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Signalling between the shoot apical meristem and developing lateral organs.

A characteristic feature of plant development is the extensive role played by cell-cell signalling in regulating patterns of growth and cell fate. This is particularly apparent in the shoot apical meristem (SAM) where signalling is involved in the maintenance of a central undifferentiated stem cell population and the formation of a regular and predictable pattern of leaves, from the meristem periphery. Although these two functions occur in different regions of the meristem, their activity must be coordinated to maintain meristem integrity. The role of signalling in the SAM was first characterised over 60 years ago by elegant surgical experiments. These studies showed that existing leaf primordia determine future sites of organ formation in adjacent regions of the SAM, a finding that laid the foundation for subsequent studies into the mechanisms controlling phyllotaxy. Recent studies have identified auxin as a likely signal promoting organ formation and shown that young primordia play an important role in determining its distribution in the SAM. These pioneering surgical experiments also revealed that signals from the meristem regulate the development of organ primordia. In this case a meristem signal promotes the formation of cell types found in the top/adaxial half of the emerging leaf. While the identity of this signal remains elusive, the recent characterisation of a small family of PHABULOSA-like (PHB-like) transcription factor genes has provided important clues to its nature. These genes, which promote adaxial cell identity, are regulated by microRNAs (miRNAs) raising the exciting possibility that the meristem signal is either a miRNA or part of a pathway regulating the distribution of miRNAs.

Body Patterning↗

ABERRANT PANICLE ORGANIZATION 1 temporally regulates meristem identity in rice.

We report a recessive mutation of rice, aberrant panicle organization 1 (apo1), which severely affects inflorescence architecture, floral organ identity, and leaf production rate. In the wild-type inflorescence, the main-axis meristem aborts after forming 10-12 primary branch primordia. However, in apo1, the main-axis meristem was converted to a spikelet meristem after producing a small number of branch primordia. In addition, the branch meristems in apo1 became spikelet meristems earlier than in wild type. Therefore, in the inflorescence, the apo1 mutation caused the precocious conversion of the meristem identity. In the apo1 flower, lodicules were increased at the expense of stamens, and carpels were formed indeterminately by the loss of meristem determinacy. Vegetative development is also affected in the apo1. Leaves were formed rapidly throughout the vegetative phase, indicating that APO1 is also involved in temporal regulation of leaf production. These phenotypes suggest that the APO1 plays an important role in the temporal regulation of both vegetative and reproductive development.

Cell Differentiation↗

WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators.

Plants continuously maintain pools of totipotent stem cells in their apical meristems from which elaborate root and shoot systems are produced. In Arabidopsis thaliana, stem cell fate in the shoot apical meristem is controlled by a regulatory network that includes the CLAVATA (CLV) ligand-receptor system and the homeodomain protein WUSCHEL (WUS). Phytohormones such as auxin and cytokinin are also important for meristem regulation. Here we show a mechanistic link between the CLV/WUS network and hormonal control. WUS, a positive regulator of stem cells, directly represses the transcription of several two-component ARABIDOPSIS RESPONSE REGULATOR genes (ARR5, ARR6, ARR7 and ARR15), which act in the negative-feedback loop of cytokinin signalling. These data indicate that ARR genes might negatively influence meristem size and that their repression by WUS might be necessary for proper meristem function. Consistent with this hypothesis is our observation that a mutant ARR7 allele, which mimics the active, phosphorylated form, causes the formation of aberrant shoot apical meristems. Conversely, a loss-of-function mutation in a maize ARR homologue was recently shown to cause enlarged meristems.

Arabidopsis↗

Conservation and diversification of meristem maintenance mechanism in Oryza sativa: Function of the FLORAL ORGAN NUMBER2 gene.

