SHALLOT-LIKE1 Is a KANADI Transcription Factor ThatModulates Rice Leaf Rolling by Regulating Leaf AbaxialCell Development W OA
Guang-Heng Zhang,a,1 Qian Xu,b,1 Xu-Dong Zhu,a Qian Qian,a and Hong-Wei Xueb,2
a State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, Zhejiang, Chinab National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
As an important agronomic trait, rice (Oryza sativa L.) leaf rolling has attracted much attention from plant biologists and
breeders. Moderate leaf rolling increases the photosynthesis of cultivars and hence raises grain yield. However, the relevant
molecular mechanism remains unclear. Here, we show the isolation and functional characterization of SHALLOT-LIKE1
(SLL1), a key gene controlling rice leaf rolling. sll1 mutant plants have extremely incurved leaves due to the defective
development of sclerenchymatous cells on the abaxial side. Defective development can be functionally rescued by
expression of SLL1. SLL1 is transcribed in various tissues and accumulates in the abaxial epidermis throughout leaf
development. SLL1 encodes a SHAQKYF class MYB family transcription factor belonging to the KANADI family. SLL1
deficiency leads to defective programmed cell death of abaxial mesophyll cells and suppresses the development of abaxial
features. By contrast, enhanced SLL1 expression stimulates phloem development on the abaxial side and suppresses
bulliform cell and sclerenchyma development on the adaxial side. Additionally, SLL1 deficiency results in increased
chlorophyll and photosynthesis. Our findings identify the role of SLL1 in the modulation of leaf abaxial cell development and
in sustaining abaxial characteristics during leaf development. These results should facilitate attempts to use molecular
breeding to increase the photosynthetic capacity of rice, as well as other crops, by modulating leaf development and rolling.
INTRODUCTION
The three-dimensional structure of the plant leaf is crucial for its
functions, including light capture, carbon fixation, and gas ex-
change for photosynthesis (Govaerts et al., 1996). Appropriate
leaf shape is an important characteristic associated with the
super-rice (a hybrid rice [Oryza sativa L.] with desirable charac-
teristics) ideotype; the rolled leaf is regarded as a critical element
for the ideal rice phenotype (Yuan, 1997; Chen et al., 2001).
Moderate leaf rolling helps to maintain the erectness of leaves,
which can increase the light transmission rate and light saturation
point without affecting the light compensation point, resulting in a
well-proportioned leaf area for photosynthesis (Duncan, 1971).
Moderate leaf rolling improves photosynthetic efficiency, accel-
erates dry-matter accumulation, increases yield, reduces the
solar radiation on leaves, and decreases leaf transpiration under
drought stress (Lang et al., 2004). Therefore, the isolation of
genes that control leaf rolling will be beneficial for breeding crops
with the desired architecture and stress tolerance.
As a polymorphic crop, rice varieties and mutants exhibit
several types of leaf rolling, including inward or outward rolling,
and these various phenotypes are regulated by complicated
developmental processes, including pattern formation, polarity
establishment, and cell differentiation (Micol and Hake, 2003;
Lang et al., 2004). Hsiao et al. (1984) reported that leaf rolling is
induced by altered osmotic pressure between internal and
external tissues. Moulia (1994) proposed that leaf rolling may
result from varying degrees of dehydration in different cross
sections of the rolled leaf. Price et al. (1997) suggested that leaf
rolling results from decreased turgidity of bulliform cells. How-
ever, the mechanism underlying leaf rolling in monocots remains
to be elucidated.
Due to its importance, many studies have been performed to
characterize the genes controlling rice leaf rolling. Up to now, 11
rice mutants with rolled leaves (rl) have been characterized, for
which six recessive genes (rl1-rl6) were mapped on rice chro-
mosomes 1, 4, 12, 3, or 7 by morphological markers (Kinoshita,
1984; Khush and Kinoshita, 1991; http://www.gramene.org/).
rl7-rl10 were mapped to chromosomes 5 (rl7; Li et al., 1998; rl8,
542 kb; Shao et al., 2005a) and 9 (rl9, 42 kb; Yan et al., 2006; rl10,
194 kb; Luo et al., 2007) by molecular markers. Rl(t), an incom-
plete recessive rolled leaf mutant, was fine-mapped to a 137-kb
region of the long arm of chromosome 2 (Shao et al., 2005b). In
addition, recent studies showed that overexpression ofOsAGO7
results in the upward curling of rice leaves (Shi et al., 2007).
In Arabidopsis thaliana, mutants with differently curved leaves
have been extensively described, including hyponastic leaves1
(Lu and Fedoroff, 2000), curly leaf (Goodrich et al., 1997), and
incurvata (Berna et al., 1999). Aside from the effects of hormones
and environmental cues, the defective establishment of leaf
polarity is one important factor that results in rolled leaves (Byrne
1 These authors contributed equally to this work.2 Address correspondence [email protected] authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Qian Qian([email protected]) and Hong-Wei Xue ([email protected]).WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.108.061457
This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been
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The Plant Cell Preview, www.aspb.org ã 2009 American Society of Plant Biologists 1 of 17
et al., 2000;Semiarti et al., 2001). Several adaxial-abaxial asymmetry-
related genes have been characterized in Arabidopsis. Members
of the class III homeodomain-leucine zipper (HD-ZIP III) family
PHABULOSA (PHB),PHAVOLUTA (PHV) (McConnell andBarton,
1998; McConnell et al., 2001), and REVOLUTA (REV; Talbert
et al., 1995; Otsuga et al., 2001) promote adaxial organ identity.
On the other hand, KANADI (KAN; Kerstetter et al., 2001) and
YABBY (YAB) family genes, including FILAMENTOUS FLOWER
(Sawa et al., 1999a, 1999b),YAB2, andYAB3 (Eshed et al., 2001),
are required to establish abaxial identity.
Other key regulators of leaf polarity include a group of func-
tional homologs, including PHANTASTICA (PHAN) inAntirrhinum
majus, ROUGH SHEATH2 in maize (Zea mays), and ASYMMET-
RIC LEAVES1 (AS1) in Arabidopsis. All are MYB domain–con-
taining transcription factors (Waites et al., 1998; Timmermans
et al., 1999; Tsiantis et al., 1999; Byrne et al., 2000; Sun et al.,
2002). ASYMMETRIC LEAVES2 (AS2), which encodes a protein
containing a leucine-zipper motif associated with AS1, is impor-
tant in promoting leaf adaxial fate (Iwakawa et al., 2002; Xu et al.,
2002). The maize gene ROLLED LEAF1 (RLD1) encodes a HD-
ZIP III family transcription factor and is involved in adaxial
specification (Juarez et al., 2004b). Recent studies showed that
the maize MILKWEED POD1 (MWP1) gene encodes a KANADI
protein that is involved in the abaxial-adaxial patterning of leaves
(Candela et al., 2008).
Posttranscriptional and posttranslational regulation are essen-
tial for proper leaf patterning (Xu et al., 2007). Transcripts of PHB,
PHV, and REV contain complementary sites for microRNAs
miR165 and miR166, which can direct their cleavage in vitro
(Reinhart et al., 2002; Tang et al., 2003). Themaize geneRLD1 has
an miRNA166 complementarity site; its defective mutant, rld1,
showed adaxialized or partially reversed axis patterns (Nelson
et al., 2002). Additionally, ASYMMETRIC LEAVES ENHANCER3
(AE3) encodes the 26Sproteasomesubunit RPN8aand is involved
in the AS1/AS2 pathway that regulates the development of leaf
polarity (Huang et al., 2006).
