Diversity of the volatilome and the fruit size and shape in European woodland strawberry (Fragaria vesca).

Woodland strawberry (Fragaria vesca subsp. vesca) is a wild relative of cultivated strawberry (F. × ananassa) producing small and typically conical fruits with an intense flavor and aroma. The wild strawberry species, F. vesca, is a rich resource of genetic and metabolic variability, but its diversity remains largely unexplored and unexploited. In this study, we aim for an in-depth characterization of the fruit complex volatilome by GC-MS as well as the fruit size and shape using a European germplasm collection that represents the continental diversity of the species. We report characteristic volatilome footprints and fruit phenotypes of specific geographical areas. Thus, this study uncovers phenotypic variation linked to geographical distribution that will be valuable for further genetic studies to identify candidate genes or develop markers linked to volatile compounds or fruit shape and size traits.

Deciphering the regulatory network of the NAC transcription factor FvRIF, a key regulator of strawberry (Fragaria vesca) fruit ripening.

The NAC transcription factor ripening inducing factor (RIF) was previously reported to be necessary for the ripening of octoploid strawberry (Fragaria × ananassa) fruit, but the mechanistic basis of RIF-mediated transcriptional regulation and how RIF activity is modulated remains elusive. Here, we show that FvRIF in diploid strawberry, Fragaria vesca, is a key regulator in the control of fruit ripening and that knockout mutations of FvRIF result in a complete block of fruit ripening. DNA affinity purification sequencing coupled with transcriptome deep sequencing suggests that 2,080 genes are direct targets of FvRIF-mediated regulation, including those related to various aspects of fruit ripening. We provide evidence that FvRIF modulates anthocyanin biosynthesis and fruit softening by directly regulating the related core genes. Moreover, we demonstrate that FvRIF interacts with and serves as a substrate of MAP kinase 6 (FvMAPK6), which regulates the transcriptional activation function of FvRIF by phosphorylating FvRIF at Thr-310. Our findings uncover the FvRIF-mediated transcriptional regulatory network in controlling strawberry fruit ripening and highlight the physiological significance of phosphorylation modification on FvRIF activity in ripening.

Non-climacteric fruit development and ripening regulation: 'the phytohormones show'.

Fruit ripening involves numerous physiological, structural, and metabolic changes that result in the formation of edible fruits. This process is controlled at different molecular levels, with essential roles for phytohormones, transcription factors, and epigenetic modifications. Fleshy fruits are classified as either climacteric or non-climacteric species. Climacteric fruits are characterized by a burst in respiration and ethylene production at the onset of ripening, while regulation of non-climacteric fruit ripening has been commonly attributed to abscisic acid (ABA). However, there is controversy as to whether mechanisms regulating fruit ripening are shared between non-climacteric species, and to what extent other hormones contribute alongside ABA. In this review, we summarize classic and recent studies on the accumulation profile and role of ABA and other important hormones in the regulation of non-climacteric fruit development and ripening, as well as their crosstalk, paying special attention to the two main non-climacteric plant models, strawberry and grape. We highlight both the common and different roles of these regulators in these two crops, and discuss the importance of the transcriptional and environmental regulation of fruit ripening, as well as the need to optimize genetic transformation methodologies to facilitate gene functional analyses.

Genome Editing by CRISPR/Cas9 in Polyploids.

CRISPR/Cas system has been widely used for genome editing in the past few years. Even though it has been performed in many polyploid species to date, its efficient accomplishment in these organisms is still a challenge. The presence of multiple homoeologous genes as targets for their editing requires more rigorous work and specific needs to assess successful genome editing. Here, we describe a general stepwise protocol to select target sites, design sgRNAs, indicate vector requirements, and screen CRISPR/Cas9-mediated genome editing in polyploid species.

Insights into transcription factors controlling strawberry fruit development and ripening.

Fruit ripening is a highly regulated and complex process involving a series of physiological and biochemical changes aiming to maximize fruit organoleptic traits to attract herbivores, maximizing therefore seed dispersal. Furthermore, this process is of key importance for fruit quality and therefore consumer acceptance. In fleshy fruits, ripening involves an alteration in color, in the content of sugars, organic acids and secondary metabolites, such as volatile compounds, which influence flavor and aroma, and the remodeling of cell walls, resulting in the softening of the fruit. The mechanisms underlying these processes rely on the action of phytohormones, transcription factors and epigenetic modifications. Strawberry fruit is considered a model of non-climacteric species, as its ripening is mainly controlled by abscisic acid. Besides the role of phytohormones in the regulation of strawberry fruit ripening, a number of transcription factors have been identified as important regulators of these processes to date. In this review, we present a comprehensive overview of the current knowledge on the role of transcription factors in the regulation of strawberry fruit ripening, as well as in compiling candidate regulators that might play an important role but that have not been functionally studied to date.

