Plant Physiol. (1998) 116: 913-921
Mortal: A Mutant of White Clover Defective in
Nodal
Root Development
Derek W.R. White*,
Derek R. Woodfield, and
John R. Caradus
Plant Molecular Genetics Laboratory (D.W.R.W.), and Plant
Improvement Group (D.R.W., J.R.C.), Grasslands Division, AgResearch,
Private Bag 11008, Palmerston North, New Zealand
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ABSTRACT |
A monogenic dominant mutant of white
clover (Trifolium repens L.), designated
Mortal, which is defective in the formation of
adventitious nodal roots, is described. Mortal plants
grown at temperatures ranging from 10 to 25°C do not initiate nodal root primordium development. However, all other aspects of plant development are normal, including the formation of lateral roots and
wound-induced adventitious roots. In some genetic backgrounds, the
Mortal mutation has a temperature-sensitive conditional
phenotype. Mortal plants shifted from growing conditions
of 20 to 30°C for 2 to 3 d form nodal root meristems. However,
new nodes that develop after plants are returned to 20°C exhibit the
mutant phenotype. The capacity to form nodal roots on cuttings placed
in water is also influenced by the genetic background of the
Mortal mutation. Genetic analysis established that the
physiological reversion of Mortal to nodal root
formation is controlled by at least two separate dominant genetic loci,
one for Nodal water response
(Now) and one for Nodal
temperature response (Not); the
Now locus has a dominant epistatic interaction with the
Not locus. The conditional nature of
Mortal should provide opportunities for the
identification of genetic and physiological mechanisms that influence
the development of nodal roots.
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INTRODUCTION |
Whereas the basic structure of angiosperms is established during
embryogenesis, most organs are formed by postembryonic development (Esau, 1977
). Generally, all of the shoot structures (leaves, nodes,
internodes, axillary shoot meristems, and flowers) are derived from the
primary shoot apical meristem. However, adventitious shoot-borne roots
are an exception because they develop endogenously from differentiated
parenchyma cells close to the vascular tissues (Lovell and White,
1986).
Little is known about the genes that control adventitious shoot-borne
root morphogenesis, despite their importance for anchorage, nutrient
acquisition, and water uptake from the soil in a wide range of plant
species. One approach to understanding the genetic mechanisms that
underlie adventitious root initiation and development is to identify
and characterize mutants altered in the process. At present, few
mutants with defects in adventitious root development are known
(Schiefelbein and Benfey, 1991). There are mutants of tomato that
produce few or no adventitious roots (Butler, 1954; Zobel, 1991) and
mutants of maize that are defective in the formation of lateral seminal
roots, crown roots, or both lateral seminal and crown roots (Jenkins,
1930; de Miranda, 1980; Hetz et al., 1996).
The general unpredictability in the formation of secondary roots on
shoots complicates the analysis of the genetic and molecular mechanisms
controlling adventitious root development. This can be minimized by
characterizing the genetic control of the formation of adventitious
nodal root primordia. In some plant species, adventitious root
primordia arise in a precise and ordered manner during node development. One such example is the nodal roots that form on the
prostrate stolons of white clover (Trifolium repens L.;
Thomas, 1987). In T. repens, the nodes of each stolon
alternate in orientation so that successive nodes produce leaves and
axillary buds on opposite sides of the stolon (Erith, 1924).
The organization of nodes and nodal roots in white clover is
illustrated in Figure 1. Root primordia
are typically absent from the first four nodes of wild-type white
clover stolons, and nodes bearing the first five leaf primordia are
enclosed within the leaf sheath of the first visible node (Erith, 1924;
Thomas, 1987). The first nodal root primordium is initiated below the axillary shoot bud of the fifth node, and a second primordium forms
above the axillary shoot of the sixth node. In the seventh node, the
lowermost of each pair of nodal root primordia matures into a root
apical meristem that grows out through both the stolon epidermis and
the stipular sheath to form a visible root, whereas development of the
uppermost nodal root meristem is normally arrested such that it remains
within the stipule. Further growth of the uppermost nodal root meristem
usually occurs only in very moist conditions.
