Tree Physiology 34, 1388–1398 doi:10.1093/treephys/tpu098
Research paper
Early physiological consequences of fire as an abiotic stressor in metabolic source and sink of young Brutian pine (Pinus brutia Ten.) Maria Alexou1 and Alexandros P. Dimitrakopoulos
Received May 5, 2014; accepted October 15, 2014; published online November 27, 2014; handling Editor Roberto Tognetti
Climatic change causes gradual deforestation, partly through forest fires. However, fire has not been seen as an oxidative stressor on surviving forest trees. In addition, discrimination of stress-induced responses from acclimation steps cannot be examined under prolonged stress. Thus, four young Brutian pine (Pinus brutia Ten.) trees, a fire-related species, were subjected to a simulation of a crown-fire event to evaluate its impact on the availability of soluble carbon (C) and nitrogen (N) and the redox status near fire-afflicted tissue. Total soluble sugars, amino acids and non-structural (NS) proteins in needles and phloem, the antioxidant ascorbic acid (AsA) and reactive oxygen species (ROS) in needles were investigated together with the phloem transport velocity. To examine the temporal progress of these parameters, samples were obtained prior to fire (pre-fire), 2 h after fire, the following day (Day 1) and the following week (Week 1). Findings were categorized into shock reactions (2 h) and acclimation steps. Phloem transport accelerated 2 h postfire by almost 30% and correlated negatively to phloem sugars. At the same time the phloem ratio of sugars/amino acids correlated negatively to needle ROS. The trees’ main response at 2 h and particularly on Day 1 was a massive increase in phloem NS proteins. The acclimation process involved also significant increases in needle NS proteins and AsA, as well as significant depletion of phloem amino acids by 65% by Week 1. The highest availability of soluble C and N was recorded on Day 1 in the phloem. Regression models explained significantly the variability of most soluble compounds postfire. Our findings suggest sink control over the source and an advanced role of phloem transport in defense processes. Keywords: abiotic stress, amino acids, ascorbic acid, biomass loss, extreme heat, non-structural proteins, phloem transport velocity, ROS, soluble metabolites, sugars.
Introduction Forest fires have become a serious threat and have been considered to be part of climatic change for some time now (IPCC 2007). Countries with a Mediterranean-type climate such as Greece, due to the combination of dry and warm climate, flammable vegetation and increased human activities, are extremely fire stricken and each year large areas of forests vanish due to summer forest fires (Dimitrakopoulos and Mitsopoulos 2006). Still, some trees survive. Fire injury causes wounds, disrupting living tissues including vascular cambium, resulting in a
loss of function. However, scorch is considered an indicator of fire occurrence, not necessarily injury (Sutherland and Smith 2000). Fire, as a stress factor, may induce metabolic changes in leaves. The prediction of how many trees will die after the fire is essential for postfire decisions (Brown et al. 2003). Knowing the extent and nature of the metabolic changes in plants is also important for the use of prescribed fire (Alonso et al. 2002). Trees surviving fire have suffered a combination of extreme heat and thermic biomass loss. The synergistic impact of
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://treephys.oxfordjournals.org/ at East Carolina University on July 11, 2015
Laboratory of Forest Protection, School of Forestry and Natural Environment, Aristotle University of Thessaloniki, P.O. Box 228, 54124 Thessaloniki, Greece; 1Corresponding author ([email protected])
Fire as abiotic stressor on pine physiology 1389 imaging (Windt et al. 2006). Thus, the transport velocity is a matter that has not been well investigated. Pinus brutia Ten. (Tsitsoni et al. 2004) is often compared with the drought-tolerant Pinus halepensis. Pinus brutia requires higher rainfalls, but tolerates a wider range of temperatures (Fady et al. 2003) and has higher genetic diversity, and thus, adaptability (Korol et al. 2002). It also has a higher phenolic load than P. halepensis (Guri et al. 2006). It is a fast-growing species of high timber quality in fire-related ecosystems of the east Mediterranean (Fady et al. 2003, Michelozzi et al. 2008). Our knowledge of the impact of fire on trees focuses more on secondary compounds in the long term. On the contrary, little is known about the early responses to fire as an oxidative stressor. For this reason, total soluble sugars, amino acids and non- structural (NS) proteins were tested in the needles and phloem of unburned twigs near fire-afflicted tissue before and after a fire simulation. The phloem transport velocity would further determine if stress or healing affects the metabolites’ flow. Soluble C and N were expected to increase to promote healing. Reactive oxygen species, indicating oxidative stress, and AsA, representative of the antioxidative capacity, were also expected to increase. Our hypothesis was that these increases would start within 2 h postfire and gradually subside as acclimation established.
