Download Is the shoot a root with a view? Philip N Benfey

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Is the shoot a root with a view?
Philip N Benfey
Recently, it has been shown that the same sets of genes act in
both root and shoot to regulate cell fate and patterning. One
gene cassette regulates epidermal cell fate, another cassette
regulates ground tissue derived cell fate and organization.
Ectopic expression and laser ablation have been used to probe
the mechanisms by which these genes perform their tissue and
organ-specific functions.
Addresses
Biology Department, New York University, 1009 Main Building, New
York, NY 10003, USA; e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:39–43
http://biomednet.com/elecref/1369526600200039
© Elsevier Science Ltd ISSN 1369-5266
Abbreviations
H
hair cells
N
non-hair cells
Introduction
Viewed in gross outline, the root and shoot could be mirror images: both are ramified structures — one exploring
the ground, the other the air. But at closer inspection their
differences seem to far outweigh their similarities. Roots
are among the simplest organs in their morphology, shoots
produce highly complex organs; roots branch from internal
differentiated tissues, shoots set aside axillary meristems
at each leaf; the root meristem is covered by the root cap,
the shoot meristem is exposed on its apical surface. Initial
genetic data have also emphasized the differences
between roots and shoots — mutations that have dramatic effects on the shoot meristem, have no apparent effect
on the root. These are only a few of the differences that
would suggest that shoots and roots are unlikely to share
common mechanisms regulating their patterning, growth
or differentiation.
Several recent findings in Arabidopsis have challenged this
view. Two independent sets of genes have now been shown
to control aspects of shoot and root cell fate and patterning.
One regulates the pattern and differentiation of epidermal
cells, the other the radial pattern of ground tissue. Here, I
will review current models of how these genes interact with
each other, then address attempts to uncover the factors
responsible for tissue-specific fate determination.
expressed preferentially in non-hair cells [1–3]. TRANSPARENT TESTA GLABRA (TTG) acts as an activator of GL2 and
thus also is a negative regulator of H fate [2,4,5•]. Mutation
of either ttg or gl2 results in ectopic root hairs. Cloning of TTG
has not yet been reported. Mutations in CAPRICE (CPC)
have the opposite phenotype to ttg and gl2 — H cell production is dramatically reduced. Genetic and molecular data
indicate that CPC is a negative regulator of GL2, making it
formally a positive regulator of H fate [6••]. CPC encodes a
myb-family protein. The current model then is that CPC and
TTG act independently on GL2: CPC represses, whereas
TTG activates it (Figure 1).
The shoot epidermis is more complex than that of the
root as it contains two specialized cell types, stomata and
trichomes. Both gl2 and ttg were first identified by the
absence of normal trichomes in the leaf [7,8]. Trichomes
are similar to root hairs in that projections are formed
from a single epidermal cell. The root phenotype of
excess root-hairs, however, appears to be opposite to the
shoot phenotype on the same mutant of loss of trichomes.
Nevertheless, the similarities in the pathways are striking. For trichome formation, TTG is a positive regulator of
GL2 [2,5•,9]. Upstream of GL2 there is also a myb- family gene, GLABRA 1 (GL1) [10,11] (Figure 1). Although
evidence for CPC expression in the shoot has not yet
been found, overexpression of CPC results in a strong
reduction in trichome number [6••]. This could be due to
it interacting with a natural target, or its myb-domain may
be sequestering GL2 in the shoot, resulting in reduced
trichome formation.
Examination of the expression pattern of GL2 in the
hypocotyl led to the discovery that the patterning of stomata in the hypocotyl is also dependent on TTG and GL2 [5•]
(Figure 1). It was found that stomata are located in the
same relative position in relation to the underlying cortex
cells as are root hairs and mutations in either gl2 or ttg
resulted in ectopic localization of stomata. Moreover, a
500 bp promoter region was shown to be essential for the
cell-specific expression pattern of GL2 in both root and
hypocotyl [5•]. This region contains a putative myb-binding site and the same region is important for trichome
expression of GL2 [12•]. In summary, both shoot and root
share a pathway for epidermal cell development that
includes the genes TTG and GL2 as well as an upstream
myb-family factor.
