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Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. The final step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genome-wide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identified ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profiles, determined during dormancy cycle under field and artificially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfillment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple.

BackgroundThe effect of global warming on dormancy and flowering patterns of crop trees threatens world-wide fruit production and food security. In Rosaceous tree species, it is believed that a group of genes encoding MADS transcription factors (TFs) controls temperature-mediated dormancy cycle. These genes are similar toSHORT VEGETATIVE PHASE(SVP) fromArabidopsis thalianaand referred asDORMANCY-ASSOCIATED MADS-BOX(DAM) genes.ResultsBy making use of apple tree (Malusxdomestica) as a model for Rosaceous species, we have investigated the function of MADS TFs during the dormancy cycle. We found that MdDAM and other dormancy related MADS TFs form multimeric complexes with MdSVPa, and that MdSVPa is essential for the transcriptional complex activity. Then, for the first time in non-model plant species, we performed sequential DNA Affinity Purification sequencing (seq-DAP-seq) to define the genome-wide binding sites of these MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained by the inducible glucocorticoid receptor expression system, and reanalyzing preexisting data related to dormancy cycle in apple trees.ConclusionWe have determined a gene regulatory network formed by MdSVPa-containing complexes that regulate the dormancy cycle in apple trees in response to environmental cues. Key genes identified with our genomic approach and the elucidated regulatory relationships provide leads for breeding fruit trees better adapted to changing climate conditions. Moreover, we provide novel molecular evidence on the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae family.

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. Made available in DSpace on 2019-06-12T00:39:12Z (GMT). No. of bitstreams: 1 fpls1000033.pdf: 2246583 bytes, checksum: 39a0aa3363675b7338cebcaa1d270aee (MD5) Previous issue date: 2019

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.

BackgroundFloral transition initiates reproductive development of plants and occurs in response to environmental and endogenous signals. InArabidopsis thaliana, this process is accelerated by several environmental cues, including exposure to long days. The photoperiod-dependent promotion of flowering involves the transcriptional induction ofFLOWERING LOCUS T(FT) in the phloem of the leaf.FTencodes a mobile protein that is transported from the leaves to the shoot apical meristem, where it forms part of a regulatory complex that induces flowering. Whether FT also has biological functions in leaves of wild-type plants remains unclear.ResultsIn order to address this issue, we first studied the leaf transcriptomic changes associated with FT overexpression in the companion cells of the phloem. We found that FT induces the transcription ofSWEET10, which encodes a bidirectional sucrose transporter, specifically in the leaf veins. Moreover,SWEET10is transcriptionally activated by long photoperiods, and this activation depends on FT and one of its earliest target genesSUPPRESSOR OF CONSTANS OVEREXPRESSION 1(SOC1). The ectopic expression ofSWEET10causes early flowering and leads to higher levels of transcription of flowering-time related genes in the shoot apex.ConclusionsCollectively, our results suggest that the FT-signaling pathway activates the transcription of a sucrose uptake/efflux carrier during floral transition, indicating that it alters the metabolism of flowering plants as well as reprogramming the transcription of floral regulators in the shoot meristem.

International audience; 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.

In perennial plants, the release of bud dormancy, with subsequent flowering, resembles the vernalization process of Arabidopsis thaliana and cereals. Especially for perennial crops from temperate regions, dormancy is an important adaptive trait for both survival and growth. Exposure to sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing periods. Here, we compile research data on mechanisms controlling the overlapping developmental processes that define dormancy induction, maintenance and release, bud burst and flowering. Recent findings highlight the relevance of genome-wide epigenetic modifications related to dormancy events, and more specifically the epigenetic regulation of DORMANCY-ASSOCIATED MADS-box, FLOWERING LOCUS C and FLOWERING LOCUS T genes, key integrators of vernalization effectors on flowering. The roles of plant growth regulators in controlling bud break and flowering are discussed in relation to epigenetic mechanisms. A growing body of knowledge demonstrates that epigenetic regulation plays a key role in these processes in perennial horticultural and forestry plants. We discuss the most relevant molecular and genomics research that contribute to better understanding of the dormancy process and pave the way to precise manipulation of dormancy-related horticultural traits, such as flowering time. Finally, some of the challenges for further research in bud dormancy and consequences in agriculture are discussed within the context of global climate change.

