Journal of Experimental Botany, Vol. 60, No. 5, pp. 1503–1513, 2009
Perspectives on Plant Development Special Issue
doi:10.1093/jxb/ern304 Advance Access publication 3 December, 2008
REVIEW PAPER
Lepidium as a model system for studying the evolution of
fruit development in Brassicaceae
Klaus Mummenhoff1, Alexander Polster1,*, Andreas Mühlhausen1 and Günter Theißen2,†
1
2
University of Osnabrück, Department of Biology, Botany, Barbarastrasse 11, D-49076 Osnabrück, Germany
Friedrich Schiller University Jena, Department of Genetics, Philosophenweg 12, D-07743 Jena, Germany
Received 12 September 2008; Revised 31 October 2008; Accepted 10 November 2008
Abstract
Fruits represent a key innovation of the flowering plants that facilitates seed dispersal. In many species of the plant
family Brassicaceae dehiscent fruits develop in which seed dispersal occurs through a process termed ‘podshatter’. In the case of dehiscence, the fruit opens during fruit maturation. Phylogeny reconstructions using
molecular markers indicate that the development of dehiscent fruits is the ancestral condition within the genus
Lepidium s.l., but that indehiscent fruits evolved independently several times from dehiscent fruits. With Lepidium
campestre and Cardaria pubescens (also known as Lepidium appelianum), very closely related taxa with dehiscent
and indehiscent fruits, respectively, were identified which constitute a well-suited model system to determine the
molecular genetic basis of evolutionary changes in fruit dehiscence. Following the rationale of evolutionary
developmental biology (‘evo-devo’) phylomimicking mutants with indehiscent fruits of the close relative Arabidopsis
have been used to define the candidate genes ALC, FUL, IND, RPL, and SHP1/2 which might be involved in the origin
of indehiscent fruits in Cardaria. Comparative expression studies in L. campestre and C. pubescens are used to
identify differentially expressed genes and thus to narrow down the number of candidate genes. Reciprocal
heterologous transformation experiments may help us to distinguish direct from indirect developmental genetic
causes of fruit indehiscence, and to assess the contribution of cis- and trans-regulatory changes.
Key words: Arabidopsis, Brassicaceae, Cardaria, character evolution, genetic regulation, Lepidium, pod-shatter, phylomimicking
mutant.
Introduction
For more than 20 years the little weed Arabidopsis thaliana
(L.) Heynh. (henceforth termed Arabidopsis) has been the
major model system for investigating developmental and
physiological phenomena in plant biology, and has led to
the identification of genes and gene regulatory networks
(GRNs) involved in numerous processes of plant life.
Arabidopsis belongs to the Brassicaceae (mustard) family,
comprising about 3700 species in 338 genera (Warwick
et al., 2006), among them many economically important ornamental and crop plants such as cabbage, canola, and oil
seed rape (Koch et al., 2003). In addition to their agronomic
importance and given the large variation in physiology and
in leaf and fruit shape within the Brassicaceae, there should
also be ample genetic material available to investigate the
molecular bases of the changes in morphology over evolutionary time (Bowman, 2006). An interesting example is difference in leaf shape, which is now being intensively
investigated using Cardamine hirsuta (which has a dissected
leaf form) in comparison with Arabidopsis (which has a
simple leaf form) (Hay and Tsiantis, 2006; Barkoulas et al.,
2008).
Indeed, recent molecular analyses have led to the knowledge that many morphological characters on which previous
systematic relationships were based are, in fact, homoplasious. Fruit structures in particular have proved to be highly
labile in evolutionary time; all molecular phylogenetic data
* Present address: Hannover Medical School, Centre for Physiology, Carl-Neuberg-Str.1, D-30625 Hannover, Germany.
y
To whom correspondence should be addressed: E-mail: [email protected]
ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1504 | Mummenhoff et al.
consistently indicate that many species with similar fruits may
be only distantly related, whereas species with dramatically
different fruits can be very closely related (Koch et al., 2003;
Mummenhoff et al., 2005; Bailey et al., 2006; Al-Shehbaz
et al., 2006, and references therein). This implies that developmental processes controlling fruit shape and structure are
extremely plastic.
A good case in point are the structural prerequisites for
mechanisms of seed dispersal in Brassicaceae. In many
species of the Brassicaceae the fruit develops into a seed pod
which opens because of mechanical forces which build up as
the fruit dries out, in a process termed dehiscence (Spence
et al., 1996). Fruit dehiscence effectively discloses the seeds
upon maturation, which can eventually be dispersed, for
example, by wind, rain or animals. Many other species of
Brassicaceae, however, develop indehiscent fruits, in which
the whole fruit is the dispersal unit, and the seeds are
released upon decomposition of the fruit valves.
