过表达MtVP1对马铃薯表型及糖代谢的影响
收稿日期: 2021-05-02
录用日期: 2021-10-13
网络出版日期: 2021-10-13
基金资助
国家自然科学基金(31660075);榆林学院高层次人才科研启动金(14GK39)
Effects of Overexpression of MtVP1 on Potato Phenotypes and Sugar Metabolism
Received date: 2021-05-02
Accepted date: 2021-10-13
Online published: 2021-10-13
I型H+-PPase参与糖异生和蔗糖分解代谢, 利用不同的糖(蔗糖、葡萄糖和果糖)饲喂拟南芥(Arabidopsis thaliana) I型H+-PPase基因不同类型的突变体, 产生的表型不一致, 因此, 推测I型H+-PPase可能存在其它影响糖代谢的机制。为进一步明确该酶对糖代谢的影响, 以过表达MtVP1的马铃薯(Solanum tuberosum)渭薯4号为研究对象, 观察不同培养条件下的表型, 监测糖含量变化, 并利用转录组测序分析转录谱。结果表明, 过表达MtVP1马铃薯表现出红色茎、紫色花和表皮毛更发达, 单株块茎数减少, 块茎变大, 块茎皱缩速度加快; 转基因马铃薯块茎中淀粉、葡萄糖和果糖含量显著下降, 芽中葡萄糖和果糖含量也显著下降。果糖饲喂导致转基因马铃薯花青素含量显著降低; 转基因马铃薯体内果糖-1,6-二磷酸酶和果糖- 2,6-二磷酸酶基因表达上调3-7倍。研究结果为进一步从糖代谢角度探究I型H+-PPase的生理功能提供参考。
关键词: I型膜结合H+-PPase; 果糖-1,6-二磷酸酶; 果糖-2,6-二磷酸酶; 糖代谢; 果糖代谢
王建武, 王文娟, 相微微, 代惠萍, 王海庆, 屈香香, 亢福仁 . 过表达MtVP1对马铃薯表型及糖代谢的影响[J]. 植物学报, 2022 , 57(2) : 197 -208 . DOI: 10.11983/CBB21074
Type I H+-PPase is involved in gluconeogenesis and sucrose catabolism. Different types of mutants of Arabidopsis thaliana type I H+-PPase gene fed with different sugars (sucrose, glucose and fructose) showed various phenotypes. Therefore, it is speculated that type I H+-PPase may contain other mechanisms that affect sugar metabolism. In order to further clarify the effect of the enzyme on sugar metabolism, phenotypes of the MtVP1 overexpressed potato Weishu 4 were observed under different culture conditions. The fluctuation of sugar content was monitored, and the transcription profile was analyzed. The results showed that the MtVP1 overexpressed potato grew red stem, purple flower, and more developed trichomes. Furthermore, tubers per plant decreased in number, but became larger in size, and shriveled up faster. However, the transgenic plant had significantly decreased starch, glucose, and fructose contents in tubers, and glucose and fructose contents in buds. At the same time, feeding fructose significantly reduced the anthocyanin, and up-regulated the gene expression of fructose-1,6-diphosphate and fructose-2,6-diphosphate by 3-7 fold in the transgenic potato. The results could shed light on further studies of the physiological function of type I H+-PPase from the perspective of sugar metabolism.
