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Cells, tissues, and organs harbour complex systems to allow communication between one another. The biological rhythms can be contrasting among organs, tissues, and cells, adjusting the physiology differently along the organism‘s regions. while also synchronising flowering and metabolism. Here, we revealed that Solanum lycopersicum manifests more balanced rhythms across the whole plant than wild tomatoes. Accordingly, the leaf development program is more coordinated in this organism than in wild species, in that young S. lycopersicum leaves develop slowly in comparison to mature leaves. Young leaves from wild tomatoes display higher photosynthetic rate than mature leaves, while large metabolite accumulations occur across plant segments. Consequently, diel metabolite levels are rather similar between young and mature leaves in the wild tomato S. pennellii, whereas the expression patterns for circadian clock genes are widely contrasting between differently aged leaves. We further demonstrated that introduction of domestication alleles into the wild tomato S. pimpinellifolium appears to synchronize the development of young and mature leaves, rendering this similar to that observed for S. lycopersicum. Collectively, the strengthening of inter-organ relationships in S. lycopersicum indicates an increased synchronization of its biology, which is probably fundamental to explain its elevated yield.

S-nitrosoglutathione (GSNO) plays a central role in nitric oxide (NO) homeostasis, and GSNO reductase (GSNOR) regulates the cellular levels of GSNO across kingdoms. Here, we investigated the role of endogenous NO in shaping shoot architecture and controlling fruit set and growth in tomato (Solanum lycopersicum). SlGSNOR silencing promoted shoot side branching and led to reduced fruit size, negatively impacting fruit yield. Greatly intensified in slgsnor knockout plants, these phenotypical changes were virtually unaffected by SlGSNOR overexpression. SlGSNOR silencing or knockout intensified protein tyrosine nitration and S-nitrosation and led to aberrant auxin production and signaling in leaf primordia and fruit-setting ovaries, besides restricting the shoot basipetal polar auxin transport stream. SlGSNOR deficiency triggered extensive transcriptional reprogramming at early fruit development, reducing pericarp cell proliferation due to restrictions on auxin, gibberellin and cytokinin production and signaling. Abnormal chloroplast development and carbon metabolism were also detected in early-developing NO-overaccumulating fruits, possibly limiting energy supply and building blocks for fruit growth. These findings provide new insights into the mechanisms by which endogenous NO fine-tunes the delicate hormonal network controlling shoot architecture, fruit set and post-anthesis fruit development, emphasizing the relevance of NO-auxin interaction for plant development and productivity.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalise the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here we identify the obv gene and the causative mutation, a non-synonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a rare polymorphism in the natural range of wild tomatoes, but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density and fruit shape, are controlled by OBV via changes in auxin signalling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR (ARF4) also results in defective BSE development, revealing an additional component of a novel genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection.One sentence summarydistribution of tomato heterobaric and homobaric leaves is controlled by a single-nucleotide polymorphism in an auxin-related transcription factor

Growing knowledge about crop domestication, combined with increasingly powerful gene-editing toolkits, sets the stage for the continual domestication of crop wild relatives and other lesser-known plant species.

The accumulation of anthocyanins is a well-known response to abiotic stresses in many plant species. However, the effects of anthocyanin accumulation on light absorbance and photosynthesis are unknown . Here, we addressed this question using a promoter replacement line of tomato constitutively expressing a MYB transcription factor (ANTHOCYANIN1, ANT1) that leads to anthocyanin accumulation. ANT1-overexpressing plants displayed traits associated with shade avoidance response: thinner leaves, lower seed germination rate, suppressed side branching, increased chlorophyll concentration, and lower photosynthesis rates than the wild type. Anthocyanin-rich leaves exhibited higher absorbance of light in the blue and red ends of the spectrum, while higher anthocyanin content in leaves provided photoprotection to high irradiance. Analyses of gene expression and primary metabolites content showed that anthocyanin accumulation produces a reconfiguration of transcriptional and metabolic networks that is consistent with, but not identical to those described for the shade avoidance response. Our results provide novel insights about how anthocyanins accumulation affects the trade-off between photoprotection and growth.

The family Cleomaceae is characterized by remarkable floral diversity and abundant nectar and pollen production. In this study, we evaluated the flower functional characteristics associated with the floral diversity of 10 Cleomaceae species, in addition to respectively seven and five accessions of Tarenaya hassleriana and T. longicarpa. The flowers were examined through five types of crosses and thus we determined the most efficient type of crossing based on the number of seeds. In addition, we evaluated pollen limitation, self-incompatibility, self-pollination and explored the possible effects on seed germination. The species can be grouped into three groups based on sexual systems: andromonoecious, hermaphroditism (chasmogamous and cleistogamous), and polygamomonoecious flowers. Gynandropsis gynandra, T. aculeata, T. diffusa, and T. microcarpa exhibited autogamy, whereas the other species did not. Overall, hand cross-pollination produced the highest seed yield, followed by open pollination. Self-pollination produced the lowest number of seeds, with no seed production noted in 16 accessions. Interestingly, the pollination type did not affect seed germination. Together, these results indicate the potential of the Cleomaceae family as a source of information regarding mechanisms involved in the determination of reproductive traits in plants.

