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Revista de biología marina y oceanografía

On-line version ISSN 0718-1957

Rev. biol. mar. oceanogr. vol.51 no.1 Valparaíso Apr. 2016

http://dx.doi.org/10.4067/S0718-19572016000100019 

 

NOTA CIENTÍFICA

Methylglyoxal metabolism in seaweeds during desiccation

Metabolismo de metilglioxal en macroalgas durante desecación

 

Camila Fierro1, Camilo López-Cristoffanini2, Nicolás Latorre1,3, Jorge Rivas4 and Loretto Contreras-Porcia1,5

1Departamento de Ecología y Biodiversidad, Facultad de Ecología y Recursos Naturales, Universidad Andres Bello, República 440, Santiago, Chile. lorettocontreras@unab.cl
2Departamento de Biología Vegetal, Facultad de Biología, Universitat de Barcelona, 08028 Barcelona, España
3Programa de Doctorado en Medicina de la Conservación, Facultad de Ecología y Recursos Naturales, Universidad Andres Bello, República 440, Santiago, Chile
4Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Llano Subercaseaux 2801, San Miguel, Chile
5Center of Applied Ecology & Sustainability (CAPES), Pontificia Universidad Católica de Chile, Avda. Libertador Bernardo O´Higgins 340, Santiago, Chile


ABSTRACT

In primary producers, diverse stressors cause an over-production of methylglyoxal (MG), which is principally detoxified by glyoxalase I (GLO1) activity. A recent proteomic study found that GLO1 was up-regulated during natural desiccation in the red seaweed Pyropia orbicularis, which inhabits the upper intertidal rocky zone and compared to other species, is highly tolerant to air exposure. To better understand and determine differential responses to desiccation stress, this study evaluated MG concentration and GLO1 activity in two species with contrasted vertical distribution, P. orbicularis and Lessonia spicata (lower distribution). Results showed that P. orbicularis successfully scavenges MG via increased GLO1 activity during desiccation. In contrast, GLO1 activity in L. spicata did not increase during desiccation, resulting in MG overproduction. This study is the first to quantify MG and GLO1 levels in seaweeds during natural stress, and partly explain the mechanisms by which P. orbicularis is dominant in the upper rocky intertidal zone.

Key words: Methylglyoxal, glyoxylase I, Pyropia, Lessonia, desiccation


INTRODUCTION

Methylglyoxal [MG; 2-oxopropanal (CH3COCHO)] is a cytotoxic compound formed by the fragmentation and elimination of phosphate from the phosphoenediolate form of glyceraldehyde-3- phosphate (GA3P) and dihydroxyacetone-phosphate (DHAP) (Thornalley 1996). It can also be produced from aminoacetone (Sartori et al. 2008) and an ozone reaction with aromatic hydrocarbons (Grosjean et al. 1996). MG is both a mutagenic and genotoxic compound, since it can degrade proteins, inactivate the antioxidant defense system, and provoke cellular death (Martins et al. 2001) by altering macromolecules through the modification of advanced glycation end products (Hossain & Teixeira 2011).

During cellular stress, the rate of glycolysis increases in diverse organisms (Wu & Juurlink 2002, Yadav et al. 2005, Singla-Pareek et al. 2006, Kumar et al. 2013), leading to an imbalance that causes a spontaneous MG overproduction (Hossain et al. 2009). In primary producers, if overproduced MG is not detoxified immediately after its production in the chloroplast, it will act as an intrinsic mediator that catalyzes the photoreduction of O2 at PS I, leading to the production of O2 •-, which generates oxidative stress (Saito et al. 2011). Efficient MG detoxification during normal physiological processes or when under various stressors is one of the adaptive stress tolerance strategies of plants (Hossain & Teixeira 2011). Indeed, most organisms protect themselves from the deleterious effects of MG by detoxifying it through the glyoxalase pathway, which is comprised of 2 enzymes: glyoxalase I (GLO1) (lactoylglutathione lyase; EC 4.4.1.5), which uses GSH as a cofactor for the conversion of MG to S-D lactoylglutathione, and glyoxalase II (GLO2) (hydroxyacyl glutathione hydrolase; EC 3.1.2.6), which gives GSH back to the system, leading to the production of D-lactate (Hossain et al. 2009).

