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一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

Published: August 25, 2018
doi:
10.3791/57800
, , , , , ,
1Brazilian Bioethanol Science and Technology Laboratory (CTBE),Brazilian Center for Research in energy and materials (CNPEM), 2University of Campinas (UNICAMP), 3University of Viçosa (UFV), 4CTBE,CNPEM, 5Brazilian Bioethanol Science and Technology Laboratory (CTBE/CNPEM),Max Planck Partner Group

Summary

一种简单、多功能、低成本的体外水培系统成功地进行了优化, 使得在无菌条件下进行大规模实验。该系统促进了化学物质在溶液中的应用, 并为分子、生物化学和生理学研究提供了有效的根系吸收。

Abstract

植物生物学的广泛研究是利用水培培养进行的。在这项工作中, 提出了一种用于评估植物对化学物质和其他物质的反应的体外培水生长系统。该系统高效地获得了 c3和 c4型拟南芥和狗尾草贝的均匀健康苗。无菌栽培避免了藻类和微生物的污染, 这是植物正常生长和栽培发展的已知限制因素。此外, 该系统具有可伸缩性, 能够大规模地收获植物材料, 具有较小的机械损伤, 而且如果需要, 还能收获植物的各个部分。详细的协议表明, 该系统有一个简单和低成本的组装, 因为它使用吸管架作为种植植物的主平台, 提供。采用拟南芥苗对该系统的可行性进行了验证, 以评价雷帕霉素 (TOR) 激酶靶化学抑制剂 AZD-8055 药物的作用。在根和芽 AZD-8055 治疗后, 早期30分钟有效检测 TOR 抑制。此外, AZD-8055-treated 植物显示预期淀粉过剩表型。我们建议这种水培系统作为植物研究人员的理想方法, 目的是监测植物诱导剂或抑制剂的作用, 以及使用同位素标记化合物评估代谢通量, 一般来说, 需要使用昂贵的试剂。

Introduction

种植植物使用水培的优势已被广泛认可, 在生产大型和均匀的植物, 使可再生的实验1,2,3。在该系统中, 营养液的组成可以在植物生长发育的各个阶段得到适当的控制和循环利用。此外, 根系不受非生物胁迫, 如土壤生长的植物, 如养分饥饿和缺水4。随着植物的生长培呈现出与土壤中培养的形态和生理特征相当相似的生物, 该系统在研究中得到了广泛的应用, 因为它允许监测根/芽的生长及其收获, 而不伤害2,5。

由于有可能改变营养液的成分和浓度, 大多数使用水培条件的研究已经完成, 以表征微-和营养素1,3 的功能。,6,7,8。然而, 该系统已证明是非常有用的广泛的应用于植物生物学, 如阐明激素和化学物质在植物中的作用。例如, 发现 strigolactones 作为一个新的类激素9和加速生长表型触发 brassinosteroid 应用10是在水培条件下进行的。此外, 该系统还可以进行标记同位素的实验 (例如, 14n/15n 和13CO2)11,12 , 以评估其纳入蛋白质和代谢物通过质谱分析。

考虑到该系统在植物研究中的重要性, 在过去几年中设计了大量的水培技术, 包括使用 (i) 将幼苗从盘子转移到水培容器3的系统, 13;(二) 西斯尔, 限制进入根系发育的早期阶段2、14、15;(iii) 聚乙烯颗粒为浮动体, 使小分子/处理的均匀应用困难16;或 (iv) 植物数量减少9,17。其中许多协议中描述的水培池体积通常很大 (体积从 1-5 升到32升)18, 这使得化学品的应用极为昂贵。虽然很少有研究表明在无菌条件下的水培栽培8,19, 系统的装配通常是相当费力的, 包括完美的调整尼龙网格成塑料或玻璃容器5,8,17,20。

由于拟南芥作为模型植物的重要性, 大多数的水培系统是为这个物种1,2,8,14,18,19,20. 尽管如此, 还有几项研究报告了其他植物的水耕生长特性, 并对种子进行预处理, 以改善它们在体外8、16 的发芽和同步速率..为了进行大规模的工作, 我们制定了一项协议, 建立一个简单和低成本的维修水培系统, 使种植植物的无菌条件, 包括一. 芥和其他物种, 如草狗尾草贝。本方法适用于不同的实验, 因为幼苗生长可以最大化, 同步, 易于监测。此外, 该系统具有以下优点: (一) 其装配简单, 可重复使用;(二) 使不同的化学品易于在液体介质中应用;(iii) 幼苗在培养基中发芽并直接生长, 不需要迁移到水培系统;(iv) 可密切监督苗根发育/生长, 并无损伤地收获幼苗;并且 (v) 它使有可能大规模地工作, 保持生理条件。

