Gracia Montilla-Bascón

Oat production in the Mediterranean region faces important yield and quality challenges, with crown rust (caused by Puccinia coronata f. sp. avenae) being one of the most important critical biotic constraints. Crown rust populations in the Mediterranean basin are reported to exhibit more virulence compared with those in central and northern Europe. Breeding oat for disease resistance is the most efficient, economic, and environmentally sustainable strategy for managing biotic stresses. However, development of resistant varieties is an ongoing challenge for breeders, as newly released varieties carrying specific resistance genes generate selection pressure on pathogen populations, leading to the rapid emergence of new races capable of overcoming these resistance mechanisms. In this work, a Mediterranean oat collection comprising over 700 Avena sativa and A. byzantina accessions was evaluated to identify durable sources of resistance against a highly virulent Mediterranean rust isolate. Phenotypic evaluations were performed at both the seedling stage under controlled conditions and the adult plant stage across three different field environments. Based on macroscopic assessments, 17 resistant accessions were selected for detailed histological analysis to dissect the cellular responses underlying the resistance observed. Microscopic evaluations revealed a diverse array of cellular responses that hampered fungal development at various stages of the infection process: prior to mesophyll penetration, at the penetration stage, and following mesophyll cell penetration. In the latter, triggering of an early or late hypersensitive response (HR) led to cell death, impeding fungal progression. In a few cases, reduced fungal colony size not associated with HR suggests the presence of posthaustorial resistance mechanisms independent of cell death. Field evaluations indicated an overall increase in resistance at the adult plant stage with A. byzantina accessions displaying a more pronounced increase in resistance compared with A. sativa. The identification of the resistant sources together with the characterization of their cellular resistance responses is of great value for breeding programs aiming to enhance resistance durability by integrating both qualitative and quantitative resistance mechanisms. Moreover, this work may contribute to unravelling the molecular basis of rust resistance in oat.

Jasmonates (JAs) are a diverse group of jasmonic acid (JA)-linked metabolites, including the biosynthetic intermediate 12-oxophytodienoic acid (OPDA). Although changes in JAs have been associated with plant responses to abiotic stress, the involvement and kinetics of specific forms such as JA, JA-Ile and OPDA require further clarification. This study analyzed jasmonate profiles in roots and leaves of two oat genotypes differing in drought tolerance. Jasmonates were quantified using UPLC-MS/MS, expression of key biosynthetic genes was assessed by qRT-PCR, and JA/OPDA treatments were applied to evaluate their effects on physiological and morphological responses to drought. Drought induced contrasting jasmonate dynamics in roots and leaves, with overall JA levels increasing in leaves and decreasing in roots, with genotype- and compound-specific differences. JA and JA-Ile ((+)-7-iso-jasmonoyl-L-isoleucine) showed similar trends, whereas OPDA displayed a distinct pattern. The tolerant genotype exhibited an early and marked reduction in root OPDA, while the susceptible one showed minimal change. Exogenous OPDA increased drought symptoms, reduced leaf relative water content and strongly decreased root length by limiting the formation of new thin roots. In contrast, JA application alleviated drought symptoms, reflected in a lower area under the drought progress curve, without affecting root length. Results suggest that under water deficit, reduced OPDA, likely due to its conversion into JA and JA-Ile, is associated with the development of small-diameter roots essential for maintaining water status in oat. Together, these results highlight tissue-specific differences in jasmonate dynamics during drought and show that OPDA and JA treatments lead to distinct drought-related responses in both leaves and roots.

