Henning Wildhagen

Upgrading Root Physiology for Stress Tolerance by Ectomycorrhizas: Insights from Metabolite and Transcriptional Profiling into Reprogramming for Stress Anticipation

Ectomycorrhizas (EMs) alleviate stress tolerance of host plants, but the underlying molecular mechanisms are unknown. To elucidate the basis of EM-induced physiological changes and their involvement in stress adaptation, we investigated metabolic and transcriptional profiles in EM and non-EM roots of gray poplar (Populus × canescens) in the presence and absence of osmotic stress imposed by excess salinity. Colonization with the ectomycorrhizal fungus Paxillus involutus increased root cell volumes, a response associated with carbohydrate accumulation. The stress-related hormones abscisic acid and salicylic acid were increased, whereas jasmonic acid and auxin were decreased in EM compared with non-EM roots. Auxin-responsive reporter plants showed that auxin decreased in the vascular system. The phytohormone changes in EMs are in contrast to those in arbuscular mycorrhizas, suggesting that EMs and arbuscular mycorrhizas recruit different signaling pathways to influence plant stress responses. Transcriptome analyses on a whole genome poplar microarray revealed activation of genes related to abiotic and biotic stress responses as well as of genes involved in vesicle trafficking and suppression of auxin-related pathways. Comparative transcriptome analysis indicated EM-related genes whose transcript abundances were independent of salt stress and a set of salt stress-related genes that were common to EM non-salt-stressed and non-EM salt-stressed plants. Salt-exposed EM roots showed stronger accumulation of myoinositol, abscisic acid, and salicylic acid and higher K+-to-Na+ ratio than stressed non-EM roots. In conclusion, EMs activated stress-related genes and signaling pathways, apparently leading to priming of pathways conferring abiotic stress tolerance.

Nitrogen nutrition of poplar trees

Many forest ecosystems have evolved at sites with growth-limiting nitrogen (N) availability, low N input from external sources and high ecosystem internal cycling of N. By contrast, many poplar species are frequent constituents of floodplain forests where they are exposed to a significant ecosystem external supply of N, mainly nitrate, in the moving water table. Therefore, nitrate is much more important for N nutrition of these poplar species than for many other tree species. We summarise current knowledge of nitrate uptake and its regulation by tree internal signals, as well as acquisition of ammonium and organic N from the soil. Unlike herbaceous plants, N nutrition of trees is sustained by seasonal, tree internal cycling. Recent advances in the understanding of seasonal storage and mobilisation in poplar bark and regulation of these processes by temperature and daylength are addressed. To explore consequences of global climate change on N nutrition of poplar trees, responses of N uptake and metabolism to increased atmospheric CO(2) and O(3) concentrations, increased air and soil temperatures, drought and salt stress are highlighted.

Seasonal nitrogen cycling in the bark of field-grown Grey poplar is correlated with meteorological factors and gene expression of bark storage proteins

Seasonal tree-internal nitrogen cycling is an important strategy for trees to achieve high efficiency in the use of nitrogen (N). Key processes of this N redistribution are autumnal leaf senescence and storage of released N as bark storage proteins (BSP) in perennial tissues. While the regulation of leaf senescence has been intensively analysed in trees, the coordination of the complementary storage processes is still poorly understood. Therefore, we ascertained relationships between physiological-level and molecular-level processes and environmental factors under natural conditions in the bark of Populus x canescens. We analysed amino-N concentrations, total soluble protein concentration and transcript abundances of BSP genes in the bark of field-grown P. x canescens harvested during two annual growth cycles. By correlation analysis and linear modelling, we assessed interactions between biological data and meteorological conditions. Day length correlated with BSP expression, and air temperature correlated strongly with total protein concentration (r = -0.92), gamma-aminobutyric acid (GABA; r = 0.76) and arginine (r = -0.70). GABA and arginine also correlated significantly with total protein concentration and transcript abundances of BSP genes. We conclude that GABA and arginine potentially contribute to adjust storage processes in the bark of poplar trees to seasonal changes in environmental conditions.

