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.     

Somatic embryogenesis from <Emphasis Type="Italic">Arabidopsis</Emphasis> shoot apical meristem mutants

Zygotic embryos of three Arabidopsis thaliana (L.) Heynh. mutants lacking an embryonic shoot apical meristem (SAM), shoot meristemless (stm), wuschel (wus) and zwille/pinhead (zll/pnh) were used as explants to establish embryogenic cell cultures. Somatic embryos of all three mutants showed the same mutant phenotypes as their zygotic equivalents. These results provide genetic evidence that the developmental program of somatic and zygotic embryos is indistinguishable. They also suggest that a functional SAM is not required for somatic embryogenic cell formation in Arabidopsis.     

The role of PENNYWISE and POUND-FOOLISH in the maintenance of the shoot apical meristem in Arabidopsis

Growth of the aerial part of the plant is dependent upon the maintenance of the shoot apical meristem (SAM). A balance between the self-renewing stem cells in the central zone (CZ) and organogenesis in the peripheral zone (PZ) is essential for the integrity, function, and maintenance of the SAM. Understanding how the SAM maintains a balance between stem cell perpetuation and organogenesis is a central question in plant biology. Two related BELL1-like homeodomain proteins, PENNYWISE (PNY) and POUND-FOOLISH (PNF), act to specify floral meristems during reproductive development. However, genetic studies also show that PNY and PNF regulate the maintenance of the SAM. To understand the role of PNY and PNF in meristem maintenance, the expression patterns for genes that specifically localize to the peripheral and central regions of the SAM were examined in Arabidopsis (Arabidopsis thaliana). Results from these experiments indicate that the integrity of the CZ is impaired in pny pnf plants, which alters the balance of stem cell renewal and organogenesis. As a result, pools of CZ cells may be allocated into initiating leaf primordia. Consistent with these results, the integrity of the central region of pny pnf SAMs can be partially restored by increasing the size of the CZ. Interestingly, flower specification is also reestablished by augmenting the size of the SAM in pny pnf plants. Taken together, we propose that PNY and PNF act to restrict organogenesis to the PZ by maintaining a boundary between the CZ and PZ.     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CKV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the "reaction-diffusion" type formalizing the role proteins CLV1, CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

A model study of the role of proteins CLV1, CLV2, CLV3, and WUS in regulation of the structure of the shoot apical meristem

In order to elucidate the role of proteins CLV1, CLV2, CLV3, and WUS in the mechanism underlying the maintenance of compartmental structure (spatial arrangement of the zones of biosynthesis of marker proteins) of the shoot apical meristem, a model of such mechanism was developed. Computational experiments led to biologically plausible solutions only when synthesis of substance W in a space between the organizing center and meristem apex was limited by the mechanism based on interaction of CLV3 with membrane receptor CLV1/CLV2 and lower boundary of the zone of W synthesis was determined by isoline of the corresponding threshold level of substance Y concentration. The model of the “reaction-diffusion” type formalizing the role proteins CLV1/CLV2, CLV3, and WUS can describe the basis of the mechanism underlying regulation of the compartmental structure of the shoot apical meristem and positioning of the organizing center in a certain site of the cell ensemble of such meristem.     

Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis

Almost all aerial parts of plants are continuously generated at the shoot apical meristem (SAM). To maintain a steady pool of undifferentiated cells in the SAM while continuously generating new organs, it is necessary to balance the rate of cell division with the rate of entrance into differentiation pathways. In the Arabidopsis meristem, SHOOT MERISTEMLESS (STM) and WUSCHEL (WUS) are necessary to keep cells undifferentiated and dividing. Here, we tested whether ectopic STM and WUS functions are sufficient to revert differentiation and activate cell division in differentiating tissues. Ectopic STM and WUS functions interacted non-additively and activated a subset of meristem functions, including cell division, CLAVATA1 expression and organogenesis, but not correct phyllotaxy or meristem self-maintenance. Our results suggest that WUS produces a non-cell autonomous signal that activates cell division in combination with STM and that combined WUS/STM functions can initiate the progression from stem cells to organ initiation.     

Out of step: The function of TALE homeodomain transcription factors that regulate shoot meristem maintenance and meristem identity

The indeterminate growth pattern displayed by shoots is mediated by the proper maintenance of the shoot meristem.Meristem maintenance is dependent upon the balance of stem cell perpetuation in the cent...     

