A horizontal view of primary metabolomes in vegetative desiccation tolerance.

Vegetative desiccation tolerance (VDT), the ability of such tissues to survive the near complete loss of cellular water, is a rare but polyphyletic phenotype. It is a complex multifactorial trait, typified by universal (core) factors but with many and varied adaptations due to plant architecture, biochemistry and biotic/abiotic dynamics of particular ecological niches. The ability to enter into a quiescent biophysically stable state is what ultimately determines desiccation tolerance. Thus, understanding the metabolomic complement of plants with VDT gives insight into the nature of survival as well as evolutionary aspects of VDT. In this study, we measured the soluble carbohydrate profiles and the polar, TMS-derivatisable metabolomes of 7 phylogenetically diverse species with VDT, in contrast with two desiccation sensitive (DS) species, under conditions of full hydration, severe water deficit stress, and desiccation. Our study confirmed the existence of core mechanisms of VDT systems associated with either constitutively abundant trehalose or the accumulation of raffinose family oligosaccharides and sucrose, with threshold ratios conditioned by other features of the metabolome. DS systems did not meet these ratios. Considerable chemical variations among VDT species suggest that co-occurring but distinct stresses (e.g., photooxidative stress) are dealt with using different chemical regimes. Furthermore, differences in the timing of metabolic shifts suggest there is not a single "desiccation programme" but that subprocesses are coordinated differently at different drying phases. There are likely to be constraints on the composition of a viable dry state and how different adaptive strategies interact with the biophysical constraints of VDT.

A review of the role of metabolites in vegetative desiccation tolerance of angiosperms.

The survival of extreme water deficit stress by tolerant organisms requires a coordinated series of responses, including those at cellular, transcriptional, translational and metabolic levels. Small molecules play a pivotal role in creating the proper chemical environment for the preservation of cellular integrity and homeostasis during dehydration. This review surveys recent insights in the importance of primary and specialised metabolites in the response to drying of angiosperms with vegetative desiccation tolerance, i.e. the ability to survive near total loss of water. Important metabolites include sugars such as sucrose, trehalose and raffinose family of oligosaccharides, amino acids and organic acids, as well as antioxidants, representing a common core mechanism of desiccation tolerance. Additional metabolites are discussed in the context of species specificity and adaptation.

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.

A Role for Allantoate Amidohydrolase (AtAAH) in the Germination of Arabidopsis thaliana Seeds.

Seed dormancy is a very complex trait controlled by interactions between genetic and environmental factors. Nitrate is inversely correlated with seed dormancy in Arabidopsis. This is explained by the fact that seed dry storage (after-ripening) reduces the need for nitrogen for germination. When nitrate is absorbed by plants, it is first reduced to nitrite and then to ammonium for incorporation into amino acids, nucleic acids and chlorophyll. Previously, we showed that ALLANTOATE AMIDOHYDROLASE (AtAAH) transcripts are up-regulated in imbibed dormant seeds compared with after-ripened seeds. AAH is an enzyme in the uric acid catabolic pathway which catalyzes the hydrolysis of allantoate to yield CO2, NH3 and S-ureidoglycine. This pathway is the final stage of purine catabolism, and functions in plants and some bacteria to provide nitrogen, particularly when other nitrogen sources are depleted. Ataah mutant seeds are more dormant and accumulate high levels of allantoate, allantoin and urea, whereas energy-related metabolites and several amino acids are lower upon seed imbibition in comparison with Columbia-0. AtAAH expression could be detected during the early stages of seed development, with a transient increase around 8 d after pollination. AtAAH expression is the highest in mature pollen. The application of exogenous potassium nitrate can partly complement the higher dormancy phenotype of the Ataah mutant seeds, whereas other nitrogen sources cannot. Our results indicate that potassium nitrate does not specifically overcome the alleviated dormancy levels in Ataah mutant seeds, but promotes germination in general. Possible pathways by which AtAAH affects seed germination are discussed.

