The Developing Xylem Vessel
by Raymond Wightman and Simon Turner
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester. M13 9PT. U.K.
The xylem vessels consist of long and narrow cylindrical cells that are interconnected to provide a conduit for the transport of water throughout the plant. At maturity, vessels undergo programmed cell death to yield hollow tubes, commonly known as tracheary elements (TEs). The transport of water through these elements exerts a large negative pressure and, for this reason, developing TEs lay down a reinforced secondary wall that contains a high proportion of cellulose. We study developing xylem vessels in order to determine the molecular mechanisms that are important for secondary wall formation which in turn may facilitate the use of these walls as a feedstock for biofuel generation.
The major problem in studying the developing xylem is its location deep within the plant, beneath several layers of tissue. Any signal from fluorescent markers need to penetrate these layers which also consist of trapped air pockets in the apoplast, resulting in loss of signal and loss of resolution. For these reasons, several research groups have made use of a differentiating cell culture which has the advantages of large cells and the maintenance of high fluorescence signal and resolution. Our focus has been upon developing xylem within the plant, specifically the thin roots of Arabidopsis seedlings [1]. Many of the problems associated with deep-tissue imaging have been overcome by the use of longer working distance water objectives and oil objectives possessing corrective collars. The focus of research efforts within the imaging field are described below:
The Cellulose Synthase Complex
In the protoxylem of the root, the secondary walls are laid down as complete hoops or spirals. The patterned distribution of secondary walls is characteristic of xylem throughout the plant. Within these walls, cellulose is deposited by a large cellulose-synthesizing machine called the cellulose synthase complex which is thought to move through the plasma membrane during the polymerization process. The composition of the secondary wall complex is different to that of the primary wall complex in that it possesses a unique set of three subunits encoded by the genes IRX1, IRX3 and IRX5 [2, 3].
The very first report of live imaging of the complex was in 2003 by Gardiner et al. [4] who looked at a fusion between the uv-excited wild-type GFP with the Irx3 subunit. Resolution was a problem, however, the researchers observed movement of intracellular compartments as well as some faint labeling of bands corresponding to the sites of secondary wall formation. Improvements in imaging hardware and optics, coupled with the use of the much brighter YFP (YFP-Irx3), allowed localization of the complex to be determined in fine detail [1]. YFP-Irx3 is seen at the cell surface as bands beneath the secondary walls and within rapidly moving intracellular compartments which include the Golgi as demonstrated by the co-localization with the xylem specific mannosidase I-CFP marker. It is yet to be determined whether the bands of YFP-Irx3 represent cellulose synthase complexes actively making cellulose.
The major problem in studying the developing xylem is its location deep within the plant, beneath several layers of tissue. Any signal from fluorescent markers need to penetrate these layers which also consist of trapped air pockets in the apoplast, resulting in loss of signal and loss of resolution. For these reasons, several research groups have made use of a differentiating cell culture which has the advantages of large cells and the maintenance of high fluorescence signal and resolution. Our focus has been upon developing xylem within the plant, specifically the thin roots of Arabidopsis seedlings [1]. Many of the problems associated with deep-tissue imaging have been overcome by the use of longer working distance water objectives and oil objectives possessing corrective collars. The focus of research efforts within the imaging field are described below:
The Cellulose Synthase Complex
In the protoxylem of the root, the secondary walls are laid down as complete hoops or spirals. The patterned distribution of secondary walls is characteristic of xylem throughout the plant. Within these walls, cellulose is deposited by a large cellulose-synthesizing machine called the cellulose synthase complex which is thought to move through the plasma membrane during the polymerization process. The composition of the secondary wall complex is different to that of the primary wall complex in that it possesses a unique set of three subunits encoded by the genes IRX1, IRX3 and IRX5 [2, 3].
The very first report of live imaging of the complex was in 2003 by Gardiner et al. [4] who looked at a fusion between the uv-excited wild-type GFP with the Irx3 subunit. Resolution was a problem, however, the researchers observed movement of intracellular compartments as well as some faint labeling of bands corresponding to the sites of secondary wall formation. Improvements in imaging hardware and optics, coupled with the use of the much brighter YFP (YFP-Irx3), allowed localization of the complex to be determined in fine detail [1]. YFP-Irx3 is seen at the cell surface as bands beneath the secondary walls and within rapidly moving intracellular compartments which include the Golgi as demonstrated by the co-localization with the xylem specific mannosidase I-CFP marker. It is yet to be determined whether the bands of YFP-Irx3 represent cellulose synthase complexes actively making cellulose.
The cytoskeleton in developing xylem vessels
Microtubules:
It has long been known that microtubules play an important part in
secondary wall formation, particularly in relation to guiding the
cellulose synthase complex. Microtubule organization was first examined
in cell culture that had been programmed to undergo xylem
differentiation [5]. The cells expressed a GFP-tubulin fusion and the
microtubules were seen to organize into thick bundles at future sites
of secondary wall formation. An interesting observation from this study
is that a thick microtubule bundle appears to split into two bundles as
the secondary wall thickens.
