Orange: a target gene for regulating carotenoid homeostasis and increasing plant tolerance to environmental stress in marginal lands

Orange: a target gene for regulating carotenoid homeostasis and increasing plant tolerance to... Abstract Carotenoids play essential roles in various light-harvesting processes in plants and help protect the photosynthetic machinery from photo-oxidative damage. Orange genes, which play a role in carotenoid accumulation, have recently been isolated from several plant species, and their functions have been intensively investigated. The Orange gene (IbOr) of sweet potato [Ipomoea batatas (L.) Lam] helps maintain carotenoid homeostasis to improve plant tolerance to environmental stress. IbOr, a protein with strong holdase chaperone activity, directly interacts with phytoene synthase, a key enzyme involved in carotenoid biosynthesis, in plants under stress conditions, resulting in increased carotenoid accumulation and abiotic stress tolerance. In addition, IbOr interacts with the oxygen-evolving enhancer protein 2-1, a member of a protein complex in photosystem II that is denatured under heat stress. Transgenic sweet potato plants overexpressing IbOr showed enhanced tolerance to high temperatures (47 °C). These findings indicate that IbOr protects plants from environmental stress not only by controlling carotenoid biosynthesis, but also by directly stabilizing photosystem II. In this review, we discuss the functions of IbOr and Or proteins in other plant species and their possible biotechnological applications for molecular breeding for sustainable development on marginal lands. Chaperone activity, heat stress, IbOr, IbPsbP, IbPSY, orange, oxidative stress, sweet potato Introduction The dramatic increase in the global population, combined with rapid industrialization in developing countries, has placed great strain on global food and energy supplies. The Food and Agriculture Organization of the United Nations (FAO) has estimated that the world population will exceed 9.1 billion in 2050 (FAO, 2009). If we use energy and food at the present rate, we will need more than 3.5 times the current energy supply and 1.7 times the current food supply in 2050. To cope with these global crises in food and energy supply, as well as environmental problems, the development of new, environmentally friendly industrial crop varieties for growth on marginal lands, including areas affected by desertification, is urgently needed to ensure sustainable development. Plant biotechnology can be used as a tool to maximize plant productivity by introducing stress-tolerance genes and genes whose products are responsible for increasing various metabolic activities in plants. Sweet potato [Ipomoea batatas (L.) Lam] is an attractive crop that could be used to help solve the world’s food and environmental problems in the 21st century. This crop species can be used as an industrial bioreactor to produce various high-value-added materials—including bio-ethanol, functional feed, and antioxidants—via molecular breeding approaches. Sweet potato plants have high water-use efficiency among the starch crops, and help reduce soil erosion. All parts of the sweet potato plant can be used for human and animal consumption. Owing to its rich nutritional content, combined with its wide adaptability to marginal lands ranging from tropical to temperate zones, sweet potato has great potential for preventing malnutrition and increasing food security in developing countries. The non-profit Center for Science in the Public Interest (CSPI) described sweet potato as one of 10 ‘superfoods’ for better health, because it contains high levels of low-molecular-weight antioxidants such as carotenoids and vitamin C, as well as dietary fiber and potassium (CPSI, 2016). Carotenoids benefit human health by acting as dietary antioxidants and helping prevent or slow the development of aging-related diseases. Carotenoids also serve as a dietary source of provitamin A, making them essential components of the human diet, since humans are unable to synthesize vitamin A. When carotenoids are ingested, they are converted to the visual pigment rhodopsin and to retinal, a precursor of retinoid acid, which regulates growth, development, and differentiation (Fraser and Bramley, 2004). Vitamin A deficiency causes night blindness, skin keratinization, dry eye syndrome, degenerative vision loss, impaired immune function, and birth defects (Rao and Rao, 2007). According to a United States Department of Agriculture (USDA) report, sweet potato can yield two to three times the level of carbohydrates as does field corn, approaching the amount that sugarcane can produce in the USA (Ziska et al., 2009). It would be worthwhile to begin pilot programs to investigate the feasibility of growing sweet potato for ethanol production on marginal lands. In addition, rational metabolic engineering of low-molecular-weight antioxidants should contribute to the development of new sweet potato cultivars with higher levels of nutritional antioxidants and abiotic stress tolerance. Carotenoids in plants Carotenoids are highly important molecules for plant growth and human health. These compounds are widespread in all photosynthetic organisms and in some non-photosynthetic bacteria and fungi. Carotenoids include carotenes and their oxidized form, xanthophylls. Carotenoids function as light-harvesting pigments and photoprotectants during photosynthesis (Niyogi, 1999; Domonkos et al., 2013). In addition, they protect plants from oxidative stress caused by excessive light by absorbing the blue-green wavelengths of light (Ledford et al., 2004). Carotenoids also serve as precursors for the biosynthesis of phytohormones, including abscisic acid (ABA) and strigolactones, and for the production of flower and fruit flavor and aroma compounds (Auldridge et al., 2006; Walter and Strack, 2011). Carotenoids can be synthesized in the membranes of nearly all plastids in the plant, except proplastids. These compounds primarily accumulate in the chloroplast and chromoplast (Howitt and Pogson 2006). Carotenoids in the chloroplast form a pigment–protein complex in the photosynthetic membrane along with chlorophyll-binding proteins (Farré et al., 2010). By contrast, carotenoids in the chromoplast form a carotenoid–lipoprotein-sequestering structure by combining with polar lipids and carotenoid-associated proteins. Consequently, these organelles maintain high levels of carotenoids (Lu and Li, 2008). Carotenoids act as strong antioxidants in plants, thereby protecting them from damage caused by various environmental stresses, including strong light, high temperature, UV, and drought (Davison et al., 2002; Götz et al., 2002; Wu et al., 2015). In addition, carotenoids directly scavenge reactive oxygen species in biological membranes (Strzałka et al., 2003). Davison et al. (2002) reported that overexpression of β-carotene hydroxylase increased stress tolerance in Arabidopsis thaliana. The authors proposed that the enhanced stress tolerance was due to the increased size of the xanthophyll cycle pool and the reversible interconversion of two carotenoids, violaxanthin and zeaxanthin, which play key photoprotective roles in plants. Overexpression of the bacterial β-carotene hydroxylase gene (crtZ), which is involved in the conversion step of β-carotene and β-cryptoxanthin to zeaxanthin, significantly enhanced UV tolerance in tobacco by increasing its zeaxanthin content (Götz et al., 2002). Silencing of the β-carotene hydroxylase gene (CHY-β) significantly increased tolerance to salt-mediated oxidative stress, as well as β-carotene levels, in transgenic sweet potato calli (Kim et al., 2012). Moreover, silencing of the gene encoding lycopene ε-cyclase (LCY-ε), which is involved in the first step of the α-branch synthesis pathway of carotenoids from lycopene, resulted in a 21-fold increase in β-carotene contents in sweet potato calli and significantly increased tolerance to salt-mediated oxidative stress (Kim et al., 2013a). Recently, Kang et al. (2017a) reported that RNAi-IbCHY-β sweet potato plants exhibited increased tolerance to salt stress with enhanced 9-cis-epoxycarotenoid dioxygenase (NCED) expression and ABA contents. The NCED gene is a key enzyme of ABA biosynthesis (Seo and Koshiba, 2002). In addition, ABA is an important phytohormone and plays a critical role in response to various stress signals (Swamy and Smith, 1999). Increased ABA levels have been shown to enhance adaptation to salt stress (Zhu, 2002). These results suggest that down-regulation of IbCHY-β in sweet potato not only increases the total carotenoid and β-carotene contents but also enhances salt-stress tolerance via increased ABA levels. However, the exact mechanism underlying how increased carotenoid contents lead to increased stress tolerance in plants remains largely unknown. Orange genes in plants The Orange (Or) genes appear to be plant specific, with homologs present in all plant species examined and in algae (see Supplementary Table S1 at JXB online). The Or gene encodes a DnaJ Cys-rich zinc finger domain-containing protein (Lu et al., 2006). The amino acid sequences of Or proteins are highly conserved among diverse plant species (Lu et al., 2006), suggesting that they play an important role in plant growth and development. However, to date, Or proteins have been functionally characterized in only a few plant species, including cauliflower, Arabidopsis, melon, sorghum, and sweet potato (Table 1). Table 1. Biological functions of homologous Or proteins from various plant species Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b View Large Table 1. Biological functions of homologous Or proteins from various plant species Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b View Large The Or gene was first discovered in an orange curd cauliflower (Brassica oleracea var. botrytis) mutant, where it was shown to enhance β-carotene accumulation (Lu et al., 2006). The expression of BoOr leads to the production of orange tissues with enhanced carotenoid contents in both white cauliflower and potato tubers (Lu et al., 2006; Lopez et al., 2008). The increased carotenoid levels in plants overexpressing BoOr are associated with the biogenesis of chromoplasts, which serve as a metabolic sink for carotenoid storage in non-photosynthetic tissues (Lopez et al., 2008; Li et al., 2012). The BoOr-induced accumulation of β-carotene in these tissues is not due to an increased capacity of the carotenoid biosynthetic pathway (Li et al., 2006). In addition to carotenoid accumulation, BoOr protein is also involved in plant growth and development. BoOr interacts with cauliflower eukaryotic release factor 1 (eRF1) protein and increases leaf petiole elongation by suppressing the expression of BoeRF1 family genes (Zhou et al., 2011b). In addition, the expression of BoOr increases the stability of potato tubers during post-harvest storage by stimulating continuous accumulation of carotenoids (Li et al., 2012). In Arabidopsis, AtOr proteins interact directly with, and post-transcriptionally regulate, phytoene synthase (PSY) to control carotenoid