Phosphate deficiency induced by infection promotes synthesis of anthracnose-resistant anthocyanin-3-O-galactoside phytoalexins in the Camellia sinensis plant

Abstract

Tea (Camellia sinensis) is a well-known beverage crop rich in polyphenols with health benefits for humans. Understanding how tea polyphenols participate in plant resistance is beneficial to breeding resistant varieties and uncovering the resistance mechanisms. Here, we report that a Colletotrichum infection-induced ‘pink ring’ symptom appeared outside the lesion, which is highly likely to occur in resistant cultivars. By identifying morphological feature-specific metabolites in the pink ring and their association with disease resistance, and analysis of the association between metabolite and gene expression, the study revealed that the accumulation of anthocyanin-3- O-galactosides, red phytotoxin compounds resistant to anthracnose, plays a pivotal role in the hypersensitive response surrounding infection sites in tea plants. The results of genetic manipulation showed that the expression of CsF3Ha, CsANSa, CsUGT78A15, CsUGT75L43, and CsMYB113, which are involved in anthocyanin biosynthesis, is positively correlated with anthracnose-resistance and the formation of the pink ring. Further phosphorus quantification and fertilization experiments confirmed that phosphate deficiency caused by anthracnose is involved in the occurrence of the pink ring. Genetic manipulation studies indicated that altering the expression levels of Pi transporter proteins (CsPHT2-1, CsPHT4;4) and phosphate deprivation response transcription factors (CsWRKY75-1, CsWRKY75-2, CsMYB62-1) enhances resistance to anthracnose and the formation of the pink ring symptom in tea plants. This article provides the first evidence that anthocyanin-3- O-galactosides are the anthracnose-resistant phytoalexins among various polyphenols in tea plants, and this presents an approach for identifying resistance genes in tea plants, where genetic transformation is challenging.

Acknowledgements

We thank the Natural Science Foundation of China (U21A20232, 32372756, 32072621), the Anhui Provincial Natural Science Foundation (2308085MC94), the National Key Research and Development Program of China (2022YFF1003103), and the Anhui Key Research and Development Program of China (202204c06020035). We are grateful to Professor Yeyun Li for providing the cultivar ‘Zhongcha108’ cuttings for 2 years with different N:P:K ratios.

Author contributions

T.L. performed the experiments and analyzed the data. S.W. and D.S. collected experimental materials. W.F. and T.J. provided help with experiments. L.Z. provided the pathogenic bacteria. Y.L. guided data analysis. T.L., L.G., and T.X. drafted the manuscript. T.L., L.G., and T.X. designed the experiments. All authors read and approved the final manuscript.

Data availability

The raw sequencing data from this study have been deposited in the Genome Sequence Archive in BIG Data Center (https://bigd.big.ac.cn/), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under the accession number CRA012468.

Conflict of interest

The authors declare no competing financial interests.

References

[1] Hayat K, Iqbal H, Malik U, et al. Tea and its consumption: benefits and risks. Crit Rev Food Sci Nutr. 2015; 55: 939-54

[2] Brody H . Tea. Nature. 2019; 566: S1

[3] Pan SY, Nie Q, Tai HC, et al. Tea and tea drinking: China’s outstanding contributions to the mankind. Chin Med. 2022; 17: 27

[4] Khan N, Mukhtar H . Tea polyphenols in promotion of human health. Nutrients. 2018; 11: 39

[5] Qian Y, Zhao X, Zhao L, et al. Analysis of stereochemistry and biosynthesis of epicatechin in tea plants by chiral phase high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2015; 1006: 1-7

[6] Zhuang J, Dai X, Zhu M, et al. Evaluation of astringent taste of green tea through mass spectrometry-based targeted metabolic profiling of polyphenols. Food Chem. 2020; 305: 125507

[7] Wang W, Zhou Y, Wu Y, et al. Insight into catechins metabolic pathways of Camellia sinensis based on genome and transcriptome analysis. J Agric Food Chem. 2018; 66: 4281-93

