Flower development is a crucial step towards the completion of the plant life cycle. Physiological processes and gene regulatory mechanisms underlying flower formation have been extensively characterized, and the implication of MADS-box transcription factors as primary regulators of flower morphology has been widely described, mainly due to the analysis of loss-of-function mutants in model species. Nevertheless, detailed characterization of allele variation in several MADS-box homologous genes from crop species remains undescribed. Here, we have characterized a tomato mutant with aberrant flower development. Mutant plants exhibit changes in petal cell identity, as well as homeotic transformations of stamens into carpelloid structures, which in most cases result in succulent organs. Molecular analysis proved that a loss-of-function mutation in the TOMATO MADS-BOX 6 (TM6) gene is responsible for this mutant phenotype. Furthermore, as a result of the loss of function of TM6, misregulation of the transcription and mRNA processing of other MADS-box genes involved in reproductive development has been detected. Our findings demonstrate that TM6 is a key player in the complex regulatory network of MADS-box genes controlling flower development and also provide a novel mutant that may be useful for generating male sterile lines in tomatoes.

Agricultural systems are currently facing significant issues, primarily due to population growth rates in the context of global climate change. Rising temperatures cause plant heat stress and impact crop yield, which in turn compromises global food production and safety. Climate change is also having a significant impact on water availability around the world, and droughts are becoming more frequent and severe in many regions. The combined effect of both heat and drought stresses increases plant damage, resulting in reduced plant development and productivity loss. Therefore, developing heat–drought-tolerant crop varieties is crucial for enhancing yield under these challenging conditions. Tomato (Solanum lycopersicum L.), a major vegetable crop highly appreciated for its nutritional qualities, is particularly sensitive to extreme temperatures, which have a significant negative impact on tomato fruit setting and cause male gametophyte abortion. In this work, a classical genetic approach was employed to identify tomato genotypes showing a resilient response to combined heat and drought stress conditions. A phenotype screening of a natural germplasm collection and an ethyl methanesulfonate (EMS) mutagenized population resulted in the identification of a significant number of tomato lines tolerant to combined heat and drought conditions, specifically 161 EMS lines and 24 natural accessions as tolerant. In addition, TILLING and Eco-TILLING analyses were used as proof-of-concept to isolate new genetic variants of genes previously reported as key regulators of abiotic stress responses in different species. The identification of these variants holds the potential to provide suitable plant material for breeding programs focused on enhancing tomato resilience to adverse climate conditions.

ABSTRACT Regulation of sodium homeostasis is crucial for plant response to salinity conditions. Here we report on the genetic and physiological characterization of two tomato allelic mutants, s odium ga therer1-2 ( sga1-2 ), which exhibit pronounced chlorosis and hyperhydration under salt stress. Mapping-by-sequencing revealed that mutant phenotype resulted from mutations in the SlRbohG gene, and CRISPR/Cas9 knockouts of this gene gave phenocopies of the sga1-2 mutants. Physiological analyses showed that sga1-2 salt hypersensitivity is linked to an increase of Na + and water transport from roots to shoots, which explains their extreme chlorosis and hyperhydration under salinity conditions. At the molecular level, SlPIP2;12 gene, an aquaporin down-regulated in the WT under salt stress, was overexpressed in the sga1-2 mutants, which could enhance water transport to the shoot. Also, sga1-2 mutants exhibited a significant reduction in the expression of key sodium transporters, thus modifying the normal distribution of Na + in tomato plant tissues. Furthermore, treatment of WT plants with the NADPH oxidase inhibitor DPI prevented H 2 O 2 production in response to salinity, resulting in elevated Na + accumulation in the shoot and reduced expression of the SlHKT1;2 gene in root. Altogether, our results show that SlRbohG plays a central role in salt tolerance through ROS-mediated signaling. HIGHLIGHT Loss of function of tomato SlRbohG gene leads hypersensibility to salt stress due to increased Na + and water transport from root to shoot.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Trichomes are specialised epidermal cells developed in the aerial surface of almost every terrestrial plant. These structures form physical barriers, which combined with their capability of synthesis of complex molecules, prevent plagues from spreading and confer trichomes a key role in the defence against herbivores. In this work, the tomato gene HAIRPLUS (HAP) that controls glandular trichome density in tomato plants was characterised. HAP belongs to a group of proteins involved in histone tail modifications although some also bind methylated DNA. HAP loss of function promotes epigenomic modifications in the tomato genome reflected in numerous differentially methylated cytosines and causes transcriptomic changes in hap mutant plants. Taken together, these findings demonstrate that HAP links epigenome remodelling with multicellular glandular trichome development and reveal that HAP is a valuable genomic tool for pest resistance in tomato breeding.

