In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria.

Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using C-13 solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Plants are one of the most fascinating and important groups of organisms living on Earth. They serve as the conduit of energy into the biosphere, provide food, and shape our environment. If we want to make headway in understanding how these essential organisms function and build the foundation for a more sustainable future, then we need to apply the most advanced technologies available to the study of plant life. In 2009, a committee of the National Academy highlighted the "understanding of plant growth" as one of the big challenges for society and part of a new era which they termed "new biology." The aim of this article is to identify how new technologies can and will transform plant science to address the challenges of new biology. We assess where we stand today regarding current technologies, with an emphasis on molecular and imaging technologies, and we try to address questions about where we may go in the future and whether we can get an idea of what is at and beyond the horizon.

Bioluminescent and fluorescent proteins are now used as tools for research in all organisms. There has been massive progress over the past 15 years in creating a palette of fluorescent proteins with a wide spectrum of specific properties. One of the big challenges is to decide which variant may be best for a certain application. A recent article by Mann et al. in BMC Biotechnology describes a new orange fluorescent protein in plants.

Organization of microtubules into ordered arrays involves spatial and temporal regulation of microtubule nucleation. Here, we show that acentrosomal microtubule nucleation in plant cells involves a previously unknown regulatory step that determines the geometry of microtubule nucleation. Dynamic imaging of interphase cortical microtubules revealed that the ratio of branching to in-bundle microtubule nucleation on cortical microtubules is regulated by the Arabidopsis thaliana B '' subunit of protein phosphatase 2A, which is encoded by the TONNEAU2/FASS (TON2) gene. The probability of nucleation from g-tubulin complexes localized at the cell cortex was not affected by a loss of TON2 function, suggesting a specific role of TON2 in regulating the nucleation geometry. Both loss of TON2 function and ectopic targeting of TON2 to the plasma membrane resulted in defects in cell shape, suggesting the importance of TON2-mediated regulation of the microtubule cytoskeleton in cell morphogenesis. Loss of TON2 function also resulted in an inability for cortical arrays to reorient in response to light stimulus, suggesting an essential role for TON2 and microtubule branching nucleation in reorganization of microtubule arrays. Our data establish TON2 as a regulator of interphase microtubule nucleation and provide experimental evidence for a novel regulatory step in the process of microtubule-dependent nucleation.

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged a-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.