Populus trichocarpa clade A PP2C protein phosphatases: their stress-induced expression patterns, interactions in core abscisic acid signaling, and potential for regulation of growth and development

  • Stephen B. Rigoulot
  • H. Earl Petzold
  • Sarah P. Williams
  • Amy M. Brunner
  • Eric P. BeersEmail author


Key message

Overexpression of the poplar PP2C protein phosphatase gene PtrHAB2 resulted in increased tree height and altered leaf morphology and phyllotaxy, implicating PP2C phosphatases as growth regulators functioning under favorable conditions.


We identified and studied Populus trichocarpa genes, PtrHAB1 through PtrHAB15, belonging to the clade A PP2C family of protein phosphatases known to regulate abscisic acid (ABA) signaling. PtrHAB1 through PtrHAB3 and PtrHAB12 through PtrHAB15 were the most highly expressed genes under non-stress conditions. The poplar PP2C genes were differentially regulated by drought treatments. Expression of PtrHAB1 through PtrHAB3 was unchanged or downregulated in response to drought, while all other PtrHAB genes were weakly to strongly upregulated in response to drought stress treatments. Yeast two-hybrid assays involving seven ABA receptor proteins (PtrRCAR) against 12 PtrHAB proteins detected 51 interactions involving eight PP2Cs and all PtrRCAR proteins with 22 interactions requiring the addition of ABA. PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 also interacted with the sucrose non-fermenting related kinase 2 proteins PtrSnRK2.10 and PtrSnRK2.11, supporting conservation of a SnRK2 signaling cascade regulated by PP2C in poplar. Additionally, PtrHAB2, PtrHAB12, PtrHAB13 and PtrHAB14 interacted with the mitogen-activated protein kinase protein PtrMPK7. Due to its interactions with PtrSnRK2 and PtrMPK7 proteins, and its reduced expression during drought stress, PtrHAB2 was overexpressed in poplar to test its potential as a growth regulator under non-stress conditions. 35S::PtrHAB2 transgenics exhibited increased growth rate for a majority of transgenic events and alterations in leaf phyllotaxy and morphology. These results indicate that PP2Cs have additional roles which extend beyond canonical ABA signaling, possibly coordinating plant growth and development in response to environmental conditions.


Abscisic acid PP2C Poplar Drought stress Leaf morphology SnRK2 



We thank Drs. Xiaoyan Sheng and Kristi DeCourcy for expert technical assistance. This work was supported by the United States Department of Energy (DOE) Office of Science (Biological and Environmental Research), Grant Nos. DE-SC0008570 to AMB and DE-FG02-07ER64449 to EPB and AMB and the United States Department of Agriculture-National Institute of Food and Agriculture (USDA-NIFA) Grant No. 2014-67013-21580 to EPB and AMB. Support was also provided by the Virginia Agricultural Experiment Station and the Hatch Program of USDA-NIFA, Project No. VA-135994.

Author contributions

SBR, HEP, AMB, and EPB designed experiments and wrote the manuscript. SBR and HEP produced vectors for Y2H and for transient and stable gene expression in plants. SBR conducted Y2H and BiFC assays, prepared and characterized transgenic poplar plants, and measured gene expression. SBR analyzed RNAseq data. SBR and SPW analyzed and documented BiFC results using confocal microscopy.

Supplementary material

11103_2019_861_MOESM1_ESM.xls (84 kb)
Supplementary material 1 (XLS 84 kb)
11103_2019_861_MOESM2_ESM.pdf (7.3 mb)
Supplementary material 2 (PDF 7519 kb)