biosynthesis (Zhou et al., 2015). AtOr is localized to plastids (Zhou et al., 2015). The expression of AtOr under the control of the endosperm-specific wheat low-molecular-weight glutenin promoter resulted in high carotenoid contents in white corn (Berman et al., 2017). The expression of AtOr also induced carotenoid accumulation in rice calli and plants (Bai et al., 2014, 2016). The overexpression of an AtOr mutant protein resulted in high β-carotene levels in Arabidopsis (Yuan et al., 2015). In melon (Cucumis melo), the presence of CmOr protein with a single amino acid change from His to Arg distinguishes orange-fleshed melon from white- or green-fleshed melon (Tzuri et al., 2015). Unlike BoOr, which has a large retrotransposon insertion and causes a stunted phenotype, the effect of the CmOr allelic variation is limited to carotenoid accumulation in fruit. Despite the important correlation between carotenoid accumulation and environmental stress tolerance in plants, only a few Or genes have been isolated and partially characterized in terms of their effect on various abiotic stresses (Table 1). It is likely that all plant species, including photosynthetic algae, have Or genes, since carotenoids are essential for photosynthesis (Supplementary Table S1). The Or gene from the green alga Chlamydomonas reinhardtii was recently isolated and transformed into C. reinhardtii, which led to increased carotenoid biosynthesis (Morikawa et al., 2017). Functional analysis of the Or gene from sweet potato In sweet potato, IbOr was initially isolated from an orange-fleshed cultivar (cv. Sinhwangmi) based on the sequence of BoOr (Kim et al., 2013b). IbOr is expressed at high levels in the leaves of sweet potatoes with various flesh colors (white, orange, and purple), but is highly expressed in storage roots only in orange-fleshed varieties. Two variants of IbOr (encoding IbOr-Wt and IbOr-Ins, which contains seven additional amino acids [KSPNPNL] inserted between residues 131 and 142 of IbOr-Wt) were transformed into non-embryogenic calli from white-fleshed sweet potato (cv. Yulmi). The average total carotenoid contents in IbOr-Ins and IbOr-Wt transgenic calli were approximately 13- and 4-fold higher, respectively, than those in control calli (Kim et al., 2013b). The levels of all carotenoids were higher in both IbOr-Wt and IbOr-Ins transgenic calli relative to the control, except for lutein in IbOr-Wt. IbOr-Ins was subsequently transformed into purple-fleshed sweet potato, leading to the production of anthocyanin and carotenoids in the same storage root (Park et al., 2015). IbOr transgenic sweet potatoes exhibit different color densities in individual transgenic lines. IbOr-201 plants are a darker purple than control plants, and IbOr plants have higher carotenoid levels (up to 7-fold) in their storage roots than control plants. Overall, the carotenoid levels in IbOr plants are positively correlated with IbOr transcript levels. Or proteins contain a Cys-rich zinc finger domain that is highly specific to DnaJ chaperone proteins. DnaJ proteins participate in essential cellular processes such as protein folding, assembly, degradation, and homeostasis under stress conditions (Wang et al., 2004; Hennessy et al., 2005). IbOr also contains a DnaJ domain and has high chaperone activity (Park et al., 2016). Among the carotenoid biosynthetic enzymes, PSY is the most important regulatory enzyme in the carotenoid biosynthesis pathway. Similar to AtOr, IbOr directly interacts with IbPSY in the chloroplast. In addition, IbPSY is protected by IbOr chaperone activity under heat and oxidative stress conditions (Park et al., 2016). Transgenic Arabidopsis plants overexpressing IbOr displayed enhanced heat-stress tolerance (Park et al., 2016). Therefore, the holdase chaperone function of IbOr is involved in carotenoid biosynthesis, as well as in environmental stress tolerance in plants, by protecting IbPSY protein (Fig. 1A). Fig. 1. View largeDownload slide Proposed model for the role of sweet potato Orange protein (IbOr) in environmental stress tolerance. (A) Stabilization of carotenoid biosynthesis-related enzymes. IbOr interacts with phytoene synthase (PSY) and carotenoid cleavage dioxygenase (CCD) 4. IbOr-mediated protection of PSY leads to increased carotenoid accumulation and stress tolerance. ABA, abscisic acid; CHY-β, β-carotene hydroxylase; CHY-ε, ε-ring hydroxylase; CRTISO, carotenoid isomerase; GGPP, geranylgeranyl pyrophosphate; LCY-β, lycopene β-cyclase; LCY-ε, lycopene ε-cyclase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; VDE, violaxanthin de-epoxidase; ZDS, f-carotene desaturase; ZEP, zeaxanthin epoxidase. (B) Stabilization of the photosynthetic machinery. IbOr-mediated protection of PsbP increases tolerance to heat stress. Fig. 1. View largeDownload slide Proposed model for the role of sweet potato Orange protein (IbOr) in environmental stress tolerance. (A) Stabilization of carotenoid biosynthesis-related enzymes. IbOr interacts with phytoene synthase (PSY) and carotenoid cleavage dioxygenase (CCD) 4. IbOr-mediated protection of PSY leads to increased carotenoid accumulation and stress tolerance. ABA, abscisic acid; CHY-β, β-carotene hydroxylase; CHY-ε, ε-ring hydroxylase; CRTISO, carotenoid isomerase; GGPP, geranylgeranyl pyrophosphate; LCY-β, lycopene β-cyclase; LCY-ε, lycopene ε-cyclase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; VDE, violaxanthin de-epoxidase; ZDS, f-carotene desaturase; ZEP, zeaxanthin epoxidase. (B) Stabilization of the photosynthetic machinery. IbOr-mediated protection of PsbP increases tolerance to heat stress. The metabolic turnover of carotenoids not only helps maintain steady levels of carotenoids in plants, but also produces important signaling and accessory apocarotenoid molecules, such as the phytohormones ABA and strigolactones (Cunningham and Gantt, 1988; Giuliano, 2014). Carotenoid cleavage dioxygenases (CCDs) generate apocarotenoids via oxidative cleavage of carotenoids (Walter and Strack, 2011). Five members of the Arabidopsis NCED family, another class of CCDs, have been implicated in ABA biosynthesis. Moreover, strigolactones, which control auxiliary branching and tillering, are synthesized by the enzymes CCD7 and CCD8 (Auldridge et al., 2006). In addition, CCD1 and CCD4 contribute to the production of apocarotenoid-derived pigments, as well as flavor and/or aroma compounds in flowers and a variety of foods, through the degradation of carotenoids (Auldridge et al., 2006; Gonzalez-Jorge et al., 2013). The loss of CCD1 or CCD4 activity results in a significant increase in carotenoid levels, indicating that both enzymes are negatively correlated with carotenoid accumulation in plants (García-Limones et al., 2008; Tanaka and Ohmiya, 2008; Campbell et al., 2010; Zhou et al., 2011a; Gonzalez-Jorge et al., 2013). Interestingly, we found that NCED, CCD1, and CCD4 were highly expressed in IbOr-overexpressing sweet potato (Park et al., 2015) and that IbOr specifically interacts with CCD4 (H.S. Kim, S.C. Park, S.S. Kwak, unpublished data). Thus, IbOr can interact not only with the carotenoid biosynthesis enzyme PSY, but also with the carotenoid degradation enzyme CCD4, suggesting that IbOr plays an important role in maintaining carotenoid homeostasis (Fig. 1A). It remains unclear whether the holdase activity of IbOr positively or negatively regulates CCD4 activity. Elucidation of the specific mechanism underlying IbOr-regulated CCD activity in the future could shed light on the regulation of carotenoid homeostasis in plants. IbOr protein normally localizes to the nucleus and chloroplasts, but it translocates from the nucleus to the chloroplasts in response to heat stress (Park et al., 2016). Furthermore, recent work has shown that transgenic sweet potato calli, Arabidopsis, alfalfa, and potato overexpressing IbOr maintain higher photosystem II (PSII) efficiency and chlorophyll content under abiotic stress compared with the wild type plants (Kim et al., 2013b; Park et al., 2015, 2016; Wang et al., 2015; Cho et al., 2016). These findings suggest that Or is a multifunctional protein that helps regulate photosynthesis. Kang et al. (2017b) recently identified proteins that are differentially expressed in response to heat stress in transgenic Arabidopsis plants overexpressing IbOr. Among these are several proteins involved in the light-dependent reaction and the Calvin cycle, suggesting that IbOr might be directly involved in regulating photosynthesis. Interestingly, IbOr interacts with oxygen-evolving enhancer protein 2-1 (PsbP), an extrinsic protein of the oxygen-evolving complex of PSII, and the holdase chaperone function of IbOr can protect PsbP from heat-induced denaturation (Kang et al., 2017b). Therefore, IbOr is a multifunctional protein that has tremendous potential for increasing carotenoid accumulation and protecting the photosynthetic machinery in plants (Fig. 1B). More studies are needed to investigate the Or genes from various plant species and non-photosynthetic organisms. Biotechnological applications of Or genes Carotenoids, especially β-carotene, are indispensable for human nutrition and provide the primary dietary source for vitamin A biosynthesis. The low levels of carotenoids in major food crops contribute to the global prevalence of vitamin A deficiency. Significant efforts have been made to generate carotenoid-enriched food crops through either biotechnology or traditional breeding strategies. By altering the levels of expression of genes encoding key enzymes involved in carotenoid biosynthesis or several enzymes in carotenoid biosynthesis mini-pathways, numerous transgenic crops with enhanced carotenoid levels have been produced (Fraser et al., 2002; Ducreux et al., 2005; Paine et al., 2005; Diretto et al., 2007a, b; Naqvi et al., 2009; Welsch et al., 2010). Overexpression of the PSY gene from bacteria or plants resulted in a significant increase in total carotenoid levels in tomato fruits (Fraser et al., 2002), potato tubers (Ducreux et al., 2005), and canola seeds (Shewmaker et al., 1999). The expression of multiple biosynthetic genes in the carotenoid biosynthetic pathway led to a profound increase in β-carotene levels in Golden Rice (Ye et al., 2000; Paine et al., 2005) and “golden” potato (Diretto et al., 2007a, b). The selection of favorable alleles that alter carotenoid metabolic flux toward β-carotene resulted in the breeding of orange maize with high β-carotene contents (Harjes et al., 2008; Yan et al., 2010). However, in some cases, the increased flux into carotenogenesis can alter or reduce flux in other competing pathways, leading to unexpected phenotypic changes. For example, the overexpression of PSY in canola and Arabidopsis seeds led not only to high levels of carotenoid accumulation, but also to delayed germination due to increased production of carotenoid-derived ABA (Shewmaker et al., 1999; Lindgren et al., 2003). The constitutive expression of PSY-1 in tomato resulted in stunted growth due to insufficient biosynthesis of gibberellins, along with pleiotropic effects such as premature pigmentation of seed coats and cotyledons (Fray et al., 