antioxidantsgroup

Plant ROS Research


Deja un comentario

Polyamines and response to salt stress in grapevine plantlets

  • José A. Hernández Cortés, Group of Fruit Biotechnology, CEBAS-CSIC (Murcia, Spain)

A recent work, carried out in our laboratory, studied the role of polyamines in the salt stress adaptation in grapevine (Vitis vinifera L.) plantlets.

Salinity is one of the most important stress factors which limits the growth and development of plants by altering their morphological, physiological and biochemical attributes. Salinity induced a water deficit as well as an ionic toxicity in the plants resulting in an alteration in the ionic homeostasis. In addition to the osmotic and toxic effects, salt stress is also manifested as an oxidative stress, contributing all these factors to the deleterious effects of salinity in plants (Hernández et al., 2001; 2003). To mitigate and repair damage initiated by ROS, plants have developed a complex antioxidant defense system. The primary components of this system include carotenoids, ascorbate, glutathione, tocopherols and enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), glutathione peroxidase (GPX, EC 1.11.1.9), peroxidases and the enzymes involved in the ascorbate-glutathione cycle (ASC-GSH cycle; Foyer and Halliwell 1976): ascorbate peroxidase (APX, EC 1.11.1.1), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and glutathione reductase (GR, EC 1.6.4.2) (Noctor and Foyer 1998).

Plant polyamines (PAs) have been suggested to play important roles in morphogenesis, growth, embryogenesis, organ development, leaf senescence, and abiotic and biotic stress responses (Kusano et al., 2008). Therefore, homeostasis of cellular PA levels is also a defensive strategy that plants have developed to cope with adverse situations (Chinnusamy et al., 2005; Groppa and Benavides, 2008). Putrescine (Put), spermidine (Spd), and spermine (Spm) are the major PA pools commonly present in higher plants and known as active oxygen scavenging compounds being considered as mediators in protective reactions against different stresses (Kovacs et al., 2010). However, PAs can also increase ROS production through its catabolism in the apoplast by the action of Cu-containing amino oxidase (CuAO) and polyamine oxidase (PAO) activities (Smith, 1985).

We studied the effect of salt stress in the presence and the absence of MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC) activity, involved in PA biosynthesis, in order to investigate the effects of both treatments on photosynthesis and oxidative metabolism providing new information about the contribution of PA metabolism to salt stress adaptation in grapevine plantlets.

 

Results

Salt stress applied in the culture medium of in vitro grapevine plantlets disturbed the growth rate. The application of MGBG, an inhibitor of SAMDC, resulted in further deterioration of plant growth, especially under salt stress conditions. Leaves from salt treated plantlets developed chlorotic symptoms in the leaf margins; this effect was more evident in the presence of both treatments (Fig. 1).Figure-1

Salt stress produced an alteration in the fluorescence chlorophyll parameters in grapevine leaves. In this sense, a decrease in the photochemical quenching parameters [qP and Y(II)] and an increase in the non-photochemical parameters (qN and NPQ) was observed (Fig 2). The presence of the inhibitor MGBG had no important effect on qN, but it decreased NPQ values, as well as qP and Y(II) (Fig. 2).The effect of NaCl and MGBG on Fv/Fm was less pronounced when the measure was performed in the middle of the leaves. However, when Fv/Fm was recorded near the chlorotic areas (in the leaves margins) the effect of NaCl and/or MGBG was more noticeable (Fig. 2).

Figure-2

NaCl and MGBG treatments induced an oxidative stress as shown by the increase in lipid peroxidation level, measured as TBARS. A synergistic effect on lipid peroxidation was observed in salt-treated plantlets grown in the presence of MGBG (Fig. 3). The increase in lipid peroxidation, and therefore the damage to membrane was parallel with ROS accumulation (H2O2 and O2.-) detected by histochemical staining with DAB, or NBT, respectively (Figs. 4 and 5).

Fig-4Fig-5

 

Salt treatment affected the PA contents in grapevine plantlets, especially the free and conjugate forms of agmatine (Agm) and Put. MGBG induced also a small rise in Agm content, whereas Put, Spd and Spm levels remained relatively unchanged in non-salinized plantlets (Fig. 6). The effect of salt-stress on Agm and Put was intensified in the presence of MGBG, mainly in their free forms. Surprisingly, the level of Spd remained unaffected by MGBG whatever its form, while, a 27% decrease in bound Spm was observed in the same conditions (Fig. 6).

Fig-6

Salt-stress induced a decrease in APX activity whereas no significant effect in MDHAR was recorded (Fig. 7). However, significant increases in SOD and POX activities were induced by NaCl (Fig. 7). The incubation of grapevine plantlets in the presence of MGBG produced no effects in APX activity, whereas significant increases in MDHAR, SOD and POX were observed, and a similar situation was recorded in the presence of both treatments (NaCl plus MGBG) (Fig. 7).