Galactinol and raffinose act together to protect dormant buds against limited availability of winter water; the apple galactinol synthases MdGolS1 and MdGolS2 are responsible for their seasonal accumulation during 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.

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.

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 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.

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. Made available in DSpace on 2022-07-26T18:20:33Z (GMT). No. of bitstreams: 1 Functional-diversification-of-the-dehydrin-gene-family-2015.pdf: 1250798 bytes, checksum: 8e044f4a7684c7c056366a414b509d35 (MD5) Previous issue date: 2015

Dormancy is an adaptive mechanism that enables plants to survive unfavorable climatic conditions. This complex process is characterized by the cessation of growth from meristems, which is often accompanied by winter bud set, extensive metabolic remodeling, an acquired high tolerance to cold and/or water deficit and, in deciduous trees, by leaf senescence and abscission. Temperate fruit crops, such as apple and pear, have great economic importance worldwide and their production is closely related to bud dormancy, given that a well-adjusted dormancy cycle is crucial for the achievement of their full genetic potential. These fruits are often classified as pipfruits because of their small hard seeds (pips) in the center of the fruit. Unlike other temperate fruit crops, such as peach and Japanese apricot, pipfruits have peculiarities related to bud dormancy, because instead of being triggered by photoperiodic changes the main regulator of this process is exposure to low temperatures. However, the recent bud dormancy models proposed to peach and poplar only takes into account the research performed in these species. Therefore, the current scenario of genetics and molecular control of bud dormancy in pipfruits is reviewed here. Progress on the identification of heritable components and candidate genes influencing the dormancy process is discussed, as well as the tools available to devise strategies for genetic and genomic analysis of pipfruits.

Key Message Cytokinin together with MdoBRR1, MdoBRR8 and MdoBRR10 genes participate in the downregulation of MdoDAM1, contributing to the transition from endo- to ecodormancy in apple buds. Abstract The fnal step of cytokinin (CK) signaling pathway culminates in the activation of type-B response regulators (BRRs), important transcriptional factors in the modulation of CK-responsive genes. In this study, we performed a genomewide analysis aiming to identify apple BRR family members and understand their involvement in bud dormancy control. The investigation identifed ten MdoBRR protein-coding genes. A higher expression of three MdoBRR (MdoBRR1, MdoBRR9 and MdoBRR10) was observed in dormant buds in comparison to other developmental stages. Interestingly, in ecodormant buds these three MdoBRR genes were upregulated in a CK-dependent manner. Transcription profles, determined during dormancy cycle under feld and artifcially controlled conditions, revealed that MdoBRR1 and MdoBRR8 played important roles in the transition from endo- to ecodormancy, probably mediated by endogenous CK stimuli. The expression of MdoBRR7, MdoBRR9, and MdoBRR10 was induced in ecodormant buds exposed to warm temperatures, indicating a putative role in growth resumption after chilling requirement fulfllment. Contrasting expression patternsin vivo between MdoBRRs and MdoDAM1, an essential dormancy establishment regulator, were observed during dormancy cycle and in CK-treated buds. Thereafter, in vivo transactivation assays showed that CK stimuli combined with transient overexpression of MdoBRR1, MdoBRR8, and MdoBRR10 resulted in downregulation of the reporter gene gusA driven by the MdoDAM1 promoter. These pieces of evidences point to the integration of CK-triggered responses through MdoBRRs that are able to downregulate MdoDAM1, contributing to dormancy release in apple. Keywords MdoBRR · Cytokinin · Bud dormancy · MdoDAM1 · Apple Made available in DSpace on 2020-12-10T09:03:34Z (GMT). No. of bitstreams: 1 10.1007-s00299-020-02595-z.pdf: 3550323 bytes, checksum: 05420c80b17b97786fe271e78e5df069 (MD5) Previous issue date: 2020