WUSCHEL-RELATED HOMEOBOX4 Is Involved in Meristem Maintenance and Is Negatively Regulated by the CLE Gene FCP1 in Rice

The shoot apical meristem is the ultimate source of the cells that constitute the entire aboveground portion of the plant body. In Arabidopsis thaliana, meristem maintenance is regulated by the negative feedback loop of WUSCHEL-CLAVATA (WUS-CLV). Although CLV-like genes, such as FLORAL ORGAN NUMBER1 (FON1) and FON2, have been shown to be involved in maintenance of the reproductive meristems in rice (Oryza sativa), current understanding of meristem maintenance remains insufficient. In this article, we demonstrate that the FON2-LIKE CLE PROTEIN1 (FCP1) and FCP2 genes encoding proteins with similar CLE domains are involved in negative regulation of meristem maintenance in the vegetative phase. In addition, we found that WUSCHEL-RELATED HOMEOBOX4 (WOX4) promotes the undifferentiated state of the meristem in rice and that WOX4 function is associated with cytokinin action. Consistent with similarities in the shoot apical meristem phenotypes caused by overexpression of FCP1 and downregulation of WOX4, expression of WOX4 was negatively regulated by FCP1 (FCP2). Thus, FCP1/2 and WOX4 are likely to be involved in maintenance of the vegetative meristem in rice.     

Isolation and characterization of a rice homebox gene, OSH15

In many eukaryotic organisms including plants, homeobox genes are thought to be master regulators that establish the cellular or regional identities and specify the fundamental body plan. We isolated and characterized a cDNA designated OSH15 (Oryza sativa homeobox 15) that encodes a KNOTTED-type homeodomain protein. Transgenic tobacco plants overexpressing the OSH15 cDNA showed a dramatically altered morphological phenotype caused by disturbance of specific aspects of tobacco development, thereby indicating the involvement of OSH15 in plant development. We analyzed the in situ mRNA localization of OSH15 through the whole plant life cycle, comparing the expression pattern with that of another rice homeobox gene, OSH1. In early embryogenesis, both genes were expressed as the same pattern at a region where the shoot apical meristem would develop later. In late embryogenesis, the expression pattern of the two genes became different. Whereas the expression of OSH1 continued within the shoot apical meristem, OSH15 expression within the shoot apical meristem ceased but became observable in a ring shaped pattern at the boundaries of some embryonic organs. This pattern of expression was similar to that observed around vegetative or reproductive shoots, or the floral meristem in mature plants. RNA in situ localization data suggest that OSH15 may play roles in the shoot organization during early embryogenesis and thereafter, OSH15 may be involved in morphogenetic events around the shoot apical meristem.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

<Emphasis Type="Italic">OsREL2</Emphasis>, a rice TOPLESS homolog functions in axillary meristem development in rice inflorescence

The morphology of the rice inflorescence, called the panicle, is determined mainly by the activities of axillary meristems including primary, secondary, and spikelet meristems. Recently, in maize, the RAMOSA1 ENHANCER LOCUS2 (REL2) gene, orthologous to the Arabidopsis shoot apical meristem fate-determining TOPLESS, was shown to be involved in the regulation of axillary meristem determinacy. In order to investigate the function of the rice REL2 homolog, we identified and characterized the rice REL2 gene (OsREL2). Compared to other rice TPL homologs, OsREL2 gene expression stayed relatively low throughout panicle development. We characterized a T-DNA insertion osrel2 mutant that showed pleiotropic phenotypic defects, such as defects in panicle heading, sterile lemma elongation, and panicle development, suggesting the OsREL2 functions in multiple developmental processes. In particular, osrel2 developed shorter axillary branches and reduced numbers of lateral organs on axillary branches in comparison to the wild-type, indicating that OsREL2 is important in axillary meristem maintenance. Interestingly, osrel2 produced more primary branches and fewer secondary branches than the wild-type. These results suggest that OsREL2 is involved in branch formation regulation, presumably by suppressing primary branch formation and promoting secondary branch formation.     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene NANA regulates cell proliferation in Arabidopsis thaliana shoot apical meristem without interaction with CLV1, CLV2, CLV3

