How deregulation, drought and increasing fire impact Amazonian biodiversity.

Biodiversity contributes to the ecological and climatic stability of the Amazon Basin1,2, but is increasingly threatened by deforestation and fire3,4. Here we quantify these impacts over the past two decades using remote-sensing estimates of fire and deforestation and comprehensive range estimates of 11,514 plant species and 3,079 vertebrate species in the Amazon. Deforestation has led to large amounts of habitat loss, and fires further exacerbate this already substantial impact on Amazonian biodiversity. Since 2001, 103,079-189,755 km2 of Amazon rainforest has been impacted by fires, potentially impacting the ranges of 77.3-85.2% of species that are listed as threatened in this region5. The impacts of fire on the ranges of species in Amazonia could be as high as 64%, and greater impacts are typically associated with species that have restricted ranges. We find close associations between forest policy, fire-impacted forest area and their potential impacts on biodiversity. In Brazil, forest policies that were initiated in the mid-2000s corresponded to reduced rates of burning. However, relaxed enforcement of these policies in 2019 has seemingly begun to reverse this trend: approximately 4,253-10,343 km2 of forest has been impacted by fire, leading to some of the most severe potential impacts on biodiversity since 2009. These results highlight the critical role of policy enforcement in the preservation of biodiversity in the Amazon.

Carotenoid sequestration protein FIBRILLIN participates in CmOR-regulated ß-carotene accumulation in melon.

Chromoplasts are plant organelles with a unique ability to sequester and store massive carotenoids. Chromoplasts have been hypothesized to enable high levels of carotenoid accumulation due to enhanced sequestration ability or sequestration substructure formation. However, the regulators that control the substructure component accumulation and substructure formation in chromoplasts remain unknown. In melon (Cucumis melo) fruit, ß-carotene accumulation in chromoplasts is governed by ORANGE (OR), a key regulator for carotenoid accumulation in chromoplasts. By using comparative proteomic analysis of a high ß-carotene melon variety and its isogenic line low-ß mutant that is defective in CmOr with impaired chromoplast formation, we identified carotenoid sequestration protein FIBRILLIN1 (CmFBN1) as differentially expressed. CmFBN1 expresses highly in melon fruit tissue. Overexpression of CmFBN1 in transgenic Arabidopsis (Arabidopsis thaliana) containing ORHis that genetically mimics CmOr significantly enhances carotenoid accumulation, demonstrating its involvement in CmOR-induced carotenoid accumulation. Both in vitro and in vivo evidence showed that CmOR physically interacts with CmFBN1. Such an interaction occurs in plastoglobules and results in promoting CmFBN1 accumulation. CmOR greatly stabilizes CmFBN1, which stimulates plastoglobule proliferation and subsequently carotenoid accumulation in chromoplasts. Our findings show that CmOR directly regulates CmFBN1 protein levels and suggest a fundamental role of CmFBN1 in facilitating plastoglobule proliferation for carotenoid sequestration. This study also reveals an important genetic tool to further enhance OR-induced carotenoid accumulation in chromoplasts in crops.

Pan-genome and multi-parental framework for high-resolution trait dissection in melon (Cucumis melo).

Linking genotype with phenotype is a fundamental goal in biology and requires robust data for both. Recent advances in plant-genome sequencing have expedited comparisons among multiple-related individuals. The abundance of structural genomic within-species variation that has been discovered indicates that a single reference genome cannot represent the complete sequence diversity of a species, leading to the expansion of the pan-genome concept. For high-resolution forward genetics, this unprecedented access to genomic variation should be paralleled and integrated with phenotypic characterization of genetic diversity. We developed a multi-parental framework for trait dissection in melon (Cucumis melo), leveraging a novel pan-genome constructed for this highly variable cucurbit crop. A core subset of 25 diverse founders (MelonCore25), consisting of 24 accessions from the two widely cultivated subspecies of C. melo, encompassing 12 horticultural groups, and 1 feral accession was sequenced using a combination of short- and long-read technologies, and their genomes were assembled de novo. The construction of this melon pan-genome exposed substantial variation in genome size and structure, including detection of ~300 000 structural variants and ~9 million SNPs. A half-diallel derived set of 300 F2 populations, representing all possible MelonCore25 parental combinations, was constructed as a framework for trait dissection through integration with the pan-genome. We demonstrate the potential of this unified framework for genetic analysis of various melon traits, including rind color intensity and pattern, fruit sugar content, and resistance to fungal diseases. We anticipate that utilization of this integrated resource will enhance genetic dissection of important traits and accelerate melon breeding.