Tomato (Solanum lycopersicum L.) is a major horticultural crop and a model species among eudicots, especially for traits related to reproductive development. Although considerable progress has been made since the tomato genome sequence project was completed, most of the genes identified remain predictions with an unknown or hypothetical function. This lack of functional characterization hampers the use of the huge amount of genomic information available to improve the quality and productivity of this crop. Reverse genetics strategies such as artificial mutagenesis and next-generation sequencing approaches build the perfect tandem for increasing knowledge on functional annotation of tomato genes. This work reports the phenotypic characterization of a tomato mutant collection generated from an EMS chemical mutagenesis program aimed to identify interesting agronomic mutants and novel gene functions. Tomato mutants were grouped into fourteen phenotypic classes, including vegetative and reproductive development traits, and the inheritance pattern of the identified mutations was studied. In addition, causal mutation of a selected mutant line was isolated through a mapping-by-sequencing approach as a proof of concept of this strategy’s successful implementation. Results support tomato mutagenesis as an essential tool for functional genomics in this fleshy-fruited model species and a highly valuable resource for future breeding programs of this crop species aimed at the development of more productive and resilient new varieties under challenging climatic and production scenarios.

Forward genetic analysis remains as one of the most powerful tools for assessing gene functions, although the identification of the causal mutation responsible for a given phenotype has been a tedious and time-consuming task until recently. Advances in deep sequencing technologies have provided new approaches for the exploitation of natural and artificially induced genetic diversity, thus accelerating the discovery of novel allelic variants. In this chapter, a mapping-by-sequencing forward genetics approach is described to identify causal mutations in tomato (Solanum lycopersicum L.), a major crop species that is also a model species for plant biology and breeding.

The domestication process of tomato (Solanum lycopersium L.) resulted in very low variability for most agronomic interesting traits. In this work we explore the variability present in an unusual segregating population obtained by selfing a second cycle hybrid to identify and mapping novel Quantitative Trait Loci (QTL) for fruit quality traits. With this aim, a molecular characterization of the population by 130 single nucleotide polymorphism designed from genome sequencing of the parental lines was performed, which resulted in a genetic map and the location of seven novel QTL associated with the inheritance of the fruit quality traits: fruit diameter (R2 ranging from 0.179 to 0.207), fruit weight (R2 = 0.171) and fruit shape index (R2 ranging from 0.179 to 0.341). The analysis through the G-statistic allowed to define the consistence of the novel QTL identified, especially those associated to fruit shape index and fruit diameter. This approach has also led to the identification of new codominant molecular markers flanking the QTL which will be useful in future genetic breeding programs.

Since membranes play essential roles in all living beings, all cells have developed mechanisms for efficient and fast repair of membrane damage. In Escherichia coli, the Phage shock stress A (PspA) protein is involved in the maintenance of the integrity of its inner membrane in response to the damage produced by exposure to stress conditions. A role in thylakoid membrane maintenance and reorganization has been proposed for Vesicle Inducing Protein in Plastid 1 (VIPP1), the putative PspA ortholog in Arabidopsis thaliana. While some membranes of plant cells have been extensively studied, the biosynthesis and maintenance of chloroplast thylakoid membrane remains poorly known. Here, we report the cloning and functional characterization of the tomato (Solanum lycopersicum L.) ortholog of Escherichia coli PspA and Arabidopsis thaliana VIPP1, which we dubbed SlVIPP1. Our genetic and molecular characterization of slvipp1, an insertional mutant, allowed us to conclude that the tomato SlVIPP1 gene is needed for development, as Arabidopsis VIPP1, but not Escherichia coli PspA. Homozygous slvipp1 tomato plants are albino and exhibit early lethality and highly aberrant chloroplast development with almost complete absence of thylakoids. The phenotype of tomato RNAi lines and that of additional slvipp1 alleles generated by CRISPR/Cas9 gene editing technology confirmed that the morphological and histological aberrations shown by slvipp1 homozygotes are caused by VIPP1 lack of function. We also found that tomato SlVIPP1 overexpression does not cause any visible effect on plant morphology and viability. Our work with slvipp1 plants evidences that SlVIPP1 is an essential gene required for tomato survival, since its function is crucial for the proper formation and/or maintenance of thylakoid membranes.

