Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

During the post-embryonic development of plants, new axes of growth emerge through lateral or adventitious organogenesis, and the reiteration of this process builds up the complex pattern of a plant body. Regulation of such lateral or adventitious organogenesis provides a flexible way for plants to alter their form and resource allocation in response to environmental changes or after injury. In this context, lateral or adventitious organogenesis plays an essential role in the post-embryonic development and survival of plants.

Among the processes of lateral and adventitious organogeneses, lateral root formation has been extensively studied by various approaches using the model plant, Arabidopsis thaliana. Lateral root formation is considered to consist of two distinct phases: lateral root initiation and the establishment of the root apical meristem (Laskowski et al., 1995; Celenza et al.,1995). The histology of both these phases have been described in detail (Malamy and Benfey,1997). During the first phase, a lateral root primordium originates from a few pericycle cells, called founder cells, that lie in the cell file adjacent to either of the xylem poles. During the second phase, the root apical meristem is established within the primordium. Thereafter, the root apical meristem is activated and becomes responsible for lateral root growth.

Accumulating pieces of physiological and genetic evidence have demonstrated a critical role of the plant growth regulator auxin, which is supplied by shoot tissues through the polar transport system, in the initiation of lateral roots (Reed et al., 1998; Celenza et al., 1995; Hobbie and Estelle, 1995; Ruegger et al., 1998; Fukaki et al., 2002; Xie et al., 2000). Formerly,the cell cycle of pericycle cells was considered to be arrested in G₂ phase and to recommence in response to auxin to initiate lateral root formation (Blakely and Evans,1979). However, recent studies have demonstrated that the actual situation is more complex. According to the work of Dubrovsky et al.(Dubrovsky et al., 2000),pericycle cells at the xylem poles continue to divide without interruption during their passage through the elongation and differentiation zones, and only some of the dividing pericycle cells are committed in a stochastic manner to the asymmetric formative division that gives rise to lateral root primordia. Beeckman et al. (Beeckman et al., 2001) proposed a slightly different but essentially similar scenario of lateral root initiation, in which pericycle cells at the xylem poles, leaving the root meristematic region, progress via S phase to G₂ phase after a transient arrest in G₁ phase, to become competent to initiate lateral roots.

There seems to be another control point for lateral root initiation apart from the developmental control point discussed above. Auxin applied exogenously to mature roots is thought to act on this later control point to induce pericycle cells arrested in G₁ phase to recommence the cell cycle. Therefore, different patterns of cell division among pericycle cells,position-dependent and stochastic determination of cell fate, and dual control points for lateral root initiation make the initial scene of lateral root formation highly complicated and difficult to access.

At the onset of adventitious root formation in tissue culture, no cells are specified to form adventitious roots. They first dedifferentiate, i.e.,acquire competence for cell proliferation and organ regeneration(Ozawa et al., 1998), then initiate cell proliferation and form adventitious root primordia. All these events can be induced reproducibly under the control of exogenous phytohormones. Therefore, adventitious root formation may provide a useful experimental system with which to study the entire process of root formation,including the pre-morphogenesis stages.

Previously, we characterized temperature-sensitive mutants of A. thaliana to dissect the process of shoot and root regeneration via callus formation (Yasutani et al.,1994; Ozawa et al.,1998; Sugiyama,2003). Here we extend this approach to elucidate the process of adventitious root formation. We report the isolation and phenotypic analysis of a series of novel mutants that are temperature-sensitive in various steps of adventitious root formation.

In the present study, the Landsberg erecta (Ler) strain of Arabidopsis thaliana (L.) Heynh. was used as the wild type. The srd2 mutant of Ler origin, which was isolated as temperature-sensitive for shoot redifferentiation(Yasutani et al., 1994) and later also shown to be defective in dedifferentiation and root development(Ozawa et al., 1998), was used together with newly isolated mutants. The scarecrow-2(scr-2) mutant in the Wassilewskija background(Scheres et al., 1995) and the microtubule organization 1-1 (mor1-1) mutant in the Columbia background (Whittington et al.,2001) were kindly provided by Dr Ben Scheres (Utrecht University,The Netherlands) and Dr Geoffrey O. Wasteneys (The Australian National University, Australia), respectively. These mutants were used for allelism tests.

