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Poplar under drought: Comparison of leaf and cambial proteomic responses Thomas C. Durand a,b,c,d , Kjell Sergeant a , Jenny Renaut a , Sébastien Planchon a , Lucien Hoffmann a , Sabine Carpin b,c , Philippe Label d , Domenico Morabito b,c,1 , Jean Francois Hausman a,⁎,1 a
CRP-Gabriel Lippmann, Department Environment and Agro-biotechnologies, 41 rue du Brill, L-4422 Belvaux, GD, Luxembourg Université d'Orléans, UFR-Faculté des Sciences, UPRES EA 1207 Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), BP 6759, F-45067 Orléans, France c Institut National de la Recherche Agronomique, USC1328 ‘Arbres et Réponses aux Contraintes Hydrique et Environnementales’ (ARCHE), F-45067 Orléans, France d Institut National de la Recherche Agronomique, UAGPF, F-45166 Olivet Cedex, France b
AR TIC LE I N FO
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Article history:
The forest ecosystem is of particular importance from an economic and ecological
Received 23 December 2010
perspective. However, the stress physiology of trees, perennial and woody plants, is far
Accepted 14 March 2011
from being fully understood. For that purpose, poplar plants were exposed to drought; the
Available online 23 March 2011
plants exhibited commonly reported drought stress traits in the different plant tissues. Leafy rooted cuttings of poplar were investigated through a proteomic approach in order to
Keywords:
compare the water constraint response of two plant tissues, namely leaf and cambium.
Cambium
Sampling was realized during the drought period at 2 time points with increased drought
Drought
intensity and 7 days after rewatering.
Poplar
Our data show that there is a difference in the moment of response to the water constraint
Abiotic stress
between the two tissues, cambium being affected later than leaves. In leaves, drought
Plant
induced a decrease in rubisco content, and an increase in the abundance of light harvesting complex proteins as well as changes in membrane-related proteins. In the cambial tissue, the salient proteome pattern change was the decrease of multiple proteins identified as bark storage proteins. After rewatering, almost all changes in cambial proteome disappeared whereas a significant number of leaf proteins appeared to be differentially regulated only during the recovery from drought. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Water deficit represents the most recurrent and the most serious environmental drawback for plants [1]. Considering
the worldwide increasing frequency of extreme weather events forecasted by climate modeling [2], intensive efforts are made to appreciate plant adjustment abilities to environmental stresses, especially drought [3].
Abbreviations: APX, ascorbate peroxidase; BSP, bark storage protein; RWC, relative water content; TCA cycle, Tricarboxylic acid cycle; TPR, tetratricopeptide repeat domain; Ψ, predawn leaf water potential. ⁎ Corresponding author. CRP-Gabriel Lippmann, Department Environment and Agro-biotechnologies, 41 rue du Brill, L-4422 Belvaux, GD, Luxembourg. E-mail address:
[email protected] (J.F. Hausman). 1 These authors contributed equally as senior author. 1874-3919/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.03.013
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The forest ecosystem is of particular importance from an economic, biological, atmospheric and hydrological perspective. At the regional scale, CO2 and water vapor exchanges between woody species and air have been demonstrated to influence the climate [4]. As harsher climate conditions threaten forest health, distribution and composition [5], it becomes important to decipher the stress-coping mechanisms implied in stress tolerance of trees. Among trees under temperate latitude, Populus species exhibit the greatest growth rates at the expense of large water requirements [6]. Although species and cultivars present a wide genetic variability in traits related to water deficit tolerance whatever the age or the environment of the plant (greenhouse, nursery, or plantation) [7–9], poplar plants are known to be among the most sensitive woody plants to water stress [10] with cavitation events beginning as soon as xylem tension reaches the range of −1 to −1.5 MPa. This is the reason why the samples used for the proteomic study where collected when the predawn leaf water potential reached −0.5 MPa and −0.9 MPa. Poplar whose genome was recently fully sequenced [11] has become a model plant for molecular studies in forestry. Its study brings insights into phenomena specific to trees such as wood formation or seasonality. Its range of reactions to biotic and abiotic factors is widely considered in the literature, especially at leaf [12,13], root [14,15] and xylem [16] levels. Hence, the use of poplar enables not only to understand specific aspects of the tree's response to each type of stress in each kind of tissue, but also the common features of the plant cell exposed to a stressing condition. Moreover, to sustain the extension of poplar cultivation from flood plains and bottomlands to uplands where soil water availability is subjected to seasonal changes, more water-use-efficient and drought tolerant hybrids are required. It is also noteworthy that over the last decades, episodes of dry weather (sometimes followed by short flooding events) have been observed in regions where poplars are