To elucidate the genetic mechanism that regulates meristem maintenance in monocots, here we have examined the function of the gene FLORAL ORGAN NUMBER2 (FON2) in Oryza sativa (rice). Mutations in FON2 cause enlargement of the floral meristem, resulting in an increase in the number of floral organs, although the vegetative and inflorescence meristems are largely normal. Molecular cloning reveals that FON2 encodes a small secreted protein, containing a CLE domain, that is closely related to CLAVATA3 in Arabidopsis thaliana. FON2 transcripts are localized at the apical region in all meristems in the aerial parts of rice plants, showing an expression pattern similar to that of Arabidopsis CLV3. Constitutive expression of FON2 causes a reduction in the number of floral organs and flowers, suggesting that both the flower and inflorescence meristems are reduced in size. This action of FON2 requires the function of FON1, an ortholog of CLV1. Constitutive expression of FON2 also causes premature termination of the shoot apical meristem in Arabidopsis, a phenotype similar to that caused by constitutive expression of CLV3. Together with our previous study of FON1, these results clearly indicate that the FON1-FON2 system in rice corresponds to the CLV signaling system in Arabidopsis and suggest that the negative regulation of stem cell identity by these systems may be principally conserved in a wide range of plants within the Angiosperms. In addition, we propose a model of the genetic regulation of meristem maintenance in rice that includes an alternative pathway independent of FON2-FON1.

Amino Acid Sequence↗

The floral organ number4 gene encoding a putative ortholog of Arabidopsis CLAVATA3 regulates apical meristem size in rice.

To understand the molecular mechanism regulating meristem development in the monocot rice (Oryza sativa), we describe here the isolation and characterization of three floral organ number4 (fon4) alleles and the cloning of the FON4 gene. The fon4 mutants showed abnormal enlargement of the embryonic and vegetative shoot apical meristems (SAMs) and the inflorescence and floral meristems. Likely due to enlarged SAMs, fon4 mutants produced thick culms (stems) and increased numbers of both primary rachis branches and floral organs. We identified FON4 using a map-based cloning approach and found it encodes a small putatively secreted protein, which is the putative ortholog of the Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) gene. FON4 transcripts mainly accumulated in the small group of cells at the apex of the SAMs, whereas the rice ortholog of CLV1 (FON1) is expressed throughout the SAMs, suggesting that the putative FON4 ligand might be sequestered as a possible mechanism for rice meristem regulation. Exogenous application of the peptides FON4p and CLV3p corresponding to the CLV3/ESR-related (CLE) motifs of FON4 and CLV3, respectively, resulted in termination of SAMs in rice, and treatment with CLV3p caused consumption of both rice and Arabidopsis root meristems, suggesting that the CLV pathway in limiting meristem size is conserved in both rice and Arabidopsis. However, exogenous FON4p did not have an obvious effect on limiting both rice and Arabidopsis root meristems, suggesting that the CLE motifs of Arabidopsis CLV3 and FON4 are potentially functionally divergent.

Amino Acid Sequence↗

NFL, the tobacco homolog of FLORICAULA and LEAFY, is transcriptionally expressed in both vegetative and floral meristems.

The homologous genes FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis regulate the formation of determinate floral meristems. Transcripts of these single-copy genes are confined to floral meristems and some floral organs as well as to the leaflike bracts that subtend Antirrhinum flowers. Based on these observations, we hypothesized that the transcription of genes homologous to FLO and LFY in tobacco, a determinate plant in which the primary shoot apex is consumed in the production of a terminal flower, would serve as a molecular marker for floral commitment. Surprisingly, transcripts of the tobacco homologs NFL1 and NFL2 (Nicotiana FLO/LFY) were found not only in floral meristems, but also in indeterminate vegetative meristems. This implies that the transcriptional expression of the FLO/LFY homologous genes in the apical meristem is not sufficient for the initiation of floral meristem development. In addition, the transcript patterns of the NFL genes identified a previously undescribed subset of cells within the shoot apical meristem that may indicate unique functional compartmentalization. This suggests that, unlike FLO and LFY, which specify determinacy only during floral development, the NFL genes act to specify determinacy in the progenitor cells for both flowers and leaves.

Amino Acid Sequence↗

FON1, an Arabidopsis gene that terminates floral meristem activity and controls flower organ number.

