Molecular identification of phenylalanine ammonia lyase-encoding genes EfPALs and EfPAL2-interacting transcription factors in Euryale ferox

Flavonoids are one of the most important secondary metabolites in plants, and phenylalanine ammonia-lyase (PAL) is the first rate-limiting enzyme for their biosynthesis. However, detailed information on the regulation of PAL in plants is still little. In this study, PAL in E. ferox was identified and functionally analyzed, and its upstream regulatory network was investigated. Through genome-wide identification, we obtained 12 putative PAL genes from E. ferox. Phylogenetic tree and synteny analysis revealed that PAL in E. ferox was expanded and mostly preserved. Subsequently, enzyme activity assays demonstrated that EfPAL1 and EfPAL2 both catalyzed the production of cinnamic acid from phenylalanine only, with EfPAL2 exhibiting a superior enzyme activity. Overexpression of EfPAL1 and EfPAL2 in Arabidopsis thaliana, respectively, both enhanced the biosynthesis of flavonoids. Furthermore, two transcription factors, EfZAT11 and EfHY5, were identified by yeast one-hybrid library assays as binding to the promoter of EfPAL2, and further luciferase (LUC) activity analysis indicated that EfZAT11 promoted the expression of EfPAL2, while EfHY5 repressed the expression of EfPAL2. These results suggested that EfZAT11 and EfHY5 positively and negatively regulate flavonoid biosynthesis, respectively. Subcellular localization revealed that EfZAT11 and EfHY5 were localized in the nucleus. Our findings clarified the key EfPAL1 and EfPAL2 of flavonoid biosynthesis in E. ferox and established the upstream regulatory network of EfPAL2, which would provide novel information for the study of flavonoid biosynthesis mechanism.

[1]  Ting Wu,et al.  MPK6‐mediated HY5 phosphorylation regulates light‐induced anthocyanin accumulation in apple fruit , 2022, Plant biotechnology journal.

[2]  Peng Wu,et al.  EfABI4 Transcription Factor Is Involved in the Regulation of Starch Biosynthesis in Euryale ferox Salisb Seeds , 2022, International journal of molecular sciences.

[3]  Xiang Li,et al.  Proteomic analysis of Euryale ferox Salisb seeds at different developmental stages. , 2022, Gene.

[4]  Yue-Ying Chen,et al.  Genome-Wide Analysis and Expression Profiling of the Phenylalanine Ammonia-Lyase Gene Family in Solanum tuberosum , 2022, International journal of molecular sciences.

[5]  Kang Zhang,et al.  The adaptive evolution of Euryale ferox to the aquatic environment through paleo‐hexaploidization , 2022, The Plant journal : for cell and molecular biology.

[6]  Li Wang,et al.  Plant flavonoids: Classification, distribution, biosynthesis, and antioxidant activity. , 2022, Food chemistry.

[7]  Xuejin Chen,et al.  Identification of PAL genes related to anthocyanin synthesis in tea plants and its correlation with anthocyanin content , 2021, Horticultural Plant Journal.

[8]  Cathie Martin,et al.  Two types of O‐methyltransferase are involved in biosynthesis of anticancer methoxylated 4′‐deoxyflavones in Scutellaria baicalensis Georgi , 2021, Plant biotechnology journal.

[9]  Peng Wu,et al.  Metabolomics and transcriptome analysis of the biosynthesis mechanism of flavonoids in the seeds of Euryale ferox Salisb at different developmental stages , 2021, Molecular Genetics and Genomics.

[10]  Ting Wu,et al.  Apple MPK4 Mediates Phosphorylation of MYB1 to Enhance Light-Induced Anthocyanin Accumulation. , 2021, The Plant journal : for cell and molecular biology.

[11]  G. Qin,et al.  The transcription factor SlHY5 regulates the ripening of tomato fruit at both the transcriptional and translational levels , 2021, Horticulture research.

[12]  Yuejin Wang,et al.  Genome-wide identification and characterisation of phenylalanine ammonia-lyase gene family in grapevine , 2021 .