Materials and methods Experimental design A forest fire simulation of a crown-fire event took place on 27 April 2010 in the suburban pine forest Kedrinos Lofos (Tsitsoni et al. 2004), Thessaloniki, Greece (40°38′36″N, 22°58′27″E), 3 days before any fire source was prohibited owing to an increased risk of forest fire. The altitude of the area ranges from 50 to 450 m. The climate is Mediterranean, with 135 dry days on average, and the dry period lasts from the middle of May to the end of September (Tsitsoni et al. 2004). The fire simulation was conducted as closely as possible to the actual fire season in the Mediterranean Basin, when most fires occur. The experiment included four young Brutian pine (P. brutia) trees (Table 1) with no dominant overstory, growing within a few meters of each other in a total area of 0.05 ha to ensure on the one hand identical site conditions and on the other hand no interaction among them. The mean temperatures in April and May 2010 were, respectively, 15.5 and 20.8 °C. The mean rainfall was, respectively, 17 and 41 mm. Table 1. Features of the treated trees on the experimental site and extent of burned crown. Tree number
Tree height (m)
Age (years)
Burned (%)
1 2 3 4
2.5 3.15 1.80 1.90
13 16 10 11
25 30 25 40
Tree Physiology Online at http://www.treephys.oxfordjournals.org
Downloaded from http://treephys.oxfordjournals.org/ at East Carolina University on July 11, 2015
such a combination cannot be easily assessed, because the first stressor can improve the plant's defense against a second one (Niinemets 2010, Christou et al. 2014). The heat shock response (HSR) is connected to stress-induced reactive oxygen species (ROS) (Foyer 2005, Pucciariello et al. 2012, Kipp and Boyle 2013). Pathways of ROS detoxification involve induction of heat shock proteins (HSP) (Baniwal et al. 2004, Pucciariello et al. 2012), even in recovery time (Nover et al. 2001), as a generalized response to stress, although different reactions against individual stressors do exist (Taylor et al. 2004). Mere mechanical wounding elicits similar, though clearly distinguishable, transcriptomic patterns to those elicited by flame wounding (Heil et al. 2012). However, every stress disturbs oxidative cell homeostasis (Elad 1992) owing to excess production of ROS creating oxidative stress conditions (Orozco-Cardenas and Ryan 1999, Apel and Hirt 2004, Suzuki and Mittler 2006). Interestingly, ROS also act as signal molecules that lead to detoxification and acclimation (Foyer 2005, Christou et al. 2014). Ascorbic acid (AsA) is often investigated under various stress conditions, e.g., heat and drought (Smirnoff 2000, Alexou 2013), owing to its major importance in controlling ROS levels. However, expressing and maintaining inducible defenses requires carbon (C) and nitrogen (N) resources (Franceschi et al. 2005, Klepzig et al. 2005), which creates sink competition within the tree (Klepzig et al. 2005, Lombardero et al. 2006). Secondary metabolites for defense include phenolics, terpenes (Alonso et al. 2002, Cannac et al. 2007) and volatile isoprenoid hormones, such as abscisic acid (ABA, Perks et al. 2002). Secondary compounds take time to accumulate but can be temporary responses (Alonso et al. 2002). Sugars have synergistic interactions with phenols to form a redox system quenching ROS, contributing to stress tolerance (Bolouri-Moghaddam et al. 2010). Amino acids also increase due to stress as a protective mechanism and build essential N and C reservoirs (Manderscheid et al. 1992, Martinelli et al. 2007). Owing to stress conditions, respiration increases (Tiwari et al. 2002). The consumption of sucrose by respiration of meristems has been postulated to sustain the gradient between the phloem and the sink cells (Lemaire and Millard 1999). The concentration of sucrose in the sink is thought to play an important role in regulating sink activity, as are the relative concentrations of C and N substrates (Lemaire and Millard 1999). Under heat stress, increase of sugars occurs at the expense of starch (Smeekens 2000, Kaplan et al. 2006, Waraich et al. 2012). Sucrose plays a significant role in C and N assimilation and also in transport. Pinus is a passive loading species, i.e., sugars move into the phloem without the use of metabolic energy by traveling down a concentration gradient from the mesophyll to the phloem (Rennie and Turgeon 2009, Jensen et al. 2013). Phloem transport is very hard to measure even with nuclear magnetic resonance flow
1390 Alexou and Dimitrakopoulos
Table 2. Field conditions on the experimental site. Field conditions
27 April
28 April
3 May
Temperature (°C) Relative humidity (%)
26 35
28 39
25 31
Table 3. Time schedule of the fire simulation on each treated tree. Samplings took place prior to fire (pre-fire), 2 h postfire (2 h), a day postfire (Day 1) and a week postfire (Week 1). 27 April
28 April
3 May
Tree
Pre-fire
2 h
Day 1
Week 1
1 2 3 4
10:55 11:15 11:45 12:15
12:00 13:20 13:45 14:30
12:40 12:45 12:46 12:50
12:20 12:23 12:25 12:30
Tree Physiology Volume 34, 2014
reaction of trees to flames and not chemical products of combustion. The final sampling date was chosen considering the time required for trees to fully enter recovery phase, the ‘aftereffect’ of stress (Alonso et al. 2002) and the low phloem transport velocity. Owing to tree size and the short duration of the experiment, to minimize biomass losses no sampling took place between Day 1 and Week 1. The temporal progress of parameters was evaluated using the respective pre-fire levels as reference points (‘repeated measures’ design).
