Shoot Meristem Development Research Articles

Characterization of a CLE processing activity

Proteins containing a conserved motif known as the CLE domain are found widely distributed across land plants. While the functions of most CLE proteins are unknown, specific CLE proteins have been shown to control shoot meristem, root and vascular development. This has been best studied for CLV3 which is required for stem cell differentiation at shoot and flower meristems. In vivo evidence indicates that the CLE domain is the functional region for CLV3, and that it is proteolytically processed from the CLV3 precursor protein. But the mechanism and activity responsible for this processing is poorly understood. Here we extend analysis of an in vitro CLE processing activity and show that in vitro cleavage occurs at Arg70, exactly matching in vivo maturation. We provide evidence that related processing activities are present in multiple tissues and species. We show that efficient protease recognition can occur with as little as four residues upstream of the CLE domain, and that the conserved arginine at position +1 and conserved acidic residues at positions -2 and/or -3 are required for efficient cleavage. Finally, we provide evidence that the N-terminal processing enzyme is a secreted serine protease while C-terminal processing may occur via a progressive carboxypeptidase.

Novel Insights from Live‐imaging in Shoot Meristem Development

Microscopic imaging of fluorescent reporters for key meristem regulators in live tissues is emerging as a powerful technique, enabling researchers to observe dynamic spatial and temporal distribution of hormonal and developmental regulators in living cells. Aided by time-lapse microphotography, new types of imaging acquisition and analysis software, and computational modeling, we are gaining significant insights into shoot apical meristem (SAM) behavior and function. This review is focused on summarizing recent advances in the understanding of SAM organization, development, and behavior derived from live-imaging techniques. This includes the revelation of mechanical forces in microtubule-controlled anisotropic growth, the role of the CLV-WUS network in the specification of peripheral zone and central zone cells, the multiple feedback loops involving cytokinin in controlling WUS expression, auxin dynamics in determining the position of new primordia, and, finally, sequence of regulatory events leading to de novo assembly of shoots from callus in culture. Future studies toward formulating "digital SAM" that incorporates multi-dimensional data ranging from images of SAM morphogenesis to a genome-scale expression map of SAM will greatly enhance our ability to understand, predict, and manipulate SAM, containing the stem cells that give rise to all above ground parts of a plant.

Toward Molecular Understanding of In Vitro and In Planta Shoot Organogenesis

Shoot organogenesis, one of the in vitro plant regeneration processes that occur during in vitro micropropagation, is used in the study of plant development. Morphological, physiological, and molecular aspects of in vitro shoot organogenesis have been extensively studied for over 50 years. Because of the research progress in plant genetics and molecular biology, our understanding of in planta and in vitro shoot meristem development, the cell cycle and cytokinin signal transduction has advanced significantly. These research advances provide useful information as well as molecular tools to study further the genetic and molecular aspects of shoot organogenesis. A number of key molecular markers, genes, and pathways have been shown to play a critical role in the process of in vitro shoot organogenesis. Furthermore, these studies reveal that in vitro shoot organogenesis, as with in planta shoot development, is a complex, well-coordinated developmental process, given that the induction of a single molecular event is likely to be insufficient to induce the entire process. Continued study is required to identify additional molecular events that trigger dedifferentiation and act as developmental switches for de novo shoot development. *This article is dedicated to the memory of Shibo Zhang, who continues to inspire our interests in this topic.

Rice OsAS2 Gene, a Member of LOB Domain Family, Functions in the Regulation of Shoot Differentiation and Leaf Development

LATERALORGAN BOUNDARIES DOMAIN (LBD) genes, a novel plant-specific family, play specific roles in plant development. Although function of ASYMMETRIC LEAVES2 (AS2), a LBD gene, was extensively studied in Arabidopsis of dicots, little is known on the role of its ortholog in rice of monocots. In this study, a LBD gene that shares higher homology with Arabidopsis AS2 gene was identified in rice and it was designated as OsAS2. Its transcripts were detected throughout predicted leaf primordia in shoot apical meristem (SAM), leaf primordia, and young leaves. Overexpression of the OsAS2 gene inhibited shoot differentiation, promoted cell division, and delayed cell differentiation in rice calli. Transgenic plants with OsAS2 gene showed the aberrant twisted leaves, which lack auricle, and leaf structure was abnormal. Furthermore, a few genes involved in shoot meristem development were upregulated in transgenic plants. Our results suggest that proper expression of the OsAS2 gene is required for shoot differentiation and leaf development in rice.

Pattern formation during de novo assembly of theArabidopsisshoot meristem

Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.

