Plant Molecular Biology 42: 151–166, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
151
Knots in the family tree: evolutionary relationships and functions of knox
homeobox genes
Leonore Reiser1,∗ , Patricia Sánchez-Baracaldo2 and Sarah Hake1
of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA (∗ author for
correspondence; e-mail [email protected]); 2 Department of Integrative Biology and University and
Jepson Herbaria, University of California, Berkeley, CA 94720, USA
1 Department
Key words: evolution, homeodomain protein, knox gene, meristem
Abstract
Knotted-like homeobox (knox) genes constitute a gene family in plants. Class I knox genes are expressed in shoot
apical meristems, and (with notable exceptions) not in lateral organ primordia. Class II genes have more diverse
expression patterns. Loss and gain of function mutations indicate that knox genes are important regulators of
meristem function. Gene duplication has contributed to the evolution of families of homeodomain proteins in
metazoans. We believe that similar mechanisms have contributed to the diversity of knox gene function in plants.
Knox genes may have contributed to the evolution of compound leaves in tomato and could be involved in the
evolution of morphological traits in other species. Alterations in cis-regulatory regions in some knox genes correlate
with novel patterns of gene expression and distinctive morphologies. Preliminary data from the analysis of class I
knox gene expression illustrates the evolution of complex patterns of knox expression is likely to have occurred
through loss and gain of domains of gene expression.
Introduction
The primary architecture of plants derives from the
shoot apical meristem, which produces leaves, internodes and axillary buds. Seemingly simple differences
in organ initiation from the meristem, such as leaf
initiation in a spiral versus decussate phyllotaxy, can
result in dramatically divergent overall morphologies.
The organization and maintenance of the meristem remains a fundamental question in plant development.
As new information emerges regarding the genetic
regulation of meristem organization and function, the
opportunity arises to explore the relationship between
gene expression in meristems during development and
in the evolution of plant form.
The establishment of phylogenetic relationships
provides a framework to analyze the evolution
of genes and gene families and to facilitate a
comparative-developmental approach. The assignment of phylogenetic relationships is necessary for
resolving questions of homology both at the molecular
and morphological level [1, 2]. Characters are considered to be homologous if they are derived from a
common ancestor [31]. Orthology and paralogy are
distinct forms of homology. Paralogous genes arise
from duplications within an organism whereas orthologous genes derive at the time of divergence between
taxa [21]. Assignment of orthology can be complicated when genes have duplicated and diverged within
an organism, making their relationships to similar
genes in other organisms difficult to establish [34]. To
distinguish between paralogous and orthologous genes
it is necessary to determine phylogenetic relationships
among all members of the gene family from the organisms being compared. Within the context of such a
phylogeny, ancestral conditions as well as trends in derived characters, such as patterns of gene expression,
can be inferred. Several recent phylogenetic analyses
of the MADS-box family of transcription factors in
plants are excellent examples of how one can look at
evolution of a gene family and assess the potential for
function in morphological evolution [19, 64, 82].
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Table 1. Some representative class I and class II knox genes from monocots and dicots. Partially sequenced genes such
as ESTs were not included. a, domiant; b, recessive; NR, not reported.
Gene
Plant
Expression
Mutant phenotype(s)
References
Class I-Monocots
kn1
maize
Apical
meristems
[27, 39, 44, 78]
rs1
maize
knox8
maize
knox4
maize
Leaf
base/internode
Roots
Apical
meristems
Leaf
base/internode
Roots
Da : knots on leaves,
blade-sheath boundary
displacement
Rb : reduced tassel
branches, fewer spikelets,
arrested shoot development.
D: displaced blade-sheath
boundary, sheath
mesophyll overgrowth
NR
[23, 24]
knox3
maize
knox5
maize
Leaf
base/internode
NRa
D (Gn1): displaced
blade-sheath boundary,
sheath mesophyll
overgrowth
NR
knox11
maize
NR
lg3
maize
NR
knox 10
OSH1
Oskn2
maize
rice
rice
Oskn3
rice
OSH15
rice
NR
Apical meristems
Apical
meristems
Leaf
base/internode
leaf
base/internode
HvKnox3
barley
Apical
meristems
Class I-Dicots
STM
Arabidopsis
ATK1/KNAT2
Arabidopsis
KNAT1
Arabidopsis
SBHI
soybean
Apical
meristems,
ovary wall
Apical
meristems
Hypocotyl,
between organ
primordia
Embryos,
meristems
[8, 72]
[39]
[39]
D (Lg4)? blade into
sheath transformation
D (Lg4)? blade into
sheath transformation
D (Lg3): blade into
sheath transformation
NR
NR
NR
[25, 44]
[44]
[55]
[63]
NR
[63]
dwarf plants with
shortened internodes,
cell identity defects
D (Hooded): ectopic inflorescences
form on awns
[67]
R: arrested shoot
development, abnormal
floral organ number
NR
[6, 20, 49, 50]
[17, 47]
NR
[16, 48]
NR
[51]
[25, 44]
[25, 44]
[60]
153
Table 1. Continued.
