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Organogenesis in vitroconsists of many aspects such as phytohormone perception, dedifferentiation of differentiated cells to acquire organogenic competence, re-entry of
quiescent cells into cell cycle, and organization of cell division to form specific organ primordia and meristems. Some of elementary processes and essential genes involved in this composite phenomenon are being identified largely through genetic analysis with various types of mutants including temperature-sensitive and activation-tagged ones.
Addresses
Botanical Gardens, Graduate School of Science, The University of Tokyo, Hakusan 3-7-1, Bunkyo-ku, Tokyo 112-0001, Japan; e-mail: sugim@hongo.ecc.u-tokyo.ac.jp
Current Opinion in Plant Biology1999, 2:61–64 http://biomednet.com/elecref/1369526600200061 © Elsevier Science Ltd ISSN 1369-5266
Abbreviations
CDK cyclin-dependent kinase
CR competent in root organogenesis and cell proliferation CSR competent in shoot and root organogenesis
IC incompetent in organogenesis and cell proliferation
Introduction
Organogenesis de novoin tissue cultures has provided use-ful systems for studying regulatory mechanisms of plant development [1]. One of the remarkable feats of earlier physiological analysis on organogenesis in vitro was the identification of a predominant role of auxin and cytokinin as chemical determinants in plant development [2]. Since this pioneer work, many other studies have been carried out with various tissue culture systems and information about organogenesis has accumulated. Molecular mecha-nisms underlying organogenesis in vitro, however, are still unknown. The present review focuses on recent findings that might lead to a greater understanding of the funda-mentals of plant organogenesis at the molecular level.
Outlines of organogenesis
in vitro
Organogenesis in vitrodepends on the application of exoge-nous phytohormones, in particular auxin and cytokinin, and also on the ability of the tissue to respond to these phyto-hormone changes during culture. Thus, the manipulatable nature of tissue culture can be exploited for the physiolog-ical dissection of organogenesis in vitro [3–8]. In general, three phases of organogenesis are recognizable, on the basis of temporal requirement for a specific balance of phytohor-mones in the control of organogenesis. In the first phase, cells in the explants acquire ‘competence’ which is defined as the ability (not capacity) to respond to hormonal signals of organ induction. The process of acquisition of organogenic competence is referred to as ‘dedifferentiation’ hereafter. The competent cells in cultured explants are
Organogenesis
in vitro
Munetaka Sugiyama
canalized and determined for specific organ formation under the influence of the phytohormone balance through the second phase. Then the morphogenesis proceeds inde-pendently of the exogenously supplied phytohormones during the third phase.
Mutants impaired in different stages of organogenesis in vitro are also useful for dissecting this phenomenon. In Arabidopsis, temperature-sensitive mutants (srd1, srd2, and srd3) that are defective for shoot redifferentiation were isolated and characterized (Figure 1; [9,10•]).
Temperature-sensitive periods of these srd mutants allowed locating the function of each SRD gene to the specific stage of organogenesis. The overall results of phenotypic analysis on various organogenic responses of the srd mutants allowed outlines of organogenesis of Arabidopsis in vitro to be drawn as follows [10•]: IC,
incompetent with respect to organogenesis and cell pro-liferation; CR, competent with respect to root Figure 1
Temperature sensitivity of adventitious organogenesis in srdmutants of
Arabidopsis. Shoot redifferentiation (a)and root redifferentiation (b)were induced from hypocotyl explants of the wild-type and srdmutants at the permissive temperature (22°C) or the restrictive temperature (27°C).
22ºC
27ºC
22ºC
27ºC (a)
(b)
Wild srd1 srd2 srd3
Wild srd1 srd2 srd3
organogenesis and cell proliferation; CSR, competent with respect to shoot and root organogenesis. Hypocotyl explants are in the IC state at the initiation of culture and enter the CSR state, via the CR state, during culture on callus-inducing medium, whereas root explants are already in the CR state at the initiation of culture. The transition from IC to CR and that from CR to CSR require the functions of SRD2 and SRD3, respectively. Explants in the CSR state can redifferentiate shoots with the aid of SRD1 and SRD2 when transferred onto shoot-inducing medium. Explants in the CR or CSR state can redifferen-tiate roots when transferred onto root-inducing medium.
Dedifferentiation
According to the requirement for the functions of SRD2 and SRD3, dedifferentiation of incompetent cells can be divided into two subphases, the first subphase from IC to CR and the second subphase from CR to CSR [10•].