Transport velocity in the phloem To determine the phloem transport velocity, 15N feeding was implemented into the bark of the treated trees based on Alexou (2006) in parallel to sampling (Table 3). In the laboratory, an aqueous solution resembling phloem sap in content and concentration had been produced and transferred frozen in 1.5-ml screw cap vials to the experimental site. Each vial contained 1 ml of the solution that consisted of 6 mM 15N glutamine (L-glutamine-a-15N, chemical purity 98%+, Cambridge Isotope Laboratories, Inc., Saarbrücken, Germany), 0.5 M sucrose, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.015 μM chloramphenicol, at pH 7, and was used as the incubation solution for separate twigs from those used for the phloem exudation samples. EDTA and chloramphenicol were included in the synthesis to prevent the formation of callose in the wounds and microbial organisms. On each treated tree, the feeding technique was applied three times (pre-fire, 2 h, Day 1). In each application, a 1.5-ml vial was fastened with tessa film onto a selected twig (
Research paper
Early physiological consequences of fire as an abiotic stressor in metabolic source and sink of young Brutian pine (Pinus brutia Ten.) Maria Alexou1 and Alexandros P. Dimitrakopoulos
Received May 5, 2014; accepted October 15, 2014; published online November 27, 2014; handling Editor Roberto Tognetti
Climatic change causes gradual deforestation, partly through forest fires. However, fire has not been seen as an oxidative stressor on surviving forest trees. In addition, discrimination of stress-induced responses from acclimation steps cannot be examined under prolonged stress. Thus, four young Brutian pine (Pinus brutia Ten.) trees, a fire-related species, were subjected to a simulation of a crown-fire event to evaluate its impact on the availability of soluble carbon (C) and nitrogen (N) and the redox status near fire-afflicted tissue. Total soluble sugars, amino acids and non-structural (NS) proteins in needles and phloem, the antioxidant ascorbic acid (AsA) and reactive oxygen species (ROS) in needles were investigated together with the phloem transport velocity. To examine the temporal progress of these parameters, samples were obtained prior to fire (pre-fire), 2 h after fire, the following day (Day 1) and the following week (Week 1). Findings were categorized into shock reactions (2 h) and acclimation steps. Phloem transport accelerated 2 h postfire by almost 30% and correlated negatively to phloem sugars. At the same time the phloem ratio of sugars/amino acids correlated negatively to needle ROS. The trees’ main response at 2 h and particularly on Day 1 was a massive increase in phloem NS proteins. The acclimation process involved also significant increases in needle NS proteins and AsA, as well as significant depletion of phloem amino acids by 65% by Week 1. The highest availability of soluble C and N was recorded on Day 1 in the phloem. Regression models explained significantly the variability of most soluble compounds postfire. Our findings suggest sink control over the source and an advanced role of phloem transport in defense processes. Keywords: abiotic stress, amino acids, ascorbic acid, biomass loss, extreme heat, non-structural proteins, phloem transport velocity, ROS, soluble metabolites, sugars.
