植物与放射性示踪剂测量矿质营养和毒物的通量
Summary
In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.
Abstract
Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.
Introduction
营养物质和有毒物质的吸收和分布强烈地影响植物生长。因此，底层传输过程对调查构成的植物生物学和研究农业科学1,2的一个主要领域，特别是在营养优化和环境压力的环境中（ 例如 ，盐胁迫，铵毒性）。对于通量植物的测量方法中，最主要是利用放射性同位素示踪物，这是显著在20世纪50年代开发的。（ 例如 ，3），一直持续到今天被广泛使用。其他方法，如养分耗竭的从根介质和/或积累在组织中，使用的离子选择性微电极的振动测量，如MIFE（微电极的离子通量的估计）和SIET（扫描离子选择性电极法），并使用离子选择性荧光染料，也被广泛应用，但在他们的检测净感能力是有限XES（ 即 ，流入和流出之间的差异）。使用放射性同位素的，另一方面，允许研究者的独特能力来分离和量化单向通量，这可以被用来解决动力学参数（ 例如 ，K M和V max）和洞察能力，热力学，机制的运输系统和调节。与放射性示踪剂制成单向磁通测量条件下在相反的方向上的磁通量是高下特别有用的，并且细胞内池的周转迅速4。此外，放射性示踪方法允许较高的底物浓度下进行测量时，与许多其它的技术（参见“讨论”，下同），因为所跟踪的同位素是针对相同的元件的另一同位素背景观察。
这里，我们提供的单向和n中的放射性同位素的测量的详细步骤矿质营养等通量和毒物的完整植株。重点是对钾（K +）通量测量，植物大量营养5，和氨/铵制成（NH 3 / NH 4 +），另一个常量营养素是，然而，中毒性时在高浓度下（ 例如本，1 10毫米）2。我们将用放射性同位素42的K +（叔2 = 12.36小时）和13 NH13分之3NH 4 +（T 2 = 9.98分钟），分别在该模型系统中的大麦的完整的苗（ 大麦 Ļ ），在两个关键协议的描述：通过示踪剂流出（CATE）直接流入（DI）和室分析。我们应该注意到，从本文简单介绍了需要执行的每个协议的步骤开始。每种技术在适当的时候，提供计算和理论的简要说明，但详细的论述，的背景和理论能够在这个问题4,6-9几个关键物品被发现。重要的是，这些协议大致上可转移到磁通的其他营养素/毒物分析（ 例如 ，24的Na +，22的Na +，86 Rb的+，13 NO 3 – ）和其它植物物种的，尽管有一些需要说明的（参见下文） 。我们还强调，放射性物质工作的所有研究者必须在通过自己的机构的电离辐射安全监管机构设置许可工作的重要性。
Protocol
Representative Results
Discussion
这表现在上面的例子中，放射性示踪方法是一个功能强大的测量营养素和有毒物质在植物中的单向磁通的装置， 图1示出的NH 3流入可超过225微摩尔克-1小时-1，这也许是达到最高善意的跨膜通量曾报道在植物中的系统13，但是，如果测定仅净通量这个磁通量的大小将是不可见的。这是因为，NH 3的大流出发生在同一时间涌入，在徒劳…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Gamma counter | Perkin Elmer | Model: Wallac 1480 Wizard 3" | |
| Geiger-Müller counter | Ludlum Measurements Inc. | Model 3 survey meter | |
| 400-mL glass beakers | VWR | 89000-206 | For pre-absorption, absorption, and desorption solutions |
| Glass funnel | VWR | 89000-466 | For efflux funnel |
| Large tubing | VWR | 529297 | For efflux funnel |
| Medium tubing | VWR | 684783 | For bundling |
| Small tubing | VWR | 63013-541 | For aeration |
| Aeration manifold | Penn Plax Air Tech | vat 5.5 | To control/distribute pressurized air into solutions |
| Glass scintillation vials | VWR | 66022-128 | For gamma counting |
| Glass centrifuge tubes | VWR | 47729-576 | For spin-drying root samples |
| Kimwipes | VWR | 470173-504 | For spin-drying root samples |
| Dissecting scissors | VWR | 470001-828 | |
| Forceps | VWR | 470005-496 | |
| Low-speed clinical centrifuge | International Equipment Co. | 76466M-4 | For spin-drying root samples |
| 1-mL pipette | Gilson | F144493 | |
| 10-mL pipette | Gilson | F144494 | |
| 1-mL pipette tips | VWR | 89079-470 | |
| 10-mL pipette tips | VWR | 89087-532 | |
| Analytical balance | Mettler toledo | PB403-S/FACT |
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