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STENOFOLIA Regulates Blade Outgrowth and Leaf VascularPatterning in Medicago truncatula and Nicotiana sylvestris C W OA
Million Tadege,a,b,1 Hao Lin,b Mohamed Bedair,a Ana Berbel,c Jiangqi Wen,a Clemencia M. Rojas,a Lifang Niu,b
Yuhong Tang,a Lloyd Sumner,a Pascal Ratet,d Neil A. McHale,e Francisco Madueno,c and Kirankumar S. Mysorea
a Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401b Department of Plant and Soil Sciences, Oklahoma State University, Stillwater, Oklahoma 74078c Instituto de Biologıa Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientıficas, Universidad Politecnica
de Valencia, CPI, 46022 Valencia, Spaind Institut des Sciences du Vegetal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette Cedex, Francee Department of Biochemistry and Genetics, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504
Dicot leaf primordia initiate at the flanks of the shoot apical meristem and extend laterally by cell division and cell expansion to
form the flat lamina, but the molecular mechanism of lamina outgrowth remains unclear. Here, we report the identification of
STENOFOLIA (STF), a WUSCHEL-like homeobox transcriptional regulator, in Medicago truncatula, which is required for blade
outgrowth and leaf vascular patterning. STF belongs to the MAEWEST clade and its inactivation by the transposable element
of Nicotiana tabacum cell type1 (Tnt1) retrotransposon insertion leads to abortion of blade expansion in the mediolateral axis
and disruption of vein patterning. We also show that the classical lam1 mutant of Nicotiana sylvestris, which is blocked in
lamina formation and stem elongation, is caused by deletion of the STF ortholog. STF is expressed at the adaxial–abaxial
boundary layer of leaf primordia and governs organization and outgrowth of lamina, conferring morphogenetic competence.
STF does not affect formation of lateral leaflets but is critical to their ability to generate a leaf blade. Our data suggest that STF
functions by modulating phytohormone homeostasis and crosstalk directly linked to sugar metabolism, highlighting the
importance of coordinating metabolic and developmental signals for leaf elaboration.
INTRODUCTION
Leaves are the principal organs for photosynthetic carbon assim-
ilation. Independent origins of the flat lamina in different plant
families provide evidence that it is an important evolutionary
adaptation of land plants for efficient capture of solar energy and
gaseous exchange. Dicot leaf primordia initiate at the flanks of the
shoot apical meristem (SAM) and extend laterally during primary
and secondary morphogenesis in which growth occurs predom-
inantly by cell division and cell expansion, respectively (Sussex,
1955; Poethig, 1997; Scarpella et al., 2010). As the primordium
extends, it asymmetrically differentiates into distinct upper (adax-
ial) and lower (abaxial) surfaces, forming a flattened lamina.
Leaf primordium initiation requires localized accumulation of
the phytohormone auxin (Reinhardt et al., 2003; Braybrook and
Kuhlemeier, 2010) and repression of Class 1 KNOTTED1-LIKE
HOMEOBOX (KNOX1) gene expression by the ASSYMMETRIC
LEAVES1 and 2 (AS1/AS2) complex (Long et al., 1996; Uchida
et al., 2007; Guo et al., 2008; Jun et al., 2010) at the initiation site.
KNOX1 genes modulate the cytokinin/gibberellin (GA) ratio in the
SAM by activating cytokinin biosynthesis and repressing GA
biosynthesis or activating GA catabolism (Jasinski et al., 2005;
Bolduc and Hake, 2009). Shortly after primordium emergence, a
distinctive band of cells along the lateralmargins differentiates into
themarginal blastozone (Hagemann andGleissberg, 1996), which
expands laterally to form the laminaor pinnae incompound leaves,
whereas cells at the central region of the primordium differentiate
to form the midrib or rachis (Poethig, 1997). The next significant
insights came from investigation of mechanisms governing leaf
dorsoventral polarity involving mutually antagonistic interplay of
MYB, class III homeodomain Leu-zipper (HD-ZIPIII), YABBY, and
KANADI class of transcription factors (Tsukaya, 2006; Husbands
et al., 2009; Braybrook and Kuhlemeier, 2010; Efroni et al., 2010).
The first described dorsoventral polarity mutant in Antirrhinum,
phantastica (phan), displays radialized abaxial leaves in severely
affected cases and at restrictive growth temperatures (Waites
and Hudson, 1995; Waites et al., 1998). PHAN encodes an MYB
transcription factor required for adaxial identity and blade out-
growth (Waites et al., 1998). This was the first demonstration that
the adaxial/abaxial identity sets a positional landmark for lateral
outgrowth of the blade. However, loss-of-function mutants of
PHAN homologs in other species have variable and less dramatic
phenotypes, including crispa in pea (Pisum sativum) (Tattersall
et al., 2005) and roughsheath2 in maize (Zea mays) (Timmermans
et al., 1999). TheArabidopsis thaliana homolog in particular, the as1
mutant (Byrne et al., 2000), displays noobviousnarrowingof lamina
1Address correspondence to million.tadege@okstate.edu.The 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: Million Tadege(million.tadege@okstate.edu) and Kirankumar S. Mysore (ksmysore@noble.org).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.111.085340
The Plant Cell, Vol. 23: 2125–2142, June 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved.
except occasionally in combination with the as2 mutant and in
certain genetic backgrounds (Xu et al., 2003). AS2 encodes a LOB
domain protein containing Leu-zipper motif and forms a complex
withAS1 to promoteadaxial cell fate (Lin et al., 2003; Xuetal., 2003)
and KNOX1 repression (Guo et al., 2008; Jun et al., 2010).
