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Secondary growth relies on precise and specialized transcriptional networks that determine cell division, differentiation, and maturation of xylem cells. We identify a novel role for the ethylene induced Populus ETHYLENE RESPONSE FACTOR ERF85 (Potri.015G023200) in balancing xylem cell expansion and secondary cell wall (SCW) formation in hybrid aspen (Populus tremula x tremuloides). Expression of ERF85 is high in phloem and cambium cells and during expansion of xylem cells, while it is low in maturing xylem tissue. Extending ERF85 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. Expression of genes associated with plant vascular development and biosynthesis of SCW chemical components such as xylan and lignin, was downregulated in the transgenic trees. Our results suggest that ERF85 activates genes related with xylem cell expansion, while preventing transcriptional activation of genes related to SCW formation. The importance of precise spatial expression of ERF85 during wood development together with the observed phenotypes in response to ectopic ERF85 expression suggests that ERF85 functions as a switch between different phases of xylem differentiation during wood formation.

Highlight Axillary buds uniquely regulate gibberellin (GA) pathway genes, enabling them to stay inhibited but simultaneously poised for growth. Decapitation promotes expression of GA-inducible 1,3-β-glucanase genes that function to reinvigorate symplasmic connections to the stem., Axillary buds (AXBs) of hybrid aspen (Populus tremula×P. tremuloides) contain a developing dwarfed shoot that becomes para-dormant at the bud maturation point. Para-dormant AXBs can grow out after stem decapitation, while dormant AXBs pre-require long-term chilling to release them from dormancy. The latter is mediated by gibberellin (GA)-regulated 1,3-β-glucanases, but it is unknown if GA is also important in the development, activation, and outgrowth of para-dormant AXBs. The present data show that para-dormant AXBs up-regulate GA receptor genes during their maturation, but curtail GA biosynthesis by down-regulating the rate-limiting GIBBERELLIN 3-OXIDASE2 (GA3ox2), which is characteristically expressed in the growing apex. However, decapitation significantly up-regulated GA3ox2 and GA4-responsive 1,3-β-glucanases (GH17-family; α-clade). In contrast, decapitation down-regulated γ-clade 1,3-β-glucanases, which were strongly up-regulated in maturing AXBs concomitant with lipid body accumulation. Overexpression of selected GH17 members in hybrid aspen resulted in characteristic branching patterns. The α-clade member induced an acropetal branching pattern, whereas the γ-clade member activated AXBs in recurrent flushes during transient cessation of apex proliferation. The results support a model in which curtailing the final step in GA biosynthesis dwarfs the embryonic shoot, while high levels of GA precursors and GA receptors keep AXBs poised for growth. GA signaling, induced by decapitation, reinvigorates symplasmic supply routes through GA-inducible 1,3-β-glucanases that hydrolyze callose at sieve plates and plasmodesmata.

SummaryResearch conductedThe phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. Here we report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation.MethodsWe applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild type and ethylene insensitive hybrid aspen trees(Populus tremula x tremuloides)and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild type and ethylene insensitive trees.Key resultsWe demonstrate that ACC and ethylene induce gelatinous-layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (ERFs, EIN3/EIL1) and wood formation.ConclusionG-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

Summary Ethylene Response Factors (ERFs) are a large family of transcription factors that mediate responses to ethylene. Ethylene affects many aspects of wood development and is involved in tension wood formation. Thus ERFs could be key players connecting ethylene action to wood development. We identified 170 gene models encoding ERFs in the Populus trichocarpa genome. The transcriptional responses of ERF genes to ethylene treatments were determined in stem tissues of hybrid aspen (Populus tremula × tremuloides) by qPCR. Selected ethylene-responsive ERFs were overexpressed in wood-forming tissues and characterized for growth and wood chemotypes by FT-IR. Fifty ERFs in Populus showed more than five-fold increased transcript accumulation in response to ethylene treatments. Twenty-six ERFs were selected for further analyses. A majority of these were induced during tension wood formation. Overexpression of ERFs 18, 21, 30, 85 and 139 in wood-forming tissues of hybrid aspen modified the wood chemotype. Moreover, overexpression of ERF139 caused a dwarf-phenotype with altered wood development, and overexpression of ERF18, 34 and 35 slightly increased stem diameter. We identified ethylene-induced ERFs that respond to tension wood formation, and modify wood formation when overexpressed. This provides support for their role in ethylene-mediated regulation of wood development.