During the first subphase, quiescent cells become ready to re-enter cell cycle, and this is considered to be correlated with expression levels of some of genes encoding compo-nents of the cell cycle engine such as cyclins and cyclin-dependent kinases (CDKs). Out of them, PSTAIRE-type CDK is most noteworthy. Plant CDKs are classified into the PSTAIRE proteins that contain a per-fectly conserved PSTAIRE domain characteristic of p34cdc2 homologs and the non-PSTAIRE proteins that contain an only partially conserved PSTAIRE domain [11]. Genes encoding PSTAIRE CDKs are expressed constitutively during cell cycle of proliferating cells whereas most of genes encoding non-PSTAIRE CDKs are expressed in a cell cycle phase-specific fashion [11,12,13•]. Histological
analysis on spatial expression patterns of cdc2aAt gene, which encodes PSTAIRE CDK of Arabidopsis, revealed that this gene is actively expressed not only in dividing cells but also in non-dividing cells of root tissues, such as the pericycle and parenchyma of the vascular cylinder [14,15]. Wounding, which usually triggers (or at least affects positively) dedifferentiation in plant tissue cultures, was found to induce rapidly cdc2aAt expression [15]. A linkage between the expression of cdc2aAt and compe-tence for cell proliferation was proposed from these findings [15,16]. Furthermore, the level of cdc2aAt expres-sion coincides with organogenic competence judged from phenotypes of the srd2mutant [10•]. Accordingly, it can be
concluded that cdc2aAT expression reflects at least some aspects of competence for cell proliferation, even though it might not be the only factor determining competence.
Expression patterns of the cell cycle genes that are not linked with cell proliferation were also reported for D-type cyclin genes of Arabidopsis, CYCD1-3[17,18]. For example, in quiescent cells starved for carbon source, auxin, and cytokinin, the transcript levels of CYCD2and CYCD3were increased by carbon source alone and by cytokinin alone, respectively, a result which indicates that these genes are expressed in response to nutritional or hormonal signals necessary but insufficient for the induction of re-entry into
cell cycle [17]. Thus, there seem to be several stages asso-ciated with active expression of a different subset of the cell cycle genes, possibly reflecting different levels of com-petence for cell proliferation. If this is true, the initial process of dedifferentiation should be characterized by activation of expression of these cell cycle genes as a pre-requisite for the commitment to cell cycle.
Root organogenesis
Cells that have become competent for root organogenesis (entered into the CR or CSR state) as a result of dediffer-entiation in vitroundergo organized cell division to form primordia of adventitious roots in the appropriate culture conditions. A semidominant mutation of tobacco, rac, is a rare example which was well characterized in relation to this process. The racmutant was originally isolated as an auxin-tolerant mutant impaired in the development of pri-mary roots [19]. Later, stem cuttings of this mutant were found to form callus instead of adventitious roots in response to exogenous auxin, which induces adventitious roots in the wild-type [20]. From detailed examination of this phenotype using molecular markers, RACis assumed to be involved in an auxin signal transduction pathway that is specific for adventitious root initiation, or in modi-fying levels of auxin sensitivity that is specifically required for adventitious root initiation [21•]. Further
investigation of the rac mutant would provide important information about molecular mechanisms that realize organized cell division to form root primordia.
Some mutations affecting adventitious root formation might be related to the competence for root organogene-sis rather than root morphogeneorganogene-sis. In rtcs mutants of maize and Mortalmutants of white clover, development of nodal roots, a kind of adventitious roots produced from the nodes, is not initiated [22,23•]. The effects of these
mutations are highly specific to nodal root formation, as all other aspects of plant development in these mutants appeared normal, including primary root development and auxin- or wound-induced formation of adventitious roots. A reasonable explanation for such a phenotype of rtcsand Mortalmutants is that cells of the nodes normally competent for root organogenesis lose competence as a result of mutations and can form adventitious roots only when dedifferentiation is artificially induced. Thus, these mutants would be useful tools for studying the control of organogenic competence during plant development.
