Expression of ZjPSY, A Phytoene Synthase Gene from Zoysia Japonica Leads to Leaf Yellowing and Plant Dwarfing in Arabidopsis Thaliana

Expression of ZjPSY, A Phytoene Synthase Gene from Zoysia Japonica Leads to Leaf Yellowing and Plant Dwarfing in Arabidopsis Thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of ZjPSY , A Phytoene Synthase Gene from Zoysia Japonica Leads to Leaf Yellowing and Plant Dwarfing in Arabidopsis Thaliana Di Dong, Ke Teng, Penghui Tan, Zhuocheng Liu, Zhuoxiong Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-349623/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Phytoene synthase (Psy) is a key limiting enzyme in the carotenoid biosynthesis pathway by regulating phytoene synthesis. In this study, ZjPSY was isolated and identified from an important lawn grass species, Zoysia japonica . ZjPSY cDNA was 1230 bp in length, corresponding to 409 amino acids. ZjPSY showed higher expression in young leaves and were down-regulated after GA3, ABA, SA, and MeJA treatments, exhibited a sensitivity to hormones. By analysis of cis-regulatory elements in ZjPSY promoter region, ZjPSY exhibited be regulated of light and multiple hormones. To investigate the functions of ZjPSY , the plant expression vector was constructed to obtain transgenic Arabidopsis thaliana . Overexpression of ZjPSY protein led to carotenoid accumulation and altered expression of genes involved in carotenoid contents. ZjPSY expressing Arabidopsis thaliana exhibited yellowing and dwarfing phenotypes and contained more carotenoids than the wild type. Yeast two-hybrid screening identified a novel interacting partner of ZjPSY , ZjJ2 ( DNAJ homologue 2 ), which encodes heat-shock protein 40 (HSP40). Taken together, this study suggests that ZjPSY plays an important role in carotenoid synthesis, leaf color development and hormone response in transgenic plants. These results broadened the understanding of carotenoid synthesis pathways and laid a foundation for the exploration and utilization of PSY gene. Plant Physiology and Morphology Plant Molecular Biology and Genetics Carotenoids Phytoene synthase ZjPSY Zoysia japonica Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction In plants, carotenoids are a class of hydrocarbons that serve as secondary metabolites of plant photosynthesis, which play classical roles in photosynthetic biological processes, including photomorphogenesis and photoprotection. Carotenoids also participate in plant color formation as pigments, which affect plant color through different levels of aggregation in the chromoplasts (Briardo Llorente and Rodriguez-Concepcion 2017). Carotenoids range in color from colorless to yellow and red and are reflected in the fruits and leaves of many plants(Nisar, et al. 2015). In addition to that, carotenoids and their oxidative and enzymatic lysates are considered to be signaling molecules for interactions of plants with environment, so carotenoids are proposed to play an important regulatory role in plant growth and development (Walter and Strack 2011). Phytoene is a precursor for the synthesis of all carotenoid substances. Carotenoids also act as precursors for gibberellin and ABA (Abscisic acid), hormones that play an important role in plant growth and development (Colasuonno, et al. 2017). Phytoene synthase ( PSY ), a key enzyme regulating carotenoid synthesis, can catalyze the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene, which is the first rate-limiting reaction in the carotenoid synthesis pathway (fig.1). PSY is a preferred candidate gene for understanding the molecular regulation of carotenoid accumulation in most species (Obrero, et al. 2015). Transgenic tobacco with overexpression of PSY significantly increased carotenoid content, and Transgenic autumn olive fruits ( Elaeagnus umbellata ) confirmed that EUTPSY played a key role in lycopene accumulation(Busch, et al. 2002; Wang, et al. 2020). Expression of PSY from Oncidium Gower Ramsey in Tobacco showed dwarfing and reduced leaf area phenotypes (Wen-Li Lee 2012). Zoysia japonica is one of the common warm-season turfgrass in the world. Due to its strong drought resistance, salt resistance and barren resistance, it has been widely used in golf courses, sports grounds and urban greening. The regulation mode of carotenoid accumulation in plant cells and the regulation mechanism of ZjPSY gene have not been studied in Zoysia japonica . In this study, ZjPSY gene of Zoysia japonica was isolated and identification to further explore. Although the genes involved in the carotenoid biosynthesis pathway have been identified, the regulation of these genes in plant growth and development and stress tolerance is not fully understood. This study could further explore the function of PSY gene, and lay a foundation for studying the role of carotene in Zoysia and improving the utilization of high-quality gene resources in turfgrass. Materials And Methods Plant Materials and Growth Conditions Zoysia japonica cultivar ‘Compadre’ seeds were purchased from the Hancock seed company (Hancock, Florida, USA). Zoysia japonica were cultivated in pots，kept at 28/23℃(day/night) with 16h (at 400 mmol/m2/s) /8h photoperiod and 50% humidity. All the Arabidopsis materials used in this study are in Col ecotype background. The Arabidopsis seeds were sown on Murashige and Skoog plates (4.43 g/L Murashige and Skoog powder, 8 g/L agar, pH 5.8), with a 16h white light (at 90 mmol/m2/s)/8h dark cycle and 50% humidity at 25°C. After the fourth leaves appeared on the seedlings, the seedlings were transferred to sterilized soil or culture medium to continue growth under the same growth conditions. Identification and Cloning of ZjPSY Total RNA was isolated from ‘Compadre’ leaves of 3-month-old with a plant RNA isolation kit (Promega, USA), and first-strand cDNA was synthesized with a reverse transcription kit (Takara, Dalian, China). Primer sequences for cloning were designed based on the sequence fragment of PSY and PCR products were ligated into the cloning vector pDM19-T for storage and further experiments. Bioinformatics analysis BLAST analysis of the NCBI database was used to identify homologs and 18 PSY protein sequences from other species were obtained. The phylogenetic tree and ZjPSY gDNA structure were constructed using MEGA version 6.0 with the neighbor-joining method and GSDS 2.0 (http://gsds.gao-lab.org/Gsds_about.php)(Tamura, et al. 2011). The molecular weights and theoretical isoelectric points were analyzed by Compute pI/MW tool (http://web.expasy.org/compute_pi/). The analysis of motif in PSY amino acids and cis-regulatory elements in promoter used MEME (http://meme-suite.org/tools/meme), PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and TBtools(Chen, et al. 2020). ZjPSY protein model was generated by using Phyre v2.0 tool (www.sbg.bio.ic.ac.uk/phyre2/). Vector construction and generation of transgenic plants The completed coding region of ZjPSY was amplified by PCR with primers ZjPSY-F and ZjPSY-R (Supplementary Table S1). The fragment containing the complete CDS was ligated to the vector plasmid pMD19-T, and stored in E. coli for long-term preservation. The 35S:: ZjPSY :YFP was constructed by fusing completed ZjPSY CDS followed by YFP with primers, 3302Y-ZjPSY-F and 3302Y-ZjPSY-R. The recombination was introduced into Agrobacterium tumefaciens GV3101, which was then used for the transformation of Arabidopsis thaliana through the floral dip method(Clough and Bent 1998). Transgenic plants were selected using 2 mg·L -1 glufosinate and verified by PCR. T 3 transgenic lines were generated by self-pollination for subsequent experiments. Stress tolerances of Transgenic Plants Three different transgenic lines with high expression levels of PSY were selected. The Wild-type and Transgenic seedlings were cultivated on 1/2 MS medium containing 500 mM mannitol or 200 mM NaCl for 30 days under conditions of 26/20℃ (day/night), 16/8 h (light/dark), and humidity of 70%. Total RNA was isolated from each sample using Plant RNA Kit (Omega Bio-tek, USA) and the first-strand cDNA was synthesized using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, China). The First-strand cDNA was used for real-time quantitative RT-PCR (qRT-PCR) analysis. Expression levels of PSY gene and pigment-related genes Quantitative real-time PCR was carried out to measure the expression levels of six pigment-related genes from different samples with the 2 - △△Ct method, following the manufacturer’s procedures of GreenTB Premix Ex Taq II (takara). The AtUBQ10(NM_116771) gene was used as an internal reference gene for evaluating transcriptional abundance in Arabidopsis. Pigment-related genes include AtCLH (AT1G19670.1), AtNYC (AT4G13250.1), AtNOL (AT5G04900.1), AtPAL (AT5G04230), AtNCED (AT1G30100) and AtZDS (AT3G04870). Relevant primer sequences used were shown in Supplementary Table S1. All experiments in the research contained three biological replicates. Quantitative real-time PCR was carried out to measure the expression levels of ZjPSY in and different developmental stages, tissues of zoysiagrass and leave subjected to normal, hormone treatment, drought, or salt stress conditions. The 2-month-old zoysiagrass were inducted with 10 μM GA 3 , 10μM ABA, 10 μM MeJA, or 0.5 mM SA. Leaves at different growth stages (young, fast-growing, and mature), roots tissues and stems tissues were also collected. RNA isolation and cDNA synthesis were performed as above. The Z. japonica beta-actin was used as the internal reference gene (GenBank accession No. GU290546). Relevant primer sequences used were shown in Supplementary Table S1. All experiments in the research contained three biological replicates, Yeast two-hybrid assay The ZjPSY ORF’s were cloned using the primers pGBKT7-ZjPSY-F/R and inserted into pGBKT7 vector, which was co-transformed into yeast strain Y2H as described in Yeastmaker™ Yeast Transformation System (Clontech, USA). The MATCHMAKER GAL4 yeast two-hybrid system was performed according to the Matchmaker™ Gold Yeast Two-Hybrid System manufacturer's instructors (Clontech, USA). Results 2.1. Identification and bioinformatic analysis of the ZjPSY Gene in Zoysia japonica According to Zoysia Genome Database, the DNA fragment containing ZjPSY Coding Sequences (CDS) was cloned and ligated into cloning