Task 1 : Benefits and drawbacks from forest investment in PSMs under natural and intensified drought conditions
popularité : 15%
Task leader : E. Ormeño
Participants : C. Fernandez, JP. Mévy, S. Greff, C. Lecareux, JP. Orts, AC. Genard, C. Boissard, J. Lathière, M. Havaux, N. Bonnaire, I. Reiter,
Postdoc - Subcontracting : H. Wortham, B. Temine (AMU), P. Délaplace (Univ Liège).
Objectives and hypothesis
Task 1 of the present project will evaluate whether investment in PSMs during periods with intensified low water resources (i) has significant detrimental consequences for forest PPMs (estimated through productivity) (sub-task 1.1), and (ii) could be beneficial for the plant in terms of oxidative pressure reduction (sub-task 1.2). For both objectives, volatile PSMs (BVOCs) and non-volatile PSMs (phenolic compounds) will be tackled.
We hypothesize that under intensified drought conditions there will be trade-offs (i.e negative correlations) between PSMs and PPMs with increasing investment in PSMs leading to lower growth rates. It is likely that this pattern is at least found during the most intense growth period (spring) since resource allocation to growth rather than to defence might endanger plant survival during the following stress summer months. We also hypothesize that BVOCs released by Q. pubescens (mostly isoprene) and non-volatile PSMs (especially flavonoids) have a protective role against damage caused by cell oxidation produced by drought conditions, but their relative efficiencies will be fully tested in this project.
In order to fully understand the mechanisms that drive the trade-offs between PSM and PPM under different water stress conditions, Task 1 will simultaneously examine the occurrence of interacting effects between water stress and plant mineral nutrition for two reasons (sub-task 1.3). First, different drought scenarios may directly affect plant nutrition which in turn is intimately correlated to plant productivity and PSMs production. Second, feedback loops between reduced plant nutrient uptake due to drought, and inhibition of decomposability of leaf litter as a consequence of higher accumulation of secondary metabolites could occur (interface between Task 1 & 2).
Sub Task 1-1 : Trade-offs between PSMs (volatile and non-volatile) and PPM (productivity) through different phenological stages
a. Indicators of plant growth and leaf traits (also see complementary part in subtask 2-1)
Investment in primary metabolic processes is traditionally studied by measuring growth, which will be estimated in both stressed and control trees all over the phenological cycle. Growth will not be studied on harvested material to maintain the site as stable as possible. Instead, a representative number of stems from different trees (control and stressed) will be selected to study the following parameters : (i) shoot elongation, (ii) leaf area, (iii) LDM : leaf dry biomass, (iv) relative growth rate of leaves (v) specific leaf area (SLA), (vi) leaf dry matter content (LDMC), (vii) leaf thickness calculated as : 1/(ρ x SLA x LDM), where ρ is the leaf density which is typically equal to 1 (Vile 2005).
Phenology will be monitored in both treatments to account for water stress induced changes. It will be evaluated by a classical method consisting of leaf phenological obser-vations (Fig. 6). Each phenological phase will be considered as representative when it will be observed at least in 5% of the entire crown of each plant and in 20% of the trees in the rain exclusion and the control plots (La Mantia et al. 2003).
c. Eco-metabolomics : Volatile and non-volatile PSMs
We will explore the metabolomics, through volatile and non-volatile PSMs, of Q. pubescens under the rain exclusion and the control plots, through the main phenological cycle (spring, summer and fall), during three consecutive years. For A. monspessulanum and C. coggygria PSMs will be monitored during the driest conditions in a summer month.
c.1. Volatile PSMs : Biogenic volatile organic compound (BVOC) emissions
BVOC emissions are quantified in combination with standard gas-exchanges (CO2, H2O) by means of dynamic enclosure chambers (Ormeño et al. 2010 ; Fares et al. 2010) (Fig. 7). Each BVOC sampling campaign, of 1 to 2 weeks, will consist of sampling BVOC emissions from at least 3 stressed and 3 control trees.
The dynamic chambers allow continuous air exchange. Environmental conditions (PAR, temperature, relative humidity) inside and outside the chamber will be recorded with appropriate probes and sensors and thermocouples wrapped around the branches and touching the leaves to measure the leaf temperatures. The dynamic chamber will also be connected to an Infrared Red Gas Analyzer (IRGA) for monitoring gas exchanges (CO2, water).