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| Figure 1.
Stolon morphology of a wild-type white clover
plant. A, Underside view of the apical portion of a stolon showing the
nodes (N), leaf stipule (S), and petiole (P). B, Schematic
representation of nodal development. SAM, Shoot apical meristem; LRP,
lower root primordium; URP, upper root primordium; LRM, lower root
meristem; URM, upper root meristem; and ASB, axillary shoot bud.
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White clover (2n = 4× = 32) is predominantly an
obligate outcrossing species with disomic inheritance. Therefore,
populations are a heterogeneous mixture of highly heterozygous
individuals. This heterogeneity and the associated plasticity in
environmental response complicates genetic analysis of some
developmental traits in white clover. However, there are dominant
self-compatible alleles of the gametophytic S locus system of sexual
incompatibility, which can be used to self plants for the genetic
analysis of traits (Williams, 1987).
To determine the genetic control of adventitious root formation in
white clover, we have identified and characterized a spontaneous mutant, designated Mortal, which is defective in nodal root
primordium initiation. When grown at 20°C, Mortal plants
lacked nodal root primordia but were normal in other aspects of shoot
and root morphology. However, in some genetic backgrounds,
Mortal was conditional, responding to either a temperature
shift to 30°C or to the placing of stolon cuttings in water, by
developing nodal roots. Here we describe Mortal and provide
a genetic model for responses of the mutant to these temperature-shift
and water treatments.
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MATERIALS AND METHODS |
A single plant of white clover (Trifolium repens L.)
with a defect in nodal root formation was identified among seedlings grown in the controlled environment rooms of the National Climate Laboratory in Palmerston North, New Zealand. This spontaneous mutation
was designated Mortal. The mutant genotype was crossed with
a wild-type genotype, and mutant plants were identified from progeny.
Three cycles of recurrent selection were conducted, in which only those
plants that exhibited the nonnodal rooting phenotype for at least 6 months were retained. Mutant and wild-type plants were grown in
individual pots containing a peat-sand mixture, under greenhouse
conditions in which temperatures ranged from 10 to 30°C.
Both mutant plants and a wild-type plant (10F) were vegetatively
propagated by rooting shoot (stolon) tip cuttings in a nutrient solution and then growing the rooted cuttings in a peat-sand mixture. The stolon cuttings, which included the first three to four visible nodes with leaves removed from all but the terminal node, were propagated by immersing the basal three nodes in one-half-strength Hoagland solution in the bottom, light-proofed portion of a two-chamber plastic container. The upper transparent portion of this chamber, containing the stolon tip and leaves, had small vent holes to allow air
exchange while maintaining high humidity. These containers were placed
in a growth cabinet (Temperzone, Temperzone Ltd., Auckland, New
Zealand) set at 20°C, with a 12-h photoperiod of 800 µE
m
2 s1 PPFD.
Histology
The first three visible nodes of wild-type and Mortal
stolons (nodes 5, 6, and 7) were excised, vacuum infiltrated with 50% ethanol, 5% acetic acid, and 3.7% formaldehyde for 15 min, and then
fixed overnight in fresh formaldehyde at atmospheric pressure. Samples
were dehydrated using ethanol, with an overnight step in 95% (v/v)
ethanol and 0.1% (w/v) eosine, cleared in Histoclear (National
Diagnostics, Atlanta, GA), and embedded in Paraplast (Oxford Labware,
St. Louis, MO), as described by Cox and Goldberg (1988). Ten-micrometer
sections were made using a rotary microtome (model RM 2045, Jung,
Nusslock, Germany) and stained with a 1% aqueous solution of
safranin-O (BDH, Dorset, UK). Whole transverse sections of nodes were
photographed using a stereomicroscope (model Wild M3Z, Leica) and color
print film (Kodak Gold III).