The development of dehiscent versus indehiscent fruits
was used for a long time as an important character in the
systematics of Brassicaceae, before the repetitive origin of
indehiscent fruits became obvious by phylogeny reconstructions employing molecular markers (Koch et al., 2003;
Mummenhoff et al., 2005; Al-Shehbaz et al., 2006, and
references therein). Fruit dehiscence in the Brassicaceae may
thus represent a system for the study of parallel or convergent evolution of reproductive characters using the
model plant Arabidopsis as a reference system.
However, extending the ideas which have arisen from the
knowledge of developmental genetic systems in Arabidopsis
to other species of the Brassicaceae will require the application of genomic technologies developed in Arabidopsis, and
the establishment of additional genetic systems and resources
in other species. Thus, it is desirable to establish model systems in which genetics can be utilised in conjunction with the
genomic resources available in Arabidopsis for the purpose of
identifying the genetic changes that have contributed to
morphological evolution.
The current paper summarizes recent literature on fruit
dehiscence/indehiscence in Arabidopsis and aims to present
a convenient model system, the genus Lepidium sensu lato
(s.l.), to shed further light on fruit evolution in wild
Brassicaceae species. Lepidium s.l. comprises Lepidium, as
traditionally delimited, and a number of nested genera, as identified by phylogeny reconstruction using molecular markers
(Fig. 1). Lepidium s.l. is among the few well-supported
monophyletic lineages found in all family-wide analyses
(Bailey et al., 2006; Beilstein et al., 2006; Al-Shehbaz et al.,
2006; Franzke et al., 2009) and is nested along with
Arabidopsis in one well-supported clade (‘lineage one’ in
Beilstein et al., 2006, 2008; Franzke et al., 2009).
The genus Lepidium L. as traditionally delimited–
Lepidium sensu strictu (s.str.)–is one of the largest genera of
the Brassicaceae, consisting of approximately 175 species. It
is distributed worldwide, primarily in temperate and subtropical regions; the genus is poorly represented in Arctic
climates and, in tropical regions, grows only in mountain
areas. Lepidium is well-known for its infrageneric systematic
difficulty, and traditional systematic concepts, mainly based
on differences in fruit shape, have been criticised as being
largely artificial (Mummenhoff et al., 2001, and references
therein). Previous molecular analyses have concentrated on
systematics (Mummenhoff et al., 2001), allopolyploidization
events (Mummenhoff et al., 2004), and the evolution of
flower structures (Bowman et al., 1999; Lee et al., 2002).
Although these studies focused on different topics and were
based on different taxon coverage, all molecular phylogenies supported three main lineages and indicated that few of
the infrageneric taxa, as delimited in the traditional
taxonomy, represent monophyletic groups.
Coronopus Zinn, Stroganowia Kar. & Kir., Winklera
Regel, Stubendorffia Schrenk ex. Fisch., and Cardaria Desv.
are among the most closely related genera of Lepidium s.str.
(Al-Shehbaz, 1986; Al-Shehbaz et al., 2006). These genera
differ from Lepidium mainly in fruit characters, especially
fruit dehiscence and inflation. Lepidium, Stroganowia, and
Winklera are characterised by dehiscent fruits (but see Fig.
1 for Winklera), Cardaria, Stubendorffia, and a fraction of
Coronopus species by indehiscent fruits, while the remaining
Coronopus species have didymous fruits. In didymous fruits,
the valve orifice that faces the replum is usually smaller than
the seed and, therefore, the seeds are not readily released,
and the fruit valves act as the dispersal unit (Al-Shehbaz
et al., 2002). Thus, from a mechanistic point of view,
didymous fruits are dehiscent but they are functionally indehiscent. Cardaria, Coronopus, and Stroganowia are hard
to distinguish morphologically from Lepidium and the distinctions of all four genera are based solely on a few fruit
characters (see above) of doubtful value. Along with preliminary molecular data it has been suggested that Cardaria,
Coronopus, and Stroganowia should be united with Lepidium (Al-Shehbaz et al., 2002). However, in the provisional
molecular analysis mentioned by Al-Shehbaz et al. (2002),
only single representatives of the genera have been included
and two other related genera, i.e. Winklera and Stubendorffia, were not considered at all.
In the current study, nuclear (ITS) and cpDNA markers
(trnT-trnL and trnL-trnF spacer, trnL intron) were used to
construct robust phylogenies in order to learn more about
fruit evolution in these taxa. Our taxon sampling of 124
species represents the full range of morphological variation
in Lepidium and the five closest related genera. Our aim was
to identify a suitable model system of closely related diploid
(sister) species which differ in fruit morphology and dehiscence. As Lepidium and related genera are quite closely
related to Arabidopsis, such a model system of diploid
species should facilitate comparative genetic studies aimed
at determining the molecular basis of evolutionary changes
in fruit structure. The analysis presented here confirms our
previous suggestions and clearly demonstrates that all related genera are well nested within Lepidium s.str.. Because
not all of these genera have been assigned to Lepidium in
previous analyses, the traditional names of all genera will be
maintained here.