N-Myristoylation Regulates the SnRK1 Pathway inArabidopsis

Cotranslational and posttranslational modifications are increasingly recognized as important in the regulation of numerous essential cellular functions. N-myristoylation is a lipid modification ensuring the proper function and intracellular trafficking of proteins involved in many signaling pathways. Arabidopsis thaliana, like human, has two tightly regulated N-myristoyltransferase (NMT) genes, NMT1 and NMT2. Characterization of knockout mutants showed that NMT1 was strictly required for plant viability, whereas NMT2 accelerated flowering. NMT1 impairment induced extremely severe defects in the shoot apical meristem during embryonic development, causing growth arrest after germination. A transgenic plant line with an inducible NMT1 gene demonstrated that NMT1 expression had further effects at later stages. NMT2 did not compensate for NMT1 in the nmt1-1 mutant, but NMT2 overexpression resulted in shoot and root meristem abnormalities. Various data from complementation experiments in the nmt1-1 background, using either yeast or human NMTs, demonstrated a functional link between the developmental arrest of nmt1-1 mutants and the myristoylation state of an extremely small set of protein targets. We show here that protein N-myristoylation is systematically associated with shoot meristem development and that SnRK1 (for SNF1-related kinase) is one of its essential primary targets.

The evolution of plant regulatory networks: what Arabidopsis cannot say for itself

Genetic and molecular analyses in the dicot model plant Arabidopsis thaliana have begun to shed some light on regulatory networks in plants. However, comparisons with other species are necessary to validate networks identified in model species on the evolutionary scale. Many key regulatory proteins are encoded by members of transcription factor gene families. Orthologous genes can be identified by phylogenetic reconstructions based on conserved protein domains and functionally substantiated by gene expression patterns and mutant analyses. Recent comparative analyses of different pathways involved in shoot meristem development reveal not only conservation from basal land plants to angiosperms but also evolutionary freedom for significant adaptations in the course of plant speciation.

A CELLULAR AUTOMATON TO MODEL THE DEVELOPMENT OF PRIMARY SHOOT MERISTEMS OF ARABIDOPSIS THALIANA

Development of organisms is a very complex process in which a lot of gene networks of different cell types are integrated. Development of a cellular automaton (Ermentrout and Edelshtein-Keshet, J Theor Biol 160:97-133, 1993) that models the morphodynamics of different cell types is the first step in understanding and analysis of the regulatory mechanisms underlying the functioning of developmental gene networks. A model of a cellular automaton has been developed, which simulates the embryonic development of shoot meristem in Arabidopsis thaliana. The model adequately describes the basic stages in development of this organ in wild and mutant types.

茎頂分裂組織と器官形成に異常を示すシロイヌナズナ変異体 gorgon の解析

茎頂分裂組織と器官形成に異常を示すシロイヌナズナ変異体 gorgon の解析

Review on molecular regulation of in vitro shoot regeneration of plants

In vitro shoot regeneration is a developmental process that multiple genes and their interactions are involved in. Initiation of somatic cells stimulated by cytokinin, division of the initiated somatic cells and succeeding shoot meristem development are three key steps in the process. Therefore, exploration of gene expression and their interactions involved in these steps will shed light on our understanding of the molecular mechanism for regulation of plant in vitro shoot regeneration. Here we reviewed the research results of molecular regulation process involved in these steps.

Maintaining the Shoot Meristem Stem Cell Population

Stem cells in plant shoot meristems give rise to stems, leaves, and flowers. One arm of the meristem stem cell maintenance pathway involves a negative feedback loop between the stem cells and the cells of the organizing center (OC) beneath the stem cells. WUSHCEL (WUS), a transcription factor present in the OC, is essential for maintenance of the stem cells, and CLAVATA3 (CLV3), which is a peptide ligand produced by the stem cells, feeds back on the OC to inhibit WUS transcription. Thus, CLV3 promotes stem cell differentiation and WUS promotes stem cell maintenance. Two reports investigate meristem stem cell signaling. Würschum et al. identified a dominant-negative mutation in the gene encoding APETALA2 (AP2) in a screen for mutations that inhibited shoot meristem development in Arabidopsis . The mutation was mapped to the first of two AP2 domains in the protein, and the plants were called l28 mutants. When l28 plants with the dominant-negative mutation in AP2 were crossed with plants with null mutations in the gene encoding AP2, the frequency of premature shoot meristem termination was increased. l28 plants also exhibited reduced expression of WUS and CLV3 . Genetic crosses indicated that termination of the shoot meristem in the l28 plants required functional CLV3, because CLV3 loss of function rescued shoot meristem development. Analysis of l28 plants crossed with wus -null plants indicated that WUS and AP2 were in the same pathway controlling shoot meristem maintenance. Because l28 mutants did not exhibit an increase in CLV3 expression, the authors propose a model in which AP2 inhibits CLV3 signaling downstream of CLV3 transcription. Ni and Clark suggest that CLV3 may be cleaved extracellularly to release the CLE domain, which is the active ligand. The Arabidopsis genome encodes several proteins with a CLE domain. Nine of thirteen intronless CLE genes rescued or partially rescued the phenotype of supernumerary flower organs in clv3-1 transgenic plants. Replacement of the CLE domain of the inactive CLE8 with the CLE domain of CLV3 resulted in complete rescue. Furthermore, replacement of all but the CLE domain of CLV3 with sequences of unrelated protein ERECTA also rescued the clv3-1 phenotype, suggesting that the CLE domain is the essential portion. When glutathione S -transferase (GST)-tagged CLV3 or CLE1, which was one of the CLE proteins that fully rescued the clv3-1 phenotype, was incubated with cauliflower protein extracts, proteolytic processing that released the CLE domain was observed. The CLE domain of CLV3 had been previously shown to alter root development, thus suggesting that this portion of the molecule may be the active region. Thus, the authors propose that CLV3 may be cleaved to release the active CLE peptide. However, it remains to be determined if CLV3 or the related CLE proteins encoded by the other members of the CLE gene family are cleaved in vivo and whether this cleavage is mediated by a specific protease involved in CLV3 signaling. T. Würschum, R. Groβ-Hardt, T. Laux, APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem. Plant Cell 18 , 295-307 (2006). [Abstract] [Full Text] J. Ni, S. E. Clark, Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain. Plant. Physiol. 140 , 726-733 (2006). [Abstract] [Full Text]

