Annals of Botany 101: 319–340, 2008
doi:10.1093/aob/mcm251, available online at www.aob.oxfordjournals.org
INVITED REVIEW
Determinate Root Growth and Meristem Maintenance in Angiosperms
S. SHISHKOVA 1 , T. L . ROST 2 and J. G. DU BROV SKY 1, *
1
Departamento de Biologı́a Molecular de Plantas, Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México,
Apartado Postal 510-3, 62250, Cuernavaca, Morelos, Mexico and 2Section of Plant Biology, College of Biological
Sciences, University of California, One Shields Avenue, Davis, CA 95616, USA
Received: 9 May 2007 Returned for revision: 9 July 2007 Accepted: 17 August 2007 Published electronically: 21 October 2007
† Background The difference between indeterminate and determinate growth in plants consists of the presence or
absence of an active meristem in the fully developed organ. Determinate root growth implies that the root apical
meristem (RAM) becomes exhausted. As a consequence, all cells in the root tip differentiate. This type of
growth is widely found in roots of many angiosperm taxa and might have evolved as a developmental adaptation
to water deficit (in desert Cactaceae), or low mineral content in the soil ( proteoid roots in various taxa).
† Scope and Conclusions This review considers the mechanisms of determinate root growth to better understand how
the RAM is maintained, how it functions, and the cellular and genetic bases of these processes. The role of the quiescent centre in RAM maintenance and exhaustion will be analysed. During root ageing, the RAM becomes smaller
and its organization changes; however, it remains unknown whether every root is truly determinate in the sense that
its RAM becomes exhausted before senescence. We define two types of determinate growth: constitutive where
determinacy is a natural part of root development; and non-constitutive where determinacy is induced usually by
an environmental factor. Determinate root growth is proposed to include two phases: the indeterminate growth
phase, when the RAM continuously produces new cells; and the termination growth phase, when cell production
gradually decreases and eventually ceases. Finally, new concepts regarding stem cells and a stem cell niche are discussed to help comprehend how the meristem is maintained in a broad taxonomic context.
Key words: Angiosperms, determinate root growth, indeterminate growth, meristem maintenance, quiescent centre, root
apical meristem, root development, stem cells, stem cell niche.
IN TROD UCT IO N
In angiosperms, root and shoot growth is maintained and
regulated through the activity of the apical meristems. A
balance between the generation of new meristematic cells,
and their transition toward differentiation, permits the maintenance of the meristem and regulates its activity. However,
in many cases the meristem is genetically programmed to
stop producing new cells at a specific developmental
stage. In these cases, the meristem is said to be determinate
(Sablowski et al., 2007). A determinate meristem usually
produces a part of the plant that has a predictable size
and form, such as the flower, whereas an indeterminate
meristem produces parts of the plant that can grow for variable periods of time, and vary in size and shape dependent
on the local environment (Sablowski et al., 2007). Thus, the
indeterminacy, or determinacy, of the meristem is directly
related to the type of growth of an organ. Edmund Sinnot
(1960) in his book Plant Morphogenesis describes indeterminate growth this way: ‘Potentially, the plant axis can
grow indefinitely in length through the activity of its
apical meristem and in width through the activity of the
vascular cambium. Actually, growth finally ceases for
various reasons, but these axial meristems are essentially
indeterminate in their activity.’ The shoot apical meristem
(SAM) develops from the plumule and in turn the axial
buds generate new SAMs of the shoot branches. If no transition to formation of generative organs occurs, the vegetative SAM may maintain its indeterminate growth for a long
* For correspondence. E-mail [email protected]
period of time. Indeterminate developmental patterns of
shoot growth are underpinned by complex mechanisms
involved in maintenance of the SAM (Bäurle and Laux,
2003; Veit, 2004; Barthélémy and Caraglio, 2007;
Sablowski, 2007). The vegetative SAM produces leaf primordia on its flanks giving rise to determinate organs, the
leaves, which take a developmental pathway for terminal
differentiation. Therefore, mature leaves do not have a meristem. However, the vegetative SAM can be transformed
into an inflorescence SAM, which can be either determinate, or indeterminate. Determinate inflorescence SAMs
form a determinate number of flower primordia.
Indeterminate inflorescences never form a terminal flower.
Even when such a plant stops growing, its SAM is still
present. Thus, either an inflorescence SAM in some
species, or a vegetative SAM in others, can be transformed
into a floral meristem that is destined to produce flower
organs, and in this way to terminate its activity. These
cases of floral and leaf meristems are clear examples of
developmental determinacy. In the underground plant
organs, clear examples are the determinate and indeterminate nitrogen-fixing root nodules of legumes. Mature determinate nodules do not have a meristem, while mature
indeterminate nodules maintain a meristem (Bauer et al.,
1997). Then, the fundamental difference between indeterminate and determinate organs is presence or absence of
an active meristem in the mature organ.
Plant roots are surprisingly complex in their growth
pattern. To the best of our knowledge, the idea of determinacy in plant roots has not been reviewed elsewhere. An
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Shishkova et al. — Determinate Root Growth and Meristem Maintenance
analysis of determinate root growth can help us understand
how the root apical meristem (RAM) is maintained and the
significance of root growth patterns on the plant life cycle.
We will analyse the biological significance, distribution and
types of determinate root growth in various plant taxa, and
the cellular and genetic mechanisms underlying the determinate developmental programme that has evolved in
roots. We will establish the role of the quiescent centre
(QC) in RAM maintenance, and show that root determinacy
is related directly to the RAM maintenance and function.
T E R M I N O LOGY A N D C L A S S I F I CAT I O N
O F T Y P E S OF D E T E R M I N AT E G ROW T H
I N ROOT S
Root determinacy as a general phenomenon
When seeds germinate, usually the primary root emerges
before the shoot. The primary root elongates for some
time and this is typically followed by emergence of either
lateral or adventitious roots, leading to the development
of the root system. In this paper, we will apply terms to
individual roots independently of their origin: primary,
lateral or adventitious. The phenomenon of root determinacy in various occasions may or may not be related to
ageing of individual root axes. In general, root life span
in plants is highly variable, from a few weeks to a few
years (Eissenstat et al., 2000). For example, fine roots of
Pinus taeda 1 mm in length can be alive up to 6 years
(Matamala et al., 2003). We do not know whether these
gymnosperm roots become determinate. The RAM could
be lost in them during the first year, but roots stayed functional for a few more years.
Thus it is important to distinguish between roots that stop
growing but remain healthy and metabolically active and
those that stop growing and die. Hereby, root ageing is
not necessarily related to root determinacy. If the RAM
remains organized, even if it is inactive, the root is not considered to be determinate. Hypothetically, roots can stop
growing while their meristem is present but not active.
Such cases are not well documented in angiosperms, but
have been described for a gymnosperm species
Libocedrus decurrens (incense cedar) roots. In this
species an individual root can become dormant and then
renew its growth (Wilcox, 1962), illustrating an unusual
case, where a root stops growth and then resumes. As we
already mentioned, the presence of the RAM, whether
active or not, is the main criterion for the indeterminate
condition.
In sterile root culture, the individual root axis can
become inactive but roots produced from this axis can
grow for many years if a subapical segment with new
lateral root tips is excised and transplanted for each subsequent passage. In this way, roots can grow for many
years [tomato (Solanum lycopersicon), White (1943);
Convolvulus arvensis, Torrey (1958)]. This indicates that
growth potential of the cultured primary or lateral root
becomes lost with time in culture and these roots may
appear to be determinate, but growth can be reestablished
by cultivation due to activity of new lateral roots
(Smirnov, 1970). This shows that root determinacy of an
individual root axis and ageing of the root system are two
separate processes.
Root growth is mainly studied not from a developmental
but rather from an ecological perspective. Typically the
behaviour and growth of the entire root mass is evaluated
(Eissenstat et al., 2000; Matamala et al., 2003; Ryser,
2006), and the development of individual root axes
during long periods of time is rarely studied. In this
review, we will focus our attention on the developmental
history of individual roots. The issue of root determinacy
is considered here irrespective of the age of the whole
root system of a plant, but rather as a developmental
phenomenon describing individual roots. Also, in this
review, although some examples of Gymnosperm roots
will be mentioned, we mainly consider angiosperms. Some
pteridophytes, like Azolla, also have determinate adventitious roots, and relevant information on determinate root
growth in ferns can be found (Webster and MacLeod, 1996).
In pea (Pisum sativum), the growth rate of the primary
root gradually increases post germination, maintains a
steady state for a period of time, and then decelerates
(Rost and Baum, 1988; Gladish and Rost, 1993). When
grown at different temperatures ranging from 15 to 32 8C,
pea seedlings reach different final root lengths as a function
of temperature; roots grown at 15 8C can exceed 20 cm of
final length, while roots grown at 32 8C stop growing, reaching about 12 cm of length (Gladish and Rost, 1993). The
length of the RAM tends to be greatest when the rate of
elongation of the primary root is at its peak, and gradually
decreases until elongation stops (Rost and Baum, 1988).
This decrease of growth rate is connected with differentiation events appearing closer to the root tip (e.g. Rost
and Baum, 1988; Soukup et al., 2002). Other studies in
cotton (Gossypium hirsutum), Arabidopsis thaliana, and
several other species representing ten different families
have demonstrated that the primary root eventually stops
growing in all species studied (Reinhardt and Rost, 1995;
Chapman, et al., 2003). Together, these observations
suggest that the primary roots in seedlings of dicotyledonous plants reach a determinate length and that this final
length may be dependent on the environment.
Determinacy in primary roots that is related to ageing is
usually accompanied by developmental changes within the
RAM. For example, RAM organization of primary roots in
six species from five families was shown to change from
closed type (defined in Von Guttenberg, 1960) to
intermediate-open type (defined in Chapman et al., 2003;
Groot et al., 2004) over a period of growth. Roots in all
of these species eventually cease elongation, reaching a
determinate age ranging from 14 to 41 d post-germination
(Chapman et al., 2003). Similar developmental changes
of the RAM organization have been well documented in
Convolvulaceae (Seago and Heimsch, 1969), Asteraceae
(Armstrong and Heimsch, 1976), Brassicaceae (Baum
et al., 2002), and other families. For example, in
A. thaliana, young roots have a closed type of RAM. As
the root grows and ages, its RAM organization changes
until at 4 weeks of seedling age the RAM becomes intermediate open and decreases in size (Baum et al., 2002).
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
The number of plasmodesmata in any given cell wall within
the primary RAM increases 1 – 2 weeks, and then decreases
to a minimum by 4 weeks post-germination (Zhu et al.,
1998). The number of plasmodesmata in cell walls of the
root cap also decreases dramatically during this time, and
cells on the periphery of the root cap undergo programmed
cell death (Zhu and Rost, 2000).
In summary, all roots have their own dynamics of ageing
and in this sense any root of annual, biennial or perennial
plants may reach determinate length by ceasing elongation
at a certain age. During this process the RAM organization
changes, it becomes smaller, cells within the RAM become
symplasmically isolated, and finally the RAM ceases to
function. Nevertheless, for most species it remains
unknown whether every root is truly determinate (which
implies its RAM reaches exhaustion), or whether it can continue to perform its functions after the meristem exhaustion.
We will present numerous data of well-documented determinate growth that in various developmental situations
takes place either in primary or lateral roots.
Root determinacy and growth phases
The growth of most individual roots can be divided into
two main phases: the phase when the growth is maintained
for an undefined period here referred to as the ‘indeterminate
growth phase’, and the ‘termination growth phase’ when
growth eventually ceases, the determinate growth phase.
During the indeterminate growth phase the RAM is continuously producing new cells. When the root reaches its determinate age, stage, and/or length, or no appropriate
conditions for growth are available, the growth can be
simply arrested. In this case, an organized RAM is still
present and the RAM cells maintain meristematic potential.
