Natural variation in response to combined water and nitrogen deficiencies in Arabidopsis.

Understanding plant responses to individual stresses does not mean that we understand real world situations, where stresses usually combine and interact. These interactions arise at different levels, from stress exposure to the molecular networks of the stress response. Here, we built an in-depth multi-omics description of plant responses to mild water (W) and nitrogen (N) limitations, either individually or combined, among five genetically different Arabidopsis (Arabidopsis thaliana) accessions. We highlight the different dynamics in stress response through integrative traits such as rosette growth and the physiological status of the plants. We also used transcriptomics and metabolomics profiling during a stage when the plant response was stabilized to determine the wide diversity in stress-induced changes among accessions, highlighting the limited reality of a 'universal' stress response. The main effect of the WxN interaction was an attenuation of the N-deficiency syndrome when combined with mild drought, but to a variable extent depending on the accession. Other traits subject to WxN interactions are often accession-specific. Multi-omics analyses identified a subset of transcript-metabolite clusters that are critical to stress responses but essentially variable according to the genotype factor. Including intra-specific diversity in our descriptions of plant stress response places our findings in perspective.

Pollinator-assisted plant phenotyping, selection, and breeding for crop resilience to abiotic stresses.

Food security is threatened by climate change, with heat and drought being the main stresses affecting crop physiology and ecosystem services, such as plant-pollinator interactions. We hypothesize that tracking and ranking pollinators' preferences for flowers under environmental pressure could be used as a marker of plant quality for agricultural breeding to increase crop stress tolerance. Despite increasing relevance of flowers as the most stress sensitive organs, phenotyping platforms aim at identifying traits of resilience by assessing the plant physiological status through remote sensing-assisted vegetative indexes, but find strong bottlenecks in quantifying flower traits and in accurate genotype-to-phenotype prediction. However, as the transport of photoassimilates from leaves (sources) to flowers (sinks) is reduced in low-resilient plants, flowers are better indicators than leaves of plant well-being. Indeed, the chemical composition and amount of pollen and nectar that flowers produce, which ultimately serve as food resources for pollinators, change in response to environmental cues. Therefore, pollinators' preferences could be used as a measure of functional source-to-sink relationships for breeding decisions. To achieve this challenging goal, we propose to develop a pollinator-assisted phenotyping and selection platform for automated quantification of Genotype × Environment × Pollinator interactions through an insect geo-positioning system. Pollinator-assisted selection can be validated by metabolic, transcriptomic, and ionomic traits, and mapping of candidate genes, linking floral and leaf traits, pollinator preferences, plant resilience, and crop productivity. This radical new approach can change the current paradigm of plant phenotyping and find new paths for crop redomestication and breeding assisted by ecological decisions.

The VIL gene CRAWLING ELEPHANT controls maturation and differentiation in tomato via polycomb silencing.

VERNALIZATION INSENSITIVE 3-LIKE (VIL) proteins are PHD-finger proteins that recruit the repressor complex Polycomb Repressive Complex 2 (PRC2) to the promoters of target genes. Most known VIL targets are flowering repressor genes. Here, we show that the tomato VIL gene CRAWLING ELEPHANT (CREL) promotes differentiation throughout plant development by facilitating the trimethylation of Histone H3 on lysine 27 (H3K27me3). We identified the crel mutant in a screen for suppressors of the simple-leaf phenotype of entire (e), a mutant in the AUX/IAA gene ENTIRE/SlIAA9, involved in compound-leaf development in tomato. crel mutants have increased leaf complexity, and suppress the ectopic blade growth of e mutants. In addition, crel mutants are late flowering, and have delayed and aberrant stem, root and flower development. Consistent with a role for CREL in recruiting PRC2, crel mutants show drastically reduced H3K27me3 enrichment at approximately half of the 14,789 sites enriched in wild-type plants, along with upregulation of many underlying genes. Interestingly, this reduction in H3K27me3 across the genome in crel is also associated with gains in H3K27me3 at a smaller number of sites that normally have modest levels of the mark in wild-type plants, suggesting that PRC2 activity is no longer limiting in the absence of CREL. Our results uncover a wide role for CREL in plant and organ differentiation in tomato and suggest that CREL is required for targeting PRC2 activity to, and thus silencing, a specific subset of polycomb targets.

