ABSTRACT: The role of bark storage proteins (BSPs) in seasonal N storage and cycling has been well documented. However, the regulatory mechanisms and pathways associated with this physiological process are poorly understood. The objective of this study is to investigate global patterns of gene expression associated with photoperiod regulated BSP accumulation and to identify candidate genes, pathways and regulatory factors governing N storage and cycling in poplar. In this study, differential expression of protease inhibitors, proteases, transcription factors, and other genes coincided with increased BSP gene expression, suggesting a complex regulatory network that might play an important role in BSP accumulation. Bark of Populus trichocarpa (Nisqually-1) was collected at weekly intervals for 6 weeks from plants treated with short-day (SD) photoperiods at 20°C as well as from long-day (LD) grown plants immediately before the SD treatment.
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.
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:Seasonal nitrogen (N) cycling in Populus, involves bark storage proteins (BSPs) that accumulate in bark phloem parenchyma in the autumn and decline when shoot growth resumes in the spring. Little is known about the contribution of BSPs to growth or the signals regulating N remobilization from BSPs. Knockdown of BSP accumulation via RNAi and N sink manipulations were used to understand how BSP storage influences shoot growth. Reduced accumulation of BSPs delayed bud break and reduced shoot growth following dormancy. Further, 13N tracer studies also showed that BSP accumulation is an important factor in N partitioning from senescing leaves to bark. Thus, BSP accumulation has a role in N remobilization during N partitioning both from senescing leaves to bark and from bark to expanding shoots once growth commences following dormancy. The bark transcriptome during BSP catabolism and N remobilization was enriched in genes associated with auxin transport and signaling, and manipulation of the source of auxin or auxin transport revealed a role for auxin in regulating BSP catabolism and N remobilization. Therefore, N remobilization appears to be regulated by auxin produced in expanding buds and shoots that is transported to bark where it regulates protease gene expression and BSP catabolism.
Project description: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:Phosphorus (P) is one of the most important macronutrients limiting plant growth and development, particularly in forest ecosystems such as temperate beech (Fagus sylvatica) forests in Central Europe. Efficient tree internal P cycling during annual growth is an important strategy of beech trees to adapt to low soil-P. Organic P (Porg) is thought to play a decisive role in P cycling, but the significance of individual compounds and processes has not been elucidated. To identify processes and metabolites involved in P cycling of beech trees, polar-metabolome and lipidome profiling was performed during annual growth with twig tissues from a sufficient (Conventwald, Con) and a low-soil-P (Tuttlingen, Tut) forest. Autumnal phospholipid degradation in leaves and P export from senescent leaves, accumulation of phospholipids and glucosamine-6-phosphate (GlcN6P) in the bark, storage of N-acetyl-D-glucosamine-6-phosphate (GlcNAc6P) in the wood, and establishing of a phospholipid "start-up capital" in buds constitute main processes involved in P cycling that were enhanced in beech trees on low-P soil of the Tut forest. In spring, mobilization of P from storage pools in the bark contributed to an effective P cycling. Due to the higher phospholipid "start-up capital" in buds of Tut beeches, the P metabolite profile in developing leaves in spring was similar in beech trees of both forests. During summer, leaves of Tut beeches meet their phosphate (Pi) needs by replacing phospholipids by galacto- and sulfolipids. Thus, several processes contribute to adequate Pi supply on P impoverished soil thereby mediating similar growth of beech at low and sufficient soil-P availability.
Project description:BACKGROUND: Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. RESULTS: We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. CONCLUSION: In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously uncharacterized NP-like proteins may function in nutrient sensing and/or signaling. These proteins are members of Group I NP-like proteins, which are widely distributed in many plant taxa. We conclude that NP-like proteins may function in plants, although this function is undefined.
Project description:Illumina HiSeq technology was used to generate mRNA profiles of bark from MIR15 compared to wildtype plants. Wild type (WT) and transgenic poplars (Populus tremula x P. alba, clone INRA 717-1B4) were grown aseptically on Woody Plant Medium. Total RNA was extracted using Tri-Reagent according to the manufacturer’s instructions. Reads of 2X100bp were generated and aligned to Populus trichocarpa v3.0 reference transcripts (http://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ptrichocarpa; Ptrichocarpa_210_transcript_primaryTranscriptOnly) using CLC Genomics Workbench 7. mRNA profiles of bark from MIR15 compared to wildtype plants were generated by paired-end (2x100bp) Illumina HiSeq2000 sequencing. Two biological replicates were sequenced for MIR15 and WT samples.
Project description:Softwood bark is an important by-product of forest industry. Currently, bark is under-utilized and mainly directed for energy production, although it can be extracted with hot water to obtain compounds for value-added use. In Norway spruce (Picea abies [L.] Karst.) bark, condensed tannins and stilbene glycosides are among the compounds that comprise majority of the antioxidative extractives. For developing feasible production chain for softwood bark extractives, knowledge on raw material quality is critical. This study examined the fate of spruce bark tannins and stilbenes during storage treatment with two seasonal replications (i.e., during winter and summer). In the experiment, mature logs were harvested and stored outside. During six-month-storage periods, samples were periodically collected for chemical analysis from both inner and outer bark layers. Additionally, bark extractives were analyzed for antioxidative activities by FRAP, ORAC, and H2O2 scavenging assays. According to the results, stilbenes rapidly degraded during storage, whereas tannins were more stable: only 5-7% of the original stilbene amount and ca. 30-50% of the original amount of condensed tannins were found after 24-week-storage. Summer conditions led to the faster modification of bark chemistry than winter conditions. Changes in antioxidative activity were less pronounced than those of analyzed chemical compounds, indicating that the derivatives of the compounds contribute to the antioxidative activity. The results of the assays showed that, on average, ca. 27% of the original antioxidative capacity remained 24 weeks after the onset of the storage treatment, while a large variation (2-95% of the original capacity remaining) was found between assays, seasons, and bark layers. Inner bark preserved its activities longer than outer bark, and intact bark attached to timber is expected to maintain its activities longer than a debarked one. Thus, to ensure prolonged quality, no debarking before storage is suggested: outer bark protects the inner bark, and debarking enhances the degradation.
Project description:The circadian nature of mood and its dysfunction in affective disorders is well recognized, but the underlying molecular mechanisms are still unclear. We showed that the circadian nuclear receptor REV-ERBa, which is associated with bipolar disorder, impacts midbrain dopamine production and mood-related behavior in mice. Genetic deletion of the Rev-erba gene or pharmacological inhibition of REV-ERBa activity in the ventral midbrain induced mania-like behavior in association with a central hyperdopaminergic state. We used microarrays to identify differentially expressed genes in the ventral midbrains of wild-type (WT) and Rev-erba knock-out (RKO) mice. Male RKO and WT mice (10-15 weeks of age) were maintained in a C57BL/6J background. Mice were housed in temperature-controlled (22-23ºC) quarters under a 12-h light-dark (LD) photoperiod (lights on at 8:00 a.m.). After entrainment for >10 days under LD conditions, mice were kept in constant darkness (DD) for 2 days starting at lights-off time. On the third day, mice were sacrificed at indicated time points by cervical dislocation.
Project description:We investigated the transcriptomes and differential gene expression at the Arabidopsis shoot meristem during flowering using INTACT reporter lines. Samples were collected in four biological replications.