Ana Ortiz-Atienza

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.

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.

The stearoyl‐ACP desaturase (SACPD) is a key enzyme in the regulation of saturated to unsaturated fatty acid ratio, playing a crucial role in regulating membrane stability and fluidity, as well as photosynthesis efficiency, which makes it an important research focus in crop species. This study reports the characterization and molecular cloning of pale dwarf (pad), a new tomato (Solanum lycopersicum L.) T‐DNA recessive mutant, which exhibits a dwarf and chlorotic phenotype. Functional studies of the T‐DNA tagged gene were conducted, including phylogenetic analysis, expression and metabolomic analyses, and generation of CRISPR/Cas9 knockout lines. The cloning of T‐DNA flanking genomic sequences and a co‐segregation analysis found the pad phenotype was caused by a T‐DNA insertion disrupting the tomato homologue of the Arabidopsis SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY 2 (SlSSI2), encoding a plastid localized isoform of SACPD. The phenotype of CRISPR/Cas9 SlSSI2 knockout lines confirmed that the morphological abnormalities in pad plants were due to SlSSI2 loss of function. Functional, metabolomic and expression analyses proved that SlSSI2 disruption causes deficiencies in 18:1 fatty acid desaturation and leads to diminished jasmonic acid (JA) content and increased salicylic acid (SA) levels. Overall, these results proved that SSI2 plays a crucial role in the regulation of polyunsaturated fatty acid profiles in tomato, and revealed that SlSSI2 loss of function results in an inhibited JA‐responsive signalling pathway and a constitutively activated SA‐mediated defence signalling response. This study lays the foundation for further research on tomato SACPDs and their role in plant performance and fitness.

Climate change exacerbates abiotic stresses like salinization, negatively impacting crop yield, so development of strategies, like using salt‐tolerant rootstocks, is crucial. The CALCINEURIN B‐LIKE 10 (SlCBL10) gene has been previously identified as a positive regulator of salt tolerance in the tomato shoot. Here, we report a different function of SlCBL10 in tomato shoot and root, as disruption of SlCBL10 only induced salt sensitivity when it was used in the scion but not in the rootstock. The use of SlCBL10 silencing rootstocks (Slcbl10 mutant and RNAi line) improved salt tolerance on the basis of fruit yield. These changes were associated with improved Na⁺ and K⁺ homoeostasis, as SlCBL10 silencing reduced the Na⁺ content and increased the K⁺ content under salinity, not only in the rootstock but also in the shoot. Improvement of Na⁺ homoeostasis in Slcbl10 rootstock seems to be mainly due to induction of SlSOS1 expression, while the higher K⁺ accumulation in roots seems to be mainly determined by expression of LKT1 transporter and SlSKOR channel. These findings demonstrate that SlCBL10 is a negative regulator of salt tolerance in the root, so the use of downregulated SlCBL10 rootstocks may provide a suitable strategy to increase tomato fruit production under salinity.

The tropical common bean (Phaseolus vulgaris L.) is an obligatory short-day plant that requires relaxation of the photoperiod to induce flowering. Similar to other crops, photoperiod-induced floral initiation depends on the differentiation and maintenance of meristems. In this study, the global changes in transcript expression profiles were analyzed in two meristematic tissues corresponding to the vegetative and inflorescence meristems of two genotypes with different sensitivities to photoperiods. A total of 3396 differentially expressed genes (DEGs) were identified, and 1271 and 1533 were found to be up-regulated and down-regulated, respectively, whereas 592 genes showed discordant expression patterns between both genotypes. Arabidopsis homologues of DEGs were identified, and most of them were not previously involved in Arabidopsis floral transition, suggesting an evolutionary divergence of the transcriptional regulatory networks of the flowering process of both species. However, some genes belonging to the photoperiod and flower development pathways with evolutionarily conserved transcriptional profiles have been found. In addition, the flower meristem identity genes APETALA1 and LEAFY, as well as CONSTANS-LIKE 5, were identified as markers to distinguish between the vegetative and reproductive stages. Our data also indicated that the down-regulation of the photoperiodic genes seems to be directly associated with promoting floral transition under inductive short-day lengths. These findings provide valuable insight into the molecular factors that underlie meristematic development and contribute to understanding the photoperiod adaptation in the common bean.

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.

Summary: CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping‐by‐sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein–protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel‐inside‐carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

Tomato cell wall-associated kinase 1 (SlWAK1) has only been studied in biotic stress response and hence its function in abiotic stress remains unknown. In a screening under salinity of an insertional mutant collection of tomato (Solanum lycopersicum L.), a mutant exhibiting lower degree of leaf chlorosis than wild type (WT) together with reduced leaf Na⁺ accumulation was selected. Genetic analysis of the mutation revealed that a single T-DNA insertion in the SlWAK1 gene was responsible of the mutant phenotype. Slwak1 null mutant reduced its shoot growth compared with WT, despite its improved Na⁺ homeostasis. SlWAK1 disruption affected osmotic homeostasis, as leaf water content was lower in mutant than in WT under salt stress. In addition, Slwak1 altered the source-sink balance under salinity, by increasing sucrose content in roots. Finally, a significant fruit yield reduction was found in Slwak1 vs. WT under long-term salt stress, mainly due to lower fruit weight. Our results show that disruption of SlWAK1 induces a higher sucrose transport from source leaf to sink root, negatively affecting fruit, the main sink at adult stage.