SlHAK20: a new player in plant salt tolerance.

The majority of crops remain sensitive to salt stress despite the steady increase in salt concentration in agricultural soil. In this issue of The EMBO Journal, Wang et al (2020) screen hundreds of tomato accessions to identify SlHAK20 as a gene accounting for quantitative differences in salt tolerance between accessions. SlHAK20 is a potassium transporter belonging to a poorly studied clade in a large family of transporters, and its mutation induces salt susceptibility both in tomato and rice.

Plasticity of maternal environment-dependent expression-QTLs of tomato seeds.

Seeds are essential for plant reproduction, survival, and dispersal. Germination ability and successful establishment of young seedlings strongly depend on seed quality and on environmental factors such as nutrient availability. In tomato (Solanum lycopersicum) and many other species, seed quality and seedling establishment characteristics are determined by genetic variation, as well as the maternal environment in which the seeds develop and mature. The genetic contribution to variation in seed and seedling quality traits and environmental responsiveness can be estimated at transcriptome level in the dry seed by mapping genomic loci that affect gene expression (expression QTLs) in contrasting maternal environments. In this study, we applied RNA-sequencing to construct a linkage map and measure gene expression of seeds of a tomato recombinant inbred line (RIL) population derived from a cross between S. lycopersicum (cv. Moneymaker) and S. pimpinellifolium (G1.1554). The seeds matured on plants cultivated under different nutritional environments, i.e., on high phosphorus or low nitrogen. The obtained single-nucleotide polymorphisms (SNPs) were subsequently used to construct a genetic map. We show how the genetic landscape of plasticity in gene regulation in dry seeds is affected by the maternal nutrient environment. The combined information on natural genetic variation mediating (variation in) responsiveness to the environment may contribute to knowledge-based breeding programs aiming to develop crop cultivars that are resilient to stressful environments.

ENO regulates tomato fruit size through the floral meristem development network.

A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/Cas9 genome editing methods, we isolated EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes.

Interaction between photoperiod and variation in circadian rhythms in tomato.

BACKGROUND: Many biological processes follow circadian rhythmicity and are controlled by the circadian clock. Predictable environmental changes such as seasonal variation in photoperiod can modulate circadian rhythms, allowing organisms to adjust the timing of their biological processes to the time of the year. In some crops such as rice, barley or soybean, mutations in circadian clock genes have altered photoperiod sensitivity, enhancing their cultivability in specific seasons and latitudes. However, how changes in circadian rhythms interact with the perception of photoperiod in crops remain poorly studied. In tomato, the appearance during domestication of mutations in EMPFINDLICHER IM DUNKELROTEN LICHT 1 (EID1, Solyc09g075080) and NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 2 (LNK2, Solyc01g068560) delayed both the phase and period of its circadian rhythms. The fact that variation in period and phase are separated in tomato provides an optimal tool to study how these factors affect the perception of photoperiod. RESULTS: Here we develop tomato near isogenic lines carrying combinations of wild alleles of EID1 and LNK2 and show that they recreate the changes in phase and period that occurred during its domestication. We perform transcriptomic profiling of these near isogenic lines under two different photoperiods, and observe that EID1, but not LNK2, has a large effect on how the tomato transcriptome responds to photoperiod. This large effect of EID1 is likely a consequence of the global phase shift elicited by this gene in tomato's circadian rhythms. CONCLUSIONS: Our study shows that changes in phase that occurred during tomato domestication determine photoperiod perception in this species, while changes in period have little effect.

SELF PRUNING 3C is a flowering repressor that modulates seed germination, root architecture, and drought responses.

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.

Arabidopsis SME1 Regulates Plant Development and Response to Abiotic Stress by Determining Spliceosome Activity Specificity.

