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Shoot Apical Meristem Size Measurement

顶端分生组织大小的测量

HC Hsuan Chou
HW Huanzhong Wang
GB Gerald A. Berkowitz

发布: 2016年12月05日第6卷第23期 DOI: 10.21769/BioProtoc.2055 浏览次数: 9494

评审: Tie LiuYuan ChenAnonymous reviewer(s)

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Abstract

The shoot apical meristem (SAM) is a collection of cells that continuously renew themselves by cell division and also provide cells to newly developing organs. It has been known that CLAVATA (CLV) 3 peptide regulates a transcription factor WUSCHEL (WUS) to keep numbers of undifferentiated cells constant and maintain the size of the SAM. The interactive feedback control of CLV3 and WUS in a non-cell autonomous signaling cascade determines stem cell fate (maintenance of pluripotency or, alternatively, differentiation into daughter cells) in the SAM. Ca2+ is a secondary messenger that plays a significant role in numerous signaling pathways. The signaling system connecting CLV3 binding to its receptor and WUS expression is not well delineated. We showed that Ca2+ is involved in CLV3 regulation of the SAM size. One of the approaches we used was measuring the size of the SAM. Here we provide a detailed protocol on how to measure Arabidopsis SAM size with Nomarski microscopy. The area of the two-dimensional dome representing the maximal ‘face’ of the SAM was used as a proxy for SAM size. Studies were done on wild type (WT) Arabidopsis in the presence and absence of a Ca2+ channel blocker Gd3+ and the CLV3 peptide, as well on genotypes that lack functional CLV3 (clv3) or a gene encoding a Ca2+-conducting ion channel (‘dnd1’).

Keywords: Arabidopsis (拟南芥) search Shoot apical meristem (顶端分生组织) search Shoot development (枝稍发育) search Cell signaling (细胞信号) search Seedlings (幼苗) search

Background

Nomarski microscopy is widely used to study Arabidopsis SAM size. Other microscopy techniques for SAM observation are time consuming and require embedding tissue in resin and then sectioning or even more sophisticated microscopy. Nomarski microscopy, along with tissue clearing techniques is fast and convenient for whole tissue imaging. Published methods on SAM size measurement with Nomarski microscopy are often briefly described. Here, we provide a modified protocol with a detailed step by step guide including steps from dissecting Arabidopsis SAM tissues, through sample preparation for Nomarski microscopy, and SAM size measurement.

Materials and Reagents

  1. 3 x 4 cell culture multi-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 150628 )
  2. Parafilm
  3. Razor blade
  4. Glass slide (75 x 38 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-550B )
  5. Coverslips (18 x 18 mm) (thickness: 0.17 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S17521 )
  6. Arabidopsis thaliana wild type (ecotype Columbia), dnd1 mutant (At5g15410), clv3 mutant (At2g27250)
  7. Synthetic CLV3 peptide RTVPhSGPhDPLHH3 (GenScript, Piscataway, NJ)
  8. Murashige and Skoog salts (MS) (Caisson Laboratories, catalog number: MSP01-10LT )
  9. Gadolinium(III) chloride (GdCl3) (Sigma-Aldrich, catalog number: 439770 )
  10. Ethanol (Sigma-Aldrich, catalog number: 459836 )
  11. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  12. MES buffer (pH 5.7) (Caisson Laboratories, catalog number: M009-100GM )
  13. Sterilized water
  14. Tris (Sigma-Aldrich, catalog number: 252859 )
  15. Acetic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: A38S-500 )
  16. Chloral hydrate (Sigma-Aldrich, catalog number: C8383 )
  17. Glycerol (Thermo Fisher Scientific, catalog number: 17904 )
  18. Plant culture medium (see Recipes)
  19. Fixing solution (see Recipes)
  20. Clearing solution (see Recipes)

Equipment

  1. Fridge
  2. Shaker
  3. Growth chamber for growing plants (100 µmol m-2 sec-1 white light for 16 h and dark for 8 h, 23 °C)
  4. Tweezers
  5. Dissecting microscope
  6. Microscope equipped with Nomarski optics (Nikon Instrument, model: MICROPHOT-FX )

Software

  1. Infinity analyze program (Lumenera, Ottawa, Canada)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Chou, H., Wang, H. and Berkowitz, G. A. (2016). Shoot Apical Meristem Size Measurement. Bio-protocol 6(23): e2055. DOI: 10.21769/BioProtoc.2055.

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分类

植物科学 > 植物发育生物学 > 形态建成

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