Heterologous activation of the Hevea PEP16 promoter in the rubber-producing laticiferous tissues of Taraxacum kok-saghyz

Hevea brasiliensis, the most abundant rubber crop, is used widely for the commercial production of natural rubber. To reduce the risk of a shortage in the supply of natural rubber that may arise from a single major rubber crop, rubber dandelion (Taraxacum kok-saghyz) has been developed as an alternative rubber-producing crop by using a transgenic approach. However, it is necessary to identify a suitable promoter for the transfer of rubber biosynthesis-related genes to the species. In this study, the promoter region of H. brasiliensis PEP16, which was isolated as a potentially important component in rubber biosynthesis, was sequenced and a pPEP16::GUS fusion construct was introduced into T. kok-saghyz. Histological and fluorometric studies using transgenic T. kok-saghyz plants indicated that the HbPEP16 promoter was highly activated in a laticiferous tissue-specific manner under normal growth conditions and that promoter activation was tightly regulated by various hormones and external signals. These findings suggested that the HbPEP16 promoter may be a useful molecular tool for the manipulation of gene expression in the laticiferous tissues of T. kok-saghyz.

Hevea brasiliensis, the most abundant rubber crop, is used widely for the commercial production of natural rubber. To reduce the risk of a shortage in the supply of natural rubber that may arise from a single major rubber crop, rubber dandelion (Taraxacum kok-saghyz) has been developed as an alternative rubber-producing crop by using a transgenic approach. However, it is necessary to identify a suitable promoter for the transfer of rubber biosynthesis-related genes to the species. In this study, the promoter region of H. brasiliensis PEP16, which was isolated as a potentially important component in rubber biosynthesis, was sequenced and a pPEP16::GUS fusion construct was introduced into T. koksaghyz. Histological and fluorometric studies using transgenic T. kok-saghyz plants indicated that the HbPEP16 promoter was highly activated in a laticiferous tissue-specific manner under normal growth conditions and that promoter activation was tightly regulated by various hormones and external signals. These findings suggested that the HbPEP16 promoter may be a useful molecular tool for the manipulation of gene expression in the laticiferous tissues of T. kok-saghyz.
Natural rubber (cis-1,4-polyisoprene) is an important raw material used in the manufacture of a wide variety of industrial products. Currently, it is sourced commercially from the Para rubber tree Hevea brasiliensis 1 , which produces an abundance of high-quality rubber that is relatively easy to harvest 2,3 . However, the decline in rubber tree plantations and the prevalence of life-threatening allergies to Hevea latex, coupled with an increase in demand, have stimulated research into the development of alternative rubber crops 4,5 . Although more than 2,000 plant species that produce natural rubber have been identified 6 , only a few of these species can produce comparable quantities of high-molecular-weight rubber to Hevea's rubber 5,7 . Among these species, the Mexican shrub guayule (Parthenium argentatum) and the rubber dandelion (Taraxacum kok-saghyz) have been examined widely as alternative rubber yielding crops.
One strategy for the development of alternative rubber crops is the identification of the key regulatory genes in the rubber biosynthesis processes and manipulate the expression of these key genes in the laticiferous tissues where natural rubber is synthesized. To induce the expression of the target genes in the tissues, a laticiferous tissue-specific promoter is required. Although several studies have previously attempted to isolate laticiferous tissue-specific promoters [8][9][10][11][12][13] , a suitable promoter for the transformation of rubber dandelion T. kok-saghyz has not yet been identified.