Temperature Treatments
Plants with 10- to 20-cm-long stolons were transferred from the
greenhouse to a growth cabinet and acclimatized for 7 to 14 d at
20°C. Elevated temperature treatments for 8 to 72 h were then
conducted by transferring plants to a second cabinet set at 30°C.
Both cabinets had 12-h photoperiods with a PPFD of 800 µE
m2 s1 (6× 375 W HPI/T
mercury iodide high-pressure lamps and 2× 1000 W tungsten halogen
lamps, Philips, Eindhoven, The Netherlands) and 50% RH. Stolon nodal
rooting response was assessed 2 d after the plants had been
returned to the cabinet set at 20°C. Effects of cumulative 8-h
periods of 30°C interspersed with 16-h periods at 20°C were tested
by growing plants in a cabinet (Conviron, Asheville, NC) with a 12-h
photoperiod, 350 µE m2
s1 PPFD (12× 115 W cool-white fluorescent
tubes, Sylvania; 8× 60 W lamps, Performer, Italy). Six temperature
cycles were completed (a total of 48 h at 30°C), followed by
2 d at 20°C, before nodal rooting frequency was recorded. Each
of the above experiments was replicated twice with four plants. At
least two stolons from each treated plant were assessed for nodal
rooting response.
Genetic Analysis
A flow chart of inheritance studies, conducted to analyze the
genetic segregation of the Mortal phenotype, is given in
Figure 2. The plants used in this study
were derived from progeny obtained from a cross between a mutant plant
(C11563/21) taken from the third cycle of recurrent selection and a
wild-type genotype containing a gene for self-compatibility (A).
Segregation of mutant and wild-type nodal rooting phenotypes were
recorded for the F1 progeny over a 4-month period
under greenhouse conditions. S1
(F2) Mortal plants 2276 and 2278 were
individuals taken from two separate S1 families that were produced by self-fertilization of F1
Mortal plants.
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| Figure 2.
Flow chart of Mortal inheritance
studies. A to E, Cross- or self-pollinated plants analyzed for genetic
segregation of the Mortal mutation and interacting
modifier loci.
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Plants with distinctive leaf markings (Brewbaker and Carnahan, 1956;
Davies, 1963) were used in the BC1 genetic
analysis (B) to guard against pollen contamination. The partially
inbred S1 mutant plant 2278, which had a
red-fleck leaf marking but no white V marking (Rf Rf v v),
was backcrossed to the original Mortal plant used in this
study (C11563/21), which had a white V leaf marking at a separate locus
but no red-flecking (rf rf V V). The recipient plant was
emasculated prior to hand pollination. Backcross progeny were
germinated and grown at 20°C in a growth cabinet and scored for both
nodal rooting and leaf markings when the stolons exceeded 10 cm in
length. This method of scoring the Mortal phenotype was
adopted for all subsequent progeny analysis. One of the
Mortal BC1 progeny plants (M4), which
did not form nodal roots in response to either temperature or water
treatments, was chosen for further genetic segregation analysis.
Mortal (M4) was outcrossed to a wild-type plant (C),
backcrossed to Mortal (2278; D), and selfed (E), all by hand
pollination. Progeny from generations B to E were all assessed for the
Mortal phenotype, and progeny from generations B, D, and E
were also scored for response to both water and temperature-shift treatments.
Auxin Treatment
Individual nodes, including 2 to 3 mm of internode on either side,
were excised from wild-type 10F and Mortal 2278 plants, incubated in Petri dishes containing 90-mm-diameter filter paper (no.
1, Whatman), and soaked in sterile water with or without 1 µm IAA, 10 µm IAA, 40 µm
indole, or 80 µm indole. Twenty nodal segments of each
genotype were tested with each solution. The dishes were placed in a
growth cabinet at 20°C for 14 d with a 12-h photoperiod and then
scored for the number of nodes forming roots and the number of roots
per node.
