CORONA, a member of the class III homeodomain leucine zipper gene family in Arabidopsis, regulates stem cell specification and organogenesis

Organogenesis at the shoot meristem requires a delicate balance between stem cell specification and differentiation. In Arabidopsis thaliana, WUSCHEL (WUS) is a key factor promoting stem cell identity, whereas the CLAVATA (CLV1, CLV2, and CLV3) loci appear to promote differentiation by repressing WUS expression. In a screen for mutations modifying clv1 mutants, we have identified a novel regulator of meristem development we term CORONA (CNA). Whereas cna single mutant plants exhibit subtle defects in meristem development, clv cna double mutants develop massively enlarged apices that display early loss of organogenesis, misexpression of WUS and CLV3, and eventual differentiation of the entire apex. The CNA gene was isolated by positional cloning and found to encode a class III homeodomain Leu zipper protein. A missense mutation resulting in the dominant-negative cna-1 allele was identified in a conserved domain of unknown function, and a likely null allele was shown to display a similar but weaker phenotype. CNA is expressed in developing vascular tissue, diffusely through shoot and flower meristems, and within developing stamens and carpels. Our analysis of WUS expression in wild-type, clv, and clv cna plants revealed that, contrary to current models, WUS is neither necessary nor sufficient for stem cell specification and that neither WUS nor CLV3 is a marker for stem cell identity. We propose that CNA functions in parallel to the CLV loci to promote organ formation.     

Further studies on the effects of different auxins and cytokinins on flower formation in thin cell layers of Nicotiana tabacum: The effects of zeatin riboside, dihydrozeatin and dihydrozeatin riboside

Organogenesis in thin cell layers of Nicotiana tabacum L. was studied in relation to the effects of natural and synthetic auxins in combination with various cytokinins. All cytokinins tested, benzyladenine (BA), kinetin, zeatin (Z), zeatin riboside (ZR), N⁶-Δ²-isopentenyl) adenine (IPA), dihydrozeatin [(diH)Z] and dihydrozeatin riboside [(diH)ZR], seem to be active in flower bud formation. In addition to the initiation of flower buds, vegetative buds or roots were also formed on the explants in the presence of BA, Z or IPA as exogenous cytokinins. Only dihydrozeatin and its riboside stimulated the initation of flower buds alone (as is known for kinetin), especially if supplemented with indole-3-acetic acid (IAA) as exogenous auxin. A high number of explants with flower buds was also found with high cytokinin/2,4-D ratios. In these conditions the presence of (diH)Z yielded the higest number of flower buds per explant.     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1 (MOC1) gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering,we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins (MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 res...     

Identification and functional analysis of the MOC1 interacting protein 1

Rice tillering is one of the most important agronomic traits that determine grain yields.Our previous study has demonstrated that the MONOCULM1(MOC1)gene is a key component that controls the formation of rice tiller buds.To further elucidate the molecular mechanism of MOC1 involved in the regulation of rice tillering.we performed a yeast-two-hybrid screening to identify MOC1 interacting proteins(MIPs).Here we reported that MIP1 interacted with MOC1 both in vitro and in vivo.The overexpression of MIP1 resulted in enhanced tillering and reduced plant height.In-depth characterization of the context of MIP1 and MOC1 would further our understanding of molecular regulatory mechanisms of rice tillering.     

Regulation of CLV3 expression by two homeobox genes in Arabidopsis

The ability of meristems to continuously produce new organs depends on the activity of their stem cell populations, which are located at the meristem tip. In Arabidopsis, the size of the stem cell domain is regulated by two antagonistic activities. The WUS (WUSCHEL) gene, encoding a homeodomain protein, promotes the formation and maintenance of stem cells. These stem cells express CLV3 (CLAVATA3), and signaling of CLV3 through the CLV1/CLV2 receptor complex restricts WUS activity. Homeostasis of the stem cell population may be achieved through feedback regulation, whereby changes in stem cell number result in corresponding changes in CLV3 expression levels, and adjustment of WUS expression via the CLV signal transduction pathway. We have analyzed whether expression of CLV3 is controlled by the activity of WUS or another homeobox gene, STM (SHOOT MERISTEMLESS), which is required for stem cell maintenance. We found that expression of CLV3 depends on WUS function only in the embryonic shoot meristem. At later developmental stages, WUS promotes the level of CLV3 expression, together with STM. Within a meristem, competence to respond to WUS activity by expressing CLV3 is restricted to the meristem apex.     

Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.     

