performed with total RNA from Jatropha male flowers using the SMARTer® RACE 5′/3′ Kit (Clontech). The primers employed for genome walking and 5′-RACE are listed in Table 1.
Construction of promoter-GUS fusions
To generate the JcAP1:GUS plasmid, XbaI and BamHI were used to digest pBI101 (Jefferson et al. 1987) and the pGEM-T Easy vector containing the 1.5 kb JcAP1 promoter. The two fragments were linked using T4 ligase (Promega). The resulting construct, JcAP1:GUS (Fig. 3b), was transferred to Agrobacterium tumefaciens EHA105 and LBA4404 via electroporation (GenePulser Xcell, Bio-Rad). Strain EHA105 harboring the construct was employed to transform Arabidopsis; strain LBA4404 harboring the construct and pBI101 (promoterless, negative control, NC) was employed to transform Jatropha.
Jatropha transformation was performed as described by Fu et al. (2015). After sterilization with 75 % (v/v) ethanol for 30 s, the mature seeds of Jatropha were sterilized with 10 % (v/v) sodium hypochlorite for 20 min. The embryos were removed from the seeds, and cotyledon explants were cut from the base of the cotyledons, leaving 3/4 of the papery cotyledons for co-cultivation with Agrobacterium. In co-cultivation experiments, 50 μM acetosyringone was added to the MS-Jc1 medium (Pan et al. 2010). After 2 days of co-cultivation, the explants were cultured in MS-Jc1 medium with 100 mg/L timentin for a 10-day recovery period and then subcultured in shoot-inducing medium (SIM, MS-Jc1 medium with 40 mg/L kanamycin and 100 mg/L timentin). After selection, the regenerated shoots were transferred to rooting medium [RM, half strength (1/2) MS medium with 0.2 mg/L IBA, 0.1 mg/L NAA and 100 mg/L timentin]. Finally, the putative Jatropha transformants were examined via TAIL-PCR, and positive transgenic plants were cultivated in soil. Arabidopsis transformation was performed through the floral dip method (Clough and Bent 1998).
Thermal asymmetric interlaced PCR (TAIL-PCR) was performed with genomic DNA isolated from the leaves of the putative Jatropha transformants. The procedure was conducted as described by Liu et al. (1995). Three specific primers were designed based on the right border sequences of the T-DNA of pBI101. Four arbitrary degenerate (AD) primers (AD1, AD2, AD3 and AD4) and the T-DNA-specific primers XK314, XK315 and XK316 were employed to examine JcAP1:GUS transformants. The primers used for these assays are listed in Table 1. After the tertiary reaction, the TAIL-PCR products were sequenced and analyzed using the Jatropha Genome Database (http://www.kazusa.or.jp/jatropha/).
Histochemical and fluorometric GUS assay
For histochemical GUS staining, various tissues of transgenic Jatropha and Arabidopsis were incubated in GUS assay buffer with 50 mM sodium phosphate (pH 7.0), 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6·3H2O, 0.5 % Triton X-100 and 1 mM X-Gluc at 37 °C overnight, then cleared in 70 % ethanol (Jefferson et al. 1987). The samples were examined via stereomicroscopy (Leica M80).
To examine the activity of the JcAP1 promoter in different tissues, a fluorometric GUS assay was performed following the protocol described by Jefferson et al. (1987), which was modified by adding 2 mM MUG to the reaction buffer. Fluorescence was examined with a Gemini XPS Microplate Spectrofluorometer (Molecular Devices Corporation). The protein concentrations of the plant extracts were measured using the Bradford method (1976).
The expression pattern of JcAP1 in Jatropha
We identified a cDNA of AP1 (GenBank accession no. KM610239) from our Jatropha flower cDNA library (Chen et al. 2014). JcAP1 encodes a MADS-box transcription factor that contains two conserved regions: a MADS-domain and a K domain (Fig. 1a). JcAP1 is highly similar to AP1 homologs from other plant species, and it is most closely related to PtAP1 from Populus trichocarpa (Fig. 1b).