vector pMD19-T. The ZjPSY cDNA sequences were deposited in the NCBI database with accession numbers KY264127.1. The full-length ZjPSY cDNA contains a 1,230 bp ORF encoding a protein of 409 amino acids, and it belongs to the Isoprenoid_Biosyn_C1 Superfamily. According to the compute pI/MW tool of Expasy, the molecular weight of ZjPSY was 46.39 kD, and the theoretical isoelectric point was 9.20. Intron/Exon organization analysis showed that ZjPSY had five exons (Fig.1A). Secondary structure analysis represents that the ZjPSY have 63% alpha helix, 1% beta strand, and 25% disorder proteins (Fig.1B). Modelled 3D Tertiary structure of ZjPSY was provide insights for molecular geometry (Fig.1C). The stereochemical structure supported the α-helical nature of the enzyme. Phylogenetic analysis of PSY proteins was performed based on the protein sequences from different species collected from NCBI database, with a neighbor-joining method (Fig. 2A). Phylogenetic analysis of the PSY protein revealed that ZjPSY has the highest homology with PSY of Zoysia matrella . Except that, it was most closely related to Salvia splendens . The PSY proteins in 18 PSY protein of different species were further analysed for the occurrence of conserved motifs (Fig. 2B). Motif sequence scan analysis showed the presence of amino-acid rich profiles, top scored three motifs was found in all 18 PSY proteins (Fig. 2C). the conserved motifs were indicated to be highly conserved. Phylogenetic analysis and motif analysis revealed that ZjPSY had a low similarity and different topology with PSY from Arabidopsis thaliana or Oryza sativa. 2.2 Prediction of cis ‑regulatory elements of ZjPSY promoter To analyze the regulatory pathway and function of ZjPSY, a 2000bp upstream sequence from the translation start site (ATG) was examined for promoter elements (Fig.3). The cis-regulatory elements were predicted using the PlantCARE database (Lescot, et al. 2002). Multiple light responsive motifs, including I-box, G-box, TCCC-motif, chs-CMA2a and CAT-box, were identified in the frequency of occurrence of cis-elements. Gibberellin responsiveness P-box, salicylic acid responsiveness TCA-element, MeJA-responsiveness TGACG-motif and CGTCA-motif were identified. In addition, several cis-acting elements involved growth and development were identified, such as endosperm expression element GCN4_motif, zein metabolism regulation element O2-site. It was also possible to recognize ARE (anaerobic induction element). 2.3 ZjPSY can bind with DNAJ homologue 2 in yeast To analyze the protein interacting of ZjPSY, yeast two-hybrid assays were performed. The screened colonies were patched onto higher stringency selective QDO/X/A plates (fig 4a). The J2 gene in zoysia was identified, and it had high homology with J2 in Arabidopsis. In Arabidopsis, J2 encodes heat-shock protein 40 (HSP40) (Barghetti, et al. 2017; Zhou, et al. 1995). The results show the plasmids pGADT7 containing ZjJ2 and the pGBKT7- ZjPSY plasmid grew well and turned blue on QDO/X/A plates, similar to the positive control. These results indicate that ZjJ2 protein interact with ZjPSY protein. 2.4 Expression pattern of ZjPSY Real-time PCR was conducted to examine the expression pattern of ZjPSY in different tissues and development stages. The expression profiles results showed that ZjPSY genes were detected in all tissues tested, and ZjPSY was differentially highly expressed in leaf(fig.5A). Its gene expression was relatively high in early leaf growth stage, and relatively small in fast-growing and mature leaves (fig.5B). ZjPSY were down-regulated after multiple hormone treatments, including GA 3 , ABA, SA, and MeJA (fig.5C-F). Treated with these four hormones, its gene expression began to decline and reached its lowest level at 3 h, then goes back to normal levels at the later stage. The expression pattern suggested that ZjPSY may play a pivotal role in leaf development and hormone response. 2.5 Chlorophyll and carotenoid contents of transgenic Arabidopsis plants and the wild-type To investigate whether ZjPSY expression affects chlorophyll and carotenoid contents in Arabidopsis thaliana , genetic transformation was conducted using Agrobacterium-mediated method. Independent transgenic lines were obtained and further verified by PCR amplification. Three positive transgenic lines designated 9#, 10#, 34# were selected for further analyses. ZjPSY transcription levels and chlorophyll and carotenoid contents were detected in the three transgenic lines and wild-type plants (fig 6). The lines with the highest gene expression showed high carotenoid content levels and low chlorophyll content levels, which indicates that alteration of expression affects carotenoid content and chlorophyll content. 2.6 ZjPSY Expression in Arabidopsis thaliana resulted in leaf yellowing and plant dwarfing In order to study the gene function of ZjPSY gene, four plants from different lines were cultured in each pot. The results showed that the ZjPSY -overexpressed plants had shorter plant height and yellowed leaves compared with the wild type (fig 7). After comparison, transgenic plants showed obvious yellowing phenotype (fig 7C). These results indicate that ZjPSY may play important roles in pigment accumulation and leaf yellowing. To further detect the transgenic plants, the three selected transgenic lines and the wild type were transplanted into MS medium. The plant height of the three transgenic lines was significantly lower than that of the wild type, and the two lines with higher ZjPSY expression showed significant dwarfed phenotypes (fig 8). The transgenic plants were less than 82% in height compared with wild type (Fig. 8B). These results indicated that expression of ZjPSY in Arabidopsis thaliana significantly increased carotenoid contents and decreased chlorophyll contents. 2.7 Regulation of pigment-related genes expression in transgenic Arabidopsis plants and the wild-type under drought treatment and salt treatment In further experiments, Differences between transgenic plants and wild types under drought and salt treatment were studied. Wild-type and transgenic lines were placed on MS agar plates containing 500 mM mannitol or 200 mM NaCl. Different from the normal condition, there was no significant dwarfing phenotype and resistance increase observed in the transgenic plants treated with salt and drought. The transgenic plants showed premature senescence under drought treatment. Under drought treatment, the chlorophyll content of the transgenic plants did not show similar performance with that of the normal treatment. The chlorophyll content of transgenic plant 34#, 10# was not significantly different from that of wild type, but the chlorophyll content of transgenic plant #10 was lower than that of wild type (fig. 9C). The carotenoid level of the transgenic plants was all lower than that of the wild type (fig. 9E). The results suggests that expression of ZjPSY may inhibit carotenoid synthesis under drought treatment. Under salt treatment, the chlorophyll content of transgenic plants 34# and 10# was higher than that of wild type (fig. 9D), the content of carotenoid in transgenic plants was significantly higher than that in wild type, which was similar to that in normal treatment (fig. 9F). In plants subjected to salt stress, expression of ZjPSY can enhance carotenoid synthesis. 2.8 Regulation of pigment-related genes expression in transgenic Arabidopsis plants and the wild-type under drought treatment and salt treatment To gain more insight into the role of ZjPSY in plant growth and development, the expression profile of six pigment-genes, including AtCLH , AtNYC , AtNOL , AtPAL , AtNCED and AtZDS , were carried out using qRT-PCR (fig 11.). Chlorophyllase ( CHL ) catalyzes the hydrolysis of ester bonds to chlorophyllide and phytol, which is the first step of chlorophyll degradation (Tsuchiya, et al. 1999). NON-YELLOWCOLORING ( NYC ) and NYC1-LIKE ( NOL ) are involved in chlorophyll band light-harvesting complex II degradation (Jia, et al. 2015; Morita, et al. 2009; Sato, et al. 2009). Phenylalanine ammonia-lyase ( PAL ), a key enzyme in the phenylalanine pathway, catalyzes the deamination of phenylalanine into trans-cinnamic acid, which is related to anthocyanin synthesis(Yu, et al. 2020). 9-cis-epoxycarotenoid dioxygenase ( NCED ), a kind of carotenoid cleavage enzyme, is a key enzyme that regulates ABA biosynthesis under stress (Espasandin, et al. 2014) . Z-carotene desaturase ( ZDS ) plays a regulatory role in the catalysis of Z-carotene to tetra-cis lycopene, and is also an important gene in the production of carotenoids (Cazzonelli 2011). Under normal conditions, the relative expression levels of several pigment-related genes measured in transgenic plants were lower than those in wild type. In addition to AtCLH and AtPAL , there was no significant difference in the relative expression levels of other measured genes between wild-type and transgenic plants under drought treatment. Discussion PSY gene regulates the first committed step in the process of carotenoid biosynthesis. In this study, ZjPSY gene was cloned from Zoysia sulcus. Bioinformatics analysis showed that the open reading frame of ZjPSY gene was 1230bp, encoding 409 amino acids. The protein belongs to the isoprenoid_biosyn_C1 superfamily. The large central cavity formed by the antiparallel α helixes and the two aspartic acid rich regions in the opposite wall constitute its catalytic sites. Structural analysis shows that ZjPSY contains lots of α helixes and its three-dimensional three-dimensional analog structure contains a large central cavity. Phylogenetic analysis of PSY proteins with a neighbor-joining method revealed that ZjPSY has the highest homology with PSY of Zoysia matrella , and ZjPSY had a low similarity with PSY in model plant Arabidopsis thaliana and Oryza sativa . The expression pattern results suggest that ZjPSY may play an important role in early leaf growth and hormone response. ZjPSY might be highly sensitive to hormone signaling, it was significantly upregulated under GA, ABA, SA and MeJA treatment. These results indicated it were involved pathways of different hormone signal response. The regulatory element analysis of the ZjPSY promoter contains of a variety of hormone responsive elements. To investigate