Plant hormones and homeoboxes:

bridging the gap?

Angela Hay, Judith Craft, and Miltos Tsiantis*
Summary
Plant hormones are signalling molecules that control
growth and development. Growth of the aerial parts of
higher plants requires the continuous activity of the
shoot apical meristem, a small mound of cells at the apex
of a plant. KNOTTED1-like HOMEOBOX(KNOX) genes are
involved in regulating meristemactivity, however, little is
known about how this regulation is mediated. Recent
evidence suggests that KNOX transcription factors may
control meristem development by regulating the balance
of activities of multiple hormones. BioEssays 26:395
404, 2004. 2004 Wiley Periodicals, Inc.
Introduction
The term hormone, from the Greek hormau meaning I
stimulate, was coined to describe the chemical messengers
that stimulate numerous processes in animal development.
Many growth and differentiation processes in animals are
controlled by protein hormones. Classical plant hormones, by
contrast, aresmall, diverse, non-proteinmolecules that control
many, if not all, aspects of plant growth and development
(Fig. 1). However, this pleiotropic nature of hormone action
has made it difficult to understand exactly how hormones act
to pattern development. Mechanisms of developmental pat-
terning require specificity, accuracy and the ability to un-
ambiguously specify and maintain developmental identities.
Such examples of developmental patterning include the action
of the homeotic selector genes in metazoan body plan
specification
(1)
and plant homeotic genes in controlling floral
organ identity.
(2)
In contrast to these conceptually elegant and
economical models of developmental patterning, hormonal
actions in plants have traditionally been considered much
more complicated and confusing, because the same molecule
may have very different effects depending on concentration
(3)
or may have diametrically opposite effects on the same pro-
cess in different species.
(4)
Last, but not least, different hor-
monal biosynthesis and sensitivity pathways are intertwined
such that one hormone can regulate the activity of a different
one, thus creating a maze of possible connections where it
is very difficult to distinguish primary from secondary
effects.
(5,6)
In the past decade, the genetic dissection of plant hormone
biosynthesis and signalling pathways has offered new
opportunities for studying the role of hormones (for example
auxin) in controlling developmental patterning.
(7,8)
Part of the
challenge of understanding hormone actions is to understand
whether (and which) developmental genes regulate hormone
activities or viceversa. Thecontrol of hormonebiosynthesis by
the KNOX class of transcription factors has recently emerged
as an example of perhaps unexpected interactions between
hormones and developmental genes.
(9,10)
This review will
discuss recent advances in howthe KNOXpathway functions,
and focus on the interconnections between KNOX genes and
hormone actions and the manner in which they regulate shoot
development.
The KNOX pathway and shoot development
Plant development depends on the continuous activity of
meristems to produce organs throughout a plants life. Embry-
ogenesis inhigher plants produces twoapical meristems at the
root and shoot that give rise to all of the below- and above-
ground organs of an adult plant, respectively. Leaf founder
cellsat the flanks of the shoot apical meristem (SAM) undergo
afundamental developmental transitionfromanindeterminate
to a determinate cell fate as they are recruited into leaves
BioEssays 26:395404, 2004 Wiley Periodicals, Inc. BioEssays 26.4 395
Plant Sciences Dept, Oxford University, Oxford.
Funding agencies: The BBSRC, Royal Society, EU and the Gatsby
Foundation.
*Correspondence to: Miltos Tsiantis, Plant Sciences Dept, Oxford
University, Oxford, OX1 3RB, UK.
E-mail: miltos.tsiantis@plants.ox.ac.uk
DOI 10.1002/bies.20016
Published online in Wiley InterScience (www.interscience.wiley.com).
