Cytokinin signaling in hybrid aspen cambial development and growth

Life on Earth is carbon based and is largely enabled by plants. As autotrophic (self-sufficient) life-forms, they fix atmospheric carbon dioxide to sugar through photosynthesis, and atmospheric oxygen is a by-product of this process. Heterotrophic life-forms such as humans are fully dependent on energy and oxygen provided by plants and cyanobacteria. Plants are thus very important for the human economy. Agriculture and forestry are large business sectors, with a global annual worth of over 3 trillion US dollars. Vascular tissues develop from the vascular cambium and serve two main functions: they give physical structure and support and distribute the water, nutrients and other substances needed for growth. Phloem transports the nutrients, and xylem is mainly responsible for conducting water and providing structural support for the plant. Trees are crucially important organisms for various ecosystems: about 31% of global terrain, over 4 billion hectares, is covered by forests. Trees contribute to carbon fixation, oxygen production, the global water cycle and soil erosion prevention. Woody biomass represents a valuable renewable source of energy and raw material for pulping and for the construction industry. Trees provide an excellent system for studying secondary vascular development in high resolution due to their massive size, which results from a wide cambial meristem. Understanding the hormonal regulation of radial growth that underlies wood development is of great importance for improving the use of tree products as a renewable resource. Detailed knowledge of these regulatory mechanisms could provide powerful tools to help tree breeders boost lignocellulosic biomass production. In the future, improved forestry and agriculture could produce the required energy, food, feed and raw biological materials using much less land, enabling the preservation of large areas of natural forest. Cytokinins are important growth regulators, but there are many other important plant hormones and growth regulators. In the work included in this thesis, we provided the first comprehensive description of the gene families for cytokinin signaling and homeostasis in black cotton wood (Populus trichocarpa). We also analyzed the hormone distribution and genome-wide expression profiles across the Populus cambial zone at an unprecedentedly high resolution. We observed increased cambial auxin concentration and auxin-responsive gene expression in cytokinin over-producing transgenic hybrid aspen (Populus) trees. Our results indicate that a graded distribution of cytokinin signaling specifies meristematic activity by influencing the amplitude of the cambial auxin gradient. It seems that cytokinins and auxin together regulate cambial developmental fate and growth rate. The two major phytohormones clearly interact in a manner which calls for continuing research. Here, we show that cytokinin and auxin display different yet partially overlapping distribution profiles across the cambium. In contrast to auxin, which is most concentrated in the actively dividing cambial cells, the greatest concentration of cytokinins is in the developing phloem. By manipulating the cytokinin concentration in the cambial zone, we could increase and decrease cambial cell division activity. Stimulation of cambial growth dramatically increased the production of lignocellulosic trunk biomass in Populus trees under greenhouse conditions.Hiilenkierto ja kasvit ovat mahdollistaneet nykymuotoisen elämän maapallolla. Kasvit ovat autotrofisia (omavaraisia) elämänmuotoja, jotka pystyvät yhteyttämisen avulla muodostamaan ilmakehän hiilidioksidista sokeria ja happea. Heterotrofiset (toisenvaraiset) elämänmuodot, kuten ihmiset, ovat täysin riippuvaisia kasvien ja syanobakteerien tuottamasta energiasta ja hapesta. Kasvit ovat myös erittäin tärkeitä ihmiskunnan taloudelle. Maa- ja metsätalous ovat suurta maailmanlaajuista liiketoimintaa, jonka arvo on yli 3 biljoonaa Yhdysvaltain dollaria. Kasvin johtosolukot kehittyvät jällestä. Johtosolukot antavat kasville sen fyysisen muodon ja tukirakenteen sekä huolehtivat kasvuun tarvittavan veden, ravinteiden ja muiden aineiden kuljetuksesta erilaisten solukoiden välillä. Nila kuljettaa lehdistä ravinteet, kuten sokerit ja aminohapot erilaisiin solukoihin, kuten esimerkiksi juuriin. Puuaines (ksyleemi) kuljettaa vettä juurista muihin solukoihin, kuten lehtiin ja antaa kasville tukirangan. Puut ovat erityisen tärkeitä eliöitä erilaisissa ekosysteemeissä. Noin 31% maapinta-alasta eli yli 4 miljardia hehtaaria on erilaisten metsien peitossa. Metsillä on merkitystä hiilen sitomisessa, hapen muodostuksessa, maailman laajuisessa veden kierrossa ja maaperän eroosion ehkäisyssä. Puubiomassa on arvokas ja uudistuva luonnonvara niin energiaksi kuin raaka-aineeksi erilaisiin tarkoituksiin. Puut ovat oiva kohde jällen yksityiskohtaiseen tutkimukseen suuren kokonsa vuoksi. Puun kehityksen ja kasvun hormonaalisen säätelyn tunteminen olisi hyvin tärkeää, jotta voitaisiin tehostaa metsien kasvua. Tehostettu metsä- ja maatalous pystyisi tulevaisuudessa tuottamaan tarvittavan määrän energiaa, ravintoa ja raaka-aineita paljon pienemmällä käytettävällä maapinta-alalla, jotta luonnontilaiset metsät voisivat olla rauhassa. Sytokiniinit ovat tärkeitä kasvun säätelijöitä, joskaan eivät ainoita. Olemme ensimmäisinä tutkijoina raportoineet sytokiniini-viestintäreitin ja kyseisen hormonin homeostasiaan vaikuttavat geenit haavan sukuisessa puussa (Populus trichocarpa). Analysoimme sytokiniini-kasvihormonin jakaumaa ja koko genomin laajuista geenien ilmentymistä aikaisempia tutkimuksia tarkemmalla erottelukyvyllä koko jälsivyöhykkeen läpi. Mielenkiintoisena, jonkinlaisena yllätyksenä havaitsimme, että lisäämällä geeniperäisesti sytokiniinien määrää myös jällen auksiinin määrä lisääntyy huomattavasti. Tulimme siihen johtopäätökseen, että ainakin sytokiniinit ja auksiini yhdessä vaikuttavat jällen kehitykseen ja puun paksuuskasvun nopeuteen ja eri hormonien yhteisvaikutuksien jatkotutkimus olisi erityisen tärkeää. Pystyimme myös osoittamaan, että sytokiniinilla ja auksiinilla on hieman toisistaan poikkeavat toiminnalliset vyöhykkeet. Sytokiniinit (ja sytokiniiniviestintä) ovat runsainta nilan puolella jälttä, kun taas auksiinivyöhyke on ksyleemin (puun) puolella jälttä (molemmat hormonit ovat voimakaasti läsnä jällessä). Olemme myös pystyneet muuttamaan (lisäämään ja vähentämään) jällen aktiivisuutta (muuttamalla sytokiniin määrää jällessä) ja siten vaikuttaneet tutkittavien puiden kasvuun. Lisäämällä geeniperäisesti jälsivyöhykkeen sytokiniinien määrää, havaitsimme huomattavaa puun paksuuskasvun lisääntymistä kasvihuoneolosuhteissa