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RESULTS |
Definitions of root primordia and root apical meristem given by
Scheres et al. (1996) were used. The initial phase of root formation
when all of the precursor cells are dividing is referred to as the root
primordium, whereas when some of the cells forming the root become
terminally differentiated, as in the formation of vascular tissue, the
remaining mitotically active cells at the root tip are termed the root
apical meristem.
Nodal Root Formation
Wild Type
Visible nodes where nodal root development occurs were numbered
proximal to the shoot apical meristem (as node 5, node 6, and node 7)
and assessed for root development. Development of the upper and lower
nodal root meristems was asynchronous in wild-type plants (Fig.
3). Whereas the lowermost root primordium
was initiated in node 5 (Fig. 3B) and developed into a root meristem at
node 6, formation of the uppermost root primordium was not initiated until node 6 (Fig. 3D). Both nodal root primordia were formed in the
cortex tissue adjacent to one of the axial vascular bundles of the
stolon and produced a vascular connection to that bundle (Fig. 3F). The
nodal root meristem also had a vascular connection to the axillary
shoot bud vascular system (not shown). Typically, the uppermost nodal
root meristem was arrested in its development and remained within the
stipular sheath (Fig. 3F).
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| Figure 3.
Nodal root development of wild-type plants.
Morphology of nodes 5 (A), 6 (C), and 7 (E), and histological
transverse sections of nodes 5 (B), 6 (D), and 7 (F). Positions of the
leaf stipule (S), lowermost nodal root primordium (LRP), lowermost
nodal root meristem (LRM), and uppermost nodal root meristem (URM) are
marked with arrowheads.
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Mortal
Plants of the Mortal genotypes 2276 and 2278 (from
separate F2 families), grown at temperatures
ranging from 10 to 25°C, were defective in nodal root primordia
formation (Fig. 4). Plants with these
genotypes had no external sign of nodal root formation on any of the
visible nodes, and serial sectioning of nodes 5 to 7 demonstrated that
even the first phase of nodal root primordium formation was absent
(Fig. 4, B, D, and F). Other aspects of stolon growth and development
did not appear to have been disrupted by the defect in nodal root
primordia initiation. In Mortal plants grown in pots under
greenhouse or growth cabinet conditions, stolon branching, leaf
emergence, and flowering were all identical to that of wild-type
plants.
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| Figure 4.
Mortal plants fail to develop
nodal root primordia. Morphology of mutant nodes 5 (A), 6 (C), and 7 (E), and histological transverse sections of nodes 5 (B), 6 (D), and 7 (F).
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To determine whether Mortal had retained the capacity to
form either wound-induced adventitious or nodal roots, an attempt was
made to root stolon tip cuttings. Stolon tip cuttings taken from
wild-type genotype 10F established roots from existing nodal root
primordia within 3 d when these cuttings were placed in nutrient solution (Table I). Some of these
cuttings also formed adventitious roots from the wounded internode of
the stolon. Surprisingly, stolon cuttings of Mortal genotype
2278 formed nodal roots after 7 to 21 d in nutrient solution
(Table I). These cuttings also formed adventitious roots from cut
internodes. Mortal genotype 2276 did not form nodal roots on
cuttings placed in solution within a 35-d period (Table I). This
genotype did, however, form adventitious roots from the wounded
internode of cuttings. The data shown in Table I indicate an influence
of genotype on the propensity of Mortal plants to form nodal
roots in water. Therefore, a single genotype (2278) that readily formed
nodal roots on stolon cuttings was selected to determine the effect of
the conditional water response on the Mortal mutation (see
"Genetic Analysis").
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Table I.
Nodal root formation on Mortal and wild-type plants
in response to water and temperature-shift treatments
Stolon cuttings (20 from each genotype) were placed in nutrient
solution at 20°C and assessed for the presence (+) or absence ( ) of
nodal and wound-induced roots. In the temperature-shift treatment,
Mortal and wild-type plants were incubated for 48 h at
30°C. Nodal root frequencies are means of the number of stolons indicated (n).
