Expression Analyses of Phosphoribulokinase Gene Induced by Stresses and Its Interacting Protein Screening in Rice (Oryza sativa)
HE Wei1,2,*, QU Meng-Yu1,2,*, WEI Yi-Dong1,2, LIAN Ling1,2, CAI Qiu-Hua1,2, ZHENG Yan-Mei1,2, WANG Ying-Heng1,2, ZHU Yong-Sheng1,2, XIE Hua-An1,2, ZHANG Jian-Fu1,2,**
1 Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350019, China; 2 State Key Laboratory for Ecological Control of Crop Pests between Fujian and Taiwan/National Engineering Laboratory of Rice/South China Research Base of State Key Laboratory of Hybrid Rice/Incubating base of State Key Laboratory of Crop Germplasm Innovation and Molecular Breeding between Fujian Province and Ministry of Science and Technology/Fuzhou Branch of National Rice Improvement Center/South China Key Laboratory of Hybrid Rice Germplasm innovation and Molecular Breeding of Ministry of Agriculture and Rural Areas/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
Abstract：Phosphoribulokinase (PRK) is a key enzyme in Calvin-Benson cycle which influences on plant growth and development. Nuclear-encoded plastid-localized PRK, coupling with other regulatory enzyme,involved in carbon assimilation in functional leaves. However, the research on RPK functions in stress response was still limited, especially in rice (Oryza sativa). In this study, target protein subcellular localization, expression characteristics analyses of rice phosphoribulokinase (OsPRK, GenBank No. LOC4330413) under abiotic stresses (drought, high salt and high temperature) and exogenous hormone treatments (abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA)), as well as MBP pull-down protein interaction screening were conducted to investigate the function of OsPRK involving in stress responses. By detecting the GFP report using laser confocal microscopy, the transient expression analysis demonstrated that OsPRK was strictly localized in chloroplast stroma of transformed rice protoplasts after 16 h protoplast culture, contrasting to control native GFP only found around the cytoplasm of protoplast. To characterize the change of the transcript abundance of OsPRK, quantitative realtime PCR (qPCR) was performed and found that the transcriptional expression of OsPRK was significantly inhibited at 0.5 and 3 h drought treatments, 1 and 6 h high salt or high temperature treatments, which implicated that OsPRK inclined to reduce transcriptional expression in drought, high salt and high temperature conditions. Beyond that, qPCR results also showed that the OsPRK transcription levels of seedlings under ABA and JA treatments were not significantly changed within 1 h, while continuously decreased when treated more than 4 h. Unlike ABA and JA treatment, the transcription level of OsPRK of SA treatment had a dramatical reduction from 0 to 8 h, and then gradually declined until 24 h. To investigate the potential OsPRK interacting proteins, OsPRK was expressed with MBP-Tag using recombinant plasmid pMAL-c5x and purified from BL21(DE3)plys prokaryotic expression system for pull-down protein interaction screening. The protein interaction experiment totally obtained 82 candidate proteins that might interact with OsPRK using MBP pull-down method. Of these, the functions of 67 candidate proteins were unknown while 15 candidate proteins were with known functions, including GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and WRKY transcription factor. This study found that OsPRK should be involved in abiotic stress and hormone response of rice seedlings. In addition, the study identified numbers of potential OsPRK interacting proteins, which can be used for further biological functions of OsPRK in stress avoidance and tolerance. All these findings accelerated the understanding of OsPRK roles in response to developmental requirements and environmental constraints.