Protocol

1. 液体和固态培养基的制备 用半强力 Murashige 和 Skoog (MS) 培养基与维生素 [0.0125 毫克/升氯化钴五水合物, 0.0125 毫克/升铜 (ii) 硫酸盐五水, 18.35 毫克/升的 ethylenediaminetetraacetate 铁钠, 3.10 毫克/升硼酸, 0.415 毫克/升的碘化钾, 硫酸锰的8.45 毫克/升, 钼酸钠的0.125 毫克/升, 硫酸锌水合物的4.30 毫克/升, 氯化钙的166.01 毫克/升, 磷酸二氢钾85毫克/升, 950 毫克/升硝酸钾, 硫酸镁90.27 毫克/升, 825 毫克/升的…

Representative Results

TOR 激酶是一种主要的调节剂, 它集成了营养和能量信号, 促进了所有真核生物的细胞增殖和生长。在植物中阐明 tor 功能的努力包括通过 RNA 干涉或人工 microRNA28、30、31, 产生含有 TOR 条件压制的拟南芥转基因线。考虑到 TOR 击倒植物的胚胎致死表型32,33<s…

Discussion

这种优化的培水结构能够成功地培养植物的体外培养。种子在吸管尖端平坦表面的固体培养基上发芽良好, 与种子浸泡在营养液中的系统相比, 有相当大的增益。该系统的一个很好的优点是在幼苗发育过程中, 根直接接触液体培养基, 而不需要迁移。此外, 化学处理可以很容易地应用于液体介质中, 减少体积。湿度保持高, 避免了养分溶液的蒸发和补给。此外, 在幼苗建立过程中, 可以很容易地?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到圣保罗研究基金会 (FAPESP) 的支持;格兰特 12/19561-0) 和普朗克社会。Araújo (FAPEMIG 14/30594), 卡罗莱纳 c. 蒙特卡罗 (FAPESP;赠款 14/10407-3), Valéria 马夫拉 (FAPESP;赠款 14/07918-6) 和薇薇安妮 (海角/CNPEM 24/2013) 感谢研究金。作者感谢 Bourgin (INRA, 凡尔赛, 法国) 的基督教迈耶, 慷慨地为 RPS6 提供抗体。作者感谢室温硫化 UNICAMP 和 Aparecido de 索萨 Manoel 在录音过程中的技术支持。

Materials

Ethanol Merck 100983
Sodium hypochlorite solution Sigma-Aldrich 425044
Polysorbate 20   Sigma-Aldrich P2287
Murashige and Skoog (MS) medium including vitamins  Duchefa Biochemie M0222
2-(N-morpholino)ethanesulfonic acid (MES) monohydrate Duchefa Biochemie M1503
Agar  Sigma-Aldrich A7921
Potassium hydroxide Sigma-Aldrich 484016
Laminar flow hood Telstar BH-100
Hotplate AREC F20510011
Growth chamber Weiss Technik HGC 1514
Glass Petri dish (150 mm x 25 mm) Uniglass 189.006
200 μL pipette tip racks  Kasvi K8-200-5 *
300 μL multichannel pipette Eppendorf 3122000060
300 μL pipette tips Eppendorf 30073088
200 μL pipette  Eppendorf 3120000054
200 μL pipette tips Eppendorf 30000870
Scissors Tramontina 25912/108
Tweezer ABC Instrumentos 702915
Scalpel blade Sigma-Aldrich S2771
Adhesive transparent tape (45mm x 50m) Scotch 3M 5803
Disposable plastic boxes, external dimensions: 353 mm (L)x 178 mm (W) x 121mm (H) Maxipac 32771
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Riferimenti

