Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

Promotion of Pylaisiella selwynii growth and gametophore formation by octopine and cytokinin

Growth, branching and gametophore formation by protonema developing from spores of the moss Pylaisiella selwynii were promoted by octopine, an unusual amino acid found in crown gall tumors. Cytokinin (0.01 μ M ) in combination with octopine (0.1 μ M to 1 m M ) increased the number of gametophores formed and decreased the time required for their development. The combined effect of these two compounds was similar to that obtained with virulent agrobacteria. At higher cytokinin concentrations, protonemal buds formed abnormal masses of cells rather than normal gametophores. Common amino acids and auxins, alone or in combination with cytokinin, had relatively little effect on the development of the moss.     

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems

Members of the class 1 knotted-like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.     

Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division

Development of Physcomitrella patens (Hedw.) B.S.G. starts with a filamentous protonema growing by apical cell division. As a developmental switch, some subapical cells produce three-faced apical cells, the so-called buds, which grow to form leafy shoots, the gametophores. Application of cytokinins enhances bud formation but no subsequent gametophore development in several mosses. We used the ipt gene of Agrobacterium tumefaciens, encoding a protein which catalyzes the rate-limiting step in cytokinin biosynthesis, to transform two developmental Physcomitrella mutants. One mutant (P24) was defective in budding (bud) and thus did not produce three-faced cells, while the other one (PC22) was a double mutant, defective in plastid division (pdi), thus possessing at the most one giant chloroplast per cell, and in gametophore development (gad), resulting in malformed buds which could not differentiate into leafy gametophores. Expression of the ipt gene rescued the mutations in budding and in plastid division but not the one in gametophore development. By mutant rescue we provide evidence for a distinct physiological difference between externally applied and internally produced cytokinins. Levels of immunoreactive cytokinins and indole-3-acetic acid were determined in tissues and in culture media of the wild-type moss, both mutants and four of their stable ipt transformants. Isopentenyl-type cytokinins were the most abundant cytokinins in Physcomitrella, whereas zeatin-type cytokinins, the major native cytokinins of higher plants, were not detectable. Cytokinin as well as auxin levels were enhanced in ipt transgenics, demonstrating a cross-talk between both metabolic pathways. In all genotypes, most of the cytokinin and auxin was found extracellularly. These extracellular pools may be involved in hormone transport in the non-vascular mosses. We suggest that both mutants are defective in signal-transduction rather than in cytokinin metabolism. Received: 24 October 1997 / Accepted: 20 March 1998     

Major transitions in the evolution of early land plants: a bryological perspective

Background Molecular phylogeny has resolved the liverworts as the earliest-divergent clade of land plants and mosses as the sister group to hornworts plus tracheophytes, with alternative topologies resolving the hornworts as sister to mosses plus tracheophytes less well supported. The tracheophytes plus fossil plants putatively lacking lignified vascular tissue form the polysporangiophyte clade. Scope This paper reviews phylogenetic, developmental, anatomical, genetic and paleontological data with the aim of reconstructing the succession of events that shaped major land plant lineages. Conclusions Fundamental land plant characters primarily evolved in the bryophyte grade, and hence the key to a better understanding of the early evolution of land plants is in bryophytes. The last common ancestor of land plants was probably a leafless axial gametophyte bearing simple unisporangiate sporophytes. Water-conducting tissue, if present, was restricted to the gametophyte and presumably consisted of perforate cells similar to those in the early-divergent bryophytes Haplomitrium and Takakia. Stomata were a sporophyte innovation with the possible ancestral functions of producing a transpiration-driven flow of water and solutes from the parental gametophyte and facilitating spore separation before release. Stomata in mosses, hornworts and polysporangiophytes are viewed as homologous, and hence these three lineages are collectively referred to as the 'stomatophytes'. An indeterminate sporophyte body (the sporophyte shoot) developing from an apical meristem was the key innovation in polysporangiophytes. Poikilohydry is the ancestral condition in land plants; homoiohydry evolved in the sporophyte of polysporangiophytes. Fungal symbiotic associations ancestral to modern arbuscular mycorrhizas evolved in the gametophytic generation before the separation of major present-living lineages. Hydroids are imperforate water-conducting cells specific to advanced mosses. Xylem vascular cells in polysporangiophytes arose either from perforate cells or de novo. Food-conducting cells were a very early innovation in land plant evolution. The inferences presented here await testing by molecular genetics.     

