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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02604
1 Department of Biological Sciences, KAIST, Daejeon 305-701, Korea.
2 Division of Molecular Biotechnology, Dong-A University, Busan 604-714,
Korea.
3 Environmental Biotechnology Research Center, Gyeongsang National University,
Jinju 660-701, Korea.
4 Faculty of Biotechnology, College of Applied Life Sciences, Cheju National
University, Jeju 690-756, Korea.
5 Laboratory of Environmental Biotechnology, Korea Research Institute of
Bioscience and Biotechnology (KRIBB), 52 Eoen-dong, Yuseong-gu, Daejeon,
305-806, Korea.
Authors for correspondence (e-mail:
gchoi{at}kaist.ac.kr;
wichung{at}kaist.ac.kr)
Accepted 30 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Polar cell elongation, Leaf development, LONGIFOLIA
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leaf morphogenesis is conceptually divided into three processes: leaf
initiation, establishment of polarity and leaf expansion. During initiation,
the leaf primordia are formed at the periphery of the shoot apical meristem
(SAM). Polarity is established with regards to the three main axes: the
proximodistal axis (also called the apicobasal axis, and herein referred to as
the leaf-length direction); the dorsoventral axis (also called the
adaxial-abaxial axis); and the left/right axis (also called the mediolateral
axis, and herein referred to as the leaf-width direction). Subsequently, the
leaves expand to their final shapes and sizes
(Sinha, 1999
;
Bowman et al., 2002;
Kim and Cho, 2006).
The final shape and size of an adult leaf is modulated by leaf expansion,
which is in turn dependent upon cell division and expansion. The importance of
cell proliferation on leaf morphology can be observed in Arabidopsis
transgenic lines expressing cyclin-dependent kinase inhibitor 1
(ICK1) or Kip-related protein 2 (KRP2), which
inhibit leaf cell proliferation through interactions with CDKA1/cyclin
complexes (Wang et al., 2000;
Verkest et al., 2005).
Overexpression of these genes has been associated with the development of
small serrated leaves that have reduced cell numbers. However, although this
suggests that general cell proliferation is crucial for the determination of
leaf shape and size, changes in cell proliferation do not always result in
altered leaf shapes. For example, overexpression of ABP1 results in
decreased cell proliferation but increased cell volume without gross changes
in leaf morphology (Jones et al.,
1998). Furthermore, studies have shown that increased cell numbers
could be compensated for by decreases in cell size, or vice versa (Hemerley et
al., 1995; De Veylder et al.,
2001; Ullah et al.,
2001; Tsukaya,
2003). In addition, other factors regulate leaf morphogenesis by
affecting polarity-dependent cell proliferation. Recently,
ROTUNDIFOLIA4 (ROT4), which encodes a membrane-bound small
peptide, was shown to inhibit cell proliferation in only the leaf-length
direction in Arabidopsis. Conversely, it was also shown that ectopic
expression of ROT4 caused short leaves
(Narita et al., 2004). Thus,
both general and specific cell proliferation can affect leaf morphology.
Cell expansion also plays an important role in the leaf expansion required
for the proper formation of a mature leaf. Genetic analyses have indicated
that cell expansion is regulated independently in the leaf-length and
leaf-width directions. Mutation of the ANGUSTIFOLIA (AN)
gene, which encodes a C-terminal binding protein (CtBP) that is responsible
for regulating the arrangement of microtubules in leaf cells, caused a defect
in cell expansion in the leaf-width direction, resulting in narrower leaves
(Kim et al., 2002). By
contrast, the mutation of ROT3, which encodes a P450 involved in
brassinosteroid (BR) biosynthesis, decreased cell expansion in the leaf-length
direction, resulting in shorter leaves
(Kim et al., 1998). This
finding of an association between ROT3 and leaf expansion provided
evidence that BR promotes cell expansion in the leaf-length direction
(Kim et al., 1998;
Kim et al., 1999;
Kim et al., 2005). Similar
findings were observed in other BR biosynthetic mutants, including
de-etiolated 2 (det2) and dwarf4 (dwf4) -
both of which show shortened leaves
(Altmann, 1998;
Fujioka et al., 1997;
Li and Chory, 1997;
Azpiroz et al., 1998;
Choe et al., 1998). Other
factors that affect cell expansion, including auxins and light, have also been
found to regulate leaf morphology (Timpte
et al., 1992; Qin et al.,
1997; Franklin et al.,
2003; Kozuka et al.,
2005)
The different leaf morphologies observed in the various
Arabidopsis ecotypes are likely to be determined by multiple factors.
