Aridity Gradients Shape Intraspecific Variability of Morphological Traits in Native Ceratonia siliqua L. of Morocco.

The carob tree (Ceratonia siliqua L.) is a significant fruit tree in the Mediterranean region with cultural, biological, and ecological importance. Despite its importance, intraspecific trait variability (ITV) in carob trees has been largely overlooked in previous studies. Understanding ITV and its relationship with environmental conditions is crucial for conservation and breeding programs. In this study, we investigated the variability of carob pod and seed-related traits across different ecological scales in 25 studied populations in Morocco. Significant differences in morphological traits were observed between carob populations at various ecological levels, and pod-related traits exhibited greater variability than seed traits. Correlation analysis revealed strong associations between carob morphological traits and environmental conditions, with altitude and aridity index playing an influential role. The aridity gradient was strongly related to changes in pod size, seed number, and size, as well as seed yield. Our findings highlight an important ITV reaching 45% at the intra-population level, 36.5% at the inter-geographic level, and 30% at the inter-population level. Overall, this study contributes valuable insights into the ecology and adaptation of carob trees, emphasizing the importance of considering intraspecific variability when studying this remarkable species. This knowledge is critical for addressing the challenges posed by climate change and human activities on the long-term survival and ecological functioning of carob populations.

Vulnerability of cocoa-based agroforestry systems to climate change in West Africa.

Previous research indicates that some important cocoa cultivated areas in West Africa will become unsuitable for growing cocoa in the next decades. However, it is not clear if this change will be mirrored by the shade tree species that could be used in cocoa-based agroforestry systems (C-AFS). We characterized current and future patterns of habitat suitability for 38 tree species (including cocoa), using a consensus method for species distribution modelling considering for the first time climatic and soil variables. The models projected an increase of up to 6% of the potential suitable area for cocoa by 2060 compared to its current suitable area in West Africa. Furthermore, the suitable area was highly reduced (14.5%) once considering only available land-use not contributing to deforestation. Regarding shade trees, 50% of the 37 shade tree species modelled will experience a decrease in geographic rate extent by 2040 in West Africa, and 60% by 2060. Hotspots of shade tree species richness overlap the current core cocoa production areas in Ghana and Côte d'Ivoire, suggesting a potential mismatch for the outer areas in West Africa. Our results highlight the importance of transforming cocoa-based agroforestry systems by changing shade tree species composition to adapt this production systems for future climate conditions.

Multiomics Molecular Research into the Recalcitrant and Orphan Quercus ilex Tree Species: Why, What for, and How.

The holm oak (Quercus ilex L.) is the dominant tree species of the Mediterranean forest and the Spanish agrosilvopastoral ecosystem, "dehesa." It has been, since the prehistoric period, an important part of the Iberian population from a social, cultural, and religious point of view, providing an ample variety of goods and services, and forming the basis of the economy in rural areas. Currently, there is renewed interest in its use for dietary diversification and sustainable food production. It is part of cultural richness, both economically (tangible) and environmentally (intangible), and must be preserved for future generations. However, a worrisome degradation of the species and associated ecosystems is occurring, observed in an increase in tree decline and mortality, which requires urgent action. Breeding programs based on the selection of elite genotypes by molecular markers is the only plausible biotechnological approach. To this end, the authors' group started, in 2004, a research line aimed at characterizing the molecular biology of Q. ilex. It has been a challenging task due to its biological characteristics (long life cycle, allogamous, high phenotypic variability) and recalcitrant nature. The biology of this species has been characterized following the central dogma of molecular biology using the omics cascade. Molecular responses to biotic and abiotic stresses, as well as seed maturation and germination, are the two main objectives of our research. The contributions of the group to the knowledge of the species at the level of DNA-based markers, genomics, epigenomics, transcriptomics, proteomics, and metabolomics are discussed here. Moreover, data are compared with those reported for Quercus spp. All omics data generated, and the genome of Q. ilex available, will be integrated with morphological and physiological data in the systems biology direction. Thus, we will propose possible molecular markers related to resilient and productive genotypes to be used in reforestation programs. In addition, possible markers related to the nutritional value of acorn and derivate products, as well as bioactive compounds (peptides and phenolics) and allergens, will be suggested. Subsequently, the selected molecular markers will be validated by both genome-wide association and functional genomic analyses.

Effect and Response of Quercus ilex subsp. ballota [Desf.] Samp. Seedlings From Three Contrasting Andalusian Populations to Individual and Combined Phytophthora cinnamomi and Drought Stresses.

