Seed mucilage in temperate grassland species is unrelated to moisture requirements.

Myxospermy, the release of seed mucilage upon hydration, plays multiple roles in seed biology. Here, we explore whether seed mucilage occurs in a suite of temperate grassland species to test if the prevalence of species producing seed mucilage is associated with habitat type or seed characteristics. Seventy plant species found in wet or dry North American temperate grasslands were tested for the presence of seed mucilage through microscopic examination of seeds imbibed with histochemical stain for mucilage. Mucilage production was compared among species with different moisture requirements and seed mass. In this study, 43 of 70 of species tested produced seed mucilage. Seed mucilage did not differ based on habitat type, species moisture requirements, or seed mass. Most seed mucilage was non-adherent and did not remain stuck to the seed after extrusion. Seed mucilage was a common trait in the surveyed temperate grassland species and was observed in 61% of evaluated species. Surprisingly, seed mucilage was more common in temperate grasslands than in previous ecological surveys from arid/semiarid systems, which found 10%-31% myxospermous species. Given the high prevalence, seed mucilage may influence seedling ecology in temperate grasslands and requires further investigation.

Overlapping Localization of MAP65-2, -6, and -7 in Arabidopsis Hypocotyl Cells.

Microtubules are essential components of eukaryotic cells. Myriad proteins associate with microtubules to facilitate the organization and operation of microtubule arrays. Various M icrotubule A ssociated P roteins (MAPs) assist the assembly and function of mitotic spindles and interphase arrays. Nine MAP65 genes exist in the genome of the acentrosomal model plant, Arabidopsis thaliana, and the function of majority of these proteins is unclear. To address this knowledge gap, we demonstrate the localization of A. thaliana MAP65-6 and MAP65-7 fusion proteins expressed from native promoters in interphase cells of developing A. thaliana seedlings. Analyses of these fusion proteins co-expressed with alpha-tubulin 6 reporters indicate that MAP65-6 and MAP65-7 bind a subset of interphase microtubules. Co-expression of GFP: MAP65-6 with mCherry: MAP65-2 from native promoters in A. thaliana showed overlapping localization patterns on interphase microtubule bundles. Collectively, these data suggested that MAP65-2 , -6, and -7 bind cortical microtubule bundles in plant interphase microtubule arrays.

Stomatal pore width and area measurements in Zea mays.

Stomatal pores are adjustable microscopic holes on the surface of photosynthetic tissues that help regulate multiple aspects of plant physiology. Stomatal pores facilitate gas exchange necessary for photosynthesis, water transport, and temperature regulation. Pore size is influenced by many intertwined environmental, molecular, cellular, and physiological cues. Accurate and precise measurements of pore size is important for understanding the mechanisms that adjust pores and plant physiology. Here we investigate whether conventional pore measurements of width are appropriate for the economically important crop plant Zea mays . Our studies demonstrate that pore area is a more sensitive measurement than width in this plant.

Appearance of microtubules at the cytokinesis to interphase transition in Arabidopsis thaliana.

Microtubule arrays drastically reorganize during the cell cycle to facilitate specific events. Many cells contain a centrosome that dictates the assembly and organization of microtubule arrays. However, plant cells and many others do not contain centrosomes or discrete microtubule organizing centers. In plants, microtubules nucleate and polymerize from gamma-tubulin-containing complexes in the interphase cell cortex. During plant cell division, microtubules nucleate near nuclei to form the mitotic spindle and plant-specific phragmoplast required for cytokinesis. Therefore, during the plant cell cycle, microtubule nucleation shifts from cell cortex to the perinuclear region. While it is unclear how this shift occurs, previous studies observed microtubules that appeared to extend from nuclei into the cortex as cells transitioned into interphase in small cells. These data led to the hypothesis that microtubule nucleation complexes move from the nuclear surface to the cortex at the transition from cytokinesis into interphase. Here we document GFP labeled microtubules in living plant cells during the transition from cytokinesis to interphase. We observed apparent groups of microtubules spanning between the nucleus and cell cortex in large, vacuolated epidermal leaf cells. We also observed microtubules in the cell cortex that appeared separate from perinuclear-associated microtubules. While these cortical microtubules were not always seen, when present they were apparent before cytokinesis was complete and/or before nuclear-associated microtubules were obvious. These data add to and deepen the knowledge of microtubule reorganization at this cell cycle transition.

Hornwort Stomata: Architecture and Fate Shared with 400-Million-Year-Old Fossil Plants without Leaves.

