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.

Dynamic shifts in primary metabolism across fruit development stages in Capsicum chinense (cv. Habanero).

The development of fleshy fruits involves changes in size and mass, followed by cell differentiation, which is associated with anatomical and histological changes. Parallel to these changes, metabolic alterations lead to the production of osmolytes and energy that modify cell turgor pressure, thereby promoting cell expansion and fruit growth. Detailed information is known about these processes in climacteric fruits (e.g. tomato); however, the regulation of metabolism and its association with anatomical changes in non-climacteric fruit development are poorly understood. In this study, we used detailed anatomical and histological analyses to define three developmental phases of chili pepper (Capsicum chinense cv. Habanero): cell division, cell expansion, and ripening. We showed that each was marked by distinct metabolic profiles, underpinning the switches in energy metabolism to support cellular processes. Interestingly, mitochondrial activity was high in the early stages of development and declined over time, with a modest increase in O2 consumption by pericarp tissues at the beginning of the ripening stage. This respiratory-like burst was associated with the degradation of starch and malate, which are the sources of energy and carbon required for other processes associated with fruit maturation.

The ORGAN SIZE (ORG) locus modulates both vegetative and reproductive gigantism in domesticated tomato.

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

Abscisic acid acts essentially on stomata, not on the xylem, to improve drought resistance in tomato.

Drought resistance is essential for plant production under water-limiting environments. Abscisic acid (ABA) plays a critical role in stomata but its impact on hydraulic function beyond the stomata is far less studied. We selected genotypes differing in their ability to accumulate ABA to investigate its role in drought-induced dysfunction. All genotypes exhibited similar leaf and stem embolism resistance regardless of differences in ABA levels. Their leaf hydraulic resistance was also similar. Differences were only observed between the two extreme genotypes: sitiens (sit; a strong ABA-deficient mutant) and sp12 (a transgenic line that constitutively overaccumulates ABA), where the water potential inducing 50% embolism was 0.25 MPa lower in sp12 than in sit. Maximum stomatal and minimum leaf conductances were considerably lower in plants with higher ABA (wild type [WT] and sp12) than in ABA-deficient mutants. Variations in gas exchange across genotypes were associated with ABA levels and differences in stomatal density and size. The lower water loss in plants with higher ABA meant that lethal water potentials associated with embolism occurred later during drought in sp12 plants, followed by WT, and then by the ABA-deficient mutants. Therefore, the primary pathway by which ABA enhances drought resistance is via declines in water loss, which delays dehydration and hydraulic dysfunction.

Does day length matter for nutrient responsiveness?

For over 2500 years, considerable agronomic interest has been paid to soil fertility. Both crop domestication and the Green Revolution shifted photoperiodism and the circadian clock in cultivated species, although this contributed to an increase in the demand for chemical fertilisers. Thus, the uptake of nutrients depends on light signalling, whereas diel growth and circadian rhythms are affected by nutrient levels. Here, we argue that day length and circadian rhythms may be central regulators of the uptake and usage of nutrients, also modulating responses to toxic elements (e.g., aluminium and cadmium). Thus, we suggest that knowledge in this area might assist in developing next-generation crops with improved uptake and use efficiency of nutrients.

Ideotype breeding and genome engineering for legume crop improvement.

Ideotype breeding is a strategy whereby traits are modeled a priori and then introduced into a model or crop species to assess their impact on yield. Thus, knowledge about the connection between genotype and phenotype is required for ideotype breeding to be deployed successfully. The growing understanding of the genetic basis of yield-related traits, combined with increasingly efficient genome engineering tools, improved transformation efficiency, and high-throughput genotyping of regenerants paves the way for the widespread adoption of ideotype breeding as a complement to conventional breeding. We briefly discuss how ideotype breeding, coupled with such state-of-the-art biotechnological tools, could contribute to knowledge-based legume breeding and accelerate yield gains to ensure food security in the coming decades.

Anthocyanins and reactive oxygen species: a team of rivals regulating plant development?

Anthocyanins are a family of water-soluble vacuolar pigments present in almost all flowering plants. The chemistry, biosynthesis and functions of these flavonoids have been intensively studied, in part due to their benefit for human health. Given that they are efficient antioxidants, intense research has been devoted to studying their possible roles against damage caused by reactive oxygen species (ROS). However, the redox homeostasis established between antioxidants and ROS is important for plant growth and development. On the one hand, high levels of ROS can damage DNA, proteins, and lipids, on the other, they are also required for cell signaling, plant development and stress responses. Thus, a balance is needed in which antioxidants can remove excessive ROS, while not precluding ROS from triggering important cellular signaling cascades. In this article, we discuss how anthocyanins and ROS interact and how a deeper understanding of the balance between them could help improve plant productivity, nutritional value, and resistance to stress, while simultaneously maintaining proper cellular function and plant growth.