GLO1 activity is not stress-specific but is activated under several environmental conditions (Blomstedt et al. 1998, Hossain et al. 2009, 2010). In pumpkin seedlings, GLO1 activity increases under conditions of drought, salinity, and white light, among others (Hossain et al. 2009). Additionally, mung beans up-regulate glyoxalase pathway enzymes through glycinebetaine- and proline-increased tolerance to cadmium stress (Hossain et al. 2010). In Sporobolus stapfianus, a desiccation-tolerant plant,GLO1 transcripts increase as relative water content diminishes (Blomstedt et al. 1998).

Desiccation is a recurrent environmental stress for seaweeds from the intertidal rocky zone, such as Pyropia orbicularis (Rhodophyta, Bangiales) commonly known as luche or nori (Ramírez et al. 2014, Guillemin et al. 2016). This species is frequently exposed to air during normal tide cycles in nature (Contreras-Porcia et al. 2011, 2012). A series of studies using physiological, biochemical, transcriptomic, and proteomic approaches have been conducted on P. orbicularis, with data revealing the mechanisms that allow this species to reside in the upper intertidal rocky zone, where desiccation stress is more severe than lower intertidal zones (Contreras-Porcia et al. 2012, 2013; Flores-Molina et al. 2014). Using a proteomic approach, López-Cristoffanini et al. (2015) found for the first time that GLO1 is up-regulated during desiccation in seaweeds, suggesting an enzymatic control of MG levels during tidal fluctuations. Given these findings, it has been hypothesized that the control of MG levels in P. orbicularis is part of the tolerance mechanisms employed by this species to grow and survive in the upper intertidal rocky zone, contrary to many other algal species (López-Cristoffanini et al. 2013, Flores-Molina et al. 2014, Guajardo et al. 2016). Therefore, the objectives of this study were to determine MG levels and GLO1 activity in the red alga P. orbicularis during the desiccation-rehydration cycle and to compare these responses with those of Lessonia spicata (Ochrophyta, Laminariales), a desiccation-sensitive species (Flores-Molina et al. 2014) with a lower intertidal distribution.

 

MATERIALS AND METHODS

Naturally hydrated fronds of P. orbicularis and L. spicata were collected and transported to laboratory as in Contreras-Porcia et al. (2011). Additionally, tolerance to the daily desiccation-rehydration cycle was assayed in vitro under 1 and 4 h of desiccation and 2 and 4 h of rehydration; and fronds were kept at -32°C until later use (Contreras-Porcia et al. 2011). For in vitro desiccation, plants were initially blotted dry and exposed to air in a growth chamber during 4 h at 12°C and with an irradiance of 70-80 µm photon m-2 s-1. Following desiccation, a subset of dehydrated fronds was immediately rehydrated in 0.22 µm of filtered seawater for 4 h to characterize the recovery from oxidative stress.

The MG levels and GLO1 activity of both species were measured in all collected fronds [natural hydration, in vitro desiccation (1 and 4 h), and rehydration (2 and 4 h)]. For MG extraction, 0.5 g of dry tissue (DT) samples, were frozen in liquid nitrogen, ground with a mortar and a pestle, homogenized on ice for 15 min with 3 mL of 0.5 M perchloric acid, and centrifuged at 11,000 x g for 10 min at 4°C. The supernatant was decolorized for 15 min at room temperature by adding 10 mg mL-1 charcoal, centrifuged at 11,000 x g for 10 min, and neutralized with a saturated solution of potassium carbonate at room temperature for 15 min, followed by centrifugation at 11,000 x g for 10 min. The supernatant was frozen at -32°C until spectrophotometric and chromatographic determinations of MG were performed.

