Selection of tree species for forests under climate change: is PSI functioning a better predictor for net photosynthesis and growth than PSII?

A chlorophyll fluorescence (ChlF) assessment was carried out on oak seedlings (Quercus ilex L., Quercus pubescens Willd., Quercus frainetto Ten.) of Italian and Greek provenance, during the years 2017 and 2018, in a common garden in central Italy planted in 2017. This trial aimed to test the relative performances of the oak species in the perspective of assisted migration as part of the actions for the adaptation of forests to climate change. The assessment of the photosynthetic performance of the tree species included the analysis of the prompt chlorophyll fluorescence (PF) transient and the modulated reflection (MR) at 820 nm, leaf chlorophyll content, leaf gas exchange (net photosynthesis, stomatal conductance), plant growth (i.e., height) and mortality rate after 2 years from the beginning of the experiment. The assessment of the performance of the three oak species was carried out 'in vivo'. Plants were generated from seeds and exposed to several environmental factors, including changing seasonal temperature, water availability, and soil biological and physical functionality. The results of PF indicate a stable functionality of the photosynthetic system PSII (expressed as FV/FM) across species and provenances and a decline in photochemistry functionality at the I-P phase (ΔVIP) in Q. frainetto, thus indicating a decline of the content of PSI in this species. This result was confirmed by the findings of MR analysis, with the speed of reduction and subsequent oxidation of PSI (VRED and VOX) strongly correlated to the amplitude of ΔVIP. The photosynthetic rates (net photosynthesis, PN) and growth were correlated with the parameters associated with PSI content and function, rather than those related to PSII. The low performance of Q. frainetto in the common garden seems to be related to early foliar senescence with the depletion of nitrogen, due to suboptimal climatic and edaphic conditions. Chlorophyll fluorescence allowed discrimination of populations of oak species and individuation of the less (or/and best) suitable species for future forest ecology and management purposes.

Elucidating Drought Stress Tolerance in European Oaks Through Cross-Species Transcriptomics.

The impact of climate change that comes with a dramatic increase of long periods of extreme summer drought associated with heat is a fundamental challenge for European forests. As a result, forests are expected to shift their distribution patterns toward north-east, which may lead to a dramatic loss in value of European forest land. Consequently, unraveling key processes that underlie drought stress tolerance is not only of great scientific but also of utmost economic importance for forests to withstand future heat and drought wave scenarios. To reveal drought stress-related molecular patterns we applied cross-species comparative transcriptomics of three major European oak species: the less tolerant deciduous pedunculate oak (Quercus robur), the deciduous but quite tolerant pubescent oak (Q. pubescens), and the very tolerant evergreen holm oak (Q. ilex). We found 415, 79, and 222 differentially expressed genes during drought stress in Q. robur, Q. pubescens, and Q. ilex, respectively, indicating species-specific response mechanisms. Further, by comparative orthologous gene family analysis, 517 orthologous genes could be characterized that may play an important role in drought stress adaptation on the genus level. New regulatory candidate pathways and genes in the context of drought stress response were identified, highlighting the importance of the antioxidant capacity, the mitochondrial respiration machinery, the lignification of the water transport system, and the suppression of drought-induced senescence - providing a valuable knowledge base that could be integrated in breeding programs in the face of climate change.

Prominent alterations of wild barley leaf transcriptome in response to individual and combined drought acclimation and heat shock conditions.

Under field conditions, drought and heat stress typically happen simultaneously and their negative impact on the agricultural production is expected to increase worldwide under the climate change scenario. In this study, we performed RNA-sequencing analysis on leaves of wild barley (Hordeum spontaneum) originated from the northern coastal region of Egypt following individual drought acclimation (DA) and heat shock (HS) treatments and their combination (CS, combined stresses) to distinguish the unique and shared differentially expressed genes (DEG). Results indicated that the number of unique genes that were differentially expressed following HS treatment exceeded the number of those expressed following DA. In addition, the number of genes that were uniquely differentially expressed in response to CS treatment exceeded the number of those of shared responses to individual DA and HS treatments. These results indicate a better adaptation of the Mediterranean wild barley to drought conditions when compared with heat stress. It also manifests that the wild barley response to CS tends to be unique rather than common. Annotation of DEG showed that metabolic processes were the most influenced biological function in response to the applied stresses.

