Myrosin idioblast cell fate and development are regulated by the Arabidopsis transcription factor FAMA, the auxin pathway, and vesicular trafficking.

Crucifer shoots harbor a glucosinolate-myrosinase system that defends against insect predation. Arabidopsis thaliana myrosinase (thioglucoside glucohydrolase [TGG]) accumulates in stomata and in myrosin idioblasts (MIs). This work reports that the basic helix-loop-helix transcription factor FAMA that is key to stomatal development is also expressed in MIs. The loss of FAMA function abolishes MI fate as well as the expression of the myrosinase genes TGG1 and TGG2. MI cells have previously been reported to be located in the phloem. Instead, we found that MIs arise from the ground meristem rather than provascular tissues and thus are not homologous with phloem. Moreover, MI patterning and morphogenesis are abnormal when the function of the ARF-GEF gene GNOM is lost as well as when auxin efflux and vesicular trafficking are chemically disrupted. Stomata and MI cells constitute part of a wider system that reduces plant predation, the so-called "mustard oil bomb," in which vacuole breakage in cells harboring myrosinase and glucosinolate yields a brew toxic to many animals, especially insects. This identification of the gene that confers the fate of MIs, as well as stomata, might facilitate the development of strategies for engineering crops to mitigate predation.

The Arabidopsis leucine-rich repeat receptor-like kinase MUSTACHES enforces stomatal bilateral symmetry in Arabidopsis.

Stomata display a mirror-like symmetry that is adaptive for shoot/atmosphere gas exchange. This symmetry includes the facing guard cells around a lens-shaped and bilaterally symmetric pore, as well as radially arranged microtubule arrays that primarily originate at the pore and then grow outwards. Mutations in MUSTACHES (MUS), which encodes a leucine-rich repeat receptor-like kinase, disrupt this symmetry, resulting in defects ranging from skewed pores and abnormally focused and depolarized radial microtubule arrays, to paired guard cells that face away from each other, or a severe loss of stomatal shape. Translational MUSproMUS:tripleGFP fusions are expressed in cell plates in most cells types in roots and shoots, and cytokinesis and cell plates are mostly normal in mus mutants. However, in guard mother cells, which divide and then form stomata, MUS expression is notably absent from new cell plates, and instead is peripherally located. These results are consistent with a role for MUS in enforcing wall building and cytoskeletal polarity at the centre of the developing stoma via signalling from the vicinity of the guard cell membrane.

Mediation of clathrin-dependent trafficking during cytokinesis and cell expansion by Arabidopsis stomatal cytokinesis defective proteins.

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.

The FOUR LIPS and MYB88 transcription factor genes are widely expressed in Arabidopsis thaliana during development.

PREMISE OF THE STUDY: The FOUR LIPS (FLP) and MYB88 transcription factors, which are closely related in structure and function, control the development of stomata, as well as entry into megasporogenesis in Arabidopsis thaliana. However, other locations where these transcription factors are expressed are poorly described. Documenting additional locations where these genes are expressed might define new functions for these genes. METHODS: Expression patterns were examined throughout vegetative and reproductive development. The expression from two transcriptional-reporter fusions were visualized with either ß-glucuronidase (GUS) or green fluorescence protein (GFP). KEY RESULTS: Both flp and myb88 genes were expressed in many, previously unreported locations, consistent with the possibility of additional functions for FLP and MYB88. Moreover, expression domains especially of FLP display sharp cutoffs or boundaries. CONCLUSIONS: In addition to stomatal and reproductive development, FLP and MYB88, which are R2R3 MYB transcription factor genes, are expressed in many locations in cells, tissues, and organs.

Regulation of cell proliferation in the stomatal lineage by the Arabidopsis MYB FOUR LIPS via direct targeting of core cell cycle genes.