In Nicotiana sylvestris, a unique lamina deletion mutant called
lam1 has been described (McHale, 1992). Morphological and
cellular studies demonstrated that mutant leaf primordia lacking
LAM1 establish normal polarity, and blade founder cells are
recruited in the correct position at the adaxial/abaxial boundary,
but lateral outgrowth of an organized lamina fails (McHale, 1992,
1993). This demonstrated that blade formation occurs in two
distinct phases. The first step involves recruitment of a loosely
organized group of blade founder cells at the adaxial/abaxial
boundary of the primordium, a process that does not require
LAM1. The second phase, lateral outgrowth and layer organiza-
tion, however, is entirely dependent on LAM1 function. Later
work on periclinal chimeras revealed that wild-type LAM1 cells in
the internal L3 domain could nonautonomously establish blade
organization and lateral outgrowth (McHale and Marcotrigiano,
1998), and that this internal organizing influence of LAM1 is an
ongoing requirement during blade expansion.
Our most recent insights on blade formation have come from
analysis of genes in theWUSCHEL (WUS)-RELATEDHOMEOBOX
(WOX) family. WUS, the founding member of WOX, is required for
stem cell maintenance in shoot and floral meristem (Laux et al.,
1996; Mayer et al., 1998).WOX genes also play important roles in
lateral organ development. Two mutants in maize, narrowsheath1
and 2 (ns1 and ns2) lead to a loss of leaf blade in the ns1 ns2double
mutant (Scanlon et al., 1996). NS1 and NS2 are duplicated WOX
genes (Nardmann et al., 2004) related to the Arabidopsis gene
PRESSED FLOWER (PRS/WOX3), the knockout of which leads to
defects in lateral sepals, petals, and stipules, but not in the leaf
blade (Matsumoto and Okada, 2001; Shimizu et al., 2009). Inter-
estingly, expression of WUS using the PRS promoter rescues the
prsphenotype, indicating somecommonmechanism inWOXgene
function (Shimizu et al., 2009). A related Arabidopsis gene, wus-
related homeobox 1 (wox1), has no visible mutant phenotype
(Haecker et al., 2004; Vandenbussche et al., 2009), but in Petunia,
mutation in WOX1-like gene, maewest (maw), has been shown
to cause a narrow lamina and defective petal fusion phenotype
(Vandenbussche et al., 2009). The maw mutation leads to stron-
ger lamina reduction when combined with the chsu mutation
(Vandenbussche et al., 2009), suggesting redundancy in regu-
lating lamina expansion. Likewise, a prs wox1 double mutant
in Arabidopsis has been shown to cause lamina reduction
(Vandenbussche et al., 2009), although the phenotype is weaker
than the maw chsu double mutant. The molecular mechanism by
which the phan single mutant or ns1 ns2 or wox1 prs ormaw chsu
double mutants restrict(s) blade outgrowth remains unknown.
Cell proliferation and cell expansion mutants of angustifolia and
rotundifolia in Arabidopsis show specific defects in mediolateral
(width direction) and proximodistal (length direction) growth, re-
spectively (Kim et al., 1998, 2002; Horiguchi et al., 2005), suggest-
ing that these two growth patterns of the lamina may bemediated
by separate mechanisms (Tsukaya, 2006). Other Arabidopsis
genes, including AINTEGUMENTA (Mizukami and Fischer,
2000), ARGOS (Hu et al., 2003), JAGGED (Dinneny et al., 2004),
and PEAPOD (White, 2006) also contribute to lamina size by
positively regulating cell proliferation, although the mechanism
remains unclear. Mutation in CINCINATA (CIN), a member of
TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family, leads to
excessive cell proliferation; CIN may make cells sensitive to the
arrest-front signal (Nath et al., 2003). TCPs are represented by a
large gene family (Martın-Trillo andCubas, 2010) and perform sev-
eral vital functions in leaf development and maturation involving
cell proliferation and differentiation (Nath et al., 2003; Palatnik et al.,
2003; Ori et al., 2007; Koyama et al., 2010; Shleizer-Burko et al.,
2011). CIN-like TCP genes negatively regulate CUP-SHAPED
COTYLEDON (CUC) genes by activating miR164, AS1 and auxin
response (Koyama et al., 2007; Koyama et al., 2010). CUC genes
are important for organ separation at boundary regions and serra-
tion of leaf margins (Aida et al., 1997; Palatnik et al., 2003; Nikovics
et al., 2006; Koyama et al., 2007; Bilsborough et al., 2011; Hasson
et al., 2011). In leaf primordia, the activity of TCP genes is regulated
by miR319a and this regulation is essential for proper leaf devel-
opment in various leaf forms (Palatnik et al., 2003; Ori et al., 2007;
Shleizer-Burko et al., 2011).
A gain-of-function mutation in blade-on-petiole (bop) or loss-of-
function in bop1 bop2 double mutants leads to formation of blade-
like outgrowthon thepetiole (Ha et al., 2007; Junetal., 2010).BOP1
and BOP2 encode BTB/POZ domain proteins that repress YABBY
and KNOX1 genes, and activate AS2 in leaf primordia (Jun et al.,
2010), suggesting that theBOP/YABBY/KNOXmodule is important
for leaf elaboration. LYRATE, the tomato homolog of JAGGED, is
shown to coordinate lateral outgrowth by interacting with KNOX1
genes and the auxin transcriptional network (David-Schwartz et al.,
2009). Modulation of KNOX1 and auxin activity also appears to be
a key component in dissected leaf morphogenesis in Cardamine
hirsuta and other leaf forms (Barkoulas et al., 2008; Hay and
Tsiantis, 2010). Together, these observations suggest a role for
auxin in lamina expansion in simple- and dissected-leafed species.