For the source materials of tissue culture, plants were grown aseptically as described in our previous paper (Ozawa et al., 1998). Seeds were surface-sterilized in a solution of about 1.0% sodium hypochlorite and 0.1% (w/v) Triton X-100 for 10 minutes. The seeds were rinsed several times with sterile water, then sown on germination medium (GMA). Plates were incubated at 22°C on a tilt of 45° under continuous light at a fluence rate of 8-14 μmol/m²/second. GMA is MS medium (Murashige and Skoog,1962) supplemented with 10 g/l sucrose, buffered with 0.5 g/l 2-(N-morpholino)ethanesulphonic acid (MES) to pH 5.7, and solidified with 1.5% agar.

Tissue culture experiments for the phenotypic characterization of mutants were performed in a similar way to that described by Ozawa et al.(Ozawa et al., 1998). Hypocotyl or root segments of 5 mm in length were excised from 12- to 14-day-old seedlings and cultured under continuous light (15-25μmol/m²/second) either at 22°C or at 28°C. As a root segment, we used a 5 mm section of primary root that was 5 mm away from the root apex. For the induction of adventitious roots and lateral roots,hypocotyl and root explants were cultured on root-inducing medium [RIM: B5 medium (Gamborg et al., 1968)supplemented with 20 g/l glucose and 0.5 mg/l indole-3-butyric acid (IBA)]. To induce callus formation, hypocotyl and root explants were cultured on callus-inducing medium [CIM: B5 medium supplemented with 20 g/l glucose, 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/l kinetin]. All tissue culture media were buffered to pH 5.7 with 0.5 g/l MES, and solidified with 2.5 g/l gellan gum.

Ler seeds were mutagenized by treatment with 0.3% ethyl methanesulfonate solution for 20 hours at room temperature. In the primary screening, about 8,000 M₂ seedlings were tested for their ability to form adventitious roots at the restrictive (28°C) and permissive(22°C) temperatures. Adventitious root formation was induced from hypocotyl segments of M₂ seedlings by culture on RIM at 28°C,and at the same time from the remaining shoots (the top part of hypocotyl plus shoot tip including cotyledons) by culture on RIM at 22°C. M₂plants that showed some defects in adventitious root formation from hypocotyl explants at 28°C but could form almost normal roots at 22°C from shoots were selected as candidate temperature-sensitive mutants. An M₃ line was constructed from the rooted shoot of each M₂candidate. Secondary screening was carried out using 4-24 hypocotyl segments of every M₃ line. One half of the segments were cultured on RIM at 22°C and the other half at 28°C. M₃ lines that exhibited temperature-dependent defects in adventitious root formation from hypocotyl explants during secondary screening were finally isolated as mutant lines. Before phenotypic analysis, they were genetically purified with three rounds of backcrosses followed by two rounds of self-reproduction.

Mutant lines were categorized into three classes on the basis of their phenotypic similarities (see Results). Complementation tests were carried out for any combinations of two mutant lines belonging to the same class, by examining the temperature sensitivity of adventitious root formation from hypocotyl explants of F₁ seedlings derived from crosses between them. To test the allelism of rid5 with mor1, F₁progenies produced by crossing rid5 with mor1-1 were tested for adventitious rooting in tissue culture and seedling morphology at 28°C. Allelism between rgd3 and scr was assessed by monitoring adventitious root growth at 28°C of F₁ progenies derived from a cross between rgd3 and scr-2.

Mutants were crossed to the Columbia strain and the resultant F₂populations were used for chromosome mapping. F₂ plants were examined for the temperature sensitivity of adventitious root formation in hypocotyl explants. Temperature-sensitive F₂ plants were judged to be homozygous for the mutant allele responsible for the defect in adventitious root formation. DNA was extracted from the cotyledons of these F₂seedlings by grinding them in 200 μl of extraction buffer [200 mM Tris-HCl(pH 7.5), 250 mM NaCl, 25 mM EDTA, 0.5% SDS]. DNA was recovered by ethanol-precipitation and subjected to simple sequence length polymorphism(SSLP) and cleaved amplified polymorphic sequence (CAPS) analyses(http://www.arabidopsis.org/aboutcaps.html).