the functions of ZjPSY in plants, the 35S:: ZjPSY :YFP was constructed and transformed to Arabidopsis thaliana through the floral dip method. The transgenic plants showed yellowing and dwarfing phenotypes. OncPSY expression has also contributed to the dwarfing of tobacco(Wen-Li Lee 2012 ).The dwarfed phenotype in transgenic plants may be caused by the conversion of large amounts of GGPP to phytoene due to the increase of active PSY protein, which leads to the suppression of gibberellin synthesis pathway (Fig. 1 .) (Fei Zhou 2020 ). The color of carotenoids in photosynthetic tissues is usually masked by chlorophyll, and carotenoids could provide bright coloration characteristic in tissue with low chlorophyll content (Bartley and Scolnik 1995 ).The content of chlorophyll and carotenoid in wild type and transgenic plants were determinated. Compared with wild type, carotenoid content of transgenic plants increased significantly, and chlorophyll content decreased significantly, which indicated ZjPSY not only changed leaf color by increasing the accumulation of carotenoid, but also affected chlorophyll. Studies have shown that plants adapt to photoprotective pigments by regulating the development of plastid structures. PSY is involved in the coordination process of carotenoid biosynthesis and storage with the molecular factors of photosynthetic development by participating in carotenogenesis (Fig. 12 .). In order to further explore the effect of ZjPSY on plant leaf color, the expression levels of six pigment-related genes, including AtCLH , AtNYC , AtNOL , AtPAL , AtNCED and AtZDS , were carried out. Under normal conditions, the expression levels of six genes were lower than those of wild-type plants. Under drought treatment, the relative expression levels of most genes had no significant difference except AtCLH and AtPAL . However, under salt treatment, the expression pattern of AtCLH was completely opposite to the normal condition. Correspondingly, the amount of carotenoid and chlorophyll synthesis was different under different treatments. Expression of ZjPSY could increase carotenoid content under normal conditions and salt treatment, but decreased under drought treatment. Expression of LbPSY in Escherichia coli and yeast cells could improve tolerance to salinity and drought stress (Cidade, et al. 2012 ). Studies on Daucus carota have confirmed that DcPSY participates in ABA-mediated salt stress tolerance through the binding of promoters and AREB transcription factors (Simpson, et al. 2018 ). In this experiment, the transgenic plants showed no significant enhanced stress tolerance, even showed premature senescence under drought treatment. Due to the low homology of PSY between model plants and Zoysia japonica , it may be caused by differences in PSY between species. Copy volume and expression level of PSY in different transgenic lines also affects the phenotype of the transgenic plants (Cidade, et al. 2012 ). In this study, Yeast two-hybrid screening identified J2 as a novel interacting partner of ZjPSY . J2 gene encode heat-shock protein 40 (HSP40) isoforms J2, molecular chaperone that play roles in preventing proteins from misfolding (Zhou, et al. 1995 ). DnaJ-like chaperone has been shown to be involved in carotenoid synthesis in several species. Expression of Orange protein that belongs to DnaJ-Like chaperones in Arabidopsis thaliana can increase the abundance of active PSY protein, but the transcription level of PSY gene does not change. It was inferred to be the chief posttranscriptional regulator of PSY in carotenoid biosynthesis (Zhou, et al. 2015 ). Expression of gene for Orange protein increased lutein and β-carotene in Chlamydomonas reinhardtii . DnaJ-like chaperone is considered to support phytoene synthase(Morikawa, et al. 2018 ). In addition, experiments have shown that defective farnesylation of HSP40 is sufficient to induce ABA hypersensitivity. It is possible that PSY and J2 work together by affecting ABA activity in plants. The pathway of carotenoid synthesis greatly affects the endogenous ABA levels (Shinjiro Yamaguchi 2007 ). In this study, ZjJ2 is confirmed as a novel interactive partner of ZjPSY , but their interaction mode needs further experimental verification. Declarations Acknowledgements The research was funded by the National Natural Science Foundation of China (No. 31971770 and 31901397). Supplementary Materials : Supplementary materials Author contribution statement Conceptualization, Y.C.; methodology, T.G., S.W. and J.Y.; software, L.X. and Y.L.; resources,T.Z.; writing—original draft preparation, T.G.; writing—review and editing, Y.C.; project administration, L.H. All authors have read and agree to the published version of the manuscript. Conflict of interest The authors declare no conflict of interest Authors and Afliations Di Dong1, Ke Teng2, Penghui Tan3, Zhuocheng Liu1, Zhuoxiong Yang1, Mengdi Wang1, Shuwen Li1, Yinreuizhi Li1, Liebao Han1*, Yuehui Chao1* Di Dong [email protected] Ke, Teng [email protected] Penghui, Tan [email protected] Zhuocheng, Liu [email protected] Zhuoxiong, Yang @qq.com Mengdi, Wang [email protected] Shuwen, Li @qq.com Yinreuizhi, Li @qq.com Liebao, Han [email protected] Yuehui, Chao [email protected] Institute of Turfgrass Science, Beijing Forestry University Beijing Academy of Agriculture and Forestry Sciences Beijing C Foreign Language School References Barghetti A, Sjögren L, Floris M, Paredes EB, Wenkel S, Brodersen P (2017) Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance. GENE DEV 31:2282–2295 Bartley GE, Scolnik PA (1995) Plant carotenoids: pigments for photoprotection, visual attraction, and human health. PLANT CELL 7:1027–1038 Bradford KJ, Nonogaki H (2007) 9 Regulation of ABA and GA levels during seed development and germination in Arabidopsis. 27:224–247 Briardo Llorente JFMC, Rodriguez-Concepcion AM (2017) Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light Briardo Llorente1, Jaime F Martinez-Garcia1,2, Claudia Stange3 and Manuel Rodriguez-Concepcion1. CURR OPIN PLANT BIOL 49–55 Busch M, Seuter A, Hain R (2002) Functional Analysis of the Early Steps of Carotenoid Biosynthesis in Tobacco. PLANT PHYSIOL 128:439–453 Cazzonelli CI (2011) Carotenoids in nature: insights from plants and beyond. FUNCT PLANT BIOL 38:833 Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020) TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. MOL PLANT 13:1194–1202 Cidade LC, de Oliveira TM, Mendes AFS, Macedo AF, Floh EIS, Gesteira AS, Soares-Filho WS, Costa MGC (2012) Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco. MOL BIOL REP 39:10201–10209 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . PLANT J 16:735–743 Colasuonno P, Lozito ML, Marcotuli I, Nigro D, Giancaspro A, Mangini G, De Vita P, Mastrangelo AM, Pecchioni N, Houston K, Simeone R, Gadaleta A, Blanco A (2017) The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments. BMC GENOMICS 18 Espasandin FD, Maiale SJ, Calzadilla P, Ruiz OA, Sansberro PA (2014) Transcriptional regulation of 9-cis-epoxycarotenoid dioxygenase (NCED) gene by putrescine accumulation positively modulates ABA synthesis and drought tolerance in Lotus tenuis plants. PLANT PHYSIOL BIOCH 76:29–35 Fei Zhou E (2020) More is better: the diversity of terpene metabolism in plants Fei Zhou and Eran Pichersky. CURR OPIN PLANT BIOL 1–10 Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N, Drake RG, Schuch W, Bramley PM (2002) Evaluation of Transgenic Tomato Plants Expressing an Additional Phytoene Synthase in a Fruit-Specific Manner. Proceedings of the National Academy of Sciences - PNAS 99:1092–1097 Jia T, Ito H, Hu X, Tanaka A (2015) Accumulation of the NON-YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana . Plant J 81:586–596 Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. NUCLEIC ACIDS RES 30:325–327 Morikawa T, Uraguchi Y, Sanda S, Nakagawa S, Sawayama S (2018) Overexpression of DnaJ-Like Chaperone Enhances Carotenoid Synthesis in Chlamydomonas reinhardtii . APPL BIOCHEM BIOTECH 184:80–91 Morita R, Sato Y, Masuda Y, Nishimura M, Kusaba M (2009) Defect in non-yellow coloring 3, an α/β hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice. Plant J 59:940–952 Nisar N, Li L, Lu S, Khin NC, Pogson BJ (2015) Carotenoid Metabolism in Plants. MOL PLANT 8:68–82 Obrero A, Gonzalez-Verdejo CI, Roman B, Gomez P, Die JV, Ampomah-Dwamena C (2015) Identification, cloning, and expression analysis of three phytoene synthase genes from Cucurbita pepo. BIOL PLANTARUM 59:201–210 Sato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M (2009) Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J 57:120–131 Shinjiro Yamaguchi YKAE (2007) 9 Regulation of ABA and GA levels during seed development and germination in Arabidopsis. Annual Plant Reviews 224–247 Simpson K, Fuentes P, Quiroz-Iturra LF, Flores-Ortiz C, Contreras R, Handford M, Stange C (2018) Unraveling the induction of phytoene synthase 2 expression by salt stress and abscisic acid in Daucus carota . J EXP BOT 69:4113–4126 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. MOL BIOL EVOL 28:2731–2739 Tsuchiya T, Ohta H, Okawa K, Iwamatsu A, Shimada H, Masuda T, Takamiya K (1999) Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: finding of a lipase motif and the induction by methyl jasmonate. Proc Natl Acad Sci U S A 96:15362–15367 Walter MH, Strack D (2011) Carotenoids and their cleavage products: biosynthesis and functions. NAT PROD REP 28:663–692 Wang T, Hou Y, Hu H, Wang C, Zhang W, Li H, Cheng Z, Yang L (2020) Functional Validation of Phytoene Synthase and Lycopene ε-Cyclase Genes for High Lycopene Content in Autumn Olive Fruit ( Elaeagnus umbellata ). J AGR FOOD CHEM 68:11503–11511 Wen-Li Lee JHAF (2012) Ectopic Expression of Oncidium Gower Ramsey Phytoene Synthase Gene Caused Pale Flower and Reduced Leaf Size in Transgenic Tobacco. Journal of International Cooperation 133–150 Yu M, Chen J, Qu J, Liu F, Zhou M, Ma Y, Xiang S, Pan X, Zhang H, Yang M (2020) Exposure to endophytic fungi quantitatively and compositionally alters anthocyanins in grape cells. PLANT PHYSIOL BIOCH 149:144–152 Zhou R, Kroczynska B, Hayman GT, Miernyk JA (1995) AtJ2, an arabidopsis homolog of Escherichia coli dnaJ. PLANT PHYSIOL 108:821–822 Zhou X, Welsch R, Yang Y, Álvarez D, Riediger M, Yuan H, Fish T, Liu