In some species, as in Libocedrus, the RAM can become
dormant but later can reinitiate its function (Wilcox, 1962),
or root growth can be arrested by drought, but the RAM
can continue to be functional (Vartanian et al., 1994).
Thus, the presence of an organized RAM at the moment of
observation is evidence of potential to resume growth, and
resume the indeterminate phase of the root growth.
Alternatively, a developmental programme leading to complete RAM exhaustion and, differentiation of root tip cells,
culminates in termination of growth. In this case, the root
has ‘determinate growth’. Determinate growth can be considered ‘constitutive’ if it occurs under any environmental
condition. The best examples here are the primary roots in
some Cactaceae and lateral roots in plants of other families,
and those in some A. thaliana mutants. Determinate growth
can also be induced under some conditions, for example,
phosphate starvation (Sánchez-Calderón et al., 2005). We
refer to this phenomenon as ‘non-constitutive’ or ‘inducible
determinate growth’.
CON S T I T U T IV E D E T E R M I N AT E RO OT
G ROW T H , I T S E CO P H Y S I O LO GY A N D RO L E
Constitutive determinate growth is found in various taxa and
represents a stable developmental programme that has
certain ecological significance. However, sometimes the
321
significance and distribution of this growth pattern within a
species is obscure (Varney and McCully, 1991). For
example, these authors found determinate growth in some
lateral roots in the maize root system (Varney and
McCully, 1991) but there is no clear understanding of a functional role of these determinate roots in maize. In this section,
we further consider, in various angiosperm taxa, cases of
determinate root growth which is characterized by a clear
developmental scenario and ecological significance.
Determinate root growth in Cactaceae and its significance
Determinate root growth of Cactaceae was first described
for lateral roots of Opuntia arenaria and O. tunicata var.
davisii (Boke, 1979). In these species, plants form first-order
determinate lateral roots that are a few centimetres long;
their RAM is active for only a limited period of time and
then these roots cease growing. New second-order lateral
roots of various lengths are formed close behind the root tip.
On these roots, third-order lateral roots develop, which are
,1 mm in length. These show determinate root growth, and
are called ‘root spurs’. The root spurs lack a root cap, the
cells at their tips become differentiated and the tips become
completely covered with root hairs. Spur roots may allow
for an increase in root surface area, presumably increasing
water uptake during infrequent rainfalls (Boke, 1979).
The determinate growth of primary roots of some
Sonoran Desert Cactaceae was first reported by
Dubrovsky (1997a, b). The species described belong to
two subfamilies (classification by Nobel, 1988):
Pachycereeae [Stenocereus thurberi, S. gummosus
(Dubrovsky, 1997a, b), S. pruinosus, S. standleyi
(Dubrovsky, 1999), Pachycereus pringlei (Dubrovsky and
Gómez-Lomelı́, 2003)] and Cactoideae [Ferocactus peninsulae (Dubrovsky, 1997b)]. A common feature of cactus
roots with determinate growth is the relatively short duration of the primary root growth and early meristem
exhaustion. For example, primary roots of F. peninsulae
and S. gummosus grow for only 2 – 3 d after the start of
seed germination and their final length does not exceed
on average 10 mm and 9 mm, respectively (Dubrovsky,
1997a, b). After the first day of growth, the RAM length
starts to decrease while RAM cells cease dividing and
undergo rapid elongation and eventually differentiation.
At the end of root growth, the RAM becomes exhausted
and no new cells are produced, while the root tip cells
elongate and differentiate. As a result of differentiation, epidermal cells form root hairs that approximate the tip of the
root, and subsequently cover it completely (Fig. 1).
The determinate developmental programme does not
necessarily start very soon after seed germination. In
P. pringlei, an indeterminate growth phase of the primary
root is extended under optimal growth conditions and the
root terminates its growth 8 – 9 d post-germination. In this
species, water deficit accelerates the determinate developmental programme resulting in termination of growth
approx. 2 d earlier (Dubrovsky and Gómez-Lomelı́,
2003). We have also shown that programmed cell death is
not involved in the RAM exhaustion of S. gummosus and
P. pringlei, although the root-cap and root-hair cells in
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Shishkova et al. — Determinate Root Growth and Meristem Maintenance
or cluster roots, dauciform roots, capillaroid roots and
cluster-like roots.
F I G . 1. Root tips of Stenocereus gummosus at 24 h (A) and 48 h (B and
C) after the start of radicle protrusion. Root hairs approach the tip (A) and
later cover the tip completely (B, arrow). (C) Close-up of the area shown
by arrow in (B). Arrow in (C) shows that only five most apical cells did
not form root hairs indicating that almost all epidermal cells differentiate
and the RAM are completely exhausted. Scale bars: A ¼ 400 mm; B ¼
100 mm; C ¼ 50 mm. Reproduced from Dubrovsky and North (2002)
with permission from University of California Press and The Regents of
the University of California.
these species can undergo programmed cell death
(Shishkova and Dubrovsky, 2005).
The common characteristic of determinate root growth in
the Cactaceae, in both the primary and lateral roots, is the
complete exhaustion of the RAM, coupled with differentiation of all previously meristematic cells, and loss of the
root cap. This developmental programme is highly stable.
Analysing thousands of plants, we have never found a
case of growth reversal of the primary root from its determinate condition. Moreover, we have shown that roots regenerated from calli in tissue culture also have determinate root
growth (Shishkova et al., 2007).
What evolutionary advantage may determinate root
growth have in the desert Cactaceae? Rapid seedling establishment in desert environments during short optimal
periods of water availability is a challenge. Successful
S. gummosus seedling establishment in the Sonoran
Desert is ,1 % (León de la Luz and Domı́nguez-Cadena,
1991). The determinate root growth in such plants was proposed to present a developmental adaptation (Dubrovsky,
1998). The beginning of RAM exhaustion correlates well
with the timing of lateral root initiation, and thus the loss
of the functional RAM is viewed as a physiological root
tip decapitation that promotes lateral root formation
(Dubrovsky, 1997a, b). Some lateral roots also have determinate growth (Dubrovsky, 1997b), resulting in a compact
and highly branched root system that permits efficient
water and mineral uptake and transport, and facilitating
rapid shoot biomass accumulation (Dubrovsky, 1998). The
accumulation of shoot biomass in this case is a critical
factor for plant survival in the harsh desert environment
(Dubrovsky, 1996). Root determinacy in these Cactaceae
species increases species fitness.
Determinate root growth in root clusters: proteoid,
dauciform and other root clusters
Root clusters of several types occur both in monocotyledonous and in dicotyledonous plants. Lambers et al. (2006)
use the term ‘root clusters’ to refer collectively to proteoid
Proteoid roots. These cluster roots consist of a large number
of determinate lateral rootlets which develop on short fragments of the main root axis, giving them a ‘bottlebrushlike’ appearance. They were described in detail in
Proteaceae species by Purnell (1960), and there are many
papers and reviews on proteoid roots (Lamont, 1982,
2003; Dinkelaker et al., 1995; Skene, 1998; Neumann and
Martinoia, 2002; Shane and Lambers, 2005; Lambers
et al., 2006). Proteoid root development is almost ubiquitous in .1800 species in the Proteaceae. However, they
also occur in members of seven other families:
Casuarinaceae,
Myricaceae,
Fabaceae,
Moraceae,
Betulaceae, Cucurbitaceae and Eleagnaceae (Skene, 1998;
Shane and Lambers, 2005, and references therein).
Proteoid roots can be ‘simple’ or ‘compound’ in Hakea
and Banksia species, respectively; the latter result from an
assemblage of simple proteiod roots (Purnell, 1960) and
are produced by only a few Proteaceae genera (Lamont,
1982). Simple cluster roots in the Proteaceae plants
usually contain many more determinate rootlets per centimetre of parent root length (up to 1000!) than those in
the Fabaceae (,50 rootlets per centimetre) (Dinkelaker
et al., 1989; Lamont, 2003). Although very few Lupinus
species produce the type of clusters that are found in
L. albus (Clements et al., 1993; Bolland, 1995, 1997;
Skene and James, 2000), other species of the family can
produce ‘cluster-like roots’, which may function in a
similar way. The rootlets of the cluster-like roots of
L. angustifolius are induced on high N with an adequate
P supply, and are sparser than those of the cluster roots of
L. luteus. In addition, they produce fewer root hairs
(Hocking and Jeffery, 2004).
Monocotyledonous families, sedges (Cyperaceae) and
rushes (Restionaceae), form root clusters termed ‘dauciform’ roots and ‘capillaroid’ roots (Shane et al., 2005;
Lambers et al., 2006). Cluster roots (e.g. Keerthisinghe
et al., 1998) and dauciform roots (Shane et al., 2005;
Playsted et al., 2006) can be also induced by P deficiency.
These root clusters markedly increase the surface area of
the root system and are adaptive for nutrient acquisition
from impoverished soils, especially for P acquisition
(Shane and Lambers, 2005; Lambers et al., 2006).
Moreover, low N supply, or limited supply of K or Fe,
may enhance cluster-root development in various species
(Shane and Lambers, 2005). Such cluster roots are ephemeral and individual cluster roots can be physiologically
active for a little more than 1 week in Lupinus albus
(Watt and Evans, 1999), and perhaps 2 –3 weeks in
Hakea species (Dinkelaker et al., 1995). Cluster roots
may show an exudative burst release of carboxylates (e.g.
citrate and malate) at very high rates (Watt and Evans,
1999), but only for a few days. Carboxylate exudates are
probably the most effective at mobilizing P, but cluster
roots can also release other compounds; e.g. acid phosphatases (Neumann et al., 1999) may contribute significantly to
P acquisition (Dinkelaker et al., 1997; Shane and Lamberts,
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
2005). Although cluster rootlets senesce and die, the main
root axis usually remains alive and active.
The development and anatomy of the determinate proteoid
rootlets of Grevillea robusta (Proteaceae) and L. albus
(Fabaceae) was described by Skene et al. (1998a, b)
and by Watt and Evans (1999). During differentiation
of the rootlet tips, some of the epidermal cells form root
hairs, which grow through the one cell-layer root cap.
Eventually, the root cap is displaced by growing root hairs.
In the RAM of mature rootlets, each cell layer differentiates
up to its initial; the two columns of xylem elements at the
rootlet apex join to form a single file of terminal xylem
cells (Skene et al., 1998a). Remarkably, even the endodermis
initial cells become differentiated (Skene et al., 1998b).
Cluster roots of L. albus develop in a similar way (Watt
and Evans, 1999). Discrete regions of closely spaced, determinate secondary rootlets emerge nearly synchronously on
the same plant grown in hydroponic culture. If on day one
after emergence the rootlets are almost entirely meristematic,
by day three, they are already approaching their final length,
the RAM is no longer present, all cells are vacuolated, and
epidermal cells around the tip are developing hairs. Root
hairs continue to develop until day six and they accumulate
around the tips of the completely differentiated rootlets
(Watt and Evans, 1999).
323
Adhesive pads of adventitious roots in the climbing fig
Climbing vines, like the climbing fig (Ficus pumila;
Moraceae), have developed a specialized structure, the
adhesive pad, a cluster of short adventitious roots
(Fig. 2A), that secrete a sticky substance permitting adherence to almost any substrate (Groot et al., 2003). This interesting structure was actually first reported by Darwin (1875)
in his book on climbing plants. Groot et al. (2003) analysed
the developmental anatomy of clustered adventitious roots.