Loss of S-nitrosoglutathione reductase disturbs phytohormone homeostasis and regulates shoot side branching and fruit growth in tomato.

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

Promoter replacement of ANT1 induces anthocyanin accumulation and triggers the shade avoidance response through developmental, physiological and metabolic reprogramming in tomato.

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

Plant domestication: setting biological clocks.

Through domestication of wild species, humans have induced large changes in the developmental and circadian clocks of plants. As a result of these changes, modern crops are more productive and adaptive to contrasting environments from the center of origin of their wild ancestors, albeit with low genetic variability and abiotic stress tolerance. Likewise, a complete restructuring of plant metabolic timekeeping probably occurred during crop domestication. Here, we highlight that contrasting timings among organs in wild relatives of crops allowed them to recognize environmental adversities faster. We further propose that connections among biological clocks, which were established during plant domestication, may represent a fundamental source of genetic variation to improve crop resilience and yield.

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.

Heterosis for capsacinoids accumulation in chili pepper hybrids is dependent on parent-of-origin effect.

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

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.

The Genetic Complexity of Type-IV Trichome Development Reveals the Steps towards an Insect-Resistant Tomato.

The leaves of the wild tomato Solanumgalapagense harbor type-IV glandular trichomes (GT) that produce high levels of acylsugars (AS), conferring insect resistance. Conversely, domesticated tomatoes (S. lycopersicum) lack type-IV trichomes on the leaves of mature plants, preventing high AS production, thus rendering the plants more vulnerable to insect predation. We hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of adult plants could be insect-resistant. We introgressed the genetic determinants controlling type-IV trichome development from S.galapagense into cv. Micro-Tom (MT) and created a line named "Galapagos-enhanced trichomes" (MT-Get). Mapping-by-sequencing revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further genetic mapping showed that S. galapagense alleles in chromosomes 1, 2, and 3 were sufficient for the presence of type-IV trichomes on adult organs but at lower densities. Metabolic and gene expression analyses demonstrated that type-IV trichome density was not accompanied by the AS production and exudation in MT-Get. Although the plants produce a significant amount of acylsugars, those are still not enough to make them resistant to whiteflies. We demonstrate that type-IV glandular trichome development is insufficient for high AS accumulation. The results from our study provided additional insights into the steps necessary for breeding an insect-resistant tomato.

Auxin-driven ecophysiological diversification of leaves in domesticated tomato.

Heterobaric leaves have bundle sheath extensions (BSEs) that compartmentalize the parenchyma, whereas homobaric leaves do not. The presence of BSEs affects leaf hydraulics and photosynthetic rate. The tomato (Solanum lycopersicum) obscuravenosa (obv) mutant lacks BSEs. Here, we identify the obv gene and the causative mutation, a nonsynonymous amino acid change that disrupts a C2H2 zinc finger motif in a putative transcription factor. This mutation exists as a polymorphism in the natural range of wild tomatoes but has increased in frequency in domesticated tomatoes, suggesting that the latter diversified into heterobaric and homobaric leaf types. The obv mutant displays reduced vein density, leaf hydraulic conductance and photosynthetic assimilation rate. We show that these and other pleiotropic effects on plant development, including changes in leaf insertion angle, leaf margin serration, minor vein density, and fruit shape, are controlled by OBV via changes in auxin signaling. Loss of function of the transcriptional regulator AUXIN RESPONSE FACTOR 4 (ARF4) also results in defective BSE development, revealing an additional component of a genetic module controlling aspects of leaf development important for ecological adaptation and subject to breeding selection.

Reduced auxin signalling through the cyclophilin gene DIAGEOTROPICA impacts tomato fruit development and metabolism during ripening.

Auxin is an important hormone playing crucial roles during fruit growth and ripening; however, the metabolic impact of changes in auxin signalling during tomato (Solanum lycopersicum L.) ripening remains unclear. Here, we investigated the significance of changes in auxin signalling during different stages of fruit development by analysing changes in tomato fruit quality and primary metabolism using mutants with either lower or higher auxin sensitivity [diageotropica (dgt) and entire mutants, respectively]. Altered auxin sensitivity modifies metabolism, through direct impacts on fruit respiration and fruit growth. We verified that the dgt mutant plants exhibit reductions in fruit set, total fruit dry weight, fruit size, number of seeds per fruit, and fresh weight loss during post-harvest. Sugar accumulation was associated with delayed fruit ripening in dgt, probably connected with reduced ethylene levels and respiration, coupled with a lower rate of starch degradation. In contrast, despite exhibiting parthenocarpy, increased auxin perception (entire) did not alter fruit ripening, leading to only minor changes in primary metabolism. By performing a comprehensive analysis, our results connect auxin signalling and metabolic changes during tomato fruit development, indicating that reduced auxin signalling led to extensive changes in sugar concentration and starch metabolism during tomato fruit ripening.