Gene
Plant
Expression
Mutant phenotype(s)
References
NTH15
tobacco
NR
[81]
Knap1
Knap2
Tkn1
apple
apple
tomato
NR
NR
NR
[88]
[88]
[32]
Tkn2/Let6
tomato
apical
meristems,
anthers
stems
stems
meristems,
leaves
meristems,
leaves, ovules
[15, 41, 62]
AmSTM
Antirrhinum
apical
meristem
D: supercompound
leaves, ectopic shoots,
reduced apical
dominance, fasciated
inflorescence
NR
[87]
Class IGymnosperms
HBK1
Picea albies
apical
meristem
NR
[79]
Class II-Monocots
knox1
knox2
maize
maize
NR
NR
[43]
[43]
knox6
maize
NR
[43]
knox7
Class II-Dicots
knat3
knat4
maize
roots
shoots, leaves,
roots, embryos
shoots, leaves,
roots, embryos
NR
NR
[43]
NR
NR
[73, 74]
[74]
knat5
Let12
Arabidopsis
tomato
NR
NR
[74]
[41]
BNHD1
Knap3
Brassica
apple
flowers
leaves, roots,
stems, etc.
highest in roots
meristems,
leaves, floral
organs, ovules
NR
NR
NR
NR
[11]
[88]
Arabidopsis
Arabidopsis
Figure 1. Diagrammatic illustration of knox gene structure. The
MEINOX (light gray box), ELK (hatched box), and homeodomain
(black box) are underscored. The open triangle represents the position of the conserved intron in class II knox genes. The closed
triangle indicates the position of the conserved intron in class I knox
genes.
Gene duplications serve as a mechanism to increase diversity at the molecular level [4, 57, 61].
Subsequent mutation of a duplicated locus might have
no morphological or physiological effect, or may be
the primary contributor to innovative developmental programs. Mutation of regulatory sequences (i.e.
tissue-specific enhancers and repressors) after gene
duplication may also contribute to diversification of
gene function [4, 18]. Changes in gene expression
rather than protein structure are expected to be the
most significant factor in the acquisition of novel func-
154
tions [57]. Because coding regions may be brought
into a new regulatory context via acquisition/deletion
of regulatory elements or as a consequence of mutations in regulatory sequences, genes can be expressed
at a new time or in a new position. In the case of
a transcription factor, expression in novel tissues can
generate new morphologies, either through induction
of a developmental program, or through interactions
with new target genes. In addition to changes in the
pattern of gene expression, mutations within protein
coding regions can result in a novel function.
The contribution of homeodomain transcription
factors to the evolution of form has been well documented in animals [36, 37, 42, 65]. The homeobox
encodes a 61 amino acid motif (the homeodomain)
that acts as a sequence-specific DNA-binding domain.
Homeobox genes are found in plants, fungi and animals and they are required for cell fate determination
as well as pattern definition and boundary specification [45, 56]. Homeodomain proteins also contain
motifs that mediate interactions with other proteins
and these interactions are known to affect their activities as transcriptional activators or repressors [52, 53].
Duplication and diversification of an ancient homeobox gene cluster is associated with alterations of the
body plan of metazoans [5, 38]. Possibly, the modular
nature of homeodomain proteins makes them particularly amenable to rearrangements, with selection for
adaptive characteristics leading to functional diversity
[4].
Knotted-like homeobox (knox) genes belong to a
larger superfamily, the Three Amino acid Loop Extension (TALE) family [13]. In addition to the conserved
ELK and homeodomains, these proteins have a conserved motif termed the MEINOX domain that may
function in protein-protein interactions (Figure 1) [9,
13, 14]. The predicted amphipathic helical region of
the ELK domain, first described for the KNOX family
of proteins, could also act as a protein-protein interaction domain [85]. TALE genes are found in plants,
animals, and fungi, indicating a common ancestral
origin [9, 13].
In plants, knox genes fall into two classes (Table 1).
Each class is distinguished by similarity of residues
within the homeodomain and in the positioning of
introns (Figure 1) [43]. Phylogenetic analysis of the
knox family in plants places class I and class II genes
into different clades (Figure 2) [9, 10]. Since both
clades contain representatives from monocots and dicots, a common ancestor of each class must have been
present before monocot and dicot lineages diverged
[10].
The phylogeny presented in Figure 2 was generated using nucleotide sequences from the homeodomain only. Other phylogenies of knox genes have
been generated using different regions of sequence
from an overlapping data set of knox genes [10]. Many
of the basic features of these phylogenies are the same,
yet there are differences in the level of resolution of
each tree and in their overall topologies. Basic patterns of gene expression of knox genes fall into groups
within the phylogenetic division that separates them
into two classes. Members of the class I group are
expressed in meristems and excluded from developing
organ primordia (with one exception discussed below). In contrast to class I genes, class II genes are
expressed in various locations throughout the plant,
including lateral organs (Table 1). Loss-of-function
mutations implicate class I knox genes in determination of cell fate and patterning in the meristem.
Gain-of-function mutations illustrate their capability
to alter plant morphology profoundly. In this review
we evaluate the knox gene family from both a developmental and evolutionary perspective. We describe
characters that define subgroups within the family of
knox genes and suggest that duplication and diversification has generated gene families whose members
share related but unique functions. We also consider
how changes in knox gene expression are manifested
in various species.