The involvement of the plant growth regulator ethylene in somatic embryo maturation of Pinus sylvestris (L.) was investigated. Genes that encoded 1-aminocyclopropane-1-carboxylate synthase (ACS), the rate-limiting enzyme in the ethylene biosynthesis pathway, were isolated and characterized. Two novel complementary DNAs (cDNAs) of PsACS1 and PsACS2 that encode ACS were isolated from embryogenic cultures (ECs) along with their polymerase chain reaction (PCR) products. The two sequences shared 69% similarity at amino acid level. PsACS1 was expressed in ECs at the proliferation and maturation stages, whereas the PsACS2 transcript was expressed in embryos only at the maturation stage. These expression levels varied among the five P. sylvestris genotypes studied. Moreover, the high levels of PsACS2 transcript abundance correlated with higher numbers of embryos at stage 3 of development. Ethylene production reached the highest levels at stage 3, and subsequently dropped. Hence, expression of PsACS2 corresponded to ethylene production during embryo development and could thus serve as a genetic marker for the early maturation stage. Both gene expression and ethylene production varied among the different genotypes. Investigating expression of ACC synthase genes further enhanced our understanding of the role of ethylene in embryo development and maturation and improved the efficiency of somatic embryogenesis of P. sylvestris.

In trees, production of intercellular signals and accessibility of signal conduits jointly govern dormancy cycling at the shoot apex. We identified 10 putative cell wall 1,3-β-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-β-glucan (callose) at pores and plasmodesmata (PD) and investigated their regulation in relation to FT and CENL1 expression. The 10 genes encode orthologs of Arabidopsis thaliana BG_ppap, a PD-associated glycosylphosphatidylinositol (GPI) lipid-anchored protein, the Arabidopsis PD callose binding protein PDCB, and a birch (Betula pendula) putative lipid body (LB) protein. We found that these genes were differentially regulated by photoperiod, by chilling (5°C), and by feeding of gibberellins GA3 and GA4. GA3 feeding upregulated all LB-associated GH17s, whereas GA4 upregulated most GH17s with a GPI anchor and/or callose binding motif, but only GA4 induced true bud burst. Chilling upregulated a number of GA biosynthesis and signaling genes as well as FT, but not CENL1, while the reverse was true for both GA3 and GA4. Collectively, the results suggest a model for dormancy release in which chilling induces FT and both GPI lipid-anchored and GA3-inducible GH17s to reopen signaling conduits in the embryonic shoot. When temperatures rise, the reopened conduits enable movement of FT and CENL1 to their targets, where they drive bud burst, shoot elongation, and morphogenesis.

The effects of the transformation of a 4-coumarate:coenzyme A ligase (4CL) gene involved in lignin biosynthesis on the interactions between transgenic plants and soil microbiota were studied in silver birch (Betula pendula Roth). Although the genetic transformation did not lead to expected changes in wood chemistry, it had significant influence on root biomass and morphology. The transgenic lines had less root biomass and lower numbers of root tips than the wild-type birch. However, the transgenic lines formed normal ectomycorrhizas with Paxillus involutus (Batsch) Fr. and no differences between the transgenic lines and the wild-type birch were detected in the colonization by mycorrhizas in vitro. In a field trial, the decomposition of the transgenic and wild-type birch litter was equally rapid, and no marked differences were found between the transgenic lines and wild-type birch in total microbial biomass (SIR) or activity (basal respiration). The effects of the genetic transformation on the ergosterol content of the leaf litter were contradictory; the litter from one transgenic line had significantly higher ergosterol content and the litters from two other transgenic lines had significantly lower ergosterol content than the litter from wild-type birch. This study shows that the 4CL antisense transformation and the pleiotropic effects of the transformation on growth characteristics of birch did not disturb the formation of ectomycorrhizas with P. involutus and had no clear influence on the biodegradability of the leaf litter.

We have used genotypic variation in birch (Betula pendula Roth) to investigate the roles of ozone (O3)-induced ethylene (ET), jasmonic acid, and salicylic acid in the regulation of tissue tolerance to O3. Of these hormones, ET evolution correlated best with O3-induced cell death. Disruption of ET perception by transformation of birch with the dominant negative mutant allele etr1-1 of the Arabidopsis ET receptor gene ETR1 or blocking of ET perception with 1-methylcyclopropene reduced but did not completely prevent the O3-induced cell death, when inhibition of ET biosynthesis with aminooxyacetic acid completely abolished O3 lesion formation. This suggests the presence of an ET-signaling-independent but ET biosynthesis-dependent component in the ET-mediated stimulation of cell death in O3-exposed birch. Functional ET signaling was required for the O3 induction of the gene encoding β-cyanoalanine synthase, which catalyzes detoxification of the cyanide formed during ET biosynthesis. The results suggest that functional ET signaling is required to protect birch from the O3-induced cell death and that a decrease in ET sensitivity together with a simultaneous, high ET biosynthesis can potentially cause cell death through a deficient detoxification of cyanide.