Taxonomic and ecological relevance of the chlorophyll a fluorescence signature of tree species in mixed European forests.

The variability of chlorophyll a fluorescence (ChlF) parameters of forest tree species was investigated in 209 stands belonging to six European forests, from Mediterranean to boreal regions. The modifying role of environmental factors, forest structure and tree diversity (species richness and composition) on ChlF signature was analysed. At the European level, conifers showed higher potential performance than broadleaf species. Forests in central Europe performed better than those in Mediterranean and boreal regions. At the site level, homogeneous clusters of tree species were identified by means of a principal component analysis (PCA) of ChlF parameters. The discrimination of the clusters of species was influenced by their taxonomic position and ecological characteristics. The species richness influenced the tree ChlF properties in different ways depending on tree species and site. Tree species and site also affected the relationships between ChlF parameters and other plant functional traits (specific leaf area, leaf nitrogen content, light-saturated photosynthesis, wood density, leaf carbon isotope composition). The assessment of the photosynthetic properties of tree species, by means of ChlF parameters, in relation to their functional traits, is a relevant issue for studies in forest ecology. The connections of data from field surveys with remotely assessed parameters must be carefully explored.

Physiological mechanisms to cope with Cr(VI) toxicity in lettuce: can lettuce be used in Cr phytoremediation?

This research aims at identifying the main deleterious effects of Cr(VI) on the photosynthetic apparatus and at selecting the most sensitive endpoints related to photosynthesis. To achieve this goal, we used lettuce (Lactuca sativa), a sensible ecotoxicological crop model. Three-week-old plants were exposed to 0, 50, 150 and 200 mg L(-1) of Cr(VI). These concentrations ranged from levels admitted in irrigation waters to values found in several Cr industry effluents and heavily contaminated environments. After 30 days of exposure, plants accumulated Cr preferably in roots and showed nutritional impairment, with decreases of K, Mg, Fe and Zn in both roots and leaves. Cr(VI)-exposed plants showed decreased levels of chlorophyll (Chl) a and anthocyanins, as well as decreased effective quantum yield of photostystem II (ΦPSII) and photochemical Chl fluorescence quenching (qp), but increases in the non-photochemical Chl fluorescence quenching (NPQ) and in the de-epoxidation state (DEP) of the xanthophyll cycle. Net CO2 assimilation rate (P N ) and RuBisCO activity were mostly impaired in the highest Cr(VI) concentration tested. Concerning the final products of photosynthesis, starch content was not affected, while soluble sugar contents increased. These alterations were accompanied by a reduction in protein content and in plant growth. Our results support that endpoints related to the photosynthesis photochemical processes (ΦPSII and the qp) and the content of anthocyanins are sensitive predictors of Cr(VI) toxicity. The advantages of using these parameters as biomarkers for Cr toxicity in plants are discussed. Finally, we report that, despite showing physiological disorders, L. sativa plants survived and accumulated high doses of Cr, and their use in environmental/decontamination studies is open to debate.

Imaging of fast chlorophyll fluorescence induction curve (OJIP) parameters, applied in a screening study with wild barley (Hordeum spontaneum) genotypes under heat stress.