Most of the identified class I knox genes are
expressed throughout the meristem but not in
lateral organs
Meristem-specific gene expression is characteristic of
all class I knox genes. Within the meristem, their
patterns are distinct as illustrated schematically in Figure 3. Expression of some class I genes, including
kn1 from maize, is detected throughout the meristem
and excluded from lateral organs (Figure 3A). kn1 is
expressed in vegetative, inflorescence and floral meristems [39]. kn1 mRNA accumulation is first detected
in the developing maize embryo at about the time of
coleoptile initiation [78]. kn1 gene expression is not
detected in determinate lateral organ primordia. kn1
is not expressed in the scutellum which is considered the grass cotyledon, nor in the coleoptile which
is considered by some to be the ensheathing base of
the scutellum [89]. Although kn1 is continuously ex-
155
Figure 2. Phylogenetic analysis of knox genes. Nucleotide sequence of the homeobox was used to align sequences for analysis which were then
analyzed using PAUP∗ 4.0 [80]. The tree represents a strict consensus of the three most parsimonious trees. We did a heuristic search. Bootstrap
analysis was performed with 100 replicates. Branches without values assigned had less than 50% bootstrap support. Accession numbers/sources
for the sequences are as follows: Ceratopteris (Hasebe and Banks, personal communication), KNAT1 (Arabidopsis thaliana, U14174), kn1 (Zea
mays, X61308), Oshkn2 (Oryza sativa, AF050180), HvKnox3 (Hordeum vulgare, AF022390), rs1 (Zea mays, L44133), OSH1 (Oryza sativa,
D16507), SBH1 (Glycine max, 13663), Tkn2/Let6 (Lycopersicon esculentum, AF000141), STM (A. thaliana, U32344), KNAT2 (A. thaliana,
U14175), POTH1 (Solanum tuberosum, U65648), Let12 (L. esculentum, AF000142), NTH23 (Nicotiana tabacum, AB004797), Knap3 (Malus
domestica, Z71980), KNAT3 (A. thaliana, X92392), KNAT4 (A. thaliana, X92393), KNAT5 (A. thaliana, X92394), Mrg1a (Mus musculus,
U68383), CEH-25 (Caenorhabditis elegans, AJ000533), X-Meis1-3 (Xenopus laevis, U68388). knox3, knox8, knox4, lg3, knox11, knox5,
knox1, knox2, knox6, knox7 Zea mays: sequences were obtained within our lab (Kerstetter, Vollbrecht, Lowe and Hake, unpublished results).
pressed in the shoot apical meristems it is negatively
regulated in some cells within the meristem where the
next leaf primordium is initiated. This region corresponds to the plastochron 0 (P0) leaf which has yet to
undergo the anticlinal divisions that form the mound
characteristic of a P1 leaf primordium [46].
All of our kn1 loss-of-function alleles display a
similar phenotype [44]. In the tassel, which bears the
male florets, fewer branches and spikelets are formed,
resulting in a sparse appearance. Within the female
reproductive structure, the ear, loss-of-function plants
make fewer branches and florets. Loss of kn1 function
also causes extra silks and abnormalities during ovule
development. Other less penetrant phenotypes are associated with recessive mutations of kn1, such as the
intitation of multiple leaves from the same node. This
phenotype has been interpreted as a failure to repress
differentiation of cells within the meristem [44]. A
more dramatic phenotype is detected in some inbred
lines where ca. 50% of the seedlings fail to progress
Figure 3. Schematic representation of the three major types of gene
expression seen for knox genes. The meristem outlined has a distichous phyllotaxy. A. kn1-like expression throughout the meristem
and excluded from P0 and the L1 layer. B. rs1-like gene expression
in the meristem is restricted to positions between the lateral organs.
C. knox gene pattern showing expression throughout the meristem
including the L1 and P0 is similar to knox expression in tomato.
beyond the coleoptile stage (Vollbrecht et al., manuscript in preparation). In the permissive inbred lines,
germinated kernels elaborate a coleoptile and cease
further development (Figure 4, compare panels A and
156
B). Instead of a meristematic dome, these arrested
shoots have a flattened area that occupies the space
where the shoot apical meristem should be (Vollbrecht
et al., manuscript in preparation). Taken together,
the expression pattern of kn1 and the loss-of-function
phenotypes indicate a function for kn1 in meristem
maintenance.
Why do presumably null alleles that lack a functional kn1 gene show such a variable, backgrounddependent phenotype? One possible explanation is
that loss of kn1 function in maize is compensated for
by the activity of a duplicate locus whose activity is
variable in different inbred lines. Maize is thought to
be an ancient allotetraploid and a large percentage of
the genome is duplicated [28, 33]. Although no duplicate factor for kn1 has been identified, the region
of chromosome 1L where kn1 resides is duplicated
on chromosome 5S [33]. An alternative explanation is
that the redundant function is supplied by one or more
related class I knox genes. It is equally probable that
the lack of an absolute requirement for kn1 function in
some backgrounds is due to the activity of a gene or
genes that are unrelated to kn1.
The SHOOTMERISTEMLESS (STM) gene of Arabidopsis shares many similarities with kn1. Like kn1,
STM is expressed specifically in meristems and is
down-regulated in the P0 [50]. STM mRNA first accumulates in a single cell of the globular embryo and
expression gradually expands to fill the space between
the cotyledon primordia [49]. STM expression remains
in the meristem throughout vegetative and floral development, and later includes the ovary wall [50].
