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, Shoot apical meristem, Shoot development, Cell signaling, Seedlings
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
Equipment
Software
Procedure
Data analysis
For comparison between SAM size of wild type, dnd1 and clv3 mutant seedlings (see Figure 1 and Figure 2A), at least 15 seedlings of each genotype were measured. For calcium channel blocker effects on endogenous CLV3 in wild type (Figure 2B) and exogenous CLV3 effects on clv3 mutant, at least 10 seedlings were measured for each treatment (Figure 2C). ANOVA analysis was used to evaluate means separation. For Figures 2A and 2B, an asterisk or two asterisks above the bar representing a genotype or treatment indicate SAM size was significantly different (at P < 0.05 or P < 0.01, respectively) than control (WT). For Figure 2C, ANOVA comparisons are indicated by brackets. Figure 2. Ca2+ signaling interacts with CLV3 control of SAM area. (a). SAM area of 7-d-old WT, dnd1 and clv3 seedlings. (b). WT seedlings grown on standard ½ MS liquid medium (Control), or medium supplemented with Gd3+ µM on day 3 or on day 0 (the day the experiment starts). (c). clv3 seedlings grown on standard medium were treated with water (control), CLV3, or CLV3 and Gd3+. CLV3 and Gd3+ were applied on day 0. ANOVA analysis was used to evaluate means separation. All seedlings shown in Figure 2 are 7-d-old seedlings. For (a) and (b), an asterisk or two asterisks above the bar representing a genotype or treatment indicate SAM size was significantly different (at P < 0.05 or P < 0.01, respectively) than control (WT). For (c), ANOVA comparisons are indicated by brackets.
Recipes
Acknowledgments
This work was supported by National Science Foundation award 1146827 (to G.A.B). This protocol was adapted and modified from Fiers et al., (2006), Ohyama et al., (2009), Carles et al., (2010) and Dr. Miguel Aguilar.
References
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