A differential tolerance to mild salt stress conditions among six Italian rice genotypes does not rely on Na+ exclusion from shoots.

Rice is very sensitive to salt stress at the seedling level, with consequent poor crop establishment. A natural variability in susceptibility to moderate saline environments was found in a group of six Italian temperate japonica rice cultivars, and the physiological determinants for salt tolerance were investigated. Cation (Na+, K+ and Mg++) levels were determined in shoots from individual rice plantlets grown in the absence or in the presence of inhibitory, yet sublethal salt levels, and at increasing time after salt treatments. Significant variations were found among genotypes, but these were unrelated to the relative tolerance, which seems to result from neither mechanism(s) for reduced Na+ translocation to the aerial part, nor its increased retrieval from the xylem mediating Na+ exclusion from leaves. Accordingly, thiobarbituric acid reactive substance levels raised in leaf tissues of salt-treated seedlings, and osmo-induced proline accumulation was found in all genotypes. Data suggest that the difference in salt tolerance most likely depends on mechanisms for osmotic adjustment and/or antioxidative defence.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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. © 2017 Society of Chemical Industry.

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 (Br2 PAMBPA), 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 Br2 PAMBPA, 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.

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 63-65 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 eight 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 100 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.

Functional characterization and expression analysis of rice δ(1)-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.

Functional properties and structural characterization of rice δ(1)-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-Šresolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.

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.

Rubrolides as model for the development of new lactones and their aza analogs as potential photosynthesis inhibitors.

Natural phytotoxins and their synthetic analogs are a potential source of new bioactive compounds for agriculture. Analogs of rubrolides, a class of γ-alkylidene-γ-lactones isolated from different ascidians, have been shown to interfere with the photosynthetic electron-transport chain, yet their activity needs to be improved. With this aim, ten 5-aryl-6-benzyl-4-bromopyridazin-3(2H)-ones were prepared in yields ranging from 44 to 88% by reaction of their correspondent γ-alkylidene-γ-lactones with NH2 NH2 . The structures of these rubrolide analogs were determined by (1) H- and (13) C-NMR, 2D-NMR (COSY and HETCOR), NOE difference, and MS techniques. These compounds were evaluated for their abilities of interfering with the light-driven reduction of ferricyanide by isolated spinach chloroplasts. Lactones with electron-withdrawing substituents in the para-position of the benzylidene ring were the most effective inhibitors. Characterization of the activity of 11b/11b' suggested a mechanism based on the interaction with the plastoquinone binding site of photosystem II. Addition of several compounds to the culture medium of a cyanobacterial model strain was found to inhibit algal growth. However, the relative effectiveness was not consistent with their activity in vitro, suggesting the occurrence of multiple targets and/or detoxyfication mechanisms. Indeed, the compounds showed differential effects on the heterotrophic growth of some crop species, Cucumis sativus and Sorghum bicolor. Pyridazin-3(2H)-ones 12e, 12i, and 12j, which have been found poorly active against the photosynthetic electron transport, were the most effective in inhibiting the growth of some weeds, Ipomoea grandifolia and Brachiaria decumbens, under greenhouse conditions.

Divergent properties and phylogeny of cyanobacterial 5-enol-pyruvyl-shikimate-3-phosphate synthases: evidence for horizontal gene transfer in the Nostocales.

As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering separately in a clade composed of diverse bacterial phyla. The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples.

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 Nimbya and Alternaria. 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 6H-benzo[c]cromen-6-ones, 5H-chromene[4,3-b]pyridin-5-one, and 5H-chromene[4,3-c]pyridin-5-one also showed inhibitory properties, and three 6H-benzo[c]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 in vitro. 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.

Synthesis and evaluation of effective inhibitors of plant δ1-pyrroline-5-carboxylate reductase.

Analogues of previously studied phenyl-substituted aminomethylene-bisphosphonic acids were synthesized and evaluated as inhibitors of Arabidopsis thaliana δ(1)-pyrroline-5-carboxylate reductase. With the aim of improving their effectiveness, two main modifications were introduced into the inhibitory scaffold: the aminomethylenebisphosphonic moiety was replaced with a hydroxymethylenebisphosphonic group, and the length of the molecule was increased by replacing the methylene linker with an ethylidene chain. In addition, chlorine atoms in the phenyl ring were replaced with various other substituents. Most of the studied derivatives showed activity in the micromolar to millimolar range, with two of them being more effective than the lead compound, with concentrations inhibiting 50% of enzyme activity as low as 50 µM. Experimental evidence supporting the ability of these inhibitors to interfere with proline synthesis in vivo is also shown.

Sublethal detergent concentrations increase metabolization of recalcitrant polyphosphonates by the cyanobacterium Spirulina platensis.

As a consequence of increasing industrial applications, thousand tons of polyphosphonates are introduced every year into the environment. The inherent stability of the C-P bond results in a prolonged half-life. Moreover, low uptake rates limit further their microbial metabolization. To assess whether low detergent concentrations were able to increase polyphosphonate utilization by the cyanobacterium Spirulina platensis, tolerance limits to the exposure to various detergents were determined by measuring the growth rate in the presence of graded levels below the critical micellar concentration. Then, the amount of hexamethylenediamine-N,N,N',N'-tetrakis(methylphosphonic acid) that is metabolized in the absence or in the presence of sublethal detergent concentrations was quantified by (31)P NMR analysis on either P-starved or P-fed cyanobacterial cultures. The strain tolerated the presence of detergents in the order: nonionic > anionic > cationic. When added to the culture medium at the highest concentrations showing no detrimental effects upon cell viability, detergents either improved or decreased polyphosphonate utilization, the anionic sodium dodecyl sulfate being the most beneficial. Metabolization was not lower in P-fed cells--a result that strengthens the possibility of using, in the future, this strain for bioremediation purposes.

Amino acid content and nectar choice by forager honeybees (Apis mellifera L.).

Dual choice feeding tests were performed to determine a preference of forager honeybees for specific amino acids. Artificial nectar containing proline was preferred over those containing only sugars. Nectar containing alanine was preferred on the first day, but preference was no longer significant thereafter. On the contrary, a negative response was found for serine. When the bees were given the choice between two nectars enriched with different compounds, proline was preferred above both alanine and serine, and alanine above serine.