Download Short root mutant of Lotus japonicus with a dramatically altered

The Plant Journal (2000) 23(1), 97±114
Short root mutant of Lotus japonicus with a dramatically
altered symbiotic phenotype
Judith Wopereis1,¶,², Eloisa Pajuelo2,¶, Frank B. Dazzo3, Qunyi Jiang4, Peter M. Gresshoff4, Frans J. de Bruijn1,3,*,
Jens Stougaard2 and Krzysztof Szczyglowski1
1
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824±1312, USA,
2
Laboratory of Gene Expression, Department of Molecular and Structural Biology, University of Aarhus, Gustav Wieds
Vej 10, DK-8000 Aarhus C, Denmark,
3
Department of Microbiology, Michigan State University, East Lansing, MI 48824±1312, USA, and
4
Department of Botany, University of Queensland, Brisbane, QLD 4072, Australia
Received 21 January 2000; revised 10 April 2000; accepted 7 May 2000.
*For correspondence: DOE-Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA (fax +1517 353-9168;
e-mail [email protected]). After July 2000: Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganisms, CNRS-INRA, 31326 CastanetTologon, Cedex, France.
²
Present address: Department of Biological Sciences, Smith College, Northampton, MA 01063, USA.
¶
These two authors made equal contributions to this work.
Summary
Legume plants carefully control the extent of nodulation in response to rhizobial infection. To examine
the mechanism underlying this process we conducted a detailed analysis of the Lotus japonicus
hypernodulating mutants, har1-1, 2 and 3 that de®ne a new locus, HYPERNODULATION ABERRANT
ROOT FORMATION (Har1), involved in root and symbiotic development. Mutations in the Har1 locus
alter root architecture by inhibiting root elongation, diminishing root diameter and stimulating lateral
root initiation. At the cellular level these developmental alterations are associated with changes in the
position and duration of root cell growth and result in a premature differentiation of har1-1 mutant root.
No signi®cant differences between har1-1 mutant and wild-type plants were detected with respect to
root growth responses to 1-aminocyclopropane1-carboxylic acid, the immediate precursor of ethylene,
and auxin; however, cytokinin in the presence of AVG (aminoetoxyvinylglycine) was found to stimulate
root elongation of the har1-1 mutant but not the wild-type. After inoculation with Mesorhizobium loti,
the har1 mutant lines display an unusual hypernodulation (HNR) response, characterized by unrestricted
nodulation (hypernodulation), and a concomitant drastic inhibition of root and shoot growth. These
observations implicate a role for the Har1 locus in both symbiotic and non-symbiotic development of
L. japonicus, and suggest that regulatory processes controlling nodule organogenesis and nodule
number are integrated in an overall mechanism governing root growth and development.
Keywords: root development, nodulation, Rhizobium, phytohormones.
Introduction
Root development in plants is a complex process
involving a high degree of morphological plasticity,
which re¯ects inherent adaptive mechanisms to highly
variable environmental conditions. Although the molecular determinants of root morphology and functioning
are only starting to be understood, classical physiological experiments have clearly implicated both local and
systemic regulatory circuits in the determination of root
plasticity. In addition to its role in selective exploitation
ã 2000 Blackwell Science Ltd
of speci®c soil domains for available nutrient and water
sources, an important role of root developmental
plasticity is to provide plants with the ability to
recognize and respond to diverse biotic signals from
soil microorganisms. Discriminative recognition of and
appropriate responses to these biotic cues are essential
for plant survival, and in the case of symbiotic plant±
microbe interactions provides an additional means for
selective exploitation of otherwise inaccessible nutrient
97
98
Judith Wopereis et al.
sources. Nitrogen-®xing symbioses of legume plants
provide an interesting example of the latter phenomenon (Vance, 1998). Upon infection with speci®c strains of
rhizobia, root cortical cells of legume plants undergo
dedifferention, initiate cell divisions, and redirect their
developmental fate towards the formation of nodule
primordia. Thereafter, through a highly organized and
controlled series of events, the nodule primordia develop into fully functional, nitrogen-®xing organs, root
nodules (for recent reviews see Hadri et al., 1998; Hirsh,
1992). Nodule organogenesis is activated in response to
speci®c lipo-chitooligosacharide signal molecules (Nod
factors) synthesized by compatible strains of rhizobia
(Hadri and Bisseling, 1998; Spaink, 1996). Structural and
functional adaptations of the root to Nod factors and
rhizobial infection is controlled by the host plant and
has been shown to be modulated by various environmental factors, including the availability of combined
nitrogen, as well as developmental cues associated with
plant growth (Caetano-Anolles and Gresshoff, 1991;
Francisco and Harper, 1995; Nutman, 1952; Parsons
et al., 1993; Streeter, 1988). One important aspect of
this control process is the plant-mediated regulation of
the extent of nodulation in response to rhizobial
infection. The plant host actively controls the number
of successful nodulation events on at least two different
levels. One level involves a premature arrest of the
majority of rhizobial infections, such that only a
restricted number of nodules are formed within a highly
speci®c susceptible zone, located just behind the
growing root tip (Vasse et al., 1993). In Medicago
truncatula, the sickle mutation has been shown to cause
a dramatic increase in the number of persistent rhizobial
infection, resulting in hypernodulation of the susceptible
zone of the mutant root (Penmetsa and Cook, 1997). The
latter phenotype was attributed to a second effect of the
same mutation, namely overall insensitivity of the
mutant plant to the hormone ethylene (Penmetsa and
Cook, 1997). These results suggest that in addition to its
well-characterized functions in plant development, ethylene is also involved in the signaling pathway controlling rhizobial nodulation of legumes. A role of the plant
hormone ethylene in several other aspects of symbiotic
development has been well documented for at least
some legume plant species (Fernandez-Lopez et al.,
1998; Grobbelaar et al., 1970; Heidstra et al., 1997; for a
recent review see Hirsch and Fang, 1994). However,
ethylene-insensitive mutants of soybean were shown to
display a wild-type nodulation pattern (Schmidt et al.,
1999). It is unclear at present whether the difference
between the effects of ethylene and/or ethylene-sensitivity on nodulation in soybean and other legume plant
species re¯ects a differential role of this hormone in
regulating nodule development.
In addition to limiting the number of persistent rhizobial
infections within the root susceptible zone, the plant also
exerts a spatial and temporal control of root susceptibility
to nodulation. This mechanism is referred to as autoregulation or feedback regulation of nodulation, and
involves inhibition of nodule formation on younger root
tissues by prior nodulation events in older root regions
(Kosslak and Bohlool, 1984; Nutman, 1952; Pierce and
Bauer, 1983). Autoregulation renders the root cells only
transiently susceptible to rhizobial infection, resulting in a
narrow zone of infection and nodule differentiation
(susceptibility zone; Bhuvaneswari et al., 1981). Plants
defective in this mechanism continue to nodulate on
newly developing roots and form a large number of
nodules over the entire root system (hypernodulation or
supernodulation phenotype). Based on experiments involving split root system and grafting between wild-type and
supernodulation mutant plants, an interplay between local
and systemic signaling events in establishing the autoregulatory control of nodulation has been postulated
(Caetano-Anolles et al., 1991; Sheng and Harper, 1997).
Similar experiments have identi®ed leaf tissues as the
major source of the systemic signal(s), implicating long
distance communication between the root and shoot in
autoregulation of nodule number. A local, non-systemic
control exerted by fully mature nodules over the outgrowth of younger nodulation events has also been
postulated (Caetano-Anolles et al., 1991; Nutman, 1952).
