Stomatal development: new signals and fate determinants.

Stomata and pavement cells are produced by a series of asymmetric divisions and progressive fate transitions within a stem cell lineage. In Arabidopsis, this process is regulated so that new lineages can be inserted between previously differentiated cells while maintaining stomatal spacing. The small peptide EPIDERMAL PATTERNING FACTOR 1 may be a positional signal secreted by stomatal precursors to modulate behavior of nearby cells. Signal-receiving cells may use TOO MANY MOUTHS and ERECTA family receptors and a MAPK pathway to regulate initiation of new lineages, promote asymmetric division, and control the plane of spacing divisions. Cell fate transitions are controlled by basic helix-loop-helix transcription factor (bHLH), MYB, and MADS-box transcription factors, and there is evidence of miRNA regulation. These results provide insight into positive and negative influences on stomatal cell transitions and suggest points of potential environmental regulation.

TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems.

Mutations in TOO MANY MOUTHS (TMM), which encodes a receptor-like protein, cause stomatal patterning defects in Arabidopsis leaves but eliminate stomatal formation in stems. Stomatal development in wild-type and tmm stems was analyzed to define TMM function. Epidermal cells in young tmm stems underwent many asymmetric divisions characteristic of entry into the stomatal pathway. The resulting precursor cells, meristemoids, appropriately expressed cell fate markers such as pTMM:GFP. However, instead of progressing developmentally by forming a guard mother cell, the meristemoids arrested, dedifferentiated, and enlarged. Thus asymmetric divisions are necessary but not sufficient for stomatal formation in stems, and TMM promotes the fate and developmental progression of early precursor cells. Comparable developmental and mature stomatal phenotypes were also found in tmm hypocotyls and in the proximal flower stalk. TMM is also a positive regulator of meristemoid division in leaves suggesting that TMM generally promotes meristemoid activity. Our results are consistent with a model in which TMM interacts with other proteins to modulate precursor cell fate and progression in an organ and domain-specific manner. Finally, the consistent presence of a small number of dedifferentiated meristemoids in mature wild-type stems suggests that precursor cell arrest is a normal feature of Arabidopsis stem development.

Microtubule arrays and Arabidopsis stomatal development.

Microtubule arrays in living cells were analysed during Arabidopsis stomatal development in order to more closely define stages in the pathway and contexts where intercellular signalling might operate. Arabidopsis stomata are patterned iteratively via the orientation of an asymmetric division in a cell located next to an existing stoma. It was found that preprophase bands of microtubules (PPBs) were correctly placed away from stomata and from two types of precursor cells. This suggests that all three cell types participate in an intercellular signalling pathway that orients the division site. These and other asymmetric divisions in the pathway were preceded by a polarized cytoplasm, with the PPB around the nucleus at one end, and the vacuole at the other. PPBs before symmetric divisions of guard mother cells (GMCs) were broader than those in asymmetric divisions, and the GMC division site was marked by unusual end-wall thickenings. This work identifies an accessible system for studying cytoskeletal function and provides a foundation for analysing the role of genes involved in stomatal development.

The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage.

The two guard cells of a stoma are produced by a single symmetric division just before terminal differentiation. Recessive mutations in the FOUR LIPS (FLP) gene abnormally induce at least four guard cells in contact with one another. These pattern defects result from a persistence of precursor cell identity that leads to extra symmetric divisions at the end of the cell lineage. FLP is likely to be required for the correct timing of the transition from cell cycling to terminal differentiation. FLP encodes a two-repeat (R2R3) MYB protein whose expression accumulates just before the symmetric division. A paralogous gene, MYB88, overlaps with FLP function in generating normal stomatal patterning. Plants homozygous for mutations in both genes exhibit more severe defects than flp alone, and transformation of flp plants with a genomic MYB88 construct restores a wild-type phenotype. Both genes compose a distinct and relatively basal clade of atypical R2R3 MYB proteins that possess an unusual pattern of amino acid substitutions in their putative DNA binding domains. Our results suggest that two related transcription factors jointly restrict divisions late in the Arabidopsis thaliana stomatal cell lineage.

Gravitropic moss cells default to spiral growth on the clinostat and in microgravity during spaceflight.

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.

SCD1 is required for cytokinesis and polarized cell expansion in Arabidopsis thaliana [corrected].