Alternation of generations – unravelling the underlying molecular mechanism of a 165‐year‐old botanical observation

Characteristically, land plants exhibit a life cycle with an ‘alternation of generations’ and thus alternate between a haploid gametophyte and a diploid sporophyte. At meiosis and fertilisation the transitions between these two ontogenies take place in distinct single stem cells. The evolutionary invention of an embryo, and thus an upright multicellular sporophyte, in the ancestor of land plants formed the basis for the evolution of increasingly complex plant morphologies shaping Earth's ecosystems. Recent research employing the moss Physcomitrella patens revealed the homeotic gene BELL1 as a master regulator of the gametophyte‐to‐sporophyte transition. Here, we discuss these findings in the context of classical botanical observations.     

A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.     

Embryology of Ceratopteris richardii (Pteridaceae, tribe Ceratopterideae), with emphasis on placental development

This comprehensive study of early embryology in Ceratopteris richardii combines light microscopy with the first ultrastructural evaluation of any pteridophyte embryo. Emphasis is placed on ontogeny of the foot and placental transfer cells. The embryology of C. richardii shares many similarities with that of other polypodiacious ferns while exhibiting distinctive division patterns. Formative embryonic stages have been reconstructed into three-dimensional models for ease of interpretation. The zygote divides perpendicular to the gametophyte plane and anterioposterior axis. This division establishes a prone embryological habit that maximizes rapid independent establishment of a leaf-root axis in a cordate gametophyte. After the formation of a globular eight-celled stage, initials of the first leaf, and root and shoot apical meristems are defined early by discrete formative divisions. Concomitantly, the foot expands and differentiates to transport nutrients from the gametophyte for the developing embryonic organs. Transfer cell wall ingrowth deposition begins in the gametophyte placental cells before the adjacent sporophyte cells just after the eight-celled stage. These observations provide an anatomical framework for future comparative developmental genetic studies of embryogenesis in free-sporing plants.     

The role of cytokinin in ovule development in Arabidopsis

The life cycle of higher plants alternates between the haploid gametophyte and diploid sporophyte. The female gametophyte (FG), surrounded by the sporophyte, develops within the ovule and orients along the chalazal/micropylar axis. This polarity is important in cell specification and development for both the ovule and FG. Previously, cytokinin was shown to act in the sporophytic tissue to regulate FG development.^1,2 In the highlighted study,³ we further showed that enriched cytokinin signaling in chalaza, the central domain of the ovule, is required for the specification of the functional megaspore, which usually occurs in the chalazal-most megaspore after meiosis. The restricted cytokinin signaling in the chalaza is achieved by localized cytokinin biosynthesis and perception. Here, we discuss the implications of this and other studies for the understanding of the role of two-component signaling in FG development and the genetic and cellular interactions between gametophytic and sporophytic cells. Further, we show that cytokinin-deficient mutants display distorted cell morphology in the inner integument and elevated mitotic activity in the maternal sporophyte. These results suggest that cytokinin negatively regulates cell proliferation in the sporophytic tissues surrounding the developing FG, consistent with previous results indicating that cytokinin deficiency causes an increase in the number of cells in the embryos and consequently an enlarged seed size.     

THE REGULATION OF ALTERNATION OF GENERATION IN FLOWERING PLANTS

The developmental changes involved in the alternation of generation represent the major gene-switching events in the life history of plants. While a large number of genes are common to both sporophyte and gametophyte, many thousand sequences are specifically expressed in each generation; indeed, certain key constituents (e.g. tubulin) are encoded by different genes in each generation, indicating that sporophyte and gametophyte are responding to different evolutionary pressures. Evidence is accumulating that major gene-switching events in plants, such as flowering, are regulated by complex control systems which ensures that development occurs only in the correct groups of cells at the appropriate time. A similar, or more sophisticated system might thus be expected to regulate alternation of generation. It is not possible to manipulate alternation of generation in a similar fashion to flowering, but study of apparent aberrations of development occurring in nature and in vitro suggests that alternation only occurs in cells which have become competent to receive particular developmental stimuli. Further, in certain cases, competent cells may be switched either into sporophytic or gametophytic developmental pathways depending upon the nature of the stimulus. Acquisition of competence seems to involve isolation of cells from the symplast, some cytoplasmic dedifferentiation, and perhaps cell cycle arrest or transition. The stimuli in vivo appear metabolic in nature, although embryogenesis may be activated by specific classes of glycoproteins. Interestingly, examination of agamospermic systems suggests that fertilization of the egg per se is not the signal which activates sporophytic development. Once competent cells have received the stimulus they start to develop, with no delay in a ‘determined’ state. Sporophytic and gametophytic development in vivo and in vitro both start with an asymmetric division, except for the female gametophyte which may arise via a range of developmental pathways, depending on the species.     

