Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.
Thaxtomin A, a phytotoxin produced by Streptomyces eubacteria, is suspected to act as a natural cellulose synthesis inhibitor. This view is confirmed by the results obtained from new chemical, molecular, and microscopic analyses of Arabidopsis thaliana seedlings treated with thaxtomin A. Cell wall analysis shows that thaxtomin A reduces crystalline cellulose, and increases pectins and hemicellulose in the cell wall. Treatment with thaxtomin A also changes the expression of genes involved in primary and secondary cellulose synthesis as well as genes associated with pectin metabolism and cell wall remodelling, in a manner nearly identical to isoxaben. In addition, it induces the expression of several defence-related genes and leads to callose deposition. Defects in cellulose synthesis cause ectopic lignification phenotypes in A. thaliana, and it is shown that lignification is also triggered by thaxtomin A, although in a pattern different from isoxaben. Spinning disc confocal microscopy further reveals that thaxtomin A depletes cellulose synthase complexes from the plasma membrane and results in the accumulation of these particles in a small microtubule-associated compartment. The results provide new and clear evidence for thaxtomin A having a strong impact on cellulose synthesis, thus suggesting that this is its primary mode of action.
Arabidopsis thaliana; callose; cellulose; defence response; isoxaben; lignin; microtubule; phytotoxin; Streptomyces; thaxtomin
Thaxtomin A (TXT) is a phytotoxin produced by all plant-pathogenic Streptomyces scabies involved in the potato scab disease. Their pathogenicity was previously correlated with the production of TXT. Calcium is known to be an essential second messenger associated with pathogen-induced plant responses and cell death. We have effectively shown that in Arabidopsis thaliana cell suspensions, TXT induces an early short lived Ca2+ influx which is involved in the cell death process and other TXT-induced responses. We extended our study to Nicotiana tabacum BY2 by monitoring cell death and changes in cytosolic calcium concentration on cells expressing the apoaequorine Ca2+ reporter protein to compare the responses to TXT of the two model plants, tobacco and A. thaliana. Our investigations show that cell death in BY2 appeared to be dose dependent with a lag of sensitivity comparing to A. thaliana. Moreover, pathway leading to cell death in BY2 does not involve calcium signaling. Our results suggest that different pathways are engaged in A. thaliana and N. tabacum BY2 to achieve the same response to TXT.
Arabidopsis thaliana; calcium; cell death; Nicotiana tabacum BY2; plant pathogen; thaxtomin A
Four highly inducible genes of poplar trees, PtdKTI5, PtdWIN4, PtdPOP3 from hybrid poplar (Populus trichocarpa × P. deltoides) and PtKTI2 from trembling aspen (Populus tremuloides Michx.) have been individually transformed into Arabidopsis thaliana for overexpression. High transcriptional level of each transgene in transgenic Arabidopsis lines was confirmed by RT-PCR analysis. The development, body weight and survivorship of cotton bollworm (Helicoverpa armigera) fed on four types of transgenic Arabidopis plants were evaluated in the laboratory. Our data indicated that these four Populus defense-related genes exhibited various degree of insectital activity on larval and postlarval development of cotton bollworm and may be utilized for herbivore resistance improvement in plant genetic engineering.
wounding-inducible gene; Populus; transgenic Arabidopsis; Helicoverpa armigera; anti-herbivore
Thaxtomin A (TA) is a phytotoxin produced by plant pathogenic Streptomyces spp. responsible for potato common scab. TA inhibits cellulose biosynthesis in expanding plant tissues and is essential for disease induction. Auxin treatment of various plant tissues has been repeatedly demonstrated to inhibit TA toxicity and to reduce common scab. This work utilises Arabidopsis thaliana mutants with resistance to cellulose biosynthesis inhibitors (CBIs) to investigate the interaction between TA, other CBIs and auxins.
