CHEN Xiao-dong,CAl Wei-jian,XlA Jin,YUAN Hua-zhao,WANG Qing-lian,PANG Fu-hua,ZHAO Mizhen

Institute of Pomology, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210000, P.R.China

Abstract Carotenoids are an important component of the human diet,and fruit is a primary source of carotenoids.The synthesis and regulation of carotenoids in fruit are important contributors to the formation of fruit quality.In China,strawberry is one of the main seasonal fruits grown in the winter.Previous studies have shown that light has a significant effect on the metabolism of anthocyanins,sugars,and polyphenols in strawberry.However,the understanding of the role of light in regulating the metabolism of carotenoids in strawberry remains limited.This study investigated the effects of blue,red,yellow-green,and white light on carotenoid metabolism in strawberry.Blue light treatment promoted the synthesis of multiple carotenoids,including lutein,compared with the other three treatment groups.The RNA sequencing data revealed that blue light treatment promoted the expression of lycopene ε-cyclase (LCYE),and the transient overexpression of LCYE in strawberry fruit promoted lutein accumulation in strawberry.Overall,the results suggest that blue light can promote the synthesis of lutein in strawberry by inducing the expression of LCYE.

Keywords: carotenoid,LED light,strawberry,lutein

1.lntroduction

Strawberry is a highly popular fruit among consumers.It is rich in vitamins,ellagic acid,and various micronutrients,making its fruits a nutritious food source for humans.Strawberry has a high production value,with a global industry value of approximately 17 billion USD (Afrinet al.2016;Warneret al.2021;Chonget al.2022).Producing strawberries with higher nutritional value can satisfy the demand for high-quality fruit and enhance the economic benefits of strawberry cultivation.

Carotenoids,a class of terpenoid pigments with C40 backbones,are beneficial to human health (Renet al.2021).Studies have revealed that the dominant carotenoids in strawberries are lutein and β-carotene (Zhuet al.2015).Lutein,a fat-soluble pigment belonging to the carotenoid family,is an important precursor of vitamin A synthesis and is the main component of human retinal macular pigment (Gazzoloet al.2021;Mitraet al.2021).In higher plants,lutein synthesis mainly occurs in plastids.In plastids,two molecules of geranylgeranyl diphosphate(GGPP) synthesize phytoene under the action of phytoene synthase (PSY) (Nisaret al.2015).Then,lycopene is gradually produced through the catalysis of phytoene desaturase (PDS),ζ-carotene desaturase (ZDS),and carotenoid isomerase (CRTISO) (Sunet al.2018).Lycopene is catalyzed by lycopene ε-cyclase (LCYE)and lycopene β-cyclase (LCYB) to produce α-carotene and β-carotene (Ronenet al.2000;Ohmiyaet al.2019).Carotene is catalyzed by β-hydroxylase (CHYb) and ε-hydroxylase (CHYe) to produce lutein (Tianet al.2003;Ohmiyaet al.2019).

Light is the most important environmental factor affecting carotenoid synthesis in plants (Sunet al.2018).Many studies have shown that light quality has a great impact on carotenoid metabolism (Stanley and Yuan 2019).Studies on carotenoid metabolism in ‘Jinli’peach have found that blue light treatment promotes the accumulation of lutein,β-cryptoxanthin,lutein,zeaxanthin,and β-carotene during storage (Caoet al.2017).It has also been found that blue light treatment can promote chlorophyll synthesis and induce regreening in the flavedo of citrus and can also promote the synthesis of lutein,β-carotene,and all-trans-violaxanthin(Maet al.2021).In China,strawberry production is primarily carried out in the winter.However,in many areas,the light in winter is insufficient for strawberry growth and development,so supplementation with artificial light is necessary.

Exogenous light supplementation can increase the yield and promote the synthesis of anthocyanins in strawberry (Zhanget al.2018).In addition,a previous study by this research group has shown that blue light can promote the synthesis of chlorogenic acid in strawberry(Chenet al.2020).However,it is still unknown whether light quality affects carotenoid synthesis in strawberry.In the present study,metabolomics and transcriptomics were used to study the effects of four light qualities on carotenoid metabolism in strawberry.These findings offer a new research avenue for exploring the role of light regulation in lutein synthesis in strawberry and provide technical support for the scientific use of LED lights in strawberry production.