The exact nature of the mechanisms involved in autoregulation is not understood and the identity of the
postulated systemic and local signaling compounds
remains unknown. However, it is tempting to speculate
that the autoregulatory response relies, at least in part, on
the mechanism of sensing and regulating cell divisions,
and thus may constitute a part of a more general
mechanism regulating plant growth. In this context it is
interesting to note that plant developmental processes
other than nodulation, such as those associated with the
generation of apical root meristems, have been shown to
in¯uence nodule formation via a mechanism that resembles autoregulation (Gresshoff et al., 1989; Nutman, 1952).
Conversely, mutations that impair the autoregulatory
response have been found to exert various pleiotropic
effects on plant growth and, almost invariably, lead to a
high nitrate-tolerant symbiosis (ef®cient nodulation in the
presence of high nitrate; nts phenotype). Nitrate (NO3±)
modulates plant growth and exerts complex effects on
root development, symbiont recognition and nodulation
(Dazzo and Brill, 1978; Gresshoff, 1993; Zhang et al., 1999).
Common factors may be involved in the mechanisms
regulating the extent of nodulation, nitrate inhibition and
other related plant growth responses (e.g. lateral root
formation). Alternatively, close interactions between speci®c regulatory pathways (e.g. autoregulatory and nitrate
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
inhibition pathways) may be suf®cient to account for the
pleiotropic effects of a single mutation in one of the
controlling elements. Understanding the nature of the
regulatory processes controlling nodule differentiation
and nodule number, and integrating them in the overall
mechanisms governing root growth and development
constitute important elements of our quest to understand
symbiotic nitrogen ®xation in legumes.
We have previously identi®ed plant mutants from the
diploid legume Lotus japonicus that de®ne a locus
controlling normal root development. The same mutations
have been found to confer an aberrant response of the
mutant plant to the challenge by symbiotic rhizobia,
resulting in hypernodulation and abnormal plant growth
phenotypes (de Bruijn et al., 1998; Schauser et al., 1998;
Szczyglowski et al., 1998a; Szczyglowski et al., 1998b).
Here we present a detailed characterization of these
mutant lines and show that the underlying mutations
affect plant development by changing the position and
duration of root cell growth.
Results
Isolation and genetic analysis of hypernodulated aberrant
root formation (Har) mutants of L. japonicus
We have previously described the isolation of two allelic
EMS-induced mutant lines of L. japonicus ecotype Gifu
(Ljsym34-1 and Ljsym34-2), which display both unusual
symbiotic (hypernodulated) as well as drastically altered
root developmental (aberrant root) phenotypes (de
Bruijn et al., 1998; Szczyglowski et al., 1998a; see Figure
1). In addition, we identi®ed an independent mutant line
from a T-DNA mutagenesis experiment (sym16) with a
highly similar phenotype, but the mutant phenotype
was found to be genetically unlinked to the T-DNA
insertion (Schauser et al., 1998). The allelic Ljsym34-1
and Ljsym34-2 mutations were found to be monogenic
recessive with regard to the aberrant root phenotype
and incomplete dominant in terms of the symbiotic
hypernodulation phenotype (Szczyglowski et al., 1998a;
Szczyglowski et al., 1998b, data not shown). The sym16
mutation was monogenic recessive for all phenotypes
(Schauser et al., 1998). Reciprocal crosses revealed that
the Ljsym34-1/2 and sym16 mutants belong to the same
complementation group (data not shown). In accordance
with the recently proposed guidelines for genetic
nomenclature for L. japonicus (Stougaard et al., 1999),
the corresponding alleles were re-named har1-1
(formerly Ljsym34-1), har1-2 (formerly Ljsym34-2), and
har1-3 (formerly sym16), and the corresponding wildtype gene was named Har1. Based on its slightly
stronger mutant phenotype, the har1-1 allele was
chosen for further detailed analysis.
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
99
The symbiotic (hypernodulated) phenotype of the har1-1
mutant
Inoculation of L. japonicus har1-1 plants with
Mesorhizobium loti strain NZP2235 resulted in an
almost total inhibition of plant growth and the unusual
hypernodulation
phenotype
previously
described
(Szczyglowski et al., 1998a; see Figure 1). Nodule-like
structures covering nearly the entire short root system
developed concomitantly with inhibition of plant growth
and deterioration of overall plant vitality (hypernodulation response, HNR, phenotype; Szczyglowski et al.,
1998a; Szczyglowski et al., 1998b). To further examine
this novel hypernodulation phenotype, a derivative of
M. loti strain NZP2235, carrying a constitutively expressed hemA::lacZ reporter gene fusion, was used to
analyze early events during the infection of wild-type
and har1-1 mutant plants. Microscopical analyses revealed that the mode of rhizobial primary entry into
har1-1 mutant roots was through infection threads
initiated within deformed root hairs, as in wild-type
plants. Other early infection events, such as root hair
deformation (Had), hair curling (Hac) and infection
thread formation (Inf) were also similar in har1-1 mutant
and wild-type plants (data not shown). However, subsequent stages of symbiotic development were found to
differ signi®cantly. In wild-type plants, the majority of
primary infection events were found to be arrested early
during symbiotic development, without advancing beyond the stage of a few cortical cell divisions, resulting
in 9±15 nitrogen-®xing nodules on the upper portion of
fully elongated 21-day-old L. japonicus wild-type roots
(see Figure 1a,d). In contrast, roots of har1-1 mutant
plants, at 11 days after inoculation with rhizobia, had
much more abundant foci of cortical cell divisions than
wild-type roots spanning almost the entire length of the
root (Figure 2 and 3a,b). These initial cortical cell
divisions gave rise to nodule primordia, and subsequently to a mass of nodules covering almost the entire
root (Figures 1e,f and 3c,d). In addition, nodule
morphogenesis on har1-1 mutant plants was found to
be insensitive to normally inhibitory concentrations of
combined nitrogen (5±15 mM NO3±). Six weeks after
inoculation with rhizobia, har1-1 mutant plants developed approximately 40±60 nodules, in the presence of
high concentrations of nitrate (5±15 mM), or ammonia
(1±3 mM). In contrast, nodule development in control
wild-type L. japonicus plants was found to be highly
inhibited by combined nitrogen sources. For example, in
the presence of 15 mM KNO3 only a few small nodulelike structures (bumps) were observed. When grown in
the presence of a low concentration of KNO3 (0.5 mM),
21-day-old nodules on har1-1 mutant plants were
signi®cantly smaller than wild-type nodules of the same
100 Judith Wopereis et al.
age (data not shown). In spite of the size difference,
light and transmission electron microscopic examination
of the infected zone of nodules formed on har1-1 plants
revealed a normal (wild-type like) cytology and histology (Figure 3e,f). Moreover, nodules formed on har1-1
mutant roots had the capacity to ®x nitrogen (reduce
acetylene) at levels comparable to wild-type nodules
when calculated on a per plant basis (data not shown).
The non-symbiotic (aberrant root formation) phenotype
of the har1-1 mutant
Uninoculated L. japonicus har1-1 mutant plants develop a
signi®cantly shortened root system and an enhanced
number of lateral roots as compared to wild-type plants
(Szczyglowski et al., 1998a). To further analyze this phenotype, we examined lateral root formation in uninoculated
har1-1 and wild-type primary roots and found no signi®cant differences in their position relative to the root tip,
as almost all LRPs were found in a region located 0.75±4.5
cm from the root apex. However, the density of lateral root
primordia and emerged lateral roots (number per unit
length of root) were at least three times higher in the har11 mutant than in wild-type plants (data not shown).
