María-Dolores Rey

The carob tree (Ceratonia siliqua L.) is a drought-tolerant species native to the Mediterranean Basin. It has been cultivated for centuries for its highly nutritious, edible pods. Genetic diversity is one of the key requirements for the effective management and utilization of plant genetic resources. In this study, we evaluated the genetic diversity and population structure of 169 seminatural carob individuals from Lebanon, Spain, and Morocco using nine expressed sequence tag-simple sequence repeat (EST-SSR) markers. A total of eight polymorphic EST-SSR loci produced 43 alleles, with Cesi_187 and Cesi_1187 identified as the most informative markers. Results of AMOVA and SAMOVA revealed that most (>80%) of the genetic variation occurred within populations, while less than 20% was attributed to variation among populations. STRUCTURE analysis indicated three genetic clusters corresponding to the sampled countries; however, some individuals from Morocco and Spain exhibited overlapping genetic structures. The PCoA and discriminant analysis of principal components complemented the STRUCTURE results and provided further insight into the genetic differentiation among countries. Our findings may improve the effectiveness of conservation and management strategies and promote the utilization of these carob genetic resources in breeding and reforestation programs.

Subtilisin-like proteases (SBTs) are a large family of serine peptidases involved in diverse biological processses, including plant stress responses, but remain poorly characterized in forest trees. In this study, we report the genome-wide identification and characterization of the SBT gene family in Quercus ilex (QiSBT), with a focus on their transcriptional organization and response to Phytophthora cinnamomi infection. A total of 87 QiSBT genes were identified, structurally annotated, and phylogenetically classified into seven subfamilies based on comparative analyses with Arabidopsis thaliana, Populus trichocarpa, and three other Quercus species (Q. lobata, Q. robur, and Q. variabilis). Analyses of conserved domains and gene structures revealed a high degree evolutionary conservation, with one subfamily enriched in intronless or intron-poor genes. Promoter analysis identified numerous stress- and hormone-responsive cis-elements, suggesting potential transcriptional regulation under environmental stress conditions. Expression profiling under P. cinnamomi infection revealed differential transcriptional responses within the QiSBT gene family. In particular, QiSBT24, QiSBT63, and QiSBT69 showed significant induction in inoculated seedlings, with approximate 3-, 8-, and 30-fold, respectively. These results provide new insights into the structure, evolution, and potential defense-related roles of SBTs in Q. ilex, offering a foundation for future functional aimed at understanding their contribution to resistance mechanisms in Mediterranean oak species.

Renewed interest in acorns from Mediterranean Quercus species is driven by their nutritional and nutraceutical potential as sustainable plant-based foods. It evaluated the phytochemical composition of eight Mediterranean species and assessed interspecific differences using integrated approaches including morphometrics, near-infrared spectroscopy, profiling of sugars, amino acids and minerals, and UHPLC–MS/MS metabolomics. Morphometric traits discriminated species and correlated positively with protein, amino acids and unsaturated fatty acids, and negatively with fiber and sugars. Principal component analysis separated acorns into two clusters: Cluster 1 (Q. suber, Q. faginea, Q. robur, Q. petraea, Q. pubescens) was enriched in amino acids, sugars, starch, protein and sodium, whereas Cluster 2 (Q. ilex subsp. ballota, Q. coccifera, Q. ilex subsp. ilex) showed higher oleic acid, fiber and energy content. This pattern reflects evolutionary relationships within Quercus. Metabolite profiling revealed conserved qualitative composition but strong quantitative differences among species, supporting the valorization of acorn for functional food applications.