Clustered adventitious roots in juvenile F. pumila vines are
initiated in pairs on either side of a vascular bundle at the
2nd to 3rd internodes of young stems. After root emergence
through the cortex and epidermis, root hairs form, which
secrete a substance that stains positively for polysaccharide
and protein. Immediately after emergence, the RAM of the
adventitious roots is short and wide (Fig. 2B). When the
roots reach their determinate length (3 –10 mm), the root
cap tends to fall off, the RAM becomes exhausted, and
its cells vacuolated (Fig. 2C). The adventitious roots and
root hairs stick together forming the adhesive pad
(Fig. 2A). If the adventitious roots fail to touch a substrate,
they usually dry up; if they touch moist soil they tend to
Dauciform roots. Dauciform root clusters of sedges
(Cyperaceae) were first described by Selivanov and
Utemova (1969) and later by Davies et al. (1973). They
were called ‘dauciform’ roots by Lamont (1974) because
of their carrot-like shape. Dauciform roots often occur in
groups of 20– 30, ranging in length from 2 mm to 12 mm
(Lamont, 1974; Shane et al., 2005), and their tips are
covered with dense, long root hairs. The dauciform roots
can be either determinate, with very long root hairs over
the tips of the mature roots, as in Cyathochaeta avenacea,
or indeterminate, with elongated non-dauciform root axes
as in other Cyathochaeta species (Shane et al., 2005). To
enhance P uptake, dauciform roots also release great
amounts of carboxylates, as well as other compounds,
during a developmentally programmed exudative burst.
They function in a very similar way to proteoid roots
(Playsted et al., 2006).
Capillaroid roots. Members of the monocotyledonous
Restionaceae family form ‘capillaroid’ root clusters,
which were also discovered and named by Lamont
(1982). The name is derived from the sponge-like properties of the clumps of rootlets, which are densely covered
with exceptionally long root hairs capable of holding soil
water (Lamont, 1982). Little is known about their structure,
development and physiology. Lamberts et al. (2006)
hypothesized that the physiology and function of capillaroid
roots is similar to that of proteoid roots.
The types of root clusters considered above are usually
those found on nutrient-poor soils. These short-lived roots
can apparently be developed in cohorts, and increase root
turnover. So, determinate growth in these roots could be
an adaptation that regulates a rapid increase in root
surface area to facilitate nutrient uptake.
F I G . 2. Adhesive pad of Ficus pumila appressed against a glass window
(A) and adventitious roots with determinate root growth at early (B) and
late (C) developmental stages. (A) The pad consists of a cluster of short
determinate adventitious roots meshed together with root hairs which
secrete a very sticky substance that holds the vine to almost any substrate
(Groot et al., 2003). (B) Section of the tip of adventitious roots that are
about to emerge from a stem. (C) Tip of adventitious root several days
after emergence. The cells of the tip have enlarged, the meristem is no
longer functional, and the root cap cells have elongated. h, root hairs
formed relatively close to the tip; arrow indicates xylem vessels developed
close to the root tip. Scale bars: A ¼ 500 mm; B, C ¼ 50 mm.
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Shishkova et al. — Determinate Root Growth and Meristem Maintenance
branch and change to a terrestrial form. A similar difference
in the type of growth between the aerial adventitious roots
and the soil-grown roots has also been observed in
Monstera deliciosa (Hinchee, 1981). Auxin treatments on
F. pumila adhesive pads suggested that auxin is not
involved in the determinate growth of clustered adventitious
roots (Groot et al., 2003). Adhesive root pads represent a
specific case of determinate root growth that has evolved,
presumably, as a plant adaptation that permits a vine to
adhere to vertical surfaces for sun exposure to increase its
photosynthetic capacity.
Determinate root growth in roots of parasite and
hemiparasite plants
There are about 3000 species of parasitic plants in 17
families, e.g. Schrophulariaceae, Cuscutaceae, Lauraceae,
Viscaceae and others (Nickrent, 2002; Riopel and Timko,
1995). Although not many of them have been extensively
studied, they are known to produce unusual structures
called ‘haustoria’, considered to be highly modified roots
(Kuijt, 1969). Haustorial roots come in two types:
primary and secondary. Primary haustorial roots are
formed at a root tip as a result of RAM cell differentiation
(Kuijt, 1969; Weber, 1987). In the genus Striga
(Scrophulariaceae), the root tip is triggered to become a
haustorium by an induction process involving a
haustoria-inducing factor (Riopel and Timko, 1995; Hood
et al., 1998). The cells of the tip cease dividing, the root
stops elongating, cortical cells near the tip enlarge, and
hairs form near the tip (Riopel and Timko, 1995). This is
followed by the new haustorium coming in contact with
the host plant where it adheres to its surface and through
a complicated developmental process penetrates the host
and connects to its vascular system, particularly the
xylem (Kuijt, 1969). This is a clear example of a determinate root growth where the root stops elongating, but the
RAM becomes transformed into a specially modified structure for absorption and transport of nutrients. An interesting
aspect of this development is that when the
haustoria-inducing factor was experimentally removed
from the parasitic seedling, the RAM re-initiated its
activity, indicating developmental plasticity (Smith et al.,
1990). Secondary haustoria can also form by localized
cell divisions in the cortex of parasitic plant roots (Riopel
and Timko, 1995) or stems, as in the case of Cuscuta
(Fahn, 1982). Since these secondary haustoria do not originate from the root pericycle, it is debatable if they are actually roots.
Interestingly, the genus Pholisma (Lennoaceae) shows
root dimorphism with long pilot roots, and short roots that
tend to grow towards a host root; when in contact with a
host, the RAM of the short root is quickly transformed
into a penetrating haustorial organ (Kuijt, 1966). The role
of plant growth regulators and development of primary
haustoria have been thoroughly studied in Triphysaria versicolor (Orobanchaceae) (Tomilov et al., 2004, 2005). In
various Cuscuta species, the tip of the haustorium penetrates the host plant and connects to the host vascular
tissue (Parker and Riches, 1993; Riopel and Timko, 1995).
Determinate root growth in parasite and hemiparasite
plants has evolved as a developmental programme that
permits the formation of haustoria due to full differentiation
of the RAM cells. As in other cases, this adaptation
increases the species fitness.
N O N - CON S T I T U T I V E D E T E R M I N AT E
RO OT GROW T H
Examples of non-constitutive determinate growth
Non-constitutive determinate root growth refers to those
cases where determinate root growth is induced by some
factor. The investigation of inducible determinate root
growth provides an ideal means to study how RAM maintenance is controlled and how determinacy is regulated
during the normal individual root life cycle.
Non-constitutive determinate growth apparently can be
caused by a physical obstacle. When wheat root axis
growth was impeded by an obstacle, growth stopped, root
hairs covered the very tip of the root, and lateral roots
were initiated closer to the main root tip (Watt et al.,
2006). This example suggests that when physical restriction
of cell division and elongation occurs, the affected RAM
cells can switch their development toward differentiation.
The primary root of the A. thaliana maintains its growth
at least for 4 (Baum et al., 2002) or 5 (Devienne-Barret
et al., 2006) weeks. It can reach 47 cm in the Shahdara
accession (Devienne-Barret et al., 2006) and 25 cm in the
Columbia-0 accession (J. G. Dubrovsky, pers. obs.). As
mentioned earlier, during the indeterminate growth phase
the RAM continuously produces new cells. At later
stages, the RAM becomes less functional, but we do not
know how the growth is terminated under optimal growth
conditions. In A. thaliana, determinate growth of the
primary root can be induced by environmental factors, particularly, when plants are grown in conditions of P
deficiency. In seedlings germinated on medium with only
1 mM of NaH2PO4, the number of cells in the RAM of the
primary root gradually decreases until no meristematic
cells can be found (Sánchez-Calderón et al., 2005). In
these roots, all RAM cells differentiate (Fig. 3) and determinacy becomes irreversible. However, if seedlings are
returned to medium with 1 mM of NaH2PO4 (optimal P
level) at the stage when meristematic cells are still
present, determinate growth can be reversible and the
roots re-establish their growth.
Similar behaviour of the RAM is found in A. thaliana
grown on medium supplemented with 50 mM or 1 mM
L-glutamate; glutamate inhibits mitotic activity of the
RAM, and the length of the growing part of the root
(RAM and elongation zone) decreases (Walch-Liu et al.,
2006). This inhibitory effect is also present when
L-glutamate is applied only to the root tip. Roots of
4-d-old seedlings transferred to medium with L-glutamate
stop growing by day three of the treatment. If seedlings
are transferred back to control medium, after treatment,
about half of them are able to re-establish root growth
within 24 h. However, none of the roots can recover if
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
325
Determinate root growth induced by fungi
F I G . 3. Inducible determinate root growth in Arabidopsis thaliana
(Col-0). Longitudinal sections of the root tips of 14-d-old plants grown
in media with 1 mM (A) and 1 mM (B) of potassium phosphate. The
figure demonstrates that phosphate starvation induces determinate root
growth. Asterisk in (A) indicates approximate position of the proximal
meristem border. (B) The root tip cells enlarged both radially and longitunally and differentiated. h, Root hairs that are formed close to the root tip.
Note that images (A) and (B) are at the same magnification. Scale bar ¼
50 mm. Reproduced from unpublished preparations done by J.G.D. with
kind permission of Dr L. Herrera-Estrella with whom this collaborative
study was done (Sánchez-Calderón et al., 2005).
seedlings are returned to control medium after day four of
treatment with this amino acid (Walch-Liu et al., 2006).
It was shown that in the RAM cells of A. thaliana glutamate triggered substantial and fast changes in cytosolic
Ca2þ, which was accompanied by a rapid transient
membrane depolarization (Dennison and Spalding, 2000;
Sivaguru et al., 2003). The GLUTAMATE RECEPTORLIKE3.3 (GLR3.3) gene of A. thaliana is a homologue of
the mammalian ionotrophic glutamate receptor, and in
two glr3.3 A. thaliana mutants the membrane depolarization response to glutamate was completely absent or very
low (Qi et al., 2006). Nevertheless, a growth or developmental phenotype was not identified in the glr3.3
mutants. On the contrary, mutation of the OsGLR3.1 gene
in rice results in a reduction in root meristem activity, a
decrease in QC size, and disorder of root cap development
(Li et al., 2006). Probably, in growth conditions that
naturally activate the GLR3.3-dependent Ca2þ-signalling
mechanism, a similar phenotype could be observed in
A. thaliana glr3.3 mutants.
These examples demonstrate that inducible determinate
growth can be reversible at the initial steps of the treatment
but becomes irreversible with more prolonged treatment.
An essential factor that defines whether the growth can be
re-established is the status of RAM cell exhaustion. If
some meristematic cells remain during the course of the
treatment, resumption of growth takes place.
It is known that 90 % of the land plant species have
associations with fungi called mycorrhizae. This symbiosis
involves about 6000 species of fungi and about 240 000
species of plants (Bonfante, 2003). The fungus provides
the plant with nutrients from the surrounding soil, and the
plant provides the fungus with sugars and other compounds
(Mauseth, 1988; Phillips and Fahey, 2006). There are two
types of mycorrhizae: endomycorrhizae and ectomycorrihzae. Endomycorrhizae are the most common type, found in
80 % of the vascular plants. They involve intracellular penetration of the fungal hyphae into the cell walls of epidermal and cortical cells, and formation of branched hyphal
structures in root tissues (Hacskaylo, 1957; Lambais,
2006). Ectomycorrhizae are found in Betulaceae, Fagaceae,
Pinaceae, Myrtaceae, and a few other families, only in
trees and shrubs (Mauseth, 1988; Burgess et al., 1994).
The infecting fungal hyphae generally do not invade the
RAM or the vascular cylinder (Hacsakaylo, 1957), but the
roots do cease their growth soon after colonization.
