time-lapse-fluorescence-imaging-arabidopsis-root-growth-with-rapid

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

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, ...

Carnegie Institution for Science

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.

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis.

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 (13)C 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.

Article	Year
Science (New York, N.Y.)\| PubMed ID: 12714675	2003
Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 11880633	2002
Plant physiology\| PubMed ID: 11950987	2002
Current opinion in plant biology\| PubMed ID: 15134757	2004
The Plant cell\| PubMed ID: 15155883	2004
Development (Cambridge, England)\| PubMed ID: 15280208	2004
Journal of fluorescence\| PubMed ID: 15617267	2004
Current opinion in plant biology\| PubMed ID: 16188489	2005
Molecular and cellular biology\| PubMed ID: 16314530	2005
Plant physiology\| PubMed ID: 16617091	2006
Science (New York, N.Y.)\| PubMed ID: 16627697	2006
Annual review of plant biology\| PubMed ID: 16669785	2006
Current opinion in plant biology\| PubMed ID: 17010658	2006
Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 17389408	2007
Plant physiology\| PubMed ID: 17911650	2007
Current biology : CB\| PubMed ID: 17964159	2007
The Plant journal : for cell and molecular biology\| PubMed ID: 17971043	2008
Journal of cell science\| PubMed ID: 18042620	2007
Current opinion in cell biology\| PubMed ID: 18243678	2008
The Plant journal : for cell and molecular biology\| PubMed ID: 18269572	2008
Plant physiology\| PubMed ID: 18583534	2008
Nature cell biology\| PubMed ID: 19525940	2009
Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20855602	2010
Plant physiology\| PubMed ID: 20921164	2010
Nature cell biology\| PubMed ID: 20935636	2010
Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis. The Plant cell\| PubMed ID: 21693695	2011
The Plant cell\| PubMed ID: 21742993	2011
The Plant cell\| PubMed ID: 22186371	2011
The Plant cell\| PubMed ID: 22366161	2012
Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22375033	2012
The Plant cell\| PubMed ID: 22395485	2012
The Plant journal : for cell and molecular biology\| PubMed ID: 22404201	2012
Molecular plant\| PubMed ID: 22492121	2012
BMC biology\| PubMed ID: 22554191	2012

David W. Ehrhardt

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