Genome-wide transcript profiling associated with metabolic regulation of Poplar N storage and cycling
ABSTRACT: Previous research has shown that glutamine and sucrose treatment of excised poplar stems induces bark storage protein (BSP) gene expression. The objective of this research is to identify changes in gene expression associated with metabolic regulation of nitrogen storage and cycling and use this information to identify potential regulatory genes. Significant, differentially expressed genes were identified in excised poplar stems incubated in solutions of glutamine, sucrose, glycine, glutamine+glucose, and glutamine+sucrose compared to incubation in a water control. Overall design: Poplar shoots with approximately 10 nodes were excised from greenhouse stock plants that were grown in LD photoperiods. The basal leaves were removed to leave only the 5 apical leaves. The basal ends of the 5-leaved shoots were pre-incubated by placing in water for 24 h in a growth chamber (20 °C, 16-light/8-h dark). Following a 24 h pre-incubation, the excised stems were then transferred to 25 mM aqueous solutions of glutamine, glucose, sucrose, glycine, glutamine+glucose or glutamine+sucrose as well as a water control and incubated for either 48 or 72 h. Solutions were replenished at 24 h intervals to maintain an approximate volume of 0.5 L. After 48 h and 72 h of incubation in the respective solutions, bark tissue was collected from each treatment and immediately frozen in liquid nitrogen. For each treatment, 3 biological replicates were collected with 5 excised stems per biological replicate. Bark from 2 pre-treatments consisting of 3 biological replicates was also collected. Bark of the 'pre1' was collected immediately after excising from the stock plants and 'pre2' bark was collected after the 24 h pre-incubation period.
Project description:Previous research has shown that glutamine and sucrose treatment of excised poplar stems induces bark storage protein (BSP) gene expression. The objectivel of this research is to identify changes in gene expression associated with metabolic regulation of nitrogen storage and cycling and use this information to identify potential regulatory genes. Significant, differentially expressed genes were identified in excised poplar stems incubated in solutions of glutamine or glutamine+glucose compared to incubation with water alone Poplar shoots with approximately 10 nodes were excised from greenhouse stock plants that were grown in LD photoperiods. The basal leaves were removed to leave only the 5 apical leaves. The basal end of the 5-leaved shoots were preincubated by placing in water for 24 h in a growth chamber (20 ◦C, 16-light/8-h dark). After 24 h pre-incubation, the trimed stems were then transfered to 25 mM aqueous solutions of glutamine, glucose, or glutamine+glucose as well as a water control and incubated for either 48 or 72 h. After 48 h and 72 h of incubation in the respective solutions, bark tissue was collected from each treatement and immediately frozen in liquid nitrogen. For each treatment, 3 biological replicates were collected with 5 excised stems per biological replicate. Bark from 2 control with 3 biological replicates were also collected. Control 1 was collected immediately after excising from the stock plant and control 2 was collected after the 24 h preincubation period.
Project description:The periderm of trees produces cork cells, whose cell walls are modified with suberin. We compared the transcriptome of outer bark (cork) vs inner bark (control containing secondary phloem and vacular meristem) to infer genes related to suberim metabolism. Overall design: mRNA profiles of periderm cells at two developmental stages of wild type (WT) poplar tree stems were generated by deep sequencing, in triplicate (young) and quadruplicate (older stem), using Illumina HiSeq2000.