Epidermal patterning in root and shoot
The root epidermis is composed of two cell types, hair cells
(H) and non-hair cells (N). Three genes have been identified
by mutation as controlling the fate of individual epidermal
cells. GLABRA 2 (GL2) is a negative regulator of H fate;
where it is active, cells take on an N fate. GL2 encodes a
homeobox-containing putative transcription factor and is
Ground tissue patterning in root and shoot
A second example of a set of genes that appears to perform
the same function in root and shoot comes from the analysis of ground tissue formation. In the root, the ground
tissue is separated into an outer cortex and an inner endodermis [13]. In both short-root (shr) and scarecrow (scr), there
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Growth and development
Figure 1
Ground Tissue
Epidermis
TTG
Leaf/Stem
Trichomes
SHR
+
SCR
GL2
Stomata
SHR
+
SCR
Endodermis
(Starch- sheath)
GL2
Root-hairs
SHR
+
SCR
Endodermis
GL2
MYB
(GL1)
Hypocotyl
TTG
?
TTG
Root
MYB
(CPC)
Endodermis
(Starch-sheath)
Gene cassettes used in root and shoot. In the
epidermis TTG, GL2 and an upstream mybfamily factor (GL1 or CPC) are used to
determine cell fate in leaf, hypocotyl (for which
the activity of a myb factor has not yet been
shown) and root. The specialized epidermal
cell type determined in each organ is different.
In the ground tissue, the genes SHORTROOT (SHR) and SCARECROW (SCR) are
required for the formation of a normal
endodermis (also known as ‘starch-sheath’) in
the stem, hypocotyl and root. It is not known if
they act in a pathway or independently.
Current Opinion in Plant Biology
is a only a single ground tissue-derived layer in the root
[14,15]. Analysis of tissue-specific markers showed that shr
is missing endodermal attributes in the remaining layer,
whereas in scr this layer has attributes of both cortex and
endodermis [14,16]. This suggests that SHR is required for
endodermal fate specification, whereas SCR is involved in
effecting the asymmetric division that the initial cell
undergoes to form the two cell layers (Figure 1). The SCR
gene encodes a protein with motifs suggestive of a transcription factor [16].
The shoot phenotype of both shr and scr mutants is a loss
of the normal gravitropic response in the hypocotyl and
inflorescence [17••]. The apparent cause is the absence of
the normal endodermis, also known as the starch sheath
which has been hypothesized to be the site of gravitropic
sensing in the shoot (Figure 1).
The hypocotyl endodermis is formed during embryogenesis concurrently with the root endodermis through a series
of divisions of the ground tissue. Embryonic analysis has
shown that SCR and SHR function are required for this
division process [15] (Y Helariutta and PN Benfey, unpublished data). Formation of the endodermis from the root
meristem, however, appears to be quite different from the
process that occurs in the shoot meristem. Fate mapping
has shown that the formation of endodermis and cortex in
the root requires two asymmetric divisions of the
cortex/endodermal initial cell. This cell is found at the root
tip where the cortex and endodermal cell files converge.
Similarly to animal stem cells, it first divides to regenerate
an initial and then the upper daughter divides to form the
progenitors of the cortex and endodermal cell files [13].
Fate mapping of the internal shoot tissues including the
endodermis has not yet been performed. Anatomical
analysis indicates that the shoot endodermis originates as a
layer of cells surrounding the vascular strands that traverse
the petiole. When these vascular strands join the stem the
endodermis forms a single layer surrounding all of the vascular bundles. Future analysis of SCR and SHR activity in
the shoot, combined with a better understanding of the
ontogeny of shoot ground tissue should determine how
similar the actual mechanism is by which the ground tissue
is patterned in both shoot and root.
Determining that a set of genes performs similar developmental tasks in more than one context (tissue, organ
etc.) is viewed as evidence for a common evolutionary
origin [18]. It has been hypothesized that present-day
shoots and roots evolved from a common ‘ancestral
meristem’ [19]. Alternatively, tissue-specific patterning
and cell fate specification may have predated the first
Is the shoot a root with a view? Benfey
meristem and thus may have been incorporated independently as the root and shoot meristems evolved.
What is not clear is how many mechanisms are shared.
Further evidence in support of at least some common
mechanisms is the isolation of the Defective embryo and
meristems (Dem) gene which is required for both shoot
and root meristem function in tomato and is expressed in
both meristems [20]. Nevertheless, mutational analysis
has also provided evidence for specialized functions that
are found in one but not the other meristem. To date no
root phenotype has been found to be associated with the
mutation of the shoot meristemless gene [21] which, as its
name suggests, results in loss of a functioning shoot
meristem, nor has one been found with the wuschel [22]
or zwille [23] mutants in which the shoot meristem is
severely compromised. It is possible that there are
homologs that perform similar functions in the root, or
that these genes are specialized for the shoot meristem’s
job of creating lateral appendages (leaves) for which
there is no equivalent in the root.