HbPEP16 (GenBank no. MN326440) is a protein attached to the rubber particles in the latex of H. brasiliensis. In our rubber biosynthesis activity assays that examined the fractions of proteins dissociated from rubber particles, the fractions that showed enhanced rubber biosynthesis activities beyond basal levels commonly contained proteins such as HbPEP16, indicating that it may play a role in rubber biosynthesis processes 14  Isolation and sequence analysis of the Hevea brasiliensis PEP16 promoter. Genomic DNA (gDNA) was isolated from the young leaves of Para rubber trees, and gDNA extraction was performed following a previously described method 15 . The proximal promoter region of the HbPEP16 gene was obtained by inverse PCR from the extracted gDNA 16 . The HincII-digested gDNA was self-ligated and used as a template for inverse PCR with primers designed from the first exon of the PEP16 gene ( Supplementary Fig. S1). The products for the first round of PCR were generated using a combination of primers (5′-GAT GAT ATC GAA TGC AGA AGC-3′ and 5′-CAA GAC ATC CTT CGC CAT GT-3′; 5′-GAT ACT GCA CCT TAT CAA CAC-3′ and 5′-CAG CAT GGA TTC GAA GCA AG-3′). The second round of PCR was performed using the 50 ×-diluted solution of the PCR products as a template, with the primers (5′-CTG AAA GTA ATC AAT CTG CAGC-3′ and 5′-GGA TCA CTA TGT TCA TCA TAG-3′). Subsequently, the second-round PCR product was cloned into a pGEM-T vector (Promega) and sequenced ( Supplementary Fig. S2). The sequence obtained was compared with previously published sequences in the NCBI database using BLASTN 17 . However, no significant sequence similarity was found. To identify cisacting regulatory elements within the PEP16 promoter, the promoter was analyzed using the PlantCARE database Fig. 1. www.nature.com/scientificreports/ plasmid construction and transformation. The pGEM-T cloned PEP16 promoter (1079 bp) was subcloned into the pGA3383 binary vector 18 . The sequence was inserted between the BamHI and HpaI sites to generate the pPEP16::GUS fusion construct Fig. 2A. The pPEP16::GUS fusion construct was verified by sequencing. Agrobacterium tumefaciens LBA4404 was transformed with the construct using the freeze-thaw method 19 .
Generation and identification of transgenic T. kok-saghyz plants. Transgenic T. kok-saghyz plants carrying pPEP16::GUS were obtained via Agrobacterium-mediated transformation 10,20 . Dandelion leaf explants were immersed in a suspension of Agrobacterium carrying pPEP16::GUS for 20 min at 25 °C and were shaken gently (50 rpm) 10 . The explants were co-cultured on regeneration medium [MS salts, 3% sucrose, 0.01 mg/L 3-indolebutyric acid (IBA), 2 mg/L benzyl aminopurine (BA), 0.3% phytagel, pH 5.8) for 3 days in the dark at 25 °C. After co-cultivation, the explants were washed with cefotaxime (500 mg/L) to remove Agrobacterium. The explants were then transferred to a regeneration medium supplemented with hygromycin B (25 mg/L) and cefotaxime (500 mg/L). After 3 weeks of culture in the dark, the tissues were maintained on the same medium for 4 weeks under continuous light conditions 10 . For rooting, explants with shoots were transferred to 1/2 MS medium containing 1.5% sucrose, 0.3% phytagel, and 25 mg/L hygromycin B at pH 5.8. In vitro rooted transgenic plants were transferred to small plastic trays containing top soil, vermiculite, and peat moss (2:1:0.5 v/v) and kept in a greenhouse.
RT-PCR analysis. RT-PCR was performed to identify GUS expression in the root tissue of transgenic T. koksaghyz plants carrying pPEP16::GUS. Total RNA was extracted from the root tissues using the Qiagen RNeasy Plant Mini Kit 21 . RNA samples were qualitatively and quantitatively analyzed using a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Approximately 1 µg of total RNA was reversetranscribed using the TOP Script RT Dry Mix (Enzynomics, South Korea) according to the manufacturer's protocol 22 . RT-PCR was performed with the prepared cDNA and GUS primers (5′-TGC AGA TAT TCG TAA TTA TGCG-3′ and 5′-CAA CAG ACG CGT GGT TAC AG-3′) 10 . As a reference gene, the T. kok-saghyz ACTIN gene was amplified using the primers 5′-CTT TTC CAT GTC GTC CCA GT-3′ and 5′-CTG GGT TTG CTG GTG ATG AT-3′. The PCR products were visualized using ethidium bromide after separation on agarose gels 10 .

Treatment of external signals and hormones.