  1. Arend M, Schnitzler JP, Ehlting B, Hansch R, Lange T, Rennenberg H, Himmelbach A, Grill E, Fromm J (2009) Expression of the Arabidopsis mutant ABI1 gene alters abscisic acid sensitivity, stomatal development, and growth morphology in gray poplars. Plant Physiol 151:2110–2119CrossRefGoogle Scholar
  2. Barrero JM, Piqueras P, Gonzalez-Guzman M, Serrano R, Rodriguez PL, Ponce MR, Micol JL (2005) A mutational analysis of the ABA1 gene of Arabidopsis thaliana highlights the involvement of ABA in vegetative development. J Exp Bot 56:2071–2083CrossRefGoogle Scholar
  3. Benschop JJ, Bou J, Peeters AJ, Wagemaker N, Guhl K, Ward D, Hedden P, Moritz T, Voesenek LA (2006) Long-term submergence-induced elongation in Rumex palustris requires abscisic acid-dependent biosynthesis of gibberellin1. Plant Physiol 141:1644–1652CrossRefGoogle Scholar
  4. Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson A, Martienssen RA (2000) Asymmetricleaves1 mediates leaf patterning and stem cell function in Arabidopsis. Nature 408:967–971CrossRefGoogle Scholar
  5. Cai S, Chen G, Wang Y, Huang Y, Marchant DB, Wang Y, Yang Q, Dai F, Hills A, Franks PJ, Nevo E, Soltis DE, Soltis PS, Sessa E, Wolf PG, Xue D, Zhang G, Pogson BJ, Blatt MR, Chen ZH (2017) Evolutionary conservation of ABA signaling for stomatal closure. Plant Physiol 174:732–747CrossRefGoogle Scholar
  6. Cerovic ZG, Ounis A, Cartelat A, Latouche G, Goulas Y, Meyer S, Moya I (2002) The use of chlorophyll fluorescence excitation spectra for the non-destructive in situ assessment of UV-absorbing compounds in leaves. Plant Cell Envir 25:1663–1676CrossRefGoogle Scholar
  7. Chan Z (2012) Expression profiling of ABA pathway transcripts indicates crosstalk between abiotic and biotic stress responses in Arabidopsis. Genomics 100:110–115CrossRefGoogle Scholar
  8. Chater C, Gray JE (2015) Stomatal closure: the old guard takes up the SLAC. Curr Biol 25:R271–R273CrossRefGoogle Scholar
  9. Chen J, Zhang D, Zhang C, Xia X, Yin W, Tian Q (2015) A putative PP2C-encoding gene negatively regulates ABA signaling in Populus euphratica. PLoS ONE 10:e0139466. CrossRefGoogle Scholar
  10. Danquah A, de Zelicourt A, Boudsocq M, Neubauer J, Frei Dit Frey N, Leonhardt N, Pateyron S, Gwinner F, Tamby JP, Ortiz-Masia D, Marcote MJ, Hirt H, Colcombet J (2015) Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana. Plant J 82:232–244CrossRefGoogle Scholar
  11. Dkhar J, Pareek A (2014) What determines a leaf’s shape? EvoDevo 5:47. CrossRefGoogle Scholar
  12. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21CrossRefGoogle Scholar
  13. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797CrossRefGoogle Scholar
  14. Filichkin SA, Meilan R, Busov VB, Ma C, Brunner AM, Strauss SH (2006) Alcohol-inducible gene expression in transgenic Populus. Plant Cell Rep 25:660–667CrossRefGoogle Scholar
  15. Filichkin SA, Hamilton M, Dharmawardhana PD, Singh SK, Sullivan C, Ben-Hur A, Reddy ASN, Jaiswal P (2018) Abiotic stresses modulate landscape of poplar transcriptome via alternative splicing, differential intron retention, and isoform ratio switching. Front Plant Sci 9:5. CrossRefGoogle Scholar
  16. Fuchs S, Grill E, Meskiene I, Schweighofer A (2013) Type 2C protein phosphatases in plants. FEBS J 280:681–693CrossRefGoogle Scholar
