Metabolic control of seed germination in legumes.

Seed development, dormancy, and germination are connected with changes in metabolite levels. Not surprisingly, a complex regulatory network modulates biosynthesis and accumulation of storage products. Seed development has been studied profusely in Arabidopsis thaliana and has provided valuable insights into the genetic control of embryo development. However, not every inference applies to crop legumes, as these have been domesticated and selected for high seed yield and specific metabolic profiles and fluxes. Given its enormous economic relevance, considerable work has contributed to shed light on the mechanisms that control legume seed growth and germination. Here, we summarize recent progress in the understanding of regulatory networks that coordinate seed metabolism and development in legumes.

Natural genetic variation in the HAIRS ABSENT (H) gene increases type-VI glandular trichomes in both wild and domesticated tomatoes.

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.

The Lanata trichome mutation increases stomatal conductance and reduces leaf temperature in tomato.

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.

De novo domestication of wild species to create crops with increased resilience and nutritional value.

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.

Genome Editing to Achieve the Crop Ideotype in Tomato.

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.

Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress.

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.

Elevated CO2 induces age-dependent restoration of growth and metabolism in gibberellin-deficient plants.

MAIN CONCLUSION: 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.

Low soil pH modulates ethylene biosynthesis and germination response of Stylosanthes humilis seeds.

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.