1 Division of Molecular Life Science, Pohang University of Science and
Technology, San 31, Hyoja-dong, Pohang, Kyungbuk, 790-784, Korea
2 National Institute for Basic Biology/Center for Integrative Bioscience,
Myodaiji-cho, Okazaki 444-8585, Japan
3 Division of Biological Science, Graduate School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-8602, Japan
* Present address: Department of Life Science and Resources, Dong-A University,
Hadan-2-dong 840, Busan, 604-714, Korea
Present address: Kumho Life and Environmental Science Laboratory, Oryong-dong,
Gwangju, 500-712, Korea
Author for correspondence (e-mail:
nam{at}bric.postech.ac.kr)
Accepted 8 October 2002
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SUMMARY |
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Key words: Arabidopsis thaliana, Meristem, Differentiation, Leaf morphogenesis, Class I knox genes, BOP1
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INTRODUCTION |
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Arabidopis provides an excellent tool to genetically dissect
developmental processes of the leaf organ owing to its simple and stable
pattern formation. Each Arabidopsis leaf can be divided into a
proximal petiole and a distal blade region. A key factor that contributes to
the control of leaf morphogenesis in Arabidopsis is the spatial and
temporal regulation of meristematic activity, which involves cellular
differentiation. Class I knox genes are believed to play crucial
roles in the specification of leaf primordia and in the control of leaf
patterning through regulation of the meristematic activities of leaf cells.
Class I knox genes include KNAT1 (for KNOTTED-like
from Arabidopsis thaliana), KNAT2, KNAT6 and
SHOOTMERISTEMLESS (STM) in Arabidopsis
(Lincoln et al., 1994;
Long et al., 1996
;
Semiarti et al., 2001
),
knotted1 and ROUGH SHEATH1 in maize
(Becraft and Freeling, 1994
;
Jackson et al., 1994
;
Schneeberger et al., 1995
),
and Nicotiana tabacum Homeobox 15 in tobacco
(Tamaoki et al., 1997
). These
genes are strongly expressed in the SAM but not in the incipient young
primordia (Jackson et al.,
1994
; Long et al.,
1996
; Tamaoki et al.,
1997
; Nishimura et al.,
1999
; Sentoku et al.,
1999
). Suppression of class I knox gene expression in
primordia and mature leaf organs is critical for the determination of leaf
cell fate. Aberrant expression of class I knox genes triggers
multiple abnormal developmental responses, including altered leaf development
and, in extreme cases, formation of ectopic meristems
(Matsuoka et al., 1993
;
Sinha et al., 1993
;
Lincoln et al., 1994
;
Schneeberger et al., 1995
;
Chuck et al., 1996
;
Hareven et al., 1996
;
Tamaoki et al., 1997
;
Sentoku et al., 2000
).
Furthermore, mutations of the Arabidopsis gene ASYMMETRIC
LEAVES1 (AS1) and AS2, which produce lobed rosette
leaves with leaflet-like organs on their petioles, result in aberrant
expression of class I knox genes
(Byrne et al., 2000
;
Ori et al., 2000
;
Semiarti et al., 2001
).
Abnormal expression of class I knox genes in leaf cells due to a lack
of negative regulators, such as AS1 and AS2, leads to the
aberrant regulation of meristematic activity and ectopic growths. AS1
encodes a Myb protein domain and is a homologue of both the ROUGH
SHEATH2 (RS2) of maize and the PHANTASTICA
(PHAN) of Antirrhinum
(Waites et al., 1998
;
Timmermans et al., 1999
;
Tsiantis et al., 1999
;
Byrne et al., 2000
).
AS2 encodes a novel protein with cysteine repeats (C-motif) and a
leucine-zipper-like sequence (Iwakawa et
al., 2002
). In addition, LEAFY PETIOLE is thought to be
involved in controlling cell division in the marginal meristem of leaves
(van der Graaff et al.,
2000
).
In spite of considerable efforts to reveal the genetic and molecular
mechanisms underlying cell fate determination in leaves, our understanding is
still largely limited (Tsukaya,
2002a; Tsukaya,
2002b
; Tsukaya,
2002c
). In this report, we describe the bop1-1 mutation
that induces abnormal expression of class I knox genes. The mutation
was originally identified by the phenotype of vigorous ectopic outgrowths
along leaf petioles. Through characterization of this mutation, we suggest
that BOP1 has a critical role in regulating meristematic activity in
leaves by modulating expression of class I knox genes.
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MATERIALS AND METHODS |
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Isolation of mutants
Approximately 40,000 seeds of the ecotype Ler were mutagenised by
soaking in a 0.33% solution of ethylmethane sulfonate (EMS) in 100 mM
phosphate buffer (pH 7.0) for 8 hours, as described previously
(Oh et al., 1996). The
M1 plants from the mutagenised seeds were grouped into 8
subpopulations and the progeny of self-fertilised M1 plants from
each group was harvested separately. A single M2 plant that
exhibited the bop1-1 phenotype was identified from more than 200,000
M2 plants. A backcross was performed using bop1-1 mutant
pollen and Ler gynoecia. Progenies of a single plant that were
derived from four backcrosses to Ler were used for genetic and
phenotypic analyses.
Mapping of the BOP1 gene
The bop1-1 mutation was initially mapped using the cleaved
amplified polymorphic sequence (CAPS) markers
(Konieczny and Ausubel, 1993).
The bop1-1 mutant was crossed with the Columbia (Col) ecotype. DNA
for mapping studies was prepared from 60 individual F2 progenies
with the mutant phenotype. In addition, we generated the bop1-1 tt5-1
double mutant and examined the segregation ratio of the mutant phenotypes in
261 F2 progenies that were derived from a cross between the double
mutant and Col. The map distance was estimated using the Kosambi mapping
function (Koorneef and Stam, 1992).
Analysis of vasculature
The vein patterns in cotyledons and rosette leaves of plants were examined
as described previously (Hamada et al.,
2000; Jun et al.,
2002
). Photographs were taken under dark-field microscopy.
Histological analysis
Plant materials for histological analysis were fixed overnight at room
temperature in a solution of 45% ethanol, 2.5% glacial acetic acid and 2.5%
formaldehyde (v/v). The samples were then dehydrated by sequential 30-minute
incubations in 50%, 60%, 70%, 80%, 90%, 95% and 99.5% (v/v) ethanol, followed
by two incubations of 1 hour each in 100% (v/v) ethanol. The dehydrated
samples were set in Technovit 7100 resin (Heraeus Kulzer, Wehrheim/Ts.,
Germany) at room temperature, once in 50% (v/v) resin and twice in 100% resin.
