Biology subjects

Granell, A.

Publications and source records attributed to Granell, A..

17 recordsLinked to original sources

bioRxiv2026

Multi-trait evaluation of a tomato MAGIC population identifies promising lines with improved nitrogen use efficiency (NUE)

Nitrogen-use efficiency (NUE) is a pivotal breeding target in tomato (Solanum lycopersicum L.) to sustain production under reduced N inputs. Here, we leveraged a recently developed tomato multi-parent advanced generation inter-cross (ToMAGIC) population to identify lines with superior performance under reduced N availability. The eight founders and a core subset of 118 ToMAGIC lines were characterized with 10,684 SNP markers and evaluated under optimal (opN, 15 mM) and suboptimal (subN, 8 mM) N supply in an experiment totalling 1,576 plants, generating 48,068 data points across 61 phenotypic variables. Under both N treatments, ToMAGIC lines exhibited transgressive segregation for most traits, confirming the value of this population as a reservoir of untapped variation. Notably, under subN conditions, harvest index (Hi) increased by 29-44%, suggesting adaptive resource redistribution toward reproductive sinks. Variance partitioning revealed that agronomic and NUE-related traits were largely under genetic control, with heritability estimates frequently above 0.80 and broadly conserved across N treatments. Multivariate trait analysis identified fruit yield N concentration (NUE component, CN,y), shoot biomass N content (NAb), and shoot growth-related traits as the main drivers of treatment differentiation. Finally, proxy traits were prioritized by integrating response magnitude, heritability, trait correlations, and treatment-discriminatory power into multi-trait selection indices. This strategy generated favorable predicted genetic gains, reaching 158% for high-performance lines and 170% for subN-adapted lines, and consistently identified lines 402, 428, 518, 800, and 816 as promising pre-breeding materials. Overall, this study supports ToMAGIC as a powerful resource for developing N-efficient cultivars suited for sustainable agriculture.

plant biology↗

bioRxiv2025

Uncovering the genetic basis of fruit volatiles in Fragaria vesca through GWAS reveals FvJMT2 as a methyl benzoate biosynthesis gene with insect-repellent function

Strawberry aroma is a key component of fruit quality, influencing consumer preferences and playing important ecological roles, including plant defense. However, the genetic basis of volatile organic compound (VOC) biosynthesis remains only partially understood, particularly in the wild woodland strawberry Fragaria vesca, which has enormous potential to uncover genetic diversity within the genus for improving the commercial strawberries. Here, we performed Genome-Wide Association Studies (GWAS) across a diverse European collection of F. vesca accessions. We identified multiple novel candidate genes involved in the biosynthesis of diverse volatile esters, lactones, terpenoids, and methyl ketones. Among them, we characterized FvJMT2, a SABATH family methyltransferase which we found associated with natural variation in benzenoid esters content. Transient expression in Nicotiana benthamiana and strawberry fruit confirmed its role in methyl benzoate biosynthesis, while enzymatic assays demonstrated that FvJMT2 encodes a promiscuous enzyme capable of methylating not only benzoic acid, but also cinnamic, salicylic, and jasmonic acids. Behavioral assays revealed that methyl benzoate at physiologically relevant concentrations significantly reduced the attraction of Drosophila suzukii flies, supporting a dual role of this VOC in both flavor and pest deterrence. Finally, natural variation analyses in wild Fragaria species and F. x ananassa cultivars showed that benzenoid esters have been largely lost in modern cultivars but retained in ancient and wild accessions. Altogether, this study provides novel insights into the genetics and ecological relevance of strawberry volatiles and identifies candidate loci and alleles for future studies.

genomics↗

bioRxiv2025

Novel substrate affinity of FaCCR1 and FaCCR1/FaOCT4 expression control the content of medium-chain esters in strawberry fruit

