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Secondary growth relies on precise and specialized transcriptional networks that determine cell division, differentiation, and maturation of xylem cells. We identified a novel role for the ethylene-induced Populus Ethylene Response Factor PtERF85 (Potri.015G023200) in balancing xylem cell expansion and secondary cell wall (SCW) formation in hybrid aspen (Populus tremula x tremuloides). Expression of PtERF85 is high in phloem and cambium cells and during the expansion of xylem cells, while it is low in maturing xylem tissue. Extending PtERF85 expression into SCW forming zones of woody tissues through ectopic expression reduced wood density and SCW thickness of xylem fibers but increased fiber diameter. Xylem transcriptomes from the transgenic trees revealed transcriptional induction of genes involved in cell expansion, translation, and growth. The expression of genes associated with plant vascular development and the biosynthesis of SCW chemical components such as xylan and lignin, was down-regulated in the transgenic trees. Our results suggest that PtERF85 activates genes related to xylem cell expansion, while preventing transcriptional activation of genes related to SCW formation. The importance of precise spatial expression of PtERF85 during wood development together with the observed phenotypes in response to ectopic PtERF85 expression suggests that PtERF85 contributes to the transition of fiber cells from elongation to secondary cell wall deposition.

Differentiation of xylem elements involves cell expansion, secondary cell wall (SCW) deposition and programmed cell death. Transitions between these phases require strict spatiotemporal control.

The function of Populus ERF139 (Potri.013G101100) in xylem differentiation was characterized in transgenic overexpression and dominant repressor lines of ERF139 in hybrid aspen (Populus tremula × tremuloides). Xylem properties, SCW chemistry and downstream targets were analyzed in both types of transgenic trees using microscopy techniques, Fourier transform‐infrared spectroscopy, pyrolysis‐GC/MS, wet chemistry methods and RNA sequencing.

Opposite phenotypes were observed in the secondary xylem vessel sizes and SCW chemistry in the two different types of transgenic trees, supporting the function of ERF139 in suppressing the radial expansion of vessel elements and stimulating accumulation of guaiacyl‐type lignin and possibly also xylan. Comparative transcriptomics identified genes related to SCW biosynthesis (LAC5, LBD15, MYB86) and salt and drought stress‐responsive genes (ANAC002, ABA1) as potential direct targets of ERF139.

The phenotypes of the transgenic trees and the stem expression profiles of ERF139potential target genes support the role of ERF139 as a transcriptional regulator of xylem cell expansion and SCW formation, possibly in response to osmotic changes of the cells.

Tension wood (TW) in hybrid aspen trees forms on the upper side of displaced stems to generate a strain that leads to uplifting of the stem. TW is characterized by increased cambial growth, reduced vessel frequency and diameter, and the presence of gelatinous, cellulose-rich (G-)fibers with its microfibrils oriented parallel to the fiber cell axis. Knowledge remains limited about the molecular regulators required for the development of this special xylem tissue with its characteristic morphological, anatomical, and chemical features. In this study, we use transgenic, ethylene-insensitive (ETI) hybrid aspen trees together with time-lapse imaging to show that functional ethylene signaling is required for full uplifting of inclined stems. X-ray diffraction and Raman microspectroscopy of TW in ETI trees indicate that, although G-fibers form, the cellulose microfibril angle in the G-fiber S-layer is decreased, and the chemical composition of S- and G-layers is altered than in wild-type TW. The characteristic asymmetric growth and reduction of vessel density is suppressed during TW formation in ETI trees. A genome-wide transcriptome profiling reveals ethylene-dependent genes in TW, related to cell division, cell wall composition, vessel differentiation, microtubule orientation, and hormone crosstalk. Our results demonstrate that ethylene regulates transcriptional responses related to the amount of G-fiber formation and their properties (chemistry and cellulose microfibril angle) during TW formation. The quantitative and qualitative changes in G-fibers are likely to contribute to uplifting of stems that are displaced from their original position.

