Are carotenoids the true colors of crop improvement?

Abstract

Carotenoids are isoprenoid compounds synthesized by all photosynthetic organisms and several heterotrophic microorganisms. In plants, carotenoids play key roles as photosynthetic pigments and structural components of the photosynthetic apparatus, reactive oxygen species (ROS) scavengers, and mediators of plant–animal interactions. Moreover, they are the precursors of bioactive apocarotenoids, such as β-cyclocitral (β-cc), zaxinone (Zax), and β-apo-11-carotenoids, and the plant hormones abscisic acid (ABA) and strigolactones (SLs; Fig. 1a), which regulate plant growth, development, stress response, and biotic interactions (Ramel et al., 2012; Wang et al., 2019; Moreno et al., 2021b; Jia et al., 2022). In mammals, carotenoids act as vitamin A precursors and antioxidants, which make them essential components of the human diet. In fact, vitamin A deficiency (VAD) is still a severe global health problem and a major reason for blindness and childhood mortality (Olson, 1996; Giuliano et al., 2003; Zheng et al., 2020). Therefore, enhancing carotenoid content, in particular provitamin A carotenoids, has been one of the preferred targets of plant genetic engineering, resulting in biofortified cereals, fruits, and tubers, including rice, tomato, and potato. However, the continuous increase in food demand of the growing world population, accompanied by climate change that negatively impacts agricultural production, makes it necessary to develop novel strategies for simultaneous improvement of stress tolerance, yield, and nutritional value of crops. Here, we briefly discuss how genetic engineering of LYCOPENE β-CYCLASE (LCYB) can be a promising strategy for crop improvement beyond carotenoid biofortification. After the key finding that PHYTOENE SYNTHASE (PSY) is the rate-limiting enzyme of the carotenoid pathway (Bird & Ray, 1991; Bramley et al., 1992; Fray et al., 1995), PSY became the preferred target for carotenoid biofortification. However, the dwarf phenotype observed upon the constitutive overexpression of PSY in tomato (Fray et al., 1995) led to the exclusion of constitutive promoters in these kinds of approaches. By contrast, tissue-specific promoters started to be widely used (Shewmaker et al., 1999; Ye et al., 2000; Paine et al., 2005; Diretto et al., 2007). The enzyme LYCOPENE β-CYCLASE (LCYB) is a further key enzyme in carotenoid biosynthesis, as it converts lycopene into β-carotene or, in combination with lycopene ε-cyclase (LCYE), into α-carotene, dividing the pathway into two branches (Fig. 1a). It was previously shown that the overexpression of LCYB from plants or bacteria modifies carotenoid content. Interestingly, growth or developmental phenotypes were not reported in any of the generated LCYB transgenic plants (Ronen et al., 2000; D'Ambrosio et al., 2004; Wurbs et al., 2007; Apel & Bock, 2009; Shi et al., 2015). However, because carotenoids, including β-carotene, are ABA precursors, several papers investigated ABA content and reported on its enhancement upon expression of carotenoid biosynthetic genes, that is, ζ-Carotene Desaturase and LCYB, in tobacco, tomato, and sweet potato. Enhanced ABA content was reflected in physiological advantages, including improved plant growth under high salt or drought conditions, but not under normal conditions (Shi et al., 2015; Li et al., 2017; Kang et al., 2018). Almost a decade ago, a study with Daucus carota (carrot) showed for the first time that constitutive overexpression of a carotenoid gene might have a positive impact on plant growth under normal conditions. This study showed that overexpressing or silencing DcLCYB1 resulted in bigger or smaller carrot roots, respectively (Moreno et al., 2013). Later work showed that constitutive DcLCYB1 expression resulted in advantageous growth and developmental phenotypes in tobacco plants grown under controlled conditions (Moreno et al., 2016). Despite the observed phenotypic changes, a key question remained open: how does the expression of a carotenogenic gene trigger growth and developmental phenotypes? The answer to that question might lie in the role of β-carotene as the common precursor of important bioactive molecules such as hormones (ABA and SLs), and the apocarotenoid growth regulators, β-cc and Zax. These molecules have different functions in photoprotection, growth, plant architecture, and stress tolerance. Therefore, it seems possible that by enhancing the level of their precursor (β-carotene), the content of these bioactive compounds can also be enhanced. However, overexpression of PSY in tobacco also led to increased β-carotene, but with a negative effect on plant growth (Busch et al., 2002). This discrepancy might be explained by the role of PSY as a key enzyme for the whole carotenoid biosynthetic pathway and by the assumption that PSY overexpression caused a redirection of the isoprenoid flux toward carotenoid biosynthesis, which led to an increase in β-carotene but also in other carotenoids and, importantly, to a depletion of the geranylgeranyl diphosphate (GGPP) pool that feeds the biosynthesis of gibberellins (GAs), chlorophylls and tocopherols, besides that of carotenoids. Considering the important biological functions of these compounds, negative side effects of constitutive PSY overexpression can be expected. For instance, reduced GA content can lead to dwarf phenotypes, as in the case of tomato and tobacco (Fray et al., 1995; Busch et al., 2002). By contrast, constitutive DcLCYB1 overexpression induced