Many of the various cellular markers are under control of a constitutive promoter such as 35S and is satisfactory for cell culture experiments but not when looking at developing xylem in the living plant. The presence of brightly labeled microtubules in cell layers above the xylem would make imaging difficult and constructs under control of promoters such as 35S are reported to express poorly within the xylem. For this reason we made use of a YFP-based microtubule reporter (A YFP fusion to the microtubule binding domain of the mouse Map4 protein) placed under the control of the IRX3 promoter to give xylem-specific labeling of microtubules in plant roots [1]. The microtubules are seen to form bundles beneath the secondary walls and, as observed in cell culture, these bundles appear to split into two as the secondary walls thicken. Bundles of hoops and spirals are seen to be interconnected by narrower microtubule bridges. Furthermore, bundles of CFP-labeled (CFP-MBD) microtubules are seen to colocalise with the bands of YFP-Irx3. Loss of the microtubule bundles, through treatment with the herbicide oryzalin, results in the loss of these YFP-Irx3 bands [1]. An interactive 3D movie showing the YFP microtubule reporter in developing xylem is shown here.
Actin: Filamentous actin has been visualized using a xylem-specific reporter based around the new bright monomeric mCherry FP fused to the actin binding domain-2 of Fimbrin 1 gene. Thick actin cables are seen to run along the length of the cell and serve as a highway for long-distance transport of intracellular organelles [1]. A movie showing actin (red) and Irx3-containing organelles (green) is shown here. Narrower actin fibers appear to exit the cables and are though to participate in delivery of the cellulose synthase complex and possibly cell wall components to sites of secondary wall formation.
Hardware requirements for imaging the developing xylem
We have found the single most important factor for observing fluorescent proteins in the developing xylem is the choice of objective lens. We use either (i) a x63 water objective with a working distance of up to 0.22 microns (Leica part no. 11506212) or (ii) a 100x oil objective with corrective collar (Leica part no. 506220). Seedlings are grown upright in the light on 0.5x MS plus 1.5% agar for 5 days. Illuminated xylem can be clearly observed approx. 0.5 mm from the root tip with secondary wall synthesis occurring at between 1 and 2 mm.
Many of the various cellular markers are under control of a constitutive promoter such as 35S and is satisfactory for cell culture experiments but not when looking at developing xylem in the living plant. The presence of brightly labeled microtubules in cell layers above the xylem would make imaging difficult and constructs under control of promoters such as 35S are reported to express poorly within the xylem. For this reason we made use of a YFP-based microtubule reporter (A YFP fusion to the microtubule binding domain of the mouse Map4 protein) placed under the control of the IRX3 promoter to give xylem-specific labeling of microtubules in plant roots [1]. The microtubules are seen to form bundles beneath the secondary walls and, as observed in cell culture, these bundles appear to split into two as the secondary walls thicken. Bundles of hoops and spirals are seen to be interconnected by narrower microtubule bridges. Furthermore, bundles of CFP-labeled (CFP-MBD) microtubules are seen to colocalise with the bands of YFP-Irx3. Loss of the microtubule bundles, through treatment with the herbicide oryzalin, results in the loss of these YFP-Irx3 bands [1]. An interactive 3D movie showing the YFP microtubule reporter in developing xylem is shown here.
Actin: Filamentous actin has been visualized using a xylem-specific reporter based around the new bright monomeric mCherry FP fused to the actin binding domain-2 of Fimbrin 1 gene. Thick actin cables are seen to run along the length of the cell and serve as a highway for long-distance transport of intracellular organelles [1]. A movie showing actin (red) and Irx3-containing organelles (green) is shown here. Narrower actin fibers appear to exit the cables and are though to participate in delivery of the cellulose synthase complex and possibly cell wall components to sites of secondary wall formation.
Hardware requirements for imaging the developing xylem
We have found the single most important factor for observing fluorescent proteins in the developing xylem is the choice of objective lens. We use either (i) a x63 water objective with a working distance of up to 0.22 microns (Leica part no. 11506212) or (ii) a 100x oil objective with corrective collar (Leica part no. 506220). Seedlings are grown upright in the light on 0.5x MS plus 1.5% agar for 5 days. Illuminated xylem can be clearly observed approx. 0.5 mm from the root tip with secondary wall synthesis occurring at between 1 and 2 mm.
FP probes targeted to the developing xylem vessel
References
1. Wightman, R., and Turner, S.R. (2008). The roles of the cytoskeleton during cellulose deposition at the secondary cell wall. Plant J 54, 794-805.
2. Taylor, N.G., Howells, R.M., Huttly, A.K., Vickers, K., and Turner, S.R. (2003). Interactions among three distinct CesA proteins essential for cellulose synthesis. Proc Natl Acad Sci U S A 100, 1450-1455.
3. Taylor, N.G., Laurie, S., and Turner, S.R. (2000). Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell 12, 2529-2540.
4. Gardiner, J.C., Taylor, N.G., and Turner, S.R. (2003). Control of cellulose synthase complex localization in developing xylem. Plant Cell 15, 1740-1748.
5. Oda, Y., Mimura, T., and Hasezawa, S. (2005). Regulation of secondary cell wall development by cortical microtubules during tracheary element differentiation in Arabidopsis cell suspensions. Plant Physiol 137, 1027-1036.
6. Wightman, R., and Turner, S.R. (2007). Severing at sites of microtubule crossover contributes to microtubule alignment in cortical arrays. Plant J 52, 742-751.
7. Nebenfuhr, A., Gallagher, L.A., Dunahay, T.G., Frohlick, J.A., Mazurkiewicz, A.M., Meehl, J.B., and Staehelin, L.A. (1999). Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121, 1127-1142.