Mutation of the Rice Narrow leaf1 Gene, Which Encodes
a Novel Protein, Affects Vein Patterning and Polar
Auxin Transport1[OA]
Jing Qi2, Qian Qian2, Qingyun Bu, Shuyu Li, Qian Chen, Jiaqiang Sun, Wenxing Liang, Yihua Zhou,
Chengcai Chu, Xugang Li, Fugang Ren, Klaus Palme, Bingran Zhao, Jinfeng Chen,
Mingsheng Chen, and Chuanyou Li*
State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (J.Q., Q.B., Q.C., J.S., W.L., S.L.,
Y.Z., C.C., J.C., M.C., C.L.); State Key Laboratory of Rice Biology, China National Rice Research Institute,
Chinese Academy of Agricultural Sciences, Hangzhou 310006, China (Q.Q.); Graduate School of Chinese
Academy of Sciences, Beijing 100049, China (J.Q., Q.C., W.L., S.L.); Institut für Biologie II und Zentrum für
Angewandte Biowissenschaften, Universität Freiburg, D–79104 Freiburg, Germany (X.L., F.R., K.P.); and
China National Hybrid Rice Research and Development Center, Changsha 410125, China (B.Z.)
The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant
leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic
phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of
longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which
the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses
demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing
that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin
transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants
and plays an important role in the control of lateral leaf growth.
The plant vascular system, a continuous network of
vascular bundles that interconnects all major plant
organs, enables long-distance transport of photoassimilates and soil nutrients and provides mechanical
support to the plant body (Esau, 1965; Dengler, 2001;
Aloni, 2004; Scarpella and Meijer, 2004). Anatomical
studies revealed that all vascular cells are differentiated from a vascular meristematic tissue, the procambium (Foster, 1952; Esau, 1965). Procambial cells are
characteristically arranged in continuous strands and
acquire their narrow shape through coordinated, oriented divisions, parallel to the axis of the emerging
strand (Nelson and Dengler, 1997; Dengler, 2001; Ye,
2002; Scarpella and Meijer, 2004). Despite extensive
1
This work was supported by grants from the Ministry of Science
and Technology of China (grant nos. 2005CB120801 and
2006AA10A101) and the National Natural Science Foundation of
China (grant nos. 30700054 and 90717007).
2
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Chuanyou Li ([email protected]).
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.118778
descriptions of different aspects of vascular development and patterning, the molecular mechanisms governing these strictly regulated processes remain to be
elucidated.
Among the various plant hormones that influence
vascular development and patterning, the role of
auxin is unique (Dengler, 2001; Aloni, 2004; Fukuda,
2004; Scarpella and Meijer, 2004; Teale et al., 2006). A
long history of elegant physiological experiments
established that the polar cell-to-cell transport of auxin
plays a crucial role in vascular tissue differentiation
and vascular patterning (Sachs, 1989; Aloni, 2004;
Fukuda, 2004; Blakeslee et al., 2005; Sauer et al., 2006;
Teale et al., 2006). The classical auxin application
studies and polar auxin transport (PAT) inhibition
studies also led to the formulation of the ‘‘auxin-flow
canalization hypothesis,’’ which suggests that auxin
flow, starting initially by diffusion, induces the formation of a polar auxin transport system and, as a consequence, promotes auxin transport and leads to
canalization of the auxin flow along a narrow file of
cells. This continuous polar transport of auxin through
cells finally results in the differentiation of vascular
strands (Sachs, 1981, 2000; Aloni, 2004; Fukuda, 2004).
Molecular genetic studies, primarily using the dicotyledon Arabidopsis (Arabidopsis thaliana) as a
model system, have boosted our understanding of
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Qi et al.
the molecular basis of PAT, PAT-mediated auxin distribution, and their association with vascular differentiation and patterning (Palme and Gälweiler, 1999;
Dengler, 2001; Friml and Palme, 2002; Ye, 2002; Aloni,
2004; Fukuda, 2004; Blakeslee et al., 2005; Paponov
et al., 2005; Teale et al., 2006). In-depth characterization
of Arabidopsis mutants defective in PAT had enabled
the identification of the putative auxin influx as well as
efflux carriers, two of the major components of the PAT
system. For example, the pin-formed1 (pin1) mutant
showed irregular vascular patterns and strongly reduced basipetal auxin transport, which resembled
plants treated with inhibitors of auxin efflux (Okada
et al., 1991; Gälweiler et al., 1998). Gene cloning and
protein localization analyses demonstrated that AtPIN1
was a member of a family of transmembrane proteins
that asymmetrically distributed at the basal end of
PAT-competent cells, supporting the hypothesis that
AtPIN1 as well as other PIN proteins were components of the auxin efflux machinery (Gälweiler et al.,
1998; Palme and Gälweiler, 1999; Friml, 2003; Aloni,
2004; Blakeslee et al., 2005; Leyser, 2005; Paponov et al.,
2005; Petrasek et al., 2006). In addition, mutations in
AtPGP1 and AtPGP19, two genes encoding putative
P-glycoprotein ABC transporters, resulted in the disruption of PAT and pleiotropic growth phenotypes,
including stunted stature and curled leaves (Noh et al.,
2001, 2003; Luschnig, 2002). Recently, heterologous
expression studies demonstrated that both AtPGP1
and AtPGP19 exhibited auxin efflux transport activity
(Geisler et al., 2005).
Through monitoring the dynamic patterns of the
polar AtPIN1 expression during critical stages of the
formation of various defined veins, recent studies
revealed a directing influence of auxin transport in
the epidermis on vein patterning (Scarpella et al., 2003;
Sauer et al., 2006). These works refined the overall
picture of the canalization hypothesis for PAT-mediated
vascular patterning (Scheres and Xu, 2006).
Although a large number of studies have established an instrumental role of PAT and PAT-mediated
auxin distribution for vascular patterning in Arabidopsis, much less is known about whether the same
paradigm also underlies vascular patterning in rice
(Oryza sativa), a model system of monocot plants.
While dicot plants show a reticulate pattern of highly
branched veins, most monocot plants, such as rice,
show parallel (striate) vascular patterns (Nelson and
Dengler, 1997; Scarpella et al., 2003; Scarpella and
Meijer, 2004). The auxin-inducible homeobox gene
Oshox1 in rice was shown recently to be a molecular
marker of a stage in vascular tissue development at
which procambial cell fate is specified but not yet
stably determined (Scarpella et al., 2000, 2005). Overexpression of Oshox1 led to reduced sensitivity to PAT
inhibitors, indicating a role of PAT in vascular tissue
differentiation in monocot species (Scarpella et al.,
2002). An association between PAT and venation was
demonstrated by characterization of the maize (Zea
mays) rough shealth2 mutant, which shows aberrant leaf
development (Tsiantis et al., 1999; Scanlon et al., 2002).