Homozygous STM mutant seedlings lack a visible
shoot apical meristem and have cotyledons with fused
petioles [6, 20]. One interpretation of the STM phenotype suggests that in the absence of STM function,
a meristem is never formed [6, 49]. Another possibility is that STM prevents precocious determination of
the meristem after it is initiated. Meristems terminate
prematurely in partial loss of function or weak alleles of STM [20]. These weak alleles are also defective
in flower development, producing fused and extra floral organs. These data also suggest a role for STM in
meristem maintenance.
In many Arabidopsis ecotypes the STM phenotype
can also be suppressed (K. Barton, personal communication) implying that a background-dependent
redundant function can compensate for loss of STM
function. Are similar genes acting to modify the loss
of knox gene function in meristems in both maize and
Arabidopsis or are the mechanisms different between
these two species? Establishing the molecular identity
of these knox gene modifiers will answer this question.
Expression patterns of other knox genes in maize
and Arabidopsis are similar to that of kn1 and STM.
knox8 expression in the maize apex overlaps completely with that of kn1 [39]. KNAT2 (also known as
AKT1) mRNA is detected in the vegetative and floral meristems of Arabidopsis [17, 48]. Based upon
analysis of promoter:: β-glucoronidase (GUS) fusions,
expression of KNAT2/AKT1 in the vegetative meristem is restricted to the L3 layer [47]. Therefore,
KNAT2/AKT1 is expressed in a subset of cells that also
express STM. These genes may share similar functions
with kn1 and STM and thus may be candidates for
redundant factors.
Based upon similarity of protein sequence and
gene expression patterns it has been suggested that
kn1 and STM are orthologues. Phylogenetic analysis
shows that STM is not the Arabidopsis orthologue of
kn1 because it resolves into a separate clade (Figure 2)
[10]. So far orthologues of STM have been only found
in dicots while orthologues of kn1 are found in monocots. Incomplete representation of gene families could
also lead to uncertainty in establishing orthology. Possibly not all of the knox genes have been identified in
Arabidopsis and maize.
The orthology of STM to the dicot genes,
Tkn2/Let6 (tomato) and SBH1 (soybean) is well supported by phylogenetic analysis (Figure 2) [10]. A
different, more limited analysis also places NTH15,
a knox gene from tobacco, in the same group with
Tkn2/Let6, STM and SBH1 [81]. STM, Tkn2/Let6 and
NTH15 are all expressed in a similar pattern in the
meristem with the exception that the tomato gene is
also expressed in leaves (see below).
Orthologues of kn1 have been identified in other
monocots. mRNA accumulation of OSH1 (rice) and
HvKnox3 (barley) is restricted to the meristem and
excluded from organ primordia [51, 55, 60]. No lossof-function mutations have been described for these
genes. Phylogenetic analysis supports the hypothesis
that OSH1 and HvKnox3 are orthologues of kn1 (Figure 2; [10]). Some grasses share extensive regions of
similarity at the molecular level within their individual
genomes and between genomes [58]. This synteny of
grass genomes provides additional evidence to support
the orthology of kn1, HvKnox3 and OSH1 [10].
157
Figure 4. kn1 loss-of-function mutant and normal sibling. A. Normal seedling 2 weeks after germination. These plants had made an average of
3 visible leaves. B. kn1-E1 seedling in W23 inbred background 2 weeks after germination. Only the coleoptile (co) emerged from the kernel.
No other apical organs were produced.
Other Class I knox genes are expressed in specific
domains within the meristem
mRNA of a second group of class I knox genes tends
to be excluded from the central region of the meristem
and instead accumulates in the periphery, between lateral organs (Figure 3B). rough sheath1 (rs1), knox4
and knox3 are all expressed in meristem cells which
appear to correspond to the internode in maize [39,
72]. Later expression in the inflorescence and floral
meristems resembles that of kn1. No recessive lossof-function mutant phenotype has been described for
either rs1, knox4 or knox3. knox4 and rs1 map to duplicated regions of the maize genome and are likely to
be duplicate factors [28, 33, 58]. If the duplication was
recent (i.e. at the time of the duplication of the maize
genome), then knox4 and rs1 may not have had time
to diverge significantly and they may share redundant
functions. In that case, a mutant phenotype may only
be apparent in the rs1/knox4 double-mutant combination. Clonal analysis of both rs1 and knox4 (Gnarley)
dominant gain-of-function mutations indicates that rs1
acts non-autonomously whereas part of knox4 action is
partially cell-autonomous which may indicate that at
least some functional divergence has occurred [8, 23].
Rice is a true diploid and therefore should have
only single copies of each knox gene. At least eight
knox genes have been isolated from rice cDNA libraries [66]. Oskn3 and OSH15, which were cloned
independently [63, 68], are identical to each other in
nucleotide sequence and very similar to rs1 [63]. In
young embryos, Oskn3/OSH15 is expressed throughout the meristem. Later in development, expression is restricted to the boundary between the lateral organs similar to the expression of rs1/knox4 in
maize. Four independent loss-of-function mutations
of OSH15 were isolated which are defective in internode elongation [67]. Within the internodes of mutant
plants, epidermal and hypodermal cellular morphology is defective; the cells are abnormally shaped and
some cell types are absent. These data suggest that
OSH15 has a role in specifying cell fate in the rice
internode. OSH15 maps to a region of the genome
that is syntenous with the maize locations of rs1 and
knox4 [3]. Oskn3/OSH15 is likely an orthologue of
knox4/rs1. If so, it should fall into the knox4/rs1
158
clade in a phylogentic analysis. Within the context
of our own phylogeny this relationship remains unresolved, possibly due to limitations of information
when only the homeodomain is used [10]. Other phylogenies clearly show that rs1/knox4 are in the same
clade, however these analyses did not include OSH15
[10]. If gene function was conserved between maize
and rice, then the rs1/knox4 loss-of-function doublemutant-phenotype might resemble that of null mutants
in OSH15.
