PLS 622, Plant Physiology I: Monday, September 25, 2006

Vegetative development: Root hair, and stomatal Development:

Objectives:

- To investigate the control of root hair development at the morphological and molecular level.

- To determine the molecular control of stomatal spacing and density.

- To construct an overview of the roles of the similar molecular players in the various developmental processes mentioned above.

Root hair development:

Root hairs have been hypothesized to expand the surface area of a root in order to enhance the water and nutrient uptake efficiency of the root. They are also the site of attachment of soil mycorhiza. Root hair development parallels trichome development in many aspects. Like trichomes root hairs arise from epidermal cells (trichoblasts) that are

over anticlinal cell walls of the underlying cortex.

Microscopically, trichoblasts can be discerned from atrichoblasts early in their development, prior to the elongation of the root hair, by having simpler plastids, larger nuclei and nucleoli, greater amounts of protein, and RNA. This gives the trichoblasts a more cytoplasmically dense appearance under normal light microscopy indicative of a delayed maturation as the cell prepares to put forth the cell extension, the root hair. The growth of the root hair has much in common with the growth of a pollen tube in that it occurs through the phenomenon of tip growth as distinguished from the diffuse growth of the vast majority of cells. In tip growth, the cell wall components required for wall synthesis to permit continued elongation are deposited at the very tip of the trichoblast by fusion of dictyosome vesicules with the plasmamembrane at the apex of the trichoblast. The newly deposited wall material is intercalated into the existing wall and is displaced towards the periphery of the cell and down along the sides of the cell extension as the trichoblast increases in length. The wall components subsequently undergo modification such as desterification of pectin as new waves of wall material deposition displace the former tip back along the trichoblast.

Root hairs elongate at a defined period of root maturation. In arabidopsis, the so called "root hair zone" of the elongating root is located about a millimeter behind the root tip. Cells in this region first commence putting forth root hairs. Root hairs can eventually attain lengths of 1.5 mm (small in relation to a cotton ovule trichome but still impressive for a single cell) elongating at a peak rate of up to 100 mm an hour!

Depending on the species, the cells that will produce root hairs (trichoblasts) are determined by a variety of means. These are summarized below and in figure 3. Please note that, at the time the fate of root hairs is being decided, the lateral root cap cells have not yet died and sloughed off the root epidermis. Hence, the epidermis in the figures is still encased in lateral root cap cells. This will not be the case later during root development when the root cap cells have died. This will be an important distinction in a later lecture when we discuss the differentiation of lateral root cap cells and epidermal cells that, in Arabidopsis, come from the same initial.

-1) plants in which the pattern of epidermal cell differentiation (trichoblast or atrichoblast) appears random.

-2) plants in which epidermal cell fate appears to be linked to asymmetric cell division with the larger daughter cell remaining an atrichoblast and the smaller developing into a trichoblast. This group includes many monocots.

-3) plants, including arabidopsis, that dictate epidermal cells positioned in the crevice between underlying cortex cells (situated over an anticlinal division of underlying cortical cells) to pursue the trichoblast pathway, while atrichoblasts form from epidermal cells positioned on top of cortical cells (situated outside a periclonal division of underlying cortical cells).

Figure 3: Determination of root hair development.

In fact, the same molecular players that determine trichome spacing and development also function to determine root hair fate, although the logic is reversed. In determining which epidermal cells of the root will be come root hairs, a similar transcriptional complex to the one we saw in trichome development decides whether the GL2 gene is up- or down-regulated. However, now, if GL2 is up-regulated the cell retains the atrichoblast fate, remaining an undifferentiated root epidermal cell. If GL2 is silenced, the cell becomes trichoblastic and a root hair develops (Fig. 4). In this instance, the identity of the bHLH protein(s) remains the same as in the trichome complexes we have studied above. Both GL3 and/or EGL3 participate in a homo- or hetero-dimer and bind to TTG1. The MYB transcription factor named WEREWOLF (WER) associates with the bHLH proteins and TTG1 in the roots to define epidermal (atrichoblast) fate. CPC (rather than TRY as we saw in the trichomes) associates with the trichoblast stimulatory complex in the roots (Fig. 4). It is rather confusing that GL1, which is part of the trichoblast stimulatory complex in trichome formation, is replaced in the root epidermal cells by a functionally identical MYB transcription factor, WER that now defines the trichoblast inhibitory complex. The story is even more complex because, the coding regions of GL1 and WER can be swapped and GL1 can be placed under the control of the WER promoter so it is only expressed in the roots. Conversely, WER can be placed under the control of the GL1 promoter so it is only expressed in the shoot. Despite the fact that the WER protein in the root directs the atrichoblast fate, in the shoot it can direct the trichoblast fate! The converse is true of GL1 in the root! So, the only determinant directing whether WER is stimulatory (shoot) or inhibitory (root) for trichoblast fate is where the gene is transcribed (positional determination). The same is true for GL1.

Of the three models in figure 3, the third, describing Arabidopsis root hair development, is depicted in figure 4. Some as yet unidentified, GL2 transcription stimulatory substance is thought to pass from the underlying cortical cells into the overlying epidermal cells (red arrows). At the edges of the cortical cells the apoplast between adjoining cells results in a region incapable of generating this message and transporting it into the overlying epidermis. Hence, epidermal cells positioned over a cell-cell junction in the cortical cells, receives less of this stimulatory substance, setting that cell up to become trichoblastic (Fig. 4).

Stomatal development:

Stomata are essential to the life of terrestrial plants. There is usually a stomata free zone surrounding each stoma in normal plants. This intervention of subsidiary (when they exist) or epidermal cells between stomata is thought to provide a pool of ions for the guard cells

and optimize the access of the underlying mesophyll cells to the external environment. Two key features combine to influence stomatal patterning; internal anatomy (cell type, position of the vasculature), and contact between stomata. Little is known respecting why stomata only rarely develop over the vasculature. Even less is known about how certain underlying cell types seem to inhibit stomata from developing over them. Hence, we will discuss the control of stomatal patterning such that stoma rarely develop side-by-side.