[1] 陈林, 吕静, 李晨羊, 等. 2020. 水稻磷酸核酮糖激酶基因OsPRK克隆、亚细胞定位与诱导表达分析[J]. 植物保护学报, 47(2): 283-291. (Chen L, Lu J, Li C Y, et al.2020. Cloning subcellular localization and expression patterns of the phosphoribulokinase gene OsPRK in the rice plant[J]. Journal of Plant Protection, 47(2): 283-291.) [2] 董明, 再吐尼古丽•库尔班, 吕芃, 等. 2019. 高梁苗期耐盐性转录组分析和基因挖掘[J]. 中国农业科学, 52(22): 3987-4001. ( Dong M, Kuerban Z, Lu P, et al.2019. Transcriptome analysis and gene mining of salt tolerance in sorghum seedlings (Sorghum bicolor L. Moench)[J]. Scientia Agricultura Sinica, 52(22): 3987-4001.) [3] 韩雪颖. 2014. 水稻类病变突变体的基因定位及蛋白质组学研究[D]. 硕士学位论文, 南京农业大学, 导师: 陈剑平, pp.7-51. (Han X Y.2014. Genetic mapping and proteomics study of rice lesion mimic mutants[D]. Thesis for M.S., Nanjing Agricultural University, Supervisor: Chen J P, pp.7-51.) [4] 何炜, 连玲, 熊海清, 等. 2012. 粳稻云引中一个受稻瘟病菌诱导的抗逆候选基因的克隆及表达分析[J]. 福建农业学报, 27(9): 924-930. (He W, Lian L, Xiong H Q, et al.2012. Functional analysis of a candidate gene in relation to stress resistance induced with Magnaporthe grisea from japonica variety Yunyin[J]. Fujian Journal of Agricultural Sciences, 27(9): 924-930.) [5] 黄幸, 丁峰, 彭宏祥, 等. 2019. 植物 WRKY 转录因子家族研究进展[J]. 生物技术通报, 35(12): 129-143. (Huang X, Ding F, Peng H X, et al.2019. Research progress on family of plant WRKY transcription factors[J]. Biotechnology Bulletin, 35(12): 129-143.) [6] 荆培培, 任红茹, 杨洪建, 等. 2020. 盐胁迫对2个不同盐敏感性水稻品种(系)叶片光合特性与产量的影响[J].作物杂志, 1: 67-75. (Jing P P, Ren H R, Yang H J, et al.2020. Effects of saline stress on leaf photosynthesis characteristics and grain yield of two rice cultivars (Lines)[J]. Crops, 1: 9) [7] 梁根云, 姬红丽, 章振羽, 等. 2007. 条锈菌侵染慢锈性小麦品种川麦107后的蛋白质组学分析[J]. 麦类作物学报, 27(2): 335-340. (Liang G Y, Ji H L, Zhang Z Y, et al.2007. Proteome analysis of slow-rusting variety Chuanmai 107 inoculated by wheat stripe rust (Puccina striiformis)[J]. Journal of Triticeae Crops, 27(2): 335-340.) [8] 刘云飞, 2014. 番茄NBS-LRR抗病基因分析及黄化曲叶病毒Ty-2抗性候选基因鉴定、表达分析[D]. 硕士学位论文, 南京农业大学, 导师: 杨悦俭, pp. 12-18. (Liu Y F, 2014. Genome-wide analysis of NBS-LRR resistance genes and in Silico inentification and expression analysis of candidate genes related to Ty-2 resistance gene in tomato[D]. Thesis for M.S., Nanjing Agricultural University, Supervisor: Yang Y J, pp. 12-18.) [9] 王亮. 2015. 基于RNA-Seq技术分析小麦矮缩病毒侵染后小麦的基因表达差异[D]. 硕士学位论文, 中国农业科学院, 导师: 刘艳, pp. 4-47. (Wang L.2015. Analysis of gene expression changes in wheat leaves in response to WDV infection using RNA-Seq[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Supervisor: Liu Y, pp. 4-47.) [10] 谢小玉, 侯爽, 冉春燕. 2020. 干旱诱导的甘蓝型油菜SSH文库及抗旱相关基因表达的分析[J]. 湖南农业大学学报(自然科学版), 46(2): 157-164. (Xie X Y, Hou S, Ran C Y, 2020. Analysis of expression of drought associated genes and SSH library of Brassica napus under drought stress[J]. Journal of Hunan Agricultural University (Natural Sciences), 46(2): 157-164.) [11] 杨德卫, 王莫, 韩利波, 等. 2019. 水稻稻瘟病抗性基因的克隆、育种利用及稻瘟菌无毒基因研究进展[J]. 植物学报, 54(2): 265-276. (Yang D W, Wang M, Han L B, et al.2019. Progress of cloning and breeding application of blast resistance genes in rice and avirulence genes in blast fungi[J]. Chinese Bulletin of Botany, 54(2): 265-276.) [12] 喻娟娟. 2011. 星星草(Puccinellia tenuiflora)叶片盐胁迫应答蛋白质组学研究[D]. 硕士学位论文, 东北农林大学, 导师: 戴绍军, pp. 20-41. (Yu J J.2011. Proteomic study on leaf response to salt stress in Puccinellia tenuiflora[D]. Thesis for M.S., Northeast Agricultural University, Supervisor: Dai S J, pp. 4-47.) [13] 张凡, 尹俊龙, 郭瑛琪, 等. 2018. WRKY转录因子的研究进展[J]. 