Toward a metabolic theory of life history.

The life histories of animals reflect the allocation of metabolic energy to traits that determine fitness and the pace of living. Here, we extend metabolic theories to address how demography and mass-energy balance constrain allocation of biomass to survival, growth, and reproduction over a life cycle of one generation. We first present data for diverse kinds of animals showing empirical patterns of variation in life-history traits. These patterns are predicted by theory that highlights the effects of 2 fundamental biophysical constraints: demography on number and mortality of offspring; and mass-energy balance on allocation of energy to growth and reproduction. These constraints impose 2 fundamental trade-offs on allocation of assimilated biomass energy to production: between number and size of offspring, and between parental investment and offspring growth. Evolution has generated enormous diversity of body sizes, morphologies, physiologies, ecologies, and life histories across the millions of animal, plant, and microbe species, yet simple rules specified by general equations highlight the underlying unity of life.

Universal rules of life: metabolic rates, biological times and the equal fitness paradigm.

Here we review and extend the equal fitness paradigm (EFP) as an important step in developing and testing a synthetic theory of ecology and evolution based on energy and metabolism. The EFP states that all organisms are equally fit at steady state, because they allocate the same quantity of energy, ~ 22.4 kJ/g/generation to the production of offspring. On the one hand, the EFP may seem tautological, because equal fitness is necessary for the origin and persistence of biodiversity. On the other hand, the EFP reflects universal laws of life: how biological metabolism - the uptake, transformation and allocation of energy - links ecological and evolutionary patterns and processes across levels of organisation from: (1) structure and function of individual organisms, (2) life history and dynamics of populations, and (3) interactions and coevolution of species in ecosystems. The physics and biology of metabolism have facilitated the evolution of millions of species with idiosyncratic anatomy, physiology, behaviour and ecology but also with many shared traits and tradeoffs that reflect the single origin and universal rules of life.

Underappreciated plant vulnerabilities to heat waves.

With climate change, heat waves are becoming increasingly frequent, intense and broader in spatial extent. However, while the lethal effects of heat waves on humans are well documented, the impacts on flora are less well understood, perhaps except for crops. We summarize recent findings related to heat wave impacts including: sublethal and lethal effects at leaf and plant scales, secondary ecosystem effects, and more complex impacts such as increased heat wave frequency across all seasons, and interactions with other disturbances. We propose generalizable practical trials to quantify the critical bounding conditions of vulnerability to heat waves. Collectively, plant vulnerabilities to heat waves appear to be underappreciated and understudied, particularly with respect to understanding heat wave driven plant die-off and ecosystem tipping points.

Underground heterosis for yield improvement in melon.

Heterosis, the superiority of hybrids over their parents, is a major genetic force associated with plant fitness and crop yield enhancement. We investigated root-mediated yield heterosis in melons (Cucumis melo) by characterizing a common variety grafted onto 190 hybrid rootstocks, resulting from crossing 20 diverse inbreds in a diallel-mating scheme. Hybrid rootstocks improved yield by more than 40% compared with their parents, and the best hybrid yield outperformed the reference commercial variety by 65% under both optimal and minimal irrigation treatments. To characterize the genetics of underground heterosis we conducted whole genome re-sequencing of the 20 founder lines, and showed that parental genetic distance was no predictor for the level of heterosis. Through inference of the 190 hybrid genotypes from their parental genomes, followed by genome-wide association analysis, we mapped multiple quantitative trait loci for root-mediated yield. Yield enhancement of the four best-performing hybrid rootstocks was validated in multiple experiments with four different scion varieties. Our grafting approach is complementary to the common roots genetic approach that focuses mainly on variation in root system architecture, and is a step towards discovery of candidate genes involved in root function and yield enhancement.

Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops.