Serial 2-µm thick sections of the plant tissues were cut with a rotary
microtome (MICROM International, Walldorf, Germany), and stained with 0.5%
Methylene Blue and 0.5% borate for 30 seconds.
Scanning electron microscopy
The samples used for scanning electron microscopy were prepared in the same
way as those for the histological analysis. After 100% ethanol treatment, the
pretreated samples were soaked overnight in a solution of amyl acetate
(Aldrich). The material was then critical point dried in liquid
CO2, coated with gold and palladium at 10-20 nm thickness, and
examined at an acceleration voltage of 10-20 kV using a scanning electron
microscope (Model 1420; LEO Electron Microscopy Ltd., Cambridge, England).
Reverse transcription polymerase chain reaction (RT-PCR)
analysis
RNA was prepared from 10-day-old plants grown on Gamborg's B-5 Medium
(GIBCOBRL) using TRI REAGENT (MCA). The cDNA molecules were synthesised from
total cellular RNA using 1 µg of oligo(dT) primer and a first-strand cDNA
Synthesis Kit (Roche). The PCR conditions for each cycle were as follows: 0.5
minutes at 94°C, 0.5 minutes at 55°C, and 1.5 minutes at 72°C. PCR
was performed over 31 cycles for the KNAT1, KNAT2 and KNAT6
cDNAs and 26 cycles for the ß-tubulin cDNA. The primers used for
amplification of the ß-tubulin cDNA
(Kim et al., 1998), KNAT1,
KNAT2 and KNAT6 cDNAs
(Semiarti et al., 2001
) were
as described previously. The amplification of ß-tubulin cDNA was used for
normalization of the RT-PCR (Kim et al.,
1998
).
ß-Glucuronidase (GUS) activity
Plants were grown on Gamborg's B-5 Medium (GibcoBRL) in a culture room
under the long day condition. Histochemical staining of the GUS activity was
performed as described earlier (Jun et
al., 2002).
Analysis of double mutants
We reciprocally crossed bop1-1 mutant plants with as1-1,
as2-2, stm-1 and bp-1 mutants. The double mutants were
identified as novel or additive phenotypes in the F2 generation.
The frequencies at which these plants arose were compatible with a Mendelian
segregation ratio of 1:16 for two genetically unlinked recessive loci.
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RESULTS |
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The mutant plants exhibited pleiotropic phenotypes other than leaf patterning. In the cotyledons and the first two leaves of the mutant, an extended, adventitious, petiole-like organ grew out from the base of the original petiole (Fig. 1B,E,F,)arrowheads; see Fig. 3 for the nature of these ectopic organs). The petiole region of the early rosette leaves of mutants frequently fused with the basal part of the petiole of a cotyledon or adjacent leaf (Fig. 1G). The leaves of the mutant exhibited extended longevity (Fig. 1H). The leaves of a 37-day-old mutant were still green and viable, whereas wild-type leaves had already turned yellow. The vegetative growth of mutant plants was extended compared to that of wild-type plants (Table 1). The average number of rosette and cauline leaves formed on the mutant plant was slightly lower than that of the wild-type plant (Table 1). Mutant flowers were often slightly distorted and had more variable numbers of sepals and petals, as evidenced by a larger standard error in these experiments (Fig. 1I, Table 2). The mutant flower also contained a slightly reduced number of stamens.
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The F1 offspring from crosses between mutant and wild-type
plants had the wild-type phenotype. The F2 offspring from these
plants segregated at a Mendelian segregation ratio of 3:1 (123 wild type: 45
mutant; 2=0.28, P>0.5) for the ectopic blade
phenotype, demonstrating that the mutant phenotype was caused by a single
recessive nuclear mutation. We named the mutation blade-on-petiole 1
(bop1). The genetic locus of the bop1 mutation was mapped to
the lower arm of chromosome 3 at approximately 7.49 cM below the AFC
CAPS locus and 7.28 cM below the TT5 locus.
Electron microscopy of mutant leaves
The leaf phenotypes of the mutant suggested that the mutation played a
critical role in leaf development and patterning. Therefore, we initially
focused our analyses on leaf development. When examined by scanning electron
microscopy, the proximal parts of the cotyledons and the first leaf of
wild-type plants had smooth surfaces without protrusions
(Fig. 2A,C, respectively). In
contrast, the adaxial side of the basal regions of the petioles of
bop1-1 cotyledons showed several outgrowths
(Fig. 2B, arrowheads). Ectopic
organogenesis was also observed on the first rosette leaf of the mutant
(Fig. 2D). The ectopic organs
(Fig. 2D, arrows) that formed
on the adaxial side of the rosette leaf resembled leaf blades with flattened
structures. These ectopic outgrowths appeared to possess dorsoventrality, with
differential growth between the two surfaces of the blade-like structures, and
trichome development on only one of the two surfaces. The abaxial side of the
first rosette leaf of bop1-1 also developed ectopic outgrowths
(Fig. 2F). However, the shapes
of these ectopic outgrowths differed from those on the adaxial side. They
never developed into blade-like structures, but remained as small,
unidentifiable outgrowths. The development of ectopic outgrowths was much more
pronounced on the third rosette leaf of a mutant than on the first rosette
leaf (Fig. 2G,H). Importantly,
numerous trichomes and leaf primordia were found in the proximal region of the
leaf (Fig. 2H). Cauline leaves
of wild-type plants appeared as smooth surfaces in the proximal region
(Fig. 2I). In contrast, ectopic
outgrowths on blade-like structures were observed at the base of the cauline
leaves in the mutant (Fig. 2J).
In addition, many filamentous outgrowths developed in the mutant on the
abaxial and proximal sides of the cauline leaves
(Fig. 2J).
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Cellular phenotypes of the ectopic organs on the petiole
The effect of the bop1-1 mutation was also examined at the
cellular level. On both cotyledons and rosette leaves of 17-day-old mutant
plants the ectopic organs were clearly seen on the adaxial surfaces
(Fig. 3B,D, respectively). The
cellular development of the ectopic outgrowths on the adaxial side resembled
that of the leaf blade. Indeed, the ectopic outgrowths had flattened
structures that resembled those of leaf blades. Methylene Blue staining
revealed clear differences in cellular arrangements between the dorsal and
ventral sides of the ectopic blade-like outgrowths. In addition, trichome
development occurred only on the adaxial side of the ectopic outgrowth that
formed on rosette leaves. The formation of secondary outgrowths on the primary
ectopic outgrowths was observed frequently
(Fig. 3D, arrows).