Project description:BACKGROUND AND AIMS: Nitrogen (N) availability in the forest soil is extremely low and N economy has a special importance in woody plants that are able to cope with seasonal periods of growth and development over many years. Here we report on the analysis of amino acid pools and expression of key genes in the perennial species Populus trichocarpa during autumn senescence. METHODS: Amino acid pools were measured throughout senescence. Expression analysis of arginine synthesis genes and cationic amino acid transporter (CAT) genes during senescence was performed. Heterologous expression in yeast mutants was performed to study Pt-CAT11 function in detail. KEY RESULTS: Analysis of amino acid pools showed an increase of glutamine in leaves and an accumulation of arginine in stems during senescence. Expression of arginine biosynthesis genes suggests that arginine was preferentially synthesized from glutamine in perennial tissues. Pt-CAT11 expression increased in senescing leaves and functional characterization demonstrated that Pt-CAT11 transports glutamine. CONCLUSIONS: The present study established a relationship between glutamine synthesized in leaves and arginine synthesized in stems during senescence, arginine being accumulated as an N storage compound in perennial tissues such as stems. In this context, Pt-CAT11 may have a key role in N remobilization during senescence in poplar, by facilitating glutamine loading into phloem vessels.
Project description:We used sector analysis to study cambium development and dynamics and to test whether fundamental developmental and functional differences exist between cambial initials as true 'stem cells' and more differentiated mother cells. In many higher plants, a cylindrical lateral meristem, the vascular cambium, forms along the plant axis. Most notably in stems of perennial tree species, this meristem gives rise to xylem (wood) towards the inside of the trunk and phloem (bark) towards the outside. As such, the vascular cambium is responsible for the production of most of the planet's forest biomass, significantly contributing to the global carbon cycle. Using the bacterial uidA reporter gene in Agrobacterium-based in vivo stem transformation experiments in poplar trees, we created 379 cambium sectors that originated from the transformation of individual cells. Results from our analysis of sector frequency and patterns are consistent with the poplar cambium featuring a single layer of true cambial initials (being able to divide both anti- and periclinally). We show that initials are frequently lost from the cambium, that such cell loss rarely occurs at mother cell level, that phloem and xylem differentiation are controlled independently, and that the frequency of mother cell replenishment is not pre-determined.
Project description:Plants have developed biochemical responses to adapt to biotic stress. To characterize the resistance mechanisms in poplar tree against Apripona germari, comprehensive metabolomic changes of poplar bark and xylem in response to A. germari infection were examined by gas chromatography time-of-flight mass spectrometry (GC-TOF/MS). It was found that, four days after feeding (stage I), A. germari infection brought about changes in various metabolites, such as phenolics, amino acids and sugars in both bark and xylem. Quinic acid, epicatechin, epigallocatechin and salicin might play a role in resistance response in bark, while coniferyl alcohol, ferulic acid and salicin contribute resistance in xylem. At feeding stages II when the larvae fed for more than one month, fewer defensive metabolites were induced, but levels of many intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were reduced, especially in xylem. These results suggested that the defense strategies against A. germari might depend mainly on the early defense responses in poplar. In addition, it was found that bark and xylem in infected trees accumulated higher levels of salicylic acid and 4-aminobutyric acid, respectively, these tissues displaying a direct and systemic reaction against A. germari. However, the actual role of the two metabolites in A. germari-induced defense in poplar requires further investigation.
Project description:Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.
Project description:Flavan-3-ols including the monomeric catechin and the polymeric proanthocyanidins (PAs) are abundant phenolic metabolites in poplar (Populus spp.) previously described to protect leaves against pathogen infection. However, it is not known whether stems are also defended in this way. Here we investigated flavan-3-ol accumulation, activity, and the regulation of formation in black poplar (P. nigra) stems after infection by a newly described fungal stem pathogen, Plectosphaerella populi, which forms canker-like lesions in stems. We showed that flavan-3-ol contents increased in P. populi-infected black poplar stems over the course of infection compared to non-infected controls. Transcripts of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes involved in the last steps of flavan-3-ol biosynthesis were also upregulated upon fungal infection indicating de novo biosynthesis. Amending culture medium with catechin and PAs reduced the mycelial growth of P. populi, suggesting that these metabolites act as anti-pathogen defenses in poplar in vivo. Among the hormones, salicylic acid (SA) was higher in P. populi-infected tissues compared to the non-infected controls over the course of infection studied, while jasmonic acid (JA) and JA-isoleucine (JA-Ile) levels were higher than controls only at the early stages of infection. Interestingly, cytokinins (CKs) were also upregulated in P. populi-infected stems. Poplar saplings treated with CK showed decreased levels of flavan-3-ols and SA in stems suggesting a negative association between CK and flavan-3-ol accumulation. Taken together, the sustained upregulation of SA in correlation with catechin and PA accumulation suggests that this is the dominant hormone inducing the formation of antifungal flavan-3-ols during P. populi infection of poplar stems.