Evolutionary Ecology of Plant-Arthropod Interactions in Light of the "Omics" Sciences: A Broad Guide.

Aboveground plant-arthropod interactions are typically complex, involving herbivores, predators, pollinators, and various other guilds that can strongly affect plant fitness, directly or indirectly, and individually, synergistically, or antagonistically. However, little is known about how ongoing natural selection by these interacting guilds shapes the evolution of plants, i.e., how they affect the differential survival and reproduction of genotypes due to differences in phenotypes in an environment. Recent technological advances, including next-generation sequencing, metabolomics, and gene-editing technologies along with traditional experimental approaches (e.g., quantitative genetics experiments), have enabled far more comprehensive exploration of the genes and traits involved in complex ecological interactions. Connecting different levels of biological organization (genes to communities) will enhance the understanding of evolutionary interactions in complex communities, but this requires a multidisciplinary approach. Here, we review traditional and modern methods and concepts, then highlight future avenues for studying the evolution of plant-arthropod interactions (e.g., plant-herbivore-pollinator interactions). Besides promoting a fundamental understanding of plant-associated arthropod communities' genetic background and evolution, such knowledge can also help address many current global environmental challenges.

The NAC transcription factor FaRIF controls fruit ripening in strawberry.

In contrast to climacteric fruits such as tomato, the knowledge on key regulatory genes controlling the ripening of strawberry, a nonclimacteric fruit, is still limited. NAC transcription factors (TFs) mediate different developmental processes in plants. Here, we identified and characterized Ripening Inducing Factor (FaRIF), a NAC TF that is highly expressed and induced in strawberry receptacles during ripening. Functional analyses based on stable transgenic lines aimed at silencing FaRIF by RNA interference, either from a constitutive promoter or the ripe receptacle-specific EXP2 promoter, as well as overexpression lines showed that FaRIF controls critical ripening-related processes such as fruit softening and pigment and sugar accumulation. Physiological, metabolome, and transcriptome analyses of receptacles of FaRIF-silenced and overexpression lines point to FaRIF as a key regulator of strawberry fruit ripening from early developmental stages, controlling abscisic acid biosynthesis and signaling, cell-wall degradation, and modification, the phenylpropanoid pathway, volatiles production, and the balance of the aerobic/anaerobic metabolism. FaRIF is therefore a target to be modified/edited to control the quality of strawberry fruits.

Allelic Variation of MYB10 Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry (Fragaria spp.) Fruit.

The fruits of diploid and octoploid strawberry (Fragaria spp) show substantial natural variation in color due to distinct anthocyanin accumulation and distribution patterns. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among which MYB10 is the main activator in strawberry fruit. Here, we show that mutations in MYB10 cause most of the variation in anthocyanin accumulation and distribution observed in diploid woodland strawberry (F. vesca) and octoploid cultivated strawberry (F ×ananassa). Using a mapping-by-sequencing approach, we identified a gypsy-transposon in MYB10 that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruited F. vesca ecotype. Two additional loss-of-function mutations in MYB10 were identified among geographically diverse white-fruited F. vesca ecotypes. Genetic and transcriptomic analyses of octoploid Fragaria spp revealed that FaMYB10-2, one of three MYB10 homoeologs identified, regulates anthocyanin biosynthesis in developing fruit. Furthermore, independent mutations in MYB10-2 are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon (FaEnSpm-2) insertion in the MYB10-2 promoter of red-fleshed accessions that was associated with enhanced expression. Our findings suggest that cis-regulatory elements in FaEnSpm-2 are responsible for enhanced MYB10-2 expression and anthocyanin biosynthesis in strawberry fruit flesh.

Characterizing the involvement of FaMADS9 in the regulation of strawberry fruit receptacle development.

FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid-1,4-lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9-specific CArG box was identified in the promoters of these genes.

TTL Proteins Scaffold Brassinosteroid Signaling Components at the Plasma Membrane to Optimize Signal Transduction in Arabidopsis.

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development, and stress responses. BRs are perceived extracellularly by plasma membrane receptor-like kinases that activate an interconnected signal transduction cascade, leading to the transcriptional regulation of BR-responsive genes. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) genes are specific for land plants, and their encoded proteins are defined by the presence of protein-protein interaction motives, that is, an intrinsic disordered region at the N terminus, six tetratricopeptide repeat domains, and a C terminus with homology to thioredoxins. TTL proteins thus likely mediate the assembly of multiprotein complexes. Phenotypic, molecular, and genetic analyses show that TTL proteins are positive regulators of BR signaling in Arabidopsis (Arabidopsis thaliana). TTL3 directly interacts with a constitutively active BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor kinase, BRI1-SUPPRESSOR1 phosphatase, and the BRASSINAZOLE RESISTANT1 transcription factor and associates with BR-SIGNALING KINASE1, BRASSINOSTEROID INSENSITIVE2 kinases, but not with BRI1-ASSOCIATED KINASE1. A functional TTL3-green fluorescent protein (GFP) shows dual cytoplasmic plasma membrane localization. Depleting the endogenous BR content reduces plasma membrane localization of TTL3-GFP, while increasing BR content causes its plasma membrane relocalization, where it strengthens the association of BR signaling components. Our results reveal that TTL proteins promote BR responses and suggest that TTL proteins may function as scaffold proteins by bringing together cytoplasmic and plasma membrane BR signaling components.