Crops for dry environments.

Climate change necessitates increased stress resilience of food crops. We describe four potential solutions, with emphasis on a relatively novel approach aiming at true tolerance of drought rather than improved water-holding capacity of crops, which is a common approach in current breeding and genome editing efforts. Some Angiosperms are known to tolerate loss of 95% of their cellular water, without dying, not dissimilar to seeds. The molecular mechanisms and their regulation underlying this remarkable ability are potentially useful to design tolerant crops. Since most crops produce desiccation tolerant seeds, genomic information for this attribute is present but inactive in vegetative parts of the plant. Based on recent evidence from both seeds and desiccation tolerant Angiosperms we address possible routes to 'flipping the switch' to vegetative desiccation tolerance in major crops.

Modulation of NF-YB genes in Ricinus communis L. in response to different temperatures and developmental stages and functional characterization of RcNF-YB8 as an important regulator of flowering time in Arabidopsis thaliana.

We have characterized the NF-YB gene family in R. communis using bioinformatics, ecotopic expression, and transcriptomics. A total of 14 RcNF-YB genes were identified in R. communis genome using the conserved NF-YB region. This number is similar to what is found in A. thaliana (13 genes) and O. sativa (11 genes), whereas it is considerably lower to what is found in P. trichocarpa (21 genes) and S. lycopersycum (29 genes). Several regulatory cis-elements were identified in the promoter region, including low temperature, defense and stress, MIC, MYB, and abscisic acid. RcNF-YB is strongly modulated by temperature and it is dependent on the stage of germination. In general, RcNF-YB genes showed higher expression levels in dry seeds and early imbibition (EI) samples as compared to later stages of seedling development. Ectopic expression of RcNF-YB8 reduced flowering time in Arabidopsis reducing the time required for the formation of the first visible bud, the time required to open the first flower, and the time required for the formation of the first visible silique. At the end of the life cycle, ectopic expression of RcNF-YB8 affected plant height (PH), silique length (SL), the total number of silique per plant, 1000-seed weight, and seed size. Our data demonstrated the role of RcNF-YB8 in flowering time, plant height and seed production, and it shows that it may constitute a key target gene for breeding superior R. communis genotypes.

Clues on an intraspecific communication system in seed-seedling transition.

How much interactivity is in a seed-seedling transition system? We hypothesize that seed-seed, seed-seedling, and seedling-seedling interactions can drive the early plant development in artificial growth systems directly due to mutual stimulation phenomena. To test the hypothesis, we performed seed germination measurements, gene expression in germination sensu stricto, water dynamics in germinating seeds, and information theory. For a biological model, we used Solanum lycocarpum A. St.-Hil. seeds. This is a neotropical species with high intraspecific variability in the seed sample. Our findings demonstrate that the dynamic and transient seed-seedling transition system is influenced by the number of individuals (seed or seedling) in the artificial system. In addition, we also discuss that: (1) the information entropy enables the quantification of system disturbance relative to individuals at the same physiological stage (seed-seed or seedling-seedling), which may be determinant for embryo growth during germination and (2) the intraspecific communication in seed-seedling transition systems formed by germinating seeds has the potential to alter the expression pattern of key genes for embryo development. Therefore, the phenomenon of mutual stimulation during the germination process can be an important aspect of seed-seedling transition, especially in laboratory conditions.

Network Analysis Prioritizes DEWAX and ICE1 as the Candidate Genes for Major eQTL Hotspots in Seed Germination of Arabidopsis thaliana.