Although distinct aspects of leaf development, including phylotac-
tic arrangement, margin serration, and vein patterning, are well
documented to involve auxin (Reinhardt et al., 2003; Scarpella
et al., 2006; Bilsborough et al., 2011), direct evidence for auxin
control of lamina outgrowth and its genetic regulation at the leaf
primordial margins is lacking.
Here, we report the identification and characterization of a novel
leaf blade mutant inMedicago truncatula called stenofolia (stf) and
a classical mutant called bladeless lam1 in N. sylvestris with very
severe defects in blade outgrowth and vein patterning. We show
thatSTF is critical for lamina outgrowth and leaf vascular patterning
in simple leaf (N. sylvestris) and compound leaf (M. truncatula)
species. Our data suggest that STF is a modulator of auxin and
cytokinin homeostasis and hormonal crosstalk that coordinates
developmental signals at the adaxial–abaxial boundary region of
leaf primordia.
RESULTS
The stfMutant Displays Multiple Defects in Leaf Lamina,
Leaf Vasculature, and Flower Development
Seven narrow leaf blademutants with identical phenotypes were
identified in forward genetics screens for bladeless mutants in
2126 The Plant Cell
5600 transposable element of Nicotiana tabacum cell type1
(Tnt1) retrotransposon tagged lines of M. truncatula genotype,
R108 (Tadege et al., 2008).These mutants were named stf from
Greek stenos for narrow. In all seven stf mutants, lamina growth
is initiated and some blade tissue is formed, but further growth in
the mediolateral axis is arrested, while growth in the proximo-
distal axis is virtually unaffected (Figures 1A to 1C).Mature leaves
of R108 have regularly serrated margins along two-thirds of the
blade distal to the petiole (Figure 1C). In stf leaves, margin
serrations are absent and adaxial–abaxial differentiation is dras-
tically reduced (Figures 1D and 1E) except in the unifoliate leaf
(first leaf), which is only partially affected (Figure 1F). The lack of
lateral expansion is also evident in stf flowers in which the outer
petal is narrow (Figures 1I and 1J), and fails to enclose the
anthers and stigma (Figures 1G and 1H), while the ovary wall fails
to close leaving ovules exposed (Figure 1K), resulting in female
sterility.
Disruption of vein patterning is another significant phenotype of
stf leaves. In R108 leaves, lateral veins radiate from the midvein at
regular intervals, extending laterally to the margin (Figure 2B). The
tip of each lateral vein corresponds to one serration in themargins
and is open ended (Figure 2D). Numerous minor veins make a
complex network of branches all over the blade (Figures 2B and
2D). In stf leaves, however, the midvein is thin and less prominent
and lateral veins are poorly developed, few in number, and do not
extend to the margin (Figures 2C and 2E). There appears to be
anothermajor vein-like structure (marginal vein), one on either side
of the midvein near the margin, extending from the base of the
blade to the tip (Figures 2C and 2E). Minor veins are reduced in
number (Figure 2E).
Examination of blade epidermal cells by light and scanning
electronmicroscopy (SEM) showed that stf leaves are affected in
cell division and cell expansion. Under the light microscope,
epidermal cells in stf appear slightly elongated and thinner, with
Figure 1. Morphological Phenotypes of stf Mutants.
(A) Adult M. truncatula genotype R108 (wild type) and stf1-2 mutant plant 9 weeks after transfer to soil.
(B) stf1-2 adult leaf showing trifoliate identity and normal proximodistal growth but drastically affected mediolateral growth.
(C) R108 adult leaf.
(D) Adaxial surface of R108 and stf adult leaves where margin serrations are absent in the mutant.
(E) Abaxial surface of R108 and stf adult leaves where major veins are not visible in the mutant.
(F) R108 and stf seedlings at the unifoliate (first leaf) stage where cotyledons are nearly wild type and the unifoliate leaf is partially affected.
(G) R108 flower before anthesis in which the anthers and stigma are enclosed by petal.
(H) stf flower in the same stage as in (G) but with anthers and stigma exposed (arrow) because of the narrow petal.
(I) stf outer petal showing the reduction in lateral expansion.
(J) R108 outer petal in the same stage as in (I).
(K) stf ovary wall failing to close and ovule protruding out (arrow). Scale bars in (I) and (J) = 1 mm and in (K) = 50 mM.
Regulation of Lamina by STENOFOLIA 2127
most cells expanding to only ;75 percent of the width of wild
type (Figures 2F and 2G); this was confirmed by SEM (see
Supplemental Figures 1A and 1B online). The total number of
cells at the equatorial plane of the stf blade is at least threefold
lower than that of wild type (see Supplemental Figures 1E and 1F
online), suggesting a major defect in cell division. SEM also
showed that elongated marginal cell files are shorter in stf blade
margins (see Supplemental Figures 1C and 1D online).
Transverse sections through the leaf blade revealed that the
mesophyll tissue is not well differentiated in stf, especially on the
adaxial side. In the wild type, palisade mesophyll cells (adaxial
side) are uniformly cylindrical in shape,whereas spongymesophyll
cells (abaxial side) are compact and irregular in shape (Figure 2H).
In stf, although cells on either side are not identical, the contrast
between palisade and spongy mesophyll cells is diminished
(Figure 2I). Transverse sections through the midvein showed that
the xylem and phloem cells maintain their relative adaxial and
abaxial positions, respectively, but fail to differentiate further and
appear difficult to distinguish in stf (Figures 2J and 2K).