In the leaf epidermis, guard mother cells undergo a stereotyped symmetric division to form the guard cells of stomata. We have identified a temperature-sensitive Arabidopsis mutant, stomatal cytokinesis-defective 1-1 (scd1-1), which affects this specialized division. At the non-permissive temperature, 22 degrees C, defective scd1-1 guard cells are binucleate, and the formation of their ventral cell walls is incomplete. Cytokinesis was also disrupted in other types of epidermal cells such as pavement cells. Further phenotypic analysis of scd1-1 indicated a role for SCD1 in seedling growth, root elongation and flower morphogenesis. More severe scd1 T-DNA insertion alleles (scd1-2 and scd1-3) markedly affect polar cell expansion, most notably in trichomes and root hairs. SCD1 is a unique gene in Arabidopsis that encodes a protein related to animal proteins that regulate intracellular protein transport and/or mitogen-activated protein kinase signaling pathways. Consistent with a role for SCD1 in membrane trafficking, secretory vesicles were found to accumulate in cytokinesis-defective scd1 cells. In addition the scd1 mutant phenotype was enhanced by low doses of inhibitors of cell plate consolidation and vesicle secretion. We propose that SCD1 functions in polarized vesicle trafficking during plant cytokinesis and cell expansion.

Stomatal development: cross talk puts mouths in place.

Stomata are crucial for the productivity and survival of land plants. Until recently, little was known about the events and molecular pathways required for stomatal development. Emerging data indicate that cell-cell signaling conveys spatial information about cell identity and location. Such information might pattern stomata by orienting the plane of asymmetric division and might control stomatal number by regulating division frequency. This pathway also provides an accessible model system for studying post-apical meristem stem cells that generate specific tissues.

Stomatal neighbor cell polarity and division in Arabidopsis.

Asymmetric divisions are key to regulating the number and patterning of stomata in Arabidopsis thaliana (L.) Heynh. Many formative asymmetric divisions take place in neighbor cells (NCs), cells adjacent to a stoma or stomatal precursor. TOO MANY MOUTHS is a receptor-like protein required for the correct plane of NC division, resulting in the placement of the new precursor distal to the pre-existing stoma. Because plant cells usually become polarized before asymmetric division, we studied whether NCs display a cytological asymmetry as a function of cell stage and of possible division behavior. Cells that divided in the developing leaf epidermis were smaller than 400 micro m(-2) in area and included NCs as well as isolated cells. All NCs in the youngest complexes divided with comparable frequencies, but divisions became restricted to the smaller and most recently produced NCs as the stomatal complex matured. The majority of developing NCs had distally located nuclei, suggesting that nuclear position is actively regulated in NCs. NC stages exhibiting distally located nuclei were the likeliest to divide asymmetrically. However, a distal nucleus did not necessarily predict an asymmetric division, because more NCs had distal nuclei than were likely to divide. No defect was detected in nuclear distribution in tmm NCs. These data suggest that TMM uses intercellular signals to control the plane of asymmetric division after or independently of nuclear positioning.

Control of stomatal distribution on the Arabidopsis leaf surface.

Stomata regulate gas exchange and are distributed across the leaf epidermis with characteristic spacing. Arabidopsis stomata are produced by asymmetric cell divisions. Mutations in the gene TOO MANY MOUTHS (TMM) disrupt patterning by randomizing the plane of formative asymmetric divisions and by permitting ectopic divisions. TMM encodes a leucine-rich repeat-containing receptor-like protein expressed in proliferative postprotodermal cells. TMM appears to function in a position-dependent signaling pathway that controls the plane of patterning divisions as well as the balance between stem cell renewal and differentiation in stomatal and epidermal development.

Stomatal development in Arabidopsis.

Stomata consist of two guard cells around a pore and act as turgor-operated valves for gas exchange. Arabidopsis stomata develop from one or more asymmetric divisions followed by the symmetric division of the guard mother cell. Stomatal number is partly a function of the availability of smaller epidermal cells that are competent to divide asymmetrically. Stomata are spaced apart from each other by at least one neighbor cell. Pattern generation may involve cell-cell signaling that transmits spatial cues used to orient specific classes of asymmetric divisions. TOO MANY MOUTHS may function in receiving or transducing these cues to orient asymmetric divisions. TMM also is a negative or positive regulator of entry into the stomatal pathway, with the direction of the response dependent on organ and location. STOMATAL DENSITY AND DISTRIBUTION1 is a negative regulator of stomatal formation throughout the shoot and encodes a processing protease that may function in intercellular communication. FOUR LIPS apparently controls the number symmetric divisions at the guard mother cell stage. In some organs, such as the hypocotyl, the placement of stomata may be coordinated with internal features and involves genes that also regulate root hair and trichome formation. Other mutations affect guard cell morphogenesis, cytokinesis, and stomatal number in response to carbon dioxide concentration. The molecular analysis of stomatal development promises advances in understanding intercellular signaling, the control of the plane and polarity of asymmetric division, the specification of cell fate, and the regulation of cell differentiation and shape.