Unfortunately, there are very few studies on the state of
the RAM in roots infected with pathogenic or mycorrhizal
fungi. In mycorrhizae roots of Ornitogalum umbellatum,
some root apices become completely inactive, the meristematic activity gradually decreases, the root tip cells become
vaculated and differentiated, and the root tip senesces
(Berta et al., 1993). In tomato roots, the pathogenic
fungus, Phytophtora nicotianae var. parasitica, induces
cell cycle arrest and subsequent differentiation of the meristematic cells in the root apex of approx. 70 % of the
adventitious and 30 % of the lateral roots. These changes
become irreversible and finally roots stop growing and die
(Fusconi et al., 1999). Interestingly, the arbuscular mycorrhizal fungus, Glomus mossae, has a protective role and
prevents the tomato root tip from necrosis. This fungus
colonizes tomato roots tips only up to the elongation zone,
but also causes an arrest in root growth by inducing differentiation of all meristematic cells in the apex (Fusconi et al.,
1999). Both, pathogenic and non-pathogenic fungi in this
case induce irreversible determinate root growth, as can be
judged from the differentiation or death of all RAM cells.
C E L L U L A R BA S E S O F M E R I S T E M
MAI NT E NA NC E
RAM organization and function has been reviewed in detail
(Clowes, 1975, 1976; Barlow, 1976a, 1994, 2002; Ivanov,
1994, 2004; Rost, 1994; Rost and Bryant, 1996; Rost
et al., 1996; Groot and Rost, 2001; Jiang and Feldman,
2005). We focus here only on how the RAM is organized
relative to cell proliferation and on how its organization
and function is maintained.
Cell proliferation and its maintenance in the RAM
Most of the cell divisions in the RAM take place in a
transverse plane (anticlinal divisions; the new cell wall is
perpendicular to the nearest root surface). Few cells
divide periclinally, forming cell walls parallel to the
nearest root surface, and increasing the number of cell
326
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
files. The cells that initiate root cell files are called ‘initial
cells’. The histogen theory of Hanstein (1870) proposed
that each cell file in each tissue represents a progeny of
an initial cell. Using analysis of sectors marked by transposon excision from the b-glucuronidase (GUS) marker gene,
Scheres et al. (1994) demonstrated the existence of relatively permanent root cell initials, independently confirming Hanstein’s histogen theory.
A division of an initial cell gives rise to two cells; one
maintains its identity as an initial cell, while the other,
called a ‘derivative cell’, gives rise to a cell file. In
A. thaliana, WS accession, and white clover (Trifolium
repens ‘Ladino’), the epidermis and peripheral root cap
develop in modules of cells derived from a single initial
cell. The root cap/epidermis initial divides first periclinally,
and then undergoes a series of anticlinal divisions to form
modules of epidermal and peripheral root cap cells,
always in multiples of eight. This indicates that cell division
within a given module is regulated by a counting mechanism (Wenzel and Rost, 2001; Wenzel et al., 2001). By
knowing the number of cells in a meristematic cell file, it
is possible to estimate the number of cycles that a derivative
cell passes to form a file of cells in the meristem. Using
equation N ¼ 2n, where N is the number of cells in a cell
file excluding the initial cell, and n is the number of cell
cycles the derivative cell passes to form a cell file
(Ivanov, 1974; López-Sáez, 1975), we can find that in
most species n ranges from 6 to 8 (Barlow, 1976a;
Ivanov, 1974). However, in thin roots n can range from 4
to 6. For example, in primary roots of A. thaliana during
their active growth phase, or primary roots of Cactaceae
before their termination of growth, N varies between 15
and 42 cells in a meristematic cell file (Fujie et al.,
1993a; Dubrovsky, 1997a, b; Dubrovsky et al., 2000;
Kidner et al., 2000; Sabatini et al., 2003). In these
examples, the similar value of n in both root types indicates
that the number of cycles within the RAM is not decisive to
define a growth pattern. After a cell derivative from an
initial passes n division cycles, its progeny starts leaving
the meristem by displacement to the elongation zone
(Fig. 4). RAM length can vary during root ontogenesis. For
example, an increase in the RAM length can be a result of
later transition of meristematic cells to elongation, while
the pace of the division of initial cells can be maintained.
Theoretically, this can happen either because cycle time of
all meristematic cells is increased, or because all cells pass
through additional rounds of division within the RAM.
The organization of cell proliferation in the RAM
(Ivanov, 1974, 1994; Barlow, 1976a) implies that the
distal cells of the cell files are initial cells for various
tissues, also called ‘functional initials’ (Barlow, 1997).
Usually, distally to the functional initials there are also
cells with lower proliferation activity, called ‘structural
initials’ (Barlow, 1997). Thus, the initial cells, or functional
initials, are located on the periphery of the group of cells
which are structural initials (Barlow, 1997). Relatively
infrequent cell division of both functional and structural
initials in the root was known since the observations
made by of the Czech botanist Bohumil Němec at the end
of the 19th century (Barlow, 1995). However, it was only
F I G . 4. Model of meristem maintenance after Ivanov (1974, 1994) and
Barlow (1976a). A peripheral cell (white boxes) of the quiescent centre
(an initial cell or a functional initial) undergoes a developmentally asymmetric division and produces a derivative cell that gives rise to a tissue cell
file. In this example, a derivative cell undergoes four proliferative divisions
producing 16 cells within the meristem. After four rounds of division, a
progeny of cells formed from a derivative cell start to be displaced into
the elongation zone, while a new derivative cell is produced at the
bottom of the file as a result of a second developmentally asymmetric division of a quiescent centre cell. Thus, due to a balance of cell division and
displacement, the length of the meristem is maintained within certain
limits. Arabic numbers and colours indicate progeny of three sequentially
generated derivative cells resulted from asymmetric divisions of a peripheral quiescent centre cell. Number of proliferative cell divisions in roots is
usually more than four.
after the experiments of F. A. L. Clowes with radioactively
labelled DNA precursors that this meristem portion became
known as the quiescent centre (QC) (Clowes, 1956). The
term was proposed by Clowes to stress the differences in
cell cycle duration of cells within this distal root zone compared with more proximal root portions. Now, .50 years
since the formulation of the QC concept, it is well established that most angiosperms have a QC.
It was proposed that, because the QC cells divide infrequently, they accumulate fewer chromosome aberrations
or other genetic lesions (Ivanov, 1974). An important function of the QC in plants is its regenerative capacity. Clowes
first demonstrated that after acute X-ray irradiation of maize
roots, the QC behaves differently to the rest of the meristem. After irradiation, proliferative activities of the dividing
meristematic and the QC cells become reversed. The QC
cells, which were originally arrested mainly in G1, remain
less damaged and start active proliferation (Clowes,
1964). As a result, the QC produces a new RAM, replacing
the damaged one. Similar behaviour of the QC is found
under other unfavourable growth conditions. At low temperature, the maize RAM becomes dormant. After transfer
to optimal growth temperature, the QC cells become
active and roots recover from dormancy (Clowes and
Stewart, 1967; Clowes and Wadekar, 1989; Kerk and
Feldman, 1994). Another type of recovery, from carbohydrate starvation, was shown in excised primary roots of
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
maize cultured in medium lacking sucrose (Webster and
Langenauer, 1973). Under these starvation conditions,
neither mitosis nor DNA synthesis takes place. However,
when the root explants that were starved for 48 h were
then transferred to medium supplemented with sucrose,
all meristematic cells, including those of the QC, started
DNA synthesis during the first day after the transfer.
During the second day of growth in the presence of
sucrose, a typical QC is detected (Webster and
Langenauer, 1973). These experiments show that when
sucrose becomes available after a starvation period, all meristematic cells including the QC cells start cycling. They
also show that the actively proliferating cells within the
meristem participate in establishment and maintenance of
the QC. Thus, the described behaviour of RAM cells in
response to X-ray, cold or carbohydrate starvation demonstrate that regeneration of RAM activity starts with activation of the QC. After re-establishment of normal
activity of the RAM, QC cells again become quiescent
and root growth resumes.
It has been proposed that the QC is a sink for auxin that
maintains an oxidized QC-internal environment, which
keeps the QC cells in the G1 cell cycle phase (Kerk and
Feldman, 1995; Jiang et al., 2003; Jiang and Feldman
2005). Transition from G1 to S (DNA synthesis) during activation of cell division in the maize QC was shown to correlate with establishment of a less-oxidized cell status
(Jiang et al., 2003). This implies that, among other
things, the redox status of cells is involved in triggering division of initial cells on the periphery of the QC. In accordance with the idea that the QC is a sink of auxin, it was
demonstrated that an auxin response maximum exists in
the A. thaliana QC and columella initials. This maximum
is instructive for tissue patterning in the root tip (Sabatini
et al., 1999). In terms of progression through the cell
cycle, a large QC in some plants, which can comprise
close to 1000 cells, is composed of an asynchronous and
heterogeneous population of cells (Clowes, 1975; Jiang
and Feldman, 2005). It was shown that after 24-h incubation
with tritiated thymidine, those cells within the maize QC
that were labelled were predominantly located in files continuous with the cells of inner cortex and outer stele regions
(Webster and Langenauer, 1974). When maize roots were
incubated for 120 h, most of the QC cells became labelled
and only a few cells located in the distal QC portion
remained unlabelled (Dubrovsky et al., 1982). This shows
that the QC is a rather dynamic structure where relatively
faster cycling cells are distributed between relatively
slower cycling cells, and where after a division of a structural initial a displacement of a daughter cell which
becomes a functional initial may occur.
These data collectively show that the QC and the rest of
the RAM are in a close interdependent relationship. As
described above, QC formation requires actively dividing
meristem cells in its vicinity, and the meristem above the
QC depends on the activity of the functional initials. This
implies that the model of meristem maintenance (see explanation in the legend to the Fig. 4) is adequate. It remains
unknown what signal(s) are involved in these cell
transitions.
327
Indeterminate growth phase and QC in Arabidopsis thaliana
A common method to identify the location and size of
the QC is to incubate roots with [3H]thymidine or 5-bromo-20 -deoxyuridine (BrdU) for a period of several
hours. The cycling cells around the QC incorporate the
label and the non-cycling cells of the QC do not. Few
studies using these techniques have been done on
A. thaliana because the roots are so small. It has been proposed that the QC in this species is composed of only four
cells (Dolan et al., 1993) (QC sensu Dolan et al., hereafter
abbreviated as QCD). These cells are located between the
provascular initial cells and the columella initials (Dolan
et al., 1993; Baum et al., 2002). Clowes did not work
with A. thaliana roots. However, the concept of the QC proposed by Clowes for other species implies that the QC in
plants comprises both internal cells (‘structural initials’ of
Barlow, see above) and initial cells (‘functional initials’
of Barlow) of the QC (Clowes, 1975, 1976). The initial
cells located at the periphery of the QC (white boxes in
the model on the Fig. 4) have extended cell cycle time
and thus are part of the QC (Ivanov, 1974; Clowes, 1975,
1976; Barlow, 1976a, 1997). Then the QC in A. thaliana
in terms of Clowes (QC sensu Clowes, hereafter abbreviated
as QCC) should be defined as the cells of the intermediate
layer together with neighbouring initial cells (Fig. 5B).
Indeed, about 70 % of A. thaliana initial cells do not incorporate BrdU during a 24 h treatment, while the majority of
the meristematic cells incorporate the label, which suggests
less-frequent cell divisions of the initial cells (Fujie et al.,
1993b). Independent time-lapse analysis demonstrated that
incidence of mitoses in initial cells is significantly lower
compared with other cells of the RAM (Campilho et al.,
2006), confirming the data of Fujie et al. (1993b). That is
why we prefer to consider the QC in this species as the
QCC, i.e. QCD together with adjacent initial cells for all
tissues except columella (Fig. 5B). Though no studies of
cell cycle duration in columella initials has been done in
A. thaliana, it is known that in most species the columella
F I G . 5. Organization of the RAM and the quiescent centre in 8-d-old
Arabidopsis thaliana plant. (A) Yellow line encloses the quiescent centre
sensu Dolan et al. (1993) (QCD) which represents a distal group of cells
in the intermediate cell layer positioned between initial cells. Initial cells
are located externally of outlined cells. e, Initial cells for epidermis and
lateral root cap; c, cortex; n, endodermis; p, pericycle; v, initial cells of
other provascular tissues; cl, columella initials of the root cap. Some
QCD cells passed anticlinal or oblique divisions. (B) Yellow line encloses
the quiescent centre sensu Clowes (QCC). By terminology of Clowes
(1975) the QC represents a group of slowly dividing cells that includes
initial cells for all tissues except for the columella.