  17. Fujita Y, Fujita M, Shinozaki K, Yamaguchi-Shinozaki K (2011) ABA-mediated transcriptional regulation in response to osmotic stress in plants. J Plant Res 124:509–525CrossRefGoogle Scholar
  18. Furumizu C, Alvarez JP, Sakakibara K, Bowman JL (2015) Antagonistic roles for KNOX1 and KNOX2 genes in patterning the land plant body plan following an ancient gene duplication. PLoS Genet 11:e1004980. CrossRefGoogle Scholar
  19. Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40:D1178–D1186CrossRefGoogle Scholar
  20. Gookin TE, Assmann SM (2014) Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors. Plant J 80:553–567CrossRefGoogle Scholar
  21. Groover AT (2005) What genes make a tree a tree? Trends Plant Sci 10:210–214CrossRefGoogle Scholar
  22. Hao Q, Yin P, Li W, Wang L, Yan C, Lin Z, Wu JZ, Wang J, Yan SF, Yan N (2011) The molecular basis of ABA-independent inhibition of PP2Cs by a subclass of PYL proteins. Mol Cell 42:662–672CrossRefGoogle Scholar
  23. Jalmi SK, Sinha AK (2016) Functional involvement of a mitogen activated protein kinase module, OsMKK3-OsMPK7-OsWRK30 in mediating resistance against Xanthomonas oryzae in rice. Sci Rep 6:37974CrossRefGoogle Scholar
  24. Klingler JP, Batelli G, Zhu JK (2010) ABA receptors: the START of a new paradigm in phytohormone signalling. J Exp Bot 61:3199–3210CrossRefGoogle Scholar
  25. Klocko AL, Lu H, Magnuson A, Brunner AM, Ma C, Strauss SH (2018) Phenotypic expression and stability in a large-scale field study of genetically engineered poplars containing sexual containment transgenes. Front Bioeng Biotechnol 6:100. CrossRefGoogle Scholar
  26. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  27. Larson PR (1975) Development and organization of the primary vascular dystem in Populus deltoides according to phyllotaxy. Am J Bot 62:1082–1099CrossRefGoogle Scholar
  28. Lee SC, Lan W, Buchanan BB, Luan S (2009) A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells. PNAS 106:21419–21424CrossRefGoogle Scholar
  29. LeNoble ME, Spollen WG, Sharp RE (2004) Maintenance of shoot growth by endogenous ABA: genetic assessment of the involvement of ethylene suppression. J Exp Bot 55:237–245CrossRefGoogle Scholar
  30. Leung J, Orfanidi S, Chefdor F, Mészaros T, Bolte S, Mizoguchi T, Shinozaki K, Giraudat J, Bögre L (2006) Antagonistic interaction between MAP kinase and protein phosphatase 2C in stress recovery. Plant Sci 171:596–606CrossRefGoogle Scholar
  31. Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930CrossRefGoogle Scholar
  32. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. CrossRefGoogle Scholar
  33. Lu Y, Sasaki Y, Li X, Mori IC, Matsuura T, Hirayama T, Sato T, Yamaguchi J (2015) ABI1 regulates carbon/nitrogen-nutrient signal transduction independent of ABA biosynthesis and canonical ABA signalling pathways in Arabidopsis. J Exp Bot 66:2763–2771CrossRefGoogle Scholar
  34. Mitula F, Tajdel M, Ciesla A, Kasprowicz-Maluski A, Kulik A, Babula-Skowronska D, Michalak M, Dobrowolska G, Sadowski J, Ludwikow A (2015) Arabidopsis ABA-activated kinase MAPKKK18 is regulated by protein phosphatase 2C ABI1 and the ubiquitin-proteasome pathway. Plant Cell Physiol 56:2351–2367CrossRefGoogle Scholar
  35. Msanne J, Lin J, Stone JM, Awada T (2011) Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta 234:97–107CrossRefGoogle Scholar
