浙江农业学报 ›› 2022, Vol. 34 ›› Issue (11): 2329-2339.DOI: 10.3969/j.issn.1004-1524.2022.11.02
水稻质体磷酸盐转运体OsPPT家族成员的功能分析
王继纯1(
), 李瑞莉2, 王姣玲3, 邵俊雯1, 赵红玉2,*()
- 1.浙江师范大学 化学与生命科学学院,浙江 金华 321004
2.中国农业科学院 农业资源与农业区划研究所,北京 100098
3.南阳师范学院 生命科学与农业工程学院,河南 南阳473061
-
收稿日期:2022-04-20出版日期:2022-11-25发布日期:2022-11-29 -
通讯作者:赵红玉 -
作者简介:*赵红玉,E-mail:zhy200869@163.com
王继纯(1997—),女,湖南郴州人,硕士研究生,研究方向为植物营养。E-mail:wangjichun930@163.com -
基金资助:国家自然科学基金青年科学基金(31801924)
Functional analysis of plastid phosphate transporter OsPPT family members in rice
WANG Jichun1(), LI Ruili2, WANG Jiaoling3, SHAO Junwen1, ZHAO Hongyu2,*()
- 1. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100098, China
3. College of Life Science and Agricultural Engineering, Nanyang Normal University, Nanyang 473061, Henan, China
-
Received:2022-04-20Online:2022-11-25Published:2022-11-29 -
Contact:ZHAO Hongyu
摘要：
磷酸烯醇式丙酮酸/磷酸盐转运体(PPT)是植物质体磷酸盐转运蛋白家族(pPTs)成员之一,介导细胞质中的磷酸烯醇式丙酮酸(PEP)进入质体基质的同时,将磷交换到细胞质中。为对水稻OsPPT基因家族进行综合分析,探索其在水稻中的潜在功能。利用水稻原生质体瞬时转化分析OsPPT的亚细胞定位,通过酵母异源表达实验分析OsPPT的磷酸盐转运能力。设置正常供磷和缺磷等非生物胁迫水培实验处理,阐明OsPPT家族成员的组织特异性表达模式,以及对非生物胁迫逆境的响应。结果表明,OsPPT基因家族4个成员均定位于叶绿体膜,而且OsPPT可以在酵母中介导磷酸盐的跨膜转运。此外,通过实时荧光定量PCR(qRT-PCR)展示了OsPPT基因家族在应对环境胁迫时表达模式上的动态变化,比如磷饥饿,以及脱落酸(abscisic acid,ABA)、水杨酸(salicylic acid,SA)、氯化钠等非生物胁迫环境。OsPPT基因家族可能参与磷酸盐在细胞质和叶绿体之间的运输,同时也可能参与植物对逆境胁迫的响应。
中图分类号:
引用本文
王继纯, 李瑞莉, 王姣玲, 邵俊雯, 赵红玉. 水稻质体磷酸盐转运体OsPPT家族成员的功能分析[J]. 浙江农业学报, 2022, 34(11): 2329-2339.
WANG Jichun, LI Ruili, WANG Jiaoling, SHAO Junwen, ZHAO Hongyu. Functional analysis of plastid phosphate transporter OsPPT family members in rice[J]. Acta Agriculturae Zhejiangensis, 2022, 34(11): 2329-2339.
