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.

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.

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 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.

Genetic variation in yield under hot ambient temperatures spotlights a role for cytokinin in protection of developing floral primordia.

Unusually hot ambient temperatures (HAT) can cause pre-anthesis abortion of flowers in many diverse species, limiting crop production. This limitation is becoming more substantial with climate change. Flower primordia of passion fruit (Passiflora edulis Sims) vines exposed to HAT summers, normally abort. Flower abortion can also be triggered by gibberellin application. We screened for, and identified a genotype capable of reaching anthesis during summer as well as controlled HAT conditions, and also more resistant to gibberellin. Leaves of this genotype contained higher levels of endogenous cytokinin. We investigated a possible connection between higher cytokinin levels and response to gibberellin. Indeed, the effects of gibberellin application were partially suppressed in plants pretreated with cytokinin. Can higher cytokinin levels protect flowers from aborting under HAT conditions? In passion fruit, flowers at a specific stage showed more resistance in response to HAT after cytokinin application. We further tested this hypothesis in Arabidopsis. Transgenic lines with high or low cytokinin levels and cytokinin applications to wild-type plants supported a protective role for cytokinin on developing flowers exposed to HAT. Such findings may have important implications in future breeding programmes as well as field application of growth regulators.

MicroRNA-resistant alleles of HOMEOBOX DOMAIN-2 modify inflorescence branching and increase grain protein content of wheat.

Plant and inflorescence architecture determine the yield potential of crops. Breeders have harnessed natural diversity for inflorescence architecture to improve yields, and induced genetic variation could provide further gains. Wheat is a vital source of protein and calories; however, little is known about the genes that regulate the development of its inflorescence. Here, we report the identification of semidominant alleles for a class III homeodomain-leucine zipper transcription factor, HOMEOBOX DOMAIN-2 (HB-2), on wheat A and D subgenomes, which generate more flower-bearing spikelets and enhance grain protein content. These alleles increase HB-2 expression by disrupting a microRNA 165/166 complementary site with conserved roles in plants; higher HB-2 expression is associated with modified leaf and vascular development and increased amino acid supply to the inflorescence during grain development. These findings enhance our understanding of genes that control wheat inflorescence development and introduce an approach to improve the nutritional quality of grain.

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement.

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding.

Comparative transcriptome analyses shed light on carotenoid production and plastid development in melon fruit.

Carotenoids, such as ß-carotene, accumulate in chromoplasts of various fleshy fruits, awarding them with colors, aromas, and nutrients. The Orange (CmOr) gene controls ß-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressing ß-carotene turnover in chromoplasts. Carotenoid isomerase (CRTISO) isomerizes yellow prolycopene into red lycopene, a prerequisite for further metabolism into ß-carotene. We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants, low-ß (Cmor) and yofi (Cmcrtiso). The Cmor mutation in low-ß caused a major transcriptomic change in the mature fruit. In contrast, the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages. These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion, but minimally by carotenoid composition in the ripe fruit. Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts. Moreover, large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-ß. CmOR family proteins were found to physically interact with light-harvesting chlorophyll a-b binding proteins, suggesting a new role of CmOR for chloroplast maintenance in melon fruit. This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.

Flower development in the passion fruit Passiflora edulis requires a photoperiod-induced systemic graft-transmissible signal.

Different organisms use gradual seasonal changes in photoperiod to correctly time diverse developmental processes, such as transition to flowering in plants. Florigen is a systemic signal formed in leaves exposed to specific environmental cues, mainly photoperiodic, and capable of triggering flower induction in several species. Here we show that in Passiflora edulis, a perennial climbing vine, flower initiation occurs throughout the year; however, without long photoperiods, flower primordia show arrested growth and differentiation at an early stage. Our results support the existence of a positive, systemic, graft-transmissible signal, produced in mature leaves under LDs, that is required for normal flower development beyond sepal formation. Our results also suggest that Gibberellin acts to inhibit flower development. We provide evidence for genetic variation in the response to short photoperiods. A genotype capable of forming developed flowers under short photoperiods produces a positive graft transmissible signal allowing normal flower development under short days in a cultivar which normally aborts flower development under these conditions. We believe these findings contribute towards discovering the chemical nature of this interesting mobile signal involved in flower development.