Stomata, which are epidermal pores surrounded by two guard cells, develop from a specialized stem cell lineage and function in shoot gas exchange. The Arabidopsis thaliana FOUR LIPS (FLP) and MYB88 genes encode closely related and atypical two-MYB-repeat proteins, which when mutated result in excess divisions and abnormal groups of stomata in contact. Consistent with a role in transcription, we show here that FLP and MYB88 are nuclear proteins with DNA binding preferences distinct from other known MYBs. To identify possible FLP/MYB88 transcriptional targets, we used chromatin immunoprecitation (ChIP) followed by hybridization to Arabidopsis whole genome tiling arrays. These ChIP-chip data indicate that FLP/MYB88 target the upstream regions especially of cell cycle genes, including cyclins, cyclin-dependent kinases (CDKs), and components of the prereplication complex. In particular, we show that FLP represses the expression of the mitosis-inducing factor CDKB1;1, which, along with CDKB1;2, is specifically required both for the last division in the stomatal pathway and for cell overproliferation in flp mutants. We propose that FLP and MYB88 together integrate patterning with the control of cell cycle progression and terminal differentiation through multiple and direct cell cycle targets. FLP recognizes a distinct cis-regulatory element that overlaps with that of the cell cycle activator E2F-DP in the CDKB1;1 promoter, suggesting that these MYBs may also modulate E2F-DP pathways.

The R2R3 MYB transcription factors FOUR LIPS and MYB88 regulate female reproductive development.

Gamete formation is an important step in the life cycle of sexually reproducing organisms. In flowering plants, haploid spores are formed after the meiotic division of spore mother cells. These spores develop into male and female gametophytes containing gametes after undergoing mitotic divisions. In the female, the megaspore mother cell undergoes meiosis forming four megaspores, of which one is functional and three degenerate. The megaspore then undergoes three mitotic cycles thus generating an embryo sac with eight nuclei. The embryo sac undergoes cellularization to form the mature seven-celled female gametophyte. Entry into and progression through meiosis is essential for megasporogenesis and subsequent megagametogenesis, but control of this process is not well understood. FOUR LIPS (FLP) and its paralogue MYB88, encoding R2R3 MYB transcription factors, have been extensively studied for their role in limiting the terminal division in stomatal development by direct regulation of the expression of cell cycle genes. Here it is demonstrated that FLP and MYB88 also regulate female reproduction. Both FLP and MYB88 are expressed during ovule development and their loss significantly increases the number of ovules produced by the placenta. Despite the presence of excess ovules, single and double mutants exhibit reduced seed set due to reduced female fertility. The sterility results at least in part from defective meiotic entry and progression. Therefore, FLP and MYB88 are important regulators of entry into megasporogenesis, and probably act via the regulation of cell cycle genes.

Stomatal development.

Stomata are cellular epidermal valves in plants central to gas exchange and biosphere productivity. The pathways controlling their formation are best understood for Arabidopsis thaliana where stomata are produced through a series of divisions in a dispersed stem cell compartment. The stomatal pathway is an accessible system for analyzing core developmental processes including position-dependent patterning via intercellular signaling and the regulation of the balance between proliferation and cell specification. This review synthesizes what is known about the mechanisms and genes underlying stomatal development. We contrast the functions of genes that act earlier in the pathway, including receptors, kinases, and proteases, with those that act later in the cell lineage. In addition, we discuss the relationships between environmental signals, stomatal development genes, and the capacity for controlling shoot gas exchange.

Role of the stomatal development regulators FLP/MYB88 in abiotic stress responses.

Stomata are vital for the adaptation of plants to abiotic stress, and in turn stomatal density is modulated by environmental factors. Less clear, however, is whether regulators of stomatal development themselves participate in the sensing or response of stomata to abiotic stress. FOUR LIPS (FLP) and its paralog MYB88 encode MYB proteins that establish stomatal patterning by permitting only a single symmetric division before stomata differentiate. Hence, flp-1 myb88 double mutants have an excess of stomata, which are often misplaced in direct contact. Here, we investigate the consequences of loss of FLP/MYB88 function on the ability of Arabidopsis plants to respond to abiotic stress. While flp-1 myb88 double mutants are viable and display no obvious aerial phenotypes under normal greenhouse growth conditions, we show that flp-1 myb88 plants are significantly more susceptible to drought and high salt, and have increased rates of water loss. To determine whether flp-1 myb88 plants are already challenged under normal growth conditions, we compared genome-wide transcript levels between flp-1 myb88 and wild-type green tissues. Unexpectedly, uninduced flp-1 myb88 plants showed a reduced accumulation of many typical abiotic stress gene transcripts. Moreover, the induction of many of these stress genes under high-salt conditions was significantly lower in flp-1 myb88 plants. Our results provide evidence for a new function of FLP/MYB88 in sensing and/or transducing abiotic stress, which is severely compromised in flp-1 myb88 mutants.