Three CBI resistant A. thaliana mutants; txr1-1 (tolerance to TA), ixr1-1 (tolerance to isoxaben - IXB) and KOR1 (cellulose deficiency), showed no altered root growth response to treatment with natural or synthetic auxins, nor with the auxin efflux transport inhibitor 2,3,5-Triiodobenzoic acid (TIBA). However, all mutants had significantly enhanced tolerance to 1-napthylphthalamic acid (NPA), another auxin efflux transport inhibitor, which blocks polar auxin transport at a site distinct from TIBA. NPA tolerance of txr1-1 and ixr1-1 was further supported by electrophysiological analysis of net H+ fluxes in the mature, but not elongation zone of roots. All three mutants showed increased tolerance to IXB, but only txr1-1 showed tolerance to TA. No mutant showed enhanced tolerance to a third CBI, dichlobenil (DCB).
We have demonstrated that plant tolerance to TA and IXB, as well as cell wall synthesis modifications in roots, have resulted in specific co-resistance to NPA but not TIBA. This suggests that CBI resistance has an impact on polar auxin efflux transport processes associated with the NPA binding protein. We also show that NPA inhibitory response in roots occurs in the mature root zone but not the elongation zone. Responses of mutants to CBIs indicate a similar, but not identical mode of action of TA and IXB, in contrast to DCB.
1-napthylphthalamic acid - NPA; 2,3,5-Triiodobenzoic acid - TIBA; Thaxtomin A; Isoxaben; Dichlobenil; Cellulose biosynthetic inhibitor; Common scab; Ion fluxes; Plasma membrane
SHORT-ROOT (SHR) is a well characterized regulator of cell division and cell fate determination in the Arabidopsis primary root. However, much less is known about the functions of SHR in the aerial parts of the plant. In this work, we cloned SHR gene from Populus trichocarpa (PtSHR1) as an AtSHR ortholog and down-regulated its expression in hybrid poplar (Populus tremula×P. tremuloides Michx-clone T89) in order to determine its physiological functions in shoot development. Sharing a 90% similarity to AtSHR at amino acid level, PtSHR1 was able to complement the Arabidopsis shr mutant. Down regulation of PtSHR1 led to a strong enhancement of primary (height) and secondary (girth) growth rates in the transgenic poplars. A similar approach in Arabidopsis showed a comparable accelerated growth and development phenotype. Our results suggest that the response to SHR could be dose-dependent and that a partial down-regulation of SHR could lead to enhanced meristem activity and a coordinated acceleration of plant growth in woody species. Therefore, SHR functions in plant growth and development as a regulator of cell division and meristem activity not only in the roots but also in the shoots. Reducing SHR expression in transgenic poplar was shown to lead to significant increases in primary and secondary growth rates. Given the current interest in bioenergy crops, SHR has a broader role as a key regulator of whole plant growth and development and SHR suppression has considerable potential for accelerating biomass accumulation in a variety of species.
Two thaumatin-like proteins (TLPs) were previously identified in phloem exudate of hybrid poplar (Populus trichocarpa × P. deltoides) using proteomics methods, and their sieve element localization confirmed by immunofluorescence. In the current study, we analyzed different tissues to further understand TLP expression and localization in poplar, and used immunogold labelling to determine intracellular localization.
Immunofluorescence using a TLP antiserum confirmed the presence of TLP in punctate, organelle-like structures within sieve elements. On western blots, the antiserum labeled two constitutively expressed proteins with distinct expression patterns. Immunogold labelling suggested that TLPs are associated with starch granules and starch-containing plastids in sieve elements and phloem parenchyma cells. In addition, the antiserum recognized TLPs in the inner cell wall and sieve plate region of sieve elements.
TLP localization in poplar cells and tissues is complex. TLP1 is expressed predominantly in tissues with a prominent vascular system such as midveins, petioles and stems, whereas the second TLP is primarily expressed in starch-storing plastids found in young leaves and the shoot apex.
Polychlorinated biphenyls (PCBs) are widely distributed persistent organic pollutants. In vitro research has shown that plant cell cultures might transform lower chlorinated congeners to hydroxylated PCBs, but there are few studies on in vivo metabolism of PCBs by intact whole plants. In this research, poplar plants (Populus deltoides × nigra, DN34) and switchgrass (Panicum vigratum, Alamo) were hydroponically exposed to 3,3′,4,4′-tetrachlorobiphenyl (CB77). Metabolism in plants occurred rapidly, and metabolites were detected after only a 24 h exposure. Rearrangement of chlorine atoms and dechlorination of CB77 by plants was unexpectedly observed. In addition, poplars were able to hydroxylate CB77 and the metabolite 6-hydroxy-3,3′,4,4′-tetrachlorobiphenyl (6-OH-CB77) was identified and quantified. Hybrid poplar was able to hydroxylate CB77, but switchgrass was not, suggesting that enzymatic transformations are plant specific. Sulfur-containing metabolites (from the action of sulfotransferases) were investigated in this study, but they were not detected in either poplar or switchgrass.
