Transcription factor CsMADS3 coordinately regulates chlorophyll and carotenoid pools in Citrus hesperidium.

Citrus, 1 of the largest fruit crops with global economic and nutritional importance, contains fruit known as hesperidium with unique morphological types. Citrus fruit ripening is accompanied by chlorophyll degradation and carotenoid biosynthesis, which are indispensably linked to color formation and the external appearance of citrus fruits. However, the transcriptional coordination of these metabolites during citrus fruit ripening remains unknown. Here, we identified the MADS-box transcription factor CsMADS3 in Citrus hesperidium that coordinates chlorophyll and carotenoid pools during fruit ripening. CsMADS3 is a nucleus-localized transcriptional activator, and its expression is induced during fruit development and coloration. Overexpression of CsMADS3 in citrus calli, tomato (Solanum lycopersicum), and citrus fruits enhanced carotenoid biosynthesis and upregulated carotenogenic genes while accelerating chlorophyll degradation and upregulating chlorophyll degradation genes. Conversely, the interference of CsMADS3 expression in citrus calli and fruits inhibited carotenoid biosynthesis and chlorophyll degradation and downregulated the transcription of related genes. Further assays confirmed that CsMADS3 directly binds and activates the promoters of phytoene synthase 1 (CsPSY1) and chromoplast-specific lycopene ß-cyclase (CsLCYb2), 2 key genes in the carotenoid biosynthetic pathway, and STAY-GREEN (CsSGR), a critical chlorophyll degradation gene, which explained the expression alterations of CsPSY1, CsLCYb2, and CsSGR in the above transgenic lines. These findings reveal the transcriptional coordination of chlorophyll and carotenoid pools in the unique hesperidium of Citrus and may contribute to citrus crop improvement.

Enzymatic isomerization of ζ-carotene mediated by the heme-containing isomerase Z-ISO.

Carotenoids are a large and diverse class of isoprenoid compounds synthesized by plants, algae, some bacteria, arthropods, and fungi. These pigments contribute to plant growth and survival by protecting plants from photooxidative stress and serving as precursors of plant hormones and other signaling compounds. In humans, carotenoids are essential components of the diet and contribute anti-oxidant and provitamin A activities. Carotenoids are synthesized in the membranes of plant plastids where phytoene is converted into all trans lycopene by a biosynthetic pathway that was only recently completed by the discovery of the new enzyme, 15-cis-ζ-carotene isomerase (Z-ISO), which controls carotenoid pathway flux to products necessary for plant development and function. Z-ISO catalysis of the cis to trans isomerization of the 15-cis double bond in 15-cis-ζ-carotene is mediated by a unique mechanism dependent on the redox-state of a heme b cofactor. This chapter describe methods for the functional analysis of Z-ISO, including complementation of Z-ISO in engineered E. coli, separation of Z-ISO enzyme substrate and products, ζ-carotene isomers, by high pressure liquid chromatography (HPLC), expression and purification of Z-ISO and in vitro enzymatic reactions.

Analysis of plant-derived carotenoids in camouflaging stick and leaf insects (Phasmatodea).

A common way to avoid predators is by use of camouflage, a strategy which the stick and leaf insects (Phasmatodea) have refined by appearing as leaves, sticks, lichen, and moss. Stick and leaf insects have perfected their camouflage by sequestering diet-based carotenoids within their exoskeleton. Visual and chemical details of such camouflage have likely been influenced through the millennia of co-evolution between these insects and the plants they mimic. It is this evolutionary struggle that has resulted in a plethora of morphological and chemical adaptations across the stick and leaf insect family tree. In this chapter we discuss prior stick and leaf insect carotenoid studies, proper rearing of specimens, and describe methods for preparation of insect exoskeleton and plant samples, carotenoid extraction and analysis.

Regulation of carotenoid and chlorophyll pools in hesperidia, anatomically unique fruits found only in Citrus.

