Essay 21.2

Cytokinin-Induced Form and Structure in Moss

Karen S. Schumaker, University of Arizona at Tucson, Dept. Plant Sciences

May, 2006

A fundamental feature of development in multicellular organisms is the constant change of the basic body form. Morphogenesis summarizes the visible expression of a number of highly complex processes that lead to these changes. In my laboratory, we study moss development to identify the processes that cause a single cell to change its pattern of growth and ultimately give rise to a multicellular organism. My goal for this web essay is to share with our studies about hormone-induced morphogenesis and the underlying cellular and molecular mechanisms. We begin by revisiting mosses and their basic developmental program.

Mosses are bryophytes in the Kingdom Plantae and the division (phylum) Bryophyta. Bryophytes are ancestral to vascular plants and are most representative of the earliest land plants. They rely on water in certain stages of their life cycle (e.g., for sperm movement during fertilization) and hold clues to understanding the innovations necessary for the transition from life in water to dry land and those that were not necessary for survival on land. These small non-seed producing plants display the beginnings of differentiation of stems and leaves, of a diploid sporophyte generation, and of an apical meristem.

Figure 1 Stages of moss development. Haploid spores (A) germinate to form a filament consisting of chloronema cells (B,C). Subsequently, light and auxin induce changes in the tip cell to give rise to caulonema cells (D). A single-celled initial (D, arrowhead) forms on the second subapical cell of the caulonema filament. In the absence of cytokinin, the initial cell will continue to grow by tip growth to form a new lateral filament (E). In the presence of cytokinin, the initial cell takes on the morphology associated with the assembly of a bud to form the leafy shoot (F,G) that eventually bears the gametangia (not shown). Following fertilization, a diploid capsule (G) forms on the leafy shoot. Ultimately, meiosis occurs within the capsule to produce haploid spores. (Reprinted with permission from the Annual Review of Plant Physiology and Plant Molecular Biology, Volume 49, ©1998, by Annual Reviews www.AnnualReviews.org.) (Click image to enlarge.)

Physcomitrella patens and Funaria hygrometrica are two species of moss used to form the basis of studies in my laboratory. The developmental changes that take place during the life cycle of the moss are defined below (Schumaker and Dietrich 1997, 1998). The germination of the haploid spore is illustrated in Figure 1. The developmental outcome of spore germination is the formation of a filamentous gametophyte that grows via tip growth as a thread-like protonema composed, at first, of a single cell type, the chloronema (Figure 2A). In response to perception of the plant hormone auxin, divisions of the most apical cell (the tip cell) give rise to a second distinct cell type, the caulonema (Figure 2B). Subsequent caulonema tip cell divisions correlate with important visible cellular and morphological changes in the second subapical cell of the filament (the third cell from the tip). Immediately after tip cell nuclear division, swelling at the apical end of the new second subapical cell begins (Figure 2B, arrow), and the nucleus and other organelles migrate to this outgrowth. Subsequent nuclear division and cytokinesis result in the formation of a fully formed initial cell (Figure 3A).

Figure 2 Light micrographs illustrating the filamentous stages of moss development. Chloronema filament (A). Caulonema filament (B), the arrow points to an outgrowth at the initial cell site. Bar in (A) = 25 µm for (A) and (B). (Reprinted with permission from the Plant Cell, Volume 9, ©1997, by the American Society of Plant Biologists.) (Click image to enlarge.)

The next stages of development are characterized by hormone-induced morphogenesis and organogenesis when a bud is assembled from the caulonema initial cell and the leafy gametophyte (shoot) develops from the bud. The caulonema initial cell has two potential fates that are developmentally distinct. In the absence of high concentrations of plant hormone cytokinin, the initial cell will continue to grow by tip growth to produce a new lateral filament (side branch), maintaining the filamentous growth habit. However, in the presence of high concentrations of this hormone, the initial cell takes on a distinct morphology associated with the assembly of a bud.

Figure 3 Light micrographs illustrating the early stages of moss bud assembly. Fully formed caulonema initial cell (A). Caulonema initial cell ~10 hr after the addition of cytokinin (B). Two-celled bud stage (C). Simple meristem (D), the arrow points to a leaf primordium. Bar in (A) = 25 µm for (A–D). (Reprinted with permission from the Plant Cell, Volume 9, ©1997, by the American Society of Plant Biologists.) (Click image to enlarge.)

Early changes during bud assembly include an altered pattern of cell expansion and elongation of the initial cell to produce the single-celled bud (Figure 3B). Later changes involve divisions within the bud (Figure 3C) to give rise to a simple meristem (Figure 3D) that produces a leafy shoot (Figure 4) that eventually bears the gametangia. Following the production of gametes and fertilization, the zygote develops into a sporophyte that remains attached to the female gametophyte and bears a single capsule containing thousands of haploid spores.

Figure 4 Light micrograph illustrating the leafy gametophyte. Mature gametophyte magnified 40 X. (Reprinted with permission from the Plant Cell, Volume 9, ©1997, by the American Society of Plant Biologists.) (Click image to enlarge.)

When the initial cell perceives cytokinin, a major developmental switch takes place; within days of germination, and the involvement of only a few cells and cell types, the pattern of growth changed dramatically from filamentous to meristematic as a leafy structure is produced on the filament. Our studies focused on understanding the very early cellular changes that take place after the initial cell perceives cytokinin. The earliest visible difference is a dramatic change in the pattern of expansion of the initial cell. While it was forming, the initial cell elongated via tip growth, an extremely polarized mode of cell expansion. As with other tip growing cells, wall extension and incorporation of new wall material is focused at a single site on the cell surface. However, once the fully formed initial cell perceives cytokinin, growth becomes diffuse——distributed across the cell surface. Whether growth is polar or diffuse, the shape of a plant cell is defined by the wall surrounding it, and is, therefore, determined by the pattern in which the wall extends as the cell grows. This pattern of wall extension, in turn, depends on the pattern of cellulose deposition, the orientation of the microtubule and microfilament cytoskeleton, and on a wide variety of wall components introduced via secretion. Vesicles containing these secreted wall components are transported to the growth site where their fusion with the plasma membrane is facilitated by a locally elevated concentration of cytoplasmic calcium.