Whole poplars in vivo, candidate species for remediation at dredged material disposal sites, are able to hydroxylate and dechlorinate CB77 with chlorine substituent rearrangements.
Hybrid poplars species are candidates for biomass production but breeding efforts are needed to combine productivity and water use efficiency in improved cultivars. The understanding of the genetic architecture of growth in poplar by a Quantitative Trait Loci (QTL) approach can help us to elucidate the molecular basis of such integrative traits but identifying candidate genes underlying these QTLs remains difficult. Nevertheless, the increase of genomic information together with the accessibility to a reference genome sequence (Populus trichocarpa Nisqually-1) allow to bridge QTL information on genetic maps and physical location of candidate genes on the genome. The objective of the study is to identify QTLs controlling productivity, architecture and leaf traits in a P. deltoides x P. trichocarpa F1 progeny and to identify candidate genes underlying QTLs based on the anchoring of genetic maps on the genome and the gene ontology information linked to genome annotation. The strategy to explore genome annotation was to use Gene Ontology enrichment tools to test if some functional categories are statistically over-represented in QTL regions.
Four leaf traits and 7 growth traits were measured on 330 F1 P. deltoides x P. trichocarpa progeny. A total of 77 QTLs controlling 11 traits were identified explaining from 1.8 to 17.2% of the variation of traits. For 58 QTLs, confidence intervals could be projected on the genome. An extended functional annotation was built based on data retrieved from the plant genome database Phytozome and from an inference of function using homology between Populus and the model plant Arabidopsis. Genes located within QTL confidence intervals were retrieved and enrichments in gene ontology (GO) terms were determined using different methods. Significant enrichments were found for all traits. Particularly relevant biological processes GO terms were identified for QTLs controlling number of sylleptic branches: intervals were enriched in GO terms of biological process like ‘ripening’ and ‘adventitious roots development’.
Beyond the simple identification of QTLs, this study is the first to use a global approach of GO terms enrichment analysis to fully explore gene function under QTLs confidence intervals in plants. This global approach may lead to identification of new candidate genes for traits of interest.
Secondary cell walls, consisting of cellulose, hemicelluloses and lignin, make up the bulk of wood biomass. It is therefore expected that dissection of the molecular mechanisms underlying secondary wall biosynthesis and its regulation will be instrumental to unravel the process of wood formation in tree species. Wood formation requires the coordinated activation of genes in the secondary wall biosynthetic program that is essential for the biosynthesis and assembly of wood components. It has recently been discovered that a group of poplar (Populus trichocarpa) wood-associated NAC domain transcription factors, PtrWNDs, which are functional orthologs of the Arabidopsis SND1, are capable of turning on the entire secondary wall biosynthetic program when expressed in Arabidopsis. In addition, two of the PtrWNDs were found to be able to activate the promoters of poplar wood biosynthetic genes and a number of other poplar wood-associated transcription factors. Further testing reveals that the promoters of these poplar wood-associated transcription factors are also activated by other PtrWNDs. It is therefore proposed that PtrWNDs are master transcriptional switches regulating a cascade of downstream transcription factors and thereby mediate the coordinated activation of wood biosynthetic genes during wood formation.
