F53D23003950006

 

 

 

Alternative strategies for improved multi-stress tolerance, yield and quality in crop plants – RESILIENCE

Descrizione

Nowadays, agriculture is threatened by adverse environmental conditions which affect plant growth and metabolism, thus severely reducing yield and quality of crop plants. In this context, genetic improvement of crops and development of new sustainable agricultural practices are essential for food and health security. The present project aims to further investigate the plant adaptation/tolerance mechanisms to abiotic and biotic stresses and to use obtained knowledge to transfer high stress tolerance, enhanced yield and improved quality traits to crop plants. In particular, the final goal of this project is to obtain new tomato (Solanum lycopersicum) varieties with high tolerance under multiple stress conditions (such as drought, salt and cold stress, and infection by Pseudomonas syringae) and with elevated fruit yield and fruit nutritional value through manipulation of genes belonging to two distinct pathways: genes involved in polyamine metabolism, and in particular in the metabolism of the recently discovered thermospermine (T-Spm), and genes encoding for a novel family of proteins named Salt Tolerance Related Proteins (STRPs).

The project is based on experimental evidence from Arabidopsis and tomato plants, which highlight the role of T-Spm and STRPs in plant growth and/or responses to biotic and abiotic stresses and suggest a beneficial effect of polyamines on fruit quality and shelf-life. This project takes advantage of techniques for plant transformation, T-DNA insertional mutant genotyping and CRISPR/Cas9-mediated gene-specific mutagenesis to obtain Arabidopsis and tomato lines with increased levels of T-Spm and STRP, alone or in combination. Comparative analysis of these lines under various stress conditions through physiological, morphological, anatomical and molecular approaches, as well as through genome-wide transcriptomic and metabolomic analyses will allow to select those with a robust stress tolerance to numerous environmental stresses, high yield and superior nutritional value and dissect underlying molecular mechanisms. In parallel, this project aims to develop new formulations of polyamine-based fertilizers with a beneficial effect on growth, yield and quality of crops even under stress conditions. This will contribute to improve sustainability of farming systems.

Altogether, this project will provide genetic resources and molecular tools to breeders, biotechnologists and plant growers for selection and development of crop varieties with high adaptability to climatic changes, yield and quality and for sustainable improvement of cultivation conditions. This study may also allow to identify polyamine- and STRP-based markers correlated to improved agronomic characteristics and useful for the screening of traditional and modern tomato varieties, thus contributing to conservation and valorisation of tomato biodiversity.

Finalità

RESILIENCE aims to evaluate whether modulation of two different pathways, one based on thermospermine (T-Spm) metabolism, and the other on the stress tolerance related proteins STRPs, can strongly interfere, alone or in combination, with plant adaptation/defence mechanisms to numerous environmental stresses. In particular, this project proposes to verify whether biotechnological and breeding strategies can be designed based on these two pathways to produce new crop varieties with improved yield and nutritional traits even under abiotic and biotic stress conditions, thus enabling them to better face climate changes and benefit consumers’ health.

In plants, polyamines are involved in several physiological, metabolic and defence processes. Polyamines exert their functions through their interaction with polyanionic molecules, thus having a protective role and a radical-scavenging capacity. Polyamines also serve as a storage pool for nitrogen and their metabolism is linked to the tricarboxylic acid cycle, the glycolysis and the urea cycle, as well as the synthesis of γ-aminobutyric acid, glutamate, 3-alanine, proline, alcaloids and various conjugates (Tavladoraki et al., 2016; Moschou et al., 2012). Moreover, H₂O₂ derived from polyamine catabolism is involved in several physiological and pathological events and acts in stress signaling (Tavladoraki et al., 2016). Polyamines are also involved in Ca²⁺ and NO homeostasis, act as signaling molecules triggering transcriptional changes and interact with various hormonal pathways, such as auxin, cytokinins, ABA and ethylene. In this context, T-Spm, the last discovered polyamine, is of particular importance since increased T-Spm levels give rise to increased stem length and reduced xylem differentiation through modulation of the auxin/cytokinin interplay. Furthermore, T-Spm is implicated to different types of stress though a systematic study is missing. These roles of polyamines make them, and in particular T-Spm, a promising target for the development of traits related to multi-stress tolerance, enhanced yield and improved quality.