BVOCs emitted by the biomass inside the enclosures will be analyzed using two tech-niques :
First, the PTR-ToF-MS (MASSALYA, LCE, Aix-Marseille Université) is used to study compounds with a higher proton affinity than that of water (166.5 kcal mol-1), including most BVOCs. This device, based on mass spectrometry (MS), allows (i) real time emission detection and quantification (i.e. on-line sampling) of BVOCs with a high time resolution (up to 1 measure.s-1), and (ii) detection of BVOCs in the order of a few ppt. The main drawback of this technique is its unsuitability to identify different volatiles with the same molecular mass (e.g. monoterpenes).
Second, in order to identify different monoterpenes we will used specific cartridges (packed with VOC-containing adsorbents such as Tenax TA or Carbotrap) for collection of BVOCs. These adsorbents will subsequently be analyzed by gas-chromatography-MS (GC-MS) in the laboratory in order to determine emission quality.
All collected data (CO2 and H2O exchanges through IRGA analyses, branch BVOC emissions through PTR-ToF-MS, light, temperature and humidity within each chamber collected by the different sensors), will automatically be monitored by means of a data system acquisition. Variations of BVOC emission, especially isoprene, recorded during this project, together with the concomitant biotic and abiotic parameters measured (phenology, growth, climatic conditions) will be analysed by LSCE using non-linear regression by means of artificial neural networks. BVOC emissions simulated by the ORCHIDEE model will be compared to the data collected to perform a long-term evaluation.
c.2. Non volatile PSMs : phenolic compounds
The phenol index (total phenol content) and single phenolics, including single flavonoids. The method for extraction and quantification of flavonoids will include total proanthocya-nidins and total and simple flavonols (Lavoir et al. submitted). All these phenolics will also be analyzed using the MULTIPLEX system based on non-invasive optical methods. This system will allow to evaluate, without leaf harvesting, (i) the phenolic production vari-ability over the entire phenological period and, (ii) differences between branches of the same tree.
Sub-Task 1-2 : Defensive role of secondary metabolites
This subtask will mainly allow to examine the role of PSMs as antioxidant compounds. Concomitantly, we will assess the role of PSMs as structuring molecules which assure membrane stability during intense drought conditions. Two approaches will be used : (a) assessment of PSMs capacity to scavenge (i.e. neutralize) oxidant molecules and, (b) comparing PSMs production and traditional (primary) antioxidant patterns.
a. Role of PSMs as scavengers of cell oxidation
a.1. Role of non-volatile PSMs
The role of non-volatile PSMs as efficient scavengers of cell oxidants will be corroborated if a negative correlation between their leaf concentration and the oxidation level is found (Hernandez et al. 2004) or if trees under the rain exclusion plot allocate a greater fraction of their resources to the production of non-volatile PSMs than naturally watered and, a result, the leaf oxidation level is limited. The oxidation level will be assayed by characterizing different types of cell oxidants :
Global lipid oxidation analyzed by autoluminescence imaging and high-temperature themoluminescence. Autoluminescence imaging provides a new, non-destructive tool to measure lipid oxidation patterns in plant tissues (Birtic et al. 2011). The origin of the signal has been demonstrated to be the spontaneous decomposition of lipid peroxides, generating light-emitting species. This phenomenon can be stimulated by heating. Heat-induced de-composition of lipid peroxides can be measured using the thermoluminescence method as a band peaking at around 130°C (Ducruet et al. 2007, Havaux 2003).
Lipid peroxidation will be assayed, via PTR-ToF-MS measurements, by monitoring the volatiles released through the lipoxygenase metabolic pathway, so called green leaf volatiles (GLV) (e.g. E,E-2,4-hexadienal, 1-hexanol, Buonaurio & Servili 1999). Recent research gives evidence that their emission denote a water stress. For example, emissions of apple trees under water stress conditions are between 5 and 310 higher than well-watered plants (Ebel et al. 1995).