In Arabidopsis, KNAT1 mRNA accumulates in
vegetative meristems at the base of the point of leaf
insertion and in the cortex of the shoot apex [48]. This
pattern of expression is similar to that of rs1/knox4
and knox3 in maize. KNAT1 RNA is not found in the
incipient lateral organ primordia, nor the inflorescence
or floral meristem [48]. Based on sequence similarity and expression patterns KNAT1 is most similar to
knox4 and rs1. However, phylogenetic analysis places
KNAT1 in a separate clade suggesting that KNAT1 represents an independent lineage (Figure 2) [10]. If this
is true, then perhaps KNAT1 and rs1/knox4 are examples of convergence in expression pattern. At this time,
we can only say that the pattern of gene expression
in the vegetative apex is similar, not that rs1/KNAT1
function in the same way. Because maize and Arabidopsis meristems are different, KNAT1 expression
may mark a different domain than that defined by
rs1/knox4 expression. Our data suggest that KNAT1
and the Ceratopteris knox gene are orthologues. In this
case, the divergence of class I and class II genes is
even more ancient than we had previously supposed.
Another phylogenetic analysis that does not include
the fern sequences indicates Tkn1 to be an orthologue
of KNAT1 [10].
Class I knox genes in tomato are expressed in
meristems and leaves
In contrast to other class I knox genes, genes from
members of the tomato family are expressed in incipient leaf primordia as well as in meristems (see
Figure 3C). Tkn1 and Tkn2/Let6 mRNAs are not
down-regulated in the P0 and continue to be expressed
in immature leaves [15]. The orthology of Tkn2/Let6
and STM is well supported while Tkn1 orthology with
KNAT1 has been indicated but is less well supported
[10]. Tkn2/Let6 and Tkn1, however, have a pattern of
gene expression unlike either STM or KNAT1.
Of all the knox gene family members, the ones in
tomato are most unique in their patterns of gene expression. What are the consequences of this pattern of
knox gene expression in tomatoes? The class I genes
Tkn1 and Tkn2/Let6 are not down-regulated in the leaf
primordia. Interestingly, the related gene NTH15 from
tobacco is negatively regulated in the P0 [81]. This
suggests that the difference in expression pattern occurred after the divergence of tobacco from the line
leading to tomato. It has been suggested that knox gene
expression in leaves may have contributed to the evolution of compound leaves in tomato [40, 75]. This
possibility can be tested by superimposing leaf shape
and the patterns of gene expression on a phylogeny
of Tkn1/Tkn2 orthologues in selected members of the
Solanaceae that have different leaf morphologies.
How has the expression of knox genes evolved?
Derived and ancestral character states can be reconstructed when these characters are superimposed upon
the phylogeny [12]. Using the related TALE homeodomain proteins BELL1 (BEL1) and ARABIDOPSIS
THALIANA HOMEOBOX 1 (ATH1) as an outgroup,
root-specific expression was mapped onto a knox
homeodomain phylogeny [10]. These results indicate
that root-specific expression was lost twice in knox
gene evolution; once in the lineage leading to class I
knox genes and once in ATH1. The analysis also shows
a gain of root specific expression in the lineage leading
to knox4/rs1.
We have mapped meristem expression patterns
onto a knox gene tree using kn1/STM-like or
KNAT1/rs1-like patterns of gene expression as character states (Figure 5). The tree represents a portion
of a larger phylogenetic analysis that included all sequenced knox genes from Arabidopsis and maize using
CEH-25, Xmeis1–3 and Mrg1a genes as the outgroup
(Sánchez-Baracaldo, unpublished results). As shown
in Figure 5, KNAT1, 2 and STM are paralogous genes
within Arabidopsis as are kn1, rs1/knox4, knox3 and
knox8 in maize. That is, each group arose from duplications that occurred after speciation. Consequently,
each group is orthologous to the other. Based upon our
phylogeny, several models could explain the evolution
of gene expression patterns in maize and Arabidopsis.
As stated above, we ascribe the similarity of expression pattern between KNAT1 and knox4/rs1 to parallel
evolution. Within the maize group, reconstruction of
character states for knox3, knox4/rs1 and knox8 is
equivocal. The knox3, knox4/rs1 pattern could have
arisen early and have been lost in the lineage leading
159
Figure 5. Character state analysis of meristem-specific patterns of expression. Only class I representatives from maize and Arabidopsis are
shown. This figure represents a branch of a larger tree consisting of only maize and Arabidopsis genes. The tree was generated from nucleotide
sequence of the homeodomain using PAUP∗ 4.0 [80], with the same outgroups as in Figure 1. This was the single and most parsimonious
tree. Character analysis was performed using MacClade (Version 3) and character states are unordered. We reconstructed the node states using
information from extant genes as there is no comparable information available for closely related genes. The most related genes are from
animals (see Bharathan et al. [10]), and other plant homeobox genes are more distantly related. We did not feel confident that including the
expression patterns of these genes in an outgroup analysis was appropriate.
to knox8. It is equally possible that knox3, knox4/rs1
patterns of expression arose independently.