Poplar Wood Rays Are Involved in Seasonal Remodeling of Tree Physiology

Understanding seasonality and longevity is a major challenge in tree biology. In woody species, growth phases and dormancy follow one another consecutively. In the oldest living individuals, the annual cycle may run for more than 1,000 years. So far, however, not much is known about the processes triggering reactivation from dormancy. In this study, we focused on wood rays, which are known to play an important role in tree development. The transition phase from dormancy to flowering in early spring was compared with the phase of active growth in summer. Rays from wood samples of poplar (Populus × canescens) were enriched by laser microdissection, and transcripts were monitored by poplar whole-genome microarrays. The resulting seasonally varying complex expression and metabolite patterns were subjected to pathway analyses. In February, the metabolic pathways related to flower induction were high, indicating that reactivation from dormancy was already taking place at this time of the year. In July, the pathways related to active growth, like lignin biosynthesis, nitrogen assimilation, and defense, were enriched. Based on “marker” genes identified in our pathway analyses, we were able to validate periodical changes in wood samples by quantitative polymerase chain reaction. These studies, and the resulting ray database, provide new insights into the steps underlying the seasonality of poplar trees.

Targeted re-sequencing of five Douglas-fir provenances reveals population structure and putative target genes of positive selection

Douglas-fir (Pseudotsuga menziesii) occurs in a coastal and an interior variety that differ in drought tolerance and other adaptive traits. To characterize genome-wide levels of genetic diversity in coding regions and to identify genes involved in local adaptation, we used targeted sequence capture to re-sequence 72 trees representing one interior and four coastal provenances. A custom NimbleGen sequence capture array was designed from 57,110 putative unique transcripts (PUTs) to enrich genomic sequencing libraries for these regions. Sequence analysis revealed that almost 100 % of target regions were captured and sequenced in at least one individual. We found 79,910 single nucleotide polymorphisms (SNPs) whose genotypes were called in all individuals. The data confirmed genetic differentiation between interior and coastal provenances and revealed little differentiation between coastal provenances. The nucleotide diversity of the total sample was estimated as π=0.0032, which is at the lower end of values observed in conifers. Outlier tests of genetic differentiation identified 58 high-confidence candidate genes for directional selection with a broad functional diversity. A priori defined genes involved in drought tolerance showed a significantly higher genetic differentiation between interior and coastal Douglas-fir suggesting a different evolution despite a low level of polymorphism. The observed data showed a reduced level of polymorphisms with low minor allele frequencies compared to standard demographic models with two populations and migration. Targeted sequence capture is an efficient method to characterize the genetic diversity of conifer trees with a complex genome.

Differential expression of specific sulphate transporters underlies seasonal and spatial patterns of sulphate allocation in trees

Sulphate uptake and its distribution within plants depend on the activity of different sulphate transporters (SULTR). In long-living deciduous plants such as trees, seasonal changes of spatial patterns add another layer of complexity to the question of how the interplay of different transporters adjusts S distribution within the plant to environmental changes. Poplar is an excellent model to address this question because its S metabolism is already well characterized. In the present study, the importance of SULTRs for seasonal sulphate storage and mobilization was examined in the wood of poplar (Populus tremula × P. alba) by analysing their gene expression in relation to sulphate contents in wood and xylem sap. According to these results, possible functions of the respective SULTRs for seasonal sulphate storage and mobilization in the wood are suggested. Together, the present results complement the previously published model for seasonal sulphate circulation between leaves and bark and provide information for future mechanistic modelling of whole tree sulphate fluxes.

Genes and gene clusters related to genotype and drought-induced variation in saccharification potential, lignin content and wood anatomical traits in Populus nigra

Abstract Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments.