Stomatal initials form by asymmetric cell division of precursor cells such that the smaller cell becomes a stomatal precursor. The stomatal initial can produce both the guard cells and ordinary epidermal cells. Considerable cell-to-cell communication takes place leading to the coordination of the plane of cell division as well as the regulation of precursor cell activity. Additionally, the timing of when a cell is competent to become a stomatal precursor and where such precursors can initiate are highly controlled.

In both dicots and monocots, the cell divisions leading to the production of stomata are highly asymmetric both in geometry and in the fate of the cells thus produced. In monocots, the first asymmetric division results in the smaller, usually rectangular cell becoming a guard mother cell which divides symmetrically producing two guard cells. In

dicots, the smaller cell is usually triangular and continues to divide after the surrounding cells have ceased to do so. Eventually, it converts into a guard mother cell and divides symmetrically to produce two guard cells (Fig. 4). Since dicot stomatal initials continue to divide after the surrounding epidermal cells have ceased they have been termed meristemoid cells to emphasize their ability to continue cell division. In dicots there are two types, primary meristemoids and secondary (satellite) meristemoids, the latter produced by an asymmetric division of a neighboring cell to the stomata.

Rectangular initials (a.k.a. short cells) in monocots comprise the three types of stomatal initial found in the angiosperms. How stomatal pattern is initiated depends on the type of initial.

Primary meristemoid positioning appears to be random, with the exception that it obeys the rule that two stomata should not be adjacent. Secondary meristemoids and stomatal initials in monocots are placed in a highly regular fashion crucial to stomatal patterning. The asymmetric division producing the stomatal initial in monocots occurs so that the initial is placed distal from the base of the leaf. Upon the formation of the initial, asymmetric divisions occur in cells adjacent to the new initial to form two subsidiary cells. Finally, the guard mother cell divides symmetrically to form two guard cells surrounding a pore (Fig. 5).

Figure 4: Dicot stomatal development.

Figure 5: Monocot stomatal development.

Primary meristemoids arise from protodermal cells. These cell undergo a series of asymmetric divisions that produces a small, triangular initial situated approximately in the middle of the future stomatal complex. The meristemoid next alters shape to an oval guard mother cell. Finally, as in monocots, the mother cell divides symmetrically and produces two guard cells.

Satellite meristemoids arise once a primary meristemoid has completed development into a stoma. One of the surrounding cells maintains meristemoid identity and undergoes asymmetric division such that the guard mother cell is formed distal from the existing stoma. Upon several asymmetric divisions, the meristemoid, now separated from the existing guard cells by epidermal cells, also converts to an oval guard mother cell and divides symmetrically.

My question to you is: what would be the outcome of conducting an experiment like the one with the transposon interrupted GUS gene used to elucidate trichome developmental patterns (Fig. 1, above) if applied to stomatal development?

Timing of stomatal development:

In arabidopsis at least, stomata develop after trichomes have formed.

Cell-to-cell communication during stomatal development:

There is a wide array of opportunities for signals to be traversing between cells thus coordinating stomatal development. Evidence for cell-to-cell communication include the inhibition of stomata to form above veins in the leaf, the formation of satellite meristemoids

situated away from existing stomata, the arrested development of meristemoids located proximally to each other or to stomata, the orientation of subsequent cell divisions to separate meristemoids when two do happened to develop beside each other and, the initiation of subsidiary cell formation in monocots.

A genetic analysis of stomatal patterning:

There are two mutants identified that violate the rule that no two stomates should be in contact. However, the four lips (flp) and too many mouths (tmm) mutations transgress by two different developmental mechanisms. tmm mutants have far greater numbers of stoma in rosette leaves, cotyledons, and abaxial sepal surface. Other tissues of tmm plants (inflorescence stem, adaxial sepal epidermis, silique tips) entirely lack stomata whereas wild type plants possess them. The flower pedicle shows an abnormal gradation of stomatal frequency from none proximal to the stem to abnormally greater numbers near the flower base. Hence, TMM is thought to control the entry of protodermal cells into the stomatal developmental process and appears to have opposite effects on meristemoids depending on the tissue (compare rosette leaves with sepal epidermis).The tmm mutation appears to increase the formation of satellite meristemoids as well as to alter the polarity of cell divisions such that meristemoids can now form next to guard cells.

The flp mutation can produce unpaired guard cells as well as result in stomatal twinning. Unlike tmm mutants, flp mutants do not exhibit increased numbers of guard cells. Analysis of stomatal development in flp mutants suggests that stomata develop normally until the guard mother cell stage. At this stage the guard mother cell undergoes a symmetric division and the resulting daughter cells divide symmetrically again to produce two, contiguous stomates. Hence FLP may control guard mother cell identity, or regulate the number of symmetric divisions a guard mother cell can undergo.

The R-558 mutation causes an increase in stomatal density throughout the plant and stomatal clustering can also occur.

References used in the preparation of these notes.

Costa S, Dolan L. 2003. Epidermal patterning genes are active during embryogenesis in Arabidopsis. Development 130, 2893-2901.

Koshino-Kimura Y, Wada T, Tachibana T, Tsugeki R, Ishiguro S and Okada K. 2005.

Regulation of CAPRICE Transcription by MYB Proteins for Root Epidermis

Differentiation in Arabidopsis. Plant Cell Physiol. 46: 817–826.

Nadeau JA, Sack FD. 2002. Control of stomatal distribution on the Arabidopsis leaf surface. Science. 296: 1697-1700.