Promotion of StHY5 in the Synthesis of SGAs during Tuber Turning-green of Potato

doi:10.13560/j.cnki.biotech.bull.1985.2024-0514

Abstract

Abstract:

【Objective】Post-harvest storage, transportation, shelving, and other factors can lead to the “turning green” of potato, resulting in a significant accumulation of SGAs in tuber. HY5 plays a crucial role as a key mediator of light signals in plants. Investigating its involvement in the turning-green of tuber can establish a molecular foundation for understanding SGAs accumulation induced by light in post-harvest potato. 【Method】Through comprehensive gene expression analysis, transcriptome profiling, targeted metabolite analysis, subcellular localization studies, yeast single hybridization and double luciferase experiments, the involvement of StHY5 in tuber turning green following light exposure was preliminarily elucidated.【Result】The StHY5 gene was expressed in both the peel and flesh of potato, but with different expression patterns. During the treatment of potato greening, the expression of the StHY5 gene in potato meat correlated with changes in SGAs content, while the expression of SGAs synthesizing genes StSGT1/GAME1 and StGAME4 was significantly up-regulated. Yeast single hybridization and double luciferase experiments demonstrated that StHY5 directly up-regulated the expression of StSGT1/GAME1 and StGAME4.【Conclusion】During the process of potato greening, the StHY5 plays a role in promoting the accumulation of SGAs by directly up-regulating the expression of StSGT1/GAME1 and StGAME4. This finding provides a solid foundation for further investigation into the function of StHY5 in potato greening.

Key words: potato, StHY5 gene, tuber turning-green induced by light, SGAs accumulation

WANG Chao, BAI Ru-qian, GUAN Jun-mei, LUO Ji-lin, HE Xue-jiao, CHI Shao-yi, MA Ling. Promotion of StHY5 in the Synthesis of SGAs during Tuber Turning-green of Potato[J]. Biotechnology Bulletin, 2024, 40(9): 113-122.

Figures/Tables 10

Table 1 Primers used in RT-qPCR

基因 Gene	正向引物序列Forward primer sequence（5'-3'）	反向引物序列Reverse primer sequence（5'-3'）
StActin	GGGATGGAGAAGTTTGGTGGTGG	CTTCGACCAAGGGATGGTGTAG
StHY5	AGATCTGGAAGCAAGGGTGAAG	CACCTGCTGTTGTGTTCTTCAG

Table 2 Eluent gradient

时间Time/min	A/%	B/%	流速Flow/（mL·min^-1）
0.00	95.00	5.00	0.800
5.00	60.00	40.00	0.800
6.00	0.00	100.00	0.800
8.00	0.00	100.00	0.800

Fig. 1 Phylogenetic tree and homologous sequence alignment of StHY5 in Solanaceae A: Phylogenetic tree analysis and homologous sequence alignment of StHY5, Solanum lycopersicum and Solanum tuberosum are marked in red; B: homologous sequence alignment of StHY5, the amino acid sequence interval（89-140 aa）marked in green is the bZIP-HY5-like domain

Fig. 2 Temporal and spatial expression analysis (A), in-situ hybridization (B) and subcellular localization analysis (C) of StHY5 in potato Different lowercases indicate significant differences at the 0.05 level. The same below

