Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

In silico study of wall-associated kinase family reveals large-scale genomic expansion potentially connected with functional diversification in Populus


The wall-associated kinases (WAKs) are a family of transmembrane proteins involved in pathogen responses and cell elongation in Arabidopsis. They belong to the major receptor-like kinase (RLK) family in plants. Given their architecture and connection to the cell wall, WAKs are thought to perceive and propagate extracellular signals. This study reports the characterization of the WAK family in a woody species based on the v3.0 genome assembly of Populus trichocarpa. In silico analysis revealed a total of 175 PtWAK sequences classified into four groups based on protein domains. Of the PtWAKs, 91.5 % were found in tandem-duplicated clusters contributing to the expansion of the family in poplar. Microarray and EST expression data mining revealed contrasting temporal and spatial expression patterns in stress treatments for several WAK members. The WAKs in poplar form the largest WAK family encountered to date in plants. The combination of phylogenetic and transcriptional data showed that members in nonexpanded clusters were mainly expressed in developmental processes, whereas PtWAKs that had evolved independently in a species-specific way were structured in clusters and were involved in resistance responses. This paper offers an overview of WAK family structure in P. trichocarpa, which will be useful for further functional analysis of the PtWAK family.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Azaiez A, Boyle B, Levée V, Séguin A (2009) Transcriptome profiling in hybrid poplar following interactions with Melampsora rust fungi. Mol Plant Microbe Interact 22:190–200

  2. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble S (2009) MEME Suite: tools for motif discovery and searching. Nucleic Acids Res 37:202–208

  3. Baluška F, Šamaj J, Wojtaszek P, Volkmann D, Menzel D (2003) Cytoskeleton–plasma membrane–cell wall continuum in plants. Emerging links revisited. Plant Physiol 133:482–491

  4. Ben Bâaziz K, Lopez D, Rabot A, Combes D, Gousset A, Bouzid S, Cochard H, Sakr S, Venisse JS (2012) Light-mediated Kleaf induction and contribution of both the PIP1s and PIP2s aquaporins in five tree species: walnut (Juglans regia) case study. Tree Physiol 32:423–434

  5. Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G (2010) A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci U S A 107:9452–9457

  6. Calendini F, Martin JF (2005) PaupUP v1.0.3.1, A free graphical frontend for Paup* Dos software

  7. Cohen D, Bogeat-Triboulot MB, Tisserant E et al (2010) Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes. BMC Genomics 11:630

  8. Decreux A, Messiaen J (2005) Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation. Plant Cell Physiol 46:268–278

  9. Decreux A, Thomas A, Spies B, Brasseur R, Van Cutsem P, Messiaen J (2006) In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis. Phytochemistry 67:1068–1079

  10. Dougherty DA (2012) The cation–π interaction. Acc Chem Res 46:885–893

  11. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

  12. Freisinger E (2011) Structural features specific to plant metallothioneins. J Biol Inorg Chem 16:1035–1045

  13. Goodstein DM, Shu S, Howson R et al (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40:1178–1186

  14. Grisel N, Zoller S, Künzli-Gontarczyk M, Lampart T, Münsterkötter M, Brunner I, Bovet L, Métraux JP, Sperisen C (2010) Transcriptome responses to aluminum stress in roots of aspen (Populus tremula). BMC Plant Biol 10:185

  15. Hamanishi ET, Raj S, Wilkins O, Thomas BR, Mansfield SD, Plant AL, Campbell MM (2010) Intraspecific variation in the Populus balsamifera drought transcriptome. Plant Cell Environ 33:1742–1755

  16. Hanada K, Zou C, Lehti-Shiu MD, Shinozaki K, Shiu SH (2008) Importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli. Plant Physiol 148:993–1003

  17. He ZH, Fujiki M, Kohorn BD (1996) A cell wall-associated, receptor-like protein kinase. J Biol Chem 271:19789–19793

  18. He ZH, He D, Kohorn BD (1998) Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response. Plant J 14:55–63

  19. He ZH, Cheeseman I, He D, Kohorn BD (1999) A cluster of five cell wall-associated receptor kinase genes, Wak1-5, are expressed in specific organs of Arabidopsis. Plant Mol Biol 39:1189–1196