1995). Thus, manipulating the formation of deposition sinks offers a new strategy for metabolic engineering of carotenoid contents in the storage tissues of various food crops. In contrast to modifying the catalytic activity of carotenoid pathway enzymes, the discovery of Or genes provides an alternative, complementary approach for increasing carotenoid levels in food crops by enhancing sink strength in storage tissues (Li and Van Eck, 2007). Overexpression of the BoOr transgene in potato tubers resulted in the production of orange tissues with enhanced carotenoid contents (Lopez et al., 2008). Moreover, the presence of Or protein promoted continuously increasing carotenoid accumulation in BoOr transgenic potato tubers during post-harvest storage (Li et al., 2012). The expression of AtOr also promoted carotenoid accumulation in transgenic corn (Berman et al., 2017). In addition to their role in increasing carotenoid accumulation, Or genes might be useful for generating transgenic plants with enhanced tolerance to environmental stress. We previously demonstrated that sweet potato plants overexpressing IbOr showed enhanced tolerance to both heat and oxidative stress (Park et al., 2015; Park et al., 2016; Kang et al., 2017b). These plants also showed enhanced drought tolerance (Supplementary Fig. S1). Further characterization of IbOr-overexpressing sweet potato plants in response to various abiotic and biotic stresses is currently under way. In addition, transgenic alfalfa and potato plants overexpressing IbOr exhibited increased tolerance to various abiotic stresses, including oxidative, salt, and drought stress, as well as increased carotenoid contents (Goo et al., 2015; Wang et al., 2015; Cho et al., 2016). Therefore, it is likely that IbOr plays a crucial role in the maintenance of photosynthesis, which confers stress tolerance to plants. As mentioned above, a single-nucleotide polymorphism (SNP) in Or underlies the differences in carotenoid contents in orange-fleshed versus green/white-fleshed melon (Cucumis melo) fruits, and is responsible for the non-orange and orange melon fruit phenotypes (Tzuri et al., 2015). Overexpression of the Arabidopsis Or protein mutagenized at the site corresponding to the melon golden SNP (Arg to His) resulted in high β-carotene levels (Yuan et al., 2015). In a preliminary experiment (H.S. Kim, S.E. Kim, S.S. Kwak, unpublished data), we found that a single amino acid substitution in IbOr protein also significantly increased carotenoid contents in sweet potato calli compared with calli harboring the original IbOr protein (Kim et al., 2013b). Transgenic sweet potato and rice plants overexpressing the IbOr-His gene are currently being generated in our laboratory. Or represents a unique class of regulatory genes that mediate carotenoid accumulation (Li and Yuan, 2013). Two natural mutations in Or promote high levels of β-carotene accumulation in plants. The first mutation is BoOr from an orange curd cauliflower mutant. BoOr contains a large retrotransposon insertion and produces three alternatively spliced transcripts, none of which alone induces massive accumulation of carotenoids (Lu et al., 2006). The pleiotropic effects of BoOr might limit its potential application in nutritional improvement of crops. The second mutation is the recently discovered CmOr gene in melon, mentioned above. CmOr protein, with a single amino acid difference (His or Arg), distinguishes orange-fleshed melon from white- or green-fleshed melon (Tzuri et al., 2015). The expression of CmOr-His suppresses the downstream metabolism of β-carotene in melon fruits; in mature CmOr-His melon fruits, only traces of lutein could be found, while other xanthophylls were undetectable (Chayut et al., 2015; Chayut et al., 2017). However, overexpression of IbOr-His in sweet potato calli induced strong accumulation of β-carotene, together with significant amounts of xanthophylls, including lutein, β-cryptoxanthin, zeaxanthin, and violaxanthin. The xanthophyll cycle (the reversible interconversion of two carotenoids, violaxanthin and zeaxanthin) plays a key photoprotective role in plants and is therefore a promising target for genetic engineering to increase stress tolerance (Yamamoto et al., 1962; Demmig-Adams and Adams, 1992; Hauvaux and Niyogi, 1999; Davison et al., 2002). Manipulation of the IbOr gene represents a promising strategy for developing crop varieties with increased tolerance to heat, salinity, and other environmental stresses, in addition to improved nutritional qualities brought about by increasing the carotenoid contents through enhancing sink strength. Furthermore, site-specific mutagenesis of IbOr combined with CRISPR-Cas9-mediated genome editing techniques could lead to significant nutritional biofortification of sweet potato. Conclusions The Or genes help maintain carotenoid homeostasis and help plants adapt to environmental stress, not only by controlling carotenoid biosynthesis, but also by directly stabilizing PSII under stress conditions (Fig. 1). Greater understanding of the Or genes will be needed in order to comprehensively elucidate the regulatory mechanisms of carotenoid accumulation under stress conditions, although the functions of these genes are already partially understood. Or genes represent a novel, useful resource for molecular breeding to increase nutritional carotenoid contents and abiotic stress tolerance in various crops, including sweet potato, potato, and rice. We anticipate that Or genes will contribute to the production of plants that function as efficient industrial bioreactors. These plants could be used to help us cope with the effects of climate change, through allowing sustainable agriculture on global marginal lands, as well as to alleviate vitamin A deficiency diseases in sub-Saharan Africa and south-east Asia. Supplementary data Supplementary data are available at JXB online. Fig. S1. Enhanced tolerance to drought stress in IbOr-overexpressing transgenic sweet potato. Table S1. List of homologous Or proteins from various plant species and algae. Abbreviations: Abbreviations: ABA Abscisic acid CCD carotenoid cleavage dioxygenases Or orange PsbP oxygen-evolving enhancer protein 2-1 PSY phytoene synthase. Acknowledgements This work was supported by grants from the Systems & Synthetic Agrobiotech Center (grant no. PJ01318401), the Next Generation BioGreen 21 Project, Rural Development Administration, Korea, the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (grant no. 2015053321), and the KRIBB Initiative Program. References Auldridge ME , McCarty DR , Klee HJ . 2006 . Plant carotenoid cleavage oxygenases and their apocarotenoid products . Current Opinion in Plant Biology 9 , 315 – 321 . Google Scholar CrossRef Search ADS PubMed Bai C , Capell T , Berman J , et al. 2016 . Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance . Plant Biotechnology Journal 14 , 195 – 205 . Google Scholar CrossRef Search ADS PubMed Bai C , Rivera SM , Medina V , et al. 2014 . An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation . Plant Journal 77 , 464 – 475 . Google Scholar CrossRef Search ADS PubMed Berman J , Zorrilla-López U , Medina V , Farré G , Sandmann G , Capell T , Christou P , Zhu C . 2017 . The Arabidopsis ORANGE (AtOR) gene promotes carotenoid accumulation in transgenic corn hybrids derived from parental lines with limited carotenoid pools . Plant Cell Reports 36 , 933 – 945 . Google Scholar CrossRef Search ADS PubMed Campbell R , Ducreux LJ , Morris WL , Morris JA , Suttle JC , Ramsay G , Bryan GJ , Hedley PE , Taylor MA . 2010 . The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato . Plant Physiology 154 , 656 – 664 . Google Scholar CrossRef Search ADS PubMed Center for Science in the Public Interest . 2016 . What to eat: 10 best foods . Retrieved from https://www.nutritionaction.com/wp-content/free-downloads/What_To_Eat_com-we-1.pdf/ Chayut N , Yuan H , Ohali S , et al. 2015 . A bulk segregant transcriptome analysis reveals metabolic and cellular processes associated with Orange allelic variation and fruit β-carotene accumulation in melon fruit . BMC Plant Biology 15 , 274 . Google Scholar CrossRef Search ADS PubMed Chayut N , Yuan H , Ohali S , et al. 2017 . Distinct mechanisms of the ORANGE protein in controlling carotenoid flux . Plant Physiology 173 , 376 – 389 . Google Scholar CrossRef Search ADS PubMed Cho KS , Han EH , Kwak SS , Cho JH , Im JS , Hong SY , Sohn HB , Kim YH , Lee SW . 2016 . Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production . Comptes Rendus Biologies 339 , 207 – 213 . Google Scholar CrossRef Search ADS PubMed Cunningham FX , Gantt E . 1998 . Genes and enzymes of carotenoid biosynthesis in plants . Annual Review of Plant Physiology and Plant Molecular Biology 49 , 557 – 583 . Google Scholar CrossRef Search ADS PubMed Davison PA , Hunter CN , Horton P . 2002 . Overexpression of beta-carotene hydroxylase enhances stress tolerance in Arabidopsis . Nature 418 , 203 – 206 . Google Scholar CrossRef Search ADS PubMed Demmig-Adams B , Adams WW . 1992 . Photoprotection and other responses of plants to high light stress . Annual Review of Plant Physiology and Plant Molecular Biology 43 , 599 – 626 . Google Scholar CrossRef Search ADS Diretto G , Al-Babili S , Tavazza R , Papacchioli V , Beyer P , Giuliano G . 2007a. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway . PLoS One 2 , e350 . Google Scholar CrossRef Search ADS PubMed Diretto G , Welsch R , Tavazza R , Mourgues F , Pizzichini D , Beyer P , Giuliano G . 2007b. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers . BMC Plant Biology 7 , 11 . Google Scholar CrossRef Search ADS PubMed Domonkos I , Kis M , Gombos Z , Ughy B . 2013 . Carotenoids, versatile components of oxygenic photosynthesis . Progress in Lipid Research 52 , 539 – 561 . Google Scholar CrossRef Search ADS PubMed Ducreux LJ , Morris WL , Hedley PE , Shepherd T , Davies HV , Millam S , Taylor MA . 2005 . Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein . Journal of Experimental Botany 56 , 81 – 89 . Google Scholar PubMed FAO . 2009 . How to feed the world in 2050 . Retrieved from http://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How_to_Feed_the_World_in_2050.pdf Farré G , Sanahuja G , Naqvi S , Bai C , Capell T , Zhu C , Christou P . 2010 . Travel advice on the road to carotenoids in plants . Plant Science 179 , 28 – 48 . Google Scholar CrossRef Search ADS Fraser PD , Bramley PM . 2004 . The biosynthesis and nutritional uses of carotenoids . Progress in Lipid Research 43 , 228 – 265 . Google Scholar CrossRef Search ADS PubMed Fraser PD , Romer S , Shipton CA , Mills PB , Kiano JW , Misawa N , Drake RG , Schuch W , Bramley PM . 2002 . Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner . Proceedings of the National Academy of Sciences, USA 99 , 1092 – 1097 . Google Scholar CrossRef Search ADS Fray RG , Wallace A , Fraser PD , Valero D , Hedden P , Bramley PM , Grierson D . 1995 . Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway . The Plant Journal 8 , 693 – 701 . Google Scholar CrossRef Search ADS García-Limones C , Schnäbele K , Blanco-Portales R , Luz Bellido M , Caballero JL , Schwab W , Muñoz-Blanco J . 