328
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
initials are the most rapidly dividing cells within the meristem. Hereby, they usually are not considered to be a part of
the QC (Ivanov, 1974; Clowes, 1975, 1976; Ivanov and
Larina, 1976) and that is why we exclude columella initials
from the QCC (Fig. 5B).
Displacement of the initial cell derivatives into the rest of
the meristem was shown by clonal analysis (Dolan et al.,
1994; Scheres et al., 1994; Kidner et al., 2000). For
example, using heat-shock inducible excision of the Dc
transposable element, it was demonstrated that cell derivatives from the QCC can replace columella and procambium
initials (including pericycle), and possibly initials of other
tissues (Kidner et al., 2000). These authors estimated that
initial cells for lateral-root cap and epidermis are displaced
every 13 d. These data are in a good agreement with anatomical observations in A. thaliana showing dynamic changes in
the RAM organization (Baum et al., 2002). Also, they
confirm extended duration of the cell cycle in initial cells.
Overall, the dynamic nature of RAM, particularly the
replacement of initial cells by derivatives of internal QCC
cells and the fact that initial derivatives are displaced into
the meristem, altogether demonstrate the strength of
the model of meristem maintenance (Fig. 4) and show
that the RAM is dependent on the QCC activity. This also
implies that without the QC the RAM maintenance would
be impossible. To prove it directly in roots with extensive
indeterminate growth phase would be difficult. However,
species having primary roots with short phase of indeterminate growth and with the termination growth phase occurring soon after germination could be useful for validation
of this hypothesis.
RAM organization and RAM maintenance in roots
with constitutive determinate growth
The RAM in cacti with determinate root growth is relatively small, with on average 15 and 24 cells per epidermal
file in S. gummosus and F. peninsulae primary roots, respectively. The RAM cells divide relatively quickly, every
10– 14 h for S. gummosus and 12– 17 h for F. peninsulae
(Dubrovsky et al., 1998). An evaluation of the steady-state
growth period (Dubrovsky, 1997a, b) and the duration of
the cell division cycle in the RAM (Dubrovsky et al.,
1998) shows that during the short steady-state period, on
average, only two cell division cycles occur in the RAM of
these species. Assuming that meristematic activity is maintained until the meristem is exhausted, the maximum
number of cell cycles in the meristem of primary roots is
four in S. gummosus and five in F. peninsulae. The final
length of the primary root varies significantly in these
species (Dubrovsky, 1997a, b); in many seedlings the indeterminate growth phase is practically absent, and root
growth becomes completed within 24 h post-germination
(J. G. Dubrovsky, personal observation).
How is the RAM organized and how do these species terminate primary root growth so rapidly? The primary root in
these species has an intermediate open-type RAM (Fig. 6)
(Rodrı́quez-Rodrı́quez et al., 2003). We demonstrated that
in the mature embryo of S. gummosus all root cell types
are well developed. However, post-germination RAM
F I G . 6. Meristem organization and lack of establishment of the quiescent
centre in the root of the Cactaceae with determinate root growth: (A)
Pachycereus pringlei 3 d after germination; (B) the same species 10 d
after germination; (C) S. gummosus 1 d after germination. The quiescent
centre (QC) is established for only short time (A), the root eventually terminates its growth (B). Also, the same happens when the QC is not established at all (C). In this species, meristem exhaustion occurs within 2 d. See
also Fig. 1. Before fixation the roots were incubated for 24 h in a medium
supplemented with 10 mM 5-bromo-20 -deoxyuridine (BrdU). The sections
were treated with primary anti-BrdU and then with secondary antibodies
labelled with fluorescein isothiocyanate (FITC). They were mounted in
glycerol supplemented with 40 ,6-diamidino-2-phenylindole (DAPI). In all
panels superposition of FITC-labelled nuclei (green) over DAPI-stained
nuclei (blue) is shown. The area outlined in (A) indicates the position of
the QC; most cells are quiescent and did not pass through DNA synthesis
during the incubation period. The area outlined in (C) shows absence of
quiescent cells at the position where the QC could be established. Scale
bar ¼ 50 mm.
Reproduced
with
minor
modifications
from
Rodrı́guez-Rodrı́guez et al. (2003) with kind permission of Springer
Science and Business Media.
initial cells have very limited or no activity
(Rodrı́quez-Rodrı́quez et al., 2003). As mentioned above,
only few division cycles take place within the RAM. Our
analysis with BrdU incorporation into cell nuclei demonstrated that the QC in S. gummosus is not established.
Thus, rapid termination of growth appears to be a direct
consequence of the lack of a QC (Rodrı́quez-Rodrı́quez
et al., 2003). Interestingly, in P. pringlei, a cactus species
with a longer phase of indeterminate growth, the QC is
established but only for 2 – 3 d. At later developmental
stages, QC cells start cell divisions and no quiescent cells
are detected. The timing of QC disappearance correlates
well with the transition to the termination growth phase
and exhaustion of the RAM (Rodrı́quez-Rodrı́quez et al.,
2003). The uniqueness of these species in the Cactaceae
is the fact that this developmental programme takes place
in the primary root, which in most other plant species has
a more extended indeterminate growth phase.
A correlation between the type of growth with absence,
or presence, of the QC was also shown in lateral roots of
Euphorbia esula. In this species, short lateral roots with
limited growth do not develop QC whereas long lateral
roots do (Raju et al., 1964). Laser ablation experiments in
A. thaliana demonstrated that QCD cells maintain the undifferentiated state of the adjacent cells (van den Berg et al.,
1997). Differentiation of neighbouring cells when a QCD
cell is ablated correlates well with differentiation of all meristematic cells in cactus roots with a dominating phase of
growth termination when QC is not established
(Rodrı́quez-Rodrı́quez et al., 2003).
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
Changes in RAM organization during meristem
exhaustion in roots with inducible determinate growth
Little research has been published on what leads to RAM
exhaustion in determinate roots (Chapman et al., 2003).
Under conditions of P deficit, the RAM in A. thaliana
becomes exhausted within 14 d after germination. During
this process, the number of cells in the elongation zone
gradually decreases from 8 to 0 by day 12. The number
of meristematic cells in the epidermis decreases steadily
from 25 to 0 by day 14 (Sánchez-Calderón et al., 2005),
indicating that meristem exhaustion is a relatively rapid
process in these conditions.
It has been shown that when cell division in the RAM is
arrested, approximately half of the cells are leaving the
meristem during a time period equal to the average cell
cycle time in the RAM (Ivanov, 1981, 1994, 1997;
Ivanov and Bystrova, 2006). If we assume the average
cycle time in the A. thaliana RAM to be 16 h [from
Beemster and Baskin (1998) and Dubrovsky et al.
(2000)], and if cell division is arrested at the beginning of
meristem exhaustion induced by P deficiency, then complete meristem exhaustion should theoretically occur in
approx. 85 h (3.5 d). When experimentally determined, this
period is extended to 14 d in A. thaliana (SánchezCalderón et al., 2005). This demonstrates that during
induced determinate growth, meristematic cells continue
their proliferation. Indeed, analysing transgenic plants expressing CycB1;1::GUS, a marker for meristem activity, it was
shown that proliferation in the RAM at P-deficit conditions
is maintained up to day 8 (Sánchez-Calderón et al., 2005).
Interestingly, a QC-specific marker QC46:GUS (Sabatini
et al., 1999) was detected in the RAM of plants under the
same conditions up to day 10 (Sánchez-Calderón et al.,
2005). This indicates that a correlation exists between the presence of cells with QCD identity and the maintenance of cell
proliferation in the RAM. Remarkably, in S. gummosus, a
species with constitutive determinate root growth, a decrease
in meristematic activity within the RAM during meristem
exhaustion is also a gradual process (Dubrovsky, 1997a),
even though in this species the QC is not established
(Rodrı́guez-Rodrı́guez et al., 2003).
There are a number of open questions in our understanding of the cellular bases of non-constitutive determinate
growth. What is the impact of the QC in this type of
growth? What cells are first targets of signals that lead to
determinate growth? What is the interaction between the
cells of the QC and those of the rest of the meristem?
Further studies in these directions are needed to uncover
the mechanisms of induced determinate root growth.
G E NET IC CON TROL O F ME RIS TEM
M A I N T E N A N C E AN D D E T E R M I N AT E
ROOT G ROW T H
Molecular mechanisms of RAM maintenance and determinate root growth in plants involve complex regulatory networks which are not well understood. The study of plant
mutants that show features of root meristem exhaustion
can help us understand the genetic control of RAM maintenance and determinate root growth. Determinate root
329
growth can also be induced as a result of overexpression
of certain genes. Although ectopic expression is not necessarily evidence of the importance of a specific gene for a
particular process, it can help reveal the molecular players
and underlying mechanisms involved. Instances where
gene overexpression leads to RAM exhaustion will be considered here. The majority of mutants in which the RAM
becomes exhausted are reported for the sole species,
A. thaliana. Therefore, in this section we refer to
A. thaliana mutants if not otherwise stated and under the
term QC we mean QCD.
Four transcription factors have been found to be important for QC specification and initial cell activity. Two of
them, PLETHORA1 (PLT1) and PLT2 belong to the
AP2/EREBR family and act redundantly (Aida et al.,
2004). Both plt1 and plt2 single mutants display a slight
but noticeable reduction in root growth rate and in the
number of cells along the meristem (Aida et al., 2004).
plt1 plt2 double mutants show much more severe defects
in the root phenotype, and their root growth rate is
reduced significantly compared with each single mutant.
Moreover, the size of the RAM of the double mutants
rapidly decreases, and eventually all cells in the root tip
differentiate soon after germination; root hairs and differentiated xylem are formed near the very tip (Aida et al.,
2004). Numerous lateral roots of the plt1 plt2 mutants are
also determinate. Using QC-specific markers, the authors
demonstrated that in the double mutants the identity of
the QC cells is changed. This study clearly shows that the
PLT genes are required for RAM maintenance in both
primary and lateral roots (Aida et al., 2004).
Two transcription factors of the GRAS family, SHORT
ROOT (SHR) and SCARECROW (SCR) (Di Laurenzio
et al., 1996; Helariutta et al., 2000), are also essential for
the QC identity. Loss of function of SHR or SCR results
in impaired asymmetric cell division of the endodermis/
cortex initial daughter cell and in the development of
short determinate primary roots (Benfey et al., 1993;
Scheres et al., 1995; Sabatini et al., 2003). Moreover, in
the src-1 mutant, cells within the QC region are aberrant
in shape. Cell-to-cell movement of the SHR protein is
essential for the promotion and maintenance of the SCR
expression. SHR mRNA is found in stele cells, while SCR
mRNA is found in the QC, the cortex/endodermis initial
cell, and the endodermis (Di Laurenzio et al., 1996;
Wysocka-Diller et al., 2000). The SHR protein moves outwards from the stele but only into the adjacent endodermis
cell layer and the QC, where it enters the nucleus and
promotes the SCR expression (Nakajima et al., 2001).
SCR was expressed in the QC region of the scr-1 mutants
using the J2341 GAL4 UAS::GFP driver line from the
enhancer trap collection established by J. Haseloff, which
shows GFP expression in the QC and columella initials in
wild-type plants (http://www.plantsci.cam.ac.uk/Haseloff;
a description of the gene transactivation method using
enhancer trap lines can also be found in Springer, 2000).