Together, these observations suggest that stf retains polarity
but has defects in cell division, cell expansion/differentiation, and
vascular patterning that severely curtail lamina outgrowth. Unlike
other lamina mutants caused by polarity defects, the stf polarity
defect appears to be a consequence rather than the cause of the
lamina phenotype since adaxial and abaxial cell types are
correctly specified both in the mesophyll and vasculature of stf,
but further differentiation of both cell types is compromised.
STFEncodesaWUS-LikeHomeodomainProteinConserved
in Dicots
We cloned STF by PCR-based genotyping of flanking sequence
tags (FST) in segregating populations (Tadege et al., 2008) and
we confirmed that the seven independent lines (stf1-1 to stf1-7)
are allelic, having Tnt1 insertions in exons one, two, and four
(Figure 3A). This was further confirmed by complementation of
the stf1-2 mutant phenotypes with a 5.3-kb genomic fragment of
STF (Figure 3B). STF encodes a 358-amino acid homeodomain
Figure 2. The stf Mutant Is Severely Defective in Leaf Vascular Patterning.
(A) Wild-type, R108, and stf1-2 mature leaves showing the regions for close-up described in (B) to (E).
(B) to (E) Leaf material observed through a light microscope after clearing with lactic acid.
(B) Major and minor veins of R108 leaf.
(C) Disorganized and poorly developed major veins in stf. Major veins are forming near the margins (one on either side of the midvein) along the
proximodistal axis (arrows).
(D) R108 major vein extends close to the margin with its tip aligned to the serration and is open ended (arrow).
(E) stf major vein poorly developed and connected to marginal vein (arrow).
(F) R108 leaf epidermal cells viewed through a light microscope.
(G) Epidermal cells of stf leaf showing narrower width.
(H) Transverse section through R108 leaf blade showing palisade mesophyll (white arrow) and spongy mesophyll (red arrow) cells. Sections were
stained with Toluidine Blue.
(I) Transverse section through stf leaf blade showing the poor distinction between palisade mesophyll (white arrow) and spongy mesophyll (red arrow)
cells.
(J) Transverse section through the midrib of R108 leaf showing xylem (yellow arrow) and phloem (orange arrow) vessels.
(K) Transverse section through stfmidrib showing poorly differentiated xylem and phloem vessels (yellow and orange arrows) and cortical tissue. Scale
bars in (B) to (E) = 500 mM, in (F) and (G) = 50 mM, and in (H) to (K) = 100 mM.
2128 The Plant Cell
transcriptional regulator with 38% amino acid identity to Arabi-
dopsisWOX1 and 45% amino acid identity to Petunia MAW. We
identified STF-like (STL) sequences from alfalfa and tobacco by
RT-PCR and from other dicot genomes by BLAST search (see
Supplemental Figures 2 and 3 online). In most of these genomes,
STF is represented by a small gene family with one or two
members. In the sequenced part of the M. truncatula genome,
there is a second partial STF with predicted 165 amino acids
but its functionality has not yet been tested. At the sequence
level, STF, MAW, and STL share conserved amino acid motifs at
the N- and C-terminal regions, in addition to the homeodomain
and WUS box, including a highly conserved 10-amino acid motif
at the 39 end (QFI/FEFLPLKN), whichwe named the STF box (see
Supplemental Figure 3B online). The region corresponding to the
STF box inWUShas been recognized as an EAR-likemotif (Ikeda
et al., 2009), but the STF box shows more similarity to the WUS
box than the WUS EAR-like motif. The WUS box confers repres-
sive functions to Arabidopsis WUS (Ikeda et al., 2009).
Phylogenetic analysis of a subset of related WOX and STL
proteins using full-length and homeodomain region amino acid
sequences showed that STF and STL group together in a
separate subclade that encompasses WOX1 but distinct from
the closely related PRS/WOX3 and WUS subclades (see Sup-
plemental Figure 2 online), in agreement with previous studies
(Vandenbussche et al., 2009; Zhang et al., 2010). In addition to
M. truncatula, tobacco, petunia, and Arabidopsis mentioned
above, for which functionality has been tested, STL sequences
were found in all eudicot species sequenced to date, suggesting
that STF is conserved in both dicot classes: rosids and asterids.
By contrast,STF homologswere not found in the genomes of rice
(Oryza sativa), maize, sorghum (Sorghum bicolor), foxtail millet
(Setaria italic), or purple false brome (Brachypodium distachyon),
or in the wheat (Triticum aestivum) and barley (Hordeum vulgare)
database of 1.5 million expressed sequence tags, as well as the
banana (Musa spp) NCBI database of 89,151 expressed se-
quence tags. On the other hand, the related gene, PRS/WOX3, is
widely conserved in angiosperms and gymnosperms (see Sup-
plemental Figures 2B and 4 online). Themolecularmechanismby
which PRS or maize NS1 and NS2 orchestrate their functions is
unknown, but the stf mutant phenotypes suggest that STF may
not be functionally redundant with PRS/NS because the strong
stf phenotype prevails in the presence of wild-type Medicago
PRS (MtWOX3). All theWUS, PRS, andMAWclades have strong
conservation in the WUS box, whereas the STF box is absent
from PRS/WOX3 genes and is modestly conserved in the WUS
clade (see Supplemental Figure 5 online). Further investigation
will be required to determine if the STF box is a functional domain
and if STLs play specific roles in dicot lamina evolution.
