Evolutionary origin and functional specialization of Dormancy-Associated MADS box (DAM) proteins in perennial crops.

BACKGROUND: Bud dormancy is a phenological adaptation of temperate perennials that ensures survival under winter temperature conditions by ceasing growth and increasing cold hardiness. SHORT VEGETATIVE PHASE (SVP)-like factors, and particularly a subset of them named DORMANCY-ASSOCIATED MADS-BOX (DAM), are master regulators of bud dormancy in perennials, prominently Rosaceae crops widely adapted to varying environmental conditions. RESULTS: SVP-like proteins from recently sequenced Rosaceae genomes were identified and characterized using sequence, phylogenetic and synteny analysis tools. SVP-like proteins clustered in three clades (SVP1-3), with known DAM proteins located within SVP2 clade, which also included Arabidopsis AGAMOUS-LIKE 24 (AthAGL24). A more detailed study on these protein sequences led to the identification of a 15-amino acid long motif specific to DAM proteins, which affected protein heteromerization properties by yeast two-hybrid system in peach PpeDAM6, and the unexpected finding of predicted DAM-like genes in loquat, an evergreen species lacking winter dormancy. DAM gene expression in loquat trees was studied by quantitative PCR, associating with inflorescence development and growth in varieties with contrasting flowering behaviour. CONCLUSIONS: Phylogenetic, synteny analyses and heterologous overexpression in the model plant Arabidopsis thaliana supported three major conclusions: 1) DAM proteins might have emerged from the SVP2 clade in the Amygdaloideae subfamily of Rosaceae; 2) a short DAM-specific motif affects protein heteromerization, with a likely effect on DAM transcriptional targets and other functional features, providing a sequence signature for the DAM group of dormancy factors; 3) in agreement with other recent studies, DAM associates with inflorescence development and growth, independently of the dormancy habit.

Characterization of the Spanish Pomegranate Germplasm Collection Maintained at the Agricultural Experiment Station of Elche to Identify Promising Breeding Materials.

Pomegranates were one of the first domesticated fruit crops, and their long history resulted in the development of local cultivars all over the world. Spain is one of the main producers and exporters of this crop in the Mediterranean Basin, but in order to maintain the competitiveness of this crop, new varieties should be developed. For this purpose, the pomegranate germplasm collection hold at the Agricultural Experiment Station of Elche, a public institution dependent on the Valencian regional government, is an interesting tool. However, the detailed characterization of any germplasm collection is a fundamental requirement to be able to make the most of these resources, allowing to identify putative promising accessions and to optimize the design of the future crosses. In this work, the genetic diversity of 94 accessions of this collection was analyzed using 19 microsatellite markers. As a result, 85 different genotypes were identified. These genetic profiles could be useful for varietal identification. Despite this genetic diversity, no clear substructure was observed, except for the ornamental accessions, that could be related to the vegetative propagation of the species. Additionally, the morphological characterization of this collection has made it possible to identify some materials that may be of interest as a source of traits for breeding. Results presented here pave the way for further genetic analyses, allowing the selection of parents to obtain segregating populations, as well as their descendants by the use of molecular assisted selection.

Regulatory circuits involving bud dormancy factor PpeDAM6.

DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression.

Structure and Expression of Bud Dormancy-Associated MADS-Box Genes (DAM) in European Plum.

Bud dormancy in temperate perennials ensures the survival of growing meristems under the harsh environmental conditions of autumn and winter, and facilitates an optimal growth and development resumption in the spring. Although the molecular pathways controlling the dormancy process are still unclear, DORMANCY-ASSOCIATED MADS-BOX genes (DAM) have emerged as key regulators of the dormancy cycle in different species. In the present study, we have characterized the orthologs of DAM genes in European plum (Prunus domestica L.). Their expression patterns together with sequence similarities are consistent with a role of PdoDAMs in dormancy maintenance mechanisms in European plum. Furthermore, other genes related to dormancy, flowering, and stress response have been identified in order to obtain a molecular framework of these three different processes taking place within the dormant flower bud in this species. This research provides a set of candidate genes to be genetically modified in future research, in order to better understand dormancy regulation in perennial species.

