Botanical Gardens, Graduate School of Science, The University of Tokyo, Hakusan 3-7-1, Bunkyo-ku, Tokyo 112-0001, Japan
* Author for correspondence (e-mail: sugiyama{at}ns.bg.s.u-tokyo.ac.jp)
Accepted 4 August 2003
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SUMMARY |
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Key words: Arabidopsis thaliana, Temperature-sensitive mutant, Adventitious root formation, Cell proliferation, Dedifferentiation, Root primordium, Root apical meristem, Auxin, microtubule, MOR1
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Introduction |
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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.
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Materials and methods |
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Plant growth conditions
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
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 (15-25
µmol/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.
Isolation of mutants
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. M2
plants 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 M2
candidate. 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.
Complementation tests
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, F1
progenies 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.
Chromosome mapping
Mutants were crossed to the Columbia strain and the resultant F2
populations 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 F2
seedlings 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).
Whole-mount preparations
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).
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Results |
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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 1 to 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.
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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 rid5 mutation 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
).
Auxin dependency of adventitious root formation in the rid5
mutant
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 rid5
mutant 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).
|
|
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 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 rid4 mutant 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).
Seedling phenotypes of the mutants
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).
|
Allelism between rid5 and mor1
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/GEM1
gene 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.
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Discussion |
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Reinitiation of cell proliferation
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.
Auxin response and microtubules
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 RID5
function, 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 in
-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/GEM1
locus: 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 mor1 mutations 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.
Root primordium development
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.
Establishment of the root apical meristem
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 SRD2
and 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.
Root growth
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.
Concluding remarks
We report on a series of temperature-sensitive mutants that affect
adventitious root formation at different stages
(Fig. 8).
|
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.
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ACKNOWLEDGMENTS |
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