STF Is Expressed at the Adaxial–Abaxial Boundary Layer in
Leaf Primordia and Shows Developmental Regulation
To investigate tissue-specific expression patterns of STF, we
performed RNA in situ hybridization in vegetative shoot apex and
flower, where the highest expression was detected by quantita-
tive RT (qRT)-PCR. In young leaf primordia at the P1 and P2
stages, STF is adaxially expressed in few cells, but absent from
theSAM (Figure 4A). In older primordia after stage P2, strongSTF
expressionwas detected inmore cells and localizes in the central
region of the adaxial–abaxial boundary layer extending from the
distal tip to the proximal base (Figure 4B). In the flower, STF
expression was detected in petal primordia and developing
petals (Figures 4C and 4E); in carpels, expression was observed
in developing margins (Figure 4C), in the placenta, at the base of
ovules (Figure 4D) and in the central region. No expression was
observed in stamens, in sepals (Figures 4D and 4E), in inflores-
cence meristem (Figure 4F), or in the floral meristem (Figure 4G),
compared with controls (Figures 4H and 4I). Fusing a 2.6-kb
region of STF promoter with a b-glucuronidase (GUS) reporter
showed a developmental gradient of GUS expression in leaves.
In very young trifoliate leaves, GUS staining was intense at the
leaf margin in the distal half, including the leaf tip (see Supple-
mental Figure 6B online). As the leaf grows, expression moves to
the proximal half and becomes progressively weaker, to an
undetectable level, in mature leaves (see Supplemental Figures
6B to 6E online). GUS staining was also detected in the unifoliate
leaf, roots, cotyledons, petals, stigma, and pods (see Supple-
mental Figures 6A and 6F to 6H online).
The lam1Mutation of N. sylvestris Is Caused by Deletion of
the STF Homolog
In the classical bladeless lam1mutant of N. sylvestris, leaf blades
are reduced to vestigial strips lacking mesophyll differentiation
(McHale and Marcotrigiano, 1998). The lam1 mutant strongly
resembles stf except that lam1 phenotypes are stronger and lack
stem elongation (Figure 5A). Based on morphological characters,
we hypothesized that stf and lam1may be caused bymutations in
homologous genes. To test this, we cloned an STF-like gene (Ns
STF1) fromwild-type N. sylvestris by RT-PCR. We also cloned the
full-length Ns STF1 genomic sequence with its promoter by
thermal asymmetric interlaced (TAIL)-PCR. Ns STF1 is similar to
Mt STF in gene structure (see Supplemental Figure 2A online) and
Figure 3. STF Encodes aWOXDomain Protein and Complements the stf
Mutant.
(A) STF gene structure showing the position of the Tnt1 insertion site in
seven independent mutant lines.
(B) stf mutant complemented with 5.3-kb genomic STF.
[See online article for color version of this figure.]
Regulation of Lamina by STENOFOLIA 2129
shares 45% amino acid identity. PCR experiments demonstrated
that Ns STF1 was deleted in the lam1 mutant. Using primers
specific to Ns STF1 (see Supplemental Table 1 online), including
2.65 kb of the promoter, we determined that at least a 5.67-kb
region of the Ns STF1 locus was deleted in lam1 (Figure 5B).
To confirm that the lam1 phenotype is due to lack of Ns STF1
function, a 5.3-kb genomic fragment ofMtSTFwas introduced into
lam1. Mt STF fully complemented the lam1 phenotypes (Figures
5C and 5D), confirming that STF function is indeed absent in the
lam1 mutant and that STF and LAM1 are functional homologs.
Transcript Profiling Identified Auxin and Multiple
Hormone-Associated Changes in stfMutant
To identify potential targets of STF and to gain insight into the
mechanism of its function, we performed transcript profiling anal-
ysis using the Medicago Affymetrix GeneChip containing 61,278
probesets.Wecomparedgeneexpression in three independent stf
mutant lines (stf1-1/2/3) with their segregating wild types in 0.5- to
0.8-mm shoot apices of 4-week-old seedlings, and identified 241
probes differentially expressedwith a twofold or more difference in
the mutant; 105 probes upregulated and 136 probes downregu-
lated. Thisanalysis identifiedgenes that are known tobe involved in
leaf development such as SCARECROW (SCR)-like, TCP3, and
indole-3-acetic acid (IAA) amidosynthase (GH3) as downregulated,
and BOP1/2 and KNAT2, KNAT6-related KNOX1 genes as upreg-
ulated, as well as changes in several phytohormone-associated
genes, especially auxin (Figure 6A, Table 1). ThePHAN andHD-ZIP
III-type adaxial polarity determinantswere not detecteddespite the
presence of probes on the array.We assessed the expression level
of PHAN, PHABULOSA (PHB), and CORONA together with eight
upregulatedand16downregulatedgenesbyqRT-PCR.Consistent
with the microarray data, polarity genes were found to be unaf-
fected (PHAN andCORONA) ormodestly reduced (PHB), while the
other genes differentially expressed in the microarray were clearly
induced or repressed in qRT-PCR (Figure 6B), confirming the
notion that polarity disruption is not the primary defect in stf. The
microarray data rather showedoverrepresentation of genes related
to phytohormones, including auxin, cytokinin, brassinosteroid,
ethylene, gibberellic acid (GA), abscisic acid, and jasmonic acid
(Table 1). After removing 11 redundant probes, at least 17.5% of
the downregulated and 17.3% of the upregulated probes were
Figure 4. STF Expression Pattern in Vegetative and Floral Apices by RNA in Situ Hybridization in R108.