Detailed examination of median longitudinal sections of
uninoculated har1-1 root samples approximately 2 cm
above the root tip revealed a signi®cantly higher level of
mitotic activity in the root pericycle layer of har1-1 versus
wild-type roots (Figure 4a,b). In 9-day-old roots of the har11 mutant, periclinal cell divisions in the pericycle, as well
as the development of two or three new cell layers, were
Figure 1. Root and nodulation phenotypes of wild-type and har1 mutant
L. japonicus plants.
Plants were grown for 21 days in the presence (a; wild-type and b; har11) or absence (c; har1-1) of Mezorhizobium loti NZP2235, with 0.5 mM
KNO3 in the watering solution. Panels (d) and (e) show a close-up of the
nodulated roots shown in (a) and (b), respectively. Panel (f) shows har1-3
grown for 3 weeks in the presence of rhizobia and for an 8 additional
weeks in nitrogen-rich Hornum growth medium containing 8 mM KNO3
and 5 mM NH4+ (Thykjaer et al., 1998).
Figure 2. Infection and nodulation events in wild-type and har1-1 plants
upon inoculation with M. loti strain NZP2235 carrying a hemA:lacZ
reporter gene fusion.
Roots were stained for b-galactosidase activity and examined using
bright®eld microscopy. Open bars denote the number of root hairs with
visible infection threads; solid bars indicate the number of nodules and
nodule primordia. Each value represents the mean of measurements
from 7 to 15 plants. Error bars represent 95% con®dence intervals.
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
101
Figure 3. Microscopic analysis of symbiotic development in wild-type and har1-1 plants.
(a,b) Bright®eld micrographs of cleared wild-type and har1-1 mutant roots 11 days after infection with M. loti NZP2235. Nodule primordia are indicated by
arrows. (c,d) Montages of the nodulation phenotypes of wild-type and har1-1 mutant plants 14 days after inoculation with M. loti NZP2235 carrying a
hemA:lacZ reporter gene fusion. Roots were stained for b-galactosidase activity and examined using bright®eld microscopy. (e,f) Transmission electron
micrographs of the central zone of wild-type and har1-1 mutant nodules showing infected host cells ®lled with bacterial endosymbionts, and a portion of
adjacent highly vacuolated uninfected cells.
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
102 Judith Wopereis et al.
Figure 4. Mitotic activity of the root pericycle in wild-type and har1-1
mutant roots.
(a,b) Median longitudinal section of segments of 9-day-old uninoculated
roots of wild-type (a) and har1-1 (b) mutant plants approximately 2 cm
above the root tip. Arrows indicate the pericycle cell layer.
detected in all sections examined. Abundant anticlinal cell
divisions in the pericycle, as well as in the neighboring
cortical cell layers, could also be detected in sections of
har1-1 mutant roots, and in several cases well-developed
lateral root primordia were observed. In contrast, wild-type
roots of a similar age exhibited only limited mitotic activity
in the pericycle, which was mostly composed of a single
cell layer (Figure 4a,b). Lateral root primordia were only
rarely observed in sections of wild-type roots.
Root and shoot phenotype of the har1-1 mutant
The short root phenotype of L. japonicus har1-1 plants has
been documented previously (de Bruijn et al., 1998;
Szczyglowski et al., 1998a; Szczyglowski et al., 1998b). To
further assess this phenotype quantitatively, the longitudinal growth of uninoculated and inoculated har1-1 and
wild-type roots was measured. An average root length of
61.3 6 0.2 mm was observed for uninoculated har1-1
plants 21 days after sowing versus 131.9 6 1.0 mm for
wild-type uninoculated control plants (Figure 5a). In
addition, the root mass of uninoculated har1-1 plants at
21 days after sowing (26 6 2 mg fresh weight) was signi®cantly smaller than that of the wild-type plants
(45 6 6 mg). The aberrant har1-1 root growth phenotype
was even more extreme when plants were inoculated with
M. loti strain NZP2235. Root growth of har1-1 plants
ceased entirely within the ®rst days after inoculation, and
Figure 5. Growth kinetics of roots and shoots of wild-type and har1-1
mutant plants in the presence or absence of M. loti.
(a,b) Root growth kinetics of uninoculated (a) and inoculated (b) wildtype and har1-1 mutant plants. (c,d) Shoot growth kinetics of
uninoculated (c) and inoculated (d) wild-type and har1-1 mutant plants.
All plants were grown in the presents of 0.5 mM KNO3. Each value
represents the mean of measurements of at least 30 plants. Error bars
represent 95% con®dence intervals.
the roots did not exceed an average length of 20 6 1.5 mm
(Figure 5b). The shoot mass of inoculated har1-1 plants
was also signi®cantly reduced in infected plants, while it
was comparable in uninoculated har1-1 versus wild-type
shoots (Figure 5c,d).
Cytology of har1-1 mutant roots
Having established that several root developmental parameters (root length/elongation, lateral root formation and
overall root mass accumulation) were signi®cantly altered
in the har1-1 mutant line, the phenotype of the har1-1
mutant roots was further analyzed. First, the cellular
organization of wild-type L. japonicus roots was examined.
Primary L. japonicus roots were found to contain an outer
single layer of epidermal cells, 3±5 irregularly shaped
cortical cell layers surrounding a single layer of endodermal cells, an innermost region consisting of a single layer
of pericycle cells enclosing the vascular cylinder, and a
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
103
Figure 6. Histology of non-symbiotic root development.
(a,b) Median longitudinal sections of the root tip regions of 9-day-old wild-type and har1-1 mutant plants showing the overall anatomy of the meristematic
region and the position of the cell elongation/vacuolation zone. (c,d) Cross-sections of roots of 22-day-old plants at approximately 600 mm above the root
tip, showing differences in the extent of cell vacuolation and root diameter. (e,f) Cross-sections of mature root regions of 6-day-old plants approximately
1 cm above the root tip.
Table 1. Morphometric measurements of root cross-sections
[area 6 SD (mm2)] at a distance of 1 cm from the root tip
Tissue
Wild type (a)
har1-1 (b)
b/a ratio
Total section
Epidermis
Cortex
Endodermis
Stele
62357 6 14772
10073 6 2250
45228 6 11283
2097 6 373
5070 6 1033
33786 6 4845
5626 6 760
24433 6 3820
1387 6 175
2655 6 513
0.54
0.56
0.54
0.66
0.52
distally located area of root cap cells. The same general
organization of root cell layers was found in har1-1 mutant
roots (Figure 6). However, several signi®cant differences
were observed. Vacuolation, which typically accompanies
cell expansion, occurred closer to the root tip in har1-1
versus wild-type roots (Figure 6a,b). In addition, the
diameter of mutant har1-1 roots (0.23 6 0.03 mm), measured using digitized micrographs of living roots at 3±
6 mm from the root tip, was signi®cantly smaller than the
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
average diameter of the corresponding region of wild-type
L. japonicus roots (0.31 6 0.02 mm; see also Figure 6c,d).
Based on these resultes, the following two hypotheses
were formulated and tested: (1) the har1-1 mutation affects
the radial organization of the L. japonicus root, and/or (2)
the decreased root length and diameter is a result of
abnormal (reduced) cell expansion.