As a result of SCR transactivation in this region, the QC
identity was restored, while the SCR-dependent separation
of ground tissue into endodermal and cortical cell layers
was not restored. Root growth of UAS::SCR-expressing
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Shishkova et al. — Determinate Root Growth and Meristem Maintenance
scr-1 plants was maintained, although growth rate and meristem length were reduced compared with the wild-type
plants (Sabatini et al., 2003). Thus, transactivation of SCR
expression in the QC region of the scr-1 mutant rescued
QC and initial cell identity and prevented consumption of
the meristem. As the QC defect in shr is not rescued by sitespecific expression of SCR in the QC, both SCR and SHR are
required for QC function (Sabatini et al., 2003). These
studies emphasize the importance of SHR, SCR and PLT
transcription factors in RAM maintenance and indeterminate
root growth. Their orthologous genes should play an essential role in root growth, at least during the indeterminate
growth phase, in other than A. thaliana species. It would
be interesting to isolate and characterize these genes in
species with constitutive determinate root growth.
Analysis of a variety of root mutants reveals a role for the
plant hormone auxin in root development starting from the
early stages of embryogenesis (reviewed in Jiang and
Feldman, 2005). PIN4, coding for a member of the PIN
FORMED (PIN) auxin efflux protein family, is expressed
in the QC and surrounding cells of developing and established RAMs (Friml et al., 2002). In pin4 mutant
embryos, a well-defined QC was replaced by cells that
divided irregularly. The expression pattern of various cell
type markers indicated that QC and surrounding cells
acquired mixed cell fates (Friml et al., 2002). Therefore,
PIN4, which encodes a putative auxin efflux carrier, is
necessary for the RAM patterning. The PIN family in
A. thaliana consists of eight members, and the defects in
pin mutants can be masked by ectopic activity of the
remaining PIN genes. Thus, although single pin1 and
pin2 mutants only show a slight reduction in root length
and RAM length, and single pin3, pin4 and pin7 mutants
display subtle cell division defects in the QC and columella
root cap, these five genes appear to collectively regulate cell
division and cell expansion in the primary root (Blilou
et al., 2005). Most double-mutant combinations show additive defects in orientation of cell division, root length and
RAM length, with pin1 pin2 double mutants and triple
and quadruple mutants containing pin2 showing the strongest defects (Friml et al., 2003; Blilou et al., 2005). PIN2
mediates auxin transport to RAM cells, which implies
that auxin transport has a critical role in regulation of
RAM length. In accordance with this, treatment with
auxins restores RAM length in pin1 pin2 double and pin2
pin3 pin7 triple mutants to that of the wild-type roots
(Blilou et al., 2005). PINOID (PID), a member of a
family of plant-specific serine-threonine kinases, is
involved in auxin signalling and transport (Christensen
et al., 2000; Benjamins et al., 2001). Although pid
mutants do not display a root phenotype, constitutive PID
overexpression results in a consumption of the primary
root meristem within a few days after germination
(Benjamins et al., 2001). All cells at the root tip become
elongated and root hairs cover the primary root tip in as
young as 4-d-old 35S::PID seedlings (Benjamins et al.,
2001). IAA levels are significantly reduced in primary-root
tips of plants overexpressing PID (Friml et al., 2004), and
treatment with the auxin efflux inhibitor naphtylphtalamic
acid prevents RAM exhaustion (Benjamins et al., 2001).
The constitutive PID overexpression leads to strong
defects in development of embryonic and seedling roots
as a result of a relocation of PIN proteins and loss of
auxin gradients (Friml et al., 2004). Clearly, the disturbance
of auxin transport and reduction of auxin content in the root
tip leads to a disturbance of RAM maintenance. On the other
hand, there is interplay between auin and transcription
factors involved in QC specification and meristem maintenance: PLT genes are induced by auxin, and the joint action of
five PIN genes has an important role in restricting the PLT
expression domain. In turn, PLT genes are required for transcription of the PIN genes in the RAM, to stabilize the auxin
response maximum at the root tip (Blilou et al., 2005).
Auxins have also been linked to changes in redox (Jiang
et al., 2003). As it was discussed above, the maize QC has a
more oxidizing environment than the adjacent meristem
cells: the ratio of the reduced and oxidized forms of ascorbate and glutathione, the two major redox couples, in the
QC is skewed in favour of the oxidized forms. In
A. thaliana, the importance of glutathione for RAM maintenance is supported by the phenotype of the rootmeristemless1 (rml1) mutant defective in glutathione biosynthesis.
The rml1 primary root is very short, with no cell division
taking place post-germination in the RAM, while embryonic development is unaffected (Cheng et al., 1995;
Vernoux et al., 2000). A similar phenotype was observed
in wild-type A. thaliana and tobacco roots treated with an
inhibitor of glutathione biosynthesis, while the rml1 phenotype could be reverted by applying glutathione to rml1
seedlings (Vernoux et al., 2000). Glutathione is shown to
be related to cell proliferation in plant and animal cells:
the oxidized forms of glutathione and ascorbate delay cellcycle progression, whereas ascorbic acid, the reduced form
of ascorbate, or its precursor activates cell divisions in
the QC of maize plants (Jiang and Feldman, 2005).
Therefore, it was proposed that auxin affects the cell
cycle in the QC via changes in redox (Jiang et al., 2003).
The rml1 mutant of A. thaliana is able to develop lateral
roots, which further suggests that glutathione is required
specifically for maintenance of cell divisions within the
RAM. Lack of glutathione does not equally affect the
SAM, probably because of differences in auxin and cytokinin requirement in SAM and RAM.
A mutation in HALTED ROOT (HLR) gene results in loss
of the QC identity after germination, while the other meristematic cells appear to retain their identity. As a result, the
cells of primary root tip of 1-month-old hlr plants differentiate. Moreover, at this age, lateral roots and primordia can
be found very close to the root tip covered with the root
hairs. HLR encodes RPT2a protein, a subunit-4 of the
26S proteasome, and it was suggested that the hlr mutant
is defective in proteasome functions (Ueda et al., 2004).
The hlr root phenotype could be explained at least in part
by the fact that auxin response is triggered by polyubiquitination of Aux/IAA transcriptional repressors by the
SCFTIR1 complex and their subsequent degradation by
26S proteasome. However, it is possible that proteasome
degradation of other proteins is also involved in the RAM
maintenance. To the best of our knowledge there is no
information available on the role of auxin or redox in
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
plants with constitutive determinate root growth. We can
hypothesize that if these factors are involved in meristem
maintenance and growth in at least the indeterminate
growth phase, they should also be involved in the determinate developmental programme.
Cytokinin negatively regulates meristem size in
A. thaliana, and it is also important for RAM maintenance.
In accordance with this, ipt3 ipt5 ipt7 triple cytokinin biosynthetic mutant that has severely reduced cytokinin levels,
has enlarged RAMs and longer roots (Dello Ioio, 2007).
Ectopic expression of cytokinin oxidase AtCKX1 driven
by promoters active in different parts of roots suggests
that cytokinins control the RAM size by acting in the site
of transition from the meristem to elongation zone (Dello
Ioio, 2007).
As we already discussed, maintenance of the RAM is
directly linked to cell proliferation and to a balance
between cell proliferation within the RAM and transition
of cells to elongation and differentiation. Modulation of
the expression of genes coding for the components of
core cell cycle machinery is apparently involved in
control of interactions between cell division and transition
to elongation and differentiation in the root apex. For
example, dominant-negative mutants of tobacco in the
cell cycle-dependent kinase has shorter roots composed of
fewer cells (Hemerly et al., 1995), while roots of
A. thaliana overexpressing a mitotic cyclin show an
increase in cell number and a significant increase in root
length (Doerner et al., 1996). In both cases, as well as in
the case of weak hobbit (hbt) mutants in the CDC27/
Nuc2 component of the anaphase-promoting complex,
only cell number, but not the organization of the RAM is
affected. Nevertheless, primary roots of strong hbt mutants
show neither QC nor differentiated columella cells. Their
RAM is mitotically inactive and the roots lack a differentiated lateral root cap (Willemsen et al., 1998). Moreover,
root hairs and lateral root primordia are formed very close
to the root tip in 7-d-old seedlings. CDC27/Nuc2 protein is
involved in the control of cell cycle progression; in
A. thaliana it is also required for the progression of cell
differentiation in root and shoot. If this protein is still functional, as in weak hbt mutant, QC is established, cell divisions occur and the RAM is still maintained. However,
cell division arrest within the RAM of the hbt loss-offunction mutants leads to cell differentiation in the root tip.
HBT activity may couple cell division and cell differentiation
by regulating cell cycle progression in the RAM, or by
restricting the response to differentiation cues. Furthermore,
HBT gene activity may influence auxin-mediated cell division and differentiation responses, as strong hbt mutants
show a reduction in expression of the DR5::GUS auxin reporter and accumulate the AXR3/IAA17 protein, a repressor of
auxin responses (Blilou et al., 2002).
Impaired maintenance of initial cells can also affect
RAM size. Modulation of expression of the
RETINOBLASTOMA-RELATED (RBR) gene in the
primary root results in changes of the RAM initial cell
number or identity (Wildwater et al., 2005). As rbr
mutation is female gametophytic lethal, RNA interference
was used to down-regulate RBR. Local reduction of RBR
331
expression in A. thaliana roots increases the number of
initial cells without affecting cell cycle duration within
the rest of the meristem. Although RBR is transcribed in
all mitotically active cells, other characteristics of the
root, e.g. root length or meristem size, are not significantly
different from wild type in plants with root-specific
RBR-RNAi. This suggests that initial cells are the more sensible target of RBR activity. To study the effect of RBR
up-regulation, the dexamethasone-inducible RBR overexpression was performed. The identity of the columella
and cortex/endodermis initial cells was lost and compromised, respectively, 24 h after induction of RBR overexpression, while neither the QC identity nor CYCB1;1::GUS
expression in the RAM changed. However, upon prolonged
dexamethasone exposure, root growth was reduced and
RAM length decreased (Wildwater et al., 2005).
Therefore, loss of initial cells identity without apparent
changes in QC identity can lead to defects in RAM maintenance. The RETINOBLASTOMA (RB) gene family in
mammals encodes related proteins that participate in cell
growth and differentiation, including cell cycle regulation
and control of gene expression. The RB regulatory
pathway is a candidate for the regulation of self-renewal
cell properties. Members of this pathway, for example,
cyclin-dependent kinases (CDKs), D cyclins, KIP-related
proteins and E2F transcription factors are found in plants.
When some of these genes of A. thaliana were overexpressed, the phenotypes of transgenic plants were in agreement with cell cycle models for the RB pathway. Moreover,
epistatic relationships between these overexpression constructs and RBR-RNAi suggest that, similarly to their
counterparts in mammals, they are involved in maintenance
of stem cells (Wildwater et al., 2005). On the other hand,
RBR activity affects initial cell maintenance downstream
of SCR, as RBR reduction on scr-4 background results in
restoration of QC function and prevention of meristem consumption. Nevertheless, transition to elongation and differentiation is compromised in these plants and, as a
consequence, root growth is not restored. Therefore, RBR
down-regulation and SCR defect seem to affect collectively
the transition of undifferentiated cells to differentiated state.
