Transcriptome profiling of Pinus radiata juvenile wood with contrasting stiffness identifies putative candidate genes involved in microfibril orientation and cell wall mechanics

<p>Abstract</p> <p>Background</p> <p>The mechanical properties of wood are largely determined by the orientation of cellulose microfibrils in secondary cell walls. Several genes and their allelic variants have previously been found to affect microfibril angle (MFA) and wood stiffness; however, the molecular mechanisms controlling microfibril orientation and mechanical strength are largely uncharacterised. In the present study, cDNA microarrays were used to compare gene expression in developing xylem with contrasting stiffness and MFA in juvenile <it>Pinus radiata </it>trees in order to gain further insights into the molecular mechanisms underlying microfibril orientation and cell wall mechanics.</p> <p>Results</p> <p>Juvenile radiata pine trees with higher stiffness (HS) had lower MFA in the earlywood and latewood of each ring compared to low stiffness (LS) trees. Approximately 3.4 to 14.5% out of 3, 320 xylem unigenes on cDNA microarrays were differentially regulated in juvenile wood with contrasting stiffness and MFA. Greater variation in MFA and stiffness was observed in earlywood compared to latewood, suggesting earlywood contributes most to differences in stiffness; however, 3-4 times more genes were differentially regulated in latewood than in earlywood. A total of 108 xylem unigenes were differentially regulated in juvenile wood with HS and LS in at least two seasons, including 43 unigenes with unknown functions. Many genes involved in cytoskeleton development and secondary wall formation (cellulose and lignin biosynthesis) were preferentially transcribed in wood with HS and low MFA. In contrast, several genes involved in cell division and primary wall synthesis were more abundantly transcribed in LS wood with high MFA.</p> <p>Conclusions</p> <p>Microarray expression profiles in <it>Pinus radiata </it>juvenile wood with contrasting stiffness has shed more light on the transcriptional control of microfibril orientation and the mechanical properties of wood. The identified candidate genes provide an invaluable resource for further gene function and association genetics studies aimed at deepening our understanding of cell wall biomechanics with a view to improving the mechanical properties of wood.</p

UHPLC-ESI/TOFMS Determination of Salicylate-like Phenolic Gycosides in Populus tremula Leaves

Associations of salicylate-like phenolic glycosides (PGs) with biological activity have been reported in Salix and Populus trees, but only for a few compounds, and in relation to a limited number of herbivores. By considering the full diversity of PGs, we may improve our ability to recognize genotypes or chemotype groups and enhance our understanding of their ecological function. Here, we present a fast and efficient general method for salicylate determination in leaves of Eurasian aspen that uses ultra-high performance liquid chromatography-electrospray ionization/time-of-flight mass spectrometry (UHPLC-ESI/TOFMS). The time required for the liquid chromatography separations was 13.5 min per sample, compared to around 60 min per sample for most HPLC protocols. In leaf samples from identical P. tremula genotypes with diverse propagation and treatment histories, we identified nine PGs. We found the compound-specific mass chromatograms to be more informative than the UV-visible chromatograms for compound identification and when quantitating samples with large variability in PG content. Signature compounds previously reported for P. tremoloides (tremulacin, tremuloidin, salicin, and salicortin) always were present, and five PGs (2'-O-cinnamoyl-salicortin, 2'-O-acetyl-salicortin, 2'-O-acetyl-salicin, acetyl-tremulacin, and salicyloyl-salicin) were detected for the first time in P. tremula. By using information about the formic acid adduct that appeared for PGs in the LTQ-Orbitrap MS environment, novel compounds like acetyl-tremulacin could be tentatively identified without the use of standards. The novel PGs were consistently either present in genotypes regardless of propagation and damage treatment or were not detectable. In some genotypes, concentrations of 2'-O-acetyl-salicortin and 2'-O-cinnamoyl-salicortin were similar to levels of biologically active PGs in other Salicaceous trees. Our study suggests that we may expect a wide variation in PG content in aspen populations which is of interest both for studies of interactions with herbivores and for mapping population structure

