Misregulation of MYB16 expression causes stomatal cluster formation by disrupting polarity during asymmetric cell divisions.

Stomatal pores and the leaf cuticle regulate evaporation from the plant body and balance the tradeoff between photosynthesis and water loss. MYB16, encoding a transcription factor involved in cutin biosynthesis, is expressed in stomatal lineage ground cells, suggesting a link between cutin biosynthesis and stomatal development. Here, we show that the downregulation of MYB16 in meristemoids is directly mediated by the stomatal master transcription factor SPEECHLESS (SPCH) in Arabidopsis thaliana. The suppression of MYB16 before an asymmetric division is crucial for stomatal patterning, as its overexpression or ectopic expression in meristemoids increased stomatal density and resulted in the formation of stomatal clusters, as well as affecting the outer cell wall structure. Expressing a cutinase gene in plants ectopically expressing MYB16 reduced stomatal clustering, suggesting that cutin affects stomatal signaling or the polarity setup in asymmetrically dividing cells. The clustered stomatal phenotype was rescued by overexpressing EPIDERMAL PATTERNING FACTOR2, suggesting that stomatal signaling was still functional in these plants. Growing seedlings ectopically expressing MYB16 on high-percentage agar plates to modulate tensile strength rescued the polarity and stomatal cluster defects of these seedlings. Therefore, the inhibition of MYB16 expression by SPCH in the early stomatal lineage is required to correctly place the polarity protein needed for stomatal patterning during leaf morphogenesis.

Using quantitative methods to understand leaf epidermal development.

As the interface between plants and the environment, the leaf epidermis provides the first layer of protection against drought, ultraviolet light, and pathogen attack. This cell layer comprises highly coordinated and specialised cells such as stomata, pavement cells and trichomes. While much has been learned from the genetic dissection of stomatal, trichome and pavement cell formation, emerging methods in quantitative measurements that monitor cellular or tissue dynamics will allow us to further investigate cell state transitions and fate determination in leaf epidermal development. In this review, we introduce the formation of epidermal cell types in Arabidopsis and provide examples of quantitative tools to describe phenotypes in leaf research. We further focus on cellular factors involved in triggering cell fates and their quantitative measurements in mechanistic studies and biological patterning. A comprehensive understanding of how a functional leaf epidermis develops will advance the breeding of crops with improved stress tolerance.

Stomatal clustering in Begonia improves water use efficiency by modulating stomatal movement and leaf structure.

Stomata are a pivotal adaptation of land plants and control gas exchange. While most plants present solitary stomata, some plant species experiencing chronic water deficiency display clustered stomata on their epidermis; for instance, limestone-grown begonias. Moreover, the membrane receptor TOO MANY MOUTHS (TMM) plays a major role in spacing stomata on the epidermis in Arabidopsis, but the function of its Begonia orthologs is unknown. We used two Asian begonias, Begonia formosana (single stomata) and B. hernandioides (clustered stomata), to explore the physiological function of stomatal clustering. We also introduced the Begonia TMMs into Arabidopsis tmm mutants to study the function of Begonia TMMs. B. hernandioides showed higher water use efficiency under high light intensity, smaller stomata, and faster pore opening than B. formosana. The short distance between stomata in a cluster may facilitate cell-to-cell interactions to achieve synchronicity in stomatal movement. Begonia TMMs function similarly to Arabidopsis TMM to inhibit stomatal formation, although complementation by TMM from the clustered species was only partial. Stomatal clustering in begonias may represent a developmental strategy to build small and closer stomata to achieve fast responses to light which provides tight support between stomatal development and environmental adaption.

Subcellular localization of biomolecules and drug distribution by high-definition ion beam imaging.

Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the "tag" and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.

Transcriptional profiling reveals signatures of latent developmental potential in Arabidopsis stomatal lineage ground cells.