A genetic screen of leaf-shape mutants from 5770 M1 EMS-mutagenized
Arabidopsis plants generated 94 complementation groups that showed
altered leaf morphology (Berna et al.,
1999). A quantitative trait loci (QTL) analysis using recombinant
inbred Ler and Col-4 lines identified 21 loci that affect six different
characteristics of the adult leaf
(Perez-Perez et al., 2002).
These previous studies suggest that leaf morphogenesis is a complex process
requiring the interplay of many different genetic components. However,
although many factors involved in leaf morphogenesis have been identified,
other genetic components remain to be examined before the determination of
leaf shape is fully understood. Here, we have screened Arabidopsis
leaf-shape mutants in an effort to discover new genetic components and to
report the identification of two new genetic components, LONGIFOLIA1
(LNG1) and LONGIFOLIA2 (LNG2), that are responsible
for regulating leaf morphology by promoting cell expansion in the leaf-length
direction in a ROT3-independent fashion.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), using Agrobacterium harboring the
activation-tagging vector pSKI1015 (Weigel
et al., 2000). The lng1-1D mutant plant was selected from
herbicide (ammonium glufosinate, known as Basta)-resistant transgenic lines.
Loss-of-function mutant alleles (lng1-2, lng1-3, lng2-1 and
lng2-2) were obtained from the SALK Institute Genomic Analysis
Laboratory (salk_107002, salk_135585, salk_067658, salk_034645 and
salk_130006, respectively). For generation of the lng1 lng2 double
homozygous mutant, homozygous lng1 plants were crossed with
homozygous lng2 plants. Homozygous lng1 lng2 double mutants
were identified by PCR analysis from the F2 population using LNG1-
and LNG2-specific primers (LBa1, 5'-TGG TTC ACG TAG TGG GCC ATC
G-3' and LBb1: 5'-GCG TGG ACC GCT TGC TGC AAC T-3').
Expression analysis
For northern analysis, total RNA was extracted from 3-week-old seedlings
using the RNeasy plant mini kit (Qiagen) and 20 µg total RNA was resolved
from each sample by 1.2% agarose gel electrophoresis. The separated RNAs were
transferred to a nylon membrane (Hybond-N; Amersham), and the filters were
prehybridized for 1-2 hours at 42°C in 50% formamide, 5xSSPE,
5xDenhardt's solution, 0.1% SDS and 100 µg/ml denatured salmon sperm
DNA. For hybridization, a 32P-dCTP-labeled probe (described below)
was added to the prehybridization buffer and the filter was hybridized
overnight. Washes were performed twice at room temperature for 10 minutes in
2xSSC and 0.1% SDS, once at 65°C for 15 minutes in 1xSSC and
0.1% SDS, and twice at 65°C for 15 minutes in 0.1xSSC and 0.1% SDS.
Results were analyzed with a scanning densitometer (Pharmacia), with
adjustments being made for small differences in RNA loading. The probes were
generated by RT-PCR from total RNA (described below) using specific primers
(At5g15580 F, 5'-TTG GTT GCC CAC TGA GCA TC-3' and R, 5'-ACA
AGT ACA TCT CAG AGA TA-3'; At5g15600 F, 5'-ATG GGG AAA GCT AGA GGA
GTT-3' and R, 5'-TCA TTT GTC AGA TCC ACA AGG-3'; At5g15610
F, 5'-CCT CTT CAA TCT ATA TAA TCT-3' and R, 5'-CTG AAA TAT
ACT GGG AGC CTT-3'). The amplified fragments were cloned into the pGEM-T
easy vector.
For RT-PCR analysis, Arabidopsis plants were grown on 1/2 MS medium and total RNA was extracted from 23-day-old plants using the total RNA Isolation System (Qiagen). RT-PCR was performed with the Access RT-PCR System (Invitrogen), according to the manufacturer's protocol. Briefly, 5 µg tRNA was reverse-transcribed to cDNA, diluted tenfold and amplified with the following specific primers: LNG1 F, 5'-ATG TCG GCG AAG CTT TTG TAT A-3' and R, 5'-GTC TCT TTC AAC TTG GCC CCT G-3'; LNG2 F, 5'-CGA CTT GAG GAG TCT AAG GTC-3' and R, 5'-GGA TCC CTG ATA ACC TTA AAA AAA TTA G-3'; actin (control) F, 5'-ATG ACT CAA ATC ATG TTT GAG ACC TTC-3' and R, 5'-ACC TTA ATC TTC ATG CTG CTT GGA GC-3'; AN F, 5'-TGA GAC GGT GCC GTG GTA TGG-3' and R, 5'-GTT GCC TAC TGG TGG ATT CC-3'; ROT3F, 5'-TGA GGC CTC GGT TGT TCT CA-3' and ROT3R, 5'-TCT CTA CGA TCT TTC CGC TG-3'; ATHB13-550R, 5'-TTC TGT TAC TGC AAG ATC CTT CAG T-3'; ATHB13-1F, 5'-ATG TCT TGT AAT AAT GGA ATG TC-3'; CYP90D1-F, 5'-GCA CAA GTT TTT GTC GGA ATC C-3'; CYP90D1-R, 5'-GTC GAT CAT ATT GTT AGC AAT C-3'.
