Rapid Assessment of Cell Wall Polymer Distribution and Surface Topology of Arabidopsis Seedlings.

The deposition and modulation of constituent polymers of plant cell walls are profoundly important events during plant development. Identification of specific polymers within assembled walls during morphogenesis and in response to stress conditions represents a major goal of plant cell biologists. Arabidopsis thaliana is a model organism that has become central to research focused on fundamental plant processes including those related to plant wall dynamics. Its fast life cycle and easy access to a variety of mutants and ecotypes of Arabidopsis have stimulated the need for rapid assessment tools to probe its wall organization at the cellular and subcellular levels. We describe two rapid assessment techniques that allow for elucidation of the cell wall polymers of root hairs and high-resolution analysis of surface features of various vegetative organs. Live organism immunolabeling of cell wall polymers employing light microscopy and confocal laser scanning microscopy can be effectively performed using a large microplate-based screening strategy (see Figs. 1 and 2). Rapid cryofixation and imaging of variable pressure scanning electron microscopy also allows for imaging of surface features of all portions of the plant as clearly seen in Fig. 3.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

A Rapid and Simple Method for Microscopy-Based Stomata Analyses.

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel.

Assessment of inequality of root hair density in Arabidopsis thaliana using the Gini coefficient: a close look at the effect of phosphorus and its interaction with ethylene.

BACKGROUND AND AIMS: Root hair density (i.e. the number of root hairs per unit root length) in Arabidopsis thaliana varies among individual plants in response to different nutrient stresses. The degree of such variation, defined as inequality, serves as a unique indicator of the uniformity of response within a plant population to nutrient availability. METHODS: Using the Gini coefficient (G) as an inequality index, the inequality of root hair density in Arabidopsis thaliana 'Columbia' was evaluated under conditions of nutrient stresses; in particular the effect of phosphorus and its interaction with ethylene. KEY RESULTS: With decreasing phosphorus concentration, root hair density increased while inequality decreased logarithmically. The addition of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) under high phosphorus increased root hair density and decreased inequality by 7-fold. Inhibition of ethylene action with 1-methylcyclopropene (MCP) and silver thiosulphate (STS) under low phosphorus decreased root hair density, and increased inequality by 9-fold and 4-fold, respectively. The ethylene action inhibitors had little effect on root hair density under high phosphorus, but inequality increased 3-fold in the presence of MCP and decreased 2-fold in the presence of STS. Compared with the control, deficiencies in S, N and K increased inequality of root hair density, whereas deficiencies in P, Ca, B, Mn, Fe, Zn, Cu and Mg decreased inequality. In particular, the inequality of root hair density increased by over 2-fold under deficiencies of N or K, but decreased 14-fold under phosphorus deficiency. CONCLUSIONS: The inequality analysis indicates a strong correlation between prevalent signals from the environment (i.e. phosphorus stress) and the response of the plant, and the role of ethylene in this response. As the environmental signals become stronger, an increasing proportion of individuals respond, resulting in a decrease in variation in responsiveness among individual plants as indicated by reduced inequality.