Interestingly, we noticed that the development of ectopic outgrowths was
associated with the formation of a new structure that resembled a vascular
system (Fig. 3B,D,
arrowheads).
The wild-type petiole has a single, round vascular system in its centre (Fig. 3A,C,E). In contrast, the vascular system of the mutant leaves had a flattened shape (Fig. 3B,D,F). The phloem-and xylem-like tissues were located on the abaxial and adaxial sides of the leaf, respectively (Fig. 3F), which is the arrangement in wild-type petioles (Fig. 3E). The flattened structure appeared to be due to the parallel development of many vascular bundles.
The newly formed, ectopic, adventitious petioles at the base of the original petioles of the cotyledons and the first two leaves of bop1-1 (Fig. 1B,E,F, arrowheads) had features that were intermediate between stems and petioles. First, the vascular system of the ectopic organs had a eustelic arrangement of vascular bundles (Fig. 3G,H), which is typical for stem bundles (see Fig. 7C,D for comparison). Furthermore, a pith-like structure was observed in the region surrounding the central vascular system (Fig. 3G,H), which is another feature of stems (see Fig. 7C,D for comparison). In cross section the ectopic organs were rounder than the wild-type petioles but not as round as the wild-type stem. In addition, the surface was not as smooth as the wild-type stem (Fig. 3G,H; see Fig. 7C for comparison). We conclude that although the ectopic organ grows from the base of the original petiole, it is not a simple petiole.
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Vein patterning in the ectopic blades of the mutant
The electron microscopic and histological analyses indicated that the
ectopic organs of bop1-1 plants had some of the characteristics of
true blades. To confirm this, we examined the venation of wild-type and
bop1-1 leaves. The wild-type cotyledons had a very simple pattern of
veins, with one central midvein (primary vein) and three or four lateral veins
that branched from the midvein (Fig.
4A), as reported previously
(Sieburth, 1999;
Jun et al., 2002
). The
wild-type rosette leaves had a central midvein (primary vein), six to eight
secondary veins that branched from the midvein, and numerous tertiary and
quaternary veins that branched from the secondary and tertiary veins,
respectively (Fig. 4B)
(Nelson and Dengler, 1997
;
Sieburth, 1999
). The
cotyledons of bop1-1 plants had a typical venation pattern of
wild-type cotyledons in the distal regions where no ectopic organogenesis was
observed (Fig. 4C). The ectopic
organ that formed on the proximal part of the mutant cotyledon developed a
reticulated venation pattern that resembled that of wild-type leaf blades.
However, the venation pattern was intermediate between those of cotyledons and
rosette leaves of the wild type in terms of complexity: the ectopic organ in
the mutant cotyledons had more complex venation than the wild-type cotyledon
and less complex venation than wild-type rosette leaves. The ectopic organs
that formed on the rosette leaves of the bop1-1 plants also developed
extensively reticulated venation systems
(Fig. 4D), which were similar
to those seen in the blades of wild-type rosette leaves
(Fig. 4B). However, unlike the
wild-type leaves, the midvein systems in the proximal region of the mutant
rosette leaves revealed an extensive vasculature with numerous parallel vein
elements. The secondary lateral veins branched from the ectopic midveins to
the ectopic outgrowths on the bop1-1 leaves.
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These results show that ectopic outgrowths formed along the petioles of bop1-1 mutants have the features of leaf blades in that they develop the higher order vascular system. The results also show that the bop1-1 mutation severely affects vein development in the proximal region of the midvein. This observation is consistent with the histological analysis of the petiole region of the mutant leaves (see above, Fig. 3B,D,F).
Ectopic meristematic activity in the bop1 mutant
To identify the origin of the abnormal features described above, the
lesions in the pattern and timing of expression of the mutant phenotypes were
examined in early seedlings of the mutant. In 2-day-old seedlings, young and
relatively undifferentiated cells with distinct nuclei were visible in both
wild-type and bop1-1 cotyledons, particularly on the adaxial sides of
cotyledons (Fig. 5A,B). At this
time, there were no noticeable differences in cellular differentiation between
the wild type and the mutant. In addition, scanning electron microscopic
examination of the 2-day-old bop1-1 mutant cotyledons did not reveal
structural differences on the surface of the cotyledon (data not shown). The
first indication of the bop1-1 phenotype was recognized in 3-day-old
seedlings (Fig. 5D,F). At this
stage, most of the cells in the petiole of wild-type cotyledon had already
differentiated (Fig. 5C,E). In
contrast, the petiole of mutant cotyledon had clusters of young cells on the
adaxial side (Fig. 5D,F). The
differentiation status of these clustered cells was evident from the large
nucleus and a lack of large vacuoles, which indicated that the cells were
relatively undifferentiated. This feature was also observed in longitudinal
sections (Fig. 5H). The mutant
cotyledons had more undifferentiated cells along the longitudinal axis than
the wild-type cotyledons. The bop1-1 cotyledonary cells near the
rosette leaf primordia had large nuclei and were stained more strongly than
the surrounding cells (Fig.
5F,H, arrowheads). At 6 days of age, the differences between
wild-type and mutant seedlings became more evident
(Fig. 5I,J). The wild-type
cells had more extensive differentiation and a more developed vascular system
than the mutant cells. The clusters of young cells in the mutant, however,
continued to divide and subsequently produced ectopic outgrowths
(Fig. 5J, arrowheads).
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Taken together, the results show that the mutant petiole cells have ectopic meristematic activities that lead to the production of ectopic organs.
Ectopic organs are also formed on flowers and stems
We investigated whether ectopic organs were formed on other parts of the
mutant plants. Initially, we examined the floral organs, which are modified
leaves. Arabidopsis has complete flowers, with four concentric whorls
of floral organs, i.e., four sepals, four petals, six stamens and two fused
carpels (Fig. 6A)
(Smyth et al., 1990). In
contrast to wild-type petals, filamentous organs of an unidentified nature
developed at the base of mutant sepals
(Fig. 6B,C). The development of
these ectopic organs was more extensive in certain flowers
(Fig. 6D, arrowheads). The
petals of bop1-1 plants had some ectopic outgrowths in their proximal
regions (Fig. 6F), in contrast
to the smooth surfaces of wild-type petals
(Fig. 6E). However, these
outgrowths did not develop into the extensive outgrowths such as the ones
observed in the mutant leaves.
