枳NLP转录因子响应干旱胁迫并与NRE顺式作用元件互作

doi:10.3864/j.issn.0578-1752.2018.17.011

Abstract

Abstract: 【Objective】The objective of this study is to investigate the changes of nitrogen content and the expression of the nitrite reductase gene (NiR) and NIN-like protein (NLP) transcription factors of Poncirus trifoliata (L.) Raf. under the drought conditions, and to analyze and confirm the binding interaction of NLP transcription factors and nitrate-responsive cis-element (NRE) of NiR promoter region. 【Method】One-year old rootstock P. trifoliata was used as the experimental material and treated with drought stress until the leaves of the plant began to wilt, and the water control was set. The total nitrogen contents in leaves and lateral roots under drought condition were measured by Kjeldahl nitrogen meter. At the same time, the gene expression of PtNiR, PtNLP2, PtNLP4, PtNLP7 and PtNLP8 under different drought conditions in leaves and lateral roots was analyzed using real-time quantitative PCR and 2^−ΔΔCT method. The NRE sequence in the promoter region of PtNiR was analyzed. The constructed vector pHIS2-4×NRE and pGADT7-Rec-NLP of Y1H were screened using Ligation-Free Cloning Kit (abm), and the interaction of P. trifoliata NLP transcription factors (PtNLP2, PtNLP4, PtNLP7 and PtNLP8) with NRE was analyzed by Y1H screening using a HIS3 reporter gene under the control of NRE (4×NRE), pHIS2-4×NRE and pGADT7-Rec-NLP plasmids were transformed into yeast Y187, and were incubated at 30℃ for 3-5 days in amino acid deficient medium SD/-Trp/-Leu (control) and SD/-His/-Trp/-Leu/+120 mmol·L^-1 3-aminotriazole (3-AT), and the relationship between NLPs transcription factor and NRE was determined by observing the growth of Y187 yeast. 【Result】The total nitrogen content in leaves of P. trifoliata increased first and then decreased, while the total nitrogen content in lateral roots decreased significantly under the drought conditions. In the water control, the contents of total nitrogen in leaves and lateral roots increased significantly. RT-qPCR analysis showed that the expression of PtNiR in leaves was up-regulated firstly and then down-regulated, while in lateral roots, it was significantly down-regulated with the increase of drought. the expression of ptNLP2, ptNLP4 and ptNLP7 in leaves and lateral roots was also up-regulated firstly and then inhibited. In contrast, the expression of ptNLP8 in leaves and lateral roots was inhibited to a certain extent by the drought. By cloning and sequencing, the NRE sequence was identified at the location of -196 to -154 in the promoter region of PtNiR. Y1H assays show that the Y187 yeast transformed into pHIS2-4×NRE-HIS3 and pGADT7-Rec-NLP vector could grow normally in the control medium (-Trp/-Leu) and the nutrient deficient medium (-His/-Trp/-Leu) containing 120 mmol·L^-1 3-AT.【Conclusion】The content of total nitrogen in lateral roots of P. trifoliata decreased significantly by drought. Compared with the control, the total nitrogen contents in leaves and lateral roots reduced relatively with the extension of drought treatment time. The expression of NiR and NLP transcription factors in leaves and lateral roots of the nitrogen metabolism pathway was responsive to the drought in varying degrees. Y1H assays show thatPtNLP2, PtNLP4, PtNLP7 and PtNLP8 transcription factors can bind to the NRE of PtNiR promoter to activate the expression of the downstream genes.

Key words: Poncirus trifoliata (L.) Raf., NIN-like protein transcription factor, drought stress, nitrate-responsive cis-element (NRE), binding

CAO XiongJun, LU XiaoPeng, XIONG Jiang, LI Jing, XIE ShenXi. The Poncirus trifoliata (L.) Raf. NIN-Like Protein Transcription Factors Responses to Drought Stress and Bind the Nitrate-Responsive Cis-element[J].Scientia Agricultura Sinica, 2018, 51(17): 3370-3378.

References

[1] 周亚洲, 徐兆林, 周利, 罗丽, 易著虎, 谢深喜. 湖南柑橘灌溉现状及节水栽培技术研究. 湖南农业科学, 2010(19): 46-48.

ZHOU Y Z, XU Z L, ZHOU L, LUO L, YI Z H, XIE S X. Status quo of irrigation and water-saving cultivation techniques of orange in hunan. Hunan Agricultural Sciences, 2010(19): 46-48. (in Chinese)

[2] 刘静, 魏开发, 高志晖, 李冰冰, 任慧波, 胡建芳, 贾文锁. 干旱胁迫下氮素营养与根信号在气孔运动调控中的协同作用. 植物学通报, 2008, 25(1): 34-40.