Relations between cytokinin level, bud development and apical control in Norway spruce, Picea abies

In conifers such as Norway spruce, the extent of shoot growth is predetermined by the size and number of embryonal organs of the buds laid down the previous year. As it is known that cytokinins have a key role in bud development a possible hypothesis is that the level of cytokinin in the buds during their formation determines their size and complexity. As a first step to test this hypothesis we compared cytokinin levels in buds of different size of annual shoots from 15- to 20-year-old trees of Picea abies (L.) Karst. Apical buds from the leaders, and from branches in lower parts of the trees, were collected in April, July and August. The difference in size of the buds and the shoots growing from them was considerable in these three positions. Extracts were purified by immunoaffinity columns, and the retained compounds were separated by high-performance liquid chromatography (HPLC). Quantification was made by enzyme-linked immunosorbent assay (ELISA), and the accuracy of this method was checked by measurements with liquid chromatography-mass spectrometry (LC-MS) and UV absorption. Zeatin riboside (ZR) was the most abundant cytokinin, but isopentenyladenosine (iPA) was also present in all samples. The large apical bud of the leader contained much higher cytokinin concentrations than the considerably smaller buds from lower positions, and during the period of secondary growth in July, similar relationships were found for annual stem tissue from different positions. The possible role of ZR as a controlling factor in bud development and apical control is discussed. Our conclusion is that the level of zeatin-type cytokinins appears to play an important role in the establishment of differences in bud size and, thereby, the architecture of the tree crown.     

Cytokinins from terminal buds of Euphoria longana during different growth stages

The presence of endogenous cytokinins were detected in the terminal buds of longan ( Euphoria longana Lam.) after purification by ion exchange and Sephadex LH-20 chromatography, and bioassay, enzymic degradation, high-performance liquid chromatography and gas chromatography-mass spectrometry. Permethylated derivatives of two highly active cytokinin glucoside compounds from dormant buds were: 6-(4-O-β-D-glucosyl-3-methyl-but-2-enylamino) purine (zeatin-O-glucoside) and 9-β-D-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino) purine (zeatin riboside-O-glucoside). Simultaneously, four active cytokinins from buds at the stages of leaf flush and flower bud initiation were identified as 6-(4-hydroxy-3-methyl-but- trans -2-enylamino) purine (zeatin), zeatin-9-β-D-ribofuranosylpurine (zeatin riboside), 6-(3-methyl-2-butenyl) aminopurine (isopentenyladenosine, 2iPA) and N-(3-methyl-2-butenyl) adenine (isopentenyladenine, 2iP). The total cytokinin levels were low at leaf flush, with the zeatin and zeatin riboside in the buds about 70% of the total. In the transition of the terminal bud from leaf flush to dormancy, the principal cytokinins were zeatin-O-glucoside and zeatin riboside-O-glucoside. However, significant decreases in the levels of zeatin-O-glucoside and zeatin riboside-O-glucoside and increases in those of zeatin, zeatin riboside, 2iPA and 2iP were observed at flower bud initiation. It is suggested that in longan, the cytokinins that are translocated to the shoots are accumulated in the buds at the dormant stage, and that later there is a considerable increase in free cytokinins during flower bud initiation, leading to the promotion of flower bud development.     

The accumulation and metabolism of plant growth regulators during organogenesis in cultures of thin cell layers of Nicotiana tabacum

The accumulation and metabolism of exogenously applied and endogenously produced auxins and cytokinins were studied in relation to organogenesis in thin cell layers of Nicotiana tabacum L. It was shown that, in order to obtain maximal flower bud formation, both exogenous auxin and cytokinin needed to be present during the first 4 days of culture (to the formation of a subepidermal meristematic zone) whereas cytokinins needed to be present for at least 4 days more (until formation of organogenic centres). Explants taken from floral branches have higher endogenous indole-3-acetic acid (IAA) levels compared with explants from the basal part of the stem which form only vegetative buds. This might be related to a different IAA metabolism in these two types of explants as was shown by the different accumulation of exogenously applied IAA. Both 'floral' and 'vegetative' cells layers contained comparable amounts of zeatin riboside (ZR) as their major cytokinin. Free bases, zeatin (Z) and dihydrozeatin [(diH)Z], given exogenously, were largely metabolised to their respective ribosides. The observation that Z was less effective than (diH)Z in the induction of flower buds could be related to (diH)ZR apparently not being a substrate for cytokinin oxidase.     

Phenylurea cytokinins assayed for induction of shoot buds in the moss Funaria hygrometrica

The induction of shoot buds from the filamentous protonema of moss is a classic bioassay for cytokinin. While a large literature documents this response in many species of moss and for a wide range of natural and synthetic cytokinins, to date only substituted adenine cytokinins have been examined in detail. This paper shows that at least some of the novel phenylurea cytokinins will induce bud formation in mosses. Funaria responds to thidiazuron much as it responds to benzyladenine. Exposure to either substance results in log-linear dose-dependent increases in bud number that reach similar maximal numbers of buds at the optimal concentration of compound. The related compound chloro-pyridyl-phenylurea (CPPU) is slightly less active, but induces buds over a wider range of concentration. Carbanilide (diphenylurea or DPU), an active cytokinin in other systems, induces very few buds in Funaria, but does so over a wide range of concentration. Bioassay of mixtures of benzyladenine and DPU finds no evidence of competition for cytokinin receptors. That result could support suggestions that the phenylurea cytokinins act indirectly, by altering endogenous cytokinin metabolism, but we favor another interpretation. Unlike other cytokinin-responsive systems, the induction of buds from moss protonema involves two cytokinin-mediated events. The number of buds is controlled by the second cytokinin-mediated event. If DPU has little or no affinity for the receptor triggering this second event, DPU treatments will produce few to no buds, and kinetic analysis using bud number would find no evidence for competition with benzyladenine. Our results would support the hypothesis that bud induction in Funaria involves two chemically distinct cytokinin receptors.     