Abbreviations: KNOX, Knotted1-like homeobox genes; SAM, shoot
apical meristem; KN1, KNOTTED1; STM, SHOOTMERISTEMLESS;
BP, BREVIPEDICELLUS; KNAT, KN1-like in Arabidopsis thaliana;
AS, ASYMMETRIC LEAVES; TALE, three amino acid loop extension;
BLH, BEL1-like homeodomain; BLR, BELLRINGER; PNY, PENNY-
WISE; ipt, isopentenyltransferase; GA, gibberellin; NTH15, Nicotiana
tabacum homeobox15; Ntc12, Nicotiana tabacum c12; AtGA20ox1,
Arabidopsis thaliana GA 20-oxidase1; TKN2, tomato KN1-like2;
LeGA20ox1, Lycopersicum esculentum GA 20-oxidase1; Me, Mouse
ears; PKL, PICKLE; CRC, CRABSCLAW; CHD3, chromodomain
helicase DNA binding protein; IAA, indole-3 acetic acid; PIN1,
PINFORMED1; PAT, polar auxin transport; CUC, CUP-SHAPED
COTYLEDON; CTR1, CONSTITUTIVE TRIPLE RESPONSE1; ETR1,
ETHYLENE RESPONSE1; EIN2, ETHYLENE INSENSITIVE2;
NtPHAN, Nicotiana tabacum PHANTASTICA.
Review articles
(Fig. 2A). For the SAM to maintain its indeterminate function
throughout the life of a plant, it is essential that cells recruited
into lateral organs be constantly replenished. KNOXtranscrip-
tion factors are on the growing list of proteins that are required
to maintain, and possibly establish, the SAM.
KNOTTED1 (KN1) defines the first homeobox gene family
to be isolated in plants, and was identified frommaize mutants
that produced knots, or outgrowths of indeterminate tissue
on the leaf.
(11)
KNOX genes can be divided into two
classes.
(12)
Class I genes share the highest degree of
sequence similarity with KN1 and are expressed in over-
lapping domains within the SAMs of both monocot and dicot
plants.
(1317)
Loss of KN1 function in maize or of the closely
related gene SHOOTMERISTEMLESS (STM) in Arabidopsis
results in failure to maintain a SAM.
(15,18,19)
The sequenced
genome of Arabidopsis contains four class I KNOX genes:
STM, BREVIPEDICELLUS (BP), KN1-like in Arabidopsis
Thaliana2 (KNAT2) and KNAT6. bp mutants have reduced
pedicel (flower stalk) length and loss of apical domi-
nance.
(20,21)
Genetic analyses demonstrate that BP acts
redundantly with STM to maintain the SAM; however, loss of
KNAT2 function has no discernible effect.
(22)
Mutations in
KNAT6 areyet to bedescribed, but its high sequencesimilarity
with KNAT2 raises the possibility that the two genes act
redundantly. It is therefore likely that disruption of both genes
will be required in order to reveal their functions.
Downregulation of KNOX expression in leaf founder cells
within the meristemmarks a change in cell fate frommeristem
to leaf.
(23)
Exclusion of KNOX expression from leaves is
important for leaf development as ectopic expression confers
indeterminate features to Arabidopsis leaves including ecto-
Figure 1. Chemical structures for the plant hormones
discussed. Cytokinins are adenine derivatives (zeatin shown
here), identified by their ability to stimulate cytokinesis.
Gibberellins are terpenoid compounds (GA
1
shown here), first
identified fromsecretions of the fungus Gibberella fujikuroi that
infects rice and promotes stem elongation. Auxins are
chemically similar to tryptophan (Indole-3 Acetic Acid shown
here), originally identified as the substance responsible for the
tropic growth of seedlings towards light. Ethylene is a small,
volatile hydrocarbon, identified as the gas released by fruit that
stimulates ripening. Other plant hormones that are not
mentioned in this review include Abscisic Acid, brassinoster-
oids, systemin and jasmonates.
Figure 2. The KNOX pathway and shoot development. A: In
situ hybridization of SHOOTMERISTEMLESS (STM) in an
Arabidopsis shoot apex. STM expression marks the shoot
apical meristem(SAM) and its absence marks the leaf founder
cells (FC) and leaves. B: Cartoon showing genetic interactions
intheshoot apex(KNOXgenesshowninwhite, hormonegenes
in black and other transcription factors in red). STM is ex-
pressed throughout the SAM (purple) but is absent from leaf
founder cells and leaves (shown in green). The expression of
ASYMMETRICLEAVES1 (AS1) and AS2 is excluded fromthe
SAM and restricted to leaves. STM negatively regulates AS1
and AS2 within the SAM, and downregulation of STMin leaves
allows AS1andAS2expression. AS1andAS2act together as a
heterodimer to negatively regulate the KNOX genes BP,
KNAT2 and KNAT6 thereby restricting their expression to the
SAM. YABBY (YAB) genes are expressed in the abaxial
domain of the leaf (shown in yellow) and repress BP and STM
expression (regulation of STM not depicted). The GA biosyn-
thetic gene AtGA20ox1 is in turn repressed by STM, restricting
its expression to the leaf. Both STM and BP can form
heterodimers with the BLH homeodomain protein BELLRIN-
GER(BLR)/PENNYWISE(PNY) in the SAM(indicated by .-.).
