Manipulation of nitric oxide levels and its impacts on tomato fruit physiology, hormonal balance and nutritional quality

Abstract

Fruit ripening involves dramatic structural and physiological changes, controlled by phytohormones and many other signaling molecules. Among phytohormones, ethylene (ET) plays a central role in the induction and maintenance of the ripening process, particularly in climacteric fruits. Recently, the free radical nitric oxide (NO) has been implicated in as regulator of numerous developmental events in plants, in most cases inhibiting the synthesis and/or signaling of ET. Although several studies have indicated that NO impairs ripening progress when exogenously applied, it is currently unknown if the internal mechanisms controlling endogenous NO levels are involved in the coordination of fleshy fruit development and ripening. Due to its high chemical reactivity, NO is intrinsically linked to the antioxidant cellular metabolism. Therefore, NO production, storage and removal appear to be under strict regulation by the plant cell. The enzyme S-nitrosoglutathione reductase (GSNOR), for instance, plays a critical role in the modulation of NO metabolism, facilitating the control of both NO endogenous levels and S-nitrosothiol abundance, the latter representing an important factor controlling the activity of many enzymes. In this scenario, the present work aims to investigate the relevance of NO metabolism for the control of tomato fruit development and ripening, particularly focusing on the importance of GSNOR for the control of the level of both NO and its derivatives. Since ET is a master switcher of the ripening process, the metabolism and signaling of this hormone will be investigated to elucidate how its interactions with NO influences the initiation and progression of tomato fruit ripening. Two main approaches will be employed: (i) Firstly, we will characterize and compare the metabolisms and signaling of NO (e.g., endogenous content, biosynthesis and degradation) and ET (e.g., emission rates, biosynthesis and conjugation) in fruit of mutants (i.e., rin, nor and NR) with altered ripening pattern. (ii) Secondly, we will generate transgenic plants with fruit-specific overexpression or silencing of the gene encoding GSNOR, thereby facilitating to characterize the impacts of fruit-specific GSNOR manipulation on tomato fruit development and ripening. Moreover, mutants defective on GSNOR will be generated via CRISPR/Cas9 system, allowing a more general characterization of the effects of the absence of this enzyme in the whole organism. The global transcriptome analysis of pericarp tissues from transgenic plants exhibiting fruit-specific overexpression or silencing of GSNOR will be performed via RNAseq in two stages of fruit development (immature and early ripening), thereby allowing the identification of genes whose expression is significantly altered in response to the fruit-specific manipulation of GSNOR levels and its consequent changes in the content of NO and its derivatives. Furthermore, immature, ripening and red ripe fruits from these transgenic and mutant plants will also be characterized regarding: (i) NO and ET metabolisms, (ii) nutritional quality (e.g., carotenoids, flavonoids, volatile compounds, tocopherols) and (iii) susceptibility to pathogen attack. These approaches may increase our current knowledge about the interaction between NO and ET during fleshy fruit development and ripening and may also promote significant biotechnological advances leading to further improvements in fruit nutritional quality and minimize economical losses due to precocious fruit deterioration during production and transportation. (AU)