Share This Article:

Genome-Wide Identification of Two-Component Signal Transduction System Genes in Melon (Cucumis melon L.)

Abstract Full-Text HTML XML Download Download as PDF (Size:1404KB) PP. 469-479
DOI: 10.4236/as.2018.94032    417 Downloads   673 Views  
Author(s)    Leave a comment
Panjing Liu1,2,3,4, Xiaoyu Yang5, Yana Zhang1,2,3,4, Shuoshuo Wang1,2,3,4, Qian Ge1,2,3,4, Qiang Li1,2,3,4, Chao Wang1,2,3,4, Qinghua Shi1,2,3,4, Zhonghai Ren1,2,3,4*, Lina Wang1,2,3,4*

ABSTRACT

Two-component system (TCS) is responsible for cytokinin signaling, which plays critical roles in plant development and physiological process. This system is generally composed of two signaling factors, a histidine kinase (HK) and a response regulator (RR) that is associated with a histidine phosphotransfer (HP) protein. In this study, we performed systematic investigation on TCS genes in melon (Cucumis melon L.). We identified 44 TCS genes in melon, including 18 HK(L)s (9 HKs and 9 HKLs), 5 HPs (4 authentic and 1 pseudo), and 21 RRs (7 Type-A, 8 Type-B, and 6 pseudo). The classification and structure of these melon TCS members were introduced in detail as well. Our results provided new insights into the characteristics of the melon TCS genes and might benefit their functional study in future.

1. Introduction

Cytokinins are essential for many of the physiological and developmental processes such as seed germination, functional root nodule establishment, lateral root development, shoot apical meristem maintenance, leaf expansion, flowering, circadian clock, nutrient mobilization, abiotic stress, and senescence [1] [2] [3] [4]. In eukaryotes such as yeast and plant, a two-component system (TCS) has been reported for the transduction of cytokinin signal [5] [6]. This TCS consists of two signaling factors, a histidine kinase (HK) gene family and a response regulator (RR) gene family [7]. HK can sense the cytokinin signals by phosphorylating its conserved histidine residues. The phosphoryl group is then transferred to a conserved asparagine residue on the receiver (Rec) domain of an RR, which modulates the activity of concerned downstream genes directly or indirectly [1] [7]. In addition, histidine phosphotransfer (HP) genes are regarded as the mediators for the transfer of the phosphoryl group between the HKs and the RRs (Figure 1) [6] [8] [9].

Melon (Cucumis melon L.) is an economically important fruit crop that originates from Asian, with an average production during the past decade more than 29 million tons per year (FAOSTAT, 2017; http://www.fao.org/faostat/en/#home). This crop is mainly cultivated in tropical and temperate countries, especially in the Asian countries, with China leading the list [10] [11]. Although melon is a eudicot of interest for its specific biological properties [10] [12] , there is still no genome-wide investigation on melon TCS genes. In this study, we examined the putative TCS genes and revealed that the melon genome contained a total of 44 members. Their classification and characteristics were also analyzed systematically. Our comprehensive analysis of the TCS genes might provide a framework for future functional dissection of TCSs in melon hormone signal transduction.

2. Materials and Methods

2.1. Data Collection

Protein sequence data of Arabidopsis AHKs, AHPs and ARRs were downloaded from the NCBI databases (https://www.ncbi.nlm.nih.gov/). Genome sequence data of melon deposited in the website of Cucurbit Genomics Data (http://cucurbitgenomics.org/organism/3) were used for TCS gene identification and analysis.

2.2. Identification of the Putative Melon TCS Genes

Previously cucumber and watermelon TCS members have been successfully identified by using Arabidopsis TCS genes [13]. So we also used Arabidopsis TCS protein sequences as queries to search for the putative counterparts in melon by BLASTP with E-value of 1e-5 [14]. The Pfam (http://pfam.janelia.org) and SMART (http://smart.embl-heidelberg.de/) tools were used to check whether these genes contained the structural characteristics and conserved domains of TCS elements, i.e., HisKA (Histidine Kinase A phosphoacceptor) domain, HATPase (histidine kinase-like ATPase) domain, Hpt (histidine-containing phosphotransfer) domain, and Rec domain. Information of abbreviation notation used in this article was listed in Table A1. Thereafter the identity of melon TCS genes with Arabidopsis was analyzed by BLASTP against Arabidopsis databases in TAIR (http://www.arabidopsis.org/). Their CDS and protein sequences

