Genesis of cells of apical meristems and realization of gametophytic apomixis in flowering plants

Based on our own and literature data on peculiarities of caryotypical variability, we concluded that gametophytic apomixis is naturally accompanied with phenomena of poly-, aneu-, and mixoploidy and that apomicts have genome instability manifesting at the level of meristematic somatic cells. In this connection, a hypothesis is substantiated that realization of this mode of seed reproduction in flowering plants is caused by modification of systems of cell cycle control, following after acts of hybridogenesis and/or polyploidization. It is concluded that instability of the seed reproduction system by gametophytic apomixis manifests not only at the stage of choice of a seed reproduction pathway (apomeiosis-euspory; apozygosis-zygosis) but also in all the cycles of reproduction of the cells of a germ line in plant ontogenesis.     

Evolution of gametophytic apomixis in flowering plants: an alternative model from Maloid Rosaceae

Gametophytic apomixis, asexual reproduction involving megagametophytes, occurs in many flowering-plant families and as several variant mechanisms. Developmental destabilization of sexual reproduction as a result of hybridization and/or polyploidy appears to be a general trigger for its evolution, but the evidence is complicated by ploidy-level changes and hybridization occurring with facultative apomixis. The repeated origins of polyploid apomictic complexes in the palaeopolyploid Maloid Rosaceae suggest a new model of evolutionary transitions that may have wider applicability. Two conjectures are fundamental to this model: (1) that as previously suggested by Rutishauser, like many sexual flowering plants the polyploid apomicts require maternal–paternal balance in the second fertilization event that gives rise to the endosperm, and (2) that the observed variation in endosperm ploidy levels relates less to flexibility late in development than to the known variation in developmental origin of the megagametophyte between mechanisms loosely categorized as diplospory and apospory. The model suggests explanations for the relative frequencies of apospory and diplospory, and for the wide but incomplete associations of apospory with a pollination requirement (pseudogamy) and of diplospory with autonomous development of the endosperm. It is suggested that pollination from other taxa may provide some adaptive advantage to pseudogamous apospory. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytogenetic peculiarities of cell genesis in apical meristems under gametophytic apomixis (using autonomous apomicts of the Asteraceae as an example)

Cytogenetic peculiarities of cell genesis in apical meristems of apomicts has been analyzed using a series of the Asteraceae species as an example. The extent to which aneu- and mixoploids are spread among plants in the investigated populations of the Asteraceae species is so high (up to 30-60% of the studied plants and their offspring), that it seems reasonable to suppose that their rise is a natural phenomenon. It has been shown that in the aposporous facultative apomict Pilosella officinarum microgametophyte is a relatively stable element of the seed reproduction system from the point of view of caryotypical variation.     

Gibberellins in apical shoot meristems of flowering and vegetative sugarcane

Gibberellins A₁, A₃, iso-A₃, A₄, A₁₉, A₂₀, and A₃₆ were identified by gas chromatography-selected ion monitoring in apices of sugarcane (Saccharum spp. hybrids). Flowering apices (i.e., 2–4 cm panicle) contained 8–9 times more (estimated by bioassay) endogenous gibberellins A and iso-GA₃ (ratio of 1:6:8, respectively; in total 51 ng g^–1 fresh weight) than vegetative apices (6.4 ng g^–1 fresh weight). Vegetative apices contained small but significant levels of GA₁₉, which could not be detected in flowering apices; vegetative apices also contained approximately four times more of a GA₃₆-like substance than flowering apices. Since the two apex types developed under the same photoperiod, the increased levels of GA and iso-GA₃ and the reduced levels of GA₁₉ and GA₃₆-like substances are correlated with the flowering state rather than with photoperiod or photoperiod changes per se. Since there were relatively high levels of C₁₉ GAs along with low levels of C₂₀ GAs in flowering apices, and since the converse is true in vegetative apices, metabolism of C₂₀ to C₁₉ GAs may be enhanced in flowering apices.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Is supernumerary chromatin involved in gametophytic apomixis of polyploid plants?

