A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development.

Multiple plant developmental processes, such as lateral root development, depend on auxin distribution patterns that are in part generated by the PIN-formed family of auxin-efflux transporters. Here we propose that AUXIN RESPONSE FACTOR7 (ARF7) and the ARF7-regulated FOUR LIPS/MYB124 (FLP) transcription factors jointly form a coherent feed-forward motif that mediates the auxin-responsive PIN3 transcription in planta to steer the early steps of lateral root formation. This regulatory mechanism might endow the PIN3 circuitry with a temporal 'memory' of auxin stimuli, potentially maintaining and enhancing the robustness of the auxin flux directionality during lateral root development. The cooperative action between canonical auxin signalling and other transcription factors might constitute a general mechanism by which transcriptional auxin-sensitivity can be regulated at a tissue-specific level.

Transcriptional regulation of PIN genes by FOUR LIPS and MYB88 during Arabidopsis root gravitropism.

PIN proteins are auxin export carriers that direct intercellular auxin flow and in turn regulate many aspects of plant growth and development including responses to environmental changes. The Arabidopsis R2R3-MYB transcription factor FOUR LIPS (FLP) and its paralogue MYB88 regulate terminal divisions during stomatal development, as well as female reproductive development and stress responses. Here we show that FLP and MYB88 act redundantly but differentially in regulating the transcription of PIN3 and PIN7 in gravity-sensing cells of primary and lateral roots. On the one hand, FLP is involved in responses to gravity stimulation in primary roots, whereas on the other, FLP and MYB88 function complementarily in establishing the gravitropic set-point angles of lateral roots. Our results support a model in which FLP and MYB88 expression specifically determines the temporal-spatial patterns of PIN3 and PIN7 transcription that are closely associated with their preferential functions during root responses to gravity.

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.

Phosphorylation of Serine 186 of bHLH Transcription Factor SPEECHLESS Promotes Stomatal Development in Arabidopsis.

The initiation of stomatal lineage and subsequent asymmetric divisions in Arabidopsis require the activity of the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). It has been shown that SPCH controls entry into the stomatal lineage as a substrate either of the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade or GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2). Here we show that three serine residues of SPCH appear to be the primary phosphorylation targets of Cyclin-Dependent Kinases A;1 (CDKA;1) in vitro, and among them Serine 186 plays a crucial role in stomatal formation. Expression of an SPCH construct harboring a mutation that results in phosphorylation deficiencies on Serine 186 residue failed to rescue stomatal defects in spch null mutants. Expression of a phosphorylation-mimic mutant SPCH(S186D) complemented stomatal production defects in the transgenic lines harboring the targeted expression of dominant-negative CDKA;1.N146. Therefore, in addition to MAPK- and BIN2-mediated phosphorylation on SPCH, phosphorylation at Serine 186 is positively required for SPCH function in regulating stomatal development.

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.

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.

Arabidopsis guard cell integrity involves the epigenetic stabilization of the FLP and FAMA transcription factor genes.

Arabidopsis guard cell (GC) fate is conferred via a transient pulse of expression of FAMA that encodes a bHLH transcription factor. Stomata often function for years, suggesting that the FAMA expression window stabilizes long-term GC identity or that additional factors operate. Transgenic lines harboring a copy of a FAMA transgene were found to induce the fate resetting of mature GCs to that of lineage-specific stem cells causing new stomata to arise within shells of the old, a Stoma-in-Stoma (SIS) phenotype. These lines disrupt the normal trimethylation on lysine 27 of histone3 (H3K27me3) on stomatal stem cell genes, a phenotype rescued by constitutive expression of the Polycomb Group (PcG) gene CURLY LEAF. Thus the stability of stomatal fate is enforced by a PcG-mediated reduction in the transcriptional accessibility of stem cell genes and by the endogenous FAMA gene itself. Moreover, a transgenic FOUR LIPS gene, which encodes a MYB protein that is not required for GC fate, also induces a SIS phenotype and disrupts H3K27 trimethylation. Thus FLP might indirectly enforce GC fate as well.

Deep functional redundancy between FAMA and FOUR LIPS in stomatal development.

Functional redundancy arises between gene paralogs as well as non-homologous genes that play a common role at a shared node. The bHLH transcription factor FAMA, along with the paralogous MYB genes, FOUR LIPS (FLP) and MYB88 all ensure that Arabidopsis stomata contain just two guard cells (GCs) by enforcing a single symmetric precursor cell division before stomatal maturity. Consistent with this function, FLP and FAMA exhibit the same expression pattern in which both translational GFP fusions emit fluorescence just before and after symmetric division; however, FAMA but not FLP is required to confer GC fate. Strikingly, swapping the genes and promoters of the FLP and FAMA genes results in the reciprocal complementation of respective loss-of-function mutants. Thus, an FLP transgene can restore GC fate to a fama mutant background. FAMA, FLP and the FLP paralog MYB88 were previously shown to influence higher order functions in stomatal development, including maintaining and stabilizing stomatal fate. Here we show that these overlapping functions are likely to also involve interactions between FLP and FAMA with the RETINOBLASTOMA-RELATED (RBR) protein.

Auxin transport and activity regulate stomatal patterning and development.