A novel gene that regulates floral meristem activity and controls floral organ number was identified in Arabidopsis and is designated FON1 (for FLORAL ORGAN NUMBER1). The fon1 mutants exhibit normal vegetative development and produce normal inflorescence meristems and immature flowers before stage 6. fon1 flowers become visibly different from wild-type flowers at stage 6, when the third-whorl stamen primordia have formed. The fon1 floral meristem functions longer than does that of the wild type: after the outer three-whorl organ primordia have initiated, the remaining central floral meristem continues to produce additional stamen primordia interior to the third whorl. Prolonged fon1 floral meristem activity also results in an increased number of carpels. The clavata (clv) mutations are known to affect floral meristem activity. We have analyzed the clv1 fon1, clv2 fon1, and clv3 fon1 double mutants. These double mutants all have similar phenotypes, with more stamens and carpels than either fon1 or clv single mutants. This indicates that FON1 and CLV genes function in different pathways to control the number of third- and fourth-whorl floral organs. In addition, to test for possible interactions between FON1 and other floral regulatory genes, we have constructed and analyzed the relevant double mutants. Our results suggest that FON1 does not interact with TERMINAL FLOWER1, APETALA1, APETALA2, or UNUSUAL FLORAL ORGAN. In contrast, normal LEAFY function is required for the expression of fon1 phenotypes. In addition, FON1 and AGAMOUS both seem to affect the domain of APETALA3 function, which also affects the formation of stamen-carpel chimera due to fon1 mutations. Finally, genetic analysis suggests that FON1 interacts with SUPERMAN, which also regulates floral meristem activity.

Arabidopsis↗

Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L).

BACKGROUND: The genetics of spikelet formation, a feature unique to grasses such as rice and maize, is yet to be fully understood, although a number of meristem and organ identity mutants have been isolated and investigated in Arabidopsis and maize. Using a two-element Ac/Ds transposon tagging system we have isolated a rice mutant, designated branched floretless 1 (bfl1) which is defective in the transition from spikelet meristem to floret meristem. RESULTS: The bfl1 mutant shows normal differentiation of the primary rachis-branches leading to initial spikelet meristem (bract-like structure equivalent to rudimentary glumes) formation but fails to develop empty glumes and florets. Instead, axillary meristems in the bract-like structure produce sequential alternate branching, thus resulting in a coral shaped morphology of the branches in the developing panicle. The bfl1 mutant harbours a single Ds insertion in the upstream region of the BFL1 gene on chromosome 7 corresponding to PAC clone P0625E02 (GenBank Acc No. message URL http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=nucleotide&list_uids=34395191&dopt=GenBank&term=ap004570AP004570). RT-PCR analyses revealed a drastic reduction of BFL1 transcript levels in the bfl1 mutant compared to that in the wild-type. In each of the normal panicle-bearing progeny plants, from occasional revertant seeds of the vegetatively-propagated mutant plant, Ds was shown to be excised from the bfl1 locus. BFL1 contains an EREBP/AP2 domain and is most likely an ortholog of the maize transcription factor gene BRANCHED SILKLESS1 (BD1). CONCLUSIONS: bfl1 is a Ds-tagged rice mutant defective in the transition from spikelet meristem (SM) to floret meristem (FM). BFL1 is most probably a rice ortholog of the maize ERF (EREBP/AP2) transcription factor gene BD1. Based on the similarities in mutant phenotypes bfl1 is likely to be an allele of the previously reported frizzy panicle locus.

Amino Acid Sequence↗

Regulation of meristem organization and cell division by TSO1, an Arabidopsis gene with cysteine-rich repeats.