The control of precursor-messenger RNA (pre-mRNA) splicing is emerging as an important layer of regulation in plant responses to endogenous and external cues. In eukaryotes, pre-mRNA splicing is governed by the activity of a large ribonucleoprotein machinery, the spliceosome, whose protein core is composed of the Sm ring and the related Sm-like 2-8 complex. Recently, the Arabidopsis (Arabidopsis thaliana) Sm-like 2-8 complex has been characterized. However, the role of plant Sm proteins in pre-mRNA splicing remains largely unknown. Here, we present the functional characterization of Sm protein E1 (SME1), an Arabidopsis homolog of the SME subunit of the eukaryotic Sm ring. Our results demonstrate that SME1 regulates the spliceosome activity and that this regulation is controlled by the environmental conditions. Indeed, depending on the conditions, SME1 ensures the efficiency of constitutive and alternative splicing of selected pre-mRNAs. Moreover, missplicing of most targeted pre-mRNAs leads to the generation of nonsense-mediated decay signatures, indicating that SME1 also guarantees adequate levels of the corresponding functional transcripts. In addition, we show that the selective function of SME1 in ensuring appropriate gene expression patterns through the regulation of specific pre-mRNA splicing is essential for adequate plant development and adaptation to freezing temperatures. These findings reveal that SME1 plays a critical role in plant development and interaction with the environment by providing spliceosome activity specificity.

Functional analysis of FRIGIDA using naturally occurring variation in Arabidopsis thaliana.

The FRIGIDA locus (FRI, AT4G00650) has been extensively studied in Arabidopsis thaliana because of its role creating flowering time diversity. The FRI protein regulates flowering induction by binding partner proteins on its N-terminus and C-terminus domains and creating a supercomplex that promotes transcription of the floral repressor FLOWERING LOCUS C (FLC). Despite the knowledge accumulated on FRIGIDA (FRI), the function of the highly conserved central domain of the protein is still unknown. Functional characterization of naturally occurring DNA polymorphisms can provide useful information about the role of a protein and the localization of its operative domains. For FRI, loss-of-function mutations are positively selected and widespread in nature, making them a powerful tool to study the function of the different domains of the protein. Here we explore natural sequence variation in the FRI locus in more than 1000 Arabidopsis accessions. We identify 127 mutations that alter the FRI protein, including 60 that had never been described before. We defined 103 different alleles of FRI and study their association with variation in flowering time. We confirmed these associations by cloning 22 different alleles and expressing them in a common null genetic background. Our analysis pinpoints two single amino acid changes in the central domain that render the protein non-functional. We show that these two mutations determine the stability and cellular localization of the FRI protein. In summary, our work makes use of natural variants at the FRI locus to help understanding the function of the central domain of the FRI protein.

Mutations in EID1 and LNK2 caused light-conditional clock deceleration during tomato domestication.

Circadian period and phase of cultivated tomato (Solanum lycopersicum) were changed during domestication, likely adapting the species to its new agricultural environments. Whereas the delayed circadian phase is mainly caused by allelic variation of EID1, the genetic basis of the long circadian period has remained elusive. Here we show that a partial deletion of the clock gene LNK2 is responsible for the period lengthening in cultivated tomatoes. We use resequencing data to phylogenetically classify hundreds of tomato accessions and investigate the evolution of the eid1 and lnk2 mutations along successive domestication steps. We reveal signatures of selection across the genomic region of LNK2 and different patterns of fixation of the mutant alleles. Strikingly, LNK2 and EID1 are both involved in light input to the circadian clock, indicating that domestication specifically targeted this input pathway. In line with this, we show that the clock deceleration in the cultivated tomato is light-dependent and requires the phytochrome B1 photoreceptor. Such conditional variation in circadian rhythms may be key for latitudinal adaptation in a variety of species, including crop plants and livestock.

Natural variation in HsfA2 pre-mRNA splicing is associated with changes in thermotolerance during tomato domestication.

Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS). We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene. We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre-mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2-II, implicated in the early stress response, at the expense of HsfA2-I which is involved in establishing short-term acclimation and thermotolerance. We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short-term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.

Pivotal Roles of Cryptochromes 1a and 2 in Tomato Development and Physiology.