Figure 1. Model of the two-component signal transduction pathway. Cytokinin responses (CREs), histidine-kinases (HKs), histidine phosphotransfer proteins (HPs) and type-B response regulators (RRs) work as a positive regulatory loop and transfer the cytokinin from plasma membrane (PM) to nucleus. Type-B RRs act as transcription factors to regulate some cytokinin targets, including type-A RRs. The type-A RRs can inhibit their own transcription, providing a negative feedback mechanism. H: phospho-accepting histidine residue. D: aspartate residue. P: phosphoric acid groups.

together with position information in melon genome were obtained from Cu

curbit Genomics Database (http://cucurbitgenomics.org/organism/3). The transmembrane domains of melon TCS proteins were analyzed by TMHMM Server v.2.0 (http://pfam.janelia.org).

2.3. Phylogenetic Analysis and Gene Structure Construction

Phylogenetic analysis of the full-length protein sequences was conducted using MEGA5 [15]. The evolutionary history was inferred using the Neighbor-Joining method with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates) [16] [17]. The DNA and cDNA sequences corresponding to each predicted genes were downloaded from the melon genome database (http://cucurbitgenomics.org/organism/3), and then the gene structures were analyzed using the Gene Structure Display Server online tool (http://gsds.cbi.pku.edu.cn/).

3. Results and Discussion

The TCS signaling is widely present in higher plant, including Arabidopsis thaliana [8] [9] , rice (Oryza sativa) [18] , lotus (Lotus japonicus) [19] , soybean (Glycine max) [14] [20] , maize (Zea maize) [21] , Physcomitrella patens [22] [23] , and wheat (Triticum aestivum L.) [24] , as well as horticultural crops such as Chinese cabbage (Brassica rapa) [20] , tomato (Solanum lycopersicum) [25] , cucumber (Cucumis sativus L.) [13] and watermelon (Citrullus lanatus) [13] (Table 1). In Arabidopsis, there are 56 TCS genes and their functions have been extensively studied [8] [9]. To find the putative TCS members in melon, we performed a BLASTP search against the melon genome database by using 56 Arabidopsis TCS protein sequences. A total of 90 genes were selected as putative TCS genes including 26 HK(L)s, 6 HPs, 58 RRs in the melon genome. To confirm these putative melon TCS genes, the amino acid sequences of all 90 genes were further filtered by Pfam and SMART based on the presence of structural and conserved TCS elements. Finally, 44 typical TCS genes including 18 HK(L)s, 5 HPs, 21 RRs were identified in melon (Table 1). To better reflect the paralogous relationship, all melon TCS members were named according to their homology with Arabidopsis counterparts.

The identified 18 putative CmHK(L)s in melon were separated as 9 CmHKs and 9 CmHKLs according to the presence or absence of conserved residues required for histidine kinase activity (Figure 2(a)). Further they were classified to four distinct gene families: the typical CmHK family (four cytokinin receptor-like CmHKs, one CKI1-like CmHK, one CKI2/AHK5-like CmHK, and one AHK1-like CmHK), the ethylene response (ETR) homolog family (two ETR1-like CmHKs, one ETR2-like CmHKLs), the phytochromes (PHY) (six PHY-like CmHKLs) and the pyruvate dehydrogenase kinase (PDK) family (two PDK-like CmHKLs) (Table 2). The protein sequences of these CmHK(L)s ranged from 352 to 1261 amino acids, indicating great variations in their structures and possible functions (Table 2).

Table 1. Summary of the TCS gene number identified in plants.

Figure 2. Phylogenetic relationship and gene structure of the Histidine kinases (a), Histidine phosphotransfer proteins (b) and Response regulators (c) in melon. The phylogenetic tree was constructed based on the Neighbor-Joining method by MEGA5. Bootstrap supports from 1000 replicates were indicated at each branch. The gene structure was analyzed using the Gene Structure Display Server online tool.

