RESUMEN
Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants, algae, certain fungi, and worms. Their biosynthesis has been found to be induced by heavy metals-both biogenic and toxic. Among all metal inducers, Cd(II) has been the most explored from a biological and chemical point of view. Although Cd(II)-induced PC biosynthesis has been widely examined, still little is known about the structure of Cd(II) complexes and their thermodynamic stability. Here, we systematically investigated glutathione (GSH) and PC2-PC6 systems, with regard to their complex stoichiometries and spectroscopic and thermodynamic properties. We paid special attention to the determination of stability constants using several complementary techniques. All peptides form CdL complexes, but CdL2 was found for GSH, PC2, and partially for PC3. Moreover, binuclear species CdxLy were identified for the series PC3-PC6 in an excess of Cd(II). Potentiometric and competition spectroscopic studies showed that the affinity of Cd(II) complexes increases from GSH to PC4 almost linearly from micromolar (log K7.4GSH = 5.93) to the femtomolar range (log K7.4PC4 = 13.39) and additional chain elongation does not increase the stability significantly. Data show that PCs form an efficient system which buffers free Cd(II) ions in the pico- to femtomolar range under cellular conditions, avoiding significant interference with Zn(II) complexes. Our study confirms that the favorable entropy change is the factor governing the elevation of phytochelatins' stability and illuminates the importance of the chelate effect in shifting the free Gibbs energy.
Asunto(s)
Cadmio/química , Complejos de Coordinación/química , Metaloproteínas/química , Fitoquelatinas/química , Glutatión/química , Concentración de Iones de Hidrógeno , Estructura Molecular , Fitoquelatinas/síntesis química , TermodinámicaRESUMEN
Phytochelatins (PCs), the heavy metal-binding peptides of plants, play a main function in heavy metal detoxification. In this study, Enhalus acoroides samples collected at six distinct seagrass beds from the coast of Khanh Hoa province, Viet Nam, were evaluated for their PCs. The contents of different PCs in each organ including leaf, rhizome, and root were determined by using HPLC analysis. Significant differences of PC2 contents among specific organs and their relation were tested by ANOVA, Tukey test, and Pearson's correlation. The results showed that higher PC2, appearance of PC3 and a strong correlation between PC2 and Pb concentration were found in the root organ collected from a Pb contaminated area. We conclude that high Pb in the sediment induce high PC2 and PC3 production in the root. This first report on in situ detection of PCs of seagrass encourages future investigation on the ability to use seagrass for phytoremediation and as a bioindicator of heavy metals based on PC contents.
Asunto(s)
Quelantes/metabolismo , Hydrocharitaceae/metabolismo , Plomo/aislamiento & purificación , Fitoquelatinas/biosíntesis , Contaminantes Químicos del Agua/aislamiento & purificación , Organismos Acuáticos , Biodegradación Ambiental , Quelantes/química , Quelantes/aislamiento & purificación , Sedimentos Geológicos/química , Hydrocharitaceae/química , Cinética , Fitoquelatinas/síntesis química , Fitoquelatinas/aislamiento & purificación , Extractos Vegetales/química , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Unión Proteica , Rizoma/química , Rizoma/metabolismoRESUMEN
A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.