Bioenergy trees: genetic and genomic strategies to improve yield

Growing energy demand, the need to reduce greenhouse gas (GHG) emissions and the move towards a low carbon economy are driving the development of non-food lignocellulosic crops to provide an alternative to fossil fuels and to support bioenergy with carbon capture and storage (CCS). Trees offer significant potential in this role. Poplar, willow and eucalyptus are suggested here as three target tree crops however, a significant yield gap (the difference between potential and observed yield) exists that may be as much as 10 tonnes ha−1y−1. New technologies offer great potential to accelerate the breeding pipeline and provide the bioeconomy with fast growing, stress tolerant and low-input bioenergy trees with higher potential yields and smaller yield gaps. These technologies include both genomic selection (GS) and genome editing, where significant progress for trees has been made in recent years. The most challenging remaining bottleneck is the accurate phenotyping of large populations of trees for traits that underpin yield; more research is required on target traits for the sustainable intensification of the production of bioenergy tree crops.

Tissue- and Cell-Specific Cytokinin Activity in Populus × canescens Monitored by ARR5::GUS Reporter Lines in Summer and Winter.

Cytokinins play an important role in vascular development. But knowledge on the cellular localization of this growth hormone in the stem and other organs of woody plants is lacking. The main focus of this study was to investigate the occurrence and cellular localization of active cytokinins in leaves, roots, and along the stem of Populus × canescens and to find out how the pattern is changed between summer and winter. An ARR5::GUS reporter construct was used to monitor distribution of active cytokinins in different tissues of transgenic poplar lines. Three transgenic lines tested under outdoor conditions showed no influence of ARR5::GUS reporter construct on the growth performance compared with the wild-type, but one line lost the reporter activity. ARR5::GUS activity indicated changes in the tissue- and cell type-specific pattern of cytokinin activity during dormancy compared with the growth phase. ARR5::GUS activity, which was present in the root tips in the growing season, disappeared in winter. In the stem apex ground tissue, ARR5::GUS activity was higher in winter than in summer. Immature leaves from tissue-culture grown plants showed inducible ARR5::GUS activity. Leaf primordia in summer showed ARR5::GUS activity, but not the expanded leaves of outdoor plants or leaf primordia in winter. In stem cross sections, the most prominent ARR5::GUS activity was detected in the cortex region and in the rays of bark in summer and in winter. In the cambial zone the ARR5::GUS activity was more pronounced in the dormant than in growth phase. The pith and the ray cells adjacent to the vessels also displayed ARR5::GUS activity. In silico analyses of the tissue-specific expression patterns of the whole PtRR type-A family of poplar showed that PtRR10, the closest ortholog to the Arabidopsis ARR5 gene, was usually the most highly expressed gene in all tissues. In conclusion, gene expression and tissue-localization indicate high activity of cytokinins not only in summer, but also in winter. The presence of the signal in meristematic tissues supports their role in meristem maintenance. The reporter lines will be useful to study the involvement of cytokinins in acclimation of poplar growth to stress.

Suitability of Illumina deep mRNA sequencing for reliable gene expression profiling in a non-model conifer species ( Pseudotsuga menziesii )

Pseudotsuga menziesii (Douglas-fir) is an ideal model system to study the effect of local adaptation and intraspecific variation in transcriptome responses to the environment. Nonetheless, the lack of genomic resources and standardized microarray platforms for gene expression profiling has been a limitation to test the hypothesis on transcriptome organization and variation. Only recently, deep mRNA sequencing has become a promising alternative to overcome the present limitations. However, information on the transcript abundance distribution is needed for unbiased gene expression profiling from mRNA sequencing data. Since this information is not available for adult conifer needle tissue, we inferred the transcript abundance distribution and tested the effect of sequencing depth on the reliable detection and quantification of transcripts from the needle tissue of 50-year-old Douglas-fir trees. We obtained a similar distribution of GO-slim categories in our mRNA-sequencing libraries and in previously published putative unique transcripts (PUTs) for Douglas-fir, that were used as alignment reference. However, the GO-slim distribution in the Douglas-fir libraries and the Douglas-fir PUTs differed from the GO-slim distributions reported from mRNA deep sequencing libraries obtained from Arabidopsis thaliana leaf tissue. Apparently, several highly abundant PUTs associated with proteins involved in photosynthesis were limiting the benefits of increased sequencing depth. Simulations and empirical data indicated that a 3-fold increase from 5 to 15 million aligned reads results in about twice the number of PUTs that surpass the 100 aligned reads threshold that was used for robust transcript quantification.