RESUMO
Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Furanos/farmacologia , Hidrolases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteólise , Piranos/farmacologia , Motivos de Aminoácidos , Proteínas de Transporte/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sequência Conservada , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Hidrolases/química , Lactonas/farmacologia , Extratos Vegetais , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Deleção de Sequência , Relação Estrutura-Atividade , Nicotiana/efeitos dos fármacosRESUMO
The vacuole is an essential organelle in plant cells, and its dynamic nature is important for plant growth and development. Homotypic membrane fusion is required for vacuole biogenesis, pollen germination, stomata opening, and gravity perception. Known components of the vacuole fusion machinery in eukaryotes include SNARE proteins, Rab GTPases, phosphoinositides, and the homotypic fusion and vacuolar protein sorting (HOPS) tethering complex. HOPS function is not well characterized in plants, but roles in embryogenesis and pollen tube elongation have been reported. Here, we show that Arabidopsis HOPS subunits VPS33 and VPS41 accumulate in late endosomes and that VPS41, but not VPS33, accumulates in the tonoplast via a wortmannin-sensitive process. VPS41 and VPS33 proteins bind to liposomes, but this binding is inhibited by phosphatidylinosiltol-3-phosphate [PtdIns(3)P] and PtdIns(3,5)P2, which implicates a nonconserved mechanism for HOPS recruitment in plants. Inducible knockdown of VPS41 resulted in dramatic vacuole fragmentation phenotypes and demonstrated a critical role for HOPS in vacuole fusion. Furthermore, we provide evidence for genetic interactions between VPS41 and VTI11 SNARE that regulate vacuole fusion, and the requirement of a functional SNARE complex for normal VPS41 and VPS33 localization. Finally, we provide evidence to support VPS33 and SYP22 at the initial stage for HOPS-SNARE interactions, which is similar to other eukaryotes. These results highlight both conserved and specific mechanisms for HOPS recruitment and function during vacuole fusion in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Membrana/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Vacúolos/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 µm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception.
Assuntos
Androstadienos/farmacologia , Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas Qb-SNARE/genética , Vacúolos/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/fisiologia , Gravitropismo/efeitos dos fármacos , Gravitropismo/fisiologia , Hipocótilo/efeitos dos fármacos , Hipocótilo/crescimento & desenvolvimento , Microscopia , Proteínas Qb-SNARE/fisiologia , Vacúolos/fisiologia , Vacúolos/ultraestrutura , WortmaninaRESUMO
In the arid and semi-arid Western U.S., access to water is regulated through a legal system of water rights. Individuals, companies, organizations, municipalities, and tribal entities have documents that declare their water rights. State water regulatory agencies collate and maintain these records, which can be used in legal disputes over access to water. While these records are publicly available data in all Western U.S. states, the data have not yet been readily available in digital form from all states. Furthermore, there are many differences in data format, terminology, and definitions between state water regulatory agencies. Here, we have collected water rights data from 11 Western U.S. state agencies, harmonized terminology and use definitions, formatted them for consistency, and tied them to a Western U.S.-wide shapefile of water administrative boundaries.
RESUMO
Retinol binding protein 4 (RBP4), a novel cytokine, is mainly secreted by hepatocytes and adipocytes. RBP4 reportedly induces insulin resistance and RBP4 secretion is increased in the adipocytes of animals or humans with type 2 diabetes, obesity, and metabolic syndrome, but its role in preadipocyte differentiation remains unclear. In this study, we investigated the effect of RBP4 on the differentiation of porcine preadipocytes into adipocytes. The results suggest that RBP4 significantly suppresses the differentiation of porcine preadipocytes into adipocytes, including those treated with the hormone cocktail methylisobutylxanthine-dexamethasone-insulin. RBP4 also weakened the activity of normal threonine 308, the phosphorylation of serine/threonine kinase AKT, and downstream insulin signaling, including the mammalian target of rapamycin (mTOR) and ß-catenin. Moreover, the activation of insulin signaling mediated by knockdown RBP4 in porcine preadipocytes was recovered in the suppression of LY294002. RBP4 also had a suppressive effect on the differentiation of porcine preadipocytes by decreasing the activation of insulin signaling pathways.