Anatomical analysis
Samples for the anatomical analysis were examined as described by Tsukaya
et al. (Tsukaya et al., 1993).
All plants were grown under identical conditions. The fifth leaves were fixed
overnight in FAA solution (5% acetic acid, 45% ethanol and 5% formaldehyde),
dried under a vacuum for 20 minutes and dehydrated in a graded ethanol series
(50, 60, 70, 80, 90, 95, 99 and 100%) at room temperature. The dehydrated
samples were incubated at room temperature in 50% Tchnovit 7100 resin (Kulzer
and Company) and 50% ethanol for 2 hours, and then in 100% resin overnight.
The resin was hardened at room temperature and the resin-impregnated leaves
were sectioned at 4 µm through the center of the broad blade for
longitudinal sections and through the widest part of the blade for transverse
sections. The slices were fixed to glass slides, dried at room temperature,
stained with 0.1% Toluidine Blue in 0.1 M phosphate buffer (pH 7.0), and
observed and photographed under a microscope.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For quantitative comparison, we measured the lengths of various organs and
structures, including primary roots, hypocotyls, petioles, leaf blades,
internodes, siliques and seeds. The fully expanded leaf blades of
lng1-1D mutants were 43% greater in length, but 29% narrower in
width, compared with the wild type, suggesting that the mutation caused
increased longitudinal leaf expansion with a concomitant decrease in
transverse expansion (see Table S1 in the supplementary material). Consistent
with the increased leaf length, the leaf petioles of lng1-1D mutants
were 19% longer than those of the wild type, and the mutant seeds were longer
(19%) and slightly narrower (6%) than those of wild type. Although there was
no significant difference in stem length, the stem diameter was noticeably
smaller (
25%) than that of wild-type plants. Taken together, the results
indicated that the dominant lng1-1D mutation affects the longitudinal
expansion of various parts of the plant, including the leaf blade, leaf
petiole, flower, cotyledon and seed.
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). To
investigate the kinetics of leaf expansion in the lng1-1D mutant, we
monitored changes over time in the lengths and widths of fifth rosette leaf
blades from at least 15 plants grown in soil under identical environmental
conditions. As shown in Fig.
2A, the expansion rate of leaf blade width was initially
comparable between lng1-1D and wild-type plants, but decreased faster
in the lng1-1D mutant, leading to narrower mature leaves in the
mutant. By contrast, the expansion rate of leaf blade length was higher in the
lng1-1D mutant from the beginning, resulting in a greater final
length of lng1-1D leaf blades compared with wild type
(Fig. 2B). Expansion of the
fifth leaves stopped around 39 days after sowing, both in wild type and
lng1-1D mutants, suggesting that the mutation altered the expansion
rate but not the timing of expansion during leaf development.
lng1-1D plants have elongated cells in their leaves, petals and siliques
Leaf expansion is determined by cell proliferation and expansion. To
investigate whether cell expansion was affected in the lng1-1D
mutant, we subjected the fifth rosette leaves of mutant and wild-type plants
to scanning electron microscopy (SEM). Magnification of the leaf midvein area
revealed that the adaxial epidermal cells in the midveins and the epidermal
cells of nearby midveins were longitudinally elongated in lng1-1D
leaves compared with wild type (Fig.
3A,B,D,E). On the adaxial side of the leaf blade, the epidermal
cells of the lng1-1D mutant plants were longitudinally elongated
compared with the wild type (Fig.
3C,F). To semi-quantify differences in cell size, we measured the
lengths and widths of more than 150 adaxial epidermal cells in mutant and
wild-type plants. The wild-type and lng1-1D cells had lengths of
88.0±11.6 µm (six plants) and 132.6±19.1 µm (five plants),
respectively, and widths of 83.0±7.4 µm (six plants) and
59.8±9.4 µm (five plants), respectively. The lng1-1D
epidermal cells were 51% longer and 28% narrower than their wild-type
counterparts, suggesting that the longer, narrower cells of the mutant largely
accounted for the differences in leaf phenotype. Similarly, the adaxial
epidermal cells in the both proximal and distal mutant petals were
longitudinally elongated and transversely narrowed in the lng1-1D
mutant compared with wild type (Fig.