[1] | 柳俊, 谢从华 (2001). 马铃薯块茎发育机理及其基因表达. 植物学通报 18, 531-539. |
[2] | 王建武, 相微微, 亢福仁 (2016). 过表达截形苜蓿液泡膜H+-PPase基因提高马铃薯的抗旱性. 分子植物育种 14, 1500-1506. |
[3] | 卫生部食品卫生监督检验所 (2003). 食品中淀粉的测定标准:GB/T 5009.9-2003. 北京: 中国标准出版社. pp. 1-3. |
[4] | Baysal C, He WS, Drapal M, Villorbina G, Medina V, Capell T, Khush GS, Zhu CF, Fraser PD, Christou P (2020). Inactivation of rice starch branching enzyme IIb triggers broad and unexpected changes in metabolism by transcriptional reprogramming. Proc Natl Acad Sci USA 117, 26503-26512. |
[5] | Bussard A, Lopez PJ (2014). Evolution of vacuolar pyrophosphatases and vacuolar H+-ATPases in diatoms. J Marine Sci Technol 22, 50-59. |
[6] | Drozdowicz YM, Rea PA (2001). Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6, 206-211. |
[7] | Duan XG, Yang AF, Gao F, Zhang SL, Zhang JR (2007). Heterologous expression of vacuolar H+-PPase enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance. Protoplasma 232, 87-95. |
[8] | Farré EM, Geigenberger P, Willmitzer L, Trethewey RN (2000). A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber. Plant Physiol 123, 681-688. |
[9] | Ferjani A, Segami S, Horiguchi G, Muto Y, Maeshima M, Tsukaya H (2011). Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis. Plant Cell 23, 2895-2908. |
[10] | Fuglsang AT, Paez-Valencia J, Gaxiola RA (2011). Plant proton pumps:regulatory circuits involving H+-ATPase and H+-PPase. In: Geisler M, Venema K, eds. Transporters and Pumps in Plant Signaling. Heidelberg: Springer. pp. 39-64. |
[11] | Gao F, Gao Q, Duan XG, Yue GD, Yang AF, Zhang JR (2006). Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance. J Exp Bot 57, 3259-3270. |
[12] | Gaxiola RA, Li JS, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001). Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci USA 98, 11444-11449. |
[13] | Gaxiola RA, Rao R, Sherman A, Grisafi P, Alper SL, Fink GR (1999). The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci USA 96, 1480-1485. |
[14] | Gaxiola RA, Regmi K, Paez-Valencia J, Pizzio G, Zhang SJ (2016). Plant H+-PPases: reversible enzymes with contrasting functions dependent on membrane environment. Mol Plant 9, 317-319. |
[15] | Gaxiola RA, Sanchez CA, Paez-Valencia J, Ayre BG, Elser JJ (2012). Genetic manipulation of a ‘vacuolar’ H+-PPase: from salt tolerance to yield enhancement under phosphorus-deficient soils. Plant Physiol 159, 3-11. |
[16] | Graus D, Konrad KR, Bemm F, Nebioglu MGP, Lorey C, Duscha K, Güthoff T, Herrmann J, Ferjani A, Cuin TA, Roelfsema MRG, Schumacher K, Neuhaus HE, Marten I, Hedrich R (2018). High V-PPase activity is beneficial under high salt loads, but detrimental without salinity. New Phytol 219, 1421-1432. |
[17] | Guo S, Yin H, Zhang X, Zhao F, Li P, Chen S, Zhao Y, Zhang H (2006). Molecular cloning and characterization of a vacuolar H+-pyrophos-phatase gene, SsVP, from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis. Plant Mol Biol 60, 41-50. |
[18] | Khuri S, Moorby J (1995). Investigations into the role of sucrose in potato cv. Estima microtuber production in vitro. Ann of Bot 75, 295-303 |
[19] | Lee CH, Pan YJ, Huang YT, Liu TH, Hsu SH, Lee CH, Chen YW, Lin SM, Huang LK, Pan RL (2011). Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. J Biol Chem 286, 11970- 11976. |
[20] | Li B, Wei AY, Song CX, Li N, Zhang JR (2008). Heterologous expression of the TsVP gene improves the drought resistance of maize. Plant Biotechnol J 6, 146-159. |
[21] | Li JS, Yang HB, Peer WA, Richter G, Blakeslee J, Bandyo padhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL, Krizek B, Murphy AS, Gilroy S, Gaxiola RA (2005). Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 310, 121-125. |
[22] | Lv SL, Zhang KW, Gao Q, Lian LJ, Song YJ, Zhang JR (2008). Overexpression of an H+-PPase gene from Thellungiella halophila in cotton enhances salt tolerance and improves growth and photosynthetic performance. Plant Cell Physiol 49, 1150-1164. |
[23] | Maeshima M (2000). Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465, 37-51. |
[24] | Maeshima M, Yoshida S (1989). Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J Biol Chem 264, 20068- 20073. |
[25] | Nakanishi Y, Saijo T, Wada Y, Maeshima M (2001). Mutagenic analysis of functional residues in putative substrate- binding site and acidic domains of vacuolar H+-pyrophosphatase. J Biol Chem 276, 7654-7660. |