2008 . Functional characterization of FaCCD1: a carotenoid cleavage dioxygenase from strawberry involved in lutein degradation during fruit ripening . Journal of Agricultural and Food Chemistry 56 , 9277 – 9285 . Google Scholar CrossRef Search ADS PubMed Giuliano G . 2014 . Plant carotenoids: genomics meets multi-gene engineering . Current Opinion in Plant Biology 19 , 111 – 117 . Google Scholar CrossRef Search ADS PubMed Gonzalez-Jorge S , Ha SH , Magallanes-Lundback M , et al. 2013 . CAROTENOID CLEAVAGE DIOXYGENASE4 is a negative regulator of β-carotene content in Arabidopsis seeds . The Plant Cell 25 , 4812 – 4826 . Google Scholar CrossRef Search ADS PubMed Goo YM , Han EH , Jeong JC , Kwak SS , Yu J , Kim YH , Ahn MJ , Lee SW . 2015 . Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato . Comptes Rendus Biologies 338 , 12 – 20 . Google Scholar CrossRef Search ADS PubMed Götz T , Sandmann G , Römer S . 2002 . Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco . Plant Molecular Biology 50 , 129 – 142 . Google Scholar CrossRef Search ADS PubMed Harjes CE , Rocheford TR , Bai L , et al. 2008 . Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification . Science 319 , 330 – 333 . Google Scholar CrossRef Search ADS PubMed Hauvaux M , Niyogi KK . 1999 . The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism . Proceedings of the National Academy of Sciences, USA 96 , 8762 – 8767 . Google Scholar CrossRef Search ADS Hennessy F , Nicoll WS , Zimmermann R , Cheetham ME , Blatch GL . 2005 . Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions . Protein Science 14 , 1697 – 1709 . Google Scholar CrossRef Search ADS PubMed Howitt CA , Pogson BJ . 2006 . Carotenoids accumulation and function in seeds and non-green tissues . Plant, Cell & Environment 29 , 435 – 445 . Google Scholar CrossRef Search ADS PubMed Kang L , Ji CY , Kim SH , et al. 2017a. Suppression of the β-carotene hydroxylase gene increases β-carotene content and tolerance to abiotic stress in transgenic sweetpotato plants . Plant Physiology and Biochemistry 117 , 24 – 33 . Google Scholar CrossRef Search ADS PubMed Kang L , Kim HS , Kwon YS , Ke Q , Ji CY , Park SC , Lee HS , Deng X , Kwak SS . 2017b. IbOr regulates photosynthesis under heat stress by stabilizing IbPsbP in sweetpotato . Frontiers in Plant Science 8 , 989 . Google Scholar CrossRef Search ADS PubMed Kim SH , Ahn YO , Ahn MJ , Lee HS , Kwak SS . 2012 . Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato . Phytochemistry 74 , 69 – 78 . Google Scholar CrossRef Search ADS PubMed Kim SH , Kim YH , Ahn YO , Ahn MJ , Jeong JC , Lee HS , Kwak SS . 2013a . Downregulation of the lycopene ε-cyclase gene increases carotenoid synthesis via the β-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli . Physiologia Plantarum 147 , 432 – 442 . Google Scholar CrossRef Search ADS Kim SH , Ahn YO , Ahn MJ , Jeong JC , Lee HS , Kwak SS . 2013b . Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures . Plant Physiology and Biochemistry 70 , 445 – 454 . Google Scholar CrossRef Search ADS Ledford HK , Baroli I , Shin JW , Fischer BB , Eggen RI , Niyogi KK . 2004 . Comparative profiling of lipid-soluble antioxidants and transcripts reveals two phases of photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas reinhardtii . Molecular Genetics and Genomics 272 , 470 – 479 . Google Scholar CrossRef Search ADS PubMed Li L , Lu S , Cosman KM , Earle ED , Garvin DF , O’Neill J . 2006 . β-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis . Phytochemistry 67 , 1177 – 1184 . Google Scholar CrossRef Search ADS PubMed Li L , Van Eck J . 2007 . Metabolic engineering of carotenoid accumulation by creating a metabolic sink . Transgenic Research 16 , 581 – 585 . Google Scholar CrossRef Search ADS PubMed Li L , Yang Y , Xu Q , et al. 2012 . The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers . Molecular Plant 5 , 339 – 352 . Google Scholar CrossRef Search ADS PubMed Li L , Yuan H . 2013 . Chromoplast biogenesis and carotenoid accumulation . Archives of Biochemistry and Biophysics 539 , 102 – 109 . Google Scholar CrossRef Search ADS PubMed Lindgren LO , Stålberg KG , Höglund AS . 2003 . Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid . Plant Physiology 132 , 779 – 785 . Google Scholar CrossRef Search ADS PubMed Lopez AB , Van Eck J , Conlin BJ , Paolillo DJ , O’Neill J , Li L . 2008 . Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers . Journal of Experimental Botany 59 , 213 – 223 . Google Scholar CrossRef Search ADS PubMed Lu S , Li L . 2008 . Carotenoid metabolism: biosynthesis, regulation, and beyond . Journal of Integrative Plant Biology 50 , 778 – 785 . Google Scholar CrossRef Search ADS PubMed Lu S , Van Eck J , Zhou X , et al. 2006 . The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation . The Plant Cell 18 , 3594 – 3605 . Google Scholar CrossRef Search ADS PubMed Men X , Sun T , Dong K , Yang Y . 2013 . Or mutation leads to photo-oxidative stress responses in cauliflower (Brassica oleracea) seedlings during de-etiolation . Journal of Plant Research 126 , 823 – 832 . Google Scholar CrossRef Search ADS PubMed Morikawa T , Uraguchi Y , Sanda S , Nakagawa S , Sawayama S . 2017 . Overexpression of DnaJ-like chaperone enhances carotenoid synthesis in Chlamydomonas reinhardtii . Applied Biochemistry and Biotechnology 184 : 80 – 91 . Google Scholar CrossRef Search ADS PubMed Naqvi S , Zhu C , Farre G , et al. 2009 . Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways . Proceedings of the National Academy of Sciences, USA 106 , 7762 – 7767 . Google Scholar CrossRef Search ADS Niyogi KK . 1999 . Photoprotection revisited: genetic and molecular approaches . Annual Review of Plant Physiology and Plant Molecular Biology 50 , 333 – 359 . Google Scholar CrossRef Search ADS PubMed Paine JA , Shipton CA , Chaggar S , et al. 2005 . Improving the nutritional value of Golden Rice through increased pro-vitamin A content . Nature Biotechnology 23 , 482 – 487 . Google Scholar CrossRef Search ADS PubMed Park S , Kim HS , Jung YJ , et al. 2016 . Orange protein has a role in phytoene synthase stabilization in sweetpotato . Scientific Reports 6 , 33563 . Google Scholar CrossRef Search ADS PubMed Park SC , Kim SH , Park S , et al. 2015 . Enhanced accumulation of carotenoids in sweetpotato plants overexpressing IbOr-Ins gene in purple-fleshed sweetpotato cultivar . Plant Physiology and Biochemistry 86 , 82 – 90 . Google Scholar CrossRef Search ADS PubMed Rao AV , Rao LG . 2007 . Carotenoids and human health . Pharmacological Research 55 , 207 – 216 . Google Scholar CrossRef Search ADS PubMed Seo M , Koshiba T . 2002 . Complex regulation of ABA biosynthesis in plants . Trends in Plant Science 7 , 41 – 48 . Google Scholar CrossRef Search ADS PubMed Shewmaker CK , Sheehy JA , Daley M , Colburn S , Ke DY . 1999 . Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects . The Plant Journal 20 , 401 – 412 . Google Scholar CrossRef Search ADS PubMed Strzałka K , Kostecka-Gugała A , Latowski D . 2003 . Carotenoids and environmental stress in plants: significance of carotenoid-mediated modulation of membrane physical properties . Russian Journal of Plant Physiology 50 , 168 – 172 . Google Scholar CrossRef Search ADS Swamy PM , Smith B . 1999 . Role of abscisic acid in plant stress tolerance . Current Science 76 , 1220 – 1227 . Tanaka Y , Ohmiya A . 2008 . Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways . Current Opinion in Biotechnology 19 , 190 – 197 . Google Scholar CrossRef Search ADS PubMed Tzuri G , Zhou X , Chayut N , et al. 2015 . A ‘golden’ SNP in CmOr governs the fruit flesh color of melon (Cucumis melo) . The Plant Journal 82 , 267 – 279 . Google Scholar CrossRef Search ADS PubMed Walter MH , Strack D . 2011 . Carotenoids and their cleavage products: biosynthesis and functions . Natural Product Reports 28 , 663 – 692 . Google Scholar CrossRef Search ADS PubMed Wang W , Vinocur B , Shoseyov O , Altman A . 2004 . Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response . Trends in Plant Science 9 , 244 – 252 . Google Scholar CrossRef Search ADS PubMed Wang Z , Ke Q , Kim MD , et al. 2015 . Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced abiotic stress tolerance . PLoS One 10 , e0126050 . Google Scholar CrossRef Search ADS PubMed Welsch R , Arango J , Bär C , et al. 2010 . Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene . The Plant Cell 22 , 3348 – 3356 . Google Scholar CrossRef Search ADS PubMed Wu J , Ji J , Wang G , Wu G , Diao J , Li Z , Chen X , Chen Y , Luo L . 2015 . Ectopic expression of the Lycium barbarum β-carotene hydroxylase gene (chyb) enhances drought and salt stress resistance by increasing xanthophyll cycle pool in tobacco . Plant Cell, Tissue and Organ Culture 121 , 559 – 569 . Google Scholar CrossRef Search ADS Yamamoto HY , Nakayama TO , Chichester CO . 1962 . Studies on the light and dark interconversions of leaf xanthophylls . Archives of Biochemistry and Biophysics 97 , 168 – 173 . Google Scholar CrossRef Search ADS PubMed Yan J , Kandianis CB , Harjes CE , et al. 2010 . Rare genetic variation at Zea mays crtRB1 increases beta-carotene in maize grain . Nature Genetics 42 , 322 – 327 . Google Scholar CrossRef Search ADS PubMed Ye X , Al-Babili S , Klöti A , Zhang J , Lucca P , Beyer P , Potrykus I . 2000 . Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm . Science 287 , 303 – 305 . Google Scholar CrossRef Search ADS PubMed Yuan H , Owsiany K , Sheeja TE , et al. 2015 . A single amino acid substitution in an ORANGE protein promotes carotenoid overaccumulation in Arabidopsis . Plant Physiology 169 , 421 – 431 . Google Scholar CrossRef Search ADS PubMed Zhou X , McQuinn R , Fei Z , Wolters AA , VAN Eck J , Brown C , Giovannoni JJ , Li . 2011a . Regulatory control of high levels of carotenoid accumulation in potato tubers . Plant, Cell & Environment 34 , 1020 – 1030 . Google Scholar CrossRef Search ADS Zhou X , Sun TH , Wang N , Ling HQ , Lu S , Li L . 2011b . The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1 . New Phytologist 190 , 89 – 100 . Google Scholar CrossRef Search ADS Zhou X , Welsch R , Yang Y , et al. 2015 . Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis . Proceedings of the National Academy of Sciences, USA 112 , 3558 – 3563 . Google Scholar CrossRef Search ADS Zhu JK . 2002 . Salt and drought stress signal transduction in plants . Annual Review of Plant Biology 53 , 247 – 273 . Google Scholar CrossRef Search ADS PubMed Ziska LH , Runion GB , Tomecek M , Prior SA , Torbet HA , Sicher R . 2009 . An evaluation of cassava, sweet potato and field corn as potential carbohydrate sources for bioethanol production in Alabama and Maryland . Biomass & Bioenergy 33 , 1503 – 1508 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Orange: a target gene for regulating carotenoid homeostasis and increasing plant tolerance to environmental stress in marginal lands

Loading next page...
 
/lp/ou_press/orange-a-target-gene-for-regulating-carotenoid-homeostasis-and-Xyp8JaI0x4
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com
ISSN
0022-0957
eISSN
1460-2431
D.O.I.
