The grapevine NIP2;1 aquaporin is a silicon channel.

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.

Sweet Cherry (Prunus avium L.) PaPIP1;4 Is a Functional Aquaporin Upregulated by Pre-Harvest Calcium Treatments that Prevent Cracking.

The involvement of aquaporins in rain-induced sweet cherry (Prunus avium L.) fruit cracking is an important research topic with potential agricultural applications. In the present study, we performed the functional characterization of PaPIP1;4, the most expressed aquaporin in sweet cherry fruit. Field experiments focused on the pre-harvest exogenous application to sweet cherry trees, cultivar Skeena, with a solution of 0.5% CaCl2, which is the most common treatment to prevent cracking. Results show that PaPIP1;4 was mostly expressed in the fruit peduncle, but its steady-state transcript levels were higher in fruits from CaCl2-treated plants than in controls. The transient expression of PaPIP1;4-GFP in tobacco epidermal cells and the overexpression of PaPIP1;4 in YSH1172 yeast mutation showed that PaPIP1;4 is a plasma membrane protein able to transport water and hydrogen peroxide. In this study, we characterized for the first time a plasma membrane sweet cherry aquaporin able to transport water and H2O2 that is upregulated by the pre-harvest exogenous application of CaCl2 supplements.

The Grapevine Uncharacterized Intrinsic Protein 1 (VvXIP1) Is Regulated by Drought Stress and Transports Glycerol, Hydrogen Peroxide, Heavy Metals but Not Water.

A MIP (Major Intrinsic Protein) subfamily called Uncharacterized Intrinsic Proteins (XIP) was recently described in several fungi and eudicot plants. In this work, we cloned a XIP from grapevine, VvXIP1, and agrobacterium-mediated transformation studies in Nicotiana benthamiana revealed that the encoded aquaporin shows a preferential localization at the endoplasmic reticulum membrane. Stopped-flow spectrometry in vesicles from the aqy-null yeast strain YSH1172 overexpressing VvXIP1 showed that VvXIP1 is unable to transport water but is permeable to glycerol. Functional studies with the ROS sensitive probe CM-H2DCFDA in intact transformed yeasts showed that VvXIP1 is also able to permeate hydrogen peroxide (H2O2). Drop test growth assays showed that besides glycerol and H2O2, VvXIP1 also transports boric acid, copper, arsenic and nickel. Furthermore, we found that VvXIP1 transcripts were abundant in grapevine leaves from field grown plants and strongly repressed after the imposition of severe water-deficit conditions in potted vines. The observed downregulation of VvXIP1 expression in cultured grape cells in response to ABA and salt, together with the increased sensitivity to osmotic stress displayed by the aqy-null yeast overexpressing VvXIP1, corroborates the role of VvXIP1 in osmotic regulation besides its involvement in H2O2 transport and metal homeostasis.

Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants.

The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.

Identification and functional characterization of grapevine transporters that mediate glucose-6-phosphate uptake into plastids.

MAIN CONCLUSION: Two grapevine glucose-6-Pi plastidial transporters differently expressed in plant organs and in response to environmental and hormonal signals are characterized. They are involved in starch accumulation in berries and canes. In grapevine, starch accumulation in the trunk is important for winter storage of carbon and in the flower for reproductive development. Berries also accumulate starch in their plastids, which are also involved in the synthesis of aroma compounds important for fruit quality. The present work characterizes two glucose-phosphate translocators (VvGPT1, VvGPT2) that control the accumulation of starch in grape amyloplasts. Three different splicing variants identified for VvGPT2 (VvGPT2α, VvGPT2ß and VvGPT2Ω) were more expressed in the leaves than in other organs. In contrast, VvGPT1 transcripts were more abundant in mature berries, canes and flowers than in the leaves. Expression of 35S-VvGPT1-GFP and 35S-VvGPT2Ω-GFP in tobacco leaf epidermal cells showed that the fusion proteins localized at the plastidial envelope. Complementation of the Arabidopsis pgi1-1 mutant impaired in leaf starch synthesis restored its ability to synthesize starch, demonstrating that VvGPT1 and VvGPT2Ω mediate the transport of glucose-6-Pi across the plastidial envelope. In grape cell suspensions, ABA, light and galactinol, together with sucrose and fructose, significantly increased the transcript abundance of VvGPT1, whereas VvGPT2Ω expression was affected only by sucrose. In addition, elicitation with methyl jasmonate strongly upregulated VvGPT1, VvGPT2Ω and VvPAL1, suggesting a role for GPTs in the production of secondary compounds in grapevine. Moreover, in grapevines cultivated in field conditions, VvGPT1 expression was higher in berries more exposed to the sun and subjected to higher temperatures. Although both VvGPT1 and VvGPT2 mediate the same function at the molecular level, they exhibit different expression levels and regulation in plant organs and in response to environmental and hormonal signals.

