Carlos Conde

Physiological, biochemical and molecular changes occurring during olive development and ripening

Since ancient times the olive tree (Olea europaea), an evergreen drought- and moderately salt-tolerant species, has been cultivated for its oil and fruit in the Mediterranean basin. Olive is unique among the commercial important oil crops for many reasons. Today, it ranks sixth in the worlds production of vegetable oils. Due to its nutritional quality, olive oil has a high commercial value compared with most other plant oils. Olive oil has a well-balanced composition of fatty acids, with small amounts of palmitate, and it is highly enriched in the moneonic acid oleate. This makes it both fairly stable against auto-oxidation and suitable for human health. Nevertheless, it is the presence of minor components, in particular phenolics, contributing for oils high oxidative stability, color and flavor, that makes olive oil unique among other oils. Moreover, as a result of their demonstrated roles in the prevention of cancer and cardiovascular diseases, olive phenolics have gained much attention during the past years. Also unique to virgin olive oil is its characteristic aroma. This results from the formation of volatile compounds, namely, aldehydes and alcohols of six carbon atoms, which is triggered when olives are crushed during the process of oil extraction. The biochemistry of the olive tree is also singular. O. europaea is one of the few species able to synthesize both polyols (mannitol) and oligosaccharides (raffinose and stachyose) as the final products of the photosynthetic CO(2) fixation in the leaf. These carbohydrates, together with sucrose, can be exported from leaves to fruits to fulfill cellular metabolic requirements and act as precursors to oil synthesis. Additionally, developing olives contain active chloroplasts capable of fixing CO(2) and thus contributing to the carbon economy of the fruit. The overall quality of table olives and olive oil is influenced by the fruit ripening stage. Olive fruit ripening is a combination of physiological and biochemical changes influenced by several environmental and cultural conditions, even if most events are under strict genetic control.

Pathways of Glucose Regulation of Monosaccharide Transport in Grape Cells

Grape (Vitis vinifera) heterotrophic suspension-cultured cells were used as a model system to study glucose (Glc) transport and its regulation. Cells transported d-[14C]Glc according to simple Michaelis-Menten kinetics superimposed on first-order kinetics. The saturating component is a high-affinity, broad-specificity H+-dependent transport system (Km = 0.05 mm). Glc concentration in the medium tightly regulated the transcription of VvHT1 (Vitis vinifera hexose transporter 1), a monosaccharide transporter previously characterized in grape berry, as well as VvHT1 protein amount and monosaccharide transport activity. All the remaining putative monosaccharide transporters identified so far in grape were poorly expressed and responded weakly to Glc. VvHT1 transcription was strongly repressed by Glc and 2-deoxy-d-Glc, but not by 3-O-methyl-d-Glc or Glc plus mannoheptulose, indicating the involvement of a hexokinase-dependent repression. 3-O-Methyl-d-Glc, which cannot be phosphorylated, and Glc plus mannoheptulose induced a decrease of transport activity caused by the reduction of VvHT1 protein in the plasma membrane without affecting VvHT1 transcript levels. This demonstrates hexokinase-independent posttranscriptional regulation. High Glc down-regulated VvHT1 transcription and Glc uptake, whereas low Glc increased those parameters. Present data provide an example showing control of plant sugar transporters by their own substrate both at transcriptional and posttranscriptional levels. VvHT1 protein has an important role in the massive import of monosaccharides into mesocarp cells of young grape berries because it was localized in plasma membranes of the early developing fruit. Protein amount decreased abruptly throughout fruit development as sugar content increases, consistent with the regulating role of Glc on VvHT1 expression found in suspension-cultured cells.

Mannitol transport and mannitol dehydrogenase activities are coordinated in olea europaea under salt and osmotic stresses

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

POLO ensures chromosome bi-orientation by preventing and correcting erroneous chromosome-spindle attachments.

Correct chromosome segregation during cell division requires bi-orientation at the mitotic spindle. Cells possess mechanisms to prevent and correct inappropriate chromosome attachment. Sister kinetochores assume a ‘back-to-back’ geometry on chromosomes that favors amphitelic orientation but the regulation of this process and molecular components are unknown. Abnormal chromosome–spindle interactions do occur but are corrected through the activity of Aurora B, which destabilizes erroneous attachments. Here, we address the role of Drosophila POLO in chromosome–spindle interactions and show that, unlike inhibition of its activity, depletion of the protein results in bipolar spindles with most chromosomes forming stable attachments with both sister kinetochores bound to microtubules from the same pole in a syntelic orientation. This is partly the result of impaired localization and activity of Aurora B but also of an altered centromere organization with abnormal distribution of centromeric proteins and shorter interkinetochore distances. Our results suggests that POLO is required to promote amphitelic attachment and chromosome bi-orientation by regulating both the activity of the correction mechanism and the architecture of the centromere.

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

AbstractMain conclusionTwo 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, VvGPT2β 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.

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001

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.

All together now: Polo joins the kinase network controlling the spindle assembly checkpoint in Drosophila

Maintenance of genomic stability during eukaryotic cell division relies on the Spindle Assembly Checkpoint (SAC), which has evolved as a surveillance mechanism that monitors kinetochore-microtubule attachment and prevents APC/C-mediated mitotic exit until all chromosomes are properly attached to the mitotic spindle. Reversible protein phosphorylation has long been accredited as a regulatory mechanism of the SAC. Nevertheless, knowledge of how several mitotic kinases act in concert within the signaling pathway to orchestrate SAC function is still emerging. In a recent study, we undertook a comprehensive dissection of the hierarchical framework controlling SAC function in Drosophila cells. We found that Polo lies at the top of the SAC pathway promoting the efficient recruitment of Mps1 to unattached kinetochores. This renders Mps1 fully active to control BubR1 phosphorylation that generates the 3F3/2 phosphoepitope at tensionless kinetochores. We have proposed that Polo is required for SAC function and that the molecular outcome of Mps1-dependent 3F3/2 formation is to promote the association of Cdc20 with BubR1 allowing proper kinetochore recruitment of Cdc20 and efficient assembly of the Mitotic Checkpoint Complex (MCC) required for a sustained SAC response.

Chromosomal localisation of five genes in Perkinsus olseni (Phylum Perkinsozoa)

The molecular karyotype of Perkinsus olseni, a pathogenic protist that infects the clam Ruditapes decussatus, comprises nine chromosomes, ranging in size from 0.15 Mb to 6.5 Mb, representing a haploid genome of about 28 Mb. In order to establish chromosome specific markers, PCR-amplified DNA sequences belonging to five conserved genes (18S rRNA, actin type I, hsp90, β-tubulin and calmodulin) were hybridised to chromosomal bands separated by pulsed-field gel electrophoresis. Three of those probes (actin type I, hsp90 and calmodulin) hybridised to only one chromosome and the remaining two (18S rRNA and β-tubulin) hybridised to two chromosomes. In the first place, the hybridisation pattern obtained serves to dispel any doubt about the nuclear location of the smallest chromosome observed in the molecular karyotype of Perkinsus olseni. Additionally, it will be a reference for further analysis of karyotype polymorphisms in the genus Perkinsus.