Jeanette A. Nadeau

Oriented Asymmetric Divisions That Generate the Stomatal Spacing Pattern in Arabidopsis Are Disrupted by the too many mouths Mutation

Wild-type stomata are spaced by intervening cells, a pattern disrupted in the Arabidopsis mutant too many mouths (tmm). To determine the mechanism of wild-type spacing and how tmm results in pattern violations, we analyzed the behavior of cells through time by using sequential dental resin impressions. Meristemoids are stomatal precursors produced by asymmetric division. We show that wild-type patterning largely results when divisions next to a preexisting stoma or precursor are oriented so that the new meristemoid is placed away. Because this placement is independent of cell lineage, these divisions may be oriented by cell–cell signaling. tmm randomizes this orientation and releases a prohibition on asymmetric division in cells at specific locations, resulting in stomatal clusters. TMM is thus necessary for two position-dependent events in leaves: the orientation of asymmetric divisions that pattern stomata, and the control of which cells will enter the stomatal pathway. In addition, our findings argue against most previous hypotheses of wild-type stomatal patterning.

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.

Stomatal Development in Arabidopsis

Abstract 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.

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.

SCD1 is required for cell cytokinesis and polarized cell expansion in Arabidopsis thaliana

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°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.

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.

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.

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

Clathrin-mediated membrane trafficking is essential for cytokinesis and cell expansion. This study shows that SCD2 and SCD1, a putative Rab GEF, which coordinate to regulate cytokinesis and cell expansion in Arabidopsis, are associated with clathrin-coated vesicles and are necessary for plasma membrane endocytosis. STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.