The grape aquaporin VvSIP1 transports water across the ER membrane.

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.

Fraturas do côndilo occipital: atualização da experiência em nosso serviço e revisão da literatura / Fractures of the occipital condyle: updated experience in our service and literature review / Fracturas del cóndilo occipital: actualización de la experiencia en nuestro servicio y revisión de la literatura

Apresentamos neste trabalho a casuística de fraturas de côndilo occipital diagnosticada e tratada em nosso serviço nos últimos 10 anos. O presente estudo dá continuidade ao levantamento epidemiológico já realizado no período de 1993 a 2000. No período de 2001 a 2011, foram diagnosticados seis casos de fraturas de côndilo occipital e todos eles foram tratados de forma conservadora, exceto um, no qual se colocou um halo craniano inicialmente. Nos cinco casos tratados de modo conservador, observaram-se bons resultados, com estabilidade do segmento nas radiografias funcionais, ausência de limitação de movimento e ausência de dor. O paciente que foi tratado com halo apresentava tetraplegia parcial FRANKEL C e fraturas associadas (C5, C6, L1 e L2) e não apresentou melhora do quadro neurológico, verificando-se limitação do movimento cervical. Destacamos ainda a importância da investigação ativa de lesões na transição occipitocervical, principalmente nos casos decorrentes de trauma de alta energia, pois além das implicações clínicas da detecção precoce da fratura, o melhor emprego dos exames subsidiários tem sido um fator relevante para o aumento da incidência de fraturas do côndilo occipital. Como é historicamente relatado, o tratamento conservador ainda é eficaz e com baixo índice de complicações, e o tratamento cirúrgico deve ser indicado em casos com instabilidade definida.

We present a series of occipital condyle fractures diagnosed and treated in our hospital during the past 10 years. The present study continues an epidemiologic survey already performed in the period 1993-2000. From 2001 to 2011, six cases of fracture of the occipital condyle were diagnosed and all of them were treated conservatively, except one, in which a halo was initially placed. In the five cases treated conservatively, there were good results, with stable segment in functional radiographs, absence of limitation of motion and no pain. The patient treated with halo had FRANKEL C partial quadriplegia and associated fractures (C5, C6, L1, and L2), showing no neurological improvement, besides observing cervical motion limitation. We also highlight the importance of active research on lesions in the occipital-cervical transition, particularly in cases resulting from high-energy trauma, because in addition to the clinical implications of early detection of fracture, the best use of ancillary tests have been an important factor for the increased incidence of fractures of the occipital condyle. As is historically reported, conservative treatment is still effective and has a low rate of complications, and surgical treatment should be indicated in cases with established instability.

Presentamos en este trabajo la casuística de fracturas de cóndilo occipital diagnosticada y tratada en nuestro servicio en los últimos 10 años. El presente estudio le da continuidad al levantamiento epidemiológico ya realizado en el período de 1993 a 2000. En el período de 2001 a 2011, fueron diagnosticados seis casos de fracturas de cóndilo occipital y todos ellos fueron tratados de forma conservadora, excepto uno, en el cual se colocó un halo craneano inicialmente. En los cinco casos tratados de modo conservador, se observaron buenos resultados, con estabilidad del segmento en las radiografías funcionales, ausencia de limitación de movimiento y ausencia de dolor. El paciente que fue tratado con halo presentaba tetraplejía parcial FRANKEL C y fracturas asociadas (C5, C6, L1 y L2) y no presentó mejora del cuadro neurológico, verificándose limitación del movimiento cervical. Destacamos además la importancia de la investigación activa de lesiones en la transición occipitocervical, principalmente en los casos provenientes de trauma de alta energía, pues además de las implicaciones clínicas de la detección precoz de la fractura, el mejor empleo de los exámenes subsidiarios ha sido un factor relevante para el aumento de la incidencia de fracturas del cóndilo occipital. Como es históricamente relatado, el tratamiento conservador aún es eficaz y con bajo índice de complicaciones, y el tratamiento quirúrgico debe ser indicado en casos con inestabilidad definida.