<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis> homologs are linked to the expression of lateral dominance in the acaulescent <Emphasis Type="Italic">Streptocarpus rexii</Emphasis> (Gesneriaceae)

Acaulescent species of Streptocarpus Lindl. show unusual patterns of growth, characterized by anisocotyly (i.e. the unequal growth of cotyledons after germination) and lack of a conventional embryonic shoot apical meristem (SAM). A SAM-like structure appears during post-embryonic development on the axis of the continuously growing cotyledon. Since we have shown previously that KNOX genes are involved in this unusual morphology of Streptocarpus rexii, here we investigated the expression pattern of WUSCHEL (WUS), which is also required for the indeterminacy of the SAM, but is expressed independently from KNOX in Arabidopsis thaliana. In A. thaliana WUSCHEL is involved in the maintenance of the stem cell fate in the organizing centre. The expression pattern of the WUS ortholog in S. rexii (SrWUS) strongly deviates from that of the model plant, suggesting a fundamentally different spatial and temporal regulation of signalling involved in meristem initiation and maintenance. In S. rexii, exogenous application of growth regulators, i.e. gibberellin (GA₃), cytokinin (CK) and a gibberellin biosynthesis inhibitor (PAC), prevents anisocotyly and relocates meristematic cells to a position of conventional SAMs; this coincides with a re-localization of the two main pathways controlling meristem formation, the SrWUS and the KNOX pathways. Our results suggest that the establishment of a hormone imbalance in the seedlings is the basis of anisocotyly, causing a lateral dominance of the macrocotyledon over the microcotyledon. The peculiar morphogenetic program in S. rexii is linked to this delicate hormone balance and is the result of crosstalk between endogenous hormones and regulatory genes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Developmental analyses of the phyllomorph formation in the rosulate species Streptocarpus rexii (Gesneriaceae)

Although some species of Streptocarpus (Gesneriaceae) do not possess a layered shoot apical meristem (SAM), but three individual meristems, the basal meristem (BM), the petiolode meristem (PM) and the groove meristem (GM) on the petiolode from which additional phyllomorphs are formed. To gain insights into the processes involved, we examined the development of seedlings from germination to the formation of the primary phyllomorph in S. rexii, a rosulate species. Our specific focus was to examine the relationship between the functional activity of the GM and meristematic activity, which was assessed by a combined analysis of toluidine blue staining of histological sections and the incorporation of BrdU into meristematic tissues. The results were integrated into 3-D graphics, which suggests a complex spatial and temporal interaction within the GM. The significance of our observations is discussed and compared to the SAM observed in most other angiosperms.     

HORMONAL REGULATION OF MORPHOGENESIS IN STREPTOCARPUS AND ITS RELEVANCE TO EVOLUTIONARY HISTORY OF THE GESNERIACEAE

Two morphogenetic patterns have contributed to phylogenetic diversification within the Gesneriaceae: accrescence of one of the paired cotyledons (anisocotyly), which serves to differentiate the subfamily Cyrtandroideae; sustained growth of the accrescent cotyledon accompanied by prolonged suppression and displacement of the embryonic apical meristem, which gives rise to an acaulescent, dorsiventral vegetative plant body (phyllomorph) and further serves to differentiate species of Cyrtandroideae found in two tribes and several genera including Streptocarpus. It was possible to prevent cotyledonary accrescence and induce caulescence at will, either by supplying exogenous GA₃ or inhibiting auxin transport in species of Streptocarpus that normally manifest an extreme, phyllomorphic morphology. It was also possible to induce sustained, phyllomorphic development of cotyledons that are normally non-accrescent with exogenous cytokinin. Therefore morphogenetic capacities previously thought to be “lost” or “lacking” in subgenus Streptocarpus and, with respect to isocotyly, the tribe Cyrtandroideae, are, in fact, present but suppressed. An hypothesis regarding the role of hormones with respect to morphogenesis and phylogeny of Streptocarpus is suggested.     

Developmental morphology of one-leaf plant Monophyllaea singularis (Gesneriaceae)

 Developmental morphology is described of the one-leaf plant Monophyllaea singularis which possesses a huge macrocotyledon, a long petiolode below it, and many small inflorescences scattered along the petiolode and midrib. Cell proliferation and basipetal differentiation occur in both cotyledons after water imbibition and germination. The basal meristem forms from a group of small, least differentiated cells at the base of a future macrocotyledon and continues blade production even at the reproductive stage. The petiolode meristem, which forms as an intercalary meristem near the base of the macrocotyledon, contributes to the elongation of the petiolode and the midrib. Although the 'groove meristem', like the groove meristem of Streptocarpus, forms between the cotyledons at the site of a shoot apical meristem, it is not involved in inflorescence production. In M. singularis, instead of the 'groove meristem', the inflorescences are initiated adventitiously from groups of cells in the dermal and subdermal layers of the petiolode and probably also of the midrib. Received October 10, 2000 Accepted August 2, 2001     