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Ectopic organ development was also found in the stem. The surface of the mutant stem, unlike the smooth surface of the wild-type stem (Fig. 7A), was rough in certain places along the longitudinal direction, and ectopic protrusions were evident in the areas of roughness (Fig. 7B). When the stems were sectioned, the eustelic bundle vasculatures were distinctively observed in the wild-type and mutant stems (Fig. 7C and D, respectively), although the number of vascular bundles was lower in the mutant stem. Furthermore, a few small ectopic outgrowths were observed on the outer surfaces (Fig. 7D,F), which was consistent with the results from electron microscopy. However, the ectopic growths on the stems were not as extensive as those on the leaves. The cellular morphology of the ectopic outgrowths on bop1-1 stems indicated that they were probably formed from outgrowths of cortical and/or epidermal cells. Trichomes often developed on these ectopic outgrowths (Fig. 7D, arrows), which indicated that the outer cells of the ectopic outgrowths resembled wild-type epidermal cells in some aspects.
Ectopic expression of knox genes in bop1-1 leaves
The most pronounced phenotypic abnormalities observed in the
bop1-1 mutant were the lobes on rosette and cauline leaves. These
abnormalities are typical of Arabidopsis plants that ectopically
express class I knox genes
(Lincoln et al., 1994;
Chuck et al., 1996
,
Ori et al., 2000
;
Semiarti et al., 2001
;
Iwakawa et al., 2002
).
Therefore, we examined the expression patterns of class I knox genes
using RNA from 10-day-old seedlings. In the wild-type plants, the KNAT1,
KNAT2, and KNAT6 transcript levels in cotyledons and leaves were
low (KNAT2 and KNAT6) or undetectable (KNAT1)
(Fig. 8A). This result was
consistent with earlier reports (Lincoln
et al., 1994
; Dockx et al.,
1995
; Byrne et al.,
2000
; Semiarti et al.,
2001
). The expression levels of these genes in the cotyledons and
rosette leaves of bop1-1 seedlings were significantly higher than
those in wild-type seedlings, except in the case of the KNAT2 that
showed only a slightly increased expression in the mutant rosette leaf. The
expression levels of all of these genes were higher in the SAM than in leaves,
as reported previously (Lincoln et al.,
1994
; Dockx et al.,
1995
; Byrne et al.,
2000
; Semiarti et al.,
2001
), but there were no notable differences between the wild-type
and mutant SAM regions in the expression of these genes. STM
transcription was not detectable in our experiment in the wild-type or mutant
cotyledons and rosette leaves (data not shown). To complement the above
results, we analysed transgenic plants that expressed the ß-glucuronidase
(GUS) reporter gene under the control of the KNAT1 promoter. In
wild-type plants, expression of KNAT1::GUS was observed in the shoot
meristem and the upper part of hypocotyl
(Fig. 8B), as reported
previously (Ori et al., 2000
).
In wild-type cotyledons and leaves, weak GUS expression was only detected at
the basal tip (Fig. 8C,D,
arrows). In bop1-1 plants, KNAT1::GUS was expressed in a
pattern similar to that in wild-type plants, but in more expanded regions
(Fig. 8E). Examination of the
expression pattern in single leaves of bop1-1 showed that, unlike in
wild-type leaves, KNAT1::GUS was detected in the petioles of
cotyledons and rosette leaves (Fig.
8F,G), suggesting that KNAT1::GUS was misexpressed in
these regions in the bop1-1. Thus, at least for the KNAT1
gene, the promoter activity observed through expression of the GUS reporter
gene is consistent with the result of the RT-PCR analysis.
|
Genetic interaction of bop1-1 with other shoot mutants
Our results suggested that the bop1-1 mutant has a lesion in
controlling expression of class I knox genes. Several mutants are
also known to have defects in the regulation of class I knox genes in
Arabidopsis (Byrne et al.,
2000; Ori et al.,
2000
; Semiarti et al.,
2001
; Iwakawa et al.,
2002
).
AS1 and AS2 promote or maintain the differentiated state
of leaf cells through negative regulation of class I knox genes
(Byrne et al., 2000;
Ori et al., 2000
;
Semiarti et al., 2001
). A
mutation in either AS1 or AS2 produces rumpled and lobed
rosette leaves with leaflet-like organs on their petioles
(Fig. 9A,B,E,F)
(Tsukaya and Uchimiya, 1997
;
Ori et al., 2000
;
Semiarti et al., 2001
).
|
The expression pattern of class I knox genes and the leaf phenotype of the bop1-1 mutant suggests that BOP1 plays a role similar to that of AS1 and AS2 in leaf development. Therefore, we examined the genetic interactions between BOP1 and AS1 and AS2 by constructing double mutants. None of the bop1-1 as1-1 and bop1-1 as2-2 double mutants showed a simple epistatic relationship. Instead, the bop1-1 as1-1 and bop1-1 as2-2 double mutants produced numerous leaflet-like structures along the fasciated leaf petioles (Fig. 9C,D,G,H). On the primary leaflet outgrowths of the double mutant, we observed smaller second- and third-degree leaflet outgrowths that made supercompound leaves. This phenomenon can be viewed as an enhancement of each mutant phenotype.
The other class I knox gene is STM. STM functions in the
formation and maintenance of the SAM in Arabidopsis
(Barton and Poethig, 1993;
Endrizzi et al., 1996
;
Long et al., 1996
). The
stm-1 mutant, a strong mutant allele, fails to establish a SAM: cells
at the site of the presumptive SAM undergo terminal differentiation
(Barton and Poethig, 1993
). As
a result, stm-1 plants lack all the organs that normally develop from
the SAM. Instead, some adventitious, rescued leaves differentiate ectopically
from a region below the fused cotyledons
(Fig. 9I). The rescued leaves
in stm-1 show a nearly wild-type appearance but with abnormal
phyllotaxy. However, in the bop1-1 stm-1 double mutant, rescued
leaves did not fully expand and were lobed
(Fig. 9J). The whole plants
showed severely fasciated morphology with a rough surface and many
leaflet-like structures. These phenotypes, thus, appear more severe than those
expected from a simple addition of the two mutant phenotypes.