Four authentic HPs and one pseudo-HP (PHP) with a pseudo-Hpt domain were identified in melon genome (Figure 2(b)). The CmHP1, CmHP2 and CmHP3 had a close relationship with AHP1 (Figure 2(b), Table 3), a positive regulators in CK signaling [26] , while the CmHP4 was close to AHP4 (Table 3), which was evolutionarily distinct from the other AHPs and functioned as a negative regulator in CK signaling [26]. CmPHP1 exhibited the longest CDS and

Table 2. Features of HK genes in melon.

Note: a. Systematic names given to genes by Cucurbit Genomics Database. b. Features indicated conserved histidine-kinase (HK) domain, diverged histidine-kinase like (HKL) domain, receiver (Rec) domain, cyclases/histidine kinases associated sensory extracellular (CHASE) domain, cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain, Per-ARNT-Sim (PAS) domain, and phytochrome (PHY) domain. c. Number of TM (transmembrane) from TMHMM Server v. 2.0 (http://pfam.janelia.org). d. The proteins belonged to which family in Arabidopsis, including cytokinin independent (CKI), Arabidopsis histidine-kinase (AHK), ethylene response (ETR), phytochrome (PHY), and pyruvate dehydrogenase kinase (PDK).

Table 3. Features of HP genes in melon.

Note: a. Systematic names given to genes by Cucurbit Genomics Database. b. Features included conserved histidine-containing phosphotransfer (HPt) domain and a pseudo-HPt domain lacking the histidine phosphorylation site. c. Number of TM (transmembrane) from TMHMM Server v. 2.0 http://pfam.janelia.org ). d. The proteins belonged to Arabidopsis histidine phosphotransfer (AHP) family.

amino acid sequence (Table 3) and had close relationship with Arabidopsis AHP6, which functioned as a competitor of other AHPs and played a negative role in CK responses by interfering with phosphorelay [27].

There were 21 protein-coding genes in the melon genome that were predicted as RRs (Table 4). Also there were 6 genes encoding RRs without the essential residues that were required for biological activity, and were thus named as pseudo-RRs (PRRs) (Table 4). Among these RRs/PRRs, we identified seven type-A RRs (CmRR1-7), each of which contained a Rec domain along with short C-terminal extension (Table 4, Figure 2(c)). Furthermore, these type-A CmRRs exhibited close relationship to their homologs, namely, ARR3, ARR5 and ARR9 in Arabidopsis (Table 4). Genetic analysis suggests that ARR3, ARR5 and ARR9 could function as negative regulators in cytokinin signaling, thus possibly

Table 4. Features of RR genes in melon.

Note: a. Systematic names given to genes by Cucurbit Genomics Database. b. Features included receiver (Rec) domain, pseudo-receiver (Pseudo-Rec) domain, Myb-like DNA binding domain and CCT (CO, COL and TOC1) motif. c. Number of TM (transmembrane) from TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/d). d. The proteins belonged to Arabidopsis response regulator (ARR) or Arabidopsis pseudo-response regulator (APRR) family.

participating in a negative feedback loop to reduce the plant sensitivity to cytokinins [28] [25].

There were eight type-B RR genes in melon, of which 6 members were transcription factors (TFs) and contained long C-terminal extensions with MYB-like DNA binding domains (Table 4). The other two type-B RRs, CmRR8 and CmRR9, only had Rec domains and their MYB-like domains might be lost during the evolution of the melon RR family (Table 4). These type-B CmRRs shared high sequence similarities to their homologs, ARR2, ARR11 and ARR12, in Arabidopsis (Table 4). It has been reported that Arabidopsis ARR2 and ARR12 play key roles in ethylene and CK signaling, respectively [29] [30] [31] [32] [33].

Six melon PRRs contained highly-diverged Rec domains (Table 4) and C-terminal extensions. Intriguingly, the CCT-domain and MYB-like domain in type-B RRs were also found in CmPRR1/CmPRR6 and CmPRR2/CmPRR3, respectively. However, the exception occurred to the CmPRR4 and CmPRR5, which lacked both the CCT and the MYB-like domains (Table 4). These great divergences should be paid more attentions on in the future study.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31401894 and 31501781), “Taishan Scholar” Foundation of the People’s Government of Shandong Province, and China Postdoctoral Science Foundation (2017M612741).