Stronger evidence supporting the notion that PAT is
involved in vascular patterning in monocot plants
came from the characterization of the classic brachytic2
(br2) mutant in maize, which shows irregular vascular
patterns in stalks (Multani et al., 2003). br2 carried
mutation in a gene that is homologous to the abovementioned Arabidopsis P-glycoproteins AtPGP1 and
AtPGP19. Similar to the Arabidopsis case, PAT was
significantly reduced in br2 mutants. The radicleless1
(ral1) mutant of rice showed distinctive embryonic and
postembryonic vascular defects, including altered
number and spacing of parallel veins, and was defective in auxin response (Scarpella et al., 2003). However,
the gene responsible for the ral1 phenotype had yet to
be identified.
Here, we report the in-depth characterization of a
previously described rice mutant named narrow leaf1
(nal1; Dong et al., 1994; Zeng et al., 2003). The nal1
mutant exhibits altered vascular patterns in leaves and
culms. Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence
showing that Nal1 is expressed preferentially in vascular tissues and that mutation of its function leads to
reduced PAT capacity. These results support the hypothesis that Nal1 plays important roles in regulating
PAT as well as vascular patterning of rice.
RESULTS
The nal1 Mutant Shows Reduced Leaf Width
One of the most characteristic phenotypes of the rice
nal1 mutant was reduced leaf width (Dong et al., 1994;
Zeng et al., 2003). The leaves of nal1 plants appeared
normal in shape but were generally smaller than those
of wild-type plants (Table I; Fig. 1C). Measurement of
leaf growth indicated that the nal1 mutation significantly affected the width of the leaf blade. Compared
with width, the length of the mutant leaf blades was
less affected. For example, the mature second leaves
(from the top of the plant) of soil-grown nal1 plants
usually attained approximately 63% of the width of
the wild-type counterparts (Table I), while the length
of the nal1 leaves was about 87% of that of wild-type
plants (Table I).
The nal1 Mutant Exhibits an Altered Internode
Elongation Pattern
In addition to narrow leaves, the nal1 mutant also
showed an altered internode elongation pattern (Fig. 1,
A, B, and D). At maturity, the height of the mutant
reached approximately 71% of the wild-type height
(Table I). We compared the internode elongation patterns between nal1 and wild-type plants. As shown in
Figure 1D and Table I, while the nal1 mutation had
little effect, if any, on the length of the panicle and the
uppermost two internodes (designated internodes I
and II, respectively), it significantly shortened the
1948
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Nal1 Affects Venation and Polar Auxin Transport
Table I. Morphometric analysis of wild-type and nal1 plants
Leaf morphometric analyses were done on mature leaves of 3-month-old plants. Blade width and
vascular pattern parameters were measured through the middle region of the leaf blade. Vascular pattern
parameters were measured in dark-field microscopic digital images of cleared leaf preparations. Numbers
of stem vascular bundles in the outer and inner rings were determined in digital microscopic images of
transverse sections through the middle part of the fourth internode of plants at the ripened inflorescence
stage. The internode IV morphometric analyses and plant height measurement were done at the mature
stage of 3-month-old plants. Results represent means 6 SD of populations of the size indicated in
parentheses. Asterisks indicate the significance of differences between wild-type and nal1 plants as
determined by Student’s t test: * 0.01 # P , 0.05, ** 0.001 # P , 0.01, *** P , 0.001.
Organ
Leaf
Blade width (cm)
Blade length (cm)
Number of large veins
Total number of small veins
Number of small veins between
two adjacent large veins
Stem
Mature plant height (cm)
Internode IV length (cm)
Number of vascular bundles in the outer ring
Number of vascular bundles in the inner ring
Total cell number of
internode IV in y axis
length of the third, the fourth, and the fifth internodes
(designated internodes III, IV, and V, respectively).
To determine whether the dwarf phenotype of the
nal1 culm results from defective cell division and/or
cell elongation, longitudinal sections of the severely
shortened internode IV of the mature mutant culm
were compared with their wild-type counterparts. As
shown in Figure 1D and Table I, the total cell number
in y axes in internode IV of nal1 was approximately
60% less than that of wild-type plants, while no
significant change in cell length was observed. These
observations suggested that the Nal1 gene may play a
role in cell division rather than cell elongation.
The nal1 Mutant Shows Defective Vascular Patterns in
Leaves and Culms
Wild-type rice leaves show a typical parallel venation pattern, in which three orders of major longitudinal veins (i.e. the midveins, large veins, and small
veins) lie parallel along the distal axis of the leaf
(Nelson and Dengler, 1997; Scarpella et al., 2003). A
wealth of anatomical studies revealed a constant relationship between leaf blade width and longitudinal
vein quantity in monocots (Russell and Evert, 1985;
Dannenhoffer et al., 1990; McHale, 1993; Nelson and
Dengler, 1997). A comparison of mature leaves revealed that the total numbers of large veins and small
veins were all reduced in the nal1 mutant (Table I).
Notably, the average number of small veins between
two large veins was reduced from approximately five
in the wild type to approximately three in the mutant
Wild Type
1.9
32.1
8.1
39.4
4.9
6
6
6
6
6
0.2
5.5
0.8
2.8
0.3
(15)
(15)
(33)
(30)
(30)
88.1
4.5
23.6
25.5
530.3
6
6
6
6
6
2.9 (20)
1.4 (20)
2.7 (23)
1.99 (23)
100.6 (16)
nal1
1.2
27.7
7.2
22.2
3.0
6
6
6
6
6
0.1
1.7
1.1
3.5
0.4
(15)***
(15)**
(42)***
(30)***
(30)***
62.8
1.2
25.2
26.5
159.1
6
6
6
6
6
1.9 (20)***
0.2 (20)***
2.5 (21)*
4.36 (21)
69.1 (16)***
(Fig. 1, E and F; Table I). These observations indicated
that the nal1 mutation affected the formation of leaf
veins, which might account for the narrow leaf phenotype of the mutant.