Shoot organogenesis
From cells that have acquired competence for shoot organogenesis (i.e. entered into the CSR state) during ded-ifferentiation, the formation of adventitious shoots can be induced by cytokinin application. The cytokinin signaling pathway leading to shoot organogenesis had been an entire black box for decades in spite of the extensive efforts of many researchers, but a component involved in this path-way was recently detected through analysis of cytokinin-independent mutants of Arabidopsis, designated
cki1-1, cki1-2, cki1-3, and cki2 [24]. These mutants were generated by activation T-DNA tagging [25], and isolated as calli that exhibited cytokinin responses such as shoot redifferentiation in the absence of exogenous cytokinin. Within a genomic DNA fragment adjacent to the T-DNA recovered from the cki1-1mutant, a gene, CKI1, responsi-ble for the cytokinin-independent phenotype was identified. Sequence analysis indicated that CKI1encodes a protein similar to the sensor kinase of the two-compo-nent regulation system. From these findings and by analogy with an ethylene receptor, ETR1, which belongs to a family of the sensor kinase [26,27], CKI1 is inferred to function as a cytokinin receptor in the process of cytokinin induction of shoot organogenesis. The cki1phenotype was explained by assuming that CKI1 overexpressed by activa-tion tagging confers the ability to sense low concentraactiva-tions of endogenous cytokinin normally below the threshold of triggering shoot organogenesis. If CKI1 determines the sensitivity of a cell to shoot-inducing signal of cytokinin, a possibility arises that the competence for shoot organogen-esis may reflect directly the activity level of CKI1.
Most recently, several genes encoding putative response regulators of the two-component system were reported from Arabidopsis [28,29•]. Among them, IBC6and IBC7are of
par-ticular interest from the viewpoint of cytokinin signaling because the mRNA levels of these genes are rapidly elevat-ed by exogenous cytokinin application [29•]. The possibility
that the products of these genes act just downstream of CKI1 as response regulators was discussed on the basis of their features [29•]. It follows, therefore, that cytokinin itself
would increase the amplitude of a response to a given con-centration of cytokinin through an autoregulatory loop of the cytokinin signaling system. Such self-amplification of cytokinin signaling, if present, might play a very important role in the control of shoot organogenesis by cytokinin.
Cytokinin signal transduced by the two-component sys-tem induces the establishment of the shoot apical meristem somehow during shoot organogenesis. For key regulators of this unknown process, KNOTTED1 (KN1) and KN1-related proteins are potential candidates. KN1, a gene responsible for the knotted leaf phenotype of a maize mutant, was isolated by transposon tagging and found to encode a homeodomain protein [30]. KN1 tran-script was abundant in the shoot apical meristem but not in the leaf primordia [31]. Close correlation between KN1 expression and the shoot meristem was also observed dur-ing axillary shoot meristem proliferation and adventitious shoot meristem formation in maize and barley [32•].
Ectopic expression of KN1 or KN1-homologs in tobacco and Arabidopsis brought about the formation of adventi-tious shoots from normally determinate organs in severe cases [33,34]. Loss-of-function mutations of KN1 caused defects in inflorescence branching and floral organogene-sis [35•]. In Arabidopsis, SHOOT MERISTEMLESS(STM)
gene, of which loss-of-function mutations result in the failure of development of a shoot meristem during
embryogenesis, was shown to encode a KN1-like home-odomain protein [36]. All these findings strongly suggest that KN1-type homeodomain proteins are involved in the establishment and maintenance of shoot apical meristems.
With respect to the role of these homeodomain proteins in shoot organogenesis in vitro, however, relatively limited information is available. Root explants from the stm-1 mutant of Arabidopsis were reported to be unable to regen-erate normal shoots but able to form abnormal shoots and leaves in tissue culture [37]. As the stm-1allele carries a nonsense mutation upstream of the homeodomain [36], induction of abnormal shoots and leaves cannot be attrib-uted to the leaky function of the mutant STM in this case. Accordingly, it is questionable whether STMis essential for triggering neo-formation of shoot apical meristems during shoot organogenesis in vitro. STM is likely to function for the establishment of shoot apical meristems only after ded-ifferentiated cells are determined for shoot organogenesis.
Conclusions
Organogenesis in vitrois generally composed of three dis-tinct phases of different dependency on exogenous phytohormones: the first phase in which cells are dedif-ferentiated to acquire organogenic competence; the second phase in which dedifferentiated cells are canal-ized and determined for specific organ formation in response to exogenous phytohormones; and the third phase in which organ morphogenesis proceeds indepen-dently of exogenous phytohormones. The first phase of dedifferentiation can be further divided into two sub-phases: the first subphase in which cells acquire competence for proliferation and root organogenesis; and the second subphase in which cells acquire competence for shoot organogenesis. Such dissection of organogenesis in vitrohas been achieved by physiological experiments and phenotypic examination of organogenesis-defective mutants. Genes that are presumed to play critical roles in each phase of organogenesis in vitroare being identified largely through genetic analysis and some of them have been already isolated. Future advances in the research on organogenesis in vitro with these genes as a scaffold would reveal important aspects of plant development. In particular, if the elementary processes essential for dedif-ferentiation were elucidated at the molecular level, it would contribute to true understanding of the flexibility characteristic of plant development.
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