Addition of nitrogen had no effect on yield and quality of apples in an high density orchard carrying a dwarf rootstock

The effect of N addition on apple yield and quality may vary according to the tree vigor. Apple trees developed over vigorous rootstocks had shown no response to N application in Brazil. In this study it was evaluated the effect of N addition to the soil on yield and quality of ´Royal Gala´ apples grafted on a dwarf rootstock (M.9). The orchard was planted in 1995 (2,857 trees ha-1) on an Oxisol containing 40 g kg-1 of organic matter and pH 6.0. The experiment was carried out from 1998 up to 2005. Treatments consisted of rates of N (0, 50, 100 and 150 kg ha-1 year-1 from 1998 to 2001, and respectively 0, 100, 200 and 300 kg ha-1 afterwards), all broadcasted within the tree row in two equal splits, at bud break and after harvest, as ammonium sulfate. Addition of N to the soil had no effect on fruit yield over the six years regardless of the applied rate. Averaged across treatments and years, fruit yield was 52.3 t ha-1. Nitrogen in the leaves (average of 24 g kg-1) or in the fruits (average of 346 mg kg-1) as well as some attributes related to fruit quality (color, firmness, acidity, soluble solids, physiological disorders) were unaffected by N addition. Some plant parameters related to tree vigor, however, grew higher with the increase on N rate. Thus, it is not necessary to apply N to deep Brazilian soils containing high organic matter in order to assure good fruit quality and yield on high-density orchards carrying dwarf rootstocks probably because the N required for tree growth and fruit production is supplied from soil organic matter decay.

The striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo.

A large number of gene products that are enriched in the striatum have ill-defined functions, although they may have key roles in age-dependent neurodegenerative diseases affecting the striatum, especially Huntington disease (HD). In the present study, we focused on Abhd11os, (called ABHD11-AS1 in human) which is a putative long noncoding RNA (lncRNA) whose expression is enriched in the mouse striatum. We confirm that despite the presence of 2 small open reading frames (ORFs) in its sequence, Abhd11os is not translated into a detectable peptide in living cells. We demonstrate that Abhd11os levels are markedly reduced in different mouse models of HD. We performed in vivo experiments in mice using lentiviral vectors encoding either Abhd11os or a small hairpin RNA targeting Abhd11os. Results show that Abhd11os overexpression produces neuroprotection against an N-terminal fragment of mutant huntingtin, whereas Abhd11os knockdown is protoxic. These novel results indicate that the loss lncRNA Abhd11os likely contribute to striatal vulnerability in HD. Our study emphasizes that lncRNA may play crucial roles in neurodegenerative diseases.

Alterations in growth and canopy architecture among dwarf, semidwarf and tall oat lines grown under northern conditions

Selostus: Korkeudeltaan eri tyyppisten kauralinjojen kasvu ja sadontuotto pohjoisissa viljelyoloissa

Mutant huntingtin impairs the post-Golgi trafficking of brain-derived neurotrophic factor but not its Val66Met polymorphism.

Brain-derived neurotrophic factor (BDNF) polymorphism is associated with the pathophysiology of several neurodegenerative disorders, including Huntington"s disease. In view ofthese data andthe involvement of huntingtin in intracellular trafficking, we examined the intracellular transport and release of Val66Val BDNF (Val-BDNF) and Val66Met BDNF (Met-BDNF) in transfected striatal knock-in cells expressing wild-type or mutant full-length huntingtin. Colocalization studies with specific markers for endoplasmic reticulum showed no differences between the Val-BDNF and Met-BDNF and were not modified by mutant huntingtin. However, post-Golgi trafficking was altered by mutant huntingtin dependent on the BDNF form. Thus, fluorescence recovery after photobleaching (FRAP) and inverse FRAP analysis showed retention of Met-BDNF inthe Golgi apparatus with respectto Val-BDNF in wild-type cells. Strikingly, mutant huntingtin diminished post-Golgi trafficking of Val-BDNF, whereas Met-BDNF was not modified. Accordingly, a reduction in the number of transport vesicles was only observed in mutant huntingtin cells transfected with Val-BDNF but not Met-BDNF. Moreover, mutant huntingtin severely affectedthe KCl-evoked release of Val-BDNF, although it had little effect on Met-BDNF regulated release. The constitutive release of Val-BDNF or Met-BDNF in mutant cells was only slightly reduced. Interestingly, mutant huntingtin only perturbed post-Golgi trafficking of proteins that follow the regulated secretory pathway (epidermal growth factor receptor or atrial natriuretic factor), whereas it did not change those that follow the constitutive pathway (p75 NTR ). We conclude that mutant huntingtin differently affects intracellular transport and release of Val-BDNF and Met-BDNF. In addition, our findings reveal a new role for huntingtin in the regulation of the post-Golgi trafficking of the regulated secretory pathway.