[13]  C. Liu,et al.  Enzymatic basis for stepwise C-glycosylation in the formation of flavonoid di-C-glycosides in sacred lotus (Nelumbo nucifera Gaertn.). , 2021, The Plant journal : for cell and molecular biology.

[14]  Xiang Yu,et al.  The role of VvMYBA2r and VvMYBA2w alleles of the MYBA2 locus in the regulation of anthocyanin biosynthesis for molecular breeding of grape (Vitis spp.) skin coloration , 2021, Plant biotechnology journal.

[15]  Kun-song Chen,et al.  Anti-diabetic effects of natural antioxidants from fruits , 2020 .

[16]  Tao Xia,et al.  Functional characterization of three flavonol synthase genes from Camellia sinensis: Roles in flavonol accumulation. , 2020, Plant science : an international journal of experimental plant biology.

[17]  Margaret H. Frank,et al.  TBtools - an integrative toolkit developed for interactive analyses of big biological data. , 2020, Molecular plant.

[18]  A. Fernie,et al.  Conservation and diversification of flavonoid metabolism in the plant kingdom. , 2020, Current opinion in plant biology.

[19]  Haiyang Wang,et al.  An R2R3 MYB transcription factor confers brown planthopper resistance by regulating the phenylalanine ammonia-lyase pathway in rice , 2019, Proceedings of the National Academy of Sciences.

[20]  Jinfang Chu,et al.  A Crucial Role of GA-Regulated Flavonol Biosynthesis in Root Growth of Arabidopsis. , 2019, Molecular plant.

[21]  S. Abou Elela,et al.  Introns are mediators of cell response to starvation , 2019, Nature.

[22]  P. Zhao,et al.  Genome-Wide Identification and Transcriptional Expression of the PAL Gene Family in Common Walnut (Juglans Regia L.) , 2019, Genes.

[23]  D. Bartel,et al.  Excised linear introns regulate growth in yeast , 2018, Nature.

[24]  A. Xiong,et al.  AgMYB2 transcription factor is involved in the regulation of anthocyanin biosynthesis in purple celery (Apium graveolens L.) , 2018, Planta.

[25]  A. Xiong,et al.  AgMYB2 transcription factor is involved in the regulation of anthocyanin biosynthesis in purple celery (Apium graveolens L.) , 2018, Planta.

[26]  Jianhua Zhu,et al.  The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato. , 2018, Plant, cell & environment.

[27]  C. Pellegrini,et al.  Dietary flavonoids as a potential intervention to improve redox balance in obesity and related co-morbidities: a review , 2018, Nutrition Research Reviews.

[28]  Zhen He,et al.  Transcriptome sequencing and analysis during seed growth and development in Euryale ferox Salisb , 2018, BMC Genomics.

[29]  Milen I Georgiev,et al.  Jasmonate-responsive MYB factors spatially repress rutin biosynthesis in Fagopyrum tataricum , 2018, Journal of experimental botany.

[30]  M. Samuel,et al.  Flavonoids and ROS Play Opposing Roles in Mediating Pollination in Ornamental Kale (Brassica oleracea var. acephala). , 2017, Molecular plant.

[31]  V. Irish,et al.  Flavonol rhamnosylation indirectly modifies the cell wall defects of RHAMNOSE BIOSYNTHESIS1 mutants by altering rhamnose flux , 2017, bioRxiv.

[32]  Jian-Ping An,et al.  The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple , 2017, Horticulture Research.

[33]  A. Fernie,et al.  Specialized Metabolites of the Flavonol Class Mediate Root Phototropism and Growth. , 2016, Molecular Plant.

[34]  J. Botto,et al.  The Multifaceted Roles of HY5 in Plant Growth and Development. , 2016, Molecular plant.

[35]  Jia Ouyang,et al.  Molecular Characterization of a Recombinant Zea mays Phenylalanine Ammonia-Lyase (ZmPAL2) and Its Application in trans-Cinnamic Acid Production from l-Phenylalanine , 2015, Applied Biochemistry and Biotechnology.

[36]  Xuebin Zhang,et al.  Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids. , 2014, Molecular plant.

[37]  Xiao-yan Xie,et al.  [Studies on chemical constituents from seeds of Euryale ferox]. , 2014, Zhong yao cai = Zhongyaocai = Journal of Chinese medicinal materials.