[8] Han Y, Huang K, Liu Y, et al. Functional analysis of two flavanone-3-hydroxylase genes from Camellia sinensis: a critical role in flavonoid accumulation. Genes (Basel). 2017; 8: 300

[9] Cui L, Yao S, Dai X, et al. Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis). J Exp Bot. 2016; 67: 2285-97

[10] Dai X, Liu Y, Zhuang J, et al. Discovery and characterization of tannase genes in plants: roles in hydrolysis of tannins. New Phytol. 2020; 226: 1104-16

[11] Yao S, Liu Y, Zhuang J, et al. Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs. Plant J. 2022; 111: 117-33

[12] Chen Y, Jiang C, Yin S, et al. New insights into the function of plant tannase with promiscuous acyltransferase activity. Plant J. 2023; 113: 576-94

[13] Jiang X, Huang K, Zheng G, et al. CsMYB5a and CsMYB5e from Camellia sinensis differentially regulate anthocyanin and proanthocyanidin biosynthesis. Plant Sci. 2018; 270: 209-20

[14] Wang P, Ma G, Zhang L, et al. A sucrose-induced MYB (SIMYB) transcription factor promoting proanthocyanidin accumulation in the tea plant (Camellia sinensis). J Agric Food Chem. 2019; 67: 1418-28

[15] He X, Zhao X, Gao L, et al. Isolation and characterization of key genes that promote flavonoid accumulation in purple-leaf tea (Camellia sinensis L.). Sci Rep. 2018; 8: 130

[16] Wei K, Wang L, Zhang Y, et al. A coupled role for CsMYB75 and CsGSTF1 in anthocyanin hyperaccumulation in purple tea. Plant J. 2019; 97: 825-40

[17] Yang Y, Zhang T . Antimicrobial activities of tea polyphenol on phytopathogens: a review. Molecules. 2019; 24: 816

[18] Naskar S, Roy C, Ghosh S, et al. Elicitation of biomolecules as host defense arsenals during insect attacks on tea plants (Camellia sinensis (L.) Kuntze). Appl Microbiol Biotechnol. 2021; 105: 7187-99

[19] Borah A, Hazarika SN, Thakur D, et al. Potentiality of actinobacteria to combat against biotic and abiotic stresses in tea [Camellia sinensis (L) O. Kuntze]. J Appl Microbiol. 2022; 133: 2314-30

[20] Lin SR, Long XP, Li ZP, et al. First report of anthracnose caused by Colletotrichum fructicola on tea in Taiwan. Plant Dis. 2020; 104: 1557

[21] Wang YC, Hao XY, Wang L, et al. Diverse Colletotrichum species cause anthracnose of tea plants (Camellia sinensis (L.) O. Kuntze) in China. Sci Rep. 2016; 6: 35287

[22] Zhang L, Li X, Zhou Y, et al. Identification and characterization of Colletotrichum species associated with Camellia sinensis anthracnose in Anhui Province, China. Plant Dis. 2021; 105: 2649-57

[23] VanEtten HD, Mansfield JW, Bailey JA, et al. Two classes of plant antibiotics: phytoalexins versus ‘phytoanticipins’. Plant Cell. 1994; 6: 1191-2

[24] Dixon RA . Natural products and plant disease resistance. Nature. 2001; 411: 843-7

[25] Westrick NM, Smith DL, Kabbage M . Disarming the host: detoxification of plant defense compounds during fungal necrotrophy. Front Plant Sci. 2021; 12: 651716

[26] Cox KL . Stronger together: ethylene, jasmonic acid, and MAPK signaling pathways synergistically induce camalexin synthesis for plant disease resistance. Plant Cell. 2022; 34: 2829-30

[27] Cheng Q, Li N, Dong L, et al. Overexpression of soybean isoflavone reductase (GmIFR) enhances resistance to Phytophthora sojae in soybean. Front Plant Sci. 2015; 6: 1024

