Validation of the effects of molecular marker polymorphisms in LcyE and CrtRB1 on provitamin A concentrations for 26 tropical maize populations

Vitamin A deficiency (VAD) compromises immune function and is the leading cause of preventable blindness in children in many developing countries. Biofortification, or breeding staple food crops that are rich in micronutrients, provides a sustainable way to fight VAD and other micronutrient malnutrition problems. Polymorphisms, with associated molecular markers, have recently been identified for two loci, LcyE (lycopene epsilon cyclase) and CrtRB1 (β-carotene hydroxylase 1) that govern critical steps in the carotenoid biosynthetic pathway in maize endosperm, thereby enabling the opportunity to integrate marker-assisted selection (MAS) into carotenoid breeding programs. We validated the effects of 3 polymorphisms (LcyE5′TE, LcyE3′Indel and CrtRB1-3′TE) in 26 diverse tropical genetic backgrounds. CrtRB1-3′TE had a two-ten fold effect on enhancing beta-carotene (BC) and total provitamin A (proA) content. Reduced-function, favorable polymorphisms within LcyE resulted in 0–30 % reduction in the ratio of alpha- to beta-branch carotenoids, and increase in proA content (sometimes statistically significant). CrtRB1-3′TE had large, significant effect on enhancing BC and total ProA content, irrespective of genetic constitution for LcyE5′TE. Genotypes with homozygous favorable CrtRB1-3′TE alleles had much less zeaxanthin and an average of 25 % less total carotenoid than other genotypes, suggesting that feedback inhibition may be reducing the total flux into the carotenoid pathway. Because this feedback inhibition was most pronounced in the homozygous favorable LcyE (reduced-function) genotypes, and because maximum total proA concentrations were achieved in genotypes with homozygous unfavorable or heterozygous LcyE, we recommend not selecting for both reduced-function genes in breeding programs. LcyE exhibited significant segregation distortion (SD) in all the eight, while CrtRB1 in five of eight digenic populations studied, with favorable alleles of both the genes frequently under-represented. MAS using markers reported herein can efficiently increase proA carotenoid concentration in maize.     

An increase of lycopene content in tomato fruit is associated with a novel Cyc-B allele isolated through TILLING technology

Tomato Cyc-B gene encodes a chromoplast-specific lycopene β-cyclase that converts lycopene to β-carotene during ripening of the fruit. By screening the tomato Red Setter mutant population with the TILLING method, we identified eight new alleles at the Cyc-B locus. Results of greenhouse phenotypic analysis revealed that the novel A949G Cyc-B allele produced modifications in the carotenoid profile and content of tomato petals and fruit. The cyc-b7 genotype, carrying the A949G Cyc-B allele, was therefore evaluated in an open field trial for standard agronomic traits as well as carotenoid content of the fruit. Results of the field trial confirmed that the induced A949G missense mutation favored the accumulation of lycopene in the fruit with no detrimental effects on the yield or on other agronomic and technological properties such as fruit firmness and Brix degree of fruit juice. On the basis of these results, it can be affirmed that the A949G Cyc-B allele constitutes a useful new genetic variant which can be used for improving carotenoid content in tomato fruit and for the development of new tomato commercial lines. Finally, the results presented here furthermore demonstrate that TILLING is a powerful methodology not only as a confirmatory system for gene functional analysis but also for selecting new gene variants useful for genetic improvement of important crops.     

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.     

Genetic architecture controlling variation in grain carotenoid composition and concentrations in two maize populations

Key message

Genetic control of maize grain carotenoid profiles is coordinated through several loci distributed throughout three secondary metabolic pathways, most of which exhibit additive, and more importantly, pleiotropic effects.