Appendix

Table A1. Information of abbreviation notation.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Liu, P. , Yang, X. , Zhang, Y. , Wang, S. , Ge, Q. , Li, Q. , Wang, C. , Shi, Q. , Ren, Z. and Wang, L. (2018) Genome-Wide Identification of Two-Component Signal Transduction System Genes in Melon (Cucumis melon L.). Agricultural Sciences, 9, 469-479. doi: 10.4236/as.2018.94032.

References

[1] Hwang, I, Sheen, J., and Müller, B. (2012) Cytokinin Signaling Networks. Annual Review of Plant Biology, 63, 353-380.
https://doi.org/10.1146/annurev-arplant-042811-105503
[2] Sasaki, T., Suzaki, T., Soyano, T., Kojima, M., Sakakibara, H., and Kawaguchi, M. (2014) Shoot-Derived Cytokinins Systemically Regulate Root Nodulation. Nature Communications, 5, 4983.
https://doi.org/10.1038/ncomms5983
[3] Nitschke, S., Cortleven, A., Iven, T., Feussner, I., Havaux, M., Riefler, M. and Schmülling, T. (2016) Circadian Stress Regimes Affect the Circadian Clock and Cause Jasmonic Acid-Dependent Cell Death in Cytokinin-Deficient Arabidopsis Plants. Plant Cell, 28, 1616-1639.
https://doi.org/10.1105/tpc.16.00016
[4] Kang, J., Lee, Y., Sakakibara, H. and Martinoia, E. (2017) Cytokinin Transporters: Go and Stop in Signaling. Trends in Plant Science, 22, 455-461.
https://doi.org/10.1016/j.tplants.2017.03.003
[5] Thomason, P. and Kay, R. (2000) Eukaryotic Signal Transduction via Histidine-Aspartate Phosphorelay. Journal of Cell Science, 113, 3141-3150.
http://jcs.biologists.org/content/joces/113/18/3141.full.pdf
[6] Urao, T., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2000) Two-Component Systems in Plant Signal Transduction. Trends in Plant Science, 5, 67-74.
https://doi.org/10.1016/S1360-1385(99)01542-3
[7] Stock, A.M., Robinson, V.L. and Goudreau, P.N. (2000) Two-Component Signal Transduction. Annual Review of Biochemistry, 69, 183-215.
https://doi.org/10.1146/annurev.biochem.69.1.183
[8] Hwang, I., Chen, H.C. and Sheen, J. (2002) Two-Component Signal Transduction Pathways in Arabidopsis. Plant Physiology, 129, 500-515.
https://doi.org/10.1104/pp.005504
[9] Schaller, G.E., Kieber, J.J. and Shiuc, S.H. (2008) Two-Component Signaling Elements and Histidyl-Aspartyl Phosphorelays. Arabidopsis Book, 6, e0112.
https://doi.org/10.1199/tab.0112
[10] Garcia-Mas, J., Benjak, A., Sanseverino, W., Bourgeois, M., Mir, G., González, V.M., Hénaff, E., Camara, F., Cozzuto, L., Lowy, E., Alioto, T., Capella-Gutiérrez, S., Blanca, J., Cañizares, J., Ziarsolo, P., Gonzalez-Ibeas, D., Rodríguez-Moreno, L., Droege, M., Du, L., Alvarez-Tejado, M., Lorente-Galdos, B., Melé, M., Yang, M., Weng, Y.Q., Navarro, A., Marques-Bonet, T., Aranda, M.A., Nuez, F., Picó, B., Gabaldón, T., Roma, G., Guigó, R., Casacuberta, J.M., Arús, P. and Puigdomènech., P. (2012) The Genome of Melon (Cucumis melo L.). PNAS, 109, 11872-11877.
https://doi.org/10.1073/pnas.1205415109
[11] Díaz, A., Martín Hernández, A.M., Dolcett-Sanjuan, R., Garcés-Claver, A., álvarez, J.M., Garcia-Mas, J., Picó, B. and Monforte, A.J. (2017) Quantitative Trait Loci Analysis of Melon (Cucumis melo L.) Domestication-Related Traits. Theoretical and Applied Genetics, 130, 1837-1856.