2.Materials and methods

2.1.Plant materials

The material used in this study was the octoploid strawberry ‘Ningyu’,which was planted in 12 cm×15 cm pots supplemented with a mixture of nutrients.The matrix was composed of peat,vermiculite,and perlite (2:1:1).The strawberry seedlings were fertilized twice a month with Yamasaki nutrient solution and watered twice a week.When the plants in the receptacles developed to the big green stage,strawberry seedlings with similar growth states were randomly selected for the light treatment experiment.Strawberries used for instantaneous transformation were cultivated in a greenhouse under natural light conditions.

2.2.Light treatment

Light intensity was maintained at 150 μmol m?2s?1,and the light quality treatments included blue (480 nm),red(660 nm),yellow (590 nm)-green (520 nm),and white light(Appendix A).The photoperiod was 10 h of light and 14 h of darkness.Completely red fruits were selected from three biological replicates (10 fruits for each replicate)of each treatment.The strawberry fruits ripened fastest under the white light treatment (about 12 days after the light treatment),and the ripening times under the red light and blue light treatments were similar (about 15 days after the light treatment).The fruit ripening time for the yellow-green light treatment group was about 26 days.The fruit was cut into small pieces,frozen immediately in liquid nitrogen,and stored at ?80°C for carotenoid and transcriptome detection.

2.3.Carotenoid determination

The plant materials were homogenized and powdered in a mill.Then,50 mg of dried powder was extracted with a mixed solution of N-hexane:acetone:ethanol (2:1:1)containing 0.01% butylated hydroxytoluene (BHT).The extract was vortexed for 20 min at room temperature(25°C),and the supernatant was collected.The supernatant was evaporated to dryness under a nitrogen gas stream and reconstituted in methyl tert-butyl ether(MTBE).The solution was filtered through a 0.22-μm filter for further spectrometric analysis.

The sample extracts were analyzed using a liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry (LC-APCI-MS/MS) system.The analytical conditions were as follows:high-performance liquid chromatography (HPLC)column,YMC C30;column temperature,28°C;injection volume,5 μL;mobile phase A,methanol:acetonitrile(3:1;0.01% BHT and 0.1% formic acid);and mobile phase B,methyl tert-butyl ether (add 0.01% BHT).The chromatographic conditions were as follows: flow rate of 300 μL min–1;0 min,100% (A);3 min,100% (A);6 min,58% (A) 42% (B);8 min,20% (A) 80% (B);9 min,5%(A) 95% (B);10 min,100% (A);and 11 min,100% (A).The effluent was alternatively connected to a triple quadrupole-linear ion trap (Q TRAP)-MS API 6500 Q TRAP LC/MS/MS system,equipped with an APCI Turbo Ion-Spray interface,operating in positive ion mode,and controlled using Analyst 1.6.3 Software (AB Sciex).The APCI source operation parameters were as follows:ion source,APCI+;source temperature,350°C;curtain gas (CUR),25.0 psi;and collision gas (CAD),medium.Declustering Potential (DP) and Collision Energy (CE) for individual multiple reaction monitoring (MRM) transitions were performed with further DP and CE optimization.A specific set of MRM transitions was monitored for each period according to the carotenoids eluted within that period.

A MetWare (http://www.metware.cn/) database was constructed based on authentic carotenoid standards(Olchemim Ltd.,Olomouc,Czech Republic;Sigma,St.Louis,MO,USA) for the qualitative analysis of mass spectrometry (MS) data.According to the retention time and ion pair information of the carotenoids,the mass spectrum peaks of each carotenoid detected in the samples were corrected.Different concentrations of a carotenoid standard solution were prepared to obtain the mass spectrum peak intensity data of the corresponding quantitative signal of each concentration standard.The standard curves of different carotenoids were drawn with the standard concentration as the abscissa and the peak area of the mass spectrum peak as the ordinate.The integral area values of the carotenoids detected in all samples were substituted into the linear equation of the standard curve for calculation,and the carotenoid content was finally obtained from the samples.

2.4.RNA-sequencing (RNA-seq)

Total RNA was extracted using a TRIzol reagent kit(Invitrogen,Carlsbad,CA,USA) according to the manufacturer’s protocol.The RNA-seq was performed using the Illumina HiSeq2500 by Gene Denovo Biotechnology Co.,Ltd.(Guangzhou,China).Reads were mapped to the cultivated octoploid strawberry genome (Fragaria×ananassaCamarosa Genome Assembly v1.0.a1) using HISAT2 (Kimet al.2015).The RNA differential expression analysis was performed using DESeq2 Software (Loveet al.2014) between two groups.Genes/transcripts with a false discovery rate (FDR)below 0.05 and absolute fold change ≥2 were considered differentially expressed genes/transcripts.