Introduction Forest fires have become a serious threat and have been considered to be part of climatic change for some time now (IPCC 2007). Countries with a Mediterranean-type climate such as Greece, due to the combination of dry and warm climate, flammable vegetation and increased human activities, are extremely fire stricken and each year large areas of forests vanish due to summer forest fires (Dimitrakopoulos and Mitsopoulos 2006). Still, some trees survive. Fire injury causes wounds, disrupting living tissues including vascular cambium, resulting in a
loss of function. However, scorch is considered an indicator of fire occurrence, not necessarily injury (Sutherland and Smith 2000). Fire, as a stress factor, may induce metabolic changes in leaves. The prediction of how many trees will die after the fire is essential for postfire decisions (Brown et al. 2003). Knowing the extent and nature of the metabolic changes in plants is also important for the use of prescribed fire (Alonso et al. 2002). Trees surviving fire have suffered a combination of extreme heat and thermic biomass loss. The synergistic impact of
© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://treephys.oxfordjournals.org/ at East Carolina University on July 11, 2015
Laboratory of Forest Protection, School of Forestry and Natural Environment, Aristotle University of Thessaloniki, P.O. Box 228, 54124 Thessaloniki, Greece; 1Corresponding author ([email protected])
Fire as abiotic stressor on pine physiology 1389 imaging (Windt et al. 2006). Thus, the transport velocity is a matter that has not been well investigated. Pinus brutia Ten. (Tsitsoni et al. 2004) is often compared with the drought-tolerant Pinus halepensis. Pinus brutia requires higher rainfalls, but tolerates a wider range of temperatures (Fady et al. 2003) and has higher genetic diversity, and thus, adaptability (Korol et al. 2002). It also has a higher phenolic load than P. halepensis (Guri et al. 2006). It is a fast-growing species of high timber quality in fire-related ecosystems of the east Mediterranean (Fady et al. 2003, Michelozzi et al. 2008). Our knowledge of the impact of fire on trees focuses more on secondary compounds in the long term. On the contrary, little is known about the early responses to fire as an oxidative stressor. For this reason, total soluble sugars, amino acids and non- structural (NS) proteins were tested in the needles and phloem of unburned twigs near fire-afflicted tissue before and after a fire simulation. The phloem transport velocity would further determine if stress or healing affects the metabolites’ flow. Soluble C and N were expected to increase to promote healing. Reactive oxygen species, indicating oxidative stress, and AsA, representative of the antioxidative capacity, were also expected to increase. Our hypothesis was that these increases would start within 2 h postfire and gradually subside as acclimation established.
Materials and methods Experimental design A forest fire simulation of a crown-fire event took place on 27 April 2010 in the suburban pine forest Kedrinos Lofos (Tsitsoni et al. 2004), Thessaloniki, Greece (40°38′36″N, 22°58′27″E), 3 days before any fire source was prohibited owing to an increased risk of forest fire. The altitude of the area ranges from 50 to 450 m. The climate is Mediterranean, with 135 dry days on average, and the dry period lasts from the middle of May to the end of September (Tsitsoni et al. 2004). The fire simulation was conducted as closely as possible to the actual fire season in the Mediterranean Basin, when most fires occur. The experiment included four young Brutian pine (P. brutia) trees (Table 1) with no dominant overstory, growing within a few meters of each other in a total area of 0.05 ha to ensure on the one hand identical site conditions and on the other hand no interaction among them. The mean temperatures in April and May 2010 were, respectively, 15.5 and 20.8 °C. The mean rainfall was, respectively, 17 and 41 mm. Table 1. Features of the treated trees on the experimental site and extent of burned crown. Tree number
Tree height (m)
Age (years)
Burned (%)
1 2 3 4
2.5 3.15 1.80 1.90
13 16 10 11
25 30 25 40
Tree Physiology Online at http://www.treephys.oxfordjournals.org
Downloaded from http://treephys.oxfordjournals.org/ at East Carolina University on July 11, 2015