Gametophytic apomixis, or unreduced embryo sac development that results in asexual reproduction through seeds, occurs in several families of angiosperms and must be polyphyletic in origin. The molecular mechanisms underlying gametophytic apomixis have not been discovered and are the subject of intense investigation. A common feature of almost all apomicts is their polyploid nature. From genetic mapping studies in both monocots and dicots, there is low genetic recombination associated with a single (rarely two), dominant locus for either aposporous or diplosporous embryo sac formation. In Pennisetum squamulatum and Cenchrus ciliaris, some DNA sequences mapping to the apospory locus are unique to apomictic genotypes and apparently hemizygous. This sequence divergence at the apomixis locus could be a consequence of genome rearrangements and isolation from genetic recombination, both of which may have contributed to the definition of a chromosomal region as supernumerary. The possible involvement of supernumerary chromatin, formed as a result of interspecific hybridization, in the origin of apomixis, is explored here. Received: 26 October 2000 / Revision accepted: 5 April 2001     

Developmental genetics of gametophytic apomixis

Some higher plants reproduce asexually by apomixis, a natural way of cloning through seeds. Apomictic plants produce progeny that are an exact genetic replica of the mother plant. The replication is achieved through changes in the female reproductive pathway such that female gametes develop without meiosis and embryos develop without fertilization. Although apomixis is a complex developmental process, genetic evidence suggests that it might be inherited as a simple mendelian trait - a paradox that could be explained by recent data derived from apomictic species and model sexual organisms. The data suggest that apomixis might rely more on a global deregulation of sexual reproductive development than on truly new functions, and molecular mechanisms for such a global deregulation can be proposed. This new understanding has direct consequences for the engineering of apomixis in sexual crop species, an application that could have an immense impact on agriculture.     

Beyond promiscuity: From sexuality to apomixis in flowering plants

Summary Little is known about the genetic basis and molecular mechanisms regulating female gametogenesis in flowering plants. In many species sexuality is replaced by apomixis, a method of asexual reproduction that circumvents female meiosis and fertilization, and culminates in the formation of clonal seeds. Using a new generation of transposon based insertional mutagenesis strategies and their resulting molecular tools, we are investigating how female meiotically derived cells (megaspores) acquire their identity. We are also determining their function and interactions, and attempting the induction of apomixis initiation in the ovule of Arabidopsis. This basic knowledge will contribute to establish the transfer of apomixis into sexual crops, a major challenge faced by plant biotechnology. The introduction of apomixis as a reproductive alternative could represent a unique opportunity to simplify breeding schemes and genetically perpetuate any desired heterozygous genotype, including hybrids.     

Cytogenetic peculiarities of the genesis of apical meristem cells in case of gametophytic apomixis (with reference to autonomous Asteraceae apomicts)

The cytogenetic peculiarities of the genesis of apical meristem cells in apomicts were analyzed using some Asteraceae species as an example. It has been revealed that the frequency of aneu- and mixoploids is so high among the plants of these species (up to 30–60% of all plants studied or their progeny) that there is every reason to say that their occurrence in apomicts is regular rather than spontaneous. It has been demonstrated that microgametophyte in aposporous facultative apomict Pilosella officinarum is a relatively stable element of the seed reproduction system in terms of the ploidy level.     

The significance of apical meristems in the phylogeny of land plants

Two principal types of shoot apex are recognized, (1) the classical pyramidal apical cell of pteridophytes which does not divide periclinally, and so never contributes directly to the inner tissues of the axis, and (2) that of seed plants, in which there are one or more superposed initials of which the innermost contributes directly to inner tissues by periclinal divisions. The occurrence of these types in the major groups of living land plants is reviewed. The first type is present in ferns,Equisetum, Psilotum, Tmesipteris, Selaginella, and bryophytes (when apical growth occurs in them). The second type occurs in all angiosperms and gymnosperms, and also inLycopodium, Phylloglossum, Isoetes, andStylites. No exceptions nor intermediate conditions occur. Both types of apex are known in Carboniferous fossils, the first type in sphenopsids and the second in pteridosperms and cordaitean gymnosperms. These apices define two evolutionary lines of land plants which may have originated separately as sporophytes from ancestors with no diploid generation. Correlation of these types with the following features is considered: sieve-element inclusions; initiation of leaves and sporangia; the primary vascular system; embryogeny, the vascular cambium; initiation of lateral roots and the type of branching. Seed plants and pteropsids differ in respect of all these features, giving support to the hypothesis that the sporophytes of these two groups arose independently. The position of sphenopsids and lycopsids is also considered.     