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Mortal Genotype 2278 Develops Nodal Roots at 30°C
During periods in the summer when the maximum daily greenhouse
temperature regularly exceeded 25°C, occasional nodal roots were
observed on plants of Mortal 2278. In subsequent cooler
periods the new nodes that formed on these plants were of the nonnodal rooting mutant phenotype. Mortal genotype 2276 did not form
nodal roots when grown under the same conditions. This observation was confirmed by determining the nodal rooting response of mutant and
wild-type genotypes treated for 2 d at 30°C (Table I). The results suggested the presence of modifier genetic loci in
Mortal genotype 2278 that might interact with the mutation
to give a temperature-sensitive phenotype. To characterize this
phenotype further, clones of Mortal 2278 were grown in a
controlled environment growth cabinet at 20°C for 2 weeks and then
treated at 30°C for 8, 16, 24, 48, or 72 h before being returned
to 20°C (Fig. 5).
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| Figure 5.
Mortal plants develop nodal root
primordia in response to a temperature-shift treatment. Mutant plants
grown at 20°C were shifted to 30°C for periods of 8 to 72 h
and were assessed for nodal root development after being returned to
growth at 20°C. Open bars, Arrested root meristems; solid bars,
normal nodal root meristems. Each experiment was replicated twice with
four plants.
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Two forms of nodal rooting response to the temperature-shift treatment
were observed: (a) fully developed nodal root apical meristems
equivalent to the lowermost meristem found in wild-type nodes 6 and 7, and (b) an arrested form of nodal root meristem that was visible from
the outside but did not protrude through the leaf stipule. The fully
formed nodal root meristems were able to develop into mature roots when
placed in water at 20°C. However, the arrested form of nodal root
meristem remained arrested. Mortal 2278 plants treated at
30°C for 8 or 16 h developed only arrested meristems, whereas
longer periods (48 and 72 h) of high-temperature induction
resulted in nodal root meristem formation. Rooting was restricted to
nodes 5 to 8, with the greatest response being at node 6. Treatment of
Mortal 2278 at 30°C for 48 or 72 h resulted in the
formation of more than one root meristem per node on node 6 (2 and 2.5 average roots/node, respectively). The 72-h treatment resulted in some
of the node 6 samples forming three root meristems. Transverse sections
taken through nodes containing three roots at a node showed that all of
the meristems were fully formed and connected to a stolon axial
vascular bundle (data not shown).
Cumulative Nodal Root Primordia Development
Mortal plants of genotype 2278 grown in the greenhouse
during the summer were not exposed to the continuous 48 h at
30°C, which was required to induce mature nodal root formation on
growth-cabinet-grown plants. Instead, the periods of exposure to
temperatures above 25°C in the greenhouse were most likely to have
been for daily durations of fewer than 6 to 8 h. When tested,
these shorter periods of elevated temperature treatment resulted only
in the formation of arrested nodal root meristems incapable of further
development at 20°C. This suggested that mature nodal root formation
on greenhouse-grown Mortal plants might occur by the
cumulative development of meristems due to successive exposure to short
periods of higher temperature interspersed with longer periods at
lower temperatures.
To test this hypothesis, Mortal 2278 plants were subjected
to six 8-h cycles at 30°C, alternated with 16 h at 20°C (i.e.
a cumulative total of 48 h at 30°C). These plants responded by
producing normal nodal roots (Fig. 6)
instead of the arrested root meristems that were obtained when the
treatment was for a single 8-h period at 30°C (Fig. 5). However, the
nodes that responded to a cumulative treatment of 48 h at 30°C,
given in periods of 8 h, differed from those that responded when
the elevated temperature was given as a continuous treatment. Nodal
root meristems formed on node 9 of the Mortal plants
receiving the cumulative treatment, whereas node 9 of plants
continuously exposed to 30°C for 48 h did not undergo any form
of nodal root primordia initiation. This result may be explained by the
ongoing growth that occurred during the prolonged cumulative treatment.