J, Thannhauser TW, Li L (2015) Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis. Proceedings of the National Academy of Sciences 112:3558–3563 Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-349623","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":20666628,"identity":"beb1c96f-71f5-4025-8a33-9799b6609788","order_by":0,"name":"Di Dong","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Dong","suffix":""},{"id":20666629,"identity":"b2366b5b-2a8d-4687-8314-cd2d07e61557","order_by":1,"name":"Ke Teng","email":"","orcid":"","institution":"Beijing Academy of Agriculture and Forestry Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Teng","suffix":""},{"id":20666630,"identity":"23831e13-5225-4519-8134-3e9035454cc4","order_by":2,"name":"Penghui Tan","email":"","orcid":"","institution":"Beijing Chaoyang foreign language school","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Penghui","middleName":"","lastName":"Tan","suffix":""},{"id":20666631,"identity":"2a64cedb-c34c-4fad-bf96-34763dc0f687","order_by":3,"name":"Zhuocheng Liu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhuocheng","middleName":"","lastName":"Liu","suffix":""},{"id":20666632,"identity":"954960d8-3567-4162-be10-19ec738f16b3","order_by":4,"name":"Zhuoxiong Yang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhuoxiong","middleName":"","lastName":"Yang","suffix":""},{"id":20666633,"identity":"30caeffb-0b82-4232-8a93-c0581c8c3d32","order_by":5,"name":"Mengdi Wang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mengdi","middleName":"","lastName":"Wang","suffix":""},{"id":20666634,"identity":"d5b9b5a3-77fc-40c3-a5fd-71d908f24b2d","order_by":6,"name":"Shuwen Li","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuwen","middleName":"","lastName":"Li","suffix":""},{"id":20666635,"identity":"63155e79-e5e3-4a15-a35a-df7e708e6e73","order_by":7,"name":"Yinreuizhi Li","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yinreuizhi","middleName":"","lastName":"Li","suffix":""},{"id":20666636,"identity":"c173c9e1-10d3-4c09-968c-a9705072cee3","order_by":8,"name":"Liebao Han","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liebao","middleName":"","lastName":"Han","suffix":""},{"id":20666637,"identity":"180e83c1-2fe7-4b8a-a4e5-037bf56d4fdf","order_by":9,"name":"Yuehui Chao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDACCQiVwMDMfODAhx+kaWFLPDizhyQtDDzGhznYiNDBP7v54WPeNps8g+M8Hw4z8DDI84sdIGDJnWPGxrxtacUGh3k3HC6wYDCcOTsBvxYDiQQzad62w4kbQFpm8DAkGNwmqCX9G1DLf6AWngeHediI0pIDsuUASAsDcVokbuQUG845l5w48zCbATCQJQj7hX9G+sYHb8rsEvvOH3784cMPG3l+aQJaQICJB8lWwspBgJGoZDIKRsEoGAUjFwAAtcpGNdz9694AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8573-6794","institution":"Turfgrass Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yuehui","middleName":"","lastName":"Chao","suffix":""}],"badges":[],"createdAt":"2021-03-21 05:40:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-349623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-349623/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":7902919,"identity":"0dfb0e2c-0239-4f8b-aa29-bd3eff878dc7","added_by":"auto","created_at":"2021-04-12 13:23:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":390077,"visible":true,"origin":"","legend":"PSY expression regulates the synthesis of carotenoids and is involved in the synthesis of hormones(Bradford and Nonogaki. 2007; Colasuonno, et al. 2017; Fraser, et al. 2002). ","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/47f189f83c498137d65863f9.png"},{"id":7902902,"identity":"1cd09001-82ff-4306-97a4-9fe19dbfbb04","added_by":"auto","created_at":"2021-04-12 13:23:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5833382,"visible":true,"origin":"","legend":"(A) Intron/Exon organization in the ZjPSY genes. Introns and exons are shown as black lines and yellow boxes, respectively. (B) secondary structure analysis of ZjPSY protein. Green helix, alpha helix; Blue arrow, beta strand; Faint lines, coil; SS confidence line, the prediction confidence. (C) Protein structure simulation of ZjPSY. ","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/8bb19c022191c74ef35d1bac.png"},{"id":7902920,"identity":"e2b5c945-16d8-443c-945d-e58636c2ce59","added_by":"auto","created_at":"2021-04-12 13:23:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3805738,"visible":true,"origin":"","legend":"(A) Phylogenetic tree analysis among 18 PSY proteins from different species. The phylogenetic tree was constructed by the alignments of amino acid sequences with the neighbor-joining method. (B) The conserved motifs in 18 PSY proteins of different species. Top scored three motifs consisting of amino acids composition were listed. (C) Protein motif analyses of PSY protein sequence. Each color show the different Conserved motif structure identified in PSY protein.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/85bf68605fd7b0cfb5d235a5.png"},{"id":7902923,"identity":"d5ebda2a-9db7-4609-ab49-3c8cab0a5fd0","added_by":"auto","created_at":"2021-04-12 13:23:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":622059,"visible":true,"origin":"","legend":"Analysis of up-stream sequence of ZjPSY gene (A) Cis acting elements contained in ZjPSY gene promoterand. (B) Predicted binding sites for the transcription factors in the ZjPSY gene promoter\n\n","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/82837a2699234b490b5bf0b9.png"},{"id":7902916,"identity":"a8c02fd9-803e-4d22-939d-e0128fbb8c99","added_by":"auto","created_at":"2021-04-12 13:23:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":715634,"visible":true,"origin":"","legend":"Screening for ZjPSY interacting proteins by yeast two-hybrid assay. (A) the screened clones patched onto QDO/X/A plates. blue clones indicate positive results, whereas white or absent clones were negative. ZjJ2 and ZjPSY were fused with activation domain of pGADT7 or DNA binding domain of pGBK-T7, respectively. (B) Interaction between ZjJ2 and ZjPSY. Yeast cells were spotted on higher stringency QDO/X/A agar plates in 10-fold serial dilutions. blue clones were positive and white or absent clones were negative. pGBKT7-53 or pGADT7-T was used as the negative control, and pGBKT7-53 and pGADT7-T were co-transformed as positive control.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/d0a32e69e68a3043d99a71c5.png"},{"id":7902914,"identity":"c0d41c29-fbe7-464f-b170-f1d39a5d7d47","added_by":"auto","created_at":"2021-04-12 13:23:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115182,"visible":true,"origin":"","legend":"Expression profiles of ZjPSY. (A) ZjPSY expression pattern in root, stem, and leaf. (B) ZjPSY expression pattern in leaves at different developmental stages. (C-F) expression pattern in leaf under 10 μM GA3 treatment (c), 10 μM ABA treatment (d), 0.5 mM SA treatment (e), and 10 μM MeJA treatment (f). Different letters above the columns indicate significant differences (p=0.05).\n\n","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/a4869f8c0f3d9e9d1d0decf0.png"},{"id":7902918,"identity":"719ce2d8-33d1-47d7-b032-f4ae15937d77","added_by":"auto","created_at":"2021-04-12 13:23:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96838,"visible":true,"origin":"","legend":"(A) qRT-PCR analysis of ZjPSY expression in wild-type plants and the transgenic lines. three individual transgenic plants expressing ZjPSY gene #34, #10, and #9 were selected to further experiment. (B) Chlorophyll content of wild-type plants and the transgenic lines. (C) Carotenoid content wild-type plants and the transgenic lines. Different letters above the columns indicate significant differences (p=0.05).","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/0de57e891f5900b81914afa3.png"},{"id":7902903,"identity":"efaf2549-0f26-445e-9ae3-59b7723b0c8d","added_by":"auto","created_at":"2021-04-12 13:23:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":8037858,"visible":true,"origin":"","legend":"Leaf yellowing and dwarfing phenotype of transgenic Arabidopsis expressing the ZjPSY gene and wild-type Arabidopsis. (A) Transgenic Arabidopsis #34 expressing ZjPSY gene and wild-type Arabidopsis of 5 days. (B) Comparison of the phenotypes of transgenic Arabidopsis and wild-type Arabidopsis at 30 days. (C) Visual appearance of WT and transgenic Arabidopsis leaves. ","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/45e7e57d62b7ccca42d6d3f9.png"},{"id":7902909,"identity":"a39025d5-e5ea-4345-b868-921bb4e1fed5","added_by":"auto","created_at":"2021-04-12 13:23:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":16378617,"visible":true,"origin":"","legend":"(A) Transgenic Arabidopsis expressing the ZjPSY gene and wild-type Arabidopsis of 45 days. (B) Plant height between different lines and wild-type. (C-D) Chlorophyll (c) and carotenoid content (d) of Transgenic Arabidopsis expressing the ZjPSY gene and wild-type Arabidopsis. ","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/10e7f6ea171dd490d3618c2d.png"},{"id":7902917,"identity":"d142b015-a837-4394-b125-b365bdbf7ada","added_by":"auto","created_at":"2021-04-12 13:23:53","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":91567251,"visible":true,"origin":"","legend":"Performance of wild-type and transgenic plants under drought treatment and salt treatment. (A) Transgenic Arabidopsis and wild-type grown in MS agar plates containing 500 mM mannitol for 45 days. (B) Transgenic Arabidopsis and wild-type grown in MS agar plates containing 200 mM NaCl for 45 days. (C-D) Chlorophyll content of Transgenic Arabidopsis expressing the ZjPSY gene and wild-type Arabidopsis under drought treatment (c) or salt treatment (d). (E-F) Carotenoid content of Transgenic Arabidopsis expressing the ZjPSY gene and wild-type Arabidopsis under drought treatment.","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/84fb3554c5d4fb45abb4097a.png"},{"id":7902915,"identity":"0f511943-2ab0-46a2-8733-34d2f4d35331","added_by":"auto","created_at":"2021-04-12 13:23:53","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":111971,"visible":true,"origin":"","legend":"Pigment-related gene expression level of wild-type and transgenic plants subjected to normal, drought and salt stress conditions. Pigment-related genes include AtCLH (a), AtNYC (b), AtNOL (c), AtPAL (d), AtNCED (e) and AtZDS (f). ","description":"","filename":"fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/02dce921636b2be90059262d.png"},{"id":7902913,"identity":"6a33cc85-98e0-4dd6-ac14-1fc838d08fad","added_by":"auto","created_at":"2021-04-12 13:23:52","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":144928,"visible":true,"origin":"","legend":"Molecular schematic of PSY's involvement in carotene accumulation and chloroplast development in plants (Briardo Llorente and Rodriguez-Concepcion 2017) . The development of chloroplasts and chromoplast differentiation are related to PSY activity and sink capacity for carotenoids. Positive regulatory factor HY5 and negative regulatory factor PIF1 coordinate PSY expression.","description":"","filename":"fig12.png","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/bae6c16a058f03dfe343a19b.png"},{"id":7903276,"identity":"cf13a749-7a10-4ebe-b46c-f7faca6f1080","added_by":"auto","created_at":"2021-04-12 13:26:44","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17803,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-349623/v1/67882e6cfcc403c90ef42c6e.