The finding that the nal1 mutation affected leaf
venation prompted us to examine the vascular system
in culms. Transverse sections of internode IV, the
length of which was significantly reduced in nal1
(Fig. 1D; Table I), were compared between wild-type
and mutant plants. In cross sections of the wild-type
internode IV, vascular bundles of inner ring and outer
ring were uniformly arranged and formed a typical
radial organization, with outer small vascular bundles
arranged concentrically below the epidermis (Fig. 1G,
left). In contrast, the distribution pattern of vascular
bundles in the nal1 mutant culm was severely disrupted (Fig. 1G, right). In addition to the altered distribution pattern, the mutant culm also contained
immature vascular bundles and an overall increased
number of vascular bundles, especially in the outer
ring (Table I; Fig. 1G, right). Furthermore, increased
numbers of parenchyma cells in the region between
the epidermis and the outer ring of small vascular
bundles could be observed (Fig. 1G, right).
Positional Cloning of Nal1
The genetic locus defined by the nal1 mutant was
previously located on the long arm of rice chromosome 4 (Dong et al., 1994; Zeng et al., 2003). For fine
mapping, the nal1 mutant in the genetic background of
Zhefu802 (an indica cultivar) was crossed to Nip-
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Qi et al.
Figure 1. The nal1 mutant shows a defective vascular system in leaves and culms. A, Gross morphology of 1-month-old wildtype (left) and nal1 (right) plants. B, Gross morphology of wild-type (left) and nal1 (right) plants at the heading stage. C, Leaf
morphology of wild-type (left) and nal1 (right) plants. Shown are the second leaf blades (from the top of the plant) of 3-month-old
plants. D, Phenotypic exhibition of the components of rice plant height in representative wild-type (left) and nal1 (right) plants at
the mature stage. P, Panicle. I to V indicate the corresponding internodes from top to bottom. E, Whole-mount clearing of the
mature fifth leaves of wild-type (WT) and nal1 plants. Shown are regions between two large veins in the middle of mature leaf
blades. lv, Large vein; sv, small vein. Bars 5 150 mm. F, Transverse sections of the mature fifth leaves in the middle part of wildtype and nal1 plants. Bars 5 20 mm. G, Transverse sections of the middle part of internode IV of wild-type (left) and nal1 (right)
plants at the mature stage. ep, Epidermis; lvb, large vascular bundles; pc, parenchyma cells; svb, small vascular bundles. The
single red arrow indicates an immature vascular bundle, and the double red arrows indicate parenchyma cell layers in the region
between the epidermis and the outer ring of small vascular bundles. Bars 5 20 mm.
ponbare (a japonica cultivar) to generate a large F2
population. Genetic analysis with 2,000 segregants
showing the nal1 mutant phenotype initially placed
the Nal1 gene between simple sequence repeat
markers RM5503 and RM3836 (Fig. 2A). Within this
region, we developed four PCR-based molecular
markers (Table II) and further delimited the target
gene to a 29-kb region defined by markers M3 and M4
on a single bacterial artificial chromosome (BAC)
clone, AL662950 (Fig. 2A). Within this 29-kb interval,
four open reading frames (ORFs), which were designated ORF1 to ORF4, were predicted (Fig. 2A). Genomic DNAs corresponding to the four putative ORFs
were PCR amplified from the nal1 mutant and sequenced. Sequence comparison revealed a 30-bp deletion in the nal1-derived ORF1 (Fig. 2B), while no
sequence difference was found in ORF2 to ORF4.
ORF1, therefore, was considered a strong candidate
for the Nal1 gene.
To test the possibility that the 30-bp deletion in nal1derived ORF1 is responsible for the mutant phenotype, we performed genetic complementation analysis.
A 12.4-kb genomic DNA fragment containing the
entire Nal1 coding region, the 2,673-bp upstream sequence, and the 1,685-bp downstream sequence was
introduced into the nal1 background by Agrobacterium
tumefaciens-mediated transformation (Hiei et al., 1994).
The 30-bp deletion in the nal1-derived ORF1 led to the
development of a PCR-based marker to determine the
mutant background in complementation tests (Fig. 2C,
bottom). Among the 24 transgenic plants confirmed by
genomic PCR, 17 primary lines (T0) showed comple-
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Nal1 Affects Venation and Polar Auxin Transport
Figure 2. Nal1 encodes a plant-specific protein with unknown biochemical function. A, Fine genetic and physical mapping of
Nal1. The target gene was initially mapped to a genetic interval between markers RM5503 and RM3836 on rice chromosome 4.
Analysis of an F2 mapping population of 2,000 plants delimited the gene to a 29-kb region between markers M3 and M4 on BAC
clone AL662950. Markers and the numbers of recombination events identified are indicated. B, Schematic representation of the
Nal1 gene structure. The nal1 allele contains a 30-bp deletion in the fourth exon. Black boxes indicate the coding sequence, white
boxes indicate the 5# and 3# untranslated regions, and lines between boxes indicate introns. The start codon (ATG) and the stop
codon (TGA) are indicated. C, Genetic complementation of nal1 (top) and molecular identification of transgenic plants (bottom).
The wild type (left), nal1 containing an empty vector (middle), and a transgenic plant containing the 12.4-kb DNA fragment of
ORF1 in the genetic background of nal1 (right) are shown. The 30-bp deletion in the nal1 was used as a PCR-based marker to
distinguish the three plants. D, Deduced amino acid sequence of Nal1. The deleted 10 amino acids are underlined, and the red
letters indicate the putative nuclear localization signal. E, Phylogeny of the Nal1 protein family. A neighbor-joining tree was built by
MEGA3 using a Poisson correction model with gaps to complete deletion. Topological robustness was assessed by bootstrap
analysis with 500 replicates. The bar is an indicator of genetic distance based on branch length. Nal1 and AK120320 are from rice;
BT014552 and BT013672 are from tomato; AC191706, AC191543, AC205276, and AC208803 are from maize; and AK248497
and AK252329 are from barley. F, Confocal image (left), transmitted light image (middle), and merged image (right) of Arabidopsis
cell culture protoplasts transfected with free GFP. Bar 5 10 mm. G, Confocal image (left), transmitted light image (middle), and
merged image (right) of Arabidopsis cell culture protoplasts transfected with Nal1-GFP fusion proteins. Bar 5 10 mm. H, Steadystate nuclear protein body patterns of 35S:Nal1-GFP in root cells from a 5-d-old transgenic Arabidopsis seedling. Confocal image
(left), transmitted light image (middle), and merged image (right) of root cortex cells. Bar 5 20 mm.
mentation of the nal1 phenotype (Fig. 2C; data not
shown). Further characterization of three of these lines
showed that the complemented phenotype was inherited in the T1 generation (data not shown). These results
demonstrated that ORF1 is equivalent to the Nal1 gene.