  1. Conn, S. J., et al. Protocol: Optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant Methods. 9, 4 (2013).
  2. Gibeaut, D. M., Hulett, J., Cramer, G. R., Seemann, J. R. Maximal Biomass of Arabidopsis thaliana Using a Simple, Low-Maintenance Hydroponic Method and Favorable Environmental Conditions. Plant Physiology. 115, 317-319 (1997).
  3. Nguyen, N. T., McInturf, S. A., Mendoza-Cózatl, D. G. Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements. Journal of Visualized Experiments. (113), e54317 (2016).
  4. Koevoets, I. T., Venema, J. H., Elzenga, J. T. M., Testerink, C. Roots Withstanding their Environment: Exploiting Root System Architecture Responses to Abiotic Stress to Improve Crop Tolerance. Frontiers in Plant Science. 7, 1335 (2016).
  5. Arteca, R. N., Arteca, J. M. A novel method for growing Arabidopsis thaliana plants hydroponically. Physiologia Plantarum. 108, 188-193 (2000).
  6. Wang, R., Okamoto, M., Xing, X., Crawford, N. M. Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose, trehalose-6-phosphate, iron, and sulfate metabolism. Plant Physiology. 132, 556-567 (2003).
  7. Hirai, M. Y., et al. Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America. 101, 10205-10210 (2004).
  8. Alatorre-Cobos, F., et al. An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions. BMC Plant Biology. 14, 69 (2014).
  9. Umehara, M., et al. Inhibition of shoot branching by new terpenoid plant hormones. Nature. 455, 195-200 (2008).
  10. Arteca, J. M., Arteca, R. N. Brassinosteroid-induced exaggerated growth in hydroponically grown Arabidopsis plants. Physiologia Plantarum. 112, 104-112 (2001).
  11. Bindschedler, L. V., Palmblad, M., Cramer, R. Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study. Phytochemistry. 69, 1962-1972 (2008).
  12. Huege, J., et al. GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling. Phytochemistry. 68, 2258-2272 (2007).
  13. Berezin, I., Elazar, M., Gaash, R., Avramov-Mor, M., Shaul, O., Asao, T. The Use of Hydroponic Growth Systems to Study the Root and Shoot Ionome of Arabidopsis thaliana. Hydroponics: A Standard Methodology for Plant Biological Researches. , 135-152 (2012).
  14. Smeets, K., et al. Critical evaluation and statistical validation of a hydroponic culture system for Arabidopsis thaliana. Plant Physiology and Biochemistry. 46, 212-218 (2008).
  15. Huttner, D., Bar-zvi, D. An improved, simple, hydroponic method for growing Arabidopsis thaliana. Plant Molecular Biology Reporter. 21, 59-63 (2003).
  16. Battke, F., Schramel, P., Ernst, D. A novel method for in vitro culture of plants: Cultivation of barley in a floating hydroponic system. Plant Molecular Biology Reporter. 21, 405-409 (2003).
  17. Negi, M., Sanagala, R., Rai, V., Jain, A. Deciphering Phosphate Deficiency-Mediated Temporal Effects on Different Root Traits in Rice Grown in a Modified Hydroponic System. Frontiers in Plant Science. 7, 550 (2016).
  18. Tocquin, P., et al. A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana. BMC Plant Biology. 3, 2 (2003).
  19. Schlesier, B., Bréton, F., Mock, H. P. A hydroponic culture system for growing Arabidopsis thaliana plantlets under sterile conditions. Plant Molecular Biology Reporter. 21, 449-456 (2003).
  20. Norén, H., Svensson, P., Andersson, B. A convenient and versatile hydroponic cultivation system for Arabidopsis thaliana. Physiologia Plantarum. 121, 343-348 (2004).
  21. Martins, P. K., Ribeiro, A. P., da Cunha, B. A. D. B., Kobayashi, A. K., Molinari, H. B. C. A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis. Biotechnology Reports. 6, 41-44 (2015).
  22. Montané, M. H., Menand, B. ATP-competitive mTOR kinase inhibitors delay plant growth by triggering early differentiation of meristematic cells but no developmental patterning change. Journal of Experimental Botany. 64, 4361-4374 (2013).
  23. Boyes, D. C., et al. Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. The Plant Cell. 13, 1499-1510 (2001).
  24. Dobrenel, T., et al. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic. Frontiers in Plant Science. 7, 1611 (2016).
  25. Lunn, J. E., Furbank, R. T. Localisation of sucrose-phosphate synthase and starch in leaves of C4 plants. Planta. 202, 106-111 (1997).
  26. Hendriks, J. H. M., Kolbe, A., Gibon, Y., Stitt, M., Geigenberger, P. ADP-Glucose Pyrophosphorylase Is Activated by Posttranslational Redox-Modification in Response to Light and to Sugars in Leaves of Arabidopsis and Other Plant Species. Plant Physiology. 133, 838-849 (2003).
  27. Stitt, M., Lilley, R. M., Gerhardt, R., Heldt, H. W., Fleischer, S., Fleischer, B. Metabolite levels in specific cells and subcellular compartments of plant leaves. Methods in Enzymology. 174, 518-552 (1989).
  28. Caldana, C., et al. Systemic analysis of inducible target of rapamycin mutants reveal a general metabolic switch controlling growth in Arabidopsis thaliana. The Plant Journal. 73, 897-909 (2013).