2.5.Quantitative reverse-transcription PCR (qRTPCR)

The qRT-PCR was conducted as previously described(Chenet al.2020).The qRT-PCR results were presented as the relative transcript levels normalized against the geometric mean ofFaACTIN(Liet al.2016) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(Chenet al.2020).The primers used for qRT-PCR are listed in Appendix B.

2.6.Bioinformatics analysis of the LCYE protein

TheArabidopsisLCYE protein sequence (Lameschet al.2011) was used as the query sequence to conduct BLASTP searches against peach (Prunuspersicav.2.1) (Verdeet al.2013),tomato (SolanumlycopersicumiTAG 2.4) (Tomato Genome Consortium 2012),apple (Malusdomesticav1.1)(Daccordet al.2017),and citrus (Citrusclementinav1.0)(Wuet al.2018) databases using Phytozome 13 (Goodsteinet al.2012).In each,the sequence with the highest score and an E-value of 0 was selected (Appendix C).Phylogenetic tree and protein domain analyses were performed as previously described (Chenet al.2020).

2.7.Gene cloning and Agrobacterium infiltration

The coding sequence ofFaLCYE(Appendix D)was inserted into the pMON530 vector to construct35Spro:LCYE.The empty vectorpMON530or the35Spro:LCYEwas transformed intoAgrobacteriumstrain GV3101.Single colonies were selected and cultured in 5 mL Luria-Bertani (LB) liquid medium (50 μg mL–1spectinomycin,20 μg mL–1gentamicin,and 20 μg mL–1rifampicin) at 28°C (200 r min–1) overnight.Bacterial solution (100 μL) was added to 100 mL LB liquid medium and cultured at 28°C (200 r min–1) for 14–16 h.The cells were collectedviacentrifugation and resuspended in infiltration buffer (10 mmol L?1MgCl2,10 mmol L?1MES,and 20 μmol L?1acetosyringone),adjusted to an optical density (OD600) of 0.8–1.0 and left to stand at room temperature for 3 h.Strawberries in the big green fruit stage were injected with 1 mL ofAgrobacteriumper fruit.After 14 days of transformation,the strawberry fruits were collected for subsequent analysis.

3.Results

3.1.Analysis of the carotenoid content in strawberry fruit under different light-quality treatments

To study the effects of different wavelengths of light on carotenoid metabolism in strawberry fruit,we evaluated the metabolomic changes in carotenoid contents under blue,red,yellow-green,and white light treatments.The ripening times of the strawberry fruits differed under different light treatments.Strawberry fruits that had turned completely red were collected for the metabolomic and transcriptomic detection of carotenoids.As shown in Table 1,16 carotenes were detected in the strawberry fruit,including β-carotene,lutein,and neoxanthin.The content of lutein was the highest,especially under the blue light treatment.The content of lutein in the strawberry fruit reached 10.627 μg g–1fresh weight (FW),which was 7.9 times that of the β-carotene content.

Table 1 Carotenoid content in strawberry under different light treatments (μg g–1 fresh weight)1)

Compared with the blue light treatment,the content of 11 carotenes in the red light treatment decreased significantly.In the yellow-green light treatment,the content of four carotenes decreased significantly,and the content of 13 carotenes decreased significantly in the white light treatment.Among them,the contents of four carotenes,namely lutein,lutein myristate,neoxanthin,and violaxanthin dibutyrate,were significantly higher under blue light treatment than under other treatments(Table 1).These results suggest that blue light treatment can promote the synthesis of various carotenoids in strawberry fruit.

3.2.Analysis of gene expression in strawberry fruit under different light-quality treatments

