Targeted transcriptomic and metabolic profiling reveals temporal bottlenecks in the maize carotenoid pathway that may be addressed by multigene engineering

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in‐depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner.     

Single‐molecule real‐time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.     

Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.     

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.     

Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin

Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm β : ε lycopene cyclase activity favours the β‐carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene β‐cyclase) and therefore accumulating extraordinary levels of β‐carotene. Here, we demonstrate that introgressing the transgenic mini‐pathway into wild‐type yellow endosperm varieties gives rise to hybrids in which the β : ε ratio is altered additively. Where the β : ε ratio in the genetic background is high, introgression of the mini‐pathway allows zeaxanthin production at an unprecedented 56 μg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.     

Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice

Transgenics for the expression of β-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and β-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T₁ progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T₁ progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T₁ progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 μg/g of polished seeds. High variation in the carotenoid accumulation was observed within the T₁ progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the β-carotene accumulation.     

Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects

A bacterial phytoene synthase (crtB) gene was overexpressed in a seed-specific manner and the protein product targeted to the plastid in Brassica napus (canola). The resultant embryos from these transgenic plants were visibly orange and the mature seed contained up to a 50-fold increase in carotenoids. The predominant carotenoids accumulating in the seeds of the transgenic plants were alpha and beta-carotene. Other precursors such as phytoene were also detected. Lutein, the predominant carotenoid in control seeds, was not substantially increased in the transgenics. The total amount of carotenoids in these seeds is now equivalent to or greater than those seen in the mesocarp of oil palm. Other metabolites in the isoprenoid pathway were examined in these seeds. Sterol levels remained essentially the same, while tocopherol levels decreased significantly as compared to non-transgenic controls. Chlorophyll levels were also reduced in developing transgenic seed. Additionally, the fatty acyl composition was altered with the transgenic seeds having a relatively higher percentage of the 18 : 1 (oleic acid) component and a decreased percentage of the 18 : 2 (linoleic acid) and 18 : 3 (linolenic acid) components. This dramatic increase in flux through the carotenoid pathway and the other metabolic effects are discussed.     

Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation

Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), ζ‐carotene desaturase (ZDS), lycopene β‐cyclase (LCY‐B), β‐ring hydroxylase (CRTR‐B) and ε‐ring hydroxylase (CRTR‐E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC‐B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased β‐carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.     

Golden bananas in the field: elevated fruit pro‐vitamin A from the expression of a single banana transgene

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro‐vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA‐biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β‐carotene equivalent (β‐CE) in the fruit. Expression of a Fe'i banana‐derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β‐CE . Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop ‘Golden Rice 2’, also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1‐aminocyclopropane‐1‐carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild‐type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate‐limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.