Pod maturation of common bean relies upon complex gene expression changes, which in turn are crucial for seed formation and dispersal. Hence, dissecting the transcriptional regulation of pod maturation would be of great significance for breeding programs. In this study, a comprehensive characterization of expression changes has been performed in two common bean cultivars (ancient and modern) by analyzing the transcriptomes of five developmental pod stages, from fruit setting to maturation. RNA-seq analysis allowed for the identification of key genes shared by both accessions, which in turn were homologous to known Arabidopsis maturation genes and furthermore showed a similar expression pattern along the maturation process. Gene-expression changes suggested a role in promoting an accelerated breakdown of photosynthetic and ribosomal machinery associated with chlorophyll degradation and early activation of alpha-linolenic acid metabolism. A further study of transcription factors and their DNA binding sites revealed three candidate genes whose functions may play a dominant role in regulating pod maturation. Altogether, this research identifies the first maturation gene set reported in common bean so far and contributes to a better understanding of the dynamic mechanisms of pod maturation, providing potentially useful information for genomic-assisted breeding of common bean yield and pod quality attributes.

Increasing evidences highlight the importance of DEAD-box RNA helicases in plant development and stress responses. In a previous study, we characterized the tomato res mutant (restored cell structure by salinity), showing chlorosis and development alterations that reverted under salt-stress conditions. Map-based cloning demonstrates that RES gene encodes SlDEAD39, a chloroplast-targeted DEAD-box RNA helicase. Constitutive expression of SlDEAD39 complements the res mutation, while the silencing lines had a similar phenotype than res mutant, which is also reverted under salinity. Functional analysis of res mutant proved SlDEAD39 is involved in the in vivo processing of the chloroplast, 23S rRNA, at the hidden break-B site, a feature also supported by in vitro binding experiments of the protein. In addition, our results show that other genes coding for chloroplast-targeted DEAD-box proteins are induced by salt-stress, which might explain the rescue of the res mutant phenotype. Interestingly, salinity restored the phenotype of res adult plants by increasing their sugar content and fruit yield. Together, these results propose an unprecedented role of a DEAD-box RNA helicase in regulating plant development and stress response through the proper ribosome and chloroplast functioning, which, in turn, represents a potential target to improve salt tolerance in tomato crops.

The screening of 862 T-DNA lines was carried out to approach the genetic dissection of indirect adventitious organogenesis in tomato. Several mutants defective in different phases of adventitious organogenesis, namely callus growth (tdc-1), bud differentiation (tdb-1, -2, -3) and shoot-bud development (tds-1) were identified and characterized. The alteration of the TDC-1 gene blocked callus proliferation depending on the composition of growth regulators in the culture medium. Calli from tds-1 explants differentiated buds but did not develop normal shoots. Histological analysis showed that their abnormal development is due to failure in the organization of normal adventitious shoot meristems. Interestingly, tdc-1 and tds-1 mutant plants were indistinguishable from WT ones, indicating that the respective altered genes play specific roles in cell proliferation from explant cut zones (TDC-1 gene) or in the organization of adventitious shoot meristems (TDS-1 gene). Unlike the previous, plants of the three mutants defective in the differentiation of adventitious shoot-buds (tdb-1, -2, -3) showed multiple changes in vegetative and reproductive traits. Cosegregation analyses revealed the existence of an association between the phenotype of the tdb-3 mutant and a T-DNA insert, which led to the discovery that the SlMAPKKK17 gene is involved in the shoot-bud differentiation process.

Pollination and therefore fruit set in tomato (Solanum lycopersicum) is very sensitive to temperature. Parthenocarpy can be a very useful trait in tomato breeding in sustainable agriculture faced with global warming. Within a collection of Moneymaker tomato mutants a monogenic and recessive mutant with high fruit number was identified in heat stress conditions and named high fruit set under stress (hfs). No morphological alterations in vegetative and reproductive organs were observed except the bigger size of ovary. hfs is parthenocarpic, pollination not being required for fruit set, although this trait was not absolute since it produced some under‐seeded fruit. When plants were grown under extreme temperatures (higher than 35°C), hfs exhibits higher fruit yield than wild‐type (WT) due to increased fruit number. Another very interesting characteristic of hfs is its improved fruit quality under heat stress, exhibiting a better sweetness/acidity balance than WT. Interestingly, hfs was also tolerant to the combination of heat and salt stress, and the positive effect on production was due to both components of yield, fruit number and fruit weight. The generation of mostly seedless fruit and the high productivity and fruit quality under extreme temperatures make hfs a very interesting mutant to obtain new breeding high‐yield lines in adverse environmental conditions.