We quantified the influence of heat stress (HS) on PSII by imaging of parameters of the fast chlorophyll fluorescence (CF) induction (OJIP) kinetic of 20 genotypes of wild barley (Hordeum spontaneum) covering a broad geographical spectrum. We developed a standardised screening procedure, allowing a repetitive fluorescence measurement of leaf segments. The impact of HS was quantified by calculating a Heat Resistance Index (HRI), derived from the decrease of the Performance Index (PI) caused by HS treatment and following recovery. For the genotype showing the lowest HRI, reduced maximum quantum yield (φP0) and increased relative variable fluorescence of the O-J phase (K-Peak) were detected after HS, whereas the basal fluorescence (F0) remained stable. An additional feature was a lowered fraction of active (QA-reducing) reaction centres (RCs). The disturbances disappeared after one day of recovery. Spatial heterogeneities of fluorescence parameters were detected, as the negative effect of HS was stronger in the leaf areas close to the leaf tip. The results of this study prove that chlorophyll fluorescence imaging (CFI) is suitable for the detection of HS symptoms and that imaging of JIP-Test parameters should be considered in future screening and phenotyping studies aiming for the characterisation of plant genotypes.

Characterization of common and distinctive adjustments of wild barley leaf proteome under drought acclimation, heat stress and their combination.

In nature, plants are often exposed to combinations of different stresses at the same time, while in many laboratory studies of molecular stress induction phenomena, single stress responses are analyzed. This study aims to identify the common (i.e. more general stress-responsive) and the stress-specific adjustments of the leaf proteome of wild barley to two often co-occurring stress phenomena, i.e. in response to (long-term) drought acclimation (DA) or to (transient) heat stress (HS). In addition, we analyzed those alterations which are specific for the combination of both stresses. Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry with a 1.5 threshold value of changes in relative protein contents. DA resulted in specific upregulation of proteins with cell detoxification functions, water homeostasis maintenance, amino acids synthesis and lipid metabolism and distinct forms of heat shock proteins (HSPs) and proteins with chaperon functions while proteins related to nitrogen metabolism were downregulated. This response was distinguished from the response to transient HS, which included upregulation of a broad range of HSP products. The common response to both stressors revealed upregulation of additional forms of HSPs and the downregulation of enzymes of the photosynthetic apparatus and chlorophyll binding proteins. The simultaneous exposure to both stress conditions resulted mostly in a combination of both stress responses and to unique abundance changes of proteins with yet unclear functions.

Comparative Analysis of Sorghum bicolor Proteome in Response to Drought Stress and following Recovery.

The adaptive response of Sorghum bicolor landraces from Egypt to drought stress and following recovery was analyzed using two-dimensional difference gel electrophoresis, 2D-DIGE. Physiological measurements and proteome alterations of accession number 11434, drought tolerant, and accession number 11431, drought sensitive, were compared to their relative control values after drought stress and following recovery. Differentially expressed proteins were analysed by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry, MALDI-TOF-MS. Alterations in protein contents related to the energy balance, metabolism (sensu Mewes et al. 1997), and chaperons were the most apparent features to elucidate the differences between the drought tolerant and sensitive accessions. Further alterations in the levels of proteins related to transcription and protein synthesis are discussed.

Phylogenetic relationship and molecular taxonomy of African grasses of the genus Panicum inferred from four chloroplast DNA-barcodes and nuclear gene sequences.

The genus Panicum s.l. comprises about 450 grass species in which the C4 and the C3 metabolic pathways of photosynthesis are realized. In the West African savannah, Panicum spp. and closely related taxa dominate the landscape, with species differentially adapted to drought conditions. We obtained four chloroplast DNA barcode sequences, rbcL, matK, ndhF and trnH-psbA intergenic region, for nine Panicum spp. with a focus on West African species, and we performed maximum likelihood analysis to infer their phylogenetic relationship. Furthermore the phylogenetic placement of five newly sequenced taxa was achieved using a published phylogeny of more than 300 Panicoids based on ndhF sequences. The comparison of the resulting phylogenetic tree constructed from a combination of all four barcode sequences with the one based on rbcL and matK showed that the latter combination of the two, is sufficient for the analysis. A tree constructed from amino acid sequences derived from isolated cDNAs of the nucleus-encoded phosphoenolpyruvate carboxylase displayed a similar topology. All ppc-sequences could be annotated to either ppc-B2 or ppc-aR. Moreover the inclusion of the West African Panicum species in an extensive dataset of Panicoids supports the proposition that within the subtribe Panicinae only the NAD-malic enzyme type of C4 photosynthesis is present.