[26] | Nel W, Terblanche SE (1992). Plant fructose-1,6-bisphosphatases: characteristics and properties. Int J Biochem 24, 1267-1283. |
[27] | Nielsen TH, Rung JH, Villadsen D (2004). Fructose-2,6- bisphosphate: a traffic signal in plant metabolism. Trends Plant Sci 9, 556-563. |
[28] | Paez-Valencia J, Patron-Soberano A, Rodriguez-Leviz A, Sanchez-Lares J, Sanchez-Gomez C, Valencia-Mayoral P, Diaz-Rosas G, Gaxiola RA (2011). Plasma membrane localization of the type I H+-PPase AVP1 in sieve element-companion cell complexes from Arabidopsis thaliana. Plant Sci 181, 23-30. |
[29] | Pizzio GA, Paez-Valencia J, Khadilkar AS, Regmi K, Patron-Soberano A, Zhang SJ, Sanchez-Lares J, Furstenau T, Li JS, Sanchez-Gomez C, Valencia-Mayoral P, Yadav UP, Ayre BG, Gaxiola RA (2015). Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning. Plant Physiol 167, 1541-1553. |
[30] | Rea PA, Poole RJ (1993). Vacuolar H+-translocating pyrophosphatase. Annu Rev Plant Physiol Plant Mol Biol 44, 157-180. |
[31] | Ruan YL (2014). Sucrose metabolism: gateway to diverse carbon use and sugar signaling. Annu Rev Plant Biol 65, 33-67. |
[32] | Rufty TW Jr, Huber SC (1983). Changes in starch formation and activities of sucrose phosphate synthase and cytoplasmic fructose-1,6-bisphosphatase in response to source-sink alterations. Plant Physiol 72, 474-480. |
[33] | Schilling RK, Tester M, Marschner P, Plett DC, Roy SJ (2017). AVP1: one protein, many roles. Trends Plant Sci 22, 154-162. |
[34] | Scott P, Kruger NJ (1995). Influence of elevated fructose- 2,6-bisphosphate levels on starch mobilization in transgenic tobacco leaves in the dark. Plant Physiol 108, 1569-1577. |
[35] | Segami S, Tomoyama T, Sakamoto S, Gunji S, Fukuda M, Kinoshita S, Mitsuda N, Ferjani A, Maeshima M (2018). Vacuolar H+-pyrophosphatase and cytosolic soluble pyrophosphatases cooperatively regulate pyrophosphate le- vels in Arabidopsis thaliana. Plant Cell 30, 1040-1061. |
[36] | Shiratake K, Kanayama Y, Maeshima M, Yamaki S (1997). Changes in H+-pumps and a tonoplast intrinsic protein of vacuolar membranes during the development of pear fruit. Plant Cell Physiol 38, 1039-1045. |
[37] | Shiroguchi K, Jia TZ, Sims PA, Xie XS (2012). Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proc Natl Acad Sci USA 109, 1347-1352. |
[38] | Solfanelli C, Poggi A, Loreti E, Alpi A, Perata P (2006). Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. Plant Physiol 140, 637-646. |
[39] | Sonnewald U (1992). Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning. Plant J 2, 571-581. |
[40] | Stitt M (1987). Fructose 2,6-bisphosphate and plant carbohydrate metabolism. Plant Physiol 84, 201-204. |
[41] | Stitt M (1990). Fructose-2,6-bisphosphate as a regulatory molecule in plants. Annu Rev Plant Physiol Plant Mol Biol 41, 153-185. |
[42] | Strand Å, Foyer CH, Gustafsson P, Gardeström P, Hurry V (2003). Altering flux through the sucrose biosynthesis pathway in transgenic Arabidopsis thaliana modifies photosynthetic acclimation at low temperatures and the development of freezing tolerance. Plant Cell Environ 26, 523-535. |
[43] | Strand Å, Zrenner R, Trevanion S, Stitt M, Gustafsson P, Gardeström P (2000). Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1,6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana. Plant J 23, 759-770. |
[44] | Teng S, Keurentjes J, Bentsink L, Koornneef M, Smeekens S (2005). Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/ PAP1 gene. Plant Physiol 139, 1840-1852. |
[45] | Wang JW, Wang HQ, Xiang WW, Chai TY (2014). A Medicago truncatula H+-pyrophosphatase gene, MtVP1, improves sucrose accumulation and anthocyanin biosynthesis in potato (Solanum tuberosum L.). Genet Mol Res 13, 3615-3626. |
[46] | Wang XL, Wang HW, Liu SX, Ferjani A, Li JS, Yan JB, Yang XH, Qin F (2016). Genetic variation in ZmVPP1 contributes to drought tolerance in maize seedlings. Nat Genet 48, 1233-1241. |
[47] | Yang HB, Knapp J, Koirala P, Rajagopal D, Peer WA, Silbart LK, Murphy A, Gaxiola RA (2007). Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+-pyrophosphatase. Plant Biotechnol J 5, 735-745. |
[48] | Zhen RG, Kim EJ, Rea PA (1997a). Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N,N'-dicyclohexylcarbodiimide. J Biol Chem 272, 22340-22348. |
[49] | Zhen RG, Kim EJ, Rea PA (1997b). The molecular and biochemical basis of pyrophosphate-energized proton translocation at the vacuolar membrane. Adv Bot Res 25, 297-337. |
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