10.1093/jxb/ery023
Publisher site
See Article on Publisher Site

Abstract

Abstract Carotenoids play essential roles in various light-harvesting processes in plants and help protect the photosynthetic machinery from photo-oxidative damage. Orange genes, which play a role in carotenoid accumulation, have recently been isolated from several plant species, and their functions have been intensively investigated. The Orange gene (IbOr) of sweet potato [Ipomoea batatas (L.) Lam] helps maintain carotenoid homeostasis to improve plant tolerance to environmental stress. IbOr, a protein with strong holdase chaperone activity, directly interacts with phytoene synthase, a key enzyme involved in carotenoid biosynthesis, in plants under stress conditions, resulting in increased carotenoid accumulation and abiotic stress tolerance. In addition, IbOr interacts with the oxygen-evolving enhancer protein 2-1, a member of a protein complex in photosystem II that is denatured under heat stress. Transgenic sweet potato plants overexpressing IbOr showed enhanced tolerance to high temperatures (47 °C). These findings indicate that IbOr protects plants from environmental stress not only by controlling carotenoid biosynthesis, but also by directly stabilizing photosystem II. In this review, we discuss the functions of IbOr and Or proteins in other plant species and their possible biotechnological applications for molecular breeding for sustainable development on marginal lands. Chaperone activity, heat stress, IbOr, IbPsbP, IbPSY, orange, oxidative stress, sweet potato Introduction The dramatic increase in the global population, combined with rapid industrialization in developing countries, has placed great strain on global food and energy supplies. The Food and Agriculture Organization of the United Nations (FAO) has estimated that the world population will exceed 9.1 billion in 2050 (FAO, 2009). If we use energy and food at the present rate, we will need more than 3.5 times the current energy supply and 1.7 times the current food supply in 2050. To cope with these global crises in food and energy supply, as well as environmental problems, the development of new, environmentally friendly industrial crop varieties for growth on marginal lands, including areas affected by desertification, is urgently needed to ensure sustainable development. Plant biotechnology can be used as a tool to maximize plant productivity by introducing stress-tolerance genes and genes whose products are responsible for increasing various metabolic activities in plants. Sweet potato [Ipomoea batatas (L.) Lam] is an attractive crop that could be used to help solve the world’s food and environmental problems in the 21st century. This crop species can be used as an industrial bioreactor to produce various high-value-added materials—including bio-ethanol, functional feed, and antioxidants—via molecular breeding approaches. Sweet potato plants have high water-use efficiency among the starch crops, and help reduce soil erosion. All parts of the sweet potato plant can be used for human and animal consumption. Owing to its rich nutritional content, combined with its wide adaptability to marginal lands ranging from tropical to temperate zones, sweet potato has great potential for preventing malnutrition and increasing food security in developing countries. The non-profit Center for Science in the Public Interest (CSPI) described sweet potato as one of 10 ‘superfoods’ for better health, because it contains high levels of low-molecular-weight antioxidants such as carotenoids and vitamin C, as well as dietary fiber and potassium (CPSI, 2016). Carotenoids benefit human health by acting as dietary antioxidants and helping prevent or slow the development of aging-related diseases. Carotenoids also serve as a dietary source of provitamin A, making them essential components of the human diet, since humans are unable to synthesize vitamin A. When carotenoids are ingested, they are converted to the visual pigment rhodopsin and to retinal, a precursor of retinoid acid, which regulates growth, development, and differentiation (Fraser and Bramley, 2004). Vitamin A deficiency causes night blindness, skin keratinization, dry eye syndrome, degenerative vision loss, impaired immune function, and birth defects (Rao and Rao, 2007). According to a United States Department of Agriculture (USDA) report, sweet potato can yield two to three times the level of carbohydrates as does field corn, approaching the amount that sugarcane can produce in the USA (Ziska et al., 2009). It would be worthwhile to begin pilot programs to investigate the feasibility of growing sweet potato for ethanol production on marginal lands. In addition, rational metabolic engineering of low-molecular-weight antioxidants should contribute to the development of new sweet potato cultivars with higher levels of nutritional antioxidants and abiotic stress tolerance. Carotenoids in plants Carotenoids are highly important molecules for plant growth and human health. These compounds are widespread in all photosynthetic organisms and in some non-photosynthetic bacteria and fungi. Carotenoids include carotenes and their oxidized form, xanthophylls. Carotenoids function as light-harvesting pigments and photoprotectants during photosynthesis (Niyogi, 1999; Domonkos et al., 2013). In addition, they protect plants from oxidative stress caused by excessive light by absorbing the blue-green wavelengths of light (Ledford et al., 2004). Carotenoids also serve as precursors for the biosynthesis of phytohormones, including abscisic acid (ABA) and strigolactones, and for the production of flower and fruit flavor and aroma compounds (Auldridge et al., 2006; Walter and Strack, 2011). Carotenoids can be synthesized in the membranes of nearly all plastids in the plant, except proplastids. These compounds primarily accumulate in the chloroplast and chromoplast (Howitt and Pogson 2006). Carotenoids in the chloroplast form a pigment–protein complex in the photosynthetic membrane along with chlorophyll-binding proteins (Farré et al., 2010). By contrast, carotenoids in the chromoplast form a carotenoid–lipoprotein-sequestering structure by combining with polar lipids and carotenoid-associated proteins. Consequently, these organelles maintain high levels of carotenoids (Lu and Li, 2008). Carotenoids act as strong antioxidants in plants, thereby protecting them from damage caused by various environmental stresses, including strong light, high temperature, UV, and drought (Davison et al., 2002; Götz et al., 2002; Wu et al., 2015). In addition, carotenoids directly scavenge reactive oxygen species in biological membranes (Strzałka et al., 2003). Davison et al. (2002) reported that overexpression of β-carotene hydroxylase increased stress tolerance in Arabidopsis thaliana. The authors proposed that the enhanced stress tolerance was due to the increased size of the xanthophyll cycle pool and the reversible interconversion of two carotenoids, violaxanthin and zeaxanthin, which play key photoprotective roles in plants. Overexpression of the bacterial β-carotene hydroxylase gene (crtZ), which is involved in the conversion step of β-carotene and β-cryptoxanthin to zeaxanthin, significantly enhanced UV tolerance in tobacco by increasing its zeaxanthin content (Götz et al., 2002). Silencing of the β-carotene hydroxylase gene (CHY-β) significantly increased tolerance to salt-mediated oxidative stress, as well as β-carotene levels, in transgenic sweet potato calli (Kim et al., 2012). Moreover, silencing of the gene encoding lycopene ε-cyclase (LCY-ε), which is involved in the first step of the α-branch synthesis pathway of carotenoids from lycopene, resulted in a 21-fold increase in β-carotene contents in sweet potato calli and significantly increased tolerance to salt-mediated oxidative stress (Kim et al., 2013a). Recently, Kang et al. (2017a) reported that RNAi-IbCHY-β sweet potato plants exhibited increased tolerance to salt stress with enhanced 9-cis-epoxycarotenoid dioxygenase (NCED) expression and ABA contents. The NCED gene is a key enzyme of ABA biosynthesis (Seo and Koshiba, 2002). In addition, ABA is an important phytohormone and plays a critical role in response to various stress signals (Swamy and Smith, 1999). Increased ABA levels have been shown to enhance adaptation to salt stress (Zhu, 2002). These results suggest that down-regulation of IbCHY-β in sweet potato not only increases the total carotenoid and β-carotene contents but also enhances salt-stress tolerance via increased ABA levels. However, the exact mechanism underlying how increased carotenoid contents lead to increased stress tolerance in plants remains largely unknown. Orange genes in plants The Orange (Or) genes appear to be plant specific, with homologs present in all plant species examined and in algae (see Supplementary Table S1 at JXB online). The Or gene encodes a DnaJ Cys-rich zinc finger domain-containing protein (Lu et al., 2006). The amino acid sequences of Or proteins are highly conserved among diverse plant species (Lu et al., 2006), suggesting that they play an important role in plant growth and development. However, to date, Or proteins have been functionally characterized in only a few plant species, including cauliflower, Arabidopsis, melon, sorghum, and sweet potato (Table 1). Table 1. Biological functions of homologous Or proteins from various plant species Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b View Large Table 1. Biological functions of homologous Or proteins from various plant species Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b Species Biological function Reference Arabidopsis (Arabidopsis thaliana) Carotenoid accumulation in rice callus and plants Bai et al., 2014; Bai et al., 2016 Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Regulators of active PSY protein Zhou et al., 2015 Carotenoid accumulation in corn Berman et al., 2017 Cauliflower (Brassica oleracea) Carotenoid accumulation in cauliflower Lu et al., 2006 Carotenoid accumulation and chromoplast formation in potato tubers Lopez et al., 2008 Petiole elongation Zhou et al., 2011b Postharvest storage in potato Li et al., 2012 Photo-oxidative responses Men et al., 2013 Melon (Cucumis melo) Carotenoid accumulation Tzuri et al., 2015 Sorghum (Sorghum bicolor) Carotenoid accumulation in Arabidopsis callus Yuan et al., 2015 Sweet potato (Ipomoea batatas) Carotenoid accumulation and salt stress tolerance in sweet potato callus Kim et al., 2013b Carotenoid accumulation in sweet potato storage roots Park et al., 2015 Carotenoid accumulation and abiotic stress tolerance in alfalfa Wang et al., 2015 Carotenoid accumulation and abiotic stress tolerance in potato Goo et al., 2015, Cho et al., 2016 Stabilization of PSY protein Park et al., 2016 Regulation of photosynthesis Kang et al., 2017b View Large The Or gene was first discovered in an orange curd cauliflower (Brassica oleracea var. botrytis) mutant, where it was shown to enhance β-carotene accumulation (Lu et al., 2006). The expression of BoOr leads to the production of orange tissues with enhanced carotenoid contents in both white cauliflower and potato tubers (Lu et al., 2006; Lopez et al., 2008). The increased carotenoid levels in plants overexpressing BoOr are associated with the biogenesis of chromoplasts, which serve as a metabolic sink for carotenoid storage in