In vitro-propagated surviving plants were grown for 2 months in a greenhouse. The transgenic plants were then exposed to cold conditions, salt, dark conditions, www.nature.com/scientificreports/ light after dark conditions, and hormones. To induce salt stress, the plant roots were gently pulled out of the soil pots and dipped in 100 mM NaCl for 12 h. For cold and dark stresses, the plants were kept in the pots and placed in a cold room for 1 day (4 °C at 12 h/12 h day/night) and 5 days in darkness at 22  Histochemical GUS activity analysis. The histochemical localization of GUS activity in transgenic T.
kok-saghyz plants was performed using the substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) with an oxidative catalyst 11,23 . The plant roots and leaves were washed with water and submerged in vials containing 1 × GUS solution (1 mM X-Gluc, 50 mM sodium phosphate buffer, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM EDTA, 0.1% Triton X-100, pH 7.0) 10 . The vials were incubated at 37 °C for 3 h in the dark. Pigments and chlorophyll were cleared by soaking the tissues in 95% ethanol. GUS staining in the longitudinal and transverse root sections was observed by using an anatomical microscope or Nikon Microphot-FXA microscope 10 . For the latex GUS assay, freshly tapped latex from the junction between the root and shoot was collected in ice-cold Eppendorf tubes containing 100 mM sodium phosphate buffer (pH 7.5) 10 . The collected latex was centrifuged (17,000 × g, 10 min, 4 °C) to separate the aqueous latex phase from the pellet 10 . Aqueous latex (60 µL) was collected and 6 µL of 10 × -concentrated GUS solution was added to a concentration of 1 × . The tubes were incubated in the dark in a 37 °C water bath for 3 h 10 .
Fluorometric GUS activity analysis. Fluorometric assays of GUS activity were conducted following the method of Jefferson et al. 23 , to quantify the levels of GUS enzyme activity in the root tissues of transgenic T. koksaghyz plants carrying pPEP16::GUS. Plant tissues were homogenized in a GUS assay buffer (50 mM potassium phosphate, 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarcosyl, 2 mM DTT, and 10 μg/mL cycloheximide), and an aliquot of the supernatant was incubated after 4-methylumbelliferyl-β-D-glucuronide (4-MUG) was added as the substrate at 37 °C for 2 h 10 . The amount of 4-methylumbelliferone (4-MU) formed by the GUS reaction was determined using a 96-well microtiter plate reader. Protein concentrations were determined following the method described by Bradford 24 , using a Coomassie protein assay kit (Bio-Rad) with BSA as the standard. Statistical analysis. All data are expressed as the mean value of at least three biological replicates. The data were analyzed using t-tests, and differences were considered statistically significant for a P value of < 0.05 22 . databases were used to identify matches in the HbPEP16 promoter to the cis-regulatory elements of other plant species Fig. 1. Several types of regulatory elements were identified in the HbPEP16 promoter. These included cis-acting elements, Box 4, chs-CMA2a, G-box, GAG that are involved in responses to light, HSE which is involved in heat stress responsiveness; ERF, consisting of TC-rich repeat elements, which is involved in the defense response; and DRE and ZAT10, which function under drought, low temperature, and high salt tolerance. Also identified were circadian elements involved in the circadian control and cis-acting elements involved in phytohormone responsiveness including the GARE-motif for responding to gibberellin, EIN3 for responding to ethylene, the TGA-element for responding to auxin, and the TGACG motif involved in the response to methyl jasmonate. A-box, which is responsive to α-amylase promoters, was identified in the promoter region. These cis-acting elements may modulate gene expression in a tissue-specific manner during growth and development.

cis-Regulatory
Generation of transgenic plants containing the pPEP16::GUS construct. The main purpose of the current study was to isolate a promoter that was specifically activated in laticiferous tissues, to provide a tool to improve the quantity and quality of rubber in rubber-producing plants by engineering the rubber biosynthetic pathways more precisely. The rubber dandelion plant, T. kok-saghyz, is a good plant model as well as an alternative rubber crop owing to its ease of planting and short maturation time. Thus, transgenic T. kok-saghyz plants were generated to express pPEP16::GUS. Using inverse PCR, 1,079 bp of the HbPEP16 promoter was isolated (Fig. S1) and sequenced (Fig. S2). The HbPEP16 promoter fragment was cloned upstream of the GUS gene of the pGA3383 vector Fig. 2A.