The adaxial-abaxial pattern of the monocot leaf is different
from that of dicot plants. In dicots, the internal mesophyll cells
are polarized; the closely packed palisade cells are adjacent to
the adaxial epidermis, and loosely packed spongy cells border
the abaxial epidermis (McConnell and Barton, 1998). In mono-
cots such asmaize, the leaf is divided into five tissue layers along
the adaxial-abaxial axis (Freeling and Lane, 1993). Its polarity
characteristics include the macrohair and bulliform cells in the
adaxial epidermis and ligule tissue on the adaxial surface of the
blade sheath boundary (Nelson et al., 2002). Additionally, the
middle vein on the maize leaf contains polarized xylem and
phloem. In contrast with those of dicots, the mesophyll cells of
some grasses, including maize and rice, have a uniform config-
uration without polarization (there is only spongy parenchyma in
leaf), termed isobilateral mesophyll (Fahn, 1990); the develop-
ment and regulation of polarity in isobilateral leaves remain to be
elucidated. In addition, there is a distinct difference between the
C3 plant rice and the C4 plant maize with regard to sclerenchy-
matous tissues. In rice, the sclerenchymatous tissues display
excessive differentiation with a characteristic distribution, and
these tissues are ubiquitous around the vascular tissues of both
adaxial and abaxial epidermis. The hypodermal sclerenchyma of
maize is rare, and, when found, it is predominantly associated
with the abaxial epidermis (Nelson et al., 2002).
In the rice lamina, programmed cell death (PCD) occurs
throughout the formation of sclerenchymatous cells, including
tracheid and vessel elements in xylem and sclerenchyma tissues
surrounding the vascular bundle (Lu et al., 1982). Along with the
formation of conducting or mechanical tissues, the tracheary
elements (TEs) or sclerenchyma tissue undergo protoplast deg-
radation and are lignified with thickened secondary cell walls,
forming the specialized cells and ultimately the nonliving cells
responsible for water transport and support (Fahn, 1990). On the
cellular level, the DNases, RNases, and proteases accumulate in
the vacuoles, regulated by specific signals. The vacuoles col-
lapse, and the cell contents of the protoplasts are degraded to
form continuous canals (Fukuda, 2000).
The sclerenchymatous cells are lignified dead cells with thick-
ened secondary cell walls. PCD is involved in the transdiffer-
entiation of mesophyll cells to sclerenchymatous cells. This
differentiation consists of three stages: dedifferentiation (stage
I), conversion of mesophyll cells to TE precursor cells (procam-
bium cells and immature xylem cells) (stage II), and TE-specific
secondary wall thickening followed by lignification and cell
death–related events (stage III) (Fukuda, 2000). Multiple genes
are involved in this process. Expression of tracheary element
dehydrogenase2 (TED2), a vascular cell-specific gene, increases
in stage II and marks the initiation of stage III (Demura and
Fukuda, 1993, 1994). Cinnamate 4-hydroxylase (C4H), which is
transcribed in stages I and III, is associated with the lignification
of secondary cell walls (Ye, 1996). Cys protease (CP), which is
transcribed at stage III, degrades the cell contents and is critical
for TE-PCD (Ye and Varner, 1996). Protease inhibitor (PI) asso-
ciates withCP to function in the TE-PCD process (Solomon et al.,
1999). Phe ammonia-lyase (PAL) and 4-coumarate-CoA ligase
(4CL) are involved in secondary wall thickening during TE-PCD
stage III (Lin and Northcote, 1990; Voo et al., 1995). However,
little is known about the roles of PCD in the formation of TEs and
sclerenchymatous cells during rice leaf development.
Here, we report the isolation and functional characterization of
a rice gene, SHALLOT-LIKE1 (SLL1), that regulates rice leaf
rolling. SLL1 deficiency leads to the flawed formation of scler-
enchymatous cells on the abaxial side of the leaf and results in
extremely inwardly rolled leaves. SLL1 is crucial in polarity
formation and helps to direct the development of the leaf abaxial
cell layer. In addition, it is involved in the transdifferentiation of
mesophyll cells to sclerenchymatous cells through the modula-
tion of PCD, highlighting a possible mechanism underlying leaf
rolling in monocot plants.
RESULTS
sll1, a Rice Mutant with Shallot-Like Leaves, Displays
Abnormal Sclerenchymatous Cell Development in the
Abaxial Cell Layers, Altered Mesophyll Cell Distribution,
Increased Amounts of Chlorophyll, and
Enhanced Photosynthesis
To investigate the molecular mechanisms of rice leaf rolling, a
rice mutant population (Oryza sativa L. ssp. japonica variety
2 of 17 The Plant Cell
Nipponbare) generated by ethyl methanesulphonate mutagene-
sis was screened. Two allelic mutants with extremely incurved
leaveswere identified, designated as sll1-1 and sll1-2 (Figure 1A).
Phenotypic observation showed that sll1 mutant plants had
narrow, extremely rolled and dark-green leaves, which appeared
during the seedling stage. These leaves became more evident
during plant growth. Observation of the cross section revealed
enlarged clear cells in the midrib of sll1 plants (Figure 1A, bottom
panel).
In contrast with thewild type,mature sll1 leaves display altered
mesophyll cell differentiation and distribution. sll1 does not form
abaxial sclerenchymatous cells in the small veins of lateral region
(Figure 1B), where the leaf starts to roll; however, the midrib
region and the margin of the blade demonstrate cellular organi-
zation similar to the wild type (Figure 1C), indicating the modu-
lated differentiation of sclerenchymatous cells in sll1. Bulliform
cells, which are normally found in the adaxial epidermis, were
found in abaxial surfaces of sll1-1 leaves (Figure 1B). Further-
more, the differences in abaxial sclerenchymatous specification
can be observed in young leaves with curved leaf sheathes
(plastochron 5), appearing after the mesophyll cells begin to
differentiate. The mesophyll cells in abaxial layers do not differ-
entiate to become sclerenchymatous cells in sll1-1 (Figure 1D).
In wild-type leaf blades, the mesophyll cells on both adaxial
and abaxial sides are interrupted by sclerenchymatous cells
connecting vascular bundles and epidermis, while those on the
abaxial side of sll1-1 leaves were replaced by mesophyll cells
(Figure 2A, top panel). Leaveswere a deeper green on the abaxial
side (Figure 2A, bottom panel), indicating increased numbers of
mesophyll cells. We also measured the chlorophyll in wild-type,
sll11-1, sll1-2, or transgenic sll1-2 plants with complemented
expression ofSLL1. Measurements were performed on the same
area of the plant (at the middle part of the second leaf from the
top of the plant) at same growth stage. Chlorophyll measure-
ments indicated that the increased numbers of mesophyll cells
resulted in a corresponding increase in the amount of chlorophyll
in sll1-1 and sll1-2 plants, and the complemented expression of
SLL1 in sll1-2 plants restores normal chlorophyll levels (Figure 2B).
Measurement of the quantumyield of photosystem II (PSII) and
the maximal quantum yield of PSII (Fv/Fm) showed that both
were slightly increased in sll1-1 or sll1-2 (Table 1), indicating
increased photosynthesis due to the increased numbers of
mesophyll cells and elevated chlorophyll content of the leaves.
SLL1 Encodes a SHAQKYF ClassMYB Transcription Factor
Genetic analysis of reciprocal crosses between the sll1-1, sll1-2,
and wild-type plants revealed that the abnormal character of sll1
was controlled by a single recessive gene. To isolate the relevant
mutant gene, SLL1 was mapped to the long arm of rice chro-
mosome 9 between markers RM1896 and RM3700. This con-
clusion is based on analysis of the F2 population from a cross
between sll1-2 and Nanjing 6 (Figure 3A). A large F2 mapping
population was then generated, allowing the fine-mapping of
SLL1 to a 29.57-kb region, using the sequence tagged site and
simple sequence repeat markers (Figure 3A; see Supplemental
Table 1 online). Three annotated candidate genes, encoding a
hypothetical protein, an En/Spm-like transposon, and a tran-
scription factor containing a MYB-like domain, respectively,
were located in this region (The Institute for Genomic Research;
http://rice.plantbiology.msu.edu/). Further amplification of the
relevant DNA fragments and sequence comparison revealed
differences in sll1-1 and sll1-2 alleles in the gene encoding the
transcription factor containing the MYB-like domain. The sll1
allele carried a single base substitution (G to A) in the intron
splicing site of both sll1-1 (the first intron) and sll1-2 (the third
intron), resulting in altered mRNA splicing and premature termi-
nation of the encoded protein (Figure 3B). To verify this, primers
located in the two open reading frames (ORFs) flanking the
mutation site were used to amplify the relevant fragments. The
longer mRNAmolecules were indeed amplified in sll1-1 and sll1-2
(Figure 3C), confirming the altered splicing of sll1 transcripts.