Hypocotyl explants were fixed overnight at 4°C in a 9:1 mixture of ethanol and acetic acid, hydrated through a graded series of ethanol, and mounted with a drop of clearing solution (a mixture of 8 g chloral hydrate, 2 ml water, and 1 ml glycerol). Root explants were cleared after fixation in 25 mM sodium phosphate buffer (pH 7.0) that contained 2% formaldehyde and 1%glutaraldehyde, or without fixation in some cases. Cleared samples were observed under a light microscope equipped with Nomarski optics (BX50-DIC;Olympus).

We screened mutagenized populations of A. thaliana for their capacity to form adventitious roots from hypocotyl explants and isolated 10 temperature-sensitive mutant lines. Temperature sensitivity was a recessive trait in all these mutants. Complementation tests (data not shown) and chromosome mapping (Table 1)indicated that the 10 mutant alleles represented nine loci.

Comparison of map positions and root phenotypes of our temperature-sensitive mutants with those of mutants known to be deficient in root development suggested possible allelism between rgd3 and scr (Scheres et al.,1995; Di Laurenzio et al.,1996). These two mutants appeared to share a part of characters such as defects in the maintenance of root meristematic activity(Sabatini et al., 2003) (see below for the rgd3 phenotype). However, adventitious root formation and root growth of the F₁ progenies derived from a cross between rgd3 and scr-2 were normal at 28°C, and sequence analysis detected no mutations in the SCR gene, including its promoter region, of the rgd3 mutant (data not shown). Accordingly, we concluded that rgd3 is not allelic to scr.

The isolated mutants showed various genetic lesions in adventitious root formation at the restrictive temperature (Figs 1, 2). They could be roughly categorized into three types: (1) defects in the initial stage and/or pre-morphogenic stage of root formation, (2) defects in the development of primordia, and (3) defects in the growth of roots after the establishment of the root apical meristem. According to this classification, the mutants were designated rid1 to 5 (root initiation defective 1to 5), rpd1 (root primordium defective 1), and rgd1 to 3 (root growth defective 1 to 3). Of these, only rpd1 had two alleles (rpd1-1 and rpd1-2). rpd1-1 showed more pronounced aberrancies than rpd1-2, and was used for subsequent analyses.

On closer observation, the rooting phenotypes of the mutants were as follows. The rid1 hypocotyl did not show any signs of cell proliferation at 28°C. At 22°C, the rid1 mutation was still effective and delayed root initiation by up to 10 days relative to the wild type (Fig. 2). The rid2,rid3, rid4 and rpd1 mutants, when observed at 6 days on RIM, had formed only young root primordia at 28°C, whereas at 22°C, they had completed adventitious root formation at this time(Fig. 2). After a longer period(16 days) of culture on RIM at 28°C, the rid2, rid3 and rid4 mutants eventually established adventitious roots although they were deformed to some extent (Fig. 1). Therefore, the main defects of rid2, rid3 and rid4 were considered to lie in the initial stages prior to the development of root primordia, and to specifically delay the timing of root primordium formation, with limited influence on the later stages. In contrast to the rid mutants, the majority of rpd1 root primordia remained undeveloped after 16 days incubation at 28°C. This suggests that the rpd1 mutation primarily affected the development of the root primordia.

The rooting phenotype of the rid5 mutant at the restrictive temperature was highly variable among explants(Fig. 1). Some explants cultured at 28°C could form adventitious roots that were almost normal in morphology and growth, whereas others gave no indication of cell division that could lead to the formation of root primordia. Thus, the rid5mutation reduced the frequency of root initiation at 28°C without affecting the later stages of root formation.