In whole plant experiments using modulators of animal cell voltage-dependent calcium channels (1,4-dihydropyridines, DHPs), we question if calcium is important for the early changes in growth that take place during bud assembly. Fully formed initial cells were treated with cytokinin in the absence (our control) or presence of nifedipine, a DHP that binds to and blocks calcium movement through these channels in animal cells (a calcium channel antagonist). We reasoned that, if calcium movement into initial cells is important for bud assembly, blocking calcium movement should prohibit bud assembly. In fact, this is what we found——increasing concentrations of nifedipine blocked the earliest visible changes in the initial cell after addition of cytokinin.

We wondered if, as the whole plant experiments suggested, there are voltage-dependent calcium channels on the moss plasma membrane that are affected by these calcium channel modulators. By monitoring passive uptake of radiolabeled calcium into moss protoplasts, we found that nifedipine prevents calcium transport while increasing concentrations of Bay K8644, a DHP that binds to and stimulates calcium movement through voltage-dependent calcium channels in animal cells (a calcium channel agonist) enhanced calcium uptake (Schumaker and Gizinski 1993). Calcium influx was stimulated by external KCl, indicating that transport is voltage-dependent. Another very interesting finding was the addition of cytokinin specifically stimulated calcium influx into moss protoplasts (Schumaker and Gizinski 1993).

To understand the molecular nature of the transport system that is affected by the channel modulators, we established conditions for binding an azido-labeled form of nifedipine (Schumaker and Gizinski 1995, 1996) and found that it bound specifically to two polypeptides in the moss plasma membrane. We are currently using tandem mass spectrometry of the partially purified DHP-binding proteins to identify the putative calcium channel and provide sequence information (Figure 5). Our ultimate goal is to understand when and where the channel is found and how it is regulated during development.

Figure 5 Tandem mass spectrometry. Regions known to contain the DHP-binding proteins are cut from the SDS-PAGE gel and are digested with the protease trypsin. The resulting peptides are introduced into the first mass spectrometer to determine the mass of each peptide. Some of these peptides proceed to a second mass spectrometer to determine their amino acid sequence. These two data sets are compared with the data predicted from the Arabidopsis thaliana database. Provided there is enough sequence similarity between Arabidopsis and Physcomitrella patens, a number of peptides should match and identify a small subset of proteins. The DHP-binding protein will be identified among this subset based on the change in mass due to the binding of the DHP. (Click image to enlarge.)

To identify other components of the mechanism underlying hormone-induced morphogenesis, we are using random insertional mutagenesis. We have characterized two mutants, both of which remain primarily filamentous (Figure 6). One mutant is impaired in a cell differentiation process, while the other is unable to respond to exogenous cytokinin. We are interested in the latter and, once we identify the site in the moss genome into which the introduced DNA has inserted (i.e., the moss gene whose function has been disrupted), we can add to our model of cellular and molecular changes that are required for the development of the bud.

Figure 6 Random insertional mutants of Physcomitrella patens. WT, wild type; mock, a transformation control (i.e., mock-transformed); 2, a mutant producing initial cells, but unable to assemble buds in response to cytokinin; 5, a mutant impaired in the differentiation of a caulonema tip cell from a chloronema tip cell. (Click image to enlarge.)

While hormone-induced morphogenesis is critical for development in all organisms, little is known about mechanisms that underlie this process in vascular plants due to the complexity of the organisms or the inaccessibility of the developmentally active cells. Moss has a number of advantages for these studies including: (i) spatial and temporal localization of developmental processes is precisely predictable, (ii) specific processes can be followed developmentally along cells of a single filament, (iii) developmental processes can be experimentally manipulated, (iv) moss is a relatively simple organism in which it is possible to identify critical genes involved in plant cell development and carry out reverse genetic (using homologous recombination), forward genetic, and detailed in vivo studies to determine the function of molecules important during development, and (v) the multicellular gametophyte enables the study of more complex development than is possible with most other haploid organisms.

As with most research, our studies leave us with many unanswered questions that will form the basis of future research. What questions come to your mind? Some of our questions include: (i) where is the cytokinin produced, (ii) where and how is it perceived, (iii) is the requirement for calcium directly linked to vesicle fusion during cell wall growth, (iv) is calcium required for other cellular changes, (v) once bud assembly has begun, what mechanisms are responsible for setting up the first cell division of the bud (an asymmetric division) and the subsequent divisions that generate the apical cell. As these questions are answered, it will be interesting to begin to define elements of development that are conserved between moss and other organisms as well as any elements that uniquely define the moss.

References

Schumaker, K. S. and Dietrich, M. A. (1997) Plant Cell 9:1099–1107.

Schumaker, K. S. and Dietrich, M. A. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 501–523.

Schumaker, K. S. and Gizinski, M. J. (1993) Proc. Natl. Acad. Sci. USA 90: 10937–10941.

Schumaker, K. S. and Gizinski, M. J. (1995) J. Biol. Chem. 270: 23461–23467.

Schumaker, K. S. and Gizinski, M. J. (1996) J. Biol. Chem. 271: 21292–21296.