Molecular ecophysiology as tool for the selection of highly stress resistant poplar species for multipurpose forests

We have developed a protocol allowing examining the proteome of poplar trees when submitted to different abiotic factors. This set of protocols allows to collect, to extract, to analyse and moreover to compare the proteomes of different series of plants submitted to abiotic stress. The originality of this methodology is that it allows a time- and dose-based comparison of the effects of the stress on the proteome of poplar leaves. Moreover, the methodology allows not only a qualitative comparison, but thanks to the use of an internal standard procedure, it allows a quantitative comparison of the changes observed real biological modifications.

Populus euphratica is a difficult to root species and therefore, difficult to propagate by cuttings. Seeds of P. euphratica are only viable for a short time. Micropropagation offers the opportunity to produce plants without limitation. We achieved the micropropagation via induction of adventitious shoots on calli of leaf explants and developed a protocol for efficient and fast rooting of the shoots including transfer of the plantlets to the greenhouse. The micropropagation protocol allows producing high numbers of vigorous plants that can be used for plantations or research.

We have developed a set of markers using endogenous levels measurements of compatible solutes. The use of these markers to assess salinity and drought tolerance in Poplar plants allowed characterizing the physiological status of the plants during the stress exposure. Combined with the other physiological and molecular markers, this set of markers was proved to be a reliable test to evaluate the stress level in trees. These markers were also proven to be helpful understanding the abiotic stress response mechanisms in trees.

We have used the normal procedures of evaluating root growth potential in controlled conditions to measure different plant variables and alometric relations, establishing a standard protocol for the studied species (P. alba, P. euphratica, Q. suber and E. globulus). The variables measured were correlated to the field performance of these species for determining the most relevant plant parameters of acclimation to drought. Results found can be useful in tree nursery production for improving seedling quality through the use of reliable predictors of field performance.

We have developed an optimal procedure for watering Populus alba in semi-arid regions. Quantities and distribution of water during the summer were optimised for survival and growth of a fast growing and drought sensitive species in field conditions. Plants were exposed to contrasting conditions of water availability (high and low water stress) through a drip irrigation system. Results found can be useful in the context of the protection, conservation and restoration of the forest ecosystems in degraded areas.

Actively growing twigs and branches of P. euphratica were sampled from selected and marked adult trees from 3 different locations in the Ein Avdat forest, according to their distance from the water stream and uphill (sites A, B and C), and thus at different degrees of salt/drought stress. This material served for in vitro propagation (cloning) for distribution to additional partners, for use in controlled salinity/drought experiments, as well as for collecting plant material for expression profiling by micro-arrays. In addition, leaves were collected from adult trees for micro-arrays, as well as for protein, ion and other physiological analyses. Callus was first initiated from one-node stems explants, subcultured, and then adventitious buds regenerated. Plantlets were established, acclimatized ex vitro, and distributed to some of the partners to be used for growth, physiological and molecular studies. Leaves and other plant parts, from both the in vitro cloned plants and from adult trees in the different field sites, were analyzed fresh (growth and development, several physiological parameters), or frozen in liquid nitrogen, then kept at -80 or lyophilized and used for protein analysis and micro-array studies. Thus, the protocol for micropropagation of P. euphratica plantlets is now well established, and an unlimited amount of clonal material can be supplied to interested researchers, to be used for assessing salt/droght tolerance and for additional expression profiling by micro-arrays. Likewise, leaves from adult trees in situ can be supplied at limited amounts.

We have tested microsatellite loci, already developed on Populus trichocarpa, Populus nigra and Populus tremuloides, and developed RAPD markers to characterize Populus alba ecotypes with different attitude in salt stress tolerance. Using 79 microsatellite loci the segregation in F1 population was detected between the progeny of tolerant and untolerant ecotypes. 54 RAPD markers were able to distinguish tolerant and untolerant ecotypes and their progeny and some of them have been developed as SCAR markers. The results obtained were 78 microsatellite primer sequences and 30 RAPD primer sequences, and the latter markers 3 potential SCAR markers have been also developed. Moreover, genomic sequences and information on degree of polymorphism in P. alba have also be obtained. Actually both type of markers are using to identify Quantitative Traits Loci related to canopy development and salt tolerance in P. alba and P. euphratica.