The har1-1 mutation results in a diminished radial
expansion of root cells
To test the ®rst hypothesis (a defect in the radial
organization of roots), a large number of root sections
were examined microscopically. No clear evidence for one
or more missing cell layers or a diminished number of root
cells in har1-1 mutant roots was found (Figure 6).
Therefore, we tested the second hypothesis (changes in
root cell expansion), by analyzing sections of the fully
differentiated region of primary roots (Figure 6e,f). The
average total cross-sectional area of har1-1 mutant roots
was almost two times smaller than that of the wild-type
104 Judith Wopereis et al.
root (Table 1). Individual cell layers (epidermis, cortex,
endodermis and root stele) of har1-1 mutant roots showed
a similar reduction in size, contributing nearly equally to
the overall decrease in root diameter (Table 1).
Subsequently, the projected cross-sectional area of individual root cells was analyzed to determine whether cell
radial expansion was altered in har1-1 mutant roots. The
radial surface areas of individual cells of the epidermis,
cortex and endodermis of roots had a frequency distribution that was con®ned to a much smaller size range for the
Figure 7. Root cell radial expansion in wild-type and har1-1 mutant plants.
The frequency distribution of the cross-sectional area of root epidermal
(a), cortical (b) and endodermal (c) cells of 6-day-old plants measured at
the location of approximately 1 cm above the root tip is shown. Ten wildtype and mutant plants were analyzed and each point represents a
frequency value for cell size range increments of 35 mm (epidermis),
200 mm (cortex) and 20 mm (endodermis); n, represents the number of
cells measured.
har1-1 mutant (Figure 7), suggesting that the har1-1
mutation limits the ability of root cells to expand.
The length of the meristematic region is shortened in
har1-1 mutant roots
To examine whether the short root phenotype of har1-1
mutant plants was caused by alteration in the primary
direction of cell expansion along the apical±basal axis, the
length of har1-1 root epidermal cells was measured. The
average length of fully expanded epidermal cells of har1-1
mutant roots (138 6 30 mm) was nearly equal to that of
wild-type roots (132 6 30 mm), indicating that the har1-1
mutation did not affect longitudinal cell expansion of the
epidermis (Figure 8a). These results suggest that the short
root phenotype of the mutant plant is unlikely to be due to
an abnormal longitudinal expansion of root cells.
Figure 8. Root cell elongation and size of meristematic region in wild-type
and har1-1 mutant plants.
(a) Epidermal cell length along the root axis. Each point represents the
mean cell length value for range increments of 250 mm (for the ®rst 4 mm
from the root tip), and 500 mm (between 4 and 11 mm from the root tip),
along the root axis. Single and double arrows indicate signi®cant
differences between the mean epidermal cell length at two consecutive
and equivalent positions in wild-type and har1-1 mutant roots. (b) Intact
roots of 14-day-old plants stained with acetocarmine. The red-stained
meristematic regions of the wild-type and har1-1 mutant roots appear as
dark areas, and their extent is indicated by the brackets.
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
However, consistent with our earlier observations (see
above), epidermal cells of the har1-1 mutant roots showed
evidence of elongation along the longitudinal axis signi®cantly closer to the root tip (hence earlier in development) than wild-type roots (Figure 8a). Transmitted
bright®eld microscopy and laser scanning microscopy of
whole cleared roots stained with acetocarmine revealed an
area of densely cytoplasmic cells constituting the root
meristematic region whose borders could be de®ned by
interactive threshholding techniques using digital image
processing. In independent experiments using both types
of microscopy, an approximately 2.6-fold reduction in the
projected area of the har1-1 mutant versus wild-type root
meristematic regions was detected (45082 6 4373 mm2;
105
n = 11 versus 121635 6 12545 mm2, n = 19; see also Figure
8b). This reduction in the size of the root meristematic
region was invariably associated with an acropetal displacement of the root cell elongation/vacuolation and
vascularization zones (Figure 8b). har1-1 mutant roots also
showed an inferior root cap structure in comparison with
the wild type, but remained gravitropic (data not shown).
In addition, the mitotic index was measured in order to
estimate the proportion of mitotic cells in the root
meristematic regions of har1-1 versus wild-type plants of
an equal age, but no signi®cant differences were found
(har1-1
mutant
MI = 3.8 6 0.8
versus
wild-type
MI = 3.96 6 0.7).
Effect of hormones on har1-1 mutant root elongation
Figure 9. Effect of light on the elongation rates of wild-type and har1-1
mutant roots.
(a) The rates of wild-type root elongation. (b) The rates of har1-1 mutant
root elongation. Each value is the mean of measurements on 20 plants.
Error bars represent 95% con®dence intervals.
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
The gross changes in har1-1 root morphology suggested
that the hormonal regulation of the mutant root development could be disturbed. In order to test this hypothesis
the effects of exogenous hormone applications on root
elongation of wild-type and har1-1 mutant roots were
investigated using a plate bioassay speci®cally developed
for this purpose. Sucrose was found to be required at a
relativey high concentration (4.5%) to support the maximal
and uniform root elongation of both wild-type and har1-1
mutant roots. The roots of wild-type plants elongated
more rapidly in the dark than in the light especially during
the ®rst 2±3 days of growth, after which time the difference
in growth rate disappeared (Figure 9a). har1-1 mutant
plants formed short roots when grown in the dark under
conditions that provided maximal wild-type root growth. A
dramatic inhibition or retardation of root elongation was
observed after 2 days of incubation in the dark.
Interestingly, the short root phenotype was partially
suppressed when har1-1 mutant plants were grown in
the light (Figure 9b).
Since har1-1 roots exhibited an abnormal pattern of
radial expansion of root cells, as well as a hypernodulation response (see Figures 1, 3d and 6), and since the
hypernodulation phenotype of a Medicago truncatula
mutant (sickle) had been correlated with a change in
ethylene sensitivity (Penmetsa and Cook, 1997), the
sensitivity of wild-type and har1-1 seedlings to the
ethylene precursor 1-aminocyclopropane1-carboxylic
acid (ACC) was examined. When grown vertically in
the dark on agar plates containing increasing concentrations of ACC, both wild-type and har1-1 mutant seedlings showed the same overall sensitivity pattern to
ethylene inhibition of root growth (Figure 10a).
However, in two independent experiments, har1-1
mutant roots displayed a slightly increased resistance
to certain concentrations of ACC as compared with wildtype roots (e.g. 1 3 10±7 to 1 3 10±6 M ACC in Figure
10a). To further evaluate the observed decrease in
106 Judith Wopereis et al.
a
sensitivity of the har1-1 mutant line to ACC, we
examined the responses of entire seedlings to exogenously applied ACC. When grown in the dark in the
presence of ACC, both wild-type and har1-1 seedlings
showed a typical triple response (Guzman and Ecker,
1990), consisting of a shortening of the hypocotyl,
inhibition of root elongation, and exaggeration of apical
hook curvature (Figure 11). Both genotypes exhibited
similar levels of sensitivity to ACC in terms of hypocotyl
length (data not shown).
wild-type
har1-1
110
100
90
80
70
60
50
40
5x
10
-
1x 7
10
-6
1x
10
-5
1x
10
-4
-7
-8
10
1x
10
5x
1x
10
10
10
5x
1x
110
-8
-9
30
20
10
0
-9
Relative root length (%)
130
120
ACC [M]
b
wild-type
har1-1
100
Relative root length (%)
90
80
70
60
50
40
30
20
10
110
-5
-6
10
1x
1x
NAA [M]
c
wild-type
har1-1
100
Figure 11. Triple response to ACC of wild-type and har1-1 mutant plants.