Quercus ilex L. is the dominant species in the Mediterranean forest and agrosilvopastoral ecosystem "dehesa." Currently, this forest species is threatened by natural and anthropogenic agents, especially by the decline syndrome, which is caused by Phytophthora cinnamomi and drought periods. Although the morphological and physiological responses of Q. ilex to combined stress (P. cinnamomi and drought) have been examined already, little is known at the molecular level. In this study, we studied the effect and response of 8-month seedlings from three contrasting Andalusian populations (Seville [Se], Granada [Gr], and Almeria [Al]) to the individual and combined stresses of P. cinnamomi and drought from morphological, physiological, biochemical, and proteomics data. Whereas, seedling damage (leaf chlorosis and necrosis) and mortality were greater under the combined stresses in the three populations, the effect of each individual stress was population-dependent. Resilient individuals were found in all the populations at different percentages. The decrease in leaf chlorophyll fluorescence, photosynthetic activity, and stomatal conductance observed in undamaged seedlings was greater in the presence of both stresses, the three populations responding similarly to drought and P. cinnamomi. Biochemical and proteomic analyses of undamaged seedlings from the two most markedly contrasting populations (Se and Al) revealed the absence of significant differences in the contents in photosynthetic pigments, amino acids, and phenolics among treatments. The Se and Al populations exhibited changes in protein profile in response to the different treatments, with 83 variable proteins in the former population and 223 in the latter. Variable proteins belonged to 16 different functional groups, the best represented among which were protein folding, sorting and degradation, carbohydrate, amino acid, and secondary metabolism, photosynthesis, and ROS scavenging. While photosynthetic proteins were mainly downaccumulated, those of stress-responsive were upaccumulated. Although no treatment-specific response was observed in any functional group, differences in abundance were especially marked under the combined stresses. The following variable proteins are proposed as putative markers for resilience in Q. ilex, namely, aldehyde dehydrogenase, glucose-6-phosphate isomerase, 50S ribosomal protein L5, and α-1,4-glucan-protein synthase [UDP-forming].

Forest and woodland replacement patterns following drought-related mortality.

Forest vulnerability to drought is expected to increase under anthropogenic climate change, and drought-induced mortality and community dynamics following drought have major ecological and societal impacts. Here, we show that tree mortality concomitant with drought has led to short-term (mean 5 y, range 1 to 23 y after mortality) vegetation-type conversion in multiple biomes across the world (131 sites). Self-replacement of the dominant tree species was only prevalent in 21% of the examined cases and forests and woodlands shifted to nonwoody vegetation in 10% of them. The ultimate temporal persistence of such changes remains unknown but, given the key role of biological legacies in long-term ecological succession, this emerging picture of postdrought ecological trajectories highlights the potential for major ecosystem reorganization in the coming decades. Community changes were less pronounced under wetter postmortality conditions. Replacement was also influenced by management intensity, and postdrought shrub dominance was higher when pathogens acted as codrivers of tree mortality. Early change in community composition indicates that forests dominated by mesic species generally shifted toward more xeric communities, with replacing tree and shrub species exhibiting drier bioclimatic optima and distribution ranges. However, shifts toward more mesic communities also occurred and multiple pathways of forest replacement were observed for some species. Drought characteristics, species-specific environmental preferences, plant traits, and ecosystem legacies govern postdrought species turnover and subsequent ecological trajectories, with potential far-reaching implications for forest biodiversity and ecosystem services.

Assessment of functional and structural changes of soil fungal and oomycete communities in holm oak declined dehesas through metabarcoding analysis.

Forest decline is nowadays a major challenge for ecosystem sustainability. Dehesas, which consists of savannah-like mediterranean ecosystems, are threatened by the holm oak decline in the south-west of Iberian Peninsula. Phytophthora cinnamomi is considered the main agent of holm oak root rot, but little is known about the relationship between diversity of soilborne microbial community and the decline syndrome of holm oak. It would be hypothesized that the changes in the structure and functionality of the soil microbiome might influence tree health status through changes in richness and diversity of beneficial organisms such as mycorrhizal species, or fungal plant pathogens such as Fusarium spp. or Alternaria spp. Total DNA of soil samples from declined oak dehesas was extracted and analyzed through metabarcoding techniques, to evaluate the specific composition and diversity of the fungal and oomycete communities and their relationship with the disease symptoms. The fungal community included a wide range of pathogens and abundance of ectomycorrhizal key taxa related with low defoliation degree. Phytophthora cinnamomi and Pythium spiculum did not appear among the most abundant oomycetes, nor were they related directly to defoliation levels. Moreover, a particular taxon belonging to the genus Trichoderma was strongly correlated with the scarcity of pathogenic Phytophthora spp. The diversity and composition of fungal and oomycete communities were related to the severity of the decline symptoms. The metabarcoding study of microbiome represents a powerful tool to develop biocontrol strategies for the management of the holm oak root rot.

Characterization of the orthodox Pinus occidentalis seed and pollen proteomes by using complementary gel-based and gel-free approaches.

