{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T03:17:26Z","timestamp":1791947846339,"version":"4.3.4"},"reference-count":46,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T00:00:00Z","timestamp":1658966400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002347","name":"German Federal Ministry of Education and Research (BMBF)","doi-asserted-by":"publisher","award":["0315972C"],"award-info":[{"award-number":["0315972C"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"German Federal Ministry of Education and Research (BMBF)","doi-asserted-by":"publisher","award":["0315972A"],"award-info":[{"award-number":["0315972A"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The cambial meristem is responsible for bark and wood formation in woody plants. The activity of the cambial meristem is controlled by various factors; one of them is the plant hormone cytokinin. Here, we have explored different approaches to genetically engineering cambial activity in poplar plants by the ectopic expression of a cytokinin biosynthesis gene with enhanced activity (named ROCK4) or of a gene encoding a constitutively active cytokinin receptor variant (ROCK3). Both genes are derived from Arabidopsis thaliana and were expressed in poplar trees under the control of their own promoter or the cambium-specific pHB8 promoter. pIPT3:ROCK4- and pHB8:ROCK4-expressing plants were smaller than wild-type plants and formed more lateral branches; pHB8:ROCK4 transgenic plants additionally showed an increased stem diameter. In contrast, pAHK3:ROCK3- and pHB8:ROCK3-expressing plants grew taller than wild type without an altered branching pattern and formed more cambial cells, leading to increased radial stem growth. The effectivity of ROCK3 when expressed in either secondary phloem cells or in cambial cells is consistent with a dual, tissue-autonomous and non-autonomous activity of cytokinin in regulating cambial activity. We propose ROCK3 as a novel gene to enhance biomass formation in woody plants.<\/jats:p>","DOI":"10.3390\/ijms23158321","type":"journal-article","created":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T20:49:28Z","timestamp":1659041368000},"page":"8321","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["A Constitutively Active Cytokinin Receptor Variant Increases Cambial Activity and Stem Growth in Poplar"],"prefix":"10.3390","volume":"23","author":[{"given":"Michael","family":"Riefler","sequence":"first","affiliation":[{"name":"Institute of Biology\/Applied Genetics, Dahlem Centre of Plant Sciences, Freie Universit\u00e4t Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9823-5630","authenticated-orcid":false,"given":"Tobias","family":"Br\u00fcgmann","sequence":"additional","affiliation":[{"name":"Thuenen Institute of Forest Genetics, Sieker Landstra\u00dfe 2, 22927 Gro\u00dfhansdorf, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9301-8581","authenticated-orcid":false,"given":"Matthias","family":"Fladung","sequence":"additional","affiliation":[{"name":"Thuenen Institute of Forest Genetics, Sieker Landstra\u00dfe 2, 22927 Gro\u00dfhansdorf, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Schm\u00fclling","sequence":"additional","affiliation":[{"name":"Institute of Biology\/Applied Genetics, Dahlem Centre of Plant Sciences, Freie Universit\u00e4t Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1016\/j.pbi.2010.03.002","article-title":"Regulation of plant biomass production","volume":"13","author":"Demura","year":"2010","journal-title":"Curr. Opin. Plant Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4119","DOI":"10.1093\/jxb\/erv081","article-title":"Molecular control of wood formation in trees","volume":"66","author":"Ye","year":"2015","journal-title":"J. Exp. Bot."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/S0167-7799(02)01983-2","article-title":"Where will the wood come from? Plantation forests and the role of biotechnology","volume":"20","author":"Fenning","year":"2002","journal-title":"Trends Biotech."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kaltschmitt, M., Hartmann, H., and Hofbauer, H. (2009). Energie aus Biomasse. Grundlagen, Techniken und Verfahren, Springer.","DOI":"10.1007\/978-3-540-85095-3"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"700928","DOI":"10.3389\/fpls.2021.700928","article-title":"Vascular cambium: The source of wood formation","volume":"12","author":"Wang","year":"2021","journal-title":"Front. Plant Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1146\/annurev-arplant-050718-100402","article-title":"The dynamics of cambial stem cell activity","volume":"70","author":"Fischer","year":"2019","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"20027","DOI":"10.1073\/pnas.0805619105","article-title":"Cytokinins are central regulators of cambial activity","volume":"105","author":"Kusumoto","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"20032","DOI":"10.1073\/pnas.0805617106","article-title":"Cytokinin signaling regulates cambial development in poplar","volume":"105","author":"Nieminen","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1111\/j.1469-8137.2011.04011.x","article-title":"Towards optimizing wood development in bioenergy trees","volume":"194","author":"Nieminen","year":"2012","journal-title":"New Phytol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1097","DOI":"10.1016\/j.semcdb.2009.09.009","article-title":"Stem cell function during plant vascular development","volume":"20","author":"Elo","year":"2009","journal-title":"Sem. Cell Dev. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1146\/annurev.arplant.57.032905.105231","article-title":"Cytokinins: Activity, biosynthesis, and translocation","volume":"57","author":"Sakakibara","year":"2006","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/j.pbi.2009.07.002","article-title":"Cytokinin action in plant development","volume":"12","author":"Werner","year":"2009","journal-title":"Curr. Opin. Plant Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1111\/j.1469-8137.2007.02240.x","article-title":"Transcript profiles of the cytokinin response regulator gene family in Populus imply diverse roles in plant development","volume":"177","author":"Morse","year":"2008","journal-title":"New Phytol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"H\u00e9ricourt, F., Larcher, M., Chefdor, F., Koudounas, K., Carqueijeiro, I., Lemos Cruz, P., Courdavault, V., Tanigawa, M., Maeda, T., and Depierreux, C. (2019). New insight into HPts as hubs in poplar cytokinin and osmosensing multistep phosphorelays: Cytokinin pathway uses specific HPts. Plants, 8.","DOI":"10.3390\/plants8120591"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00425-019-03297-x","article-title":"Characterization of five CHASE-containing histidine kinase receptors from Populus\u2009\u00d7\u2009canadensis cv. obusta sensing isoprenoid and aromatic cytokinins","volume":"251","author":"Jaworek","year":"2019","journal-title":"Planta"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1093\/pcp\/pce112","article-title":"Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP\/ADP isopentenyltransferases","volume":"42","author":"Kakimoto","year":"2001","journal-title":"Plant Cell Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"26405","DOI":"10.1074\/jbc.M102130200","article-title":"Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana","volume":"276","author":"Takei","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2532","DOI":"10.1105\/tpc.014928","article-title":"Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in regulating shoot and root meristem activity","volume":"15","author":"Werner","year":"2003","journal-title":"Plant Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"652","DOI":"10.3389\/fpls.2016.00652","article-title":"Tissue- and cell-specific cytokinin activity in Populus \u00d7 canescens monitored by ARR5::GUS reporter lines in summer and winter","volume":"7","author":"Paul","year":"2016","journal-title":"Front. Plant Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"613488","DOI":"10.3389\/fpls.2021.613488","article-title":"The cytokinin status of the epidermis regulates aspects of vegetative and reproductive development in Arabidopsis thaliana","volume":"12","author":"Werner","year":"2021","journal-title":"Front. Plant Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"10487","DOI":"10.1073\/pnas.171304098","article-title":"Regulation of plant growth by cytokinins","volume":"98","author":"Werner","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1105\/tpc.110.079079","article-title":"Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana","volume":"23","author":"Bartrina","year":"2011","journal-title":"Plant Cell"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1990","DOI":"10.1016\/j.cub.2016.05.053","article-title":"Cytokinin and auxin display distinct but interconnected distribution and signaling profiles to stimulate cambial activity","volume":"26","author":"Immanen","year":"2016","journal-title":"Curr. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1476","DOI":"10.1111\/nph.17255","article-title":"Cytokinin signaling localized in phloem non-cell-autonomously regulates cambial activity during secondary growth of Populus stems","volume":"230","author":"Fu","year":"2021","journal-title":"New Phytol."},{"key":"ref_25","unstructured":"Jensen, H. (2014). Molecular Characterisation of Dominant Repressors of the Cytokinin Deficiency Syndrome. [Ph.d. Thesis, Freie Universit\u00e4t Berlin]. Available online: http:\/\/dx.doi.org\/10.17169\/refubium-6507."