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation

Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars

HLA Genes, Islet Autoantibodies and Residual C-Peptide at the Clinical Onset of Type 1 Diabetes Mellitus and the Risk of Retinopathy 15 Years Later

HLA genes, islet autoantibodies and residual C-peptide were studied to determine the independent association of each exposure with diabetic retinopathy (DR), 15 years after the clinical onset of type 1 diabetes in 15-34 year old individuals.The cohort was identified in 1992 and 1993 by the Diabetes Incidence Study in Sweden (DISS), which investigates incident cases of diabetes for patients between 15 and 34 years of age. Blood samples at diagnosis were analyzed to determine HLA genotype, islet autoantibodies and serum C-peptide. In 2009, fundus photographs were obtained from patient records. Study measures were supplemented with data from the Swedish National Diabetes Registry.The prevalence of DR was 60.2% (148/246). Autoantibodies against the 65 kD isoform of glutamate decarboxylase (GADA) at the onset of clinical diabetes increased the risk of DR 15 years later, relative risk 1.12 for each 100 WHO units/ml, [95% CI 1.02 to 1.23]. This equates to risk estimates of 1.27, [95% CI 1.04 to 1.62] and 1.43, [95% CI 1.06 to 1.94] for participants in the highest 25(th) (GADA>233 WHO units/ml) and 5(th) percentile (GADA>319 WHO units/ml) of GADA, respectively. These were adjusted for duration of diabetes, HbA(1c), treated hypertension, sex, age at diagnosis, HLA and C-peptide. Islet cell autoantibodies, insulinoma-antigen 2 autoantibodies, residual C-peptide and the type 1 diabetes associated haplotypes DQ2, DQ8 and DQ6 were not associated with DR.Increased levels of GADA at the onset of type 1 diabetes were associated with DR 15 years later. These results, if confirmed, could provide additional insights into the pathogenesis of the most common microvascular complication of diabetes and lead to better risk stratification for both patient screenings and DR treatment trials

Impaired Growth and Force Production in Skeletal Muscles of Young Partially Pancreatectomized Rats: A Model of Adolescent Type 1 Diabetic Myopathy?

This present study investigated the temporal effects of type 1 diabetes mellitus (T1DM) on adolescent skeletal muscle growth, morphology and contractile properties using a 90% partial pancreatecomy (Px) model of the disease. Four week-old male Sprague-Dawley rats were randomly assigned to Px (n = 25) or Sham (n = 24) surgery groups and euthanized at 4 or 8 weeks following an in situ assessment of muscle force production. Compared to Shams, Px were hyperglycemic (>15 mM) and displayed attenuated body mass gains by days 2 and 4, respectively (both P<0.05). Absolute maximal force production of the gastrocnemius plantaris soleus complex (GPS) was 30% and 50% lower in Px vs. Shams at 4 and 8 weeks, respectively (P<0.01). GP mass was 35% lower in Px vs Shams at 4 weeks (1.24±0.06 g vs. 1.93±0.03 g, P<0.05) and 45% lower at 8 weeks (1.57±0.12 vs. 2.80±0.06, P<0.05). GP fiber area was 15–20% lower in Px vs. Shams at 4 weeks in all fiber types. At 8 weeks, GP type I and II fiber areas were ∼25% and 40% less, respectively, in Px vs. Shams (group by fiber type interactions, P<0.05). Phosphorylation states of 4E-BP1 and S6K1 following leucine gavage increased 2.0- and 3.5-fold, respectively, in Shams but not in Px. Px rats also had impaired rates of muscle protein synthesis in the basal state and in response to gavage. Taken together, these data indicate that exposure of growing skeletal muscle to uncontrolled T1DM significantly impairs muscle growth and function largely as a result of impaired protein synthesis in type II fibers