(A) STF expression in 12-d-old vegetative shoot apex viewed in longitudinal sections. At very early stages, STF is adaxially expressed in few cells (black
arrows), but absent from the central zone of SAM (white arrow).
(B) STF expression in older leaf primordia showing localization at the adaxial–abaxial boundary layer (arrow).
(C) STF expression in young flower showing a strong signal in petal primordia and developing petal (red arrows) and developing carpel (black arrow).
(D) STF expression in mature flower showing strong localization in the placenta at the base of the ovules (arrow).
(E) STF expression in mature flower showing expression in the petal lobe (arrow).
(F) STF expression in inflorescence apex showing activity in floral organ primordia, but no detection in the inflorescence meristem (white arrow).
(G) STF expression in inflorescence apex showing no detection in the floral meristem (white arrow).
(H) PIM (AP1) expression in inflorescence apex shown here as positive control for expression in floral meristem (arrow).
(I) RNA in situ hybridization in the inflorescence apex using STF sense probe as negative control.
2130 The Plant Cell
found to be directly related to genes associated with hormones,
especially auxin (Table 1). This represents a 10-fold enrichment
(17.4% compared with a random 0.74%) for the seven hormones
mentioned here. This enrichment is 17-fold for auxin-associated
genes alone just by querying for the word “auxin.” For example, for
most altered auxin genes in the array, 11 of the 48 small auxin-
upregulated RNA (SAUR) probes present on the GeneChip were
induced (Table 1), accounting for 4.78% of the altered genes
instead of the expected 0.08% for unbiased change. Small auxin-
upregulated RNA genes are known to negatively regulate auxin
biosynthesis and auxin response (Kant et al., 2009). This transcrip-
tional change in auxin-associated genes is consistent with the stf
mutant phenotype and the anticipated role of auxin in lamina
outgrowth because auxin has been implicated in mechanisms
controlling leaf vascular patterning, leaf margin serrations, repro-
ductive organ development, and blade outgrowth although the
association with blade outgrowth is circumstantial. These obser-
vations prompted us to think that the function of STF may be
connected to auxin, and the other hormone responses may have
been altered as a result of hormonecrosstalk.We followed this lead
and evaluated the auxin connection further.
stf and lam1Mutants Accumulate Less FreeAuxin in Leaves
To investigate the role of auxin in stf/lam1 phenotype, we
measured free auxin (IAA) directly by gas chromatography-
mass spectrometry (GC-MS) in 4-week-old leaves of lam1 and
two alleles of stf (stf1-2 and sft1-3). We found that stf accumu-
lates;68% and lam1 accumulates 50% of the respective wild-
type IAA levels (Figure 6C), indicating that the mutants have an
auxin deficiency. Because N. sylvestris is faster to transform and
is known to be more sensitive to hormones than M. truncatula,
we focused the next experiments solely on lam1.To indepen-
dently confirm the reduced auxin content by a different method,
we transformed lam1 and its wild type with the auxin responsive
DR5:GUS construct. GUS staining in young leaves from 3- to
4-week-old plants was faintly detectable in DR5:GUS-expressing
lam1 leaves, whereas strong GUS staining was observed in DR5:
GUS-expressing wild-type leaves (Figures 6D and 6E), confirming
that lam1 indeed accumulates reduced auxin.
The reduced steady state level of free auxin could be caused
by a defect in auxin biosynthesis, signaling, transport, homeo-
stasis, or a combination thereof. To distinguish among these
possibilities, we applied exogenous auxins to lam1 shoots and
roots. Foliar spraying of 2-week-old seedlings with naphthalene
acetic acid (NAA) showed that mutant and wild-type leaves
respond in the same way by epinastic curling and tolerated up to
100 mM NAA concentrations (see Supplemental Figures 7A and
7B online). Application of 10 mM NAA or IAA with lanolin to the
shoot apex of lam1 at the 4 leaf stage or adult plants resulted in
ectopic lateral bumps and branches on the leaf blade (see
Supplemental Figures 7D and 7E online). Similarly, regenerating
new plants from lam1 leaf explants in tissue culture via somatic
organogenesis in the presence of auxin (0.53 mM NAA) and
cytokinin (4.44 mM benzyl amino purine [BAP]), frequently pro-
duced variously branched and bifurcated lam1 leaves that have
restricted and irregular flattening on the main leaf axis (see
Supplemental Figure 7C online). These observations suggest
that the lam1 mutant is not blocked in auxin response. The
addition of NAA, 2,4-dichlorophenoxyacetic acid (2,4-D), IAA, or
the polar auxin transport inhibitor N-1-naphthylphthalamic acid
(NPA) to Murashige and Skoog (MS) medium affected lam1 and
wild-type root elongation in a similar manner (see Supplemental
Figure 7F online), although both roots tolerated much higher
levels of the natural auxin IAA compared with the synthetic
auxins. NAA and 2,4-D are known to be substrates of efflux and
influx carriers, respectively (Yamada et al., 2009). The absence of
a differential response to both or to NPA suggests that efflux and
influx processes and transport are not significantly affected in
lam1. However, growing mutant and wild-type plants on MS
medium containing the auxin biosynthetic intermediate Trp,
indicated that lam1 roots elongated slightly better (P < 0.05)
than wild-type roots (see Supplemental Figure 7F online), sug-
gesting that lam1 could be partly affected in auxin biosynthesis
upstream of Trp.
We next performed metabolite profiling in leaf extracts by GC-
MS to identify any accumulated intermediates of auxin biosyn-
thesis that would indicate a metabolic blockage point in lam1.