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1783","DOI":"10.1104\/pp.16.01903","article-title":"Gain-of-function mutants of the Arabidopsis cytokinin receptors AHK2 and AHK3 regulate plant organ size, flowering time and plant longevity","volume":"173","author":"Bartrina","year":"2017","journal-title":"Plant Physiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1046\/j.1365-313X.2003.01945.x","article-title":"Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: Tissue specificity and regulation by auxin, cytokinin, and nitrate","volume":"37","author":"Miyawaki","year":"2004","journal-title":"Plant J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4171","DOI":"10.1242\/dev.121.12.4171","article-title":"The expression of the Athb-8 homeobox gene is restricted to provascular cells in Arabidopsis thaliana","volume":"121","author":"Baima","year":"1995","journal-title":"Development"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Wang, J., Kucukoglu, M., Zhang, L., Chn, P., Decker, D., Nilsson, O., Jones, B., Sandberg, G., and Zheng, B. (2013). The Arabidopsis LRR-RLK, PXC1, is a regulator of secondary wall formation correlated with the TDIF-PXY\/TDR-WOX4 signaling pathway. BMC Plant Biol., 13.","DOI":"10.1186\/1471-2229-13-94"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"8821","DOI":"10.1073\/pnas.0402887101","article-title":"In planta functions of the Arabidopsis cytokinin receptor family","volume":"101","author":"Higuchi","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1111\/j.1365-313X.2011.04584.x","article-title":"The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors","volume":"67","author":"Stolz","year":"2011","journal-title":"Plant J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1016\/j.pbi.2005.07.013","article-title":"Cytokinin signaling","volume":"8","author":"Ferreira","year":"2005","journal-title":"Curr. Opin. Plant Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1038\/s41586-018-0839-y","article-title":"Mobile PEAR transcription factors integrate positional cues to prime cambial growth","volume":"565","author":"Miyashima","year":"2019","journal-title":"Nature"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1111\/nph.14631","article-title":"WUSCHEL-RELATED HOMEOBOX4 (WOX4)-like genes regulate cambial cell division activity and secondary growth in Populus trees","volume":"215","author":"Kucukoglu","year":"2017","journal-title":"New Phytol."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Oles, V., Panchenko, A., and Smertenko, A. (2017). Modeling hormonal control of cambium proliferation. PLoS ONE, 12.","DOI":"10.1101\/101550"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1078","DOI":"10.1104\/pp.18.00199","article-title":"Root engineering in barley: Increasing cytokinin degradation produces a larger root system, mineral enrichment in the shoot and improved drought tolerance","volume":"177","author":"Ramireddy","year":"2018","journal-title":"Plant Physiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7146","DOI":"10.1093\/jxb\/eraa419","article-title":"Cytokinin regulates the activity of the inflorescence meristem and components of seed yield in oilseed rape","volume":"71","author":"Schwarz","year":"2020","journal-title":"J. Exp. Bot."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1007\/BF00232166","article-title":"Transgenic poplars: Expression of chimeric genes using four different constructs","volume":"11","author":"Leple","year":"1992","journal-title":"Plant Cell Rep."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Schmidt, E.R., and Hankeln, T. (1996). Gene transfer in aspen. Transgenic Organisms and Biosafety, Springer.","DOI":"10.1007\/978-3-642-61460-6"},{"key":"ref_40","first-page":"453","article-title":"Woody plant medium (WPM)\u2014A mineral nutrient formulation for microculture of woody plant species","volume":"16","author":"McCown","year":"1981","journal-title":"HortScience"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.1104\/pp.107.106989","article-title":"Recombinational cloning with plant Gateway vectors","volume":"145","author":"Karimi","year":"2007","journal-title":"Plant Physiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/S1360-1385(02)02251-3","article-title":"GATEWAY\u2122 vectors for Agrobacterium-mediated plant transformation","volume":"7","author":"Karimi","year":"2002","journal-title":"Trends Plant Sci."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1023\/A:1018421620040","article-title":"Genetic transformation of Populus genotypes with different chimeric gene constructs: Transformation efficiency and molecular analysis","volume":"6","author":"Fladung","year":"1997","journal-title":"Transgenic Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/978-1-4939-8778-8_12","article-title":"Poplar transformation","volume":"1864","author":"Bruegmann","year":"2019","journal-title":"Meth. Mol. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3901","DOI":"10.1002\/j.1460-2075.1987.tb02730.x","article-title":"GUS fusions: \u00df-glucuronidase as a sensitive and versatile gene fusion marker in higher plants","volume":"6","author":"Jefferson","year":"1987","journal-title":"EMBO J."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"821","DOI":"10.2144\/04365ST06","article-title":"Isolation of high-quality RNA from gymnosperm and angiosperm trees","volume":"36","author":"Kolosova","year":"2004","journal-title":"Biotechniques"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8321\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:57:58Z","timestamp":1760140678000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/15\/8321"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,28]]},"references-count":46,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2022,8]]}},"alternative-id":["ijms23158321"],"URL":"https:\/\/doi.org\/10.3390\/ijms23158321","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,7,28]]}}}