Nal1 Encodes a Plant-Specific Protein with Unknown
Biochemical Function
Sequence comparison between genomic DNA and
cDNA revealed that the Nal1 gene was composed
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Qi et al.
Table II. PCR-based molecular markers used for genetic mapping
Primer Pairsa
Fragment Sizeb
Restriction Enzyme
F, 5#-ACAGTACCTTCATTCAGCCTCTTC-3#;
R, 5#-GATGCGTTCGTCTCACCAGTT-3#
F, 5#-CAACTAAGGCTGGGGATGAAAAG-3#;
R, 5#-ACCGAGTCTGGCGTCTACACAA-3#
F, 5#-TGGGGGTGGAGGACAGGT-3#;
R, 5#-TTTTTTGCGGGGAGATGGAG-3#
F, 5#-GGACAGCCAAAAGTAGTAGTAGTAG-3#;
R, 5#-GGTAAATAATAGGGGGAAATA-3#
368
FokI
911
PstI
Marker
M1
M2
M3(STS)
M4
a
F, Forward primer; R, reverse primer.
fragments.
433
313
NsiI
b
Numbers indicate the size (in base pairs) of amplified
of five exons and four introns (Fig. 2B) that encoded
a protein with 582 amino acids (Fig. 2D). In nal1,
the 30-bp deletion, which existed in exon 4 of the
mutated Nal1 gene, resulted in the in-frame absence
of 10 amino acids (Fig. 2, B and D).
A survey of the published rice genome database
(http://www.gramene.org/) identified the existence
of another gene named Nal1-like (AK120820), which
was predicted to encode a protein that shares greater
than 70% amino acid sequence identity with Nal1 (Fig.
2E). BLAST searches of the DNA and protein databases also identified Nal1-like genes from other plant
species, including monocots and dicots (Fig. 2E). Notably, three putative homologous genes, At2g35155
(AY059774), At5g45030 (AY099637), and At3g12950
(BT012224), which shared around 60% amino acid
sequence identity with the Nal1 protein, were identified in the Arabidopsis genome (Fig. 2E). Putative Nal1
homologous genes were also identified from the genomes of tomato (Solanum lycopersicum), maize, and
barley (Hordeum vulgare; Fig. 2E). Database searches
failed to identify yeast or animal proteins showing
significant similarity to Nal1. The existence of Nal1-like
genes in different plant species might suggest conserved biochemical function of the Nal1 protein family.
However, the Nal1 sequence did not match any protein
of known biochemical function in the public databases.
Subcellular Localization of Nal1
Using the WoLF PSORT programs (http://wolfpsort.
org/), it was predicted that the Nal1 protein contains a
putative nuclear localization signal (PKRLRSD, amino
acid residues 567–573; Fig. 2D). To determine the
subcellular localization of Nal1, GFP was fused to
the C terminus of Nal1 under the control of the 35S
promoter and the fusion gene was transformed into
Arabidopsis protoplast (Meskiene et al., 2003). The
Nal1-GFP chimeric protein was localized to the nucleoplasm and cytoplasm and concentrated in discrete
structures (Fig. 2G), which appeared similar to nuclear
protein bodies. However, nuclear protein body formation was not detected in control cells, which expressed
GFP alone under the control of the 35S promoter (Fig.
2F). Similarly, our parallel experiment indicated that
At3g12950, one of the Arabidopsis homologs of Nal1,
also can form nuclear protein bodies in Arabidopsis
protoplast (data not shown). To substantiate these
observations, we generated transgenic Arabidopsis
plants containing the 35S:Nal1-GFP fusion gene. Confocal observation of the root segments of the transgenic seedlings indicated that the Nal1-GFP fusion
protein form nuclear protein bodies and that the
number, size, and brightness of the nuclear protein
bodies show high variation in different cells (Fig. 2H;
data not shown).
Expression Pattern of Nal1
Reverse transcription (RT)-PCR analysis indicated
that Nal1 was expressed in organs such as leaf sheaths,
leaves, culms, and young panicles (Fig. 3A). The nal1
mutation led to irregular venation patterns in leaves
and culms, suggesting that the Nal1 gene might act in
vascular tissues. To test this, we generated transgenic
rice plants expressing a 1,918-bp fragment of the Nal1
promoter fused with the GUS reporter gene. GUS
staining of transgenic plants indicated that Nal1 was
preferentially expressed in vascular tissues of etiolated
coleoptiles (Fig. 3, B and C). In a cross section of culm
at heading stage, strong GUS staining was found in the
phloem of vascular bundles (Fig. 3D). RNA in situ
hybridization with cross sections of 1-month-old seedlings provided further evidence that Nal1 was richly
expressed in vascular tissues (Fig. 3, E–I). The finding
that Nal1 was preferentially expressed in vascular tissues together with the fact that the nal1 mutant showed
defective vascular patterns in leaves and culms implied
that Nal1 might be involved in rice vascular patterning.
The nal1 Mutant Shows Reduced Polar Auxin
Transport Activity
The phytohormone auxin plays a pivotal role in
different aspects of vascular tissue development and
patterning (Ye, 2002; Aloni, 2004; Fukuda, 2004; Scarpella
and Meijer, 2004; Teale et al., 2006). Our finding that
the nal1 mutation led to defective vascular development in leaves and culms raised the interesting pos-
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Nal1 Affects Venation and Polar Auxin Transport
Figure 3. Nal1 expression pattern. A, RTPCR analysis of Nal1 expression. Total
RNA was isolated from leaf sheaths (S),
leaves (L), culms (C), and panicles (P) of
wild-type plants. Amplification of the rice
ACTIN1 gene was used as a control. B to D,
Nal1 expression revealed by GUS staining
in Nal1 promoter-GUS transgenic rice
plants. B, Nal1 promoter-GUS expression
patterns in 5-d-old dark-grown coleoptiles
showing intense GUS staining in vascular
tissues. C, Transverse section of the middle
part of the coleoptile in B showing intense
expression of GUS in the phloem. D, Transverse section of a fourth internode at the
heading stage showing the expression of
Nal1 promoter-GUS in the phloem of vascular bundles. ph, Phloem; v, vascular
bundle. Bars 5 400 mm in B, 20 mm in C,
and 100 mm in D. E to I, Nal1 expression
patterns revealed by RNA in situ hybridization. E, Transverse section of a 1-monthold seedling showing intense expression of
Nal1 in vascular tissues of leaves and leaf
sheaths. F, Magnified image of the boxed
region in E showing the strong expression
of Nal1 in developing vascular bundles of
leaf sheath. G, Transverse section of a
young culm. H, Magnified image of the
boxed region in G showing the strong
expression of Nal1 in vascular bundles. I,
Background control. No hybridization signal was observed with the sense probe. X,
Xylem. Bars 5 200 mm in B, 130 mm in E,
G, and I, 70 mm in F and H, 50 mm in C,
and 20 mm in D.
