Expression of knotted1 marks shoot meristem formation during maize embryogenesis

The formation of shoot and root meristems that ultimately give rise to all tissues of the plant body occurs for the first time during embryogenesis. Meristem formation has traditionally been defined in terms of the appearance of histological features of meristems; this approach has led to varying interpretations of the timing of meristem formation relative to other events in embryogenesis. Markers that would provide more objective criteria for the analysis of meristem formation have not been widely available. The maize homeobox gene, knotted1 (kn1), is expressed in shoot meristems throughout postembryonic stages of shoot development. In order to determine whether this gene is expressed in the shoot meristem from its earliest inception, we examined the expression of kn1 in embryos at a series of stages by in situ hybridization to kn1 mRNA and immunolocalization of KN1 protein. Our results show that the onset of kn1 expression is temporally and spatially coincident with the earliest histologically recognizable signs of shoot meristem formation in the embryo, and thus provides a valuable marker for this process. © 1995 Wiley-Liss, Inc.     

Shoot apical meristem: In vitro regeneration and morphogenesis in wheat (Triticum aestivum L.)

Summary We report a less genotype-dependent in vitro regeneration system capable of producing multiple shoot clumps and whole plants in four different wheat genotypes. Shool apical meristems from 7-d-old-seedlings produced axillary and adventitious shoots and somatic embryos on media containing N⁶-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). All four genotypes responded positively to shoot multiplication depending upon media composition. Scanning electron microscopies of cultures showed a proliferating budding state that gave rise to adventitious shoots and somatic embryos on further multiplication. The percentage of relative shoot apical meristem multiplication was 80–90%, and the average number of shoot meristems per multiplied shoot was 40–50 in all genotypes. Among different concentrations of phytohormones, 2 and 4 mgl⁻¹ BA (8.8 and 17.7 μM) in combination with 0.5 mg l⁻¹ 2,4-D (2.26 μM) gave the best results. Actively multiplying shoot clumps were recovered with high frequency among 3-mo.-old cultures. These shoot clumps regenerated normally and produced fertile plants containing viable seeds. This in vitro system might prove useful for the production of transgenic plants of wheat in a relatively genotype-independent manner.     

Strategy for shoot meristem proliferation in plants

Shoot apical meristem (SAM) of plants harbors stem cells capable of generating the aerial tissues including reproductive organs. Therefore, it is very important for plants to control SAM proliferation and its density as a survival strategy. The SAM is regulated by the dynamics of a specific gene network, such as the WUS-CLV interaction of A. thaliana. By using a mathematical model, we previously proposed six possible SAM patterns in terms of the manner and frequency of stem cell proliferation. Two of these SAM patterns are predicted to generate either dichotomous or axillary shoot branch. Dichotomous shoot branches caused by this mechanism are characteristic of the earliest vascular plants, such as Cooksonia and Rhynia, but are observed in only a small minority of plant species of the present day. On the other hand, axillary branches are observed in the majority of plant species and are induced by a different dynamics of the feedback regulation between auxin and the asymmetric distribution of PIN auxin efflux carriers. During evolution, some plants may have adopted this auxin-PIN system to more strictly control SAM proliferation.     

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.     

Cyclin‐dependent kinase activity maintains the shoot apical meristem cells in an undifferentiated state

As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration between cell division rates and fate specification is essential for normal organ formation and plant development. However, the inter‐dependence of cell‐cycle machinery and meristem‐organizing genes is still poorly understood. To investigate this mechanism, we specifically inhibited the cell‐cycle machinery in the shoot apex by expression of a dominant negative allele of the A‐type cyclin‐dependent kinase (CDK) CDKA;1 in meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven into the differentiation pathway, as indicated by premature cell expansion and onset of endo‐reduplication. Although the meristem structure and expression patterns of the meristem identity genes were maintained in most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a threshold model in which the effect of the cell‐cycle machinery on meristem organization is determined by the level of CDK activity.     