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExpression of \u003cem\u003eZjPSY\u003c/em\u003e, A Phytoene Synthase Gene from \u003cem\u003eZoysia Japonica\u003c/em\u003e Leads to Leaf Yellowing and Plant Dwarfing in \u003cem\u003eArabidopsis Thaliana\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn plants, carotenoids are a class of hydrocarbons that serve as secondary metabolites of plant photosynthesis, which play classical roles in photosynthetic biological processes, including photomorphogenesis and photoprotection. Carotenoids also participate in plant color formation as pigments, which affect plant color through different levels of aggregation in the chromoplasts (Briardo Llorente and Rodriguez-Concepcion 2017). Carotenoids range in color from colorless to yellow and red and are reflected in the fruits and leaves of many plants(Nisar, et al. 2015). In addition to that, carotenoids and their oxidative and enzymatic lysates are considered to be signaling molecules for interactions of plants with environment, so carotenoids are proposed to play an important regulatory role in plant growth and development (Walter and Strack 2011).\u003c/p\u003e\n\u003cp\u003ePhytoene is a precursor for the synthesis of all carotenoid substances. Carotenoids also act as precursors for gibberellin and ABA (Abscisic acid), hormones that play an important role in plant growth and development (Colasuonno, et al. 2017).\u003cem\u003ePhytoene synthase \u003c/em\u003e(\u003cem\u003ePSY\u003c/em\u003e), a key enzyme regulating carotenoid synthesis, can catalyze the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene, which is the first rate-limiting reaction in the carotenoid synthesis pathway (fig.1). \u003cem\u003ePSY\u003c/em\u003e is a preferred candidate gene for understanding the molecular regulation of carotenoid accumulation in most species (Obrero, et al. 2015). Transgenic tobacco with overexpression of \u003cem\u003ePSY\u003c/em\u003e significantly increased carotenoid content, and Transgenic autumn olive fruits (\u003cem\u003eElaeagnus umbellata\u003c/em\u003e) confirmed that \u003cem\u003eEUTPSY\u003c/em\u003e played a key role in lycopene accumulation(Busch, et al. 2002; Wang, et al. 2020). Expression of \u003cem\u003ePSY\u003c/em\u003e from \u003cem\u003eOncidium\u003c/em\u003e Gower Ramsey in Tobacco showed dwarfing and reduced leaf area phenotypes (Wen-Li Lee 2012).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eZoysia japonica\u003c/em\u003e is one of the common warm-season turfgrass in the world. Due to its strong drought resistance, salt resistance and barren resistance, it has been widely used in golf courses, sports grounds and urban greening. The regulation mode of carotenoid accumulation in plant cells and the regulation mechanism of \u003cem\u003eZjPSY\u003c/em\u003e gene have not been studied in \u003cem\u003eZoysia japonica\u003c/em\u003e. In this study, \u003cem\u003eZjPSY\u003c/em\u003e gene of \u003cem\u003eZoysia japonica \u003c/em\u003ewas isolated and identification to further explore. Although the genes involved in the carotenoid biosynthesis pathway have been identified, the regulation of these genes in plant growth and development and stress tolerance is not fully understood. This study could further explore the function of \u003cem\u003ePSY\u003c/em\u003e gene, and lay a foundation for studying the role of carotene in Zoysia and improving the utilization of high-quality gene resources in turfgrass.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant Materials and Growth Conditions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eZoysia japonica\u003c/em\u003e cultivar \u0026lsquo;Compadre\u0026rsquo; seeds were purchased from the Hancock seed company (Hancock, Florida, USA). \u003cem\u003eZoysia japonica\u003c/em\u003e were cultivated in pots，kept at 28/23℃(day/night) with 16h (at 400 mmol/m2/s) /8h photoperiod and 50% humidity. All the Arabidopsis materials used in this study are in Col ecotype background. The Arabidopsis seeds were sown on Murashige and Skoog plates (4.43 g/L Murashige and Skoog powder, 8 g/L agar, pH 5.8), with a 16h white light (at 90 mmol/m2/s)/8h dark cycle and 50% humidity at 25\u0026deg;C. After the fourth leaves appeared on the seedlings, the seedlings were transferred to sterilized soil or culture medium to continue growth under the same growth conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification and Cloning of ZjPSY \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from \u0026lsquo;Compadre\u0026rsquo; leaves of 3-month-old with a plant RNA isolation kit (Promega, USA), and first-strand cDNA was synthesized with a reverse transcription kit (Takara, Dalian, China). Primer sequences for cloning were designed based on the sequence fragment of PSY and PCR products were ligated into the cloning vector pDM19-T for storage and further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBLAST analysis of the NCBI database was used to identify homologs and 18 PSY protein sequences from other species were obtained. The phylogenetic tree and ZjPSY gDNA structure were constructed using MEGA version 6.0 with the neighbor-joining method and GSDS 2.0 (http://gsds.gao-lab.org/Gsds_about.php)(Tamura, et al. 2011).\u003c/p\u003e\n\u003cp\u003eThe molecular weights and theoretical isoelectric points were analyzed by Compute pI/MW tool (http://web.expasy.org/compute_pi/). The analysis of motif in PSY amino acids and cis-regulatory elements in promoter used MEME (http://meme-suite.org/tools/meme), PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and TBtools(Chen, et al. 2020). ZjPSY protein model was generated by using Phyre v2.0 tool (www.sbg.bio.ic.ac.uk/phyre2/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVector construction and generation of transgenic plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe completed coding region of \u003cem\u003eZjPSY\u003c/em\u003e was amplified by PCR with primers ZjPSY-F and ZjPSY-R (Supplementary Table S1). The fragment containing the complete CDS was ligated to the vector plasmid pMD19-T, and stored in \u003cem\u003eE. coli\u003c/em\u003e for long-term preservation. The 35S::\u003cem\u003eZjPSY\u003c/em\u003e:YFP was constructed by fusing completed \u003cem\u003eZjPSY\u003c/em\u003e CDS followed by YFP with primers, 3302Y-ZjPSY-F and 3302Y-ZjPSY-R. The recombination was introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101, which was then used for the transformation of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e through the floral dip method(Clough and Bent 1998). Transgenic plants were selected using 2 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e glufosinate and verified by PCR. T\u003csub\u003e3\u003c/sub\u003e transgenic lines were generated by self-pollination for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStress tolerances of Transgenic Plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree different transgenic lines with high expression levels of \u003cem\u003ePSY\u003c/em\u003e were selected. The Wild-type and Transgenic seedlings were cultivated on 1/2 MS medium containing 500 mM mannitol or 200 mM NaCl for 30 days under conditions of 26/20℃ (day/night), 16/8 h (light/dark), and humidity of 70%. Total RNA was isolated from each sample using Plant RNA Kit (Omega Bio-tek, USA) and the first-strand cDNA was synthesized using the PrimeScript\u003csup\u003eTM\u003c/sup\u003e II 1st Strand cDNA Synthesis Kit (TAKARA, China). The First-strand cDNA was used for real-time quantitative RT-PCR (qRT-PCR) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression levels of \u003cem\u003ePSY\u003c/em\u003e gene and pigment-related genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative real-time PCR was carried out to measure the expression levels of six pigment-related genes from different samples with the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△Ct \u003c/sup\u003emethod, following the manufacturer\u0026rsquo;s procedures of GreenTB Premix Ex Taq II (takara). The AtUBQ10(NM_116771) gene was used as an internal reference gene for evaluating transcriptional abundance in Arabidopsis. Pigment-related genes include \u003cem\u003eAtCLH\u003c/em\u003e (AT1G19670.1), \u003cem\u003eAtNYC\u003c/em\u003e (AT4G13250.1), \u003cem\u003eAtNOL\u003c/em\u003e (AT5G04900.1), \u003cem\u003eAtPAL\u003c/em\u003e (AT5G04230), \u003cem\u003eAtNCED\u003c/em\u003e (AT1G30100) and \u003cem\u003eAtZDS\u003c/em\u003e (AT3G04870). Relevant primer sequences used were shown in Supplementary Table S1. All experiments in the research contained three biological replicates.