Determining the composition of lignins in different tissues of Silber Birch

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Differential regulation of auxin and cytokinin during the secondary vascular tissue regeneration in Populus trees

Tissue regeneration upon wounding in plants highlights the developmental plasticity of plants. Previous studies have described the morphological and molecular changes of secondary vascular tissue (SVT) regeneration after large-scale bark girdling in trees. However, how phytohormones regulate SVT regeneration is still unknown. Here, we established a novel in vitro SVT regeneration system in the hybrid aspen (Populus tremula x Populus tremuloides) clone T89 to bypass the limitation of using field-grown trees. The effects of phytohormones on SVT regeneration were investigated by applying exogenous hormones and utilizing various transgenic trees. Vascular tissue-specific markers and hormonal response factors were also examined during SVT regeneration. Using this in vitro regeneration system, we demonstrated that auxin and cytokinin differentially regulate phloem and cambium regeneration. Whereas auxin is sufficient to induce regeneration of phloem prior to continuous cambium restoration, cytokinin only promotes the formation of new phloem, not cambium. The positive role of cytokinin on phloem regeneration was further confirmed in cytokinin overexpression trees. Analysis of a DR5 reporter transgenic line further suggested that cytokinin blocks the re-establishment of auxin gradients, which is required for the cambium formation. Investigation on the auxin and cytokinin signalling genes indicated these two hormones interact to regulate SVT regeneration. Taken together, the in vitro SVT regeneration system allows us to make use of various molecular and genetic tools to investigate SVT regeneration. Our results confirmed that complementary auxin and cytokinin domains are required for phloem and cambium reconstruction.Peer reviewe