Allelic variation in the CETS (CENTRORADIALIS, TERMINAL FLOWER 1, SELF PRUNING) gene family controls agronomically important traits in many crops. CETS genes encode phosphatidylethanolamine-binding proteins that have a central role in the timing of flowering as florigenic and anti-florigenic signals. The great expansion of CETS genes in many species suggests that the functions of this family go beyond flowering induction and repression. Here, we characterized the tomato SELF PRUNING 3C (SP3C) gene, and show that besides acting as a flowering repressor it also regulates seed germination and modulates root architecture. We show that loss of SP3C function in CRISPR/Cas9-generated mutant lines increases root length and reduces root side branching relative to the wild type. Higher SP3C expression in transgenic lines promotes the opposite effects in roots, represses seed germination, and also improves tolerance to water stress in seedlings. These discoveries provide new insights into the role of SP paralogs in agronomically relevant traits, and support future exploration of the involvement of CETS genes in abiotic stress responses.

Aluminium (Al), a limiting factor for crop productivity in acidic soils (pH ≤ 5.5), imposes drastic constraints for food safety in developing countries. The major mechanisms that allow plants to cope with Al involve manipulations of organic acids metabolism and DNA-checkpoints. When assumed individually both approaches have been insufficient to overcome Al toxicity. On analysing the centre of origin of most cultivated plants, we hypothesised that day-length seems to be a pivotal agent modulating Al tolerance across distinct plant species. We observed that with increasing distance from the Equator, Al tolerance decreases, suggesting a relationship with the photoperiod. We verified that long-day (LD) species are generally more Al-sensitive than short-day (SD) species, whereas genetic conversion of tomato for SD growth habit boosts Al tolerance. Reduced Al tolerance correlates with DNA-checkpoint activation under LD. Furthermore, DNA-checkpoint-related genes are under positive selection in Arabidopsis accessions from regions with shorter days, suggesting that photoperiod act as a selective barrier for Al tolerance. A diel regulation and genetic diversity affect Al tolerance, suggesting that day-length orchestrates Al tolerance. Altogether, photoperiodic control of Al tolerance might contribute to solving the historical obstacle that imposes barriers for developing countries to reach a sustainable agriculture.

Glandular trichomes produce and exude secondary metabolites, conferring insect resistance in many crop species. Whereas some of its wild relatives are insect-resistant, tomato (Solanum lycopersicum) is not. Identifying the genetic changes that altered trichome development and biochemistry during tomato domestication would contribute to breeding for insect resistance. A mutation in the HAIRS ABSENT (H) gene, which encodes a C2H2 zinc finger protein (ZFP8), leads to reduced trichome density. Several geographic accessions of S. pimpinellifolium, the wild ancestor of domesticated tomato, have glabrous organs that resemble the phenotype caused by h. Here, we investigated allelic diversity for H in tomato and S. pimpinellifolium accessions and their associated trichome phenotypes. We also evaluated how the developmental stage can affect trichome development in glabrous and non-glabrous plants. We found that glabrous accessions of S. pimpinellifolium have different ZFP8 nucleotide sequence changes, associated with altered trichome development and density. We also found that while the glabrous appearance of h mutants is caused by a lower density of long trichomes, the density of type-VI glandular trichomes is increased, particularly in the adult stages of plant development. These insights on the genetic control of trichome development may contribute to breeding for insect resistance in tomatoes and other crops.

Heterosis for agronomic traits is a widespread phenomenon that underpins hybrid crop breeding. However, heterosis at the level of cellular metabolites has not yet been fully explored. Some metabolites are highly sought after, like capsaicinoids found in peppers of theCapsicumgenus, which confer the characteristic pungent (‘hot’) flavour of the fruits. We analysed the metabolic profile of the fruit placenta and pericarp of inter- and intra-specific hybrids of two species ofCapsicumpeppers,C. chinense(cv. Habanero and cv. Biquinho) andC. annuumvar.annuum(cv. Jalapeño and cv. Cascadura Ikeda) in complete diallel crosses with reciprocals. The parents and hybrids were grown in a glasshouse and the profile of primary metabolites (sugars, amino acids and organic acids) and capsaicinoids was generated via gas chromatography–time of flight-mass spectrometry (GC–TOF-MS) and ultra-performance liquid chromatography coupled to a mass spectrometer (UPLC-MS), respectively. We found considerable heterotic effects specifically for capsaicinoids accumulation in the fruit placenta of the hybrids, including those derived from non-pungent parents. Furthermore, a large fraction of fruit primary metabolism was influenced by the specific cross combination, with marked parent-of-origin effects, i.e. whether a specific genotype was used as the pistillate or pollen parent. The differences in metabolite levels between the hybrids and their parents provide a snapshot of heterosis for primary and secondary metabolites and may contribute to explain the manifestation of whole-plant heterotic phenotypes.