The rgd mutants showed neither an apparent delay nor a reduction in frequency in adventitious root formation even at 28°C(Fig. 2). In these mutants,however, the subsequent growth of adventitious roots was strongly inhibited at 28°C (Fig. 1).

The srd2 mutant, which was originally isolated as temperature-sensitive for shoot redifferentiation(Yasutani et al., 1994),exhibited a very similar phenotype to that of rid1 with respect to adventitious root formation (Figs 1, 2). Lack of cell proliferation at 28°C in srd2 is consistent with its previously reported defects in the process of cell proliferation during the dedifferentiation of hypocotyl explants (Ozawa et al.,1998).

As described in the previous paragraph, the rid5 mutation specifically influenced the initiation of adventitious roots at 28°C. In tissue culture, the rate of adventitious rooting generally depends on the concentration of exogenous auxin, which is required for dedifferentiation and subsequent root initiation. Taking this into consideration, we examined the effects of IBA on the rooting rate of wild type and rid5(Fig. 3). In our standard RIM,the IBA concentration is set to 0.5 mg/l. The rooting rate of the wild type was gradually reduced as IBA concentrations were lowered. In the wild type,the IBA concentration required for maximal rooting was a little higher at 22°C than at 28°C. Compared with the wild type, the rid5mutant showed clear temperature sensitivity in auxin-dependent rooting. The IBA-response curve for the rooting rate of rid5 was similar to that of the wild type at 22°C, but was shifted greatly at 28°C. These results imply that the rid5 mutation is detrimental to auxin signaling that induces root formation. Similar experiments using the other mutants did not detect such a shift in the auxin-response curve with an increase in temperature (data not shown).

Root explants of the mutants were cultured on RIM at 22°C and 28°C to induce lateral root formation, and were observed after 16 days in culture. srd2, rid1, rid2 and rid4 exhibited aberrant lateral roots morphology under restrictive conditions(Fig. 4). The rid1mutant showed the most severe phenotype, with lateral root primordia developing into massive structures. In the root explants of the rid4mutant, RIM culture at 28°C induced fasciated lateral roots at a high frequency (data not shown).

To examine the effects of these mutations on cell proliferation independently of tissue organization, we tested the temperature sensitivity of callus formation in these mutants by culturing hypocotyl and root explants on CIM for 24 days at either 22°C or 28°C.

Callus formation was completely blocked in the hypocotyls of the srd2 and rid1 mutants at 28°C(Fig. 5). Root explants of both mutants, however, formed calli at 28°C that were similar to those formed at 22°C (Fig. 6). The srd2 and rid1 mutations thus interfere with cell proliferation from hypocotyl explants, but not with cell proliferation from root explants.

Callus formation from hypocotyl explants of the rid2 mutant was highly temperature-sensitive and severely inhibited at 28°C(Fig. 5). Root explants of rid2 could form calli at 28°C, but their appearance was unusual(Fig. 6Z). In this case, calli formed at intervals where no cell proliferation was observed. This is quite different from normal callus formation, which takes place all over the explant. The discontinuous callusing phenotype of the rid2 mutant may be attributable to a deficiency in the initiation of cell proliferation from root pericycle cells that are competent but quiescent (see Discussion for details).

Callus formation by the rid3 mutant appeared to be temperature-insensitive both in hypocotyl explants and in root explants after 24 days in culture on CIM (Figs 5, 6). When observed on day 4 in culture, however, callus formed at 28°C from hypocotyl explants was markedly smaller than that formed at 22°C (data not shown), suggesting leaky defects of the rid3 mutation in the initial stage of cell proliferation or in the dedifferentiation stage that precedes cell proliferation.

Callus formation in both hypocotyl and root explants of the rid4mutant showed temperature sensitivity (Figs 5, 6). At 28°C, variable suppression of cell proliferation was observed among explants. In some explants, cell proliferation was strongly inhibited, whereas it was only partially inhibited in others. Such phenotypes could be explained by assuming that the defect in the rid4 mutant during callus formation is restricted to the initial step of cell proliferation and that the rid4 explants are able to develop calli once they pass through this step by chance.