Functional analysis of the TM6 MADS-box gene in the octoploid strawberry by CRISPR/Cas9-directed mutagenesis.

The B-class of MADS-box transcription factors has been studied in many plant species, but remains functionally uncharacterized in Rosaceae. APETALA3 (AP3), a member of this class, controls petal and stamen identities in Arabidopsis. In this study, we identified two members of the AP3 lineage in cultivated strawberry, Fragaria × ananassa, namely FaAP3 and FaTM6. FaTM6, and not FaAP3, showed an expression pattern equivalent to that of AP3 in Arabidopsis. We used the CRISPR/Cas9 genome editing system for the first time in an octoploid species to characterize the function of TM6 in strawberry flower development. An analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed highly efficient genome editing already present in the T0 generation. Phenotypic characterization of the mutant lines indicated that FaTM6 plays a key role in anther development in strawberry. Our results validate the use of the CRISPR/Cas9 system for gene functional analysis in F. × ananassa as an octoploid species, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.

Genome Editing as a Tool for Fruit Ripening Manipulation.

Over the last few years, a series of tools for genome editing have been developed, allowing the introduction of precise changes into plant genomes. These have included Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, which is so far the most successful and commonly used approach for targeted and stable editing of DNA, due to its ease of use and low cost. CRISPR/Cas9 is now being widely used as a new plant breeding technique to improve commercially relevant crop species. Fruit ripening is a complex and genetically controlled developmental process that is essential for acquiring quality attributes of the fruit. Although the number of studies published to date using genome editing tools to molecularly understand or improve fruit ripening is scarce, in this review we discuss these achievements and how genome editing opens tremendous possibilities not only for functional studies of genes involved in fruit ripening, but also to generate non-transgenic plants with an improved fruit quality.

Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa).

RNA-seq has been used to perform global expression analysis of the achene and the receptacle at four stages of fruit ripening, and of the roots and leaves of strawberry (Fragaria × ananassa). About 967 million reads and 191 Gb of sequence were produced, using Illumina sequencing. Mapping the reads in the related genome of the wild diploid Fragaria vesca revealed differences between the achene and receptacle development program, and reinforced the role played by ethylene in the ripening receptacle. For the strawberry transcriptome assembly, a de novo strategy was followed, generating separate assemblies for each of the ten tissues and stages sampled. The Trinity program was used for these assemblies, resulting in over 1.4 M isoforms. Filtering by a threshold of 0.3 FPKM, and doing Blastx (E-value < 1 e-30) against the UniProt database of plants reduced the number to 472,476 isoforms. Their assembly with the MIRA program (90% homology) resulted in 26,087 contigs. From these, 91.34 percent showed high homology to Fragaria vesca genes and 87.30 percent Fragaria iinumae (BlastN E-value < 1 e-100). Mapping back the reads on the MIRA contigs identified polymorphisms at nucleotide level, using FREEBAYES, as well as estimate their relative abundance in each sample.

Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering.

Plants can produce organs throughout their entire life from pluripotent stem cells located at their growing tip, the shoot apical meristem (SAM). At the time of flowering, the SAM of Arabidopsis thaliana switches fate and starts producing flowers instead of leaves. Correct timing of flowering in part determines reproductive success, and is therefore under environmental and endogenous control. How epigenetic regulation contributes to the floral transition has eluded analysis so far, mostly because of the poor accessibility of the SAM. Here we report the temporal dynamics of the chromatin modifications H3K4me3 and H3K27me3 and their correlation with transcriptional changes at the SAM in response to photoperiod-induced flowering. Emphasizing the importance of tissue-specific epigenomic analyses we detect enrichments of chromatin states in the SAM that were not apparent in whole seedlings. Furthermore, our results suggest that regulation of translation might be involved in adjusting meristem function during the induction of flowering.

A SAM oligomerization domain shapes the genomic binding landscape of the LEAFY transcription factor.