Global agriculture is dominated by a handful of species that currently supply a huge proportion of our food and feed. It additionally faces the massive challenge of providing food for 10 billion people by 2050, despite increasing environmental deterioration. One way to better plan production in the face of current and continuing climate change is to better understand how our domestication of these crops included their adaptation to environments that were highly distinct from those of their centre of origin. There are many prominent examples of this, including the development of temperate Zea mays (maize) and the alteration of day-length requirements in Solanum tuberosum (potato). Despite the pre-eminence of some 15 crops, more than 50 000 species are edible, with 7000 of these considered semi-cultivated. Opportunities afforded by next-generation sequencing technologies alongside other methods, including metabolomics and high-throughput phenotyping, are starting to contribute to a better characterization of a handful of these species. Moreover, the first examples of de novo domestication have appeared, whereby key target genes are modified in a wild species in order to confer predictable traits of agronomic value. Here, we review the scale of the challenge, drawing extensively on the characterization of past agriculture to suggest informed strategies upon which the breeding of future climate-resilient crops can be based.

The selection of pure lines followed by crosses to create superior hybrids is one of the oldest strategies in crop breeding. However, in domesticated species of the Capsicum genus hybrid breeding has lagged, in part due to a lack of detailed information about the phenotypic and metabolic consequences of hybridization. Here, we performed reciprocal crosses between four inbred varieties of two species of cultivated C. chinense (cv. Habanero and Biquinho) and C. annuum var. annuum (cv. Jalapeño and cv. Cascadura Ikeda). These varieties were specifically selected for their highly divergent traits, including plant growth habit, fruit size, shape and pungency: Habanero and Jalapeño peppers are highly pungent forms, particularly popular in Mexico. The Biquinho cultivar of C. chinense and the Cascadura Ikeda bell pepper are traditional sweet cultivars from Brazil. From the parental genotypes and from the progeny of the reciprocal crosses, we measured 28 phenotypic traits, including plant growth, and yield, 32 fruit shape parameters, and 50 fruit pericarp and placenta metabolites, including capsaicinoids. We found large differences for agronomic and metabolic traits between the genotypes, including heterosis for pungency and reciprocal effects for agronomic traits. We also show that the strong association between fruit shape and pungency can be broken in intraspecific hybrids, paving the way for the precision breeding of novel varieties.Once sentence summaryHybrids of Capsicum peppers display heterosis and reciprocal effects for agronomic traits and fruit pungency

SummaryGlandular trichomes are involved in the production of food- and medicine-relevant chemicals in plants, besides being associated with pest resistance. In some wild Solanum species closely related to the cultivated tomato (S. lycopersicum), the presence of type-IV glandular trichomes leads to the production of high levels of insecticide acylsugars (AS). Conversely, low AS production observed in the cultivated tomato is attributed to its incapacity to develop type-IV trichomes in adult organs. Therefore, we hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of mature plants can be pest resistant. We introgressed into the tomato cultivar Micro-Tom (MT) the capability of S. galapagense to maintain the development of type-IV trichomes throughout all plant stages, thus creating a line named “Galapagos enhanced trichomes” (MT-Get). Mapping-by-sequencing of MT-Get revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further mapping reveled that S. galapagense alleles on chromosomes 1, 2 and 3 are sufficient for the presence of type-IV trichomes, but in lower densities. GC-MS, LC-MS, and gene expression analyses demonstrated that the increased density of type-IV trichomes was not accompanied by high AS production and exudation in MT-Get. Moreover, MT-Get did not differ from MT in its susceptibility to whitefly (Bemisia tabaci). Our findings demonstrates that type-IV glandular trichome development along with AS production and exudation are partially uncoupled at the genetic level. The MT-Get genotype represents a valuable resource for further studies involving the biochemical manipulation of type-IV trichome content through either genetic introgression or transgenic approaches.Significance StatementThis work identified loci in the tomato genome that control the heterochronic development of type-IV glandular trichomes and uncoupled the genetic control of this type of trichome ontogeny from acylsugar biosynthesis and accumulation, revealing a higher than anticipated genetic complexity of acylsugar-based insect resistance. The findings reported herein will contribute to further dissect the genetics of trichome development in tomato as well as to introgress broad and durable insect resistance in tomato and other Solanaceae.

A major issue in modern agriculture is water loss through stomata during photosynthetic carbon assimilation. In water-limited ecosystems, annual plants have strategies to synchronize their growth and reproduction to the availability of water. Some species or ecotypes of flowers are early to ensure that their life cycles are completed before the onset of late season terminal drought ("drought escape"). This accelerated flowering correlates with low water-use efficiency (WUE). The molecular players and physiological mechanisms involved in this coordination are not fully understood. We analyzed WUE using gravimetry, gas exchange, and carbon isotope discrimination in florigen deficient (sft mutant), wild-type (Micro-Tom), and florigen over-expressing (SFT-ox) tomato lines. Increased florigen expression led to accelerated flowering time and reduced WUE. The low WUE of SFT-ox was driven by higher stomatal conductance and thinner leaf blades. This florigen-driven effect on WUE appears be independent of abscisic acid (ABA). Our results open a new avenue to increase WUE in crops in an ABA-independent manner. Manipulation of florigen levels could allow us to produce crops with a life cycle synchronized to water availability.