The Cucurbitaceae family (cucurbit) includes several economically important crops, such as melon, cucumber, watermelon, pumpkin, squash and gourds. During the past several years, genomic and genetic data have been rapidly accumulated for cucurbits. To store, mine, analyze, integrate and disseminate these large-scale datasets and to provide a central portal for the cucurbit research and breeding community, we have developed the Cucurbit Genomics Database (CuGenDB; http://cucurbitgenomics.org) using the Tripal toolkit. The database currently contains all available genome and expressed sequence tag (EST) sequences, genetic maps, and transcriptome profiles for cucurbit species, as well as sequence annotations, biochemical pathways and comparative genomic analysis results such as synteny blocks and homologous gene pairs between different cucurbit species. A set of analysis and visualization tools and user-friendly query interfaces have been implemented in the database to facilitate the usage of these large-scale data by the community. In particular, two new tools have been developed in the database, a 'SyntenyViewer' to view genome synteny between different cucurbit species and an 'RNA-Seq' module to analyze and visualize gene expression profiles. Both tools have been packed as Tripal extension modules that can be adopted in other genomics databases developed using the Tripal system.

High-density NGS-based map construction and genetic dissection of fruit shape and rind netting in Cucumis melo.

Melon is an important crop that exhibits broad variation for fruit morphology traits that are the substrate for genetic mapping efforts. In the post-genomic era, the link between genetic maps and physical genome assemblies is key for leveraging QTL mapping results for gene cloning and breeding purposes. Here, using a population of 164 melon recombinant inbred lines (RILs) that were subjected to genotyping-by-sequencing, we constructed and compared high-density sequence- and linkage-based recombination maps that were aligned to the reference melon genome. These analyses reveal the genome-wide variation in recombination frequency and highlight regions of disrupted collinearity between our population and the reference genome. The population was phenotyped over 3 years for fruit size and shape as well as rind netting. Four QTLs were detected for fruit size, and they act in an additive manner, while significant epistatic interaction was found between two neutral loci for this trait. Fruit shape displayed transgressive segregation that was explained by the action of four QTLs, contributed by alleles from both parents. The complexity of rind netting was demonstrated on a collection of 177 diverse accessions. Further dissection of netting in our RILs population, which is derived from a cross of smooth and densely netted parents, confirmed the intricacy of this trait and the involvement of major locus and several other interacting QTLs. A major netting QTL on chromosome 2 co-localized with results from two additional populations, paving the way for future study toward identification of a causative gene for this trait.

The multi-allelic APRR2 gene is associated with fruit pigment accumulation in melon and watermelon.

Color and pigment contents are important aspects of fruit quality and consumer acceptance of cucurbit crops. Here, we describe the independent mapping and cloning of a common causative APRR2 gene regulating pigment accumulation in melon and watermelon. We initially show that the APRR2 transcription factor is causative for the qualitative difference between dark and light green rind in both crops. Further analyses establish the link between sequence or expression level variations in the CmAPRR2 gene and pigment content in the rind and flesh of mature melon fruits. A genome-wide association study (GWAS) of young fruit rind color in a panel composed of 177 diverse melon accessions did not result in any significant association, leading to an earlier assumption that multiple genes are involved in shaping the overall phenotypic variation in this trait. Through resequencing of 25 representative accessions and allelism tests between light rind accessions, we show that multiple independent single nucleotide polymorphisms in the CmAPRR2 gene are causative of the light rind phenotype. The multi-haplotypic nature of this gene explains the lack of detection power obtained through genotyping by sequencing-based GWAS and confirms the pivotal role of this gene in shaping fruit color variation in melon. This study demonstrates the power of combining bi- and multi-allelic designs with deep sequencing, to resolve lack of power due to high haplotypic diversity and low allele frequencies. Due to its central role and broad effect on pigment accumulation in fruits, the APRR2 gene is an attractive target for carotenoid bio-fortification of cucurbit crops.

Distinct Mechanisms of the ORANGE Protein in Controlling Carotenoid Flux.

ß-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, ß-carotene accumulation is governed by the Orange gene (CmOr) golden single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowß) that lowered fruit ß-carotene levels with impaired chromoplast biogenesis. Cmor-lowß exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high ß-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of ß-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (yofi), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The yofi F2 segregants accumulated prolycopene independently of the golden SNP Moreover, Cmor-lowß was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of ß-carotene to dramatically affect both carotenoid content and plastid fate.

Metabolic heat production and thermal conductance are mass-independent adaptations to thermal environment in birds and mammals.