Fruit flavor and aroma are influenced by the sugars-to-acid ratio and most importantly by the specific profile and abundance of volatile organic compounds (VOCs). While over 550 VOCs have been identified in strawberry fruit, fewer than 20 are believed to be key for aroma, being esters the major components. A major and stable quantitative trait locus (QTL) explaining a large percentage (40%) of phenotypic variation for several medium-chain esters (MCE) was mapped at about 19 Mb in chromosome 6A using both a biparental population and genome-wide association studies (GWAS). Using a combination of genetic mapping, global transcriptomic studies of bulked contrasting lines, and in vivo and in silico functional analyses, we identified the cinnamoyl Co-A reductase FaCCR1(6A) and the organic cation/carnitine transporter FaOCT4(6A) as underlying genes. Subcellular localization experiments indicated that FaCCR1(6A) and FaOCT4(6A) localize at the cytosol and tonoplast membrane respectively. Transient overexpression of FaCCR1(6A) and FaOCT4(6A) in fruits resulted in a significant increase in MCE content. Despite the well-described function of CCRs in the biosynthesis of monolignols, in vitro enzymatic assays and molecular docking revealed that FaCCR1(6A) also presents high affinity for MCE precursors, indicating a previously unidentified function of this protein in volatile biosynthesis. Based in our findings, we have developed a predictive KASP assay in FaOCT4(6A) for selecting superior cultivars with enhanced fruit aroma.

plant biology↗

bioRxiv2025

Precision RNAi in Tomato Using Synthetic Trans-Acting Small Interfering RNAs Derived From Minimal Precursors

RNA interference (RNAi) is a highly conserved gene silencing mechanism regulating gene expression at transcriptional and post-transcriptional levels in plants. Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) have emerged as powerful tools for highly specific and efficient gene silencing. However, their application in crops has been constrained by the need for transgene integration and the relatively long length of TAS-derived precursors. Here, we developed a novel syn-tasiRNA platform for Solanum lycopersicum (tomato) based on minimal precursors targeted by endogenous SlmiR482b microRNA. These minimal precursors, comprising only a 22-nt miRNA target site, an 11-nt spacer and the syn-tasiRNA sequence(s), effectively produced functional syn-tasiRNAs in both transgenic and transient virus-induced gene silencing (syn-tasiR-VIGS) systems. To facilitate their broader application, we engineered a series of vectors for high-throughput cloning and efficient syn-tasiRNA expression from SlmiR482b-based minimal precursors in tomato. Our results show that minimal precursors induce robust gene silencing of endogenous tomato genes and confer antiviral resistance to the economically important tomato spotted wilt virus. Furthermore, we show that syn-tasiR-VIGS can be applied in a transgene-free manner through crude extract delivery, leading to efficient silencing of endogenous genes. This study establishes minimal syn-tasiRNA precursors as a versatile and efficient tool for precision RNAi in tomato, with applications in functional genomics and crop improvement.

plant biology↗

bioRxiv2025

High throughput isolation of male gametophyte cells of Solanum lycopersicum var. Micro-Tom by fluorescence-activated cell sorting.

Efficient isolation of male gametes has enabled unprecedented advances in omics research, crucial for elucidating the molecular mechanisms governing male gametogenesis and fertilization. In this study, we developed a method for isolating generative and sperm cells from the economically important crop Solanum lycopersicum. A double fluorescent marker line was generated in the tomato variety Micro-Tom, employing mTurquoise and mScarlet-I fluorescent proteins under the control of promoters exhibiting preferential activity in generative and sperm cells, respectively. Then we developed a protocol that combines male gamete release from pollen of this line and SYTOX Red live/dead cell stain to obtain viable cells by Fluorescence-activated cell sorting. This allows the isolation of generative cells from mature pollen grains, and of sperm cells from pollen tubes after semi-in vivo growth, both in high quantity and purity. Additionally, an unexpected mScarlet-I signal in the vegetative nucleus, that persists until the sperm cells are formed, allows the sorting of vegetative nuclei. We anticipate that our novel double-marker line will accelerate research into tomato male gametogenesis, thereby enhancing efforts to improve the resilience of fertilization processes to climate change.

plant biology↗

bioRxiv2025

Editing strigolactone biosynthesis genes in tomato reveals novel phenotypic effects and highlights D27 as a key target for parasitic weed resistance