Our phylogenetic analysis allowed a convenient model
system for the study of fruit evolution to be identified. All
Fruit evolution in Brassicaceae | 1505
Fig. 1. Phylogenetic relationships among Lepidium species and related genera based on ITS sequence analysis. These genera are
nested within and should be transferred to a broader defined Lepidium s.l.. For a better understanding of a broader readership, however,
the traditional names of these genera are maintained in this study. Shown is the Bayesian 50% majority rule consensus tree with mean
posterior probability values (clade credibilities) above branches, the distribution of species by continents and the assignment of fruit
characters among the genera. Roman numerals I, II, and III refer to the main phylogenetic lineages. In Winklera, fruits may open but they
stay indehiscent for a longer period and probably not all fruits finally open. In W. silaifolia, opening fruits are didymous. Species used for
1506 | Mummenhoff et al.
Cardaria species with indehiscent fruits were found to be
closely related to Lepidium species with dehiscent fruits. The
diploid species Cardaria pubescens C.A. Meyer (Jarmolenko)¼Lepidium appelianum Al-Shehbaz with indehiscent fruits
and Lepidium campestre (L.) R.Br. with dehiscent fruits were
selected for anatomical analysis of the fruit. It has been
possible to demonstrate that the anatomy of dehiscent
Lepidium and indehiscent Cardaria fruits agrees completely
with the pattern of Arabidopsis wild-type (dehiscent) and
mutant (indehiscent) fruit types, respectively, suggesting
hypotheses for the developmental genetic basis of the
morphological difference that can be experimentally tested.
starting from different randomly chosen trees and with
four independent chains with a heating temperature set to
0.2. Each chain was run for 2 500 000 update cycles and
the chain states were sampled every 100th cycle. The first
6250 samples were discarded as the ‘burn-in’ of the
Markov chain. The values sampled for different parameters were examined using TRACER 1.3 (Rambaut and
Drummond, 2003). Maximum likelihood searches were
conducted using a fast maximum likelihood ratchet
approach (Morrison, 2007) as implemented in PRAP
v.2.0 (Müller, 2004). PRAP-generated command files were
handed over to PAUP (Swofford, 2002) as described by
Wall et al. (2008).
Study group
Ninety-seven Lepidium L. species from all continents representing all infrageneric taxonomic entities (Thellung, 1906)
were analysed. Furthermore, species were included of the
most closely related genera of Lepidium, i.e. Coronopus
(eight species representing each of the four sections; AlShehbaz, 1986), Cardaria (two species, one with two
subspecies), Winklera (both species; Schulz, 1936; Czerepanov, 1995), Stroganowia (ten species of each of the two sections; Botschantsev, 1984), and Stubendorffia (four species
of the two sections; Bush, 1939). Thus, our taxon sampling
of 124 species represents the full range of morphological
variation in Lepidium and related genera, and the entire
geographic distribution of these taxa on all continents.
Following Mummenhoff et al. (2001), Hornungia petraea
(L.) Reichenbach was used as an outgroup. Sources of plant
material are given in Table S1 in the Supplementary data at
JXB online.
Methods for DNA extraction, PCR, and direct sequencing of ITS and non-coding cpDNA (trnT/trnL spacer, trnL
intron, trnL/trnF spacer) have been presented elsewhere
(Bowman et al., 1999; Mummenhoff et al., 2001). The DNA
sequences were aligned by hand, and regions of ambiguous
alignment eliminated. Insertions/deletions (indels) were
treated as missing data from the data matrix.
Phylogenetic analyses
The model of nucleotide substitution for the Bayesian analysis of phylogeny that best fitted the data was determined
using MrModelTest 2.2 software (Posada and Crandall,
1998) and the Akaike information criterion (AIC). Bayesian inference of phylogeny was performed using MrBayes
3.1 (Huelsenbeck and Ronquist, 2003). Following MrModelTest, the symmetrical model of sequence evolution
(SYM) was used in MrBayes, with an allowance for
a gamma (G) distribution of rates and a proportion of
invariant sites. Two separated runs were conducted, each
Anatomical analysis of fruits
Infructescences from L. campestre with dehiscent fruits and
C. pubescens (also known as L. appelianum) with indehiscent fruits were harvested from plants grown from seeds of
known wild origin and stored in 70% (v/v) ethanol until
further use. Fruits of both species were taken at different
ontogenetic stages. The floral morphogenesis and fruit development of the model plant Arabidopsis has been described
in detail (Spence et al., 1996). Smyth et al. (1990) divided
the early flower development of Arabidopsis from initiation
of the flower until anthesis into 13 stages, and this study has
been used as a reference for the description of many mutants
and the characterization of numerous Arabidopsis genes.
Ferrándiz et al. (1999) divided post-fertilisation fruit
development of Arabidopsis into seven further ontogenetic
stages. In the current study, this approach has been followed and fruits of L. campestre and Cardaria pubescens
have been harvested from stage 14 (beginning of fruit
development subsequent to pollination) to stage 19 (fruit
valves begin to separate from the replum).