In the cases of the rpd1, rgd1 and rgd2 mutants, both hypocotyl and root explants could initiate callus formation at 28°C but there were defects in callus development or growth (Figs 5, 6). The rid5 and rgd3 mutants did not show apparent temperature sensitivity in callus formation from hypocotyl and root explants (Figs 5, 6).

To assess the effect of the mutations on other developmental processes, the whole-plant phenotypes of seedlings grown on vertically placed GMA at 22°C or 28°C for 12 days were observed (Fig. 7). At 22°C, the mutant seedlings looked almost normal except rid1 and rid4. The primary roots of these two mutants were short relative to the wild type and the other mutants. At 28°C, the primary roots of all the mutants were much shorter than those formed at 22°C (Fig. 7A,B). The shoot phenotypes of the mutants at 28°C varied somewhat between repeated experiments. Formation of linear true leaves, lacking leaf lamina in severe cases, was reproducibly and frequently observed in the rid3 seedlings(Fig. 7L,M).

rid5 mutant seedlings exhibited peculiar temperature-dependent abnormalities. Their primary root grew leftward at 28°C(Fig. 7B, inset). Left-handed helical growth was induced at 28°C in epidermal cell files of the hypocotyls, as well as the primary roots(Fig. 7F,J). Moreover, the root hairs of rid5 often branched and the branching was more pronounced at the higher temperature (Fig. 7J).

Helical growth similar to those observed in the rid5 seedling at 28°C have been reported for several microtubule-related mutants and wild-type plants treated with microtubule-depolymerizing or -stabilizing drugs(Rutherford and Masson, 1996; Marinelli et al., 1997; Furutani et al., 2000; Whittington et al., 2001; Thitamadee et al., 2002; Sedbrook et al., 2002). This similarity suggested a possible relationship between rid5 and the microtubule system. Since rid5 was mapped near the MOR1/GEM1gene encoding a microtubule-associated protein(Whittington et al., 2001; Twell et al., 2002), we tested whether rid5 is an allele of this gene. F₁ seedlings produced by crossing rid5 and mor1-1 all exhibited left-handed helical growth at 28°C (data not shown). Adventitious root formation from the F₁ hypocotyls on RIM containing 0.25 mg/l IBA at 28°C was very poor as compared to the wild-type explants (data not shown). These results confirmed the allelic relationship of rid5 to mor1-1. Sequence analysis of the MOR1/GEM1 gene of rid5 identified a mutation of TGC to TAC, which causes an amino acid substitution of Tyr for Cys at residue 96 in the gene product.

We isolated nine novel temperature-sensitive mutants of A. thaliana that are defective in adventitious root formation. On the basis of their phenotypes at the restrictive temperature, we roughly classified these mutants into three groups: (1) the rid mutants, defective in the initial stage or the pre-morphogenic stage of adventitious root formation;(2) the rpd1 mutant, arrests during the development of root primordia, and (3) the rgd mutants, which can establish adventitious roots but fail to maintain their growth. We note that the mutations, except for rgd3, seem to interfere not with tissue organization per se but with mechanisms involved in cell proliferation-related events during adventitious root formation.

Adventitious root formation from hypocotyl segments starts with the recommencement of cell division in quiescent hypocotyls. Earlier observations on the srd2 mutant revealed that, at the restrictive temperature,hypocotyl explants did not reinitiate cell proliferation, whereas root explants could reinitiate cell proliferation and form calli(Ozawa et al., 1998). This suggested that `reinitiation of cell proliferation' from hypocotyl explants entails two distinct stages: the acquisition of competence for cell proliferation and the resumption of the cell cycle. In our view, the non-dividing cells of root explants are equivalent to the cells of hypocotyl explants that have acquired the competence for cell proliferation. Among the novel series of mutants reported here, the rid1 phenotype was very similar to that of srd2. Hypocotyl explants of rid1 could not reinitiate cell proliferation at the restrictive temperature but their root explants could (Figs 5, 6). This feature suggests that both the SRD2 and RID1 genes are involved in the same process of the acquisition of competence for cell proliferation during dedifferentiation of hypocotyl cells, and that their functions are not required for the resumption and subsequent progression of the cell cycle.