Expression of SP1 related proteins in P. euphratica plants growing in hydroponics (lab exp.) and under greenhouse (pots) salt stress conditions was recorded. Six EST sequences homologous to the P. tremula SP1 protein were identified in the P. euphratica EST database POPEST. All the ESTs have exactly the same ORF showing 70.5% similarity and 61% identity to P. tremula SP1. SP1 proteins were extracted from leaves of P. euphratica plants and sequenced. The sequence shows 100 % homology to P. tremula SP1. Internal sequencing of SP1-related protein from leaves of P. euphratica plants growing under greenhouse conditions showed 100 % homology to P. tremula SP1. Significant increment in SP1 levels was not observed upon salt stress in leaves, shoots or roots. Polyclonal anti-SP1 antibodies (P. tremula) were affinity purified against the P. euphratica SP protein. Western blot analysis revealed the specificity of the affinity purified antibody and are being used for sub-cellular in situ localization (immuno-gold labelling) of SP1 proteins in plant tissue exposed to salt stress. Preliminary results indicate specific accumulation of P. euphratica SP1 in cell membranes in response to salt stress. sp1 transcript was down-regulated under stress conditions in both P. tremula and P. euphratica, but was more pronounce at increasing salt concentrations in P. euphratica. Hybridization of RNA extracted from salt experiments (hydroponics) was carried out on micro-arrays slides containing 350 stress related genes previously selected by all the participants. Among the analyzed genes, 3% were up-regulated on P. tremula plants in 150mM NaCl while no differences were observed in P. euphratica plants. On the other hand, 6% were severely down-regulated in P. tremula when plants grown in 150mM NaCl. Further experiments will be done as biological and technical repeats.

We have identified that highly salt tolerant Populus euphratica growing in extremely saline soils are highly arbuscular mycorrhizal. We have partially identified the species of mycorrhizas. Cultivation of these mycorrhizas would allow them to be used in improving growth of crops in saline conditions. Salinity is a major problem in semi-arid regions. In most cases the state of the art treatment requires hard engineering, mycorrhizas would offer a soft engineering option. Us of mycorrhizas from high saline environments would greatly improve the state of the art. A weaknesses is that the persistence of introduced mycorhizas is unknown.

On the basis of climatic conditions of native areas we selected three Populus alba genotypes from an ex situ Italian germplasm collection: 6K3 (North Italy, 400m above sea level, no salt and drought stress conditions), 14P11 (South Italy, about 20m from the coastline, drought stress with the probable occurrence of saline water intrusion) and 2AS11 (inland area of South Italy, 250m above sea level, drought intermediate conditions respect to the others two genotypes, no salt stress conditions). We set up a reproducible micropropagation protocol from node explants. MS medium added with 30g/l of sucrose and 7g/l of agar was used for multiplication, while for rooting macro and microelements were reduced to ½ MS with the exception of Na2EDTA and FeSO4-7H2O. Sucrose concentration was 10 g/l. At the beginning BAP concentration ranges from 0.1 (6K3) to 0.5mg/l (2AS11 and 14P11). Roots induction was obtained with an immersion in liquid IBA solution for 24 hours; rooting percentage was high (around100%). Acclimation period (1 month) were carried out in controlled ambient.Conserving Populus alba genetic resources to screen germplasm pool for selection and breeding strategy. Micropropagation protocol could help the exchange of genotypes between different research laboratories.

The degree of drought tolerance of the poplar Populus euphratica was tested by imposing a controlled water shortage to micro-propagated rooted cuttings grown in pots. Two main lines of evidence showed that this species, despite its wide distribution in arid regions of Central Asia, displays only a very low drought tolerance: - Its xylem, whether in leaves, in roots and in stems, is very prone to cavitation and embolism. Cavitation n induced losses of conductivity occurred already at still high levels of leaf water potential (above 1 MPa), which makes this species one of the most vulnerable species among all those tested until now. Among the three poplar species tested, it was the most vulnerable one. This high vulnerability resulted in the induction of significant losses of conductivity during drought stress in situ, which is a seldom occurrence among forest trees. - During water shortage, we observed very rapidly (at still high levels of water potential) growth cessation, and stomatal closure. Nevertheless, despite these rapid and severe responses, all tested plants recovered from stress after rewatering. This species therefore may be suited for afforestation under semi arid or even arid conditions, only in the case of the presence of a water table at reasonable depth. The ability of the P euphratica trees to reach such tables and to efficiently consume the available water, is the main explanation for its survival under such conditions. Anyway, afforestation with this species is probably a difficult task that may require heavy irrigation during the first years until the roots reach the water table. Aged populations of this species probably only little use sexual reproduction (seedlings may suffer drought and die) but rely on vegetative propagation based on root suckers that may develop and rely on the water supplied by roots from larger and adjacent trees. This information may be crucial for the success of afforestation program with this species.