STRPs share several common features with LEA (a large family of proteins with relevant role in plant responses to biotic and abiotic stresses) and were reported to be involved in both salt and cold stress tolerance mechanisms (Fiorillo et al., 2020; Du et al., 2008; Liu et al., 2017; Aziz et al., 2021). However, STRP contribution to these mechanisms is still unknown. As other intrinsically disordered proteins lacking a well-defined three-dimensional fold, STRPs may bind to different specific targets, thus playing a role in a wide range of cellular processes, such as gene transcription, signal transduction, chaperon action, and regulation of assembly of multi-protein complexes. Interestingly, Arabidopsis STRP (AtSTRP) preferentially localizes into the nucleus under stress conditions and associates to chromatin, suggesting a possible role in chromatin stabilization under stress conditions and/or regulation of expression of stress responsive genes (Fiorillo et al., 2020). These considerations make STRPs another promising target to improve plant tolerance towards multiple stresses.

This study is focused on both the model plant Arabidopsis thaliana and the crop Solanum lycopersicum (tomato) to better dissect mechanisms and obtain information on traits regarding stress tolerance, yield and quality. Tomato (variety Moneymaker) has been chosen for the present study since it is a major crop growing all over the world and has an enormous socio-economic importance being an important dietary source of vitamins and carotenoids. Furthermore, tomato is susceptible to numerous abiotic and biotic stresses which limit vegetative growth, compromise reproduction, inhibit seed development and affect fruit growth and nutrient composition. In addition, tomato has become an established model system for the study of the physiology and biochemistry of crop plants for understanding fleshy fruit development.

Target genes of RESILIENCE are AtPAO1 and AtPAO5 (genes encoding for T-Spm catabolic enzymes), ACL5 (a gene involved in T-Spm biosynthesis) and AtSTRP, as well as their tomato homologues (SlPAO1, SlPAO2, SlPAO3, SlPAO4, SlACL5-1, SlACL5-2, SlACL5-3, SlSTRP). Susceptibility/tolerance to three different abiotic stresses (drought, salt and cold stress) and a biotic one (Pseudomonas syringae) are assessed within this project. Specific objectives of this project are shown in Figure 1.

Risultati attesi

-Information on the regulation of AtPAO1, AtPAO5, ACL5 and AtSTRP gene expression.

-Development and characterization of Arabidopsis and tomato plants overexpressing ACL5 (ACL5over) and AtSTRP (AtSTRPover), as well as of Arabidopsis ACL5over-atpao5, ACL5over-atpao1atpao5, AtSTRPover–atpao5 and AtSTRPover–atpao1atpao5 plants.

-Development and characterization of tomato slpao2, slpao4, slpao2slpao3, slpao3slpao4 mutants.

-Selection of tomato genotypes with increased tolerance to different abiotic (drought, salt stress, cold stress) and biotic (Pseudomonas syringae) stresses.

-Information about the mechanisms underlying T-Spm and STRP mediated stress tolerance.

-Information about the contribution of polyamine metabolism in tomato yield and nutritional value.

-Information about the effect of exogenous application of T-Spm on tomato growth, stress tolerance and fruit quality.

-Development of new formulations of polyamine-based fertilizers with a beneficial effect on growth, yield and quality of crops even under stress conditions.

 

Risultati raggiunti

-Data on the regulation of AtPAO1, AtPAO5, ACL5 and AtSTRP gene expression under stress conditions.

-Development of Arabidopsis and tomato plants overexpressing ACL5 (ACL5over) and AtSTRP (AtSTRPover), as well as of Arabidopsis ACL5over-atpao5 and AtSTRPover-atpao5 plants.

-Development and molecular characterization of slpao2, slpao3 and slpao4 tomato mutants, as well as slpao2slpao3 and slpao3slpao4 mutants.

-Phenotypic characterization of slpao2, slpao3 and slpao4 tomato mutants under normal conditions.

-Characterization of atpao5 mutants and ACL5over Arabidopsis plants under cold stress conditions.

-Characterization of atpao5, AtSTRover and AtSTRPover-atpao5 Arabidopsis plants under drought stress conditions.

-Selection of tomato slpao3 mutants with increased tolerance to drought stress.

 

Stato dell’arte

Plants live in constantly changing environments that are often unfavorable for growth and development and threaten food security. Plants have evolved complex and integrated processes to perceive stress signals and survive in challenging environments. The understanding of the plant responses to abiotic and biotic stresses and the development of crop varieties that can adapt well under adverse environmental conditions have been major objectives of breeders and biotechnologists since a long time. However, despite significant discoveries, the challenge still persists highlighting the complexity of these responses and the need to establish novel approaches to improve plant adaptation/defence mechanisms to environmental stresses.