https://doi.org/10.1007/s00122-017-2928-y
[12] Chang, C.W., Wang, Y.H. and Tung, C.W. (2017) Genome-Wide Single Nucleotide Polymorphism Discovery and the Construction of a High-Density Genetic Map for Melon (Cucumis melo L.) Using Genotyping-by-Sequencing. Frontiers in Plant Science, 8, 125.
https://doi.org/10.3389/fpls.2017.00125
[13] He, Y.J., Liu, X., Zou, T., Pan, C.T., Qin, L., Chen, L.F. and Lu, G. (2016) Genome-Wide Identification of Two-Component System Genes in Cucurbitaceae Crops and Expression Profiling Analyses in Cucumber. Frontiers in Plant Science, 7, 899.
https://doi.org/10.3389/fpls.2016.00899
[14] Mochida, K., Yoshida, T., Sakurai, T., Yamaguchi-Shinozaki, K., Shinozaki, K. and Tran, L.P. (2010) Genome-Wide Analysis of Two-Component Systems and Prediction of Stress-Responsive Two-Component System Members in Soybean. DNA Research, 17, 303-324.
https://doi.org/10.1093/dnares/dsq021
[15] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-2739.
https://doi.org/10.1093/molbev/msr121
[16] Saitou, N. and Nei, M. (1987) The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees. Molecular Biology and Evolution, 4, 406-425.
[17] Li, Q., Zhao, P., Li, J., Zhang, C., Wang, L. and Ren Z. (2014) Genome-Wide Analysis of the WD-Repeat Protein Family in Cucumber and Arabidopsis. Molecular Genetics and Genomics, 289, 103-124.
https://doi.org/10.1007/s00438-013-0789-x
[18] Pareek, A., Singh, A., Kumar, M., Kushwaha, H.R., Lynn, A.M. and Singla-Pareek, S.L. (2006) Whole-Genome Analysis of Oryza sativa Reveals Similar Architecture of Two-Component Signaling Machinery with Arabidopsis. Plant Physiology, 142, 380-397.
https://doi.org/10.1104/pp.106.086371
[19] Ishida, K., Niwa, Y., Yamashino, T. and Mizuno, T. (2009) A Genome-Wide Compilation of the Two-Component Systems in Lotus japonicus. DNA Research, 16, 237-247.
https://doi.org/10.1093/dnares/dsp012
[20] Liu, Z., Zhang, M., Kong, L., Lv, Y., Zou, M., Lu, G., Cao, J.S. and Yu, X.L. (2014) Genome Wide Identification, Phylogeny, Duplication, and Expression Analyses of Two Component System Genes in Chinese Cabbage (Brassica rapa ssp. pekinensis). DNA Research, 21, 379-396.
https://doi.org/10.1093/dnares/dsu004
[21] Chu, Z.X., Ma, Q., Lin, Y.X., Tang, X.L., Zhou, Y.Q., Zhu, S.W., Fan, J. and Cheng, B.J. (2011) Genome-Wide Identification, Classification, and Analysis of Two-Component Signal System Genes in Maize. Genetics and Molecular Research, 10, 3316-3330.
https://doi.org/10.4238/2011.December.8.3
[22] Ishida, K., Yamashino, T., Nakanishi, H. and Mizuno, T. (2010) Classification of the Genes Involved in the Two-Component System of the Moss Physcomitrella patens. Bioscience, Biotechnology, and Biochemistry, 74, 2542-2545.
https://doi.org/10.1271/bbb.100623
[23] Satbhai, S.B., Yamashino, T., Okada, R., Nomoto, Y., Mizuno, T., Tezuka, Y., Itoh, T., Tomita, M., Otsuki, S. and Aoki, S. (2011) Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella Patens: Insights into the Evolution of the PRR Family in Land Plants. DNA Research, 18, 39-52.
https://doi.org/10.1093/dnares/dsq033