3G,H,K,L), as were the cells of the siliques
(Fig. 3I,J,M,N).
LNG1 and LNG2 encode novel proteins that activate longitudinal plant organ expansion
We performed a plasmid rescue experiment to identify the gene responsible
for the lng1-1D mutant phenotypes and identified a single T-DNA
insertion at 5477 bp upstream of At5g15880
(Fig. 4A). Since the T-DNA used
included multiple copies of the 35S enhancer element, we tested whether the
insertion caused the increased expression of nearby genes. Northern blot
analysis indicated that the expression of one gene (At5g15880) was higher in
mutant plants compared with wild type (Fig.
4B). This gene encodes a 927 amino acid protein containing a
putative nuclear localization signal. BLAST searching identified no homologous
proteins with known function, but did identify one closely related
Arabidopsis homolog (At3g02170, 63% identical) of unknown function
and a few related proteins from Arabidopsis and rice (22-27%
identical; Fig. 4C). Based on
the sequence similarity and our functional analysis, we designated At5g15580
as LNG1 and At3g02170 as LNG2. Amino acid sequence alignment
indicated that several regions are well conserved among LNG1, LNG2
and the related rice proteins (see Fig. S1 in the supplementary material). One
of the conserved regions is serine rich, but none shows any similarity to
known motifs.
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As LNG proteins have not been characterized in previous studies, we
experimentally determined the subcellular localization of LNG1 in transgenic
Arabidopsis plants expressing a GFP-LNG1 fusion protein and in onion
epidermis that had been bombarded with GFP-LNG1 fusion constructs. Bombardment
with vectors encoding GFP alone caused green fluorescence in both the cytosol
and nucleus (data not shown), with the nuclear localization likely to be due
to the low molecular weight of GFP alone
(von Arnim et al., 1998). The
GFP-LNG1 fusion protein showed similar localizations in both the cytosol and
nucleus (Fig. 5A-C). However,
since the molecular weight of the fusion protein is 131 kDa, the nuclear
localization of the fusion protein is likely to reflect the actual nuclear
localization of LNG1 protein. To confirm this finding, we generated transgenic
plants expressing GFP-LNG1 fusion proteins under the control of the CAMV 35S
promoter. The transgenic plants showed phenotypes similar to those of the
lng1-1D mutants, including very elongated and serrated leaves (data
not shown), suggesting that the GFP-LNG1 fusion protein is functional. The GFP
signal, however, was relatively weak and was detected only faintly in the root
tip cells of transgenic plants (Fig.
5D-G). Consistent with the subcellular localization in bombarded
onion epidermal cells, GFP signals were detected in both the cytosol and
nucleus. It is, however, possible that the cytosolic GFP signals were the
result of partially degraded GFP-LNG1 proteins.
To further investigate the physiological functions of LNG1 and
LNG2, we determined the expression patterns of these genes in
promoter:GUS transgenic plants. Promoter sequences (2 kb) from
the LNG1 and LNG2 genes were used to drive expression of
GUS. The ProLNG1:GUS and
ProLNG2:GUS transgenes were expressed in various parts of
adult transgenic plants, including the petioles, leaf blades, floral organs
and roots (Fig. 6). In roots,
GUS staining was mainly detected in the lateral roots. GUS was strongly
expressed in and at the base of the petioles
(Fig. 6B,F). In leaves, GUS
staining was observed in all parts, with stronger staining in the veins
(Fig. 6C,G). Consistent with
these observations, microarray analysis data compiled in Genevestigator
(https://www.genevestigator.ethz.ch/)
also indicate that LNG1 and LNG2 are expressed widely in various organs. These
results collectively suggest that LNG1 and LNG2 are
expressed in various Arabidopsis tissues, where they appear to
regulate cell elongation.