Transverse fruit sections approximately 10 lm in thickness
were cut by hand using a single-sided razor blade. Each section was immersed for 1 min in phloroglucinol (mixed as
described by Braune et al., 1994) and afterwards in diluted
hydrochloric acid. Thickened and lignified cell walls were
stained red. Transverse sections were analysed by a Leica
DM/LS light microscope. A Sony F717 digital camera was
used for photographic documentation.
Phylogeny of Lepidium and related genera as
revealed by molecular markers
A comprehensive molecular systematic analysis has been
performed in order to identify an appropriate model system
for the analysis of the regulation of fruit dehiscence/
indehiscence in wild species. After eliminating regions with
anatomical fruit analysis, i.e. Cardaria pubescens and Lepidium campestre, are underlined and printed in bold type. Abbreviations: EA,
Eurasia; AF, Africa; Aus/NZ, Australia/New Zealand; NA/ZA, North America/Central America; SA, South America; Pa, Pacific Islands
(Hawaii); Did, didymous. Hornungia petraea was used as the outgroup.
Fruit evolution in Brassicaceae | 1507
ambiguous alignment, the aligned ITS data matrix consisted
of 482 characters of which 285 were variable and 197
potentially informative. For the cpDNA marker, 1605 nucleotide positions were available for phylogenetic analysis, of
which 563 characters were variable, and 235 potentially
informative.
The Bayesian tree generated from cpDNA or ITS data set
was an almost exact match of the respective maximum–
likelihood tree, with the exception of slight differences in
branching among some terminal nodes. These differences,
however, did not have any impact on the phylogenetic conclusions drawn in this study and, therefore, only the
Bayesian tree for the cpDNA- and ITS data is shown. Both
phylogenies consistently detect three main phylogenetic
lineages, I–III. The grouping in lineage II generally follows
the geographic distribution of the species by continents
(Fig. 1). The minor incongruence of species relationships
within lineage II based on two separate molecular markers,
one of which is maternally inherited (cpDNA) and the other
biparentally inherited, but with a potential for concerted
evolution (ITS) (Wendel, 2000; Soltis et al., 2008), and the
large number of polyploid species both suggest that the
evolutionary history of Lepidium is reticulate (Mummenhoff
et al., 2001, 2004; Lee et al., 2002). This was explicitly
shown for the origin and evolution of the Australian/New
Zealand endemic Lepidium species (Mummenhoff et al.,
2004). The incongruence in the position of a group of three
Stubendorffia and one Stroganowia species in the cpDNA(within lineage II) and ITS tree (within lineage I) also
indicate a reticulate history, but will not be discussed
further here. Both phylogenies clearly demonstrate that the
genera suggested to be closely related to Lepidium, i.e.
Cardaria, Coronopus, Stroganowia, Stubendorffia, and Winklera, are all well nested within the three main lineages of
a broader defined Lepidium, and only one of these related
genera (Winklera) is monophyletic. Instead of being separate from Lepidium, species of these genera group along
with Lepidium species of the same continental distribution
(Fig. 1). To accommodate a broader readership we have,
however, maintained the traditional names of these genera
here.
In the traditional classification systems, these taxa have
been recognised as separate genera mainly based on differences in fruit morphology. Therefore, fruit characters have
been mapped onto the phylogenetic tree based on molecular
markers (Fig. 1). The evolution of different fruit types was
detected within one lineage (indehiscent and dehiscent fruits
in Lepia) and parallel or convergent evolution of the same
fruit type in different lineages (indehiscent fruits in Lepia
and different clades of Lepidium s.str.; Fig. 1). Numerous
studies (summarised in Koch and Mummenhoff, 2006) have
amply demonstrated that morphological characters in the
Brassicaceae, especially fruit characters, are highly homoplasious. So the questions arise: exactly how fluid is
evolution of the fruit structure in Brassicaceae? Why is fruit
form so variable in the Brassicaceae, and what is the
underlying genetic basis for this variation? It has previously
been hypothesised that dramatic differences in morphology
may result from mutations of so-called major morphogenetic genes, not accompanied by adequate change in the
molecular marker system (Kadereit, 1994; Theißen et al.,
2000; Bateman and DiMichele, 2002). The idea that major
genes might be important in evolution is not new and has
already been expressed by de Vries (1901) and Goldschmidt
(1940). In the last few years developmental geneticists
provided experimental support that the genetic basis of
dramatic change in morphology might be relatively simple.
A good example is the teosinte branched 1 (tb1) gene in
maize and teosinte. Differences in the expression of tb1
result in very different morphotypes (Doebley, 2004).
The distribution of dehiscent/indehiscent fruits was used
as a key diagnostic character in traditional Brassicaceae
classification to separate, for instance, Cardaria and Coronopus from Lepidium. In our phylogenetic tree, all Cardaria
species with indehiscent fruits are sister to Lepidium species
with dehiscent fruits (e.g. L. campestre). C. pubescens and L.
campestre have been selected for further anatomical analysis
of the fruit for the following reasons: both species are
closely related but they differ in fruit morphology. The two
species of this model system are diploid (2n¼2x¼16) and
they are found in the same major phylogenetic lineage as
Arabidopsis (Beilstein et al., 2006) facilitating comparative
genetic studies.