the expression of key genes in the carotenoid (PSY), MEP (DXS), GGPP (GGPPS), GA (GA20ox and KS), and chlorophyll (CHL) biosynthetic pathways, resulting in an increase in GAs (GA1 and GA4), chlorophylls, and carotenoids (Fig. 1a,b) (Moreno et al., 2020). Enhanced GA content led to the emergence of a series of phenotypes, such as enhanced leaf area and plant height, early flowering and longer internodes, redesigning plant architecture. This modification of plant architecture, that is, taller stems with less and bigger leaves, which was caused by the transgene, together with higher carotenoid, xanthophyll and chlorophyll content resulted in enhanced photosynthetic efficiency under fluctuating light conditions, giving rise to 23% and 32% higher plant biomass and seed yield, respectively (Fig. 1c,d). In addition, DcLCYB1 tobacco lines showed enhanced photoprotection and tolerance to high salinity and H2O2 (Moreno et al., 2021a). These positive effects may be related to higher xanthophyll and ABA content and to the upregulation of key transcription factors, such as bZIP12, NAC002, SCL14, GIF3, GRF1, which are involved in growth and stress tolerance. Altogether, these results suggest that expressing the LCYB gene under the control of a constitutive promoter can positively influence plant physiology, plant development, and stress tolerance simultaneously, besides enhancing carotenoid content. Interestingly, tomato plants expressing LCYBs from different sources also showed growth advantages (Mi et al., 2022). Overexpression of LCYB from daffodil, tomato, and the bacterium Pantoea ananatis (formerly Erwinia uredovora) in three different tomato cultivars caused changes in carotenoid and hormone contents in shoots and fruits, leading to biomass partitioning, increased seed yield, high light, salt and drought stress tolerance, and enhanced shelf life (Fig. 2a,b) (Mi et al., 2022). This study revealed common effects of LCYB overexpression and specific ones that depend on the origin of the overexpressed LCYB gene and the integration compartment of the gene, that is, in nucleus or plastid. For instance, plant LCYBs showed a stronger impact on carotenoid profiles in shoots and fruits, leading to an eightfold increase in β-carotene content in transgenic fruits (orange color; Fig. 2b), than in the bacterial LCYB (Mi et al., 2022). Consistent with these increments, levels of seven species of nonhydroxylated β-apocarotenoids, including retinal that was recently identified as root growth regulator (Dickinson et al., 2021), were increased between five- and 50-fold. Besides having enhanced contents of isoprenoid-derived hormones ABA, GA, and cytokinins, these lines showed changes in the homeostasis of auxins, jasmonic acid (JA), and JA-Ile (Fig. 2a), which are involved in stress response. For instance, higher ABA, JA, JA-Ile, and/or IAA contents enhance salt and drought tolerance in Arabidopsis, rice, and white clover (Sharma et al., 2013; Yoshida et al., 2014; Kazan, 2015; Shani et al., 2017; Hazman et al., 2019; Zhang et al., 2020). Although the mechanisms underlying the abiotic stress tolerance of the LCYB-overexpressing tomato lines are not fully understood, the observed change in at least one of the abovementioned hormones may contribute to the observed abiotic stress tolerance. In addition, the content of important primary metabolites triggered the emergence of unexpected phenotypes in the transgenic tomato lines. An example is the enhanced leaf and fruit content of osmoprotectants, such as glucose, fructose, trehalose, and myo-inositol, which contribute to abiotic stress tolerance. In addition, the transgenic lines also showed accumulation of ornithine and putrescine, which together, with ABA, extend fruit shelf life (Perez-Vicente et al., 2002; Valero et al., 2002; Diretto et al., 2019). In summary, increasing LCYB expression and β-carotene content can trigger the emergence of a series of beneficial phenotypes by modulating the content of hormones, growth regulators, and osmoprotectants in tobacco and tomato (Moreno et al., 2020, 2021a; Kossler et al., 2021; Mi et al., 2022). Besides being a tool for provitamin A biofortification, the effect of constitutive overexpression of LCYB on the composition of bioactive metabolites revealed a promising alternative for improving crop productivity and resilience. We plead for exploring this option in other crops, particularly in cereals to increase the carotenoid content in their endosperm (which contains low levels of these pigments) and enhance their yield and resilience. It would be also very interesting to investigate the effect of specific LCYB overexpression in root tissues. Would higher LCYB activity in roots lead to increased level of the root growth regulator β-cc (or β-cyclocitric acid)? Would it also affect ABA content? Would it give rise to enhanced root growth and abiotic stress tolerance? And is it possible that those changes affect rhizospheric interactions? Answering these and other questions related to LCYB metabolic impact will significantly increase our knowledge of the formation of bioactive metabolites in crops and may contribute to increased food security. We are thankful to Dr Manuel Rodriguez-Concepcion and Dr Yagiz Alagoz for their helpful comments. We apologize to colleagues whose work could not be cited due to space constraints. None declared. JCM took photographs, prepared figures, and wrote the manuscript and SA-B edited the manuscript and provided feedback and comments. There are no new data reported in this letter.