We then characterized T-DNA-inserted putative loss-of-function LNG1 (lng1-2 and lng1-3) and LNG2 (lng2-1 and lng2-2) mutants, which were obtained from the ABRC (Arabidopsis Biological Resource Center; Fig. 7A). To determine whether the T-DNA insertion lines were true loss-of-function mutants, we tested their expression levels of LNG1 and LNG. We observed weak expression of LNG1 in lng1-2 plants, but no such expression in lng1-3, indicating that lng1-2 was a weak mutant allele and that lng1-3 was a strong mutant allele (data not shown). No detectable expression of LNG2 was observed in the lng2-1 and lng2-2 mutants, indicating that both lng2-1 and lng2-2 were strong mutant alleles. To determine whether LNG1 and LNG2 regulate each other, we examined the reciprocal expression levels in each line. As shown in Fig. 7B, loss-of-function of LNG1 or LNG2 did not affect the expression of the other gene. In addition, overexpression of LNG1 (observed in the lng1-1D mutant) did not alter the expression of LNG2.
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15% shorter in
both single mutants, and almost 30% shorter in the double mutant, indicating
that LNG1 and LNG2 additively regulate the leaf-length
expansion. The additivity of LNG1 and LNG2 in leaf-length
expansion was also observed in other organs. Petiole length did not
significantly differ between the lng1-3 and lng2-1 single
mutants and wild-type plants, but was 37% shorter in the double mutant
compared with wild type. Similarly, the silique length of the double mutant
was about 40% shorter (see Table S1 in the supplementary material;
Fig. 7C). In addition to the
decreased leaf-length expansion of these organs, we observed that the stem
diameters of the double mutant plants were increased by almost 60% compared
with the wild type. Taken together, these data suggest that LNG1 and
LNG2 additively promote leaf-length expansion in various organs,
while inhibiting the expansion of stem thickness.
LNG1 and LNG2 additively promote longitudinal polar cell elongation in the leaf
SEM analysis of lng1-1D mutant plants suggested that LNG1
promotes longitudinal polar cell elongation. To further investigate the role
of LNG1 and LNG2 in polar cell elongation, we determined
cell sizes in transverse and longitudinal leaf sections
(Table 1; see Fig. S2 in the
supplementary material). In longitudinal sections, the palisade cells were 46%
longer in the lng1-1D mutant than wild type, but 24% shorter in the
lng1-3 lng2-1 double mutant (Table
1; see Fig. S2 in the supplementary material). In transverse
sections (representing width), however, we did not observe significant
differences among the different lines
(Table 1). Interestingly, cell
proliferation did not appear to be significantly altered in the mutants. Both
transverse and longitudinal sections showed similar numbers of palisade and
mesophyll cells in wild-type, lng1-1D, lng1-3, lng2-1 and lng1-3
lng2-1 plants (Table 1).
These results indicate that the altered leaf blade sizes in these plants are
not due to altered cell proliferation, and that LNG1 and
LNG2 regulate leaf expansion by promoting longitudinal polar cell
elongation, not by regulating cell proliferation.
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;
Kim et al., 1999;
Kim et al., 2002), whereas AN
and ATHB13 regulate leaf expansion by promoting transverse cell expansion
(Tsuge et al., 1996;
Hanson et al., 2001;
Kim et al., 2002). As our data
indicate that cell expansion is also regulated by LNG1 and LNG2, we tested for
functional relationships among these factors. We first tested for
interrelation of their expression levels. Our results revealed that the
expression levels of ROT3, CYP90D1 (a ROT3 homolog),
AN and ATHB13 were not altered in lng1-1D or
lng1-3 lng2-1 double mutant plants
(Fig. 8A), and that
LNG1 and LNG2 expression levels were unchanged in the
rot3-1 mutant (Fig.
8B), compared to wild type. These results indicate that
LNG1 and LNG2 do not regulate expression of ROT3,
CYP90D1, AN or ATHB13. Similarly, ROT3 does not appear
to regulate expression of LNG1 or LNG2.
The functional relationship among these genes was further examined by
analysis of a lng1-3 lng2-1 rot3-1 triple mutant and of a lng1-1D
rot3-1 double mutant. The petiole length was shorter in the triple mutant
compared with the lng1-3 lng2-1 double mutant and rot3-1
single mutant, both of which had shorter petioles than wild type
(Fig. 8D). This difference in
petiole length was mainly due to longitudinal shortening of the cells (see
Table S2 in the supplementary material). By contrast, the leaf blade size of
the triple mutant was similar to that of the lng1-3 lng2-1 double
mutant (Fig. 8C), and the blade
cells were similar in size among the mutants
(Fig. 8G; see Table S3 in the
supplementary material). The leaf blades were already short in the lng1-3
lng2-1 and rot3-1 mutants, perhaps masking an additive
shortening effect. Kozuka et al. suggested that the mechanism of petiole
elongation differs from that of leaf blade elongation
(Kozuka et al., 2005).