生物技术通报, 34(1): 40-48. (Zhang F, Yin J L, Guo Y Q, et al.2018. Research advances on WRKY transcription factors[J]. Biotechnology Bulletin, 34(1): 40-48.) [14] 张明菊, 朱莉, 夏启中. 2021. 植物激素对胁迫反应调控的研究进展[J].湖北大学学报(自然科学版), 43(3): 242-253. (Zhang M J, Zhu L, Xia Q Z.2021. Research progress on the regulation of plant hormones to stress responses[J] . Journal of Hubei University (Natural Science), 43(3): 242-253.) [15] 周梦迪, 胡志程, 付秋实, 等. 2020. NaCl胁迫对甜瓜生理指标及相关基因表达的影响[J].中国蔬菜, 2: 30-39. (Zhou M D, Hu Z C, Fu Q S, et al.2020. Effects of NaCl stress on physiology index and related gene expression in melon[J]. 2: 30-39.) [16] 祖艳群, 梅馨月, 闵强, 等. 2016. 砷胁迫对三七皂苷和黄酮含量、关键酶活性的影响及其蛋白质组分析[J]. 应用生态学报, 27(12): 4013-4021. (Zu Y Q, Mei X Y, Min Q, et al.2016. Effects of As stress on contents of saponin and flavonoid, key enzymes activities of Panax notoginseng and its proteomic analysis[J]. Chinese Journal of Applied Ecology, 27(12): 4013-4021.) [17] Cai W W, Yang S, Wu R, et al.2021. Pepper NAC-type transcription factor NAC2c balances the trade-off between growth and defense responses[J]. Plant Physiology, 186: 2169-2189. [18] Chen X F, Yu T, Xiong J H, et al.2005. Molecular cloning and expression analysis of rice phosphoribulokinase gene that is regulated by environmental stresses[J]. Molecular Biology Reports, 31(4): 249-255. [19] Elena L C P, Amani O A, Tracy L, et al.2017. Arabidopsis CP12 mutants have reduced levels of phosphoribulokinase and impaired function of the Calvin-Benson cycle[J]. Journal of Experimental Botany, 68(9): 2285-2298. [20] Groszmann M, Bayon R G, Lyons R L, et al.2015. Hormone-regulated defense and stress response networks contribute to heterosis in Arabidopsis F1 hybrids[J]. PNAS, 112(46): 6397-6406. [21] Guo L, Devaiah S P, Narasimhan R, et al.2012. Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dδ to transduce hydrogen peroxide signals in the Arabidopsis responseto stress[J]. The Plant Cell, 24(5): 2200-2212. [22] Habash D Z, Parry M A J, Parmar S, et al.1996. The regulation of component processes of photosynthesis in transgenic tobacco with decreased phosphoribulokinase activity[J]. Photosynthesis Research, 49(2): 159-167. [23] Han S J, Wang Y, Zheng X Y, et al.2015. Cytoplastic gyceraldehyde-3-phosphate dehydrogenases interact with ATG3 to negatively regulate alltophagy and immunity in Nicotiana benthamiana[J]. Plant Cell, 27(4): 1316-1331. [24] Hariharan T, Johnson P J, Cattolico R A.1998. Purification and characterization of phosphoribulokinase from the marine chromophytic alga Heterosigma carterae[J]. Plant Physiology, 117(1): 321-329. [25] Henry E, Nicholas F, Liu J, et al.2015. Beyond Glycolysis: GAPDHs are multi-functional enzymes involved in regulation of ROS, autophagy, and plant immune responses[J]. Plos Genetics, 11(4): e1005199. [26] Hildebrandt T, Knuesting J, Berndt C, et al.2015. Cytosolic thiol switches regulating basic cellular functions: GAPDH as an information hub?[J]. Biological Chemistry, 396(5): 523-537. [27] Horsnell P R, Raines C A.1991. Nucleotide sequence of a cDNA clone encoding chloroplast fructose-1,6-bisphosphatase from Arabidopsis thaliana[J]. Plant Molecular Biology, 17(1): 183-184. [28] Hu X L, Wu X L, Li C H, et al.2012. Abscisic acid refines the synthesis of chloroplast proteins in maize (Zea mays) in response to drought and light[J]. PLOS ONE, 7(11): e49500. [29] Jiang Y Q, Yang B, Harris N S, et al.2007. Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots[J]. Journal of Experimental Botany, 8(13): 3591-3607. [30] Lim C, Kang K, Shim Y, et al.2022. Inactivating transcription factor OsWRKY5 enhances drought tolerance through abscisic acid signaling pathways[J]. Plant Physiology, 188(4): 1900-1916. [31] Livak K J, Schmittgen T D .2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,25(4): 402-408. [32] Marri L, Sparla F, Pupillo P, et al.2004. Co-ordinated gene expression of photosynthetic glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, and CP12 in Arabidopsis thaliana[J]. Journal of Experimental Botany, 56(409): 73-80. [33] Michels A K, Wedel N, Kroth P G.2005. Diatom plastids possess a phosphoribulokinase with an altered regulation and no oxidative pentose phosphate pathway[J]. Plant Physiology, 137(3): 911-920. [34] Mitra S, Baldwin I T.2014. RuBPCase activase (RCA) mediates growth-defense trade-offs: silencing RCA redirects jasmonic acid (JA) flux from JA-isoleucine to methyl jasmonate (MeJA) to attenuate induced defense responses in Nicotiana attenuate[J]. The New Phytologist , 201(4): 1385-1395. [35] Paul M J, Driscoll S P, Andralojc P J, et al.2000. Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: Definition of the light environment under which phosphoribulokinase is not in large excess[J]. Planta, 211(1): 112-119. [36] Paul M J, Knight J S, Habash D, et al.1995. Reduction in phosphoribulokinase activity by antisense RNA in transgenic tobacco: Effect on CO2 assimilation and growth in low irradiance[J]. The Plant Journal, 7(4): 535-542. [37] Porter M A, Milanez S, Stringer C D, et al.1986. Purification and characterization of ribulose-5-phosphate kinase from spinach[J]. Archives of Biochemistry and Biophysics, 245(1): 14-23. [38] Verma V, Ravindran P, Kumar P P.2016. Plant hormone-mediated regulation of stress responses[J]. BMC Plant Biology, 16(1): 86. [39] Wang J, Zheng C F, Shao X Q, et al.2020. Transcriptomic and genetic approaches reveal an essential role of the NAC transcription factor SlNAP1 in the growth and defense response of tomato[J]. Horticulture Research, 7(1):209-219. [40] Wang Q, Yu F F, Xie Q.2020. Balancing growth and adaptation to stress: Crosstalk between brassinosteroid and abscisic acid signaling[J]. Plant Cell Environment, 43(10): 2325-2335. [41] Wawer I, Bucholc M, Astier J, et al.2010. Regulation of Nicotiana tabacum osmotic stress activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity[J]. The Biochemical Journal, 429(1):73-83. [42] Xie Y, Jiang S, Li L, et al.2020. Single-cell RNA sequencing efficiently predicts transcription factor targets in plants[J]. Frontiers in Plant Science, 11: 603302. [43] Yang S, Cai W W, Shen L, et al.2022. Solanaceous plants switch to cytokinin-mediated immunity against Ralstonia solanacearum under high temperature and high humidity[J]. Plant Cell Environment, 45(2): 459-478. [44] Zhang Y, Su J B, Duan S, et al.2011. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes[J]. Plant Methods, 7(1): 30-43. [45] Züst T, Agrawal A A.2017. Trade-offs between plant growth and defense against insect herbivory: An emerging mechanistic synthesis[J]. Annual Review of Plant Biology, 68(1): 513-534.