Requirement of homeobox gene STIMPY/WOX9 for Arabidopsis meristem growth and maintenance

Most organs of flowering plants develop postembryonically from groups of pluripotent cells called meristems [1]. The shoot apical meristem (SAM) is specified by two complementary pathways [2-4]. SHOOT MERISTEMLESS (STM; [5]) defines the entire SAM region [6]. WUSCHEL (WUS), on the other hand, functions in a more restricted set of cells to promote stem-cell fate and is regulated by the CLAVATA genes in a negative feedback loop [7-10]. In contrast, little is known about how the growth of the SAM, which increases in size during vegetative development [11], is regulated. We have characterized STIMPY (STIP; also called WOX9 [12]), a homeobox gene required for the growth of the vegetative SAM, in part by positively regulating WUS expression. In addition, STIP is required in several other aerial organs and the root. What sets STIP apart from STM and WUS is that stip mutants can be fully rescued by stimulating the entry into the cell cycle with sucrose. Therefore, STIP is likely to act in all these tissues by maintaining cell division and preventing premature differentiation. Taken together, our findings suggest that STIP identifies a new genetic pathway integrating developmental signals with cell-cycle control.     

POLTERGEIST functions to regulate meristem development downstream of the CLAVATA loci

Mutations at the CLAVATA loci (CLV1, CLV2 and CLV3) result in the accumulation of undifferentiated cells at the shoot and floral meristems. We have isolated three mutant alleles of a novel locus, POLTERGEIST (POL), as suppressors of clv1, clv2 and clv3 phenotypes. All pol mutants were nearly indistinguishable from wild-type plants; however, pol mutations provided recessive, partial suppression of meristem defects in strong clv1 and clv3 mutants, and nearly complete suppression of weak clv1 mutants. pol mutations partially suppressed clv2 floral and pedicel defects in a dominant fashion, and almost completely suppressed clv2 phenotypes in a recessive manner. These observations, along with dominant interactions observed between the pol and wuschel (wus) mutations, indicate that POL functions as a critical regulator of meristem development downstream of the CLV loci and redundantly with WUS. Consistent with this, pol mutations do not suppress clv3 phenotypes by altering CLV1 receptor activation.     

FASCIATA genes for chromatin assembly factor-1 in arabidopsis maintain the cellular organization of apical meristems

Postembryonic development of plants depends on the activity of apical meristems established during embryogenesis. The shoot apical meristem (SAM) and the root apical meristem (RAM) have similar but distinct cellular organization. Arabidopsis FASCIATA1 (FAS1) and FAS2 genes maintain the cellular and functional organization of both SAM and RAM, and FAS gene products are subunits of the Arabidopsis counterpart of chromatin assembly factor-1 (CAF-1). fas mutants are defective in maintenance of the expression states of WUSCHEL (WUS) in SAM and SCARECROW (SCR) in RAM. We suggest that CAF-1 plays a critical role in the organization of SAM and RAM during postembryonic development by facilitating stable maintenance of gene expression states.     

拟南芥WUSCHEL基因在转基因烟草中的超表达

WUSCHEL(WUS)是近年报道的一个重要的干细胞调控基因.本实验用RT-PCR技术从拟南芥(Arabidopsisthaliana L.)中克隆到其cDNA并构建了双增强的CaMV3 5S启动子驱动的超表达载体pBKB.借助农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum L.),获得转基因植株.PCR和RT-PCR鉴定分别证明,外源WUS已整合到烟草基因组并已表达.转基因烟草地上部分出现大量异位增生的突起,扫描电镜观察表明:突起部分的细胞与分生组织细胞相似,部分突起能够发育为叶芽、花芽,表明WUS超表达引起烟草细胞异常分裂并在已分化组织中重新启动了器官形成.茎尖和花的内两轮器官没有上述变化.结合拟南芥的有关研究,推测烟草中可能也存在类似拟南芥WUS和其阻抑蛋白CLAVATA3、AGAMOUS间的反馈调节机制.转基因烟草叶发育表型变化明显,与生长素极性运输受抑制引起的表型相似,因此,作为生长点调控基因,WUS可能通过生长素对叶的发育进行调控.本研究为WUS基因的功能分析和有关生物技术应用提供了有意义的信息.