To further evaluate the mechanism by which light quality influences carotenoids in strawberry fruit,the gene expression in strawberry fruits under different light-quality treatments was investigated using RNA-seq.Blue light treatment resulted in the upregulation of 81 genes and the downregulation of 100 genes compared to the red light treatment (Fig.1-A;Appendix E).Compared to the yellow-green light treatment,the blue light treatment increased the expression of 223 genes and decreased the expression of 318 genes (Fig.1-B;Appendix E).Compared to the white light treatment,the blue light treatment resulted in the upregulation of 996 genes and the downregulation of 626 genes (Fig.1-C;Appendix E).To study the mechanism by which blue light promotes the synthesis of various carotenoids,the upregulated and downregulated genes in the three groups were analyzed using a Venn diagram (Hulsenet al.2008).The results showed that there were 25 upregulated and 15 downregulated genes across three groups,respectively(Fig.1-D and E;Appendix F).We further conducted KEGG enrichment analysis on these co-upregulated and downregulated genes.In KEGG map00906(CAROTENOID BIOSYNTHESIS),LCYE(maker-Fvb1-4-augustus-gene-34.46-mRNA-1) encoding lycopene cyclase was specifically overexpressed in the blue light treatment group (Fig.1-F).Studies have shown that LCYE catalyzes the transformation of lycopene to lutein in the carotenoid metabolic pathway (Hermannset al.2020).

Fig.1 Summary of the RNA-seq data and FaLCYE identification.A–C,volcano plot of differentially expressed genes (DEGs) in a comparison between R-vs.-B (A),YG-vs.-B (B) and W-vs.-B (C).B,R,YG,and W represent blue,red,yellow-green,and white light,respectively.FC,fold change;FDR,false discovery rate.The upregulated,downregulated,and unchanged unigenes are dotted in red,blue,and grey,respectively.D and E,Venn diagram analysis of upregulated (D) and downregulated (E) genes among the three different groups.F,detailed diagram of carotenoid biosynthesis.Enzymes with enhanced expression during the blue light treatment are shown in red.G,genetic evolution analysis of six LCYE proteins retrieved from Arabidopsis thaliana (At),Prunus persica (Pp),Solanum lycopersicum (Sl),Malus domestica (Md),Citrus clementina (Cit) and Fragaria ananassa (Fa).The protein motifs of LCYE are shown below the graph and are denoted by rectangles of different colors.The sequences of these motifs are shown in Appendix G.

This work further analyzed the conserved domain and genetic evolution of the LCYE protein in seven different species.The LCYE proteins in strawberry,apple,and peach,all of which belong to the Rosaceae family,were closely related (Fig.1-G).MEME (Baileyet al.2015)was used to analyze the domains of the LCYE proteins in different species.Seven protein domains were identified in the LCYE proteins of these different species,indicating that the LCYE protein is highly conserved in different species (Fig.1-G).

3.3.FaLCYE expression was induced by blue light

The transcriptome results showed that the expression ofFaLCYEin strawberry fruit under blue light treatment was significantly higher than that under the red,yellow-green,and white light treatments (Fig.2-A).To further validate the transcriptome analysis results,qRT-PCR was used to verify the expression ofFaLCYEin strawberry.The results were highly consistent with the transcriptome data.FaLCYEexpression under the blue light treatment was 1.9,1.7,and 1.7 times higher than that in the red,yellowgreen,and white light treatments,respectively (Fig.2-B).

Fig.2 Effects of blue,red,yellow-green,and white light on FaLCYE expression in strawberry.A,schematic diagram of carotenoid metabolism and FaLCYE expression based on RNA-seq data represented by a heat map.B,the quantitative reverse transcription PCR (qRT-PCR) analysis of FaLCYE expression under different light-quality treatments.B,R,YG,and W represent blue,red,yellow-green,and white light,respectively.Data represent the mean±standard error (n=3).＊＊,P<0.01 in a two-sided Student’s t-test with the control.

3.4.FaLCYE overexpression promoted lutein synthesis in strawberry

To study the biological function ofFaLCYEin strawberry,FaLCYEwas transiently overexpressed in the strawberry fruit.The qRT-PCR results showed that the expression ofFaLCYEin the transiently transformed strawberry fruit35Spro:LCYE-OE#1and35Spro:LCYE-OE#2was 219 and 184 times higher,respectively,than that of the control(Fig.3-A).Furthermore,the lutein content in these transiently transformed strawberry fruits increased by 3.9 and 2.4 times for35Spro:LCYE-OE#1and35Spro:LCYEOE#2,respectively (Fig.3-B).These results indicate thatFaLCYEcan promote lutein synthesis in strawberry fruit.

Fig.3 Effects of FaLCYE overexpression on lutein metabolism.A,expression level of FaLCYE in transiently transformed fruit.B,analysis of the lutein content in transiently transformed fruit.Data represent the mean±standard error (n=3).The individual values are indicated by black squares.＊＊,P<0.01 in a two-sided Student’s t-test with the control.