The Role of Carotenogenic Metabolic Flux in Carotenoid Accumulation and Chromoplast Differentiation: Lessons From the Melon Fruit.

Carotenoids have various roles in plant physiology. Plant carotenoids are synthesized in plastids and are highly abundant in the chromoplasts of ripening fleshy fruits. Considerable research efforts have been devoted to elucidating mechanisms that regulate carotenoid biosynthesis, yet, little is known about the mechanism that triggers storage capacity, mainly through chromoplast differentiation. The Orange gene (OR) product stabilizes phytoene synthase protein (PSY) and triggers chromoplast differentiation. OR underlies carotenoid accumulation in orange cauliflower and melon. The OR's 'golden SNP', found in melon, alters the highly evolutionary conserved Arginine108 to Histidine and controls ß-carotene accumulation in melon fruit, in a mechanism yet to be elucidated. We have recently shown that similar carotenogenic metabolic flux is active in non-orange and orange melon fruit. This flux probably leads to carotenoid turnover but known carotenoid turnover products are not detected in non-orange fruit. Arrest of this metabolic flux, using chemical inhibitors or mutations, induces carotenoid accumulation and biogenesis of chromoplasts, regardless of the allelic state of OR. We suggest that the 'golden SNP' induces ß-carotene accumulation probably by negatively affecting the capacity to synthesize downstream compounds. The accumulation of carotenoids induces chromoplast biogenesis through a metabolite-induced mechanism. Carotenogenic turnover flux can occur in non-photosynthetic tissues, which do not accumulate carotenoids. Arrest of this flux by the 'golden SNP' or other flux-arrest mutations is a potential tool for the biofortification of agricultural products with carotenoids.

Shielding Flowers Developing under Stress: Translating Theory to Field Application.

Developing reproductive organs within a flower are sensitive to environmental stress. A higher incidence of environmental stress during this stage of a crop plants' developmental cycle will lead to major breaches in food security. Clearly, we need to understand this sensitivity and try and overcome it, by agricultural practices and/or the breeding of more tolerant cultivars. Although passion fruit vines initiate flowers all year round, flower primordia abort during warm summers. This restricts the season of fruit production in regions with warm summers. Previously, using controlled chambers, stages in flower development that are sensitive to heat were identified. Based on genetic analysis and physiological experiments in controlled environments, gibberellin activity appeared to be a possible point of horticultural intervention. Here, we aimed to shield flowers of a commercial cultivar from end of summer conditions, thus allowing fruit production in new seasons. We conducted experiments over three years in different settings, and our findings consistently show that a single application of an inhibitor of gibberellin biosynthesis to vines in mid-August can cause precocious flowering of ~2-4 weeks, leading to earlier fruit production of ~1 month. In this case, knowledge obtained on phenology, environmental constraints and genetic variation, allowed us to reach a practical solution.

Evolutionary, genetic, environmental and hormonal-induced plasticity in the fate of organs arising from axillary meristems in Passiflora spp.

Tendrils can be found in different plant species. In legumes such as pea, tendrils are modified leaves produced by the vegetative meristem but in the grape vine, a same meristem is used to either form a tendril or an inflorescence. Passiflora species originated in ecosystems in which there is dense vegetation and competition for light. Thus climbing on other plants in order to reach regions with higher light using tendrils is an adaptive advantage. In Passiflora species, after a juvenile phase, every leaf has a subtending vegetative meristem, and a separate meristem that forms both flowers and a tendril. Thus, flowers are formed once a tendril is formed yet whether or not this flower will reach bloom depends on the environment. For example, in Passiflora edulis flowers do not develop under shaded conditions, so that tendrils are needed to bring the plant to positions were flowers can develop. This separate meristem generally forms a single tendril in different Passiflora species yet the number and position of flowers formed from the same meristem diverges among species. Here we display the variation among species as well as variation within a single species, P. edulis. We also show that the number of flowers within a specific genotype can be modulated by applying Cytokinins. Finally, this separate meristem is capable of transforming into a leaf-producing meristem under specific environmental conditions. Thus, behind what appears to be a species-specific rigid program regarding the fate of this meristem, our study helps to reveal a plasticity normally restrained by genetic, hormonal and environmental constraints.