Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins.

Eukaryotic genome replication depends on thousands of DNA replication origins (ORIs). A major challenge is to learn ORI biology in multicellular organisms in the context of growing organs to understand their developmental plasticity. We have identified a set of ORIs of Arabidopsis thaliana and their chromatin landscape at two stages of post-embryonic development. ORIs associate with multiple chromatin signatures including transcription start sites (TSS) but also proximal and distal regulatory regions and heterochromatin, where ORIs colocalize with retrotransposons. In addition, quantitative analysis of ORI activity led us to conclude that strong ORIs have high GC content and clusters of GGN trinucleotides. Development primarily influences ORI firing strength rather than ORI location. ORIs that preferentially fire at early developmental stages colocalize with GC-rich heterochromatin, but at later stages with transcribed genes, perhaps as a consequence of changes in chromatin features associated with developmental processes. Our study provides the set of ORIs active in an organism at the post-embryo stage that should allow us to study ORI biology in response to development, environment, and mutations with a quantitative approach. In a wider scope, the computational strategies developed here can be transferred to other eukaryotic systems.

The Functional Topography of the Arabidopsis Genome Is Organized in a Reduced Number of Linear Motifs of Chromatin States.

Chromatin is of major relevance for gene expression, cell division, and differentiation. Here, we determined the landscape of Arabidopsis thaliana chromatin states using 16 features, including DNA sequence, CG methylation, histone variants, and modifications. The combinatorial complexity of chromatin can be reduced to nine states that describe chromatin with high resolution and robustness. Each chromatin state has a strong propensity to associate with a subset of other states defining a discrete number of chromatin motifs. These topographical relationships revealed that an intergenic state, characterized by H3K27me3 and slightly enriched in activation marks, physically separates the canonical Polycomb chromatin and two heterochromatin states from the rest of the euchromatin domains. Genomic elements are distinguished by specific chromatin states: four states span genes from transcriptional start sites (TSS) to termination sites and two contain regulatory regions upstream of TSS. Polycomb regions and the rest of the euchromatin can be connected by two major chromatin paths. Sequential chromatin immunoprecipitation experiments demonstrated the occurrence of H3K27me3 and H3K4me3 in the same chromatin fiber, within a two to three nucleosome size range. Our data provide insight into the Arabidopsis genome topography and the establishment of gene expression patterns, specification of DNA replication origins, and definition of chromatin domains.

Ethylene is involved in strawberry fruit ripening in an organ-specific manner.

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.

Metabolic engineering of aroma components in fruits.

Plants have the ability to produce a diversity of volatile metabolites, which attract pollinators and seed dispersers and strengthen plant defense responses. Selection by plant breeders of traits such as rapid growth and yield leads, in many cases, to the loss of flavor and aroma quality in crops. How the aroma can be improved without affecting other fruit attributes is a major unsolved issue. Significant advances in metabolic engineering directed at improving the set of volatiles that the fruits emit has been aided by the characterization of enzymes involved in the biosynthesis of flavor and aroma compounds in some fruits. However, before this technology can be successfully applied to modulate the production of volatiles in different crops, further basic research is needed on the mechanisms that lead to the production of these compounds in plants. Here we review the biosynthesis and function of volatile compounds in plants, and the attempts that have been made to manipulate fruit aroma biosynthesis by metabolic engineering. In addition, we discuss the possibilities that molecular breeding offers for aroma enhancement and the implications of the latest advances in biotechnological modification of fruit flavor and aroma.

Eugenol production in achenes and receptacles of strawberry fruits is catalyzed by synthases exhibiting distinct kinetics.

