The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation

In higher plants, molecular mechanisms regulating shoot apical meristem (SAM) formation and organ separation are largely unknown. The CUC1 (CUP-SHAPED COTYLEDON1) and CUC2 are functionally redundant genes that are involved in these processes. We cloned the CUC1 gene by a map-based approach, and found that it encodes a NAC-domain protein highly homologous to CUC2. CUC1 mRNA was detected in the presumptive SAM during embryogenesis, and at the boundaries between floral organ primordia. Surprisingly, overexpression of CUC1 was sufficient to induce adventitious shoots on the adaxial surface of cotyledons. Expression analyses in the overexpressor and in loss-of-function mutants suggest that CUC1 acts upstream of the SHOOT MERISTEMLESS gene.     

Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation

Overall shoot architecture in higher plants is highly dependent on the activity of embryonic and axillary shoot meristems, which are produced from the basal adaxial boundaries of cotyledons and leaves, respectively. In Arabidopsis thaliana, redundant functions of the CUP-SHAPED COTYLEDON genes CUC1, CUC2, and CUC3 regulate embryonic shoot meristem formation and cotyledon boundary specification. Their functional importance and relationship in postembryonic development, however, is poorly understood. Here, we performed extensive analyses of the embryonic and postembryonic functions of the three CUC genes using multiple combinations of newly isolated mutant alleles. We found significant roles of CUC2 and CUC3, but not CUC1, in axillary meristem formation and boundary specification of various postembryonic shoot organs, such as leaves, stems, and pedicels. In embryogenesis, all three genes make significant contributions, although CUC3 appears to possess, at least partially, a distinct function from that of CUC1 and CUC2. The function of CUC3 and CUC2 overlaps that of LATERAL SUPPRESSOR, which was previously shown to be required for axillary meristem formation. Our results reveal that redundant but partially distinct functions of CUC1, CUC2, and CUC3 are responsible for shoot organ boundary and meristem formation throughout the life cycle in Arabidopsis.     

Cellular parameters of the shoot apical meristem in Arabidopsis.

The shoot apical meristem (SAM) is a small group of dividing cells that generate all of the aerial parts of the plant. With the goal of providing a framework for the analysis of Arabidopsis meristems at the cellular level, we performed a detailed morphometric study of actively growing inflorescence apices of the Landsberg erecta and Wassilewskija ecotypes. For this purpose, cell size, spatial distribution of mitotic cells, and the mitotic index were determined in a series of optical sections made with a confocal laser scanning microscope. The results allowed us to identify zones within the inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that was four to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type with the enlarged meristems of two mutants, clavata3-1 (clv3-1) and mgoun2 (mgo2). One of the proposed functions of the CLV genes is to limit cell division rates in the center of the meristem. Our data allowed us to reject this hypothesis, because the mitotic index was reduced in the inflorescence meristem of the clv3-1 mutant. We also observed a large zone of slowly dividing cells in meristems of clv3-1 seedlings. This zone was not detectable in the wild type. These results suggest that the central area is increased in size in the mutant meristem, which is in line with the hypothesis that the CLV3 gene is necessary for the transition of cells from the central to the peripheral zone. Genetic and microscopic analyses suggest that mgo2 is impaired in the production of primordia, and we previously proposed that the increased size of the mgo2 meristem could be due to an accumulation of cells at the periphery. Our morphometric analysis showed that mgo2 meristems, in contrast to those of clv3-1, have an enlarged periphery with high cell proliferation rates. This confirms that clv3-1 and mgo2 lead to meristem overgrowth by affecting different aspects of meristem function.     

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.     

Abnormal development and altered hormone profile and sensitivity in Arabidopsis plants ectopically expressing Brassica shoot apical meristem genes

The previously isolated Brassica genes homologous to the Arabidopsis SHOOT MERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL) were characterized during embryonic and postembryonic development in vivo. Ectopic expression of the Brassica genes in Arabidopsis caused profound phenotypic deviations from the WT. While the over-expression of BnCLV1 produced smaller embryonic shoot apical meristems (SAMs) with delayed activation at germination, the introduction of Brassica STM enhanced the structure of the SAM and accelerated meristem reactivation. These opposite behaviors were related to differential levels of endogenous cytokinins and abscisic acid (ABA), as well as the expression of genes regulating meristem activity. Low levels of ABA and increased accumulation of the cytokinins trans-zeatin-O-glucoside (t-ZOG), cis-zeatin-O-glucoside (c-ZOG), trans-zeatin riboside (t-ZR), and isopentenyladenosine (iPA) were measured in seedlings of Arabidopsis plants over-expressing the Brassica STM. This was in contrast to BnCLV1 over-expressors which had very low levels of cytokinins. During the early phases of meristem reactivation the expression of the Arabidopsis AtKNAT6, AtWUSCHEL, and AtCUPSHAPED COTYLEDON-1 was induced by the introduction of the Brassica STM whereas that of AtCLAVATA 3 was inhibited. An opposite expression profile was measured in lines ectopically expressing BnCLV1. Other phenotypic abnormalities observed in Arabidopsis plants over-expressing the Brassica STM included lobed leaves, ectopic meristems, and increased number of reproductive organs, i.e. flowers and siliques. The introduction of BnZLL-1 and -2 did not cause major developmental abnormalities.     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.     

Formation and maintenance of the shoot apical meristem

Development in higher plants is characterized by the reiterative formation of lateral organs from the flanks of shoot apical meristems. Because organs are produced continuously throughout the life cycle, the shoot apical meristem must maintain a pluripotent stem cell population. These two tasks are accomplished within separate functional domains of the apical meristem. These functional domains develop gradually during embryogenesis. Subsequently, communication among cells within the shoot apical meristem and between the shoot apical meristem and the incipient lateral organs is needed to maintain the functional domains within the shoot apical meristem.     

Pattern formation during de novo assembly of the Arabidopsis shoot 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.     

Characterization of an Arabidopsis gene that mediates cytokinin signaling in shoot apical meristem development

Cytokinins are adenine derivatives that regulate numerous plant growth and developmental processes, including apical and floral meristem development, stem growth, leaf senescence, apical dominance, and stress tolerance. However, not much is known about how cytokinin biosynthesis and metabolism is regulated. We identified a novel Arabidopsis gene, ALL, encoding an aldolase-like enzyme that regulates cytokinin signaling. An Arabidopsis mutant, all-1D, in which ALL is activated by the nearby insertion of the 35S enhancer, exhibited extreme dwarfism with rolled, dark-green leaves and reduced apical dominance, symptomatic of cytokinin-overproducing mutants. Consistent with this, ARR4 and ARR5, two representative primary cytokinin-responsive genes, were significantly induced in all-1D. Whereas SHOOT MERISTEMLESS (STM) and KNAT1, which regulate meristem development, were also greatly induced, expression of REV and PHV that regulate lateral organ polarity was inhibited. ALL encodes an aldolase-like enzyme that belongs to the HpcH/HpaI aldolase family in prokaryotes and is down-regulated by exogenous cytokinin, possibly through a negative feedback pathway. We propose that ALL is involved in cytokinin biosynthesis or metabolism and acts as a positive regulator of cytokinin signaling during shoot apical meristem development and determination of lateral organ polarity.