의 측면 루트 유도 성 시스템<em> 애기 장대</em>과 옥수수
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
이 토양에서 앵커리지 및 물 섭취와 영양분을 보장 이후 루트 시스템은 식물 성장에 중요하다. 루트 시스템의 팽창은 주로 측면 뿌리의 생산에 사용하기 때문에, 그 개시 및 형성은 널리 연구되고있다. 측면 뿌리는 설립자 세포 1이라고 pericycle 세포의 특정 부분 집합으로 시작됩니다. 옥수수와 같은 외떡잎에, 그들은 사부 극 3에서 발견되는 반면, 같은 애기 장대와 같은 대부분의 dicots, 이러한 세포, protoxylem 기둥 2에 있습니다. 설립자 세포는 특정 세포주기 유전자의 발현에 이어 증가 옥신 반응 (4)에 의해 표시된다 (예를 들어, 사이클린 B1 1 / CYCB1는 1), 그 후 그들이 비대칭 배사 부문 (5)의 첫 번째 라운드를 겪는다. 조정 배사와 periclinal 부문의 일련의 후 측면 루트 primordium 최종적으로 부상 할 것으로 형성된다utonomous 측면 루트. 이러한 이벤트가 풍부한이나 동기화도 있기 때문에 위치 및 측면 루트 개시 타이밍은, 그러나 예측하지 않습니다. 여기에는이 과정을 연구하기 transcriptomics 분자 접근법의 사용을 방해한다.
이를 해결하기 위해, 횡 루트 유도 성 시스템 (LRIS)는, 6 개발되었다 (7).이 시스템에서는, 묘 먼저 따라서 횡 루트 개시를 막고, 옥신 전송 및 축적을 억제하는 N -1- naphthylphthalamic 산 (NPA)로 처리 8. 이어서 합성 옥신 -1- 나프탈렌 초산 (NAA)를 함유하는 배지에 종묘를 전송함으로써, 전체 pericycle 층함으로써 대규모 고가 옥신 레벨 유도부 횡 루트 개시 세포 분열 (6)에 응답한다. 이와 같이,이 시스템은 후반의 특정 스테이지에 대해 농후 루트 샘플의 수집을 용이하게, 고속 동기 및 광범위한 측면 뿌리 시작 (initiation)에 이르게RAL 루트 개발. 이어서, 이들 샘플은 횡 루트 형성 동안 게놈 전체의 발현 프로필을 결정하기 위해 사용될 수있다. LRIS 다른 식물 종에이 시스템을 적용하는 측면 루트 애기 장대의 시작과 옥수수 9-13,하지만 필요에 대해 이미 상당한 지식을 나왔고 더 게놈 서열대로보다 명확하게하고 경제적 중요한에게 지식을 전달하는 관심이 높아지고있다 종.
여기, 애기 장대와 옥수수 LRISs의 상세한 프로토콜이 제공됩니다. 다음, 시스템의 사용의 예는 옥수수 LRIS 얻은 transcriptomics 데이터가 다른 식물 종에 걸쳐 횡 루트 개시 동안 보존 된 기능을 가질 기능적 동족체를 식별하기 위해 사용할 수있는 방법을 예시하여 제공된다. 다른 종의 식물이 제안에 대해 마지막으로, 지침 LRIS을 최적화합니다.
Protocol
Representative Results
Discussion
애기 LRIS 프로토콜에서, 오직 완전히 NPA 함유 증식 배지와 접촉 성장한 묘목을 전송하는 것이 중요하다. 이 측면 루트 개시는 전체 루트 길이에 걸쳐 차단되는 것을 보장한다. 전송 중에 묘목 부상 방지하기 위해, 만곡 된 포셉 팔 종묘의 자엽 하에서 후크 될 수있다. 전송시, 묘목의 뿌리가 NAA 함유 한천 배지와 충분한 접촉에 있는지 확인하십시오. 이것은 작은 거리에 걸쳐 한천 표면에 뿌?…
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 |
References
- Van Norman, J. M., Xuan, W., Beeckman, T., Benfey, P. N. To branch or not to branch: the role of pre-patterning in lateral root formation. Development. 140, 4301-4310 (2013).
- Dolan, L., et al. Cellular organisation of the Arabidopsis thaliana root. Development. 119, 71-84 (1993).
- Bell, J. K., McCully, M. E. A histological study of lateral root initiation and development in Zea mays. Protoplasma. 70, 179-205 (1970).
- Benkova, E., et al. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell. 115, 591-602 (2003).