NAC domain transcription factor; Populus trichocarpa; secondary wall biosynthesis; transcriptional regulation; wood formation
Enterobacter sp. 638 is an endophytic plant growth promoting gamma-proteobacterium that was isolated from the stem of poplar (Populus trichocarpa×deltoides cv. H11-11), a potentially important biofuel feed stock plant. The Enterobacter sp. 638 genome sequence reveals the presence of a 4,518,712 bp chromosome and a 157,749 bp plasmid (pENT638-1). Genome annotation and comparative genomics allowed the identification of an extended set of genes specific to the plant niche adaptation of this bacterium. This includes genes that code for putative proteins involved in survival in the rhizosphere (to cope with oxidative stress or uptake of nutrients released by plant roots), root adhesion (pili, adhesion, hemagglutinin, cellulose biosynthesis), colonization/establishment inside the plant (chemiotaxis, flagella, cellobiose phosphorylase), plant protection against fungal and bacterial infections (siderophore production and synthesis of the antimicrobial compounds 4-hydroxybenzoate and 2-phenylethanol), and improved poplar growth and development through the production of the phytohormones indole acetic acid, acetoin, and 2,3-butanediol. Metabolite analysis confirmed by quantitative RT–PCR showed that, the production of acetoin and 2,3-butanediol is induced by the presence of sucrose in the growth medium. Interestingly, both the genetic determinants required for sucrose metabolism and the synthesis of acetoin and 2,3-butanediol are clustered on a genomic island. These findings point to a close interaction between Enterobacter sp. 638 and its poplar host, where the availability of sucrose, a major plant sugar, affects the synthesis of plant growth promoting phytohormones by the endophytic bacterium. The availability of the genome sequence, combined with metabolome and transcriptome analysis, will provide a better understanding of the synergistic interactions between poplar and its growth promoting endophyte Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar as an energy feedstock on marginal, non-agricultural soils using endophytic bacteria as growth promoting agents.
Poplar is considered as the model tree species for the production of lignocellulosic biomass destined for biofuel production. The plant growth promoting endophytic bacterium Enterobacter sp. 638 can improve the growth of poplar on marginal soils by as much as 40%. This prompted us to sequence the genome of this strain and, via comparative genomics, identify functions essential for the successful colonization and endophytic association with its poplar host. Analysis of the genome sequence, combined with metabolite analysis and quantitative PCR, pointed to a remarkable interaction between Enterobacter sp. 638 and its poplar host with the endophyte responsible for the production of a phytohormone, and a precursor for another that poplar is unable to synthesize, and where the production of the plant growth promoting compounds depended on the presence of plant synthesized compounds, such as sucrose, in the growth medium. Our results provide the basis to better understanding the synergistic interactions between poplar and Enterobacter sp. 638. This information can be further exploited to improve establishment and sustainable production of poplar on marginal, non-agricultural soils using endophytic bacteria such as Enterobacter sp. 638 as growth promoting agents.
Identifying genetic sequences underlying insect associations on forest trees will improve the understanding of community genetics on a broad scale. We tested for genomic regions associated with insects in hybrid poplar using quantitative trait loci (QTL) analyses conducted on data from a common garden experiment. The F2 offspring of a hybrid poplar (Populus trichocarpa x P. deltoides) cross were assessed for seven categories of insect leaf damage at two time points, June and August. Positive and negative correlations were detected among damage categories and between sampling times. For example, sap suckers on leaves in June were positively correlated with sap suckers on leaves (P<0.001) but negatively correlated with skeletonizer damage (P<0.01) in August. The seven forms of leaf damage were used as a proxy for seven functional groups of insect species. Significant variation in insect association occurred among the hybrid offspring, including transgressive segregation of susceptibility to damage. NMDS analyses revealed significant variation and modest broad-sense heritability in insect community structure among genets. QTL analyses identified 14 genomic regions across 9 linkage groups that correlated with insect association. We used three genomics tools to test for putative mechanisms underlying the QTL. First, shikimate-phenylpropanoid pathway genes co-located to 9 of the 13 QTL tested, consistent with the role of phenolic glycosides as defensive compounds. Second, two insect association QTL corresponded to genomic hotspots for leaf trait QTL as identified in previous studies, indicating that, in addition to biochemical attributes, leaf morphology may influence insect preference. Third, network analyses identified categories of gene models over-represented in QTL for certain damage types, providing direction for future functional studies. These results provide insight into the genetic components involved in insect community structure in a fast-growing forest tree.
There is an increasing demand for renewable resources to replace fossil fuels. However, different applications such as the production of secondary biofuels or combustion for energy production require different wood properties. Therefore, high-throughput methods are needed for rapid screening of wood in large scale samples, e.g., to evaluate the outcome of tree breeding or genetic engineering. In this study, we investigated the intra-specific variability of lignin and energy contents in extractive-free wood of hybrid poplar progenies (Populus trichocarpa × deltoides) and tested if the range was sufficient for the development of quantitative prediction models based on Fourier transform infrared spectroscopy (FTIR). Since lignin is a major energy-bearing compound, we expected that the energy content of wood would be positively correlated with the lignin content.
