Overexpression of a carrot BCH gene, DcBCH1, improves tolerance to drought in Arabidopsis thaliana

Background Carrot (Daucus carota L.), an important root vegetable, is very popular among consumers as its taproot is rich in various nutrients. Abiotic stresses, such as drought, salt, and low temperature, are the main factors that restrict the growth and development of carrots. Non-heme carotene hydroxylase (BCH) is a key regulatory enzyme in the β-branch of the carotenoid biosynthesis pathway, upstream of the abscisic acid (ABA) synthesis pathway. Results In this study, we characterized a carrot BCH encoding gene, DcBCH1. The expression of DcBCH1 was induced by drought treatment. The overexpression of DcBCH1 in Arabidopsis thaliana resulted in enhanced tolerance to drought, as demonstrated by higher antioxidant capacity and lower malondialdehyde content after drought treatment. Under drought stress, the endogenous ABA level in transgenic A. thaliana was higher than that in wild-type (WT) plants. Additionally, the contents of lutein and β-carotene in transgenic A. thaliana were lower than those in WT, whereas the expression levels of most endogenous carotenogenic genes were significantly increased after drought treatment. Conclusions DcBCH1 can increase the antioxidant capacity and promote endogenous ABA levels of plants by regulating the synthesis rate of carotenoids, thereby regulating the drought resistance of plants. These results will help to provide potential candidate genes for plant drought tolerance breeding.

DcPAR1 is Required for Development and Carotenoid Synthesis in the Dark-Grown Carrot Taproot

Light stimulates carotenoid synthesis in plants during photomorphogenesis through the expression of PHYTOENE SYNTHASE (PSY), a key gene in carotenoid biosynthesis. The orange Daucus carota (carrot) synthesizes and accumulates high amounts of carotenoids in the taproot that grows underground. Contrary to other organs, light impairs carrot taproot development and represses the expression of carotenogenic genes such as DcPSY1 and DcPSY2 reducing carotenoid accumulation. By means of an RNA-seq, in previous analysis we observed that carrot PHYTOCHROME RAPIDLY REGULATED 1 (DcPAR1) is more expressed in the underground grown taproot respect to those grown in light. PAR1 is a transcriptional cofactor with a negative role in the shade avoidance syndrome regulation in Arabidopsis thaliana through the dimerization with PHYTOCHROME INTERACTING FACTORs (PIFs), allowing a moderate synthesis of carotenoids. Here we show that overexpressing AtPAR1 in carrot produces an increment of carotenoids in taproots grown underground as well as higher DcPSY1 expression. The high identity of AtPAR1 and DcPAR1 let us to suggest a functional role of DcPAR1 that was verified through the in vivo binding to AtPIF7 and the overexpression in Arabidopsis, where it increments AtPSY expression and carotenoid accumulation together with a photomorphogenic phenotype. Finally, DcPAR1 antisense carrot lines presented a dramatic decrease in carotenoids levels and in the relative expression of key carotenogenic genes as well as impairment in taproot development. These results let us to propose that DcPAR1 is a key factor for secondary root development, plastid differentiation and carotenoid synthesis in carrot taproot grown underground.One-sentence summaryDcPAR1 is a key factor for secondary root development, plastid differentiation and carotenoid synthesis in carrot taproot grown underground.

Transcriptomic and metabolic analysis uncovers the role of light quality in carotenoid accumulation of grapefruit during ripening

