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Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation

['Angélica M. Jaramillo', 'Harvestplus', 'C O The Alliance Of Bioversity International', 'The International Center For Tropical Agriculture', 'Ciat', 'Cali', 'Santiago Sierra', 'The Alliance Of Bioversity International', 'Paul Chavarriaga-Aguirre', 'Diana Katherine Castillo']

Date: 2022-02 

Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

Introduction

Vitamin A is essential for vision and cell differentiation and its deficiency is the main cause of preventable blindness, cause development disorders, and impairs the immune system [1, 2]. Vitamin A deficiency persists as a major public health problem globally, and disproportionately affects preschool children and pregnant women in low- and middle-income countries [3–5]. Thus, the first and most important step to prevent primary vitamin A deficiency is the regular consumption of vitamin A-rich foods [6]. Plant foods provide vitamin A precursors, also known as provitamin A (pVA) carotenoids. Carotenoids are natural lipophilic isoprenoids that are involved in photosynthesis, provide pigmentation to a wide range of plant tissues, and serve as precursors for the formation of phytohormones and signaling precursors essential for plant homeostasis [7, 8]. β-Carotene is one of the most abundant carotenoids in nature and along with α-carotene and β-cryptoxanthin are the main carotenoids in plants with pVA function [9, 10].

Cassava (Manihot esculenta Crantz) is a native crop to Central and South America [11]. It is highly appreciated for its ease of agronomic handling, high productivity, and tolerance to poor soils and drought [12], thus it is a major crop in low- and middle-income countries in the tropics [13, 14]. The leaves and roots are used for human consumption, livestock feed, and starch production [15–17]. In Africa, cassava is the second most important staple food in terms of calories supply [18]. However, white cassava roots are a poor source of micronutrients such as iron, zinc, and pVA carotenoids, and only very few yellow-root cultivars synthesize and store carotenoids [19]. Thus, there is a need to increase the content of pVA carotenoids for enhancing the nutritional quality of agronomically preferred cassava varieties that can be incorporated into diets and contribute to alleviating vitamin A deficiency.

Biofortification is the process of increasing minerals and vitamins in food crops through conventional breeding, genetic engineering, and agronomic practices [20]. It is a complementary strategy to other interventions such as the promotion of a diverse diet, food fortification, or supplementation. Biofortification seeks to mitigate nutritional deficiencies through the consumption of staple food crops [21]. It is considered economically cost-effective and sustainable, in addition to having the capacity to reach people with limited access to other nutritional interventions [22, 23].

To increase the content of pVA carotenoids in cassava, conventional breeding has focused on QTLs related to the carotenoids biosynthesis pathway [24]. Phytoene synthase (PSY) is the first specific enzyme in the carotenoid biosynthesis pathway and is considered as rate-limiting for carotenoid biosynthesis. Cassava has three PSY genes, of which PSY1 and PSY2 are highly abundant in leaves while PSY2 transcripts dominate in roots [25]. PSY3 expression is absent in all tissues analyzed so far but suggested to be involved in carotenoid biosynthesis for apocarotenoid signal compounds as shown for the tomato homologue [26]. PSY1 is more responsive to abiotic stress than PSY2 while PSY2 shows higher abundance in petals and roots, suggesting some specificity for carotenoid biosynthesis in non-green tissues. However, there is no association between the expression levels of any cassava PSY paralog and carotenoid accumulation in cassava varieties [27] while a SNP in PSY2 explained most of the carotenoid variations. In fact, it was shown that this polymorphism increased the enzymatic activity of recombinant PSY enzymes by 3-fold and suggested that PSY activity determined the total carotenoid content in cassava roots. Accordingly, white cassava genotypes transformed with the bacterial PSY gene CRTB produced increases of up to 22 μg/g (DW) of total carotenoids and 7 μg/g (DW) of β-carotene, the most abundant carotenoid in cassava roots [27]. However, the fact that mainly carotene intermediates like phytoene accumulated, unraveled carotene desaturation as a subsequent rate-limitation for β-carotene formation. When transformed with CRTB gene alone or in combination with the bacterial phytoene desaturase gene (CRTI) or with the upstream gene 1-deoxyxylulose 5-phosphate synthase (DXS), the highest levels achieved were 60 μg/g (DW) of total carotenoids in cassava roots [28, 29].