[28] Ibraheem F, Gaffoor I, Chopra S . Flavonoid phytoalexin-dependent resistance to anthracnose leaf blight requires a functional yellow seed1 in Sorghum bicolor. Genetics. 2010; 184: 915-26

[29] Nicholson RL, Kollipara SS, Jeffrey R, et al. Phytoalexin synthesis by the sorghum mesocotyl in response to infection by pathogenic and nonpathogenic fungi. Proc Natl Acad Sci USA. 1987; 84: 5520-4

[30] Wang Y, Hao X, Lu Q, et al. Transcriptional analysis and histochemistry reveal that hypersensitive cell death and H₂O₂ have crucial roles in the resistance of tea plant (Camellia sinensis (L.) O. Kuntze) to anthracnose . Hortic Res. 2018; 5: 18

[31] Snyder BA, Nicholson RL . Synthesis of phytoalexins in sorghum as a site-specific response to fungal ingress. Science. 1990; 248: 1637-9

[32] Li X, Ahammed GJ, Zhang XN, et al. Melatonin-mediated regulation of anthocyanin biosynthesis and antioxidant defense confer tolerance to arsenic stress in Camellia sinensis L. J Hazard Mater. 2021; 403: 123922

[33] Mei Y, Xie H, Liu S, et al. Metabolites and transcriptional profiling analysis reveal the molecular mechanisms of the anthocyanin metabolism in the "Zijuan" tea plant (Camellia sinensis var. assamica). J Agric Food Chem. 2021; 69: 414-27

[34] Wang P, Liu Y, Zhang L, et al. Functional demonstration of plant flavonoid carbocations proposed to be involved in the biosynthesis of proanthocyanidins. Plant J. 2020; 101: 18-36

[35] Wang P, Zhang L, Jiang X, et al. Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis. Planta. 2018; 247: 139-54

[36] Shi Y, Jiang X, Chen L, et al. Functional analyses of flavonol synthase genes from Camellia sinensis reveal their roles in anther development. Front Plant Sci. 2021; 12: 753131

[37] Devaiah BN, Madhuvanthi R, Karthikeyan AS, et al. Phosphate starvation responses and gibberellic acid biosynthesis are regulated by the MYB62 transcription factor in Arabidopsis. Mol Plant. 2009; 2: 43-58

[38] Meng J, Wang H, Chi R, et al. The eTM-miR858-MYB62-like module regulates anthocyanin biosynthesis under low-nitrogen conditions in Malus spectabilis. New Phytol. 2023; 238: 2524-44

[39] Devaiah BN, Karthikeyan AS, Raghothama KG . WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol. 2007; 143: 1789-801

[40] Cong L, Qu Y, Sha G, et al. PbWRKY75 promotes anthocyanin synthesis by activating PbDFR, PbUFGT, and PbMYB10b in pear. Physiol Plant. 2021; 173: 1841-9

[41] Wang Y, Wang F, Lu H, et al. Phosphate uptake and transport in plants: an elaborate regulatory system. Plant Cell Physiol. 2021; 62: 564-72

[42] Khusnutdinov E, Sukhareva A, Panfilova M, et al. Anthocyanin biosynthesis genes as model genes for genome editing in plants. Int J Mol Sci. 2021; 22: 8752

[43] Chaves-Silva S, Santos AL, Chalfun-Júnior A, et al. Understanding the genetic regulation of anthocyanin biosynthesis in plants - tools for breeding purple varieties of fruits and vegetables. Phytochemistry. 2018; 153: 11-27

[44] Liu Y, Tikunov Y, Schouten RE, et al. Anthocyanin biosynthesis and degradation mechanisms in solanaceous vegetables: a review. Front Chem. 2018; 6: 52

[45] Iwashina T. Contribution to flower colors of flavonoids including anthocyanins: a review. Nat Prod Commun. 2015; 10: 529-44