Comparative analysis of barley leaf proteome as affected by drought stress.

The adaptive response of Egyptian barley land races to drought stress was analyzed using difference gel electrophoresis (DIGE). Physiological measurements and proteome alterations of accession number 15141, drought tolerant, and accession number 15163, drought sensitive, were compared. Differentially expressed proteins were subjected to MALDI-TOF-MS analysis. Alterations in proteins related to the energy balance and chaperons were the most characteristic features to explain the differences between the drought-tolerant and the drought-sensitive accessions. Further alterations in the levels of proteins involved in metabolism, transcription and protein synthesis are also indicated.

Photosynthetic responses of a C(3) and three C(4) species of the genus Panicum (s.l.) with different metabolic subtypes to drought stress.

Young plants of Panicum bisulcatum (C(3)), Zuloagaea bulbosa [NADP-malic enzyme (ME)-C(4)], P. miliaceum (NAD-ME-C(4)) and Urochloa maxima [phosphoenolpyruvate carboxykinase (PCK)-C(4)] were subjected to drought stress (DS) in soil for 6 days. The C(3) species showed severe wilting symptoms at higher soil water potential (-1.1 MPa) and relative leaf water content (77 %) than in the case of the C(4) species (-1.5 to -1.7 MPa; 58-64 %). DS decreased photosynthesis, both under atmospheric and under saturating CO(2). Stomatal limitation of net photosynthesis (P(N)) in the C(3), but not in the C(4) species was indicated by P(N)/C(o) curves. Chlorophyll fluorescence of photosystem II, resulting from different cell types in the four species, indicated NADPH accumulation and non-stomatal limitation of photosynthesis in all four species, even under high CO(2). In the NAD-ME-C(4) and the PCK-C(4) species, DS plants showed increased violaxanthin de-epoxidase rates. Biochemical analyses of carboxylating enzymes and in vitro enzyme activities of the C(4) enzymes identified the most likely non-stomatal limiting steps of photosynthesis. In P. bisulcatum, declining RubisCO content and activity would explain the findings. In Z. bulbosa, all photosynthesis enzymes declined significantly; photosynthesis is probably limited by the turnover rate of the PEPC reaction. In P. miliaceum, all enzyme levels remained fairly constant under DS, but photosynthesis can be limited by feedback inhibition of the Calvin cycle, resulting in asp inhibition of PEPC. In U. maxima, declines of in vivo PEPC activity and feedback inhibition of the Calvin cycle are the main candidates for non-stomatal limitation of photosynthesis under DS.

A competent extraction method of plant proteins for 2-D gel electrophoresis.

The efficient extraction of high-quality proteins is a key factor for a successful proteomic analysis approach. In the method suggested here, absolute ethanol containing 10 mM DTT was used to precipitate the proteins in plant tissue homogenates followed by their resuspension in a urea-/thiourea- and NP-40-containing solution. Protein profiles were examined on pH 3-11 non-linear IEF strips and SDS-PAGE and compared with extracts using the established method of acetone-10% TCA/0.07% 2-mercaptoethanol precipitation (V. Méchin et al., Methods Mol. Biol. 2006, 355, 1-8). In addition to protein profile similarity for the two extracts, the acidic part of the acetone containing 10% TCA/0.07% 2-mercaptoethanol extraction showed protein spots with high molecular weight in the range of 250-150 kDa, while the ethanol containing 10 mM DTT extracts indicated extra proteins spots at the basic part of the gels with molecular weights in the range of 25-15 kDa. The MALDI-TOF-MS of differential spots from acetone containing 10% TCA/0.07% 2-mercaptoethanol precipitation method and absolute ethanol containing 10mM DTT indicated no similarity, ruling out the possibility that the two clusters shown represent identical proteins. The described method is easy in implementation, chemicals used are less toxic and proteins are easier to resuspend therefore presents an additional choice to implement towards finding the optimum method for extraction.