Seed germination is characterized by a constant change of gene expression across different time points. These changes are related to specific processes, which eventually determine the onset of seed germination. To get a better understanding on the regulation of gene expression during seed germination, we performed a quantitative trait locus mapping of gene expression (eQTL) at four important seed germination stages (primary dormant, after-ripened, six-hour after imbibition, and radicle protrusion stage) using Arabidopsis thaliana Bay x Sha recombinant inbred lines (RILs). The mapping displayed the distinctness of the eQTL landscape for each stage. We found several eQTL hotspots across stages associated with the regulation of expression of a large number of genes. Interestingly, an eQTL hotspot on chromosome five collocates with hotspots for phenotypic and metabolic QTL in the same population. Finally, we constructed a gene co-expression network to prioritize the regulatory genes for two major eQTL hotspots. The network analysis prioritizes transcription factors DEWAX and ICE1 as the most likely regulatory genes for the hotspot. Together, we have revealed that the genetic regulation of gene expression is dynamic along the course of seed germination.

Detection of QTLs for genotype × environment interactions in tomato seeds and seedlings.

Seed quality and seedling establishment are the most important factors affecting successful crop development. They depend on the genetic background and are acquired during seed maturation and therefor, affected by the maternal environment under which the seeds develop. There is little knowledge about the genetic and environmental factors that affect seed quality and seedling establishment. The aim of this study is to identify the loci and possible molecular mechanisms involved in acquisition of seed quality and how these are controlled by adverse maternal conditions. For this, we used a tomato recombinant inbred line (RIL) population consisting of 100 lines which were grown under two different nutritional environmental conditions, high phosphate and low nitrate. Most of the seed germination traits such as maximum germination percentage (Gmax ), germination rate (t50 ) and uniformity (U8416 ) showed ample variation between genotypes and under different germination conditions. This phenotypic variation leads to identification of quantitative trait loci (QTLs) which were dependent on genetic factors, but also on the interaction with the maternal environment (QTL × E). Further studies of these QTLs may ultimately help to predict the effect of different maternal environmental conditions on seed quality and seedling establishment which will be very useful to improve the production of high-performance seeds.

Arabidopsis in the Wild-The Effect of Seasons on Seed Performance.

Climate changes play a central role in the adaptive life histories of organisms all over the world. In higher plants, these changes may impact seed performance, both during seed development and after dispersal. To examine the plasticity of seed performance as a response to environmental fluctuations, eight genotypes known to be affected in seed dormancy and longevity were grown in the field in all seasons of two years. Soil and air temperature, day length, precipitation, and sun hours per day were monitored. We show that seed performance depends on the season. Seeds produced by plants grown in the summer, when the days began to shorten and the temperature started to decrease, were smaller with deeper dormancy and lower seed longevity compared to the other seasons when seeds were matured at higher temperature over longer days. The performance of seeds developed in the different seasons was compared to seeds produced in controlled conditions. This revealed that plants grown in a controlled environment produced larger seeds with lower dormancy than those grown in the field. All together the results show that the effect of the environment largely overrules the genetic effects, and especially, differences in seed dormancy caused by the different seasons were larger than the differences between the genotypes.

Intertwined signatures of desiccation and drought tolerance in grasses.

Grasses are among the most resilient plants, and some can survive prolonged desiccation in semiarid regions with seasonal rainfall. However, the genetic elements that distinguish grasses that are sensitive versus tolerant to extreme drying are largely unknown. Here, we leveraged comparative genomic approaches with the desiccation-tolerant grass Eragrostis nindensis and the related desiccation-sensitive cereal Eragrostis tef to identify changes underlying desiccation tolerance. These analyses were extended across C4 grasses and cereals to identify broader evolutionary conservation and divergence. Across diverse genomic datasets, we identified changes in chromatin architecture, methylation, gene duplications, and expression dynamics related to desiccation in E. nindensis It was previously hypothesized that transcriptional rewiring of seed desiccation pathways confers vegetative desiccation tolerance. Here, we demonstrate that the majority of seed-dehydration-related genes showed similar expression patterns in leaves of both desiccation-tolerant and -sensitive species. However, we identified a small set of seed-related orthologs with expression specific to desiccation-tolerant species. This supports a broad role for seed-related genes, where many are involved in typical drought responses, with only a small subset of crucial genes specifically induced in desiccation-tolerant plants.