When grown for 50 d at 20°C in a growth room, both wild-type and
Mortal plants had a node production rate of approximately
0.4 nodes per day (D.W.R. White and B. Campbell, unpublished data).
Plants produced approximately 0.45 nodes per day when grown for 14 d at 30°C.
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| Figure 6.
Effect of cumulative temperature-shift treatments
on nodal root development in Mortal plants. Mutant
plants grown at 20°C were treated with six cycles of 30°C for
8 h, interspersed with 20°C for 16 h, and then scored for
nodal root development. Each experiment was replicated twice with four
plants. Open bars, Arrested root meristems; solid bars, normal nodal
root meristems.
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Mortal Cannot Be Rescued by Auxin Treatment
Plant growth regulators, particularly auxins, influence the
initiation, growth, and development of secondary roots. To determine whether the defect in nodal root primordia initiation was due to an
inadequate supply of auxin, excised nodal segments of mutant plant 2278 and wild-type plant 10F were treated with either water, IAA, or the IAA
precursor indole. Incubation of Mortal node explants in
water, IAA (1 or 10 µm), or indole (40 or 80 µm) for 14 d did not stimulate development of root
primordia, whereas soaking wild-type nodal segments in water, IAA, or
indole resulted in the outgrowth of the existing lowermost and
uppermost root meristems. Therefore, the defect in nodal root primordia
initiation caused by the Mortal mutation is not due to a
shortage of auxin or an auxin precursor.
Genetic Analysis
Genetic analysis of the Mortal mutation was initially
based on segregation for the presence or absence of nodal roots among F1 and BC1 progeny (Table
II). The 1:1 segregation ratio observed in the F1 generation indicated that the mutation
was possibly monogenic and dominant. To test this hypothesis, a
Mortal F2 (S1) plant (2278) was backcrossed to the original Mortal
(C11563/21) parent plant (Fig. 2). The 3:1 segregation of the mutant
phenotype observed in the BC1 generation is
consistent with the hypothesis. To confirm this conclusion, we
identified a true-breeding genotype of Mortal (M4) from
among the BC1 progeny plants. When
Mortal M4 was outcrossed to wild type, selfed, or
backcrossed to Mortal 2278, all of the progeny examined had
the mutant phenotype. Some of the progeny obtained from the inbred
generations D and E were stunted and slow to develop. It is likely that
these stunted phenotypes were due to inbreeding depression. Such plants
were not included in the analysis. In summary, the results of the
segregation of the mutant phenotype among progeny, as described in
Table II, indicate that Mortal is inherited as a monogenic,
dominant trait.
The genetic background of the Mortal mutation appeared to
influence the conditional nodal rooting response obtained from both temperature-shift and water treatments (Table I). To examine the
possibility that this conditional nodal rooting response of Mortal was due to the presence of one or more modifier
genes, the progenies of generations B, D, and E (Fig. 2) were tested for their response to water and temperature-shift treatments. The 1:2:1
segregation of nodal water response among the BC1
mutant progeny (Table III) indicated that
this trait was controlled by a dominant modifier gene, which we have
designated Now (for Nodal water
response). This 1:2:1 segregation ratio would be expected if both
parents were heterozygous for the locus (Now/now) and homozygous progeny had a greater penetrance for the character than
heterozygous progeny. The modifier locus that appeared to control the
conditional temperature shift was designated Not (for Nodal temperature response).
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Table III.
Genetic segregation of nodal water response
phenotype among Mortal progeny
Six stolon cuttings from each genotype were tested. Three classes of
response, all (All Now), some (Partial Now), or
no (now) cuttings forming nodal roots, were observed.