Oat (Avena sativa L.) is increasingly recognized as a functional food due to its unique profile of antioxidant and health-promoting compounds. Beyond its traditional role, our study reveals that Mediterranean landraces and related species harbour exceptional variability in both their nutritional and bioactive traits, offering untapped potential for functional food development. We analysed 126 genotypes, including landraces and cultivars, from 18 Mediterranean and European countries, quantifying the β-glucans, arabinoxylans, phenolic acids (soluble and cell wall bound), avenanthramides (AVAs: A, B, and C), carbon (C), nitrogen (N), C:N ratio, and protein content. The protein levels ranged from 9.5% to 18.5%, with several genotypes exceeding 17%, far above typical oat averages. The β-glucans reached clinically relevant thresholds (>5%) in multiple accessions, while the arabinoxylans surpassed 2% in selected genotypes. The phenolic acids and avenanthramides showed striking diversity, with some landraces accumulating more than 2000 µg/g of total AVAs. The species and phenology strongly influenced the grain composition. Thus, A. strigosa exhibited the highest β-glucan and avenanthramide contents, while early-heading genotypes had doubled avenanthramide levels compared to late-heading ones. A correlation analysis revealed synergistic patterns among the β-glucans, avenanthramides, and proteins, suggesting multi-component interactions that could enhance antioxidant functionality. These findings underscore the strategic value of Mediterranean oat germplasm for breeding programs targeting high-protein, fibre-rich, and antioxidant-enhanced cultivars. By exploiting this diversity, oats could play a pivotal role in preventing chronic diseases and advancing sustainable, health-oriented food systems.

Drought stress significantly impacts crop productivity and plant physiology. Nitric oxide (NO) signalling is essential for drought tolerance. This study explores the relationship between in vivo NO levels, mediated by NO scavenging phytoglobin (encoded by Pgb , non‐symbiosis associated hemoglobin), and drought tolerance in oat ( Avena sativa ). Real‐time in vivo NO measurements suggested increased production under moderate to high water stress in the susceptible cultivar Flega compared to the resistant Patones. This elevated NO correlated with increased senescence in Flega. Conversely, the resistant cultivar Patones showed a marked increase in Pgb gene expression, which correlated with reduced NO levels in vivo. This suggested that Pgb acts as a protective mechanism against NO‐induced stress. Water stress‐induced NO increases fed into the polyamine pathway, leading to a significant rise in arginine decarboxylase ( ADC ) expression, leading to putrescine accumulation in the susceptible cultivar, whereas the resistant Patones maintained lower ADC expression and polyamine levels. Elevated in vivo ethylene production was also observed in the susceptible cultivar Flega, correlating with severe drought‐induced senescence symptoms and linked to the naturally high NO levels in this cultivar. Assessment of other oat genotypes confirmed a negative correlation between Pgb expression and drought symptoms. These results underscore an important role of phytoglobins in modulating NO levels to counter drought in oat and suggest a potential target for genetic improvement of oat for drought tolerance.

Key message: Genomic analysis of Mediterranean oats reveals high genetic diversity and three loci for adaptation to this environment. This information together with phenotyping and passport data, gathered in an interactive map, will be a vital resource for oat genetic improvement. During the twentieth century, oat landraces have increasingly been replaced by modern cultivars, resulting in loss of genetic diversity. However, landraces have considerable potential to improve disease and abiotic stress tolerance and may outperform cultivars under low input systems. In this work, we assembled a panel of 669 oat landraces from Mediterranean rim and 40 cultivated oat varieties and performed the first large-scale population genetic analysis of both red and white oat types of Mediterranean origin. We created a public database associated with an interactive map to visualize information for each accession. The oat collection was genotyped with 17,288 single-nucleotide polymorphism (SNP) loci to evaluate population structure and linkage disequilibrium (LD); to perform a genome-wide association study (GWAs) for heading date, a key character closely correlated with performance in this drought-prone area. Population genetic analysis using both structure and PCA distinguished two main groups composed of the red and white oats, respectively. The white oat group was further divided into two subgroups. LD decay was slower within white lines in linkage groups Mrg01, 02, 04, 12, 13, 15, 23, 33, whereas it was slower within red lines in Mrg03, 05, 06, 11, 21, 24, and 28. Association analysis showed several significant markers associated with heading date on linkage group Mrg13 in white oats and on Mrg01 and Mrg08 in red oats.