Intra and Inter-specific Variability of Salt Tolerance Mechanisms in Diospyros Genus.

Saline stress is one of most important problems that agriculture must face in the context of climate change. In the Mediterranean basin, one of the regions most affected, persimmon production can be compromised by this effect, due to the limited availability of salt tolerant rootstocks. Seedlings coming from four populations from the Diospyros genus have been exposed to salt stress in order to identify salt tolerance genotypes within these populations. Morphological, physiological, and transcriptomic approaches have revealed different mechanisms of tolerance among the population studied. An HKT1-like gene has been shown to have different root expression related to the salt tolerance phenotypes among and within populations. Additionally, we have observed differences in salt-responsive expression among PIP aquaporin genes. Genetic variability for salt tolerance can be generated in Diospyros species through crossings and used for overcome salt stress. Furthermore, differences in water use efficiency (WUE) have been obtained between and within populations. The information gathered at transcriptomic and physiological level demonstrated natural and heritable variability among Diospyros genus which is the key for salt-tolerant rootstock breeding programs.

MBW complexes impinge on anthocyanidin reductase gene regulation for proanthocyanidin biosynthesis in persimmon fruit.

MBW protein complexes containing MYB, bHLH and WD40 repeat factors are known transcriptional regulators of secondary metabolites production such as proanthocyanidins and anthocyanins, and developmental processes such as trichome formation in many plant species. DkMYB2 and DkMYB4 (MYB-type), DkMYC1 (bHLH-type) and DkWDR1 (WD40-type) factors have been proposed by different authors to take part of persimmon MBW complexes for proanthocyanidin accumulation in immature fruit, leading to its characteristic astringent flavour with important agronomical and ecological effects. We have confirmed the nuclear localization of these proteins and their mutual physical interaction by bimolecular fluorescence complementation analysis. In addition, transient expression of DkMYB2, DkMYB4 and DkMYC1 cooperatively increase the expression of a persimmon anthocyanidin reductase gene (ANR), involved in the biosynthesis of cis-flavan-3-ols, the structural units of proanthocyanidin compounds. Collectively, these data support the presence of MBW complexes in persimmon fruit and suggest their coordinated participation in ANR regulation for proanthocyanidin production.

Modulation of Dormancy and Growth Responses in Reproductive Buds of Temperate Trees.

During autumn perennial trees cease growth and form structures called buds in order to protect meristems from the unfavorable environmental conditions, including low temperature and desiccation. In addition to increased tolerance to these abiotic stresses, reproductive buds modulate developmental programs leading to dormancy induction to avoid premature growth resumption, and flowering pathways. Stress tolerance, dormancy, and flowering processes are thus physically and temporarily restricted to a bud, and consequently forced to interact at the regulatory level. We review recent genomic, genetic, and molecular contributions to the knowledge of these three processes in trees, highlighting the role of epigenetic modifications, phytohormones, and common regulatory factors. Finally, we emphasize the utility of transcriptomic approaches for the identification of key structural and regulatory genes involved in bud processes, illustrated with our own experience using peach as a model.

Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes.

BACKGROUND: Plum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes. RESULTS: Here, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as Prunus armeniaca PPVres MATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1. CONCLUSIONS: Accordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.

Chromatin-associated regulation of sorbitol synthesis in flower buds of peach.

KEY MESSAGE: PpeS6PDH gene is postulated to mediate sorbitol synthesis in flower buds of peach concomitantly with specific chromatin modifications. Perennial plants have evolved an adaptive mechanism involving protection of meristems within specialized structures named buds in order to survive low temperatures and water deprivation during winter. A seasonal period of dormancy further improves tolerance of buds to environmental stresses through specific mechanisms poorly known at the molecular level. We have shown that peach PpeS6PDH gene is down-regulated in flower buds after dormancy release, concomitantly with changes in the methylation level at specific lysine residues of histone H3 (H3K27 and H3K4) in the chromatin around the translation start site of the gene. PpeS6PDH encodes a NADPH-dependent sorbitol-6-phosphate dehydrogenase, the key enzyme for biosynthesis of sorbitol. Consistently, sorbitol accumulates in dormant buds showing higher PpeS6PDH expression. Moreover, PpeS6PDH gene expression is affected by cold and water deficit stress. Particularly, its expression is up-regulated by low temperature in buds and leaves, whereas desiccation treatment induces PpeS6PDH in buds and represses the gene in leaves. These data reveal the concurrent participation of chromatin modification mechanisms, transcriptional regulation of PpeS6PDH and sorbitol accumulation in flower buds of peach. In addition to its role as a major translocatable photosynthate in Rosaceae species, sorbitol is a widespread compatible solute and cryoprotectant, which suggests its participation in tolerance to environmental stresses in flower buds of peach.