Cryptochromes are flavin-containing blue/UVA light photoreceptors that regulate various plant light-induced physiological processes. In Arabidopsis (Arabidopsis thaliana), cryptochromes mediate de-etiolation, photoperiodic control of flowering, entrainment of the circadian clock, cotyledon opening and expansion, anthocyanin accumulation, and root growth. In tomato (Solanum lycopersicum), cryptochromes are encoded by a multigene family, comprising CRY1a, CRY1b, CRY2, and CRY3 We have previously reported the phenotypes of tomato cry1a mutants and CRY2 overexpressing plants. Here, we report the isolation by targeting induced local lesions in genomes, of a tomato cry2 knock-out mutant, its introgression in the indeterminate Moneymaker background, and the phenotypes of cry1a/cry2 single and double mutants. The cry1a/cry2 mutant showed phenotypes similar to its Arabidopsis counterpart (long hypocotyls in white and blue light), but also several additional features such as increased seed weight and internode length, enhanced hypocotyl length in red light, inhibited primary root growth under different light conditions, anticipation of flowering under long-day conditions, and alteration of the phase of circadian leaf movements. Both cry1a and cry2 control the levels of photosynthetic pigments in leaves, but cry2 has a predominant role in fruit pigmentation. Metabolites of the sterol, tocopherol, quinone, and sugar classes are differentially accumulated in cry1a and cry2 leaves and fruits. These results demonstrate a pivotal role of cryptochromes in controlling tomato development and physiology. The manipulation of these photoreceptors represents a powerful tool to influence important agronomic traits such as flowering time and fruit quality.

Mild drought in the vegetative stage induces phenotypic, gene expression, and DNA methylation plasticity in Arabidopsis but no transgenerational effects.

There is renewed interest in whether environmentally induced changes in phenotypes can be heritable. In plants, heritable trait variation can occur without DNA sequence mutations through epigenetic mechanisms involving DNA methylation. However, it remains unknown whether this alternative system of inheritance responds to environmental changes and if it can provide a rapid way for plants to generate adaptive heritable phenotypic variation. To assess potential transgenerational effects induced by the environment, we subjected four natural accessions of Arabidopsis thaliana together with the reference accession Col-0 to mild drought in a multi-generational experiment. As expected, plastic responses to drought were observed in each accession, as well as a number of intergenerational effects of the parental environments. However, after an intervening generation without stress, except for a very few trait-based parental effects, descendants of stressed and non-stressed plants were phenotypically indistinguishable irrespective of whether they were grown in control conditions or under water deficit. In addition, genome-wide analysis of DNA methylation and gene expression in Col-0 demonstrated that, while mild drought induced changes in the DNA methylome of exposed plants, these variants were not inherited. We conclude that mild drought stress does not induce transgenerational epigenetic effects.

Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites.

Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics, and 13C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2 Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl-CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity.

Allele-specific expression and genetic determinants of transcriptomic variations in response to mild water deficit in tomato.

Characterizing the natural diversity of gene expression across environments is an important step in understanding how genotype-by-environment interactions shape phenotypes. Here, we analyzed the impact of water deficit onto gene expression levels in tomato at the genome-wide scale. We sequenced the transcriptome of growing leaves and fruit pericarps at cell expansion stage in a cherry and a large fruited accession and their F1 hybrid grown under two watering regimes. Gene expression levels were steadily affected by the genotype and the watering regime. Whereas phenotypes showed mostly additive inheritance, ~80% of the genes displayed non-additive inheritance. By comparing allele-specific expression (ASE) in the F1 hybrid to the allelic expression in both parental lines, respectively, 3005 genes in leaf and 2857 genes in fruit deviated from 1:1 ratio independently of the watering regime. Among these genes, ~55% were controlled by cis factors, ~25% by trans factors and ~20% by a combination of both types of factors. A total of 328 genes in leaf and 113 in fruit exhibited significant ASE-by-watering regime interaction, among which ~80% presented trans-by-watering regime interaction, suggesting a response to water deficit mediated through a majority of trans-acting loci in tomato. We cross-validated the expression levels of 274 transcripts in fruit and leaves of 124 recombinant inbred lines (RILs) and identified 163 expression quantitative trait loci (eQTLs) mostly confirming the divergences identified by ASE. Combining phenotypic and expression data, we observed a complex network of variation between genes encoding enzymes involved in the sugar metabolism.