UNLABELLED: This work presents an analysis of Pinus occidentalis pollen and seed proteomes, in which both gel-based and gel-free approaches have been used. Proteins were extracted from P. occidentalis seeds and pollen by using the TCA/acetone/phenol precipitation protocol, and protein extracts were subjected to 1- and 2-DE coupled to MALDI-TOF-TOF as well as to shotgun (nLC-LTQ-Orbitrap) analysis. All bands (1-DE) and the most abundant spots (2-DE) were excised, trypsin digested and the resulting peptides analyzed by MALDI TOF/TOF. In order to increase the proteome coverage, a gel free approach was used. Proteins were identified from mass spectra by using three different databases, including UniProtKB, NCBI and a Pinus spp. custom database [2]. The gel-based approach resulted in 42 (seeds) and 94 (pollen) protein identifications, while the shotgun approach permitted the identification of 187 (seed) and 960 (pollen) proteins. Proteins were classified based on their corresponding functional categories. In seeds, storage proteins were the most abundant ones, and some allergens and proteases were also identified. In pollen proteins related to general metabolism were the most predominant. Data are compared and discussed from a methodological and biological point of view, taking into account the particularities of the seed and pollen organs. BIOLOGICAL SIGNIFICANCE: In this work we characterized P. occidentalis proteome with seeds and pollen samples implementing two complementary approaches for the analysis. We found a high content of storage protein, stress response and metabolism related proteins in the seed proteome. Similarly, in the pollen proteome we found predominant groups of proteins related to metabolism and stress response.

2-DE proteomics analysis of drought treated seedlings of Quercus ilex supports a root active strategy for metabolic adaptation in response to water shortage.

Holm oak is a dominant tree in the western Mediterranean region. Despite being well adapted to dry hot climate, drought is the main cause of mortality post-transplanting in reforestation programs. An active response to drought is critical for tree establishment and survival. Applying a gel-based proteomic approach, dynamic changes in root proteins of drought treated Quercus ilex subsp. Ballota [Desf.] Samp. seedlings were followed. Water stress was applied on 20 day-old holm oak plantlets by water limitation for a period of 10 and 20 days, each followed by 10 days of recovery. Stress was monitored by changes in water status, plant growth, and electrolyte leakage. Contrary to leaves, holm oak roots responded readily to water shortage at physiological level by growth inhibition, changes in water status and membrane stability. Root proteins were extracted using trichloroacetate/acetone/phenol protocol and separated by two-dimensional electrophoresis. Coomassie colloidal stained gel images were analyzed and spot intensity data subjected to multivariate statistical analysis. Selected consistent spots in three biological replicas, presenting significant changes under stress, were subjected to MALDI-TOF mass spectrometry (peptide mass fingerprinting and MS/MS). For protein identification, combined search was performed with MASCOT search engine over NCBInr Viridiplantae and Uniprot databases. Data are available via ProteomeXchange with identifier PXD002484. Identified proteins were classified into functional groups: metabolism, protein biosynthesis and proteolysis, defense against biotic stress, cellular protection against abiotic stress, intracellular transport. Several enzymes of the carbohydrate metabolism decreased in abundance in roots under drought stress while some related to ATP synthesis and secondary metabolism increased. Results point at active metabolic adjustment and mobilization of the defense system in roots to actively counteract drought stress.

A method to quantify infection and colonization of holm oak (Quercus ilex) roots by Phytophthora cinnamomi.

Phytophthora cinnamomi Rands. is an important root rot pathogen widely distributed in the north hemisphere, with a large host range. Among others diseases, it is known to be a principal factor in the decline of holm oak and cork oak, the most important tree species in the "dehesa" ecosystem of south-western Spain. Previously, the focus of studies on P. cinnamomi and holm oak have been on molecular tools for identification, functional responses of the host, together with other physiological and morphological host variables. However, a microscopic index to describe the degree of infection and colonization in the plant tissues has not yet been developed. A colonization or infection index would be a useful tool for studies that examine differences between individuals subjected to different treatments or to individuals belonging to different breeding accessions, together with their specific responses to the pathogen. This work presents a methodology based on the capture and digital treatment of microscopic images, using simple and accessible software, together with a range of variables that quantify the infection and colonization process.

Proteomic analysis of Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) pollen.