In many developmental contexts, cell lineages have variable or flexible potency to self-renew. What drives a cell to exit from a proliferative state and begin differentiation, or to retain the capacity to divide days or years later is not clear. Here we exploit the mixed potential of the stomatal lineage ground cell (SLGC) in the Arabidopsis leaf epidermis as a model to explore how cells might balance potential to differentiate with a reentry into proliferation. By generating transcriptomes of fluorescence-activated cell sorting-isolated populations that combinatorically define SLGCs and integrating these data with other stomatal lineage datasets, we find that SLGCs appear poised between proliferation and endoreduplication. Furthermore, we found the RNA polymerase II-related mediator complex interactor DEK and the transcription factor MYB16 accumulate differentially in the stomatal lineage and influence the extent of cell proliferation during leaf development. These findings suggest that SLGC latent potential is maintained by poising of the cell cycle machinery, as well as general and site-specific gene-expression regulators.

Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases.

Signal transduction from a cell's surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. Plant development, defense, and homeostasis rely on plasma membrane receptor-like kinases to perceive endogenous and environmental signals, but little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry, and live-cell imaging, we show that the VAP-RELATED SUPPRESSOR OF TMM (VST) family is required for ERECTA-mediated signaling in growth and cell-fate determination and reveal a role for ERECTA-LIKE2 in modulating signaling by its sister kinases. We show that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, thereby potentially influencing the organization of the membrane milieu to promote efficient and differential signaling from the ERECTA-family members to their downstream intracellular targets.

Arabidopsis microtubule-associated protein MAP65-3 cross-links antiparallel microtubules toward their plus ends in the phragmoplast via its distinct C-terminal microtubule binding domain.

Plant cytokinesis is brought about by the phragmoplast, which contains an antiparallel microtubule (MT) array. The MT-associated protein MAP65-3 acts as an MT-bundling factor that specifically cross-links antiparallel MTs near their plus ends. MAP65 family proteins contain an N-terminal dimerization domain and C-terminal MT interaction domain. Compared with other MAP65 isoforms, MAP65-3 contains an extended C terminus. A MT binding site was discovered in the region between amino acids 496 and 588 and found to be essential for the organization of phragmoplast MTs. The frequent cytokinetic failure caused by loss of MAP65-3 was not rescued by ectopic expression of MAP65-1 under the control of the MAP65-3 promoter, indicating nonoverlapping functions between the two isoforms. In the presence of MAP65-3, however, ectopic MAP65-1 appeared in the phragmoplast midline. We show that MAP65-1 could acquire the function of MAP65-3 when the C terminus of MAP65-3, which contains the MT binding site, was grafted to it. Our results also show that MAP65-1 and MAP65-3 may share redundant functions in MT stabilization. Such a stabilization effect was likely brought about by MT binding and bundling. We conclude that MAP65-3 contains a distinct C-terminal MT binding site with a specific role in cross-linking antiparallel MTs toward their plus ends in the phragmoplast.

Characterization of the Arabidopsis augmin complex uncovers its critical function in the assembly of the acentrosomal spindle and phragmoplast microtubule arrays.

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim γ-tubulin 3, plays a critical role in γ-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of γ-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.

Interaction of antiparallel microtubules in the phragmoplast is mediated by the microtubule-associated protein MAP65-3 in Arabidopsis.

In plant cells, microtubules (MTs) in the cytokinetic apparatus phragmoplast exhibit an antiparallel array and transport Golgi-derived vesicles toward MT plus ends located at or near the division site. By transmission electron microscopy, we observed that certain antiparallel phragmoplast MTs overlapped and were bridged by electron-dense materials in Arabidopsis thaliana. Robust MT polymerization, reported by fluorescently tagged End Binding1c (EB1c), took place in the phragmoplast midline. The engagement of antiparallel MTs in the central spindle and phragmoplast was largely abolished in mutant cells lacking the MT-associated protein, MAP65-3. We found that endogenous MAP65-3 was selectively detected on the middle segments of the central spindle MTs at late anaphase. When MTs exhibited a bipolar appearance with their plus ends placed in the middle, MAP65-3 exclusively decorated the phragmoplast midline. A bacterially expressed MAP65-3 protein was able to establish the interdigitation of MTs in vitro. MAP65-3 interacted with antiparallel microtubules before motor Kinesin-12 did during the establishment of the phragmoplast MT array. Thus, MAP65-3 selectively cross-linked interdigitating MTs (IMTs) to allow antiparallel MTs to be closely engaged in the phragmoplast. Although the presence of IMTs was not essential for vesicle trafficking, they were required for the phragmoplast-specific motors Kinesin-12 and Phragmoplast-Associated Kinesin-Related Protein2 to interact with MT plus ends. In conclusion, we suggest that the phragmoplast contains IMTs and highly dynamic noninterdigitating MTs, which work in concert to bring about cytokinesis in plant cells.

Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis.

In higher plant cells, microtubules (MTs) are nucleated and organized in a centrosome-independent manner. It is unclear whether augmin-dependent mechanisms underlie spindle MT organization in plant cells as they do in animal cells. When AUGMIN subunit3 (AUG3), which encodes a homolog of animal dim γ-tubulin 3/human augmin-like complex, subunit 3, was disrupted in Arabidopsis thaliana, gametogenesis frequently failed due to defects in cell division. Compared with the control microspores, which formed bipolar spindles at the cell periphery, the mutant cells often formed peripheral half spindles that only attached to condensed chromosomes or formed elongated spindles with unfocused interior poles. In addition, defective cells exhibited disorganized phragmoplast MT arrays, which caused aborted cytokinesis. The resulting pollen grains were either shrunken or contained two nuclei in an undivided cytoplasm. AUG3 was localized along MTs in the spindle and phragmoplast, and its signal was pronounced in anaphase spindle poles. An AUG3-green fluorescent protein fusion exhibited a dynamic distribution pattern, similar to that of the γ-tubulin complex protein2. When AUG3 was enriched from seedlings by affinity chromatography, AUG1 was detected by immunoblotting, suggesting an augmin-like complex was present in vivo. We conclude that augmin plays a critical role in MT organization during plant cell division.

Microtubule Reorganization during Mitosis and Cytokinesis: Lessons Learned from Developing Microgametophytes in Arabidopsis Thaliana.

In angiosperms, mitosis and cytokinesis take place in the absence of structurally defined microtubule-organizing centers and the underlying mechanisms are largely unknown. In the spindle and phragmoplast, microtubule reorganization depends on microtubule-interacting factors like the γ-tubulin complex. Because of their critical functions in cell division, loss-of-function mutations in the corresponding genes are often homozygous or sporophytic lethal. However, a number of mutations like gem1, gcp2, and nedd1 can be maintained in heterozygous mutants in Arabidopsis thaliana. When mutant microspores produced by a heterozygous parent undergo pollen mitosis I, they are amenable for phenotypic characterization by fluorescence microscopy. The results would allow us to pinpoint at specific functions of particular proteins in microtubule reorganization that are characteristic to specific stages of mitosis and cytokinesis. Conclusions made in the developing microgametophytes can be extrapolated to somatic cells regarding mechanisms that regulate nuclear migration, spindle pole formation, phragmoplast assembly, and cell division plane determination.

Evaluating the microtubule cytoskeleton and its interacting proteins in monocots by mining the rice genome.

BACKGROUND: Microtubules (MTs) are assembled by heterodimers of alpha- and beta-tubulins, which provide tracks for directional transport and frameworks for the spindle apparatus and the phragmoplast. MT nucleation and dynamics are regulated by components such as the gamma-tubulin complex which are conserved among eukaryotes, and other components which are unique to plants. Following remarkable progress made in the model plant Arabidopsis thaliana toward revealing key components regulating MT activities, the completed rice (Oryza sativa) genome has prompted a survey of the MT cytoskeleton in this important crop as a model for monocots. SCOPE: The rice genome contains three alpha-tubulin genes, eight beta-tubulin genes and a single gamma-tubulin gene. A functional gamma-tubulin ring complex is expected to form in rice as genes encoding all components of the complex are present. Among proteins that interact with MTs, compared with A. thaliana, rice has more genes encoding some members such as the MAP65/Ase1p/PRC1 family, but fewer for the motor kinesins, the end-binding protein EB1 and the mitotic kinase Aurora. Although most known MT-interacting factors have apparent orthologues in rice, no orthologues of arabidopsis RIC1 and MAP18 have been identified in rice. Among all proteins surveyed here, only a few have had their functions characterized by genetic means in rice. Elucidating functions of proteins of the rice MT cytoskeleton, aided by recent technical advances made in this model monocot, will greatly advance our knowledge of how monocots employ their MTs to regulate their growth and form.