Therefore, it is also possible that the LNG genes and ROT3 may
regulate petiole length additively, while regulating blade length in a
collaborative fashion. To further clarify the relationships between LNG1,
LNG2 and ROT3, we measured the leaf blade length of an
lng1-1D rot3-1 double mutant. The blade lengths of lng1-1D
rot3-1 double mutant plants were intermediate between those of the
lng1-1D overexpression line and the rot3-1 loss-of-function
mutant plants (Fig. 8C). The
intermediate blade length of the lng1-1D rot3-1 double mutant was due
to cells of intermediate length, not to a change in cell number
(Fig. 8G; see Table S3 in the
supplementary material). Independent regulation of longitudinal elongation by
LNG1 and ROT3 can also be seen in silique length (see Table S4 in the
supplementary material). The lng1-1D had longer siliques than wild
type, whereas the lng1-3 lng2-1 double mutant and the rot3-1
single mutant had shorter siliques. In support of independent regulation of
longitudinal elongation by LNG1 and ROT3, the lng1-3 lng2-1 rot3-1
triple mutant had even shorter siliques than the lng1-3 lng2-1 double
mutant or rot3-1 single mutant, whereas the lng1-1D rot3-1
double mutant had intermediate siliques compared with lng1-1D and
rot3-1 mutants. As LNG1 does not regulate the expression of
ROT3 or of its close homolog CYP90D1
(Fig. 8A), these results
collectively suggest that LNG1 and ROT3 independently
regulate longitudinal organ size (Fig.
8C-E).
The relationship between LNG and ROT3 is less clear in terms of regulating leaf-width expansion. The lng1-1D mutant had a slightly narrower leaf blade compared with wild type, whereas the rot3-1 mutant and the lng1-1D rot3-1 double mutant showed similar phenotypes to each other, with a marginally wider leaf blade. The latter observation may suggest that rot3-1 is epistatic to lng1-1D for leaf-width expansion. However, because the leaf blades of the lng1-1D rot3-1 double mutants were shorter than those of the lng1-1D mutants, it is unclear whether the marginally wider leaf blade of the double mutant is caused by rot3-1, or is instead associated with the shortening of the leaf length.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LNG1 and LNG2 promote longitudinal polar cell elongation in a ROT3-independent fashion
Characterization of a dominant Arabidopsis mutant with unusually
long petioles, leaves, petals and siliques allowed us to identify a gene
involved in longitudinal expansion of aerial plant organs. We designated this
gene LNG1 and identified a homolog in Arabidopsis, which we
designated LNG2. LNG1 and LNG2 play positive roles in
longitudinal organ expansion in length direction, as evidenced by the
elongated organs of the lng1-1D mutant and transgenic lines
overexpressing LNG1 or LNG2
(Fig. 4F,G), or the shortened
organs of plants harboring loss-of-function mutations in LNG1 and
LNG2. Consistent with their amino acid sequence similarity (63%),
analysis of the lng1 lng2 double loss-of-function mutant revealed
that LNG1 and LNG2 are functionally overlapping genes.
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The roles of LNG1 and LNG2 in longitudinal cell expansion
appear similar to that of the previously characterized ROT3. For
example, ROT3-overexpression lines have longer leaf cells compared
with the wild type (Kim et al.,
1999), whereas rot3-1 loss-of-function mutants have
shorter organs comprising shorter cells
(Tsuge et al., 1996). Although
these similar phenotypes could indicate that these genes are members of the
same pathway, several lines of evidence suggest that LNG1 and
LNG2 regulate longitudinal cell expansion independently of
ROT3. First, our expression analysis revealed that altering the
expression levels of LNG1 and LNG2 did not alter the
expression levels of ROT3 or its homolog, CYP90D1
(Fig. 8A). Second, the
lng1-3 lng2-1 rot3-1 triple loss-of-function mutant showed additive
shortening of cell length, whereas the lng1-1D rot3-1 double mutant
showed an intermediate cell length, particularly in the case of petiole cells
(see Table S2 in the supplementary material). Third, the lng1-1D
rot3-1 double mutant showed longer leaf blades and petioles compared with
the rot3-1 single mutant, but shorter leaf blades and petioles
compared with the lng1-1D single mutant. These data indicate that the
two classes of genes do not have an epistatic relationship with regard to
longitudinal cell expansion, suggesting that longitudinal cell expansion is
regulated by at least two independent pathways in Arabidopsis: one
involving ROT3, and the other involving LNG1 and
LNG2. As ROT3 encodes an enzyme required for BR biosynthesis
(Kim et al., 2005), these data
further suggest that Arabidopsis contains a BR-independent pathway
that regulates longitudinal polar cell elongation.