non-photosynthetic tissues (Lopez et al., 2008; Li et al., 2012). The BoOr-induced accumulation of β-carotene in these tissues is not due to an increased capacity of the carotenoid biosynthetic pathway (Li et al., 2006). In addition to carotenoid accumulation, BoOr protein is also involved in plant growth and development. BoOr interacts with cauliflower eukaryotic release factor 1 (eRF1) protein and increases leaf petiole elongation by suppressing the expression of BoeRF1 family genes (Zhou et al., 2011b). In addition, the expression of BoOr increases the stability of potato tubers during post-harvest storage by stimulating continuous accumulation of carotenoids (Li et al., 2012). In Arabidopsis, AtOr proteins interact directly with, and post-transcriptionally regulate, phytoene synthase (PSY) to control carotenoid biosynthesis (Zhou et al., 2015). AtOr is localized to plastids (Zhou et al., 2015). The expression of AtOr under the control of the endosperm-specific wheat low-molecular-weight glutenin promoter resulted in high carotenoid contents in white corn (Berman et al., 2017). The expression of AtOr also induced carotenoid accumulation in rice calli and plants (Bai et al., 2014, 2016). The overexpression of an AtOr mutant protein resulted in high β-carotene levels in Arabidopsis (Yuan et al., 2015). In melon (Cucumis melo), the presence of CmOr protein with a single amino acid change from His to Arg distinguishes orange-fleshed melon from white- or green-fleshed melon (Tzuri et al., 2015). Unlike BoOr, which has a large retrotransposon insertion and causes a stunted phenotype, the effect of the CmOr allelic variation is limited to carotenoid accumulation in fruit. Despite the important correlation between carotenoid accumulation and environmental stress tolerance in plants, only a few Or genes have been isolated and partially characterized in terms of their effect on various abiotic stresses (Table 1). It is likely that all plant species, including photosynthetic algae, have Or genes, since carotenoids are essential for photosynthesis (Supplementary Table S1). The Or gene from the green alga Chlamydomonas reinhardtii was recently isolated and transformed into C. reinhardtii, which led to increased carotenoid biosynthesis (Morikawa et al., 2017). Functional analysis of the Or gene from sweet potato In sweet potato, IbOr was initially isolated from an orange-fleshed cultivar (cv. Sinhwangmi) based on the sequence of BoOr (Kim et al., 2013b). IbOr is expressed at high levels in the leaves of sweet potatoes with various flesh colors (white, orange, and purple), but is highly expressed in storage roots only in orange-fleshed varieties. Two variants of IbOr (encoding IbOr-Wt and IbOr-Ins, which contains seven additional amino acids [KSPNPNL] inserted between residues 131 and 142 of IbOr-Wt) were transformed into non-embryogenic calli from white-fleshed sweet potato (cv. Yulmi). The average total carotenoid contents in IbOr-Ins and IbOr-Wt transgenic calli were approximately 13- and 4-fold higher, respectively, than those in control calli (Kim et al., 2013b). The levels of all carotenoids were higher in both IbOr-Wt and IbOr-Ins transgenic calli relative to the control, except for lutein in IbOr-Wt. IbOr-Ins was subsequently transformed into purple-fleshed sweet potato, leading to the production of anthocyanin and carotenoids in the same storage root (Park et al., 2015). IbOr transgenic sweet potatoes exhibit different color densities in individual transgenic lines. IbOr-201 plants are a darker purple than control plants, and IbOr plants have higher carotenoid levels (up to 7-fold) in their storage roots than control plants. Overall, the carotenoid levels in IbOr plants are positively correlated with IbOr transcript levels. Or proteins contain a Cys-rich zinc finger domain that is highly specific to DnaJ chaperone proteins. DnaJ proteins participate in essential cellular processes such as protein folding, assembly, degradation, and homeostasis under stress conditions (Wang et al., 2004; Hennessy et al., 2005). IbOr also contains a DnaJ domain and has high chaperone activity (Park et al., 2016). Among the carotenoid biosynthetic enzymes, PSY is the most important regulatory enzyme in the carotenoid biosynthesis pathway. Similar to AtOr, IbOr directly interacts with IbPSY in the chloroplast. In addition, IbPSY is protected by IbOr chaperone activity under heat and oxidative stress conditions (Park et al., 2016). Transgenic Arabidopsis plants overexpressing IbOr displayed enhanced heat-stress tolerance (Park et al., 2016). Therefore, the holdase chaperone function of IbOr is involved in carotenoid biosynthesis, as well as in environmental stress tolerance in plants, by protecting IbPSY protein (Fig. 1A). Fig. 1. View largeDownload slide Proposed model for the role of sweet potato Orange protein (IbOr) in environmental stress tolerance. (A) Stabilization of carotenoid biosynthesis-related enzymes. IbOr interacts with phytoene synthase (PSY) and carotenoid cleavage dioxygenase (CCD) 4. IbOr-mediated protection of PSY leads to increased carotenoid accumulation and stress tolerance. ABA, abscisic acid; CHY-β, β-carotene hydroxylase; CHY-ε, ε-ring hydroxylase; CRTISO, carotenoid isomerase; GGPP, geranylgeranyl pyrophosphate; LCY-β, lycopene β-cyclase; LCY-ε, lycopene ε-cyclase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; VDE, violaxanthin de-epoxidase; ZDS, f-carotene desaturase; ZEP, zeaxanthin epoxidase. (B) Stabilization of the photosynthetic machinery. IbOr-mediated protection of PsbP increases tolerance to heat stress. Fig. 1. View largeDownload slide Proposed model for the role of sweet potato Orange protein (IbOr) in environmental stress tolerance. (A) Stabilization of carotenoid biosynthesis-related enzymes. IbOr interacts with phytoene synthase (PSY) and carotenoid cleavage dioxygenase (CCD) 4. IbOr-mediated protection of PSY leads to increased carotenoid accumulation and stress tolerance. ABA, abscisic acid; CHY-β, β-carotene hydroxylase; CHY-ε, ε-ring hydroxylase; CRTISO, carotenoid isomerase; GGPP, geranylgeranyl pyrophosphate; LCY-β, lycopene β-cyclase; LCY-ε, lycopene ε-cyclase; NCED, 9-cis-epoxycarotenoid dioxygenase; NXS, neoxanthin synthase; PDS, phytoene desaturase; VDE, violaxanthin de-epoxidase; ZDS, f-carotene desaturase; ZEP, zeaxanthin epoxidase. (B) Stabilization of the photosynthetic machinery. IbOr-mediated protection of PsbP increases tolerance to heat stress. The metabolic turnover of carotenoids not only helps maintain steady levels of carotenoids in plants, but also produces important signaling and accessory apocarotenoid molecules, such as the phytohormones ABA and strigolactones (Cunningham and Gantt, 1988; Giuliano, 2014). Carotenoid cleavage dioxygenases (CCDs) generate apocarotenoids via oxidative cleavage of carotenoids (Walter and Strack, 2011). Five members of the Arabidopsis NCED family, another class of CCDs, have been implicated in ABA biosynthesis. Moreover, strigolactones, which control auxiliary branching and tillering, are synthesized by the enzymes CCD7 and CCD8 (Auldridge et al., 2006). In addition, CCD1 and CCD4 contribute to the production of apocarotenoid-derived pigments, as well as flavor and/or aroma compounds in flowers and a variety of foods, through the degradation of carotenoids (Auldridge et al., 2006; Gonzalez-Jorge et al., 2013). The loss of CCD1 or CCD4 activity results in a significant increase in carotenoid levels, indicating that both enzymes are negatively correlated with carotenoid accumulation in plants (García-Limones et al., 2008; Tanaka and Ohmiya, 2008; Campbell et al., 2010; Zhou et al., 2011a; Gonzalez-Jorge et al., 2013). Interestingly, we found that NCED, CCD1, and CCD4 were highly expressed in IbOr-overexpressing sweet potato (Park et al., 2015) and that IbOr specifically interacts with CCD4 (H.S. Kim, S.C. Park, S.S. Kwak, unpublished data). Thus, IbOr can interact not only with the carotenoid biosynthesis enzyme PSY, but also with the carotenoid degradation enzyme CCD4, suggesting that IbOr plays an important role in maintaining carotenoid homeostasis (Fig. 1A). It remains unclear whether the holdase activity of IbOr positively or negatively regulates CCD4 activity. Elucidation of the specific mechanism underlying IbOr-regulated CCD activity in the future could shed light on the regulation of carotenoid homeostasis in plants. IbOr protein normally localizes to the nucleus and chloroplasts, but it translocates from the nucleus to the chloroplasts in response to heat stress (Park et al., 2016). Furthermore, recent work has shown that transgenic sweet potato calli, Arabidopsis, alfalfa, and potato overexpressing IbOr maintain higher photosystem II (PSII) efficiency and chlorophyll content under abiotic stress compared with the wild type plants (Kim et al., 2013b; Park et al., 2015, 2016; Wang et al., 2015; Cho et al., 2016). These findings suggest that Or is a multifunctional protein that helps regulate photosynthesis. Kang et al. (2017b) recently identified proteins that are differentially expressed in response to heat stress in transgenic Arabidopsis plants overexpressing IbOr. Among these are several proteins involved in the light-dependent reaction and the Calvin cycle, suggesting that IbOr might be directly involved in regulating photosynthesis. Interestingly, IbOr interacts with oxygen-evolving enhancer protein 2-1 (PsbP), an extrinsic protein of the oxygen-evolving complex of PSII, and the holdase chaperone function of IbOr can protect PsbP from heat-induced denaturation (Kang et al., 2017b). Therefore, IbOr is a multifunctional protein that has tremendous potential for increasing carotenoid accumulation and protecting the photosynthetic machinery in plants (Fig. 1B). More studies are needed to investigate the Or genes from various plant species and non-photosynthetic organisms. Biotechnological applications of Or genes Carotenoids, especially β-carotene, are indispensable for human nutrition and provide the primary dietary source for vitamin A biosynthesis. The low levels of carotenoids in major food crops contribute to the global prevalence of vitamin A deficiency. Significant efforts have been made to generate carotenoid-enriched food crops through either biotechnology or traditional breeding strategies. By altering the levels of expression of genes encoding key enzymes involved in carotenoid biosynthesis or several enzymes in carotenoid biosynthesis mini-pathways, numerous transgenic crops with enhanced carotenoid levels have been produced (Fraser et al., 2002; Ducreux et al., 2005; Paine et al., 2005; Diretto et al., 2007a, b; Naqvi et al., 2009; Welsch et al., 2010). Overexpression of the PSY gene from bacteria or plants resulted in a significant increase in total carotenoid levels in tomato fruits (Fraser et al., 2002), potato tubers (Ducreux et al., 2005), and canola seeds (Shewmaker et al., 1999). The expression of multiple biosynthetic genes in the carotenoid biosynthetic pathway led to a profound increase in β-carotene levels in Golden Rice (Ye et al., 2000; Paine et al., 2005) and “golden” potato (Diretto et al., 2007a, b). The selection of favorable alleles that alter carotenoid metabolic flux toward β-carotene resulted in the breeding of orange maize with high β-carotene contents (Harjes et al., 2008; Yan et al., 2010). However, in some cases, the increased flux