Review articles
396 BioEssays 26.4
pic meristems and a dramatic change in leaf shape.
(14,24)
At
least two mechanisms exist to repress KNOX expression in
Arabidopsis leaves (Fig. 2B). The myb transcription factor
ASYMMETRIC LEAVES1 (AS1) and AS2, a member of the
LATERAL ORGAN BOUNDARIES family of transcriptional
regulators, act together as a heterodimer to repress expres-
sion of BP, KNAT2 and KNAT6 in the leaf.
(2529)
Activity of the
YABBYfamily of putative transcription factors also contributes
to the exclusion of STM and BP expression from leaves, in
addition to the role that they play in specifying dorsoventral
polarity of the leaf.
(30)
Negative interactions also exist between
STM and AS1/2 such that AS1/2 expression is excluded from
the meristem (Fig. 2B and Refs. 22,26).
The transcriptional context in which KNOX proteins act is
another important aspect of their function. KNOX proteins
belong to the TALE superclass of homeodomain proteins,
(31)
and interact with a second group of TALE proteins, the BEL1
homeodomain (BLH) family, in both monocot and dicot
plants.
(3235)
These interactions are known to be selective
between specific protein family members in both maize and
Arabidopsis,
(34,35)
suggesting that different combinations of
KNOX/BLH transcription factors may regulate different down-
stream genes. Genetic interactions between mutations in the
BLH gene BELLRINGER (BLR)
(36)
/PENNYWISE (PNY)
(35)
and stm indicate that the BLR-STM heterodimer may play a
role in maintaining the SAM(Fig. 2Band Ref. 36). BPmaintains
the SAMinas1;stmdouble mutants and BLRis required for this
maintenance, suggesting that BLR also functions as a
heterodimer with BP in the SAM (Fig. 2B and Ref. 36).
The developmental functions of KNOXtranscription factors
are likely to be mediated by targets that include hormone
biosynthetic genes. This idea is based on altered hormone
levels observed in plants overexpressing KNOX genes.
(37)
However, given that KNOX overexpression renders the leaf
tissue more meristematic, it is critical to establish that changes
in hormone levels are not simply an indirect consequence of
these tissue transformations. Further, it is not known whether
hormones mediate KNOX activity in the natural domain of
KNOXexpressionthe SAM. The following sections describe
our current knowledge of the interactions between KNOX
transcription factors and hormone pathways in the processes
of shoot meristem and leaf development.
KNOX and cytokinins
The first tentative link between hormones and KNOX proteins
emerged from observations that overexpression of ipt, which
encodes the cytokinin biosynthetic enzyme isopentenyltrans-
ferase, confers a suite of phenotypes very similar to KNOX
overexpression in tobacco, including ectopic shoot meristem
formation on leaves.
(38,39)
It was later shown that KNOX-
overexpressing plants do indeed contain elevated cytokinin
levels.
(37)
This suggested that one role of KNOX proteins is to
activate cytokinin biosynthesis. This hypothesis was tested
by expressing the maize kn1 gene under the control of a
senescence-inducible promoter, resulting in expression of
KN1 late in leaf development (at senescence) when the leaf is
no longer competent to develop ectopic shoot meristems.
(40)
Cytokinin levels in leaves overexpressing KN1 were in-
creased 15-fold relative to control plants and leaf senescence
was delayed, similar to leaves of cytokinin-overproducing
plants.
(40)
These results suggest that kn1 expression is
sufficient to induce cytokinin biosynthesis in the develop-
mental context of mature leaves. Cytokinin accumulation, as
determined by immunolocalisation, predicts the initiation of
ectopic leaves in lettuce plants overexpressing BP, supporting
the idea that KNOX genes act by inducing local changes in
cytokinin content.