Alteration of HbPEP16 promoter activity by environmental signals. Analysis of the HbPEP16
promoter sequence revealed a few cis-elements for light, low temperature, and salt-based signals. To test this possibility, the HbPEP16 promoter activity in transgenic T. kok-saghyz plants carrying pPEP16::GUS was investigated in response to dark, light, salt, and cold conditions. Histochemical staining showed that the HbPEP16 promoter as highly activated in the laticiferous tissues of untreated control transgenic plants under normal growth conditions Fig. 4A. However, when exposed to the salt (100 mM NaCl) treatment, GUS expression was dramatically lower in the laticiferous tissues of the treated plants than in those of control plants under normal growth conditions Fig. 4B. The fluorometric assay revealed that the quantitative results of GUS activity were consistent with those of histochemical studies Fig. 5A. In response to the cold treatment, GUS expression was also lower than that in the control Fig. 4C. The fluorometric assay showed lower GUS activity under cold treatment, but this was not significant Fig. 5A. GUS expression was greatly reduced following the 5-day dark condition Fig. 4D, left), but resumed slightly at 12 h Fig. 4D, middle) and completely at 60 h Fig. 4D, right) after the plants were Regulation of the HbPEP16 promoter by hormonal signals. Analysis of the PEP16 promoter sequence revealed cis-elements for hormones such as ethylene, auxin, GA 3 , MeJA, and ABA (drought). To evaluate the hormonal regulation activity of the HbPEP16 promoter in transgenic T. kok-saghyz plants carrying pPEP16::GUS, GUS activity was measured after the roots of transgenic plants were treated with the different hormones. By fluorometric analysis, the highest GUS activity was measured in the ABA treatment Fig. 5C. MeJA enhanced GUS activity to levels comparable with those achieved by ABA. A significant reduction in GUS activity was observed after auxin (NAA) treatment. However, treatments with GA3 and ethylene-releasing ethephon did not affect GUS expression significantly, although a slight decrease or increase in the activity was observed, respectively Fig. 5C.

Discussion
Owing to the increased demand for natural rubber, and the risk of collapse of the current major rubber crop, Hevea, an alternative source of rubber is required. T. kok-saghyz is grown widely in various temperate zones and may be a good alternative source of natural rubber, as the rubber quality of T. kok-saghyz is similar to that of H. brasiliensis 1 . To be competitive as a commercial rubber crop, T. kok-saghyz needs to be more productive and have a higher rubber content. To enhance the rubber yield in T. kok-saghyz, transgenic approaches have been studied 1 . To express the transferred genes in the laticiferous tissues of T. kok-saghyz, where rubber biosynthesis occurs, molecular tools, such as tissue-specific promoters, are required. In a previous study with Taraxacum brevicorniculatum 10 , we reported a laticifer tissue-specific promoter, the Hevea SRPP gene promoter. However, the HbSRPP promoter activity is only weakly detected under normal growth conditions and strongly activated under cold conditions, which are not favorable for rubber biosynthesis. In addition, the ToPPO-1 promoter also showed weak activity in the GUS staining of transgenic dandelion plants grown at normal temperatures 11 . In this study, we isolated the promoter of the HbPEP16 gene, which is strongly activated under normal growth conditions but inactivated under stress conditions, including salt and darkness. Whether the expression levels are desirable or sufficient may be dependent on target genes, as the required expression level differs according www.nature.com/scientificreports/ to their cellular functions. It is also important to consider whether the promoter activity is modulated in parallel with the activity of the specific target metabolism, for example, rubber biosynthesis. The transgenic T. kok-saghyz plants were generated to carry pPEP16::GUS because HbPEP16 was identified in our preliminary studies as a potential component of the rubber biosynthesis process. Strong GUS activity driven by the HbPEP16 promoter was observed in root sections of the transgenic plants. The HbPEP16 promoter activity was observed in dot-like concentric ring structures in the secondary phloem regions of the transversely sectioned T. kok-saghyz roots. Strong GUS activity driven by the HbPEP16 promoter was also pronounced in the latex of T. kok-saghyz. In contrast, the control (WT) plants showed no GUS activity and a low level of GUS staining was detected only in the main vein of the leaf tissue of the transgenic plants, where some latex was also present. This clearly showed the expression of the HbPEP16 promoter in the laticiferous tissues of T. kok-saghyz. Notably, the transgenic T. kok-saghyz plants showed no abnormality in photosynthetic efficiency compared with the WT. The values of F0, Fm and Fv/Fm were not significantly different between WT and the T. kok-saghyz transgenic plants (Supplementary Fig. S3).