Parasitic weed infestations pose an increasing threat to agriculture worldwide, especially in the Mediterranean region. Phelipanche ramosa and P. aegyptiaca (broomrapes) cause severe damage to field-grown tomato (Solanum lycopersicum L.). Strigolactones (SLs), apocarotenoid phytohormones, play a critical role in plant physiology and development, and are also the primary signals that trigger the germination of parasitic weed seeds. We generated CRISPR/Cas9 tomato knock-out lines for the SlD27 gene, as well as three other key genes involved in SL biosynthesis (SlCCD7, SlCCD8, SlMAX1), all within the same genetic background. The edited lines exhibited a marked reduction in SL content in root exudates, along with impaired broomrape seed germination. A comprehensive analysis of morphological, reproductive, and fruit-related traits revealed gene-specific effects on plant phenotype, including vegetative traits, fruit set, fruit development, and volatilome. Specifically, the knock-out of two CCDs and the MAX1 had a specific impact not only on plant development but also on the production of volatile organic compounds during fruit ripening. In contrast, the Sld27 lines, produced for the first time in this study, displayed a phenotype similar to the control non-edited plants, suggesting that the D27 gene holds promise as a breeding target for enhancing resistance to parasitic weeds in tomato. HighlightThe characterization of tomato CRISPR/Cas9-edited lines for the four core genes involved in strigolactone biosynthesis revealed gene-specific effects on plant phenotype, with D27 emerging as a potential target for resistance to parasitic weeds.

genetics↗

bioRxiv2024

New QTLs involved in the control of stigma position in tomato

The tomato mating system was strongly affected by domestication events. Mutations disrupting self-incompatibility paralleled by changes retracting the stigma position (SP) within the staminal cone conferred strict autogamy and self-fertility to the cultivated forms. Although major genes affecting these changes have been identified, SP control in domesticated forms retaining a non-inserted or a heat-inducible SP needs elucidation. To widen the possibility of identifying SP genetic determinants, we analysed the trait in four populations (two germplasm collections, a multiparental recombinant inbred and a biparental progeny) under different environmental conditions (normal and heat stressed). Overall, 36 markers significantly associated with the trait were discovered. Several co-localizations were found, both among regions firstly reported in this work and among them and previously reported positions. This supported the reliability of the analysis. Three of such regions, in the long arms of chromosome 1, 8 and 11, were validated in an independent segregating population and candidate genes in confidence intervals were identified among transcription factors and hormone, stress and cell-wall-related genes. In conclusion, the work supported the hypothesis that the SP phenotype is controlled by different key-genes in tomato, paving the way to identifying novel players and novel mechanisms involved in the regulation of herkogamy. HighlightThe study of three germplasm collections in tomato allowed the identification new significant markers for the stigma position trait while confirmed several previously reported QTLs.

plant biology↗

bioRxiv2023

RNA virus-mediated gene editing for tomato trait breeding

Virus-induced genome editing (VIGE) is a flexible and robust technology that relies on viral vectors for the transient delivery of CRISPR-Cas components into plants. Tomato (Solanum lycopersicum L.) is a mayor horticultural crop grown worldwide; despite its economic importance, little is known about VIGE applicability in this species. This study presents the successful use of VIGE in tomato for fruit color breeding. We report (i) the generation of a transgenic Cas9-expressing line of tomato cv. Micro-Tom (MT-Cas9), (ii) the use of pLX-PVX, an enhanced RNA viral vector, for single-guide RNA (sgRNA) delivery into MT-Cas9 plants, (iii) heritable, proof-of-concept VIGE of PHYTOENE DESATURASE and recovery of albino progeny, and (iv) the recovery of progeny with recolored green-flesh fruits by VIGE of STAYGREEN 1, thus confirming the successful breeding of tomato fruit color. Altogether, our results indicate that the presented VIGE approach can be readily applied for accelerated functional genomics of tomato variation, as well as for precision breeding of tomato traits with horticultural interest. HIGHLIGHTSO_LIGeneration of a transgenic Cas9-expressing line of tomato cv. Micro-Tom C_LIO_LIUse of PVX for sgRNA delivery into Micro-Tom Cas9 plants C_LIO_LIHeritable proof-of-concept VIGE of tomato PHYTOENE DESATURASE (PDS) C_LIO_LIRecovery of green-flesh fruits by VIGE of tomato STAYGREEN 1 (SGR1) C_LI

plant biology↗

bioRxiv2022

Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically-engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly, and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild type levels), Valine (9-fold), Isoleucine (3-fold), and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

bioengineering↗

bioRxiv2022

Computer generation of fruit shapes from DNA sequence

The generation of realistic plant and animal images from marker information could be a main contribution of artificial intelligence to genetics and breeding. Since morphological traits are highly variable and highly heritable, this must be possible. However, a suitable algorithm has not been proposed yet. This paper is a proof of concept demonstrating the feasibility of this proposal using decoders, a class of deep learning architecture. We apply it to Cucurbitaceae, perhaps the family harboring the largest variability in fruit shape in the plant kingdom, and to tomato, a species with high morphological diversity also. We generate Cucurbitaceae shapes assuming a hypothetical, but plausible, evolutive path along observed fruit shapes of C. melo. In tomato, we used 353 images from 129 crosses between 25 maternal and 7 paternal lines for which genotype data were available. In both instances, a simple decoder was able to recover expected shapes with large accuracy. For the tomato pedigree, we also show that the algorithm can be trained to generate offspring images from their parents shapes, bypassing genotype information. Data and code are available at https://github.com/miguelperezenciso/dna2image.

