Jose M. Garcia-Mina

Design, Synthesis, and Biological Evaluation of Phosphoramide Derivatives as Urease Inhibitors

The design, synthesis, and biological evaluation of phosphoramide derivatives as urease inhibitors to reduce the loss of ammonia has been carried out. Forty phosphorus derivatives were synthesized and their inhibitory activities evaluated against that of jack bean urease. In addition, in vivo assays have been carried out. All of the compounds were characterized by IR, (1)H NMR, MS, and elemental microanalysis. In some cases, detailed molecular modeling studies were carried out, and these highlighted the interaction between the enzyme active center and the compounds and also the characteristics related to their activity as urease inhibitors. According to the IC(50) values for in vitro inhibitory activity, 12 compounds showed values below 1 microM and 8 of them represent improvements of activity in comparison to the commercial urease inhibitor N-n-butylthiophosphorictriamide (NBPT) (100 nM) (AGROTAIN). On the basis of the activity results and the conclusions of the molecular modeling study, a structural model for new potential inhibitors has been defined.

The root application of a purified leonardite humic acid modifies the transcriptional regulation of the main physiological root responses to Fe deficiency in Fe-sufficient cucumber plants

The aim of this study is to investigate the effect of a well-characterized purified humic acid (non-measurable concentrations of the main plant hormones were detected) on the transcriptional regulation of the principal molecular agents involved in iron assimilation. To this end, non-deficient cucumber plants were treated with different concentrations of a purified humic acid (PHA) (2, 5, 100 and 250 mg of organic carbonL(-1)) and harvested 4, 24, 48, 76 and 92 h from the onset of the treatment. At harvest times, the mRNA transcript accumulation of CsFRO1 encoding for Fe(III) chelate-reductase (EC 1.16.1.7); CsHa1 and CsHa2 encoding for plasma membrane H+-ATPase (EC 3.6.3.6); and CsIRT1 encoding for Fe(II) high-affinity transporter, was quantified by real-time RT-PCR. Meanwhile, the respective enzyme activity of the Fe(III) chelate-reductase and plasma membrane H+-ATPase was also investigated. The results obtained indicated that PHA root treatments affected the regulation of the expression of the studied genes, but this effect was transient and differed (up-regulation or down-regulation) depending on the genes studied. Thus, principally the higher doses of PHA caused a transient increase in the expression of the CsHa2 isoform for 24 and 48 h whereas the CsHa1 isoform was unaffected or down-regulated. These effects were accompanied by an increase in the plasma membrane H+-ATPase activity for 4, 48 and 96 h. Likewise, PHA root treatments (principally the higher doses) up-regulated CsFRO1 and CsIRT1 expression for 48 and 72 h; whereas these genes were down-regulated by PHA for 96 h. These effects were associated with an increase in the Fe(III) chelate-reductase activity for 72 h. These effects were not associated with a significant decrease in the Fe root or leaf concentrations, although an eventual effect on the Fe root assimilation pattern cannot be ruled out. These results stress the close relationships between the effects of humic substances on plant development and iron nutrition. However, further studies are needed in order to elucidate if these effects at molecular level are caused by mechanisms involving hormone-like actions and/or nutritional factors.

Complementary multianalytical approach to study the distinctive structural features of the main humic fractions in solution: Gray humic acid, brown humic acid, and fulvic acid

Previous studies have indicated that the main fractions of humic substances (HS), gray humic acid (GHA), brown humic acid (BHA), and fulvic acid (FA), present different molecular patterns in water solution that are probably associated with specific structural features. However, the techniques used in these previous studies did not permit clarification of the principal qualitative characteristics of these structures. To study more in depth this subject several GHA, BHA, and FA have been analyzed through the complementary use of UV-visible and FTIR spectroscopy, (13)C NMR, thermogravimetry, and pyrolysis GC-MS. The results indicate that the studied humic fractions have different and distinctive structural features. Thus, large and nonpolar structural units (paraffins, olefins, terpenes) and aliphatic structures seem to accumulate in the gray fraction, whereas the smallest and more polar (furfural, phenols) and simpler structural units (sugar- and amino acid-related structures) are present in the fulvic one. BHA has a higher content in polycyclic aromatic moieties, S-containing compounds and aromatic structures, thus suggesting the presence of more condensed aromatic rings. Likewise, differences in both the presence of polar groups and the apparent molecular size explain the pattern of solubility as a function of pH and ionic strength (I) that defines each HS fraction. These results also indicate that the structural differences among the HS fractions are not only quantitative (the presence of the same type of structures differing in size and the concentration of functional groups) but also qualitative, because each fraction presented different and distinctive structural domains. These structural domains explain the molecular patterns associated with each HS fraction. Thus, the presence of smaller and more O-functionalized structural units including aromatic domains in FA explain their tendency to form molecular aggregates (hydrogen bridges, metal bridges, and hydrophobic interactions) in solution. This fact could also explain the presence of molecular aggregates in BHA, although to a lesser extent than in FA. Finally, the dominant aliphatic and less functionalized character of GHA may justify its lower tendency to form aggregates in solution at neutral and alkaline pH. Likewise, the results also indicate that the different structural domains associated with these fractions may be the consequence of diverse biosynthetic pathways involving different precursors.

Structural Characteristics of Phosphoramide Derivatives as Urease Inhibitors. Requirements for Activity

Taking as a reference the structural characteristics of a set of compounds that act as jack bean ( Canavalia ensiformis) urease inhibitors, namely, phenylphosphorodiamidate (PPD), N- n-butylthiophosphorictriamide (NBPT), and N- n-butylphosphorictriamide (NBPTO), we have studied the structure-activity relationships of a series of phosphoramide derivatives for which the activity as urease inhibitors in both in vitro and in vivo assays is known. Molecular modeling studies were carried out, and the results highlighted the relevance of characteristics such as the presence of intramolecular hydrogen bonds, the volume of the fragment involved in the enzyme interaction, and the degree of conformational freedom as well as the HOMO orbital and atomic orbital contributions to the HOMO orbital, electron density, and PEM distributions on the activity of these compounds as urease inhibitors. These data, along with the preliminary docking study carried out, allow us to propose a union mode to the active site of the enzyme for these compounds.

Screening of Spanish Medicinal Plants for Antioxidant and Antifungal Activities

Abstract Dichloromethane, ethyl acetate, methanol, and aqueous extracts obtained from 16 Spanish medicinal plants were screened for their antioxidant and antifungal activities. The radical scavenging capacity was evaluated by the DPPH method using a rapid screening by TLC and a spectrophotometric assay. Polar extracts obtained from Jasonia glutinosa L. (Lamiaceae), Tanacetum parthenium (L.) Schultz (Lamiaceae), Equisetum telmateia Ehrh. (Equisetaceae), Verbena officinalis L. (Verbenaceae), and Lythrum salicaria L. (Lythraceae) showed high antioxidant properties. Among them, the methanol extract of Lythrum salicaria showed the strongest antiradical capacity with an IC50 value similar to the positive control ascorbic acid. On the contrary, the best antifungal properties against Rhizopus stolonifer were produced by ethyl acetate or dichloromethane extracts from Anthemis arvensis L. subsp. arvensis (Asteraceae), Tanacetum parthenium, Santolina chamaecyparissus L. subsp. squarrosa Nyman (Asteraceae), Anagallis arvensis L. (Primulaceae) and the methanol extract of Anagallis foemina Miller (Primulaceae). The dichloromethane extract of Anthemis arvensis subsp. arvensis was the best inhibitor of fungus growth.

Changes in the C/N balance caused by increasing external ammonium concentrations are driven by carbon and energy availabilities during ammonium nutrition in pea plants: the key roles of asparagine synthetase and anaplerotic enzymes

An understanding of the mechanisms underlying ammonium (NH(4)(+)) toxicity in plants requires prior knowledge of the metabolic uses for nitrogen (N) and carbon (C). We have recently shown that pea plants grown at high NH(4)(+) concentrations suffer an energy deficiency associated with a disruption of ionic homeostasis. Furthermore, these plants are unable to adequately regulate internal NH4(+) levels and the cell-charge balance associated with cation uptake. Herein we show a role for an extra-C application in the regulation of C-N metabolism in NH(4)(+) -fed plants. Thus, pea plants (Pisum sativum) were grown at a range of NH(4)(+) concentrations as sole N source, and two light intensities were applied to vary the C supply to the plants. Control plants grown at high NH(4)(+) concentration triggered a toxicity response with the characteristic pattern of C-starvation conditions. This toxicity response resulted in the redistribution of N from amino acids, mostly asparagine, and lower C/N ratios. The C/N imbalance at high NH(4)(+) concentration under control conditions induced a strong activation of root C metabolism and the upregulation of anaplerotic enzymes to provide C intermediates for the tricarboxylic acid cycle. A high light intensity partially reverted these C-starvation symptoms by providing higher C availability to the plants. The extra-C contributed to a lower C4/C5 amino acid ratio while maintaining the relative contents of some minor amino acids involved in key pathways regulating the C/N status of the plants unchanged. C availability can therefore be considered to be a determinant factor in the tolerance/sensitivity mechanisms to NH(4)(+) nutrition in plants.