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Segregation analyses of both the nodal water response and the nodal
temperature-response phenotypes among the mutant plants in the progeny
of generations B, D, and E are given in Table
IV. Mortal progeny from the
backcross generation Mortal 2278 × Mortal C11563/21 segregated 12:3:1 for the phenotypes, nodal water and temperature response:temperature response only:no response to either
treatment. The segregation ratio observed indicated a possible epistatic interaction between two dominant modifier loci. To obtain the
observed 12:3:1 segregation ratio, both parents would have to be
heterozygous for both the Now and Not genetic
loci. To confirm this interpretation of the data, mutant progeny from
the backcross Mortal M4 × Mortal 2278 were
also analyzed for segregation of the Now and Not
loci. Segregation analysis indicated that both modifier loci were
absent from the M4 parent used in this cross. The 2:1:1 segregation
ratio of Now/Not to now/Not to now/not
observed among the mutant progeny of generation D (Table IV) supports
the hypothesis that Now has a dominant epistatic interaction
with the not allele of the nodal temperature response locus
(i.e. in the presence of Now and the absence of
Not Mortal plants formed nodal roots in response
to a temperature-shift treatment).
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Table IV.
Genetic segregation of modifier loci among Mortal
progeny
Mortal progeny plants were scored for the formation of nodal
roots in response to a temperature-shift (Not) treatment or
the response of three stolon cuttings to a water (Now)
treatment.
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DISCUSSION |
We report here the identification and initial characterization of
a white clover mutant defective in the formation of nodal root
primordium. To our knowledge, the only other defect specifically disrupting nodal root formation that has been described in detail is
the recessive rtcs mutation of maize (Hetz et al., 1996).
The Mortal mutation had no apparent pleiotropic effects on
the shoot morphology of white clover plants. Furthermore, the
Mortal mutation exclusively affected nodal root primordium
formation; neither lateral root development nor wound-induced
adventitious rooting on cuttings was altered. Smith and Fedoroff (1995)
postulated that the paucity of mutants exclusively affecting secondary
root development is due to the genes involved having duplicate
functions.
It is noteworthy that the HRGPnt3 gene of tobacco and the
LRP1 gene isolated from Arabidopsis, which are both
expressed during the early phases of lateral root primordium
development, are also expressed in adventitious root primordia (Vera et
al., 1994; Smith and Fedoroff, 1995). However, the presence of the
Mortal phenotype of white clover and the rtcs
mutant phenotype in maize indicates that there are genetic aspects of
secondary root primordium initiation and development that are unique to
the node. Because nodal root primordia are regularly initiated in an
invariant pattern on the stolons of wild-type white clover (Thomas,
1987), we were able to determine by serial sectioning of nodes that the
blockage in development caused by the Mortal mutation
prevented the initial divisions that contribute to nodal root
primordium development. The rcts mutation in maize also
prevents the initiation of nodal root primordia (Hetz et al., 1996).
An unusual feature of Mortal in some genetic backgrounds is
its response to a shift in growing temperature from 20 to 30°C, which
rescues nodal root primordium development. It is more common for
temperature-sensitive mutants to adopt the mutant phenotype when
shifted to a higher temperature rather than revert from mutant to
normal development. An example is the temperature-sensitive mutants of
Arabidopsis isolated by Baskin et al. (1992), which are normal when
grown at 18°C but have radial swelling of the root apex when
transferred to 31°C. There are only a few cases in which expression
of a mutation in plant development occurs at low temperature and
development is normal at high temperature. The recessive Arabidopsis
mutant fab2, which overproduces the fatty acid stearate
(Lightner et al., 1994), recessive sweetclover mutants defective in
chlorophyll production (Bevins et al., 1993), and a recessive,
temperature-dependent shooty mutant of tobacco (Samuelsen et al.,
1997) are specific examples.