Desiccation Tolerance: Avoiding Cellular Damage During Drying and Rehydration.

Desiccation of plants is often lethal but is tolerated by the majority of seeds and by vegetative tissues of only a small number of land plants. Desiccation tolerance is an ancient trait, lost from vegetative tissues following the appearance of tracheids but reappearing in several lineages when selection pressures favored its evolution. Cells of all desiccation-tolerant plants and seeds must possess a core set of mechanisms to protect them from desiccation- and rehydration-induced damage. This review explores how desiccation generates cell damage and how tolerant cells assuage the complex array of mechanical, structural, metabolic, and chemical stresses and survive.Likewise, the stress of rehydration requires appropriate mitigating cellular responses. We also explore what comparative genomics, both structural and responsive, have added to our understanding of cellular protection mechanisms induced by desiccation, and how vegetative desiccation tolerance circumvents destructive, stress-induced cell senescence.

Structural Plasticity of Intrinsically Disordered LEA Proteins from Xerophyta schlechteri Provides Protection In Vitro and In Vivo.

Late embryogenesis abundant (LEA) proteins are essential to the ability of resurrection plants and orthodox seeds to protect the subcellular milieu against irreversible damage associated with desiccation. In this work, we investigated the structure and function of six LEA proteins expressed during desiccation in the monocot resurrection species Xerophyta schlechteri (XsLEAs). In silico analyses suggested that XsLEAs are hydrophilic proteins with variable intrinsically disordered protein (IDP) properties. Circular dichroism (CD) analysis indicated that these proteins are mostly unstructured in water but acquire secondary structure in hydrophobic solution, suggesting that structural dynamics may play a role in their function in the subcellular environment. The protective property of XsLEAs was demonstrated by their ability to preserve the activity of the enzyme lactate dehydrogenase (LDH) against desiccation, heat and oxidative stress, as well as growth of Escherichia coli upon exposure to osmotic and salt stress. Subcellular localization analysis indicated that XsLEA recombinant proteins are differentially distributed in the cytoplasm, membranes and nucleus of Nicotiana benthamiana leaves. Interestingly, a LEA_1 family protein (XsLEA1-8), showing the highest disorder-to-order propensity and protective ability in vitro and in vivo, was also able to enhance salt and drought stress tolerance in Arabidopsis thaliana. Together, our results suggest that the structural plasticity of XsLEAs is essential for their protective activity to avoid damage of various subcellular components caused by water deficit stress. XsLEA1-8 constitutes a potential model protein for engineering structural stability in vitro and improvement of water-deficit stress tolerance in plants.

The interaction between genotype and maternal nutritional environments affects tomato seed and seedling quality.

Seed and seedling traits are affected by the conditions of the maternal environment, such as light, temperature, and nutrient availability. In this study, we have investigated whether different maternally applied nitrate and phosphate concentrations affect the seed and seedling performance of two closely related tomato species: Solanum lycopersicum cv. Money maker and Solanum pimpinellifolium accession CGN14498. We observed large differences for seed and seedling traits between the two species. Additionally, we have shown that for nitrate most of the seed and seedling traits were significantly affected by genotype-environment interactions (G×E). The effect of the maternal environment was clearly visible in the primary metabolites of the dry seeds. For example, we could show that the amount of γ-aminobutyric acid (GABA) in Money maker seeds was affected by the differences in the maternal environments and was positively correlated with seed germination under high temperature. Overall, compared with phosphate, nitrate had a larger effect on seed and seedling performance in tomato. In general, the different responses to the maternal environments of the two tomato species showed a major role for G×E in shaping seed and seedling traits.

Induction of desiccation tolerance in desiccation sensitive Citrus limon seeds.