Lignin contents of extractive-free poplar wood samples determined by the acetyl bromide method ranged from 23.4% to 32.1%, and the calorific values measured with a combustion calorimeter varied from 17260 to 19767 J g-1. For the development of calibration models partial least square regression and cross validation was applied to correlate FTIR spectra determined with an attenuated total reflectance (ATR) unit to measured values of lignin or energy contents. The best models with high coefficients of determination (R2 (calibration) = 0.91 and 0.90; R2 (cross-validation) = 0.81 and 0.79) and low root mean square errors of cross validation (RMSECV = 0.77% and 62 J g-1) for lignin and energy determination, respectively, were obtained after data pre-processing and automatic wavenumber restriction. The calibration models were validated by analyses of independent sets of wood samples yielding R2 = 0.88 and 0.86 for lignin and energy contents, respectively.
These results show that FTIR-ATR spectroscopy is suitable as a high-throughput method for lignin and energy estimations in large data sets. Our study revealed that the intra-specific variations in lignin and energy contents were unrelated to each other and that the lignin content, therefore, was no predictor of the energy content. Employing principle component analyses we showed that factor loadings for the energy content were mainly associated with carbohydrate ring vibrations, whereas those for lignin were mainly related to aromatic compounds. Therefore, our analysis suggests that it may be possible to optimize the energy content of trees without concomitant increase in lignin.
Bioenergy; heat value; intraspecific variation; lignin; high throughput method; FTIR spectroscopy
The habituation of cell cultures to cellulose biosynthesis inhibitors constitutes a valuable method for learning more about the plasticity of plant cell wall composition and structure. The subculture of habituated cells in the absence of an inhibitor (dehabituation) offers complementary information: some habituation-associated modifications revert, whereas others remain, even after longterm (3–5 years) dehabituation processes. However, is dehabituation simply the opposite to the process of habituation, in the same way that the cloth woven by Penélope during the day was unwoven during the night? Principal Component Analysis applied to Fourier Transformed Infrared (FTIR) spectra of cell walls from dichlobenil-habituated and dehabituated bean cell lines has shown that dehabituation follows a different pathway to that of habituation. Principal component loadings show that dehabituated cells have more pectins, but that these display a lower degree of methyl-esterification, than those of habituated ones. Further analysis of cell walls focusing on the first steps of habituation would serve to identify which specific modifications in pectins are responsible to the fine modulation of cell wall architecture observed during the habituation/dehabituation process.
cell-wall; cellulose; dichlobenil; habituation-dehabituation; Fourier transform infrared spectroscopy; principal component analysis
The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3′ of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as 1H- and 13C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(l-4-nitrotryptophyl-l-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.
Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula×alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 nl l−1). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.
Cellulose; lignin; ozone; poplar; tension wood
The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F1 individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, β-glucuronidase, cellobiohydrolase, β-glucosidase, β-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner.
Heterosis; heritability; host genetic control; Laccaria bicolor; poplar; secreted enzymes
During the last decades the importance of the genus Populus increased because the poplar genome has been sequenced and molecular tools for basic research have become available. Poplar species occur in different habitats and harbor large genetic variation, which can be exploited for economic applications and for increasing our knowledge on the basic molecular mechanisms of the woody life style. Poplars are, therefore, employed to unravel the molecular mechanisms of wood formation, stress tolerance, tree nutrition and interaction with other organisms such as pathogens or mycorrhiza. The basis of these investigations is the reproducible production of homogeneous plant material. In this method paper we describe techniques and growth conditions for the in vitro propagation of different poplar species (Populus × canescens, P. trichocarpa, P. tremula, and P. euphratica) and ectomycorrhizal fungi (Laccaria bicolor, Paxillus involutus) as well as for their co-cultivation for ectomycorrhizal synthesis. Maintenance and plant preparation require different multiplication and rooting media. Growth systems to cultivate poplars under axenic conditions in agar and sand cultures with and without mycorrhizal fungi are described. Transfer of the plants from in vitro to in situ conditions is critical and hardening is important to prevent high mortality. Growth and vitality of the trees in vitro and outdoors with and without ectomycorrhizas are reported.