Neither of the analyses described above is proof
of the ancestral state for knox gene expression patterns. What they do provide are hypotheses that can
be tested as new sequence and expression pattern data
emerge. One hypothesis based upon our character
reconstruction is that kn1-type meristem-specific expression represents the ancestral state. We need to fill
in the gaps for expression pattern and sequence in the
model systems Arabidopsis and maize as well as more
distantly related taxa.
Recent work has begun to address the gaps in our
understanding of knox genes in non-flowering plants.
A knox gene from spruce (HBK1) was recently cloned
and is similar to class I knox genes suggesting that the
divergence of class I and class II genes occurred before
the split between gymnosperms and angiosperms [79].
In situ localization of HBK1 mRNA in the meristem
showed expression in the internal cell layers and not in
the needles. Three knox genes that are similar to kn1
have been cloned from the fern Ceratopteris (Hasebe
and Banks, personal communication). Based on RNA
gel blots, two of these knox genes are highly expressed
in the shoot apical region of the sporophyte and moderately expressed in immature leaves (fiddleheads). A
truncated form of one Ceratopteris knox gene is also
expressed in the gametophyte (Hasebe and Banks, per-
160
sonal communication). We have used an antibody to
KN1 to immunolocalize KNOX proteins in representatives of non-flowering plant groups. Our preliminary
results show that KNOX proteins are found throughout
the apical regions of both Gnetum and Lycopodium
and do not appear to be restricted to specific cell
types (Sánchez-Baracaldo, unpublished results). We
are using PCR to clone knox genes from Gnetum and
Lycopodium in order to determine their phylogenetic
relationships and patterns of gene expression with
gene-specific probes.
Class II knox genes in maize and Arabidopsis have
a more general pattern of expression
The pattern of mRNA accumulation for a number of
class II knox genes has been determined. In general,
class II genes have a more diverse pattern of gene
expression than class I genes (see Table 1). Gene
expression of knox1, -6 and -7 was assayed with
northern blots [43]. knox6 and -7 are expressed in
leaves, stems, inflorescences, meristems and roots
while knox1 is expressed only in roots. In Arabidopsis,
three class II genes, KNAT3, 4 and 5, have been identified to date, and are expressed in a wide variety of
tissues [74]. mRNA accumulation was determined by
northern analysis. KNAT3 and KNAT4 are expressed
in most tissues while KNAT5 is primarily found in
roots. Analysis of a KNAT3 promoter-GUS fusion indicates that KNAT3 is expressed in lateral organs, but
not in meristems [73]. The sequence of tomato Let12 is
most similar to KNAT3 and KNAT4 [41]. Like KNAT4,
Let12 mRNA accumulation appears to be ubiquitous at
the organ level. In situ localization of mRNA in floral
organs indicates Let12 is expressed in all four floral
whorls and the ovule [41]. No mutant phenotypes have
been associated with loss of function of any class II
knox genes.
The variation in gene expression patterns for class
II genes suggests diverse roles for members of this
family. Perhaps our inability to identify a mutation
in any of these genes is due to redundant functions
shared by members of this class that mask a loss of
function phenotype. Alternatively, loss of function of
some class II genes may have either a subtle effect
or such a strong effect on development that they are
lethal.
Effects of gain of function of knox genes on plant
morphology
Dominant mutations of knox genes in maize leaves
are characterized by ectopic expression of the mutant gene, resulting in perturbation of the blade-sheath
boundary. Maize leaves are divided into the distal
blade and proximal sheath region. The ligule (an epidermal outgrowth) and auricle are positioned at the
boundary between the blade and sheath. Kn1, Rs1,
Liguleless3 (Lg3) and Gnarley1 (Gn1) mutations share
a characteristic displacement of sheath and ligule [8,
23]. Other features distinguish each class of mutation. Kn1 mutations are characterized by vein clearing
and knot formation over lateral veins which is interpreted as another shift of sheath character onto the
blade (see Figure 6A) [27]. All dominant Kn1 mutations are associated with ectopic expression of kn1
mRNA and protein along the vasculature in young
leaves [30, 77]. In addition to alterations of the bladesheath boundary, Rs1 mutants have reduced stature
and excessive proliferation of auricle mesophyll cells
[8]. In contrast to their normal siblings, rs1 mRNA is
detectable in leaf primordia as early as P2 (the second visible leaf primordium) in Rs1-O mutant plants
[72]. Transposon-induced revertants of Rs1-O were
obtained and mapped, but the genetic nature of the
dominant phenotype is not known. Gn1 mutants, like
Rs1, display shortening of the internodes, proliferation of sheath mesophyll cells as well as displacement
of the blade-sheath boundary (Figure 6B). Gn1 maps
to the same position as knox4, the putative duplicate
factor for rs1 [22]. Gn1 ectopically expresses knox4
mRNA and protein in P2–P8/9 leaves suggesting that
a mutation in knox4 is responsible for the dominant
Gn1 phenotype [24]. A polymorphism 50 to the knox4
gene cannot be genetically uncoupled from the Gn1
mutant phenotype [24]. Another dominant leaf mutation, Lg3-O, is a class I knox gene [43]. Lg3-O also
transforms blade, auricle and ligule tissue into sheathlike tissue [25, 26, 59]. Lg3 is most closely related to
the duplicated loci knox5 and knox11 (Figure 1).