Project description:The Gram-negative bacterium Lonsdalea populi causes a lethal disease known as bark canker on Populus × euramericana in China and Europe. Typical symptoms of bark canker include an abundant white-colored fluid, which oozes from the infected tissues. The availability of the genomic sequence of the bacterium provided the necessary resource to launch genome-scale investigations into the mechanisms fundamental to pathogenesis. Functional analyses of a diverse group of genes encoding virulence factors and components of signaling pathways indicate that successful bark infection depends on specific responses by the pathogen to various stresses, including oxidative stress. Although physiology of resistance is well studied, the molecular processes underlying the defense responses and the genetic basis of resistance to L. populi and in other poplar species remain largely unknown. Control of the disease has relied on chemical measures. Due to the genetic amenability of Lonsdalea and poplar, this pathosystem will become an important model system to unravel molecular mechanisms of bacterial pathogenicity on woody plants. Increased understanding of pathogenesis and signaling in the interaction will facilitate the management of this kind of poplar canker.
Project description:BACKGROUND: Riverine ecosystems, highly sensitive to climate change and human activities, are characterized by rapid environmental change to fluctuating water levels and siltation, causing stress on their biological components. We have little understanding of mechanisms by which riverine plant species have developed adaptive strategies to cope with stress in dynamic environments while maintaining growth and development. RESULTS: We report that poplar (Populus spp.) has evolved a systems level "stress proteome" in the leaf-stem-root apoplast continuum to counter biotic and abiotic factors. To obtain apoplast proteins from P. deltoides, we developed pressure-chamber and water-displacement methods for leaves and stems, respectively. Analyses of 303 proteins and corresponding transcripts coupled with controlled experiments and bioinformatics demonstrate that poplar depends on constitutive and inducible factors to deal with water, pathogen, and oxidative stress. However, each apoplast possessed a unique set of proteins, indicating that response to stress is partly compartmentalized. Apoplast proteins that are involved in glycolysis, fermentation, and catabolism of sucrose and starch appear to enable poplar to grow normally under water stress. Pathogenesis-related proteins mediating water and pathogen stress in apoplast were particularly abundant and effective in suppressing growth of the most prevalent poplar pathogen Melampsora. Unexpectedly, we found diverse peroxidases that appear to be involved in stress-induced cell wall modification in apoplast, particularly during the growing season. Poplar developed a robust antioxidative system to buffer oxidation in stem apoplast. CONCLUSION: These findings suggest that multistress response in the apoplast constitutes an important adaptive trait for poplar to inhabit dynamic environments and is also a potential mechanism in other riverine plant species.
Project description:Seasonal nitrogen (N) storage and reuse is important to the N-use efficiency of temperate deciduous trees. In poplar, bark storage proteins (BSPs) accumulate in protein storage vacuoles of the bark parenchyma and xylem ray cells in the fall. During spring growth, N from stored BSPs is remobilized and utilized by growing shoots. The goal of this study is to investigate global gene expression changes in the bark during BSP remobilization and shoot regrowth under long-day conditions. Long-day (LD) grown poplar (Populus trichocarpa, Nisqually-1) plants were transferred to short-day (SD) for 8 weeks at 20°C followed by an addition 12 weeks of SD at 10°C (day) and 4°C (night). Following this treatment plants were then moved to LD and 20°C for 3 weeks for regrowth. Bark samples were collected from plants released from dormancy just prior to transfer to LD and at weekly intervals for 3 weeks after exposure to LD at 20°C.