Intraspecific Variability of Floral Nectar Volume and Composition in Rapeseed (Brassica napus L. var. oleifera)

Numerous angiosperms rely on pollinators to ensure efficient flower fertilization, offering a reward consisting of nourishing nectars produced by specialized floral cells, known as nectaries. Nectar components are believed to derive from phloem sap that is enzymatically processed and transformed within nectaries. An increasing body of evidence suggests that nectar composition, mainly amino acids, may influence pollinator attraction and fidelity. This notwithstanding, little is known about the range of natural variability in nectar content for crop species. Sugar and amino acid composition of nectar harvested from field-grown plants at the 63 to 65 phenological stage was determined for a set of 44 winter genotypes of rapeseed, a bee-pollinated crop. Significant differences were found for solute concentrations, and an even higher variability was evident for nectar volumes, resulting in striking differences when results were expressed on a single flower basis. The comparison of nectar and phloem sap from a subset of 8 varieties pointed out qualitative and quantitative diversities with respect to both sugars and amino acids. Notably, amino acid concentration in phloem sap was up to one hundred times higher than in nectar. Phloem sap showed a much more uniform composition, suggesting that nectar variability depends mainly on nectary metabolism. A better understanding of the basis of nectar production would allow an improvement of seed set efficiency, as well as hive management and honey production

Evolution of plant δ(1)-pyrroline-5-carboxylate reductases from phylogenetic and structural perspectives

Proline plays a crucial role in cell growth and stress responses, and its accumulation is essential for the tolerance of adverse environmental conditions in plants. Two routes are used to biosynthesize proline in plants. The main route uses glutamate as a precursor, while in the other route proline is derived from ornithine. The terminal step of both pathways, the conversion of δ(1)-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by P5C reductase (P5CR) using NADH or NADPH as a cofactor. Since P5CRs are important housekeeping enzymes, they are conserved across all domains of life and appear to be relatively unaffected throughout evolution. However, global analysis of these enzymes unveiled significant functional diversity in the preference for cofactors (NADPH vs. NADH), variation in metal dependence and the differences in the oligomeric state. In our study we investigated evolutionary patterns through phylogenetic and structural analysis of P5CR representatives from all kingdoms of life, with emphasis on the plant species. We also attempted to correlate local sequence/structure variation among the functionally and structurally characterized members of the family

Functional characterization and expression analysis of rice δ¹-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism

While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.publishe

Different FeSOD levels are associated with different Cr(VI) tolerance in two strains of Scenedesmus acutus

Despite being chromium tolerance not related to the induction of phytochelatins, the tolerance to this metal seems strictly connected to sulfur metabolism (Pereira et al., 2008; Schiavon et al., 2008). In the freshwater green alga Scenedesmus acutus sulfur starvation induces a transient increase of Cr(VI) tolerance both in the wild type and in a Cr-tolerant strain (Gorbi et al 2007). This transient increase is lost after 2-day recovery in standard medium. Studies based on microarray analysis showed that in Chlamydomonas reinhardtii sulfur starvation induces the expression of numerous genes, among which plasma membrane sulfate transporters, many enzymes of the sulfate assimilation pathway and enzymes involved in the antioxidative response, such as superoxide dismutase (SOD), GPX, FSD and SHMT (Zhang et al 2004). In order to verify if differences in SOD expression are involved in the observed transient increase in Cr-tolerance, as well in the differential sensitivity shown by the two S. acutus strains, we analysed SOD protein levels after different time of Cr(VI) exposure. No changes was observed in MnSOD or in Cu/ZnSOD following Cr treatment. On the contrary, immunoassays showed in the Cr-tolerant strain treated with Cr(VI) 1mg/l significantly higher levels of FeSOD than in the wild type. A 3-day sulfur starvation enhances FeSOD levels in both strains, though remaining significantly higher in the Cr-tolerant strain. At the present, it is not clear if the enhancement of FeSOD levels could be directly involved in the transient tolerance induced by S-sarvation. Nonetheless, these results suggest the existence of a different threshold of oxidative stress perception that may induce a prompter response of the Cr-tolerant strain to Cr exposure. Gorbi et al., 2007, Aquat. Toxicol. 84:457-464 Pereira et al., 2008, Toxicol Sci 106(2): 400-412 Schiavon et al., 2008, J. Environ. Qual. 37: 1536-1545 Zhang et al. 2004 Eukariotic Cell Vol.3 , No.5: 1331-134

Functional properties and structural characterization of rice δ¹-pyrroline-5-carboxylate reductase

The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ(1)-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP(+) were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP(+) ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.publishe

Phytotoxicity of aminobisphosphonates targeting both δ1-pyrroline-5-carboxylate reductase and glutamine synthetase