We found that metabolites of the shikimate pathway, including
shikimic acid, Tyr, and Phe, as well as five- and six-carbon
sugars of sugar metabolism, including Glc and Fru were signif-
icantly reduced in the mutant (P < 0.001), indicating a defect in
sugar metabolism. No significant change (P < 0.05) in 12-carbon
sugars, including Suc and galactinol were observed, however,
glucitol, Gln, Ser, Gly, and Prowere increased (P < 0.001) by 2- to
15-fold in lam1 (Figure 7), which also indicated a defect in sugar
metabolism. Hexoses limit erythrose and pyruvate availability
Figure 5. The N. sylvestris lam1 Mutation Is Caused by Deletion of the
Ns STF1 Gene.
(A) Ten-week-old adult lam1 mutant plant.
(B) Genomic PCR showing deletion of the Ns STF1 locus in the lam1
mutant. Primers F1+R2 amplify the complete CDS plus the 39 UTR, and
primers F2+R3 amplify the promoter, the 59 UTR, plus part of the CDS,
and together span 5.67 kb of the Ns STF1 region. * = 3 kb. WT, wild type.
(C)Untransformed lam1mutant and lam1 complementedwithM. truncatula
5.3-kb genomic STF regenerating in nonselective tissue culture media.
(D) Complemented lam1 in (C) 4 weeks after transfer to soil.
Regulation of Lamina by STENOFOLIA 2131
and could account for the downregulation of the shikimate
pathway, including auxin biosynthesis. These observations sug-
gest that the reduced auxin levels measured in lam1 leaves may
be caused by a broader defect in sugar metabolism rather than a
single block in the IAA biosynthetic pathway.
Ectopic Expression of STF Leads to Auxin and Cytokinin
Overproduction Phenotypes
To evaluate if increased STF activity leads to auxin overproduction,
we ectopically expressed Mt STF and Nicotiana benthamiana STF
(Nb STF1) in N. sylvestris using the 35S promoter. IAA was
increased by approximately twofold in transgenic lines com-
pared with controls (Figure 8A), indicating a direct relationship
between STF expression and steady state level of free auxin
accumulation. Transgenic plants expressing either of the genes
showed a range of auxin and cytokinin overproduction phenotypes
that have been previously observed in tobacco (Eklof et al., 2000),
including leaf upward curling (Figures 8B and 8C). Eight of the
strongest STF expressers among 27 independent transgenic lines
displayed extreme dwarf and nonflowering phenotypes with de-
formed shoots, roots, and leaves (Figure 8E; see Supplemental
Figure 8A online). Interestingly, these severely affected lines pro-
duced one or more tumors on the roots or at the crown (Figure 8F;
see Supplemental Figure 8A online). Because tumor formation
requires overproduction of auxin and cytokinin (Zambryski et al.,
1989), these results suggest that STF expressors are also over-
producing cytokinin. Consistent with this, exogenous application of
cytokinin alone with lanolin to lam1 shoot apex frequently produced
leaf branches, and rarely shoots, while application of auxin and
cytokinin together partially rescued the lam1 blade phenotype
(Figures 9B, 9D, and 9F), suggesting that STF may directly or
indirectly affect cytokinin biosynthesis. The application of cytokinin
or cytokinin and auxin together to wild-type shoots has an inhibitory
Figure 6. Microarray Analysis and Auxin Quantification in stf and lam1 Mutants.
(A) A heat map showing differentially expressed genes in three stf mutant lines compared with wild type R108 in 4-week-old shoot apices.
Representative genes that are downregulated (green) and upregulated (red) with twofold or more difference are shown. ABA, abscisic acid.
(B) Validation of relative gene expression of selected genes in the stfmutant compared with the wild type by qRT-PCR. Wild-type expression level was
arbitrarily set to 1.0. Green, downregulated genes; red, upregulated genes; blue, genes not detected by the microarray. Values are the mean and SE of
three biological replicates.
(C) Free IAA content in 4-week-old leaves of stf and lam1 mutants compared with their wild type (WT). Values are the mean and SE of five experiments
(***P < 0.001, **P < 0.01).
(D) GUS staining in DR5:GUS-transformed wild-type N. sylvestris leaf.
(E) GUS staining in DR5:GUS-transformed lam1 leaf showing reduced auxin.
2132 The Plant Cell
effect on leaf growth (Figures 9A, 9C, and 9E), but the application of
cytokinin alone rarely induces shoot regeneration on leaves similar
to its effect on lam1 shoots. These data suggest that the function of
STF in regulating lamina outgrowthmay be connected to the auxin:
cytokinin ratio.
WUS activates cytokinin signaling by A-type ARR repression
(Leibfried et al., 2005). To explore the possibility that cytokinin
signaling is disrupted in lam1 mutant primordia, we transformed
lam1 explants with STF:WUS. We observed complementation of
the lamina and venation defects of lam1 (Figure 10), with this
construct suggesting that cytokinin signaling is in fact a major
component of LAM1 function. This is consistent with the STF
overexpression phenotypes and the combined auxin and cytoki-
nin treatments. Together, our data support amodel inwhich auxin-
and cytokinin-mediated signaling modulated by STF/LAM1 at the
leaf primordial margins regulates vein patterning and blade out-
growth in the simple- and dissected-leafed eudicots.