Comparison with the corresponding genomic sequence re-
vealed that the SLL1 gene consists of six exons and five introns.
The gene encodes a 377–amino acid MYB family transcription
factor (Figure 3B). Homologous analysis showed that SLL1
shares high similarity with the KAN family members in Arabidop-
sis (Table 2). The Arabidopsis KAN1 is involved in the polarity
regulation of lateral organs, and three other KAN genes (KAN2,
KAN3, and KAN4) also redundantly specify abaxial fate (Eshed
et al., 1999, 2001; Kerstetter et al., 2001; Hawker and Bowman
2004). This finding suggested the possible effects of SLL1 in
polarity identity. Neighbor-joining phylogenetic tree analysis
showed that among the KANADI members in rice, maize, and
Arabidopsis, SLL1 is closest tomaizeMWP1 (MILKWEEDPOD1,
which has been shown to be involved in adaxial/abaxial pattern-
ing) (Candela et al., 2008), two other KANADI members (KAN1
and KAN3), and rice KANADI member KAN2 (Figure 4A). SLL1 is
closest to At KAN1 among the Arabidopsis KANADI members
(Figure 4A, Table 2), and structural organization analysis showed
that all KANADI proteins share a highly conserved GARP domain
(Figure 4B). The GARP domain is a plant-specific DNA binding
domain of the helix-loop-helix superfamily that is distantly related
to the MYB domain (GARP is an acronym for the founding
members of the family: maize Golden 2, ARR [Arabidopsis
response regulators], and Psr1 [phosphorous stress response1
from Chlamydomonas]). Studies have shown that the GARP
domain is crucial for regulating the transcription of downstream
genes (Kerstetter et al., 2001). The shared identity for this GARP
domain in the KANADI family is higher than 85% (the overall
protein identity is lower than 43%; Table 2).
Although both mutant alleles show rolled leaves, leaf rolling
index (LRI) analysis showed that sll1-1 has more severe rolling
(Figure 4C), which is consistentwith the fact thatmutation of sll1-1
affects theGARPdomain,while that of sll1-2doesnot (Figure 4B).
These phenotypic differences indicate the importance of the
GARP domain to the function of SLL1, probably because of its
central role regulating gene transcription.
The Expression Pattern of SLL1
Quantitative real-time RT-PCR (qRT-PCR) analysis revealed the
expression of SLL1 in various tissues, including roots, stems,
leaves, flowers, and seedlings, with relatively high expression in
leaf and seedlings (Figure 5A, top panel). With an emphasis on
the leaf, analysis by qRT-PCR showed that SLL1 is ubiquitously
SLL1 Controls Rice Leaf Rolling 3 of 17
Figure 1. sll1 Has Extremely Incurved Leaves, with Deficiency of Sclerenchymatous Cells at the Abaxial Side.
(A)Morphology of wild-type and sll1-1 plants and leaves. Mature wild-type and sll1-1 mutant plants were observed. The clear cells of the midrib (white
box) are highlighted with arrows. Bars = 5 cm (top panel) or 2 mm (bottom panel).
(B) sll1-1 leaf blades display altered cellular organization. Bulliform cells on the abaxial surfaces of sll1-1 leaves are highlighted (top panel, arrows). The
altered differentiation and distribution of mesophyll cells are highlighted (blue box) in the bottom panel. LE, lower epidermis; SC (ab), abaxial
sclerenchyma; B, bulliform cells; P, phloem; X, xylem; MS, mestome sheath; VBS, vascular bundle sheath; M, mesophyll cells; SC (ad), adaxial
sclerenchyma; UE, upper epidermis. Bars = 100 mm (top panel) or 20 mm (bottom panel). The broken line indicates the defective sclerenchyma at the
abaxial epidermis. Sections (;20 mm) of rice leaf blade were stained in Ruthenium red solution.
(C) The defective sclerenchymatous cells on the abaxial side of sll1-1 leaves. The defect is mainly in the small veins where the curl occurs (2; broken
lines). Cellular organization at the midrib region (1) and the margin of the blade (3) in sll1-1 is normal. Bar = 50 mm.
(D) Transverse section of young seedlings at 5 d after germination. Differentiated sclerenchymatous cells on the wild-type abaxial epidermis are
highlighted (left panel, arrows) but still lack a thickened secondary wall. These are deficient in sll1-1 (broken arrows). Bars = 100 mm.
4 of 17 The Plant Cell
expressed in shoot apical meristem (SAM), leaf blade, or leaf
sheath at different developmental stages (Figure 5A, bottom
panel).
To assess the expression pattern comprehensively, b-glucu-
ronidase (GUS) activity was examined histochemically in trans-
genic plants carrying an SLL1 promoter-GUS reporter gene.
Results showed thatSLL1was transcribed instem (Figure5B,panels
1 and 2), anthers of young or mature flowers (Figure 5B, panels 3
to 5), pistil tip (Figure 5B, panel 6), glume (Figure 5B, panel 7),
vascular tissues of mature seeds (Figure 5B, panels 8 and 9),
coleoptile and embryonic root of germinating seedlings (Figure
5B, panel 10), and root vascular tissues (Figure 5B, panel 11). In
addition, SLL1 is highly transcribed in leaf veins and leaf sheath
(Figure 5C, panels 1 to 3), guard cells, and tracheal elements
(Figure 5C, panels 4 and 5).
We further examined the spatial and temporal localization of
SLL1 during leaf development by in situ hybridization analysis.
The mRNA expression of SLL1 was detected throughout the
young leaf primordium (plastochrons 1 to 3) and was more
intense in abaxial cell layer through leaf development (plasto-
chrons 4 and 5) (Figure 5D, panel 2). However, the SLL1 tran-
script did not demonstrate apical/basal polarity and did not
accumulate at the apex of the meristem (Figure 5D, panel 3).
Cross-section analysis of the shoot apex showed that SLL1 was
more highly expressed in the abaxial cell layer, including the
epidermis and vasculature of the early leaf blade (Figure 5D,
panels 5 and 6). In the mature leaf, SLL1 mainly accumulated at
the abaxial epidermis, abaxial mesophyll cells, and vasculature
(Figure 5D, panels 8 and 9). In contrast with the sense probe
(Figure 5D, panels 4 and 7), SLL1 was transcribed at a relatively
low level throughout other leaf positions including the adaxial
epidermis (Figure 5D, panels 5 and 6).
Complemented Expression of SLL1 Rescued the Rolled
Leaves of sll1
The physiological effect of SLL1 in leaf rolling was further
confirmed by genetic complementation analysis. A construct
carrying the 7.8-kb genomic fragment containing the entire
promoter region and SLL1 ORF (excised from BAC B1040D06
and subcloned into vector pCAMBIA1300) was transformed into
sll1-2 plants (sll1-1 plants are almost sterile). Independent trans-
genic lines were identified through qRT-PCR analysis of SLL1
expression using primers matching the sequence of normal
transcript after correct splicing. The transcript in the sll1-2
mutant could not be amplified with these primers due to the
altered splicing (Figure 6A). Phenotypic observation confirmed
that complementary SLL1 expression of the normal splicing
product rescued leaf rolling, with restoration of normal leaf shape
at different developmental stages (3 or 6 weeks; Figure 6B). In
addition, observations of leaf cross sections indicated the normal
formation of sclerenchymatous cells at the abaxial epidermis
(Figures 6C and 6D).
SLL1 Modulates Leaf Rolling by Regulating Abaxial
Mesophyll Cell PCD
Defective formation of sclerenchymatous cells in the curve
region of sll1 leaves (Figure 1C) suggested that loss of
Figure 2. SLL1 Deficiency Results in Increased Chlorophyll Content.
