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Salt stress simultaneously causes ionic toxicity, osmotic stress and oxidative stress, which directly impact plant growth and development. Plants have developed numerous strategies to adapt to saline environments. Whereas some of these strategies have been investigated and exploited for crop improvement, much remains to be understood, including how salt stress is perceived by plants and how plants coordinate effective responses to the stress. It is, however, clear that the plant cell wall is the first contact point between external salt and the plant. In this context, we have achieved significant advances in our understanding of halotropism, cell wall synthesis and integrity surveillance, as well as salt-related cytoskeletal rearrangements. Indeed, molecular mechanisms underpinning some of these processes have recently been elucidated. In this review, we aim to provide insights into how plants respond and adapt to salt stress, with a special focus on primary cell wall biology in the model plant Arabidopsis thaliana.

The preparation of biological samples, especially for live-cell microscopy, remains a major experimental challenge in the lab despite technological advances. In addition, high-resolution microscopy techniques require higher sample quality and uniformity, which is difficult to ensure during manual preparation while maintaining "ideal" growth conditions. In this protocol, we provide a way out by growing Arabidopsis thaliana seedlings directly in an imaging chamber, which eliminates invasive sample preparation directly before imaging. This method hinges on the precise placement of seeds into imaging chambers, which can be grown in conventional climate chambers. We detail three methods to grow hypocotyls, cotyledons, leaves, and roots for high-resolution and long-term imaging of the plant cytoskeleton. Furthermore, we show that the growth and development of seedlings inside the chambers can be externally manipulated by the addition of chemicals.

Rice (Oryza sativa) is one of our main food crops, feeding ∼3.5 billion people worldwide. An increasing number of studies note the importance of the cytoskeleton, including actin filaments and microtubules, in rice development and environmental responses. Yet, reliable in vivo cytoskeleton markers are lacking in rice, which limits our knowledge of cytoskeletal functions in living cells. Therefore, we generated bright fluorescent marker lines of the actin and microtubule cytoskeletons in rice, suitable for live-cell imaging in a wide variety of rice tissues. Using these lines, we show that actin bundles and microtubules engage and co-function during pollen grain development, how the cytoskeletal components are coordinated during root cell development, and that the actin cytoskeleton is robust and facilitates microtubule responses during salt stress. Hence, we conclude that our cytoskeletal marker lines, highlighted by our findings of cytoskeletal associations and dynamics, will substantially further future investigations in rice biology.

Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields.

Cytoskeletal motor proteins are essential to the function of a wide range of intra-cellular mechano-systems. The biophysical characterization of the movement of motor proteins along their filamentous tracks is therefore of large importance. Towards this end, in vitro stepping motility assays are commonly used to determine the motor’s velocities and runlengths. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental setups, the experimental conditions and the data analysis methods. This work describes a novel unified method to evaluate traces of fluorescently-labeled, processive dimeric motor proteins and proposes an algorithm to correct the measurements for finite filament length as well as photobleaching. Statistical errors of the proposed evaluation method are estimated by a bootstrap method. Numerical simulation and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules was used to verify the proposed approach and it was shown (i) that the velocity distribution should be fitted by a t location-scale probability density function rather than a normal distribution, (ii) that the temperature during the experiments should be controlled with a precision well below 1 K, (iii) that the impossibility to measure events shorter than the image acquisition time needs to be accounted for, (iv) that the motor’s runlength can be estimated independent of the filament length distribution, and (v) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. This allows for a better statistical comparison of motor proteins influenced by other external factors e.g. ionic strength, ATP concentration, or post-translational modifications of the filaments. In this context, the described method was then applied to experimental data to investigate the influence of the nucleotide state of the microtubule on the motility behavior of the kinesin-1 motor