Dual regulation of water retention and cell growth by a stress-associated protein (SAP) gene in Prunus.

We have identified a gene (PpSAP1) of Prunus persica coding for a stress-associated protein (SAP) containing Zn-finger domains A20 and AN1. SAPs have been described as regulators of the abiotic stress response in plant species, emerging as potential candidates for improvement of stress tolerance in plants. PpSAP1 was highly expressed in leaves and dormant buds, being down-regulated before bud dormancy release. PpSAP1 expression was moderately induced by water stresses and heat in buds. In addition, it was found that PpSAP1 strongly interacts with polyubiquitin proteins in the yeast two-hybrid system. The overexpression of PpSAP1 in transgenic plum plants led to alterations in leaf shape and an increase of water retention under drought stress. Moreover, we established that leaf morphological alterations were concomitant with a reduced cell size and down-regulation of genes involved in cell growth, such as GROWTH-REGULATING FACTOR (GRF)1-like, TONOPLAST INTRINSIC PROTEIN (TIP)-like, and TARGET OF RAPAMYCIN (TOR)-like. Especially, the inverse expression pattern of PpSAP1 and TOR-like in transgenic plum and peach buds suggests a role of PpSAP1 in cell expansion through the regulation of TOR pathway.

Induced parthenogenesis by gamma-irradiated pollen in loquat for haploid production.

Successful haploid induction in loquat (Eriobotrya japonica (Thunb.) Lindl.) through in situ-induced parthenogenesis with gamma-ray irradiated pollen has been achieved. Female flowers of cultivar 'Algerie' were pollinated using pollen of cultivars 'Changhong-3', 'Cox' and 'Saval Brasil' irradiated with two doses of gamma rays, 150 and 300 Gy. The fruits were harvested 90, 105 and 120 days after pollination (dap). Four haploid plants were obtained from 'Algerie' pollinated with 300-Gy-treated pollen of 'Saval Brasil' from fruits harvested 105 dap. Haploidy was confirmed by flow cytometry and chromosome count. The haploids showed a very weak development compared to the diploid plants. This result suggests that irradiated pollen can be used to obtain parthenogenetic haploids.

Epigenetic regulation of bud dormancy events in perennial plants.

Release of bud dormancy in perennial plants resembles vernalization in Arabidopsis thaliana and cereals. In both cases, a certain period of chilling is required for accomplishing the reproductive phase, and several transcription factors with the MADS-box domain perform a central regulatory role in these processes. The expression of DORMANCY-ASSOCIATED MADS-box (DAM)-related genes has been found to be up-regulated in dormant buds of numerous plant species, such as poplar, raspberry, leafy spurge, blackcurrant, Japanese apricot, and peach. Moreover, functional evidence suggests the involvement of DAM genes in the regulation of seasonal dormancy in peach. Recent findings highlight the presence of genome-wide epigenetic modifications related to dormancy events, and more specifically the epigenetic regulation of DAM-related genes in a similar way to FLOWERING LOCUS C, a key integrator of vernalization effectors on flowering initiation in Arabidopsis. We revise the most relevant molecular and genomic contributions in the field of bud dormancy, and discuss the increasing evidence for chromatin modification involvement in the epigenetic regulation of seasonal dormancy cycles in perennial plants.

Genomic analysis reveals MATH gene(s) as candidate(s) for Plum pox virus (PPV) resistance in apricot (Prunus armeniaca†L.).