LIU J, WEI K F, GAO Z H, LI B B, REN H B, HU J F, JIA W S. Nitrate as an enhancer of root signal in the regulation of stomatal movement in plants under drought stress. Chinese Bulletin of Botany, 2008, 25(1): 34-40. (in Chinese)

[3] LAWLOR D W, LEMAIRE G, GASTAL F. Nitrogen, plant growth and crop yield//Plant nitrogen. Springer, 2001: 343-367.

[4] CRAWFORD N M. Nitrate: nutrient and signal for plant growth. The plant cell, 1995, 7(7): 859-869.

[5] XIE S X, CAO S Y, LIU Q, XIONG X Y, LU X P. Effect of water deficit stress on isotope ¹⁵N uptake and nitrogen metabolism of newhall orange and Yamasitaka mandarin seedling. Journal of Life Sciences, 2013, 7(11): 1170-1178.

[6] JU H, LIN E D, WHEELER T, CHALLINOR A, JIANG S. Climate change modelling and its roles to Chinese crops yield. Journal of Integrative Agriculture, 2013, 12(5): 892-902.

[7] 潘根兴, 高民, 胡国华, 魏钦平, 杨晓光, 张文忠, 周广胜, 邹建文. 气候变化对中国农业生产的影响. 农业环境科学学报, 2011, 30(9): 1698-1706.

PAN G X, GAO M, HU G H, WEI Q P, YANG X G, ZHANG W Z, ZHOU G S, ZOU J W. Impacts of climate change on agricultural production of China. Journal of Agro-Environment Science, 2011, 30(9): 1698-1706. (in Chinese)

[8] 李振轮, 谢德体. 柑橘生长与生态因子的关系研究进展. 中国农学通报, 2003, 19(6): 181-184, 189.

LI Z L, XIE D T. A review of relationship between citrus growth and environment. Chinese Agricultural Science Bulletin, 2003, 19(6): 181-184, 189. (in Chinese)

[9] 周静, 崔键, 梁家妮. 土壤水分对柑橘叶片生长及多胺代谢的影响. 土壤, 2009, 41(5): 796-800.

ZHOU J, CUI J, LIANG J N. Effects of soil water content on growth and polyamines’ content for citrus leaves. Soils, 2009, 41(5): 796-800. (in Chinese)

[10] 樊卫国, 李庆宏, 吴素芳. 长期干旱环境对柑橘生长及养分吸收和相关生理的影响. 中国生态农业学报, 2012, 20(11): 1484-1493.

FAN W G, LI Q H, WU S F. Effect of perennial drought environments on Citrus tangerina Hort. cv. “Chuanju” growth, nutrient absorption and physiology. Chinese Journal of Eco-Agriculture, 2012, 20(11): 1484-1493. (in Chinese)

[11] 张规富, 谢深喜. 柑橘对水分胁迫的反应特点及研究方向. 天津农业科学, 2012, 18(6): 5-8.

ZHANG G F, XIE S X. Characteristics and research direction in response to citrus under water stress. Tianjin Agricultural Sciences, 2012, 18(6): 5-8. (in Chinese)

[12] TAYLOR S H, RIPLEY B S, WOODWARD F I, OSBORNE C P. Drought limitation of photosynthesis differs between C₃ and C₄ grass species in a comparative experiment. Plant, cell & environment, 2011, 34(1): 65-75.

[13] 谢深喜, 张秋明, 熊兴耀, 邓子牛, LOVATT C J. 水分胁迫对柑橘叶片和根系细胞超微结构的影响. 湖南农业大学学报 (自然科学版), 2008, 34(2): 168-172.

XIE S X, ZHANG Q M, XIONG X Y, DENG Z N, LOVATT C J. Effect of water stress on leaf and root cell ultra-structure of Citrus. Journal of Hunan Agricultural University (Natural Sciences), 2008, 34(2): 168-172. (in Chinese)

[14] 谢深喜, 刘强, 熊兴耀, 张秋明, LOVATT C J. 水分胁迫对枳壳和枳橙光合作用及细胞超微结构的影响. 江西农业大学学报, 2011, 33(2): 234-238.

XIE S X, LIU Q, XIONG X Y, ZHANG Q M, LOVATT C J. Influences of water stress on citrus photosynthesis characteristic and cell ultra-structure. Acta Agriculturae Universitatis Jiangxiensis, 2011, 33(2): 234-238. (in Chinese)

[15] 谢深喜, 刘强, 熊兴耀, 张秋明, LOVATT C J. 水分胁迫对柑橘光合特性的影响. 湖南农业大学学报 (自然科学版), 2010, 36(6): 653-657.

XIE S X, LIU Q, XIONG X Y, ZHANG Q M, LOVATT C J. Effect of water stress on citrus photosynthesis characteristic. Journal of Hunan Agricultural University (Natural Sciences), 2010, 36(6): 653-657. (in Chinese)

[16] 马文涛, 樊卫国. 干旱胁迫对实生红橘、甜橙和柚叶中营养元素含量的影响. 西南农业学报, 2007, 20(4): 630-633.