WM5: Isolation and characterisation of a gene expressed during early meiosis and shoot meristem development in wheat.

Wheat Meiosis 5 (WM5), isolated from an early meiosis anther cDNA library of wheat by cDNA subtraction encodes a novel glycine-serine-proline-alanine-rich protein. The corresponding homologous genes are located on the short arms of chromosomes 3A, 3B and 3D of allohexaploid wheat (Triticum aestivum L.). The copy on 3DS is located within the region deleted in the wheat mutant ph2a that displays increased homoeologous chromosome pairing in crosses with alien species. While WM5 is expressed primarily in young flower buds during early meiosis it is also expressed in shoot meristems, thus indicating functional roles in both meiosis and meristem development. Overall, the WM5 amino acid sequence shares no significant similarity with other known proteins in the NCBI database. However, the carboxyl-terminal region does have similarity with the Arabidopsis PDF1 (Protodermal Factor 1) protein. Comparing WM5 and PDF1 reveals that the two proteins share 33% identity and have similar hydropathy plots and predicted secondary structures. In situ immuno-staining locates the protein to the nuclei of pollen mother cells undergoing meiosis and the epidermal layer of the shoot and flower meristem, including the cell wall and cuticle. We propose that the WM5 protein has a role in shoot and flower development within this economically important cereal crop.

Influence of plant growth regulators, carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock `Colt' (Prunus avium � P. pseudocerasus)

The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of 4 mg l−1 of kinetin and 2% of maltose under illumination stimulated shoot development and, subsequently, whole plants have been recovered by applying 1.5 mg l−1 kinetin to the rooting medium. Although numerous treatments have been tested involving both embryogenic masses and whole embryos, normal embryo germination was observed sporadically. Cold treatment was effective in stimulating secondary somatic embryogenesis with embryo development to the cotyledonary stage, but did not promote their germination. Similarly, a higher concentration (44–55 mg l−1) of chelated iron than that commonly used in tissue culture media (36.7 mg l−1) produced, after 3 weeks in culture, almost a 50% increase in the number of embryos at the cotyledonary stage per embryogenic mass. Among the cytokinins tested, 1 mg l−1 of 6-benzylaminopurine and 0.1 mg l−1 of thidiazuron were effective in inducing secondary somatic embryogenesis; however, each of them expressed highest efficiency with specific medium and environmental conditions. Furthermore, application of 1 mg l −1 thidiazuron reverted morphogenic callus to non-morphogenic callus, particularly in medium containing 2% sucrose. Finally, hormone free medium with 2% maltose enhanced maturation of the emb-ryos to the normal cotyledonary stage. This paper has improved knowledge of embryo culture and plant production in this important genotype, opening the way for genetic improvement by biotechnological techniques, mainly with the aim of modifying the growth pattern of the canopy of sweet cherry grafted on it.

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.

Identifying novel regulators of shoot meristem development

New organs are initiated throughout the life span of higher plants. This process occurs at the shoot meristem, which is initiated during embryogenesis and is later responsible for generating the above-ground portion of the plant. The shoot meristem can be thought of as having two zones, a central zone containing meristematic cells in an undifferentiated state, and a surrounding peripheral zone where cells enter a specific developmental pathway toward a differentiated state. Recent advances have revealed several genes that specifically regulate meristem development inArabidopsis. However, extensive mutagenesis by several labs have identified only a handful, of loci that appear to specifically regulate shoot meristem development. We have undertaken an enhancer/suppressor mutagenesis of an existing meristem mutant (clv1) and have identified novel regulators of meristem development.

