Functional characterization of peach GOLVEN-like peptides in model systems

Peach (Prunus persica L. Batsch) produces climacteric fleshy fruits; their ripening is strictly regulated and relies mainly on the action of two hormones, auxin and ethylene. A peptide belonging to GOLVEN family, CTG134, was identified some years ago as a possible candidate to regulate the interaction between the two hormones. CTG134 was previously characterized in model systems Arabidopsis thaliana and Nicotiana tabacum, where it demonstrated to influence regulative processes in which above-mentioned hormones are involved. The goal of my PhD project was to test the function of GOLVEN peptides in the regulation of peach fruit ripening. During this thesis work, genes belonging to GOLVEN family were identified, through the use of bioinformatic tools. Among them, genes transcribed during ripening stages, crucial for fruit development, were determined. Another peptide shared the CTG134 expression profile, CTG512. Both were transcribed during climacteric stage and were induced by auxin and not by ethylene. To complete its functional characterization, CTG134 was expressed in Solanum lycopersicum under the control of a fruit specific promoter. Despite no evident phenotype was detected, fruit sampling allowed to test the expression system used by reporter gene GUS, and transcriptomic analysis allowed to verify that CTG134 influences transcription of ethylene related genes possibly inducing its synthesis by inducing expression on an involved gene and increasing tissue sensitivity by partial inhibition of a receptor transcription. To perform preliminary functional characterization, CTG512 was expressed in Arabidopsis e tobacco. Siliques presented ovule and embryo abortion, correlated to transgene expression level. This phenotype is associated to auxin and ethylene action, which are necessary for ovule and embryo development. Results obtained uphold the hypothesis that GOLVEN peptides act as hormone peptides, and together with auxin and ethylene, they participate to regulate fruit developmental steps fundamental for its quality

The Peach RGF/GLV Signaling Peptide pCTG134 Is Involved in a Regulatory Circuit That Sustains Auxin and Ethylene Actions

In vascular plants the cell-to-cell interactions coordinating morphogenetic and physiological processes are mediated, among others, by the action of hormones, among which also short mobile peptides were recognized to have roles as signals. Such peptide hormones (PHs) are involved in defense responses, shoot and root growth, meristem homeostasis, organ abscission, nutrient signaling, hormone crosstalk and other developmental processes and act as both short and long distant ligands. In this work, the function of CTG134, a peach gene encoding a ROOT GROWTH FACTOR/GOLVEN-like PH expressed in mesocarp at the onset of ripening, was investigated for its role in mediating an auxin-ethylene crosstalk. In peach fruit, where an auxin-ethylene crosstalk mechanism is necessary to support climacteric ethylene synthesis, CTG134 expression peaked before that of ACS1 and was induced by auxin and 1-methylcyclopropene (1-MCP) treatments, whereas it was minimally affected by ethylene. In addition, the promoter of CTG134 fused with the GUS reporter highlighted activity in plant parts in which the auxin-ethylene interplay is known to occur. Arabidopsis and tobacco plants overexpressing CTG134 showed abnormal root hair growth, similar to wild-type plants treated with a synthetic form of the sulfated peptide. Moreover, in tobacco, lateral root emergence and capsule size were also affected. In Arabidopsis overexpressing lines, molecular surveys demonstrated an impaired hormonal crosstalk, resulting in a re-modulated expression of a set of genes involved in both ethylene and auxin synthesis, transport and perception. These data support the role of pCTG134 as a mediator in an auxin-ethylene regulatory circuit and open the possibility to exploit this class of ligands for the rational design of new and environmental friendly agrochemicals able to cope with a rapidly changing environment