In higher plants, meristem organization and cell division regulation are two fundamentally important and intimately related biological processes. Identifying and isolating regulatory genes in these processes is essential for understanding higher plant growth and development. We describe the molecular isolation and analyses of an Arabidopsis gene, TSO1, which regulates both of these processes. We previously showed that tso1 mutants displayed defects in cell division of floral meristem cells including partially formed cell walls, increased DNA content, and multinucleated cells (Liu, Z., Running, M. P. and Meyerowitz, E. M. (1997). Development 124, 665-672). Here, we characterize a second defect of tso1 in influorescence meristem development and show that the enlarged influorescence in tso1 mutants results from repeated division of one inflorescence meristem into two or more influorescence meristems. Using a map-based approach, we isolated the TSO1 gene and found that TSO1 encodes a protein with cysteine-rich repeats bearing similarity to Drosophila Enhancer of zeste and its plant homologs. In situ TSO1 mRNA expression pattern and the nuclear localization of TSO1-GFP are consistent with a regulatory role of TSO1 in floral meristem cell division and in influorescence meristem organization.

Amino Acid Sequence↗

Origin and development of adventitious shoot meristems initiated on plant chimeras.

Most studies concerning the initiation and development of the shoot apical meristem have been performed on meristems that developed during embryogenesis. We characterized the in situ formation of adventitious shoots originating from cells located near leaf axils in a series of six interspecific periclinal tobacco chimeras. Shoots were generated by decapitating the plants and removing all of the axillary buds. Eighty-four of the 413 shoots regenerated were chimeral. Many of the shoots were complex mericlinal chimeras, with several of their axillary buds possessing meristems arranged as periclinal chimeras. The adventitious shoots originated from derivatives of only the second and third cell layers in the meristem of the source plant since derivatives of the outermost meristem layer were removed with the removal of the axillary buds. With time, nearly all adventitious shoot apices that were initially chimeral became nonchimeral or stabilized as periclinal chimeras. A statistical analysis indicated that the arrangement of genetically distinct tissues in the cell layers of adventitious meristems was influenced by competition between cell types. Our method for generating these shoots can be used to create small genetically distinct sectors analogous to radiation-induced sectors as well as a complete series of periclinical chimeras, both of which have potential for use in determining tissue-to-tissue interactions. An analysis of sector length and position on chimeral shoots indicates that the first one to three leaves of adventitious shoots do not arise as derivatives of the shoot apical initials of a meristem proper.

Cell Communication↗

Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca).

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.

Germination↗

Control of cell fate in plant meristems.

In contrast to animals, plants do not stop to initiate organs with the end of embryogenesis. Instead, most of the growth and development of higher plants will take place during later phases following germination of the seed. Plant development depends on the activity of two meristems, the root meristem and the shoot apical meristem (SAM), that are located at opposite ends of the plant embryo. These meristems serve as a source of pluripotent stem cells and, in case of the SAM, provide a centre for repetitive organ initiation. In order to maintain the SAM throughout plant life, the cells that are lost from the meristem through organ initiation and differentiation have to be replaced from the stem cell population. In this paper, we will discuss recent results indicating that the fate of stem cells in plant meristems is controlled by directional signalling systems.

Animals↗

Coordination of cell proliferation and cell fate decisions in the angiosperm shoot apical meristem.

A unique feature of flowering plants is their ability to produce organs continuously, for hundreds of years in some species, from actively growing tips called apical meristems. All plants possess at least one form of apical meristem, whose cells are functionally analogous to animal stem cells because they can generate specialized organs and tissues. The shoot apical meristem of angiosperm plants acts as a continuous source of pluripotent stem cells, whose descendents become incorporated into organ primordia and acquire different fates. Recent studies are unveiling some of the molecular pathways that specify stem cell fate in the center of the shoot apical meristem, that confer organ founder cell fate on the periphery, and that connect meristem patterning elements with events at the cellular level. The results are providing important insights into the mechanisms through which shoot apical meristems integrate cell fate decisions with cellular proliferation and global regulation of growth and development.

Animals↗

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA.

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.

AGAMOUS Protein, Arabidopsis↗

Root-specific CLE19 overexpression and the sol1/2 suppressors implicate a CLV-like pathway in the control of Arabidopsis root meristem maintenance.