Quince 'CPP': new dwarf rootstock for pear trees on organic and high density planting

In Brazil, pear production presents the same incipient situation over the last 15 years, due mostly to low production technology. In this context, this study aimed to evaluate the development, growth and production of the pear tree cultivars Cascatense, Tenra and Hosui grafted on 'CPP' quince rootstock, using 'FT' pear as interstem. This trial was carried out in Guarapuava, State of Paraná, Southern region of Brazil, by five productive cycles. The pear trees were planted in September of 2004, spaced at 1.0 x 4.0 m (2,500 trees ha-1), trained to the modified central leader, on a Four-wire trellis, with drip irrigation and cultivated under organic production system. The following variables were evaluated: sprouting, anthesis, yield, fruit weight, soluble solids content, titratable acidity, pulp firmness, canopy area per plant and per hectare and trunk diameter. The pear tree cv. Tenra was outstanding most of the years for fruit yield, and, consequently, showed the highest accumulated yield over the period (51.6 t ha-1), followed by the cultivars Cascatense (39.7 t ha-1) and Hosui (18.7 t ha-1). All pear cultivars presented suitable physical-chemical characteristics for commercial purposes, with minimal average soluble solids content of 11% at harvest. The maximum canopy area per hectare was attained for cv. Cascatense (3063.2 m²), that was considered insufficient for a high yield. These results suggest the needs for studies with higher density planting and other training systems, searching optimize canopy volume. One of the most limiting factors in the organic pear orchard was the incidence of pear dieback caused by Botriosphaeria dothidea, severe more often in pear trees cv. Hosui.

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining tilling mutant populations.

Background: TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results: Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions: This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

Targeted Therapy for Patients with BRAF-Mutant Lung Cancer: Results from the European EURAF Cohort.

INTRODUCTION: Approximately 2% of lung adenocarcinomas have BRAF (v-Raf murine sarcoma viral oncogene homolog B) mutations, including V600E and other types. Vemurafenib, dabrafenib, and sorafenib as BRAF inhibitors are currently tested in clinical trials, but access for patients is limited. The aim of this study was to document the clinical course of patients treated outside of clinical trials. METHODS: We conducted a retrospective multicenter cohort study in Europe of patients with advanced BRAF-mutant lung cancer treated with known BRAF inhibitors. Data were anonymized and centrally assessed for age, gender, smoking, histology, stage, local molecular diagnostic results, systemic therapies, and survival. Best response was assessed locally by RECIST1.1. RESULTS: We documented 35 patients treated in 17 centers with vemurafenib, dabrafenib, or sorafenib. Median age was 63 years (range 42-85); gender was balanced; 14 (40%) were never smokers; all (100%) had adenocarcinoma; 29 (83%) had V600E; 6 (17%) had other mutations; one of them had a concomitant KRAS mutation. Thirty (86%) patients had chemotherapy in the first line. Overall survival with first-line therapy was 25.3 months for V600E and 11.8 months for non-V600E. Thirty-one patients received one BRAF inhibitor, and four received a second inhibitor. Overall response rate with BRAF therapy was 53%, and disease control rate was 85%. Median progression-free survival with BRAF therapy was 5.0 months, and overall survival was 10.8 months. CONCLUSIONS: These results confirm the activity of targeted therapy in patients with BRAF-mutant lung adenocarcinoma. Further trials are warranted to study combination therapies and drug resistance mechanisms.

Characterization and genetic mapping of eceriferum-ym (cer-ym), a cutin deficient barley mutant with impaired leaf water retention capacity.

The cuticle covers the aerial parts of land plants, where it serves many important functions, including water retention. Here, a recessive cuticle mutant, eceriferum-ym (cer-ym), of Hordeum vulgare L. (barley) showed abnormally glossy spikes, sheaths, and leaves. The cer-ym mutant plant detached from its root system was hypersensitive to desiccation treatment compared with wild type plants, and detached leaves of mutant lost 41.8% of their initial weight after 1 h of dehydration under laboratory conditions, while that of the wild type plants lost only 7.1%. Stomata function was not affected by the mutation, but the mutant leaves showed increased cuticular permeability to water, suggesting a defective leaf cuticle, which was confirmed by toluidine blue staining. The mutant leaves showed a substantial reduction in the amounts of the major cutin monomers and a slight increase in the main wax component, suggesting that the enhanced cuticle permeability was a consequence of cutin deficiency. cer-ym was mapped within a 0.8 cM interval between EST marker AK370363 and AK251484, a pericentromeric region on chromosome 4H. The results indicate that the desiccation sensitivity of cer-ym is caused by a defect in leaf cutin, and that cer-ym is located in a chromosome 4H pericentromeric region.