Los tricomas son protuberancias de la epidermis que participan en la resistencia a estreses abioticos y actuan como barrera mecanica y quimica frente a estreses bioticos como las plagas. El tomate cultivado muestra una gran diversidad fenotipica para caracteres relacionados con el desarrollo reproductivo, pero, como consecuencia del proceso de domesticacion, es susceptible a numerosas plagas y enfermedades que provocan cuantiosas perdidas economicas. Con el fin de incrementar la variabilidad genetica de S. lycopersicum, iniciamos un programa de mutagenesis en el que hemos identificado al mutante hairplus (hap), que presenta una elevada densidad de tricomas glandulares tipo I en todos los organos de la parte aerea de la planta, lo que incrementa su resistencia a plagas. El tomate cultivado (Solanum lycopersicum L.) es uno de los cultivos de mayor importancia economica entre la familia de las solanaceas, ya que presenta caracteres agronomicos muy favorables, como por ejemplo una elevada tolerancia a estreses abioticos como la salinidad y el estres hidrico. Sin embargo, como resultado de un exhaustivo proceso de domesticacion, la variabilidad genetica del tomate es escasa, sobre todo para caracteres relacionados con la respuesta a estreses bioticos. Tradicionalmente, los genes de resistenciaa plagas y enfermedades han sido introgresados en la especie cultivada a partir de especies silvestres. Sin embargo, los hibridos interespecificos muestran numerosos rasgos agronomicos indeseables y se requieren numerosas generaciones de retrocruzamiento para recuperar los rasgos favorables del tomate cultivado

Common bean (Phaseolus vulgaris L.) is an important grain legume domesticated independently in Mexico and Andean South America approximately 8000 years ago. Wild forms are obligate short-day plants, and relaxation of photoperiod sensitivity was important for expansion to higher latitudes and subsequent global spread. To better understand the nature and origin of this key adaptation, we examined its genetic control in progeny of a wide cross between a wild accession and a photoperiod-insensitive cultivar. We found that photoperiod sensitivity is under oligogenic control, and confirm a major effect of the Ppd locus on chromosome 1. The red/far-red photoreceptor gene PHYTOCHROME A3 (PHYA3) was identified as a strong positional candidate for Ppd, and sequencing revealed distinct deleterious PHYA3 mutations in photoperiod-insensitive Andean and Mesoamerican accessions. These results reveal the independent origins of photoperiod insensitivity within the two major common bean gene pools and demonstrate the conserved importance of PHYA genes in photoperiod adaptation of short-day legume species.

Arlequin (Alq) is a gain-of-function mutant whose most relevant feature is that sepals are able to become fruit-like organs due to the ectopic expression of the ALQ-TAGL1 gene. The role of this gene in tomato fruit ripening was previously demonstrated. To discover new functional roles for ALQ-TAGL1, and most particularly its involvement in the fruit set process, a detailed characterization of Alq yield-related traits was performed. Under standard conditions, the Alq mutant showed a much higher fruit set rate than the wild type. A significant percentage of Alq fruits were seedless. The results showed that pollination-independent fruit set in Alq is due to early transition from flower to fruit. Analysis of endogenous hormones in Alq suggests that increased content of cytokinins and decreased level of abscisic acid may account for precocious fruit set. Comparative expression analysis showed relevant changes of several genes involved in cell division, gibberellin metabolism, and the auxin signalling pathway. Since pollination-independent fruit set may be a very useful strategy for maintaining fruit production under adverse conditions, fruit set and yield in Alq plants under moderate salinity were assessed. Interestingly, Alq mutant plants showed a high yield under saline conditions, similar to that of Alq and the wild type under unstressed conditions.