Eugenol is a volatile that serves as an attractant for pollinators of flowers, acts as a defense compound in various plant tissues, and contributes to the aroma of fruits. Its production in a cultivated species such as strawberry (Fragaria × ananassa), therefore, is important for the viability and quality of the fruit. We have identified and functionally characterized three strawberry complementary DNAs (cDNAs) that encode proteins with high identity to eugenol synthases from several plant species. Based on a sequence comparison with the wild relative Fragaria vesca, two of these cDNAs, FaEGS1a and FaEGS1b, most likely correspond to transcripts derived from allelic gene variants, whereas the third cDNA, FaEGS2, corresponds to a different gene. Using coniferyl acetate as a substrate, FaEGS1a and FaEGS1b catalyze the in vitro formation of eugenol, while FaEGS2 catalyzes the formation of eugenol and also of isoeugenol with a lower catalytic efficiency. The expression of these genes is markedly higher in the fruit than in other tissues of the plant, with FaEGS1a and FaEGS1b mostly expressed in the green achenes, whereas FaEGS2 expression is almost restricted to the red receptacles. These expression patterns correlate with the eugenol content, which is highest in the achene at the green stage and in the receptacle at the red stage. The transient expression of the corresponding cDNAs in strawberry fruit and the subsequent volatile analyses confirm FaEGSs as genuine eugenol synthases in planta. These results provide new insights into the diversity of phenylpropene synthases in plants.

Proteomic analysis of strawberry achenes reveals active synthesis and recycling of L-ascorbic acid.

Although the commonly named strawberry fruit (Fragaria×ananassa) is the sum of achenes and receptacles, the true fruit in the botanical sense is the achene. Here we report the protein changes occurring in the achene when developing from immature to mature stage. We have used 2-DE followed by image analysis, and protein identification by PMF combined with MS/MS, to investigate the protein variations associated to this transition. From a total of 331 spots analyzed, the corresponding 315 proteins have been identified. Differentially accumulated proteins between immature and mature achenes mostly reflect the physiological events associated to seed development and maturation, with only a few changes related to the development of the dry pericarp. We have focused our attention on vitamin C biosynthesis. Interestingly, GDP-mannose 3',5'-epimerase, a key enzyme in the l-ascorbate biosynthesis pathway, and ascorbate peroxidase, involved in l-ascorbic acid oxidation, accumulate in immature achenes. The higher amount of these enzymes found in the green achene is coincident with a higher content of l-ascorbate, and higher expression levels of these and other gene encoding enzymes of the l-ascorbic acid biosynthesis pathway. Altogether our results suggest an important role of l-ascorbic acid at the early developmental stage of the achene. BIOLOGICAL SIGNIFICANCE: In this manuscript we report the identification of the most abundant proteins in strawberry (F.×ananassa) achenes at early and late stages of development, thus providing a proteomic view of the events that occur during the development of this organ. Despite the importance of strawberry as a commercial fruit, the molecular changes governing its growth and ripening processes are largely unknown. The lack of information is even greater in the case of the achenes, which are the true fruit and play a critical role in the developmental process of the receptacle. Our original proteomic study reported here, restricted to the achenes, completes the previous transcriptomic (very limited) and metabolomic maps of this organ, adding clarity to the role of the achene in the global ripening process. The results obtained not only complement the previous "omics" studies significantly, but also open new key questions that deserve further research (role of hormones). We finally focus on the biosynthesis of l-ascorbic acid, which appears to be tightly regulated by some specific pathways, and whose content is important in the achene. The information provided here will be of interest not only for the groups studying strawberry, but also for many other groups interested in the fruit ripening process, as well as for groups studying the regulation of l-ascorbic acid content in different plant tissues.

Demethylation of oligogalacturonides by FaPE1 in the fruits of the wild strawberry Fragaria vesca triggers metabolic and transcriptional changes associated with defence and development of the fruit.

Ectopic expression of the strawberry (Fragaria×ananassa) gene FaPE1 encoding pectin methyl esterase produced in the wild species Fragaria vesca partially demethylated oligogalacturonides (OGAs), which conferred partial resistance of ripe fruits to the fungus Botrytis cinerea. Analyses of metabolic and transcriptional changes in the receptacle of the transgenic fruits revealed channelling of metabolites to aspartate and aromatic amino acids as well as phenolics, flavanones, and sesquiterpenoids, which was in parallel with the increased expression of some genes related to plant defence. The results illustrate the changes associated with resistance to B. cinerea in the transgenic F. vesca. These changes were accompanied by a significant decrease in the auxin content of the receptacle of the ripe fruits of transgenic F. vesca, and enhanced expression of some auxin-repressed genes. The role of these OGAs in fruit development was revealed by the larger size of the ripe fruits in transgenic F. vesca. When taken together these results show that in cultivated F. ananassa FaPE1 participates in the de-esterification of pectins and the generation of partially demethylated OGAs, which might reinforce the plant defence system and play an active role in fruit development.