Chemical profiles of birch and alder bark by ambient mass spectrometry

Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that allows the analysis of both polar and nonpolar compounds directly from the surfaces of various sample types. Here, DAPPI was used to study the chemical profiles in different parts of birch and alder tree barks. Four distinct fractions of Betula pendula (silver birch) bark were collected from three different developmental stages of the stem, after which the chemical profiles of the different tissue types were measured. Of special interest were triterpenoids, a class of important defensive substances, which are found in the bark of the silver birch. Additionally, the chemical profiles of lenticels and the surrounding surfaces in the phellem of B. pendula (silver birch), Alnus glutinosa (black alder), and Alnus incana (gray alder) were screened with DAPPI. Another ambient MS technique, laser ablation atmospheric pressure photoionization (LAAPPI), was further used for the mass spectrometry imaging of lenticels on the B. pendula phellem. All the studied birch bark fractions showed individual chemical profiles in DAPPI. The mass spectra from the young apical stem and the transition zone resembled each other more than the mature stem. Instead, the phellem was found to contain a high amount of triterpenoids in all the developmental stages of the stem. The most intense peaks in the DAPPI mass spectra of the birch bark fractions were those of betulin and lupeol. Betulinic and betulonic acid peaks were intense as well, and these compounds were detected especially in the lenticels of the tree samples.Peer reviewe

Cytokinin and Auxin Display Distinct but Interconnected Distribution and Signaling Profiles to Stimulate Cambial Activity.

Despite the crucial roles of phytohormones in plant development, comparison of the exact distribution profiles of different hormones within plant meristems has thus far remained scarce. Vascular cambium, a wide lateral meristem with an extensive developmental zonation, provides an optimal system for hormonal and genetic profiling. By taking advantage of this spatial resolution, we show here that two major phytohormones, cytokinin and auxin, display different yet partially overlapping distribution profiles across the cambium. In contrast to auxin, which has its highest concentration in the actively dividing cambial cells, cytokinins peak in the developing phloem tissue of a Populus trichocarpa stem. Gene expression patterns of cytokinin biosynthetic and signaling genes coincided with this hormonal gradient. To explore the functional significance of cytokinin signaling for cambial development, we engineered transgenic Populus tremula × tremuloides trees with an elevated cytokinin biosynthesis level. Confirming that cytokinins function as major regulators of cambial activity, these trees displayed stimulated cambial cell division activity resulting in dramatically increased (up to 80% in dry weight) production of the lignocellulosic trunk biomass. To connect the increased growth to hormonal status, we analyzed the hormone distribution and genome-wide gene expression profiles in unprecedentedly high resolution across the cambial zone. Interestingly, in addition to showing an elevated cambial cytokinin content and signaling level, the cambial auxin concentration and auxin-responsive gene expression were also increased in the transgenic trees. Our results indicate that cytokinin signaling specifies meristematic activity through a graded distribution that influences the amplitude of the cambial auxin gradient.J.I., K.N., J.A.S. and Y.H. were funded by ERC, Fibic EffFibre, Academy of Finland (by Centre of Excellence and other programs) and Tekes. O.P.S., L.P. and P.A. were funded by Academy of Finland. The hormone analysis was supported by Japan Advanced Plant Science Network. R.P.B. was funded by grants from Berzili, TC4F and FUTURE trees.This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.cub.2016.05.05

Characterization of cytokinin signaling and homeostasis gene families in two hardwood tree species : Populus trichocarpa and Prunus persica

Peer reviewe

Tissue-specific study across the stem reveals the chemistry and transcriptome dynamics of birch bark.

Tree bark is a highly specialized array of tissues that plays important roles in plant protection and development. Bark tissues develop from two lateral meristems; the phellogen (cork cambium) produces the outermost stem-environment barrier called the periderm, while the vascular cambium contributes with phloem tissues. Although bark is diverse in terms of tissues, functions and species, it remains understudied at higher resolution. We dissected the stem of silver birch (Betula pendula) into eight major tissue types, and characterized these by a combined transcriptomics and metabolomics approach. We further analyzed the varying bark types within the Betulaceae family. The two meristems had a distinct contribution to the stem transcriptomic landscape. Furthermore, inter- and intraspecies analyses illustrated the unique molecular profile of the phellem. We identified multiple tissue-specific metabolic pathways, such as the mevalonate/betulin biosynthesis pathway, that displayed differential evolution within the Betulaceae. A detailed analysis of suberin and betulin biosynthesis pathways identified a set of underlying regulators and highlighted the important role of local, small-scale gene duplication events in the evolution of metabolic pathways. This work reveals the transcriptome and metabolic diversity among bark tissues and provides insights to its development and evolution, as well as its biotechnological applications.peerReviewe

AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins

Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism.Peer reviewe

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.Peer reviewe