Anatomical basis of dehiscence in the
Arabidopsis fruit
Our first aim was to compare the anatomical pattern during
fruit development of dehiscent and indehiscent fruit types in
Arabidopsis (wild-type and mutant plants) and the Cardaria/
Lepidium model system. The development of the fruit in the
model plant, Arabidopsis, provides an excellent system for
analysing the mechanism that patterns a plant organ
because of the distinctive morphological landmarks and the
availability of reporter lines that mark specialised tissues
(Dinneny et al., 2005). The region of the fruit that encloses
the seeds is divided into three zones: the fruit valve, the
replum, and the fruit valve margin (Fig. 2A). The valves are
the seed pod walls and they encircle the developing seeds.
The valves are connected with the replum, which forms the
middle ridge of the fruit. A specialised strip of tissue, the
valve margin, forms at the valve–replum boundary which is
specialised for fruit opening, i.e. the detachment of the
valves from the replum. The valve margin consists of two
different cell types. On the replum side of the valve margin,
the separation layer (sl) forms, which is responsible for the
detachment of the valve from the replum through cell–cell
separation. On the valve side of the margin, a layer of rigid
lignified cells forms (lignified valve margin cells: vmc) which
is connected with an adjacent layer of lignified tissue at the
inner side of the fruit valves, termed the endocarp layer
b (enb). The region of lignified valve margin cells, together
with the separation layer is termed the dehiscence zone. A
spring-like tension within the drying fruit eventually results
in fruit opening and fruit valves detach from the replum by
1508 | Mummenhoff et al.
Fig. 2. Anatomy (cross-sections) of dehiscent and indehiscent fruits of Arabidopsis wild-type (A, B) and mutant plants (D, E), Lepidium
campestre (C), and Cardaria pubescens (F). (A) Diagrammatic view of Arabidopsis wild-type gynoeceum after fertilization (left) and during
fruit dehiscence together with factors contributing to fruit opening (right). (B) Section of dehiscence zones at the fruit valve margin of an
Arabidopsis wild-type stage 17B fruit. Lignified cell walls have been traced in pink for clarity, and the fracture surface in the separation
layer is noted by a blue line. (C) The valve margin region of L. campestre fruits at stage 19. Lignified tissue is selectively stained red by
phloroglucinol treatment. As in the Arabidopsis wild-type fruit (B), the dehiscence zone is clearly visible, consisting of a strip of lignified
valve margin cells (vmc), in addition to the lignified endocarp layer b (enb) and the vascular bundles of the replum. Cell–cell separation
within the separation layer indicating the detachment of the fruit valves from the replum is marked by arrowheads. (D) Arabidopsis
35S::FUL mutant fruit (stage 17). Due to ectopic expression of FUL in the fruit valve margin, the dehiscence zone (sl, vmc) is not properly
established resulting in indehiscence. (E) In Arabidopsis ind ful fruits (stage 17), the dehiscence zone is not formed. In addition, ectopic
valve lignification does not occur. (F) In indehiscent fruit of C. pubescens (stage 17B) and Arabidopsis mutants (D, E), the dehiscence
zone is not formed. Instead, a continuous strip of lignified cells (marked by an arrowhead) is stretching from one replar vascular bundle to
the other one at the opposite side of the fruit. For genetic regulation of fruit valve margin differentiation see Fig. 3 and text. dz,
dehiscence zone; ena, endocarp layer a; enb, endocarp layer b; r, replum; sl, separation layer; v, fruit valve; vb, vascular bundle; vmc,
lignified valve margin cells. Scale bars: 100 lm. Reprinted and modified with permission from Macmillan Publishers Ltd, Liljegren et al.
(2000) SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 404, 766–769 (A), Oxford University Press,
Ferrándiz (2002) Regulation of fruit dehiscence in Arabidopsis. Journal of Experimental Botany 53, 2031–2038 (B), American Association
for the Advancement of Science, Ferrándiz et al. (2000) Negative regulation of the SHATTERPROOF genes by FRUITFULL during
Arabidopsis fruit development. Science 289, 436–438 (D), Elsevier, Liljegren et al. (2004) Control of fruit patterning in Arabidopsis by
INDEHISCENT. Cell 116, 843–853 (E).
Fruit evolution in Brassicaceae | 1509
autolysis of middle-lamellae within the separation layer
(Fig. 2A).
knocked out, or the ectopic expression of other genes in the
valve margin suppresses the expression of valve margin
determinating genes (for details, see below).