Assuntos
Adipócitos/citologia , Adipogenia , Insulina/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular , Cromonas/farmacologia , Dexametasona/administração & dosagem , Inativação Gênica , Insulina/administração & dosagem , Resistência à Insulina , Lentivirus/genética , Morfolinas/farmacologia , Transdução de Sinais , Suínos , Serina-Treonina Quinases TOR/metabolismo , Xantina/administração & dosagemRESUMO
We examine long-run trends in surface water quality in Texas, USA, with a focus on nutrient pollution and its potential economic impacts. Using >2 million observations of total nitrogen, total phosphorous, dissolved oxygen, and chlorophyll a concentrations from water quality monitors in the state's 23 river sub-basins, we find that nutrient pollution may be a growing problem that is essentially statewide in scope. In addition, because economic impacts of nutrient pollution depend not just on observed water quality, but also on the typical uses of surface water resources that people value, we quantify the share of the state's surface water resources that does not meet common definitions of quality suitable for boating, fishing, swimming, and drinking, as well as the share that does not meet state regulatory standards for their designated uses. This analysis indicates that water quality improvements relative to human uses have stagnated over the last three decades and that nutrient pollution represents a much greater relative threat to attainment of designated uses than it did in the 1970s. We conclude that nutrient pollution is likely taking a toll on the economic value of Texas' water resources.
RESUMO
Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mutação/genética , Vacúolos/metabolismo , Androstadienos/farmacologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Epiderme Vegetal/citologia , Epiderme Vegetal/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Imagem com Lapso de Tempo , WortmaninaRESUMO
Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vti11 mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase (PI3K). We provide evidence that Phosphatidylinositol 3-Phosphate (PtdIns(3)P) regulates vacuole fusion in vti11 mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements.
Assuntos
Proteínas de Arabidopsis/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Qb-SNARE/metabolismo , Vacúolos/metabolismo , Androstadienos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Cromonas/farmacologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , WortmaninaRESUMO
A large body of evidence has linked retinol-binding protein 4 (RBP4) to systemic insulin resistance, but little is known about its function in fat deposition. This study aimed to confirm the involvement of RBP4 in inguinal fat deposition and insulin by intraperitoneal injection of adenovirus-mediated RBP4 to mice. Intraperitoneal injection of adenoviral vectors was validated as an efficient gene manipulation tool for over-expressing recombinant proteins in vivo. Ectopic expression of RBP4 decelerated inguinal fat deposition by decreasing the size of adipocytes. Moreover, the introduction of exogenous RBP4 blunted the response of inguinal adipocytes to insulin signals. These findings suggest that RBP4 impaired in vivo adipogenesis, partly through the repression of the insulin pathway.
Assuntos
Tecido Adiposo/metabolismo , Insulina/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Animais , Resistência à Insulina , CamundongosRESUMO
Transforming growth factor-ß and related growth factors are essential regulators for the development of follicles. Bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI) was reported as a key factor participating in the transforming growth factor-ß signal pathway. To investigate the role of BAMBI in porcine granulosa cells, the full length of the BAMBI was cloned from porcine ovarian cDNA. The results of bioinformatics analyses showed that the signaling peptide was located in between positions 20 and 21. The results of online prediction on phosphorylation sites indicate that the sites of Ser, Thr, and Tyr are 9, 1, and 1, respectively. In addition, BAMBI was highly homologous in rodent and livestock. Real-time quantitative polymerase chain reaction (qPCR) indicated that BAMBI was widely expressed in porcine tissues. Immunofluorescence showed that BAMBI was located in both nucleus and cytoplasm. Stimulating the granulosa cells with FSH in vitro could alter BAMBI expression level in a time-dependent manner. Moreover, the expression level declined after treatment with FSH. These results indicated that BAMBI is an FSH-repressed gene in porcine luteinizing granulosa cells and it may be involved in the regulation of ovarian follicle development and oocyte maturation.