(A) Distribution of mesophyll cells on the abaxial side of wild-type and
sll1-1 leaves. The broader distribution of mesophyll cells on the abaxial
side of sll1-1 leaves is highlighted (arrows), rendering the deeper green
color of sll1-1 abaxial epidermis. Sections (;20 mm) of rice leaf blades
were stained in aniline blue solution. Bars = 20 mm (top panel) or 2 mm
(bottom panel).
(B) Chlorophyll levels in wild-type, sll1-1, sll1-2, and sll1-2 plants with
complemented expression of SLL1. To measure chlorophyll levels, we
collected samples from an area from the middle part of the second leaf
from the top of wild-type, sll11-1, sll1-2, or transgenic sll1-2 plants with
complemented expression of SLL1 (at the same growth stage). Exper-
iments are biological replicates. Error bar represents SD (n > 30 in each
measurement), and statistical analysis by heteroscedastic t test indi-
cates the significant differences (* P < 0.05; ** P < 0.01).
Table 1. The Photosynthetic Efficiency of Wild-Type, sll1-1, and sll1-2
Plants
Wild Type sll1-1 sll1-2
Yield 0.739 6 0.017 0.761 6 0.02** 0.747 6 0.018
Fv/Fm 0.799 6 0.017 0.817 6 0.006* 0.808 6 0.019
The quantum yield of PSII and maximal quantum yield of PSII (Fv/Fm)
were measured and statistically analyzed. For measurements, the
middle parts of the 8th to 10th rice leaves (at the same developmental
stage) were used. Data are presented as means 6 SE (n > 10).
Heteroscedastic t test indicates the significant differences in sll1-1
(* P < 0.05; ** P < 0.01) compared with the wild type.
SLL1 Controls Rice Leaf Rolling 5 of 17
mechanical strength in abaxial sclerenchymatous cells leads to
inward rolling and that SLL1 is required for the normal formation
of sclerenchymatous cells. Previous studies have shown the
involvement of PCD in the differentiation of sclerenchymatous
cells and that multiple genes are involved in this process. We
performed qRT-PCR analysis to determine the expression of
rice genes that share high homology with TE-PCD marker
genes in Zinnia or other species. These genes include TED2,
C4H, CP, PI, PAL, 4CL, and Dirigent-1 (involved in lignin
assembly rather than as amarker gene of the TE-PCD process,
as determined by the finding that proteins harboring arrays of
monolignol radical binding sites are involved in macromolec-
ular lignin assembly; Davin and Lewis, 2005). The expression
ofCP, PI, PAL, and 4CL genes were suppressed in sll1-2, while
TED2, C4H, and Dirigent-1 were not obviously altered (Figure
7). The only known marker gene of stage II is TED2, which did
Figure 3. Map-Based Cloning of SLL1, Which Encodes an MYB Transcription Factor.
(A)Mapping of SLL1. SLL1was mapped primarily to the long arm of rice chromosome 9 between markers RM1896 and RM3700 and then to a 29.57-kb
region in a large F2mapping population. Amplification of relevant DNA fragments and sequence comparison revealed that the sll1 alleles resulted from a
single base substitution (G to A) in the intron-splicing site of both sll1-1 (the first intron) and sll1-2 (the third intron).
(B) SLL1 encodes an MYB transcription factor. Nucleotide mutations result in altered splicing of mRNA and premature termination of the protein. The
boxed region in the amino acid sequence represents the MYB domain. The red triangles demarcate the start point of nucleotide mutations and altered
transcription. The red or blue markings indicate the mutated amino acid sequences of sll1-1 (red) or sll1-2 (blue).
(C) Altered splicing of sll1-1 (left panel) and sll1-2 (right panel) transcripts. mRNAmolecules were amplified with primers located in two ORFs flanking the
mutation site.
6 of 17 The Plant Cell
not show obvious changes in the presence of SLL1 deficiency.
Several genes marking stage III, such as CP, PI, PAL and 4CL,
are evidently altered. This indicates that SLL1 mainly modu-
lates the stage III events of PCD during the transdifferentiation
of mesophyll cells to sclerenchymatous cells. The absence of
an alteration in Dirigent-1 expression further confirmed that
SLL1 specifically regulated the PCD in abaxial sclerenchyma
development.
sll1 Leaves Showed Adaxialized Trends in Abaxial
Epidermis, while Enhanced SLL1 Expression Suppresses
the Adaxial Characteristics and Stimulates Abaxial
Leaf Features
As mentioned above, a distinguishing characteristic of the
monocot leaf is that microhairs or ligules are present only at
the adaxial surface. In sll1-1, the microhairs can be observed on
Table 2. Amino Acid Identities (%) among Arabidopsis and Rice KANADI Proteins and Maize MWP1
SLL1 MWP1 KAN2_rice KAN3_rice KAN4_rice KAN1_At KAN2_At KAN3_At KAN4_At
SLL1 – 58 42 26 19 23 24 20 17
MWP1 100 – 49 27 18 24 25 19 16
KAN2_rice 96 96 – 28 18 22 22 18 16
KAN3_rice 93 93 90 – 20 22 26 25 20
KAN4_rice 93 93 92 89 – 18 19 16 27
KAN1_At 96 96 96 92 92 – 29 24 19
KAN2_At 96 96 93 92 93 96 – 41 18
KAN3_At 89 89 85 85 89 85 89 – 19
KAN4_At 92 92 89 89 90 90 90 89 –
The upper numbers (above the dashes) indicate identities among the entire proteins and the lower numbers indicate the identities among GARP
domains.
Figure 4. SLL1 Has a Close Relationship with KANADI Family Members.
(A) Phylogenetic relationships among the KANADI family members. KANADI proteins from Arabidopsis (KAN1, 2, 3, and 4), rice (SLL1, KAN2, 3, 4, and
5), and Z. mays (MWP1, KAN1, 3, 4, and 5) are analyzed. The numbers at each node represent the bootstrap support (percentage). The scale bar
indicates genetic distance based on branch length.
(B) Alignment analysis of KANADI proteins. This analysis revealed the conservation of a GARP domain among the KANADI proteins. The conserved
amino acid residues are highlighted in the bottom line in blue.
(C) LRI of sll1 mutant plants. sll1-1 has a more evident LRI than sll1-2 at different developmental stages (40 or 75 d). Error bars represent SD (n > 30).
SLL1 Controls Rice Leaf Rolling 7 of 17
Figure 5. SLL1 Expression Pattern Analysis.
(A) qRT-PCR analysis of SLL1 expression in various tissues. Expression of SLL1 in roots (from 10-d-old seedlings), stems, leaves (middle part of 4th
leaf), flowers, and 10-d-old seedlings (whole plants including roots and shoots) (top panel) in the SAM, shoot (whole plant without roots), and the 4th leaf
of 10-d-old seedlings and in the SAM, 10th leaf, and leaf sheath of plants at the tillering stage (bottom panel) are analyzed. For SAM samples, the
precisely defined intact SAM and young leaf primordia (without leaf sheath) were collected. Experiments are biological replicates.
(B) Promoter-reporter gene (GUS) fusion studies on the expression of SLL1. Expression of SLL1 in stem (1 and 2), anthers (3 to 5), pistil tip (6), seed
vascular tissues (7 to 9), coleoptile and embryonic roots of germinating seeds (10), and root vascular tissues (11) is analyzed. Bars = 1 mm (4 and 6), 2
mm (1, 2, and 7 to 10), or 100 mm (3, 5, and 11).
(C) Promoter-GUS fusion studies of expression of SLL1 in leaf. Expression of SLL1 in young leaf (middle part of 2nd leaf of 10-d-old seedling; 1), mature
leaf and leaf vein (2), leaf sheath (3), guard cells (4), and vascular tissues (5) is analyzed. Bars = 1 mm (1), 5 mm (2 and 3), or 50 mm (4 and 5).
8 of 17 The Plant Cell
both epidermal surfaces of some plants (Figure 8A), and the
ligules exist on the abaxial side of the joint (Figure 8B). This
suggests an adaxialized trend in the abaxial epidermis of sll1-1
and indicates the involvement of SLL1 in the development of
polarity throughout the leaf abaxial epidermis.