- Beeckman, T., Burssens, S., Inzé, D. The peri-cell-cycle in Arabidopsis. J. Exp. Bot. 52, 403-411 (2001).
- Himanen, K., Boucheron, E., Vanneste, S., de Almeida Engler, J., Inzé, D., Beeckman, T. Auxin-mediated cell cycle activation during early lateral root initiation. Plant Cell. 14, 2339-2351 (2002).
- Jansen, L., Parizot, B., Beeckman, T. Inducible system for lateral roots in Arabidopsis thaliana and maize. Methods Mol Biol. 959, 149-158 (2013).
- Casimiro, I., et al. Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell. 13, 843-852 (2001).
- Himanen, K., et al. Transcript profiling of early lateral root initiation. Proc. Natl. Acad. Sci. USA. 101, 5146-5151 (2004).
- De Smet, I., et al. Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science. 322, 594-597 (2008).
- Vanneste, S., et al. Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana. Plant Cell. 17, 3035-3050 (2005).
- De Rybel, B., et al. A role for the root cap in root branching revealed by the non-auxin probe naxillin. Nat. Chem. Biol. 8, 798-805 (2012).
- Jansen, L., et al. Comparative transcriptomics as a tool for the identification of root branching genes in maize. Plant Biotechnol. J. 11, 1092-1102 (2013).
- Parizot, B., et al. Diarch symmetry of the vascular bundle in Arabidopsis root encompasses the pericycle and is reflected in distich lateral root initiation. Plant Physiol. 146, 140-148 (2008).
- Murashige, T., Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962).
- Bellini, C., Pacurar, D. I., Perrone, I. Adventitious roots and lateral roots: similarities and differences. Annu Rev Plant Biol. 65, 639-666 (2014).
- Grunewald, W., Parizot, B., Inzé, D., Gheysen, G., Beeckman, T. Developmental biology of roots: One common pathway for all angiosperms?. Int. J. Plant Dev. Biol. 1, 212-225 (2007).
- Parizot, B., Beeckman, T., Crespi, M. Genomics of root development. Root Genomics and Soil Interactions. , 3-28 (2012).
- Bargmann, B. O., Birnbaum, K. D. Fluorescence activated cell sorting of plant protoplasts. Journal of visualized experiments : JoVE. , (2010).
- Nakazono, M., Qiu, F., Borsuk, L. A., Schnable, P. S. Laser-capture microdissection, a tool for the global analysis of gene expression in specific plant cell types: identification of genes expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell. 15, 583-596 (2003).
- Ludwig, Y., Hochholdinger, F. Laser microdissection of plant cells. Methods Mol Biol. 1080, 249-258 (2014).
- Beeckman, T., Engler, G. An easy technique for the clearing of histochemically stained plant tissue. Plant Mol. Biol. Rep. 12, 37-42 (1994).
- Ditengou, F. A., et al. Mechanical induction of lateral root initiation in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. 105, 18818-18823 (2008).
- Lucas, M., Godin, C., Jay-Allemand, C., Laplaze, L. Auxin fluxes in the root apex co-regulate gravitropism and lateral root initiation. J. Exp. Bot. 59, 55-66 (2008).
- Wang, S., Taketa, S., Ichii, M., Xu, L., Xia, K., Zhou, X. Lateral root formation in rice (Oryza Sativa. L.): differential effects of indole-3-acetic acid and indole-3-butyric acid. Plant Growth Regul. 41, 41-47 (2003).
- Martinez-de la Cruz, E., Garcia-Ramirez, E., Vazquez-Ramos, J. M., Reyes de la Cruz, H., Lopez-Bucio, J. Auxins differentially regulate root system architecture and cell cycle protein levels in maize seedlings. J Plant Physiol. 176, 147-156 (2015).
- Beyer, E. M., Morgan, P. W. Effect of ethylene on uptake, distribution, and metabolism of indoleacetic acid-1-14C and -2-14C and naphthaleneacetic acid-1-14C. Plant Physiol. 46, 157-162 (1970).
- Delbarre, A., Muller, P., Imhoff, V., Guern, J. Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta. 198, 532-541 (1996).
- Bailey-Serres, J. Microgenomics: genome-scale, cell-specific monitoring of multiple gene regulation tiers. Annu Rev Plant Biol. 64, 293-325 (2013).
- Perez-Torres, C. A., et al. Phosphate availability alters lateral root development in Arabidopsis by modulating auxin sensitivity via a mechanism involving the TIR1 auxin receptor. Plant Cell. 20, 3258-3272 (2008).