Despite these high levels using transgenic approaches, those obtained by means of conventional breeding methods were not surpassed (70 μg/g DW) [30]. However, these varieties did not meet characteristics such as optimal size, cooking time, safety, and texture properties among others to make them available to the general population for their adoption [31] Hitherto, the highest levels of total pVA carotenoids reached by biofortified varieties with favorable adoption characteristics were up to 50 μg/g (DW) [32].

Carotenoids are subjected to enzymatic and more importantly to oxidative non-enzymatic degradation [33–35]. In cassava, postharvest processes such as storage conditions can produce significant losses of carotenoids of 20–50% [36, 37], while 5–95% may be lost upon cooking with different methods [19, 38–40]. Considering the only moderate carotenoid content achieved so far in edible cassava genotypes, and the losses caused by postharvest processes, the retention of pVA carotenoids can be low. The Estimated Average Requirement (EAR) of vitamin A for children and women of reproductive age is 275 μg and 500 μg according to the Institute Of Medicine [41]. Thus, even though considering a high conversion rate of pVA carotenoids into vitamin A of 5:1 in cassava, the final contribution of biofortified cassava varieties to the EAR could be modest at best [36, 40].

The lack of efficient molecular tools for carotenoid accumulation in cassava has hindered the development of cassava varieties with superior content of pVA carotenoids, as in carrot [42, 43], maize [44], or sweet potato [45, 46]. Thus, the identification of novel genes in cassava that can boost carotenoid accumulation and increase protection against degradation is critical to assuring a sufficient pVA supply by cassava to the diet.

ORANGE (OR) protein has gained increasing interest in recent years as it has several functions related to carotenoid accumulation and stabilization [47, 48]. The OR gene is not part of the carotenoid biosynthetic pathway and it was found originally from an orange-curd cauliflower (Brassica oleracea) mutant with a high content of carotenoids [49]. An increase in carotenoid content was observed alongside a higher storage capacity, but with no significant change in the expression of OR or PSY genes [50, 51]. In the cauliflower OR mutant, a mutation in the OR gene caused by a retrotransposon produces three in-frame splicing variants with partially altered molecular properties [51, 52]. In orange-fleshed melon, however, a SNP in the OR is responsible for large amounts of carotenoids accumulation [53], similarly in carrot [54].

OR is a holdase chaperone highly conserved among plants and can be localized in both the chloroplasts and the nucleus [55–58]. It is considered to induce chromoplast differentiation and carotenoid crystals formation, thus generating a sink for carotenoid storage in potato, Arabidopsis and melon [59–61]. It can also attenuate β-carotene metabolism, although it is yet unknown whether the expression of specific genes such as β-carotene hydroxylase (BCH) are altered to lead to ∝-carotene accumulation [44, 62–64]. Likewise, OR shown to physically interact with PSY in Arabidopsis, sweet potato, and cauliflower, leading to the post-translational regulation of this protein as well as mutual co-regulation with a consequent increase in the production of carotenoids [52, 56, 65]. The OR gene can also improve abiotic stress tolerance in potato and sweet potato possibly altering the abscisic acid (ABA) signaling pathway [65–67]. Additionally, OR in Arabidopsis can interact in the nucleus with transcription factors involved in chloroplast biogenesis and interacts with plastid division factor to interfere with chromoplast division [57, 68]. Some plants such as Arabidopsis and melon harbor OR-like proteins, which are homologous OR proteins with unique but partially redundant functions [56, 69].

The relevance of OR in the accumulation of carotenoids has been reported extensively upon the overexpression of the wild type or mutagenized OR genes from Arabidopsis, cauliflower, sweet potato, or melon in potato [60, 66], Arabidopsis [70], rice [71], maize [44], tomato [72], sweet potato [65, 67], and cauliflower [52], among other plants [73, 74]. However, sparse information is available on the effect of the OR on the accumulation of carotenoids in yellow cassava [29]. The aim of this study was to identify OR genes in cassava, investigate their correlation with carotenoids accumulation, and explore their potential to increase and stabilize carotenoid levels in cassava, using cassava in vitro tissues as a model system.

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