Furthermore, transgenic rice lines overexpressing SLL1 ge-
nomic DNA (driven by its own native promoter) were generated
on a wild-type background (Figure 9A, top panel). Observations
of leaf growth showed that SLL1 overexpression resulted in
dwarf plants with twisted and abnormal inner rolled leaves
(Figure 9A, bottom panel). We also observed enlarged phloem
in the midrib as well as large and small veins (Figure 9B, Table 3),
revealing the enhanced abaxial features of leaves following SLL1
overexpression.
Our observations of the bulliform cells formed on the adaxial
epidermis revealed the loss of the normal fan-shaped anatomical
structure. The adaxial sclerenchymatous cells proximal to the
vascular bundle were defective in some transgenic lines (Figure
9C), indicating the suppression of adaxial characteristics and
demonstrating that SLL1 is important in controlling the specifi-
cation of abaxial epidermis.
SLL1 Controls Multiple Developmental Processes
SLL1 affected developmental processes in multiple develop-
mental stages, in accordance with its expression patterns. Aside
from altered leaf morphology, other tissues, including seeds,
anthers, and roots, displayed abnormal development in sll1
mutants. In seeds, both the palea and outer glume had a
defective shape and failed to close; the seeds were smaller
and exposed (Figure 10A). With regard to anther development,
negligible pollen was generated and the generated pollen fell in a
scattered fashion. Extremely few pollen grains were active when
analyzed by fluorescein diacetate (FDA) staining (Figure 10B). In
addition, abnormal root growth was observed in SLL1-deficient
plants. In comparison with the wild type, the lengths of primary
roots and lateral roots were shortened and the number of
adventitious roots was significantly decreased (Figure 10C).
DISCUSSION
Sclerenchymatous tissues present different characteristic distri-
butions in C3 and C4 plants. This study expands our knowledge
of rice leaf development, especially the roles of leaf sclerenchy-
matous cells. Although the rice plant has an equifacial leaf, our
results support independent regulation of differentiation in the
adaxial and abaxial mesophyll cell layers. This modulates the
shape of rice leaves as well as photosynthesis efficiency by
regulating the development of polarity.
SLL1Arrests Normal SclerenchymatousCell Formation and
Controls Leaf Rolling
Multiple factors are involved in the control of leaf form, including
axis-determining leaf genes (Eshed et al., 2001; Bowman et al.,
2002), miRNAs and phytohormones (Juarez et al., 2004a; Kinder
and Martienssen, 2004), as well as factors controlling the estab-
lishment of leaf polarity. The form of the dicot leaf is determined
strongly by the mesophyll cell type; altered polarity in spongy
tissue or palisade tissues will result in leaf rolling. In the monocot
leaves, alteration in the distribution of leaf polarity characteris-
tics, such as ligules and epidermal hairs (microhairs), does not
lead to leaf rolling. Indeed, the cell types on the adaxial and
abaxial epidermal surfaces are very similar in some grasses (but
not in dicots). Although the sll1 mutants showed altered leaf
polarity characteristics in the abaxial epidermis (presence of
bulliform cells in the abaxial epidermis [Figure 1B] andmicrohairs
at the blade sheath boundary of the abaxial epidermis [Figure
6A]), this change in epidermal markers of leaf polarity is not
observed in all rolled leaf mutants. By contrast, because scler-
enchymatous cells contribute continuousmechanical strength to
maintain normal leaf shape, the lack of sclerenchymatous cells
on the abaxial surface in regions of leaf curving in sll1 mutants
indicates the critical role of sclerenchyma in controlling leaf
rolling in monocots and the role of SLL1 in schlerenchyma
formation that influences leaf rolling.
In wild-type rice plants, the specification of mesophyll cells
occurs during the early stages of leaf development, when the
young leaf remains wrapped inside the sheath in a crimped state.
At plastochron 5, the differentiation of sclerenchymatous cells is
visible; however, the mechanical strength is not yet developed,
requiring the initiation of PCD and thickening of the secondary
cell walls. These processes progress gradually, which is crucial
for leaf flattening. The defective differentiation of sclerenchyma-
tous cells in sll1 results in deficient mechanical strength and,
hence, the rolled leaf. Throughout tissue differentiation, SLL1
accumulates in the abaxial cell layer (plastochrons 4 and 5). This
pattern of expression is more distinct during the early stages of
leaf development (plastochrons 1 to 3), consistent with the role of
SLL1 in the differentiation of sclerenchymatous cells.
The structure, number, and distribution of sclerenchymatous
cells differ between C3 and C4 plants, and sclerenchyma is
critical to determining leaf form in both C3 and C4 plants. The
maize leaf shows polarization of a few sclerenchymatous cells on
the abaxial side, while rice has sclerenchymatous cells on both
Figure 5. (continued).
(D) In situ hybridization analysis of expression of SLL1 in the abaxial cell layer and in tracheal elements throughout leaf development. The SLL1 signal
was detected at the shoot apical meristem and leaf primordia (2; enlarged in 3, arrows). Cross section of the shoot apex showed higher expression of
SLL1 in the abaxial cell layer, including the epidermis and vascular system of early leaf blades (5; enlarged in 6, arrows). In the mature leaf, there is an
accumulation of SLL1 in the region of the developing abaxial sclerenchymatous cells and tracheal elements (8 and 9, arrow). Arrows highlight the more
intense accumulation of SLL1 at the abaxial cell layer. The sense probe was hybridized and used as control (1, 4, and 7). Bars = 200 mm (1 to 4 and 6 to
8), 505 mm (5), or 100 mm (9).
SLL1 Controls Rice Leaf Rolling 9 of 17
Figure 6. SLL1 Functionally Complements the Defective Sclerenchymatous Cells on the Abaxial Side of sll1-2.
(A) qRT-PCR analysis on the expression of SLL1. Normal splicing of SLL1 in wild-type or transgenic sll1-2 plants with complemented expression of
SLL1 following expression of the SLL1 genomic fragment is detected, but cannot be detected in sll1-2. Experiments are biological replicates.
(B) Morphology of leaves of wild-type, sll1-1, sll1-2, and transgenic sll1-2 plants with complemented expression of SLL1. Leaf shape of these plants at
different developmental stages (3 or 6 weeks, respectively) is observed. The squared leaf regions (white box) are enlarged and shown above the plants
to highlight the leaf shape.
(C) Recovered formation of sclerenchymatous cells in sll1-2 with complemented expression of SLL1. Cross-sectional analysis is performed using the
5th leaf from 6-week-old seedlings, and formation of sclerenchyma at the abaxial epidermis of sll1-2 after complemented expression of SLL1 is
observed. Bars = 50 mm.
(D) Enlarged squared regions from (C). Arrows highlight the positions of sclerenchymatous cells on the abaxial side, lacking in sll1-2.
the abaxial and adaxial sides (corresponding to vascular posi-
tions). Notably,mutantswith defective abaxial/adaxial patterning
show varying sclerenchyma specification. We compared two
abaxial specification defective mutants (rld1 and sll1). Maize
RLD1 promotes adaxial identity, and the abaxial misexpression
of which results in ectopic switch sclerenchyma cells extending
from the abaxial side to the adaxial side along a subset of veins
(due to the partially reversed D/V patterning). By contrast, sll1
forms sclerenchyma cells on the adaxial sides, but these plants
exhibit defective abaxial sclerenchyma development, indicating
defective abaxial specification or partially abaxialized develop-
ment without reverse D/V patterning. These findings suggest the
possibility of differential roles for sclerenchymatous cells in the
control of leaf development in C3 compared with C4 plants.