The RID2 gene may play some essential roles in the second stage of the reinitiation of cell proliferation, i.e., the resumption of the cell cycle in competent but quiescent cells. The root explants of rid2 formed calli at intervals under the restrictive conditions(Fig. 6Z). One possible explanation for this phenotype is that the rid2 mutation specifically inhibits the reentry of non-dividing cells into the cell cycle but does not affect callus formation by dividing cells, such as those of the lateral root primordia present at intervals in the root segments. This hypothesis can also account for the temperature-sensitive initiation of callus and adventitious roots from hypocotyl explants of the rid2 mutant. Further characterization of the rid2 mutant is in progress to test this hypothesis.

The threshold level of auxin required for adventitious root initiation became higher in the rid5 mutant at the restrictive temperature(Fig. 3). This finding suggests that the RID5 gene functions somewhere, directly or indirectly, in the auxin signaling pathway that stimulates cells to proliferate to form adventitious roots. The auxin signaling machinery may require RID5function, either for the acquisition of competence or for resumption of the cell cycle, or for both.

In the rid5 seedlings cultured at the restrictive temperature, the primary roots grew leftward, and the epidermal cell files of the hypocotyls and primary roots formed left-handed helices(Fig. 7). Similar phenotypes have been reported for several microtubule-related mutants(Furutani et al., 2000; Whittington et al., 2001; Thitamadee et al., 2002). Cortical microtubule arrays of spr mutants, which display right-handed helical growth (their primary roots grow rightward), are skewed into left-handed obliques (Furutani et al.,2000). Recently, mutants with left-handed helical growth, lefty1 and lefty2, were found to have mutations inα-tubulin genes (Thitamadee et al.,2002). The lefty mutant tubulins were incorporated into microtubule polymers, producing right-handed cortical arrays. These studies indicate that the orientation of the cortical microtubule arrays are critical in the handedness of helical growth. In light of these findings, the temperature-sensitive helical growth of the rid5 mutant very probably results from some alterations in cortical microtubules. Root-hair branching of the rid5 mutant may also be related to cortical microtubules.

Temperature-sensitive mutants, mor1-1 and mor1-2, of a MAP215 family microtubule-associated protein, MOR1/GEM1, have been described in a recent report (Whittington et al.,2001). They exhibited aberrant cortical microtubule organization and left-handed helical growth at the restrictive temperature. A complementation test and sequence analysis showed that rid5 is a new mutant allele of the MOR1/GEM1 gene.

So far, four mutant alleles have been reported for the MOR1/GEM1locus: mor1-1, mor1-2, gem1-1 and gem1-2. The mor1-1 and mor1-2 mutations, both of which result in a single amino acid substitutions of the amino-terminal HEAT repeat of the MOR1/GEM1 protein, affect cortical microtubule arrays only in interphase(Whittington et al., 2001). The gem1-1 and gem1-2 mutations, both of which result in a carboxy-terminal truncation, cause a severe defect in cytokinesis and consequently lethality (Twell et al.,2002).

Our new mutant allele, rid5, results in an amino acid substitution at the amino-terminal region outside of and adjacent to the HEAT repeat. Hence it is possible that the rid5 mutation specifically interferes with the interphase organization of cortical microtubules like the mor1mutations and unlike the gem1 mutations. This view is consistent with the rid5 phenotypes, particularly in the left-handed helical growth and the negligible effect on cell proliferation at the restrictive temperature.

Phytohormones, including auxin, function in modulating cyclic changes in the orientations of microtubule arrays between transverse and longitudinal via an oblique orientation (Shibaoka,1994; Takesue and Shibaoka,1999). Interestingly, Marinelli et al.(Marinelli et al., 1997)reported a digenic mutant that showed right-handed helical growth and enhanced sensitivity to auxin with respect to its inhibitory effect on primary root elongation. Taken together with the reduced responsiveness to auxin and presumed alterations in the cortical microtubules of the rid5 mutant,we surmise that the auxin signaling pathway in adventitious root formation is mediated by modulation of the orientation of the microtubule arrays, which involves the MOR1/GEM1 function.