Regeneration of whole plants from apical meristems of Pinus radiata

A methodology to regenerate whole plants of Pinus radiata from apical meristems of 3- and 7-year-old trees was developed. Meristematic domes with two or three leaf primordia were excised from surface-sterilized branch tips of field-grown plants and cultured in LP medium with half strength macronutrients (1/2 LP) and full strength micronutrients. The early growth of meristems required approximately 12 weeks, including a recovery stage during the first 2 weeks. After 8 weeks, some meristems developed abnormal phenotypes and died during the subsequent stages of development. However, healthy meristems elongated and formed shoots when they were transferred to LP medium supplemented with MS vitamins, 30 mg l^–1 casein hydrolysate, and 0.4 g l^–1 agar plus 2.85 g l^–1 Gelrite. Meristems that developed vigorous shoots were used for rooting experiments when they were 2 cm in length. Whole plants were obtained after 5 days of root induction in water-agar medium containing 8.2 M IBA and 5.4 M NAA and 1 month culture in LP medium with 10 g l^–1 sucrose. Plants regenerated from meristems were further propagated by rooting of cuttings. Of the rooted cuttings, 10% were morphologically juvenile.     

Plant stem cells and their regulations in shoot apical meristems

Stem cells in plants, established during embryogenesis, are located in the centers of the shoot apical meristem (SAM) and the root apical meristem (RAM). Stem cells in SAM have a capacity to renew themselves and to produce new organs and tissues indefinitely. Although fully differentiated organs such as leaves do not contain stem cells, cells in such organs do have the capacity to re-establish new stem cells, especially under the induction of phytohormones in vitro. Cytokinin and auxin are critical in creating position signals in the SAM to maintain the stem cell organizing center and to position the new organ primordia, respectively. This review addresses the distinct features of plant stem cells and focuses on how stem cell renewal and differentiation are regulated in SAMs.     

Volume-based pollen size analysis: an advanced method to assess somatic and gametophytic ploidy in flowering plants

Pollen size is often used as a biological parameter to estimate the ploidy and viability of mature pollen grains. In general, pollen size quantification is performed one- or two-dimensionally using image-based diameter measurements. As these approaches are elaborate and time consuming, alternative approaches that enable a quick, reliable analysis of pollen size are highly relevant for plant research. In this study, we present the volume-based particle size analysis technique as an alternative method to characterize mature pollen. Based on a comparative assay using different plant species (including tomato, oilseed rape, kiwifruit, clover, among others), we found that volume-based pollen size measurements are not biased by the pollen shape or position and substantially reduce non-biological variation, allowing a more accurate determination of the actual pollen size. As such, volume-based particle size techniques have a strong discriminative power in detecting pollen size differences caused by alterations in the gametophytic ploidy level and therefore allow for a quick and reliable estimation of the somatic ploidy level. Based on observations in Arabidopsis thaliana gametophytic mutants and differentially reproducing Boechera polyantha lines, we additionally found that volume-based pollen size analysis provides quantitative and qualitative data about alterations in male sporogenesis, including aneuploid and diploid gamete formation. Volume-based pollen size analysis therefore not only provides a quick and easy methodology to determine the somatic ploidy level of flowering plants, but can also be used to determine the mode of reproduction and to quantify the level of diplogamete formation.     