| 基因Gene | 上游引物Forward primer(5'→3') | 下游引物Reverse primer(5'→3') |
|---|---|---|
| OsPPT1 | TTCCAATTGTTGGTGGTGTG | AGGTGACATTTGAGGCCATT |
| OsPPT2 | GCATCTCTTACTGAGGCTTCCT | ATTTGAAGCCATTGCACTCC |
| OsPPT3 | TCTACTTCAACATCTACAACAAGCAG | GATGACGAAGGAGCCGAAC |
| OsPPT4 | TCAATCGCGAAATGTTTTCA | GGTTTATATCATCCAATGTTTCCTC |
表1 qRT-PCR引物
Table 1 Primers for qRT-PCR
| 基因Gene | 上游引物Forward primer(5'→3') | 下游引物Reverse primer(5'→3') |
|---|---|---|
| OsPPT1 | TTCCAATTGTTGGTGGTGTG | AGGTGACATTTGAGGCCATT |
| OsPPT2 | GCATCTCTTACTGAGGCTTCCT | ATTTGAAGCCATTGCACTCC |
| OsPPT3 | TCTACTTCAACATCTACAACAAGCAG | GATGACGAAGGAGCCGAAC |
| OsPPT4 | TCAATCGCGAAATGTTTTCA | GGTTTATATCATCCAATGTTTCCTC |
| 基因Gene | 上游引物Forward primer(5'→3') | 下游引物Reverse primer(5'→3') |
|---|---|---|
| OsPPT1 | CGCGGATCCATGCAGAGCGCGGCGGCCGT | ACGCGTCGACGGCAGTCTTGGGCTTGGGTTT |
| OsPPT2 | CGCGGATCCATGCAGAGCGCCGCGGCGGCGTT | ACGCGTCGACCGCAGCCTTGGGCTTGGGCTT |
| OsPPT3 | CGCGGATCCATGCAGCGCGCGGCGGCGGCCT | ACGCGTCGACGGCATTCTTTGGTTTTGTTCTCTT |
| OsPPT4 | CGCGGATCCATGCAGGCCGTGGCGGCGGCGA | ACGCGTCGACTGCAGTCTTAGCCTTTGGTTTAGC |
表2 扩增引物
Table 2 Primers used for fragment amplification
| 基因Gene | 上游引物Forward primer(5'→3') | 下游引物Reverse primer(5'→3') |
|---|---|---|
| OsPPT1 | CGCGGATCCATGCAGAGCGCGGCGGCCGT | ACGCGTCGACGGCAGTCTTGGGCTTGGGTTT |
| OsPPT2 | CGCGGATCCATGCAGAGCGCCGCGGCGGCGTT | ACGCGTCGACCGCAGCCTTGGGCTTGGGCTT |
| OsPPT3 | CGCGGATCCATGCAGCGCGCGGCGGCGGCCT | ACGCGTCGACGGCATTCTTTGGTTTTGTTCTCTT |
| OsPPT4 | CGCGGATCCATGCAGGCCGTGGCGGCGGCGA | ACGCGTCGACTGCAGTCTTAGCCTTTGGTTTAGC |
| 引物名称Primer name | 引物序列Forward primer(5'→3') |
|---|---|
| OsPPT1-BamHI-F | GCAGCCCGGGGGATCCATGCAGAGCGCGGCGGCC |
| OsPPT1-SacI-R | GGGAACAAAAGCTGGAGCTCTCAGGCAGTCTTGGGCTTGGGT |
| OsPPT2-BamHI-F | GCAGCCCGGGGGATCCATGCAGAGCGCCGCGGCG |
| OsPPT2-SacI-R | GGGAACAAAAGCTGGAGCTCTTACGCAGCCTTGGGCTTGG |
| OsPPT3-BamHI-F | GCAGCCCGGGGGATCCATGCAGCGCGCGGCGGCG |
| OsPPT3-SacI-R | GGGAACAAAAGCTGGAGCTCTCAGGCATTCTTTGGTTTTGTTCTC |
| OsPPT4-BamHI-F | GCAGCCCGGGGGATCCATGCAGGCCGTGGCGGCG |
| OsPPT4-SacI-R | GGGAACAAAAGCTGGAGCTCTCATGCAGTCTTAGCCTTTGG |
表3 酵母载体构建所用引物
Table 3 Primers for yeast vector construction
| 引物名称Primer name | 引物序列Forward primer(5'→3') |
|---|---|
| OsPPT1-BamHI-F | GCAGCCCGGGGGATCCATGCAGAGCGCGGCGGCC |
| OsPPT1-SacI-R | GGGAACAAAAGCTGGAGCTCTCAGGCAGTCTTGGGCTTGGGT |
| OsPPT2-BamHI-F | GCAGCCCGGGGGATCCATGCAGAGCGCCGCGGCG |
| OsPPT2-SacI-R | GGGAACAAAAGCTGGAGCTCTTACGCAGCCTTGGGCTTGG |
| OsPPT3-BamHI-F | GCAGCCCGGGGGATCCATGCAGCGCGCGGCGGCG |
| OsPPT3-SacI-R | GGGAACAAAAGCTGGAGCTCTCAGGCATTCTTTGGTTTTGTTCTC |
| OsPPT4-BamHI-F | GCAGCCCGGGGGATCCATGCAGGCCGTGGCGGCG |
| OsPPT4-SacI-R | GGGAACAAAAGCTGGAGCTCTCATGCAGTCTTAGCCTTTGG |
图1 PPT家族系统进化树分析 不同的颜色代表不同的质体磷酸盐转运体家族:红色代表GPT和XPT家族;灰色代表TPT家族;绿色代表PPT家族。英文字母代表不同物种的名字:Os,水稻;AT,拟南芥;ATR,无油樟;Bradi,二穗短柄草;Cre,莱茵衣藻;Mapoly,地钱;Potri,毛果杨;Pp,小立碗藓;SMO,江南卷柏;Solyc,番茄;Zm,玉米。