D-Root: a system for cultivating plants with the roots in darkness or under different light conditions

In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark-Root (D-Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D-Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D-Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.

The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium

Ammonium can result in toxicity symptoms in many plants when supplied as a sole nitrogen source. Nitrate reduces the negative effects caused by ammonium and promotes plant growth. In order to explore the mechanism responsible of this beneficial effect, we investigated whether nitrate application causes significant changes in the indoleacetic acid (IAA)- and cytokinin-plant distribution and abscisic acid (ABA) accumulation in wheat (Triticum aestivum L.) plants grown with ammonium. Two different doses of nitrate were supplied to ammonium-fed plants (100 microM and 5mM), to determine whether the effects of nitrate require significant doses (nutritional character), or can be promoted by very low doses (signal effect). The results showed that the presence of NO(3)(-) was associated with clear increases in the active forms of cytokinins (zeatine (Z), trans-zeatine riboside (tZR), isopentenyl adenosine (IPR)) and reduction of the levels of the lower active forms (cis-zeatine riboside (cZR)), independently of the dose applied. Likewise, the presence of nitrate also enhanced IAA shoot content, which correlated with higher cytokinin levels and a tendency toward lower ABA concentration. This study presents further evidence that the possible signal effect of NO(3)(-) involved in its beneficial effect on the growth of wheat plants fed with NH(4)(+) could be mediated by a coordinated action of the levels of cytokinins, IAA and ABA in the shoot.

Nitrate supply induces changes in polyamine content and ethylene production in wheat plants grown with ammonium

In order to explore the mechanisms of nitrates beneficial effect on ammonium-grown plants, we investigated the effects of nitrate on free and conjugated polyamine plant content and ethylene biosynthesis in wheat (Triticum aestivum L.) plants grown with ammonium nutrition. Two different doses of nitrate (100 microM and 5 mM) were supplied to ammonium-fed plants, in order to determine whether the effects of nitrate require significant doses (nutritional character), or can be promoted by very low doses (pseudo-hormonal character). Our results showed that nitrates effects on putrescine, spermidine and spermine contents of ammonium-grown plants tended to follow the pattern associated with strict nitrate nutrition. Both low (100 microM) and high (5 mM) nitrate doses caused a rapid and significant increase in free spermidine content in roots and shoots, which was well correlated with reduced root ethylene production. In shoots, this increase in free spermidine was correlated with changes in the conjugation pattern, while in roots these changes appear to be due to alternative mechanisms. On the other hand, no clear relationship between the supply of a lower dose of nitrate (100 microM) and a reduction of free putrescine content was observed. With higher doses of nitrate (5 mM) we observed a reduction of free putrescine content that was well correlated with increases in its conjugated forms. In conclusion, nitrates effects on putrescine, spermidine and spermine contents of ammonium-fed plants tended to follow the pattern associated with strict nitrate nutrition, corroborating its beneficial effect.

Regulation of hormonal responses of sweet pepper as affected by salinity and elevated CO2 concentration.

This study examines the extent to which the predicted CO2 -protective effects on the inhibition of growth, impairment of photosynthesis and nutrient imbalance caused by saline stress are mediated by an effective adaptation of the endogenous plant hormonal balance. Therefore, sweet pepper plants (Capsicum annuum, cv. Ciclón) were grown at ambient or elevated [CO2] (400 or 800 µmol mol(-1)) with a nutrient solution containing 0 or 80 mM NaCl. The results show that, under saline conditions, elevated [CO2] increased plant dry weight, leaf area, leaf relative water content and net photosynthesis compared with ambient [CO2], whilst the maximum potential quantum efficiency of photosystem II was not modified. In salt-stressed plants, elevated [CO2 ] increased leaf NO3(-) concentration and reduced Cl(-) concentration. Salinity stress induced ABA accumulation in the leaves but it was reduced in the roots at high [CO2], being correlated with the stomatal response. Under non-stressed conditions, IAA was dramatically reduced in the roots when high [CO2] was applied, which resulted in greater root DW and root respiration. Additionally, the observed high CK concentration in the roots (especially tZR) could prevent downregulation of photosynthesis at high [CO2], as the N level in the leaves was increased compared with the ambient [CO2], under salt-stress conditions. These results demonstrate that the hormonal balance was altered by the [CO2], which resulted in significant changes at the growth, gas exchange and nutritional levels.