DNA methylation is a critical factor influencing plant growth, adaptability, and phenotypic plasticity. While extensively studied in model and crop species, it remains relatively unexplored in holm oak and other non-domesticated forest trees. This study conducts a comprehensive in-silico mining of DNA methyltransferase and demethylase genes within the holm oak genome to enhance our understanding of this essential process in these understudied species. The expression levels of these genes in adult and seedling leaves, as well as embryos, were analysed using quantitative real-time PCR (qRT-PCR). Global DNA methylation patterns were assessed through methylation-sensitive amplified polymorphism (MSAP) techniques. Furthermore, specific methylated genomic sequences were identified via MSAP sequencing (MSAP-Seq). A total of 13 DNA methyltransferase and three demethylase genes were revealed in the holm oak genome. Expression levels of these genes varied significantly between organs and developmental stages. MSAP analyses revealed a predominance of epigenetic over genetic variation among organs and developmental stages, with significantly higher global DNA methylation levels observed in adult leaves. Embryos exhibited frequent demethylation events, while de novo methylation was prevalent in seedling leaves. Approximately 35% of the genomic sequences identified by MSAP-Seq were methylated, predominantly affecting nuclear genes and intergenic regions, as opposed to repetitive sequences and chloroplast genes. Methylation was found to be more pronounced in the exonic regions of nuclear genes compared to their promoter and intronic regions. The methylated genes were predominantly associated with crucial biological processes such as photosynthesis, ATP synthesis-coupled electron transport, and defence response. This study opens a new research direction in analysing variability in holm oak by evaluating the epigenetic events and mechanisms based on DNA methylation. It sheds light on the enzymatic machinery governing DNA (de)methylation, and the changes in the expression levels of methylases and demethylases in different organs along the developmental stages. The expression level was correlated with the DNA methylation pattern observed, showing the prevalence of de novo methylation and demethylation events in seedlings and embryos, respectively. Several methylated genes involved in the regulation of transposable element silencing, lipid biosynthesis, growth and development, and response to biotic and abiotic stresses are highlighted. MSAP-seq integrated with whole genome bisulphite sequencing and advanced sequencing technologies, such as PacBio or Nanopore, will bring light on epigenetic mechanisms regulating the expression of specific genes and its correlation with the phenotypic variability and the differences in the response to environmental cues, especially those related to climate change.

Holm oak (Quercus ilex) is considered to be one of the major structural elements of Mediterranean forests and the agrosilvopastoral Spanish “dehesa”, making it an outstanding example of ecological and socioeconomic sustainability in forest ecosystems. The exotic Phytophthora cinnamomi is one of the most aggressive pathogens of woody species and, together with drought, is considered to be one of the main drivers of holm oak decline. The effect of and response to P. cinnamomi inoculation were studied in the offspring of mother trees from two Andalusian populations, Cordoba and Huelva. At the two locations, acorns collected from both symptomatic (damaged) and asymptomatic (apparently healthy) trees were sampled. Damage symptoms, mortality, and chlorophyll fluorescence were evaluated in seedlings inoculated under humid and drought conditions. The effect and response depended on the population and were more apparent in Huelva than in Cordoba. An integrated proteomic and metabolomic analysis revealed the involvement of different metabolic pathways in response to the pathogen in both populations, including amino acid metabolism pathways in Huelva, and terpenoid and flavonoid biosynthesis in Cordoba. However, no differential response was observed between seedlings inoculated under humid and drought conditions. A protective mechanism of the photosynthetic apparatus was activated in response to defective photosynthetic activity in inoculated plants, which seemed to be more efficient in the Cordoba population. In addition, enzymes and metabolites of the phenylpropanoid and flavonoid biosynthesis pathways may have conferred higher resistance in the Cordoba population. Some enzymes are proposed as markers of resilience, among which glyoxalase I, glutathione reductase, thioredoxin reductase, and cinnamyl alcohol dehydrogenase are candidates.

Molecular plant biology is the study of the molecular basis of plant life

The holm oak (Quercus ilex subsp. ballota) is the most representative species of the Mediterranean Basin and the agrosylvopastoral Spanish “dehesa” ecosystem. Being part of our life, culture, and subsistence since ancient times, it has significant environmental and economic importance. More recently, there has been a renewed interest in using the Q. ilex acorn as a functional food due to its nutritional and nutraceutical properties. However, the holm oak and its related ecosystems are threatened by different factors, with oak decline syndrome and climate change being the most worrying in the short and medium term. Breeding programs informed by the selection of elite genotypes seem to be the most plausible biotechnological solution to rescue populations under threat. To achieve this and other downstream analyses, we need a high-quality and well-annotated Q. ilex reference genome. Here, we introduce the first draft genome assembly of Q. ilex using long-read sequencing (PacBio). The assembled nuclear haploid genome had 530 contigs totaling 842.2 Mbp (N50 = 3.3 Mbp), of which 448.7 Mb (53%) were repetitive sequences. We annotated 39,443 protein-coding genes of which 94.80% were complete and single-copy genes. Phylogenetic analyses showed no evidence of a recent whole-genome duplication, and high synteny of the 12 chromosomes between Q. ilex and Quercus lobata and between Q. ilex and Quercus robur. The chloroplast genome size was 142.3 Kbp with 149 protein-coding genes successfully annotated. This first draft should allow for the validation of omics data as well as the identification and functional annotation of genes related to phenotypes of interest such as those associated with resilience against oak decline syndrome and climate change and higher acorn productivity and nutraceutical value.