A constancy of stem cell pool in shoot apical meristem of Arabidopsis thaliana is provided by a genetic regulation system with negative feedback loop based on the interaction of the gene WUS, which maintains indeterminate state of cells, with CLV genes, which restrict the level of WUS expression and stem cell pool size. clv mutations lead to an increase in the pool of stem cells in the apical and floral meristems and wus mutation leads to the opposite effect. Mutation na (nana), like wus mutation, causes premature termination of shoot apical meristem function, although it does not affect the activity of the flower meristem. To elucidate the role of NA in the control of shoot apical meristem functioning, the interaction of NA with CLV genes were investigated. Additive phenotype of double mutants na clv1, na clv2-1, and na clv3-2 indicates that the NA gene makes an independent contribution to the functioning of the shoot apical meristem. It is assumed that the NA gene controls apical meristem cell proliferation during the transition to the reproductive phase of plant development, acting much later and independently of the genes WUS-CLV.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Expression of SHOOT MERISTEMLESS, WUSCHEL, and ASYMMETRIC LEAVES1 Homologs in the Shoots of Podostemaceae: Implications for the Evolution of Novel Shoot Organogenesis

Podostemaceae (the river weeds) are ecologically and morphologically unusual angiosperms. The subfamily Tristichoideae has typical shoot apical meristems (SAMs) that produce leaves, but Podostemoideae is devoid of SAMs and new leaves arise below the base of older leaves. To reveal the genetic basis for the evolution of novel shoot organogenesis in Podostemaceae, we examined the expression patterns of key regulatory genes for shoot development (i.e., SHOOT MERISTEMLESS (STM), WUSCHEL (WUS), and ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) orthologs) in Tristichoideae and Podostemoideae. In the SAM-mediated shoots of Tristichoideae, like in model plants, STM and WUS orthologs were expressed in the SAM. In the SAM-less shoots of Podostemoideae, STM and WUS orthologs were expressed in the initiating leaf/bract primordium. In older leaf/bract primordia, WUS expression disappeared and STM expression became restricted to the basal part, whereas ARP was expressed in the distal part in a complementary pattern to STM expression. In the reproductive shoots of Podostemoideae with a normal mode of flower development, STM and WUS were expressed in the floral meristem, but not in the floral organs, similar to the pattern in model plants. These results suggest that the leaf/bract of Podostemoideae is initiated as a SAM and differentiates into a single apical leaf/bract, resulting in the evolution of novel shoot-leaf mixed organs in Podostemaceae.     

TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation via the WOX4 Homeobox Gene in Arabidopsis

The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell–cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.     

ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression

Auxin and cytokinin direct cell proliferation and differentiation during the in vitro culture of plant cells, but the molecular basis of these processes, especially de novo shoot regeneration, has not been fully elucidated. Here, we describe the regulatory control of shoot regeneration in Arabidopsis thaliana (L.) Heynh, based on the interaction of ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and WUSCHEL (WUS). The major site of ARR12 expression coincided with the location where the shoot apical meristem (SAM) initiated. The arr12 mutants showed severely impaired shoot regeneration and reduced responsiveness to cytokinin; consistent with this, the overexpression of ARR12 enhanced shoot regeneration. Certain shoot meristem specification genes, notably WUSCHEL (WUS) and CLAVATA3, were significantly downregulated in the arr12 explants. Chromatin immunoprecipitation (ChIP) and transient activation assays demonstrated that ARR12 binds to the promoter of WUS. These observations indicate that during shoot regeneration, in vitro, ARR12 functions as a molecular link between cytokinin signaling and the expression of shoot meristem specification genes.