CLAUSA Is a MYB Transcription Factor That Promotes Leaf Differentiation by Attenuating Cytokinin Signaling.

Leaf morphogenesis and differentiation are highly flexible processes, resulting in a large diversity of leaf forms. The development of compound leaves involves an extended morphogenesis stage compared with that of simple leaves, and the tomato (Solanum lycopersicum) mutant clausa (clau) exposes a potential for extended morphogenesis in tomato leaves. Here, we report that the CLAU gene encodes a MYB transcription factor that has evolved a unique role in compound-leaf species to promote an exit from the morphogenetic phase of tomato leaf development. We show that CLAU attenuates cytokinin signaling, and that clau plants have increased cytokinin sensitivity. The results suggest that flexible leaf patterning involves a coordinated interplay between transcription factors and hormones.

Prominent alterations of wild barley leaf transcriptome in response to individual and combined drought acclimation and heat shock conditions.

Under field conditions, drought and heat stress typically happen simultaneously and their negative impact on the agricultural production is expected to increase worldwide under the climate change scenario. In this study, we performed RNA-sequencing analysis on leaves of wild barley (Hordeum spontaneum) originated from the northern coastal region of Egypt following individual drought acclimation (DA) and heat shock (HS) treatments and their combination (CS, combined stresses) to distinguish the unique and shared differentially expressed genes (DEG). Results indicated that the number of unique genes that were differentially expressed following HS treatment exceeded the number of those expressed following DA. In addition, the number of genes that were uniquely differentially expressed in response to CS treatment exceeded the number of those of shared responses to individual DA and HS treatments. These results indicate a better adaptation of the Mediterranean wild barley to drought conditions when compared with heat stress. It also manifests that the wild barley response to CS tends to be unique rather than common. Annotation of DEG showed that metabolic processes were the most influenced biological function in response to the applied stresses.

A quantitative genetic basis for leaf morphology in a set of precisely defined tomato introgression lines.

Introgression lines (ILs), in which genetic material from wild tomato species is introgressed into a domesticated background, have been used extensively in tomato (Solanum lycopersicum) improvement. Here, we genotype an IL population derived from the wild desert tomato Solanum pennellii at ultrahigh density, providing the exact gene content harbored by each line. To take advantage of this information, we determine IL phenotypes for a suite of vegetative traits, ranging from leaf complexity, shape, and size to cellular traits, such as stomatal density and epidermal cell phenotypes. Elliptical Fourier descriptors on leaflet outlines provide a global analysis of highly heritable, intricate aspects of leaf morphology. We also demonstrate constraints between leaflet size and leaf complexity, pavement cell size, and stomatal density and show independent segregation of traits previously assumed to be genetically coregulated. Meta-analysis of previously measured traits in the ILs shows an unexpected relationship between leaf morphology and fruit sugar levels, which RNA-Seq data suggest may be attributable to genetically coregulated changes in fruit morphology or the impact of leaf shape on photosynthesis. Together, our results both improve upon the utility of an important genetic resource and attest to a complex, genetic basis for differences in leaf morphology between natural populations.

Comparative transcriptomics reveals patterns of selection in domesticated and wild tomato.

Although applied over extremely short timescales, artificial selection has dramatically altered the form, physiology, and life history of cultivated plants. We have used RNAseq to define both gene sequence and expression divergence between cultivated tomato and five related wild species. Based on sequence differences, we detect footprints of positive selection in over 50 genes. We also document thousands of shifts in gene-expression level, many of which resulted from changes in selection pressure. These rapidly evolving genes are commonly associated with environmental response and stress tolerance. The importance of environmental inputs during evolution of gene expression is further highlighted by large-scale alteration of the light response coexpression network between wild and cultivated accessions. Human manipulation of the genome has heavily impacted the tomato transcriptome through directed admixture and by indirectly favoring nonsynonymous over synonymous substitutions. Taken together, our results shed light on the pervasive effects artificial and natural selection have had on the transcriptomes of tomato and its wild relatives.