This project aims to develop alternative strategies to confer crop plants improved tolerance to multiple stresses, both biotic and abiotic ones, maintaining at the same time high yield and nutritional value. In particular, this project proposes to study in detail the contribution to the plant defence mechanisms, as well as to yield and quality traits of two distinct classes of proteins: enzymes involved in polyamine metabolism (Tavladoraki et al., 2016) and a novel family of proteins named Salt Tolerance Related Proteins (STRPs; Fiorillo et al., 2020).

In plants, the polyamines putrescine, spermidine (Spd), spermine (Spm) and the newly discovered thermospermine (T-Spm) play a critical role in a range of developmental and defence processes and are linked various metabolic pathways, including those involved in the synthesis of fruit nutrients (Alcázar et al., 2020; Gao et al., 2021; Gerlin et al., 2021). The different polyamines exhibit common physiological roles, but also specific functions. In particular, T-Spm specifically participates in the control of stem elongation and xylem differentiation interfering with the auxin and cytokinin signaling pathways (Cai et a., 2016; Yoshimoto et al., 2016) and up-regulates defense-related genes (Sagor et al., 2016). Furthermore, Arabidopsis mutants for thermospermine synthase gene (ACL5) presents growth defects, altered morphology of the vascular bundles and hypersensitivity to salt stress, while ACL5 over-expression confers increased resistance to salt stress and the pathogen Pseudomonas viridiflava (Marina et a., 2013; Shinohara et al., 2019).

Polyamine oxidases (PAOs) are FAD-dependent enzymes which oxidize Spd, Spm and T-Spm and have an important role in the control of polyamine levels and cellular redox homeostasis (Tavladoraki et al., 2016; Salvi and Tavladoraki, 2020). In Arabidopsis, five PAOs (AtPAO1-AtPAO5) are present with distinct characteristics and physiological roles (Fincato et al., 2011; Fincato et al., 2012). Among them, AtPAO1 and AtPAO5 are cytosolic enzymes which specifically oxidize Spm and T-Spm. AtPAO1 is expressed in the root meristematic region in an abscisic acid (ABA)-dependent way and AtPAO5 in xylem tissues (Ahou et al., 2014). Furthermore, atpao5 mutants present, together with increased T-Spm levels, increased stem length, reduced xylem differentiation, and improved tolerance to drought and salt stress as compared to the wild-type plants (Sagor et al., 2016; Ahou et al., 2014; Zarza et al., 2016; Alabdallah et al., 2017). In tomato, three AtPAO5 homologues are present (SlPAO2, SlPAO3 and SlPAO4) and data indicate that slpao3 mutants present reduced number of xylem vessels, increased growth rate and improved adaptation to drought stress (D’Incà et al., 2024).

STRPs were first identified in Arabidopsis (AtSTRP) and initially proposed to be involved in the plant response to salt stress based on the hypersensitivity to salt stress displayed by the loss-of-function atstrp Arabidopsis mutant (Du et al., 2008). More recently, STRPs were suggested to play a role also in plant responses to cold stress, since AtSTRP rapidly accumulates during a short-term cold exposure due to increased protein stability and atstrp mutants are more susceptible to cold stress and less responsive to ABA, as compared to wild-type plants, suggesting that STRPs may be additionally involved in the ABA transduction pathway (Fiorillo et al., 2020; Rocco et al., 2013). AtSTRP shares common features with the Late Embryogenesis Abundant (LEA) proteins (a family of extremely hydrophilic and intrinsically disordered proteins identified in a wide range of organisms) which in plants accumulate at high levels in embryos before seed desiccation and in vegetative tissues under various stress conditions and were proposed to play a protective role by stabilizing cellular structures. Similarly to the LEA, AtSTRP is a hydrophilic and largely unstructured protein which remains highly soluble after boiling and is able to prevent enzyme inactivation after freezing (Fiorillo et al., 2020). AtSTRP localizes at the plasma membrane, the cytosol and the nucleus and cold stress determines an increase in AtSTRP nuclear content promoting its association to chromatin (Fiorillo et al., 2020). AtSTRP was further found to associate with DEK3, a protein involved in chromatin remodelling processes in response to salt stress (Waidmann et al., 2014), which suggests the involvement of AtSTRP in chromatin remodelling processes modulating the expression of stress-related genes.

 

References

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Riferimento: PRIN 2022 – Codice progetto: PRIN 20227MFA8Z – CUP: F53D23003950006

Investimento totale del progetto: 229.672 €

Partner/proponente: Paraskevi Tavladoraki

Coordinatore dell’UdR Università degli Studi Roma Tre: Paraskevi Tavladoraki