  20. Hou X, Tong H, Selby J, Dewitt J, Peng X, He ZH (2005) Involvement of a cell wall-associated kinase, WAKL4, in Arabidopsis mineral responses. Plant Physiol 139:1704–1716

  21. Humphrey TV, Bonetta DT, Goring DR (2007) Sentinels at the wall: cell wall receptors and sensors. New Phytol 176:7–21

  22. Johnson WE, Li C, Rabinovic A (2007) Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8:118–127

  23. Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–329

  24. Jourez B, Riboux A, Leclercq A (2001) Anatomical characteristics of tension wood and opposite wood in young inclined stems of poplar (Populus euramericana cv ‘ghoy’). IAWA J 22:133–157

  25. Kishino H, Hasegawa M (1989) Evaluation of the maximum likelihood estimate of the evolutionnary tree topologies from DNA sequences data, and the branching order in Hmonoidea. J Mol Evol 29:170–179

  26. Kohler A, Rinaldi C, Duplessis S, Baucher M, Geelen D, Duchaussoy F, Meyers BC, Boerjan W, Martin F (2008) Genome-wide identification of NBS resistance genes in Populus trichocarpa. Plant Mol Biol 66:619–636

  27. Kohorn BD, Kobayashi M, Johansen S, Friedman HP, Fischer A, Byers N (2006) Wall-associated kinase 1 (WAK1) is crosslinked in endomembranes, and transport to the cell surface requires correct cell-wall synthesis. J Cell Sci 119:2282–2290

  28. Kohorn BD, Johansen S, Shishido A, Todorova T, Martinez R, Defeo E, Obregon P (2009) Pectin activation of MAP kinase and gene expression is WAK2 dependent. Plant J 60:974–982

  29. Kohorn BD, Kohorn SL, Todorova T, Baptiste G, Stansky K, McCullough M (2012) A dominant allele of Arabidopsis pectin-binding wall-associated kinase induces a stress response suppressed by MPK6 but not MPK3 mutations. Mol Plant 5:841–851

  30. Lally D, Ingmire P, Tong HY, He ZH (2001) Antisense expression of a cell wall-associated protein kinase, WAK4, inhibits cell elongation and alters morphology. Plant Cell 13:1317–1331

  31. Lan T, Yang ZL, Yang X, Liu YJ, Wang XR, Zeng QY (2009) Extensive functional diversification of the Populus glutathione S-transferase supergene family. Plant Cell 21:3749–3766

  32. Lehti-Shiu MD, Zou C, Hanada K, Shiu SH (2009) Evolutionary history and stress regulation of plant Receptor-Like Kinase/Pelle genes. Plant Physiol 150:12–26

  33. Letunic I, Bork P (2011) Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39:475–478

  34. Levée V, Major I, Levasseur C, Tremblay L, MacKay J, Séguin A (2009) Expression profiling and functional analysis of Populus WRKY23 reveals a regulatory role in defense. New Phytol 184:48–70

  35. Li H, Zhou SY, Zhao WS, Su SC, Peng YL (2009) A novel wall-associated receptor-like protein kinase gene, OsWAK1, plays important roles in rice blast disease resistance. Plant Mol Biol 69:337–346

  36. Lopez D, Bronner G, Brunel N et al (2012) Insights into Populus XIP aquaporins: evolutionary expansion, protein functionality, and environmental regulation. J Exp Bot 63:2217–2230

  37. Marchler-Bauer A, Lu S, Anderson JB et al (2011) CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res 39:225–229

  38. Meier S, Ruzvidzo O, Morse M, Donaldson L, Kwezi L, Gehring C (2010) The Arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes. PLoS One 5:e8904

  39. Moore RC, Purugganan MD (2005) The evolutionary dynamics of plant duplicate genes. Curr Opin Plant Biol 8:122–128

  40. Morgan WD, Birdsall B, Frenkiel TA, Gradwell MG, Burghaus PA, Syed SEH, Uthaipibull C, Holder AA, Feeney J (1999) Solution structure of an EGF module pair from the Plasmodium falciparum merozoite surface protein 1. J Mol Biol 289:113–122

  41. Paterson AH, Freeling M, Tang H, Wang X (2010) Insights from the comparison of plant genome sequences. Annu Rev Plant Biol 61:349–372

  42. Petre B, Major I, Rouhier N, Duplessis S (2011) Genome-wide analysis of eukaryote thaumatin-like proteins (TLPs) with an emphasis on poplar. BMC Plant Biol 11:33