The role of ethylene (ET) signaling in the responses of two hybrid aspen (Populus tremula L. × P.tremuloides Michx.) clones to chronic ozone (O3; 75 nL L−1) was investigated. The hormonal responses differed between the clones; the O3-sensitive clone 51 had higher ET evolution than the tolerant clone 200 during the exposure, whereas the free salicylic acid concentration in clone 200 was higher than in clone 51. The cellular redox status, measured as glutathione redox balance, did not differ between the clones suggesting that the O3 lesions were not a result of deficient antioxidative capacity. The buildup of salicylic acid during chronic O3 exposure might have prevented the up-regulation of ET biosynthesis in clone 200. Blocking of ET perception with 1-methylcyclopropene protected both clones from the decrease in net photosynthesis during chronic exposure to O3. After a pretreatment with low O3 for 9 d, an acute 1.5-fold O3 elevation caused necrosis in the O3-sensitive clone 51, which increased substantially when ET perception was blocked. The results suggest that in hybrid aspen, ET signaling had a dual role depending on the severity of the stress. ET accelerated leaf senescence under low O3, but under acute O3 elevation, ET signaling seemed to be required for protection from necrotic cell death.

The rate-limiting enzyme in the ethylene biosynthesis pathway is 1-aminocyclo-propane-1 -carboxylate (ACC) synthase. We have investigated an ozone (O 3 )-induced ACC synthase gene in Arabidopsis thaliana. Four previously reported ACC synthase genes from A. thaliana (ACS1. ACS2. ACS4 and ACS5) were not induced in response to O 3 stress. An O 3 -induced 1098-bp cDNA encoding ACC synthase was isolated from foliage of A. thaliana; this sequence was identical to that of ACS6 which could be induced by multiple stimuli including touch. When plants were treated with 350 nl l -1 of O 3 for 2 or 6 h, the evolution of ethylene became maximal after 1.5 h and gradually decreased thereafter. Similarly, the mRNA transcript of O 3 -induced ACC synthase was detected after 30 min of exposure, attained a maximum level after 1 h, and then declined. Visible damage in O 3 -treated plants was observed at the end of the 2-h exposure. This ACC synthase gene in A. thaliana provides a useful tool for studying O 3 -induced responses related to ethylene production at the molecular and genetic levels.

The plant hormone ethylene is an important signal in plant growth responses to environmental cues. In vegetative growth, ethylene is generally considered as a regulator of cell expansion, but a role in the control of meristem growth has also been suggested based on pharmacological experiments and ethylene-overproducing mutants. In this study, we used transgenic ethylene-insensitive and ethylene-overproducing hybrid aspen ( Populus tremula × tremuloides ) in combination with experiments using an ethylene perception inhibitor [1-methylcyclopropene (1-MCP)] to demonstrate that endogenous ethylene produced in response to leaning stimulates cell division in the cambial meristem. This ethylene-controlled growth gives rise to the eccentricity of Populus stems that is formed in association with tension wood.

Background The phytohormone ethylene (ET) has the potential to regulate secondary growth of plants and wood formation in trees. Application of exogenous ethylene or its in planta precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), to wood forming tissues of hybrid aspen (Populus tremula x Populus tremuloides) enhances xylem growth [1]. In the same study it was demonstrated that stimulation of enhanced xylem formation (tension wood, TW) at the upper side of leaning stems is mediated by endogenous ET. The production of endogenous ET in TW forming tissues is further supported by the increase of ACC oxidase gene transcript and enzyme activity on the TW side [2]. The ET perception and signal transmission cascade in Arabidopsis has been linked to the transcriptional activation of Ethylene Response Factors (ERFs) [3,4]. As transcription factors, ERFs regulate the expression of various specific downstream target genes by binding to cis-elements in their promoters [5]. We hypothesize that ERFs participate in xylem development through ethylene signaling and that they are involved in ET responses during TW formation.