Figure-7

Salt-stress slightly affected the reduced ASC contents, although a strong accumulation in oxidized ascorbate (DHA) was recorded. This effect resulted in a strong decrease in the redox state of ascorbate in NaCl-treated plants (Table 1). No effect in the reduced ASC contents was observed when grapevine plantlets were incubated with MGBG. However, a significant decrease was noticed after simultaneous incubation with NaCl and MGBG (Table 1). Surprisingly, in plants treated with MGBG, in absence or presence of NaCl, no accumulation of DHA was noticed. Even a decrease in DHA in relation to control plants occurred, and accordingly, an increase in the redox state of ascorbate (Table 1). Salt-stress also produced a decrease in reduced glutathione (GSH) both in the absence and in the presence of MGBG (Table 1). In contrast, the treatment with MGBG alone had no effect in GSH contents. No significant change in oxidised glutathione (GSSG) was produced, but due to the negative effect of NaCl in GSH, a decrease in the redox state of glutathione was observed in salt-stressed grapevine plantlets (Table 1).

 

Table 1

Results showed that MGBG treatment contribute to the deleterious effect of oxidative stress in grapevine plantlets grown in presence of NaCl, affecting different physiological and biochemical processes, including plant growth, PA levels,  photosynthesis and redox state of the cells, highlighting a possible protecting role of PA homeostasis in plants subjected to salt stress.

These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

 

 

References

  • Chinnusamy V, Jagendorf A, Zhu JK. (2005) Understanding and improving salt tolerance in plants. Crop Sci 45:437–448.
  • Foyer CH, Halliwell B (1976) Presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133: 21-25.
  • Groppa MD, Benavides MP. (2008) Polyamines and abiotic stress: recent advances. Amino Acids 2008; 34:35–45.
  • Hernández JA, Ferrer MA, Jiménez A, Ros-Barceló A, Sevilla F. (2001) Antioxidant systems and O2.-/H2O2 production in the apoplast of Pisum sativum L. leaves: its relation with NaCl-induced necrotic lesions in minor veins. Plant Physiol 127:817-831.
  • Hernández JA, Aguilar A, Portillo B, López-Gómez E, Mataix Beneyto J, García-Legaz MF. (2003) The effect of calcium on the antioxidant enzymes from salt-treated loquat and anger plants. Funct Plant Biol 30:1127-1137.
  • Kovacs Z, Simon-Sarkadi L, Szücs A, Kocsy G. (2010) Different effects of cold, osmotic stress and abscisic acid on polyamine accumulation in wheat. Amino Acids 38: 623–631.
  • Kusano T, Yamaguchi K, Barberich T, Takahashi Y. (2007) The polyamine spermine rescues Arabidopsis from salinity and drought stresses. Plant Signal Behav 2:250-251.
  • Noctor G, Foyer CH. (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49: 249-279.
  • Smith TA.  (1985) The di- and poly-amine oxidases of higher plants. Biochem Soc Trans 13:319-322.

 

For more information, please consult:

Ikbal FE, Hernández JA, Barba-Espín G, Koussa T, Aziz A, Faize M, Diaz-Vivancos P. (2014) Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants. J Plant Physiol. 2014, 171:779-88. doi: 10.1016/j.jplph.2014.02.006.

 

 

 


Deja un comentario

Leaf Gas Exchange Musings

Simon P Driscoll, scientist and jazz musician

Plants are sessile and must adapt to their immediate environment so there is no running away if conditions become bad. This means they have to survive a wide range of sub optimal conditions in order to produce their progeny; these conditions can be either biotic or abiotic. We were interested in water use efficiency, and as a starting point we looked at maize. We had custom built gas exchange equipment which measured photosynthesis rate by measuring the rate of carbon dioxide uptake. Commercial gas exchange systems typically have one leaf chamber and enclose one leaf analysing both leaf surfaces simultaneously. Producing data for maize like our Figure 1.

Simon

Figure 1 shows carbon dioxide on the x axis and carbon dioxide uptake on the Y axis .As we would expect from a C4 species the initial slope is very steep and then the system saturates for the given light intensity. However we built a leaf chamber which measures carbon dioxide uptake on each leaf surface, top and bottom, independently (Figure 2).

Now we can see that the two leaf surfaces are responding to the carbon dioxide increase very differently (Figure 2). Almost all the gas exchange is being accomplished by the bottom surface. The top surface value is much lower rises to a maximum and then falls almost to zero. In maize the ratio of stomata top to bottom is 0.7-0.8 so this difference in carbon dioxide uptake is much greater than the difference in stomata numbers. Having different carbon dioxide uptake and hence different water loss rates (transpiration ) does not make sense if there is free mixing of gases inside the leaf because the control at the surface would achieve nothing internally .We looked at carbon dioxide flux versus a wide range of concentration gradients (Figure 3).

