shallot-like1 is a kanadi transcription factor that modulates

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

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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.


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.


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.


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).


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).


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).


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.


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.


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).


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.


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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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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