[46] Sinopoli A, Calogero G, Bartolotta A . Computational aspects of anthocyanidins and anthocyanins: a review. Food Chem. 2019; 297: 124898

[47] Naing AH, Kim CK . Abiotic stress-induced anthocyanins in plants: their role in tolerance to abiotic stresses. Physiol Plant. 2021; 172: 1711-23

[48] Ma Y, Ma X, Gao X, et al. Light induced regulation pathway of anthocyanin biosynthesis in plants. Int J Mol Sci. 2021; 22: 11116

[49] Jezek M, Zorb C, Merkt N, et al. Anthocyanin management in fruits by fertilization. J Agric Food Chem. 2018; 66: 753-64

[50] Gao HN, Jiang H, Cui JY, et al. Review: the effects of hormones and environmental factors on anthocyanin biosynthesis in apple. Plant Sci. 2021; 312: 111024

[51] Kangatharalingam N, Pierce ML, Bayles MB, et al. Epidermal anthocyanin production as an indicator of bacterial blight resistance in cotton. Physiol Mol Plant Pathol. 2002; 61: 189-95

[52] Becker L, Bellow S, Carré V, et al. Correlative analysis of fluorescent phytoalexins by mass spectrometry imaging and fluorescence microscopy in grapevine leaves. Anal Chem. 2017; 89: 7099-106

[53] Wang Y-C, Qian WJ, Li NN, et al. Metabolic changes of caffeine in tea plant (Camellia sinensis (L.) O. Kuntze) as defense response to Colletotrichum fructicola. J Agric Food Chem. 2016; 64: 6685-93

[54] Nielsen KA, Gotfredsen CH, Buch-Pedersen MJ, et al. Inclusions of flavonoid 3-deoxyanthocyanidins in Sorghum bicolor self-organize into spherical structures. Physiol Mol Plant Pathol. 2004; 65: 187-96

[55] Lopez A, Lucas J . A cytological study of compatible and incompatible interactions between Sorghum bicolor and Colletotrichum sublineolum. New Phytol. 1996; 43: 25-34

[56] Walters DR, Bingham IJ . Influence of nutrition on disease development caused by fungal pathogens: implications for plant disease control. Ann Appl Biol. 2007; 151: 307-24

[57] Dong Z, Li W, Liu J, et al. The rice phosphate transporter protein OsPT8 regulates disease resistance and plant growth. Sci Rep. 2019; 9: 5408

[58] Baker A, Ceasar SA, Palmer AJ, et al. Replace, reuse, recycle: improving the sustainable use of phosphorus by plants. J Exp Bot. 2015; 66: 3523-40

[59] Misson J, Raghothama KG, Jain A, et al. A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proc Natl Acad Sci USA. 2005; 102: 11934-9

[60] Bustos R, Castrillo G, Linhares F, et al. A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet. 2010; 6: e1001102

[61] Liu F, Chang XJ, Ye Y, et al. Comprehensive sequence and whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice. Mol Plant. 2011; 4: 1105-22

[62] Campos-Soriano L, Bundó M, Bach-Pages M, et al. Phosphate excess increases susceptibility to pathogen infection in rice. Mol Plant Pathol. 2020; 21: 555-70

[63] Kc S, Long L, Zhang Q, et al. Effect of interactions between phosphorus and light intensity on metabolite compositions in tea cultivar Longjing43. Int J Mol Sci. 2022; 23: 15194

[64] Zhou Y, Deng R, Xu X, et al. Isolation of mesophyll protoplasts from tea (Camellia sinensis) and localization analysis of enzymes involved in the biosynthesis of specialized metabolites. Beverage Plant Res. 2021; 1: 1-9

[65] Ding Y, Lawrence CE . A statistical sampling algorithm for RNA secondary structure prediction. Nucleic Acids Res. 2003; 31: 7280-301