Abscisic acid (ABA) signalling apparently is also
involved in the control of RAM maintenance. Allelic
abi8/eld1/kob1 mutants have been identified in screens for
ABA response defects or dwarf plants (Cheng et al.,
2000; Pagant et al., 2002; Lertpiriyapong and Sung, 2003;
Brocard-Gifford et al., 2004). Strong and null alleles abscisic acid insensitive 8 (abi8) (Brocard-Gifford et al., 2004)
and elongation defective 1 (eld1) (Cheng et al., 2000)
mutants possess similar pleiotropic growth defects resulting
in determinate root growth, severely dwarfed phenotype or
death, while two alleles of kobito1 (kob1) display weaker
phenotypes (Pagant et al., 2002). Nevertheless, primary
roots of all mutants are impaired in elongation and cell
differentiation. Cell elongation was also impaired in every
organ, and the dwarf phenotype could not be rescued by
treatment with any of the known hormones or their inhibitors. Although the root tip of recently germinated eld1 seedlings had cells with meristematic characteristics, only few
or no cell divisions were found in the RAM of the eld1
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Shishkova et al. — Determinate Root Growth and Meristem Maintenance
mutant post-germination, while meristematic cells continued transition to the elongation zone, resulting in meristem
consumption. Soon after germination, eld1 root apex cells
had differentiated, as was evidenced by the presence
of root hairs immediately above the root cap, and the
differentiation of vascular cells close to the columella
(Cheng et al., 2000).
The plant-specific ABI8/ELD1/COB1 protein has no
domains of known function (Pagant et al., 2002;
Lertpiriyapong and Sung, 2003; Brocard-Gifford et al.,
2004). Genetic analysis places its action in a network of signalling factors (Brocard-Gifford et al., 2004). Other
ABA-insensitive mutants, e.g. abi4 and abi5, display a
glucose-insensitive phenotype (Arenas-Huertero et al.,
2000), suggesting that ABA has an essential role in
glucose signalling. abi8 mutants were also resistant to
glucose levels that induce developmental arrest of wild-type
seedlings. Moreover, glucose treatment restored root growth
and vascular differentiation (but not cell elongation). This
indicates that the abi8 mutation may alter the ability of
roots to produce or respond to signals promoting growth.
On the other hand, abi8/eld1/kob1 roots have reduced
expression of cdc2a (Cheng et al., 2000), a CDK whose
activity is maintained in all cell proliferation-competent
tissues (Hemerly et al., 1993), but can be inhibited by
ABA-mediated induction of CDK inhibitor, ICK1 (Wang
et al., 1998).
Another indication that ABA is involved in RAM maintenance is the fact that it rescues the RAM defects of the
lateral root organ defective (latd) mutant of Medicago truncatula. When grown in a medium without ABA, the latd
RAM organization in both primary and lateral roots is irregular and long root hairs are formed close to the apex,
which suggest that the meristem is exhausted. When
grown in a medium supplemented with ABA, the roots of
latd plants show normal RAM structure and short root
hairs start to appear approximately at the same distance
from the root tip as in wild-type plants (Liang et al.,
2007). These studies imply that ABA can be involved in
RAM maintenance and that transition from indeterminate
to determinate growth may be ABA dependent.
It is well known that WUSCHEL (WUS) – CLAVATA3
(CLV3) interaction is responsible for SAM maintenance.
CLAVATA3 (CLV3) is a putative ligand that interacts
with a CLV1/CLV2 receptor kinase complex to restrict
the size of the SAM. clv1 and clv3 mutant SAMs are
larger than wild-type SAMs. Conversely, in the SAM of
strong wus mutants, no self-maintaining stem cells are
established, rather the cells at the SAM apex differentiate.
WUS encodes a homeodomain transcription factor. With
WUS-inducing CLV3 expression, the WUS – CLV3 interaction establishes a negative feedback loop with the potential to control the SAM size (for details see, for example,
Bowman and Eshed, 2000; Bäurle and Laux, 2003). In contrast to the SAM, however, there is no clear evidence to
support the involvement of CLV-type proteins in the formation and maintenance of the RAM. However, several
papers reported the loss of the RAM as a result of CLV3/
ESR (CLE) overexpression (Hobe et al., 2003; Fiers
et al., 2005). Members of the CLE protein family in
A. thaliana contain a putative secretion signal and a conserved 14-amino acid motif. No CLE genes specifically
expressed in the root tip were found, although the
expression domain of some of them, such as CLE40 and
CLE19, includes the root (Casamitjana-Martinez et al.,
2003; Hobe et al., 2003). Constitutive overexpression of
several CLE genes (CLV3, CLE19 and CLE40) induces
striking developmental phenotypes in the root and shoot
in A. thaliana, including root meristem consumption and
differentiation of root tip cells (Hobe et al., 2003; Fiers
et al., 2005). A similar root phenotype was observed for
transformants specifically overexpressing CLE19 in the
root meristem (Casamitjana-Martı́nez et al., 2003). In
vitro application of synthetic 14-amino acid peptides,
CLV3p, CLE19p and CLE40p, corresponding to the conserved CLE motif, imitated the overexpression phenotype.
These peptides represent the major active domain of the
corresponding CLE proteins (Fiers et al., 2005). Together
these results suggest that a CLV1-like signal transduction
pathway may also be involved in RAM maintenance.
Despite of the absence of clear evidence of involvement
of CLV-like genes in RAM maintenance, the
WUSCHEL-RELATED HOMEOBOX 5 (WOX5) gene does
express specifically in the QC starting from early embryogenesis. wox5 mutants show defects in the QC and columella initial cells but do not affect initial cells of other
tissues and the RAM. Nevertheless, meristem exhaustion
and termination of the primary root growth in wox5 scr
and wox5 shr double mutants as well as in wox5 plt1 plt2
triple mutant occurs much earlier than in the scr, shr and
plt1 plt2 mutants (Sarkar et al., 2007). Therefore, WOX5
participates redundantly in meristem maintenance. Other
homologues of the WUS, QUIESCENT-CENTERSPECIFIC HOMEOBOX (QHB) gene of rice, also
expresses in the QC but not in the shoot apex (Kamiya
et al., 2003). No phenotype was observed in rice transformants expressing the antisense or RNAi QHB; however,
transgenic rice plants with the most severe phenotype that
constitutively overexpress QHB or WUS did not develop
crown roots (Kamiya et al., 2003). On the other hand,
ectopic WUS expression in A. thaliana roots resulted in a
different phenotype, namely, it caused induction of shoot
stem cell identity and leaf development (Gallois et al.,
2004). These observations further suggest the existence of
differences in the mechanisms underlying meristem maintenance between SAM and RAM, as well as among
plants species. Moreover, the fact that WUS-type genes
are expressed in the QC of dicot and monocot plants
permits the speculation that CLV– WUS-like signalling
could be involved in RAM maintenance in angiosperms
in general.
In summary, the majority of mutations in genes mentioned above result in incorrect specification of either the
QC or initial cells, which leads to early termination of
primary root growth. Apparently, only a small fraction of
genes involved in QC identity and meristem maintenance
can be discovered by mutational analysis, because a high
degree of functional redundancy in the QC was suggested
by Nawy et al. (2005). In this work, the authors have
found several genes encoding transcription factors enriched
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
333
F I G . 7. Genetic control of the RAM maintenance in Arabidopsis thaliana. Cellular compartments are depicted in squares and not drawn to scale, and
some major cellular processes are shown in ovals. Collective activity of PIN auxin efflux carriers is essential for the stabilization of auxin response
maximum in the root tip, which is necessary for QC establishment. Transcription factor genes SCR, SHR, PLT1, PLT2 and WOX5 are involved in the
establishment and conservation of QC identity. The RBR pathway participates in control of initial cell activity. Cell proliferation in the rest of the
RAM is maintained by cell cycle machinery: its defects, for example, the hbt mutations, results in differentiation of the root tip cells. Glutathione is
specifically required for cell divisions in the RAM, as evidenced by the phenotype of the rml1 mutant. A phenotype of hlr mutant evidences an importance
for the RAM maintenance of 26S proteasome-mediated protein degradation, which could be in part explained by its involvement in auxin response.
Possible participation of ABA signal transduction pathway in RAM maintenance is suggested from the phenotype of abi8 mutants. Cell differentiation
is inhibited in proliferating cells. Cytokinins control the RAM size by promoting transition of cells from proliferation to elongation specifically at the distal
part of the elongation zone (transition zone). Loss-of-function mutations in genes essential for QC identity and cell proliferation lead to RAM exhaustion
and determinate root growth.
in QC and have analysed mutants affected in these genes.
However, although promoter regions of these genes did
confer QC-specific expression, the mutants did not have a
root phenotype (Nawy et al., 2005). As we have reviewed
here, many genes are involved in RAM maintenance.
They can be roughly classified in three major groups:
(1) the genes involved in pattern formation, QC and
initial cell maintenance; (2) the genes involved in maintenance of cell cycle and cell differentiation; and (3) the genes
involved in metabolism and signal transduction pathways
of auxins, cytokinins and abscisic acid. A summary of
the data discussed here is presented in the Fig. 7. Our
knowledge of the role of these factors in various taxa is
limited. However, the examples indicate that the general
regulatory mechanisms that control RAM maintenance
may exist in angiosperms.
D E T E R M I N AT E G ROW T H , S T E M C E L L S
A ND S T E M C E L L N I C HE
By definition, the stem cell is a cell that ‘continuously produces unaltered daughters and also has the ability to
produce daughter cells that have different, more restricted
properties’ (Smith, 2006). After discovery of self-renewing
properties in mouse bone marrow cells (Becker et al., 1963;
Siminovitch et al., 1963), the stem cell concept was
developed and used mainly in animal studies. As in thick
roots, like in maize, the large QC represents a population
of self-renewing cells, this concept became popular in
plant studies (Ivanov, 1974; Barlow, 1976b, 1997) even
though there are obviously some general differences in
stem cell properties between plant and animal organisms
(Ivanov, 2003, 2004; Laux, 2003).
In relation to A. thaliana, it was proposed that, in the
root, stem cells are initial cells (Sabatini et al., 2003).
Self-renewing properties of cells are important for RAM
formation and maintenance and, in this section, we first
consider how the stem cell concept is applied to arabidopsis
and then to other angiosperms. Experiments on laser
ablation of only one QCD cell showed that integrity of
QCD is essential to maintain self-renewal properties of
surrounding initial cells (van den Berg et al., 1997). As
discussed above, elegant experiments of the same group
made with the aid of a GAL4-transactivation system
demonstrated that SCR expression is required for the QCD
maintenance and thus for stem cell activity and root
growth maintenance (Sabatini et al., 2003).
In the literature, however, we can find some data that
show that the stem cells in A. thaliana comprise both
QCD and the initial cells. In other words, QCC (QC sensu
Clowes) and columella initials represent a stem cell population. We know that the QCD behaves as a dynamic population over plant ontogenesis. Periclinal (Fig. 3A), anticlinal
and/or oblique (Fig. 5) divisions within the QCD cells of
wild-type roots (see also Ishikawa and Evans, 1997,
fig. 1; Baum et al., 2002, figs 4 –5; Werner et al.,
2003, fig. 8C; Wildwater et al., 2005, fig. 2M; Campilho
et al., 2006, fig. 5B; Sarkar et al., 2007, fig. 1c), as well
as transition from closed type of RAM to an intermediate
open type (Baum et al., 2002; Chapman, et al., 2003)
altogether reveal that both structural and functional initial
cells within the QCC behave as one cell population, and
the daughters of the structural initials can become functional initials. Detailed clonal analysis also demonstrated
that QCD cells divide mainly asymmetrically, producing
one daughter that replaces an initial and another daughter
that is retained in the QCD (Kidner et al., 2000). In terms
of cell proliferation, all QCC cells have increased cycle
times compared with that in the rest of the meristem
(Dolan et al., 1993; Fujie et al., 1993b; Campilho et al.,
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Shishkova et al. — Determinate Root Growth and Meristem Maintenance
2006). There are also some molecular data supporting that
the QCC is a population of cells with common properties.