such a combination cannot be easily assessed, because the first stressor can improve the plant's defense against a second one (Niinemets 2010, Christou et al. 2014). The heat shock response (HSR) is connected to stress-induced reactive oxygen species (ROS) (Foyer 2005, Pucciariello et al. 2012, Kipp and Boyle 2013). Pathways of ROS detoxification involve induction of heat shock proteins (HSP) (Baniwal et al. 2004, Pucciariello et al. 2012), even in recovery time (Nover et al. 2001), as a generalized response to stress, although different reactions against individual stressors do exist (Taylor et al. 2004). Mere mechanical wounding elicits similar, though clearly distinguishable, transcriptomic patterns to those elicited by flame wounding (Heil et al. 2012). However, every stress disturbs oxidative cell homeostasis (Elad 1992) owing to excess production of ROS creating oxidative stress conditions (Orozco-Cardenas and Ryan 1999, Apel and Hirt 2004, Suzuki and Mittler 2006). Interestingly, ROS also act as signal molecules that lead to detoxification and acclimation (Foyer 2005, Christou et al. 2014). Ascorbic acid (AsA) is often investigated under various stress conditions, e.g., heat and drought (Smirnoff 2000, Alexou 2013), owing to its major importance in controlling ROS levels. However, expressing and maintaining inducible defenses requires carbon (C) and nitrogen (N) resources (Franceschi et al. 2005, Klepzig et al. 2005), which creates sink competition within the tree (Klepzig et al. 2005, Lombardero et al. 2006). Secondary metabolites for defense include phenolics, terpenes (Alonso et al. 2002, Cannac et al. 2007) and volatile isoprenoid hormones, such as abscisic acid (ABA, Perks et al. 2002). Secondary compounds take time to accumulate but can be temporary responses (Alonso et al. 2002). Sugars have synergistic interactions with phenols to form a redox system quenching ROS, contributing to stress tolerance (Bolouri-Moghaddam et al. 2010). Amino acids also increase due to stress as a protective mechanism and build essential N and C reservoirs (Manderscheid et al. 1992, Martinelli et al. 2007). Owing to stress conditions, respiration increases (Tiwari et al. 2002). The consumption of sucrose by respiration of meristems has been postulated to sustain the gradient between the phloem and the sink cells (Lemaire and Millard 1999). The concentration of sucrose in the sink is thought to play an important role in regulating sink activity, as are the relative concentrations of C and N substrates (Lemaire and Millard 1999). Under heat stress, increase of sugars occurs at the expense of starch (Smeekens 2000, Kaplan et al. 2006, Waraich et al. 2012). Sucrose plays a significant role in C and N assimilation and also in transport. Pinus is a passive loading species, i.e., sugars move into the phloem without the use of metabolic energy by traveling down a concentration gradient from the mesophyll to the phloem (Rennie and Turgeon 2009, Jensen et al. 2013). Phloem transport is very hard to measure even with nuclear magnetic resonance flow
1390 Alexou and Dimitrakopoulos
Table 2. Field conditions on the experimental site. Field conditions
27 April
28 April
3 May
Temperature (°C) Relative humidity (%)
26 35
28 39
25 31
Table 3. Time schedule of the fire simulation on each treated tree. Samplings took place prior to fire (pre-fire), 2 h postfire (2 h), a day postfire (Day 1) and a week postfire (Week 1). 27 April
28 April
3 May
Tree
Pre-fire
2 h
Day 1
Week 1
1 2 3 4
10:55 11:15 11:45 12:15
12:00 13:20 13:45 14:30
12:40 12:45 12:46 12:50
12:20 12:23 12:25 12:30
Tree Physiology Volume 34, 2014
reaction of trees to flames and not chemical products of combustion. The final sampling date was chosen considering the time required for trees to fully enter recovery phase, the ‘aftereffect’ of stress (Alonso et al. 2002) and the low phloem transport velocity. Owing to tree size and the short duration of the experiment, to minimize biomass losses no sampling took place between Day 1 and Week 1. The temporal progress of parameters was evaluated using the respective pre-fire levels as reference points (‘repeated measures’ design).
Transport velocity in the phloem To determine the phloem transport velocity, 15N feeding was implemented into the bark of the treated trees based on Alexou (2006) in parallel to sampling (Table 3). In the laboratory, an aqueous solution resembling phloem sap in content and concentration had been produced and transferred frozen in 1.5-ml screw cap vials to the experimental site. Each vial contained 1 ml of the solution that consisted of 6 mM 15N glutamine (L-glutamine-a-15N, chemical purity 98%+, Cambridge Isotope Laboratories, Inc., Saarbrücken, Germany), 0.5 M sucrose, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.015 μM chloramphenicol, at pH 7, and was used as the incubation solution for separate twigs from those used for the phloem exudation samples. EDTA and chloramphenicol were included in the synthesis to prevent the formation of callose in the wounds and microbial organisms. On each treated tree, the feeding technique was applied three times (pre-fire, 2 h, Day 1). In each application, a 1.5-ml vial was fastened with tessa film onto a selected twig (