(41)
The mode by which KN1 activates cytokinin levels can now
be investigated as biosynthetic and catabolic genes have
recently been identified as well as a number of cytokinin re-
ceptors and signal transduction components, with similarity
to prokaryotic two-component response pathways.
(4246)
Expression analysis of these genes in altered KNOX back-
grounds and characterization of genetic interactions between
cytokinin signalling components and KNOX mutants, will
reveal the extent to which cytokinin mediates KNOX function.
An interesting twist in the KNOXcytokinin story is that
ectopic ipt expression results in increased accumulation of BP
and STM transcripts.
(47)
One interpretation of these results is
that cytokinin acts not only downstream but also upstream of
KNOX genes such that a feedback-loop is established
between cytokinin and KNOX expression. Alternatively,
because reduced cytokinin results in a smaller SAM,
(46)
it is
possible that ectopic ipt expression results in an enlarged
SAM, thus increasing the proportion of meristematic tran-
scripts detected. This potential feedback between cytokinin
and KNOXwas investigated in tobaccowhereit was found that
elevated cytokinin levels did not induce the KNOX gene
NTH15.
(40)
Recent genome-wide expression analysis in
Arabidopsis also failed to detect an increase in BP or STM
expression following cytokinin application.
(48)
However, these
results do not exclude the possibility that cytokinin may
activate KNOX expression specifically in the context of the
SAM (Fig. 5).
KNOX and gibberellins
Gibberellin (GA) homeostasis was linked to KNOX function in
a series of reports showing that overexpression of rice or
tobaccoKNOXgenes ledtoadecreaseinGAlevels.
(9,37,4951)
Two recent reports present biochemical and genetic evidence
that repression of the biosynthetic gene, GA 20-oxidase, is an
important component of KNOX action both in the SAM and
upon ectopic expression in leaves.
(9,10)
Induction of the
tobacco STMorthologue, NTH15, resulted in rapid repression
of GA biosynthesis and transcript levels of the GA 20-oxidase
gene, Ntc12.
(9)
Recombinant NTH15 protein was also shown
Review articles
BioEssays 26.4 397
to bind specifically to a sequence in the first intron of Ntc12.
Expression patterns of NTH15 and Ntc12 are mutually
exclusive in the shoot apex and this mutual exclusion depends
on the binding of NTH15 to its target cis element in the Ntc12
intron (Fig. 3ADandandRef. 9). Thus, oneof thefunctions of
KNOX proteins is to directly repress the synthesis of bioactive
GAs in the SAM.
Similar to the results in tobacco, induction of KN1 activity in
Arabidopsis resulted in rapid repression of the AtGA20ox1
transcript.
(10)
Constitutive GA signalling by the spindly muta-
tion suppressed the lobed leaves resulting from KNOX over-
expression, giving a more wild-type leaf shape (Fig. 4C and
Ref. 10). Inaddition, constitutiveGAsignallingenhancedweak
stmphenotypes, resulting in shootless plants that fail to forma
meristem (Fig. 3EH and Ref. 10). In stm mutants,
AtGA20ox1was expressedat highlevels inthenormal domain
of STMexpression, reinforcing the idea that STMexcludes GA
biosynthesis from the SAM and that a reduced GA regime is
favourable for meristematic activity.
(10)
Conversely, these observations suggest that high local GA
levels may promote the differentiation of leaves. The
mechanistic basis for this may involve the regulation of
cytoskeletal dynamics. GA is known to promote transverse
cell divisions and longitudinal cell expansion, as occurs in
differentiating leaf cells, by the reorientation of cytoskeletal
components.
(52)
Thus, low GA levels may allow random cell
division patterns in thecorpus of theSAM, whilehighGAlevels
induce an ordered arrangement of cell divisions that favour
determinate growth in the leaf.
(9)
A similar antagonistic relationship exists between the
KNOX gene TKN2 and the LeGA20ox1 gene in the dissected
leaves of tomato that express KNOX genes. Overexpression
of TKN2 in the Mouse ears (Me) and Curl mutants results in
reduced LeGA20ox1 transcript.
(10)
Constitutive GA signalling
by the procera mutation suppresses the level of dissection of
the tomato leaf, and, moreover, suppresses the increased leaf
dissection seen in Me (Fig. 4D and Ref. 10). Thus the KNOX/
GA interaction seems to be a component of a developmental
module controlling indeterminacy. This module likely functions
to maintain the SAM of most species and also to regulate leaf
shape of certain dissected leaf species.