Lepidium campestre versus Cardaria
pubescens: anatomical analysis of dehiscent
versus indehiscent fruits
Evolutionary developmental genetics of fruit
dehiscence: general aspects
Following Fig. 2A, a sketch-map allowing orientation
within the photographic illustrations of fruit transverse sections was created. Each photograph contains a complete,
idealised, and exactly adjusted transverse section of the fruit
under study, with cell types coloured as in Fig. 2A. Tissues
shown in the respective photographic illustrations of fruit
cross-sections are boxed in the corresponding sketch-map in
the right corner of each photograph. Transverse sections of
the replum-valve region of fruits of these two taxa demonstrate clear differences, although L. campestre and C.
pubescens are closely related (Fig. 1). The anatomy of the
replum-valve region of L. campestre dehiscent fruits (Fig.
2C) closely resembles the dehiscent wild-type fruit anatomy
of Arabidopsis (Fig. 2B) (Liljegren et al., 2004). Welldeveloped dehiscence zones are apparent on both sides of
the replar vascular bundles. Unlignified and, with increasing
fruit ripening, successively lignified valve margin cells and
endocarp layer b cells are discernible; at ontogenetic stage
19, lignification of endocarp layer b and the valve margin
cells is completed and thus both tissues are connected to
form a lignified strip of cells around both sides of the
replum. Xylem cells of the replar vascular bundle lignify
concomitantly with valve margin cells and endocarp layer
b (Fig. 2B, C).
Although the corresponding species are very closely
related, transverse sections of indehiscent fruits of
C. pubescens do not exhibit typical traits of the dehiscent L.
campestre fruits. There are neither lignified valve margin
cells nor the typical separation layer, although the replum is
visible on the outside (Fig. 2F). One process associated with
fruit evolution within the Brassicaceae is the loss of dehiscence through non-development of a separation tissue
(Zohary, 1948). This is clearly seen in those indehiscent
fruits in which the replum and the valves are still well
marked. Furthermore, in Cardaria fruits, there is a continuous strip of lignified cells stretching from one replar
vascular bundle to the other at the opposite side of the
fruit, not interrupted by a dehiscence zone as in the
dehiscent Lepidium fruits.
It is interesting to note that the anatomy of indehiscent C.
pubescens fruits (Fig. 2F) strongly resembles the anatomical
pattern of indehiscent fruits of some Arabidopsis loss-offunction mutants (e.g. Fig. 2E). As in some Arabidopsis
mutant (indehiscent) fruits, no dehiscence zone is formed.
Instead, a continuous strip of lignified cells (enb) stretches
from one replar vascular bundle to the other at the opposite
side of the fruit (Fig. 2F). In the Arabidopsis mutant fruits,
the dehiscence zone is not properly formed because the
genes responsible for their proper establishment are
An important goal of contemporary evolutionary developmental biology (evo-devo, for short) is to identify the
molecular genetic changes that led to evolution at the
morphological level. One question of general importance
from a heuristic point of view is whether mutations causing
one taxon to mimic another (‘phylomimicking mutations’)
define evolutionary relevant loci (Haag and True, 2001).
Only if this is true in many cases, are candidate gene
approaches effective strategies to clone genes of evolutionary importance.
It is argued here that the genus Lepidium s.l. (with related
genera included), for a number of reasons, is well-suited to
address this question, using valve margin development and
fruit dehiscence as exemplary characters. Firstly, Lepidium
s.l. is closely related to Arabidopsis, which facilitates the
transfer of knowledge and tools from one system to the
other. Secondly, some Arabidopsis mutants develop indehiscent fruits that resemble the indehiscent fruits of the wildtype in some Lepidium s.l. species down to the cellular
details (Fig. 2), making it conceivable that the genes which
mutated during the evolution of fruit (in)dehiscence in
Lepidium s.l. are orthologues of the phylomimicking Arabidopsis genes. Thirdly, dehiscent fruits are ancestral in
Lepidium s.l and fruit indehiscence evolved several times
independently within Lepidium s.l., enabling the study of the
molecular mechanisms behind parallel or convergent evolution.
Typical evo-devo approaches employ detailed knowledge
about a developmental process of interest from a wellstudied model system, i.e. in this case Arabidopsis.
Regulation of valve margin development in
Arabidopsis
Based on mutant analysis, much has been learned in recent
years about the molecular basis of fruit patterning in
Arabidopsis (Fig. 3). Genes which control the differentiation
of tissues during fruit development can be divided into two
classes, those that promote valve-margin development and
those that repress it (Dinneny and Yanofsky, 2004). The
functionally redundant genes SHATTERPROOF1 and 2
(SHP1, 2), encoding MADS-domain proteins, are on top of
a gene regulatory network composed of transcription
factors that specify valve margin identity (Liljegren et al.,
2000). Double loss-of-function mutants in which SHP
activity is eliminated (shp1 shp2) develop indehiscent fruits
that lack lignified valve margin cells and separation layer
development. Both downstream and in parallel to SHP act
INDEHISCENT (IND) and ALCATRAZ (ALC), two genes
1510 | Mummenhoff et al.