Sharka disease, caused by Plum pox virus (PPV), is the most important viral disease affecting Prunus species. A major PPV resistance locus (PPVres) has been mapped to the upper part of apricot (Prunus armeniaca) linkage group 1. In this study, a physical map of the PPVres locus in the PPV-resistant cultivar 'Goldrich' was constructed. Bacterial artificial chromosome (BAC) clones belonging to the resistant haplotype contig were sequenced using 454/GS-FLX Titanium technology. Concurrently, the whole genome of seven apricot varieties (three PPV-resistant and four PPV-susceptible) and two PPV-susceptible apricot relatives (P. sibirica var. davidiana and P. mume) were obtained using the Illumina-HiSeq2000 platform. Single nucleotide polymorphisms (SNPs) within the mapped interval, recorded from alignments against the peach genome, allowed us to narrow down the PPVres locus to a region of ∼196 kb. Searches for polymorphisms linked in coupling with the resistance led to the identification of 68 variants within 23 predicted transcripts according to peach genome annotation. Candidate resistance genes were ranked combining data from variant calling and predicted functions inferred from sequence homology. Together, the results suggest that members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing proteins are the most likely candidate genes for PPV resistance in apricot. Interestingly, MATHd proteins are hypothesized to control long-distance movement (LDM) of potyviruses in Arabidopsis, and restriction for LDM is also a major component of PPV resistance in apricot. Although the PPV resistance gene(s) remains to be unambiguously identified, these results pave the way to the determination of the underlying mechanism and to the development of more accurate breeding strategies.

A non-targeted approach unravels the volatile network in peach fruit.

Volatile compounds represent an important part of the plant metabolome and are of particular agronomic and biological interest due to their contribution to fruit aroma and flavor and therefore to fruit quality. By using a non-targeted approach based on HS-SPME-GC-MS, the volatile-compound complement of peach fruit was described. A total of 110 volatile compounds (including alcohols, ketones, aldehydes, esters, lactones, carboxylic acids, phenolics and terpenoids) were identified and quantified in peach fruit samples from different genetic backgrounds, locations, maturity stages and physiological responses. By using a combination of hierarchical cluster analysis and metabolomic correlation network analysis we found that previously known peach fruit volatiles are clustered according to their chemical nature or known biosynthetic pathways. Moreover, novel volatiles that had not yet been described in peach were identified and assigned to co-regulated groups. In addition, our analyses showed that most of the co-regulated groups showed good intergroup correlations that are therefore consistent with the existence of a higher level of regulation orchestrating volatile production under different conditions and/or developmental stages. In addition, this volatile network of interactions provides the ground information for future biochemical studies as well as a useful route map for breeding or biotechnological purposes.

Chilling-dependent release of seed and bud dormancy in peach associates to common changes in gene expression.

Reproductive meristems and embryos display dormancy mechanisms in specialized structures named respectively buds and seeds that arrest the growth of perennial plants until environmental conditions are optimal for survival. Dormancy shows common physiological features in buds and seeds. A genotype-specific period of chilling is usually required to release dormancy by molecular mechanisms that are still poorly understood. In order to find common transcriptional pathways associated to dormancy release, we analyzed the chilling-dependent expression in embryos of certain genes that were previously found related to dormancy in flower buds of peach. We propose the presence of short and long-term dormancy events affecting respectively the germination rate and seedling development by independent mechanisms. Short periods of chilling seem to improve germination in an abscisic acid-dependent manner, whereas the positive effect of longer cold treatments on physiological dwarfing coincides with the accumulation of phenylpropanoids in the seed.

Physical mapping of a pollen modifier locus controlling self-incompatibility in apricot and synteny analysis within the Rosaceae.