Organ and tissue-specific cell fate
The idea has been presented that the shoot and root use
the same gene cassettes to perform similar functions.
Although the MYB/TTG/GL2 cassette has been shown to
regulate the placement and fate of specialized epidermal
cells in the root, hypocotyl and leaf, the resulting cell type
is different for each of these organs. Moreover, these
genes negatively regulate the specialized epidermal cells
in the root and hypocotyl, and positively regulate trichome fate in the leaf. What can account for the
differences in activity of the same genes in different
organs? Part of the answer lies in the fact that some of the
genes in the cassette are transcription factors which could
interact with different downstream targets, activating the
genes required for trichome initiation in leaves and
repressing the genes for root hair or stomatal initiation in
root and hypocotyl.
One approach to identifying which of the genes in the
pathway controls organ- and tissue-specific cell fate
determination is to ectopically express each of the genes
and ask if this is sufficient to produce the cell type in an
abnormal location. As mentioned above, CPC ectopic
expression results in severely reduced trichome numbers
and ectopic root hairs [6••]. Because there is no change in
specialized cell type in either shoot or root (i.e. root hairs
aren’t produced on leaves), nor does ectopic expression
cause a change in the tissue or organ-specificity of trichome formation in the shoot, it is unlikely that CPC
plays a role in determining organ- or tissue-specificity. It
has been shown that the maize myb-domain containing R
gene, which is involved in anthocyanin biosynthesis, can
rescue the ttg mutant phenotype indicating that it can
substitute for the Arabidopsis TTG gene. Overexpression
of R results in reduced root hair numbers and trichome
formation on shoot organs on which trichomes are not
normally found [24].
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This suggests that TTG regulates the organ specificity of
trichome formation in the shoot, but that it does not control
the type of specialized cell made in the shoot versus in the
root. Initial results indicated that ectopic expression of GL1
causes a reduction in trichome number [10], which is contrary to what would be expected of an activator of GL2.
Recently, ectopic expression of GL1 has been analyzed in
plants in which the tryptichon gene (a negative regulator of
trichome formation) was mutated. This resulted in additional leaf-borne trichomes as well as trichome formation in
the sub-epidermal layers of the leaf with similar patterning
to that found in a normal leaf epidermis [25••]. This indicates that GL1 regulates the tissue-specific formation of
trichomes. Unexpectedly, these plants also had trichomes
on several stem derived organs (pistils, stamens, flower
stalks) which normally do not have trichomes [25••].
Because previous work pointed to TTG as the prime regulator of shoot organ-specificity, it has been suggested that
overexpression of GL1 might induce TTG in these organs
[25••]. Nevertheless, in both of the GL1 overexpression situations (with or without mutant tryptichon), no alteration in
root hair number or placement was observed [10,25••].
These results present the following picture of the activities
of each of these genes: GL1 is normally expressed only in
the epidermis and is present in many shoot organs at low
levels but not in roots [10,11,12•,25••]; TTG is expressed
only in specific shoot organs (e.g. leaves and hypocotyls)
and is limiting for trichome formation. Overexpression of
GL1 can induce TTG expression in shoot organs in which it
is not normally expressed. Because overexpression of GL1
has no effect on root epidermal cell fate, and overexpression of a TTG substitute or CPC only alters the amount but
not the type of cell (root-hair versus trichome), other, as yet
unidentified, factors must be present to distinguish the
root from the shoot pathways. These results indicate that
the decision to make a specialized epidermal cell in the
root and shoot involves multiple steps. As the molecules
are identified that regulate this pathway, it should become
clear to what extent these decisions are made in a hierarchical manner (for example: epidermis, then shoot versus
root, then which shoot organ) as opposed to overlapping
inputs that are integrated by the downstream targets of
each of these genes.
Signaling between cells
An alternative approach to identifying the factors responsible
for tissue-specific cell fate determination is to ask where signals originate that influence cell fate. In both root and shoot,
fate mapping has shown that position relative to other cells is
the primary determinant of cell fate. For root epidermal cells,
clonal analysis was used to show that the decision to become
a hair cell versus a non-hair cell is determined entirely by the
epidermal cell’s position relative to the underlying cortex
cells [26••]. Hair cells in Arabidopsis normally form over the
cleft between two underlying cortex cells, whereas cells that
contact only a single underlying cell become non-hair cells.