Fig. 3. Genetic pathway controlling fruit development in Arabidopsis. The differentiation of tissues is controlled by two sets of
genes that either promote valve margin development, i.e. SHP1,
2 (MADS-box genes), IND and ALC (basic helix-loop-helix-type
transcription factor genes) or restrict it from surrounding regions
(FUL, RPL). Proper establishment of the dehiscence zone is
controlled by SHP1, 2, IND, and ALC. IND, and ALC appear to act
largely downstream of SHP1,2. SHP1, 2 and IND control the
separation layer and lignified valve margin cell development, while
ALC is controlling only the separation layer. The expression of
these four genes is restricted to the valve margin through
repression by FUL (MADS-box gene) in the valves, and by RPL
(BELL-subfamily homeobox gene) in the replum. JAG, FIL, and
YAB3 function redundantly to promote the expression of the valve
margin identity genes and FUL. These activities are negatively
regulated by RPL in the replum (Dinneny and Yanofsky, 2004;
Dinneny et al., 2005). dz, dehiscence zone; enb, endocarp layer b;
sl, separation layer; vmc, lignified valve margin cells. Reproduced
and modified by permission of the Company of Biologists: Dinneny
et al. (2005) Development 132, 4687-4696.
which encode basic helix-loop-helix-type (bHLH) transcription factors. IND and ALC also specify valve margin identity, with IND playing a crucial role in both lignified and
separation layer development, while the function of ALC is
restricted to controlling separation layer development
(Rajani and Sundaresan, 2001; Liljegren et al., 2004). The
expression of these ‘valve margin identity genes’ is restricted
to the valve margin because of repression by FRUITFULL
(FUL), another MADS-domain transcription factor, in the
valves, and REPLUMLESS (RPL), a BELL-family homeodomain transcription factor, in the replum (Gu et al., 1998;
Ferrándiz et al., 2000; Roeder et al., 2003). In ful loss-offunction mutants, the valve margin identity genes become
ectopically expressed in the valves, which leads to a valve
margin-like development, including the ectopic formation of
lignified and separation layer-like cell types (Ferrándiz
et al., 2000). Ectopic expression of FUL in the fruit valve
margin in transgenic plants (35S::FUL) also results in
indehiscent fruits (Fig. 2D) because FUL prevents the
expression of SHP1, SHP2, IND, and ALC genes which
are responsible for the proper establishment of the dehiscence zone. In rpl loss-of-function mutants, ectopic
expression of SHP in the replum causes the valve margins
to coalesce into a single strip of tissues in place of the
replum, leading to partially indehiscent fruits. Interestingly,
the MADS-box genes SHP and FUL have ancestral
functions in ovule and inflorescence meristem development,
respectively, and have been recruited for derived functions
during fruit development more recently (Theißen, 2000,
2001).
Comparatively little is known about how the patterns of
valve margin identity genes are initially established. However, it could already be demonstrated that SHP gene expression is activated by AGAMOUS, a MADS-box gene
which specifies carpel identity (Savidge et al., 1995).
Moreover, it was shown that FILAMETOUS FLOWER
(FIL) and YABBY3 (YAB3), two YABBY genes which encode transcription factors, control the non-overlapping
expression patterns of FUL and SHP (Dinneny et al.,
2005). FIL and YAB3 activate the valve margin identity
genes redundantly with JAGGED (JAG), a gene which encodes a C2H2 zinc-finger transcription factor that promotes
leaf blade growth, suggesting that several genetic pathways
converge to regulate these genes (Dinneny et al., 2005). The
activities of FIL, YAB3, and JAG are negatively regulated
by RPL, which divides FIL/JAG activity, thus generating
two distinct strips of valve margin. YABBY genes control
the polarity of tissues in lateral organs thus revealing a functional link between polarity establishment and the regulation of tissue identity in the developing Arabidopsis fruit
(Dinneny et al., 2005).
Analysing the genetic basis of the origin of
indehiscent fruits in Lepidium s.l.
To further our understanding of the molecular mechanisms
behind the transition from dehiscent to indehiscent fruits in
the lineage that led to C. pubescens, the orthologues of six
candidate genes, ALC, FUL, IND, RPL, SHP1, and SHP2,
have been cloned from both L. campestre and C. pubescens.
These genes have been chosen, because they are well-known
to be involved in the differentiation of the dehiscence zone in
the Arabidopsis fruit, and because the position of these genes
within the gene regulatory network specifying valve margin
identity is quite well established (Fig. 3). Moreover, anatomical analysis of the fruits of C. pubescens (Fig. 2F) revealed
striking similarities to Arabidopsis mutants or transgenics in
which the expression of these genes is changed, including
shp1 shp2, ind, and ind ful (Fig. 2E) loss-of-function mutants
as well as 35S::FUL transgenic plants (Fig. 2D) (Ferrándiz
et al., 2000; Liljegren et al., 2000, 2004). Particularly because
of the close relationship between Arabidopsis and Lepidium
s.l. these findings strongly increase the likelihood that the
candidate genes are involved in the origin of fruit indehiscence in Cardaria.