The effect of elevated [CO2] on the growth of tomato plants with reduced gibberellin content is influenced by developmental stage. The impact of increased atmospheric carbon dioxide (CO2) on plants has aroused interest in the last decades. Signaling molecules known as plant hormones are fundamental controllers of plant growth and development. Elevated CO2 concentration ([CO2]) increases plant growth; however, whether plant hormones act as mediators of this effect is still an open question. Here, we show the response to elevated [CO2] in tomato does not require a functional gibberellin (GA) biosynthesis pathway. We compared growth and primary metabolism between wild-type (WT) and GA-deficient mutant (gib-1) plants transferred from ambient (400 ppm) to elevated (750 ppm) [CO2] at two different growth stages (either 21 or 35 days after germination, DAG). Growth, photosynthetic parameters and primary metabolism in the stunted gib-1 plants were restored when they were transferred to elevated [CO2] at 21 DAG. Elevated [CO2] also stimulated growth and photosynthetic parameters in WT plants at 21 DAG; however, only minor changes were observed in the level of primary metabolites. At 35 DAG, on the other hand, elevated [CO2] did not stimulate growth in WT plants and gib-1 mutants showed their characteristic stunted growth phenotype. Taken together, our results reveal that elevated [CO2] enhances growth only within a narrow developmental window, in which GA biosynthesis is dispensable. This finding could be relevant for breeding crops in the face of the expected increases in atmospheric CO2 over the next century.

Selenium modulates the formation of primary and lateral roots through alterations in auxin and ethylene, leading to new patterns of root architecture in rice seedlings. Selenium (Se) at low concentrations can control root growth through interaction with hormone biosynthesis. Auxin and ethylene have been shown to control the root architecture, with most of the information obtained from the eudicots such Arabidopsis and Nicotiana tabacum. Here, we presented the effects of Se on auxin and ethylene pathways and examined their impact on primary metabolism and root system architecture in rice (Oryza sativa L.) seedlings. Se treatment increased elongation of primary root, but decreased the number and length of lateral roots. Se led to decreased expression of genes associated with the biosynthesis of auxin and ethylene, concomitantly with reduced production of these hormones by the roots. Moreover, Se decreased the abundance of transcripts encoding auxin transport proteins. Indole-3-acetic acid (IAA) treatment overrode the repressive effect of Se on lateral root growth. The ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine (AVG) increased elongation of primary root, whereas the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) resulted in the opposite effect. Soluble sugars accumulate in roots of rice seedlings under Se treatment. Thus, Se modulates the formation of primary and lateral roots through alterations in auxin and ethylene, leading to new patterns of root architecture in rice seedlings.

Gigantism is a key component of the domestication syndrome, a suite of traits that differentiates crops from their wild relatives. Allometric gigantism is strongly marked in horticultural crops, causing disproportionate increases in the size of edible parts such as stems, leaves or fruits. Tomato (Solanum lycopersicum) has attracted attention as a model for fruit gigantism, and many genes have been described controlling this trait. However, the genetic basis of a corresponding increase in size of vegetative organs contributing to isometric gigantism, has remained relatively unexplored. Here, we identified a 0.4 Mbp region on chromosome 7 in introgression lines (ILs) from the wild speciesSolanum pennelliiin two different tomato genetic backgrounds (cv. M82 and cv. Micro-Tom) that controls vegetative and reproductive organ size in tomato. The locus, namedORGAN SIZE(ORG), was fine-mapped using genotype-by-sequencing. A survey of literature revealed thatORGoverlaps with previously mapped QTLs controlling tomato fruit weight during domestication. Alleles from the wild species led to reduced cell number in different organs, which was partially compensated by greater cell expansion in leaves but not in fruits. The result was a proportional reduction in leaf, flower and fruit size in the ILs harbouring the wild alleles. These findings suggest that selection for large fruit during domestication also tends to select for increases in leaf size by influencing cell division. Since leaf size is relevant for both source-sink balance and crop adaptation to different environments, the discovery ofORGcould allow fine-tuning of these parameters.One sentence summaryA locus that controls isometric size increase in vegetative and reproductive organs of tomato through changes in cell division

Gibberellin has been proposed to increase leaf elongation in radish (Raphanus sativus L.) plants, which is associated with decreased tuber growth. Since light intensity can control growth through interaction with gibberellin, investigation of the effect of gibberellin levels on the growth of radish plants would be a step forward towards unraveling factors that underlie biomass accumulation and allocation in response to irradiance levels. Here, we report that the gibberellin biosynthesis inhibitor paclobutrazol (PAC) decreased petiole elongation, but not lamina growth of radish plants grown under full sunlight. However, shading promoted an increase in shoot elongation, while in plants treated with PAC the petiole and leaf lamina fail to elongate. Plants treated with PAC allocated proportionally more biomass to their tubers and less to shoot compared to control under shade. Moreover, PAC decreased the abundance of transcripts encoding cell wall expansion proteins in leaf lamina and petiole of plants grown under shade, which was positively correlated with sugar consumption by the tuber, thereby increasing the mass fraction and concentrations of minerals for tuber. Thus, allocation of biomass during the growth of radish plants and nutritional quality of tubers depend on gibberellin and light intensity.