Fig.1 Phylogenetic tree analysis of PPT family members The different colours indicated different plastidic phosphate translocators family: red, GPT and XPT; grey, TPT; green, PPT. Letters in the codes represented species names as follows: Os, Oryza sativa; AT, Arabidopsis thaliana; ATR, Amborella trichopoda; Bradi, Brachypodium distachyon; Cre, Chlamydomonas reinhardtii; Mapoly, Marchantia polymorpha; Potri, Populus trichocarpa; PP, Physcomitrella patens; SMO, Selaginella moellendorffii; Soly, Solanum lycopersicum; ZM, Zea mays.
图2 OsPPT基因家族成员在各组织中的表达模式 VS,营养生长阶段,生长21 d的水稻;FS,开花时期,生长 48 d的水稻;GFS,种子灌浆阶段,生长60 d的水稻。数据为3次统计的平均值±标准误。Leaf1~Leaf7代表的叶片是从下面开始数,Leaf1是指从下面数第2片叶(第1片叶已经脱落),Leaf7是指从下面数第8片叶(剑叶)。不同的字母代表显著性差异,P<0.05。
Fig.2 Expression pattern of OsPPT family members in various tissues in rice VS, Vegetative stage, 21-day-old plants; FS, Flowering stage, 48-day-old plants; GFS, Grain filling stage, 60-day-old plants. Data are x -±s(n=3). Leaf1-Leaf7 represents the number of leaves from below, leaf1 refers to the number of the second leaf from below (the first leaf has fallen off), and leaf7 refers to the number of the eighth leaf from below (flag leaf). Different letters represent significant differences, P<0.05.
图3 OsPPT1~OsPPT4蛋白在水稻原生质体中的亚细胞定位 绿色信号表示绿色荧光蛋白(GFP)荧光信号,红色信号表示叶绿素自发荧光信号。Bright,明场;标尺=10 μm。
Fig.3 Subcellular localization of OsPPT1-OsPPT4 proteins in rice protoplasts The green signals indicated green fluorescent protein (GFP), and the red signals indicated autofluorescence of chlorophyll. Bright, bright area; Scale bar=10 μm.
图4 OsPPT基因家族在酵母突变体中能够介导磷酸盐的运输 PHO84是高亲和磷酸转运体,作为阳性对照。转空载体酵母为阴性对照。
Fig.4 OsPPT genes can confer phosphate transport in yeast PHO84 was a high-affinity phosphate transporter and served as a positive control, and empty vector was a negative control.
图5 OsPPT基因家族成员在缺磷期间地上部转录水平的动态变化
Fig.5 Dynamic changes in transcript levels of OsPPT gene family members during phosphate deprivation in shoots of rice seeding
图6 OsPPT基因家族成员在NaCl、SA、ABA胁迫条件下转录水平的动态变化 水稻苗在正常营养液中培养14 d,然后用不同的化学试剂处理24 h。 ABA, 100 μmol·L-1; NaCl, 100 mmol·L-1; SA, 500 μmol·L-1。数据为3次统计的平均值±标准误。*,P<0.05;**,P<0.01;***,P<0.001。
Fig.6 Dynamic changes of transcript levels of OsPPT gene family members in response to NaCl, SA and ABA stresses of rice seedings Fourteen-day-old seedlings grown under normal conditions were exposed to different chemical treatments for 24 h. ABA, 100 μmol·L-1; NaCl, 100 mmol·L-1; SA, 500 μmol·L-1. Data were x -±s(n=3). *, P<0.05; **, P<0.01; ***, P<0.001.
| [1] |
LÓPEZ-ARREDONDO D L, LEYVA-GONZÁLEZ M A, GONZÁLEZ-MORALES S I, et al. Phosphate nutrition: improving low-phosphate tolerance in crops[J]. Annual Review of Plant Biology, 2014, 65: 95-123.
