Plant cell biology in the new millennium: new tools and new insights

Abstract

The highly regulated structural components of the plant cell form the basis of its function. It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary metabolism to the "transducon" complexes of signal transduction elements that allow for the highly efficient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of the plant cell. First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell biologists to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell biologist now has available a vast array of fluorescent probes that will report cell parameters as diverse as the pH of the cytosol, the oxygen level in a tissue, or the dynamics of the cytoskeleton. The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio‐temporal imaging of F‐actin dynamics in roots

“…Although immunofluorescence microscopy of fixed plant material continues to be an integral tool for studies on the plant cytoskeleton, the fact that the cytoskeleton is a highly dynamic entity makes it essential to observe the organization of these structural proteins within the context of the living cell. The discovery of green fluorescent protein (GFP) from the jellyfish Aequoria victoria has opened a new dimension in studies of the plant cytoskeleton since it allowed the observation of cytoskeletal dynamics in living, functioning plant cells without the technical difficulties associated with microinjection [Blancaflor and Gilroy, 2000;Ehrhardt, 2004]. GFP was first used in studies of the plant cytoskeleton through its fusion with the microtubule-binding domain (MBD) of MAP4 (GFP-MBD).…”

Metabolons involving plant cytochrome P450s

“…Traditional biochemical assays, including isotope dilution, substrate feeding, and sub-cellular fractionation, while valuable, are limited in their ability to provide direct evidence of enzyme interactions. Co-immunoprecipitation, protein cross-linking and affinity purifications are best suited for stable interactions (Blancaflor and Gilroy 2000); and yeast two-hybrid assays are not applicable for membrane bound P450s. Therefore, newer technologies, such as multiphoton confocal co-localization (Blancaflor and Gilroy 2000), FRET and fluorescence lifetime imaging microscopy (FLIM) analysis (Wallrabe and Periasamy 2005), split ubiquitin assays (Thaminy et al 2004), protein fragment complementation assays (Subramaniam et al 2001), and surface plasmon resonance assays (Magee et al 2002) may provide direct and in vivo enzyme interaction analysis.…”

Epi-illumination microscopy coupled to in situ hybridization and its utility in the study of evolution and development in non-model species

“…In alternative techniques to SEM, for example, cryo-SEM, environmental SEM, and laser scanning confocal microscopy, the tissue preparation methods themselves are not inherently damaging or incompatible with downstream gene expression analyses. The microscopy itself, however, is often destructive to tissue, precluding downstream RNA in situ hybridization (Blancaflor and Gilroy 2000;Lemon and Posluszny 1998).…”