侧根诱导系统<em>拟南芥</em>和玉米
Summary
The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.
Abstract
Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.
Introduction
根系统是植物生长的关键，因为它确保锚固的水分吸收和养分从土壤。因为根系统的扩展主要是依赖于生产侧根，其起始和形成已被广泛研究。侧根开始在柱鞘细胞的特定子集，称为创始人电池 1。在大多数双子叶植物，如拟南芥 ，这些细胞都位于原生木质部极2，而在单子叶植物，如玉米，它们被发现在韧皮部磁极3。创办人细胞通过增加生长素应答4标记，接着通过特定的细胞周期基因表达（例如， 细胞周期蛋白B1; 1 / CYCB1; 1），之后，它们经历了第一轮的不对称背斜司5。经过一系列的协调背斜与平周分裂，一侧根原基形成的最终将成为一个一utonomous侧根。的位置和侧根起始定时是但是不可预见的，因为这些事件既不丰富也不同步。这妨碍了使用分子的方法，例如转录研究这一过程。
为了解决这个问题，一个侧根诱导系统（LRIS）已经开发6,7。在这个系统中，幼苗首先用N – 1-naphthylphthalamic酸（NPA），其抑制生长素运输和积累，从而阻断侧根开始8。通过随后转移至苗含有合成植物生长素-1-萘乙酸（NAA）培养基中，整个柱鞘层响应该升高的生长素水平从而大规模诱导侧根启动细胞分裂6。因此，这一制度导致了快速，同步和广泛的侧根灌顶，方便收集富含晚期的特定阶段根样本拉尔根系发育。随后，这些样品可以被用来确定在侧根形成的全基因组表达谱。该LRIS取得了约侧根开始在拟南芥和玉米9-13，但需要已经显著的知识，这个系统应用到其他植物物种变得更基因组被测序更加明显，并且有越来越多的兴趣将知识传授给经济的重要种类。
这里， 拟南芥和玉米LRISs的详细方案给出。接着，使用该系统的一个例子是提供，通过说明如何从玉米LRIS获得转录数据可用于识别具有在不同的植物物种侧根发起期间的保守功能功能同系物。最后，指导方针，以优化LRIS其他植物物种提出了建议。
Protocol
Representative Results
Discussion
在拟南芥 LRIS协议，它只能传输已经变得完全在与含NPA生长培养基接触的幼苗是重要的。这确保侧根开始被阻止在整个根长度。为了防止伤人传输过程中的小植株，弯曲钳子的臂能下的幼苗的子叶钩住。转让时，请确保幼苗根在与含NAA琼脂培养基充分接触。这可以通过掠过根在琼脂表面上一个小的距离来实现。这将确保一个有效的同步感应侧根萌生过的总根长。的根源可以种植没有他们的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Davy Opdenacker for technical assistance and photography. We greatly thank Dr. Annick Bleys for helpful suggestions to improve the manuscript. This work was financed by the Interuniversity Attraction Poles Programme IUAP P7/29 ‘MARS’ from the Belgian Federal Science Policy Office, by the FWO grant G027313N and by the Agency for Innovation by Science and Technology, IWT (IR).