Aluminium is a limiting factor for crop productivity in acidic soils (pH ≤ 5.5). Since acid soil distribution on Earth cannot adequately explain the differential Al tolerance across the plant kingdom, we investigated photoperiod effects on plant Al tolerance. We observed that with increasing distance from the equator, Al tolerance disappears, suggesting a relationship with the photoperiod. Long-day (LD) species are generally more Al-sensitive than short-day (SD) species, whereas genetic conversion of tomato for SD growth boosts Al tolerance. Reduced Al tolerance correlates with DNA-checkpoint activation under LD. DNA-checkpoint-related genes are under positive selection in Arabidopsis accessions from regions with shorter days, suggesting photoperiod acts as a selective barrier for Al tolerance. Our findings revealed that diel regulation and genetic diversity affect Al tolerance, suggesting that day-length orchestrates Al tolerance.One-Sentence SummaryAluminum is a major constraint for crop yield worldwide. We reveal that photoperiod acts as a barrier for Al tolerance in plants.

Ethylene is a gaseous hormone with a well-established role in the regulation of plant growth and development. However, its role in the modulation of carbon assimilation and central metabolism remains unclear. Here, we investigated the morphophysiological and biochemical responses of tomato plants (Solanum lycopersicum) following the application of ethylene in the form of ethephon (CEPA - 2-chloroethylphosphonic acid), forcing the classical triple response phenotype. CEPA-treated plants were characterized by growth inhibition, as revealed by significant reductions in both shoot and root dry weights, coupled with a reduced number of leaves and lower specific leaf area. Growth inhibition was associated with a reduction in carbon assimilation due to both lower photosynthesis rates and stomatal conductance, coupled with impairments in carbohydrate turnover. Furthermore, exogenous ethylene led to the accumulation of cell wall compounds (i.e., cellulose and lignin) and phenolics, indicating that exposure to exogenous ethylene also led to changes in specialized metabolism. Collectively, our findings demonstrate that exogenous ethylene disrupts plant growth and leaf structure by affecting both central and specialized metabolism, especially that involved in carbohydrate turnover and cell wall biosynthesis, ultimately leading to metabolic responses that mimic stress situations.

For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type ("ideotype"). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.

For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type ("ideotype"). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.

Fruit pungency is caused by the accumulation of capsaicinoids, secondary metabolites whose relation to primary metabolism remains unclear. We have selected ten geographically diverse accessions of Capsicum chinense Jacq with different pungency levels. A detailed metabolic profile was conducted in the fruit placenta and pericarp at 20, 45, and 60 days after anthesis aiming at increasing our understanding of the metabolic changes in these tissues across fruit development and their potential connection to capsaicin metabolism. Overall, despite the variation in fruit pungency among the ten accessions, the composition and metabolite levels in both placenta and pericarp were uniformly stable across accessions. Most of the metabolite variability occurred between the fruit developmental stages rather than among the accessions. Interestingly, different metabolite adjustments in the placenta were observed among pungent and non-pungent accessions, which seem to be related to differences in the genetic background. Furthermore, we observed high coordination between metabolites and capsaicin production in C. chinense fruits, suggesting that pungency in placenta is adjusted with primary metabolism.

Creating crops with resistance to drought, soil salinity and insect damage, that simultaneously have higher nutritional quality, is challenging to conventional breeding due to the complex and diffuse genetic basis of those traits. Recent advances in gene editing technology, such as base editors and prime-editing, coupled with a deeper understanding of the genetic basis of domestication delivered by the analysis of crop 'pangenomes', open the exciting prospect of creating novel crops via manipulation of domestication-related genes in wild species. A de novo domestication platform may allow rapid and precise conversion of crop wild relatives into crops, while retaining many of the valuable resilience and nutritional traits left behind during domestication and breeding. Using the Solanaceae family as case in point, we discuss how such a knowledge-driven pipeline could be exploited to contribute to food security over the coming decades.