MA W T, FAN W G. Effect of drought stress on mineral nutrient contents in leaves of seedling plant of C. tangerina Hort., C. sinensis Osbeck and C. grandis Osbeck. Southwest China Journal of Agricultural Sciences, 2007, 20(4): 630-633. (in Chinese)

[17] BARKATAKY S, MORGAN K T, EBEL R C, ROKA F M. Effects of short-term drought stress and mechanical harvesting on sweet orange tree health, water uptake and yield. Acta Horticulturae, 2012, 965: 73-81.

[18] FAROOQ M, HUSSAIN M, WAHID A, SIDDIQUE K. Drought stress in plants: An overview//AROCA R. An Overview, Plant Responses to Drought Stress. Springer, 2012: 1-33.

[19] 吴翔宇, 许志茹, 曲春浦, 李蔚, 孙琦, 刘关君. 毛果杨NLP基因家族生物信息学分析与鉴定. 植物研究, 2014, 34(1): 37-43.

WU X Y, XU Z R, QU C P, LI W, SUN Q, LIU G J. Genome-wide identification and characterization of NLP gene family in Populus trichocarpa. Bulletin of Botanical Research, 2014, 34(1): 37-43. (in Chinese)

[20] KONISHI M, YANAGISAWA S. Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nature Communications, 2013, 4(3): 1617.

[21] KONISHI M, YANAGISAWA S. Emergence of a new step towards understanding the molecular mechanisms underlying nitrate-regulated gene expression. Journal of experimental botany, 2014, 65(19): 5589-5600.

[22] CHARDIN C, GIRIN T, ROUDIER F, MEYER C, Krapp A. The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development. Journal of experimental botany, 2014, 65(19): 5577-5587.

[23] 曹雄军, 卢晓鹏, 熊江, 李静, 吴倩, 周芳芳, 谢深喜. 枳NLP转录因子克隆及其在不同水分条件下的表达. 中国农业科学, 2016, 49(2): 381-390.

CAO X J, LU X P, XIONG J, LI J, WU Q, ZHOU F F, XIE S X. Cloning and expression of Poncirus trifoliata (L.) Raf. NIN-like transcription factors under different water conditions. Scientia Agricultura Sinica, 2016, 49(2): 381-390. (in Chinese)

[24] CASTAINGS L, CAMARGO A, POCHOLLE D, GAUDON V, TEXIER Y, BOUTET-MERCEY S, TACONNAT L, RENOU J P, DANIEL-VEDELE F, FERNANDEZ E, Meyer C, Krapp A. The nodule inception-like protein 7 modulates nitrate sensing and metabolism in Arabidopsis. The Plant Journal, 2009, 57(3): 426-435.

[25] YAN J w, YUAN F r, LONG G y, QIN L, DENG Z n. Selection of reference genes for quantitative real-time RT-PCR analysis in citrus. Molecular biology reports, 2012, 39(2): 1831-1838.

[26] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. methods, 2001, 25(4): 402-408.

[27] BI Y M, WANG R L, ZHU T, ROTHSTEIN S J. Global transcription profiling reveals differential responses to chronic nitrogen stress and putative nitrogen regulatory components in Arabidopsis. BMC genomics, 2007, 8: 281.

[28] FORDE B G. Local and long-range signaling pathways regulating plant responses to nitrate. Annual Review of Plant Biology, 2002, 53(1): 203-224.

[29] HACHIYA T, MIZOKAMI Y, MIYATA K, THOLEN D, WATANABE C K, NOGUCHI K. Evidence for a nitrate-independent function of the nitrate sensor NRT1.1 in Arabidopsis thaliana. Journal of plant research, 2011, 124(3): 425-430.

[30] KROUK G, MIROWSKI P, LECUN Y, SHASHA D E, CORUZZI G M. Predictive network modeling of the high-resolution dynamic plant transcriptome in response to nitrate. Genome Biology, 2010, 11(12): R123.

[31] SUZUKI W, KONISHI M, YANAGISAWA S. The evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes may involve a loss of nitrate responsiveness of the NIN transcription factor. Plant signaling & behavior, 2013, 8(10): e25975.

[32] MARCHIVE C, ROUDIER F, CASTAINGS L, BRÉHAUT V, BLONDET E, COLOT V, MEYER C, KRAPP A. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants. Nature communications, 2013, 4(2): 1713.

[33] KONISHI M, YANAGISAWA S. Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response. The Plant Journal, 2010, 63(2): 269-282.

[34] KONISHI M, YANAGISAWA S. An NLP-binding site in the 3′ flanking region of the nitrate reductase gene confers nitrate-inducible expression in Arabidopsis thaliana (L.) Heynh. Soil Science and Plant Nutrition, 2013, 59(4): 612-620.

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The Poncirus trifoliata (L.) Raf. NIN-Like Protein Transcription Factors Responses to Drought Stress and Bind the Nitrate-Responsive Cis-element

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