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

Angélica M Jaramillo,Tianhu Sun,Diana Katherine Castillo,Santiago Sierra,Li Li,Erick Boy,Juan Pablo Arciniegas,Daniel Álvarez,Luis Augusto Becerra López-Lavalle,Ralf Welsch,Anestis Gkanogiannis,Paul Chavarriaga-Aguirre,Hiroshi Ezura

doi:10.1371/journal.pone.0262412

Abstract

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.

Highlights

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]
Five putative ORANGE protein (OR) genes were found in the Phytozome database, three on chromosome 14, one on chromosome 6 (MeOR_X2), and one on chromosome 9 (MeOR_X4; Table 1)
The corresponding molecular weight (MW) of the proteins was predicted and an alignment in BLAST was performed to determine their identity with the OR proteins from melon, cauliflower, Arabidopsis, and sweet potato

Summary

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]. Plant foods provide vitamin A precursors, known as provitamin A (pVA) carotenoids. 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], it is a major crop in low- and middle-income countries in the tropics [13, 14]. 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

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