Functional characterization of peach GOLVEN-like peptides in model systems

Peach (Prunus persica L. Batsch) produces climacteric fleshy fruits; their ripening is strictly regulated and relies mainly on the action of two hormones, auxin and ethylene. A peptide belonging to GOLVEN family, CTG134, was identified some years ago as a possible candidate to regulate the interaction between the two hormones. CTG134 was previously characterized in model systems Arabidopsis thaliana and Nicotiana tabacum, where it demonstrated to influence regulative processes in which above-mentioned hormones are involved. The goal of my PhD project was to test the function of GOLVEN peptides in the regulation of peach fruit ripening. During this thesis work, genes belonging to GOLVEN family were identified, through the use of bioinformatic tools. Among them, genes transcribed during ripening stages, crucial for fruit development, were determined. Another peptide shared the CTG134 expression profile, CTG512. Both were transcribed during climacteric stage and were induced by auxin and not by ethylene. To complete its functional characterization, CTG134 was expressed in Solanum lycopersicum under the control of a fruit specific promoter. Despite no evident phenotype was detected, fruit sampling allowed to test the expression system used by reporter gene GUS, and transcriptomic analysis allowed to verify that CTG134 influences transcription of ethylene related genes possibly inducing its synthesis by inducing expression on an involved gene and increasing tissue sensitivity by partial inhibition of a receptor transcription. To perform preliminary functional characterization, CTG512 was expressed in Arabidopsis e tobacco. Siliques presented ovule and embryo abortion, correlated to transgene expression level. This phenotype is associated to auxin and ethylene action, which are necessary for ovule and embryo development. Results obtained uphold the hypothesis that GOLVEN peptides act as hormone peptides, and together with auxin and ethylene, they participate to regulate fruit developmental steps fundamental for its quality.Il pesco (Prunus persica L. Batsch) produce frutti carnosi e climaterici, la cui maturazione è un processo altamente regolato che dipende principalmente dall’azione di due ormoni, auxina ed etilene. Un peptide appartenente alla famiglia GOLVEN, CTG134, è stato identificato alcuni anni fa come possibile candidato alla regolazione dell’interazione tra i due ormoni. CTG134 è stato precedentemente caratterizzato nei sistemi modello Arabidopsis thaliana e Nicotiana tabacum, dove ha dimostrato di influire in processi regolativi in cui sono coinvolti i due ormoni sopra citati. L’obiettivo del mio progetto di dottorato era di caratterizzare funzionalmente i peptidi GOLVEN e identificare il loro ruolo durante la maturazione della pesca. Durante questo lavoro di tesi sono stati identificati i geni presenti nel genoma di pesco appartenenti alla famiglia GOLVEN, avvalendosi di strumenti bioinformatici. Tra questi sono stati determinati i geni trascritti nelle fasi climateriche, cruciali per la maturazione del frutto. Oltre a CTG134 un altro peptide condivideva il suo stesso profilo trascrizionale, CTG512. Entrambi sono trascritti durante la fase climaterica e vengono indotti da auxina, ma non da etilene. CTG134 è stato espresso in Solanum lycopersicum sotto il controllo di un promotore frutto specifico in modo da proseguire la sua caratterizzazione funzionale. Nonostante non siano stati riscontrati fenotipi evidenti, il campionamento dei frutti di pomodoro ci ha permesso di testare il funzionamento del sistema di espressione attraverso il gene reporter GUS, e l’analisi trascrittomica ha permesso di verificare che CTG134 influisce su geni correlati all’etilene presumibilmente in modo da indurne la sintesi mediante l’induzione trascrizionale di un gene coinvolto in essa, ed allo stesso tempo aumentare la sensibilità dei tessuti inibendo parzialmente la trascrizione di un recettore. CTG512 invece è stato espresso in Arabidopsis e tabacco per effettuare una caratterizzazione funzionale preliminare. Le silique presentavano aborti ovulari ed embrionali correlati al livello di transgene espresso. Questo fenotipo è associato all’azione di auxina ed etilene che sono necessari per lo sviluppo di ovulo ed embrione. I risultati ottenuti supportano l’ipotesi che i peptidi della famiglia GOLVEN agiscano come peptidi ormonali, ed in concerto con auxina ed etilene, intervengano nella regolazione delle fasi della maturazione del frutto fondamentali per lo sviluppo delle qualità organolettiche