cultivated or naturally distributed. Poplar drought stress responses at the root level [17], the leaf level [18], or both [19] have been extensively documented. Given that these organs are responsible for the water dynamics inside the whole plant, they must react rapidly to a stress in order to allow plant survival. On the long run, a successful reaction should result in acclimation, which requires that each part of the organism fine-tunes to suboptimal conditions. The perception of the constraint largely depends upon the position, the age and physiological stage of the organ [20,21]. Among the tree tissues, some further investigation is needed to unravel the cambium response to stress, as this specific tissue is responsible for perennial life of trees through secondary growth and wood formation, an exclusive histological trait of woody plant species [22]. Knowing the functioning of cambium during drought stress is critical for an integrative comprehension of the plant response which relies on multiple and complex processes. Cambium during water deficit has been studied in a few articles [12,23], but seldom within a proteomic approach [19]. Since gene transcription level is not automatically correlated to the actual abundance of active proteins, and given that the poplar genome, although sequenced, is not fully annotated yet, untargeted approaches such as proteomic
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studies keep on constituting effective tools for the identification and for time-dynamics assessment of biological functions affected by a constraint. The present research paper aims at characterizing the physiological state of poplar plants at 2 time points of a gradually imposed water constraint, mild and severe stress, and after a recovery period. A quantitative proteomic analysis was undertaken for the identification of proteome changes in leaf and cambium in response to the constraint. The hybrid poplar genotype INRA 717-1B4 was chosen because of its rapid growth and its use as a forest model species [24]. The other reason is that this genotype can be genetically transformed with Agrobacterium and regenerated efficiently into transgenic trees within 6–10 months [25]. Thus it will be easy in the future to use transgenic plants from this genotype to demonstrate protein function.
2.
Material and methods
2.1.
Plant material and water constraint
Rooted cuttings of Populus tremula L. × P. alba L. (Populus × canescens (Aiton) Smith) genotype INRA 717-1B4 were placed in 10 dm3 pots filled with a sand–peat moss soil mixture (25:75, v/v, pH 6.9) in a growth chamber. Control conditions were set at 22 °C, 70% relative humidity, and irradiance of 1000 μmol m− 2 s− 1 provided during 16 h per day. The branches collected from the nursery and used to obtain the rooted cuttings were divided in 15 cm length cuttings made up of two or three buds. During the rooting period before the drought period we have favored the growth of a single stem per cutting in order to produce stable continuous growth and to minimize the variability potentially induced by different numbers of stem per plant (as described in [26]). At the beginning of the stress period, the rooted plantlets were three months old and had a total leaf area of 1725± 125 cm² in average. The plants were divided into two plots; the first one was used to measure the physiological parameters, the second one was used to collect the samples for the proteomic analysis. Control plants were watered to field capacity every second day. Water stress was induced by withholding water for 12 days. Once the plants reached a predawn leaf water potential equivalent to −0.9 MPa they were re-watered to field capacity for 7 days.
2.2.
Predawn leaf water potential (Ψ)
This parameter was measured using one mature leaf of 4 different plants per treatment. Soil water availability was estimated by measuring the predawn leaf water potential (Ψwp, MPa) with a Scholander-type pressure chamber (PMS670; PMS Instrument, Albany, OR, USA) as previously reported [26]. This parameter was used to assess the intensity of the constraint so as to perform the samplings at the appropriate moment and to start the rewatering period. In poplar it has been demonstrated that cavitation events begin as soon as xylem tension reaches the range of −1 to −1.5 MPa and could lead to the plant death [27]. Therefore the time course of soil water potential is a more appropriate parameter to determine the survival of poplar than the duration of withholding water.
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Gas exchange measurement
Gas exchange was measured using the last fully expanded leaf of 4 different plants per treatment. The same leaf level was chosen for the proteomic analysis. The stomatal conductance (gs, mmol H2O m− 2 s− 1) and the photosynthetic rate (PN, μmol CO2 m− 2 s− 1) were measured using a portable photosynthesis system (Ciras-2, PP Systems, Hitchin, Herts, UK). Temperature in the photosynthetic leaf chamber (PLC) was controlled at 23 °C ± 1 °C. The CO2 concentration of the air flowing at a rate of 200 mL min− 1 over the 2.5 cm2 section of leaf clamped inside the PLC was 400 μmol mol− 1. The light intensity during measurements was 1000 μmol m− 2 s− 1. We considered that the stomatal conductance is the parameter the most closely correlated with the beginning of the water stress, and is therefore the first symptom of stress.
2.4.