The novel proteins LNG1 and LNG2 have homologs in other plant species
Analysis of the predicted protein sequences of LNG1 and LNG2 indicated that
they are novel proteins of unknown function. However, BLAST analysis
identified homologous protein sequences in Arabidopsis and rice. The
Arabidopsis genome contains two additional LNG-like proteins showing
overall sequence similarity and a few proteins that show partial sequence
similarity. In rice, we identified three proteins showing 22-27% overall
sequence similarity to LNG1 (Fig.
4C; see Fig. S1 in the supplementary material). Multiple sequence
alignment of the four Arabidopsis proteins and the three rice
proteins allowed us to identify several regions that are well conserved among
the family members. One conserved region is highly enriched with serine
residues, but overall these regions overall lack known motifs. Taken together,
these results suggest that LNG family members are novel plant-specific
proteins that promote longitudinal polar cell elongation through unknown
mechanisms.
The presence of LNG-like genes in other plant species raises the
possibility that these genes might play important roles in shaping various
plant forms. In Arabidopsis, different ecotypes display different
leaf shapes, ranging from rot3-like round leaves to an-like
narrow leaves. Previous QTL analysis using recombinant inbred lines identified
21 QTL responsible for the natural variations found in adult leaves of the Ler
and Col-4 ecotypes (Perez-Perez et al.,
2002). Notably, one of the reported QTL loci (ad-LSI2) overlaps
with the chromosome position of LNG1. According to the QTL analysis,
ad-LSI2 was responsible for about 5% of the variance found in the recombinant
inbred lines. Further experiments are needed to prove that LNG1
corresponds to ad-LSI2.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/21/4305/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Altmann, T. (1998). Recent advances in brassinosteroid molecular genetics. Curr. Opin. Plant Biol. 1,378 -383.[CrossRef][Medline]
Azpiroz, R., Wu, Y., LoCascio, J. C. and Feldmann, K. A.
(1998). An Arabidopsis brassinosteroid-dependent mutant is
blocked in cell elongation. Plant Cell
10,219
-230.
Berna, G., Robles, P. and Micol, J. L. (1999).
A mutational analysis of leaf morphogenesis in Arabidopsis thaliana.Genetics 152,729
-742.
Bowman, J. L., Eshed, Y. and Baum, S. F. (2002). Establishment of the polarity in angiosperm lateral organs. Trends Genet. 18,134 -141.[CrossRef][Medline]
Choe, S., Dilkes, B. P., Fujioka, S., Takatsuto, S., Sakurai, A.
and Feldmann, K. A. (1998). The DWF4 gene of Arabidopsis
encodes a cytochrome P450 that mediates multiple 22alpha-hydroxylation steps
in brassinosteroid biosynthesis. Plant Cell
10,231
-243.
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
De Veylder, L., Beeckman, T., Beemster, G. T., Krols, L.,
Terras, F., Landrieu, I., van der Schueren, E., Maes, S., Naudts, M. and Inze,
D. (2001). Functional analysis of cyclin-dependent kinase
inhibitors of Arabidopsis. Plant Cell
13,1653
-1668.
Franklin, K. A., Davis, S. J., Stoddart, W. M., Vierstra, R. D.
and Whitelam, G. C. (2003). Mutant analyses define multiple
roles for phytochrome C in Arabidopsis photomorphogenesis. Plant
Cell 15,1981
-1989.
Fujioka, S., Li, J., Choi, Y. H., Seto, H., Takatsuto, S., Noguchi, T., Watanabe, T., Kuriyama, H., Yokota, T., Chory, J. et al. (1997). The Arabidopsis deetiolated2 mutant is blocked early in brassinosteroid biosynthesis. Plant Cell 9,1951 -1962.[Abstract]
Hanson, J., Johannesson, H. and Engstrom, P. (2001). Sugar-dependent alterations in cotyledon and leaf development in transgenic plants expressing the HDZhdip gene ATHB13. Plant Mol. Biol. 45,247 -262.[CrossRef][Medline]
Hemerly, A., Engler Jde, A., Bergounioux, C., Van Montagu, M., Engler, G., Inze, D. and Ferreira, P. (1995). Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development. EMBO J. 15,3925 -3936.
Jones, A. M., Im, K. H., Savka, M. A., Wu, M. J., DeWitt, N. G., Shillito, R. and Binns, A. N. (1998). Auxin-dependent cell expansion mediated by overexpressed auxin-binding protein 1. Science 6,1114 -1117.