In the Arabidopsis shoot apical meristem, an organizing center signals in a non-cell-autonomous manner to specify the overlying stem cells. Stem cells express the small, secreted protein CLAVATA3 (CLV3; ) that activates the CLV1-CLV2 receptor complex, which negatively controls the size of the organizing center. Consistently, CLV3 overexpression restricts shoot meristem size. The root meristem also contains a stem cell organizer, and here we show that localized overexpression in roots of CLE19, encoding a CLV3 homolog, restricts the size of the root meristem. This suggests that CLE19 acts by overactivating an endogenous CLV-like pathway involved in root meristem maintenance. Surprisingly, CLE19 restricts meristem size without directly interfering with organizer and stem cell specification. We isolated mutations in two loci, SOL1 and SOL2, which suppress the CLE19 overexpression phenotype. sol2 plants display floral phenotypes reminiscent of clv weak alleles; these phenotypes suggest that components of a CLV pathway are shared in roots and shoots. SOL1 encodes a putative Zn(2+)-carboxypeptidase, which may be involved in ligand processing.

Amino Acid Sequence↗

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.

Meristem↗

Transient decreases in methylation at 5'-cCGG-3' sequences in potato (Solanum tuberosum L.) meristem DNA during progression of tubers through dormancy precede the resumption of sprout growth.

The 5-methylcytosine (5mC) content in DNA of tuber meristems isolated from field-grown potatoes (Solanum tuberosum L.) was determined during a 7-month storage period at 3 degrees C for three growing/postharvest seasons. No significant changes in 5mC levels were noted genome-wide or within 5'-CG-3' dinucleotide sequences, 5'-CG-3' islands or 5'-CA(T)G-3' trinucleotide sequences during storage. However, a consistent but transient 50-70% decrease in methylation at both cytosines within 5'-CCGG-3' sequences was detected that peaked 112-194 days after harvest. This result was corroborated by methylation-sensitive amplified fragment length polymorphism analysis of meristem DNA. Similar to tuber meristems undergoing progression through natural dormancy, premature chemical termination of dormancy resulted in rapid, transient 5'-CCGG-3' demethylation in meristem DNA. Minimum methylation levels at this sequence preceded initiation of high levels of de novo DNA synthesis by two days. Cytosine methylation status was also followed in in vitro-generated potato microtubers during 7 months of post-harvest storage. As in DNA from tuber bud meristems, no changes in genome-wide 5mC content or methylation at 5'-CA(T)G-3' or 5'-CG-3' island sequences were noted in microtuber DNA. However, there was a transient 46% drop in methylation at 5'-CG-3' dinucleotides concomitant with minimum levels of 5'-CCGG-3' methylation (30-60% below those in dormant microtubers) 57-98 days after harvest. As microtubers exited dormancy, there were sustained three- and seven-fold increases in RNA and DNA synthesis rates, peaking on or after 98 days of storage, respectively. Together, these data demonstrate that demethylation of 5'-CCGG-3' sequences occurs independently of tuber age during dormancy progression and precedes transcriptional activation of genes leading to cell division and meristem growth in potatoes.

5-Methylcytosine↗

Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases.

Meristems require a myriad of intercellular signaling pathways for coordination of cell division within and between functional zones and clonal cell layers. This control of cell division ensures a constant availability of stem cells throughout the life span of the meristem while limiting overproliferation of meristematic cells and maintaining the meristem structure. We have undertaken a genetic screen to identify additional components of meristem signaling pathways. We identified pluripetala (plp) mutants based on their dramatically larger meristems and increased floral organ number. PLURIPETALA encodes the alpha-subunit shared between protein farnesyltransferase and protein geranylgeranyltransferase-I. plp mutants also have altered abscisic acid responses and overall much slower growth rate. plp is epistatic to mutations in the beta-subunit of farnesyltransferase and shows a synergistic interaction with clavata3 mutants. plp mutants lead to insights into the mechanism of meristem homeostasis and provide a unique in vivo system for studying the functional role of prenylation in eukaryotes.

Abscisic Acid↗