GA2 and GA20-oxidase expressions are associated with the meristem position in Streptocarpus rexii (Gesneriaceae)

We examined genes involved in the regulatory pathway of gibberellin (GA) in meristems of Streptocarpus rexii. The plants do not possess a typical shoot apical meristem (SAM) and form unique meristems: the basal meristem extends the lamina area of one cotyledon to produce anisocotylous seedlings; the groove meristem forms new leaves at the base of the macrocotyledon. Exogenous application of GA significantly suppresses the basal meristem activity in developing cotyledons and the seedlings remain isocotyl. To examine the role of endogenous GA on these meristems in vivo, we isolated homologs of GA2-oxidase responsible for degrading active GAs (SrGA2ox), and GA20-oxidase regulating the rate limiting step of active GA synthesis (SrGA20ox). During embryogenesis, while first partly overlapping, the expression of SrGA2ox and SrGA20ox became more differentiated and mutually exclusive, ending with SrGA2ox being expressed solely in the adaxial–proximal domain of the embryo in regions with meristem activity, whereas SrGA20ox was restricted to the fork between the two cotyledons. The latter may be responsible for suppressing the formation of an embryonic SAM in S. rexii. In developing seedlings, SrGA2ox expression also followed the centers of meristem activity, where SrGA20ox expression was excluded. Our results suggest that low levels of GA are required in S. rexii meristems for their establishment and maintenance. Thus, the meristems in S. rexii share similar regulatory pathways suggested for the SAM in model plants, but that in S. rexii evolutionary modifications involving a lateral transfer of function, from shoot to leaves, is implicated in attaining the unusual morphology of the plants.     

Modulating zucchini cotyledon plate meristem activity by interactions between the cycline-dependent kinase inhibitor roscovitine and cytokinins

The inhibitors of cytokinin N-glucosylation are known to influence the growth of some plant objects including cotyledons. The use of the plate meristem of zucchini cotyledon as an experimental system allowed us to study for the first time the way in which the changes in the cell division are integrated in this growth reaction. Roscovitine, a potent inhibitor of cytokinin N-glucosylation and cycline-dependent kinases, did not show to have an effect on the meristem activity when applied in 100 μM to cultivated zucchini cotyledons, and acted as an inhibitor in concentrations higher than 400 μM. A 200 μM roscovitine stimulated both palisade cell division and growth. In different seed batches, 400 μM roscovitine acted as a stimulator or an inhibitor. A much stronger stimulating effect on growth and cell division was observed after application of benzyladenine (BA, 10 μM). In contrast to BA, roscovitine provoked a formation of principally flat lamina. In combined treatments, it lowered the stimulating effect of BA; 400 μM roscovitine combined with BA severely suppressed the growth and division activity. This cellular behavior and changes in cotyledon growth could be due to the roscovitine-provoked changes in endogenous cytokinin levels via the inhibition of cytokinin N-glucosylation. Roscovitine-caused stimulation of cell growth and division is stronger in the marginal meristem than that registered in central regions of the cotyledon blade. In this region it also changed the pattern of cell division and lowered the adhesion between the clusters, which enhanced the appearance of local ruptures of the cotyledon edges. The first palisade layer of the plate meristem of cultured zucchini cotyledons, the natural mono-layer of proliferating palisade cells, may be used for screening the inhibitors of cycline-dependent kinases and cytokinin N-glucosylation with regard to their effects on cell division and growth.     

EVOLUTION OF MORPHOLOGICAL NOVELTY: A PHYLOGENETIC ANALYSIS OF GROWTH PATTERNS IN STREPTOCARPUS (GESNERIACEAE)

Abstract.— Streptocarpus shows great variation in vegetative architecture. In some species a normal shoot apical meristem never forms and the entire vegetative plant body may consist of a single giant cotyledon, which may measure up to 0.75 m (the unifoliate type) or with further leaves arising from this structure (the rosulate type). A molecular phylogeny of 87 taxa (77 Streptocarpus species, seven related species, and three outgroup species) using the internal transcribed spacers and 5.8S region of nuclear ribosomal DNA suggests that Streptocarpus can be divided into two major clades. One of these broadly corresponds to the caulescent group (with conventional shoot architecture) classified as subgenus Streptocarpella, whereas the other is mainly composed of acaulescent species with unusual architecture (subgenus Streptocarpus). Some caulescent species (such as S. papangae) are anomalously placed with the acaulescent clade. Available cytological data are, however, completely congruent with the two major clades: the caulescent clade is x = 15 and the acaulescent clade (including the caulescent S. papangae) is x = 16 (or polyploid multiples of 16). The genera Linnaeopsis, Saintpaulia, and Schizoboea are nested within Streptocarpus. The sequenced region has evolved, on average, 2.44 times faster in the caulescent clade than in the acaulescent clade and this is associated with the more rapid life cycle of the caulescents. Morphological variation in plant architecture within the acaulescent clade is homoplastic and does not appear to have arisen by unique abrupt changes. Instead, rosulate and unifoliate growth forms have evolved several times, reversals have occurred, and intermediate architectures are found. An underlying developmental plasticity seems to be a characteristic of the acaulescent clade and is reflected in a great lability of form.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Embryonic shoot apical meristem formation in higher plants