Wild-type and har1-1 mutant seeds were germinated on MS medium in
the dark at 28°C in the absence (0) or presence of increasing
concentations (1±100 mM) of ACC. The photograph was taken 6 days after
incubation in the dark. The triple response is characterized by shortened
hypocotyls, roots and exaggerated apical hook formation.
90
80
70
60
50
40
30
20
10
-4
1x
10
-5
1x
10
-6
1x
10
-7
-7
5x
10
1x
10
-8
5x
10
-8
0
1x
10
Relative root length (%)
10
-7
5x
10
-7
1x
10
-8
5x
10
-8
10
-9
1x
10
1x
5x
10
-1
0
0
Figure 10. Wild-type and har1-1 root growth in the presence of increasing
concentrations of exogenously applied plant hormones.
The relative elongation of wild-type and har1-1 mutant roots in the
presence of (a) 1-aminocyclopropane1-carboxilic acid (ACC); (b) anaphthalene-acetic acid (NAA); or 6-benzylaminopurine (BA) is shown.
Each value is the mean of measurements on 20 plants. Error bars
represent 95% con®dence intervals. The mean value of 100% root growth
in (a) wild-type, 44.5 6 3.4 mm; har1-1 21.8 6 1.2 mm; in (b) wild-type,
56.9 6 2.4 mm; har1-1, 25.8 6 1.6 mm; in (c) wild-type 37.1 6 1.9; har1-1,
21.6 6 1.1 mm.
BA [M]
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
Subsequently, the effect of the auxin a-naphthaleneacetic acid (NAA) on root growth was examined. Wild-type
and har1-1 mutant roots were found to display a similar
overall NAA sensitivity pattern but, as had been observed
with ACC, roots of har1-1 mutant plants showed a slight
NAA-insensitive phenotype at higher concentrations of
NAA (Figure 10b).
In Arabidopsis, cytokinin inhibits root elongation in
light- and dark-grown seedlings due to the stimulation of
endogenous ethylene production (Cary et al., 1995).
Therefore, the sensitivity of wild-type and har1-1 mutant
roots to exogenously applied cytokinin (thus endogenously produced ethylene) was also examined. The presence of even very low concentrations (10±50 nM) of the
synthetic cytokinin, 6-benzylaminopurine (BA), signi®cantly reduced root growth in both genotypes. However,
again the har1-1 mutant roots exhibited a moderately
higher resistance to a wide range of BAP concentrations
then wild-type roots (Figure 10c). This slightly elevated
resistance of har1-1 mutant root could be due to either an
altered ethylene-independent response to cytokinin,
diminished cytokinin-stimulated ethylene production,
and/or an attenuated response to endogenously produced
ethylene. To distinguish between these possibilities, the
in¯uence of exogenously added cytokinin on inhibition of
root growth was examined in the presence silver ions,
applied as silver thiosulfate, to inhibit ethylene binding
(Beyer, 1979), or aminoetoxyvinylglycine (AVG; Yang and
Hoffman, 1984) to inhibit ethylene biosynthesis. Since 50
nanomolar BA almost maximaly inhibited root elongation
Figure 12. Effect of BA on elongation of the
wild-type and har1-1 mutant roots in the
presence of ethylene perception/synthesis
inhibitors.
The relative elongation of wild-type and
har1-1 mutant roots in the presence of (a)
Ag+; (b) BA plus Ag+; (c) AVG; and (d) BA
plus AVG is shown. Mean value of 100%
root growth in (a) and (b) wild-type,
41.6 6 3.0 mm; har1-1, 23.3 6 1.2 mm; in (c)
and (d) wild-type, 47.5 6 4.0 mm; har1-1,
23.2 6 1.6 mm. For further details see legend
to Figure 10.
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
107
in both genotypes, we used this concentration of cytokinin
in combination with variable concentrations of inhibitors.
In the absence of BA, Ag+ only had a limited stimulatory
effect on the growth/elongation of wild-type and har1-1
mutant roots (Figure 12a). Five mM Ag+ was suf®cient to
overcome all the inhibitory effects of cytokinin on har1-1
mutant roots, whereas 5 mM Ag+ restored wild-type root
growth to a level of about 60% of that of untreated control
roots (Figure 12b).
A similar set of experiments was conducted with an
inhibitor of ethylene synthesis, AVG. In control experiments, an AVG concentration equal to or lower than
0.1 mM had no measurable effect on root growth in both
genotypes (Figure 12c), whereas higher concentrations
were strongly inhibitory (data not shown). In contrast to
the silver-mediated phenotypic suppression, a low
concentration of AVG (0.1 mM) was found to relieve all
of the inhibition caused by cytokinin, and to restore a
normal long-root phenotype to wild-type plants (Figure
12d). However, the har1-1 mutant plants responded
differently to the same treatment, not only by recovering a short root phenotype, but also by an additional
stimulation of root growth. The latter effect resulted in
the mutant plants developing long roots that elongated
at the same rate as wild-type roots. However, a
prolonged incubation (more then 10 days) of both wildtype and mutant har1-1 plants in the presence of
cytokinin and AVG resulted in the total arrest of their
root growth. This effect was found to be associated
with a terminal differentiation of the root meristem, thus
108 Judith Wopereis et al.
precluding experiments with longer treatment times
(data not shown).
Discussion
We have shown here that the Har1 locus of L. japonicus is
critical for both root and symbiotic development by
analyzing three mutant alleles at this locus, designed
har1-1, har1-2 and har1-3. The har1-1 allele has the most
pronounced phenotype and therefore was examined in
most detail. However, a similar range of de®ned mutant
phenotypes was found during a parallel analysis of har1-3,
and also a partial analysis of har1-2 mutant lines (data not
shown). Under axenic growth conditions, the most
extreme phenotypic effects of har1 mutations are the
restriction of root elongation and the promotion of lateral
root growth. In the presence of rhizobia, har1 mutants
display an unusual hypernodulation (HNR) response,
characterized by unrestricted nodulation (hypernodulation), and a concomitant drastic inhibition of root and
shoot growth (Figure 1). Other legume symbiotic mutants
with a pleiotropic phenotype have been described suggesting that at least in some cases the corresponding
genes may function in both symbiotic and non-symbiotic
processes (Delves et al., 1986; Duc and Messager, 1989;
Kneen et al., 1994; Lee and Larue, 1992; Penmetsa and
Cook, 1997).
Since the har1 mutant root phenotype is observed in the
absence of rhizobia, we postulate that a primary role of the
Har1 locus is to control root development. A speci®c defect
in the root developmental program, such as the one
caused by the har1-1 mutation, may indirectly affect the
ability of the mutant plant to control nodule organogenesis
by, for example, antagonizing one or more functions by
which the host plant autoregulates nodulation.
Alternatively, the L. japonicus Har1 gene product may
play a direct role in mechanism(s) controlling nodulation,
and thus could represent a common regulatory element
for both root and symbiotic development.