Table 3 Cis-acting elements of StHY5 promoter in potato

名称 Name	序列 Sequence	数量 Account	功能 Function
MBS	CAACTG	1	参与干旱诱导的MYB结合位点MYB binding site involved in drought-inducibility
LTR	CCGAAA	1	参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness
GC-motif	CCCCCG	1	参与缺氧特异性诱导的增强剂Enhancer-like element involved in anoxic specific inducibility
P-box	CCTTTTG	1	赤霉素反应元件Gibberellin-responsive element
GT1-motif	GGTTAA	1	光敏元件Light-sensitive element
TCCC-motif	TCTCCCT	1	光响应元件的一部分Part of a light responsive element
TCA-element	CCATCTTTTT/TCAGAAGAGG	3	参与水杨酸反应的顺式作用元件Cis-acting element involved in salicylic acid responsiveness
ABRE	ACGTG/GCAACGTGTC	3	参与脱落酸反应的顺式作用元件Cis-acting element involved in the abscisic acid responsiveness
ARE	AAACCA	2	厌氧诱导所需的顺式作用元件Cis-acting regulatory element essential for the anaerobic induction
G-Box	CACGTT	3	参与光反应的顺式作用元件Cis-acting regulatory element involved in light responsiveness
CGTCA-motif	CGTCA	5	茉莉酸甲酯响应元件Cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	TGACG	5	茉莉酸甲酯响应元件Cis-acting regulatory element involved in the MeJA-responsiveness

Fig. 3 SGAs content and StHY5 expression in green turning treatment A-B: CIP183 SGAs intensity of potato peel and flesh. C-D: StHY5 expression of CIP183 potato peel and flesh. E-F: CIP150 SGAs intensity of potato peel and flesh.G-H: StHY5 expression in CIP150 potato peel and flesh

Fig. 4 Transcriptome analysis of potato tuber CIP150 before and after flesh turning green A: Volcanic map analysis. B: KEGG enrichment analysis. C: GO enrichment analysis

Fig. 5 Differential expression of SGAs synthetic genes in potato tuber CIP150 before and after flesh turning green StAACT to St7-DR2 is the synthesis pathway of cholesterol, StGAME7 to StSGT3 is the synthesis pathway of SGAs, the arrow indicates the sequence of genes involved in the SGAs synthesis pathway

Fig. 6 Yeast single hybridization analysis between StHY5 and StSGT1/GAME1 and StGAME4 promoters L, W and H indicate Leu, Trp and His respectively, and 10-1, 10-2 and 10-3 indicate the point plate concentration of yeast OD600 when it is adjusted to 0.1, 0.01 and 0.001, respectively

Fig. 7 Double luciferase analysis between StHY5 and StS-GT1/GAME1 and StGAME4 promoters **P<0.01, ***P<0.001

