The production of temperate fruit crops depends on plant developmental processes, primarily the shift from the juvenile phase to the reproductive phase, dormancy transitions and flowering. Apple tree (Malus ×domestica Borkh.) development is regulated by chilling temperatures, which are required for bud dormancy progression. The apple cultivar Castel Gala is a spontaneous mutation of "Gala Standard". "Castel Gala" is characterized by a 50 % decrease in the chilling requirement (CR) for dormancy release, which results in an earlier budbreak. This work explores the contrasting phenotypes of these cultivars using suppression subtractive hybridization (SSH). From 1,019 unigenes identified by SSH, we selected 28 candidate genes putatively associated with dormancy cycling. Reverse transcription-quantitative polymerase chain reaction was used to validate the differential expression profiles and to transcriptionally characterize these genes in three distinct apple cultivars ("Castel Gala", "Royal Gala" and "Fuji Standard") during a cycle comprising growth to dormancy. Of the 28 candidate genes analyzed, 17 confirmed the differences in expression predicted by SSH. Seasonal transcript accumulation during the winter was observed for several genes, with higher steady-state mRNA levels maintained longer in cultivars with a high CR. The transcription profiles suggest that these genes may be associated with dormancy establishment and maintenance. Of the 17 candidate genes, transcripts coding for dormancy-associated MADS-box (DAM), dehydrins, GAST1, LTI65, NAC, HTA8, HTA12 and RAP2.12-like proteins displayed major differences in gene expression between cultivars through the winter. These genes were therefore considered good candidates for key roles in the dormancy process in apple trees. Keywords Apple . Bud dormancy . Gene expression . Malus ×domestica . RT-qPCR . Suppression subtractive hybridization Abbreviations ABA Abscisic acid AP2 APETALA2 ARC5 Accumulation and replication of chloroplast 5 ARP6 Actin-related protein 6 CAMTA1 Calmodulin-binding transcription activator 1 CBF C-repeat binding factor CO CONSTANS COR Cold-regulated CR Chilling requirement CRT C-repeat DAM Dormancy-associated MADS-box DHN Dehydrin DRE Dehydration-responsive element DREB Dehydration-responsive element binding protein EST Expressed sequence tag FT FLOWERING LOCUS T GAST1 GA stimulated transcript 1 GO Gene ontologyThe nucleotide sequences reported in this paper have been submitted to GenBank with the accession numbers JZ480898 to JZ482228.

The molecular control of bud dormancy establishment and release is still not well understood, although some genes have already been demonstrated to play important roles in this process. The dormancy-associated MADS-box (DAM) genes were first identified in the peach EVERGROWING locus and are considered the main regulators of bud dormancy control. In this work, the apple (Malus × domestica Borkh.), a perennial plant adapted to temperate climates that displays cycles of growth and bud dormancy, was screened for the presence of DAM genes. The candidate genes retrieved were characterized in comparison to DAM genes from other species. Four of them (MdDAM1-4) are structurally very similar to the reported DAM genes. When apple genomic segments containing these candidates were compared to the peach EVERGROWING locus, a highly conserved noncoding region was detected inside their largest intron. Similar sequences were also identified inside introns of apricot and pear DAM genes. Organ expression patterns revealed that MdDAM1-4 are mainly expressed in dormant buds and seeds, with low transcript accumulation in vegetative structures. In addition, the MdDAM genes showed seasonally oscillating patterns of steady-state messenger RNA (mRNA) levels and were downregulated by artificial chilling. Motif analyses in the promoter and in the intronic conserved region of the MdDAM genes disclosed some clues to the regulation of the expression patterns observed. Possible roles for the conserved intronic sequence in dormancy regulation are discussed.

The Dof (DNA-binding with one finger) protein family spans a group of plant transcription factors involved in the regulation of several functions, such as plant responses to stress, hormones and light, phytochrome signaling and seed germination. Here we describe the Dof-like gene family in grapevine (Vitis vinifera L.), which consists of 25 genes coding for Dof. An extensive in silico characterization of the VviDofL gene family was performed. Additionally, the expression of the entire gene family was assessed in 54 grapevine tissues and organs using an integrated approach with microarray (cv Corvina) and real-time PCR (cv Pinot Noir) analyses. The phylogenetic analysis comparing grapevine sequences with those of Arabidopsis, tomato, poplar and already described Dof genes in other species allowed us to identify several duplicated genes. The diversification of grapevine DofL genes during evolution likely resulted in a broader range of biological roles. Furthermore, distinct expression patterns were identified between samples analyzed, corroborating such hypothesis. Our expression results indicate that several VviDofL genes perform their functional roles mainly during flower, berry and seed development, highlighting their importance for grapevine growth and production. The identification of similar expression profiles between both approaches strongly suggests that these genes have important regulatory roles that are evolutionally conserved between grapevine cvs Corvina and Pinot Noir.

Chilling requirement (CR) for bud dormancy completion determines the time of bud break in apple (Malus × domestica Borkh.). The molecular control of bud dormancy is highly heritable, suggesting a strong genetic control of the trait. An available Infinium II SNP platform for genotyping containing 8,788 single nucleotide polymorphic markers was employed, and linkage maps were constructed in a F1 cross from the low CR M13/91 and the moderate CR cv. Fred Hough. These maps were used to identify quantitative trait loci (QTL) for bud break date as a trait related to dormancy release. A major QTL for bud break was detected at the beginning of linkage group 9 (LG9). This QTL remained stable during seven seasons in two different growing sites. To increase mapping efficiency in detecting contributing genes underlying this QTL, 182 additional SNP markers located at the locus for bud break were used. Combining linkage mapping and structural characterization of the region, the high proportion of the phenotypic variance in the trait explained by the QTL is related to the coincident positioning of Arabidopsis orthologs for ICE1, FLC, and PRE1 protein-coding genes. The proximity of these genes from the most explanatory markers of this QTL for bud break suggests potential genetic additive effects, reinforcing the hypothesis of inter-dependent mechanisms controlling dormancy induction and release in apple trees.

Seedlessness in grapes is a desirable trait, especially for in natura consumption. Previously, we showed that VviAGL11 is the main responsible gene for seed morphogenesis in grapevine. Here we tested the function of this gene in grapevine with the use of plant plasmids. VviAGL11 was cloned into silencing and overexpression versions of p28iIR plasmid. Reproductive grapevine bunches from different seeded and seedless cultivars were separately treated with VviAGL11-harboring plasmids, along with controls. Plasmids were detected in leaves after a month of treatment, and berries, leaves, stems and seeds were analyzed for ectopic gene expression by RT-qPCR after 90 days of plasmid injection. Fruits from the seedless 'Linda' treated with the VviAGL11-overexpression plasmid showed high expression levels of VviAGL11 and exhibited small seeds that were not found in the untreated control samples. Mature grapes from seeded 'Italia' and 'Ruby' bunches treated with the VviAGL11-silencing plasmid showed decreased VviAGL11 expression, reduced number of seeds and increased number of seed traces. The present study confirms that VviAGL11 is a key master regulator of seed morphogenesis in grapevine and corroborates with the applicability of plant plasmids as promising biotechnological tools to functionally test genes in perennial plants in a rapid and confident way.