A fundamental plant evolutionary problem: the origin of land-plant sporophyte; is a new hypothesis possible?

The origin of the sporophyte in land plants represents a fundamental phase in the plant evolution. Today this subject is controversial and, in my opinion, scarcely considered in our textbooks and journals of botany, in spite of its importance. There are two conflicting theories concerning the origin of the alternating generations in land plants: the "antithetic" and the "homologous" theory. These have never been fully resolved. The antithetic theory maintains that the sporophyte and gametophyte generations are fundamentally dissimilar and that the sporophyte originated in an ancestor organism with haplontic cycle by the zygote dividing mitotically rather than meiotically, and with a developmental pattern not copying the developmental events of the gametophyte. The sporophyte generation was an innovation of critical significance for the land-plant evolution. By contrast, the homologous theory simply stated that a mass of cells forming mitotically from the zygote adopted the same developmental plan of the gametophyte, but giving origin to a diploid sporophyte. In this context, a very important question concerns the possible ancestor or ancestors of the land plants. Considerable evidences at morphological, cytological, ultrastructural, biochemical and, especially, molecular level, strongly suggest that the land plants or Embryophyta (both vascular and non-vascular) evolved from green algal ancestor(s), similar to those belonging to the genus Coleochaete, Chara and Nitella, living today. Their organism is haploid for most of their life cycle, and diploid only in the zygote phase (haplontic cycle). On the contrary, the land plants are characterized by a diplo-haplontic life cycle. Several questions are implied in these theories, and numerous problems remain to be solved, such as, for example, the morphological difference between gametophyte and sporophyte (heteromorphism, already present in the first land plants, the bryophytes), and the strong gap existing between these last with a sporophyte dependent on the gametophyte, and the pteridophytes having the gametophyte and sporophyte generations independent. On the ground of all of the evidences on the ancestors of the land plants, the antithetic theory is considered more plausible than the homologous theory. Unfortunately, no phylogenetic relationship exists between some green algae with diplontic life cycle and the land plants. Otherwise, perhaps, it should be possible to hypothesize another scenario in which to place the origin of the alternating generations of the land plants. In this case, could the gametophyte be formed by gametes produced from the sporophyte, through their mitoses or a delayed fertilization process?     

Inhibition of cell division in a moss protonema by some cytokinin isomers

Summary The cytokinin isomers, 1- and 9-isopentenyladenine, are very active inhibitors of cell division in the protonema of the moss Ceratodon purpureus. They also abolish the N⁶-isopentenyladenine-induced stimulation of cell divisions in this material whereas they are inactive on the induction of gametophore buds in moss protonemata by cytokinins.This work was supported by the Polish Academy of Sciences within the project 09.3.1.     

岩蕨属植物配子体发育初报

本文为岩蕨属植物心岩蕨、岩蕨和耳羽岩蕨配子体个体发育和比较形态学初步研究。报道了用无机琼脂培养基由孢子培养配子体的方法。由孢子萌发到配子体成熟分四个时期:萌发期,原丝体发育期,过渡期和原叶体发育期。大多数原丝体发育到七个细胞,与顶端细胞相隔的原丝体细胞开始胞间细胞纵向分裂,进入过渡期。同时顶端细胞的横分裂受到抑制,直至原叶体顶端细胞形成开始原叶体发育期。岩蕨成熟配子体精子器盖细胞不分裂,耳羽岩蕨分裂为一个圆形细胞和一个镰刀形细胞,而心岩蕨两种情况兼而有之,配子体比较形态特征支持心岩蕨可能是以岩蕨和耳羽岩蕨为双亲的异源双二倍体的假设[5]。