S-locus products (S-RNase and F-box proteins) are essential for the gametophytic self-incompatibility (GSI) specific recognition in Prunus. However, accumulated genetic evidence suggests that other S-locus unlinked factors are also required for GSI. For instance, GSI breakdown was associated with a pollen-part mutation unlinked to the S-locus in the apricot (Prunus armeniaca L.) cv. 'Canino'. Fine-mapping of this mutated modifier gene (M-locus) and the synteny analysis of the M-locus within the Rosaceae are here reported. A segregation distortion loci mapping strategy, based on a selectively genotyped population, was used to map the M-locus. In addition, a bacterial artificial chromosome (BAC) contig was constructed for this region using overlapping oligonucleotides probes, and BAC-end sequences (BES) were blasted against Rosaceae genomes to perform micro-synteny analysis. The M-locus was mapped to the distal part of chr.3 flanked by two SSR markers within an interval of 1.8 cM corresponding to ~364 Kb in the peach (Prunus persica L. Batsch) genome. In the integrated genetic-physical map of this region, BES were mapped against the peach scaffold_3 and BACs were anchored to the apricot map. Micro-syntenic blocks were detected in apple (Malus × domestica Borkh.) LG17/9 and strawberry (Fragaria vesca L.) FG6 chromosomes. The M-locus fine-scale mapping provides a solid basis for self-compatibility marker-assisted selection and for positional cloning of the underlying gene, a necessary goal to elucidate the pollen rejection mechanism in Prunus. In a wider context, the syntenic regions identified in peach, apple and strawberry might be useful to interpret GSI evolution in Rosaceae.

Histone modifications and expression of DAM6 gene in peach are modulated during bud dormancy release in a cultivar-dependent manner.

• Bud dormancy release in many woody perennial plants responds to the seasonal accumulation of chilling stimulus. MADS-box transcription factors encoded by DORMANCY ASSOCIATED MADS-box (DAM) genes in peach (Prunus persica) are implicated in this pathway, but other regulatory factors remain to be identified. In addition, the regulation of DAM gene expression is not well known at the molecular level. • A microarray hybridization approach was performed to identify genes whose expression correlates with the bud dormancy-related behaviour in 10 different peach cultivars. Histone modifications in DAM6 gene were investigated by chromatin immunoprecipitation in two different cultivars. • The expression of DAM4-DAM6 and several genes related to abscisic acid and drought stress response correlated with the dormancy behaviour of peach cultivars. The trimethylation of histone H3 at K27 in the DAM6 promoter, coding region and the second large intron was preceded by a decrease in acetylated H3 and trimethylated H3K4 in the region of translation start, coinciding with repression of DAM6 during dormancy release. • Analysis of chromatin modifications reinforced the role of epigenetic mechanisms in DAM6 regulation and bud dormancy release, and highlighted common features with the vernalization process in Arabidopsis thaliana and cereals.

Narrowing down the apricot Plum pox virus resistance locus and comparative analysis with the peach genome syntenic region.

Sharka disease, caused by the Plum pox virus (PPV), is one of the main limiting factors for stone fruit crops worldwide. Only a few resistance sources have been found in apricot (Prunus armeniaca L.), and most studies have located a major PPV resistance locus (PPVres) on linkage group 1 (LG1). However, the mapping accuracy was not sufficiently reliable and PPVres was predicted within a low confidence interval. In this study, we have constructed two high-density simple sequence repeat (SSR) improved maps with 0.70 and 0.68 markers/cm, corresponding to LG1 of 'Lito' and 'Goldrich' PPV-resistant cultivars, respectively. Using these maps, and excluding genotype-phenotype incongruent individuals, a new binary trait locus (BTL) analysis for PPV resistance was performed, narrowing down the PPVres support intervals to 7.3 and 5.9 cm in 'Lito' and 'Goldrich', respectively. Subsequently, 71 overlapping oligonucleotides (overgo) probes were hybridized against an apricot bacterial artificial chromosome (BAC) library, identifying 870 single BACs from which 340 were anchored onto a map region of approximately 30-40 cm encompassing PPVres. Partial BAC contigs assigned to the two allelic haplotypes (resistant/susceptible) of the PPVres locus were built by high-information content fingerprinting (HICF). In addition, a total of 300 BAC-derived sequences were obtained, and 257 showed significant homology with the peach genome scaffold_1 corresponding to LG1. According to the peach syntenic genome sequence, PPVres was predicted within a region of 2.16 Mb in which a few candidate resistance genes were identified.