As one of the earliest plant groups to evolve stomata, hornworts are key to understanding the origin and function of stomata. Hornwort stomata are large and scattered on sporangia that grow from their bases and release spores at their tips. We present data from development and immunocytochemistry that identify a role for hornwort stomata that is correlated with sporangial and spore maturation. We measured guard cells across the genera with stomata to assess developmental changes in size and to analyze any correlation with genome size. Stomata form at the base of the sporophyte in the green region, where they develop differential wall thickenings, form a pore, and die. Guard cells collapse inwardly, increase in surface area, and remain perched over a substomatal cavity and network of intercellular spaces that is initially fluid filled. Following pore formation, the sporophyte dries from the outside inwardly and continues to do so after guard cells die and collapse. Spore tetrads develop in spore mother cell walls within a mucilaginous matrix, both of which progressively dry before sporophyte dehiscence. A lack of correlation between guard cell size and DNA content, lack of arabinans in cell walls, and perpetually open pores are consistent with the inactivity of hornwort stomata. Stomata are expendable in hornworts, as they have been lost twice in derived taxa. Guard cells and epidermal cells of hornworts show striking similarities with the earliest plant fossils. Our findings identify an architecture and fate of stomata in hornworts that is ancient and common to plants without sporophytic leaves.

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.

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.

Progressive transverse microtubule array organization in hormone-induced Arabidopsis hypocotyl cells.

The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell's midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces.

MAP65-1 and MAP65-2 promote cell proliferation and axial growth in Arabidopsis roots.

We investigated the role of the Arabidopsis microtubule associated proteins 65-1 and 65-2 (MAP65-1 and MAP65-2) in the control of axial root growth. Transgenic plants expressing fluorescent fusion proteins from native promoters indicated exactly overlapping accumulation of MAP65-1 and MAP65-2 in the root tip and elongation zone. Nearly identical protein accumulation patterns were observed when MAP65-1 and MAP65-2 were expressed behind a constitutive CaMV 35S promoter, suggesting a level of post-transcriptional control that restricts these proteins to rapidly growing portions of the root. Co-expression of MAP65-1 and MAP65-2 fusion proteins showed precise co-localization to interphase and cytokinetic microtubule arrays. In interphase root tip cells, the fluorescent protein fusions labeled microtubules that were organized into a variety of different array patterns. In the rapidly growing cells of the root elongation zone, we found MAP65-1 and MAP65-2 co-localized exclusively to the lateral faces of cells that were axially extending. Genetic analysis showed that MAP65-1 and MAP65-2 are coordinately required for proper root elongation. Double map65-1-1 map65-2-2 mutant roots from dark-grown plants contained 50% fewer cells per file than wild-type roots, but we found no evidence that cytokinesis was disrupted. We additionally discovered that cell length was significantly shorter in the mature regions of the root beyond the zone where MAP65-1 and MAP65-2 accumulated. Our data indicate that MAP65-1 and MAP65-2 play a critical role in root growth by promoting cell proliferation and axial extension.

Microtubule-associated proteins MAP65-1 and MAP65-2 positively regulate axial cell growth in etiolated Arabidopsis hypocotyls.

The Arabidopsis thaliana MAP65-1 and MAP65-2 genes are members of the larger eukaryotic MAP65/ASE1/PRC gene family of microtubule-associated proteins. We created fluorescent protein fusions driven by native promoters that colocalized MAP65-1 and MAP65-2 to a subset of interphase microtubule bundles in all epidermal hypocotyl cells. MAP65-1 and MAP65-2 labeling was highly dynamic within microtubule bundles, showing episodes of linear extension and retraction coincident with microtubule growth and shortening. Dynamic colocalization of MAP65-1/2 with polymerizing microtubules provides in vivo evidence that plant cortical microtubules bundle through a microtubule-microtubule templating mechanism. Analysis of etiolated hypocotyl length in map65-1 and map65-2 mutants revealed a critical role for MAP65-2 in modulating axial cell growth. Double map65-1 map65-2 mutants showed significant growth retardation with no obvious cell swelling, twisting, or morphological defects. Surprisingly, interphase microtubules formed coaligned arrays transverse to the plant growth axis in dark-grown and GA(4)-treated light-grown map65-1 map65-2 mutant plants. We conclude that MAP65-1 and MAP65-2 play a critical role in the microtubule-dependent mechanism for specifying axial cell growth in the expanding hypocotyl, independent of any mechanical role in microtubule array organization.

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.

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.