\u003c/p\u003e\n\u003cp\u003eQuantitative real-time PCR was carried out to measure the expression levels of ZjPSY in and different developmental stages, tissues of zoysiagrass and leave subjected to normal, hormone treatment, drought, or salt stress conditions. The 2-month-old zoysiagrass were inducted with 10 \u0026mu;M GA\u003csub\u003e3\u003c/sub\u003e, 10\u0026mu;M ABA, 10 \u0026mu;M MeJA, or 0.5 mM SA. Leaves at different growth stages (young, fast-growing, and mature), roots tissues and stems tissues were also collected. RNA isolation and cDNA synthesis were performed as above. The Z. japonica beta-actin was used as the internal reference gene (GenBank accession No. GU290546). Relevant primer sequences used were shown in Supplementary Table S1. All experiments in the research contained three biological replicates,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast two-hybrid assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ZjPSY ORF\u0026rsquo;s were cloned using the primers pGBKT7-ZjPSY-F/R and inserted into pGBKT7 vector, which was co-transformed into yeast strain Y2H as described in Yeastmaker\u0026trade; Yeast Transformation System (Clontech, USA). The MATCHMAKER GAL4 yeast two-hybrid system was performed according to the Matchmaker\u0026trade; Gold Yeast Two-Hybrid System manufacturer's instructors (Clontech, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2.1. Identification and bioinformatic analysis of the \u003cem\u003eZjPSY\u003c/em\u003e Gene in \u003cem\u003eZoysia japonica\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Zoysia Genome Database, the DNA fragment containing \u003cem\u003eZjPSY\u003c/em\u003e Coding Sequences (CDS) was cloned and ligated into cloning vector pMD19-T. The ZjPSY cDNA sequences were deposited in the NCBI database with accession numbers KY264127.1. The full-length \u003cem\u003eZjPSY\u003c/em\u003e cDNA contains a 1,230 bp ORF encoding a protein of 409 amino acids, and it belongs to the Isoprenoid_Biosyn_C1 Superfamily. According to the compute pI/MW tool of Expasy, the molecular weight of ZjPSY was 46.39 kD, and the theoretical isoelectric point was 9.20. Intron/Exon organization analysis showed that \u003cem\u003eZjPSY \u003c/em\u003ehad five exons (Fig.1A). Secondary structure analysis represents that the ZjPSY have 63% alpha helix, 1% beta strand, and 25% disorder proteins (Fig.1B). Modelled 3D Tertiary structure of ZjPSY was provide insights for molecular geometry (Fig.1C). The stereochemical structure supported the \u0026alpha;-helical nature of the enzyme.\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of PSY proteins was performed based on the protein sequences from different species collected from NCBI database, with a neighbor-joining method (Fig. 2A).\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of the PSY protein revealed that ZjPSY has the highest homology with PSY of \u003cem\u003eZoysia matrella\u003c/em\u003e. Except that, it was most closely related to \u003cem\u003eSalvia splendens\u003c/em\u003e. The PSY proteins in 18 PSY protein of different species were further analysed for the occurrence of conserved motifs (Fig. 2B). Motif sequence scan analysis showed the presence of amino-acid rich profiles, top scored three motifs was found in all 18 PSY proteins (Fig. 2C). the conserved motifs were indicated to be highly conserved. Phylogenetic analysis and motif analysis revealed that ZjPSY had a low similarity and different topology with PSY from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e or \u003cem\u003eOryza sativa. \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Prediction of cis\u003c/strong\u003e\u003cstrong\u003e‑regulatory elements of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eZjPSY\u003c/em\u003e promoter\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the regulatory pathway and function of ZjPSY, a 2000bp upstream sequence from the translation start site (ATG) was examined for promoter elements (Fig.3). The cis-regulatory elements were predicted using the PlantCARE database (Lescot, et al. 2002). Multiple light responsive motifs, including I-box, G-box, TCCC-motif, chs-CMA2a and CAT-box, were identified in the frequency of occurrence of cis-elements. Gibberellin responsiveness P-box, salicylic acid responsiveness TCA-element, MeJA-responsiveness TGACG-motif and CGTCA-motif were identified.\u003c/p\u003e\n\u003cp\u003eIn addition, several cis-acting elements involved growth and development were identified, such as endosperm expression element GCN4_motif, zein metabolism regulation element O2-site. It was also possible to recognize ARE (anaerobic induction element).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 ZjPSY can bind with DNAJ homologue 2 in yeast\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo analyze the protein interacting of ZjPSY, yeast two-hybrid assays were performed. The screened colonies were patched onto higher stringency selective QDO/X/A plates (fig 4a). The \u003cem\u003eJ2\u003c/em\u003e gene in zoysia was identified, and it had high homology with \u003cem\u003eJ2\u003c/em\u003e in Arabidopsis. In Arabidopsis, \u003cem\u003eJ2\u003c/em\u003e encodes heat-shock protein 40 (HSP40) (Barghetti, et al. 2017; Zhou, et al. 1995). The results show the plasmids pGADT7 containing \u003cem\u003eZjJ2\u003c/em\u003e and the pGBKT7-\u003cem\u003eZjPSY\u003c/em\u003e plasmid grew well and turned blue on QDO/X/A plates, similar to the positive control. These results indicate that ZjJ2 protein interact with ZjPSY protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Expression pattern of ZjPSY \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReal-time PCR was conducted to examine the expression pattern of \u003cem\u003eZjPSY\u003c/em\u003e in different tissues and development stages. The expression profiles results showed that ZjPSY genes were detected in all tissues tested, and\u003cem\u003e ZjPSY\u003c/em\u003e was differentially highly expressed in leaf(fig.5A). Its gene expression was relatively high in early leaf growth stage, and relatively small in fast-growing and mature leaves (fig.5B). \u003cem\u003eZjPSY\u003c/em\u003e were down-regulated after multiple hormone treatments, including GA\u003csub\u003e3\u003c/sub\u003e, ABA, SA, and MeJA (fig.5C-F). Treated with these four hormones, its gene expression began to decline and reached its lowest level at 3 h, then goes back to normal levels at the later stage. The expression pattern suggested that \u003cem\u003eZjPSY\u003c/em\u003e may play a pivotal role in leaf development and hormone response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Chlorophyll and carotenoid contents of transgenic Arabidopsis plants and the wild-type\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether \u003cem\u003eZjPSY\u003c/em\u003e expression affects chlorophyll and carotenoid contents in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, genetic transformation was conducted using Agrobacterium-mediated method. Independent transgenic lines were obtained and further verified by PCR amplification. Three positive transgenic lines designated 9#, 10#, 34# were selected for further analyses. \u003cem\u003eZjPSY\u003c/em\u003e transcription levels and chlorophyll and carotenoid contents were detected in the three transgenic lines and wild-type plants (fig 6). The lines with the highest gene expression showed high carotenoid content levels and low chlorophyll content levels, which indicates that alteration of expression affects carotenoid content and chlorophyll content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 ZjPSY Expression in Arabidopsis thaliana resulted in leaf yellowing and plant dwarfing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to study the gene function of \u003cem\u003eZjPSY\u003c/em\u003e gene, four plants from different lines were cultured in each pot. The results showed that the \u003cem\u003eZjPSY\u003c/em\u003e-overexpressed plants had shorter plant height and yellowed leaves compared with the wild type (fig 7). After comparison, transgenic plants showed obvious yellowing phenotype (fig 7C). These results indicate that \u003cem\u003eZjPSY\u003c/em\u003e may play important roles in pigment accumulation and leaf yellowing.\u003c/p\u003e\n\u003cp\u003eTo further detect the transgenic plants, the three selected transgenic lines and the wild type were transplanted into MS medium. The plant height of the three transgenic lines was significantly lower than that of the wild type, and the two lines with higher \u003cem\u003eZjPSY\u003c/em\u003e expression showed significant dwarfed phenotypes (fig 8). The transgenic plants were less than 82% in height compared with wild type (Fig. 8B). These results indicated that expression of \u003cem\u003eZjPSY\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e significantly increased carotenoid contents and decreased chlorophyll contents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Regulation of pigment-related genes expression in transgenic Arabidopsis plants and the wild-type under drought treatment and salt treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn further experiments, Differences between transgenic plants and wild types under drought and salt treatment were studied. Wild-type and transgenic lines were placed on MS agar plates containing 500 mM mannitol or 200 mM NaCl. Different from the normal condition, there was no significant dwarfing phenotype and resistance increase observed in the transgenic plants treated with salt and drought. The transgenic plants showed premature senescence under drought treatment. Under drought treatment, the chlorophyll content of the transgenic plants did not show similar performance with that of the normal treatment. The chlorophyll content of transgenic plant 34#, 10# was not significantly different from that of wild type, but the chlorophyll content of transgenic plant #10 was lower than that of wild type (fig. 9C). The carotenoid level of the transgenic plants was all lower than that of the wild type (fig. 9E). The results suggests that expression of \u003cem\u003eZjPSY\u003c/em\u003e may inhibit carotenoid synthesis under drought treatment. Under salt treatment, the chlorophyll content of transgenic plants 34# and 10# was higher than that of wild type (fig. 9D), the content of carotenoid in transgenic plants was significantly higher than that in wild type, which was similar to that in normal treatment (fig. 9F). In plants subjected to salt stress, expression of \u003cem\u003eZjPSY \u003c/em\u003ecan enhance carotenoid synthesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Regulation of pigment-related genes expression in transgenic Arabidopsis plants and the wild-type under drought treatment and salt treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain more insight into the role of ZjPSY in plant growth and development, the expression profile of six pigment-genes, including \u003cem\u003eAtCLH\u003c/em\u003e, \u003cem\u003eAtNYC\u003c/em\u003e, \u003cem\u003eAtNOL\u003c/em\u003e, \u003cem\u003eAtPAL\u003c/em\u003e, \u003cem\u003eAtNCED\u003c/em\u003e and \u003cem\u003eAtZDS\u003c/em\u003e, were carried out using qRT-PCR (fig 11.). \u003cem\u003eChlorophyllase\u003c/em\u003e (\u003cem\u003eCHL\u003c/em\u003e) catalyzes the hydrolysis of ester bonds to chlorophyllide and phytol, which is the first step of chlorophyll degradation (Tsuchiya, et al. 1999). \u003cem\u003eNON-YELLOWCOLORING\u003c/em\u003e (\u003cem\u003eNYC\u003c/em\u003e) and \u003cem\u003eNYC1-LIKE\u003c/em\u003e (\u003cem\u003eNOL\u003c/em\u003e) are involved in chlorophyll band light-harvesting complex II degradation (Jia, et al. 2015; Morita, et al. 2009; Sato, et al. 2009). \u003cem\u003ePhenylalanine ammonia-lyase\u003c/em\u003e (\u003cem\u003ePAL\u003c/em\u003e), a key enzyme in the phenylalanine pathway, catalyzes the deamination of phenylalanine into trans-cinnamic acid, which is related to anthocyanin synthesis(Yu, et al. 2020). \u003cem\u003e9-cis-epoxycarotenoid dioxygenase\u003c/em\u003e (\u003cem\u003eNCED\u003c/em\u003e), a kind of carotenoid cleavage enzyme, is a key enzyme that regulates ABA biosynthesis under stress (Espasandin, et al. 2014) . \u003cem\u003eZ-carotene desaturase\u003c/em\u003e (\u003cem\u003eZDS\u003c/em\u003e) plays a regulatory role in the catalysis of Z-carotene to tetra-cis lycopene, and is also an important gene in the production of carotenoids (Cazzonelli 2011). Under normal conditions, the relative expression levels of several pigment-related genes measured in transgenic plants were lower than those in wild type. In addition to \u003cem\u003eAtCLH\u003c/em\u003e and \u003cem\u003eAtPAL\u003c/em\u003e, there was no significant difference in the relative expression levels of other measured genes between wild-type and transgenic plants under drought treatment.\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003e \u003cem\u003ePSY\u003c/em\u003e gene regulates the first committed step in the process of carotenoid biosynthesis. In this study, \u003cem\u003eZjPSY\u003c/em\u003e gene was cloned from Zoysia sulcus. Bioinformatics analysis showed that the open reading frame of \u003cem\u003eZjPSY\u003c/em\u003e gene was 1230bp, encoding 409 amino acids. The protein belongs to the isoprenoid_biosyn_C1 superfamily. The large central cavity formed by the antiparallel α helixes and the two aspartic acid rich regions in the opposite wall constitute its catalytic sites. Structural analysis shows that ZjPSY contains lots of α helixes and its three-dimensional three-dimensional analog structure contains a large central cavity. Phylogenetic analysis of PSY proteins with a neighbor-joining method revealed that ZjPSY has the highest homology with PSY of \u003cem\u003eZoysia matrella\u003c/em\u003e, and ZjPSY had a low similarity with PSY in model plant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe expression pattern results suggest that \u003cem\u003eZjPSY\u003c/em\u003e may play an important role in early leaf growth and hormone response. \u003cem\u003eZjPSY\u003c/em\u003e might be highly sensitive to hormone signaling, it was significantly upregulated under GA, ABA, SA and MeJA treatment. These results indicated it were involved pathways of different hormone signal response. The regulatory element analysis of the \u003cem\u003eZjPSY\u003c/em\u003e promoter contains of a variety of hormone responsive elements.\u003c/p\u003e \u003cp\u003eTo investigate the functions of \u003cem\u003eZjPSY\u003c/em\u003e in plants, the 35S::\u003cem\u003eZjPSY\u003c/em\u003e:YFP was constructed and transformed to \u003cem\u003eArabidopsis thaliana\u003c/em\u003e through the floral dip method. The transgenic plants showed yellowing and dwarfing phenotypes. \u003cem\u003eOncPSY\u003c/em\u003e expression has also contributed to the dwarfing of tobacco(Wen-Li Lee \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).The dwarfed phenotype in transgenic plants may be caused by the conversion of large amounts of GGPP to phytoene due to the increase of active PSY protein, which leads to the suppression of gibberellin synthesis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.) (Fei Zhou \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe color of carotenoids in photosynthetic tissues is usually masked by chlorophyll, and carotenoids could provide bright coloration characteristic in tissue with low chlorophyll content (Bartley and Scolnik \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).The content of chlorophyll and carotenoid in wild type and transgenic plants were determinated. Compared with wild type, carotenoid content of transgenic plants increased significantly, and chlorophyll content decreased significantly, which indicated \u003cem\u003eZjPSY\u003c/em\u003e not only changed leaf color by increasing the accumulation of carotenoid, but also affected chlorophyll. Studies have shown that plants adapt to photoprotective pigments by regulating the development of plastid structures. \u003cem\u003ePSY\u003c/em\u003e is involved in the coordination process of carotenoid biosynthesis and storage with the molecular factors of photosynthetic development by participating in carotenogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003e.).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn order to further explore the effect of \u003cem\u003eZjPSY\u003c/em\u003e on plant leaf color, the expression levels of six pigment-related genes, including \u003cem\u003eAtCLH\u003c/em\u003e, \u003cem\u003eAtNYC\u003c/em\u003e, \u003cem\u003eAtNOL\u003c/em\u003e, \u003cem\u003eAtPAL\u003c/em\u003e, \u003cem\u003eAtNCED\u003c/em\u003e and \u003cem\u003eAtZDS\u003c/em\u003e, were carried out. Under normal conditions, the expression levels of six genes were lower than those of wild-type plants. Under drought treatment, the relative expression levels of most genes had no significant difference except \u003cem\u003eAtCLH\u003c/em\u003e and \u003cem\u003eAtPAL\u003c/em\u003e. However, under salt treatment, the expression pattern of \u003cem\u003eAtCLH\u003c/em\u003e was completely opposite to the normal condition. Correspondingly, the amount of carotenoid and chlorophyll synthesis was different under different treatments. Expression of \u003cem\u003eZjPSY\u003c/em\u003e could increase carotenoid content under normal conditions and salt treatment, but decreased under drought treatment. Expression of \u003cem\u003eLbPSY\u003c/em\u003e in Escherichia coli and yeast cells could improve tolerance to salinity and drought stress (Cidade, et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Studies on \u003cem\u003eDaucus carota\u003c/em\u003e have confirmed that \u003cem\u003eDcPSY\u003c/em\u003e participates in ABA-mediated salt stress tolerance through the binding of promoters and AREB transcription factors (Simpson, et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this experiment, the transgenic plants showed no significant enhanced stress tolerance, even showed premature senescence under drought treatment. Due to the low homology of PSY between model plants and \u003cem\u003eZoysia japonica\u003c/em\u003e, it may be caused by differences in PSY between species. Copy volume and expression level of \u003cem\u003ePSY\u003c/em\u003e in different transgenic lines also affects the phenotype of the transgenic plants (Cidade, et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, Yeast two-hybrid screening identified \u003cem\u003eJ2\u003c/em\u003e as a novel interacting partner of \u003cem\u003eZjPSY\u003c/em\u003e. \u003cem\u003eJ2\u003c/em\u003e gene encode heat-shock protein 40 (HSP40) isoforms J2, molecular chaperone that play roles in preventing proteins from misfolding (Zhou, et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). DnaJ-like chaperone has been shown to be involved in carotenoid synthesis in several species. Expression of Orange protein that belongs to DnaJ-Like chaperones in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e can increase the abundance of active PSY protein, but the transcription level of \u003cem\u003ePSY\u003c/em\u003e gene does not change. It was inferred to be the chief posttranscriptional regulator of PSY in carotenoid biosynthesis (Zhou, et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Expression of gene for Orange protein increased lutein and β-carotene in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. DnaJ-like chaperone is considered to support phytoene synthase(Morikawa, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, experiments have shown that defective farnesylation of HSP40 is sufficient to induce ABA hypersensitivity. It is possible that \u003cem\u003ePSY\u003c/em\u003e and \u003cem\u003eJ2\u003c/em\u003e work together by affecting ABA activity in plants. The pathway of carotenoid synthesis greatly affects the endogenous ABA levels (Shinjiro Yamaguchi \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In this study, \u003cem\u003eZjJ2\u003c/em\u003e is confirmed as a novel interactive partner of \u003cem\u003eZjPSY\u003c/em\u003e, but their interaction mode needs further experimental verification.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e The research was funded by the National Natural Science Foundation of China (No. 31971770 and 31901397).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Materials\u003c/strong\u003e: Supplementary materials\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e Conceptualization, Y.C.; methodology, T.G., S.W. and J.Y.; software, L.X. and Y.L.; resources,T.Z.; writing\u0026mdash;original draft preparation, T.G.; writing\u0026mdash;review and editing, Y.C.; project administration, L.H.\u003c/p\u003e\n\u003cp\u003eAll authors have read and agree to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest \u003c/strong\u003eThe authors declare no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Afliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDi Dong1, Ke Teng2, Penghui Tan3, Zhuocheng Liu1, Zhuoxiong Yang1, Mengdi Wang1, Shuwen Li1, Yinreuizhi Li1, Liebao Han1*, Yuehui Chao1*\u003c/p\u003e\n\u003cp\u003eDi Dong\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eKe, Teng\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003ePenghui, Tan\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eZhuocheng, Liu\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eZhuoxiong, Yang\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eMengdi, Wang\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eShuwen, Li\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eYinreuizhi, Li\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eLiebao, Han\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eYuehui, Chao\u003c/p\u003e\n\u003cp\[email protected]\u003c/p\u003e\n\u003cp\u003eInstitute of Turfgrass Science, Beijing Forestry University\u003c/p\u003e\n\u003cp\u003eBeijing Academy of Agriculture and Forestry Sciences\u003c/p\u003e\n\u003cp\u003eBeijing C Foreign Language School\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eBarghetti A, Sj\u0026ouml;gren L, Floris M, Paredes EB, Wenkel S, Brodersen P (2017) Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance. GENE DEV 31:2282\u0026ndash;2295\u003c/p\u003e\n\u003cp\u003eBartley GE, Scolnik PA (1995) Plant carotenoids: pigments for photoprotection, visual attraction, and human health. PLANT CELL 7:1027\u0026ndash;1038\u003c/p\u003e\n\u003cp\u003eBradford KJ, Nonogaki H (2007) 9 Regulation of ABA and GA levels during seed development and germination in Arabidopsis. 27:224\u0026ndash;247\u003c/p\u003e\n\u003cp\u003eBriardo Llorente JFMC, Rodriguez-Concepcion AM (2017) Illuminating colors: regulation of carotenoid biosynthesis and accumulation by light Briardo Llorente1, Jaime F Martinez-Garcia1,2, Claudia Stange3 and Manuel Rodriguez-Concepcion1. CURR OPIN PLANT BIOL 49\u0026ndash;55\u003c/p\u003e\n\u003cp\u003eBusch M, Seuter A, Hain R (2002) Functional Analysis of the Early Steps of Carotenoid Biosynthesis in Tobacco. PLANT PHYSIOL 128:439\u0026ndash;453\u003c/p\u003e\n\u003cp\u003eCazzonelli CI (2011) Carotenoids in nature: insights from plants and beyond. FUNCT PLANT BIOL 38:833\u003c/p\u003e\n\u003cp\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020) TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. MOL PLANT 13:1194\u0026ndash;1202\u003c/p\u003e\n\u003cp\u003eCidade LC, de Oliveira TM, Mendes AFS, Macedo AF, Floh EIS, Gesteira AS, Soares-Filho WS, Costa MGC (2012) Ectopic expression of a fruit phytoene synthase from \u003cem\u003eCitrus paradisi\u003c/em\u003e Macf. promotes abiotic stress tolerance in transgenic tobacco. MOL BIOL REP 39:10201\u0026ndash;10209\u003c/p\u003e\n\u003cp\u003eClough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. PLANT J 16:735\u0026ndash;743\u003c/p\u003e\n\u003cp\u003eColasuonno P, Lozito ML, Marcotuli I, Nigro D, Giancaspro A, Mangini G, De Vita P, Mastrangelo AM, Pecchioni N, Houston K, Simeone R, Gadaleta A, Blanco A (2017) The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments. BMC GENOMICS 18\u003c/p\u003e\n\u003cp\u003eEspasandin FD, Maiale SJ, Calzadilla P, Ruiz OA, Sansberro PA (2014) Transcriptional regulation of 9-cis-epoxycarotenoid dioxygenase (NCED) gene by putrescine accumulation positively modulates ABA synthesis and drought tolerance in Lotus tenuis plants. PLANT PHYSIOL BIOCH 76:29\u0026ndash;35\u003c/p\u003e\n\u003cp\u003eFei Zhou E (2020) More is better: the diversity of terpene metabolism in plants Fei Zhou and Eran Pichersky. CURR OPIN PLANT BIOL 1\u0026ndash;10\u003c/p\u003e\n\u003cp\u003eFraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N, Drake RG, Schuch W, Bramley PM (2002) Evaluation of Transgenic Tomato Plants Expressing an Additional Phytoene Synthase in a Fruit-Specific Manner. Proceedings of the National Academy of Sciences - PNAS 99:1092\u0026ndash;1097\u003c/p\u003e\n\u003cp\u003eJia T, Ito H, Hu X, Tanaka A (2015) Accumulation of the NON-YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Plant J 81:586\u0026ndash;596\u003c/p\u003e\n\u003cp\u003eLescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. NUCLEIC ACIDS RES 30:325\u0026ndash;327\u003c/p\u003e\n\u003cp\u003eMorikawa T, Uraguchi Y, Sanda S, Nakagawa S, Sawayama S (2018) Overexpression of DnaJ-Like Chaperone Enhances Carotenoid Synthesis in \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e. APPL BIOCHEM BIOTECH 184:80\u0026ndash;91\u003c/p\u003e\n\u003cp\u003eMorita R, Sato Y, Masuda Y, Nishimura M, Kusaba M (2009) Defect in non-yellow coloring 3, an \u0026alpha;/\u0026beta; hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice. Plant J 59:940\u0026ndash;952\u003c/p\u003e\n\u003cp\u003eNisar N, Li L, Lu S, Khin NC, Pogson BJ (2015) Carotenoid Metabolism in Plants. MOL PLANT 8:68\u0026ndash;82\u003c/p\u003e\n\u003cp\u003eObrero A, Gonzalez-Verdejo CI, Roman B, Gomez P, Die JV, Ampomah-Dwamena C (2015) Identification, cloning, and expression analysis of three phytoene synthase genes from Cucurbita pepo. BIOL PLANTARUM 59:201\u0026ndash;210\u003c/p\u003e\n\u003cp\u003eSato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M (2009) Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll\u0026emsp;b and light-harvesting complex\u0026emsp;II degradation during senescence in rice. Plant J 57:120\u0026ndash;131\u003c/p\u003e\n\u003cp\u003eShinjiro Yamaguchi YKAE (2007) 9 Regulation of ABA and GA levels during seed development and germination in Arabidopsis. Annual Plant Reviews 224\u0026ndash;247\u003c/p\u003e\n\u003cp\u003eSimpson K, Fuentes P, Quiroz-Iturra LF, Flores-Ortiz C, Contreras R, Handford M, Stange C (2018) Unraveling the induction of phytoene synthase 2 expression by salt stress and abscisic acid in \u003cem\u003eDaucus carota\u003c/em\u003e. J EXP BOT 69:4113\u0026ndash;4126\u003c/p\u003e\n\u003cp\u003eTamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. MOL BIOL EVOL 28:2731\u0026ndash;2739\u003c/p\u003e\n\u003cp\u003eTsuchiya T, Ohta H, Okawa K, Iwamatsu A, Shimada H, Masuda T, Takamiya K (1999) Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: finding of a lipase motif and the induction by methyl jasmonate. Proc Natl Acad Sci U S A 96:15362\u0026ndash;15367\u003c/p\u003e\n\u003cp\u003eWalter MH, Strack D (2011) Carotenoids and their cleavage products: biosynthesis and functions. NAT PROD REP 28:663\u0026ndash;692\u003c/p\u003e\n\u003cp\u003eWang T, Hou Y, Hu H, Wang C, Zhang W, Li H, Cheng Z, Yang L (2020) Functional Validation of Phytoene Synthase and Lycopene \u0026epsilon;-Cyclase Genes for High Lycopene Content in Autumn Olive Fruit (\u003cem\u003eElaeagnus umbellata\u003c/em\u003e). J AGR FOOD CHEM 68:11503\u0026ndash;11511\u003c/p\u003e\n\u003cp\u003eWen-Li Lee JHAF (2012) Ectopic Expression of Oncidium Gower Ramsey Phytoene Synthase Gene Caused Pale Flower and Reduced Leaf Size in Transgenic Tobacco. Journal of International Cooperation 133\u0026ndash;150\u003c/p\u003e\n\u003cp\u003eYu M, Chen J, Qu J, Liu F, Zhou M, Ma Y, Xiang S, Pan X, Zhang H, Yang M (2020) Exposure to endophytic fungi quantitatively and compositionally alters anthocyanins in grape cells. PLANT PHYSIOL BIOCH 149:144\u0026ndash;152\u003c/p\u003e\n\u003cp\u003eZhou R, Kroczynska B, Hayman GT, Miernyk JA (1995) AtJ2, an arabidopsis homolog of Escherichia coli dnaJ. PLANT PHYSIOL 108:821\u0026ndash;822\u003c/p\u003e\n\u003cp\u003eZhou X, Welsch R, Yang Y, \u0026Aacute;lvarez D, Riediger M, Yuan H, Fish T, Liu J, Thannhauser TW, Li L (2015) Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis. Proceedings of the National Academy of Sciences 112:3558\u0026ndash;3563\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carotenoids, Phytoene synthase, ZjPSY, Zoysia japonica","lastPublishedDoi":"10.21203/rs.3.rs-349623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-349623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhytoene synthase (Psy) is a key limiting enzyme in the carotenoid biosynthesis pathway by regulating phytoene synthesis. In this study, \u003cem\u003eZjPSY\u003c/em\u003e was isolated and identified from an important lawn grass species, \u003cem\u003eZoysia japonica\u003c/em\u003e. \u003cem\u003eZjPSY\u003c/em\u003e cDNA was 1230 bp in length, corresponding to 409 amino acids. \u003cem\u003eZjPSY\u003c/em\u003e showed higher expression in young leaves and were down-regulated after GA3, ABA, SA, and MeJA treatments, exhibited a sensitivity to hormones. By analysis of cis-regulatory elements in \u003cem\u003eZjPSY\u003c/em\u003e promoter region, ZjPSY exhibited be regulated of light and multiple hormones. To investigate the functions of \u003cem\u003eZjPSY\u003c/em\u003e, the plant expression vector was constructed to obtain transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Overexpression of \u003cem\u003eZjPSY\u003c/em\u003e protein led to carotenoid accumulation and altered expression of genes involved in carotenoid contents. \u003cem\u003eZjPSY\u003c/em\u003e expressing Arabidopsis thaliana exhibited yellowing and dwarfing phenotypes and contained more carotenoids than the wild type. Yeast two-hybrid screening identified a novel interacting partner of \u003cem\u003eZjPSY\u003c/em\u003e, \u003cem\u003eZjJ2\u003c/em\u003e (\u003cem\u003eDNAJ homologue 2\u003c/em\u003e), which encodes heat-shock protein 40 (HSP40). Taken together, this study suggests that ZjPSY plays an important role in carotenoid synthesis, leaf color development and hormone response in transgenic plants. These results broadened the understanding of carotenoid synthesis pathways and laid a foundation for the exploration and utilization of \u003cem\u003ePSY\u003c/em\u003e gene.\u003c/p\u003e","manuscriptTitle":"Expression of ZjPSY, A Phytoene Synthase Gene from Zoysia Japonica Leads to Leaf Yellowing and Plant Dwarfing in Arabidopsis Thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-12 13:23:38","doi":"10.21203/rs.3.rs-349623/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d806cbd0-f11c-47f5-ba84-978ec4685be1","owner":[],"postedDate":"April 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3569309,"name":"Plant Physiology and Morphology"},{"id":3569310,"name":"Plant Molecular Biology and Genetics"}],"tags":[],"updatedAt":"2021-05-19T08:15:37+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-12 13:23:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-349623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-349623","identity":"rs-349623","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}