When the effects of SLL1 are compared with the effects of the
maize MWP1 (the closest KANADI protein to SLL1, involved in
the abaxial-adaxial patterning of leaves), both sll1 and mwp1
show adaxialized sectors of cells in the sheath. However, it is not
yet clear whether the phenotypic differences between sll1 and
mwp1 are due to the difference between the influence of KANADI
proteins on C3 and C4 leaf anatomies or because different
KANADI proteins have different functions in determining abaxial
polarity of different cell types in different species. There are five
KANADI proteins in rice, and it is possible that other KANADI
proteins contribute to the polarity determination of rice leaves,
and SLL1 has a specialized role in schlerenchyma formation.
SLL1 Controls the Establishment of Rice Leaf Polarity
Arabidopsis AS1, which encodes an R2-R3 MYB transcription
factor, is closely related to PHAN in Antirrhinum and ROUGH
SHEATH2 in maize. This gene negatively regulates the expres-
sion of KNOX, a marker of founder cell recruitment into promor-
dium (Smith et al., 1992; Jackson et al., 1994), thus modulating
the establishment of leaf polarity (Waites et al., 1998; Byrne et al.,
2000; Semiarti et al., 2001). However, our studies showed that
the function of SLL1 is not similar to the functions of these factors
in regulating founder cell identity. SLL1 contains a SHAQKYF
class Myb-like DNA binding domain, and analysis of homologs
showed that SLL1 was similar to Arabidopsis KANADI1 family
members sharing a highly conserved GARP domains (Kerstetter
et al., 2001). SLL1 was involved in the establishment of leaf
Figure 7. Altered Expression of Genes Encoding Proteins Related to TE-
PCD in sll1-1 and sll1-2.
qRT-PCR analysis of rice gene expression. These genes include (A)
TED2, C4H, PAL, dirigent-1 (macromolecular lignin assembly protein),
and 4CL; and (B) dirigent-1, PI, and CP in sll1-1, sll1-2, or transgenic
sll1-2 plants with complemented expression of SLL1. The experiments
were repeated three times using biological replicates; the data were
statistically analyzed and presented as means 6 SD. Heteroscedastic
t test indicated the significant differences (* P < 0.05; ** P < 0.01).
Figure 8. Sll1 Alters Adaxial-Abaxial Pattern Formation.
(A) Altered epidermal features of sll1-2 leaves. Scanning electron mi-
croscopy analysis showed that the microhairs can be observed at the
abaxial epidermis of sll1-2, highlighted in the bottom panel (arrows) or
enlarged (blue box, arrows). Bars = 50 mm.
(B) The ectopic ligules in sll1-1 mutants. The ligules, which are normally
found on the adaxial side of wild-type leaves (left), can be observed on
the abaxial side at the junction of the leaf blade (lb) and leaf sheath (ls) of
sll1-1 mutants. Arrows indicate the positions of ligules. Bars = 1 cm.
SLL1 Controls Rice Leaf Rolling 11 of 17
polarity, and recently studies showed that maizemwp1 (the gene
most homologous to SLL1) displayed adaxialized sectors in the
sheath and proximal part of the leaf (Candela et al., 2008),
confirming the conserved function of KANADI family members in
monocots.
In dicots, KANADI and YABBY are two primary determinants in
establishing abaxial identity (Sawa et al., 1999a, 1999b; Siegfried
et al., 1999; Kerstetter et al., 2001). Eshed et al. (2004) proposed
that, in Arabidopsis, initial asymmetric leaf development is reg-
ulated primarily by KANADI activity. KANADI translation modu-
lates the polar expression of YAB, contributing to abaxial cell
fate. By contrast, in monocots, Yamaguchi et al. (2004) observed
that YAB genes may direct midrib cell division but are not
sufficient for the control of dorsoventrality. YAB1 is expressed
throughout the rice leaf and functions in gibberellic acid regula-
tion (Dai et al., 2007), and YAB4 is expressed in developing
vascular tissue andmay regulate the development of vasculature
in rice (Liu et al., 2007). Indeed, the unaltered expression of YAB
in sll1 (see Supplemental Figure 1 online) supports the idea that
the rice YAB genes have different functions to the dicot genes,
underpinning a view that the molecular mechanisms for the es-
tablishment of leaf polarity differ between monocots and dicots.
Although rice YAB1 and YAB2 are both expressed in precursor
cells of certain specific cell types, including abaxial scleren-
chyma, which suggests that these genes may be involved in leaf
cell differentiation (Toriba et al., 2007), there is no direct evidence
that SLL1 regulates abaxial sclerenchyma formation and func-
tions in the PCD process through sustaining the normal expres-
sion of rice YAB in abaxial sclerenchyma.
Although the PCDprocess during sclerenchymadifferentiation
is altered in sll1, we suggest that SLL1 functions primarily as a
key factor in specifying polarity during adaxial/abaxial pattern-
ing. Abnormal sclerenchyma formation resulted in defective
PCD, affecting adaxial/abaxial patterning; thus, SLL1 probably
regulates PCD indirectly.
In addition, the monocot mesophyll cells show a uniform
configuration without polarization, in contrast with the adaxial-
palisade cells or abaxial-spongy mesophyll cells in dicots, indi-
cating differential controls for the establishment of leaf polarity. In
dicots, altered adaxial-abaxial pattern formation often results in
rolled leaves; while the leaf rolling of sll1 is due to the defective
differentiation of sclerenchymatous cells at the abaxial surface.
However, the differentiation of sclerenchymatous cells on theFigure 9. Enhanced Expression of SLL1 Stimulated Phloem Develop-
ment in the Abaxial Cell Layer and Suppressed the Formation of
Sclerenchymatous Cells in the Adaxial Cell Layer.
(A) qRT-PCR analysis confirmed the enhanced expression of SLL1 (top
panel). The transgenic plants (3-week-old seedlings) revealed a pheno-
type with upper-rolled leaves (bottom panel, bar = 5 cm). The enlarged
squared regions highlight leaf rolling compared with control. Experi-
ments are biological replicates.
(B) Enhanced development of phloem of SLL1-overexpressing plants.
Cross-section analyses were performed to analyze the development of
phloem in the midrib (the squared regions are enlarged) and the large and
small veins of wild-type and SLL1-overexpressing plants. P, phloem.
Bars = 50 mm.
(C) Defective formation of sclerenchymatous cells in the adaxial cell layer
of SLL1-overexpressing plants. Broken arrows indicate the regions
where sclerenchymatous cells would have formed in normal leaf (bottom
panel). Bars = 100 mm.
Table 3. Measurement of the Vascular Elements in Wild-Type and
SLL1-Overexpressing Plants
Phloem (mm2) Phloem/Xylem Phloem/Vascular Bundle
Wild Type 495 6 148 1.25 6 0.24 0.18 6 0.02
pSLL1:SLL1 1700 6 477** 2.18 6 0.39** 0.39 6 0.03**
The areas of phloem, xylem, and whole vascular bundles in large veins
were measured, as well as the ratios of phloem/xylem and phloem/
vascular bundle. The large veins for measurement were selected from
10 rolled leaves of three independent transgenic lines. The data are
statistically analyzed and presented as means 6 SD (n > 30). Hetero-
scedastic t test indicated the significant differences (** P < 0.01)
compared with the wild type.
12 of 17 The Plant Cell
adaxial surface is unaffected in sll1. These findings not only
indicate the specific role of SLL1 in controlling abaxial identity
and determining abaxial mesophyll cell fate in rice but also
suggest that the mesophyll cells of monocot plants do not really
have isobilateral mesophyll and, in fact, have polarity. Alterna-
tively, the specification of mesophyll cells on either the adaxial or
the abaxial side may rely on distinct signals.
SLL1 Modulates Photosynthesis by Regulating Cell Fate
The rolled leaf is an important agronomic trait, directly related to
photosynthesis and crop yield. The erect, half-rolled rice leaf
results in enhanced light capture capacity and increased chlo-
rophyll contents and, hence, increased photosynthesic effi-
ciency and yield. Previous studies in maize, sugarcane, and
grain sorghum showed that the arrangement of leaf vascular
bundles and mesophyll cells is closely related to photosynthesic
efficiency. In maize, the developed vascular bundle contains
more chlorophyll and compact mesophyll cells, resulting in a
higher photosynthetic capacity. SLL1 deficiency suppresses the
specification of sclerenchymatous cells in the abaxial mesophyll
and may thus help to increase the chlorophyll content through
increasing the proportion of mesophyll cells, consequently en-
hancing photosynthesis and providing a useful tool for rice
breeders. Accordingly, the photosynthetic efficiency of sll1-2 mu-
tants was slightly increased (;3%), consistent with the increased
amounts of chlorophyll. However, photosynthetic efficiency is
affected by multiple factors, and other photosynthesis-related
processes are suppressed in sll1-2, which may explain why the
increased chlorophyll content does not result in a marked im-
provement of photosynthetic efficiency as compared with sll1.