4.Discussion

The light quality,light intensity,and photoperiod have important effects on plant growth and secondary metabolism (Alrifaiet al.2019;Warneret al.2021).To reveal the effects of different LED lights on the growth and development of strawberry,we conducted a series of experiments to examine the effects of light quality on the metabolism of strawberry.Previous studies have found that blue light treatment promoted chlorogenic acid synthesis in strawberry (Chenet al.2020).The present study compared the differential metabolites and transcripts in the blue light treatment with the other three treatment groups (red light,yellow-green light,and white light),which found that blue light treatment induced lutein synthesis in strawberry.

Lutein has antioxidant effects,can reduce the occurrence of skin wrinkles or pigmentation,and can prevent age-related macular degeneration and Alzheimer’s disease (Bhat and Mamatha 2021).Lutein can accumulate in the human retina,filter blue light,and protect vision (Renet al.2021).Lutein metabolism in plants is regulated by a variety of environmental factors(Renet al.2021).Blue light treatment can promote lutein synthesis in peach and microgreens (Caoet al.2017;Samuolien?et al.2017),which is consistent with our findings in strawberry.These studies suggest that the promotional effect of blue light on lutein synthesis is present in a variety of plant species.

The expression of many genes involved in the carotenoid metabolic pathway is regulated by light(Hermannset al.2020).In this study,the expression ofFaLCYEwas induced by blue light.Studies on peach and citrus have also revealed that blue light treatment can induceLCYEexpression (Caoet al.2017;Maet al.2021).These studies suggest that the promotion ofLCYEexpression by blue light may be a conserved mechanism.

Previous studies have shown that multiple members of the light-signaling pathway can regulate carotenoid metabolism (Hermannset al.2020).Phytochrome interacting factors (PIFs) are basic helix-loop-helix (bHLH)family transcription factors that interact with photoreceptor phytochrome proteins (Paiket al.2017).Studies inArabidopsisand tomato have shown that PIF proteins bind to the G-box element of thePSYpromoter of the carotenoid metabolic pathway and inhibitPSYexpression (Toledo-Ortizet al.2014;Llorenteet al.2016;Bianchettiet al.2018).In addition to the PIF protein,ELONGATED HYPOCOTYL 5(HY5),a positive regulator of the light-signaling pathway,can bind to the G-box element of thePSYgene promoter and promote the expression of thePSYgene (Toledo-Ortizet al.2014;Stanley and Yuan 2019).HY5 and PIFs are key factors of the light signal transduction pathway,among which HY5 is a positive regulator and PIFs act as negative regulators (Paiket al.2017;Balcerowicz 2020).Studies have shown that blue light treatment can promote the expression ofHY5and inhibit the protein accumulation of PIFs (Paiket al.2017;Xiaoet al.2022).These studies indicate that different light qualities can regulate carotenoid synthesis by regulating its downstream signal pathway members.However,research on the regulation of lycopene cyclase (LCYE and LCYB) coding genes by members of the light signaling pathway is limited.

LCYE is the key enzyme of lutein metabolism and mediates the cyclization of lycopene to produce α-carotene,which is subsequently converted into lutein(Ohmiyaet al.2019;Hermannset al.2020).In tomato andChlamydomonasreinhardtii,LCYEoverexpression promotes lutein synthesis (Tokunagaet al.2021;Yuanet al.2022).The downregulation ofLCYEin apples dramatically decreases the lutein content (Ampomah-Dwamenaet al.2012).In strawberry,lutein accumulation has been associated with a high transcriptional level ofLCYE(Zhuet al.2015).Transiently overexpressingLCYEin strawberry fruit promoted lutein synthesis,suggesting that lutein metabolism can be regulated byLCYEexpression.This provides a theoretical basis for cultivating lutein-rich strawberry varieties in the future.

5.Conclusion

This study found that blue light promoted lutein synthesis andFaLCYEexpression,compared with red,yellowgreen,and white light.The transient overexpression ofFaLCYEin strawberry fruit promoted lutein synthesis.These results suggest that blue light can promote lutein synthesis by inducingFaLCYEexpression.This research revealed the mechanism of blue light regulation of lutein synthesis and provided a theoretical basis for the design of efficient LED lighting for use in strawberry production.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31901996),the Natural Science Foundation of Jiangsu Province,China (BK20190264),and the Major Agricultural New Varieties Creation Project of Jiangsu Province,China (PZCZ201721).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.04.002