Apical meristem formation during zygotic embryo development of white spruce

The first notable sign of shoot and root meristem development in zygotic embryos of white spruce (Picea glauca (Moench) Voss) was the appearance of starch in the respective poles of the embryo. Starch granules gradually accumulated in the subapical cells of the shoot pole and were soon followed by vacuolation in the subapical cells. Vacuolation reached its highest degree in these cells at the early embryo stage. With the formation of the large vacuolated cells, the surface cell layer at the summit of the shoot pole enlarged and differentiated into the surface initials of the shoot meristem. These cells were large with distinct nuclei. As the embryo matured, the large vacuoles within the subapical cells were replaced by small ones with a concomitant increase in the cytoplasmic density of the subapical cells. After germination, the surface initials remained distinct; however, the subapical cells had different fates depending on their location. The subapical cells located next to the surface initials became the central mother cells of the shoot meristem and those located near the cotyledon junction divided periclinally and gave rise to the epicotyl rib meristem. The remaining subapical cells near the procambium became part of the pith. In the root pole, starch could be found in the cap region terminating underneath the developing procambium at the club-shaped stage of embryo development. Two layers of root initials appeared at the junction between the developing procambium and the root cap. The initials were distinct as they were larger in size than surrounding cells. Cell division activity could not be detected in the layer of initials immediately next to the procambium, while mitotic activity could be seen in the adjoining layer next to the root cap. As the embryo matured, cells surrounding the root initials also took on structural characteristics similar to the root meristem initials. As a result, the region of the root meristem initials expanded in size, and the bilayered configuration became obscure. After germination, mitotic figures could be found in the root initials. As seedling continued to grow, fewer mitotic figures could be found in the root meristem cells adjacent to the procambium pole. In roots of mature plants, a group of isodiametric cells could be discerned between the procambium and the root cap. Mitotic activity was not readily detected within this group of isodiametric cells but mitotic figures could be found in surrounding cells.Key words: apical meristems, Picea glauca, white spruce, zygotic embryogenesis.

The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis

The CLAVATA (CLV1 and CLV3) and SHOOT MERISTEMLESS (STM) genes specifically regulate shoot meristem development in Arabidopsis. CLV and STH appear to have opposite functions: c1v1 and Clv3 mutants accumulate excess undifferentiated cells in the shoot and floral meristem, while stm mutants fail to form the undifferentiated cells of the shoot meristem during embryonic development. We have identified a weak allele of stm (stm-2) that reveals STM is not only required for the establish- ment of the shoot meristem, but is also required for the continued maintenance of undifferentiated cells in the shoot meristem and for proper proliferation of cells in the floral meristem. We have found evidence of genetic interactions between the CLV and STM loci. clv1 and c1v3 mutations partially suppressed the stm-1 and stm-2 phenotypes, and were capable of suppression in a dominant fashion. clv stm double mutants and plants homozygous for stm but heterozygous for clv, while still lacking an embryonic shoot meristem, exhibited greatly enhanced postembryonic shoot and floral meristem development. Although stm phenotypes are recessive, stm mutations dominantly suppressed clv homozygous and heterozygous phenotypes. These results indicate that the stm phenotype is sensitive to the levels of CLV activity, while the clv phenotype is sensitive to the level of STM activity. We propose that these genes play related but opposing roles in the regulation of cell division and/or cell differentiation in shoot and floral meristems.

CLAVATA3 is a specific regulator of shoot and floral meristem development affecting the same processes as CLAVATA1

ABSTRACT We have previously described the phenotype of Arabidopsis thaliana plants with mutations at the CLAVATA1 (CLV1) locus (Clark, S. E., Running, M. P. and Meyerowitz, E. M. (1993) Development 119, 397-418). Our investigations demonstrated that clv1 plants develop enlarged vegetative and inflorescence apical meristems, and enlarged and indeterminate floral meristems. Here, we present an analysis of mutations at a separate locus, CLAVATA3 (CLV3), that disrupt meristem development in a manner similar to clv1 mutations. clv3 plants develop enlarged apical meristems as early as the mature embryo stage. clv3 floral meristems are also enlarged compared with wild type, and maintain a proliferating meristem throughout flower development. clv3 root meristems are unaffected, indicating that CLV3 is a specific regulator of shoot and floral meristem development. We demonstrate that the strong clv3-2 mutant is largely epistatic to clv1 mutants, and that the semi-dominance of clv1 alleles is enhanced by double heterozygosity with clv3 alleles, suggesting that these genes work in the same pathway to control meristem development. We propose that CLV1 and CLV3 are required to promote the differentiation of cells at the shoot and floral meristem.

Development of white spruce somatic embryos: II. Continual shoot meristem development during germination

This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.

Optimization of somatic embryogenesis and embryo germination in soybean

Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container.