If repression of GA biosynthesis is a critical component of
KNOX function to specify and maintain the SAM, and GA is a
diffusible molecule, then what prevents diffusion of GA from
young leaves into the SAM? One mechanism to control the
domain of GAaction is to place both synthesis and breakdown
under the control of KNOX transcription factors. A 2-oxidase
gene involved in GA breakdown in rice is localized to a ring
surrounding the SAM, consistent with breakdown of bioactive
GAs such that they do not enter the SAM.
(53)
In Arabidopsis,
a 2-oxidase gene is rapidly activated by induction of STM
expression (J.C., J. Martinez & M.T. unpublished observa-
tions). Thus, two potential targets of KNOX transcription
factors in Arabidopsis are genes that control GA homeostasis
via synthesis and breakdown.
However, the role that GA plays in BP function is difficult
to discern from the semidwarf phenotype of bp plants.
(20,21)
Because ectopic BP expression in leaves results in reduced
AtGA20ox1 transcript,
(10)
one may expect that bp mutants
would have a tall, spindly phenotype due to increased
AtGA20ox1 expression. It is somewhat surprising then that
bp mutants are semidwarfed, a phenotype often caused by a
reduction in GA levels. This observation may reflect diver-
gencein thefunction of BPandSTM, suchthat unlikeSTM, BP
does not regulate GA biosynthesis in vivo. In this case,
overexpression of BP results in repression of GA biosynthesis
Figure 3. KNOX directly represses GA 20 oxidase expres-
sion in the SAM, thus allowing normal shoot development.
Increased GA activity enhances the effect of decreased KNOX
function, thus preventing shoot development. AD (adapted
from Sakamoto T et al., Genes and Development, 2001,15:
581590 with the permission of Cold Spring Harbor Laboratory
Press.): Mutually exclusive expression patterns of NTH15 (A)
and Ntc12 (B) mRNA in a tobacco shoot apex. Mutation in the
NTH15-binding sequence expands the wild-type Ntc12 ex-
pression pattern (C: Ntc12-w.t.) to the corpus of the SAM (D:
Ntc12-m2). EH (adapted from Hay A et al., Current Biology
2002,12,15571565 with permission of Elsevier): Constitutive
GAsignalling in an stm-2 background results in failure to initiate
a SAM. Aerial view of Arabidopsis plants of the following
genotypes: E: spy-5, F: stm-2, G: wild type, H: stm-2;spy-5.
The spy mutation
(97)
is used in these experiments to confer
constitutive GA signalling to the plant but may affect additional
processes that have not yet been described.
Review articles
398 BioEssays 26.4
due to similarities between the BP and STM proteins that
allow promiscuous repression of STM targets such as
AtGA20ox1. Another possibility is that BP acts as a repressor
of GA biosynthesis in some tissues, such as the SAM, but as
an activator in others, such as the inflorescence stem,
depending on the transcriptional context in which the BP
protein functions. However, it is also possible that the dwarf
phenotype of bp is unrelated to GA activity. Dwarfismand loss
of apical dominance are typical of reduced auxin signalling;
(54)
therefore, other hormones may be mediating BP function.
KNOX, PICKLE and GA
The pickle (pkl) mutation was identified by ectopic embryo
development on roots, the penetrance of which is increas-
ed by growth on GA biosynthesis inhibitors.
(55)
It was
independently isolated as an enhancer of crabsclaw (crc),
identified by ectopic ovule development on carpels.
(56)
It is
therefore likely that PKL functions as a general repressor of
indeterminacy, a role consistent with it encoding a CHD3
chromatin-remodeling factor predicted to be involved in the
repression of transcription.
(56,57)
The pkl mutation is interest-
ingbecauseit behaves as anegativeregulator of GAsignalling
and also enhances the KNOX misexpression phenotypes of
the as1 and as2 mutations.
(25,55)
pkl mutants show reduced
levels of AtGA20ox1 in leaves yet show no misexpression of
KNOX genes.
(10)
Regulation of AtGA20ox1 may therefore
represent a point of convergence of two distinct pathways that
control determinacya KNOX and a PKL-defined pathway.