Introduction The oxylipin methyl jasmonate (MeJA) is a plant hormone active in response signalling and defence against herbivores. Although MeJA is applied experimentally to mimic herbivory and induce plant defences, its downstream effects on the plant metabolome are largely uncharacterized, especially in the context of primary growth and tissue-specificity of the response. Objectives We investigated the effects of MeJA-simulated and real caterpillar herbivory on the foliar metabolome of the wild plant Brassica nigra and monitored the herbivore-induced responses in relation to leaf ontogeny. Methods As single or multiple herbivory treatments, MeJA- and mock-sprayed plants were consecutively exposed to caterpillars or left untreated. Gas chromatography (GC) and liquid chromatography (LC) time-of-flight mass-spectrometry (TOF-MS) were combined to analyse foliar compounds, including central primary and specialized defensive plant metabolites. Results Plant responses were stronger in young leaves, which simultaneously induced higher chlorophyll levels. Both MeJA and caterpillar herbivory induced similar, but not identical, accumulation of tricarboxylic acids (TCAs), glucosinolates (GSLs) and phenylpropanoids (PPs), but only caterpillar feeding led to depletion of amino acids. MeJA followed by caterpillars caused higher induction of defence compounds, including a three-fold increase in the major defence compound allyl-GSL (sinigrin). When feeding on MeJA-treated plants, caterpillars gained less weight indicative of the reduced host-plant quality and enhanced resistance. Conclusions The metabolomics approach showed that plant responses induced by herbivory extend beyond the regulation of defence metabolism and are tightly modulated throughout leaf development. This leads to a new understanding of the plant metabolic potential that can be exploited for future plant protection strategies.

Thickening of tree stems is the result of secondary growth, accomplished by the meristematic activity of the vascular cambium. Secondary growth of the stem entails developmental cascades resulting in the formation of secondary phloem outwards and secondary xylem (i.e., wood) inwards of the stem. Signaling and transcriptional reprogramming by the phytohormone ethylene modifies cambial growth and cell differentiation, but the molecular link between ethylene and secondary growth remains unknown. We addressed this shortcoming by analyzing expression profiles and co-expression networks of ethylene pathway genes using the AspWood transcriptome database which covers all stages of secondary growth in aspen (Populus tremula) stems. ACC synthase expression suggests that the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is synthesized during xylem expansion and xylem cell maturation. Ethylene-mediated transcriptional reprogramming occurs during all stages of secondary growth, as deduced from AspWood expression profiles of ethylene-responsive genes. A network centrality analysis of the AspWood dataset identified EIN3D and 11 ERFs as hubs. No overlap was found between the co-expressed genes of the EIN3 and ERF hubs, suggesting target diversification and hence independent roles for these transcription factor families during normal wood formation. The EIN3D hub was part of a large co-expression gene module, which contained 16 transcription factors, among them several new candidates that have not been earlier connected to wood formation and a VND-INTERACTING 2 (VNI2) homolog. We experimentally demonstrated Populus EIN3D function in ethylene signaling in Arabidopsis thaliana. The ERF hubs ERF118 and ERF119 were connected on the basis of their expression pattern and gene co-expression module composition to xylem cell expansion and secondary cell wall formation, respectively. We hereby establish data resources for ethylene-responsive genes and potential targets for EIN3D and ERF transcription factors in Populus stem tissues, which can help to understand the range of ethylene targeted biological processes during secondary growth.

The woody tissues serve to stabilise plants, store nutrients and translocate water and minerals. The formation of wood, or ’secondary xylem’, follows a well-defined developmental gradient which is initiated by cell division activity in the vascular cambium. The ’xylem cambial derivatives’ then expand before deposition of the secondary cell wall (SCW), which is where most of the biomass of wood originates. After this, some cells of the xylem typically undergo programmed cell death (PCD). Cellulose and lignin are chemical components of the SCW that provide structural support and water impermeability, respectively. The chemical composition of the SCWs is also important economically since it affects the efficiency of wood processing during pulping and enzymatic hydrolysis. Two dominant xylem cell types of angiosperm tree species like Populus are the fibers and the vessel elements. Fibers are important for the mechanical strength of the wood and provide the majority of the wood biomass. Vessel elements join endwise to form hollow tubes, or vessels, for the purposes of water and solute transport in the stem.

Formation of wood is a complex process, subject to multiple levels of regulation. Plant hormones are important for wood formation, and ethylene signalling has been shown to stimulate cambial activity, affect the ratio between fibers and vessel elements, as well as the expansion of the cambial derivatives. Ethylene is also involved in the ‘tension wood’ response of stems that are displaced from their original vertical position. Formation of ’tension wood’ generates a force that lifts the stem back to the upright growing position. What remains unknown is the molecular link between ethylene signalling and wood formation. The work in this thesis focuses on providing this link using the model tree species hybrid aspen (Populus tremula x tremuloides).