DOI URL |
| [2] |
NIKITHA M S T, SADHANA E U B R B, VANI S S. Phosphorous and phosphate solubilising bacteria and their role in plant nutrition[J]. International Journal of Current Microbiology and Applied Sciences, 2017, 6(4): 2133-2144.
DOI URL |
| [3] |
RUAN W Y, GUO M N, XU L, et al. An SPX-RLI1 module regulates leaf inclination in response to phosphate availability in rice[J]. The Plant Cell, 2018, 30(4): 853-870.
DOI PMID |
| [4] |
VERONICA N, SUBRAHMANYAM D, VISHNU KIRAN T, et al. Influence of low phosphorus concentration on leaf photosynthetic characteristics and antioxidant response of rice genotypes[J]. Photosynthetica, 2017, 55(2): 285-293.
DOI URL |
| [5] |
FOYER C, SPENCER C. The relationship between phosphate status and photosynthesis in leaves[J]. Planta, 1986, 167(3): 369-375.
DOI URL |
| [6] |
SHARKEY T D. Is triose phosphate utilization important for understanding photosynthesis?[J]. Journal of Experimental Botany, 2019, 70(20): 5521-5525.
DOI PMID |
| [7] | FURBANK R T, FOYER C H, WALKER D A. Inhibition of photophosphorylation by ribulose-1, 5-bisphosphate carboxylase[J]. Biochimica et Biophysica Acta (BBA) -Bioenergetics, 1986, 852(1): 46-54. |
| [8] |
CARSTENSEN A, HERDEAN A, SCHMIDT S B, et al. The impacts of phosphorus deficiency on the photosynthetic electron transport chain[J]. Plant Physiology, 2018, 177(1): 271-284.
DOI PMID |
| [9] | TCHERKEZ G. The mechanism of rubisco-catalysed oxygenation[J]. Plant, Cell & Environment, 2016, 39(5): 983-997. |
| [10] |
MARCUS Y, GUREVITZ M. Activation of cyanobacterial RuBP-carboxylase/oxygenase is facilitated by inorganic phosphate via two independent mechanisms[J]. European Journal of Biochemistry, 2000, 267(19): 5995-6003.
PMID |
| [11] | FREDEEN A L, RAAB T K, RAO I M, et al. Effects of phosphorus nutrition on photosynthesis in Glycine max(L.) Merr[J]. Planta, 1990, 181(3): 399-405. |
| [12] | KESSEL-VIGELIUS S K. Toward intracellular membrane transport-characterization of the mitochondrial carrier family in plants[D]. Dusseldorf: Heinrich Heine University, 2013. |
| [13] |
VERSAW W K, HARRISON M J. A chloroplast phosphate transporter, PHT2;1, influences allocation of phosphate within the plant and phosphate-starvation responses[J]. The Plant Cell, 2002, 14(8): 1751-1766.
DOI URL |
| [14] |
LIU X L, WANG L, WANG X W, et al. Mutation of the chloroplast-localized phosphate transporter OsPHT2;1 reduces flavonoid accumulation and UV tolerance in rice[J]. The Plant Journal: for Cell and Molecular Biology, 2020, 102(1): 53-67.
DOI URL |
| [15] |
WANG G Y, ZHANG C, BATTLE S, et al. The phosphate transporter PHT4;1 is a salicylic acid regulator likely controlled by the circadian clock protein CCA1[J]. Frontiers in Plant Science, 2014, 5: 701.
DOI PMID |
| [16] |
IRIGOYEN S, KARLSSON P M, KURUVILLA J, et al. The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis[J]. Plant Physiology, 2011, 157(4): 1765-1777.
DOI URL |
| [17] |
MIYAJI T, KUROMORI T, TAKEUCHI Y, et al. AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis[J]. Nature Communications, 2015, 6: 5928.