BREVIPEDICELLUS (BP) encodes the homeodomain protein KNAT1
(Douglas et al., 2002;
Venglat et al., 2002
). A
loss-of-function mutation in BP (KANT1) is characterized by
compact floral internodes, short pedicles and downward-pointing siliques
(Fig. 9K). The vegetative
rosette leaves of the bop1-1 bp-1 double mutant exhibited a lobed
morphology characteristic for bop1-1. The reproductive shoot of the
double mutant produced a compact inflorescence meristem, short pedicles and
downward-oriented siliques, which are characteristic for bp-1
(Fig. 9L). The phenotypes of
the double mutant, thus, appear additive.
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DISCUSSION |
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These observations show that, following mutation of the BOP1 gene, the petiole cells do not undergo correct developmental specification and can be diverted towards other developmental fates. Thus, BOP1 may function to determine or maintain the fate of petiole cells.
BOP1 regulates the meristematic activity of leaf cells in a
proximodistal manner
The bop1-1 mutant plants developed extensive ectopic outgrowths in
the petiole region as a result of defective cellular differentiation.
Normally, petiole cells undergo cellular determination upon growth. However,
in the mutant plants, some cells escaped determination and remained as
clusters of young cells. These young cells continued dividing and produced
ectopic outgrowths. Thus, the wild-type BOP1 gene is involved in the
control of meristematic activity in the petiole region. However, the
meristematic cells in the mutant petiole did not develop into a new shoot
meristem. Instead, a leaflet-like structure appeared in the early
developmental stages of the mutant cotyledons. This result appears to be
consistent with previous reports that the formation of leaf primordia can be
uncoupled from shoot meristem formation
(Jackson et al., 1994;
Long et al., 1996
;
Tamaoki et al., 1997
;
Nishimura et al., 1999
;
Sentoku et al., 1999
).
Moreover, the ectopic outgrowth of leaf blades was not limited to the
petiole regions of cotyledons and rosette leaves. Ectopic outgrowth of blades
was also observed in cauline leaves that lacked the petiole. This means that
BOP1 is involved in mechanisms other than petiole cell
differentiation. Indeed, the effect of the bop1-1 mutation was seen
in all leaves, including cotyledons, rosettes and cauline leaves. A common
feature found among all of these leaves in the mutant was that the proximal
region produced ectopic blades. Thus, the bop1-1 defect in leaves may
be explained in terms of the disruption of cellular differentiation along the
proximodistal axis. In Arabidopsis, the leaves mature basipetally,
from the tip to the base of the leaf (Pyke
et al., 1991; Donnelly et al.,
1999
). Thus, cells of the proximal petiole region differentiate
later and have a developmental competence that is different from that of
distal blade cells. The combination of a less differentiated proximal region
and bop1-1 mutation may have caused the mutant leaf phenotype to
produce ectopic blades in the petioles of the cotyledons and rosette leaves
and in the proximal regions of the cauline leaves. Developmental lesions were
even observed in petals. Although the ectopic outgrowths in petals were not
extensive, they appeared primarily in the proximal regions. Therefore, we
speculate that BOP1 controls meristematic activity in a proximodistal
manner in a broad range of leaf types.
BOP1 functions within a specific developmental context
The meristematic cells that developed on the petiole of the mutant had
limited developmental fates and were not totipotent. The ectopic outgrowths
that developed on the adaxial side of the petiole were leaf blades. The base
of the original petiole in the mutant cotyledon and first two rosette leaves
had some stem features, particularly with respect to the morphology of the
vascular system. Other structures, such as a new shoot or inflorescence, did
not develop.
Furthermore, the ectopic organs on the abaxial side of the mutant petiole had different characteristics than those on the adaxial side. While the outgrowths on the adaxial side of the petiole developed mainly into blades, those that formed on the abaxial side did not develop beyond small unidentifiable protrusions. This observation implies that the dorsoventrality of the petiole region of the mutant was maintained. This phenomenon was also observed in the ectopic blades that were generated on the adaxial side of the mutant petioles. The ectopic blades maintained dorsoventrality, as evidenced by trichome formation on the adaxial side only. The ectopic vascular system of the petiole also showed dorsoventrality, such that the xylem cells were oriented towards the adaxial side and the phloem cells were oriented towards the abaxial side. Thus, BOP1 appears to be minimally, if at all, involved in the control of dorsoventrality.
There were also some position-dependent differences in the developmental
defects seen in the leaves. A clear example of this was the extended
petiole-like region that occurred exclusively in the cotyledons and the first
two leaves. The degree of ectopic organ formation also differed depending on
the leaf position, which may be due to the heteroblastic nature of leaf
development. In most plant species, including Arabidopsis, the leaves
that form early in shoot development (juvenile leaves) are morphologically and
physiologically different from the leaves that appear later (adult leaves)
(Telfer et al., 1997;
Kerstetter and Poethig, 1998
;
Tsukaya et al., 2000
). Thus,
the production processes for juvenile and adult leaves are modulated by
different developmental programs
(Kerstetter and Poethig,
1998
). The extended petiole-like outgrowth of the bop1-1
mutant may have been caused by a combination of prolonged and ectopic
meristematic activity that is unique to the developmental program of juvenile
leaves.
As mentioned above, the mutation did not completely disrupt the developmental fate of the leaf cells, although it remains possible that this is a characteristic of this specific allele. Thus, although BOP1 regulates the meristematic activity of leaf cells, it functions within a given developmental context that encompasses dorsoventrality, proximodistality and heteroblasty. In other words, BOP1 controls the developmental fate of leaf cells in association with other factors that are involved in leaf development.
Ectopic leaf blade formation on the petiole, which was the most prominent phenotype observed for the bop1-1 mutant, may be explained as follows. The mutation caused a disturbance in cellular specification along the proximodistal axis. The mutant petiole (essentially the proximal part of the leaf) displayed prolonged meristematic activity while escaping the differentiation program. Thus, these meristematic cells produced leaf blades by a developmental program that was specified in the leaf.
BOP1 functions in locations other than leaf organs
The mutant phenotypes of the bop1-1 plants was apparent in organs
other than leaves, such as stems and floral organs. Ectopic outgrowths were
observed in the proximal parts of petals and ectopic filamentous organs
developed at the base of the sepals. Ectopic outgrowths were also observed on
the surface of the stem. The morphology of these ectopic outgrowths did not
resemble that of any wild-type organ, and they appeared to be simply
unidentifiable overgrowths of cells. Thus, BOP1 also functions in the
developmental control of organs other than leaves. The defects in these other
organs are probably due to the ectopic meristematic activities of the mutant
cells, as is the case in leaves.