Using a state-of-the-art transcriptomic database that spans all phases of xylem differentiation in hybrid aspen wood, from cell division through xylem cell expansion to xylem maturation (SCW deposition and PCD), the expression of the ethylene pathway related genes was investigated during normal wood formation. The analyses reveal ethylene perception and transcriptional reprogramming is possible across all zones of wood formation. Previously uncharacterised components were identified that may be important contributors to wood formation. Furthermore, although ethylene is known to affect the ratio between the abundance of the vessel elements and the fibers, genetic evidence is lacking. Using the tension wood response and transgenic trees modified in ethylene signalling, it was shown that ethylene is a negative regulator of vessel formation and important for a functional tension wood response. Furthermore, characterisation of two transcription factors (TFs), belonging to the ethylene response factor (ERF) gene family, suggests that aspects of xylem cell division, expansion and subsequent SCW formation, including lignification, can be affected by ERF85 and ERF139 in an ethylene-dependent manner. Phase transitions during wood formation need to be controlled spatiotemporally, and transcriptional regulation by these ERFs seems to be part of such control to establish correct transitions between cell expansion, secondary cell wall formation and lignification. The work presented here also identifies promising additions to the toolkit available for forest tree biotechnology and molecular breeding programmes.

Växternas ledningsvävnad består till stora delar av vedartad vävnad som bidrar till att stabilisera plantan, lagra näringsämnen och transportera vatten och mineraler. Bildandet av veden, eller "det sekundära xylemet", är en väldefinierad process som börjar med celldelningsaktiviteten i det vaskulära kambiet.  Celler som bildas i kambiet differentierar genom att först expandera och sedan bilda en tjock sekundär cellvägg (SCV) som ger upphov till största delen av biomassa i veden. Efter detta, vissa celler i xylemet dör genom ett genetiskt styrt celldödsprogram.  SCV innehåller kemiska komponenter, så som cellulosa och lignin, som är viktiga för funktionen av hela plantan. Cellulosa utformar skelettet i SCV och lignin ger en hydrofobisk yta som behövs för vattentransport. Sammansättningen och mängden av SCV komponenter är viktiga också för industriella tillämpningar. Cellulosa är värdeful som råvara för produktion av pappersmassa och olika bioprodukter medans lignin oftast är en störande komponent som måste avlägsnas i dessa processer. De vanligaste xylem celltyper i trädslag som poplar och aspar är ’kärlelement’ och ’fibrer’. Fibrer ger mekanisk hållfasthet och största delen av vedbiomassan. Kärlelement bildar ihåliga rör, eller ’kärl’, som transporterar vatten och mineraler i stammen.

Vedbildning är en komplicerad process  some regleras av olika faktorer så som växthormoner. En är de viktigaste växthormoner i vedbildning är etylen. Den har visats stimulera kambieaktiviteten, påverka förhållandet mellan fibrer och kärlelement, och expansion av xylemceller. Etylen är också involverad i bildandet av såkallad ”dragved” som sker när trädstammen flyttas från sitt ursprungliga vertikala läge p.g.a. till exempel vind. Bildandet av dragved genererar en kraft som lyfter stammen tillbaka till den upprätt växande ställningen. Det som fortfarande är okänt är den molekylära kopplingen mellan etylensignalering och vedbildning. Arbetet i denna avhandling har haft en målsättning att avslöja åtminstone delar av denna koppling i hybrid asp (Populus tremula x tremuloides).

En databas över genutryck användes för att visa att etylensignalering pågår i flera olika faser av xylemutveckling. Det är därför troligt att etylen kan också reglera flera olika processer som sker under differentiering av xylemceller.  Flera nya komponenter kunde också identifieras som verkar delta i reglering av vedbildning. Farmakologiska experiment och genetisk modifiering av etylensignalering avslöjade att etylen reglerar negativt bildning av nya kärlelement i veden. Ökat antal kärlelement i etylenokänsliga, genetiskt modifierade hybrid asp träd korrelerade negativt med  trädens kapacitet att nå upprätt växande ställning efter att de hade placerats i en vågrätt ställning, vilket tyder på att andelen kärlelement är central för funktionen av dragved.  Vidare studerades två transkriptionsfaktorer som hör till ETYLEN RESPONS FAKTOR (ERF) genfamilj. ERF85 och ERF139 är både uttryckta i xylemet, och deras genuttryck kräver funktionell etylen signalering. Globala genuttrycksstudier i hybrid asp träd som var genetiskt modifierade antingen för ERF85 eller ERF139 avslöjade att de har olika molekylära mekanismer men att de båda verkade influera övergången mellan olika faser av xylem differentiering. Detta bekräftades också genom analys av xylem differentiering och vedkemi i de genetiskt modifierade träden. Det samlade data från arbeten som presenteras i denna avhandling identifierar också lovande tillägg till den verktygslåda som finns för biotekniska initiativ och molekylär avel i skogsträd.