Figure 3.

Figure 3.

C4 plants like maize have the Kranz structure which means they are more internally organised than C3 plants. It was therefore a surprise when we looked at carbon dioxide flux in tobacco leaves and found a carbon dioxide flux of 2.5 ppm for a concentration gradient of almost 2500 ppm. We looked at other C4 plants like Paspalum and found a similar pattern so it looked that assimilation of carbon dioxide was being maximised whilst water loss was being minimised. However when we looked at wheat a different story emerged (Figure 4).

Simon 4

Figure 4. Gas exchange in wheat.

In wheat almost all the gas exchange is via the top surface which is a very different strategy to every other plant we looked at why wheat is different I do not know. Having most gas exchange happening via the lower surface makes sense to me. I cannot see the advantage in having the top surface perform almost all the gas exchange. Also if you invert wheat leaf in the leaf chamber the surface which was the bottom immediately takes over the uptake of carbon dioxide. This shows it is a function of leaf orientation not a surface phenomenon. When the leaf is inverted the surface which was the bottom takes over the gas exchange (Figure 4).

Figure 5 shows that in wheat the water use efficiency is very different for the two surfaces with the top surface being more efficient following the same trend ass the assimilation.

Figura 5.

Figura 5. Wheat water use efficiency

In Maize although the surfaces have different assimilation rates they have similar water use efficiency rates which is higher than in C3 species. What is interesting is that it looks tightly controlled as the Figure 6 shows a straight line with both surfaces falling on the same line. I do not know the mechanism for this I am receptive to any ideas.

Figure 6.

Figure 6. Maize water use efficiency


4 comentarios

Fluorescencia de clorofilas

José A. Hernández, Grupo de Biotecnología de Frutales (CEBAS-CSIC)

La fluorescencia es un fenómeno foto-físico de las moléculas de clorofila que permite estudiar la función del fotosistema II (PSII) durante el transporte electrónico en la fotosíntesis y la sensibilidad del PSII al daño que puede sufrir por efecto de diferentes estreses, y las consecuencias que esto tiene en el proceso global de la fotosíntesis (Figura 1).

Por tanto, la fluorescencia de clorofilas es una técnica muy útil que permite hacer un seguimiento al proceso de fotosíntesis en general. Se emplea en diferentes estudios:

  •  Fisiología de la fotosíntesis
  • Ecofisiología
  • Biología Marina y Acuática
  • Horticultura
  • Agricultura
  • Fisiología de Post-cosecha
  • Mejora Vegetal
  • Genética

¿Qué es la fluorescencia?

Los electrones que forman parte de un átomo o una molécula tienden a permanecer en un estado de menor energía (estado fundamental). Si un átomo absorbe un fotón con suficiente energía, un electrón puede saltar a un orbital de mayor energía. Este estado de mayor energía es más reactivo que el estado fundamental y puede participar en reacciones químicas que son imposibles para el estado fundamental. Esto es muy importante para la fotosíntesis. Incluso en ausencia de reacciones, el estado excitado es inestable y puede volver a sus estado fundamental por diferentes vías, incluido la emisión de un fotón. El fotón emitido es la fluorescencia.

Figura 1. Esquema en Z de la cadena de transporte de electrones en el cloroplasto.

Figura 1. Esquema en Z de la cadena de transporte de electrones en el cloroplasto.

La energía luminosa absorbida por las moléculas de clorofila en la hoja tiene tres posibles destinos: La mayor parte se va a usar en fotosíntesis (energía fotoquímica). Una pequeña parte de la energía, la que no puede emplearse en fotosíntesis, se disipa en forma de calor o bien puede ser re-emitida como luz (en forma de fluorescencia) con el fin de que el exceso de energía no dañe a los fotosistemas. La cantidad de energía emitida como fluorescencia es muy pequeña (1-2% del total de luz absorbida) (Figura 2).

Figura 2. Esquema mostrando el uso de la energía luminosa en condiciones fisiológicas. La mayor parte se va a usar en fotosíntesis y una pequeña parte de la energía, la que no puede emplearse en fotosíntesis, se disipa en forma de calor o bien puede ser re-emitida como luz (en forma de fluorescencia).

Figura 2. Esquema mostrando el uso de la energía luminosa en condiciones fisiológicas. La mayor parte se va a usar en fotosíntesis y una pequeña parte de la energía, la que no puede emplearse en fotosíntesis, se disipa en forma de calor o bien puede ser re-emitida como luz (en forma de fluorescencia).