Kim, G. T. and Cho, K. H. (2006). Recent advances in the genetic regulation of the shape of simple leaves. Physiol. Plantarum 126,494 -502.
Kim, G. T., Tsukaya, H. and Uchimiya, H.
(1998). The ROTUNDIFOLIA3 gene of Arabidopsis
thaliana encodes a new member of the cytochrome P-450 family that is
required for the regulated polar elongation of leaf cells. Genes
Dev. 12,2381
-2391.
Kim, G. T., Tsukaya, H., Saito, Y. and Uchimiya, H.
(1999). Changes in the shapes of leaves and flowers upon
overexpression of cytochrome P450 in Arabidopsis. Proc. Natl. Acad.
Sci. USA 96,9433
-9437.
Kim, G. T., Shoda, K., Tsuge, T., Cho, K. H., Uchimiya, H., Yokoyama, R., Nishitani, K. and Tsukaya, H. (2002). The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation. EMBO J. 21,1267 -1279.[CrossRef][Medline]
Kim, G. T., Fujioka, S., Kozuka, T., Tax, F. E., Takatsuto, S., Yoshida, S. and Tsukaya, H. (2005). CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana. Plant J. 41,710 -721.[CrossRef][Medline]
Kozuka, T., Horiguchi, G., Kim, G. T., Ohgishi, M., Sakai, T.
and Tsukaya, H. (2005). The different growth responses of the
Arabidopsis thaliana leaf blade and the petiole during shade
avoidance are regulated by photoreceptors and sugar. Plant Cell
Physiol. 46,213
-223.
Li, J. and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90,929 -938.[CrossRef][Medline]
Narita, N. N., Moore, S., Horiguchi, G., Kubo, M., Demura, T., Fukuda, H., Goodrich, J. and Tsukaya, H. (2004). Overexpression of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana. Plant J. 38,699 -713.[CrossRef][Medline]
Perez-Perez, J. M., Serrano-Cartagena, J. and Micol, J. L.
(2002). Genetic analysis of natural variations in the
architecture of Arabidopsis thaliana vegetative leaves.
Genetics 162,893
-915.
Qin, M., Kuhn, R., Moran, S. and Quail, P. H. (1997). Overexpressed phytochrome C has similar photosensory specificity to phytochrome B but a distinctive capacity to enhance primary leaf expansion. Plant J. 12,1163 -1172.[CrossRef][Medline]
Sinha, N. (1999). Leaf development in angiosperms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50,419 -446.[CrossRef][Medline]
Timpte, C. C., Wilson, A. K. and Estelle, M. (1992). Effects of the axr2 mutation of Arabidopsis on cell shape in hypocotyl and inflorescence. Planta 188,271 -278.[CrossRef]
Tsuge, T., Tsukaya, H. and Uchimiya, H. (1996). Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development 122,1589 -1600.[Abstract]
Tsukaya, H. (2003). Organ shape and size: a lesson from studies of leaf morphogenesis. Curr. Opin. Plant Biol. 6,57 -62.[CrossRef][Medline]
Tsukaya, H., Naito, S., Rédei, G. P. and Komeda, Y. (1993). A new class of mutations in Arabidopsis thaliana, acaulis1, affecting the development of both inflorescences and leaves. Development 118,751 -764.[Abstract]
Ullah, H., Chen, J. G., Young, J. C., Im, K. H., Sussman, M. R. and Jones, A. M. (2001). Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science 15,2066 -2069.
Verkest, A., Manes, C. L., Vercruysse, S., Maes, S., Van Der
Schueren, E., Beeckman, T., Genschik, P., Kuiper, M., Inze, D. and De Veylder,
L. (2005). The cyclin-dependent kinase inhibitor KRP2
controls the onset of the endoreduplication cycle during Arabidopsis leaf
development through inhibition of mitotic CDKA;1 kinase complexes.
Plant Cell 17,1723
-1736.
von Arnim, A. G., Deng, X. W. and Stacey, M. G. (1998). Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene 221, 35-43.[CrossRef][Medline]
Wang, H., Zhou, Y., Gilmer, S., Whitwill, S. and Fowke, L. C. (2000). Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology. Plant J. 24,613 -623.[CrossRef][Medline]
Weigel, D., Ahn, J. H., Blazquez, M. A., Borevitz, J. O.,
Christensen, S. K., Fankhauser, C., Ferrandiz, C., Kardailsky, I.,
Malancharuvil, E. J., Neff, M. M. et al. (2000). Activation
tagging in Arabidopsis. Plant Physiol.
122,1003
-1013.
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