The symbiotic phenotype of the har1-1 mutant indicates
a defect in the autoregulatory mechanism controlling
nodulation
M. loti induces the HNR response on L. japonicus har1-1
mutant plants. The most striking phenotypic effect associated with this response is an almost total inhibition of
shoot and root growth that results in an overall small
stature of inoculated har1-1 mutant plants. On a cytological level, this rapid growth arrest appears to coincide with
the initiation of abundant cortical cell divisions and the
formation of numerous nodule primordia (Figure 3). The
hypothesis that the HNR is directly linked to nodulation is
further strengthened by the identi®cation of a partially
epistatic mutation in the har1-2 line (LjEMS40;
Szczyglowski et al., 1998a; Szczyglowski et al., 1998b). In
this double mutant line (LjEMS40), the second unlinked
allele (Ljsym22-2) confers a non-nodulating (Nod±) phenotype and suppresses the HNR, without affecting root
morphology. Introduction of the Ljsym22-2 allele into a
har1-1 genetic background suppresses both nodulation
and the HNR responses, but does not alter the root
morphology phenotype of the har1-1 mutant
(Szczyglowski et al., 1998a; K. Szczyglowski et al., unpublished data). Although the exact mechanism responsible for HNR remains unknown, it appears likely that the
abundant early nodulation events in har1-1 mutant roots
are directly responsible for triggering the HNR.
Reduced plant growth has been previously observed in
supernodulating/hypernodulating mutants of soybean and
other legume plant species, although the overall growth
retardation was never as extreme as that observed in
L. japonicus har1-1 plants (Carroll et al., 1985; Day et al.,
1986; Gremaud and Harper, 1988; Novak et al., 1997; Sheng
and Harper, 1997). Unlike the HNR response of L. japonicus
har1-1 mutant plants, and in spite of the abundant level of
nodulation, the initial growth rate of the soybean supernodulating nts384 mutant is greater than that of the
parental soybean line cv. Bragg. Only at a later stage,
when nts384 roots become even more extensively nodulated, does a signi®cant reduction in plant growth rate and
size occur.
In Vicia sativa subsp., nigra infection with Rhizobium
leguminosarum induces the formation of thick and short
roots (Tsr phenotype; Van Brussel et al., 1982; Van Brussel
et al., 1986), somewhat resembling the short root phenotype of inoculated har1-1 plants. However, the Tsr
response has been shown to be associated with an
ethylene-related increase in root cell radial expansion
and to be accompanied by the inhibition of nodulation
on primary roots and restricted nodule formation on lateral
roots (Van Brussel et al., 1982; Van Brussel et al., 1986). In
contrast, the primary roots of har1-1 mutant plants, as well
as infrequently formed lateral roots, remain fully susceptible to rhizobial infection. The latter characteristic strongly
suggests that the har1-1 mutants may be impaired in the
mechanism(s) that autoregulate nodulation. This is further
supported by the observation that har1-1 mutants, similar
to other previously described mutants defective in autoregulation of nodulation, is able to be nodulated in the
presence of high nitrate concentrations. The latter conditions support better root elongation and more frequent
lateral root formation in har1-1 mutant plants in spite of
the presence of rhizobia. These new root tissues remain
susceptible to rhizobial infection and nodulation, regardless of the presence of mature nodules on older parts of
har1-1 mutant roots (Figure 1f). Nitrate represents a major
environmental factor that negatively regulates nodulation
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
in legumes, including L. japonicus (Carroll and Mathews,
1990; Streeter, 1988). A connection between the mechanisms underlying nitrate inhibition and autoregulation of
nodule development has been hypothesized previously
(Delves et al., 1986; Gresshoff, 1993). Thus, the observed
coexistence of nitrate-tolerance and hypernodulation (lack
of autoregulation) phenotypes in the har1-1 mutant line
lends additional support to this hypothesis. Results of
preliminary grafting experiments have revealed that it is
shoots and not roots that determine the altered symbiotic
phenotype of the har1-1 mutant plants (data not shown).
Altogether, these observations strongly suggest that the
har1-1 mutation results in an impairment of the mechanism(s) responsible for systemic regulation of nodule
number in L. japonicus.
The har1-1 mutation affects root growth by changing the
position and duration of root cell growth
Changes in root morphology have been associated
previously with hypernodulating (supernodulating) mutations in legumes, but have not been analyzed in detail. In
the case of the soybean supernodulating mutant nts382,
early (prior to nodule emergence) lateral root formation is
increased, independent of the application of rhizobia and/
or nitrate (Day et al., 1986). The pea hypernodulating
mutant nod3 also displays an altered root morphology
phenotype. This mutant line shows a reduced tap root
growth pattern, forms more lateral roots, and during
growth on nitrate produces more tertiary roots than the
wild-type plant (Jacobsen and Feenstra, 1984).
A primary function of the L. japonicus Har1 locus in the
mechanism(s) regulating root development is suggested
by two independent observations. First, the mutations in
this locus signi®cantly alter root development in the
absence of rhizobia. Under the same growth conditions,
in the absence of rhizobia, the aerial portions of har1-1
plants continue to have a wild-type appearance. Second,
nodulation of har1-1 mutant plants can be speci®cally
suppressed by the presence of additional unlinked mutations (e.g. the Ljsym22-2 allele) without a noticeable effect
on the altered root morphology phenotype (see above).
These results clearly show that the Har1 gene product is
involved in the non-symbiotic development of L. japonicus
roots. All three har1 mutations described above drastically
diminish elongation of both main and lateral roots,
decrease root diameter, and promote lateral root differentiation. The latter phenomenon is particularly intriguing
since development of lateral roots also involves the
initiation of new meristems (Steeves and Sussex, 1989).
It has been suggested that lateral root formation in
Arabidopsis involves at least two distinct stages. During
the ®rst stage, the lateral root primordium (LRP) is formed
by stimulation of dedifferentiation and proliferation of
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
109
cells in the root pericycle layer. This is followed by the
second phase, which involves the organization of an
autonomous lateral root meristem that is capable of
perpetuating LRP structures and producing lateral roots
(Celenza Jr et al., 1995; Cheng et al., 1995; Laskowski et al.,
1995; Malamy and Benfey, 1997). The development of
LRPs appears to be signi®cantly stimulated in har1-1
mutant versus wild-type roots. The activated pericycle in
har1-1 mutant roots forms an uninterrupted ®le of two to
three cell layers suggestive of abundant lateral root
formation events (Figure 4). Assuming that all these
pericycle cell divisions indeed re¯ect initiation of LRPs in
har1 mutant root, the approximately threefold increase in
the number of emerged lateral roots suggests that in the
har1-1 mutant line a certain amount of control of lateral
root differentiation and growth still exists. har1 mutants do
not show enhanced proliferation of adventitious roots
(roots derived from hypocotyl) or distortion of root and/or
hypocotyl structures, phenotypes associated with increased lateral root development in the Arabidopsis rooty
mutant (Boerjan et al., 1995; Celenza Jr et al., 1995; King
et al., 1995; Lehman et al., 1996). har1 mutants are also
phenotypically different from other previously reported
root mutants of Arabidopsis that display alterations either
in the radial organization of the root (e.g. shortroot mutant;
Scheres et al., 1995) or root cell expansion (e.g. CORE
mutants; Hauser et al., 1995; see also Nicol and Hofte,
1998). Unlike shortroot, which lacks an endodermis, har1
mutants have the same root cell layers as wild-type plants
(Figure 6). In contrast to the CORE mutants, har1 mutations
result in a reduction, rather then increase, in root cell radial
expansion, and are not associated with a defect in the
orientation of root cell growth (Figures 7 and 8). The
primary direction of growth of har1-1 mutant root cells
remains longitudinal, along the apical±basal axis, and the
extent of cell elongation does not appear to be affected by
the har1-1 mutation. However, the position at which root
cells start to elongate and become vacuolated is signi®cantly altered, being closer to the root tip in the har1-1
mutant than in wild-type roots. This is accompanied by a
signi®cant reduction in the size of the area of the
cytoplasmically dense cells in har1-1 mutant roots
(Figure 8).
