Cytokinin signalling in the regulation of cambial development

Secondary growth of plants is of pivotal importance in terrestrial ecosystems, providing a significant carbon sink in the form of wood. As plant biomass accumulation results largely from the cambial growth, it is surprising that quite little is known about the hormonal or genetic control of this important process in any plant species. The central aim of my thesis studies was to explore the function of cytokinin in the regulation of cambial development. Since their discovery as regulators of plant cell divisions, cytokinins have been assumed to participate in the control of cambial development. Evidence for this action was deduced from hormone treatment experiments, where exogenously applied cytokinin was shown to enhance cambial cell divisions in diverse plant organs and species. In my thesis work, the conservation of cytokinin signalling and homeostasis genes between a herbaceous plant, Arabidopsis, and a hardwood tree species, Populus trichocarpa. Presumably reflecting the ancient origin of cytokinin signalling system, the Populus genome contains orthologs for all Arabidopsis cytokinin signalling and homeostasis genes. Thus, genes belonging to five main families of isopentenyl transferases (IPTs), cytokinin oxidases (CKXs), two-component receptors, histidine containing phosphotransmitters (HPts) and response regulators (RRs) were identified from the Populus genome. Three subfamilies associated with cytokinin signal transduction, the CKI1-like family of two-component receptors, the AHP4-like HPts, and the ARR22-like atypical RRs, were significantly larger in Populus genome than in Arabidopsis. Potential contribution to the extensive secondary development of Populus by the members of these considerably expanded gene families will be discussed. Representatives of all cytokinin signal transduction elements were expressed in the Populus cambial zone, and most of the expressed genes appeared to be slightly more abundant on the phloem side of the meristem. The abundance of cytokinin related genes in the cambium emphasizes the important role of this hormone in the regulation of the extensive secondary growth characteristic of tree species. The function of the pseudo HPts in primary vascular development was studied in Arabidopsis root vasculature. It was demonstrated that the pseudo HPt AHP6 has a role in locally inhibiting cytokinin signalling in the protoxylem position in the Arabidopsis root, thus enabling differentiation of the protoxylem cell file. The possible role of pseudo HPts in cambial development will be discussed. The expression peak of cytokinin signalling genes in the tree cambial zone strongly indicates that cytokinin has a role in the regulation of this meristem function. To address whether cytokinin signalling is required for cambial activity, transgenic Populus trees with modified cytokinin signalling were produced. These trees were expressing a cytokinin catabolic gene from Arabidopsis, CYTOKININ OXIDASE 2, (AtCKX2) under the promoter of a Betula CYTOKININ RECEPTOR 1 (BpCRE1). The pBpCRE1::CKX2 transgenic Populus trees showed a reduced concentration of a biologically active cytokinin, correlating with their impaired cytokinin response. Furthermore, the radial growth of these trees was compromised, as illustrated by a smaller stem diameter than in wild-type trees of the same height. Moreover, the level of cambial cytokinin signalling was down-regulated in these thin-stemmed trees. The reduced signalling correlated with a decreased number of meristematic cambial cells, implicating cytokinin activity as a direct regulator of cambial cell division activity. Together, the results of my study indicate that cytokinins are major hormonal regulators required for cambial development.

Väitöskirjatyöni käsittelee erään kasvihormonin, sytokiniinin, tehtävää puun paksuuskasvun säätelyssä. Puut kasvavat paksuutta jällen solujen jakautumisen kautta. Jälsi tuottaa ulospäin nilaa, joka kuljettaa yhteyttämistuotteita lehdistä muualle kasviin, ja sisäänpäin puuta, joka kuljettaa rungossa vettä juurista lehtiin. Tutkimuksessani havaittiin sytokiniini-hormonin reseptoreiden ilmentyvän runsaasti jällen jakaantuvissa soluissa. Tämä havainto osoitti, että sytokiniini-hormoni toimii jällen soluissa. Tutkiakseni jällen sytokiniinin merkitystä puun paksuuskasvulle, tuotin siirtogeenisiä hybridihaapoja, joissa jällen sytokiniini-pitoisuutta oli alennettu. Siirtogeenisten puiden sytokiniini-määrän vähentäminen oli toteutettu lisäämällä tätä hormonia hajottavan entsyymin ilmentymistä jälsisolukossa. Näitä puita tutkittaessa havaittiin, että jällen jakautuvien solujen määrä oli alentunut. Tämä johti vähentyneeseen uuden puun ja nilan tuottoon, joten sytokiniini-pitoisuudeltaan alentuneet siirtogeeniset puut olivat rungoltaan tavanomaista ohuempia. Näiden havaintojen perusteella voin todeta sytokiniini-kasvihormonin olevan keskeinen jällen toimintaa ja puiden paksuuskasvua säätelevä tekijä. Tutkimuksestani saatua tietoa voidaan jatkossa hyödyntää kokeissa, joissa on tavoitteena jällen toiminnan ja siten puun paksuuskasvun nopeuttaminen. Puiden lisääntyneestä biomassan tuotannosta saattaa tulevaisuudessa olla merkittävää kansantaloudellista hyötyä.