AGROFORESTRY AND PLANT BIOCHEMISTRY, PROTEOMICS AND SYSTEMS BIOLOGY. AGR-164

Abstract:
The present proposal arises from the risk situation in which the dehesa and the Mediterranean forest currently find themselves, reflected in the increasing death of holm oak forests, which, associated with the drought syndrome, has been observed in recent decades. This situation can worsen in a climate change scenario. On the other hand, there is also a renewed interest in the acorn and its food use as nuts, flour or derivatives. It proposes the implementation of holm oak improvement programs based on the selection of elite genotypes resilient to drought, climate change and with high nutritional value. These genotypes may be used in holm oak forest restoration programs and reforestation of marginal or abandoned areas.
It is a continuation of previous work by the group in which: i) the variability in the response to stresses in holm oak populations and individuals has been analyzed using morphometric, physiological, biochemical and molecular biology (-omics) approaches; ii) the chemical composition of acorn has been determined, pointing to a high nutritional value, identifying, on the one hand, bioactive compounds, and, on the other, the presence of allergens. As a result of these works, highly tolerant/resistant individuals to drought and Phytophthora cinnamomi have been selected and some genes and gene products associated with these phenotypes have been identified.
Recently, the group has sequenced the oak genome, which makes possible analyzes of genomic variability through “Genome-Wide Association Study” studies for the identification of SNPs (changes in nucleotides), in an attempt to identify key genes in the resilient character, genes and gene products (transcripts, proteins) that can be used as markers in breeding programs and traceability. The phenotypic variability and plasticity that characterizes holm oaks has a cause not only genetic (genes and gene variants), but also epigenetic. Epigenetic processes and mechanisms as a source of variability in forest species are, to a large extent, unknown, with few studies in this field. In this direction, the project proposes studies of gene variability based on DNA methylation, in an attempt to identify epigenetic mechanisms involved in resilience and memory associated with stress tolerance.

Abstract:
The present project proposes two novel biotechnological approaches for the conservation, management, and reforestation of holm oak (Quercus ilex), being of relevance against the decline syndrome and climate change. Holm oak is the predominant and most representative species of the Andalusian geography, and the defining element of the agrosylvopastoral system dehesa, playing a key role in our environment and economy, especially in rural areas, as well as a source of a wide variety of goods and services for humans and animals. Q. ilex is considered a good candidate for afforestation due to its high adaptability to our climate conditions, drought tolerance and plasticity of response to variable edaphic conditions. However, it is an established fact that the holm oak, and therefore the dehesa, is facing serious problems that jeopardize their conservation and use. Factors such as tree ageing, overexploitation and poor regeneration,
loss of subsidies and the effect of biotic and abiotic stresses, including soil pathogens (such as Phytophthora cinnamomi), high temperatures and long periods of drought, are threatening the species, contributing, to a greater or lesser extent, to the so-called decline syndrome. The current proposal, included within the research programme about holm oak started 20 years back at the Agroforestry and Plant Biochemistry, Proteomics and Systems Biology research group led by Prof. Jesús V. Jorrín Novo, is a continuation of previous financed projects (AGL2009-12243-C02-02, 2009-2012; BIO2015-64737-R, 2015-2018; PID2019-109038RB-100, 2020-2023; UCO_FEDER-18-12575795R, 2020- 2022). The group has made an important effort in the in the development and optimisation of methodologies and protocols that allow a multidisciplinary approach to investigate on this forest species from physiological to molecular analysis, including field and greenhouse experiments, with the main objective related to evaluation of the decline syndrome in Andalusia. A double objective is proposed in the project: the identification and validation of candidate genes and gene products associated to the resilient phenotype for its employment in breeding programs, and the use of biostimulants or plant activators that promote growth and induce defence responses against biotic and abiotic stresses. The integration of the data generated (Forestry, Physiology, and Molecular Biology) will allow us to know in deep the biology of holm oak, to understand and exploit its great diversity and plasticity, and to face actual challenges and contribute to the conservation, sustainable management, reforestation, and exploitation of forest ecosystems.

Abstract:

This proposal deals with two biotechnological strategies for the conservation, management and reforestation of the Mediterranean forest and the “dehesa”, focusing on the holm oak (Quercus ilex).

It addresses the current problem of tree mortality associated to root rot and drought stresses, the main causal agents of the decline syndrome, whose incidence is predicted to be worse in a climatic change scenario.

The first approach is based on molecular marked assisted selection of elite, resilient, genotypes, and the second one on elicitor priming of defence responses.

The work proposed is included within the research program on holm oak and other forest species started 17 years ago by the research groups “Agroforestry and Plant Biochemistry, Proteomics and Systems Biology”, and “Assessment and Restoration of Agricultural and Forest Systems” at the University of Córdoba. Both groups have made an important effort in the evaluation of the decline syndrome in Andalusia, and in the development and optimisation of methodologies and protocols that allow a multidisciplinary approach to investigate on this species, from remote sensing to physiological and molecular analysis, including field and greenhouse experiments.