This paper presents an analysis of Holm oak pollen proteome, together with an evaluation of the potentiality that a proteomic approach may have in the provenance variability assessment. Proteins were extracted from pollen of four Holm oak provenances, and they were analyzed by gel-based (1- and 2-DE in combination with MALDI-TOF/TOF) and gel-free (nLC-LTQ Orbitrap MS) approaches. A comparison of 1- and 2-DE protein profiles of the four provenances revealed significant differences, both qualitative and quantitative, in abundance (18 bands and 16 spots, respectively). Multivariate statistical analysis carried out on bands and spots clearly showed distinct associations between provenances, which highlight their geographical origins. A total of 100 spots selected from the 402 spots observed on 2-DE gels were identified by MALDI-TOF/TOF. Moreover, a complementary gel-free shotgun approach was performed by nLC-LTQ Orbitrap MS. The identified proteins were classified according to biological processes, and most proteins in both approaches were related to metabolism and defense/stress processes. The nLC-LTQ Orbitrap MS analysis allowed us the identification of proteins belonging to the cell wall and division, transport and translation categories. Besides providing the first reference map of Holm oak pollen, our results confirm previous studies based on morphological observations and acorn proteomic analysis. Moreover, our data support the valuable use of proteomic techniques as phylogenetic tool in plant studies.

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.) through acorn protein profile analysis.

Studies of variability in Holm oak (Quercus ilex subsp. ballota [Desf.] Samp.), the dominant tree species in the typical Mediterranean forest, have been carried out by using electrophoresis-based proteomic analysis of acorns. Ten populations distributed throughout the Andalusia region have been surveyed. Acorns were sampled from individual trees and proteins extracted from seed flour by using the TCA-acetone precipitation protocol. Extracts were subjected to SDS-PAGE and 2-DE for protein separation, gel images captured, spot or bands quantified, and subjected to statistical analysis (ANOVA, SOM and clustering). Variable bands or spots among populations were subjected to MALDI-TOF/TOF and LC-MS/MS for identification. The protein yield of the used protocol varied among populations, and it was in the 2.92-5.92 mg/g dry weight range. A total of 23 bands were resolved by SDS-PAGE in the 3-35 kDa Mr range, with 8 and 12, out of the total, showing respectively qualitative and quantitative statistically significant differences among populations. Data allowed grouping populations, with groups being correlated according to geographical location and climate conditions, to northern and southern, as well as the discrimination of both mesic and xeric groups. Acorn flour extracts from the most distant populations were analyzed by 2-DE, and 56 differential spots were proposed as markers of variability. Identified proteins were classified into two principal categories; storage and stress/defense protein. Besides providing the first reference map of mature acorn seeds, the use of SDS-PAGE and proteomics in characterizing natural biodiversity in forest trees will be discussed.

Clones identification of Sequoia sempervirens (D. Don) Endl. in Chile by using PCR-RAPDs technique.

A protocol of polymerase chain reaction-random amplified polymorphic DNAs (PCR-RAPDs) was established to analyse the gene diversity and genotype identification for clones of Sequoia sempervirens (D. Don) Endl. in Chile. Ten (out of 34) clones from introduction trial located in Voipir-Villarrica, Chile, were studied. The PCR-RAPDs technique and a modified hexadecyltrimethylammonium bromide (CTAB) protocol were used for genomic DNA extraction. The PCR tests were carried out employing 10-mer random primers. The amplification products were detected by electrophoresis in agarose gels. Forty nine polymorphic bands were obtained with the selected primers (BG04, BF07, BF12, BF13, and BF14) and were ordered according to their molecular size. The genetic similarity between samples was calculated by the Jaccard index and a dendrogram was constructed using a cluster analysis of unweighted pair group method using arithmetic averages (UPGMA). Of the primers tested, 5 (out of 60) RAPD primers were selected for their reproducibility and high polymorphism. A total of 49 polymorphic RAPD bands were detected out of 252 bands. The genetic similarity analysis demonstrates an extensive genetic variability between the tested clones and the dendrogram depicts the genetic relationships among the clones, suggesting a geographic relationship. The results indicate that the RAPD markers permitted the identification of the assayed clones, although they are derived from the same geographic origin.

Postfire regeneration in Pinus pinea L. and Pinus pinaster aiton in Andalucia (Spain).

The objective of this study was to examine postfire regeneration of tree, shrub, and dwarf shrub species, in relation to levels of damage in four planted pine forests (Pinus pinea, Pinus pinaster) in Andalusia. A prefire vegetation map was used for detailing species composition, vertical structure, and density and another for detailing the extent and intensity of fire damage. Between 3 and 7 years after the fires, an inventory was made of the vegetation in each area, using the step-point method. The information thus obtained was used to determine the amount of cover in the dwarf/shrub and tree layers, the frequency of species in each of the layers, floristic richness, and diversity (Shannon index). The botanical composition of the dwarf and shrub layer was analyzed using TWINSPAN. Variables were poorly correlated with level of fire damage, which suggests that the forests in this study followed the autosuccession model. Because of the artificial origin or seminatural condition, regeneration of the dominant tree species is poor, and it seems unlikely that forests will recover to their prefire state. Therefore action is recommended to restore these ecosystems.