To explore the expression pattern of JcAP1 in Jatropha, qRT-PCR was performed with total RNAs extracted from various tissues. The results indicated that JcAP1 is a flower-specific gene that is predominantly expressed in floral tissues, especially in inflorescence buds (Fig. 2). Because most AP1 genes are involved in the development of floral organs, we also examined JcAP1 expression in each organ of male and female Jatropha flowers. As shown in Fig. 2, JcAP1 was highly expressed in the sepals, while the levels in the petals and stamens were low in male flowers; in female flowers, the highest level was also found in the sepals, which was approximately two times that in the petals, and the expression level in the pistils was very low. The expression pattern of JcAP1 in floral organs was consistent with that of A-class genes in other plants (Coen and Meyerowitz 1991; Litt and Kramer 2010).
Isolation and sequence analysis of the JcAP1 promoter
On the basis of expression data, a 1.5 kb JcAP1 promoter fragment (−1313/+150) (GenBank accession no. KM610240) was isolated from Jatropha by genome walking (Siebert et al. 1995). The transcription start site, determined by 5′-RACE, was located 150 nucleotides upstream of the translation start codon of JcAP1 (Fig. 3a). The putative cis-acting elements were analyzed using the PLACE database (Higo et al. 1999). The JcAP1 promoter sequence and putative plant regulatory elements are shown in Fig. 3a. The analysis revealed that the JcAP1 promoter contains a CArG box, which acts as a binding site for MADS-box proteins and is an important element mediating the regulatory effect on MADS-box genes (Tilly et al. 1998). Another important element of the promoter is a binding site (CCAATGT) for the LEAFY protein, which is a transcriptional factor that activates AP1 expression in Arabidopsis (Wagner et al. 1999). This promoter also contained some pollen-specific elements, including the POLLEN1LELAT52 motif and the GTGANTG10 motif, which are essential for the pollen-specific expression of tomato LAT52 and tobacco g10, respectively (Twell et al. 1991; Muschietti et al. 1994; Rogers et al. 2001).
Characterization of JcAP1 promoter activity in transgenic Arabidopsis
To test the activity of the promoter, JcAP1:GUS was transformed into Arabidopsis for preliminary analysis. GUS staining was examined in the T2 generation of five independent transgenic lines. The results showed that GUS activity was first detectable in the inflorescence buds and then in the anthers where pollen staining was intense (Fig. 4). The staining results in transgenic Arabidopsis indicated that the activity of the JcAP1 promoter was confined to flowers; however, the observed activity was not consistent with JcAP1 expression in Jatropha, which was abundant in the sepals rather than the stamens (Fig. 2). This result led us to hypothesize that the pollen-specific elements present in the JcAP1 promoter region may be hyperactive in the heterogeneous expression system.
Deletion analysis of the JcAP1 promoter in transgenic Arabidopsis
A series of 5′ deletions of the JcAP1 promoter (Fig. 5a) were generated to analyze the regulatory effect of different regions of the promoter. After a histochemical GUS assay, we found that compared with the full-length promoter, GUS activity could not be detected in the inflorescence buds of all five deletions (D1–D5), whereas staining was still observed in the anthers, except in the D5 (Fig. 5b). The disappearance of GUS activity in the inflorescence buds of plants transformed with the D1 construct indicated that the −1313/−1057 region is essential for inflorescence bud specificity.
To further investigate the regions of the JcAP1 promoter involved in the regulation of anther-specific activity, we detected GUS expression in the anthers of plants transformed with constructs containing the full-length sequence and deletions of the JcAP1 promoter. As shown in Fig. 5a, removing the −1313/−1057 region resulted in an increase in GUS expression for all of the deletions except for the shortest (D5), and D1 exhibited the highest expression level. This result indicated that the −1313/−1057region represses the activity of the JcAP1 promoter in anthers. Deletion of the −1057/−796 region from D1 to produce D2 led to a reduction of the GUS expression level, while further deletion to position −521 (D3) resulted in almost the same expression level observed for D2. Removal of the −521/−242 fragment from D3 to produce D4 caused a further decrease in the GUS expression level. Consistent with the GUS staining results, a fluorometric GUS assay revealed very low GUS activity for D5 (Fig. 5a, right panel). These results suggested that the −1057/−796 and −521/+19 regions are capable of inducing JcAP1 promoter activity in anthers, while the −796/−521 region shows no significant effects.
Characterization of JcAP1 promoter activity in transgenic Jatropha
Following evaluation in Arabidopsis, the JcAP1:GUS construct was transformed into Jatropha to characterize the activity of the JcAP1 promoter. Transformed plantlets were generated via kanamycin selection and validated through PCR amplification. Then, the PCR-positive transformants were examined via TAIL-PCR to confirm stable transgene integration. The T-DNA right border insert from pBI101 was used for integration analysis. The sequences of the T-DNA integration sites (i.e., the T-DNA/Jatropha genomic DNA junction regions) obtained from four transformant events (B1, B5, B12, and B14) selected randomly are listed below the sequence of the T-DNA right border of pBI101 in Table 2. The results showed that either the entire T-DNA right border sequence (B1 and B5) or part of it (B12 and B14) had been lost and that the transgenes were integrated into the genome of Jatropha transformants. In addition, the integration sites in the Jatropha genome were different between different insertion events, indicating that the plants were independent lines for the transformation event. The transgenic plantlets were grown in soil for further analysis.
A histochemical GUS assay was first performed on regenerated transgenic shoots, and no GUS staining was observed (data not shown). After the transgenic plantlets had grown into the adult plants, various tissues, including roots, stems, young and mature leaves, shoot apices, inflorescence buds, female and male flowers, fruits at 12 DAP and seeds at 25 DAP, were collected for histochemical GUS assay. The results (Fig. 6) showed that GUS staining was strong in the shoot apices, inflorescence buds, male flowers and seeds; weak in the stems and female flowers, and absent from the roots, leaves and pericarps. No GUS staining was detected in the negative control. In male flowers, the anther was also stained (Fig. 6), which is consistent with the results from transgenic Arabidopsis (Fig. 4c). Thus, the activity of the JcAP1 promoter in anthers was not caused by the previously described heterogeneous expression. This result demonstrated that the JcAP1 promoter isolated here is active in anthers. Furthermore, the JcAP1 promoter is active in shoot apices and seeds, differing from the expectation of expression restricted to inflorescence buds and flowers.
Next, we compared JcAP1 promoter activity in different tissues of adult Jatropha plants via a fluorometric GUS assay. Tissues from five independent lines of the T0 generation were used for detection, including the roots, stems, young and mature leaves, shoot apices, inflorescence buds, female and male flowers, fruits at 12 DAP, pericarps, and seeds at 25 DAP. As shown in Fig. 7, the highest GUS expression level was detected in inflorescence buds, followed by the seeds at 25 DAP. The expression levels in shoot apices and male flowers were approximately equal but were less than half of the levels observed in seeds at 25 DAP. Although all four tissues exhibited clearly visible GUS staining, activity differed from the tissues. Consistent with the GUS staining results, fluorometric GUS activity in the stems, female flowers and fruits (12 DAP) was lower than in the shoot apices and male flowers. These results indicated that the JcAP1 promoter is primarily active in inflorescence buds and seeds (25 DAP).
AP1 is a MADS-box transcription factor that plays an important role in the initiation of flowering and floral organ formation (Kotoda et al. 2002; Murai et al. 2003; Huang et al. 2014). AP1 is first expressed in floral meristems and continues to be expressed in the outer whorls of the flower. In maize, ZAP1 expression is restricted to the inflorescences and non-reproductive organs of male and female flowers (Mena et al. 1995). Similar to ZAP1, a high expression level of JcAP1 was first detected in inflorescence buds and then in male and female sepals (Fig. 2). However, the expressions of other AP1 orthologs exhibit different patterns. CsAP1 from Citrus sinensis is expressed not only in the outer whorls of the sepal and petal, but also in the inner whorls of the stamen and carpel (Pillitteri et al. 2004). A hybrid aspen LAP1 was found to be expressed at a high level in the apex and may function mainly in seasonal growth rather than flowering (Azeez et al. 2014). In this study, because JcAP1 was shown to be flower-specific, we isolated its 5′-flanking region as a tissue-specific promoter and characterized this region in transgenic Arabidopsis and Jatropha.