The bHLH protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis

During post-embryonic shoot development, new meristems are initiated in the axils of leaves. They produce secondary axes of growth that determine morphological plasticity and reproductive efficiency in higher plants. In this study, we describe the role of the bHLH-protein-encoding Arabidopsis gene REGULATOR OF AXILLARY MERISTEM FORMATION (ROX), which is the ortholog of the branching regulators LAX PANICLE1 (LAX1) in rice and barren stalk1 (ba1) in maize. rox mutants display compromised axillary bud formation during vegetative shoot development, and combination of rox mutants with mutations in RAX1 and LAS, two key regulators of axillary meristem initiation, enhances their branching defects. In contrast to lax1 and ba1, flower development is unaffected in rox mutants. Over-expression of ROX leads to formation of accessory side shoots. ROX mRNA accumulates at the adaxial boundary of leaf and flower primordia. However, in the vegetative phase, axillary meristems initiate after ROX expression has terminated, suggesting an indirect role for ROX in meristem formation. During vegetative development, ROX expression is dependent on RAX1 and LAS activity, and all three genes act in concert to modulate axillary meristem formation.     

Effects of auxin and light treatments of donor plants on shoot production fromindica-type rice (Oryza sativa L.)

An efficient clonal propagation procedure for a Brazilianindica rice subspecies was developed with shoot apex explants. Shoot apices were excised from 4-d-old seedlings and cultured on MS medium supplemented with 8.9 μM 6-benzyladenine. The efficiency of shoot production was influenced by growth regulators and light treatments to the donor plant. Explants derived from seedlings growth in the presence of 10.7 μM naphthaleneacetic acid and in the absence of light showed significantly increased regeneration capacity as compared to control explants. Anatomical analysis of the new shoot meristems revealed that they originated from preexisting apical and axillary meristem as well as from the mesocotyl parenchyma.     

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.     

Development of maize plants from cultured shoot apices

Excised shoot apices of maize (Zea mays L.), comprising the apical meristem and one or two leaf primordia, have been cultured and can form rooted plantlets. The plantlets, derived from meristems that had previously formed 7–10 nodes, develop into mature, morphologically normal plants with as many nodes as seed-grown plants. These culture-derived plants exhibited the normal pattern of development, with regard to the progression of leaf lengths along the plant and position of axillary buds and aar shoots. Isolation of the meristem from previously formed nodes reinitiates the pattern and number of nodes formed in the new plant. Thus, cells of the meristem of a maize plant at the seedling stage are not determined to form a limited number of nodes.     

The origin, initiation and development of axillary shoot meristems in Lotus japonicus

BACKGROUND AND AIMS: Lotus japonicus 'Gifu' develops multiple axillary shoots in the cotyledonary node region throughout the growth of the plant. The origin, initiation and development of these axillary meristems were investigated. METHODS: Morphological, histological and mRNA in situ analyses were done to characterize the ontogeny of cotyledonary axillary shoot meristems in Lotus. Morphological characterization of a putative Lotus shoot branching mutant (super-accessory branches) sac, is presented. KEY RESULTS: By using expression of an L. japonicus STM-like gene as a marker for meristematic tissues, it was demonstrated that groups of cells maintained in the meristematic state at the cotyledonary axil region coincide with the sites where additional axillary meristems (accessory meristems) form. A Lotus shoot branching mutant, sac, is a putative Lotus branching mutant characterized by increased proliferation of accessory shoots in all leaf axils including the cotyledons. CONCLUSION: In Lotus, axillary shoot meristems continually develop at the cotyledonary node region throughout the growth of the plant. These cotyledonary primary and accessory axillaries arise from the position of a meristematic zone of tissue at the cotyledonary node axil region.     