BACKGROUND: Dual-target inhibitors may contribute to the management of herbicide-resistant weeds and avoid or delay the selection of resistant biotypes. Some aminobisphosphonates inhibit the activity of both glutamine synthetase and δ1-pyrroline-5-carboxylate (P5C) reductase in vitro, but the relevance of the latter in vivo has yet to be proven. This study aimed at demonstrating that these compounds can also block proline synthesis in planta. RESULTS: Two aminophosphonates, namely 3,5-dichlorophenylamino-methylenebisphosphonic acid and 3,5-dibromophenylaminomethylenebis phosphonic acid (Br2PAMBPA), showed inverse effectiveness against the two partially purified target enzymes from rapeseed. The compounds showed equipotency in inhibiting the growth of rapeseed seedlings and cultured cells. The analysis of amino acid content in treated cells showed a strong reduction in glutamate and glutamate-related amino acid pools, but a milder effect on free proline. In the case of Br2PAMBPA, toxic P5C levels accumulated in treated seedlings, proving that the inhibition of P5C reductase takes place in situ. CONCLUSIONS: Phenyl-substituted aminobisphosphonates may be regarded as true dual-target inhibitors. Their use to develop new active principles for crop protection could consequently represent a tool to address the problem of target-site resistance among weeds. © 2016 Society of Chemical Industry

Synthesis, theoretical studies, and effect on the photosynthetic electron transport of trifluoromethyl arylamides

The photosynthetic apparatus is targeted by various herbicides, including several amides such as diuron and linuron. Considering the need for the discovery of new active ingredients to cope with weed resistance, the synthesis of a series of trifluoromethyl aryl amides is herein described whose inhibitory properties were assessed in vitro on the photosynthetic electron transport chain, and in vivo on the growth of a model cyanobacterial strain. Theoretical studies were also carried out. Starting with 1-fluoro-2-nitro-4-(trifluoromethyl) benzene, the preparation of the amides was achieved via a three-step sequence, namely nucleophilic aromatic substitution, reduction with SnCl2/HCl, and acylation reactions. The measurement of ferricyanide reduction by functionally intact spinach chloroplasts showed that several derivatives are capable of inhibiting the photosynthetic apparatus. The most active amides presented IC50 values close to 1 μmol L−1, and showed the presence of a 4-bromophenyl group as a common structural feature. The addition of these brominated amides to the culture medium of a model cyanobacterial strain, Synechococcus elongatus PCC 6301, caused various degrees of growth inhibition. Theoretical studies (molecular modeling and quantitative structure–activity relationship) of all amides and their comparison with some known herbicides confirmed these experimental findings and provided more in-depth information about the possible molecular target of these compounds. Trifluoromethyl amides herein described, which were shown to act at the PSII level, may represent a novel scaffold to be exploited aiming at the development of new active ingredients for weed control.731123602371CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE MINAS GERAIS - FAPEMIGSem informação448562/2014-6Sem informaçã

Synthesis, theoretical studies, and effect on the photosynthetic electron transport of trifluoromethyl arylamides

The photosynthetic apparatus is targeted by various herbicides, including several amides such as diuron and linuron. Considering the need for the discovery of new active ingredients to cope with weed resistance, the synthesis of a series of trifluoromethyl aryl amides is herein described whose inhibitory properties were assessed in vitro on the photosynthetic electron transport chain, and in vivo on the growth of a model cyanobacterial strain. Theoretical studies were also carried out. Starting with 1‐fluoro‐2‐nitro‐4‐(trifluoromethyl) benzene, the preparation of the amides was achieved via a three‐step sequence, namely nucleophilic aromatic substitution, reduction with SnCl2/HCl, and acylation reactions. The measurement of ferricyanide reduction by functionally intact spinach chloroplasts showed that several derivatives are capable of inhibiting the photosynthetic apparatus. The most active amides presented IC50 values close to 1 μmol L−1, and showed the presence of a 4‐bromophenyl group as a common structural feature. The addition of these brominated amides to the culture medium of a model cyanobacterial strain, Synechococcus elongatus PCC 6301, caused various degrees of growth inhibition. Theoretical studies (molecular modeling and quantitative structure–activity relationship) of all amides and their comparison with some known herbicides confirmed these experimental findings and provided more in‐depth information about the possible molecular target of these compounds. Trifluoromethyl amides herein described, which were shown to act at the PSII level, may represent a novel scaffold to be exploited aiming at the development of new active ingredients for weed control

The Fungal Phytotoxin Alternariol 9‑Methyl Ether and Some of Its Synthetic Analogues Inhibit the Photosynthetic Electron Transport Chain

Alternariol and monomethylalternariol are natural phytotoxins produced by some fungal strains, such as <i>Nimbya</i> and <i>Alternaria</i>. These substances confer virulence to phytopathogens, yet no information is available concerning their mode of action. Here we show that in the micromolar range alternariol 9-methyl ether is able to inhibit the electron transport chain (IC₅₀ = 29.1 ± 6.5 μM) in isolated spinach chloroplasts. Since its effectiveness is limited by poor solubility in water, several alternariol analogues were synthesized using different aromatic aldehydes. The synthesized 6<i>H</i>-benzo­[<i>c</i>]­cromen-6-ones, 5<i>H</i>-chromene­[4,3-<i>b</i>]­pyridin-5-one, and 5<i>H</i>-chromene­[4,3-<i>c</i>]­pyridin-5-one also showed inhibitory properties, and three 6<i>H</i>-benzo­[<i>c</i>]­cromen-6-ones were more effective (IC₅₀ = 12.8–22.8 μM) than the lead compound. Their addition to the culture medium of a cyanobacterial model strain was found to inhibit algal growth, with a relative effectiveness that was consistent with their activity <i>in vitro</i>. In contrast, the growth of a nonphotosynthetic plant cell culture was poorly affected. These compounds may represent a novel lead for the development of new active principles targeting photosynthesis