Many economically important perennial species bear recalcitrant seeds, including tea, coffee, cocoa, mango, citrus, rubber, oil palm and coconut. Orthodox seeds can be dried almost completely without losing viability, but so-called recalcitrant seeds have a very limited storage life and die upon drying below a higher critical moisture content than orthodox seeds. As a result, the development of long-term storage methods for recalcitrant seeds is compromised. Lowering this critical moisture content would be very valuable since dry seed storage is the safest, most convenient and cheapest method for conserving plant genetic resources. Therefore, we have attempted to induce desiccation tolerance (DT) in the desiccation sensitive seeds of Citrus limon. We show that DT can be induced by paclobutrazol (an inhibitor of gibberellin biosynthesis) and we studied its associated transcriptome to delineate the molecular mechanisms underlying this induction of DT. Paclobutrazol not only interfered with gibberellin related gene expression but also caused extensive changes in expression of genes involved in the biosynthesis and signaling of other hormones. Paclobutrazol induced a transcriptomic switch encompassing suppression of biotic- and induction of abiotic responses. We hypothesize that this is the main driver of the induction of DT by paclobutrazol in C. limon seeds.

Dissecting the Genomic Diversification of Late Embryogenesis Abundant (LEA) Protein Gene Families in Plants.

Late embryogenesis abundant (LEA) proteins include eight multigene families that are expressed in response to water loss during seed maturation and in vegetative tissues of desiccation tolerant species. To elucidate LEA proteins evolution and diversification, we performed a comprehensive synteny and phylogenetic analyses of the eight gene families across 60 complete plant genomes. Our integrated comparative genomic approach revealed that synteny conservation and diversification contributed to LEA family expansion and functional diversification in plants. We provide examples that: 1) the genomic diversification of the Dehydrin family contributed to differential evolution of amino acid sequences, protein biochemical properties, and gene expression patterns, and led to the appearance of a novel functional motif in angiosperms; 2) ancient genomic diversification contributed to the evolution of distinct intrinsically disordered regions of LEA_1 proteins; 3) recurrent tandem-duplications contributed to the large expansion of LEA_2; and 4) dynamic synteny diversification played a role on the evolution of LEA_4 and its function on plant desiccation tolerance. Taken together, these results show that multiple evolutionary mechanisms have not only led to genomic diversification but also to structural and functional plasticity among LEA proteins which have jointly contributed to the adaptation of plants to water-limiting environments.

NADP-MALIC ENZYME 1 Affects Germination after Seed Storage in Arabidopsis thaliana.

Aging decreases the quality of seeds and results in agricultural and economic losses. The damage that occurs at the biochemical level can alter the seed physiological status. Although loss of viability has been investigated frequently, little information exists on the molecular and biochemical factors involved in seed deterioration and loss of viability. Oxidative stress has been implicated as a major contributor to seed deterioration, and several pathways are involved in protection against this. In this study, we show that seeds of Arabidopsis thaliana lacking a functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Seeds of the NADP-ME1 loss-of-function mutant display higher levels of protein carbonylation than those of the wild type. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH. Upon seed imbibition, malate and amino acids accumulate in embryos of aged seeds of the NADP-ME1 loss-of-function mutant compared with those of the wild type. NADP-ME1 expression is increased in imbibed aged as compared with non-aged seeds. NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. In seedlings of aged seeds, NADP-ME1 is specifically active in the root meristematic zone. We propose that NADP-ME1 activity is required for protecting seeds against oxidation during seed dry storage.

Genome-level responses to the environment: plant desiccation tolerance.

Plants being sessile organisms are well equipped genomically to respond to environmental stressors peculiar to their habitat. Evolution of plants onto land was enabled by the ability to tolerate extreme water loss (desiccation), a feature that has been retained within genomes but not universally expressed in most land plants today. In the majority of higher plants, desiccation tolerance (DT) is expressed only in reproductive tissues (seeds and pollen), but some 135 angiosperms display vegetative DT. Here, we review genome-level responses associated with DT, pointing out common and yet sometimes discrepant features, the latter relating to evolutionary adaptations to particular niches. Understanding DT can lead to the ultimate production of crops with greater tolerance of drought than is currently realized.