Cell-lineage patterns in the shoot apical meristem of the germinating maize embryo

A fate map for the shoot apical meristem of Zea mays L. at the time of germination was constructed by examining somatic sectors (clones) induced by -rays. The shoot apical meristem produced stem, leaves, and reproductive structures above leaf 6 after germination and the analysis here concerns their formation. On 160 adult plants which had produced 17 or 18 leaves, 277 anthocyanin-deficient sectors were scored for size and position. Sectors found on the ear shoot or in the tassel most often extended into the vegetative part of the plant. Sectors ranged from one to six internodes in length and some sectors of more than one internode were observed at all positions on the plant. Single-internode sectors predominated in the basal internodes (7,8,9) while longer sectors were common in the middle and upper internodes. The apparent number of cells which gave rise to a particular internode was variable and sectors were not restricted to the lineage unit: a leaf, the internode below it, and the axillary bud and prophyll at the base of the internode. These observations established two major features of meristem activity: 1) at the time of germination the developmental fate of any cell or group of cells was not fixed, and 2) at the time of germination cells at the same location in a meristem could produce greatly different amounts of tissue in the adult plant. Consequently, the developmental fate of specific cells in the germinating meristem could only be assigned in a general way.Abbreviations ACN apparent cell number - LI, LII, LI-LII sectors restricted to the epidermis, the subepidermis, or encompassing epidermis and subepidermis - PCN progenitor cell     

Shoot meristem differentiation in shoot primordia culture (SPC) obtained from root primordia culture (RPC) of Solanum lycopersicoides Dun.

We present a unique example of conversion of the morphogenesis type (from rhisogenesis to shoot organogenesis) in in vitro cultures of Solanum lycopersicoides. Liquid shoot primordia cultures (SPC) were obtained from root primordia culture (RPC) on two kinds of MS-based media with BA. The first SMS₈, contained a full set of organic compounds; the second, ²SMS₈, was devoid of organic compounds except sucrose and edamine. Two weeks after passage of RPC onto both the media, disintegration of root primordia and cell aggregates began. After 8 weeks of cultivation on SMS₈ and ²SMS₈ media, SPC aggregates developed from meristematic cells located near the vascular tissue of disintegrating RPC aggregates. Initiation of shoot meristems started from meristematic cells centers which were localised under the surface of the newly formed SPC aggregates. The change in the type of morphogenesis occurred faster on medium SMS₈, but the SPC on medium ²SMS₈ was characterized by more frequent formation of shoots and plants (45% of aggregates, in the case of SPC on medium SMS₈, and 90% in the case of SPC on medium ²SMS₈). This fact was correlated with the structural organization of the meristematic centers. Our results indicate that an RPC system has high morphogenetic potential due to the continual presence of meristematic cells. The change in the type of morphogenesis was followed by a rebuilding of the aggregate structure on the basis of the meristematic cells already existing in RPC, which gave rise to SPC aggregates from which shoot meristems developed.     

Isolation of an Arabidopsis homologue of the maize homeobox Knotted-1 gene

The shoot apical meristem (SAM) is responsible for forming most of the above-ground portion of the plant. We sought to isolate regulatory genes expressed in the Arabidopsis SMA by screening a Brassica oleracea (cauliflower) meristem cDNA library with the homeobox fragment from the maize Knotted-1 (Kn1) gene. We isolated and characterized the corresponding clone, Merihb1, from Arabidopsis. Analysis shows that the predicted MERIHB1 protein exhibits strong homology to KN1 and RS1 from maize, SBH1 from soybean, and KNAT1 and KNAT2 from Arabidopsis. Merihb1 is highly expressed in mRNA from cauliflower meristems and also accumulates in stem and flower mRNA. Based on the similarity of the Merihb1 and Kn1 sequences, expression patterns, and in situ hybridizations, we suggest that Merihb1 represents an Arabidopsis homologue of the maize Kn1 gene.     

Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems

The primary shoot apical meristem of angiosperm plants is formed during embryogenesis. Lateral shoot apical meristems arise postembryonically in the axils of leaves. Recessive mutations at the PINHEAD locus of Arabidopsis interfere with the ability of both the primary shoot apical meristem as well as lateral shoot apical meristems to form. However, adventitious shoot apical meristems can form in pinhead mutant seedlings from the axils of the cotyledons and also from cultred root explants. In this report, the phenotype of pinhead mutants is described, and a hypothesis for the role of the wild-type PINHEAD gene product in shoot meristem initiation is presented. © 1995 Wiley-Liss, Inc.     

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.     

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.