ABSTRACT

Dormancy is one of the most important mechanism developed by perennial plants in order to survive in an unfavourable environment that consequently also conditions the stability of the production for the next years. The study of the genes regulated during dormancy release is crucial for a better understanding of the process with the final objective of the development of new varieties with a better adaptation to certain environments; and this is particularly important considering the increasing economical weight of fruitculture in low and medium chilling regions as in the Mediterranean area. The incomplete accumulation of chilling still represents a challenge for peach production in temperate and warm climates because the use of chemicals as well as agronomical practices showed great limits. Also chilling requirement measured with climatic models is still highly dependent on the year and location; becoming evident that just a better understood of the molecular mechanisms beneath dormancy release, and their integration into climatic models, will improve their prediction of chilling release. 
The general objective of this tesis is to understand the molecular and physiological mechanisms underlying the maintenance and release of seasonal dormancy in peach. In order to achieve it we first used suppression subtractive hybridization (SSH) to identify genes expressed in dormant and dormancy-released buds in two cultivars with different chilling requirements, ‘Zincal-5’ and ‘Springlady’ and successively validated their differential expression with a microarray platform cDNAs enriched in flower buds of peach. Additionally, we carried out a genome-wide search of peach genes related to dormancy release by hybridizing the previous cDNA microarray with mRNA samples from 10 cultivars showing different dormancy behaviour, followed by an expression correlation analysis.
Among the identified genes from this two first works, the peach transcriptional models ppa011123m, ppa010822m and ppa010714m, corresponding to MADS-box genes DAM4, DAM5 and DAM6, were the most interesting also because other groups, working independently, observed their variations in expression during the season from peach buds (Jimenez et al., 2010b; Yamane et al., 2011) as well from other species (Mazzitelli et al., 2007; Yamane et al., 2008; Druart et al., 2007; Horvath et al., 2008). The central role of DAM genes in dormancy regulation has also been confirmed by additional functional approaches from the secuence annotation of the evg locus in the mutant (Bielenberg et al., 2008), QTL analysis (Fan et al., 2010a) and transgenic approaches (Sasaki et al., 2011; Fan et al., 2010b).
In our second work we focused on the molecular mechanisms of DAM6 down-regulation concomitant with dormancy release in flower buds. A ChIP analysis of DAM6 promoter and structural gene revealed chromatin modification events similar to those observed in vernalization of Arabidopsis and cereals. We showed that the DAM6 is transcriptionally active in dormant peach flower buds collected in October, when a short chromatin region around its ATG is trimethylated at H3K4 and acetylated at the H3. Concomitantly with DAM6 repression, H3K4 became demethylated and H3 deacetylated. Later H3K27 is trimethylated along a genomic region larger than 4kb, including promoter, coding sequence and intron. Due to their relevance in dormancy regulation, DAM genes could be utilized as expression markers to assess the dormancy stage of an individual plant and to evaluate the chilling requirements of new cultivars. In fact, we have shown in this work that the expression pattern of DAM5, together with other transcripts (BD396, DB247, SB280 and PpB63), correlates well with chilling requirements values of five different varieties (‘Big Top’, ‘Catherina’, ‘Fergold’, ‘Maruja’ and ‘Springlady’) measured following Utah and Dynamic models.
Some of the genes identified in the transcriptomic experiments using flower buds, as DAM1, DAM5 and DAM6, were also regulated during the cold stratification of peach seeds, suggesting the presence of common regulatory pathways in the dormancy process of buds and seeds. These similarities between bud and seed dormancy have possible important implications in the evaluation of bud chilling requirements of early and late flowering genotypes directly at seed level greatly reducing the time needed for evaluating plant material in breeding programs. Among others, a significant number of genes identified in this work were homologous to ABA and drought related genes from other species. ABA, in fact, has been proposed to promote and maintain bud dormancy (Arora et al., 2003; Horvath et al., 2003; Rohde & Balherao, 2007) although few molecular data support this prediction. Our results also contributed to the identification of peach genes regulated by ABA, drought and salt stress as ppa006974m codes for a protein similar to ABA-INSENSITIVE5 (ABI5) binding protein (AFP) involved in signal transduction in Arabidopsis that binds and promotes the degradation of ABI5. ABI5 is a basic leucine zipper (bZIP) transcription factor that regulates ABA-dependent genes by binding to the ABA-responsive element, ABRE (Lopez-Molina et al., 2003). Additionally, drought stress modulates gene expression through the dehydration-responsive element (DRE) and their DRE-binding proteins (DREB; Liu et al., 1998), similar to the transcript model ppa007606m encoding a DREB2C-like factor. The product of ppa008849m is similar to calcium binding annexins involved in ABA and osmotic stress signal transduction in Arabidopsis (Lee et al., 2004). Among others we identified ppa008979m (AtMYB44-like), ppa008651m (LEA), ppa012373m (A20/AN1 zinc-finger), ppa005514m (dehydrin) and ppa012188m (AWPM-19-like), involved in cold hardening and frost tolerance.