References 41

[1]	Hardigan MA, Laimbeer FPE, Newton L, et al. Genome diversity of tuber-bearing Solanum uncovers complex evolutionary history and targets of domestication in the cultivated potato[J]. Proc Natl Acad Sci USA, 2017, 114(46): E9999-E10008.
[2]	Menoble C. Research and comparative analysis about potato production situation between China and continents in the world[C]. Agricultural Engineering, 2011.
[3]	Begum SS, Das D, Gour NK, et al. Computational modelling of nanotube delivery of anti-cancer drug into glutathione reductase enzyme[J]. Sci Rep, 2021, 11(1): 4950. doi: 10.1038/s41598-021-84006-1 pmid: 33654109
[4]	何虎翼, 唐洲萍, 杨鑫, 等. 马铃薯淀粉合成与降解研究进展[J]. 生物技术通报, 2019, 35(4): 101-107. doi: 10.13560/j.cnki.biotech.bull.1985.2018-0829
	He HY, Tang ZP, Yang X, et al. Research progress on potato starch synthesis and degradation[J]. Biotechnol Bull, 2019, 35(4): 101-107.
[5]	聂碧华, 谢从华, 聂先舟. 马铃薯抗病毒机制研究进展[J]. 园艺学报, 2012, 39(9): 1703-1714.
	Nie BH, Xie CH, Nie XZ. Progress in research on the resistance mechanism against viruses in potatoes[J]. Acta Hortic Sin, 2012, 39(9): 1703-1714.
[6]	邓仁菊, 邓宽平, 何天久, 等. 马铃薯主要病害抗性育种研究进展[J]. 西南农业学报, 2014, 27(3):1337-1342.
	Deng RJ, Deng KP, He TJ, et al. Progresses of main diseases resistance breeding in potato[J]. Southwest China J Agric Sci, 2014, 27(3): 1337-1342.
[7]	Wang T, He WS, Jiang HG, et al. The effects of nitrogen, phosphorus and potassium application on yield and starch content of potato plants[J]. Soil Fertil Sci China, 2016(3): 80-86.
[8]	Dhalsamant K, Singh CB, Lankapalli R. A review on greening and glycoalkaloids in potato tubers: potential solutions[J]. J Agric Food Chem, 2022, 70(43): 13819-13831.
[9]	Percival G, Dixon GR. Glycoalkaloid concentrations in aerial tubers of potato(Solanum tuberosum L.)[J]. J Sci Food Agric, 1996, 70(4): 439-448.
[10]	Bamberg J, Moehninsi, Navarre R, et al. Variation for tuber greening in the diploid wild potato Solanum microdontum[J]. Am J Potato Res, 2015, 92(3): 435-443.
[11]	Baur S, Bellé N, Hausladen H, et al. Quantitation of toxic steroidal glycoalkaloids and newly identified saponins in post-harvest light-stressed potato(Solanum tuberosum L.) varieties[J]. J Agric Food Chem, 2022, 70(27): 8300-8308.
[12]	Ostreikova TO, Kalinkina OV, Bogomolov NG, et al. Glycoalkaloids of plants in the family solanaceae(Nightshade)as potential drugs[J]. Pharm Chem J, 2022, 56(7): 948-957.
[13]	Itkin M, Heinig U, Tzfadia O, et al. Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes[J]. Science, 2013, 341(6142): 175-179. doi: 10.1126/science.1240230 pmid: 23788733
[14]	Akiyama R, Umemoto N, Mizutani M. Recent advances in steroidal glycoalkaloid biosynthesis in the genus Solanum[J]. Plant Biotechnol, 2023, 40(3): 185-191. doi: 10.5511/plantbiotechnology.23.0717b pmid: 38293253
[15]	Yang LW, Jiang ZM, Jing YJ, et al. PIF1 and RVE1 form a transcriptional feedback loop to control light-mediated seed germination in Arabidopsis[J]. J Integr Plant Biol, 2020, 62(9): 1372-1384.
[16]	Zhou P, Song MF, Yang QH, et al. Both PHYTOCHROME RAPIDLY REGULATED1(PAR1)and PAR2 promote seedling photomorphogenesis in multiple light signaling pathways[J]. Plant Physiol, 2014, 164(2): 841-852.
[17]	Yu JW, Rubio V, Lee NY, et al. COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability[J]. Mol Cell, 2008, 32(5): 617-630.
[18]	Jiao YL, Lau OS, Deng XW. Light-regulated transcriptional networks in higher plants[J]. Nat Rev Genet, 2007, 8(3): 217-230. doi: 10.1038/nrg2049 pmid: 17304247
[19]	Gangappa SN, Botto JF. The BBX family of plant transcription factors[J]. Trends Plant Sci, 2014, 19(7): 460-470. doi: 10.1016/j.tplants.2014.01.010 pmid: 24582145
[20]	Koornneef M, Rolff E, Spruit CJP. Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana(L.) heynh[J]. Z Für Pflanzenphysiol, 1980, 100(2): 147-160.