To narrow down the number of promising candidate
genes, their expression will be compared during development
of the dehiscent fruits of L. campestre and the indehiscent
fruits of C. pubescens using diverse techniques, including in
situ hybridization analyses. The results may provide us with
Fruit evolution in Brassicaceae | 1511
clues as to whether changes in the temporal or spatial
expression patterns of some genes contributed to the
evolution of indehiscence. Only genes that are differentially
expressed in L. campestre and C. pubescens will be selected
for the next series of experiments.
To distinguish direct from indirect developmental genetics causes, and to assess the contribution of cis- and transregulatory changes, reciprocal heterologous transformation
experiments will be carried out. If transformation protocols
for L. campestre and C. pubescens can be established, preferably by the ‘floral dip’ method, that works for a number
of Brassicaceae already (Clough and Bent, 1998; Bartholmes
et al., 2008), transformation experiments will include these
species.
General strategies to identify the genetic causes of morphological evolution have been reviewed by Baum (2002)
and were successfully applied to the analysis of rosette
flowering in diverse Brassicaceae taxa by Yoon and Baum
(2004).
Specifically, once promising candidate genes have been
identified which are differentially expressed in the ancestral
(L. campestre) and derived species (C. pubescens) in a way
that makes their involvement in the evolution of fruit
dehiscence conceivable, the genomic loci, including putative
regulatory regions upstream and downstream of the coding
regions (such as promoters), will be cloned from the derived
species and transformed into the ancestral species and into
Arabidopsis.
To understand the rationale behind these experiments
better, let us consider one example. Let us assume that gene
expression studies suggest that heterotopic expression of the
FUL orthologue of C. pubescens (CpFUL) in the valve
margin is involved in the origin of indehiscent fruits. In that
case, it appears likely (even though not certain) that CpFUL
exerts a dominant and gain-of-function effect when brought
into Arabidopsis or L. campestre. So the CpFUL locus
(including regulatory sequences) would be transformed into
the ful mutant of Arabidopsis. As controls, the FUL locus of
L. campestre (LcFUL) and of Arabidopsis (FUL) would also
be transformed into the ful mutant. If Arabidopsis ful FUL
and LcFUL transgenics and their descendants show wildtype phenotypes, this would indicate that Arabidopsis and
Lepidium s.l. are not too distantly related for these kinds of
complementation experiments. If, then, Arabidopsis ful
mutants carrying the CpFUL transgene develop indehiscent
fruits as do C. pubescens, this would indicate that changes in
cis-regulatory sites of the CpFUL gene caused the transition
from dehiscent to indehiscent fruits. If Arabidopsis ful
CpFUL plants develop dehiscent wild-type fruits, however,
this would suggest that changes in trans-acting factors
caused the changes in the expression (and possibly function)
of the CpFUL gene. If possible, CpFUL should also be
transformed into L. campestre (or a lcful mutant generated,
for example, by RNAi), which may result in indehiscent
fruits if changes in cis-regulatory sites of the CpFUL gene
caused the transition from dehiscent to indehiscent fruits. In
this case, transformation of the ancestral LcFUL into the
wild-type C. pubescens should not bring about a mutant
phenotype, but C. pubescens cpful LcFUL plants should
develop dehiscent fruits like L. campestre. If, however,
changes in trans-acting factors caused the transition from
dehiscent to indehiscent fruits, C. pubescens cpful LcFUL
plants should develop indehiscent fruits like C. pubescens
wild-type plants do.
Fruit indehiscence in Lepidium s.l. to study
parallel and convergent evolution
Indehiscent fruits evolved independently several times
within Brassicaceae (Appel and Al-Shehbaz, 2003; Fig. 1).
In general, the repeated evolution of specific morphologies
could be due to parallel or convergent evolution. According
to the definition reviewed by Yoon and Baum (2004),
parallelism refers to the independent evolution of the same
derived trait via the same developmental changes, whereas
convergence refers to superficially similar traits that have
a distinct developmental basis. Thus, if the molecular
genetic basis of fruit indehiscence is analysed for additional
pairs of closely related species with indehiscent and dehiscent fruits, it will also be possible to determine whether
the independent evolution of indehiscent fruits represent
cases of parallel or convergent evolution.
Supplementary data
Supplementary data are available at JXB online.
Supplementary Table S1. Lepidum (Brassicaceae) as
a model system for studying the evolution of fruit development in wild species.
Acknowledgements
We thank Ulrike Coja for technical assistance, numerous
herbaria and collectors for providing plant material, the
staff of the Botanical Garden Osnabrück for plant cultivation, Andreas Franzke for critical discussion, and Lucille
Schmieding for correcting style and grammar. Many thanks
also to Teresa Lenser, Pia Nutt, and Diana Lobbes for their
contributions to the fruit dehiscence project. Work in the
authors’ laboratories on the evo-devo of fruit dehiscence in
Brassicaceae is supported by grants from the Deutsche
Forschungsgemeinschaft (DFG) to KM (MU 1137/8-1) and
GT (TH 417/6-1).
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