Calpain-Mediated Positional Information Directs Cell Wall Orientation to Sustain Plant Stem Cell Activity, Growth and Development.

Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

Spores before sporophytes: hypothesizing the origin of sporogenesis at the algal-plant transition.

Fossil spores from mid-Ordovician deposits (475 million yr old) are the first indication of plants on land and predate megafossils of plants by 30-50 million yr. Sporopollenin-walled spores distinguish land plants from algae, which typically have heavy-walled zygotes that germinate via meiosis into motile or protonemal cells. All land plants are embryophytes with spores produced by the sporophyte generation. It is generally assumed that retention of the zygote and delay in meiosis led to matrotrophic embryo development and intercalation of the diploid sporophyte before spore production. However, new data on the cell biology of sporogenesis in extant bryophytes suggest that spores were produced directly from zygotes in protoembryophytes. The mechanism of wall transfer from zygote to meiospores was a three-phase heterochrony involving precocious initiation of cytokinesis, acceleration of meiosis, and concomitant delay in wall deposition. In bryophyte sporogenesis, cytokinesis is typically initiated in advance of meiosis, and quadrilobing of the cytoplasm is followed by development of a bizarre quadripolar spindle that assures coordination of nuclear distribution with predetermined spore domains. This concept of the innovation of sporogenesis at the onset of terrestrialization provides a new perspective for interpreting fossil evidence and understanding the evolution of land plants.

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.

Dividing without centrioles: innovative plant microtubule organizing centres organize mitotic spindles in bryophytes, the earliest extant lineages of land plants.

BACKGROUND AND AIMS: As remnants of the earliest land plants, the bryophytes (liverworts, mosses and hornworts) are important in understanding microtubule organization in plant cells. Land plants have an anastral mitotic spindle that forms in the absence of centrosomes, and a cytokinetic apparatus comprised of a predictive preprophase band (PPB) before mitosis and a phragmoplast after mitosis. These microtubule arrays have no counterpart in animal cells and the nature of the plant microtubule organizing centre (MTOC) remained an enigma for many years until antibodies to γ-tubulin, an essential component of the MTOC in all eukaryotes, became available for tracing the origin of microtubule arrays. METHODOLOGY: We used immunofluorescence techniques to colocalize γ-tubulin, microtubules and chromosomes in mitotic cells of a representative liverwort, moss and hornwort to study the organization of microtubules during mitotic cell division. PRINCIPAL RESULTS: THE FUTURE DIVISION SITE IS MARKED BY A PPB IN ALL TAXA BUT THE MTOCS INITIALLY GENERATING THE HALF SPINDLES DIFFER: polar organizers in the liverwort, plastid MTOCs in the hornwort, and nuclear envelope-associated MTOCs in the moss. By mid-prophase, the forming spindles become more similar as γ-tubulin begins to spread around the polar regions of the nuclear envelope. CONCLUSIONS: Regardless of origin, mature metaphase spindles are identical and indistinguishable from the typical anastral spindle of higher plants with broad polar regions consisting of numerous subsets of converging microtubules. A curious phenomenon of plant spindles, true of bryophytes as well as higher plants, is the movement of γ-tubulin into the metaphase spindle itself. The bipolar arrays of phragmoplast microtubules are organized by diffuse γ-tubulin located at proximal surfaces of reforming nuclear envelopes. Phragmoplast development appears similar in the three taxa and to vascular plants as well.

Diversity in meiotic spindle origin and determination of cytokinetic planes in sporogenesis of complex thalloid liverworts (Marchantiopsida).

As the earliest divergent land plants, bryophytes (mosses, hornworts, and liverworts) provide insight into the evolution of the unique plant process of sporogenesis by which meiosis results in heavy walled spores. New immunohistochemical data on microtubules and gamma-tubulin in four genera of complex thalloid liverworts combined with previously published data on another four genera demonstrate grades in the evolution of spindle organization in meiosis. We have discovered that all recognized forms of microtubule organizing centers (MTOCs) in plant cells (plastid MTOCs, spheroid cytoplasmic MTOCs, polar organizers, and nuclear envelope MTOCs) occur in organization of the meiotic spindle of complex thalloid liverworts. In addition, all aspects of pre-meiotic preparation for quadripartitioning of the sporocyte into a tetrad of spores occur, with the exception of pre-meiotic wall precursors found in certain simple thalloids. The preparation includes morphogenetic plastid migration, cortical bands of microtubules that mark future cytokinetic planes in pre-meiosis, quadrilobing of the cytoplasm during meiotic prophase, and quadripolar microtubule systems that are transformed into functionally bipolar metaphase I spindles. Quadripolar spindle origin is typical of bryophyte sporogenesis even though the MTOCs involved may differ. However, in certain crown taxa of complex thalloids the spindle develops with no traces of quadripolarity and placement of intersporal walls is determined after meiosis, as is typical of higher plants.

Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta).

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of gamma-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from gamma-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.

Microtubules in early development of the megagametophyte of Ginkgo biloba.