The Mortal mutation is distinct because the
temperature-sensitive response requires the presence of a separate,
dominant-modifier genetic locus. The temperature-shift effect on
Mortal was rapidly reversible because further development of
the partially formed primordia or meristems induced by an 8-h treatment
at 30°C was blocked when the plants were returned to growth at
20°C. This arrest may parallel development of the uppermost nodal
root meristem of wild-type plants, which is interrupted before
maturity, and further examination of conditions that lead to continued
development of the uppermost nodal root meristem of wild-type plants
may provide insight into the normal function of the Mortal
gene. It is noteworthy that some of the nodes on wild-type plants
treated at 30°C for 48 h responded by outgrowth of the uppermost
nodal root meristem (Table I). Also, further development of the
uppermost nodal root meristem can be induced when the intact stolon is
immersed in water. Both inhibition and reactivation of nodal meristem
development may therefore be a normal feature of uppermost nodal root
formation in white clover.
Numerous experimental results indicate that phytohormones play an
important role in the regulation of secondary root primordium development. There are secondary root development mutants that have
either elevated levels of auxin (Boerjan et al., 1995; King et al.,
1995) or can be rescued by an exogenous supply of auxin (Celenza et
al., 1995). Haissig (1972) demonstrated that both the level of
endogenous auxin and applied GA3 influence the
number of cells in developing nodal root primordia of brittle willow. However, the lack of a response to the addition of IAA or indole to
nodal explants suggests that the Mortal mutation is not due to a defect in auxin biosynthesis. This conclusion does not eliminate the possibility that altered auxin homeostasis may be involved in the
disruption of nodal rooting.
The genetic background of the Mortal mutation also
influenced the ability of a genotype to form nodal roots on cuttings
placed in nutrient solution (Table I). This genetic background effect on both temperature and water responses indicates that modifier genes
activate a signaling pathway between environmental conditions and
Mortal gene function. Genetic analysis determined that the defect in nodal root development designated Mortal was due
to a monogenic dominant mutation. Furthermore, results from the genetic analysis support a model in which physiological reversion of
Mortal to nodal root primordium development is determined by
at least two independently segregating, naturally occurring, dominant
modifier loci (Now for water response and Not for
the temperature-shift response). The presence of the Now
locus is sufficient to allow a nodal rooting response on mutant plants
to both water and temperature-shift treatments, but the Not
locus confers nodal rooting only in response to the temperature-shift
treatment. This dominant epistatic interaction between the modifier
loci suggests that a complex signaling pathway controls nodal root
development and maturation in white clover.
Any model to explain the Mortal mutation has to include a
dominant loss-of-function alteration and an inhibition of development that can occur at any phase between nodal root primordium initiation and formation of the meristem. Our working hypothesis is that Mortal is due to the expression of a product that inhibits
both the initiation and continued division of those cells that would normally constitute the nodal root primordium.
The conditional nature of Mortal will provide a means of
identifying the genetic and molecular mechanisms that control the development of adventitious root meristems. Because mutant plants can
be treated to provide material arrested at various stages of nodal root
primordium development, methods of mRNA comparison can be used to
identify gene expression specific to different phases of adventitious
root development. Furthermore, Mortal plants provide useful
material with which to study the ecological implications of nodal root
formation for plants such as white clover, which have a clonal
vegetative growth form, and to deduce the signaling mechanisms that
control nodal root development.
| |
FOOTNOTES |
*
Corresponding author; e-mail whited{at}agresearch.cri.nz; fax
64-6-351-8042.
Received September 10, 1997;
accepted November 21, 1997.
1
This work was supported by the New Zealand
Foundation for Research, Science and Technology (grant no. C10-405 to
D.W.R.W. and grant no. C10-310 to J.R.C. and D.R.W.).
| |
ACKNOWLEDGMENTS |
The authors would like to thank Roy Meeking and Thomas Berryman
of the D.W.R. White laboratory for their technical contributions and
Greg Cousins of the Plant Improvement Group for some of the crossing of
mutant white clover plants. We are grateful to Warren Williams and
Bruce Campbell for comments on the manuscript.
| |
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Abstract
Introduction