  36. Nakashima K, Fujita Y, Kanamori N, Katagiri T, Umezawa T, Kidokoro S, Maruyama K, Yoshida T, Ishiyama K, Kobayashi M, Shinozaki K, Yamaguchi-Shinozaki K (2009) Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol 50:1345–1363CrossRefGoogle Scholar
  37. Nath U, Crawford BC, Carpenter R, Coen E (2003) Genetic control of surface curvature. Science 299:1404–1407CrossRefGoogle Scholar
  38. Papacek M, Christmann A, Grill E (2017) Interaction network of ABA receptors in grey poplar. Plant J 92:199–210CrossRefGoogle Scholar
  39. Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, ZhaoY Lumba S, Santiago J, Rodrigues A, Chow TF et al (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324:1068–1071Google Scholar
  40. Reeves WM, Lynch TJ, Mobin R, Finkelstein RR (2011) Direct targets of the transcription factors ABA-insensitive (ABI) 4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors. Plant Mol Biol 75:347–363CrossRefGoogle Scholar
  41. Rodgers-Melnick E, Mane SP, Dharmawardhana P, Slavov GT, Crasta OR, Strauss SH, Brunner AM, Difazio SP (2012) Contrasting patterns of evolution following whole genome versus tandem duplication events in Populus. Genome Res 22:95–105CrossRefGoogle Scholar
  42. Saez A, Apostolova N, Gonzalez-Guzman M, Gonzalez-Garcia MP, Nicolas C, Lorenzo O, Rodriguez PL (2004) Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling. Plant J 37:354–369CrossRefGoogle Scholar
  43. Saez A, Robert N, Maktabi MH, Schroeder JI, Serran R, Rodriguez PL (2006) Enhancement of abscisic acid sensitivity and reduction of water consumption in Arabidopsis by combined inactivation of the protein phosphatases type 2C ABI1 and HAB1. Plant Physiol 141:1389–1399CrossRefGoogle Scholar
  44. Soma F, Mogami J, Yoshida T, Abekura M, Takahashi F, Kidokoro S, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2017) ABA-unresponsive SnRK2 protein kinases regulate mRNA decay under osmotic stress in plants. Nat Plants 3:16204. CrossRefGoogle Scholar
  45. Song X, Ohtani M, Hori C, Takebayasi A, Hiroyama R, Rejab NA, Suzuki T, Demura T, Yin T, Yu X, Zhuge Q (2015) Physical interaction between SnRK2 and PP2C is conserved in Populus trichocarpa. Plant Biotechnol 32:337–341CrossRefGoogle Scholar
  46. Soon FF, Ng LM, Zhou XE, West GM, Kovach A, Tan MH, Suino-Powell KM, He Y, Xu Y, Chalmers MJ, Brunzelle JS, Zhang H, Yang H, Jiang H, Li J, Yong EL, Cutler S, Zhu JK, Griffin PR, Melcher K, Xu HE (2012) Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases. Science 335:85–88CrossRefGoogle Scholar
  47. Sorce C, Giovannelli A, Sebastiani L, Anfodillo T (2013) Hormonal signals involved in the regulation of cambial activity, xylogenesis and vessel patterning in trees. Plant Cell Rep 32:885–898CrossRefGoogle Scholar
  48. Szostkiewicz I, Richter K, Kepka M, Demmel S, Ma Y, Korte A, Assaad FF, Christmann A, Grill E (2010) Closely related receptor complexes differ in their ABA selectivity and sensitivity. Plant J 62:25–35CrossRefGoogle Scholar
  49. Tajdel M, Mitula F, Ludwikow A (2016) Regulation of Arabidopsis MAPKKK18 by ABI1 and SnRK2, components of the ABA signaling pathway. Plant Signal Behav 11:e1139277. CrossRefGoogle Scholar