  29. Livak, K. J., Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 25, 402-408 (2001).
  30. Dobrenel, T., et al. Sugar metabolism and the plant target of rapamycin kinase: a sweet operaTOR?. Frontiers in Plant Science. 4, 93 (2013).
  31. Moreau, M., et al. Mutations in the Arabidopsis homolog of LST8/GβL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days. The Plant Cell. 24, 463-481 (2012).
  32. Deprost, D., et al. The Arabidopsis TOR kinase links plant growth, yield, stress resistance and mRNA translation. EMBO Reports. 8, 864-870 (2007).
  33. Menand, B., et al. Expression and disruption of the Arabidopsis TOR (target of rapamycin) gene. Proceedings of the National Academy of Sciences of the United States of America. 99, 6422-6427 (2002).
  34. Mahfouz, M. M., Kim, S., Delauney, A. J., Verma, D. P. Arabidopsis TARGET OF RAPAMYCIN Interacts with RAPTOR, Which Regulates the Activity of S6 Kinase in Response to Osmotic Stress Signals. The Plant Cell. 18, 477-490 (2006).
  35. Zhang, R., et al. ScFKBP12 bridges rapamycin and AtTOR in Arabidopsis. Plant Signaling & Behavior. 8, e26115 (2013).
  36. Schepetilnikov, M., et al. TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h. The EMBO Journal. 32, 1087-1102 (2013).
  37. Schepetilnikov, M., et al. Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation. The EMBO Journal. 30, 1343-1356 (2011).
  38. Xiong, Y., et al. Glucose-TOR signalling reprograms the transcriptome and activates meristems. Nature. 496, 181-186 (2013).
  39. Creff, A., Sormani, R., Desnos, T. The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent. Plant Molecular Biology. 73, 533-546 (2010).
  40. Turck, F., Zilbermann, F., Kozma, S. C., Thomas, G., Nagy, F. Phytohormones participate in an S6 kinase signal transduction pathway in Arabidopsis. Plant Physiology. 134, 1527-1535 (2004).
  41. Gibon, Y., et al. Adjustment of diurnal starch turnover to short days: Depletion of sugar during the night leads to a temporary inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the followin. Plant Journal. 39, 847-862 (2004).
  42. Smith, A. M., Stitt, M. Coordination of carbon supply and plant growth. Plant, Cell & Environment. 30, 1126-1149 (2007).
  43. Smith, A. M., Zeeman, S. C., Smith, S. M. Starch Degradation. Annual Review of Plant Biology. 56, 73-98 (2005).
  44. Orzechowski, S. Starch metabolism in leaves. Acta Biochimica Polonica. 55, 435-445 (2008).
  45. Gibon, Y., et al. Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods. Plant, Cell & Environment. 32 (7), 859-874 (2009).
  46. Kim, J. B., Kang, J. Y., Soo, Y. K. Over-expression of a transcription factor regulating ABA-responsive gene expression confers multiple stress tolerance. Plant Biotechnology Journal. 2, 459-466 (2004).
  47. Vishwakarma, K., et al. Abscisic Acid Signaling and Abiotic Stress Tolerance in Plants: A Review on Current Knowledge and Future Prospects. Frontiers in Plant Science. 8, 161 (2017).
  48. Yoshida, T., et al. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress. Plant, Cell & Environment. 38, 35-49 (2015).
  49. Koch, K. E. Carbohydrate-Modulated Gene Expression in Plants. Annual Review of Plant Physiology and Plant Molecular Biology. 47, 509-540 (1996).
  50. Price, J., Laxmi, A., St Martin, S. K., Jang, J. C. Global transcription profiling reveals multiple sugar signal transduction mechanisms in Arabidopsis. The Plant Cell. 16, 2128-2150 (2004).
  51. Thimm, O., et al. mapman: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. The Plant Journal. 37, 914-939 (2004).
  52. Bläsing, O. E., et al. Sugars and Circadian Regulation Make Major Contributions to the Global Regulation of Diurnal Gene Expression in Arabidopsis. The Plant Cell. 17, 3257-3281 (2005).
  53. Osuna, D., et al. Temporal responses of transcripts, enzyme activities and metabolites after adding sucrose to carbon-deprived Arabidopsis seedlings. The Plant Journal. 49, 463-491 (2007).
  54. Yadav, U. P., et al. The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P. Journal of Experimental Botany. 65, 1051-1068 (2014).
  55. Brutnell, T. P., et al. Setaria viridis: A Model for C4 Photosynthesis. The Plant Cell. 22, 2537-2544 (2010).
  56. Altman, N., Krzywinski, M. Points of Significance: Clustering. Nature Methods. 14, 545-546 (2017).
  57. Pratelli, R., Boyd, S., Pilot, G. Analysis of amino acid uptake and translocation in Arabidopsis with a low-cost hydroponic system. Journal of Plant Nutrition and Soil Science. 179, 286-293 (2016).

Tags

Hydroponic SystemIn VitroSterile ConditionsPlant ResponsesSmall MoleculesArabidopsis ThalianaMS MediumLaminar Flow HoodSterilizationUV LightSolid MediumLiquid Medium

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Monte-Bello, C. C., Araujo, E. F., Martins, M. C., Mafra, V., da Silva, V. C., Celente, V., Caldana, C. A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions. J. Vis. Exp. (138), e57800, doi:10.3791/57800 (2018).
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