Domesticated citrus varieties are woody perennials and interspecific hybrid crops of global economic and nutritional importance. The citrus fruit "hesperidium" is a unique morphological innovation not found in any other plant lineage. Efforts to improve the nutritional quality of the fruit are predicated on understanding the underlying regulatory mechanisms responsible for fruit development, including temporal control of chlorophyll degradation and carotenoid biosynthesis. Here, we investigated the molecular basis of the navel orange (Citrus sinensis) brown flavedo mutation, which conditions flavedo that is brown instead of orange. To overcome the limitations of using traditional genetic approaches in citrus and other woody perennials, we developed a strategy to elucidate the underlying genetic lesion. We used a multi-omics approach to collect data from several genetic sources and plant chimeras to successfully decipher this mutation. The multi-omics strategy applied here will be valuable in driving future gene discovery efforts in citrus as well as in other woody perennial plants. The comparison of transcriptomic and genomic data from multiple genotypes and plant sectors revealed an underlying lesion in the gene encoding STAY-GREEN (SGR) protein, which simultaneously regulates carotenoid biosynthesis and chlorophyll degradation. However, unlike SGR of other plant species, we found that the carotenoid and chlorophyll regulatory activities could be uncoupled in the case of certain SGR alleles in citrus and thus we propose a model for the molecular mechanism underlying the brown flavedo phenotype. The economic and nutritional value of citrus makes these findings of wide interest. The strategy implemented, and the results obtained, constitute an advance for agro-industry by driving opportunities for citrus crop improvement.

Building the Synthetic Biology Toolbox with Enzyme Variants to Expand Opportunities for Biofortification of Provitamin A and Other Health-Promoting Carotenoids.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene ß-cyclase (LCYB) to ß-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene ß-cyclase (CsLCYb2a) produced "golden" transgenic tomato fruits with 9.3-fold increased levels of ß-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of ß-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYB genes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYB genes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

Improved Expression and Purification of the Carotenoid Biosynthetic Enzyme Z-ISO.

Carotenoids are a large class of pigments that are essential for survival of plants and other species that consume these plant-derived compounds and their bioactive derivatives. The plant biosynthetic pathway is nuclear-encoded and localized in plastids. The pathway enzymes had been known for many years, except for a recently discovered isomerase, 15-cis-ζ-carotene isomerase (Z-ISO) which utilizes a novel mechanism to mediate isomerization in response to the redox state of its heme b cofactor. To further study this enzyme, a protocol is described which maximizes purification of a fusion between Maltose Binding Protein and Zea mays (maize) Z-ISO (MBP::Z-ISO) expressed in E. coli treated with heme biosynthesis precursors which were used to increase heme available for loading into the expressed protein. Further enrichment of the protein was accomplished by improved sonication to release membranes containing Z-ISO, an integral membrane protein, and collection of the membrane fraction which was subjected to Nickel affinity chromatography. The fusion protein bound to the column through a His-tag. The MBP::Z-ISO protein was released using histidine, and not imidazole which binds heme and would interfere with enzyme recovery. Purification of the 75.46 kD MBP::Z-ISO expressed in E. coli was accomplished with fivefold improvement of yield and doubled heme content compared to the previously published method Beltrán et al. (Nat Chem Biol 11(8):598-605, 2015). The newer protocol will yield, per liter of culture, 5-6 mg MBP::Z-ISO protein with ~1:1 heme to Z-ISO ratio.

Elucidating Carotenoid Biosynthetic Enzyme Localization and Interactions Using Fluorescent Microscopy.

Carotenoids are essential for survival of all plants, where these colorful pigments and derivatives are biosynthesized, as well as for humans and other species that obtain plant-derived carotenoids in their diets and rely upon them for vitamin biosynthesis or antioxidant actions. The plant carotenoid biosynthetic pathway consists of nuclear encoded enzymes that are imported into chloroplasts and other plastids. The pathway structural genes are known and have been targeted for metabolic engineering to improve carotenoid profiles or content. However, results are not always as expected because there remain fundamental gaps in understanding how the pathway is physically organized. Many of the enzymes have been found in high molecular weight complexes which are poorly described. Elucidation of enzyme localization as well as enzyme interactions in vivo are needed for advancing the carotenoid field and facilitating our understanding of the three-dimensional organization of this important pathway. Fluorescent protein fusions with carotenoid enzymes can provide in vivo information when these fusions are introduced and transiently expressed in plant cells. Current advances in fluorescent microscopy, especially confocal microscopy, provide the resolution needed to localize fluorescently tagged carotenoid enzymes within suborganellar locations of plastids. Interactions between carotenoid biosynthetic enzymes can be determined using bimolecular fluorescence complementation (BiFC), a method whereby genes of interest are fused with sequences encoding nonfluorescent N- and C-terminal halves of YFP (yellow fluorescent protein), and then introduced into plant protoplasts to allow expression and visualization by fluorescence microscopy. The YFP fluorescence is restored only if the N and C-terminal regions are brought together by interacting fusion partners. Here we describe the methodology, with extensive tips and notes, for determining in vivo carotenoid enzyme localization and enzyme interactions by transient expression of enzyme-fluorescent protein fusions.