Nitrogen promotes changes in SLA through metabolism and anatomical traits in Capsicum plants. Specific leaf area (SLA) is a key trait influencing light interception and light use efficiency that often impacts plant growth and production. SLA is a key trait explaining growth variations of plant species under different environments. Both light and nitrogen (N) supply are important determinants of SLA. To better understand the effect of irradiance level and N on SLA in Capsicum chinense, we evaluated primary metabolites and morphological traits of two commercial cultivars (Biquinho and Habanero) in response to changes in both parameters. Both genotypes showed increased SLA with shading, and a decrease in SLA in response to increased N supply, however, with Habanero showing a stable SLA in the range of N deficiency to sufficient N doses. Correlation analyses indicated that decreased SLA in response to higher N supply was mediated by altered amino acids, protein, and starch levels, influencing leaf density. Moreover, in the range of moderate N deficiency to N sufficiency, both genotypes exhibited differences in SLA response, with Biquinho and Habanero displaying alterations on palisade and spongy parenchyma, respectively. Altogether, the results suggest that SLA responses to N supply are modulated by the balance between certain metabolites content and genotype-dependent changes in the parenchyma cells influencing leaf thickness and density.

The tomato mutant Never ripe (Nr), a loss-of-function for the ethylene receptor SlETR3, shows enhanced growth, associated with increased carbon assimilation and a rewiring of the central metabolism. Compelling evidence has demonstrated the importance of ethylene during tomato fruit development, yet its role on leaf central metabolism and plant growth remains elusive. Here, we performed a detailed characterization of Never ripe (Nr) tomato, a loss-of-function mutant for the ethylene receptor SlETR3, known for its fruits which never ripe. However, besides fruits, the Nr gene is also constitutively expressed in vegetative tissues. Nr mutant showed a growth enhancement during both the vegetative and reproductive stage, without an earlier onset of leaf senescence, with Nr plants exhibiting a higher number of leaves and an increased dry weight of leaves, stems, roots, and fruits. At metabolic level, Nr also plays a significant role with the mutant showing changes in carbon assimilation, carbohydrates turnover, and an exquisite reprogramming of a large number of metabolite levels. Notably, the expression of genes related to ethylene signaling and biosynthesis are not altered in Nr. We assess our results in the context of those previously published for tomato fruits and of current models of ethylene signal transduction, and conclude that ethylene insensitivity mediated by Nr impacts the whole central metabolism at vegetative stage, leading to increased growth rates.

Much attention has been given to the enhancement of photosynthesis as a strategy for the optimization of crop productivity. As traditional plant breeding is most likely reaching a plateau, there is a timely need to accelerate improvements in photosynthetic efficiency by means of novel tools and biotechnological solutions. The emerging field of synthetic biology offers the potential for building completely novel pathways in predictable directions and, thus, addresses the global requirements for higher yields expected to occur in the 21st century. Here, we discuss recent advances and current challenges of engineering improved photosynthesis in the era of synthetic biology toward optimized utilization of solar energy and carbon sources to optimize the production of food, fiber, and fuel., This review summarizes the progress in the emerging field of synthetic biology applied to photosynthesis. The authors also discuss recent strategies used for manipulating photosynthetic efficiency and their potential biotechnological applications.

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

Background and aimsTrichomes are epidermal structures with an enormous variety of ecological functions and economic applications. Glandular trichomes produce a rich repertoire of secondary metabolites, whereas non-glandular trichomes create a physical barrier against biotic and abiotic stressors. Intense research is underway to understand trichome development and function and enable breeding of more resilient crops. However, little is known on how enhanced trichome density would impinge on leaf photosynthesis, gas exchange and energy balance.MethodsPrevious work has compared multiple species differing in trichome density, instead here we analyzed monogenic trichome mutants in a single tomato genetic background (cv. Micro-Tom). We determined growth parameters, leaf spectral properties, gas exchange and leaf temperature in thehairs absent(h),Lanata(Ln) andWoolly(Wo) trichome mutants.Key resultsShoot dry mass, leaf area, leaf spectral properties and cuticular conductance were not affected by the mutations. However, theLnmutant showed increased carbon assimilation (A) possibly associated with higher stomatal conductance (gs), since there were no differences in stomatal density or stomatal index between genotypes. Leaf temperature was furthermore reduced inLnin the early hours of the afternoon.ConclusionsWe show that a single monogenic mutation can increase glandular trichome density, a desirable trait for crop breeding, whilst concomitantly improving leaf gas exchange and reducing leaf temperature.HIGHLIGHTA monogenic mutation in tomato increases trichome density and optimizes gas exchange and leaf temperature

Photosynthesis is the basis of primary productivity on the planet. Crop breeding has sustained steady improvements in yield to keep pace with population growth increases. Yet these advances have not resulted from improving the photosynthetic process per se, but rather from altering the way carbon is partitioned within the plant. Given that the pathways of photosynthesis and respiration catalyze partially opposing processes, it follows that their relative activities must be carefully regulated within plant cells. Exciting recent developments in efforts to understanding the interaction between respiration and photosynthesis during the last decades and the potential mechanisms linking mitochondrial function and photosynthetic efficiency will be reviewed. We additionally emphasize the challenges and opportunities to further understand the complex interplay between photosynthesis and related metabolic processes that has limited success in the manipulation and improvement of photosynthesis. We will also summarize recent advances of alternative approaches for the manipulation and enhancement of photosynthesis and their possible application for crop improvement.