into carotenogenesis can alter or reduce flux in other competing pathways, leading to unexpected phenotypic changes. For example, the overexpression of PSY in canola and Arabidopsis seeds led not only to high levels of carotenoid accumulation, but also to delayed germination due to increased production of carotenoid-derived ABA (Shewmaker et al., 1999; Lindgren et al., 2003). The constitutive expression of PSY-1 in tomato resulted in stunted growth due to insufficient biosynthesis of gibberellins, along with pleiotropic effects such as premature pigmentation of seed coats and cotyledons (Fray et al., 1995). Thus, manipulating the formation of deposition sinks offers a new strategy for metabolic engineering of carotenoid contents in the storage tissues of various food crops. In contrast to modifying the catalytic activity of carotenoid pathway enzymes, the discovery of Or genes provides an alternative, complementary approach for increasing carotenoid levels in food crops by enhancing sink strength in storage tissues (Li and Van Eck, 2007). Overexpression of the BoOr transgene in potato tubers resulted in the production of orange tissues with enhanced carotenoid contents (Lopez et al., 2008). Moreover, the presence of Or protein promoted continuously increasing carotenoid accumulation in BoOr transgenic potato tubers during post-harvest storage (Li et al., 2012). The expression of AtOr also promoted carotenoid accumulation in transgenic corn (Berman et al., 2017). In addition to their role in increasing carotenoid accumulation, Or genes might be useful for generating transgenic plants with enhanced tolerance to environmental stress. We previously demonstrated that sweet potato plants overexpressing IbOr showed enhanced tolerance to both heat and oxidative stress (Park et al., 2015; Park et al., 2016; Kang et al., 2017b). These plants also showed enhanced drought tolerance (Supplementary Fig. S1). Further characterization of IbOr-overexpressing sweet potato plants in response to various abiotic and biotic stresses is currently under way. In addition, transgenic alfalfa and potato plants overexpressing IbOr exhibited increased tolerance to various abiotic stresses, including oxidative, salt, and drought stress, as well as increased carotenoid contents (Goo et al., 2015; Wang et al., 2015; Cho et al., 2016). Therefore, it is likely that IbOr plays a crucial role in the maintenance of photosynthesis, which confers stress tolerance to plants. As mentioned above, a single-nucleotide polymorphism (SNP) in Or underlies the differences in carotenoid contents in orange-fleshed versus green/white-fleshed melon (Cucumis melo) fruits, and is responsible for the non-orange and orange melon fruit phenotypes (Tzuri et al., 2015). Overexpression of the Arabidopsis Or protein mutagenized at the site corresponding to the melon golden SNP (Arg to His) resulted in high β-carotene levels (Yuan et al., 2015). In a preliminary experiment (H.S. Kim, S.E. Kim, S.S. Kwak, unpublished data), we found that a single amino acid substitution in IbOr protein also significantly increased carotenoid contents in sweet potato calli compared with calli harboring the original IbOr protein (Kim et al., 2013b). Transgenic sweet potato and rice plants overexpressing the IbOr-His gene are currently being generated in our laboratory. Or represents a unique class of regulatory genes that mediate carotenoid accumulation (Li and Yuan, 2013). Two natural mutations in Or promote high levels of β-carotene accumulation in plants. The first mutation is BoOr from an orange curd cauliflower mutant. BoOr contains a large retrotransposon insertion and produces three alternatively spliced transcripts, none of which alone induces massive accumulation of carotenoids (Lu et al., 2006). The pleiotropic effects of BoOr might limit its potential application in nutritional improvement of crops. The second mutation is the recently discovered CmOr gene in melon, mentioned above. CmOr protein, with a single amino acid difference (His or Arg), distinguishes orange-fleshed melon from white- or green-fleshed melon (Tzuri et al., 2015). The expression of CmOr-His suppresses the downstream metabolism of β-carotene in melon fruits; in mature CmOr-His melon fruits, only traces of lutein could be found, while other xanthophylls were undetectable (Chayut et al., 2015; Chayut et al., 2017). However, overexpression of IbOr-His in sweet potato calli induced strong accumulation of β-carotene, together with significant amounts of xanthophylls, including lutein, β-cryptoxanthin, zeaxanthin, and violaxanthin. The xanthophyll cycle (the reversible interconversion of two carotenoids, violaxanthin and zeaxanthin) plays a key photoprotective role in plants and is therefore a promising target for genetic engineering to increase stress tolerance (Yamamoto et al., 1962; Demmig-Adams and Adams, 1992; Hauvaux and Niyogi, 1999; Davison et al., 2002). Manipulation of the IbOr gene represents a promising strategy for developing crop varieties with increased tolerance to heat, salinity, and other environmental stresses, in addition to improved nutritional qualities brought about by increasing the carotenoid contents through enhancing sink strength. Furthermore, site-specific mutagenesis of IbOr combined with CRISPR-Cas9-mediated genome editing techniques could lead to significant nutritional biofortification of sweet potato. Conclusions The Or genes help maintain carotenoid homeostasis and help plants adapt to environmental stress, not only by controlling carotenoid biosynthesis, but also by directly stabilizing PSII under stress conditions (Fig. 1). Greater understanding of the Or genes will be needed in order to comprehensively elucidate the regulatory mechanisms of carotenoid accumulation under stress conditions, although the functions of these genes are already partially understood. Or genes represent a novel, useful resource for molecular breeding to increase nutritional carotenoid contents and abiotic stress tolerance in various crops, including sweet potato, potato, and rice. We anticipate that Or genes will contribute to the production of plants that function as efficient industrial bioreactors. These plants could be used to help us cope with the effects of climate change, through allowing sustainable agriculture on global marginal lands, as well as to alleviate vitamin A deficiency diseases in sub-Saharan Africa and south-east Asia. Supplementary data Supplementary data are available at JXB online. Fig. S1. Enhanced tolerance to drought stress in IbOr-overexpressing transgenic sweet potato. Table S1. List of homologous Or proteins from various plant species and algae. Abbreviations: Abbreviations: ABA Abscisic acid CCD carotenoid cleavage dioxygenases Or orange PsbP oxygen-evolving enhancer protein 2-1 PSY phytoene synthase. Acknowledgements This work was supported by grants from the Systems & Synthetic Agrobiotech Center (grant no. PJ01318401), the Next Generation BioGreen 21 Project, Rural Development Administration, Korea, the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (grant no. 2015053321), and the KRIBB Initiative Program. References Auldridge ME , McCarty DR , Klee HJ . 2006 . Plant carotenoid cleavage oxygenases and their apocarotenoid products . Current Opinion in Plant Biology 9 , 315 – 321 . Google Scholar CrossRef Search ADS PubMed Bai C , Capell T , Berman J , et al. 2016 . Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor-product balance . Plant Biotechnology Journal 14 , 195 – 205 . Google Scholar CrossRef Search ADS PubMed Bai C , Rivera SM , Medina V , et al. 2014 . An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation . Plant Journal 77 , 464 – 475 . Google Scholar CrossRef Search ADS PubMed Berman J , Zorrilla-López U , Medina V , Farré G , Sandmann G , Capell T , Christou P , Zhu C . 2017 . The Arabidopsis ORANGE (AtOR) gene promotes carotenoid accumulation in transgenic corn hybrids derived from parental lines with limited carotenoid pools . Plant Cell Reports 36 , 933 – 945 . Google Scholar CrossRef Search ADS PubMed Campbell R , Ducreux LJ , Morris WL , Morris JA , Suttle JC , Ramsay G , Bryan GJ , Hedley PE , Taylor MA . 2010 . The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato . Plant Physiology 154 , 656 – 664 . Google Scholar CrossRef Search ADS PubMed Center for Science in the Public Interest . 2016 . What to eat: 10 best foods . Retrieved from https://www.nutritionaction.com/wp-content/free-downloads/What_To_Eat_com-we-1.pdf/ Chayut N , Yuan H , Ohali S , et al. 2015 . A bulk segregant transcriptome analysis reveals metabolic and cellular processes associated with Orange allelic variation and fruit β-carotene accumulation in melon fruit . BMC Plant Biology 15 , 274 . Google Scholar CrossRef Search ADS PubMed Chayut N , Yuan H , Ohali S , et al. 2017 . Distinct mechanisms of the ORANGE protein in controlling carotenoid flux . Plant Physiology 173 , 376 – 389 . Google Scholar CrossRef Search ADS PubMed Cho KS , Han EH , Kwak SS , Cho JH , Im JS , Hong SY , Sohn HB , Kim YH , Lee SW . 2016 . Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production . Comptes Rendus Biologies 339 , 207 – 213 . Google Scholar CrossRef Search ADS PubMed Cunningham FX , Gantt E . 1998 . Genes and enzymes of carotenoid biosynthesis in plants . Annual Review of Plant Physiology and Plant Molecular Biology 49 , 557 – 583 . Google Scholar CrossRef Search ADS PubMed Davison PA , Hunter CN , Horton P . 2002 . Overexpression of beta-carotene hydroxylase enhances stress tolerance in Arabidopsis . Nature 418 , 203 – 206 . Google Scholar CrossRef Search ADS PubMed Demmig-Adams B , Adams WW . 1992 . Photoprotection and other responses of plants to high light stress . Annual Review of Plant Physiology and Plant Molecular Biology 43 , 599 – 626 . Google Scholar CrossRef Search ADS Diretto G , Al-Babili S , Tavazza R , Papacchioli V , Beyer P , Giuliano G . 2007a. Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacterial mini-pathway . PLoS One 2 , e350 . Google Scholar CrossRef Search ADS PubMed Diretto G , Welsch R , Tavazza R , Mourgues F , Pizzichini D , Beyer P , Giuliano G . 2007b. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers . BMC Plant Biology 7 , 11 . Google Scholar CrossRef Search ADS PubMed Domonkos I , Kis M , Gombos Z , Ughy B . 2013 . Carotenoids, versatile components of oxygenic photosynthesis . Progress in Lipid Research 52 , 539 – 561 . Google Scholar CrossRef Search ADS PubMed Ducreux LJ , Morris WL , Hedley PE , Shepherd T , Davies HV , Millam S , Taylor MA . 2005 . Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein . Journal of Experimental Botany 56 , 81 – 89 . Google Scholar PubMed FAO . 2009 . How to feed the world in 2050 . Retrieved from http://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/How_to_Feed_the_World_in_2050.pdf Farré G , Sanahuja G , Naqvi S , Bai C , Capell T , Zhu C , Christou P . 2010 . Travel advice on the road to carotenoids in plants . Plant Science 179 , 28 – 48 . Google Scholar CrossRef Search ADS Fraser PD , Bramley PM . 2004 . The biosynthesis and nutritional uses of carotenoids . Progress in Lipid Research 43 , 228 – 265 . Google Scholar CrossRef Search ADS PubMed Fraser PD , Romer S , Shipton CA , Mills PB , Kiano JW , Misawa N , Drake RG , Schuch W , Bramley PM . 2002 . Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner . Proceedings of the National Academy of Sciences, USA 99 , 1092 – 1097 . Google Scholar CrossRef Search ADS Fray RG , Wallace A , Fraser PD , Valero D , Hedden P , Bramley PM , Grierson D . 1995 . Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway . The Plant Journal 8 , 693 – 701 . Google Scholar CrossRef Search ADS García-Limones C , Schnäbele K , Blanco-Portales R , Luz Bellido M , Caballero JL , Schwab W , Muñoz-Blanco J . 2008 . Functional characterization of FaCCD1: a carotenoid cleavage dioxygenase from strawberry involved in lutein degradation during fruit ripening . Journal of Agricultural and Food Chemistry 56 , 9277 – 9285 . Google Scholar CrossRef Search ADS PubMed Giuliano G . 2014 . Plant carotenoids: genomics meets multi-gene engineering . Current Opinion in Plant Biology 19 , 111 – 117 . Google Scholar CrossRef Search ADS PubMed Gonzalez-Jorge S , Ha SH , Magallanes-Lundback M , et al. 2013 . CAROTENOID CLEAVAGE DIOXYGENASE4 is a negative regulator of β-carotene content in Arabidopsis seeds . The Plant Cell 25 , 4812 – 4826 . Google Scholar CrossRef Search ADS PubMed Goo YM , Han EH , Jeong JC , Kwak SS , Yu J , Kim YH , Ahn MJ , Lee SW . 2015 . Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato . Comptes Rendus Biologies 338 , 12 – 20 . Google Scholar CrossRef Search ADS PubMed Götz T , Sandmann G , Römer S . 2002 . Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco . Plant Molecular Biology 50 , 129 – 142 . Google Scholar CrossRef Search ADS PubMed Harjes CE , Rocheford TR , Bai L , et al. 2008 . Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification . Science 319 , 330 – 333 . Google Scholar CrossRef Search ADS PubMed Hauvaux M , Niyogi KK . 1999 . The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism . Proceedings of the National Academy of Sciences, USA 96 , 8762 – 8767 . Google Scholar CrossRef Search ADS Hennessy F , Nicoll WS , Zimmermann R , Cheetham ME , Blatch GL . 2005 . Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions . Protein Science 14 , 1697 – 1709 . Google Scholar CrossRef Search ADS PubMed Howitt CA , Pogson BJ . 2006 . Carotenoids accumulation and function in seeds and non-green tissues . Plant, Cell & Environment 29 , 435 – 445 . Google Scholar CrossRef Search ADS PubMed Kang L , Ji CY , Kim SH , et al. 2017a. Suppression of the β-carotene hydroxylase gene increases β-carotene content and tolerance to abiotic stress in transgenic sweetpotato plants . Plant Physiology and Biochemistry 117 , 24 – 33 . Google Scholar CrossRef Search ADS PubMed Kang L , Kim HS , Kwon YS , Ke Q , Ji CY , Park SC , Lee HS , Deng X , Kwak SS . 2017b. IbOr regulates photosynthesis under heat stress by stabilizing IbPsbP in sweetpotato . Frontiers in Plant Science 8 , 989 . Google Scholar CrossRef Search ADS PubMed Kim SH , Ahn YO , Ahn MJ , Lee HS , Kwak SS . 2012 . Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato . Phytochemistry 74 , 69 – 78 . Google Scholar CrossRef Search ADS PubMed Kim SH , Kim YH , Ahn YO , Ahn MJ , Jeong JC , Lee HS , Kwak SS . 2013a . Downregulation of the lycopene ε-cyclase gene increases carotenoid synthesis via the β-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli . Physiologia Plantarum 147 , 432 – 442 . Google Scholar CrossRef Search ADS Kim SH , Ahn YO , Ahn MJ , Jeong JC , Lee HS , Kwak SS . 2013b . Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures . Plant Physiology and Biochemistry 70 , 445 – 454 . Google Scholar CrossRef Search ADS Ledford HK , Baroli I , Shin JW , Fischer BB , Eggen RI , Niyogi KK . 2004 . Comparative profiling of lipid-soluble antioxidants and transcripts reveals two phases of photo-oxidative stress in a xanthophyll-deficient mutant of Chlamydomonas reinhardtii . Molecular Genetics and Genomics 272 , 470 – 479 . Google Scholar CrossRef Search ADS PubMed Li L , Lu S , Cosman KM , Earle ED , Garvin DF , O’Neill J . 2006 . β-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis . Phytochemistry 67 , 1177 – 1184 . Google Scholar CrossRef Search ADS PubMed Li L , Van Eck J . 2007 . Metabolic engineering of carotenoid accumulation by creating a metabolic sink . Transgenic Research 16 , 581 – 585 . Google Scholar CrossRef Search ADS PubMed Li L , Yang Y , Xu Q , et al. 2012 . The Or gene enhances carotenoid accumulation and stability during post-harvest storage of potato tubers . Molecular Plant 5 , 339 – 352 . Google Scholar CrossRef Search ADS PubMed Li L , Yuan H . 2013 . Chromoplast biogenesis and carotenoid accumulation . Archives of Biochemistry and Biophysics 539 , 102 – 109 . Google Scholar CrossRef Search ADS PubMed Lindgren LO , Stålberg KG , Höglund AS . 2003 . Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid . Plant Physiology 132 , 779 – 785 . Google Scholar CrossRef Search ADS PubMed Lopez AB , Van Eck J , Conlin BJ , Paolillo DJ , O’Neill J , Li L . 2008 . Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers . Journal of Experimental Botany 59 , 213 – 223 . Google Scholar CrossRef Search ADS PubMed Lu S , Li L . 2008 . Carotenoid metabolism: biosynthesis, regulation, and beyond . Journal of Integrative Plant Biology 50 , 778 – 785 . Google Scholar CrossRef Search ADS PubMed Lu S , Van Eck J , Zhou X , et al. 2006 . The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation . The Plant Cell 18 , 3594 – 3605 . Google Scholar CrossRef Search ADS PubMed Men X , Sun T , Dong K , Yang Y . 2013 . Or mutation leads to photo-oxidative stress responses in cauliflower (Brassica oleracea) seedlings during de-etiolation . Journal of Plant Research 126 , 823 – 832 . Google Scholar CrossRef Search ADS PubMed Morikawa T , Uraguchi Y , Sanda S , Nakagawa S , Sawayama S . 2017 . Overexpression of DnaJ-like chaperone enhances carotenoid synthesis in Chlamydomonas reinhardtii . Applied Biochemistry and Biotechnology 184 : 80 – 91 . Google Scholar CrossRef Search ADS PubMed Naqvi S , Zhu C , Farre G , et al. 2009 . Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways . Proceedings of the National Academy of Sciences, USA 106 , 7762 – 7767 . Google Scholar CrossRef Search ADS Niyogi KK . 1999 . Photoprotection revisited: genetic and molecular approaches . Annual Review of Plant Physiology and Plant Molecular Biology 50 , 333 – 359 . Google Scholar CrossRef Search ADS PubMed Paine JA , Shipton CA , Chaggar S , et al. 2005 . Improving the nutritional value of Golden Rice through increased pro-vitamin A content . Nature Biotechnology 23 , 482 – 487 . Google Scholar CrossRef Search ADS PubMed Park S , Kim HS , Jung YJ , et al. 2016 . Orange protein has a role in phytoene synthase stabilization in sweetpotato . Scientific Reports 6 , 33563 . Google Scholar CrossRef Search ADS PubMed Park SC , Kim SH , Park S , et al. 2015 . Enhanced accumulation of carotenoids in sweetpotato plants overexpressing IbOr-Ins gene in purple-fleshed sweetpotato cultivar . Plant Physiology and Biochemistry 86 , 82 – 90 . Google Scholar CrossRef Search ADS PubMed Rao AV , Rao LG . 2007 . Carotenoids and human health . Pharmacological Research 55 , 207 – 216 . Google Scholar CrossRef Search ADS PubMed Seo M , Koshiba T . 2002 . Complex regulation of ABA biosynthesis in plants . Trends in Plant Science 7 , 41 – 48 . Google Scholar CrossRef Search ADS PubMed Shewmaker CK , Sheehy JA , Daley M , Colburn S , Ke DY . 1999 . Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects . The Plant Journal 20 , 401 – 412 . Google Scholar CrossRef Search ADS PubMed Strzałka K , Kostecka-Gugała A , Latowski D . 2003 . Carotenoids and environmental stress in plants: significance of carotenoid-mediated modulation of membrane physical properties . Russian Journal of Plant Physiology 50 , 168 – 172 . Google Scholar CrossRef Search ADS Swamy PM , Smith B . 1999 . Role of abscisic acid in plant stress tolerance . Current Science 76 , 1220 – 1227 . Tanaka Y , Ohmiya A . 2008 . Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways . Current Opinion in Biotechnology 19 , 190 – 197 . Google Scholar CrossRef Search ADS PubMed Tzuri G , Zhou X , Chayut N , et al. 2015 . A ‘golden’ SNP in CmOr governs the fruit flesh color of melon (Cucumis melo) . The Plant Journal 82 , 267 – 279 . Google Scholar CrossRef Search ADS PubMed Walter MH , Strack D . 2011 . Carotenoids and their cleavage products: biosynthesis and functions . Natural Product Reports 28 , 663 – 692 . Google Scholar CrossRef Search ADS PubMed Wang W , Vinocur B , Shoseyov O , Altman A . 2004 . Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response . Trends in Plant Science 9 , 244 – 252 . Google Scholar CrossRef Search ADS PubMed Wang Z , Ke Q , Kim MD , et al. 2015 . Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced abiotic stress tolerance . PLoS One 10 , e0126050 . Google Scholar CrossRef Search ADS PubMed Welsch R , Arango J , Bär C , et al. 2010 . Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene . The Plant Cell 22 , 3348 – 3356 . Google Scholar CrossRef Search ADS PubMed Wu J , Ji J , Wang G , Wu G , Diao J , Li Z , Chen X , Chen Y , Luo L . 2015 . Ectopic expression of the Lycium barbarum β-carotene hydroxylase gene (chyb) enhances drought and salt stress resistance by increasing xanthophyll cycle pool in tobacco . Plant Cell, Tissue and Organ Culture 121 , 559 – 569 . Google Scholar CrossRef Search ADS Yamamoto HY , Nakayama TO , Chichester CO . 1962 . Studies on the light and dark interconversions of leaf xanthophylls . Archives of Biochemistry and Biophysics 97 , 168 – 173 . Google Scholar CrossRef Search ADS PubMed Yan J , Kandianis CB , Harjes CE , et al. 2010 . Rare genetic variation at Zea mays crtRB1 increases beta-carotene in maize grain . Nature Genetics 42 , 322 – 327 . Google Scholar CrossRef Search ADS PubMed Ye X , Al-Babili S , Klöti A , Zhang J , Lucca P , Beyer P , Potrykus I . 2000 . Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm . Science 287 , 303 – 305 . Google Scholar CrossRef Search ADS PubMed Yuan H , Owsiany K , Sheeja TE , et al. 2015 . A single amino acid substitution in an ORANGE protein promotes carotenoid overaccumulation in Arabidopsis . Plant Physiology 169 , 421 – 431 . Google Scholar CrossRef Search ADS PubMed Zhou X , McQuinn R , Fei Z , Wolters AA , VAN Eck J , Brown C , Giovannoni JJ , Li . 2011a . Regulatory control of high levels of carotenoid accumulation in potato tubers . Plant, Cell & Environment 34 , 1020 – 1030 . Google Scholar CrossRef Search ADS Zhou X , Sun TH , Wang N , Ling HQ , Lu S , Li L . 2011b . The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1 . New Phytologist 190 , 89 – 100 . Google Scholar CrossRef Search ADS Zhou X , Welsch R , Yang Y , et al. 2015 . Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis . Proceedings of the National Academy of Sciences, USA 112 , 3558 – 3563 . Google Scholar CrossRef Search ADS Zhu JK . 2002 . Salt and drought stress signal transduction in plants . Annual Review of Plant Biology 53 , 247 – 273 . Google Scholar CrossRef Search ADS PubMed Ziska LH , Runion GB , Tomecek M , Prior SA , Torbet HA , Sicher R . 2009 . An evaluation of cassava, sweet potato and field corn as potential carbohydrate sources for bioethanol production in Alabama and Maryland . Biomass & Bioenergy 33 , 1503 – 1508 . Google Scholar CrossRef Search ADS © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Experimental BotanyOxford University Press

Published: Feb 7, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off