The extent to which different kinds of organisms have adapted to environmental temperature regimes is central to understanding how they respond to climate change. The Scholander-Irving (S-I) model of heat transfer lays the foundation for explaining how endothermic birds and mammals maintain their high, relatively constant body temperatures in the face of wide variation in environmental temperature. The S-I model shows how body temperature is regulated by balancing the rates of heat production and heat loss. Both rates scale with body size, suggesting that larger animals should be better adapted to cold environments than smaller animals, and vice versa. However, the global distributions of ∼9,000 species of terrestrial birds and mammals show that the entire range of body sizes occurs in nearly all climatic regimes. Using physiological and environmental temperature data for 211 bird and 178 mammal species, we test for mass-independent adaptive changes in two key parameters of the S-I model: basal metabolic rate (BMR) and thermal conductance. We derive an axis of thermal adaptation that is independent of body size, extends the S-I model, and highlights interactions among physiological and morphological traits that allow endotherms to persist in a wide range of temperatures. Our macrophysiological and macroecological analyses support our predictions that shifts in BMR and thermal conductance confer important adaptations to environmental temperature in both birds and mammals.

A 'golden' SNP in CmOr governs the fruit flesh color of melon (Cucumis melo).

The flesh color of Cucumis melo (melon) is genetically determined, and can be white, light green or orange, with ß-carotene being the predominant pigment. We associated carotenoid accumulation in melon fruit flesh with polymorphism within CmOr, a homolog of the cauliflower BoOr gene, and identified CmOr as the previously described gf locus in melon. CmOr was found to co-segregate with fruit flesh color, and presented two haplotypes (alleles) in a broad germplasm collection, one being associated with orange flesh and the second being associated with either white or green flesh. Allelic variation of CmOr does not affect its transcription or protein level. The variation also does not affect its plastid subcellular localization. Among the identified single nucleotide polymorphisms (SNPs) between CmOr alleles in orange versus green/white-flesh fruit, a single SNP causes a change of an evolutionarily highly conserved arginine to histidine in the CmOr protein. Functional analysis of CmOr haplotypes in an Arabidopsis callus system confirmed the ability of the CmOr orange haplotype to induce ß-carotene accumulation. Site-directed mutagenesis of the CmOr green/white haplotype to change the CmOR arginine to histidine triggered ß-carotene accumulation. The identification of the 'golden' SNP in CmOr, which is responsible for the non-orange and orange melon fruit phenotypes, provides new tools for studying the Or mechanism of action, and suggests genome editing of the Or gene for nutritional biofortification of crops.

A Kelch Domain-Containing F-Box Coding Gene Negatively Regulates Flavonoid Accumulation in Muskmelon.

The flavonoids are phenylpropanoid-derived metabolites that are ubiquitous in plants, playing many roles in growth and development. Recently, we observed that fruit rinds of yellow casaba muskmelons (Cucumis melo 'Inodorous Group') accumulate naringenin chalcone, a yellow flavonoid pigment. With RNA-sequencing analysis of bulked segregants representing the tails of a population segregating for naringenin chalcone accumulation followed by fine mapping and genetic transformation, we identified a Kelch domain-containing F-box protein coding (CmKFB) gene that, when expressed, negatively regulates naringenin chalcone accumulation. Additional metabolite analysis indicated that downstream flavonoids are accumulated together with naringenin chalcone, whereas CmKFB expression diverts the biochemical flux toward coumarins and general phenylpropanoids. These results show that CmKFB functions as a posttranscriptional regulator that diverts flavonoid metabolic flux.

A Single Amino Acid Substitution in an ORANGE Protein Promotes Carotenoid Overaccumulation in Arabidopsis.

Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression of OR from Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression of AtOR(His) (R90H) or SbOR(His) (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtOR(Ala) (R90A) functioned similarly to AtOR(His) to promote carotenoid overproduction. Neither AtOR nor AtOR(His) greatly affected carotenogenic gene expression. AtOR(His) exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtOR(His) triggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtOR(His) in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates OR(His/Ala) as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying OR(His)-regulated carotenoid accumulation.

A bulk segregant transcriptome analysis reveals metabolic and cellular processes associated with Orange allelic variation and fruit ß-carotene accumulation in melon fruit.