Impaired auxin signaling increases vein and stomatal density but reduces hydraulic efficiency and ultimately net photosynthesis.

Auxins are known to regulate xylem development in plants, but their effects on water transport efficiency are poorly known. Here we used tomato plants with the diageotropica mutation (dgt), which has impaired function of a cyclophilin 1 cis-trans isomerase involved in auxin signaling, and the corresponding wild type (WT) to explore the mutation's effects on plant hydraulics and leaf gas exchange. The xylem of the dgt mutant showed a reduced hydraulically weighted vessel diameter (Dh) (24-43%) and conduit number (25-58%) in petioles and stems, resulting in lower theoretical hydraulic conductivities (Kt); on the other hand, no changes in root Dh and Kt were observed. The measured stem and leaf hydraulic conductances of the dgt mutant were lower (up to 81%), in agreement with the Kt values; however, despite dgt and WT plants showing similar root Dh and Kt, the measured root hydraulic conductance of the dgt mutant was 75% lower. The dgt mutation increased the vein and stomatal density, which could potentially increase photosynthesis. Nevertheless, even though it had the same photosynthetic capacity as WT plants, the dgt mutant showed a photosynthetic rate c. 25% lower, coupled with a stomatal conductance reduction of 52%. These results clearly demonstrate that increases in minor vein and stomatal density only result in higher leaf gas exchange when accompanied by higher hydraulic efficiency.

A long and stressful day: Photoperiod shapes aluminium tolerance in plants.

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

Bringing more players into play: Leveraging stress in genome wide association studies.

In order to meet the demand of the burgeoning human population as well as to adapt crops to the enhanced abiotic and biotic stress caused by the global climatic change, breeders focus on identifying valuable genes to improve both crop stress tolerance and crop quality. Recently, with the development of next-generation sequencing methods, millions of high quality single-nucleotide polymorphisms (SNPs) have been made available and genome-wide association studies (GWAS) are widely used in crop improvement studies to identify the associations between genetic variants of genomes and relevant crop agronomic traits. Here, we review classic cases of use of GWAS to identify genetic variants associated with valuable traits such as geographic adaptation, crop quality and metabolites. We discuss the power of stress GWAS to identify further associations including those with genes that are not, or only lowly, expressed during optimal growth conditions. Finally, we emphasize recent demonstrations of the efficiency and accuracy of time-resolved dynamic stress GWAS and GWAS based on genomic gene expression and structural variations, which can be applied to resolve more comprehensively the genetic regulation mechanisms of complex traits.

Increased branching independent of strigolactone in cytokinin oxidase 2-overexpressing tomato is mediated by reduced auxin transport.

Tomato production is influenced by shoot branching, which is controlled by different hormones. Here we produced tomato plants overexpressing the cytokinin-deactivating gene CYTOKININ OXYDASE 2 (CKX2). CKX2-overexpressing (CKX2-OE) plants showed an excessive growth of axillary shoots, the opposite phenotype expected for plants with reduced cytokinin content, as evidenced by LC-MS analysis and ARR5-GUS staining. The TCP transcription factor SlBRC1b was downregulated in the axillary buds of CKX2-OE and its excessive branching was dependent on a functional version of the GRAS-family gene LATERAL SUPPRESSOR (LS). Grafting experiments indicated that increased branching in CKX2-OE plants is unlikely to be mediated by root-derived signals. Crossing CKX2-OE plants with transgenic antisense plants for the strigolactone biosynthesis gene CAROTENOID CLEAVAGE DIOXYGENASE (CCD7-AS) produced an additive phenotype, indicating independent effects of cytokinin and strigolactones on increased branching. On the other hand, CKX2-OE plants showed reduced polar auxin transport and their bud outgrowth was reduced when combined with auxin mutants. Accordingly, CKX2-OE basal buds did not respond to auxin applied in the decapitated apex. Our results suggest that tomato shoot branching depends on a fine-tuning of different hormonal balances and that perturbations in the auxin status could compensate for the reduced cytokinin levels in CKX2-OE plants.