Presumable role of plasmodesmata in floral signal transduction in shoot apical meristems of rudbeckia and perilla plants

The number of plasmodesmata was calculated per 1 μm of cell wall length in the central and medullar zones of shoot apical meristems (SAM) in the course of floral transition in a long-day (LD) plant Rudbeckia bicolor Nutt. and a short-day plant Perilla nankinensis Lour. Under the day length unfavorable for flowering (control), the numbers of plasmodesmata differed in the central and medullar zones of SAM, which produce the reproductive organs and stems, respectively. Besides, the numbers of plasmodesmata in the central zone of perilla SAM considerably differed between the anticlinal and periclinal cell walls of the first and second cell layers. Following the photoperiodic induction (PI) with eight LD in rudbeckia and twelve SD in perilla favorable for floral transition, the numbers of plasmodesmata considerably increased in the anticlinal and periclinal cell walls of the first and second cell layers of the central zone; meanwhile in the medullar zone, the numbers of plasmodesmata dropped down following PI. These data show that floral transition presumably involves the activation of cell-to-cell interactions and enhances the signal transduction in SAM.     

Stem cell maintenance in shoot apical meristems

Stem cell homeostasis in shoot apical meristems of higher plants is regulated through a dynamic balance between spatial regulation of gene expression, cell growth patterns and patterns of differentiation. Cell-cell communication mediated by both the local factors and long-range signals have been implicated in stem cell homeostasis. Here we have reviewed recent developments on spatio-temporal regulation of cell-cell communication processes with an emphasis on how ubiquitously utilized signals such as plant hormones function with local factors in mediating stem cell homeostasis. We also provide a brief overview of how the activity of ubiquitously utilized epigenetic regulators are modulated locally to orchestrate gene expression.     

Hetrogeneity of nuclear volumes and interphase nuclear growth in cells of root apical meristems

Nuclear volume distributions from proliferative- and stationary-phase meristems of pea roots have been compared and shown to be almost identical. A wide range of nuclear volumes is also found within cells of the same interphase age, specifically early G1 cells and late G2 cells, further indicating that nuclear volume cannot be used to estimate interphase age. The average increase in nuclear volume is approx. 2.5-fold, and all nuclei of the meristem, whatever their starting volume, increase by the same factor. Sister nuclei have very similar volumes in early G1, indicating that asymmetric mitoses within the meristem do not contribute significantly to the heterogeneity found.     

Cell lineage dynamics in stratified shoot apical meristems

We devise a stochastic and spatially explicit model for the dynamics of the initials cells in a stratified shoot apical meristem (SAM). The meristem is composed of three layers with seven initials per layer. We investigate the probability and number of divisions for a mutant lineage to either reach fixation or becoming purged through selection or drift. In contrast to previous studies our results show that the functional organization of the initials in stratified SAMs acts as an efficient purging mechanism particularly of deleterious mutations. All mutants are rapidly purged when deleterious. The probability of fixation for mutants with a higher fitness than the wild type increases linearly up to 70%. The median number of divisions to fixation of both genotypes is insensitive to the mutant's fitness. The median number of divisions to wild-type fixation is less than 100, with the upper quartile below 200. The largest number of divisions to wild-type fixation are in the order of 100 000 divisions. Our results indicate that the spatial organization of SAM enables the efficient purging of mutant lineages, particularly if they are deleterious. On the other hand, long-lived chimeric stages are common when mutant lineages succeed to overcome the initial numerical disadvantage.     

Specific proteins in stem meristems during transition to flowering

Immunodiffusion tests were used for studying protein composition of apical buds ofRudbeckia bicolor andPerilla nankinensis during their transition from vegetative to reproductive state under inductive photoperiodic conditions or GA₃ treatment. In both species the induced buds differ from the vegetative ones in the presence of specific proteins (P): P1, P2, P3 appear inRudbeckia apical buds 2, 8, 16 d after the start of inductive treatment; P4 appears inPerilla apical buds 6 d after inductive treatment. P1, P2, P4 are revealed in induced buds in the early period of apex development when morphogenetic changes are not yet present. The similarity between antigenic spectra of induced buds and of those treated by GA₃ appears only inRudbeckia. These observations support the hypothesis of a change in gene expression at floral evocation. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Cryopreservation of in vitro-grown apical meristems of lily by vitrification

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige & Skoog (1962) medium - PVS2 vitrification solution