It was shown that a reduction of RBR copy numbers from
three to one (using the heat-shock promoter-driven Cre
recombinase) affects stem cell maintenance (Wildwater
et al., 2005). Reduced RBR activity in sectors within or
close to the QCD leads to increased cell proliferation
within the entire area corresponding to the QCC
(Wildwater et al., 2005, fig. 4F). In this case, RAM organization becomes similar to a RAM of an intermediate open
type. This supports the functional connection of all cells in
the QCC. Various molecular markers exist that define QCD
cells (Sabatini et al., 1999, 2003). This does not contradict
the notion that QCC is a cell population with common properties. It is simply composed of two domains: one of structural initials (for which molecular markers are known) and
another of functional initials. Hypothetically, molecular
markers should exist that define all QCC cells. PLT1 can
be considered to be one such marker. In situ hybridization
of PLT1 in wild-type plant roots shows positive signals in
both the QCD and in the initial cells, i.e. in the QCC
(Aida et al., 2004, fig. 1E). A similar expression pattern
was shown for pPLT1::CFP (CYAN FLOURESCENT
PROTEIN) line (Xu et al., 2006). These data additionally
indicate that the QCC can be considered as a stem cell population with common properties.
In other angiosperms it is definite that peripheral cells
(initial cells) of the QC give rise to tissue. Even in roots
with a large QC as in maize, the QC cell population is
dynamic, and the daughter cells of structural initials can
become functional initials. This fits well with the definition
of Smith (2006) given for animal cells that stem cells are
those that ‘continuously produce unaltered daughters’.
The experiments mentioned earlier on re-establishment of
the RAM after X-ray radiation, cold treatment or carbohydrate starvation support the idea that dividing QC cells
either become functional initials or maintain their own
population. Therefore, apparently, in other angiosperms,
all QC cells can function as stem cells. The RAM cannot
be maintained without stem cells as, in this case, no
source of new meristematic cells would exist. In the
Cactaceae roots which lack a QC, the RAM cannot
be maintained (Rodrı́guez-Rodrı́guez et al., 2003). This
example illustrates the importance of stem cells for RAM
maintenance and function and for defining whether the
root would be terminating or continuing growth. It also
suggests that in angiosperms all QC cells are cells with
stem cell-like properties.
The cellular microenvironment providing support,
stimuli or conditions necessary to maintain self-renewal
properties of stem cells is defined as stem cell niche
(Spradling et al., 2001; Smith, 2006). The concept of
stem cell niche was applied to plant RAMs relatively
recently by Scheres and co-authors (Sabatini et al., 2003).
They concluded that the QCD represents a stem cell niche
required for stem cell (initial cell) activity. In later works
these authors also considered that the QCD and the adjacent
functional initial cells altogether comprise the stem cell
niche (Wildwater et al., 2005). This definition of stem
cell niche differs from that suggested for animal systems
(Spradling et al., 2001; Smith, 2006), because they consider
the initial cells as stem cells and niche cells at the same
time.
We have another view of the definition of the stem cell
niche in the RAM of plants. One approach to identify a
stem cell niche is to remove the stem cells and analyse
whether the niche persists in the absence of stem cells
(Spradling et al., 2001). In roots with a large QC such as
in maize, microsurgery can be performed in the RAM.
This approach demonstrated that not the QC, but rather
more proximal portions of the RAM fulfil the requirement
for a stem cell niche. Feldman (1976) removed the QC in
maize roots and only 36 h after the surgery a small QC
could be detected with the aid of [3H]thymidine (supplemented during last 8 or 12 h before root fixation) as a
small group of unlabelled cells positioned within the rest
of the RAM with labelled cell nuclei. Moreover, when
[3H]thymidine was applied also to maize roots during
24– 48 h following excision of the root tip ( portion about
120 mm above the root body – root cap junction) and the
roots were left to regenerate their tips for 120 h after excision and then fixed, a mirror image of the regenerated QC
with labelled cell nuclei within the rest of the RAM with
the unlabelled cell nuclei was obtained (Arzee et al.,
1977). Thus, the proliferatively inert cells of the QC originated from the actively proliferating cells of the proximal
meristem close to the excised surface. In most cases, the
maize QC can be re-established when no more than 25 %
of the meristem length is excised. If the excised portion is
greater, the RAM cells decrease proliferation activity,
roots stop growing and all cells at the tip finally differentiate (Feldman, 1976; Ivanov, 1987). In this case root
growth becomes determinate due to experimental loss of
the RAM. The niche does not persist in such cases when
a critical number of the RAM non-stem cells above the
QC (stem cells) are excised. Interestingly, when the distal
200– 300 mm of the RAM is excised repeatedly in the
same root two or three times, with inter-excision intervals
equal to 3 – 5 d, the maize root maintains its growth
similar to that in intact plants (Ivanov and Larina, 1983).
However, when inter-excision intervals were reduced to
2 d, the root growth rate was significantly decreased
(Ivanov and Larina, 1983), presumably because the regenerated QC was too small. These experiments altogether
suggest that a portion of the proximal root meristem
above the QC that permits maintenance of root growth,
and whose experimental removal does not abolish root
growth, can be considered a stem cell niche. The stem
cell niche thus defined creates a specific microenvironment
for the stem cells of the RAM and is critical for stem cell
maintenance and eventually for maintenance of the indeterminate phase of root growth.
To the best of our knowledge, there have been no reports
on experimental removal of QCD together with initial cells
(or, of the entire QCC) in the A. thaliana root. These experiments would be required to experimentally establish if
RAM cells fulfil the function of stem cell niche.
Nevertheless, results with laser ablation of QCD are available, and they support our view that the root stem cell
niche in this species is also outside the QC. Laser ablation
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
of all QCD cells in A. thaliana leads to rapid regeneration of
these cells from provascular (stele) cells (van den Berg
et al., 1997). Unfortunately, in this work it was not reported
what particular provascular cells were involved in QCD
regeneration. However, in other very interesting studies
of this group, pWOX::GFP, pSHR::SHR::GFP and
pSCR::H2B::YFP reporter lines were used to monitor cell
identity during the laser ablation. It was demonstrated,
that when the QCD was ablated, a new QCD regenerated
not from initial cells, but from the second or third cell
located above a provascular initial cell (Xu et al., 2006).
This means, regeneration took place not from remaining
QCC cells, but one or two cells above the region of QCC.
Again, although only QCD cells were ablated, all QCC
cells behaved as one population, and none of these cells
participated in stem cell restoration. Then, according to
the criterion for stem cell niche identification proposed by
Spradling et al. (2001), cells located in the proximal meristem above the A. thaliana QCC behave as a stem cell niche
because these cells, when they persist, can lead to stem cell
regeneration.
From a developmental perspective this analysis of stem
cells and stem cell niche helps in understanding not only
how the meristem is maintained during the indeterminate
growth phase, but also how termination of growth is
achieved. We hypothesize that in roots with an indeterminate growth phase of limited duration, and especially
when this phase is very short (S. gummosus, Cactaceae),
stem cells are not established as a result of some intrinsic
properties of the RAM cells. One such property can be
accelerated transition of cells to differentiation possibly
due to increased cytokinin synthesis or increased sensitivity
to this hormone. As a result the proximal meristem cannot
become a niche for stem cells and the stem cells are not
established. In the roots with a relatively long indeterminate
growth phase, when these roots become determinate,
intrinsic RAM changes can occur as a result of
hormonal changes in ageing plants. Insufficient auxin
and abscisic acid synthesis or their altered signalling can
lead to decreased cell proliferation within the RAM,
thus affecting the stem cell niche properties and,
eventually, the self-renewing properties of stem cells can
be lost. In this way loss of the RAM could be coupled to
plant ageing.
CON CL U S I ON S
The mechanism of root growth in vascular plants is one of
the most important but poorly understood topics in plant
development. It is not well known how individual roots
which have an extended indeterminate phase finally terminate their growth. Numerous works cited in this review
illustrate that in many plant species, under various conditions, determinate growth represents a stable developmental programme. This programme operates as a
developmental adaptation to deficit of water (in desert
Cactaceae), minerals ( proteoid roots in various taxa) or
organic substances (hemiparasitic plants) and ageing. In
other cases it has a specific role in plant anchoring to
increase photosynthetic capacity (roots of adhesive pads
335
in climbing fig). Yet in other cases, the biological
meaning of this developmental programme is still to be
uncovered. For example, it is unclear why some maize
lateral roots loose their meristem during normal root
system development (Varney and McCully, 1991). For
many plant taxa we do not know how common root determinacy is, but it is clear that the determinate programme
evolved in angiosperm roots and that it has important
ecological significance.
Another reason for our interest in determinate root
growth is to uncover the mechanisms underpinning RAM
maintenance in roots with an extended phase of indeterminate growth. In this case, determinate root growth can be
considered a model that becomes useful to investigate the
mechanisms of meristem maintenance. Most advanced
studies in this respect are those using A. thaliana mutants
and primary roots of some species in the Cactaceae. It
has long been proposed, but until recently unproven, that
the QC is comprised of a cell population crucial for the
maintenance of the RAM and root growth. Experiments
on A. thaliana scr mutants (Sabatini et al., 2003) and
Cactaceae with determinate root growth (Rodrı́guezRodrı́guez et al., 2003) demonstrated that no establishment,
or only temporary establishment, as well as improper
identity of the QC post-germination is responsible for the
determinate growth pattern.
The research on both constitutive and non-constitutive
determinate growth demonstrates that a QC is required for
meristem maintenance, that a decrease in the number of
meristematic cells during meristem exhaustion is a
gradual process and that during meristem exhaustion, meristematic cells switch into a differentiation path. These
observations validate the previously proposed models of
meristem maintenance by providing evidence that the QC
functions as a pool of non-differentiated self-renewing
(stem) cells, that the derivatives of the QC are displaced
into the RAM, and also that the meristematic cells
undergo few cell division cycles within the RAM after
which they are displaced into the transition zone of the
root tip.
Cellular, molecular and genetic tools are very important
in current research, and the discovery of two sets of transcription factors from the GRAS family and AP family in
A. thaliana has made a significant advancement in understanding meristem maintenance. It is now clear that activity
of SCR, SHR, PLT1, PLT2 and normal auxin signalling are
required for QC identity and thus for meristem maintenance
and root growth (Sabatini et al., 2003; Aida et al., 2004).
Cytokinins are involved in regulation of RAM length and
control of transition of meristematic cells to elongation
(Dello Ioio et al., 2007). Other elements of possible regulatory networks are mostly unknown. Although we can
hypothesize that the number of cycles through which stem
cell derivatives pass in the meristem is fixed within
certain limits, it is undefined how these limits are established. Regulation of transition of meristematic cells to
elongation may be also dependent on the time that meristematic cells spent in the meristem (Ivanov, 1974; Ivanov and
Bystrova, 2006), but how and by what factors this process is
controlled is yet another enigma.
336
Shishkova et al. — Determinate Root Growth and Meristem Maintenance
ACK N OW L E D G E M E N T S
This work is dedicated to Prof. V. B. Ivanov of the Russian
Academy of Sciences on the occasion of his 70th birthday.
We apologize to those whose work could not be cited due to
space constraints. The authors thank M. Ivanchenko,
A. Soukup and two anonymous reviewers for their valuable
comments on previous versions of this paper, L. Koppel for
help with obtaining of an old Russian publication,
S. Napsucialy-Mendivil and J. R. Ciria for their excellent
technical help, and N. Doktor for her help with art work.
The research in the corresponding author’s laboratory was
partially supported by Programa de Apoyo a Proyectos de
Investigación e Innovación Tecnológica (PAPIIT),
Universidad Nacional Autónoma de México, UNAM,
Projects IN227206 (SS) and IN225906 (JGD) and by
Mexican Council for Science and Technology, CONACyT,
Grants 52476 (SS) and 49267 (JGD).
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