Interestingly, the number of floral organs in mature flowers was different in bop1-1 plants than in wild-type plants. The morphological defects in bop1-1 mutant flowers mainly appeared in the organs of the first and second whorl. Therefore, BOP1 may have a role in the development of floral organs in addition to controlling the meristematic activities of lateral organs and stems.
A possible mechanism for BOP1 function
Our results suggest that BOP1 is crucial for cellular
differentiation by controlling meristematic activity in various organs and
during certain developmental stages. How does BOP1 control this
process?
The bop1-1 mutation led to a wide range of defects that were
reminiscent of transgenic Arabidopsis lines that overexpress class I
knox genes (Lincoln et al.,
1994; Chuck et al.,
1996
; Pautot et al.,
2001
). Expression of the three class I knox genes,
KNAT1, KNAT2 and KNAT6, was increased in the cotyledons and
rosette leaves of the mutant, in accordance with the phenotype. Thus,
BOP1 is involved in controlling the expression of class I
knox genes in these organs. Plants with defective expression of class
I knox genes exhibit developmental abnormalities along the
proximodistal axis, such as displacement of the proximal features of leaves
(stipule, sheath and petiole) to more distal tissues (blades)
(Becraft and Freeling, 1994
;
Jackson et al., 1994
;
Lincoln et al., 1994
;
Schneeberger et al., 1995
;
Chuck et al., 1996
;
Tamaoki et al., 1997
;
Ori et al., 2000
). These
features are similar to those of the bop1-1 mutant. In fact it would
seem that the distal lamina of the leaf is now displaced to more proximal
regions. This leads us to suggest that the ectopic outgrowths in
bop1-1 mutants are attributable to the failure of BOP1 to
perform spatial and temporal regulation of class I knox genes. This
proposition is supported by the synergistic genetic interaction of the
bop1-1 mutation with the mutations in other knox gene
regulators, AS1 and AS2.
The bop1 mutation was found to be recessive in terms of the ectopic outgrowth phenotype. Thus, the mutant phenotype is probably the result of a functionally defective wild-type protein. In this regard, BOP1 may negatively regulate the expression of class I knox genes and the resulting meristematic activity of leaf cells. However, molecular analysis of the allele is needed to confirm this proposition.
Although BP is misexpressed in the bop1-1 mutant, the
analysis of the bop1-1 bp-1 double mutant showed a rather simple
additive genetic interaction. This suggests that misexpression of BP
may be neither responsible nor sufficient to induce the bop1-1
phenotypes. One possible explanation would be that the lobed-leaf phenotypes
of the bop1-1 mutant are caused by misexpression of class I
knox genes other than BP or by combined misexpression of
three class I knox genes, KNAT1, KNAT2 and KNAT6,
as has been suggested for the as1 and as2 mutants
(Byrne et al., 2002). In this
regard, it is notable that the bop1-1 stm-1 double mutant shows some
degree of synergistic mutant phenotype.
The morphology of the leaf organs and the misregulation of class I knox genes in the as1 and as2 mutants were similar to those found in the bop1-1 mutant. This indicates that these genes may function in related pathways that control leaf morphogenesis and regulate class I knox gene expression. Furthermore, the double mutants (bop1-1 as1-1 and bop1-1 as2-2) showed synergistic mutant phenotypes. Although it is difficult to precisely define genetic relationships based on the morphological phenotypes of the double mutants, it is clear that they interact both genetically and functionally to control leaf development. An interesting hypothesis is that both combinations of BOP1 and AS1 and/or BOP1 and AS2 control the expression of class I knox genes in a synergistic manner and thereby control leaf development.
The bop1 mutants had some leaf phenotypes that were not observed
in the as1 or as2 mutants. Unlike the case of bop1,
no lobed structures were observed on the cotyledons or the first two leaves of
as1 and as2 mutant plants at early stages of development
(Tsukaya and Uchimiya, 1997;
Ori et al., 2000
;
Semiarti et al., 2001
). The
occurrence of petiole-like outgrowths at the base of cotyledons and the first
two leaves was also not observed in the as1 and as2 mutants.
In addition, as1 se and as2 se double mutants had phenotypes
that were very similar to those of KNAT1 overexpressers
(Ori et al., 2000
). In
contrast, the bop1 mutation did not appear to interact with the
se mutation (data not shown). Thus, BOP1 may have functions
that are distinct from those of AS1 and AS2 as well as the
functions that are shared among them in controlling leaf development.
The known regulators of class I knox genes in higher plants
include the RS2 of Zea mays, the AS1 gene of
Arabidopsis and PHAN gene of Antirrhinum majus
(Schneeberger et al., 1998;
Byrne et al., 2000
;
Ori et al., 2000
;
Semiarti et al., 2001
;
Waites et al., 1998
;
Timmermans et al., 1999
;
Tsiantis et al., 1999
;
Byrne et al., 2000
). The lesion
in the rs2 and as1 mutants is characterized by alterations
in their proximodistal identity
(Schneeberger et al., 1995
;
Timmermans et al., 1999
;
Tsiantis et al., 1999
;
Ori et al., 2000
). The
as1 mutants also exhibit some defects in the dorsoventral identity
(Ori et al., 2000
). The
phan mutation can be explained by defects in proximodistal and
dorsoventral character (Waites and Hudson,
1995
; Waites et al.,
1998
). In addition to these genes, KANADY (KAN)
also plays a major role in the promotion of abaxial cell fates
(Kerstetter et al., 2001
;
Eshed et al., 2001
). In
kan1 kan2 double mutants, ectopic outgrowths develop on their abaxial
side. Although bop1-1 mutants developed some ectopic outgrowths in
the abaxial side of the leaf, mostly in the proximal region, we suggest that
the effect of the bop1-1 mutation on dosoventrality is minimal, if
any. This is supported by the normal cellular and vascular arrangement and by
trichome formation only on the adaxial side in ectopic young leaves.
The cotyledons of the bop1-1 embryos did not exhibit any distinct
morphological defects prior to germination. Instead, the mutant cotyledons
produced ectopic blades on their petioles only a few days after germination,
at which stage cell expansion and differentiation were taking place in the
wild-type petioles (Tsukaya et al.,
1994). In addition, a clear difference in cellular morphology,
i.e., the appearance of meristematic clusters of cells at the bases of
cotyledonary petioles, was observed in 3-day-old seedlings of bop1-1.