  50. Tang S, Dong Y, Liang D, Zhang Z, Ye CY, Shuai P, Han X, Zhao Y, Yin W, Xia X (2014) Analysis of the drought stress-responsive transcriptome of black cottonwood (Populus trichocarpa) using deep RNA sequencing. Plant Mol Biol Rep 33:424–438CrossRefGoogle Scholar
  51. Tischer SV, Wunschel C, Papacek M, Kleigrewe K, Hofmann T, Christmann A, Grill E (2017) Combinatorial interaction network of abscisic acid receptors and coreceptors from Arabidopsis thaliana. PNAS 114:10280–10285CrossRefGoogle Scholar
  52. Tylewicz S, Petterle A, Marttila S, Miskolczi P, Azeez A, Singh RK, Immanen J, Mähler N, Hvidsten TR, Eklund DM, Bowman JL, Helariutta Y, Bhalerao RP (2018) Photoperiodic control of seasonal growth is mediated by ABA acting on cell-cell communication. Science 360:212–215CrossRefGoogle Scholar
  53. Vandenbussche M, Horstman A, Zethof J, Koes R, Rijpkema AS, Gerats T (2009) Differential recruitment of WOX transcription factors for lateral development and organ fusion in Petunia and Arabidopsis. Plant Cell 21:2269–2283CrossRefGoogle Scholar
  54. Walhout AJ, Vidal M (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24:297–306CrossRefGoogle Scholar
  55. Wang K, He J, Zhao Y, Wu T, Zhou X, Ding Y, Kong L, Wang X, Wang Y, Li J, Song CP, Wang B, Yang S, Zhu JK, Gong Z (2018a) EAR1 negatively regulates ABA signaling by enhancing 2C protein phosphatase activity. Plant Cell 30:815–834CrossRefGoogle Scholar
  56. Wang P, Zhao Y, Li Z, Hsu CC, Liu X, Fu L, Hou YJ, Du Y, Xie S, Zhang C, Gao J, Cao M, Huang X, Zhu Y, Tang K, Wang X, Tao WA, Xiong Y, Zhu JK (2018b) Reciprocal regulation of the TOR kinase and ABA receptor balances plant growth and stress response. Mol Cell 69:100–112CrossRefGoogle Scholar
  57. Wang YG, Fu FL, Yu HQ, Hu T, Zhang YY, Tao Y, Zhu JK, Zhao Y, Li WC (2018c) Interaction network of core ABA signaling components in maize. Plant Mol Biol 96:245–263CrossRefGoogle Scholar
  58. Williams SP, Rangarajan P, Donahue JL, Hess JE, Gillaspy GE (2014) Regulation of sucrose non-fermenting related kinase 1 genes in Arabidopsis thaliana. Front Plant Sci 5:324Google Scholar
  59. Xiang Y, Song B, Nee G, Kramer K, Finkemeier I, Soppe WJ (2016) Sequence polymorphisms at the REDUCED DORMANCY5 pseudophosphatase underlie natural variation in Arabidopsis dormancy. Plant Physiol 171:2659–2670Google Scholar
  60. Xie J, Yang X, Song Y, Du Q, Li Y, Chen J, Zhang D (2017) Adaptive evolution and functional innovation of Populus-specific recently evolved microRNAs. New Phytol 213:206–219CrossRefGoogle Scholar
  61. Xu J, Zhang S (2015) Mitogen-activated protein kinase cascades in signaling plant growth and development. Trends Plant Sci 20:56–64CrossRefGoogle Scholar
  62. Yang JC, Zhang JH, Wang ZQ, Zhu QS, Liu LJ (2003) Involvement of abscisic acid and cytokinins in the senescence and remobilization of carbon reserves in wheat subjected to water stress during grain filling. Plant, Cell Environ 26:1621–1631CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.School of Plant and Environmental SciencesVirginia TechBlacksburgUSA
  2. 2.Department of Forest Resources and Environmental ConservationVirginia TechBlacksburgUSA
  3. 3.Department of BiochemistryVirginia TechBlacksburgUSA
  4. 4.Department of Plant SciencesUniversity of TennesseeKnoxvilleUSA
  5. 5.Department of BiologyCollege of William and MaryWilliamsburgUSA

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