Because leaf rolling may be subject to mechanical regulation
(the maintenance of longitudinal rolling requires a continuous
mechanical force), the modulation of sclerenchymatous cell
formation will provide new insights into the regulation of leaf
rolling. Our studies elucidate the control of leaf polarity estab-
lishment. This information may help to increase area yield by
increasing the number of photosynthetic cells in the leaf and thus
enhancing overall photosynthesis.
METHODS
Plant Material and Growth Conditions
The sll1 ricemutantwas isolated from thepopulationofOryza sativaL. ssp.
japonica cultivar Nipponbare treated with a 1% ethyl methanesulfonate
solution. Nipponbare plants represent the wild type. Mutant sll1 was
crossedwith an indica rice variety, Nanjing6 (with flat leaf); the resultant F1
plantswereselfedtoproducetheF2seedsforconstructingtheF2mapping
population.Riceplantswerecultivated inanexperimental fieldat theChina
National Rice Research Institute under natural growing conditions; field
management adhered to normal agricultural practice. For transgenic rice
growth, rice seeds were germinated in sterilized water and then grown in
pots in a phytotron with a 12-h-light (268C) and 12-h-dark (188C) cycle.
Map-Based Cloning of SLL1
SLL1 was mapped primarily with simple sequence repeat (http://www.
gramene.org/microsat/ssr.html) and sequence tagged site (see Supple-
mental Table 1 online) markers using 152 F2 mutant plants. The SLL1
locus was further localized within a 29.57-kb region between markers
T5904-7 and T5904-9 using 711 F2 mutant plants. New molecular
markers (see Supplemental Table 1 online) were developed by comparing
original or cleaved amplified polymorphic sequences between indica
variety 9311 and Nipponbare, according to data published at http://www.
ncbi.nlm.nih.gov.
Figure 10. SLL1 Affected Multiple Developmental Processes in Rice.
(A) Altered seed development and maturation of sll1. Bars = 2 mm (left
panel) or 1.5 cm (right panel).
(B) Reduced pollen grains of sll1-1 mutant. Bars = 200 mm (wild type) or
100 mm (sll1-1).
(C) Abnormal root development of sll1 seedlings. Phenotypic observa-
tion (left panel, bars = 1 cm) and measurement analysis (right panel, n >
30 in each experiment, and experiments are biological replicates)
showed that sll1 had shortened primary roots and reduced numbers of
lateral and adventitious roots. The boxed region was enlarged to high-
light the abnormal roots of sll1-1 seedlings (left panel, bars = 1 cm).
SLL1 Controls Rice Leaf Rolling 13 of 17
To define molecular lesions, 29.57-kb genomic DNA from the sll1 and
relevant wild-type variety (Nipponbare) were amplified by PCR. All PCR
products were sequenced and the candidate gene was amplified from
both sll1 and Nipponbare genomic DNAs using different primers (see
Supplemental Table 2 online). Obtained sequences were analyzed with
DNAMAN software (version 5.2.2).
Histology and Microscopy Observation
Fresh hand-cut sections (;20 mm) of rice leaf blade were stained in
Ruthenium red solution (0.2mg/mL) or in aniline blue solution (5mg aniline
blue in 50% ethanol). Treated samples were transferred into water. The
sectionsweremicroscopically examined (LeicaDMR) and photographed.
Leaves at certain developmental stages were sampled for resin section-
ing, intenerated by hydrofluoric acid, fixed in 2.5%glutaraldehyde (16 to 48
h, 488C), and dehydrated through a graded ethanol series. The samples
wereembedded inEpon812 resin (Fluka) andpolymerizedat608C.Sections
(3mm)were cut and stained with filtered 1% toluidine blue, microscopically
examined (LeicaDMR), andphotographed.Areameasurementsof vascular
elements were performed using Leica Qwin software.
FDAwas used to determine the viability of pollen grains.Mature flowers
before opening were harvested, and picked anthers were crushed into a
fine powder and treated with FDA solution (10 mg FDA dissolved in 1 mL
acetone as stock, diluted with 7% sucrose to a concentration of 100 mg/
mL for use) for 5 min and then observed under UV light (Leica DMR).
Pollen grains emitting green fluorescence were regarded as viable pollen.
Measurement of Chlorophyll Content in Leaves, Photosynthetic
Efficiency, and Leaf Rolling Index
The second leaf from the top of wild-type, sll1-1, sll1-2, or transgenic
sll1-2 plants with complemented expression of SLL1 (at the same growth
stage) was collected using a hole-puncher to cut three circles in the
center area of the leaf beside the midvein. Before sampling, the paper
circle was cut using the same hole-puncher to calculate the areas of leaf
samples. The collected leaf samples were incubated in 5 mL 80% ace-
tone (v/v) for 24 h under darkness. Chlorophyll contents were determined
by measuring the absorbance at 652 nm according to Arnon (1949).
The middle part of the 8th to 10th rice leaves in wild-type, sll1-1, and
sll1-2 plants at the tilling stage were collected and used to measure the
photosynthetic efficiency. The TEACHIG-PAM-2100 chlorophyll fluorom-
eter was used to detect the quantum yield of PSII and the maximal
quantum yield of PSII, Fv/Fm, through the RUN1 and RUN2 programs
(Schreiber, 1997).
The widths of 4th to 5th or 10th to 11th leaves in 40-d-old or 75-d-old
sll1-1 and sll1-2 plants were measured under either the natural (Ln) or
unfolding state (Lw). The LRI was calculated as LRI = (Lw 2 Ln)/Lw.
Phylogenetic Analysis
The BLAST search program (http://www.ncbi.nlm.gov/BLASTp/) was
used to identify the homologous sequence of SLL1 using its amino acid
sequence as query in searches of the National Center for Biotechnology
Information and The Institute for Genomics Research. All the resulting
sequences were aligned using ClustalX1.83 multiple alignment mode,
and the neighbor-joining phylogenetic tree was generated using MEGA
4.0 (Tamura et al., 2007). The bootstrap values for nodes in the phylo-
genetic tree are from 1000 replications. The handing gap option was
pairwise deletion, and the numbers at the branching points indicated the
bootstrap values. The peptide identities among the different proteins
were calculated using Genedoc software.
Promoter-Reporter Gene Fusion Studies
For promoter-GUS fusion studies, a 2.1-kb genomic DNA fragment
containing the promoter region of the SLL1 gene was amplified by PCR
using primers 59-GCGTCGACTTTTGGCTCTGGCTAACTTA-39 and
59-GCTCTAGACACCTCCTTCTCCACCTC-39 and then subcloned into
vector pCAMBIA1301 (Cambia), resulting in fusion of the promoter and
the GUS reporter gene. The positive construct was transformed into wild-
type plants as described above. About 20 independent transgenic lines
were obtained after screening, and GUS activities were histochemically
detected as described (Jefferson et al., 1987).
In Situ Hybridization Analysis
Wild-type leaves from two developmental stages (4th seedling leaf and
10th mature leaf) were fixed in a formaldehyde solution (4%), dehydrated
through an ethanol series, embedded in paraffin (Sigma-Aldrich), and
sectioned at 8mmusing an YL3-A rotarymicrotome (Shanghai Instrument
Factory). A 343-bp gene-specific region of SLL1 cDNA, amplified by PCR
reaction (primers 59-CCGTGAAGAGCACTGACAAGCC-39 and 59-GAA-
GACAATCCTACGGAGCGATG-39) and a 528-bp gene-specific region of
rice YAB1 cDNA, amplified by PCR reaction (primers 59-GCCAA-
AAGGGTCAAGAGG-39 and 59-GGACGTATAGGTGACAC-39), were
used as the probes. The amplified DNA fragments were subcloned into
a pGEM-T easy vector (Promega) in two orientations; the sense and
antisense probes were synthesized and used to generate the RNA probe.