Generally, the growth rate of a plant organ is regulated
by the combined activity of two linked processes, cell
production and cell expansion (Beemster and Baskin,
1998). Kinematic methods have been used recently to
show that the root meristem of Arabidopsis, de®ned on
the basis of distribution of mitotic events in the root apical
region, extends well into the region of rapid cell elongation
(Beemster and Baskin, 1998). Assuming a similar situation
in L. japonicus, the size of such meristematic regions
appears to be reduced in har1-1 mutant roots. Therefore,
we postulate that the short root phenotype of har1-1 plants
110 Judith Wopereis et al.
is likely to be due to a decrease or modi®cation of root
meristem activity. One attractive possibility is that the Har1
gene product is a component of the mechanism(s) that
control(s) meristematic activity and/or orchestrates(s) cell
division and cell differentiation in the root meristem. The
reduction of the har1-1 mutant root meristematic area, as
well as an acropetal displacement of the cell elongation
zone, indeed suggest an impairment in these processes,
resulting in an inhibition of har1-1 mutant root elongation.
The latter may result in the generation of a physiological
signal to stimulate lateral development, in a manner
similar to the promotion of lateral root formation upon
physical removal of the root tip (Steeves and Sussex,
1989), or inhibition of root meristematic activity by a
genetic ablation of root cap cells (Tsugeki and Fedoroff,
1999). Increased lateral root development, in turn, may
exert an additional negative effect on the elongation of
har1-1 mutant roots.
The decreased radial expansion of har1-1 mutant roots
may also be a direct consequence of the reduction of har11 mutant root elongation. It is generally assumed that once
root cells are present in the zone of rapid elongation, they
cease radial expansion (Pysh and Benfey, 1998). Thus, the
acropetal displacement of the rapid cell elongation zone in
har1-1 mutant roots may limit the duration of the period of
cell expansion, resulting in an overall smaller diameter of
har1-1 mutant roots.
Of course, alternative scenarios can also be envisaged.
For example, the observed deregulation of lateral growth
may be a primary, rather then secondary effect of the har11 mutation. Some of the developmental aberrations
observed in har1-1 mutant roots may also be due to
independent effects of the har1-1 mutation. In any case,
the har1-1 mutation clearly affects root elongation and
promotes lateral root initiation. Therefore, it is tempting to
speculate that nodule formation represents an alternative
type of lateral root growth, and therefore the physiological
conditions that promote lateral root formation during nonsymbiotic development in har1-1 mutant roots may be the
same as those stimulating nodule formation, while
antagonizing autoregulation of nodulation. Thus, the
Har1 gene product appears to be an essential component
of the regulatory mechanism responsible for maintaining
root growth rates and suppressing lateral root and nodule
formation.
Physiological aspects of the har1-1 mutant phenotype
It has been established previously that plant growth
regulators affect both root development and nodulation.
For example, ethylene is a potent inhibitor of cortical cell
division in roots and nodule formation (Hirsch and Fang,
1994; Lee and LaRue, 1992). Ethylene also exerts a strong
inhibitory effect on elongation of primary roots, which is
usually accompanied by an increase in radial root cell
expansion (Jackson et al., 1992). As mentioned above, a
recessive mutation in M. truncatula (sickle) results in overall
plant ethylene insensitivity, as well as hyperinfection by
rhizobia, resulting in a signi®cant increase in the number of
root nodules formed (Penmetsa and Cook, 1997).
Moreover, Caba et al. (1999) have suggested recently that
the sensitivity of nodulation to ethylene might be speci®cally decreased in soybean supernodulation mutants,
although a signi®cant difference in ethylene perception
between wild-type and supernodulating mutants on a
whole-plant basis was not found. No signi®cant differences
in sensitivity to exogenously applied ACC were observed in
har1-1 mutant versus wild-type L. japonicus plants (Figure
10). Although a slightly elevated resistance of har1-1 roots
to particular concentrations of ACC was observed, this
effect was not speci®c for ACC since similar differences
were observed with auxin. Inhibitors of ethylene perception
(Ag+) or ethylene synthesis (AVG) were found to have a
limited stimulatory effect on elongation of both har1-1 and
wild-type roots. In addition, 0.1 mM AVG did not signi®cantly
affect root growth, nodulation or the development of the
HNR response in inoculated har1-1 mutant plants (data not
shown). The latter result suggests that, unlike the tsr
phenotype (Zaat et al., 1989; see also above) and the
recently described inhibitor growth responses of white
clover (Trifolium repens L.) roots to the external application
of diglycosyl diacylglycerol membrane glycolipids derived
from Rhizobium leguminosarum bv. trifolii (Orgambide
et al., 1994), the HNR response is ethylene-independent.
Cytokinin appears to stimulate root growth of the darkgrown uninoculated har1-1 mutant plants in an ethyleneindependent manner, such that the length of the main har11 roots is almost identical to that of the wild-type roots after
10 days of incubation in the presence of 0.05 mM cytokinin
and 0.1 mM AVG (Figure 12). Interestingly, light also partially
restores the elongation of the har1-1 mutant root (Figure 9).
Cytokinin, together with auxin, regulate cell division and
differentiation processes (D'Agostino and Kieber, 1999). In
addition, cytokinin promotes cell expansion and exerts an
inhibitory effect on pericycle cell division (Cary et al., 1995;
Coenen and Lomax, 1997). All these functions can be
pertinent to the observed stimulatory effect of cytokinin on
har1-1 mutant root elongation. In fact, we observed a strong
inhibition of pericycle cell divisions in the har1-1 and wildtype roots when incubated in the presence of 0.05 mM
cytokinin and 0.1 mM AVG (data not shown).
The Har1 locus has been mapped to the linkage group
LG2 of an L. japonicus F2 genetic map derived from a cross
between homozygous har1-1 (Gifu) and the polymorphic
L. japonicus ecotype B-581 Funakura (data not shown).
Molecular cloning and studies on the expression of the
Har1 locus in response to applied cytokinin are in progress
and will help to uncover its function in the cytokininã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
Root and symbiotic mutant of Lotus japonicus
mediated control of root growth and nodulation in
legumes.
Experimental procedures
Plant material and growth conditions
The har1-1 (Ljsym34-1) and har1-2 (Ljsym34-2) mutant alleles
were generated by ethylmethanesulfonate (EMS) mutagenesis of
L. japonicus seeds (Gifu ecotype), as previously described
(Szczyglowski et al., 1998a). The har1-3 (sym16) mutant allele
was isolated from a population of L. japonicus (ecotype Gifu)
containing T-DNA insertions, but the T-DNA insertion was found
to be unlinked to the har1-3 mutation (Schauser et al., 1998).
L. japonicus seeds were surface sterilized and germinated as
described previously (Szczyglowski et al., 1998a). For root and
stem growth measurments, 7-day-old axenic seedlings were
transferred to pots containing a 6 : 1 mixture of vermiculite and
sand, and grown as described by Kapranov et al. (1997). At given
time-points, plants were carefully uprooted, washed, blotted dry,
and the masses of the whole root and shoot were determined.