Food storage tissue in the seeds of gymnosperms is female gametophyte (megagametophyte) that develops before fertilization, whereas, in seeds of angiosperms, food is stored as endosperm initiated by double fertilization. The megagametophyte is haploid, and endosperm is usually triploid, at least initially. Despite differences in origin, ploidy level, and developmental trigger, the early events of female gametophyte development in ginkgo are very similar to nuclear endosperm development in the seeds of angiosperms. In both, development begins as a single cell that undergoes multiple mitoses without cytokinesis, to produce a large syncytium. This study provided evidence that microtubule involvement in organization of the syncytium into nuclear cytoplasmic domains (NCDs) via nuclear-based radial microtubule systems is a critical developmental feature in the ginkgo megagametophyte, as it is in endosperm. Once the initial anticlinal walls have been deposited at the boundaries of NCDs, cellularization proceeds by the process of alveolation. Continued unidirectional growth of the alveolar walls is an outstanding example of polar cytokinesis. Ginkgo megagametophyte development appears to occur uniformly throughout the entire chamber, whereas nuclear type endosperm usually exhibits distinct developmental domains. These observations suggest that there is a fundamental pathway for the development and cellularization of syncytia in seed development.

Subcellular localization and functional domain studies of DEFECTIVE KERNEL1 in maize and Arabidopsis suggest a model for aleurone cell fate specification involving CRINKLY4 and SUPERNUMERARY ALEURONE LAYER1.

DEFECTIVE KERNEL1 (DEK1), which consists of a membrane-spanning region (DEK1-MEM) and a calpain-like Cys proteinase region (DEK1-CALP), is essential for aleurone cell formation at the surface of maize (Zea mays) endosperm. Immunolocalization and FM4-64 dye incubation experiments showed that DEK1 and CRINKLY4 (CR4), a receptor kinase implicated in aleurone cell fate specification, colocalized to plasma membrane and endosomes. SUPERNUMERARY ALEURONE LAYER1 (SAL1), a negative regulator of aleurone cell fate encoding a class E vacuolar sorting protein, colocalized with DEK1 and CR4 in endosomes. Immunogold localization, dual-axis electron tomography, and diffusion of fluorescent dye tracers showed that young aleurone cells established symplastic subdomains through plasmodesmata of larger dimensions than those connecting starchy endosperm cells and that CR4 preferentially associated with plasmodesmata between aleurone cells. Genetic complementation experiments showed that DEK1-CALP failed to restore wild-type phenotypes in maize and Arabidopsis thaliana dek1 mutants, and DEK1-MEM also failed to restore wild-type phenotypes in Arabidopsis dek1-1 mutants. Instead, ectopic expression of DEK1-MEM under the control of the cauliflower mosaic virus 35S promoter gave a dominant negative phenotype. These data suggest a model for aleurone cell fate specification in which DEK1 perceives and/or transmits a positional signal, CR4 promotes the lateral movement of aleurone signaling molecules between aleurone cells, and SAL1 maintains the proper plasma membrane concentration of DEK1 and CR4 proteins via endosome-mediated recycling/degradation.

Regulation of seed size by hypomethylation of maternal and paternal genomes.

DNA methylation is an epigenetic modification of cytosine that is important for silencing gene transcription and transposons, gene imprinting, development, and seed viability. DNA METHYLTRANSFERASE1 (MET1) is the primary maintenance DNA methyltransferase in Arabidopsis (Arabidopsis thaliana). Reciprocal crosses between antisense MET1 transgenic and wild-type plants show that DNA hypomethylation has a parent-of-origin effect on seed size. However, due to the dominant nature of the antisense MET1 transgene, the parent with a hypomethylated genome, its gametophyte, and both the maternal and paternal genomes of the F(1) seed become hypomethylated. Thus, the distinct role played by hypomethylation at each generation is not known. To address this issue, we examined F(1) seed from reciprocal crosses using a loss-of-function recessive null allele, met1-6. Crosses between wild-type and homozygous met1-6 parents show that hypomethylated maternal and paternal genomes result in significantly larger and smaller F(1) seeds, respectively. Our analysis of crosses between wild-type and heterozygous MET1/met1-6 parents revealed that hypomethylation in the female or male gametophytic generation was sufficient to influence F(1) seed size. A recessive mutation in another gene that dramatically reduces DNA methylation, DECREASE IN DNA METHYLATION1, also causes parent-of-origin effects on F(1) seed size. By contrast, recessive mutations in genes that regulate a smaller subset of DNA methylation (CHROMOMETHYLASE3 and DOMAINS REARRANGED METHYLTRANSFERASES1 and 2) had little effect on seed size. Collectively, these results show that maternal and paternal genomes play distinct roles in the regulation of seed size in Arabidopsis.

DNA methylation is critical for Arabidopsis embryogenesis and seed viability.

DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.

Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally.