En condiciones normales, la fotosíntesis predomina sobre los otros procesos, pero en condiciones de estrés, la planta no puede trabajar a pleno rendimiento y el exceso de energía debe disiparse. Como consecuencia, los procesos no fotoquímicos aumentan.

Para un análisis de fluorescencia es conveniente adaptar a la planta a condiciones de oscuridad durante unos 10-15 minutos. Cuando una hoja se transfiere desde la oscuridad a la luz, los centros de reacción del PSII se van cerrando progresivamente. Esto da lugar a un aumento en el rendimiento de la fluorescencia de las clorofilas. A partir de este momento, los niveles de fluorescencia disminuyen de nuevo. Este fenómeno se conoce como quenching y se explica  de dos maneras: Primero, se produce un incremento en la tasa de transporte de electrones fuera del PSII. Esto es debido a la activación mediada por luz de los enzimas implicados en el metabolismo del carbono y en la apertura de los estomas. Este tipo de quenching se denomina “quenching fotoquímico”. Al mismo tiempo, se produce un aumento de la eficiencia en la que la energía se convierte en calor. Este último proceso se denomina “quenching no fotoquímico” (NPQ).

Para el análisis de la fluorescencia de clorofilas se han definido y calculado diferentes coeficientes para cuantificar el quenching fotoquímico y no fotoquímico. Para los procesos fotoquímicos, el parámetro más útil para medir la eficiencia del PSII es el rendimiento cuántico del PSII (ØPSII o Y(II)), que mide la proporción de luz absorbida por la clorofila asociada al PSII  que es usada en procesos fotoquímicos. Otro parámetro ampliamente usado es el quenching fotoquímico (qP).  Aunque es muy similar al ØPSII , el significado del qP es algo diferente. En este caso, el qP hace referencia a la proporción de centros de reacción del PSII que están abiertos. ØPSII y qP están interrelacionados con un tercer parámetro, Fv/Fm, que mide la eficiencia del PSII, es decir, mide el rendimiento cuántico si todos los centros de reacción del PSII estuviesen abiertos.

Los procesos no fotoquímicos (NPQ) están relacionados con la disipación de calor, y su escala varía desde 0 hasta el infinito. El NPQ tiene varios componentes, pero el más importante es el denominado qN (coeficiente del quenching no fotoquímico). Este parámetro varía en una escala desde 0 a 1 y está relacionado con la disipación de calor mediante el ciclo de las xantofilas (Fig 3). NPQ y qN son indicadores de estrés y han demostrado ser parámetros muy sensibles para la detección temprana de condiciones de estrés mediante imagen de fluorescencia. En este sentido se pueden usar para valorar situaciones de estrés abiótico como biótico, pudiendo analizar el efecto de estreses ambientales en el cloroplasto, incluso antes de que se observen señales de síntomas en las hojas (Fig 4).

Figura 3. Esquema del ciclo de las Xantofilas. La interconversión de anteroxantina en zeaxantina lleva asociada una disipación de energía en forma de calor.

Figura 3. Esquema del ciclo de las Xantofilas. La interconversión de anteroxantina en zeaxantina lleva asociada una disipación de energía en forma de calor.

Figura 4. Efecto del estrés hídrico sobre los parámetros qP y qN. Las plantas 1,2 y 3 se sometieron a un periodo de falta de riego de 15 días. Las imágenes muestran cómo el estrés reduce el valor de qP, pero de forma más dramática en plantas 1 y 2. Por el contrario, la sequía aumenta la disipación de calor (qN) en las plantas 1 y 2 con el fin de poder minimizar daños por exceso de energía luminosa. La planta 3 presenta sólo una pequeña variación en qN.

Figura 4. Efecto del estrés hídrico sobre los parámetros qP y qN. Las plantas 1,2 y 3 se sometieron a un periodo de falta de riego de 15 días. Las imágenes muestran cómo el estrés reduce el valor de qP, pero de forma más dramática en plantas 1 y 2. Por el contrario, la sequía aumenta la disipación de calor (qN) en las plantas 1 y 2 con el fin de poder minimizar daños por exceso de energía luminosa. La planta 3 presenta sólo una pequeña variación en qN.

Para más información:

  • Maxwell K, Johnson GN (2000) Chlorophyll fluorescence – a practical guide. J. Exp. Bot. 51: 659-668.
  • Pérez-Bueno ML, Ciscato M, vandeVen M, Gacía-Luque I, Valcke R, Barón M (2006) Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus. Photosyntesis Research 90:111–123.
  • Taiz L, Zeiger E (2010) Plant  Physiology, Fifth Edition, Sinauer Associates Inc., Publishers, Sinderland, Massachusetts, USA