Light, a crucial environmental signal, is involved in the regulation of secondary metabolites. To understand the mechanism by which light influences carotenoid metabolism, grapefruits were bagged with four types of light-transmitting bags that altered the transmission of solar light. We showed that light-transmitting bagging induced changes in carotenoid metabolism during fruit ripening. Compared with natural light, red light (RL)-transmittance treatments significantly increased the total carotenoid content by 142%. Based on weighted gene co-expression network analysis (WGCNA), ‘red’, ‘darkred’, ‘yellow’, ‘brown’ and ‘midnightblue’ modules were remarkably associated with carotenoid metabolism under different light treatment. Transcriptome analysis identified the transcription factors (TFs) bHLH74/91/122, NAC56/78/90/100, MYB/MYB308, WRKY7/55, MADS29/AGL61, ERF043/118 as being involved in the regulation of carotenoid metabolism in response to RL. Under RL treatment, these TFs regulated the accumulation of carotenoids by directly modulating the expression of carotenogenic genes, including PSY, Z-ISO2, ZDS6, LCYB, LCYE, CHYB, CCD1-1/1-3, CCD4-2 and NCED2/3. Based on these results, a network of the regulation of carotenoid metabolism by light in citrus fruits was preliminarily proposed. These results showed that RL treatments have great potential to improve coloration and nutritional quality of citrus fruits.

Evaluation of Carotenoids Accumulation and Biosynthesis in Two Genotypes of Pomelo (Citrus maxima) during Early Fruit Development

Pomelo is rich in bioactive compounds (carotenoids, phenolics and essential oil) in the early stage of fruit development, but it is often wasted in the cultivation and management process. To gain an insight into the carotenoid metabolism pathway in pomelo, the carotenoid profiles and the expression patterns of carotenogenic genes were investigated in two genotypes of pomelo during early fruit development. The results showed that a higher carotenoid content was observed in honey pomelo as compared with golden pomelo, which may be related to different gene regulation mechanisms. Lutein, a-carotene, and β-carotene were the main carotenoids in pomelo young fruit, and lutein was the highest one. The accumulation of carotenoids during fruit early development in honey pomelo is related to the transcriptional regulation of ZISO and LUT5. In golden pomelo, the rate-limiting gene for carotenoids is PDS and ZDS. In addition, the expression of seven genes except CRTISO in honey pomelo was higher than that in golden pomelo. The results are helpful to further clarify the regulatory mechanism of carotenoid accumulation during early fruit development and provide a direction for the high-value utilization of young fruits in pomelo.

A fruit ripening-associated transcription factor CsMADS5 positively regulates carotenoid biosynthesis in citrus

Carotenoids in citrus contribute the quality of the fruit, but the transcriptional regulatory mechanism is still limitedly known. Here, we characterized a citrus FUL-like MADS gene, CsMADS5, that was ripening-inducible and acted as a nucleus-localized trans-activator. Transient overexpression of CsMADS5 in citrus induced fruit coloration and enhanced carotenoid contents. The expression levels of carotenogenic genes including phytoene synthase (PSY), phytoene desaturase (PDS), and lycopene β-cyclase 1 (LCYb1) were significantly increased in the peel of fruits overexpressing CsMADS5. Similar results were observed from stable overexpression of CsMADS5 in tomato fruits and citrus calli, even though the effect of CsMADS5 on the carotenoid metabolism in transgenic citrus calli was limited. Further biochemical analyses demonstrated that CsMADS5 activated the transcription of PSY, PDS, and LCYb1 by directly binding to their promoters. It is concluded that CsMADS5 positively regulates carotenoid biosynthesis in fruits by directly activating the transcription of carotenogenic genes. Moreover, CsMADS5 physically interacted with a positive regulator CsMADS6, indicating that CsMADS5 may form an enhancer complex with CsMADS6 to synergistically promote carotenoid accumulation. These findings expand our understanding of the complex transcriptional regulatory hierarchy for carotenoid biosynthesis during fruit ripening.

A citrus phosphate starvation response factor CsPHL3 negatively regulates carotenoid metabolism

Carotenoids provide precursors for the biosynthesis of strigolactones (SLs), which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was up-regulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene β-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents, elevated expression of LCYb genes but downregulation of other key carotenogenic genes including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

Metabolic engineering of Saccharomyces cerevisiae for production of β-carotene from hydrophobic substrates

β-Carotene is a yellow-orange-red pigment used in food, cosmetics, and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB, and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained on mineral medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.