bioinformatics↗

bioRxiv2021

European Vintage tomatoes galore: a result of farmers combinatorial assorting/swapping of a few diversity rich loci

A comprehensive collection of 1,254 tomato accessions corresponding to European heirlooms and landraces, together with modern varieties, early domesticates and wild relatives, were analyzed by genotyping by sequencing. A continuous genetic gradient between the vintage and modern varieties was observed. European vintage tomatoes displayed very low genetic diversity, with only 298 loci out of 64,943 variants being polymorphic at the 95% threshold. European vintage tomatoes could be classified in several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed a higher genetic diversity than the rest varieties, suggesting that these regions might be independent secondary centers of diversity and with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a GWAS with fruit morphological traits in the European vintage collection, and the corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise diversity-poor genome suggests a history of balancing selection, in which tomato farmers maintained the morphological variation by applying a high selective pressure within different varietal types. HighlightThe high phenotypic diversity observed among European vintage varieties was created by traditional farmers by combining very few polymorphic loci subjected to balancing selection.

plant biology↗

bioRxiv2021

Custom-made design of metabolite composition in N. benthamiana leaves using CRISPR activators.

Transcriptional regulators based on CRISPR architecture expand our ability of reprogramming endogenous gene expression in plants. One of their potential applications is the customization of plant metabolome through the activation of selected enzymes in a given metabolic pathway. Using the previously described multiplexable CRISPR activator dCasEV2.1, we assayed the selective enrichment in Nicotiana benthamiana leaves of four different flavonoids, namely naringenin, eriodictyol, kaempferol and quercetin. After careful selection of target genes and guide RNAs combinations, we created successful activation programs for each of the four metabolites, each program activating between three and seven genes, and with individual gene activation levels ranging from 4- to 1500-fold. Metabolic analysis of the flavonoid profiles of each multigene activation program showed a sharp and selective enrichment of the intended metabolites and their glycosylated derivatives. Remarkably, principal component analysis of untargeted metabolic profiles clearly separated samples according to their activation treatment, and hierarchical clustering separated the samples in five groups, corresponding to the expected four highly enriched metabolite groups, plus an un-activated control. These results demonstrate that dCasEV2.1 is a powerful tool for re-routing metabolic fluxes towards the accumulation of metabolites of interest, opening the door for custom-made design of metabolic contents in plants.

synthetic biology↗

bioRxiv2021

GABA plays a key role in plant acclimation to a combination of high light and heat stress

Plants are frequently subjected to different combinations of abiotic stresses, such as high light intensity and elevated temperatures. These environmental conditions pose an important threat to agriculture production, affecting photosynthesis and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources towards stress responses and suppressing growth. Here we show that the primary metabolic response of Arabidopsis thaliana plants to high light or heat stress is different than that of plants subjected to a combination of high light and heat stress. We further demonstrate that a combination of high light and heat stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids, coupled with decreased levels of metabolites participating in the tricarboxylic acid (TCA) cycle. Among the amino acids exclusively accumulated during a combination of high light and heat stress, we identified the non-proteinogenic amino acid {gamma}-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis, in particular two independent alleles of glutamate decarboxylase 3 (gad3), reveal that GABA plays a key role in the acclimation of plants to a combination of high light and heat stress. Taken together, our findings identify a new role for GABA in regulating plant responses to stress combination. One sentence summaryThe non-proteinogenic amino acid {gamma}-aminobutyric acid (GABA) is required for plant acclimation to a combination of high light and heat stress in Arabidopsis.

plant biology↗

bioRxiv2020

Edition of complex gene families in tobacco with GoldenBraid 4.0, a multipurpose web-based platform for plant genome engineering

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL mutants harboring up to 9 biallelic mutations in a single generation. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and parts collection, GB4.0 turns an unprecedentedly comprehensive open platform for plant genetic engineering.