sibility that the polar auxin flow in the nal1 mutant
may be altered. Given that the etiolated cereal coleoptile is used as a model system to study auxin-mediated
growth and development (Haga et al., 2005; Li et al.,
2007), together with our anatomical investigations
showing that the overall structures and vasculatures
of etiolated coleoptiles between the wild type and nal1
were similar (Fig. 4, D–G), we compared the PAT
activities in etiolated coleoptiles of wild-type and
mutant plants. We first examined the uptake of tritiumlabeled indole-3-acetic acid ([3H]IAA) using 2-mm
coleoptile fragments and found that the uptake capacity of [3H]IAA in nal1 was comparable to that of the
wild type (Fig. 4A). To measure basipetal transport of
IAA, [3H]IAA was applied to the apical tips of darkgrown coleoptile fragments, and the basipetal auxin
transport activity in nal1 coleoptiles was reduced to
approximately 33% of that of wild-type plants (Fig.
4B). As a control, acropetal transport was also measured through feeding [3H]IAA to the basal tips of
coleoptile fragments and found to be much lower than
basipetal transport, but it was similar between nal1
and the wild type (Fig. 4B). The PAT activities of nal1
and wild-type plants were also examined in the presence of N-1-naphthylphthalamic acid (NPA), an inhibitor of PAT (Morris et al., 2004). As shown in Figure 4B,
in the presence of 20 mM NPA, the PAT activity in wildtype coleoptiles was reduced to approximately 36% of
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Qi et al.
Figure 4. The nal1 mutant shows defective PAT activity. A, Comparison of auxin uptake between wild-type (WT) and nal1 plants
in dark-grown coleoptiles. Values represent means 6 SD of six independent assays. B, Comparison of auxin transport between
wild-type and nal1 plants in dark-grown coleoptiles. Values represent means 6 SD of six independent assays. The asterisk
indicates that the difference between the wild type and nal1 is significant (Student’s t test, P , 0.001). C, IAA efflux of dark-grown
coleoptile segments. Five coleoptile segments were used in each assay, and values are given as means 6 SD of three independent
assays. The difference between the wild type and nal1 is significant (Student’s t test, P , 0.05). D and E, Cross sections of darkgrown wild-type (D) and nal1 (E) shoots showing that the vasculature of nal1 coleoptile is similar to that of the wild type. Bars 5
50 mm. F, Magnified image of the boxed region in D. Bar 5 50 mm. G, Magnified image of the boxed region in E. Bar 5 50 mm. H
to K, Immunohistochemical analysis of etiolated wild-type and nal1 seedlings using the anti-AtPIN1 antibodies as probe. H,
Transverse section through the middle part of a 5-d-old dark-grown wild-type seedling. I, Bright-field image of K. J, Transverse
section through the middle part of a 5-d-old dark-grown nal1 seedling. K, Bright-field image of J. Bars 5 50 mm. L, Protein gelblot analysis using the anti-AtPIN1 antibodies as probe. Membrane protein extracts from wild-type and nal1 coleoptiles were
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Nal1 Affects Venation and Polar Auxin Transport
that of buffer control. In the case of nal1, the PAT
activity was already low and application of NPA led to
only a little reduction in PAT. In order to compare the
efflux rates of auxin transport between the wild type
and mutant, coleoptile segments were incubated sequentially in the [3H]IAA-containing solution and in auxinfree buffer, and then the amount of auxin retained in
the coleopitle segments was measured. As shown in
Figure 4C, in the absence of NPA, the [3H]IAA retained
in nal1 coleoptiles was approximately 32% higher than
that of the wild type. In the presence of 2 mM NPA, the
amount of auxin retained in wild-type coleoptiles was
increased by 140%, whereas that in nal1 coleoptiles
was only increased by 24%. Together, these results
indicated that mutation of Nal1 led to defective basipetal PAT activity and rendered the nal1 mutant less
sensitive than the wild type to NPA-mediated inhibition of polar auxin flow.
The nal1 Mutation Might Affect the Abundance of a
Deduced Rice Homolog of AtPIN1, an Auxin Efflux
Facilitator in Arabidopsis
The findings that nal1 shows reduced PAT activity in
the shoot and decreased sensitivity to NPA-mediated
PAT inhibition raised the interesting possibility that
the nal1 mutation might affect auxin efflux. Considering the established roles of the Arabidopsis PIN family
proteins in the regulation of auxin efflux, together with
the existence of highly conserved PIN proteins in rice
(Gälweiler et al., 1998; Palme and Gälweiler, 1999;
Paponov et al., 2005; Xu et al., 2005), we conducted
immunohistochemical analysis in 5-d-old dark-grown
seedlings using antibodies raised against the AtPIN1
protein, a well-characterized auxin efflux carrier in
Arabidopsis. As shown in Figure 4, H to K, the AtPIN1
antibodies detected signals in the vascular tissues of
both mutant and wild-type seedlings and, interestingly, the signals detected in nal1 was weaker than
those in wild-type plants. To validate this observation,
membrane proteins were extracted from both nal1 and
wild-type coleoptiles and subjected to protein gel-blot
analysis using the same AtPIN1 antibodies as probe.
As shown in Figure 4L, a specific band with comparable size could be detected in both nal1 and the wild
type. However, the abundance of the signal in nal1 was
significantly reduced over that in the wild type (Fig.
4L). These results suggested that the nal1 mutation
affected the abundance of a deduced rice homolog of
AtPIN1, an auxin efflux facilitator in Arabidopsis.
Among the three PIN1 family genes in rice, OsPIN1
(AF056027) shows the highest sequence similarity to
AtPIN1 (Xu et al., 2005). Our quantitative real-time RTPCR assays indicated that the OsPIN1 expression
levels in dark-grown coleoptiles of nal1 were significantly
reduced compared with those in the wild type (Fig. 4M),
suggesting that the nal1 mutation affects the OsPIN1
abundance and activities at the transcription level.
DISCUSSION
Our results demonstrated that the nal1 mutation
affects vascular patterns in leaves and culms of rice.