Abstract

The genetic basis for the variation in maize grain carotenoid concentrations was investigated in two F_2:3 populations, DEexp × CI7 and A619 × SC55, derived from high total carotenoid and high β-carotene inbred lines. A comparison of grain carotenoid concentrations from population DEexp × CI7 grown in different environments revealed significantly higher concentrations and greater trait variation in samples harvested from a subtropical environment relative to those from a temperate environment. Genotype by environment interactions was significant for most carotenoid traits. Using phenotypic data in additive, environment-specific genetic models, quantitative trait loci (QTL) were identified for absolute and derived carotenoid traits in each population, including those specific to the isomerization of β-carotene. A multivariate approach for these correlated traits was taken, using carotenoid trait principal components (PCs) that jointly accounted for 97 % or more of trait variation. Component loadings for carotenoid PCs were interpreted in the context of known substrate-product relationships within the carotenoid pathway. Importantly, QTL for univariate and multivariate traits were found to cluster in close proximity to map locations of loci involved in methyl-erythritol, isoprenoid and carotenoid metabolism. Several of these genes, including lycopene epsilon cyclase, carotenoid cleavage dioxygenase1 and beta-carotene hydroxylase, were mapped in the segregating populations. These loci exhibited pleiotropic effects on α-branch carotenoids, total carotenoid profile and β-branch carotenoids, respectively. Our results confirm that several QTL are involved in the modification of carotenoid profiles, and suggest genetic targets that could be used for the improvement of total carotenoid and β-carotene in future breeding populations.     

Heterologous biosynthesis of lutein in S. cerevisiae enabled by temporospatial pathway control

The market-expanding lutein is currently mainly supplied by plant extraction, with microbial fermentation using engineered cell factory emerging as a promising substitution. During construction of lutein-producing yeast, α-carotene formation through asymmetric ε- and β-cyclization of lycopene was found as the main limiting step, attributed to intra-pathway competition of the cyclases for lycopene, forming β-carotene instead. To solve this problem, temperature-responsive expression of β-cyclase was coupled to constitutive expression of ε-cyclase for flux redirection to α-carotene by allowing ε-cyclization to occur first. Meanwhile, the ε-cyclase was engineered and re-localized to the plasma membrane for further flux reinforcement towards α-carotene. Finally, pathway extension with proper combination of carotenoid hydroxylases enabled lutein (438 μg/g dry cells) biosynthesis in S. cerevisiae. The success of heterologous lutein biosynthesis in yeast suggested temporospatial pathway control as a potential strategy in solving intra-pathway competitions, and may also be applicable for promoting the biosynthesis of other natural products.     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

沉默lycB基因对雨生红球藻类胡萝卜素合成代谢的影响

雨生红球藻是一种淡水浮游单细胞绿藻, 逆境条件下可积累大量的类胡萝卜素。番茄红素是类胡萝卜素中的一种, 是类胡萝卜素合成代谢中的一个重要中间产物。番茄红素β-环化酶(LycB)是催化番茄红素形成β-胡萝卜素的关键酶。文章以杜氏盐藻lycB基因为干扰序列, 构建了含卡那霉素与阿特拉津双抗性的RNAi载体p1301-BS-RNAi。将其电转化进雨生红球藻细胞, 经抗性筛选、基因组PCR及RT-PCR筛选, 获得了16个独立的干扰株系。选取生长良好的7个进行高光诱导, 发现其番茄红素含量增加了99.4%, β-胡萝卜素含量减少了48.4%, 即通过异源的lycB-RNAi基因沉默可抑制番茄红素向β-胡萝卜素的转化。对比分析发现, 番茄红素增加量仅是β-胡萝卜素减少量的5%, 表明因lycB-RNAi抑制而产生的番茄红素的95%又被其他通路转换成了其他代谢产物, 因此要实现雨生红球藻番茄红素含量的大幅增长, 需协同调控其他代谢通路。     

Lutein Accumulation in the Absence of Zeaxanthin Restores Nonphotochemical Quenching in the Arabidopsis thaliana npq1 Mutant

Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid ΔpH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and α-carotene than the wild type. szl1 contains a point mutation in the lycopene β-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene β-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.     

Functional analysis of multiple carotenogenic genes from <Emphasis Type="Italic">Lycium barbarum</Emphasis> and <Emphasis Type="Italic">Gentiana lutea</Emphasis> L. for their effects on β-carotene production in transgenic tobacco

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, β-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into β-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and δ-carotene desaturase gene, lycopene β-cyclase, lycopene ε-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced β-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.