Recently, phenotyping has become one of the main bottlenecks in plant breeding and fundamental plant science. This is particularly true for plant disease assessment, which has to deal with time-consuming evaluations and the subjectivity of visual assessments. In this work, we have developed an open source Robust, User-friendy Script Tool (RUST) for semi-automated evaluation of leaf rust diseases. RUST runs under the free Fiji imaging software (developed from ImageJ), which is a well-recognized software among the scientific community. The script enables the evaluation of leaf rust diseases using a color transformation tool and provides three different automation modes. The script opens images sequentially and records infection frequency (pustules per area) (semi-)automatically for high-throughput analysis. Furthermore, it can manage several scanned leaf segments in the same image, consecutively selecting the desired segments. The script has been validated with nearly 900 samples from 80 oat genotypes ranging from resistant to susceptible and from very light to heavily infected leaves showing a high accuracy with a Lin’s concordance correlation coefficient of 0.99. The analysis show a high repeatability as indicated by the low variation coefficients obtained when repeating the measurement of the same samples. The script also has optional steps for calibration and training to ensure accuracy, even in low-resolution images. This script can evaluate efficiently hundreds of leaves facilitating the screening of novel sources of resistance to this important cereal disease.

Salicylic acid (SA) is involved in several plant processes including responses to abiotic stresses. Although SA is thought to interact with other regulatory molecules in a complex way, currently, little information is available regarding its molecular mechanisms of action in response to abiotic stresses. In a previous work, we observed that drought-resistant oat plants significantly increased their SA levels as compared with a susceptible cultivar. Furthermore, exogenous SA treatment alleviated drought symptoms. Here, we investigated the interaction between SA and polyamine biosynthesis during drought responses in oat and revealed that SA regulated polyamine biosynthesis through changes in polyamine gene expression. Overall, SA treatment decreased the levels of putrescine under drought conditions while increased those of spermine. This correlates with the downregulation of the ADC gene and upregulation of the AdoMetDC gene. Based on the presented results, we propose that SA modulates drought responses in oat by regulating polyamine content and biosynthesis.

Oat ( Avena sativa L.) has a high concentration of oils, comprised primarily of healthful unsaturated oleic and linoleic fatty acids. To accelerate oat plant breeding efforts, we sought to identify loci associated with variation in fatty acid composition, defined as the types and quantities of fatty acids. We genotyped a panel of 500 oat cultivars with genotyping-by-sequencing and measured the concentrations of ten fatty acids in these oat cultivars grown in two environments. Measurements of individual fatty acids were highly correlated across samples, consistent with fatty acids participating in shared biosynthetic pathways. We leveraged these phenotypic correlations in two multivariate genome-wide association study (GWAS) approaches. In the first analysis, we fitted a multivariate linear mixed model for all ten fatty acids simultaneously while accounting for population structure and relatedness among cultivars. In the second, we performed a univariate association test for each principal component (PC) derived from a singular value decomposition of the phenotypic data matrix. To aid interpretation of results from the multivariate analyses, we also conducted univariate association tests for each trait. The multivariate mixed model approach yielded 148 genome-wide significant single-nucleotide polymorphisms (SNPs) at a 10% false-discovery rate, compared to 129 and 73 significant SNPs in the PC and univariate analyses, respectively. Thus, explicit modeling of the correlation structure between fatty acids in a multivariate framework enabled identification of loci associated with variation in seed fatty acid concentration that were not detected in the univariate analyses. Ultimately, a detailed characterization of the loci underlying fatty acid variation can be used to enhance the nutritional profile of oats through breeding.

Nitric oxide (NO) is a key messenger in plant stress responses but its exact role in drought response remains unclear. To investigate the role of NO in drought response we employed transgenic barley plants (UHb) overexpressing the barley non-symbiotic hemoglobin gene HvHb1 that oxidizes NO to NO₃⁻. Reduced NO production under drought conditions in UHb plants was associated with increased drought tolerance. Since NO biosynthesis has been related to polyamine metabolism, we investigated whether the observed drought-related NO changes could involve polyamine pathway. UHb plants showed increases in total polyamines and in particular polyamines such as spermidine. These increases correlated with the accumulation of the amino acid precursors of polyamines and with the expression of specific polyamine biosynthesis genes. This suggests a potential interplay between NO and polyamine biosynthesis during drought response. Since ethylene has been linked to NO signaling and it is also related to polyamine metabolism, we explored this connection. In vivo ethylene measurement showed that UHb plants significantly decrease ethylene production and expression of aminocyclopropane-1-carboxylic acid synthase gene, the first committed step in ethylene biosynthesis compared with wild type. These data suggest a NO-ethylene influenced regulatory node in polyamine biosynthesis linked to drought tolerance/susceptibility in barley.