In the course of adventitious root formation, after reinitiation of cell proliferation cells undergo several rounds of division to develop the root primordia in which the apical meristem subsequently arises. At the restrictive temperature, adventitious root primordia of the rpd1 mutant were arrested during development, prior to the formation of recognizable apical meristems (Fig. 2). This phenotype suggests the involvement of the RPD1 gene in the construction of the root apical meristem. However, the rpd1 mutation also inhibits callus development at early stages. Thus, the RPD1 gene may participate in the maintenance of the cell proliferation required for both root primordium and callus development.

When lateral roots were induced from root explants at the restrictive temperature, the srd2, rid1 and rid2 mutants generated deformed laterals, indicating defects in establishing the root apical meristem(Fig. 4). The acquisition of competence for cell proliferation, which was affected by the srd2 and rid1 mutations during adventitious root formation and callus formation from hypocotyl explants, is not required for lateral root formation from root explants that are initially competent. Therefore, the failure to establish apical meristems in lateral root primordia in the srd2 and rid1 mutants may suggest a direct involvement of the SRD2and RID1 functions. In the case of the rid2 mutant,misorganization of the root apical meristems might be a secondary consequence of its leaky deficiency in reinitiation of cell proliferation from the root pericycle.

Both adventitious root growth and callus growth were inhibited in the rgd1 and rgd2 mutants at the restrictive temperature. The rgd3 mutant, however, was defective in adventitious root growth but not in callus growth (Figs 1, 5, 6). These results may indicate that RGD1 and RGD2 function in general aspects of root and callus growth, and the RGD3 function is required for a mechanism specific to root growth, e.g., the maintenance of the root apical meristem. Alternatively, the rgd3 mutant allele may be weak and therefore not showing the full gene function.

We report on a series of temperature-sensitive mutants that affect adventitious root formation at different stages(Fig. 8).

Hypocotyl explants of Arabidopsis thaliana are initially incompetent for cell proliferation. Upon culture on RIM, they become competent. The srd2 and rid1 mutants are defective at this stage. Hypocotyl explants that have acquired competence, which are comparable to root explants, recommence cell division. This step is defective in rid2. The rid5 mutation affects adventitious root formation somewhere in the signaling pathway from auxin to the reinitiation of cell proliferation probably via alterations of cortical microtubule arrays. Proliferating cells undergo cycles of cell division to form the root primordia. The rpd1 mutant is unable to maintain cell proliferation of the root primordia. The root apical meristem is subsequently established. At this stage, the srd2 and rid1 mutants are again affected. The growth of adventitious roots requires the functions of at least three genes, RGD1, RGD2 and RGD3. We include the process of callus formation in this scheme to address the effects of mutations in the RPD1,RGD1 and RGD2 genes in callus formation.

Owing to the temperature-sensitive nature of our series of mutants, they are not likely to represent full loss-of-function alleles. Therefore we cannot assess whether the corresponding genes are required only at the steps affected in the mutants, or whether they serve a more general role. Further analysis of these mutants should nevertheless identify elementary components of the mechanisms underlying adventitious root formation.

We thank Dr Geoffrey O. Wasteneys for providing the mor1-1 seeds and Dr Ben Scheres for providing the scr-2 seeds and valuable comments. We also thank Dr Takashi Hashimoto for helpful suggestions and Yoko Tanaka-Fukuda for technical assistance in genetic purification. This work was supported by Grants-in-Aid for the `Research for the Future' program from the Japan Society for the Promotion of Science and from the Ministry of Sports,Culture, Science and Technology of Japan (RFTF97L00601 and RFTF00L01605), by Grants-in-Aid for Scientific Research from the Ministry of Education, Sports,Culture, Science and Technology of Japan (no. 10182101 and no. 14036209), and by the Sasakawa Scientific Research Grant from the Japan Science Society (no. 15196).