Pure MG showed low radiation absorption, as determined by spectrophotometry. Therefore, a derivatization with 1,2-diaminobenzene was performed in order to obtain a high absorbance molecule, 2-methylquinoxaline, as described in Yadav et al. (2005). For this, different concentrations (5-100 µmol L-1) of pure MG (Sigma-Aldrich, USA) were derivatized, and 2-methylquinoxaline was measured by spectrophotometry with a UV/Visible Smartspect 3000 (BioRad, USA) at a wavelength range of 200-700 nm to determine the maximum value in its absorption spectra. A 2 mL reaction mixture containing 0-100 µmol L-1 MG, 1.98 mmol L-1 1,2-diaminobenzene, 0.5 mol L-1 perchloric acid, and double-distilled water was used. Additionally, MG concentration was assessed by high performance liquid chromatography coupled to UV detection (HPLC/UV) (Shimadzu, Kioto, Japan), and determined using a wide range standard curve (0.2-50 µmol L-1) of pure MG that obeys the Beer-Lambert Law. The 2 mL reaction mixture contained 0.2-50 µmol L-1 MG or the algal extracts, 1.98 mM 1,2-diaminobenzene, 0.5 M perchloric acid, and double-distilled water. A 20 µL aliquot was injected onto an HPLC column (Inertsil ODS-3 RP-C18 4.6 x 250 mm, 5 µm, GL Sciences, USA) using an isocratic mobile phase consisting of 25 mM ammonium formate buffer (pH 3.4) and methanol 60:40% v/v, with a 1 mL min-1 flow. Derivated MG was detected at 334 nm.

To quantify GLO1 activity, proteins from algal tissue samples were extracted and quantified according to Contreras et al. (2005). GLO1 activity was measured by spectrophotometry following the initial rate of increase in absorbance at 240 nm, as described in Arai et al. (2014) with slight modifications. Briefly, a reaction mixture containing 0.7 mM glutathione (GSH), 0.7 mM MG, and 50 mM sodium phosphate buffer (pH 6.6) was incubated for 10 min at 37ºC. Subsequently, 40 µg of protein extract was added to complete a total volume of 2 mL, and absorbance was continuously monitored for 5 min at 240 nm. For each sample, a non-enzymatic control was included, and GLO1 activity was calculated using the change in molar extinction coefficient between the enzymatically generated s-lactoylglutathione and the spontaneous reaction between MG and GSH that forms a hemithioacetal (e= 2.86 mM-1cm-1).

 

RESULTS AND DISCUSSION

Since MG in algal tissue samples could not be directly identified by spectrophotometric methods due to low radiation absorption, it was derivatized to a quinoxaline with a strong UV absorption band, as described by Yadav et al. (2005). This substantially improved quantitative determinations by spectrophotometry at low MG concentrations. Yadav et al. (2005) propose that the maximum absorption of derivated methylglyoxal detected by HPLC/UV is at 320 nm, but previous spectrophometric assays have shown variable absorbance data (Cordeiro & Freire 1996). Therefore, an absorbance curve between 200-700 nm was performed, and maximum absorption at a low concentration (10 µM) was determined to be at 334 nm (Fig. 1). This wavelength was used to quantify MG in the seaweeds subjected to desiccation stress. Furthermore, HPLC/UV and spectrophotometric methods showed acceptable selectivity since extracts without MG did not show detectable signals (results not shown). Additionally, HPLC methodology was optimized to determine MG content in algal extracts (absolute calibration curve of 1-50 µmol L-1) (Fig. 2), where the detection and quantification limits were 6 x 10-4 µmol L-1 and 2 x 10-4 µmol L-1, respectively, with low error.