The shoot apical meristem (SAM) is essential for organ formation in higher plants. How the SAM is formed during plant development is poorly understood, however. In this review, we focus on several recent studies that provide new insights into the mechanism of SAM formation during embryogenesis. Recently, positive and negative regulators of the class I KNOX genes, which are thought to be necessary for SAM formation, have been identified; the Arabidopsis CUP-SHAPED COTYLEDON (CUC) genes are required for the expression of a class I KNOX gene, SHOOT MERISSTEMLES (STM) during embryogenesis, and the Arabidopsis ASYMMETRIC LEAVES1 (AS1), AS2, and several other genes negatively regulate KNOX gene expression in cotyledon primordia. Also, several genes that are involved in the formation of the adaxial–abaxial axis of cotyledons seem to regulate embryonic SAM formation. Electronic Publication     

<Emphasis Type="Italic">MIR166</Emphasis>/<Emphasis Type="Italic">165</Emphasis> genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The miR166/165 group and its target genes regulate diverse aspects of plant development, including apical and lateral meristem formation, leaf polarity, and vascular development. We demonstrate here that MIR166/165 genes are dynamically controlled in regulating shoot apical meristem (SAM) and floral development in parallel to the WUSCHEL (WUS)-CLAVATA (CLV) pathway. Although miR166 and miR165 cleave same target mRNAs, individual MIR166/165 genes exhibit distinct expression domains in different plant tissues. The MIR166/165 expression is also temporarily regulated. Consistent with the dynamic expression patterns, an array of alterations in SAM activities and floral architectures was observed in the miR166/165-overproducing plants. In addition, when a MIR166a-overexpressing mutant was genetically crossed with mutants defective in the WUS-CLV pathway, the resultant crosses exhibited additive phenotypic effects, suggesting that the miR166/165-mediated signal exerts its role via a distinct signaling pathway.     

Is the wild oat embryo monocotylous?

The embryogeny of the wild oat (Avena fatua L.) was studied in detail. The pattern of embryo development was observed to be similar to the other investigated grass taxa, conforming to thePoa variation of the Asterad type. The embryogeny and anatomy of young seedlings showed that the embryo of the wild oat was not monocotylous, but dicotylous. The scutellum of the embryo, as reported for other grasses, was regarded as the first cotyledon, and the first leaf primordium, which developed later into a photosynthesizing leaf and situated opposite the scutellum, was interpreted as the second cotyledon. Observations indicated that the cotyledons of the embryo were placed lateral to the shoot apical meristem, which was terminal in position. The cotyledons were found to be dimorphic in structure and function. The scutellum, a modified cotyledon, functioned as a suctorial organ, transporting nutrients from the endosperm to the embryo axis. The second cotyledon or the first true leaf supplied nutrients directly to the embryo axis through the process of photosynthesis.     

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic <Emphasis Type="Italic">6b</Emphasis> gene of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.     

Pepper (<Emphasis Type="Italic">capsicum annuum</Emphasis> L.) regenerants obtained by direct somatic embryogenesis fail to develop a shoot

Summary Three auxin-type herbicides, namely 2.4-dichlorophenoxyacetic acid (2,4-D), (4-chlorophenoxy)acetic acid 2-(dimethylamino)ethyl ester (centrophenoxine), and quinolinecarboxylic acid (quinclorac) induced direct somatic embryogenesis in seed-derived zygotic embryo explants of sweet pepper (Capsicum annuum L.) when added to Murashige and Skoog medium with 200 mM sucrose. Optimum concentrations for embryogenesis induction were 0.40–0.45 mM and 1.15–1.30 μM for 2.4-D and centrophenoxine, respectively (in the presence of 5.0 gl⁻¹ activated charcoal), or 40 μM for quinclorac (in medium without activated charcoal). Somatic embryos emerged from the epidermal and subepidermal tissues and developed on the surface of the explant. Centrophenoxine- or 2.4-D-mediated embryogenesis was accomplished from 95% of the explants in about 3 wk and, on average, six embryos were formed per explant. Induction efficieney was lower for quinelorac. Centrophenoxine-mediated embryognesis was possible in 10 pepper cultivars, the extent of the reponse-being genotype-dependent. embryos detached from the explant and transplanted onto a growth regulator-free medium germinated; however, the recovered regenerants were without a shoot, and some of them bore a single deformed cotyledon while others had no cotyledons. Regenerants lacking a shoot were generated irrespective of the auxin type applied and across all responsive genotypes investigated. Absence of a shoot, resulting from a failure in the establishment of a normal functioning apical shoot meristem, was the principal developmental disorder that precluded regeneration of normal plants via direct somatic embryogenesis. Since stem cells of the shoot meristem become established in globular and heart-stage embryos, we deduce that the absence of a shoot in germinating embryos could orginate from deviant differentiation at these early stages of embryogeny.     

