Lycopene ε-cyclase mediated transition of α-carotene and ß-carotene metabolic flow in carrot fleshy root.

The accumulation of carotenoids, such as xanthophylls, lycopene, and carotenes, is responsible for the color of carrot (Daucus carota subsp. sativus) fleshy roots. The potential role of DcLCYE, encoding a lycopene ε-cyclase associated with carrot root color, was investigated using cultivars with orange and red roots. The expression of DcLCYE in red carrot varieties was significantly lower than that in orange carrots at the mature stage. Furthermore, red carrots accumulated larger amounts of lycopene and lower levels of α-carotene. Sequence comparison and prokaryotic expression analysis revealed that amino acid differences in red carrots did not affect the cyclization function of DcLCYE. Analysis of the catalytic activity of DcLCYE revealed that it mainly formed ε-carotene, while a side activity on α-carotene and γ-carotene was also observed. Comparative analysis of the promoter region sequences indicated that differences in the promoter region may affect the transcription of DcLCYE. DcLCYE was overexpressed in the red carrot 'Benhongjinshi' under the control of the CaMV35S promoter. Lycopene in transgenic carrot roots was cyclized, resulting in the accumulation of higher levels of α-carotene and xanthophylls, while the ß-carotene content was significantly decreased. The expression levels of other genes in the carotenoid pathway were simultaneously upregulated. Knockout of DcLCYE in the orange carrot 'Kurodagosun' by CRISPR/Cas9 technology resulted in a decrease in the α-carotene and xanthophyll contents. The relative expression levels of DcPSY1, DcPSY2, and DcCHXE were sharply increased in DcLCYE knockout mutants. The results of this study provide insights into the function of DcLCYE in carrots, which could serve as a basis for creating colorful carrot germplasms.

Advances in engineering the production of the natural red pigment lycopene: A systematic review from a biotechnology perspective.

BACKGROUND: Lycopene is a natural red compound with potent antioxidant activity that can be utilized both as pigment and as a raw material in functional food, and so possesses good commercial prospects. The biosynthetic pathway has already been documented, which provides the foundation for lycopene production using biotechnology. AIM OF REVIEW: Although lycopene production has begun to take shape, there is still an urgent need to alleviate the yield of lycopene. Progress in this area can provide useful reference for metabolic engineering of lycopene production utilizing multiple approaches. KEY SCIENTIFIC CONCEPTS OF REVIEW: Using conventional microbial fermentation approaches, biotechnologists have enhanced the yield of lycopene by selecting suitable host strains, utilizing various additives, and optimizing culture conditions. With the development of modern biotechnology, genetic engineering, protein engineering, and metabolic engineering have been applied for lycopene production. Extraction from natural plants is the main way for lycopene production at present. Based on the molecular mechanism of lycopene accumulation, the production of lycopene by plant bioreactor through genetic engineering has a good prospect. Here we summarized common strategies for optimizing lycopene production engineering from a biotechnology perspective, which are mainly carried out by microbial cultivation. We reviewed the challenges and limitations of this approach, summarized the critical aspects, and provided suggestions with the aim of potential future breakthroughs for lycopene production in plants.

Differential hydroxylation efficiency of the two non-heme carotene hydroxylases: DcBCH1, rather than DcBCH2, plays a major role in carrot taproot.

Carotene hydroxylase plays an important role in catalyzing the hydroxylation of carotene to xanthopylls, including two types: non-heme carotene hydroxylase (BCH type) and heme-containing cytochrome P450 hydroxylase (P450 type). Two BCH-encoding genes were annotated in the carrot genome. However, the role of BCHs and whether there are functional interactions between the duplicated BCHs in carrot remains unclear. In this study, two BCH encoding genes, DcBCH1 and DcBCH2, were cloned from carrot. The relative expression level of DcBCH1 was much higher than that of DcBCH2 in carrot taproots with different carotene accumulation levels. Overexpression of DcBCH1 in 'KRD' (high carotene accumulated) carrot changed the taproot color from orange to yellow, accompanied by substantial reductions in α-carotene and ß-carotene. There was no obvious change in taproot color between transgenic 'KRD' carrot overexpressing DcBCH2 and control carrot. Simultaneously, the content of α-carotene in the taproot of DcBCH2-overexpressing carrot decreased, but the content of ß-carotene did not change significantly in comparison with control carrot. Using the CRISPR/Cas9 system to knock out DcBCH1 in 'KRD' carrot lightened the taproot color from orange to pink-orange; the content of α-carotene in the taproot increased slightly, while the ß-carotene content was still significantly decreased, compared with control carrot. In DcBCH1-knockout carrot, the transcript level of DcBCH2 was significantly increased. These results indicated that in carrot taproot, DcBCH1 played the main function of BCH enzyme, which could hydroxylate α-carotene and ß-carotene; DcBCH1 and DcBCH2 had functional redundancy, and these two DcBCHs could partially compensate for each other.