Reduction of AtGA20ox1 transcript in pkl is somewhat
counterintuitive, as mutations that block GA signalling tend to
result in elevated levels of AtGA20ox1 expression due to
feedback regulation of the GA biosynthetic pathway.
(58)
However, it is not yet clear how PKL is involved in regulating
GA signalling or how GA-dependent targets relate to other
target genes of PKL,
(59)
so the full significance of this ob-
servation requires further experimentation. For example, it is
possible that PKL regulates aspects of GA biosynthesis in
addition to signalling, accounting for the fact that certain pkl
phenotypes are suppressed in the presence of exogenous
GA.
(55)
It is also possible that pkl disrupts aspects of GA
feedback regulation, or defines a GAsignalling pathway that is
not subject to feedback regulation.
KNOX and auxin
A key feature of auxin action is the polar transport of IAA
through plant tissues. In Arabidopsis, members of the
Figure 4. KNOXrepressesGAactivityintheleaf. A: IncreasedKNOXexpressionintheleaf represses GAactivity, resultinginalobedleaf
shape in Arabidopsis (A.t) and a super-dissected leaf shape in tomato (L.e). B: Increased GA activity suppresses the lobed leaf shape in
Arabidopsis (A.t) and suppresses the super-dissected leaf shape in tomato (L.e). C: Arabidopsis leaves from left to right: WT, spy-1
(increased GA), spy-1;35S:BP (increased GA, increased KNOX) and 35S:BP (increased KNOX). D: Tomato leaves fromleft to right: WT,
pro (increased GA), Me/;pro (increased GA, increased KNOX) and Me/ (increased KNOX). Figure adapted from Hay A et al., Current
Biology 2002;12:15571565 with permission of Elsevier.
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BioEssays 26.4 399
PINFORMED protein family are putative auxin efflux carriers
that account for the directional transport of IAA.
(60)
The AtPIN
gene family was identified from mutant phenotypes indicative
of impaired polar auxin transport (PAT) and encode mem-
brane proteins that are localized in a polar manner.
(8,6166)
Classical physiological experiments have suggested that
auxin gradients emanating from developing leaves influence
thepositionof emergenceof younger leavesat theperipheryof
the SAM, thus specifying phyllotactic pattern.
(67)
Microappli-
cation of IAA to the leafless SAMs of plants treated with PAT
inhibitors or pin1 mutants, resulted in a new primordium
initiating at the site of application, supporting a role for auxin in
leaf initiation.
(68)
Localisation of PIN1 and the putative auxin
influx carrier AUXIN RESISTANT1 indicate that auxin is
transported toward the SAM through the outermost cell
layer.
(69)
Induction of PIN1 expression at the sites of incipient
and young leaf primordia indicates that existing leaves act as
auxin sinks. Therefore, auxin accumulates at a certain
distance away from existing leaves, thus defining the position
of future primordia.
(69)
The site of leaf initiation in the SAM is
therefore defined by both an auxin-dependent process and
downregulation of KNOX expression.
Whether these two processes are connected has been
investigated by culturing maize SAMs on PAT inhibitors.
(70)
These experiments demonstrated that PAT inhibitors induce
both thearrest of leaf initiation andthe failureto exclude KNOX
proteins from leaf founder cells in the SAM. Subsequent
transfer of cultured apices to inhibitor-free media resulted in
the restoration of normal KNOX expression patterns and the
resumption of leaf initiation. Therefore, alterations in auxin
gradients at the leaf meristem boundary result in a failure to
downregulate KNOX expression and a consequent failure to
initiate leaves. This suggests that KNOX genes may respond
to an auxin gradient, such that downregulation of KNOX
expression occurs at the point in the SAM where the auxin
gradient predicts leaf initiation (Fig. 5).
Connections between auxin and the regulation of KNOX
expression have also been made based on similarities
between mutants deficient in PAT and mutations in the
ROUGHSHEATH2 (RS2) myb transcription factor in
maize.
(71)
rs2 mutants accumulate KNOX inappropriately in
leaves and have a dwarf stature and twisted leaves with
abnormal vasculature, similar to PAT-deficient mutants.
(7274)
rs2 mutants have been shown to be deficient in PAT, as have
semaphore1 mutants which, like rs2, misexpress KNOX
genes in the maize leaf.