Formation, Maintenance and Function of the Shoot Apical Meristem in Rice

In higher plants, the process of embryogenesis establishes the plant body plan (body axes). On the basis of positional information specified by the body axes, the shoot apical meristem (SAM) and root apical meristem (RAM) differentiate at fixed positions early in embryogenesis. After germination, SAM and RAM are responsible for the development of the above-ground and below-ground parts, respectively, of the plant. Because of the importance of SAM function in plant development, the mechanisms of SAM formation during embryogenesis and of SAM maintenance and function in post-embryonic development are priority questions in plant developmental biology. Recent advances in molecular and genetic analysis of morphogenetic mutations in Arabidopsis have revealed several components required for SAM formation, maintenance and function. Although these processes are fundamental to the life cycle of every plant, conservation of the components does not explain the diversity of plant morphologies. Rice is used as a model plant of the grass family and of monocots because of the progress in research infrastructure, especially the collection of unique mutations and genome information. In comparison with the dicot Arabidopsis, rice has many unique organs or processes of development. This review summarizes what is known of the processes of SAM formation, maintenance and function in rice.     

Inflorescence and floral development in Streptocarpus and Saintpaulia (Gesneriaceae) with particular reference to the impact of bracteole suppression

Floral development and inflorescence structure within Streptocarpus and Saintpaulia were investigated using Scanning Electron Microscopy (SEM). We discuss the structure and development of the pair-flowered cyme and the floral ontogeny found in the Gesneriaceae in a phylogenetic context with particular reference to an East African clade of Streptocarpus and Saintpaulia. Current phylogenetic hypotheses divide the caulescent East African Streptocarpus species into two distinct clades, in relation to which the position of Saintpaulia is not yet clear. Variation in the branching of the inflorescence showed phylogenetic significance and included dichasial, monochasial and unbranched patterns. In four of the East African Streptocarpus species sampled a single lateral bracteole was present on the first to third axes, after which the inflorescence was ebracteolate. Our results indicate that there may be some link between bracteole suppression and an alteration in the order of sepal initiation. The loss or suppression of lateral bracteoles also appears to result in the precocious development of the lateral cyme meristem.     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

SHOOT MERISTEMLESS trafficking controls axillary meristem formation,meristem size and organ boundaries in Arabidopsis

The shoot stem cell niche, contained within the shoot apical meristem (SAM) is maintained in Arabidopsis by the homeodomain protein SHOOT MERISTEMLESS (STM). STM is a mobile protein that traffics cell‐to‐cell, presumably through plasmodesmata. In maize, the STM homolog KNOTTED1 shows clear differences between mRNA and protein localization domains in the SAM. However, the STM mRNA and protein localization domains are not obviously different in Arabidopsis, and the functional relevance of STM mobility is unknown. Using a non‐mobile version of STM (2xNLS‐YFP‐STM), we show that STM mobility is required to suppress axillary meristem formation during embryogenesis, to maintain meristem size, and to precisely specify organ boundaries throughout development. STM and organ boundary genes CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 regulate each other during embryogenesis to establish the embryonic SAM and to specify cotyledon boundaries, and STM controls CUC expression post‐embryonically at organ boundary domains. We show that organ boundary specification by correct spatial expression of CUC genes requires STM mobility in the meristem. Our data suggest that STM mobility is critical for its normal function in shoot stem cell control.     

One-leaf plants in the Gesneriaceae: Natural mutants of the typical shoot system

The aerial part of seed plants is called the shoot, which is composed of stems, leaves, and axial buds. These are produced by indeterminate activity in the shoot apical meristem (SAM), whereas the morphogenesis of leaves depends on determinate activity of leaf meristems. However, one-leaf plants in the Gesneriaceae family (eudicots) do not have a typical SAM and do not produce new organs when in the vegetative phase. Instead, they have one cotyledon whose growth is indeterminate. This peculiar development is supported by the groove meristem, which corresponds to the canonical SAM, and the basal meristem, which corresponds to the typical leaf meristem. However, the former does not produce any organ and the latter is active indeterminately. Gene expression and physiological analyses have been conducted in an effort to determine the molecular nature of this peculiar organogenesis. This review summarizes the current understanding of the development of one-leaf plants to provide future perspectives in this field of research.