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.

Variable timing of developmental progression in the stomatal pathway in Arabidopsis cotyledons.

• Stomatal production depends on the rates of precursor cell formation (e.g. the formation of meristemoids by asymmetric division), and of developmental progression (e.g. the division of guard mother cells). It is not known whether these rates follow steady-state kinetics or are variable. • The timing of development was scored in Arabidopsis cotyledons in fixed and living tissue using the dental resin impression method. • Cotyledons exhibited much less of a longitudinal gradient in stomatal formation than leaves. The timing of the appearance of stomatal and precursor cells during cotyledon development varied between individual plants. Precursor cell formation ceased much earlier in the adaxial than in the abaxial epidermis. Meristemoids are precursors that form guard mother cells. The ratio of these cell types varied greatly in different plants as well as in the same epidermis through time. There was also considerable variability in the duration of the meristemoid stage between individual cell lineages. • Precursor cell production follows non-steady-state kinetics. Early steps in the pathway are not necessarily synchronized, but later steps, such as the conversion of meristemoids to guard mother cells, sometimes appear to be coordinated.

Diversity in spindle morphology in Arabidopsis root tip.

A primary function of the spindle apparatus is to segregate chromosomes into two equal sets in a dividing cell. It is unclear whether spindles in different cell types play additional roles in cellular regulation. As a first step in revealing new functions of spindles, we investigated spindle morphology in different cell types in Arabidopsis roots in the wild-type and the cytokinesis defective1 (cyd1) mutant backgrounds. cyd1 provides cells larger than those of the wild type for testing the cell size effect on spindle morphology. Our observations indicate that cell type (shape), not cell size, is likely a factor affecting spindle morphology. At least three spindle types were observed, including small spindles with pointed poles in narrow cells, large barrel-shaped spindles (without pointed poles) in wide cells, and spindles intermediate in pole focus and size in other cells. We hypothesize that the cell-type-associated spindle diversity may be an integral part of the cell differentiation processes.

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.

Gravitropism in the starch excess mutant of Arabidopsis thaliana.

Amyloplasts are hypothesized to play a key role in the cellular mechanisms of gravity perception in plants. While previous studies have examined the effects of starch deficiency on gravitropic sensitivity, in this paper, we report on gravitropism in plants with a greater amount of starch relative to the normal wild type. Thus, we have studied the sex1 (starch excess) mutant of Arabidopsis thaliana, which accumulates extra starch because it is defective in a protein involved in the regulation of starch mobilization. Compared to the wild type (WT), sex1 seedlings contained excess starch in cotyledons, hypocotyls, the root-hypocotyl transition zone, the body of the root, root hairs, and in peripheral rootcap cells. Sedimented amyloplasts were found in both the WT and in sex1 in the rootcap columella and in the endodermis of stems, hypocotyls, and petioles. In roots, the starch content and amyloplast sedimentation in central columella cells and the gravitropic sensitivity were comparable in sex1 and the WT. However, in hypocotyls, the sex1 mutant was much more sensitive to gravity during light-grown conditions compared to the WT. This difference was correlated to a major difference in size of plastids in gravity-perceiving endodermal cells between the two genotypes (i.e., sex1 amyloplasts were twice as big). These results are consistent with the hypothesis that only very large changes in starch content relative to the WT affect gravitropic sensitivity, thus indicating that wild-type sensing is not saturated.

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.

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.

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.

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.

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 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.