The tomato mutant Never ripe ( Nr ), a loss-of-function for the ethylene receptor Sl ETR3, shows enhanced growth, associated with increased carbon assimilation and a rewiring of the central metabolism. Compelling evidence has demonstrated the importance of ethylene during tomato fruit development, yet its role on leaf central metabolism and plant growth remains elusive. Here, we performed a detailed characterization of Never ripe (Nr) tomato, a loss-of-function mutant for the ethylene receptor SlETR3, known for its fruits which never ripe. However, besides fruits, the Nr gene is also constitutively expressed in vegetative tissues. Nr mutant showed a growth enhancement during both the vegetative and reproductive stage, without an earlier onset of leaf senescence, with Nr plants exhibiting a higher number of leaves and an increased dry weight of leaves, stems, roots, and fruits. At metabolic level, Nr also plays a significant role with the mutant showing changes in carbon assimilation, carbohydrates turnover, and an exquisite reprogramming of a large number of metabolite levels. Notably, the expression of genes related to ethylene signaling and biosynthesis are not altered in Nr. We assess our results in the context of those previously published for tomato fruits and of current models of ethylene signal transduction, and conclude that ethylene insensitivity mediated by Nr impacts the whole central metabolism at vegetative stage, leading to increased growth rates.

Breeding of crops over millennia for yield and productivity1 has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost2. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, our engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Our results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants.

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp. Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.

The ideotype is a theoretical model of an archetypal cultivated plant. Recent progress in genome editing is aiding the pursuit of this ideal in crop breeding. Breeding is relatively straightforward when the traits in question are monogenic in nature and show Mendelian inheritance. Conversely, traits with a diffuse, polygenic basis such as abiotic stress resistance are more difficult to harness. In recent years, many genes have been identified that are important for plant domestication and act by increasing yield, grain or fruit size or altering plant architecture. Here, we propose that (a) key monogenic traits whose physiology has been unveiled can be molecularly tailored to achieve the ideotype; and (b) wild relatives of crops harboring polygenic stress resistance genes or other traits of interest could be de novo domesticated by manipulating monogenic yield-related traits through state-of-the-art gene editing techniques. An overview of the genomic and physiological challenges in the world’s main staple crops is provided. We focus on tomato and its wild Solanum (section Lycopersicon) relatives as a suitable model for molecular design in the pursuit of the ideotype for elite cultivars and to test de novo domestication of wild relatives.

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and ABA content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. cat1, Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

Coordination between structural and physiological traits is key to plants’ responses to environmental fluctuations. In heterobaric leaves, bundle sheath extensions (BSEs) increase photosynthetic performance (light-saturated rates of photosynthesis, Amax) and water transport capacity (leaf hydraulic conductance, Kleaf). However, it is not clear how BSEs affect these and other leaf developmental and physiological parameters in response to environmental conditions. The obscuravenosa (obv) mutation, found in many commercial tomato varieties, leads to absence of BSEs. We examined structural and physiological traits of tomato heterobaric and homobaric (obv) near-isogenic lines (NILs) grown at two different irradiance levels. Kleaf, minor vein density and stomatal pore area index decreased with shading in heterobaric but not in homobaric leaves, which show similarly lower values in both conditions. Homobaric plants, on the other hand, showed increased Amax, leaf intercellular air spaces and mesophyll surface area exposed to intercellular airspace (Smes) in comparison with heterobaric plants when both were grown in the shade. BSEs further affected carbon isotope discrimination, a proxy for long-term water-use efficiency. BSEs confer plasticity in traits related to leaf structure and function in response to irradiance levels and might act as a hub integrating leaf structure, photosynthetic function and water supply and demand.Summary statementThe presence of bundle sheath extension (BSEs) defines leaves as heterobaric, as opposed to homobaric leaves that lack them. Multiple functions have been proposed for BSEs, but their impact on different environmental conditions is still unclear. Here, we compared a tomato (Solanum lycopersicum) homobaric mutant lacking BSEs with its corresponding heterobaric wild-type, grown under two irradiance conditions. We show that the presence of BSEs differentially alters various physiological and anatomical parameters in response to growth irradiance. We propose that BSEs could act as hubs coordinating leaf plasticity in response to environmental factors.Article typeResearch article

Trichomes are epidermal structures with a large variety of ecological functions and economic applications. Glandular trichomes produce a rich repertoire of secondary metabolites, whereas non-glandular trichomes create a physical barrier on the epidermis: both operate in tandem against biotic and abiotic stressors. A deeper understanding of trichome development and function would enable the breeding of more resilient crops. However, little is known about the impact of altered trichome density on leaf photosynthesis, gas exchange and energy balance. Previous work has compared multiple, closely related species differing in trichome density. Here, we analysed monogenic trichome mutants in the same tomato genetic background (Solanum lycopersicum cv. 'Micro-Tom'). We determined growth parameters, leaf spectral properties, gas exchange and leaf temperature in the hairs absent (h), Lanata (Ln) and Woolly (Wo) trichome mutants. Shoot dry weight, leaf area, leaf spectral properties and cuticular conductance were not affected by the mutations. However, the Ln mutant showed increased net carbon assimilation rate (An), associated with higher stomatal conductance (gs), with no differences in stomatal density or stomatal index between genotypes. Leaf temperature was furthermore reduced in Ln in the hottest, early hours of the afternoon. We show that a single monogenic mutation that modifies trichome density, a desirable trait for crop breeding, concomitantly improves leaf gas exchange and reduces leaf temperature.