Growth measurement
The primary growth was calculated from the measurement of all the leaves of 6 different plants per treatment. Primary growth of plants was estimated on the basis of the total leaf area (cm²), which was calculated according to the formula given by Brignolas et al. [28]. The cutting diameter growth was monitored on 4 plants per treatment using an automatic point dendrometer described in Morabito et al. [29].
2.5.
Water contents
The water content of leaf and cambial zone was calculated from 4 different plants per treatment. The water content of the stem was calculated as WC (%) = (fresh weight − dry weight) × 100/fresh weight. In the case of the leaves, the calculation of the relative water content (RWC) was performed as follow. RWC (%) = (fresh weight − dry weight) × 100 / (saturated weight − dry weight).
2.6.
Tissue sampling for protein extraction
Sampling was realized on day 0-8-12 and 19, from stressed and control plants. For each sample, five replicates each one corresponding to an independent plant were realized. Three fully expanded leaves per plant were collected at the same level as the one used to measure the gas exchanges. The cells from the cambial zone were collected as described in Durand et al. [30] Tissues were immediately frozen in liquid N2 and stored at − 80 °C prior to protein extraction.
2.7.
Protein extraction and separation
Proteins were extracted from ground tissues using a TCAacetone protocol described in Durand et al. [30]. The extracted proteins were labeled with 240 pmol CyDyes™ (GE Healthcare, Little Chalfont, UK) per 30 μg of proteins. Cy2 was used for the labeling of the internal standard consisting of a mix of equal amounts of each sample. For control and treated plant samples, a dye swap was used between Cy3 and Cy5 to avoid problems associated with preferential labeling. The separation of the proteins was performed by bidimensional electrophoresis. The isoelectrofocusing was done using an IPGphor
system on 24 cm strips 3–11 non linear pH range (GE healthcare) at 20 °C with the following program: 60 V for 2 h; gradient from 60 to 1000 V for 3 h; hold 1000 V for 1 h, gradient from 1000 to 8000 V for 3 h; hold 8000 V until 85,000 Vh, with a maximum current setting of 50 mA/strip. The strips were then equilibrated in two steps. First, proteins were reduced by 1% (w/v) DTT for 15 min and, second, alkylated by 2.5% (w/v) iodoacetamide for 15 min. The SDS-PAGE was performed on 12.5% (w/v) acrylamide-bisacrylamide (37.5/1) gels. A power of 2W per gel was applied. After migration and fixing of proteins in the gel, images were captured using a Typhoon Variable Mode Imager 9400 (GE Healthcare).
2.8.
Protein relative quantification and identification
Images of gels were analyzed using DeCyder v.6.05.11 software (GE Healthcare). The automatic matching was manually confirmed for spots selected for further study and identification. The relative quantification of proteins, based on the normalized volume of spots, allowed the statistical comparison of their abundance. A spot was validated if present in at least 3 gels out of the 4 replicates. The statistical treatment based on the spot volume was performed by the DeCyder software with a level of 5%. We selected ratios above 1.3 or below −1.3 (or 0.76). Spots of interest were picked from the preparative gel and digested by trypsin for 6 h at 37 °C using an Ettan Dalt Spot Handling Workstation (GE Healthcare) before acquisition of MS and MS/MS spectra with a MALDI-TOF-TOF analyser (4800 Applied Biosystems, Foster City, CA, USA). MS–MS analysis was performed using the GPS Explorer Version 3.6 software of Applied Biosystems. During the extraction of data from the spectrum and the creation of peaks lists for submission to database searches a filtering was applied; only peaks with Signal/Noise ratio higher than 10 within the mass range of 800 to 4000 Da were used in peptide mass fingerprint analysis, furthermore excluding known masses from common contaminants such as keratin and trypsin. For MS–MS analysis only peaks with a Signal/Noise ratio higher than 10, and a mass in the range from 60 Da to 20 Da below the precursor mass were used. Furthermore the peak density was limited to 50 peaks per 200 Da. The filter was completed with a mass exclusion list containing the trypsin fragment masses. The NCBI poplar Expressed Sequence Tags (EST) database used for the interrogation contained 419,944 poplar sequences and was downloaded from the NCBI database on 06/11/2009. A Viridiplantae protein database was also used, that was downloaded on 02/17/2009 and contained 1,214,000 sequences. Detailed search criteria were described in Durand et al. [30]. Each spectrum was furthermore manually verified, as was the final protein identification.
2.9.
Statistics
Physiological data were compared with Student t-test; difference between means were considered significant for a p-value inferior to 0.05. A Student t-test based on spot volumes was performed inside the DeCyder in order to determine differentially expressed proteins with a variation factor of at least 1.3 in abundance (up and down) and a p-value of