A T-DNA insertion in the Arabidopsis thaliana DEK1 gene, encoding a calpain-like cysteine proteinase with a predicted membrane anchor, causes unorganized embryo development displaying irregular mitotic divisions in the embryo proper and suspensor. Embryo development is arrested at the globular stage, and the embryo proper lacks a defined protoderm. In the endosperm, the aleurone-like peripheral cell layer is partly or completely lacking. The Arabidopsis DEK1 wild-type transcript is expressed evenly throughout the endosperm and the embryo in developing seed as determined using in situ hybridization. The conclusion that the observed phenotype is caused by a T-DNA insertion in the Arabidopsis DEK1 gene is confirmed by complementation with the Arabidopisis DEK1 genomic sequence, as well as analysis of a second T-DNA insertion allele. Over-expression of the Arabidopsis DEK1 gene coding sequence under the control of the 35S promoter causes a number of developmental phenotypes, including a global lack of trichomes, leaves exhibiting improper dorsiventral symmetry and aberrant cell organization in flowers. We interpret the data to suggest a role for DEK1 in providing cells with positional clues for an appropriate developmental context within plant tissues.

gamma-Tubulin, microtubule arrays, and quadripolarity during sporogenesis in the hepatic Aneura pinguis (Metzgeriales).

This is the first report on the organization of a quadripolar microtubule system (QMS) in polyplastidic meiosis of a hepatic with polar organizers (POs). Unlike the monoplastidic sporocytes of mosses and hornworts, in which meiotic quadripolarity can be traced to plastid division and migration, sporocytes of Aneura pinguis are polyplastidic and tetrahedrally lobed before the QMS is organized. Whereas the QMS in mosses and hornworts is plastid-based, the QMS of A. pinguis is focused at four POs where gamma tubulin (gamma-tubulin) is concentrated. An aster of microtubules emanates from each PO centered in the four cytoplasmic lobes and the opposing radial microtubules interact to form the QMS that envelops the nucleus. A functionally bipolar spindle is gradually formed as the four poles converge in pairs on either side of opposite cleavage furrows. The resulting spindle remains quadripolar. Although gamma-tubulin is most concentrated in the deeply concave poles straddling cleavage furrows, it also extends into the spindle itself. Telophase groups of chromosomes curve around the polar cleavage furrows and a phragmoplast that originates in the interzonal region guides a cell plate that extends to the equatorial cleavage furrows. Discrete POs are reformed at opposite tips of the elongated dyad nuclei in prophase II and microtubules radiating from them give rise to the spindles of second meiosis. Spindles remain sharply focused and gamma-tubulin extends into distal portions of the spindle. Interzonal phragmoplasts that expand to join with pre-established cleavage furrows mediate cytokinesis resulting in a tetrad of spores. Each young tetrad member has a radial microtubule system emanating from the nucleus.

Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers.

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.

The microtubule cycle during successive mitotic waves in the syncytial female gametophyte of ginkgo.

Plant morphogenesis is driven by a surprising number of microtubule arrays. The four arrays of vegetative tissues are hoop-like cortical, preprophase band (PPB), spindle, and phragmoplast. When syncytia occur during the reproductive phase of the plant life cycle, neither hoop-like corticals nor PPBs are present, and functional phragmoplasts fail to form following the proliferative mitoses that give rise to the multinucleate cytoplasm. Instead, the interphase microtubules are radial microtubule systems (RMSs) that emanate from the nuclei. These RMSs organize the cytoplasm into nascent cells and ultimately trigger phragmoplast formation at their boundaries. During investigations of the syncytial stage that initiates development of the female gametophyte in gymnosperms, we studied the large (3-4 mm) female gametophyte of Ginkgo biloba. Here we describe the microtubule cycle correlated with successive mitotic waves and discuss the importance of this system in studying the acentrosomal nucleation and organization of cycling microtubule arrays.

MONOPLASTIDIC CELL DIVISION IN LOWER LAND PLANTS.

In many bryophytes and vascular cryptogams mitosis and/or meiosis takes place in cells containing a single plastid. In monoplastidic cell division plastid polarity assures that nuclear and plastid division are infallibly coordinated. The two major components of plastid polarity are morphogenetic plastid migration and microtubule organization at the plastids. Before nuclear division the plastid migrates to a position intersecting the future division plane. This morphogenetic migration is a reliable marker of division polarity in cells with and without a preprophase band of microtubules (PPB). The PPB, which predicts the future division plane before mitosis, is a characteristic feature of land plants and its insertion into the cytokinetic apparatus marks the evolution of a cortical microtubule system and a commitment to meristematic growth. Microtubule systems associated with plastid division, the axial microtubule system (AMS) in mitosis and the quadripolar microtubule system (QMS) in meiosis, contribute to predictive positioning of plastids and participate directly in spindle ontogeny. Division polarity in monoplastidic sporocytes is remarkable in that division sites are selected prior to the two successive nuclear divisions of meiosis. Plastid arrangement prior to meiosis determines the future spore domains in monoplastidic sporocytes, whereas in polyplastidic sporocytes the spore nuclei play a major role in claiming cytoplasmic domains. It is hypothesized that predivision microtubule systems associated with monoplastidic cell division are early forming components of the mitotic apparatus that serve to orient the spindle and insure equal apportionment of nucleus and plastids. "Can it be supposed that cytoplasm would be intrusted with so important a task as the preparation of a chloroplast for each of the four nuclei that are later to preside over the spores before there is any indication that such nuclear division is to take place?" Bradley Moore Davis, 1899.