Although a yield ceiling has been reached for several major crops, enhancements are required to keep up with the demands of the increasing population. Here, traits which could help tackle this issue are discussed.The rates of increase in yield of the main commercial crops have been steadily falling in many areas worldwide. This generates concerns because there is a growing demand for plant biomass due to the increasing population. Plant yield should thus be improved in the context of climate change and decreasing natural resources. It is a major challenge which could be tackled by improving and/or altering light-use efficiency, CO2 uptake and fixation, primary metabolism, plant architecture and leaf morphology, and developmental plant processes. In this review, we discuss some of the traits which could lead to yield increase, with a focus on how natural genetic variation could be harnessed. Moreover, we provide insights for advancing our understanding of the molecular aspects governing plant growth and yield, and propose future avenues for improvement of crop yield. We also suggest that knowledge accumulated over the last decade in the field of molecular physiology should be integrated into new ideotypes.

High salinity in the soil hampers crop performance. In this context, elevated atmospheric CO2 concentration can alter plant responses to excess salinity, as shown by studies exposing plants to high concentration of NaCl (osmotic shock) in controlled conditions under constant irradiance and temperature. These experiments, however, may not reflect the conditions that occur in natural environments. Here, we studied the impact of progressively imposed salt stress on tomato plant biomass, hormone biosynthesis and primary metabolism under elevated CO2 concentration with fluctuating irradiance and temperature. Na+ concentration and Na+: K+ ratios in plant tissues were increased by high salinity under both ambient and elevated CO2 concentration. Elevated CO2 led to enhanced growth in tomato plants under salt stress through stimulation of photosynthesis and reduced concentrations of abscisic acid and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in both leaves and roots. Furthermore, whereas high salinity reduced the concentration of Krebs Cycle intermediates and increased the concentration of photorespiratory metabolites glycine and serine, all were restored to control levels by elevated CO2. Our results demonstrate a beneficial effect of rising atmospheric CO2 concentration for crops and reveal new interactions in the physiological network controlling salinity stress responses.

Although the chemical characteristics of selenomethionine (SeMet) are similar to those of methionine (Met), the physiological activity of SeMet apparently differs in its ability to stimulate ethylene production in plant tissues. Since selenium alters root architecture of rice seedlings by modifying ethylene production, the investigation of the effect of SeMet and Met on rice growth would be a step forward towards unraveling factors that underlie selenium toxicity. Here, we report that SeMet increased concentrations of reactive oxygen species (ROS), inhibiting auxin and increasing ethylene production in rice seedlings. The effect of SeMet on seedlings was mediated by the inhibition of the abundance of transcripts encoding auxin transport and cell expansion proteins. Moreover, SeMet led to increased seedling respiration, which was positively correlated with organic acids consumption, but negatively with sugars consumption, thereby decreasing seedling growth. In contrast with SeMet treatment, Met did not affect ROS production, hormone biosynthesis and seedling growth, indicating an exclusive selenium effect. The singlet oxygen scavenger, 1,4-diazabicyclooctane, overrode the repressive effect of SeMet in seedling growth. Our results demonstrate a phytotoxic effect of SeMet for rice seedlings and reveal a relationship between reactive oxygen species, hormone homeostasis and carbon availability, which regulates growth responses.

Yield-related traits of Capsicum chinense are highly dependent on coordination between vegetative and reproductive growth, since the formation of reproductive tissues occurs iteratively in new sympodial bifurcations. In this study, we used two C. chinense cultivars (Biquinho and Habanero), contrasting for fruit size and fruit set, to investigate the responses of nitrogen (N) deficiency and excess on growth, photosynthesis, carbon (C) and N metabolisms as well as yield-related traits. Both cultivars increased biomass allocation to leaves in conditions of higher N supply and exhibited a parabolic behavior for fruit biomass allocation. Plants growing under N-deficiency produced a lower number of flowers and heavier fruits. Contrarily, plants under high N condition tended to decrease their CO2 assimilation rate, harvest index and fruit weight. Biquinho, the cultivar with lower fruit size and higher fruit set, was initially less affected by excess of N due to its continuous formation of new reproductive sinks in relation to Habanero (which has lower fruit set and higher fruit size). The results suggest that N amount influences sucrose supply to different organs and can differentially affect yield-related traits between Capsicum cultivars with contrasting source-sink relations.