To determine where the positional information originates,
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Growth and development
laser ablation was used to isolate epidermal cells from their
epidermal neighbors or from the underlying cortex cells. The
surprising finding was that there was no change in cell fate
with either type of ablation [26••]. As it was not technically
feasible to completely isolate an epidermal cell from both
cortex and epidermal neighbors, it is possible that redundant
pathways exist. An alternative possibility is that positional
information is not maintained by cell–cell communication at
all, but rather comes from factors found in the cell walls
underneath the cells. Support for this hypothesis comes from
analysis of the first division of the brown alga, Fucus, in
which cell wall factors were shown to determine cell fate
[27]. A recent analysis of later stages of Fucus development,
however, indicates that cell wall factors do not play a part in
fate determination [28], suggesting that this is not a general
mechanism in plants.
Conclusions
The finding that gene cassettes are providing a similar
developmental function in shoot and root should not come
as a surprise to plant evolutionary biologists for whom it
has always appeared likely that the root meristem evolved
from an ancestral shoot. Developmental geneticists, on
the other hand, have generally separated the plant into
root and shoot processes and until recently these have
appeared to be quite different. The notable exceptions
have been that in shoot and root, cell fate is determined
by positional information and that the core function of
both meristems is to maintain an undifferentiated population of cells from which new tissues can be formed. Future
work should include determining how many other conserved gene cassettes act in both shoot and root. A central
focus should be on defining the mechanisms by which
these genes act and are acted upon to determine patterning and cell fate in different developmental contexts.
These are exciting times, as we are rapidly closing in on
the molecular mechanisms that regulate the most fundamental processes of plant development.
Acknowledgements
I would like to thank Ben Scheres and Jocelyn Malamy for very helpful
discussions and comments. Support for work in my laboratory on radial
pattern formation is provided by a grant from the NIH(GM#R01-43772) and
from the Human Frontiers Science Program.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Rerie WG, Feldmann KA, Marks MD: The GLABRA2 gene encodes
a homeo domain protein required for normal trichome
development in Arabidopsis. Genes Dev 1994, 8:1388-1399.
2.
Di Cristina M, Sessa G, Dolan L, Linstead P, Baima S, Ruberti I,
Morelli G: The Arabidopsis Athb-10 (GLABRA2) is an HD-Zip
protein required for regulation of root hair development. Plant J
1996, 10:393-402.
3.
Masucci JD, Rerie WG, Foreman DR, Zhang M, Galway ME, Marks
MD, Schiefelbein JW: The homeobox gene GLABRA2 is required
for position-dependent cell differentiation in the root epidermis of
Arabidopsis thaliana. Development 1996, 122:1253-1260.
4.
Galway ME, Masucci JD, Lloyd AM, Walbot V, Davis RW, Schiefelbein:
The TTG gene is required to specify epidermal cell fate and cell
patterning in the Arabidopsis root. Dev Biol 1994, 166:740-754.
5.
•
Hung CY, Lin Y, Zhang M, Pollock S, Marks MD, Schiefelbein J:
A common position-dependent mechanism controls cell-type
patterning and GLABRA2 regulation in the root and hypocotyl
epidermis of Arabidopsis. Plant Physiol 1998, 117:73-84.
This paper is of interest because it provides the first evidence that stomatal patterning in the hypocotyl is under the same control of the TTG/GL2
cassette as root hair patterning.
6.
••
Wada T, Tachibana T, Shimura Y, Okada K: Epidermal cell
differentiation in Arabidopsis determined by a Myb homolog, CPC.
Science 1997, 277:1113-1116.
This paper presents a major breakthrough in our understanding of root hair
formation through the cloning and characterization of a new myb-family gene,
CPC which is shown to be an upstream regulator of root-hair formation and
GL2 expression.
7.
Koornneef M, Dellaert SWM, van der Veen JH: EMS- and radiationinduced mutation frequencies at individual loci in Arabidopsis
thaliana (L) Heynh. Mutat Res 1982, 93:109-123.
8.
Koornneef M: The complex syndrome of ttg mutants. Arabidopsis
Inf Serv 1981, 18: 45-51.
9.
Hulskamp M, Misra S, Jürgens G: Genetic dissection of trichome
cell development in Arabidopsis. Cell 1994, 76:555-566.
10. Larkin JC, Oppenheimer DG, Lloyd AM, Paparozzi ET, Marks MD:
Roles of the glabrous1 and transparent testa glabra genes in
Arabidopsis development. Plant Cell 1994, 6:1065-1076.
11. Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD: A
myb gene required for leaf trichome differentiation in Arabidopsis
is expressed in stipules. Cell 1991, 67:483-493.