Rice perception of symbiotic arbuscular mycorrhizal fungi requires the karrikin receptor complex.

In terrestrial ecosystems, plants take up phosphate predominantly via association with arbuscular mycorrhizal fungi (AMF). We identified loss of responsiveness to AMF in the rice (Oryza sativa) mutant hebiba, reflected by the absence of physical contact and of characteristic transcriptional responses to fungal signals. Among the 26 genes deleted in hebiba, DWARF 14 LIKE is, the one responsible for loss of symbiosis . It encodes an alpha/beta-fold hydrolase, that is a component of an intracellular receptor complex involved in the detection of the smoke compound karrikin. Our finding reveals an unexpected plant recognition strategy for AMF and a previously unknown signaling link between symbiosis and plant development.

Reversal of a full-length mutant huntingtin neuronal cell phenotypeby chemical inhibitors of polyglutamine-mediated aggregation

Background: Huntington's disease (HD) is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotypephenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expressionof mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results: We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1¿171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 < 15 ¿M, including gossypol, gambogic acid, juglone, celastrol, sanguinarine and anthralin. Of these, both juglone and celastrol were effective in reversing the abnormal cellular localization of full-length mutant huntingtin observed in mutant HdhQ111/Q111 striatal cells. Conclusions: At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that alter in vitro aggregation is a viable approach for defining potential therapeutic compounds that may act on the deleterious conformational property of full-length mutant huntingtin.

A chromosomal insertion toolbox for promoter probing, mutant complementation and pathogenicity studies in Ralstonia solanacearum

We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors the Ralstonia chromosome (pRC) series that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).

Profound hyperglycemia in knockout mutant mice identifies novel function for POU4F2/Brn-3b in regulating metabolic processes.

The POU4F2/Brn-3b transcription factor has been identified as a potentially novel regulator of key metabolic processes. Loss of this protein in Brn-3b knockout (KO) mice causes profound hyperglycemia and insulin resistance (IR), normally associated with type 2 diabetes (T2D), whereas Brn-3b is reduced in tissues taken from obese mice fed on high-fat diets (HFD), which also develop hyperglycemia and IR. Furthermore, studies in C2C12 myocytes show that Brn-3b mRNA and proteins are induced by glucose but inhibited by insulin, suggesting that this protein is itself highly regulated in responsive cells. Analysis of differential gene expression in skeletal muscle from Brn-3b KO mice showed changes in genes that are implicated in T2D such as increased glycogen synthase kinase-3β and reduced GLUT4 glucose transporter. The GLUT4 gene promoter contains multiple Brn-3b binding sites and is directly transactivated by this transcription factor in cotransfection assays, whereas chromatin immunoprecipitation assays confirm that Brn-3b binds to this promoter in vivo. In addition, correlation between GLUT4 and Brn-3b in KO tissues or in C2C12 cells strongly supports a close association between Brn-3b levels and GLUT4 expression. Since Brn-3b is regulated by metabolites and insulin, this may provide a mechanism for controlling key genes that are required for normal metabolic processes in insulin-responsive tissues and its loss may contribute to abnormal glucose uptake.

Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

Avaliação de danos causados pelo Barley yellow dwarf virus: PAV em cultivares de trigo no Brasil

Com o objetivo de avaliar os danos causados pelo Barley yellow dwarf virus - PAV (BYDV-PAV), em cinco cultivares de trigo (BRS 177, BRS 179, BRS 194, BRS Camboatá e BRS Angico), foi conduzido um experimento no telado da Embrapa-Trigo (Passo Fundo RS) no ano de 2005. Os danos induzidos pelo BYDV-PAV foram determinados por meio de análise de características agronômicas (estatura das plantas e massa de matéria seca) e do rendimento (número de afilhos, espigas e grãos por planta; massa de mil grãos). Os dados foram submetidos à análise de variância e as médias ao teste de Tukey a 5%. Danos significativos em função da infecção viral foram observados em todas as variáveis avaliadas. A característica agronômica mais afetada foi a massa de matéria seca, que variou de 26,1% (BRS 177) a 51,4% (BRS 179). Para estatura de plantas foram observadas reduções de 12,5% (BRS 177) a 15,5% (BRS Camboatá). O rendimento total de grãos foi o mais afetado pela infecção viral, sendo que, danos significativos foram observados em todas as cultivares, cuja redução variou de 34,2% (BRS Camboatá) a 60,8% (BRS 179). No número médio de afilhos por planta, apenas as cultivares BRS Angico e BRS 179 apresentaram reduções de 20% e 26,6%, respectivamente. A redução do número médio de grãos variou de 26,1% (BRS Camboatá) a 54,3% (BRS 179). Também ocorreu diminuição no peso de mil grãos com redução que variou de 16,9% (BRS Camboatá) a 38,4% (BRS 194).