Human adenosine deaminase as an allosteric modulator of human A(1) adenosine receptor: abolishment of negative cooperativity for [H](R)-pia binding to the caudate nucleus

It has been shown that adenosine deaminase (ADA; EC 3.5.4.4) behaves as an ecto-enzyme anchored to membrane proteins, among them A(1) adenosine receptors (A(1)Rs). Bovine ADA interacts with A(1)Rs from many species and regulates agonists binding to receptors in an activity-independent form. However, it was not known whether human ADA exerted any effect on the agonist binding to human A(1)Rs, because of both technical difficulties in obtaining pure human ADA and tissues containing human A(1)Rs. In this study, human ADA was purified to homogeneity. Taking in consideration that A(1)Rs form homodimers and taking advantage of a new procedure to fit binding data to receptors dimers, which allows to calculate ligand dissociation constants and the degree of cooperativity between the two subunits in the dimer, here it is demonstrated that human ADA markedly enhances the agonist and antagonist affinity and abolishes the negative cooperativity on agonist binding to human striatal A(1)Rs. ADA also increases the ability of the agonist to decrease the forskolin-induced cAMP levels. The results show that human ADA, apart from reducing the adenosine concentration and thus preventing A(1)R desensitization, binds to A(1)R behaving as an allosteric effector that markedly enhances agonist affinity and increases receptor functionality. The physiological role of the interaction is to make receptors more sensitive to adenosine. This powerful regulation has important implications for the physiology and pharmacology of neuronal A(1)Rs.     

Hormonal Modulation of Citrus Responses to Flooding

In this work, variations in endogenous levels of several hormones were measured in citrus under conditions of continuous flooding following a time-course design. The use of three genotypes differing in their ability to tolerate waterlogging has allowed the discrimination between common and specific hormonal responses. Data suggest an essential involvement of the aerial part in the regulation of tolerance to flooding, whereas in roots more general responses were detected. The progressive increase in leaf abscisic acid (ABA) correlating with the different tolerance of genotypes confirms the involvement of this hormone in plant responses to stress. The late increase in 1-aminocyclopropane-1-carboxylic acid, concomitant with severe leaf injury, points to ethylene as a promoter of leaf senescence in citrus. Leaf putrescine increased in all flooded genotypes, suggesting a general protective role, whereas a higher protective ability of spermidine and spermine was enforced by their exclusive accumulation in the sensitive genotype. Leaf jasmonic acid (JA) increased rapidly and transiently under flooding, suggesting a role for this hormone in triggering downstream responses. In stressed roots, while indole-3-acetic acid increased, JA and ABA levels rapidly decreased to reach almost complete depletion in all flooded citrus genotypes. This suggests that not only should the increase in the so-called stress hormones be considered a signal but also their reduction. The results contribute to the understanding of the intricate set of connections between plant hormones that regulate physiologic responses to stress.     

Detection of heteromerization of more than two proteins by sequential BRET-FRET

Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. We combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. We describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, we identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level.     

Antioxidant enzymatic activity is linked to waterlogging stress tolerance in citrus

Soil flooding constitutes a seasonal factor that negatively affects plant performance and crop yields. In this work, the relationship between oxidative damage and flooding sensitivity was addressed in three citrus genotypes with different abilities to tolerate waterlogging. We examined leaf visible damage, oxidative damage in terms of malondialdehyde (MDA) concentration, leaf proline concentration, leaf and root ascorbate and glutathione contents and the antioxidant enzyme activities superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.8.1.7). No differences in the extent of oxidative damage relative to controls were found among genotypes. However, a different ability to delay the apparition of oxidative damage was associated to a higher tolerance to waterlogging. This ability was linked to an enhanced activated oxygen species' scavenging capacity in terms of an increased antioxidant enzyme activity and higher content in polar antioxidant compounds. Therefore, the existence of a direct relationship between stress sensitivity and the early accumulation of MDA is proposed. In addition, data indicate that the protective role of proline has to be considered minimal as its accumulation was inversely correlated with tolerance to the stress. The positive antioxidant response in Carrizo citrange ( Poncirus trifoliata L. Raf. ×  Citrus sinensis L. Osb.) and Citrumelo CPB 4475 ( Poncirus trifoliata L. Raf. ×  Citrus paradisi L. Macf.) might be responsible for a higher tolerance to flooding stress, whereas in Cleopatra mandarin ( Citrus reshni Hort. Ex Tan.), the early accumulation of MDA seems to be associated to an impaired ability for H₂O₂ scavenging.     

Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory

Arabidopsis (Arabidopsis thaliana) defenses against herbivores are regulated by the jasmonate (JA) hormonal signaling pathway, which leads to the production of a plethora of defense compounds. Arabidopsis defense compounds include tryptophan-derived metabolites, which limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, we used Arabidopsis mutants disrupted in the synthesis of tryptophan-derived secondary metabolites to identify phytochemicals involved in the defense against T. urticae. We show that of the three tryptophan-dependent pathways found in Arabidopsis, the indole glucosinolate (IG) pathway is necessary and sufficient to assure tryptophan-mediated defense against T. urticae. We demonstrate that all three IGs can limit T. urticae herbivory, but that they must be processed by myrosinases to hinder T. urticae oviposition. Putative IG breakdown products were detected in mite-infested leaves, suggesting in planta processing by myrosinases. Finally, we demonstrate that besides IGs, there are additional JA-regulated defenses that control T. urticae herbivory. Together, our results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple IGs, specific myrosinases, and additional JA-dependent defenses.

Three IGs and specific myrosinases help protect Arabidopsis thaliana against herbivory by the two-spotted spider mite T. urticae.     

A relationship between tolerance to dehydration of rice cell lines and ability for ABA synthesis under stress.

Plant dehydration is commonly caused by some adverse environmental conditions such as salinity, drought and freezing. As the plant hormone abscisic acid (ABA) is involved in responses to water stress, we studied its putative relationship with the degree of tolerance to these abiotic stresses. For this purpose we used cell lines that had been established from mature embryos of rice (Oryza sativa L, cvs. Bahia and Bomba), and selected by their high (L-T) or low (L-S) levels of tolerance to each type of stress. Tolerance of rice calli to either osmotic, saline, or freezing stress was generally improved by a previous treatment with ABA. This ABA effect was evident in those callus lines with low tolerance (L-S), as their ability to recover from stress increased up to three fold. Independent of the cultivar used, there were no significant differences in the endogenous ABA contents between untreated L-T and L-S lines. However, upon stress, the increase in endogenous ABA was higher in L-T than in L-S lines. These results, together with those obtained by using Fluridone, an inhibitor of ABA synthesis, show that differences in the level of cell tolerance to osmotic, saline and freezing stress are related to their different capacity of ABA synthesis under stress conditions.     

Interaction of drought and ozone exposure on isoprene emission from extensively cultivated poplar

The combined effects of ozone (O₃) and drought on isoprene emission were studied for the first time. Young hybrid poplars (clone 546, Populus deltoides cv. 55/56 x P. deltoides cv. Imperial) were exposed to O₃ (charcoal‐filtered air, CF, and non‐filtered air +40 ppb, E‐O₃) and soil water stress (well‐watered, WW, and mild drought, MD, one‐third irrigation) for 96 days. Consistent with light‐saturated photosynthesis (A_sat), intercellular CO₂ concentration (C_i) and chlorophyll content, isoprene emission depended on drought, O₃, leaf position and sampling time. Drought stimulated emission (+38.4%), and O₃ decreased it (?40.4%). Ozone increased the carbon cost per unit of isoprene emission. Ozone and drought effects were stronger in middle leaves (13th–15th from the apex) than in upper leaves (6th–8th). Only A_sat showed a significant interaction between O₃ and drought. When the responses were up‐scaled to the entire‐plant level, however, drought effects on total leaf area translated into around twice higher emission from WW plants in clean air than in E‐O₃. Our results suggest that direct effects on plant emission rates and changes in total leaf area may affect isoprene emission from intensively cultivated hybrid poplar under combined MD and O₃ exposure, with important feedbacks for air quality.