(71,75)
rs2 mutants can be phenocopied
by wild-type maize seedlings grown on PAT inhibitors, but
KNOX expression was not detected in the leaves of these
seedlings, suggesting that KNOXgenes act upstreamof auxin
transport.
(71)
However, ectopic accumulation of KNOXprotein
was observed in the aberrant leaves that developed from
apices cultured on PAT inhibitors, consistent with auxin-
dependent processes also acting upstream of KNOX gene
regulation.
(70)
Given the role of pin genes in auxin transport, it
will beinterestingtodeterminewhether auxintransport andthe
connection with KNOX regulation involves the function of pin-
like genes in maize.
Genetic interactions reveal that auxin transport is required
together with KNOX gene function to correctly pattern the
Arabidopsis shoot apex. During embryogenesis, initiation of
the SAMand separation of the two cotyledons requires activity
of STM and the putative transcription factors CUP-SHAPED
COTYLEDON1 (CUC1) and CUC2.
(15,76,77)
In a similar
manner to stm and cuc mutants, mutations in pin1 affect
growth and separation of the cotyledons as well as their
position and number in Arabidopsis seedlings.
(78)
Bilateral
symmetry of the embryo is maintained in these different
mutants, however double mutant combinations between pin1
and stm or pin1;cuc1;cuc2 fail to establish bilateral sym-
metry.
(79)
This suggests that CUC1/CUC2 or STM may
be regulated by auxin distribution. Presence of an auxin-
responsive element in CUC2 provides an attractive mechan-
ismfor this regulation.
(79)
Since CUC1 and CUC2 are required
to promote STM expression,
(77)
it is possible that auxin
regulates STM via CUC gene activity.
Figure 5. Regulation of hormone activities by KNOX proteins
in the shoot apex. This cartoon represents a longitudinal viewof
a shoot apex. KNOX expression in the SAM (shown in pink)
promotes cytokinin synthesis (shown as a purple gradient) and
cytokinin may in turn promote KNOX expression in the SAM
(dashed arrow). KNOX represses GA synthesis (and may
promote breakdown of GA) such that GA activity is confined to
leaves (shown in green). Auxin (IAA) gradients predict the site of
leaf inception (shown as a yellow arrowhead) and promote
primordium outgrowth and vascular differentiation (shown as
yellow arrows). KNOX expression may be repressed in regions
of high IAA; this would confine KNOXactivity to the SAM, and/or
KNOX may inhibit IAA transport thus confining it to leaves
(dashed line).
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400 BioEssays 26.4
KNOX and ethylene
Ethylene-insensitive and constitutive response mutants have
been readily identified in Arabidopsis using a growth assay
whereby dark-grown wild-type seedlings exposed to ethylene
display a triple response of growth inhibition. The success
of this approach has resulted in extensive characterisation of
the ethylene-signalling pathway.
(80)
Overexpression of KNOX
genes affects shoot initiation and senescence, two processes
that are promoted by cytokinins and inhibited by ethy-
lene.
(38,40,8183)
It was therefore investigated whether KNOX
genes and ethylene interact antagonistically or whether this
antagonism is mediated by cytokinins.
(84)
Investigations into
the interactions of KNOX genes and ethylene showed that the
KNOX gene KNAT2 acts antagonistically to ethylene when
overexpressed and that ethylene regulates KNAT2 reporter
expression in the SAM.
(84)
This interaction may be relevant to
SAMfunction as increased ethylene levels reduce the number
of cells in the SAM and this defect is rescued by induction of
KNAT2 activity. The constitutive ethylene response mutant
ctr1 is also sufficient to reduce cell numbers in the SAM but
ethylene-insensitive mutants (etr1 and ein2) have a normal
SAM, indicating that ethylene is not necessary to limit cell
numbers.
(84)
To further investigate the role of ethylene sig-
nalling in the SAM, it will be interesting to assess whether ctr1
enhances the meristem defect of weak stm mutations and
whether ethylene acts antagonistically to the other class1
KNOX genes.
KNOX proteins: jacks of all trades?