Reactive oxygen species (hydrogen peroxide H202 and the superoxide anion O2-) will be assayed through ‘Functional Imaging’. The method, already set-up in our laboratory (Ait Samir 2011) relies in the use of confocal microscopy and the use of fluorophores, especially ionophores. This technique not only reveals ROS dynamics in vivo at a high spatial resolu-tion.
a.2. Role of volatile PSMs
Demonstration of the scavenging ability of volatile PSMs (mostly isoprene) requires - in addition to isoprene and cell oxidation estimation - other technical considerations since isoprene is highly volatile. As a result, its presence is not necessarily related to a defence function but could also be a volatilization phenomenon, which will be unavoidably favoured by the high temperatures reached in trees receiving low precipitation.
Volatile PSMs may also function as signal molecules. Recently, the CEA-Cadarache group has found that volatile compounds, such as a-ionone or a-cyclocitral, derived from the oxidation of carotenoids have a signalling function, inducing the expression of a large variety of genes related to stress defence and repressing genes related to growth and development. It has been suggested that those volatile compounds are stress signals that mediate gene response to ROS, especially singlet oxygen. In the frame of this ANR project, we will study the levels and functions of this type of volatile carotenoid derivatives.
b. Comparing secondary and primary (universal) antioxidants
The second approach that will be undertaken to validate that PSMs (volatile or not) play an antioxidant role for the plant that produces them, consists of comparing the production of PSMs to that of traditional (primary or universal) antioxidants. A positive correlation between PSMs and at least one traditional antioxidant should be found to validate our hypothesis. Primary non-enzymatic and enzymatic antioxidants will be analyzed :
b.1. Non-enzymatic universal antioxidants
Non-enzymatic antioxidants will be spectrophotometrically assayed using the methods developed by De Pinto et al. (1999), Zhang & Kirkham (1996) and Morris et al. (2004) :
Ascorbate is the most important non-enzymatic antioxidant in plants (Blokhina et al. 2003) as it is present in every cellular compartment. Moreover, the ratio between its re-duced and oxidized forms is a reliable indicator of the level of oxidative stress undergone by a biological system (Noctor et al. 1998).
Glutathione reduced and oxidized forms will be analyzed. Reduced forms are highly abundant under intense drought conditions, resulting in a low ratio between its reduced and oxidized forms and induce the synthesis of the reduced form. The accumulation of the oxidized form of glutathione could lead to the inhibition of both the cell cycle and the protein biosynthesis (Foyer et al. 2005).
Total carotenoids, important antioxidants that limit the concentrations of free radicals in the plant membranes (Howitt & Pogson 2005), will be measured after acetone extraction by using a mean extinction coefficient at 450 nm. Single carotenoids will be analyzed by HPLC, as described previously (Havaux et al. 2005).
Tocopherols, the major antioxidant molecules present in the thylakoid membranes of chloroplasts (Mène-Saffrané & Della Penna 2010), will be analyzed, together with related antioxidants (plastoquinone, plastochromanol) by HPLC with UV-visible detection combined with fluorescence detection (Eugeni-Piller et al. 2011).
b.2. Enzymatic universal antioxidants
Ascorbate peroxidase (APX) and catalase (CAT) activities that detoxify ROS will be meas-ured based on the protocols of Nakano & Asada (1981) and Claiborne (1985) respectively. The very high affinity of APX for ROS suggests that this enzyme is involved in the regulation of low levels of ROS, e.g. in a signal context (Mittler 2002). APX activity is calculated by measuring the absorbance decrease of an ascorbate solution in the presence of H2O2 and the sample. Catalase activity increases in case of acute oxidative stress (Feierabend 2005) and is assayed by measuring the decrease of ROS absorbance in the presence of the sample.
Sub-Task 1-3 : Impact of plant mineral nutrition on PPM and PSM trade-offs under naturally and intensified water stressed plants
In parallel to foliar and litter secondary metabolite analysis, we will characterise leaf macronutrient contents (N, P, K) of 3 woody species (Q. pubescens, A. monspessulanum and C. coggygria trees) in control and rain exclusion zones during the main phenological stages until leaf senescence of 3 growing seasons. Foliar and stem N, P, K contents will be investigated using a CHN analyzer, spectrophotometer and ionic chromatography.
Correlations between nutrient concentrations and secondary metabolite contents in the rain exclusion plot and the plot receiving natural precipitation will allow to account for interacting effects between water stress and mineral nutrition.