High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms

Gene regulatory network inference : connecting plant biology and mathematical modeling

Plant responses to environmental and intrinsic signals are tightly controlled by multiple transcription factors (TFs). These TFs and their regulatory connections form gene regulatory networks (GRNs), which provide a blueprint of the transcriptional regulations underlying plant development and environmental responses. This review provides examples of experimental methodologies commonly used to identify regulatory interactions and generate GRNs. Additionally, this review describes network inference techniques that leverage gene expression data to predict regulatory interactions. These computational and experimental methodologies yield complex networks that can identify new regulatory interactions, driving novel hypotheses. Biological properties that contribute to the complexity of GRNs are also described in this review. These include network topology, network size, transient binding of TFs to DNA, and competition between multiple upstream regulators. Finally, this review highlights the potential of machine learning approaches to leverage gene expression data to predict phenotypic outputs

Arabidopsis Homologs of the Petunia HAIRY MERISTEM Gene Are Required for Maintenance of Shoot and Root Indeterminacy

Maintenance of indeterminacy is fundamental to the generation of plant architecture and a central component of the plant life strategy. Indeterminacy in plants is a characteristic of shoot and root meristems, which must balance maintenance of indeterminacy with organogenesis. The Petunia hybrida HAIRY MERISTEM (HAM) gene, a member of the GRAS family of transcriptional regulators, promotes shoot indeterminacy by an undefined non-cell-autonomous signaling mechanism(s). Here, we report that Arabidopsis (Arabidopsis thaliana) mutants triply homozygous for knockout alleles in three Arabidopsis HAM orthologs (Atham1,2,3 mutants) exhibit loss of indeterminacy in both the shoot and root. In the shoot, the degree of penetrance of the loss-of-indeterminacy phenotype of Atham1,2,3 mutants varies among shoot systems, with arrest of the primary vegetative shoot meristem occurring rarely or never, secondary shoot meristems typically arresting prior to initiating organogenesis, and inflorescence and flower meristems exhibiting a phenotypic range extending from wild type (flowers) to meristem arrest preempting organogenesis (flowers and inflorescence). Atham1,2,3 mutants also exhibit aberrant shoot phyllotaxis, lateral organ abnormalities, and altered meristem morphology in functioning meristems of both rosette and inflorescence. Root meristems of Atham1,2,3 mutants are significantly smaller than in the wild type in both longitudinal and radial axes, a consequence of reduced rates of meristem cell division that culminate in root meristem arrest. Atham1,2,3 phenotypes are unlikely to reflect complete loss of HAM function, as a fourth, more distantly related Arabidopsis HAM homolog, AtHAM4, exhibits overlapping function with AtHAM1 and AtHAM2 in promoting shoot indeterminacy

Deciphering Strawberry Ripening by Tissue Specific Gene Regulatory Networks

During ripening, fruits undergo a number of metabolic and physiological changes leading to softening and improvement of characters such as flavor and palatability. Insights into transcriptome changes during strawberry fruit ripening have been reported, but always using either complete fruits in the analysis or separating achenes and the fleshy part (receptacle). However, the receptacle is composed of heterogeneous cell types, each of them with different characteristics and functions. Hence, transcriptomic studies performed so far may have lost important regulatory elements which expression is low but important in a specific cell-type specific. In our study, we use Laser Capture Microdissection (LCM) technique for the isolation of cells from specific tissue types such as the epidermis, vascular bundles, cortex, and pith. Transcriptome profiling of these tissue types was performed by RNAseq. A gene co-expression analysis was performed by Weighted Correlation Network Analysis (WGCNA). Ontology analysis of each module showed wax biosynthesis as the main biological pathway enriched at the red epidermis specific module. In order to elucidate the putative regulatory elements that control the synthesis of waxes in this tissue, a Gene Regulatory Network (GRN) was generated using GENIST (de Luis Balaguer, 2017). As a result, we have identified a set of transcription factors that might regulate the expression of eceriferum genes and a fatty acid elongase necessary for wax biosynthesis in ripe epidermis. Ultimately, our results open the possibility of implementing novel targeted breeding approaches. Moreover, this work shows that LCM followed by RNAseq is a powerful tool that can be used to clarify the regulatory scenario of tissue-specific biological processes during strawberry ripening.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Dose-Duration Reciprocity for G protein activation: Modulation of kinase to substrate ratio alters cell signaling