The length of primary roots was determined by direct measurements. The Mesorhizobium loti wild-type strain NZP2235 was
used in nodulation experiments, as described previously
(Szczyglowski et al., 1997). For microscopical studies, L. japonicus
seedlings were grown in nylon `pillow systems', as described
previously by Szczyglowski et al. (1998a). For hormone treatments, surface sterilized wild-type and har1-1 mutant seeds were
germinated on ®lter papers soaked with sterile H20 for 2 days
under light, and sown onto 100 cm2 agar plates (®ve seedlings per
plate) containing 1/2 strength Gamborg's B5 basal medium with
minimal organics (Sigma), 0.8% phytagel (Sigma), 2.5 mM 2-Nmorpholinoethanesufonic acid (MES) and 4.5% sucrose. The pH
of the medium was adjusted to 6.0 with NaOH. The plates were
incubated in the dark at 22°C for 10 days, after which time the
change in main root length was measured. Root growth was
expressed as relative growth (i.e. the ratio of root length in the
presence and absence of hormones 3 100).
Complementation and genetic segregation analyses
Complementation analyses and dominance tests were conducted
by cross-pollinating homozygous plants and analyzing both root
and symbiotic phenotypes of the F1 and F2 seedlings. In addition,
homozygous har1-1 (Gifu) plants were crossed with polymorphic
L. japonicus B-581 Funakura plants to produced F1 seeds. A
mapping population of 242 F2 plants was established. Preliminary
linkage analysis was performed using randomly chosen, representative markers from the skeletal F2 map derived from a cross
between L. japonicus ecotype B129-S9 Gifu and B-581 Funakura
(Jiang and Gresshoff, 1997), utilizing the MAPMAKER version 1.0
Program.
Microscopic examination of symbiotic development
Primary root infections were examined by bright®eld microscopy,
using M. loti strain NZP2235 carrying a hemA::lacZ reporter gene
fusion. Seven-day-old wild-type and mutant L. japonicus seedlings were inoculated and transferred to the nylon pillow systems.
Seven to 11 days after inoculation wild-type and har1-1 mutant
plants were vacuum in®ltrated for 15 min with ®xative solution
(1.25% v/v glutaraldehyde solution buffered with 0.2 M sodium
ã Blackwell Science Ltd, The Plant Journal, (2000), 23, 97±114
111
cacodylate pH 7.2), and incubated in the same buffer for an
additional 1 h. Fixed specimens were rinsed twice with 0.2 M
sodium cacodylate pH 7.2 for 15 min, and stained for b-galactosidase activity using a staining solution that contained 800 ml of
sodium cacodylate (0.2 M, pH 7.2), 50 ml of 100 mM K3[Fe(CN)6],
50 ml of 100 mM K4[Fe(CN)6], and 40 ml of 5-bromo-4-chloro-3indolyl-b-D-galactoside (2%) substrate in N,N-dimethylformamide. After overnight incubation at room temperature, the roots
were rinsed successively with 0.2 M sodium cacodylate buffer
(pH 7.2; 3 3 10 min), distilled water (2 3 5 min), and cleared
following the protocol described by Malamy and Benfey (1997).
Specimens were examined by bright®eld light microscopy. Color
micrographs were digitized and images processed into montages
using Adobe Photoshop 5.0 software.
Foci of cortical cell divisions and nodule primordia were
examined by bright®eld microscopy of whole roots 7±11 days
after inoculation with wild-type M. loti, as described previously
(Szczyglowski et al., 1998a). Histology and cytology of nodules
harvested 21 days after inoculation with M. loti strain NZP2235
were examined by combined light and transmission electron
microscopy, as described previously (Szczyglowski et al., 1998a).
Microscopic analysis of non-symbiotic plant development
In order to evaluate their non-symbiotic root phenotype, wild-type
and har1-1 mutant L. japonicus plants were grown in the absence
of symbiotic bacteria and in the presence of a low concentration
of combined nitrogen (0.5±1.0 mM KNO3). Root specimens were
examined by bright®eld light microscopy to document their
morphological and developmental characteristics.
The distribution of events of lateral root development, including
pericycle cell divisions, formation of lateral root primordia, and
emerged lateral roots, were evaluated using Clorox-cleared
L. japonicus roots as previously described (Szczyglowski et al.,
1998a).
For cytological and histological examination of L. japonicus
wild-type and har1-1 mutant roots, 4 mm long root segments
were vacuum in®ltrated for 2 h in 2.5% glutaraldehyde, 50 mM
sodium cacodylate buffer (pH 6.8), rinsed for 30 min in 50 mM
sodium cacodylate buffer (pH 6.8; 3 3 15 min) and post®xed for 2 h
in 1% osmium tetroxide in the same buffer. After washing in
50 mM sodium cacodylate (pH 6.8; 3 3 15 min) and dH2O
(3 3 5 min), the root segments were dehydrated in an acetone
series and embedded in Spurr's epoxy resin. Two mm semi-thin
sections were stained with 0.02% toluidine blue and examined by
light microscopy. A series of micrographs from longitudinal
sections derived from either the meristematic region of the root or
a mature portion located 2 cm above the root tip were reconstructed into montages using Adobe Photoshop software. Root
cell layers, cell numbers and projected cell areas were measured
using micrographs of semi-thin cross-sections taken from the
differentiated zone of the root (1 cm above the root tip) by image
analysis using Bioquant System IV program (Dazzo and Petersen,
1989). The diameter of the living root was measured by image
analysis of a series of micrographs covering a distance up to
approximately 1 cm from the root tip. The length of root
epidermal cells was measured from digitized videoprint micrographs of unstained wet mounts of whole roots using CMEIAS
ImageTool software (Liu et al. 2000).
The size of the root meristematic region was estimated using
intact 10-day-old roots stained with acetocarmine (1% carmine in
45% acetic acid). The projected areas of the red-stained meristematic regions of wild-type and har1-1 mutant roots were
measured from digitized videoprint micrographs using CMEIAS/
112 Judith Wopereis et al.
ImageTool software. The mitotic index (MI) was measured using
squashed segments of root tips excised from 7-day-old
L. japonicus plants. Two mm segments were ®xed for 24 h in a
3 : 1 mixture of ethanol:acetic acid at 4°C, washed three times with
dH2O, incubated in 1N HCl for 30 min, washed three times with
dH2O, and stored at 4°C until further use. Root segments were
transferred to a drop of 45% acetic acid and the region covering
the root meristem was dissected under the stereomicroscope. A
drop of acetocarmine stain was added, and root samples were
gently squashed in between the microscope slide and cover slip.
The mitotic ®gures were scored using bright®eld light microscopy. More than 600 cells from each dissected sample of root
meristem were analyzed from 10 wild type and har1-1 mutant
roots.
Acknowledgements
We would like to thank Dr Joanne Whallon from the Center for
Electron Optics at Michigan State University and Dr Philip Benfey
at New York University for their help and stimulating discussions
during the course of work and Kurt Stepnitz, Marlene Cameron
and Ryan Williams for their help in preparing the ®gures. The
research described in this paper was supported by grant no. NSF09630189 and NSF-IBN9904908 from the National Science Foundation (to F.dB. and K.S.), and by grant no. DE-FG02±91ER20021
from the Department of Energy (to F.dB.). E.P. was supported by
the Danish Research Council Postdoctoral Program and an EU
Marie Curie fellowship. The research at the University of Aarhus
was further supported by a grant from the Danish Biotechnology
Program and the Ministry of Food and Agriculture (to J.S.).
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