The cis-acting elements responding to abiotic stresses such as light, salt, and cold were predicted in the HbPEP16 promoter sequence. Therefore, it was expected that the HbPEP16 promoter was regulated by these external signals. The histochemical data indicated that the HbPEP16 promoter was greatly downregulated in response to external signals such as salt and dark stresses. The fluorometric assay showed quantitative results that were consistent with those obtained with the histochemical GUS expression assay. In contrast, the light signal was found to positively regulate the HbPEP16 promoter immediately in plants subjected to darkness. This may be due to the presence of multiple cis-acting elements, namely Box-4, chs-CMA2a, G-box, and GAG motif, which are responsive to light. www.nature.com/scientificreports/ In addition to abiotic stress regulatory elements, cis elements responding to hormones such as ABA, ethephon, MeJA, GA 3 , and NAA were also identified in the promoter region of HbPEP16. ABA responds to various environmental stressors 28,29 . Following ABA treatment of the root tissues of the transgenic T. kok-saghyz plants, GUS expression was enhanced in the root laticiferous tissues compared with the control, indicating that the promoter activity was enhanced by the ABA hormone. MeJA plays an important role in plant defense reactions 30 . Upon treatment of the transgenic T. kok-saghyz plant root tissues with MeJA, increased GUS expression was observed in the root tissues, to a similar extent as that observed with ABA treatment. Previous studies have shown that exposure to exogenous MeJA and wounding can stimulate natural rubber production in rubber trees 31,32 . Furthermore, JA is believed to be a general inducer of natural rubber biosynthesis and activator of some genes related to it 33,34 . Therefore, these results support the hypothesis that the HbPEP16 promoter is activated in a synchronized manner during rubber biosynthesis activity. Ethylene, a ripening hormone, plays an important role in the defense mechanism against pathogen attack/invasion. Ethephon treatment of the root tissues did not significantly affect the promoter activity. It was previously reported that ethylene stimulated latex production, but had little direct effect on the acceleration of rubber biosynthesis in H. brasiliensis when applied as ethephon 35 . The phytohormone gibberellin (GA 3 ) is a significant growth regulator in promoting plant growth 36 and the antagonistic interactions between ABA and GA responsive elements have been reported 37,38 . Auxin, another plant growth-promoting hormone, downregulated the promoter activity to a greater extent. In line with this, the exogenous auxin treatment of plant root tissues has been found to inhibit primary root growth 39 .
Both external and internal signals influenced HbPEP16 gene promoter activity. In rubber plants, rubber is produced from the stored photosynthetic products 1 and it has been proposed that rubber content per latex volume varies diurnally owing to the diurnal variation in HMG-CoA reductase activity 40 . The reduced HbPEP16 activity under prolonged darkness observed in this study may be due to the absence or reduction in the major photosynthetic components involved in rubber biosynthesis, namely acetyl-CoA, NADPH, and ATP. Decreased GUS expression under salt or dark stress conditions denotes the downregulation of the HbPEP16 promoter in unfavorable environments. Among plant hormones, cis elements responding to ABA and MeJA positively affected the HbPEP16 promoter when the root tissues were directly exposed to the hormones. It should be noted that the effects of the hormones on HbPEP16 promoter activity in the root tissues may be different when the hormones are treated on the leaf tissues. This remains to be further investigated in the future.
In conclusion, we identified and characterized the HbPEP16 gene promoter, which can drive tissue-specific gene expression during the growth and development of T. kok-saghyz plants. This study demonstrates that HbPEP16 is a laticiferous tissue-specific promoter, which is highly activated under normal growth conditions and is tightly regulated by several internal and external stimuli. This may facilitate the appropriate spatial and temporal expression of transfer genes in transgenic plants of the rubber-producing dandelion.