In animal cells, activation of heterotrimeric G protein signaling generally occurs when the system’s cognate signal exceeds a threshold, whereas in plant cells, both the amount and the exposure time of at least one signal, D-glucose, are used toward activation. This unusual signaling property called Dose-Duration Reciprocity, first elucidated in the genetic model Arabidopsis thaliana, is achieved by a complex that is comprised of a 7-transmembrane REGULATOR OF G SIGNALING (RGS) protein (AtRGS1), a Gα subunit that binds and hydrolyzes nucleotide, a Gβγ dimer, and three WITH NO LYSINE (WNK) kinases. D-glucose is one of several signals such as salt and pathogen-derived molecular patterns that operates through this protein complex to activate G protein signaling by WNK kinase transphosphorylation of AtRGS1. Because WNK kinases compete for the same substrate, AtRGS1, we hypothesize that activation is sensitive to the AtRGS1 amount and that modulation of the AtRGS1 pool affects the response to the stimulant. Mathematical simulation revealed that the ratio of AtRGS1 to the kinase affects system sensitivity to D-glucose, and therefore illustrates how modulation of the cellular AtRGS1 level is a means to change signal-induced activation. AtRGS1 levels change under tested conditions that mimic physiological conditions therefore, we propose a previously-unknown mechanism by which plants react to changes in their environment

Down-regulation of Fra a 1.02 in strawberry fruits causes transcriptomic and metabolic changes compatible with an altered defense response

The strawberry Fra a 1 proteins belong to the class 10 Pathogenesis-Related (PR-10) superfamily. In strawberry, a large number of members have been identified, but only a limited number is expressed in the fruits. In this organ, Fra a 1.01 and Fra a 1.02 are the most abundant Fra proteins in the green and red fruits, respectively, however, their function remains unknown. To know the function of Fra a 1.02 we have generated transgenic lines that silence this gene, and performed metabolomics, RNA-Seq, and hormonal assays. Previous studies associated Fra a 1.02 to strawberry fruit color, but the analysis of anthocyanins in the ripe fruits showed no diminution in their content in the silenced lines. Gene ontology (GO) analysis of the genes differentially expressed indicated that oxidation/reduction was the most represented biological process. Redox state was not apparently altered since no changes were found in ascorbic acid and glutathione (GSH) reduced/oxidized ratio, but GSH content was reduced in the silenced fruits. In addition, a number of glutathione-S-transferases (GST) were down-regulated as result of Fra a 1.02-silencing. Another highly represented GO category was transport which included a number of ABC and MATE transporters. Among the regulatory genes differentially expressed WRKY33.1 and WRKY33.2 were down-regulated, which had previously been assigned a role in strawberry plant defense. A reduced expression of the VQ23 gene and a diminished content of the hormones JA, SA, and IAA were also found. These data might indicate that Fra a 1.02 participates in the defense against pathogens in the ripe strawberry fruits

DOF2.1 Controls Cytokinin-Dependent Vascular Cell Proliferation Downstream of TMO5/LHW

To create a three-dimensional structure, plants rely on oriented cell divisions and cell elongation. Oriented cell divisions are specifically important in procambium cells of the root to establish the different vascular cell types [1, 2]. These divisions are in part controlled by the auxin-controlled TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) transcription factor complex [3-7]. Loss-of-function of tmo5 or lhw clade members results in strongly reduced vascular cell file numbers, whereas ectopic expression of both TMO5 and LHW can ubiquitously induce periclinal and radial cell divisions in all cell types of the root meristem. TMO5 and LHW interact only in young xylem cells, where they promote expression of two direct target genes involved in the final step of cytokinin (CK) biosynthesis, LONELY GUY3 (LOG3) and LOG4 [8, 9] Therefore, CK was hypothesized to act as a mobile signal from the xylem to trigger divisions in the neighboring procambium cells [3, 6]. To unravel how TMO5/LHW-dependent cytokinin regulates cell proliferation, we analyzed the transcriptional responses upon simultaneous induction of both transcription factors. Using inferred network analysis, we identified AT2G28510/DOF2.1 as a cytokinin-dependent downstream target gene. We further showed that DOF2.1 controls specific procambium cell divisions without inducing other cytokinin-dependent effects such as the inhibition of vascular differentiation. In summary, our results suggest that DOF2.1 and its closest homologs control vascular cell proliferation, thus leading to radial expansion of the root.Peer reviewe

Identificación y caracterización de genes implicados en la maduración y la calidad de la fresa

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project (TRANSFR-Q, Starting Grant-ERC), we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size, and resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance.ERC Starting Grant ERC-2014-StG 63813

Transcriptional regulatory network controlling strawberry fruit ripening and quality

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project, we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size or resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Study of Transcriptional Regulatory Network Controlling Strawberry Fruit Ripening and Quality

Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits. In our project we aim at the identification and characterization of key transcription factors involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. On the other hand, we are carrying out a Genome-Wide Association Study using a germplasm collection of the woodland strawberry (Fragaria vesca) in order to identify loci involved in important traits such as aroma, fruit size, and resistance to pathogens. Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec