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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.

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.

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 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.

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.

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.

In Stylosanthes humilis, salt stress tolerance is associated with ethylene production by the seeds, however, how salt stress controls seed germination and ethylene production is poorly understood. Here, we studied the hormonal and metabolic changes triggered by salt stress on germination of S. humilis seeds. Salt stress led to decreased seed germination and ethylene production, concomitantly with higher abscisic acid (ABA) production by seeds. Treatment with NaCl and ABA promoted distinct changes in energy metabolism, allowing seeds to adapt to salt stress conditions. Treatment with the ABA biosynthesis inhibitor fluridone or ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) reversed the effects of salt stress on seed germination and ethylene production. Moreover, ethylene concentration was decreased by increasing the pH of the salt solution. High pH, however, did not influence concentration of ABA in seeds under salt stress. We conclude that biosynthesis of ABA and ethylene in response to salt stress constitutes a point of convergence that provides flexibility to regulate energy metabolism and embryo growth potential of S. humilis seeds within a given pH condition.

Stylosanthes humilis is known to exhibit high persistence in acid soils, however, how low soil pH controls seed germination as well as root and hypocotyl growth remains unknown. This study was carried out to evaluate the hormonal and metabolic alterations induced by low soil pH on seed germination behavior of S. humilis. Seeds of S. humilis were sown in acid soil samples or sand soaked in buffer solution with pH ranging from 4.0 to 7.0. Concentrations of indole-3-acetic acid, ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), primary metabolite profile and final seed germination were evaluated after four days. Low soil pH led to increased final seed germination, concomitantly with higher root penetration into the soil as well as higher ACC and ethylene production by seedlings. Treatment with the ethylene biosynthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine (AVG) greatly reduced final seed germination under acidic conditions. Final seed germination of seeds treated with AVG was increased by exogenous ethylene application in a dose-dependent manner. Furthermore, low soil pH promoted distinct changes in IAA concentrations, and in carbon and nitrogen metabolism in hypocotyl and roots. Low soil pH increases the final germination of S. humilis seeds through alterations in ethylene metabolism, allowing root penetration into the soil.

Glandular trichomes are structures with widespread distribution and deep ecological significance. In the Solanum genus, type-IV glandular trichomes provide resistance to insect pests. The occurrence of these structures is, however, poorly described and controversial in cultivated tomato (Solanum lycopersicum). Optical and scanning electron microscopy were used to screen a series of well-known commercial tomato cultivars, revealing the presence of type-IV trichomes on embryonic (cotyledons) and juvenile leaves. A tomato line overexpressing the microRNA miR156, known to promote heterochronic development, and mutants affecting KNOX and CLAVATA3 genes possessed type-IV trichomes in adult leaves. A re-analysis of the Woolly (Wo) mutant, previously described as enhancing glandular trichome density, showed that this effect only occurs at the juvenile phase of vegetative development. Our results suggest the existence of at least two levels of regulation of multicellular trichome formation in tomato: one enhancing different types of trichomes, such as that controlled by the WOOLLY gene, and another dependent on developmental stage, which is fundamental for type-IV trichome formation. Their combined manipulation could represent an avenue for biotechnological engineering of trichome development in plants.

Naturally occurring genetic variation in plants can be very useful to dissect the complex regulation of primary metabolism as well as of physiological traits such as photosynthesis and photorespiration. The physiological and genetic mechanisms underlying natural variation in closely related species or accessions may provide important information that can be used to improve crop yield. In this chapter we describe in detail the use of a population of introgression lines (ILs), with the Solanum pennellii IL population as a study case, as a tool for the identification of genomic regions involved in the control of photosynthetic efficiency.

The tropical forage legume Stylosanthes humilis is naturally distributed in the acidic soils of the tropics. However, data concerning the role of low soil pH in the control of S. humilis seed germination remains limited. Recently, we have demonstrated that acidic soil triggers increased ethylene production during germination of S. humilis seeds, concomitantly with higher root penetration into the soil. Our finding points an important role of low soil pH as a signal allowing penetration of root in the soil through interaction with the ethylene signalling pathway. Herein, we discuss how low soil pH induces changes on seed hormonal physiology.