Compared with their wild-type counterparts, mature
leaves of the nal1 mutants showed distinctive vascular
defects, including a reduced number of parallel veins,
especially small veins (Fig. 1, E and F). On the other
hand, transverse sections of the shortened internodes
in the nal1 mutant also exhibited disturbed vascular
patterns, in which the number and arrangement of the
vascular bundles were altered (Fig. 1G). Gene cloning
studies indicated that Nal1 encodes a novel protein
that was preferentially expressed in vascular tissues
(Fig. 3, B–I).
It had been established that auxin, especially its
characteristic polar transport, plays a key role during
pattern formation of the vascular system in both dicot
and monocot plants (Tsiantis et al., 1999; Dengler, 2001;
Scanlon et al., 2002; Aloni, 2004; Fukuda, 2004; Scarpella
and Meijer, 2004; Teale et al., 2006). Our findings that
the nal1 mutant showed abnormal vascular patterns in
leaves and culms suggested that the nal1 phenotypes
might be associated with auxin-dependent deficiencies.
A series of analyses indicated that the nal1 mutant is
defective in polar auxin transport. Supporting evidence
for this notion came from measurements of [3H]IAA in
etiolated coleoptiles, showing that the basipetal PAT
activity in nal1 was significantly decreased (Fig. 4B)
whereas the auxin retention in nal1 was significantly
increased (Fig. 4C). Given that nal1 plants show abnormal vascular patterns in leaves and culms, the reduced
PAT activity in nal1 could be due to defective vascular
development in coleoptiles. Our investigation of 5-dold dark-grown coleoptiles indicated that the morphology, vascular bundle number, and vascular structure
between nal1 and the wild type were similar (Fig. 4,
D–G). These observations rule out the possibility that
the observed difference in IAA transport arises from
structure differences between nal1 and the wild type.
Our auxin transport assay also indicated that the
nal1 mutant was less sensitive than the wild type to the
Figure 4. (Continued.)
probed with anti-AtPIN1 antibodies. The signal (arrowhead) abundance detected in nal1 was significantly reduced from that in
the wild type. The numbers at left denote the molecular masses of marker proteins in kilodaltons. Coomassie blue staining of the
protein samples was used as a loading control (bottom gel). M, Quantitative real-time RT-PCR analysis comparing OsPIN1
relative expression levels in 5-d-old coleoptiles of the wild type and nal1. Values represent means 6 SD of three independent
assays. The difference between the wild type and nal1 is significant (Student’s t test, P , 0.05).
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Qi et al.
inhibitory effect of NPA on basipetal PAT (Fig. 4, B and
C). It was proposed that the action of NPA to inhibit
auxin transport was achieved through interacting with
the so-called NPA-binding protein, which has long
been considered part of the auxin efflux carrier complex (Dixon et al., 1996; Ruegger et al., 1997; Blakeslee
et al., 2005). On the other hand, a characteristic feature
of NPA action is that auxin accumulates in treated
cells, and the effect of NPA on PAT was generally
interpreted as an inhibition of auxin efflux (Morris
et al., 2004). In this scenario, the findings that the nal1
mutant exhibited decreased PAT activity and altered
responses to NPA led us to the hypothesis that the nal1
mutation might affect, directly or indirectly, auxin
efflux. To test this, we looked at Arabidopsis, a model
system in which the auxin efflux apparatus is relatively well understood. Among the molecular components involved in polar auxin transport in Arabidopsis
identified to date, the best characterized AtPIN1 protein was believed to play an important role in the
control of auxin efflux (Palme and Gälweiler, 1999).
The existence of a family of highly conserved PIN
proteins in the rice genome (Paponov et al., 2005),
together with recent experimental evidence showing
that one member of the rice PIN protein family, named
OsPIN1, plays a role in auxin-dependent development
(Xu et al., 2005), suggested that the function of the PIN
proteins in regulating auxin efflux is conserved between these two species (Paponov et al., 2005; Xu et al.,
2005). Therefore, available antibodies raised against
the auxin efflux carrier AtPIN1 were used as probes to
conduct immunohistochemical and protein gel-blot
analyses in nal1 and wild-type plants. In both assays,
the AtPIN1 antibodies could detect specific signals in
vascular tissues (Fig. 4, H–K). Furthermore, signals
detected by the AtPIN1 antibodies in the nal1 mutant
were shown to be significantly weaker than those in
wild-type plants (Fig. 4L). Gene expression analysis
suggested that the nal1 mutation might affect the
abundance and activities of OsPIN1 (Fig. 4M). Together, these results led us to the following speculations that need to be explored in our ongoing and
future studies. First, the signals detected by the AtPIN1
antibodies might represent the rice homolog(s) of the
AtPIN1 protein, which presumably acts as an auxin
efflux carrier in rice. Second, reduced abundance of
the rice homolog(s) of AtPIN1 might account for the
PAT deficiency in the nal1 mutant. The results presented here demonstrated that mutation of the Nal1
function affects both auxin transport and vascular
patterns; however, further studies are required to
address whether the nal1 aberrations in vascular patterns are the result of its deficient auxin transport.
Another significant aspect of the nal1 phenotype is
defective cell division. For example, the total cell
number of the longitudinal sections of internode IV
in nal1 mutants decreased to around one-third of that
of wild-type plants (Fig. 1D; Table I). Given the
established role of auxin in the control of cell division
(Meijer and Murray, 2001; Morris et al., 2004; Woodward
and Bartel, 2005), it is reasonable to generally associate
the reduced cell number of the nal1 internode IV with
its defective PAT-mediated auxin distribution. However, the detailed molecular mechanism by which
Nal1 exerts its action on the cell cycle needs to be
explored in future studies.
Subcellular localization studies using Arabidopsis
protoplast or transgenic plants indicated that the Nal1GFP fusion protein appears to form discrete nuclear
protein bodies (Fig. 2, G and H). Accumulating evidence in Arabidopsis indicated that nuclear protein
bodies are the sites of regulated protein degradation
mediated by the ubiquitin/26S proteasome. For example, a Rac GTPase stimulates the recruitment of
GFP-labeled AUXIN/IAAs from the nucleoplasm into
nuclear protein bodies to be degraded by the ubiquitin/26S proteasome apparatus (Tao et al., 2005). AFP, a
negative regulator of abscisic acid signaling, colocalizes with ABI5, a positive regulator of abscisic acid
signaling, in nuclear protein bodies (Lopez-Molina
et al., 2003). Abscisic acid also induces a heterogeneous nuclear ribonucleoprotein-type protein named
AKIP1 to form nuclear protein bodies (Ng et al., 2004).