Expression of proximal characters in the distal leaf
blade can be interpreted in a variety of ways [30,
59]. A consistent theme is the failure to acquire a
specific developmental fate. That is, ectopic expression of knox genes interferes with normal processes of
differentiation.
161
Figure 6. Phenotypes of plants misexpressing knox genes. A. Leaf blade of Kn1 plant showing knots forming on the lamina. B. Gn1 mutant leaf.
Excessive cellular proliferation of the sheath mesophyll causes buckling and contortions of the sheath(s). The blade (b) is relatively unaffected.
C. Flower from a Hooded barley plant showing ectopic floral meristems (arrowheads) forming on the awn. D. Leaf lobing and ectopic meristems
(arrowheads) on leaves of Arabidopsis plants expressing 35S::KNAT1. E. Main axis of a tobacco plant expressing a 35S::KNAT2 transgene.
Leaves are very reduced and malformed. F. Tobacco leaf from a transgenic plant overexpressing KNAT1 with ectopic meristems. G. A single
leaf from a 35S::kn1 tomato plant showing many additional orders of compounding.
Ectopic expression of the Hvknox3 gene in barley
alters floral development
Hooded, a dominant mutation of barley, is caused
by the duplication of intron sequences in the class I
knox gene Hvknox3 [60]. Ectopic Hvknox3 expression in the lemma, which is the first lateral organ of
the floret, causes formation of florets on the awn (an
outgrowth of the lemma; see Figure 6C). A Hooded
phenocopy is induced when kn1 expression in barley
is introduced under the control of a ubiquitin promoter
[91]. Williams-Carrier suggested that, in response to
ectopic knox gene expression, the upper leaf zone of
the lemma forms an ectopic inflorescence from which
the flowers are produced [91]. In this regard overexpression of kn1 in barley resembles that of other knox
gene overexpressors that make ectopic meristems on
leaves (see below). Why barley forms ectopic meristems in the presence of HvKnox3 is unknown but could
be dependent upon interactions with genes expressed
in the awn.
162
Phenotypes due to knox gene mutations and
overexpression in dicots are different from
monocots
The effects of knox gene overexpression in dicots are
distinct from those in monocots and may be attributable to differences in competence of tissues to respond
to, or modulate the function of, KNOX proteins [90].
Transgenic Arabidopsis plants that constitutively express knox genes have highly lobed leaves and can
have ectopically placed meristems in the sinuses of
these lobes (Figure 6D) [16, 48]. Ectopic expression in tobacco of several knox genes driven by the
constitutive cauliflower mosaic virus (CaMV) 35S
promoter result in leaf phenotypes such as rumpling,
reduced lamina, and formation of ectopic shoots on
the leaves (Figure 6E, F) [55, 63, 69, 76, 81]. A
dramatic response to ectopic knox gene expression is
seen in tomato. Transgenic plants overexpressing both
endogenous and heterologous gene constructs make
supercompound leaves (Figure 6G). Other phenotypes
in tomato include reduced apical dominance, reduced
laminae, and ectopic shoot development [32, 40].
The way in which genes are misexpressed may
affect the phenotype. The tomato dominant mutation
Curl (Cu) is characterized by the formation of supercompound leaves and epiphyllic shoots among other
phenotypes [62]. The Mouse ears (Me) mutation induces changes in leaf and overall architecture that are
similar to but distinct from Cu [62]. Cu and Me are
both genetically linked to Tkn2. Tkn2 mRNA shows
a five-fold increase in expression in Cu mutant plants
compared to normal sibs suggesting that levels rather
than the pattern of Tkn2 gene expression are altered
[62]. Two labs have shown a correlation between
the Me phenotype and altered spatial regulation of
Tkn2/Let6 [15, 62]. In Me, the Tkn2 coding region
is fused to a constitutively expressed gene. The result is high-level expression of a fusion mRNA that
is ectopically localized [15, 62]. It was suggested that
ectopic expression versus overexpression contributes
to the differences in phenotype between Me and Cu
[62].
Within each species there are often a range of phenotypes in the transgenic plants expressing different
knox transgenes. This could be due to variation in
transgene expression or differences in genetic backgrounds of the transformants. Differences might also
reflect unique specificities of individual knox genes
[40, 90]. Each KNOX protein may interact with dif-
ferent protein partners and thus target distinct DNA
sequences.
What makes certain tissues competent to respond
to knox genes? Why do ectopic meristems form along
the midvein of the lamina in tobacco, in the sinuses
of lobed leaves in Arabidopsis and on the awn of barley flowers? Why does kn1 make knots in maize but
not barley leaves? Other genes must influence the effect of ectopic knox gene expression. For example,
CaMV 35S::Tkn1 does not correct the phenotypes of
Lanceolate, solanifolia and other tomato leaf shape
mutants indicating that overexpression of knox genes
alone is not sufficient to induce the formation of
compound leaves in tomato [32].