DOI URL |
| [18] |
LI R L, WANG J L, XU L, et al. Functional analysis of phosphate transporter OsPHT4 family members in rice[J]. Rice Science, 2020, 27(6): 493-503.
DOI |
| [19] |
BOCKWOLDT M, HEILAND I, FISCHER K. The evolution of the plastid phosphate translocator family[J]. Planta, 2019, 250(1): 245-261.
DOI PMID |
| [20] |
FISCHER K, ARBINGER B, KAMMERER B, et al. Cloning and in vivo expression of functional triose phosphate/phosphate translocators from C3-and C4-plants: evidence for the putative participation of specific amino acid residues in the recognition of phosphoenolpyruvate[J]. The Plant Journal: for Cell and Molecular Biology, 1994, 5(2): 215-226.
DOI URL |
| [21] |
SCHNEIDER A, HÄUSLER R E, KOLUKISAOGLU Ü, et al. An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished[J]. The Plant Journal, 2002, 32(5): 685-699.
DOI URL |
| [22] |
TOYOTA K, TAMURA M, OHDAN T, et al. Expression profiling of starch metabolism-related plastidic translocator genes in rice[J]. Planta, 2006, 223(2): 248-257.
PMID |
| [23] | HEINEKE D, KRUSE A, FLÜGGE U I, et al. Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants[J]. Planta, 1994, 193(2): 174-180. |
| [24] |
KAMMERER B, FISCHER K, HILPERT B, et al. Molecular characterization of a carbon transporter in plastids from heterotrophic tissues: the glucose 6-phosphate/phosphate antiporter[J]. The Plant Cell, 1998, 10(1): 105-117.
DOI URL |
| [25] |
ZIMMERMANN P, HIRSCH-HOFFMANN M, HENNIG L, et al. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox[J]. Plant Physiology, 2004, 136(1): 2621-2632.
DOI URL |
| [26] |
NIEWIADOMSKI P, KNAPPE S, GEIMER S, et al. The Arabidopsis plastidic glucose 6-phosphate/phosphate translocator GPT1 is essential for pollen maturation and embryo sac development[J]. The Plant Cell, 2005, 17(3): 760-775.
DOI URL |
| [27] |
ZHANG M M, XU X W, ZHENG Y P, et al. Expression of a plastid-localized sugar transporter in the suspensor is critical to embryogenesis[J]. Plant Physiology, 2020, 185(3): 1021-1038.
DOI PMID |
| [28] |
QU A L, XU Y, YU X X, et al. Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1 (OsGPT1) in rice[J]. Journal of Genetics and Genomics, 2021, 48(8): 695-705.
DOI PMID |
| [29] |
EICKS M, MAURINO V, KNAPPE S, et al. The plastidic pentose phosphate translocator represents a link between the cytosolic and the plastidic pentose phosphate pathways in plants[J]. Plant Physiology, 2002, 128(2): 512-522.
PMID |
| [30] |
HILGERS E J A, SCHÖTTLER M A, METTLER-ALTMANN T, et al. The combined loss of triose phosphate and xylulose 5-phosphate/phosphate translocators leads to severe growth retardation and impaired photosynthesis in Arabidopsis thaliana tpt/xpt double mutants[J]. Frontiers in Plant Science, 2018, 9: 1331.
DOI URL |
| [31] | FISCHER K, KAMMERER B, GUTENSOHN M, et al. A new class of plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/phosphate antiporter[J]. The Plant Cell, 1997, 9(3): 453-462. |
| [32] |
HERRMANN K M. The shikimate pathway: early steps in the biosynthesis of aromatic compounds[J]. The Plant Cell, 1995, 7(7): 907-919.
PMID |
| [33] |
STREATFIELD S J, WEBER A, KINSMAN E A, et al. The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression[J]. The Plant Cell, 1999, 11(9): 1609-1621.
DOI URL |
| [34] |
KNAPPE S, LÖTTGERT T, SCHNEIDER A, et al. Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis: AtPPT1 may be involved in the provision of signals for correct mesophyll development[J]. The Plant Journal: for Cell and Molecular Biology, 2003, 36(3): 411-420.
DOI URL |
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