Phytochrome B moves to the nucleus and forms nuclear protein bodies in response to different light
conditions (Chen et al., 2003). Similarly, the E3 ligase
COP1 has been observed to form nuclear protein
bodies with several regulators of light signaling, including HY5 (Saijo et al., 2003), LAF1 (Seo et al., 2003),
and HFR1 (Jang et al., 2005; Yang et al., 2005). In this
context, our results showing that Nal1-GFP forms
nuclear protein bodies link the Nal1 protein to the
proteolysis process. As a first step to elucidate the
biochemical and molecular mechanisms underlying
the Nal1 actions, we are conducting experiments to
determine whether Nal1 is involved in the degradation of other proteins or Nal1 itself is subjected to
degradation by the 26S proteasome.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
A rice (Oryza sativa) mutant line showing a characteristic phenotype of
narrow leaves was kindly provided by Dr. G.S. Khush at the International Rice
Research Institute in the Philippines. The original mutant was back-crossed to
the indica cultivar Zhefu802 for 10 successive generations, and the resulting
mutant line was renamed nal1 (Dong et al., 1994; Zeng et al., 2003). nal1 and
Zhefu802 were therefore considered to be a pair of nearly isogenic lines, and
Zhefu802 was used as the wild-type line for all experiments involving
comparisons of mutant and wild-type plants in this study. Rice plants were
cultivated in the experimental field at the China National Rice Research
Institute in the natural growing season.
Phenotypic Characterization of the nal1 Mutant
To investigate the morphology of the vascular bundles of the leaf blade and
the internode, tissues at indicated stages were fixed in 5% formaldehyde, 5%
glacial acetic acid, and 63% ethanol and dehydrated in a graded ethanol series.
The samples were embedded in Paraplast Plus chips (Sigma). Microtome
sections (8 mm thick) were affixed to poly-Lys-coated slides (Sigma), dewaxed
in xylene, rehydrated through a graded ethanol series, and dried overnight
before staining with toluidine blue for light microscopy.
1956
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Nal1 Affects Venation and Polar Auxin Transport
Map-Based Cloning of Nal1
To identify the Nal1 gene using a positional cloning approach, nal1 was
crossed to Nipponbare, a japonica cultivar, to generate a large F2 mapping
population. For fine mapping, PCR-based markers were developed based on
the sequence difference between japonica variety Nipponbare and indica variety
9311 (http://www.gramene.org/resources/). The primer sequences of the
molecular markers used are listed in Table II. For RT-PCR, we used total
RNA isolated from young seedlings as template. The full-length cDNA of Nal1
was amplified using the primer pair 5#-CGCTTTCGGCATTCGTTATCTACC-3#
and 5#-GGGATCCGGCAATGGTGTATATCA-3#, sequenced, and cloned into
pBluescript SK1 vector (Stratagene).
Complementation Test
A 12.4-kb genomic DNA fragment containing the entire Nal1 coding
region, the 2,673-bp upstream sequence, and the 1,685-bp downstream sequence was cut from BAC AL662950 with the restriction enzyme XbaI. This
fragment was cloned into the XbaI sites of the binary vector pCAMBIA2300 to
generate the transformation plasmid for the complementation test. The
resulting transformation plasmid, as well as the empty pCAMBIA2300 vector
as control, was introduced into the nal1 mutant by Agrobacterium tumefaciensmediated transformation according to a published protocol (Hiei et al., 1994).
The 30-bp deletion of the putative Nal1 allele in the mutant, which was
confirmed in both cDNA and genomic sequences between nal1 and wild-type
plants, could be detected with PCR using the primer pair 5#-GATGACTTTGACATTTCCACCGT-3# and 5#-GAGTGATTCATTGGTAATGATAA-3#.
Phylogenetic Analysis
Homologous sequences of Nal1 were obtained through National Center for
Biotechnology Information BLAST search. Maize (Zea mays) coding sequences
were predicted by Fgenesh using homologous BAC clone sequences as input.
Others were directly from a homologous cDNA clone. The collected protein
sequences were then aligned by DIALIGN 2. Note that the program DIALIGN
2 employs the BLOSUM62 amino acid substitution matrix but does not use
gap penalty. Instead, it assembles global alignments from gap-free local pairwise alignments. The alignment was revised critically and used as input for
MEGA3 to construct a phylogenetic tree. A neighbor-joining tree was built by
MEGA3 using a Poisson correction model with gaps to complete deletions.
Topological robustness was assessed by bootstrap analysis with 500 replicates.
Gene Expression Analysis
For RT-PCR analysis, total RNA was extracted from various tissues using
TRIZOL reagent (Invitrogen). After RNase-free DNase (Takara) treatment, 2.5
mg of RNA was reverse transcribed using oligo(dT) primer and SuperScript III
(Invitrogen). An appropriate amount of the products was further applied for
a 20-mL PCR amplification using the following gene-specific primer pairs:
5#-GATGACTTTGACATTTCCACCGT-3# and 5#-GAGTGATTCATTGGTAATGATAA-3# for Nal1 (40 cycles of 15 s at 94°C, 15 s at 60°C, and 30 s at 72°C)
and 5#-CATCTTGGCATCTCTCAGCAC-3# and 5#-AACTTTGTCCACGCTAATGAA-3# for ACTIN1 (25 cycles of 15 s at 94°C, 15 s at 60°C, and 30 s at 72°C).
For quantitative real-time RT-PCR, first-strand cDNAs were synthesized
by reverse transcription from 3 mg of total RNA. The cDNAs were then used as
templates in real-time PCR using the SYBR Green PCR Master Mix (Applied
Biosystems) according to the manufacturer’s instructions. The amplification
of the target genes was analyzed using the ABI Prism 7000 sequence detection
system and software (PE Applied Biosystems). The relative expression levels
of each transcript were obtained by normalization to the UBIQUITIN5 (UBQ5)
gene. The following gene-specific primers were used for real-time PCR
analysis: 5#-CGCCGTGCCGCTGCTGTCGTTCC-3# and 5#-TCCCCCGGCGGCTGAGGTG-3# for OsPIN1 and 5#-ACCACTTCGACCGCCACTACT-3# and
5#-ACGCCTAAGCCTGCTGGTT-3# for UBQ5. Three independent RNA isolations were used for cDNA synthesis, and each cDNA sample was subjected
to real-time PCR analysis in triplicate.