Although we cannot completely rule out the possibility that the gene functions
at an earlier stage, it appears that the major effect of BOP1 is on
post-embryonic development.
The data presented here strongly suggest that BOP1 is a key component in the differentiation of cellular states and in the specification of spatially and temporally regulated developmental patterns in plant leaves. Molecular analyses of the nature and function of the gene product should provide further insights into this important phenomenon.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Barton, M. K. and Poethig, R. S. (1993).
Formation of the shoot apical meristem in Arabidopsis thaliana: an
analysis of development in the wild type and in the shootmeristemless
mutant. Development 119,823
-831.
Becraft, P. W. and Freeling, M. (1994). Genetic
analysis of Rough sheath1 developmental mutants of maize.
Genetics 136,295
-311.
Byrne, M. E., Barley, R., Curtis, M., Arroyo, J. M., Dunham, M., Hudson, A and Martienssen, R. A. (2000). Asymmetric leaves mediates leaf patterning and stem cell function in Arabidopsis. Nature 408,967 -971.[CrossRef][Medline]
Byrne, M. E., Simorowski, J. and Martienssen, R. A.
(2002). ASYMMETRIC LEAVES1 reveals knox gene
redundancy in Arabidopsis. Development
129,1957
-1965.
Chuck, G., Lincoln, C. and Hake, S. (1996).
KNAT1 induces lobed leaves with ectopic meristems when overexpressed
in Arabidopsis. Plant Cell
8,1277
-1289.
Dockx, J., Quaedvlieg, N., Keultjes, G., Kock, P., Weisbeek, P. and Smeekens, S. (1995). The homeobox gene ATK1 of Arabidopsis thaliana is expressed in the shoot apex of the seedling and in flowers and inflorescence stems of mature plants. Plant Mol. Biol. 28,723 -737.[Medline]
Donnelly, P. M., Bonetta, D., Tsukaya, H., Dengler, R. E. and Dengler, N. G. (1999). Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev. Biol. 215,407 -419.[CrossRef][Medline]
Douglas, S. J., Chuck, G., Denger, R. E., Pelecanda, L. and
Riggs, C. D. (2002). KNAT1 and ERECTA
regulate inflorescence architecture in Arabidopsis. Plant
Cell 14,1
-13.
Endrizzi, K., Moussian, B., Haecker, A., Levin, J. Z. and Laux, T. (1996). The SHOOT MERISTEMLESS gene is required for maintenance of undifferentiated cells in Arabidopsis shoot and floral meristems and acts at a different regulatory level than the meristem genes WUSCHEL and ZWILLE. Plant J. 10,101 -113.[CrossRef]
Eshed, Y., Baum, S. F., Perea, J. V. and Bowman, J. L. (2001). Establishment of polarity in lateral organs of plants. Curr. Biol. 11,1251 -1260.[CrossRef][Medline]
Hamada, S., Onouchi, H., Tanaka, H., Kudo, M., Liu, Y. G., Shibata, D., MacHida, C. and Machida, Y. (2000). Mutations in the WUSCHEL gene of Arabidopsis thaliana result in the development of shoots without juvenile leaves. Plant J. 24,91 -101.[CrossRef][Medline]
Hareven, D., Gutfinger, T., Parnis, A., Eshed, Y. and Lifschitz, E. (1996). The making of a compound leaf: Genetic manipulations of leaf architecture in tomato. Cell 84,735 -744.[Medline]
Iwakawa, H., Ueno, Y., Semiarti, E., Onouchi, H., Kojima, S.,
Tsukaya, H., Hasebe, M., Soma, T., Ikezaki, M., Machida, C. and Machida,
Y. (2002). The ASYMMETRIC LEAVES2 gene of
Arabidopsis thaliana, required for formation of a symmetric flat leaf
lamina, encodes a member of a novel family of proteins characterized by
cysteine repeats and a leucine zipper. Plant Cell
Physiol. 43,467
-478.
Jackson, D., Veit, B. and Hake, S. (1994).
Expression of maize KNOTTED1 related homeobox genes in the shoot
apical meristem predicts patterns of morphogenesis in the vegetative shoot.
Development 120,405
-413.
Jun, J. H., Ha, C. M. and Nam, H. G. (2002).
Involvement of the VEP1 Gene in vascular strand development in
Arabidopsis thaliana. Plant Cell Physiol.
43,323
-330.
Kerstetter, R. A. and Poethig, R. S. (1998). The specification of leaf identity during shoot development. Annu. Rev. Cell Dev. Biol. 14,373 -398.[CrossRef][Medline]
Kerstetter, R. A., Bollman, K., Taylor, R. A., Bomblies, K. and Poethig, R. S. (2001). KANADI regulates organ polarity in Arabidopsis. Nature 411,706 -709.[CrossRef][Medline]
Kim, G.-T., Tsukaya, H. and Uchimiya, H.
(1998). The ROTUNDIFOLIA3 gene of Arabidopsis
thaliana encodes a new member of the cytochrome P450 family that is
required for the regulated polar elongation of leaf cells. Genes
Dev. 12,2381
-2391.
Konieczny, A. and Ausubel, F. M. (1993). A procedure for mapping Arabidopsis mutations using codominant ecotype-specific PCR-based markers. Plant J. 4, 403-410.[CrossRef][Medline]
Koornneef, M. and Stam, P. (1992). Genetic Analysis. In Methods in Arabidopsis Research (ed. C. Koncz, N.-H. Chua and J. Schell), pp. 85-99. Singapore: World Scientific.
Lincoln, C., Long, J., Yamaguchi, J., Serikawa, K. and Hake,
S. (1994). A Knotted1-like homeobox gene in
Arabidopsis is expressed in the vegetative meristem and dramatically
alters leaf morphology when overexpressed in transgenic plants.
Plant Cell 6,1859
-1876.
Long, J. A., Moan, E. I., Medford, J. I. and Barton, M. K. (1996). A member of the KNOTTED class of homeodomain proteins encoded by the SHOOTMERISTEMLESS gene of Arabidopsis.Science 379,66 -69.[CrossRef]
Matsuoka, M., Ichikawa, H., Saito, A., Tamda, Y., Fujimura, T.
and Kano Murakami, Y. (1993). Expression of a rice homeobox
gene causes altered morphology of transgenic plants. Plant
Cell 5,1039
-1048.
Nelson, T. and Dengler, N. (1997). Leaf
vascular pattern formation. Plant Cell
9,1121
-1135.