Materials
| ARABIDOPSIS LRIS | |||
| Seeds | |||
| Arabidopsis seeds | Col-0 ecotype | ||
| Gas sterilization of seeds | |||
| micro-centrifuge tubes 1.5 ml | SIGMA-ALDRICH | 0030 125.215 | Eppendorf microtubes 3810X, PCR clean |
| micro-centrifuge tubes 2 ml | SIGMA-ALDRICH | 0030 120.094 | Eppendorf Safe-Lock microcentrifuge tubes |
| hydrochloric acid | Merck KGaA | 1,003,171,000 | 37% (fuming) for analysis EMSURE ACS,ISO,Reag. Ph Eu |
| glass desiccator | SIGMA-ALDRICH | Pyrex | |
| glass beaker | |||
| plastic micro-centrifuge tubes box or holder | |||
| Bleach sterilization of seeds | |||
| ethanol | Chem-Lab nv | CL00.0505.1000 | Ethanol, abs. 100% a.r. dilute to 70% |
| sodium hypochlorite (NaOCl) | Carl Roth | 9062.3 | 12% |
| Tween 20 | SIGMA-ALDRICH | P1379 | |
| sterile water | |||
| Growth medium | |||
| Murashige and Skoog salt mixture | DUCHEFA Biochemie B.V. | M0221-0050 | |
| myo-inositol | SIGMA-ALDRICH | I5125-100G | |
| 2-(N-morpholino)ethanesulfonic acid (MES) | DUCHEFA Biochemie B.V. | M1503.0100 | |
| sucrose | VWR, Internation LLC | 27483.294 | D(+)-Sucrose Ph. Eur. |
| KOH | Merck KGaA | 1050211000 | pellets for analysis (max. 0.002% Na) EMSURE ACS,ISO,Reag. Ph Eur |
| Plant Tissue Culture Agar | LabM Limited | MC029 | |
| Lateral root induction chemicals | |||
| N-1-naphthylphthalamic acid (NPA) | DUCHEFA Biochemie B.V. | No. N0926.0250 | 10 µM (Arabidopsis) |
| 1-naphthalene acetic acid (NAA) | DUCHEFA Biochemie B.V. | No. N0903.0050 | 10 µM (Arabidopsis) |
| dimethylsulfoxide (DMSO) | SIGMA-ALDRICH | 494429-1L | |
| Making a mesh for transfer | |||
| nylon mesh | Prosep byba | Synthetic nylon mesh 20 µm | |
| Sowing and seedling handling | |||
| square petri dish plates | GOSSELIN | BP124-05 | 12 x 12 cm |
| 50 ml DURAN tubes | SIGMA-ALDRICH | CLS430304 | Corning 50 mL centrifuge tubes |
| drigalski | Carl Roth | K732.1 | |
| pipette | |||
| cut pipette tips | Daslab | 162001X | Universal 200, cut off 5 mm of tip before autoclaving |
| breathable tape | 3M Deutschland GmbH | cat. no. 1530-1 | |
| tweezers | Fiers nv/sa | K342.1; K344.1 | Dumont tweezers type a nr 5; Dumont tweezers type e nr 7 |
| Growth conditions | |||
| growth room | 21 °C, continuous light | ||
| Materials | Company | Catalog | Comments |
| MAIZE LRIS | |||
| Seeds | |||
| Maize kernels | B-73 | ||
| Bleach sterilization of kernels | |||
| glass beaker | |||
| magnetic stirrer | Fiers nv/sa | C267.1 | |
| sodium hypochlorite (NaOCl) | Carl Roth | 9062.3 | 12% |
| sterile water | |||
| Lateral root induction chemicals | |||
| N-1-naphthylphthalamic acid (NPA) | DUCHEFA Biochemie B.V. | No. N0926.0250 | 50 µM (maize primary root), 25 µM (maize adventitious root) |
| 1-naphthalene acetic acid (NAA) | DUCHEFA Biochemie B.V. | No. N0903.0050 | 50 µM (maize) |
| dimethylsulfoxide (DMSO) | SIGMA-ALDRICH | 494429-1L | |
| Sowing and seedling handling | |||
| paper hand towels | Kimberly-Clark Professional* | 6681 | SCOTT Hand Towels – Roll / White; sheet size (24 x 46 cm) |
| seed germination paper | Anchor Paper Company | 10 X 15 38# seed germination paper | |
| tweezers | Fiers nv/sa | K342.1; K344.1 | Dumont tweezers type a nr 5; Dumont tweezers type e nr 7 |
| 250 ml (centrifuge) tubes | SCHOTT DURAN | 2160136 | approx. 5.6 cm diameter and 14.7 cm height |
| 700 ml tubes | DURAN GROUP | 213994609 | cylinders, round foot tube, D 60 x 250 |
| rack | for maize tubes, home made | ||
| sterile water | |||
| Growth conditions | |||
| growth cabinet | 27 °C, continuous light, 70% relative humidity |
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