For promoter-GUS assay, a 1,918-bp genomic fragment upstream of the
Nal1 gene translation start codon was PCR amplified with the primers
5#-AAGTCGACTCCGAACCAAACACCAACACAC-3# and 5#-AAGAATTCACAGTTTGCGAACCTATTATA-3#. The DNA fragment was cloned into the
SalI and EcoRI sites of the vector pCAMBIA1391Z. The resulting plasmid was
transformed into rice, and the resulted transgenic plants were analyzed by
GUS staining assay as described (Scarpella et al., 2003).
RNA in situ hybridization was performed as described by Schwarzacher
and Heslop-Harrison (2000). The 3# end of Nal1 was amplified from wild-type
plants with the following primers: 5#-CACCAACCTGAACAACCCCT-3# and
5#-GGTCTTTTGATCTACTACTA-3#. The PCR product was subcloned into
pGEM-T Easy vector (Promega) and used as a template to generate RNA
probes. Transverse sections (8 mm thick) were probed with digoxigeninlabeled antisense probes (DIG Northern Starter Kit; Roche). The slides were
observed with a microscope (Leica) and photographed using a 3CCD color
video camera (Apogee Instruments).
Subcellular Localization of Nal1
The Nal1 cDNA was amplified from wild-type rice plants with the primers
5#-AAGTCGACATGAAGCCTTCGGACGATAA-3# and 5#-AGCCATGGTCTCCAGGTCAAGGCTTGAT-3# and cloned into the SalI/NcoI sites of the
CaMV35S:GFP (S65T)-NOS-30 cassette vector (Niwa et al., 1999; Li et al.,
2003). The resulting 35S:Nal1-GFP construct was introduced into Arabidopsis
(Arabidopsis thaliana) protoplast as described (Meskiene et al., 2003).
The 35S:Nal1-GFP construct was digested with HindIII/EcoRI and cloned
into the binary vector pCAMBIA1300 (http://www.cambia.org.au) to transform Arabidopsis plants using the floral dip method (Clough and Bent, 1998).
Progeny of an identified T3 transgenic plant homozygous for the transgene
were used to investigate the Nal1-GFP localization. The root cells of the
5-d-old transgenic seedlings grown on Murashige and Skoog (1962) agar
plates were analyzed. Confocal images were collected using a Zeiss LSM 510
Meta confocal laser scanning microscope. GFP fluorescence was excited
with a 488-nm argon laser, and images were collected in the 500- to 530-nm
range. In all cases, transmissible light images were collected simultaneously.
At least 20 seedlings were observed, and a representative image is
presented.
Auxin Influx and Efflux Assays
Auxin uptake assays were conducted according to a method described
previously (Dai et al., 2006) with some modifications. The upper ends (0.5 cm
away from the tip) of 3-d-old dark-grown coleoptiles were harvested and
allowed to equilibrate in a 1.5-mL microcentrifuge tubes containing 1/2 MS
buffer (5 mM MES and 1% [w/v] Suc, pH 5.8) for 2 h. The coleoptile fragments
(0.2 cm in length) were then incubated in 1/2 MS buffer containing 100 nM
[3H]IAA (25 Ci/mmol; Amersham) for 10 min. Samples were then transferred
into a new 1.5-mL microcentrifuge tube and rinsed in 1 mL of ice-cold 1/2 MS
buffer three times. After rinsing, the samples were incubated in scintillation
liquid for 18 h and the radioactivity was counted with a liquid scintillation
counter (1450 MicroBeta TriLux; Perkin-Elmer). For each assay, five coleoptiles
were measured.
The polar auxin transport assays were performed using coleoptiles of 5-dold dark-grown rice seedlings as described previously (Okada et al., 1991; Dai
et al., 2006; Li et al., 2007) with minor modifications. In each assay, five apical
coleoptile segments each (0.2 cm away from the tip) of 2.0-cm length were
preincubated in 1/2 MS medium with shaking at 100 rpm for 2 h. The
coleoptile segments were then placed in a 1.5-mL microcentrifuge tube with
one end submerged in 10 mL of 1/2 MS liquid medium containing 500 nM
[3H]IAA plus 500 nM free IAA in the dark at room temperature for 2 h. NPA
(Chem Service) was added to the medium as indicated. The unsubmerged
ends of the segments (0.5 cm in length) were excised and washed three times
in 1/2 MS liquid medium. After rinsing, the segments were incubated in 2.0
mL of universal scintillation fluid for 18 h and the radioactivity was counted
with a liquid scintillation counter (1450 MicroBeta TriLux; Perkin-Elmer).
Based on the orientation of the coleoptile segments submerged in the buffer,
basipetal or acropetal auxin transport was measured, with the acropetal auxin
transport as control.
IAA efflux of dark-grown coleoptile segments was assayed as described
previously (Dai et al., 2006). In each assay, five apical dark-grown coleoptile
segments (2.0 mm in length) were put into 50 mL of 1/2 MS medium buffer
containing 500 nM [3H]IAA plus 500 nM free IAA and incubated at room
temperature for 2 h with shaking at 100 rpm. NPA (2 mM) was added to the
medium as indicated. The segments were then rinsed and incubated for
another 2 h in the same buffer without IAA to allow the IAA to be
transported out. After washing twice, the segments were then incubated in
2.0 mL of universal scintillation fluid for 18 h, and the radioactivity was
counted with a liquid scintillation counter (1450 MicroBeta TriLux; PerkinElmer).
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Qi et al.
Immunohistochemical Localization and Protein
Gel-Blot Analysis
Immunohistochemical localizations and microscopy were conducted as
described previously (Noh et al., 2003). The antibody used was anti-AtPIN1
(Gälweiler et al., 1998) at a final dilution of 1:200. Fluorescein isothiocyanateconjugated antibody was used as a secondary antibody. A confocal laser
scanning microscope (Zeiss LSM 510 Meta) was used for immunofluorescence
imaging.
One gram of coleoptiles from 5-d-old dark-grown seedlings was used
for membrane protein extraction using the ProteoExtract kit (M-PEK;
Calbiochem). Membrane proteins were separated by 10% SDS-PAGE and
probed with anti-AtPIN1 (1:200) antibodies.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: Nal1 (EU093963), putative
OsPIN1 (AF056027).
ACKNOWLEDGMENTS
We thank Dr. Jiayang Li (Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences) for critical reading of the manuscript. We
thank Dr. Gurdev Singh Khush (International Rice Research Institute) for
providing the original nal1 mutant, Dr. Bin Han (National Center for Gene
Research, China) for providing the BAC clone AL662950, and Dr. Kang
Chong (Institute of Botany, Chinese Academy of Sciences) for assistance in
RNA in situ hybridization.
Received March 6, 2008; accepted June 10, 2008; published June 18, 2008.
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