The multidisciplinary approach employed and the integration of all data (geospatial, forestry, physiology and molecular biology) will allow us to know in deep the biology of the species, to understand and exploit its great diversity and plasticity, to transfer the knowledge to the productive sector, and, ultimately, to face actual challenges and contribute to the conservation, sustainable management, reforestation and exploitation of forest ecosystems.

The application is a follow up of two previous financed projects: variability, cataloguing, responses to stresses and clonal propagation of holm oak, and study of variability and responses to stress in holm oak by a multi-omic approach. Those projects have complemented with “Detección temprana de procesos de decaimiento de Quercus ilex a partir de la integración de datos hiperespectrales y ecofisiológicos”, and “Patrones espaciales de eficiencia fotosintética y balance hídrico analizados desde el espacio en ecosistemas mediterráneos” projects.

The two proposed strategies are novel in Q. ilex research programs:

1. The first one pretends a deeper characterization of the genetic and epigenetic variability among healthy, escape, individuals in surveyed areas affected by the decline syndrome, the characterization of resistance mechanisms, and the selection of molecular markers to be used in breeding programs.
2. The second strategy is based on the priming phenomenon and treatment with inductors of defence reactions as a control strategy against Phytophthora, accompanied by a molecular analysis using omics approaches, to determine induced defence genes.

It is pretended to transfer the generated knowledge to the productive sector, to keep a high publication level (44 published related papers in WoS indexed journals, 39 Q1), and to offer a niche for the academic and scientific formation of graduate, master, and PhD students. It will also allow us to keep international collaborations and integration in worldwide referent forums on forestry research.

Apart from the principal researches, 2 senior scientists, 2 young postdoc, 2 PhD students, will take part of the research working team.

Abstract:

The progressive death of trees in the meadows and the lack of regeneration due to La Seca syndrome, determined a response from the Ministry of Agriculture, Fisheries and Food through the creation of a Working Group in 2017, where the Ministry itself as coordinator, the autonomous communities most affected, and the research organizations and universities that have been studying this syndrome for a long time, are represented.

The group’s objective is to deepen knowledge and promote concrete and practical actions to put a stop to the problem. For this, three subgroups have been created:

1. Inventory and monitoring of areas with “Seca”
2. Genetic and physiological improvement
3. Management

Our research group is included, along with other public entities, in working subgroup 2.

In this context, one of the tasks contemplated and for which our group is responsible is the search for molecular markers of response and resistance/tolerance to Phytophthora cinnamomi in holm oak and cork oak, both of the SSR type and from -omic approaches, which will allow determining the mechanisms involved in the resistance / tolerance response to P. cinnamomi, as well as establishing molecular markers associated with resistant phenotypes.

Abstract:

The main objective of this project is to sequence the first draft of the holm oak genome (Quercus ilex) to identify and characterize abiotic and biotic resistance genes related to Quercus decline and the climate change.

Despite its environmental (predominant species of the Mediterranean-forest ecosystem) and economic interest (part of “dehesa”), the holm oak is an orphan species, with molecular studies almost absent.

The functional caracterización will carry out by –omics approaches, Transcriptomics and Proteomics, considering that our research group has experience and international prestige in the proposed topics and methodologies.

This project is included in a research line started fourteen years ago, which has been funded by 7 research projects, giving a total of 21 research papers, 5 doctoral thesis and 8 thesis of degrees and master. Due to the recent incorporation of the Dr. Rey to the research group, who will lead the present project, we can propose several objectives based on cytogenetic techniques.

In total, we include four objectives:

1. First draft of holm oak genome, its functional annotation and identification of desirable characters such as stress resistant genes or related to pathogens, among others
2. Karyotype of the holm oak genome and identification of population variability
3. Selection of resistant genotypes to Phytophthora cinnamomi, considering one of the main causes of the Quercus decline and characterization its response by proteomic and transcriptomic approaches
4. Increase the data base in holm oak which is in progress currently. This data base includes information about both phenotypic and -omics data, together with molecular data of the variability observed in Andalusia

Both the PAIDI AGR-164 research group led by Prof. Jesús V. Jorrín Novo, and international and national collaborations included in this proposal will allow to obtain an optimal development of this project. This research group has worked hardly to develop molecular techniques in the analysis of species, mainly in forest species, which show quite problems as an experimental system.

In order to continue in the direction of systems biology, we will implement techniques widely used in our laboratories (forestry, ecophysiology, microscopy, classical biochemistry, transcriptomics, proteomics and bioinformatics) with those of genomics, genetics and cytogenetics. We expect to start a new research line in our research group to complete the holm oak genome sequencing and its karyotype. Also, we pretend to continue working in the establishment of the phenotypic and molecular basis for the selection of elite individuals, those presenting the highest adaptability and resistance to stresses, such as those associated with the decline syndrome.

The project fits within the scientific standards required by the social challenges of Andalusia: internationalization (involvement of two European groups), publications in high impact journals, holm oak molecular analysis and international collaboration to expand the holm oak biology. Moreover, we pretend to translate it to the academia, as a way to promote forestry research in degree, master and thesis programs.