poplar; mycorrhiza; fungi; laboratory protocols; in vitro; plant growth; micropropagation
Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we cloned and identified three salt overly sensitive 2 (SOS2) genes in the woody plant Populus trichocarpa, designated as PtSOS2.1, PtSOS2.2, and PtSOS2.3, which were transformed into hybrid poplar clone T89 (Populus tremula× Populus tremuloides Michx clone T89) mediated by Agrobacterium tumefaciens. Southern and northern blot analyses verified that the three genes integrated into the plant genome, and were expressed at a stable transcription level. Meanwhile, overexpression of all three PtSOS2 genes did not retard the growth of plants under normal conditions. Instead, it promoted growth in both agar-medium and soil conditions in response to salinity stress. Under salt stress, overexpression of PtSOS2.1, PtSOS2.2, and PtSOS2.3 increased the concentrations of proline and photosynthetic pigments, and the relative water content (RWC), and the activity of antioxidant enzymes, and decreased the malondialdehyde (MDA) concentrations in transgenic lines compared to the control. These results suggest that overexpression of PtSOS2 plays a significant role in improving the salt tolerance of poplars, reducing the damage to membrane structures, and enhancing osmotic adjustment and antioxidative enzyme regulation under salt stress.
Electronic supplementary material
The online version of this article (doi:10.1007/s11105-013-0640-x) contains supplementary material, which is available to authorized users.
Populus; PtSOS2; Salt tolerance; SnRK; Overexpression
Poplars are extensively cultivated worldwide, and their susceptibility to the leaf rust fungus Melampsora larici-populina leads to considerable damages in plantations. Despite a good knowledge of the poplar rust life cycle, and particularly the epidemics on poplar, the perennial status of the plant host and the obligate biotrophic lifestyle of the rust fungus are bottlenecks for molecular investigations. Following the completion of both M. larici-populina and Populus trichocarpa genome sequences, gene families involved in poplar resistance or in rust fungus virulence were investigated, allowing the identification of key genetic determinants likely controlling the outcome of the interaction. Specific expansions of resistance and defense-related genes in poplar indicate probable innovations in perennial species in relation with host-pathogen interactions. The genome of M. Larici-populina contains a strikingly high number of genes encoding small secreted proteins (SSPs) representing hundreds of candidate effectors. Transcriptome analyses of interacting partners in compatible and incompatible interactions revealed conserved set of genes involved in poplar defense reactions as well as timely regulated expression of SSP transcripts during host tissues colonisation. Ongoing functional studies of selected candidate effectors will be achieved mainly on the basis of recombinant protein purification and subsequent characterisation.
Hopanoids are triterpenoic, pentacyclic compounds that are structurally similar to sterols, which are required for normal cell function in eukaryotes. Hopanoids are thought to be an important component of bacterial cell membranes because they control membrane fluidity and diminish passive diffusion of ions, and a few taxons modulate their hopanoid content in response to environmental stimuli. However, to our knowledge, mutational studies to assess the importance of hopanoids in bacterial physiology have never been performed. Genome sequencing of the potato scab pathogen, Streptomyces scabies 87-22, revealed a hopanoid biosynthetic gene cluster (HBGC) that is predicted to synthesize hopene and aminotrihydroxybacteriohopane products. Hopene was produced by fully sporulated cultures of S. scabies on solid ISP4 (International Streptomyces Project 4) medium as well as by submerged mycelia grown in liquid minimal medium. The elongated hopanoid aminotrihydroxybacteriohopane was not detected under either growth condition. Transcription of the S. scabies HBGC was upregulated during aerial growth, which suggests a link between hopanoid production and morphological development. Functional analysis of the S. scabies Δhop615-1 and Δhop615-7 mutant strains, the first hopanoid mutants created in any bacterial taxon, revealed that hopanoids are not required for normal growth or for tolerance of ethanol, osmotic and oxidative stress, high temperature, or low pH. This suggests that hopanoids are not essential for normal streptomycete physiology.