Figure 1. Absorption spectra for a range of concentrations of MG
Figura 1. Espectro de absorción para un rango de concentraciones de MG


Figure 2. Absolute standard calibration curve obtained for determination of
2-methylquinoxaline, derivatized MG, by HPLC /UV at 334 nm.
Values are mean ± SD of 5 replicates
Figura 2. Curva de calibración estándar absoluta obtenida para la determinación
del derivado de 2-metilquinoxalina de MG por HPLC/UV a 334 nm.
Los valores son la media ± DS de 5 réplicas

 

At a biological level, a slight increase of MG in P. orbicularis occurred during desiccation (60 nmol g-1 DT); however, these levels were not significant (ANOVA, T= 0.420, P= 0.992) in comparison with naturally hydrated plants (55 nmol g-1 DT) (Fig. 3A). During rehydration (2 and 4 h), the MG levels were lower than in desiccated tissue, but similar (T= 2.53, P= 0.160; T= 2.45, P= 0.179, respectively) to those registered during maximum natural hydration (higher after 8 h of high tide hydration). In contrast, the MG levels in L. spicata increased significantly (T= 5.74, P= 0.0014) during desiccation, from 37 nmol g-1 DT registered during hydration to 100 nmol g-1 DT after 4 h of desiccation (Fig. 3A). While these levels dropped 45-50% during rehydration, the values registered during maximum hydration were not recovered (T= 6.613, P < 0.001) due a low MG detoxification.


 

Figure 3. MG concentration (expressed in nmol g-1 DT) (A) and specific activity of GLO1
(expressed as S-D-lactoylglutathione pg min-1 µg-1 protein) (B) in P. orbicularis and
L. spicata
during a desiccation (D)-rehydration (Rh) cycle. The asterisk
above the histogram indicates significant differences with hydration at
P
< 0.05. DT, dry tissue. Values are mean ± SD of 5 replicates
Figura 3. Concentración de MG (expresada en nmol g-1 DT) (A) y la actividad específica de GLO 1
(expresada como proteína S-D-lactoilglutationa pg min-1 µg-1) (B) en P. orbicularis y
L. spicata
durante un ciclo de desecación (D)-rehidratación (Rh). El asterisco arriba
del histograma indica diferencias significativas con hidratación P < 0,05.
DT, tejido seco. Los valores son la media ± DS de 5 réplicas

 

GLO1 activity increased significantly during desiccation in comparison to hydration (0.5 pg min-1 µg-1 protein) in P. orbicularis (Fig. 3B), with maximum specific activity after 4 h of desiccation (4 pg min-1 µg-1 protein) (T= 15.99, P < 0.001). This activity decreased by 38% after 4 h of rehydration, but did not reach basal levels (1.5 pg min-1 µg-1 protein; T= 4.97, P= 0.004). In L. spicata, a slight increase in GLO1 activity was determined after 1 h of desiccation, going only from 0.3 pg min-1 µg-1 protein during hydration to 1 pg min-1 µg-1 protein during desiccation (T= 9.63, P < 0.001). No increased activity was observed during rehydration (Fig. 3B).

These data provide evidence of an over-production of MG in the desiccation-sensitive species L. spicata, concomitant with low GLO1 activation. In contrast, GLO1 had active participation during desiccation stress in P. orbicularis, which explains the attenuated MG production that occurs during the natural desiccation-rehydration cycle. The high levels of MG in L. spicata demonstrate a low scavenging of this toxic compound, suggestive of high cellular oxidation. In fact, higher lipid and protein oxidation occurs in L. spicata tissue during desiccation than in P. orbicularis (Contreras-Porcia et al. 2011, Flores-Molina et al. 2014, Guajardo et al. 2016), which impedes cellular stability during extended air exposure. To our knowledge, this is the first study showing GLO1 activity in a seaweed species, and the high activity of GLO1 observed in the desiccation-stressed fronds of P. orbicularis provides additional insights of desiccation tolerance of seaweeds to inhabit the upper intertidal rocky zone.

 

ACKNOWLEDGMENTS

This work was supported by FONDECYT 1120117 and DI-501-14/R (Universidad Andrés Bello, Proyectos Regulares Internos) to LC-P. We are especially grateful to J. Zapata, F. Castañeda and A. Meynard for field assistance.

 

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Received 7 October 2015 and accepted 6 January 2016
Editor: Claudia Bustos

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