Stomata are two-celled valves that control epidermal pores whose spacing optimizes shoot-atmosphere gas exchange. They develop from protodermal cells after unequal divisions followed by an equal division and differentiation. The concentration of the hormone auxin, a master plant developmental regulator, is tightly controlled in time and space, but its role, if any, in stomatal formation is obscure. Here dynamic changes of auxin activity during stomatal development are monitored using auxin input (DII-VENUS) and output (DR5:VENUS) markers by time-lapse imaging. A decrease in auxin levels in the smaller daughter cell after unequal division presages the acquisition of a guard mother cell fate whose equal division produces the two guard cells. Thus, stomatal patterning requires auxin pathway control of stem cell compartment size, as well as auxin depletion that triggers a developmental switch from unequal to equal division.

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.

FOUR LIPS and MYB88 conditionally restrict the G1/S transition during stomatal formation.

Consistent with their valve-like function in shoot-atmosphere gas exchange, guard cells are smaller than other epidermal cells and usually harbour 2C DNA levels in diploid plants. The paralogous Arabidopsis R2R3 MYB transcription factors, FOUR LIPS and MYB88, ensure that stomata contain just two guard cells by restricting mitosis. The loss of both FLP and MYB88 function in flp myb88 double mutants induces repeated mitotic divisions that lead to the formation of clusters of stomata in direct contact. By contrast, CYCLIN DEPENDENT KINASE B1 function is required for the symmetric division that precedes stomatal maturation. It was found that blocking mitosis by chemically disrupting microtubules or by the combined loss of FLP/MYB88 and CDKB1 function, causes single (undivided) guard cells (sGCs) to enlarge and attain mean DNA levels of up to 10C. The loss of both FLP and CDKB1 function also dramatically increased plastid number, led to the formation of multiple nuclei in GCs, altered GC and stomatal shape, and disrupted the fate of lineage-specific stem cells. Thus, in addition to respectively restricting and promoting symmetric divisions, FLP and CDKB1 together also conditionally restrict the G1/S transition and chloroplast and nuclear number, and normally maintain fate and developmental progression throughout the stomatal cell lineage.

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.

Polar development of preprophase bands and cell plates in the Arabidopsis leaf epidermis.

Preprophase bands are belts of cortical microtubules that appear at the end of interphase and predict where cell plates will fuse with parental walls during division. Phragmoplasts are microtubule-rich arrays that orchestrate the growth and guidance of cell plates during cytokinesis. Descriptions of the development of these arrays often assume non-polar formation, with preprophase bands developing more or less simultaneously around the cell circumference. Phragmoplasts are often described as initiating at the cell center and then expanding evenly outwards until fusion with parent cell walls. We analyzed the spatio-temporal development of both arrays because initial observations of array growth in the Arabidopsis leaf epidermis revealed directional variability. Almost all preprophase bands formed in a polar fashion, with initiation and maturation occurring first in the cell cortex near the inside of the leaf, and later in the outer cell cortex. A similar polarity developed in phragmoplasts and cell plates, raising the possibility that polarized division is common in plants. Together, these findings identify additional polar features of the epidermis, and thereby provide a visually accessible system for identifying new proteins and subcellular components involved in the development of cell division and the previously formed division site.

Developmental regulation of CYCA2s contributes to tissue-specific proliferation in Arabidopsis.

In multicellular organisms, morphogenesis relies on a strict coordination in time and space of cell proliferation and differentiation. In contrast to animals, plant development displays continuous organ formation and adaptive growth responses during their lifespan relying on a tight coordination of cell proliferation. How developmental signals interact with the plant cell-cycle machinery is largely unknown. Here, we characterize plant A2-type cyclins, a small gene family of mitotic cyclins, and show how they contribute to the fine-tuning of local proliferation during plant development. Moreover, the timely repression of CYCA2;3 expression in newly formed guard cells is shown to require the stomatal transcription factors FOUR LIPS/MYB124 and MYB88, providing a direct link between developmental programming and cell-cycle exit in plants. Thus, transcriptional downregulation of CYCA2s represents a critical mechanism to coordinate proliferation during plant development.

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.

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.

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.

MOS11: a new component in the mRNA export pathway.

Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS)-mediated nuclear protein import, Nuclear Export Signal (NES)-dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107-160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1, 11 (mos11). Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly(A) mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol.

The Arabidopsis callose synthase gene GSL8 is required for cytokinesis and cell patterning.

Cytokinesis is the division of the cytoplasm and its separation into two daughter cells. Cell plate growth and cytokinesis appear to require callose, but direct functional evidence is still lacking. To determine the role of callose and its synthesis during cytokinesis, we identified and characterized mutants in many members of the GLUCAN SYNTHASE-LIKE (GSL; or CALLOSE SYNTHASE) gene family in Arabidopsis (Arabidopsis thaliana). Most gsl mutants (gsl1-gsl7, gsl9, gsl11, and gsl12) exhibited roughly normal seedling growth and development. However, mutations in GSL8, which were previously reported to be gametophytic lethal, were found to produce seedlings with pleiotropic defects during embryogenesis and early vegetative growth. We found cell wall stubs, two nuclei in one cell, and other defects in cell division in homozygous gsl8 insertional alleles. In addition, gsl8 mutants and inducible RNA interference lines of GSL8 showed reduced callose deposition at cell plates and/or new cell walls. Together, these data show that the GSL8 gene encodes a putative callose synthase required for cytokinesis and seedling maturation. In addition, gsl8 mutants disrupt cellular and tissue-level patterning, as shown by the presence of clusters of stomata in direct contact and by islands of excessive cell proliferation in the developing epidermis. Thus, GSL8 is required for patterning as well as cytokinesis during Arabidopsis development.