Nishimura, A., Tamaoki, M., Sato, Y. and Matsuoka, M. (1999). The expression of tobacco knotted1-type class 1 homeobox genes correspond to regions predicted by the cytohistological zonation model. Plant J. 18,337 -347.[CrossRef][Medline]
Oh, S. A., Lee, S. Y., Chung, I. K., Lee, C. H. and Nam, H. G. (1996). A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. Plant Mol. Biol. 30,739 -754.[Medline]
Ori, N., Eshed, Y., Chuck, G., Bowman, J. L. and Hake, S.
(2000). Mechanisms that control knox gene expression in
the Arabidopsis shoot. Development
127,5523
-5532.
Pautot, V., Dockx, J., Hamant, O., Kronenberger, J., Grandjean,
O., Jublot, D. and Traas, J. (2001). KNAT2: evidence for a
link between knotted-like genes and carpel development.
Plant Cell 13,1719
-1734.
Pyke, K. A., Marrison, J. L. and Leech, R. M. (1991). Temporal and spatial development of the cells of the expanding first leaf of Arabidopsis thaliana (L.) Heynh. J. Exp. Bot. 42,1407 -1416.
Schneeberger, R. G., Becraft, P. W., Hake, S. and Freeling, M. (1995). Ectopic expression of the knox homeo box gene rough sheath1 alters cell fate in the maize leaf. Genes Dev. 9,2292 -2304.[Abstract]
Schneeberger, R., Tsiantis, M., Freeling, M. and Langdale, J.
A. (1998). The rough sheath2 gene negatively
regulates homeobox gene expression during maize leaf development.
Development 125,2857
-2865.
Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H., Machida, C.
and Machida, Y. (2001). The ASYMMETRIC LEAVES2 gene
of Arabidopsis thaliana regulates formation of a symmetric lamina,
establishment of venation and repression of meristem-related homeobox genes in
leaves. Development 128,1771
-1783.
Sentoku, N., Sato, Y., Kurata, N., Ito, Y., Kitano, H. and
Matsuoka, M. (1999). Regional expression of the rice
kn1-type homeobox gene family during embryo, shoot, and flower
development. Plant Cell
11, 1651-1664. in
monocot and dicot plants. Science 284, 154-156.
Sentoku, N., Sato, Y. and Matsuoka, M. (2000). Overexpression of rice OSH genes induces ectopic shoots on leaf sheaths of transgenic rice plants. Dev. Biol. 220,358 -364.[CrossRef][Medline]
Sieburth, L. E. (1999). Auxin is required for
leaf vein pattern in Arabidopsis. Plant Physiol.
121,1179
-1190.
Sinha, N. R., Williams, R. E. and Hake, S. (1993). Overexpression of the maize homeobox gene, KNOTTED-1, causes a switch from determinate to indeterminate cell fates. Genes Dev. 7,787 -795.[Abstract]
Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M.
(1990). Early flower development in Arabidopsis. Plant
Cell 2,755
-767.
Steeves, T. A. and Sussex, I. M. (1989). Patterns in Plant Development. Cambridge: Cambridge University Press.
Tamaoki, M., Kusaba, S., Kano-Murakami, Y. and Matsuoka, M. (1997). Ectopic expression of a tobacco homeobox gene, NTH15, dramatically alters leaf morphology and hormone levels in transgenic tobacco. Plant Cell Physiol. 38,917 -927.[Medline]
Telfer, A., Bollman, K. M. and Poethig, R. S.
(1997). Phase change and the regulation of trichome distribution
in Arabidopsis thaliana. Development
124,645
-654.
Timmermans, M. C., Hudson, A., Becraft, P. W. and Nelson, T.
(1999). ROUGH SHEATH2: a Myb protein that represses
knox homeobox genes in maize lateral organ primordia.
Science 284,151
-153.
Tsiantis, M., Schneeberger, R., Golz, J. F., Freeling, M. and
Langdale, J. A. (1999). The maize rough sheath2 gene
and leaf development programs in monocot and dicot plants.
Science 284,154
-156.
Tsukaya, H., Tsuge, T. and Uchimiya, H. (1994). The cotyledon: a superior system for studies of leaf development. Planta 195,309 -312.
Tsukaya, H. and Uchimiya, H. (1997). Genetic analysis of the formation of the serrated margin of leaf blades in Arabidopsis: combination of a mutational analysis of leaf morphogenesis with the characterization of a specific marker gene expressed in hydathodes and stipules. Mol. Gen. Genet. 256,231 -238.[CrossRef][Medline]
Tsukaya, H. (1998). Genetics evidence for polarities that regulate leaf morphogernesis. J. Plant Res. 111,113 -119.
Tsukaya H., Shoda K., Kim G. T. and Uchimiya H. (2000). Heteroblasty in Arabidopsis thaliana (L.) Heynh. Planta 210,536 -542.[CrossRef][Medline]
Tsukaya, H. (2002a). Interpretation of mutants in leaf morphology: genetic evidence for a compensatory system in leaf morphogenesis that provides a new link between Cell and Organismal theory. Int. Rev. Cytol. 217,1 -39.[Medline]
Tsukaya, H. (2002b). The leaf index:
heteroblasty, natural variation, and the genetic control of polar processes of
leaf expansion. Plant Cell Physiol.
43,372
-378.
Tsukaya, H. (2002c). Leaf Development In The Arabidopsis Book (ed. C. R. Somerville and E. M. Meyerowitz) American Society of Plant Biologists (http://www.aspb.org/downloads/arabidopsis/tsukayafinal.pdf).
van der Graaff, E., Dulk-Ras, A. D., Hooykaas, P. J. and Keller,
B. (2000). Activation tagging of the LEAFY PETIOLE
gene affects leaf petiole development in Arabidopsis thaliana.Development 127,4971
-4980.
Venglat, S. P., Dumonceaux, T., Rozwadowski, K., Parnell, L.,
Babic, V., Keller, W., Martienssen, R., Selvaraj, G. and Datla, R.
(2002). The homeobox gene BREVIPEDICELLUS is a key
regulator of inforescence architecture in Arabidopsis. Proc. Natl.
Acad. Sci. USA 99,4730
-4735.
Waites, R. and Hudson, A. (1995).
phantastica: a gene required for dorsoventrality of leaves in
Antirrhinum majus. Development
121,2143
-2154.
Waites, R., Selvadurai, H. R. N., Oliver, I. R. and Hudson, A. (1998). The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum. Cell 93,779 -789.[Medline]