Deciphering the mechanisms directing transcription factors (TFs) to specific genome regions is essential to understand and predict transcriptional regulation. TFs recognize short DNA motifs primarily through their DNA-binding domain. Some TFs also possess an oligomerization domain suspected to potentiate DNA binding but for which the genome-wide influence remains poorly understood. Here we focus on the LEAFY transcription factor, a master regulator of flower development in angiosperms. We have determined the crystal structure of its conserved amino-terminal domain, revealing an unanticipated Sterile Alpha Motif oligomerization domain. We show that this domain is essential to LEAFY floral function. Moreover, combined biochemical and genome-wide assays suggest that oligomerization is required for LEAFY to access regions with low-affinity binding sites or closed chromatin. This finding shows that domains that do not directly contact DNA can nevertheless have a profound impact on the DNA binding landscape of a TF.

DNA-Binding Factor Target Identification by Chromatin Immunoprecipitation (ChIP) in Plants.

Chromatin immunoprecipitation (ChIP) allows the precise identification of genomic loci that physically interact with a protein of interest, whether that protein is a transcription factor, a core polymerase, a histone, or other chromatin-associated protein. In short, tissue is first cross-linked to freeze a population of DNA-protein interactions at a stage of interest. Chromatin is then extracted, fragmented, and incubated with a specific antibody against the protein of interest. Next, the resultant DNA-protein complexes are immunoprecipitated and captured using beads that bind to the antibody constant region. Samples are finally reverse cross-linked to separate the bound fragments and the DNA is purified. This DNA is analyzed by quantitative PCR for enrichment of genomic regions expected to be bound by the protein under study. The protocol detailed in this chapter has been successfully applied in the identification of target genes for seven transcriptional regulators of diverse classes involved in Arabidopsis thaliana floral transition.

Modulation of Ambient Temperature-Dependent Flowering in Arabidopsis thaliana by Natural Variation of FLOWERING LOCUS M.

Plants integrate seasonal cues such as temperature and day length to optimally adjust their flowering time to the environment. Compared to the control of flowering before and after winter by the vernalization and day length pathways, mechanisms that delay or promote flowering during a transient cool or warm period, especially during spring, are less well understood. Due to global warming, understanding this ambient temperature pathway has gained increasing importance. In Arabidopsis thaliana, FLOWERING LOCUS M (FLM) is a critical flowering regulator of the ambient temperature pathway. FLM is alternatively spliced in a temperature-dependent manner and the two predominant splice variants, FLM-ß and FLM-δ, can repress and activate flowering in the genetic background of the A. thaliana reference accession Columbia-0. The relevance of this regulatory mechanism for the environmental adaptation across the entire range of the species is, however, unknown. Here, we identify insertion polymorphisms in the first intron of FLM as causative for accelerated flowering in many natural A. thaliana accessions, especially in cool (15°C) temperatures. We present evidence for a potential adaptive role of this structural variation and link it specifically to changes in the abundance of FLM-ß. Our results may allow predicting flowering in response to ambient temperatures in the Brassicaceae.

A quantitative and dynamic model of the Arabidopsis flowering time gene regulatory network.

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.

Control of flowering by ambient temperature.

The timing of flowering is a crucial decision in the life cycle of plants since favourable conditions are needed to maximize reproductive success and, hence, the survival of the species. It is therefore not surprising that plants constantly monitor endogenous and environmental signals, such as day length (photoperiod) and temperature, to adjust the timing of the floral transition. Temperature in particular has been shown to have a tremendous effect on the timing of flowering: the effect of prolonged periods of cold, called the vernalization response, has been extensively studied and the underlying epigenetic mechanisms are reasonably well understood in Arabidopsis thaliana. In contrast, the effect of moderate changes in ambient growth temperature on the progression of flowering, the thermosensory pathway, is only starting to be understood on the molecular level. Several genes and molecular mechanisms underlying the thermosensory pathway have already been identified and characterized in detail. At a time when global temperature is rising due to climate change, this knowledge will be pivotal to ensure crop production in the future.

Temperature-dependent regulation of flowering by antagonistic FLM variants.

The appropriate timing of flowering is crucial for plant reproductive success. It is therefore not surprising that intricate genetic networks have evolved to perceive and integrate both endogenous and environmental signals, such as carbohydrate and hormonal status, photoperiod and temperature. In contrast to our detailed understanding of the vernalization pathway, little is known about how flowering time is controlled in response to changes in the ambient growth temperature. In Arabidopsis thaliana, the MADS-box transcription factor genes FLOWERING LOCUS M (FLM) and SHORT VEGETATIVE PHASE (SVP) have key roles in this process. FLM is subject to temperature-dependent alternative splicing. Here we report that the two main FLM protein splice variants, FLM-ß and FLM-δ, compete for interaction with the floral repressor SVP. The SVP-FLM-ß complex is predominately formed at low temperatures and prevents precocious flowering. By contrast, the competing SVP-FLM-δ complex is impaired in DNA binding and acts as a dominant-negative activator of flowering at higher temperatures. Our results show a new mechanism that controls the timing of the floral transition in response to changes in ambient temperature. A better understanding of how temperature controls the molecular mechanisms of flowering will be important to cope with current changes in global climate.