  43. Punta M, Coggill PC, Eberhardt RY et al (2012) The Pfam protein families database. Nucleic Acids Res 40:290–301

  44. R Development Core Team (2008). R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

  45. Rao Z, Handford P, Mayhew M, Knott V, Brownlee GG, Stuart D (1995) The structure of a Ca2+-binding epidermal growth factor-like domain: its role in protein-protein interactions. Cell 82:131–141

  46. Saeed A, Sharov V, White J et al (2003) TM4: A free, open-source system for microarray data management and analysis. Biotechniques 34:374–378

  47. Schug J, Schuller WP, Kappen C, Salbaum JM, Bucan M, Stoeckert CJ (2005) Promoter features related to tissue specificity as measured by Shannon entropy. Genome Biol 6:R33

  48. Shiu SH, Bleecker AB (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci U S A 98:10763–10768

  49. Shiu SH, Bleecker AB (2003) Expansion of the Receptor-Like Kinase/Pelle gene family and Receptor-Like Proteins in Arabidopsis. Plant Physiol 132:530–543

  50. Sivaguru M, Ezaki B, He ZH, Tong H, Osawa H, Baluška F, Volkmann D, Matsumoto H (2003) Aluminum-induced gene expression and protein localization of a cell wall-associated receptor kinase in Arabidopsis. Plant Physiol 132:2256–2266

  51. Sjödin A, Street NR, Sandberg G, Gustafsson P, Jansson S (2009) The Populus Genome Integrative Explorer (PopGenIE): a new resource for exploring the Populus genome. New Phytol 182:1013–1025

  52. Stenflo J, Stenberg Y, Muranyi A (2000) Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions. Biochim Biophys Acta 1477:51–63

  53. Swofford DL (2000) PAUP*: phylogenetic Analysis Using Parsimony (and Other Methods) Version 4. Sinauer Associates, Sunderland

  54. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

  55. Telewski FW (2006) A unified hypothesis of mechanoperception in plants? Am J Bot 93:1466–1476

  56. Tocquard K, Lopez D, Decourteix M, Thibaut B, Julien JL, Label P, Leblanc-Fournier N, Roeckel-Drevet P (2014) The molecular mechanisms of reaction wood induction. In: Gardiner B, Barnett J, Saranpää P, Gril J (eds) Biol. React. Wood, Springer, Berlin Heidelberg, pp 107–138

  57. Tuskan GA, Difazio S, Jansson S et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604

  58. Verica JA, He ZH (2002) The cell Wall-Associated Kinase (WAK) and WAK-Like kinase gene family. Plant Physiol 129:455–459

  59. Verica JA, Chae L, Tong H, Ingmire P, He ZH (2003) Tissue-specific and developmentally regulated expression of a cluster of tandemly arrayed cell wall- associated kinase-like kinase genes in Arabidopsis. Plant Physiol 133:1732–1746

  60. Wagner TA, Kohorn BD (2001) Wall-associated kinases are expressed throughout plant development and are required for cell expansion. Plant Cell 13:303–318

  61. Wilkins O, Nahal H, Foong J, Provart NJ, Campbell MM (2009a) Expansion and diversification of the R2R3-MYB family of transcription factors. Plant Physiol 149:981–993

  62. Wilkins O, Waldron L, Nahal H, Provart NJ, Campbell MM (2009b) Genotype and time of day shape the Populus drought response. Plant J 60:703–715

  63. Wolf S, Hématy K, Höfte H (2012) Growth control and cell wall signaling in plants. Annu Rev Plant Biol 63:381–407

  64. Wouters MA, Rigoutsos I, Chu CK, Feng LL, Sparrow DB, Dunwoodie SL (2005) Evolution of distinct EGF domains with specific functions. Protein Sci 14:1091–1103

  65. Wrzaczek M, Brosché M, Salojärvi J, Kangasjärvi S, Idänheimo N, Mersmann S, Robatzek S, Karpiński S, Karpińska B, Kangasjärvi J (2010) Transcriptional regulation of the CRK/DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis. BMC Plant Biol 10:95

  66. Yang X, Kalluri UC, DiFazio SP, Wullschleger SD, Tschaplinski TJ, Cheng ZM, Tuskan GA (2009) Poplar genomics: state of the science. Crit Rev Plant Sci 28:285–308

  67. Zhang S, Chen C, Li L, Meng L, Singh J, Jiang N, Deng XW, He ZH, Lemaux P (2005) Evolutionary expansion, gene structure, and expression of the rice wall-associated kinase gene family. Plant Physiol 139:1107–1124

Download references


This work was supported by the French Ministery of Higher Education and Research (doctoral contract). Editing and proofreading were done by Auvergne Traduction Technique.