Hormonal Profile in Vegetative and Floral Buds of Azalea: Levels of Polyamines, Gibberellins, and Cytokinins

The floral transition includes a complex system of factors that interact and involve various biochemical signals, including plant growth regulators. The physiological signals involved in the control of the floral transition have been sparsely studied and mainly in plant species whose genetics are poorly known. In this work, the role of polyamines, gibberellins, and cytokinins was investigated by analyzing their endogenous content in vegetative and floral buds of azalea. The results showed that there is a clear distinction between floral and vegetative buds with respect to the levels of these plant hormones, with floral buds containing higher amounts of conjugated polyamines, gibberellins (GAs) from the non-13-hydroxylation pathway (GA₉, GA₇, and GA₄), and cytokinins (particularly isopentenyl-type species), and vegetative buds containing higher amounts of free polyamines and gibberellins from the early 13-hydroxylation pathway and fewer cytokinins. In conclusion, there is a specific pattern of endogenous hormone profiles in both vegetative and floral bud development in azalea, which may be relevant for future research on the control of flowering by exogenous hormone applications.     

植物干细胞决定基因WUS的研究进展

WUS(WUSCHEL)基因编码一转录因子,它的存在使周围细胞具有干细胞的特征,与之相关的信号系统近年逐步被阐明.在茎尖分生组织内WUS和CLV(CLAVATA)之间形成一个反馈调节环,使得干细胞保持自我更新,维持茎尖的顶端优势.在胚胎分生组织内,CLV3的表达只依赖于WUS的存在,然而在胚以后的发育中,CLV3的表达受到WUS和STM(SHOOTMERISTEMLESS)的双重调节,启动器官发生.在花分生组织中,WUS和LFY(LEAFY)共同激活AG(AGAMOUS)基因的表达,WUS受AG的反馈抑制.由WUS建立的信号体系还参与胚珠的发育.当WUS蛋白和生长素共存时,可以高效启动体细胞胚的发生.细胞对WUS信号的感应性与细胞所处的微环境有关,WUS在不同环境条件下可以启动不同的下游基因表达.     

In vitro regeneration in chile pepper (Capsicum annuum L.) from ‘half-seed explants’

An in vitro regeneration protocol has been developed from half-seed explants of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.Abbreviations BA benzylamino purine - IAA indole-3-acetic acid - 2iP 6--dimethyl (allyl) amino purine - KIN kinetin - MS Murashige and Skoog medium - NAA napthaleneacetic acid - ZEA zeatin     

Cytokinins in Vegetative and Reproductive Buds of Pseudotsuga menziesii

Immunoaffinity techniques using columns of immobilized antibodies raised against zeatin riboside and isopentenyladenosine were found to be effective in isolating cytoklnins from vegetative, female, and male buds of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The purified cytokinins were separated by reverse phase high performance liquid chromatography and analyzed by radioimmunoassay. Confirmation of cytokinin identities was by gas chromatography-mass spectrometry. Immediately prior to bud burst, all bud types contained three major cytokinins: isopentenyladenosine, zeatin riboside, and a hexose conjugate of zeatin riboside (not zeatin riboside O-glucoside). Zeatin-type cytokinins were present in relatively high concentration in vegetative and female buds. In male buds, however, relatively high levels of isopentenyladenosine were found together with low levels of zeatin-type cytokinins.     

The WUSCHEL and SHOOTMERISTEMLESS genes fulfil complementary roles in Arabidopsis shoot meristem regulation

Continuous organ formation from the shoot apical meristem requires the integration of two functions: a set of undifferentiated, pluripotent stem cells is maintained at the very tip of the meristem, while their daughter cells in the periphery initiate organ primordia. The homeobox genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) encode two major regulators of meristem formation and maintenance in Arabidopsis, yet their interaction in meristem regulation is presently unclear. Here, we have addressed this question using loss- and gain-of-function approaches. We show that stem cell specification by WUS does not require STM activity. Conversely, STM suppresses differentiation independently of WUS and is required and sufficient to promote cell division. Consistent with their independent and distinct phenotypic effects, ectopic WUS and STM activities induce the expression of different downstream target genes. Finally, the pathways regulated by WUS and STM appear to converge in the suppression of differentiation, since coexpression of both genes produced a synergistic effect, and increased WUS activity could partly compensate for loss of STM function. These results suggest that WUS and STM share labour in the shoot apical meristem: WUS specifies a subset of cells in the centre as stem cells, while STM is required to suppress differentiation throughout the meristem dome, thus allowing stem cell daughters to be amplified before they are incorporated into organs.