In situ hybridization was performed as described (Luo et al., 1996).
Confirmation of SLL1 Mutation in sll1-1 and sll1-2 Plants
Total RNA was extracted from the 4th or 5th leaf blade of wild-type and
sll1 mutant lines using TRIzol reagent (Invitrogen) according to the
manufacturer’s protocol. The isolated RNA was reverse-transcribed
(SuperScript preamplification system) and used as templates for analysis.
The SLL1 transcripts in sll1-1 or sll1-2 mutants were amplified through
RT-PCRwith primers for sll1-1 (59-AGGCTCAACGGCATGCTCTC-39 and
59-GGCTCTTGACATGCGCTAGC-39) or for sll1-2 (59-TGTATCGCACC-
GTGAAGAGCA-39 and 59-TTGAGGAGTTGCTCCACCTGG-39).
Constructs and Rice Transformation
A 7.8-Kb genomic DNA fragment containing the entire SLL1 gene (coding
region, 1600-bp upstream sequence and 900-bp downstream sequence)
was cut from BAC B1040D06 and subcloned into vector pCAMBIA1300.
The construct obtained was introduced into the Agrobacterium tumefa-
ciens strain EHA105 (Hood et al., 1993) by electroporation; positive
clones were used for transformation of the wild type and sll1-2 using
immature embryos as materials.
Real-Time qRT-PCR Analysis
Real-time qRT-PCR analysis was performed to examine the expression
pattern of SLL1 in various tissues and different stages of leaf growth to
identify the transgenic lines with enhanced or complemented SLL1
expression as well as to identify altered transcripts of PCD-related genes
in the wild type compared with sll1-1 or sll1-2 plants.
Total RNAwas extracted using TRIzol solution and reverse-transcribed
according to the manufacturer’s instructions (SuperScript preamplifica-
tion system) and then quantitatively analyzed on the Rotor-Gene real-time
thermocycler R3000 (Corbett Research) with real-time PCR Master Mix
(Toyobo). For the analysis, a linear standard curve was generated using a
series of dilutions for each PCR product; the levels of the transcript in all
unknown samples were determined according to the standard curve. The
rice ACTIN gene (Os03g50890, amplified by primers 59-TCCATCTTGG-
CATCTCTCAG-39 and 59-GTACCCGCATCAGGCATCTG-39) was ampli-
fied and used as an internal standard to normalize the expression of SLL1
and the other tested genes.
14 of 17 The Plant Cell
To test the expressions of the PCD-related genes and rice YAB genes,
leaf samples were collected from themiddle part of the 7th leaf from differ-
ent lines. Each sample was duplicated independently to ensure validity
with biological replicates. All experiments were repeated three times; the
data were statistically analyzed and presented as means plus SD.
The primers used to test the expression of SLL1 were as follows: in
various tissues, 59-CAGGTGTCCAACCATGAGC-39 and 59-GCCTCTG-
TGATTGCCATCTAAT-39; in transgenic plants, 59-CTCTTCAGGGCC-
GGCGGA-39 and 59-TGAGGAGTTGCTCCACCT-39. To test the expres-
sion of PCD-related genes, the following genes were examined:Dirigent1
(Os11g42500, 59-CCTACAGTTGTACAGATGCAATCG-39 and 59-GATG-
GAGGAATGGATTGATGGT-39), PI (Os05g06780, 59-ATCAAGCCGG-
AGGTCGCCATC-39 and 59-CAAGGCAGCGTGTAATCTCC-39), TED2
(Os10g41170, 59-AAAGCAATCCAAGCAGCCGAAGACG-39 and 59-CCA-
CGCGGCATCAGACACTCCATTG-39), PAL (Os05g35290, 59-TACACC-
GACCACCTCACCCACA-39 and 59-CGAGCCTCTTCGCCTCCTTCAT-39),
C4H (Os05g25640, 59-GGCGAGATCAACCACGACAACG-39 and
59-GCAACCGCAGCGTCTCCTTCA-39), 4CL (Os06g44620, 59-GTCGT-
CGCCCTCCCTTACTCCT-39 and 59-AACAGCGGCAGCAAGCACAGC-39),
and CP (Os08g44270, 59-CCAACAGGTTCGCCGACCTCAC-39 and
59-CCGCACGAGCCTTGGTCCTTGA-39). To test the expression of Os
YAB genes, the primers used were as follows: Os YAB1 (Os07g06620,
59-GCCAACCAGTCAGCAGCAAGTGTCA-39 and 59-CAAAATGGAGCC-
GGGGAAGATGAG-39), Os YAB2 (Os03g44710, 59-CGGCGCCGGAG-
CATGTGTG-39 and 59-TGGCCACAACGGACGGTCACGA-39), and Os
YAB6 (Os12g42610, 59-GCCTCCTGCTGCTGCTGCCATGG-39 and 59-
AATGGATGTTTGGATAATGT-39).
Scanning Electron Microscopy
The 4th leaves of the wild type and sll1-2 were collected and fixed in
formalin:glacial acetic acid:50% ethanol (2:1:17, v:v:v) overnight. After
dehydration in a graded ethanol series, the sections were dried for 4 h
(Hitachi critical point dryer), surface-sprayed with gold powder, and
examined under a scanning electron microscope (Hitachi S-450).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
database under the following accession numbers: rice SLL1
(Os09g23200), ACTIN (Os03g50890), Dirigent1 (Os11g42500), PI
(Os05g06780), TED2 (Os10g41170), PAL (Os05g35290), C4H
(Os05g25640), 4CL (Os06g44620), CP2 (Os08g44270), KAN2
(Os08g33050), KAN3 (Os08g06370), KAN4 (Os03g55760), and KAN5
(Os02g46940);Arabidopsis KAN1 (At5g16560),KAN2 (At1g32240),KAN3
(At4g17695), KAN4 (At5g42630); maize KAN1 (ABB89931), KAN3
(ACF87413), KAN4 (ACG26785), KAN5 (NP_001132886), and MWP1
(ACG26785); and grape KAN1 (CAO38744) and KAN2 (CAO63351).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Unaltered Expression of rice YABBY Genes
in sll1-1.
Supplemental Table 1. New Developed Polymorphic STS and SSR
Markers Used for Map-Based Cloning of SLL1.
Supplemental Table 2. Primers Used to Amplify the Genomic DNA
Fragment of Candidate Gene from sll1 and Wild-Type Variety.
Supplemental Data Set 1. Alignment Analysis of the KANADI
Proteins, Including Those from Arabidopsis (KAN1, 2, 3, and 4),
rice (SLL1 and KAN2, 3, 4, and 5), and Zea mays (MWP1 and KAN1,
3, 4, and 5).
ACKNOWLEDGMENTS
The study was supported by the State Key Project of Basic Research
(2005CB120803 and 2006AA10A102), the National Science Foundation
of China (30425034 and 30623006), and the National Key Laboratory of
Plant Molecular Genetics. We thank Shu-Ping Xu for help with rice
transformation, Wei Huang for in situ hybridization, and Hui-Qiong
Zheng and Xiao-Yan Gao for the leaf cross-section technique.
Received June 16, 2008; revised February 10, 2009; accepted February
27, 2009; published March 20, 2009.
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SLL1 Controls Rice Leaf Rolling 17 of 17
DOI 10.1105/tpc.108.061457; originally published online March 20, 2009;Plant Cell
Guang-Heng Zhang, Qian Xu, Xu-Dong Zhu, Qian Qian and Hong-Wei XueRegulating Leaf Abaxial Cell Development
SHALLOT-LIKE1 Is a KANADI Transcription Factor That Modulates Rice Leaf Rolling by
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