KNOXproteins appear to regulatemeristemdevelopment in at
least two distinct ways. Firstly, they establish a hormonal
regime favourable for meristemfunction by regulating GA and
cytokinin activities (Fig. 5). Secondly, they allow develop-
mental patterning of the shoot apex via negative regulatory
interactions with the AS1-AS2 transcription factors that
promote leaf fate (Fig. 2B). Thus, KNOX proteins integrate
fields of hormone activities with the activity of transcription
factors that provide the coordinates for shoot development.
Further research will determine the exact points at which auxin
and ethylene interface with KNOX-dependent processes. If
the bulk of KNOX function is mediated via hormone activities,
then creating the appropriate hormonal regime in the pertinent
cell types should recapitulate KNOX overexpression pheno-
types or rescue KNOX mutants.
Cell division and differentiation
a way to talk?
These interactions between developmental regulators such
as KNOX genes and hormone pathways also provide a
means of integrating developmental patterning signals with
cell division and differentiation. Cytokinins activate cell divi-
sion in Arabidopsis through induction of a D-type cyclin,
(85)
whereas GA controls growth via regulation of cell elonga-
tion.
(86)
GA appears to promote cell elongation by changing
the orientation of cortical microtubules from longitudinal to
transverse.
(52)
KNOX transcription factors may regulate both
cytokinin and GA gradients in the shoot apex, thereby
providing input into cell division pathways via control of cyclin
genes, and cellular-differentiation pathways via controlling
cytoskeletal dynamics (Fig. 6). Additionally, recent evidence
suggests that KNOX proteins directly suppress cellular
differentiation by repressing lignin biosynthetic genes, which
can be considered terminal differentiation genes in plant
cells (Fig. 6 and Ref. 87). A class of proteins called expansins
also regulates cell wall structure.
(88)
Expansins are expres-
sedinleaf founder cells andaresufficient toinduceprimordium
outgrowth when applied to SAMs.
(89,90)
Both GA and
auxin
(91,92)
induce expansin gene expression; therefore
KNOX control of hormone homeostasis may indirectly
regulate cell wall loosening (Fig. 6). Thus KNOX proteins
provide multiple inputs into the complex networks controlling
cell divisionanddifferentiation. Thechallengenowis toidentify
further components of the cross-talk machinery between
developmental patterning and cell division/cell differentiation.
Finally, evidence suggests that cell division activity may
feed back to regulate KNOX gene expression (Fig. 6).
Changing cell division frequency and orientation in a group
of cells within the SAM, by induction of phragmoplastin gene
expression, decreased expression of the KNOX gene NTH15
andincreasedthat of NTPHAN, thus mimickingtheexpression
of thesegenes inleaf founder cells.
(93)
Theauthors proposean
interesting mechanism for this feedback whereby a change in
cell division patterns alters the function of plamodesmata-
gated channels in plant cell walls that traffic regulatory proteins
and RNAs.
(94)
KNOX and hormones: a quest for specificity?
Connections between KNOX proteins and hormones have
begun to be mapped out but the precise relationships are still
unclear. Evidence of direct regulation of hormone gene ex-
pression by KNOX transcription factors, as exists for GA
biosynthesis,
(9)
is required to understand these relationships.
Studies on auxin have also illustrated the importance of
understanding the spatial distribution of a hormones activity.
Thus, detailed spatiotemporal expression analysis of all com-
ponents that contribute to the activity of a hormone (synthesis,
breakdown, signalling, conjugation and transport) in response
to altered KNOX expression and vice versa will be pivotal to
understanding how KNOX activity interconnects with that of
hormones. Further, if gradients of hormone activity and inte-
gration of multiple hormonal inputs are critical for eliciting
developmental responses, then what are the integrating
factors? Where short-range signalling is likely to be important,
as for example at the leaf meristem boundary, additional
molecules may translate hormonal gradients into short-range
specific messages. Good candidates for this type of signalling
Review articles
BioEssays 26.4 401
are small peptidessome of which have previously been
implicated in hormone signalling.
(95,96)
Whether other shoot
development genes emerge as regulators of hormone acti-
vities or whether this control is a KNOX gene-specific function
remain to be seen.
Acknowledgments
We thank S. Hake, P. Hedden, A. Phillips and J. Martinez for
collaboration on the KNOX/GA projects. We thank J. Baker
for photography and S. McCormick for comments on the
manuscript.
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