Gain and loss of function of knox genes can affect
overall plant stature, leaf shape, and floral development in a variety of species. From these data, we can
infer potential sites where changes in knox gene expression may have influenced the evolution of plant
developmental processes. Dramatic effects of knox
expression on leaf morphology led to a hypothesis
that knox genes may be involved in the evolution of
compound leaves in tomato [32, 75]. How general is
the effect of knox expression in developing leaves?
Compound leaves arose multiple times via different
mechanisms. Therefore, not all cases of compound
leaves would necessarily be attributable to changes in
knox gene expression. For example, in pea, the degree of leaf compounding is controlled by unifoliata,
a leafy homologue [35].
The dominant mutations of knox genes in maize
and tomato are good examples of how mutations can
alter expression domains and consequently affect morphogenesis. For example, the Kn1-O allele is a tandem
duplication of kn1 in which a second coding region is
associated with a novel 50 -upstream region [54, 84].
Many of the other Kn1 alleles characterized to date
are associated with insertions of transposable elements
into the large third intron [29]. These transposon insertions point to at least two potential regulatory domains
within the kn1 genomic region that determine kn1
expression in leaves. Based upon the phenotype of certain dominant and revertant Kn1 alleles we suspect the
presence of leaf silencing elements at the 50 end of the
gene and in the large third intron [29, 54]. Mutation
of either of these regulatory domains or of second-site
loci that interact with these domains could alter the
spatial or temporal pattern of kn1 gene expression.
But what does the analysis of knox gene expression evolution tell us about morphological evolution in
plants? While the cis-regulatory changes such as those
163
described for knox genes in maize and tomato clearly
alter plant morphology, there is no firm evidence
implicating knox genes in morphological evolution.
Changes in the regulation of knox gene expression may
or may not be due to specific changes at the knox locus
which alter its regulation. Baum made the very important point that ectopic expression of a specific gene
does not by itself define the innovative force in the
evolution of form [7]. It is first necessary to have phylogenetic evidence that observed differences in gene
expression are meaningful in terms of morphological
evolution. Then it is appropriate to identify cis or trans
effects on gene expression to provide mechanistic explanations for a change that defines the proximate
cause of novelty in gene expression.
Mechanisms to repress knox gene expression may
be similar in monocots and dicots
An emerging theme in the expression of class I knox
genes is their negative regulation in the meristem prior
to organ initiation. To date, with the exception of
tomato, all class I knox genes are excluded from lateral
organ primordia. A significant feature of all the ectopic
expression studies is that despite the fact that the transgenes are presumably under the control of constitutive
promoters, the transgene is not detected uniformly
throughout the plants. While it is possible that these
differences are a consequence of transgene silencing
in specific domains, the most probable explanation is
post-transcriptional regulation of knox accumulation
[91].
The recessive rough sheath2 mutation in maize has
a phenotype similar to that of dominant knox gene
mutations [71]. Ectopic expression of rs1, knotted1,
and liguleless3 can be detected as early as P1 in rs2
mutant leaf primoridia. Therefore, rs2 acts to negatively regulate knox genes in immature leaves [71].
However, ectopic knox gene expression is not seen in
P0 leaves and is not uniform throughout the leaves.
The ability to negatively regulate knox genes in the P0
and later stages of development in rs2 mutants may
be due to the presence and function of a duplicate
factor for rs2. Other loci may be required, such as narrowsheath (ns) and leafbladeless (lbl), both of which
are thought to participate in founder cell recruitment
[70, 83]. Double mutants between ns and rs2 were
additive, indicating that these genes act in separate
pathways to restrict knox gene expression in the leaf
primordia [71]. rs2 was cloned and the predicted pro-
tein encodes a myb-like transcription factor similar to
the phantastica (phan) gene from Antirrhinum [71, 83,
87]. Lateral organs in phan mutants are abaxialized
and have radial symmetry [86]. The phan expression
domain complements that of an Antirrhinum kn1-like
gene, consistent with a role in delimiting the parameters of knox gene expression [87]. These data
suggest that a common mechanism to regulate knox
gene expression exists in Antirrhinum and maize.
Concluding remarks
Knox genes are known to play important roles in
meristems and are likely to function in other tissues.
In situ localization shows differences in patterns of
gene expression that likely reflect novel functions for
duplicated genes. Class II genes have more diverse
patterns of expression suggesting that members of this
group have functions outside of the meristem. Cis- and
trans-regulatory changes in knox gene expression are
correlated with alterations in plant morphology. Duplications and diversification of knox genes have likely
contributed to changes in organization of meristems
and features of lateral organs.
Acknowledgements
We thank Neelima Sinha for providing the image of
the 35S::kn1 tomato plant leaf in Figure 6G and Geeta
Bharathan for allowing us to preview unpublished results. We are indebted to Toby Kellogg and the anonymous reviewers whose comments greatly enhanced
this review. Our thanks to Brent Mishler for many
discussions and invaluable assistance with the phylogenetic analysis and to D.L. Swofford for the beta test
version of PAUP ∗ 4.0. We are grateful to Paul Bethke,
George Chuck, Naomi Ori, Mark Running, and Kristen Shepard for comments on this manuscript. Work
in S.H.’s lab on knox genes is funded by USDA grant
9701255. L.R. is supported by NIH post-doctoral fellowship 18330-03. P.S-B. is funded by NSF GRANT
DEB-9801245 and a Rimo Bacigalupi Fellowship.
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