Background: Tomato mutants altered in leaf morphology are usually identified in the greenhouse, which demands considerable time and space and can only be performed in adequate periods. For a faster but equally reliable scrutiny method we addressed the screening in vitro of 971T-DNA lines. Leaf development was evaluated in vitro in seedlings and shoot-derived axenic plants. New mutants were characterized in the greenhouse to establish the relationship between in vitro and in vivo leaf morphology, and to shed light on possible links between leaf development and agronomic traits, a promising field in which much remains to be discovered. Results: Following the screening in vitro of tomato T-DNA lines, putative mutants altered in leaf morphology were evaluated in the greenhouse. The comparison of results in both conditions indicated a general phenotypic correspondence, showing that in vitro culture is a reliable system for finding mutants altered in leaf development. Apart from providing homogeneous conditions, the main advantage of screening in vitro lies in the enormous time and space saving. Studies on the association between phenotype and nptII gene expression showed co-segregation in two lines (P > 99%). The use of an enhancer trap also allowed identifying gain-of-function mutants through reporter expression analysis. These studies suggested that genes altered in three other mutants were T-DNA tagged. New mutants putatively altered in brassinosteroid synthesis or perception, mutations determining multiple pleiotropic effects, lines affected in organ curvature, and the first tomato mutant with helical growth were discovered. Results also revealed new possible links between leaf development and agronomic traits, such as axillary branching, flower abscission, fruit development and fruit cracking. Furthermore, we found that the gene tagged in mutant 2635-MM encodes a Sterol 3-beta-glucosyltransferase. Expression analysis suggested that abnormal leaf development might be due to the lack-off-function of this gene. Conclusion: In vitro culture is a quick, efficient and reliable tool for identifying tomato mutants altered in leaf morphology. The characterization of new mutants in vivo revealed new links between leaf development and some agronomic traits. Moreover, the possible implication of a gene encoding a Sterol 3-beta-glucosyltransferase in tomato leaf development is reported.

Los tricomas son celulas epidermicas especializadas presentes en la superficie de casi todas las plantas y una de sus funciones es la defensa frente a plagas, actuando como barrera fisica o secretando sustancias nocivas o deterrentes. Durante la caracterizacion de una coleccion de mutantes de tomate para fenotipos relacionados con la resistencia a estreses bioticos, se identifico el mutante hairplus (hap), que presenta una elevada densidad de tricomas tipo I. Se han generado dobles mutantes obtenidos del cruzamiento de hap con todos los mutantes alterados en la densidad y la identidad de tricomas de tipo I descritos, lo que nos ha permitido identificar dos rutas geneticas que controlan la formacion de tricomas, asi como que los mutantes con mas tricomas son mas resistentes a las plagas. El tomate (Solanum lycopersicum L.) pertenece a la familia Solanaceae,entre la que destaca como uno de los cultivos de mayor impacto economico.El proceso de domesticacion de esta especie ocurrio en dos etapas y ha ocasionado una reduccion considerable en su variabilidad genetica, sobre todo para caracteres relacionados con la resistencia a estreses bioticos. Con el objetivo de aumentar la variabilidad genetica de tomate, nuestro grupo de investigacion ha iniciado un programa de mutagenesis quimica empleando el mutageno quimico etil metil sulfonato (EMS) en plantas del cultivar Moneymaker de tomate. Durante la caracterizacion de la coleccion seleccionamos el mutante hairplus (hap),denominado asi por presentar una elevada densidad de tricomas tipo I, especialmente en tallos vegetativos y tallos de la inflorescencia

Background: The res (restored cell structure by salinity) mutant, recently identified as the first tomato mutant accumulating jasmonate in roots under non-stressful conditions, exhibits a remarkable growth inhibition and morphological alterations in roots and leaves, which are suppressed when the mutant plants are exposed to salinity. In order to understand the molecular basis of the phenotype recovery induced by salt stress in the res mutant, we carried out a comparative transcriptomic analysis in roots and leaves of wild-type and res plants in absence of stress (control) and when the phenotypic recovery of res mutant began to be observed upon salt stress (5 days of 200 mM NaCl). Results: The number of differentially expressed genes was three times greater in roots than in leaves of res vs WT plants grown in control, and included the down-regulation of growth-promoting genes and the up-regulation of genes involved in Ca2+ signalling, transcription factors and others related to stress responses. However, these expression differences were attenuated under salt stress, coinciding with the phenotypic normalisation of the mutant. Contrarily to the attenuated response observed in roots, an enhanced response was found in leaves under salt stress. This included drastic expression changes in several circadian clock genes, such as GIGANTEA1, which was down-regulated in res vs WT plants. Moreover, the higher photosynthetic efficiency of res leaves under salt stress was accompanied by specific salt-upregulation of the genes RUBISCO ACTIVASE1 and ALTERNATIVE OXIDASE1A. Very few genes were found to be differentially expressed in both tissues ( root and leaf) and conditions (control and salt), but this group included SlWRKY39 and SlMYB14 transcription factors, as well as genes related to protein homeostasis, especially protease inhibitors such as METALLOCARBOXYPEPTIDASE INHIBITOR, which also seem to play a role in the phenotype recovery and salt tolerance of res mutant. Conclusions: In summary, in this study we have identified genes which seem to have a prominent role in salt tolerance. Moreover, we think this work could contribute to future breeding of tomato crops with increased stress tolerance.