BACKGROUND: Melon fruit flesh color is primarily controlled by the "golden" single nucleotide polymorhism of the "Orange" gene, CmOr, which dominantly triggers the accumulation of the pro-vitamin A molecule, ß-carotene, in the fruit mesocarp. The mechanism by which CmOr operates is not fully understood. To identify cellular and metabolic processes associated with CmOr allelic variation, we compared the transcriptome of bulks of developing fruit of homozygous orange and green fruited F3 families derived from a cross between orange and green fruited parental lines. RESULTS: Pooling together F3 families that share same fruit flesh color and thus the same CmOr allelic variation, normalized traits unrelated to CmOr allelic variation. RNA sequencing analysis of these bulks enabled the identification of differentially expressed genes. These genes were clustered into functional groups. The relatively enriched functional groups were those involved in photosynthesis, RNA and protein regulation, and response to stress. CONCLUSIONS: The differentially expressed genes and the enriched processes identified here by bulk segregant RNA sequencing analysis are likely part of the regulatory network of CmOr. Our study demonstrates the resolution power of bulk segregant RNA sequencing in identifying genes related to commercially important traits and provides a useful tool for better understanding the mode of action of CmOr gene in the mediation of carotenoid accumulation.

Systems approach for exploring the intricate associations between sweetness, color and aroma in melon fruits.

BACKGROUND: Melon (Cucumis melo) fruits exhibit phenotypic diversity in several key quality determinants such as taste, color and aroma. Sucrose, carotenoids and volatiles are recognized as the key compounds shaping the above corresponding traits yet the full network of biochemical events underlying their synthesis have not been comprehensively described. To delineate the cellular processes shaping fruit quality phenotypes, a population of recombinant inbred lines (RIL) was used as a source of phenotypic and genotypic variations. In parallel, ripe fruits were analyzed for both the quantified level of 77 metabolic traits directly associated with fruit quality and for RNA-seq based expression profiles generated for 27,000 unigenes. First, we explored inter-metabolite association patterns; then, we described metabolites versus gene association patterns; finally, we used the correlation-based associations for predicting uncharacterized synthesis pathways. RESULTS: Based on metabolite versus metabolite and metabolite versus gene association patterns, we divided metabolites into two key groups: a group including ethylene and aroma determining volatiles whose accumulation patterns are correlated with the expression of genes involved in the glycolysis and TCA cycle pathways; and a group including sucrose and color determining carotenoids whose accumulation levels are correlated with the expression of genes associated with plastid formation. CONCLUSIONS: The study integrates multiple processes into a genome scale perspective of cellular activity. This lays a foundation for deciphering the role of gene markers associated with the determination of fruit quality traits.

The macroecology of sustainability.

The discipline of sustainability science has emerged in response to concerns of natural and social scientists, policymakers, and lay people about whether the Earth can continue to support human population growth and economic prosperity. Yet, sustainability science has developed largely independently from and with little reference to key ecological principles that govern life on Earth. A macroecological perspective highlights three principles that should be integral to sustainability science: 1) physical conservation laws govern the flows of energy and materials between human systems and the environment, 2) smaller systems are connected by these flows to larger systems in which they are embedded, and 3) global constraints ultimately limit flows at smaller scales. Over the past few decades, decreasing per capita rates of consumption of petroleum, phosphate, agricultural land, fresh water, fish, and wood indicate that the growing human population has surpassed the capacity of the Earth to supply enough of these essential resources to sustain even the current population and level of socioeconomic development.

Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit.

Sulfur-containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur-containing and other volatiles. L-methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C- and ²H-labeled L-methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an L-methionine aminotransferase and preserves the main carbon skeleton of L-methionine. The second route apparently involves the action of an L-methionine-γ-lyase activity, releasing methanethiol, a backbone for formation of thiol-derived aroma volatiles. Exogenous L-methionine also generated non-sulfur volatiles by further metabolism of α-ketobutyrate, a product of L-methionine-γ-lyase activity. α-Ketobutyrate was further metabolized into L-isoleucine and other important melon volatiles, including non-sulfur branched and straight-chain esters. Cell-free extracts derived from ripe melon fruit exhibited L-methionine-γ-lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing L-methionine-γ-lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co-segregated with the levels of sulfur-containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that L-methionine is a precursor of both sulfur and non-sulfur aroma volatiles in melon fruit.