molecular biology↗

bioRxiv2019

A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

Plant synthetic biology aims to contribute to global food security by engineering plants with new or improved functionalities. In recent years, synthetic biology has rapidly advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits able to provide organisms with novel functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to their complex multicellular nature and the lack of reliable DNA devices that allow an accurate design of more sophisticated biological circuits. Among these basic devices, gene switches are crucial to deploying new layers of regulation into the engineered organisms. Of special interest are bistable genetic toggle switches, which allow a living organism to exist in two alternative states and switch between them with a minimal metabolic burden. Naturally occurring toggle switches control important decision-making processes such as cell fate and developmental events. We sought to engineer whole plants with an orthogonal genetic toggle switch to be able to regulate artificial functions with minimal interference with their natural pathways. Here we report a bistable toggle memory switch for whole plants based on the phage PhiC31 serine integrase and its cognate recombination directionality factor (RDF). This genetic device was designed to control the transcription of two genes of interest by inversion of a central DNA regulatory element. Each state of the device is defined by one transcriptionally active gene of interest, while the other one remains inactive. The state of the switch can be reversibly modified by the action of the recombination actuators, which were administered externally (e.g. via agroinfiltration), or produced internally in response to an inducible chemical stimulus. We extensively characterized the kinetics, memory, and reversibility of this genetic switch in Nicotiana benthamiana through transient and stable transformation experiments using transgenic plants and hairy roots. Finally, we coupled the integrase expression to an estradiol-inducible promoter as a proof of principle of inducible activation of the switch.

synthetic biology↗

bioRxiv2019

Identification of transgene-free CRISPR edited plants of rice and tomato by monitoring DsRED fluorescence in dry seeds

CRISPR/Cas technology for specific and precise modification of genomes has transformed molecular biology. However, quick elimination of the transgene remains a challenge in plant biotechnology after genome edition, especially for crops due to their long life cycle and multiploidy, not only to avoid transgene position effects and to minimize the probability of off-target mutation appearance, but also to deliver end users with edited plants free of the recombinant gene editing machinery. Counter selection based on resistance marker genes are inconvenient in the case of CRISPR/Cas applications because plants lacking the transgene cannot survive the selection, and thus two more generations must be screened to evaluate the presence of the transgene. In the case of some crops, generations can last between 6 months and a few years, and the workload may be a limiting factor because transgene detection by PCR requires germination of seeds, and selected plants must be grown until a new generation can be harvested. The expression of fluorescent proteins as selective marker has been successfully used in Arabidopsis thaliana (Stuitje et al., 2003) as a fast method for transgene presence detection prior to seed germination. Despite this clear advantage, it has not been tested in other species yet, because of the special requirements of in vitro transformation protocols. To overcome the above mentioned inconvenient, we have adapted fluorescence-dependent monitoring of transgene to genome editing approaches in tomato and rice with the goal of obtaining transgene-free homozygous edited crop plants in two generations.

plant biology↗

bioRxiv2018

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator.

Synthetic Biology (SynBio) aims at rewiring plant metabolic and developmental programs with orthogonal regulatory circuits. This endeavour requires new molecular tools able to interact with endogenous factors in a potent yet at the same time highly specific manner. A promising new class of SynBio tools that could play this function are the synthetic transcriptional activators based on CRISPR/Cas9 architecture, which combine autonomous activation domains (ADs) capable of recruiting the cells transcription machinery, with the easily customizable DNA-binding activity of nuclease-inactivated Cas9 protein (dCas9), creating so-called Programmable Transcriptional Activators (PTAs). In search for optimized dCas9-PTAs we performed a combinatorial analysis with seven different ADs arranged in four different protein/RNA architectures. This analysis resulted in the selection of a new dCas9-PTA with improved features as compared with previously reported activators. The new synthetic riboprotein, named dCasEV2.1, combines EDLL and VPR ADs using a multiplexable mutated version (v2.1) of the previously described aptamer-containing guide RNA2.0. We show here that dCasEV2.1 is a strong and wide spectrum activator, displaying variable activation levels depending on the basal activity of the target promoter. Maximum activation rates reaching up to 10000 fold were observed when targeting the NbDFR gene. Most remarkably, RNAseq analysis of dCasEV2.1-transformed N. benthamiana leaves revealed that the topmost activation capacity of dCasEV2.1 on target genes is accompanied with strict genome-wide specificity, making dCasEV2.1 an attractive tool for rewiring plant metabolism and regulatory networks.

synthetic biology↗