[38]  D. Yun,et al.  ZAT11, a zinc finger transcription factor, is a negative regulator of nickel ion tolerance in Arabidopsis , 2014, Plant Cell Reports.

[39]  Shuzhen Wang,et al.  Molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (NnPAL1) from Nelumbo nucifera: novel insight into the evolution of the PAL family in angiosperms , 2014, BMC Evolutionary Biology.

[40]  Rongcheng Lin,et al.  Antagonistic Basic Helix-Loop-Helix/bZIP Transcription Factors Form Transcriptional Modules That Integrate Light and Reactive Oxygen Species Signaling in Arabidopsis[W] , 2013, Plant Cell.

[41]  C. Dong,et al.  Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon (Citrullus lanatus) , 2013, Planta.

[42]  P. Casati,et al.  Flavonoids: biosynthesis, biological functions, and biotechnological applications , 2012, Front. Plant Sci..

[43]  C. Dong,et al.  Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L. , 2012, Planta.

[44]  C. Dong,et al.  Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L. , 2012, Planta.

[45]  Chenglei Li,et al.  Cloning, Characterization and Functional Analysis of a Phenylalanine Ammonia-lyase Gene (FtPAL) from Fagopyrum tataricum Gaertn , 2012, Plant Molecular Biology Reporter.

[46]  F. Hou,et al.  Isolation and identification of compounds responsible for antioxidant capacity of Euryale ferox seeds. , 2011, Journal of agricultural and food chemistry.

[47]  S. Park,et al.  Differential expression of flavonoid biosynthesis genes and accumulation of phenolic compounds in common buckwheat (Fagopyrum esculentum). , 2010, Journal of agricultural and food chemistry.

[48]  B. Fan,et al.  Functional Analysis of the Arabidopsis PAL Gene Family in Plant Growth, Development, and Response to Environmental Stress1[W][OA] , 2010, Plant Physiology.

[49]  R. Dixon,et al.  Phenylalanine ammonia-lyase (PAL) from tobacco (Nicotiana tabacum): characterization of the four tobacco PAL genes and active heterotetrameric enzymes. , 2009, The Biochemical journal.

[50]  R. Nazar,et al.  Tomato Phenylalanine Ammonia-Lyase Gene Family, Highly Redundant but Strongly Underutilized* , 2008, Journal of Biological Chemistry.

[51]  G. Choi,et al.  PIF3 regulates anthocyanin biosynthesis in an HY5-dependent manner with both factors directly binding anthocyanin biosynthetic gene promoters in Arabidopsis. , 2007, The Plant journal : for cell and molecular biology.

[52]  Yuanlei Hu,et al.  Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis , 2006, Plant Cell Reports.

[53]  D. Treutter.  Significance of Flavonoids in Plant Resistance and Enhancement of Their Biosynthesis , 2005, Plant biology.

[54]  J. V. Van Beeumen,et al.  Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism , 2004, The Plant Cell Online.

[55]  Jeroen Raes,et al.  Genome-Wide Characterization of the Lignification Toolbox in Arabidopsis1[w] , 2003, Plant Physiology.

[56]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[57]  D. Twell,et al.  Novel anther-specific myb genes from tobacco as putative regulators of phenylalanine ammonia-lyase expression. , 2001, Plant physiology.

[58]  Xing Wang Deng,et al.  Targeted destabilization of HY5 during light-regulated development of Arabidopsis , 2000, Nature.

[59]  R. Dixon,et al.  Stress-Induced Phenylpropanoid Metabolism. , 1995, The Plant cell.

[60]  M. Parniske,et al.  Modes of expression and common structural features of the complete phenylalanine ammonia-lyase gene family in parsley. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[61]  M. Bevan,et al.  A flower‐specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. , 1994, The EMBO journal.

[62]  R. Dixon,et al.  Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues. , 1989, The Journal of biological chemistry.

[63]  L. Hsieh,et al.  Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii , 2010, Molecular Biology Reports.

[64]  Thomas D. Schmittgen,et al.  Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 2 DD C T Method , 2022 .