[24] Gahlaut, V., Mathur, S., Dhariwal, R., Khurana, J.P., Tyagi, A.K., Balyan, H.S. and Gupta, P.K. (2014) A Multi-Step Phosphorelay Two-Component System Impacts on Tolerance against Dehydration Stress in Common Wheat. Functional & Integrative Genomics, 14, 707-716.
https://doi.org/10.1007/s10142-014-0398-8
[25] He, Y.J., Liu, X., Ye, L., Pan, C.T., Chen, L.F., Zou, T. and Lu, G. (2016) Genome-Wide Identification and Expression Analysis of Two-Component System Genes in Tomato. International Journal of Molecular Sciences, 17, 1204.
https://doi.org/10.3390/ijms17081204
[26] Hutchison, C.E., Li, J., Argueso, C., Gonzalez, M., Lee, E., Lewis, M.W., Maxwell, B.B., Perdue, T.D., Schaller, G.E., Alonso, J.M., Ecker, J.R. and Kieber, J.J. (2006) The Arabidopsis Histidine Phosphotransfer Proteins Are Redundant Positive Regulators of Cytokinin Signaling. Plant Cell, 18, 3073-3087.
https://doi.org/10.1105/tpc.106.045674
[27] Mähönen, A.P., Bishopp, A., Higuchi, M., Nieminen, K.M., Kinoshita, K., Törmäkangas, K., Ikeda, Y., Oka, A., Kakimoto, T. and Helariutta, Y. (2006) Cytokinin Signaling and Its Inhibitor AHP6 Regulate Cell Fate during Vascular Development. Science, 311, 94-98.
https://doi.org/10.1126/science.1118875
[28] To, J.P., Haberer, G., Ferreira, F.J., Deruere, J., Mason, M.G., Schaller, G.E., Alonso, J.M., Ecker, J.R. and Kieber, J.J. (2004) Type-A Arabidopsis Response Regulators Are Partially Redundant Negative Regulators of Cytokinin Signaling. Plant Cell, 16, 658-671.
https://doi.org/10.1105/tpc.018978
[29] To, J.P.C., Deruere, J., Maxwell, B.B., Morris, V.F., Hutchison, C.E., Ferreira, F.J., Schaller, G.E. and Kieber, J.J. (2007) Cytokinin Regulates Type-A Arabidopsis Response Regulator Activity and Protein Stability via Two-Component Phosphorelay. Plant Cell, 19, 3901-3914.
https://doi.org/10.1105/tpc.107.052662
[30] Mason, M.G., Li, J., Mathews, D.E., Kieber, J.J. and Schaller, G.E. (2004) Type-B Response Regulators Display Overlapping Expression Patterns in Arabidopsis. Plant Physiology, 135, 927-937.
https://doi.org/10.1104/pp.103.038109
[31] Yokoyama, A., Yamashino, T., Amano, Y., Tajima, Y., Imamura, A., Sakakibara, H. and Mizuno, T. (2007) Type-B ARR Transcription Factors, ARR10 and ARR12, Are Implicated in Cytokinin-Mediated Regulation of Protoxylem Differentiation in Roots of Arabidopsis thaliana. Plant and Cell Physiology, 48, 84-96.
https://doi.org/10.1093/pcp/pcl040
[32] Ishida, K., Yamashino, T., Yokoyama, A. and Mizuno, T. (2008) Three Type-B Response Regulators, ARR1, ARR10, and ARR12, Play Essential But Redundant Roles in Cytokinin Signal Transduction throughout the Life Cycle of Arabidopsis thaliana. Plant and Cell Physiology, 49, 47-57.
https://doi.org/10.1093/pcp/pcm165
[33] Hass, C., Lohrmann, J., Albrecht, V., Sweere, U., Hummel, F., Yoo, S.D., Hwang, I., Zhu, T., Schafer, E., Kudla, J. and Harter, K. (2004) The Response Regulator 2 Mediates Ethylene Signalling and Hormone Signal Integration in Arabidopsis. The EMBO Journal, 23, 3290-3302.
https://doi.org/10.1038/sj.emboj.7600337

  
comments powered by Disqus

Copyright © 2019 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.