The major polysaccharides in dicot wood biomass are cellulose and xylan. Although wood-associated cellulose synthase genes responsible for cellulose biosynthesis have been characterized, wood-associated xylan synthase genes have not been biochemically identified. A recent report by Lee et al. (2012) provides the first biochemical evidence that two functionally non-redundant Arabidopsis GT43 members are xylosyltransferases (XylTs) that function cooperatively in the elongation of the xylan backbone. We further extend this finding in the current report demonstrating that two poplar (Populus trichocarpa) GT43 glycosyltransferases, PtrGT43B and PtrGT43C, are xylan XylTs involved in wood formation. We show that microsomes from transgenic tobacco BY2 cells coexpressing PtrGT43B and PtrGT43C exhibited a high XylT activity capable of generating β-(1,4)-linked xylooligosaccharides, whereas little XylT activity was detected in microsomes with expression of PtrGT43B or PtrGT43C alone. These findings indicate that poplar GT43 members are XylTs that act cooperatively in catalyzing the successive transfer of xylosyl residues during xylan backbone biosynthesis, which provides further support of the hypothesis that the biochemical functions of GT43 members in vascular plants are evolutionarily conserved.
GT43; poplar; wood formation; xylan; xylosyltransferase
Variation in xylem structure and function has been extensively studied across different species with a wide taxonomic, geographical, and ecological coverage. In contrast, our understanding of how xylem of a single species can adjust to different growing condition remains limited. Here phenotypic and developmental plasticity in xylem traits of hybrid poplar (Populus trichocarpa×deltoides) was studied. Clonally propagated saplings were grown under experimental drought, nitrogen fertilization, and shade for >30 d. Xylem hydraulic and anatomical traits were subsequently examined in stem segments taken from two different vertical positions along the plant’s main axis. The experimental treatments affected growth and development and induced changes in xylem phenotype. Across all treatments, the amount of leaf area supported by stem segments (AL) scaled linearly with stem native hydraulic conductivity (K
native), suggesting that the area of assimilating leaves is constrained by the xylem transport capacity. In turn, K
native was mainly driven by the size of xylem cross-sectional area (AX). Moreover, the structural and functional properties of xylem varied significantly. Vulnerability to cavitation, measured as the xylem pressure inducing 50% loss of conductivity (P50), ranged from –1.71MPa to –0.15MPa in saplings subjected to drought and nitrogen fertilization, respectively. Across all treatments and stem segment positions, P50 was tightly correlated with wood density. In contrast, no relationship between P50 and xylem-specific conductivity (K
S) was observed. The results of this study enhance our knowledge of plant hydraulic acclimation and provide insights into common trade-offs that exist in xylem structure and function.
Cavitation; hydraulic conductivity; phenotypic plasticity; vessels; wood density; xylem embolism.
Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L) 6-benzylaminopurine and (0.08 mg/L) naphthaleneacetic acid, we have achieved the highest frequency (90%) for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0) and an infection time (20–30 min). According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP) marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30%) than older leaves (10%).
regeneration; transformation; leaf age; poplar; Agrobacterium
Populus species are currently being domesticated through intensive time- and resource-dependent programs for utilization in phytoremediation, wood and paper products, and conversion to biofuels. Poplar leaf rust disease can greatly reduce wood volume. Genetic resistance is effective in reducing economic losses but major resistance loci have been race-specific and can be readily defeated by the pathogen. Developing durable disease resistance requires the identification of non-race-specific loci. In the presented study, area under the disease progress curve was calculated from natural infection of Melampsora ×columbiana in three consecutive years. Association analysis was performed using 412 P. trichocarpa clones genotyped with 29,355 SNPs covering 3,543 genes. We found 40 SNPs within 26 unique genes significantly associated (permutated P<0.05) with poplar rust severity. Moreover, two SNPs were repeated in all three years suggesting non-race-specificity and three additional SNPs were differentially expressed in other poplar rust interactions. These five SNPs were found in genes that have orthologs in Arabidopsis with functionality in pathogen induced transcriptome reprogramming, Ca2+/calmodulin and salicylic acid signaling, and tolerance to reactive oxygen species. The additive effect of non-R gene functional variants may constitute high levels of durable poplar leaf rust resistance. Therefore, these findings are of significance for speeding the genetic improvement of this long-lived, economically important organism.