[21]	Oyama T, Shimura Y, Okada K. The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl[J]. Genes Dev, 1997, 11(22): 2983-2995.
[22]	Jing YJ, Zhang D, Wang X, et al. Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation[J]. Plant Cell, 2013, 25(1): 242-256.
[23]	Gangappa SN, Botto JF. The multifaceted roles of HY5 in plant growth and development[J]. Mol Plant, 2016, 9(10): 1353-1365. doi: S1674-2052(16)30134-4 pmid: 27435853
[24]	Wang WH, Wang PW, Li XJ, et al. The transcription factor SlHY5 regulates the ripening of tomato fruit at both the transcriptional and translational levels[J]. Hortic Res, 2021, 8(1): 83.
[25]	Wang CC, Meng LH, Gao Y, et al. Manipulation of light signal transduction factors as a means of modifying steroidal glycoalkaloids accumulation in tomato leaves[J]. Front Plant Sci, 2018, 9: 437.
[26]	Itkin M, Rogachev I, Alkan N, et al. GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato[J]. Plant Cell, 2011, 23(12): 4507-4525.
[27]	Wan LB, Gao HD, Gao HL, et al. Selective extraction and determination of steroidal glycoalkaloids in potato tissues by electromembrane extraction combined with LC-MS/MS[J]. Food Chem, 2022, 367: 130724.
[28]	Zhu GT, Wang SC, Huang ZJ, et al. Rewiring of the fruit metabolome in tomato breeding[J]. Cell, 2018, 172(1/2): 249-261.e12.
[29]	Jakoby M, Weisshaar B, Dröge-Laser W, et al. bZIP transcription factors in Arabidopsis[J]. Trends Plant Sci, 2002, 7(3): 106-111. doi: 10.1016/s1360-1385(01)02223-3 pmid: 11906833
[30]	Kurihara Y, Makita Y, et al. Time-course transcriptome study reveals mode of bZIP transcription factors on light exposure in Arabidopsis[J]. Int J Mol Sci, 2020, 21(6): 1993.
[31]	Agarwal P, Baranwal VK, Khurana P. Genome-wide analysis of bZIP transcription factors in wheat and functional characterization of a TabZIP under abiotic stress[J]. Sci Rep, 2019, 9(1): 4608. doi: 10.1038/s41598-019-40659-7 pmid: 30872683
[32]	Chattopadhyay S, Ang LH, Puente P, et al. Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression[J]. Plant Cell, 1998, 10(5): 673-683. doi: 10.1105/tpc.10.5.673 pmid: 9596629
[33]	Sa QL, Li WB, Sun YR. Transcriptional regulation of the G-box and G-box-binding proteins in plant gene expression[J]. Plant Physiol Commun, 2003, 39(1): 89-92.
[34]	McCue KF, Allen PV, Shepherd LVT, et al. Potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis[J]. Phytochemistry, 2007, 68(3): 327-334. doi: 10.1016/j.phytochem.2006.10.025 pmid: 17157337
[35]	McCue KF, Allen PV, Shepherd LVT, et al. The primary in vivo steroidal alkaloid glucosyltransferase from potato[J]. Phytochemistry, 2006, 67(15): 1590-1597. pmid: 16298403
[36]	McCue KF, Shepherd LVT, Allen PV, et al. Metabolic compensation of steroidal glycoalkaloid biosynthesis in transgenic potato tubers: using reverse genetics to confirm the in vivo enzyme function of a steroidal alkaloid galactosyltransferase[J]. Plant Sci, 2005, 168(1): 267-273.
[37]	Shakya R, Navarre DA. LC-MS analysis of solanidane glycoalkaloid diversity among tubers of four wild potato species and three cultivars(Solanum tuberosum)[J]. J Agric Food Chem, 2008, 56(16): 6949-6958.
[38]	Iijima Y, Watanabe B, Sasaki R, et al. Steroidal glycoalkaloid profiling and structures of glycoalkaloids in wild tomato fruit[J]. Phytochemistry, 2013, 95: 145-157. doi: 10.1016/j.phytochem.2013.07.016 pmid: 23941899
[39]	Cárdenas PD, Sonawane PD, Heinig U, et al. The bitter side of the nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism[J]. Phytochemistry, 2015, 113: 24-32. doi: 10.1016/j.phytochem.2014.12.010 pmid: 25556315
[40]	Sawai S, Ohyama K, Yasumoto S, et al. Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato[J]. Plant Cell, 2014, 26(9): 3763-3774.
[41]	Cárdenas PD, Sonawane PD, Pollier J, et al. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway[J]. Nat Commun, 2016, 7: 10654. doi: 10.1038/ncomms10654 pmid: 26876023