Correction: The bZIP transcription factor AREB3 mediates FT signalling and floral transition at the Arabidopsis shoot apical meristem.

[This corrects the article DOI: 10.1371/journal.pgen.1010766.].

Characterization of the nucellus-specific dehydrin MdoDHN11 demonstrates its involvement in the tolerance to water deficit.

KEY MESSAGE: MdoDHN11 acts in the nucellus layer to protect the embryo and the endosperm from limited water availability during apple seed development. Dehydrins (DHNs) are protective proteins related to several plant developmental responses that involve dehydration such as seed desiccation and abiotic stresses. In apple (Malus × domestica Borkh.), the seed-specific MdoDHN11 was suggested to play important roles against dehydration during seed development. However, this hypothesis has not yet been evaluated. Within this context, several experiments were performed to functionally characterize MdoDHN11. In situ hybridization analysis during apple seed development showed that MdoDHN11 expression is confined to a maternal tissue called nucellus, a central mass of parenchyma between the endosperm and the testa. The MdoDHN11 protein was localized in the cytosol and nucleus. Finally, transgenic Arabidopsis plants expressing MdoDHN11 were generated and exposed to a severe water-deficit stress, aiming to mimic a situation that can occurs during seed development. All transgenic lines showed increased tolerance to water deficit in relation to wild-type plants. Taken together, our results provide evidences that MdoDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from limited water availability, and the mechanism of action probably involves the interaction of MdoDHN11 with proteins and other components in the cell.

Evolutionary diversification of galactinol synthases in Rosaceae: adaptive roles of galactinol and raffinose during apple bud dormancy.

Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply.

Functional diversification of the dehydrin gene family in apple and its contribution to cold acclimation during dormancy.

Dehydrins (DHN) are proteins involved in plant adaptive responses to abiotic stresses, mainly dehydration. Several studies in perennial crops have linked bud dormancy progression, a process characterized by the inability to initiate growth from meristems under favorable conditions, with DHN gene expression. However, an in-depth characterization of DHNs during bud dormancy progression is still missing. An extensive in silico characterization of the apple DHN gene family was performed. Additionally, we used five different experiments that generated samples with different dormancy status, including genotypes with contrasting dormancy traits, to analyze how DHN genes are being regulated during bud dormancy progression in apple by real-time quantitative polymerase chain reaction (RT-qPCR). Duplication events took place in the diversification of apple DHN family. Additionally, MdDHN genes presented tissue- and bud dormant-specific expression patterns. Our results indicate that MdDHN genes are highly divergent in function, with overlapping levels, and that their expressions are fine-tuned by the environment during the dormancy process in apple.

Target enrichment sequencing coupled with GWAS identifies MdPRX10 as a candidate gene in the control of budbreak in apple.

The timing of floral budbreak in apple has a significant effect on fruit production and quality. Budbreak occurs as a result of a complex molecular mechanism that relies on accurate integration of external environmental cues, principally temperature. In the pursuit of understanding this mechanism, especially with respect to aiding adaptation to climate change, a QTL at the top of linkage group (LG) 9 has been identified by many studies on budbreak, but the genes underlying it remain elusive. Here, together with a dessert apple core collection of 239 cultivars, we used a targeted capture sequencing approach to increase SNP resolution in apple orthologues of known or suspected A. thaliana flowering time-related genes, as well as approximately 200 genes within the LG9 QTL interval. This increased the 275 223 SNP Axiom® Apple 480 K array dataset by an additional 40 857 markers. Robust GWAS analyses identified MdPRX10, a peroxidase superfamily gene, as a strong candidate that demonstrated a dormancy-related expression pattern and down-regulation in response to chilling. In-silico analyses also predicted the residue change resulting from the SNP allele associated with late budbreak could alter protein conformation and likely function. Late budbreak cultivars homozygous for this SNP allele also showed significantly up-regulated expression of C-REPEAT BINDING FACTOR (CBF) genes, which are involved in cold tolerance and perception, compared to reference cultivars, such as Gala. Taken together, these results indicate a role for MdPRX10 in budbreak, potentially via redox-mediated signaling and CBF gene regulation. Moving forward, this provides a focus for developing our understanding of the effects of temperature on flowering time and how redox processes may influence integration of external cues in dormancy pathways.

An efficient protocol for functional studies of apple transcription factors using a glucocorticoid receptor fusion system.

PREMISE: We report a protocol for studying the function of apple (Malus ×domestica) transcription factors based on the glucocorticoid receptor (GR) system, which allows the dexamethasone (DEX)-mediated activation of plant transcription factors to monitor the expression levels of their potential target genes. METHODS AND RESULTS: Apple leaves are transformed with a vector that allows the expression of the studied transcription factor (i.e., FLOWERING LOCUS C [MdFLC]) fused to GR. Calli derived from the transformed leaves are treated with DEX and cycloheximide, a protein synthesis inhibitor. Compared with other methods, combining the GR system with cycloheximide treatments enables the differentiation between direct and indirect transcription factor target genes. Finally, the expression levels of putative MdFLC target genes are quantified using quantitative reverse transcription PCR. CONCLUSIONS: We demonstrate the efficiency of our GR system to study the function of apple transcription factors. This method is accessible to any laboratory familiar with basic molecular cloning procedures and the apple leaf-mediated agro-transformation technique.

Spring Is Coming: Genetic Analyses of the Bud Break Date Locus Reveal Candidate Genes From the Cold Perception Pathway to Dormancy Release in Apple (Malus × domestica Borkh.).

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.

I Want to (Bud) Break Free: The Potential Role of DAM and SVP-Like Genes in Regulating Dormancy Cycle in Temperate Fruit Trees.

Bud dormancy is an adaptive process that allows trees to survive the hard environmental conditions that they experience during the winter of temperate climates. Dormancy is characterized by the reduction in meristematic activity and the absence of visible growth. A prolonged exposure to cold temperatures is required to allow the bud resuming growth in response to warm temperatures. In fruit tree species, the dormancy cycle is believed to be regulated by a group of genes encoding MADS-box transcription factors. These genes are called DORMANCY-ASSOCIATED MADS-BOX (DAM) and are phylogenetically related to the Arabidopsis thaliana floral regulators SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE 24. The interest in DAM and other orthologs of SVP (SVP-like) genes has notably increased due to the publication of several reports suggesting their role in the control of bud dormancy in numerous fruit species, including apple, pear, peach, Japanese apricot, and kiwifruit among others. In this review, we briefly describe the physiological bases of the dormancy cycle and how it is genetically regulated, with a particular emphasis on DAM and SVP-like genes. We also provide a detailed report of the most recent advances about the transcriptional regulation of these genes by seasonal cues, epigenetics and plant hormones. From this information, we propose a tentative classification of DAM and SVP-like genes based on their seasonal pattern of expression. Furthermore, we discuss the potential biological role of DAM and SVP-like genes in bud dormancy in antagonizing the function of FLOWERING LOCUS T-like genes. Finally, we draw a global picture of the possible role of DAM and SVP-like genes in the bud dormancy cycle and propose a model that integrates these genes in a molecular network of dormancy cycle regulation in temperate fruit trees.