The JcAP1 promoter showed marked activity in inflorescence buds in both transgenic Arabidopsis (Fig. 4) and Jatropha (Fig. 7), which was consistent with the JcAP1 expression pattern in Jatropha (Fig. 2). Through deletion analysis, we found that the −1313/−1057 region is essential for JcAP1 promoter activity in inflorescence buds. In addition, we identified a CArG box in this region, which is an important element mediating the regulatory effect of MADS-box proteins. In Arabidopsis, APETALA3 (AP3) is a MADS-box gene that functions in the control of petal and stamen development (Kramer et al. 1998). Three CArG boxes in the AP3 promoter mediate both positive and negative effects on the establishment and maintenance of the AP3 expression pattern (Tilly et al. 1998). Therefore, the absence of the CArG box in deletion constructs of the JcAP1 promoter (D1–D5, Fig. 5a) may completely abolish inflorescence bud activity. In floral organs, JcAP1 was expressed highly in sepals but was expressed at low levels in stamens. However, GUS staining was not observed in either Arabidopsis (Fig. 4c) or Jatropha sepals (Fig. 6); on the contrary, the anthers were stained, and Arabidopsis pollen showed intense staining (Fig. 4c). We also noted that the JcAP1 promoter contained some pollen-specific elements, including six POLLEN1LELAT52 motifs and five GTGANTG10 motifs, and promoter activity in the anthers decreased with the step-down numbers of these elements. In particular, deleting the −1057/−796 region, containing two GTGANTG10 motifs, and the −521/+19 region, containing five POLLEN1LELAT52 motifs, caused considerable loss of activity in the anthers (Fig. 5). Thus, we assumed that these elements confer the pollen-specific activity of the JcAP1 promoter. Moreover, when the −1313/−1057 region was removed, JcAP1 promoter activity in the anthers increased until the pollen-specific elements had been completely deleted; suggesting that the CArG box in the −1313/−1057 region repressed the pollen-specific elements. Similarly, the third CArG box (CArG3) in the Arabidopsis AP3 promoter is required to maintain a low level of gene expression during early floral stages, and the GUS expression level was found to increase when CArG3 was mutated (Tilly et al. 1998).
In addition, the JcAP1 promoter was unexpectedly active in regions beyond the flowers, showing a high expression level in the seeds of transgenic Jatropha. This result is consistent with seed-specific elements being present in this promoter, such as the E box (Kawagoe and Murai 1992; Stålberg et al. 1996), the DOF core sequence (Yanagisawa and Schmidt 1999), and the SEF motif (Lessard et al. 1991). The JcAP1 expression, however, was not detected in Jatropha seeds (Fig. 2). Similarly, the 920 bp potato ubi3 promoter was shown to be insufficient to achieve wound- and ethylene-dependent activation although native ubi3 expression in tubes is induced by wounding and ethylene treatments (Garbarino and Belknap 1994). Moreover, the gene internal sequences may also take part in the regulation of gene expression. The parsley 4CL-1 gene expression was developmentally regulated by light and stresses, such as pathogen infection, UV-irradiation and wounding. But the 4CL-1 promoter alone was only sufficient to direct gene cell-specific expression. Actually, the exonic sequences were required, in addition to the promoter, for the 4CL-1 gene expression induced by fungal elicitor or light treatment (Douglas et al. 1991). The Arabidopsis ACT2 promoter could not confer the strong expression of ACT2 throughout the vegetative tissues unless it was associated with the first intron (Jeong et al. 2009). Hence, because an isolated promoter may not reveal the full expression pattern of a gene, it is necessary to evaluate promoter activity in target plant species.
Taken together, our findings indicate that the cooperation of multiple cis-acting elements in the JcAP1 promoter is required to confer accurate activity in transgenic plants. Although the 1.5 kb JcAP1 promoter did not exhibit the same expression pattern in transgenic Arabidopsis and Jatropha as was observed for JcAP1 mRNA in Jatropha plants, this region could be used as a reproductive tissue-specific promoter in transgenic studies of Jatropha.
Author contribution statement
Y-B Tao and Z-F Xu designed research and wrote the paper, Y-B Tao performed research, L-L He helped in data analysis, and L-J Niu helped in collection of plant materials.
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This work was supported by funding from the National Natural Science Foundation of China (31370595), the CAS 135 program (XTBG-T02), and the Top Science and Technology Talents Scheme of Yunnan Province (2009CI123). The authors gratefully acknowledge the Central Laboratory of the Xishuangbanna Tropical Botanical Garden for providing research facilities.
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Tao, YB., He, LL., Niu, L. et al. Isolation and characterization of the Jatropha curcas APETALA1 (JcAP1) promoter conferring preferential expression in inflorescence buds. Planta 244, 467–478 (2016). https://doi.org/10.1007/s00425-016-2519-1
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