12. Szymanski DB, Jilk RA, Pollock SM, Marks MD: Control of GL2
•
expression in Arabidopsis leaves and trichomes. Development
1998, 125:1161-1171.
Further evidence that shoot and root use the same pathway comes from the
finding that the same promoter region responsible for root and hypocotyl tissue-specific expression is required for trichome-specific expression.
13. Dolan L, Janmaat K, Willemsen V, Linstead P, Poethig S, Roberts K,
Scheres B: Cellular organization of the Arabidopsis thaliana root.
Development 1993, 119:71-84.
14. Benfey PN, Linstead PJ, Roberts K, Schiefelbein JW, Hauser M-T,
Aeschbacher RA: Root development in Arabidopsis: four mutants
with dramatically altered root morphogenesis. Development 1993,
119:57-70.
15. Scheres B, Di Laurenzio L, Willemsen V, Hauser M-T, Janmaat K,
Weisbeek P, Benfey PN: Mutations affecting the radial organisation
of the Arabidopsis root display specific defects throughout the
radial axis. Development 1995, 121:53-62.
16. Di Laurenzio L, Wysocka-Diller J , Malamy JE, Pysh L, Helariutta Y,
Freshour G, Hahn MG, Feldmann KA, Benfey PN: The SCARECROW
gene regulates an asymmetric cell division that is essential for
generating the radial organization of the Arabidopsis root. Cell
1996, 86:423-433.
17.
••
Fukaki H, Wysocka-Diller J, Kato T, Fujisawa H, Benfey PN, Tasaka M:
Genetic evidence that the endodermis is essential for shoot
gravitropism in Arabidopsis thaliana. Plant J 1998, 14:425-430.
Addressing the long-standing question of the site of gravitropic sensing in
the shoot led to the discovery that mutations in the root radial pattern regulators, SCARECROW and SHORT-ROOT also cause the loss of a normal
endodermis in the hypocotyl and shoot inflorescence.
18. Tautz D: Evolutionary biology. Debatable homologies. Nature 1998,
395:17-19.
19. Steeves TA, Sussex IM: Patterns in Plant Development. Cambridge:
Cambridge University Press, 1991.
20. Keddie JS, Carroll BJ, Thomas CM, Reyes ME, Klimyuk V, Holtan H,
Gruissem W, Jones JDG: Transposon tagging of the Defective
embryo and meristems gene of tomato. Plant Cell 1998,
10:877-888.
21. Long JA, Moan EI, Medford JI, Barton MK: A member of the
KNOTTED class of homeodomain proteins encoded by the STM
gene of Arabidopsis. Nature 1996, 379:66-69.
22. Laux T, Mayer KF, Berger J, Jurgens G: The WUSCHEL gene is
required for shoot and floral meristem integrity in Arabidopsis.
Development 1996, 122:87-96.
Is the shoot a root with a view? Benfey
23. Moussian B, Schoof H, Haecker A, Jurgens G, Laux T: Role of the
ZWILLE gene in the regulation of central shoot meristem cell fate
during Arabidopsis embryogenesis. EMBO J 1998, 17:1799-1809.
24. Lloyd AM, Walbot V, Davis RW: Arabidopsis and Nicotiana
anthocyanin production activated by maize regulators R and C1.
Science 1992, 258:1773-1775.
25. Schnittger A, Jurgens G, Hulskamp M: Tissue layer and organ
•• specificity of trichome formation are regulated by GLABRA1 and
TRIPTYCHON in Arabidopsis. Development 1998, 125:2283-2289.
This paper reports the surprising finding that when ectopically expressed in
a tryptichon mutant background, GL1 is able to cause the formation of trichomes on various shoot organs and in the subepidermal tissue of leaves.
Nevertheless, no alteration of root epidermal cell fate is observed.
43
26. Berger F, Haseloff J, Schiefelbein J, Dolan L: Positional information
•• in root epidermis is defined during embryogenesis and acts in
domains with strict boundaries. Curr Biol 1998, 8:421-430.
Counter to expectation, this paper reports that laser ablation of the cells surrounding root epidermal cells results in no detectable change in cell fate,
leading the authors to postulate that factors in the underlying cell walls are
responsible for fate determination.
27.
Berger F, Taylor AR, Brownlee C: Cell fate determination by the
cell wall in early Fucus development. Science 1994,
263:1421-1423.
28. Bouget FY, Berger F, Brownlee C: Position dependent control of
cell fate in the Fucus embryo: role of intercellular communication.
Development 1998, 125:1999-2008.