[QTL mapping for contents of main carotenes and lycopene in carrot (Daucus carota L.)].

An F2 population derived from two carrot inbred lines, P50006 and HCM A.C. with high carotene accumulation, was developed and used to map and analyze quantitative trait locus (QTL) associated with the accumulation of alpha and beta-carotene, total carotene and lycopene. Broad-sense heritabilities of these traits were 0.75, 0.50, 0.31, and 0.93, respectively. A genetic map with 91 SRAP (Sequence-related amplified polymorphism) markers was developed, which spanned 502.9 cM in 9 linkage groups with a mean marker interval of 5.5 cM. Mixed-model-based composite interval mapping was performed to analyze QTL and epistasis effects. One major QTL each for beta-carotene, total carotene and lycopene accumulation were detected which can explain 12.79%, 12.87%, and 14.61% of total phenotypic variations, respectively. Additive genetic variance was primarily responsible for genetic variability in all three major QTL. In addition, a pair of epistasis QTL for beta-carotene and lycopene accumulation was detected, which were able to explain 15.1% and 6.5% of total phenotypic variation, respectively. The dominant x additive and dominant x dominant interaction variance were primary epistasis effect for beta-carotene and lycopene. These SRAP markers linked to QTL could be used in selection or QTL pyramiding for high carotene and lycopene content in carrot breeding.

The dual role of phytoene synthase genes in carotenogenesis in carrot roots and leaves.

Carrot (Daucus carota L.) is an important food crop and is useful for studying carotenogenesis due to the quantity and diversity of carotenoids in its roots. Phytoene synthase catalyzes the first committed step in the carotenoid biosynthesis pathway, and its overexpression is the main driving force in the orange phenotype. At present, we lack fundamental knowledge of the role of these genes and their effects on carotenoid accumulation in leaves. In the present study, three backcross inbred lines (BC2S4) with different colored roots derived from a cross between the orange inbred line (Af) and related wild species were used to investigate the role of the duplicated DcPSY genes in root carotenogenesis. Promoter analysis showed that DcPSY genes have diverged substantially in their regulatory sequences after gene duplication. Expression levels of DcPSY1 and DcPSY2 were generally positively correlated with carotenoid content during root development. In mature leaves, total carotenoid content was higher than that in the roots, DcPSY1 expression increased extremely higher than DcPSY2 expression compared with roots, and DcPSY1 was more sensitive than DcPSY2 during leaf de-etiolation under sunlight. These results suggest that DcPSY1 seems to make an important contribution to carotenoid accumulation in the leaves and is important for photosynthesis and photoprotection, but they are not the determining factors of root color. This expands our understanding of the regulation of carotenoid biosynthesis in carrot.

Cucumis monosomic alien addition lines: morphological, cytological, and genotypic analyses.

Cucumis hystrix Chakr. (HH, 2n=24), a wild relative of the cultivated cucumber, possesses several potentially valuable disease-resistance and abiotic stress-tolerance traits for cucumber ( C. sativus L., CC, 2n=14) improvement. Numerous attempts have been made to transfer desirable traits since the successful interspecific hybridization between C. hystrix and C. sativus, one of which resulted in the production of an allotriploid (HCC, 2n=26: one genome of C. hystrix and two of C. sativus). When this genotype was treated with colchicine to induce polyploidy, two monosomic alien addition lines (MAALs) (plant nos. 87 and 517: 14 CC+1 H, 2n=15) were recovered among 252 viable plants. Each of these plants was morphologically distinct from allotriploids and cultivated cucumbers. Cytogenetic and molecular marker analyses were performed to confirm the genetic constitution and further characterize these two MAALs. Chromosome counts made from at least 30 meristematic cells from each plant confirmed 15 nuclear chromosomes. In pollen mother cells of plant nos. 87 and 517, seven bivalents and one univalent were observed at diakinesis and metaphase I; the frequency of trivalent formation was low (about 4-5%). At anaphase I and II, stochastic and asymmetric division led to the formation of two gamete classes: n=7 and n=8; however, pollen fertility was relatively high. Pollen stainability in plant no. 87 was 86.7% and in plant no. 517 was 93.2%. Random amplified polymorphic DNA analysis was performed using 100 random 10-base primers. Genotypes obtained with eight primers (A-9, A-11, AH-13, AI-19, AJ-18, AJ-20, E-19, and N-20) showed a band common to the two MAAL plants and C. hystrix that was absent in C. sativus, confirming that the alien chromosomes present in the MAALs were derived from C. hystrix. Morphological differences and differences in banding patterns were also observed between plant nos. 87 and 517 after amplification with primers AI-5, AJ-13, N-12, and N-20, suggesting that these plants may contain different C. hystrix chromosomes.