In the search for salt tolerant genotypes it is important to apply a fast and efficient screening system to test plant performance under salt stress. We developed a salt stress protocol that efficiently tests a high number of plants within a short time frame for salt resistance. Plants are grown in hydroponics, which allows to easily applying salt to the nutrient solution. In contrast to stress treatments on soil, hydroponics allow keeping the concentration of salt in the nutrient solution constant at all phases of the experiment. The performance of plants under salt stress was tested by measuring chlorophyll fluorescence and water potential.

We have generated a collection of 13000 P. euphratica EST clones in pSPORT and pGEMT vectors available as glycerol or plasmid stocks. Part of the collection has been re-sequenced to verify the identity of each clone. The EST clones can be used as probes for hybridization, identification of cDNAs and gene discovery; and construction of macro- and microarrays.

The following methodology to determine the leaf epidermal cell adaptation to the increasing salt stress levels was set up. At the beginning, before starting with salt stress experiment and at every change of salt concentration we marked last leaf in order to analyse only leaves formed in the following tested salt level. We observed the distension process with daily measurement of lamina length and decided that it could be completed when for five time we found the same value. So, we picked up these leaves and at once we took away with scotch-tape the hair layer that cover the lower surface sprayed with a commercial removable glue; then, we cover the central middle part of the lower surface with a transparent nail-polish in order to obtain after drying an imprint of it. Using an image analyse software combined with a light microscope we measured the area surface of stomata and epidermal cells and their density in order to calculate stomata index parameter. For all the tested genotype we found a good correlation with increasing salt stress levels and the reduction of stomata area; so this parameter could be considered a good morphological marker related to salt stress condition in Populus alba species.

We have sequenced 19584 ESTs from 17 different control and stress enriched P. euphratica libraries which after sequence curation yielded 13553 ESTs. The sequence information has been used to identify potential genes involved in salt stress tolerance of P. euphratica. The sequence information has also been collated together with all availabe Popoulus ESTs and used for annotation and gene finding in the sequenced genome of Populus trichocarpa.

We have PCR-amplified a P. euphratica uni-gene set consisting of 7800 ESTs and spotted it together with controls onto microarrays. The microarrays have been used to identify genes regulated by various abiotic stress treatments (salt, drought and ozone). A new version of the microarray will be printed at the Finnish national microarray facility in Turku: (http://www.btk.utu.fi/Research_Groups/Finnish_DNA_Microarray_Centre/finnish_dna_microarray_centre.html)These array will be available to buy for academic researchers across Europe.

Estimating the degree of cavitation and embolism in small tree organs like secondary roots, small twigs or petioles, is not possible wit conventional methods, based on hydraulic conductivity measurements. A new technique was therefore developed, using following steps: - Pressurize the given organ in a pressure chamber under the target pressure; - Rapidly freeze the sample in liquid nitrogen; - Observe and count the number of empty vessels in cross sections of these organs with a variable pressure electron microscope. This procedure has proven very efficient and has been calibrated against traditional technique, yielding a perfect calibration. It was already successfully applied to study the vulnerability to cavitation in a range of organs of several poplar species.

Abiotci stress causes increased production of activated oxygen species able to inactivate enzymes and damage cellular components. H2O2 functions in a concentration-dependent manner - as a signal at low concentrations and phytotoxically at higher. MDA an end product of lipid peroxidation is used extensively as indicator for membrane injuries and production of free radicals.Short-term salt stress in vitro led to decrease of H2O2 in tolerant P. euphratica and P. alba 2AS poplars. Longer stress resulted in H2O2 increase in sensitive P. tremula x tremuloides, P. alba x tremula and P. alba 6K3. H2O2 levels continued to be significantly lower in tolerant clones.Sensitive poplars showed increased MDA levels while the tolerant were with significantly reduced MDA. Under salt stress in pots similar patterns of H2O2 were found in poplars on normal and salty soils. P. euphratica showed particularly low levels of H2O2. Salt sensitive genotypes were with significantly higher MDA levels during stress. No differences between control and stressed tolerant plants were found. In our experiments, the salt tolerance of P. euphratica and P. alba 2AS appears to be related to maintenance of H2O2 and MDA levels similar or lower to those under normal conditions.

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