Abstract
Root development in vascular plants is innately tied to the environment. However, relatively little attention has been paid toward understanding the spatial scales at which the root perceives and responds to external stimuli. While some environmental signals elicit global responses that affect root system architecture, others may have more localized effects. We have observed that various developmental processes can be induced or suppressed along the circumference of the main root depending on local contact with available water in a process termed hydropatterning (Bao et al., 2014). Our studies of hydropatterning indicate that the root can detect and respond to certain external stimuli at the resolution of the diameter of a single organ. In order to characterize developmental patterning at this spatial scale, we developed a procedure to vary environmental inputs across the circumferential axis of the root in vitro using agar media. Roots are grown between two blocks of agar media in a “sandwich”. Local environmental conditions can be varied depending on the composition of the media on either side. Stimuli that act locally can be distinguished from those that act systemically based on the developmental response of the root. Here we describe the overall method and provide an example of how it can be used to analyze lateral root patterning in Zea mays (maize) in response to an external water potential gradient. We also discuss how the method can be used more broadly for other plant species and environmental treatments.
Keywords: Plant-environment interactions, Root development, Lateral roots, Agar sandwich, Water stress
Materials and Reagents
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Data analysis
See Figure 6A for an example graph of average lateral root densities in different agar sandwich conditions. Table 1 contains the output from an ANOVA conducted in R. Figure 6B summarizes results of post-hoc testing after observation of a significant cis-condition:trans-condition interaction term in the ANOVA. Table 1. Output from analysis of variance in R. The cis-condition had a significant effect on lateral root density (P < 0.05), which supports the hypothesis that the treatment has a local effect on development. The trans-condition and the cis-condition:trans-condition interaction term were also significant, indicating that the treatment also had a systemic effect on development. All terms which contain “side” as a variable are not significant (P > 0.05), indicating that the effects of the treatment did not depend on the method of application in the agar sandwich (block or plate agar media). numDF and denDF denote numerator and denominator degrees of freedom, respectively. Figure 6. Representative data and statistical analysis. A. Maize seedlings were treated with either control or mannitol agar media in 4 agar sandwich conditions. Average lateral root densities on each side of the root are shown. The Air Gap density was 0 in each condition. Error bars = standard error, n = 6 plants per condition. B. Summary of data from post-hoc testing. Since there was a significant effect of the cis-condition:trans-condition term on lateral root density and no significant effect of the side of the sandwich (block vs plate) (see Table 1), all pairwise comparisons between the different treatment categories were performed using the "multcomp" package in R. Similar groups are denoted with the same letter (P < 0.05 for significant difference). The response to control media was independent of the treatment on the other side of the root, indicating that the root responded locally to this condition. Contrastingly, the response to mannitol differs depending on whether the other side of the root is treated with mannitol or control media, indicating that mannitol can have systemic effects on development. Error bars = standard error, n = 12 observations per category.
Notes
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Acknowledgments
This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1147470. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.
References
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