Data archiving statement

A full list of the Phytozome gene sequences used for this paper is included in Online ressource 1. Data used for transcriptomic analyses are referenced on GEO database (accession numbers: GSE13990, GSM412653, GSE9673, GSE17223, GSE15242, GSE17226, GSE17230, GSE21171, GSE19297, and GSE13109).

Author information

Correspondence to Patricia Roeckel-Drevet.

Additional information

Communicated by J. L. Wegrzyn

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online resource 1

P. trichocarpa WAKs. PtWAKs were classified based on their chromosomal location. Alias names of v1 and v2 of poplar genome annotation are indicated. Domain structure, EST names and numbers per sequence, and WAK genomic, transcript, CDS, and protein sequences are given (PDF 29 kb)

Online resource 2

Alignment of EGF-Ca2+ domains (PDF 51 kb)

Online resource 3

Phylogenetic tree of PtWAKs containing the WAKassoc domain. The phylogenetic tree of PtWAKs using conserved WAKassoc domain is represented. The tree obtained by the BI method is represented, and posterior probability and bootstrap values of >50 are indicated at nodes as BI/NJ/MP (PDF 33 kb)

Online resource 4

Heatmap representation of all PtWAK expression patterns among different poplar tissues/environmental conditions/genotypes. PtWAK v3.0 gene model names are indicated. For each tissue/environmental condition/genotype, mean relative expression values (three to four replicates) are displayed. Sample and WAK, relative expression value was calculated by dividing the absolute value (RMA normalized) by the median of absolute values of all samples, except for the apex assay. In this case, relative expression was obtained by dividing each array-normalized expression value by the median of absolute values of all samples, and was then log2-transformed. An asterisk indicates significant tissue/environmental condition/genotype effect on PtWAK expression according to Welch’s t test/one-way ANOVA for two or more samples followed by adjusted Bonferroni correction (*p < 0.05). Blue-, red-, and green-shaded PtWAK names belong to PtWAK-RLK, PtWAK-RLCK, and PtWAK-RLP gene groups, respectively (PDF 144 kb)

Online resource 5

Expression pattern comparison of PtWAK and randomly selected genes;Seventy-two PtWAK genes or randomly selected genes were used for this analysis. a Box-plot representation of PtWAK and random gene expression level distribution. Expression values are RMA-normalized data from all experiments performed in this work (see “Methods”). The significant PtWAK effect on expression level was detected with Welch’s t test (***p < 0.001). For the Shannon entropy calculation, see “Methods.” b Tissue specificity of PtWAK and random genes as measured by Shannon entropy distribution. Low entropy values indicate high tissue specificity. χ2 value is given (not significant, p = 0.064). c Condition specificity of PtWAK and random genes as measured by Shannon entropy distribution. Low entropy values indicate high condition specificity. χ2 value is given (**p < 0.01). d Genotype specificity of PtWAK and random genes as measured by Shannon entropy distribution. Low entropy values indicate high genotype specificity. χ2 value is given (*p < 0.05) (PDF 55 kb)

Online resource 6

PtWAKs whose apex expression is affected depending on water availability. The mean relative expression values (three samples per condition) and their standard errors shown here were significantly affected by water availability variations according to one-way ANOVA followed by adjusted Bonferroni correction (*p < 0.05). Relative expression was obtained by dividing each array-normalized expression value by the median of absolute values of all samples for the PtWAK, and was then log2-transformed. Values for control (black charts), drought (grey charts) and rewatering after drought (white charts) are given (PDF 24 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tocquard, K., Lafon-Placette, C., Auguin, D. et al. In silico study of wall-associated kinase family reveals large-scale genomic expansion potentially connected with functional diversification in Populus . Tree Genetics & Genomes 10, 1135–1147 (2014).

Download citation


  • WAK
  • Populus
  • Phylogeny
  • Microarray data mining
  • Abiotic biotic stress