Progetto Small Peptides in peachs

Peach is a climacteric fruit but its ripening largely depends also on the action of auxin, which, by inducing the expression of 1-aminocyclopropane-
1-carboxylic acid synthase 1 (ACS1) triggers the synthesis of the ethylene that is necessary for the ripening to occur quickly and dramatically (Tatsuki et al., 2013; Trainotti et al., 2007), as it is typical of many so-called melting flesh cultivars. This peculiar ripening physiology makes it difficult to store fruit for prolonged time leading to rapid fruit decay after harvest and may be related to the development of chilling-induced postharvest diseases. The relationships between auxin and ethylene during peach ripening are poorly understood, but preliminary data collected in our laboratory seem to place apoplastic peptide hormones (PHs) similar to Arabidopsis Root Growth Factor (Matsuzaki et al., 2010) at the crossroads between the two hormones (Busatto, 2012).
PHs comprise several families with conserved characteristics, as the short length of the translated propeptide (usually less that 100 amino acids), the poor conservation of the sequence outside the part encompassing the mature PH, which, on the contrary, is strongly conserved, the possibility of post-translational modifications at the level of the PH residues and others (Matsubayashi and Sakagami, 2006). The short length allow to group HPs with short interfering peptides (siPEPs) that are nuclear localized and exert their biological function by dimerizing with transcription factors (TFs), thus altering their activities (Seo et al., 2011). The short open reading frames (sORFs) of both PHs and siPEPs make it challenging their prediction and ab initio discovery in sequenced genomes.
RNAseq transcriptomics and reads mapping on the reference genome strongly improves the possibilities to identify short and poorly conserved genes coding for small peptides. This approach will be taken also in this project (see WP1: Fruit transcriptomics by RNAseq and identification of peptide hormones and small interfering peptides), as the extremely good peach genome sequence, available since 2010 (Sosinski et al., 2010;
Verde et al., 2013), needs anyway improvements at this level (Trainotti et al., 2012). New gene predictions and annotations of short peptides and related receptors/TFs and the determination of expression profiles of selected best candidates are among the expected projects results.
The spreading of ripening trough the fruit parts is due to gradients of sugars, hormones, metabolites and gene expression profiles that are still poorly understood. Activities planned within WP2 (Cytological and histological localization of peptide hormones and small interfering peptides) will provide information on possible gradients between PH production and perception and on how this perception may be linked to ethylene synthesis.
PHs are relatively new in plant biology but their discovery opens the possibility to design new agrochemicals that, by enhancing or inhibiting their action, will have positive effects on agriculture. This rational design needs knowledge on how the interactions between the hormones and their receptors occur at the structural level. The activities planned within WP3 (Functional characterization of a peptide hormone) aim to gain such knowledge for at least one HP/LRR-RK couple. In silico modeling will guide the selection for the correct matching of the most probable HP/LRR-RK couples as preliminary data indicate that several PH and LRR-RK genes are co-expressed in ripening peach. Heterologous expression and chemical synthesis of receptor proteins and PHs will be used to test these predictions.
Altogether, the project aims to shed light on the auxin-ethylene cross-talk that occurs at the onset of peach ripening, on how this cross-talk may be mediated by PHs and on how this new class of signaling molecules may be exploited to design new rational and environmentally friendly agrochemicals.