DISCUSSION
STF Controls Blade Outgrowth at the Adaxial–Abaxial
Boundary Layer
We have described the leaf lamina mutants, stf and lam1, repre-
senting rosids and asterids, respectively. In several adaxial polarity
mutants, including phan (Waites and Hudson, 1995) and phb/phv
Table 1. Hormone-Associated Genes Differentially Expressed in stf Shoot Apex
Probe Sets Putative Annotation P Valuea Fold Changeb Putative Functionc
Mtr.18769.1.S1_at Mt HOMEOBOX PROTEIN1 0 0.31 Abscisic acid/auxin signaling
Mtr.24418.1.S1_at Abscisic acid-89-hydroxylase 0 0.35 Abscisic acid catabolism
Mtr.1887.1.S1_at GA20 oxidase like 0 0.36 GA biosynthesis
Mtr.40263.1.S1_at GH3/IAA amidosynthase 0 0.36 Auxin homeostasis
Mtr.43236.1.S1_at UDP/cytokinin glucosyltransferase 0 0.37 Putative cytokinin homeostasis
Mtr.4206.1.S1_at MYB 94 transcription factor 0 0.31 Multiple hormone response
Mtr.2065.1.S1_at Ent-kaurenoic acid oxidase 4.4409E-16 0.39 GA biosynthesis
Mtr.11553.1.S1_at AHP-like phosphotransfer protein 2.15E-269 0.39 Cytokinin signal transduction
Mtr.25950.1.S1_at 1-aminocyclopropane-1-carboxylic
acid oxidase
0 0.41 Ethylene biosynthesis
Mtr.1108.1.S1_at MYB 77-like tf - auxin signaling 5.266E-48 0.43 Multiple hormone response
Mtr.10192.1.S1_at TINY-like tf – cell growth 6.7714E-57 0.44 Ethylene response
Mtr.37279.1.S1_at Xanthoxin dehydrogenase-like 3.4884E-23 0.44 Putative abscisic acid biosynthesis
Mtr.25341.1.S1_at Auxin:hydrogen symporter 4.3091E-07 0.45 Auxin transport
Mtr.27392.1.S1_at Auxin-induced protein 5NG4 2.1832E-09 0.47 Auxin response
Mtr.42075.1.S1_at Abscisic acid hydroxylase 6.7002E-39 0.48 Abscisic acid catabolism
Mtr.29279.1.S1_at Lipoxygenase 1.922E-33 0.48 Jasmonic acid biosynthesis
Mtr.9203.1.S1_at SERK1-like protein 3.9199E-79 0.49 Brassinosteroid signaling
Mtr.19928.1.S1_at Auxin-responsive SAUR70 1.9E-19 2.01 Auxin signaling
Mtr.19898.1.S1_x_at Auxin-responsive SAUR76 3.09E-07 2.03 Auxin signaling
Mtr.12959.1.S1_s_at Auxin-response factor 6 1.22E-07 2.1 Auxin signaling
Mtr.14486.1.S1_at Auxin-responsive SAUR83 3.25E-14 2.11 Auxin signaling
Mtr.13212.1.S1_at Jasmonate methyltransferase 1.0493E-12 2.15 Methyl jasmonate biosynthesis
Mtr.19891.1.S1_s_at Auxin-responsive SAUR81-like 2.95E-45 2.18 Auxin signaling
Mtr.697.1.S1_at Auxin-responsive SAUR70-like 9.4E-105 2.22 Auxin signaling
Mtr.4357.1.S1_at Auxin-responsive SAUR82-like 0 2.25 Auxin signaling
Mtr.33772.1.S1_at UDP-glucuronosyltransferase 0 2.27 Putative cytokinin/auxin homeostasis
Mtr.19925.1.S1_x_at Auxin-responsive SAUR69-like 0 2.33 Auxin signaling
Mtr.40821.1.S1_at Abscisic acid-activated kinase 1.6015E-07 2.33 Abscisic acid signaling
Mtr.19880.1.S1_at Auxin-responsive SAUR80 6.76E-20 2.33 Auxin signaling
Mtr.38122.1.S1_at EREB factor 1.5283E-10 2.38 Ethylene signaling
Mtr.49767.1.S1_x_at Auxin-responsive SAUR69 4.62E-15 2.39 Auxin signaling
Mtr.12648.1.S1_at GA-induced ovary protein 1.79E-22 2.44 GA response
Mtr.7260.1.S1_at Ethylene-induced esterase 3.43E-42 2.53 Ethylene response
Mtr.702.1.S1_at Auxin-responsive SAUR81-like2 1.65E-101 2.56 Auxin signaling
Mtr.50766.1.S1_at BTB/POZ;NPH3 protein 3.42E-35 2.66 Light/auxin signaling
Mtr.38765.1.S1_at LOB domain protein 38 7.6E-185 2.73 Auxin signaling
Mtr.49400.1.S1_at Auxin-responsive SAUR29 1.5983E-92 2.88 Auxin signaling
Mtr.37975.1.S1_at CBL-interacting protein kinase 2.577E-125 3 Abscisic acid/glucose signaling
Mtr.35796.1.S1_at GAST-like gene product 8.01E-14 3.23 GA response
Mtr.45080.1.S1_at Abscisic acid receptor-like kinase 6.8378E-25 3.68 Abscisic acid signaling
aP value is obtained from Associative Analysis (Dozmorov and Centola, 2003).bRelative abundance of transcript in stf shoot apex/R108 shoot apex.cCategory of predicted gene function.
Regulation of Lamina by STENOFOLIA 2133
double mutants (McConnell et al., 2001), there is not only loss of
adaxial identity but also the appearance of abaxial characteristics
in the adaxial domain. The abaxialization of severely affected phan
mutants led Waites and Hudson (1995) to propose that the juxta-
position of adaxial and abaxial cells is a prerequisite for blade
outgrowth. In the stfmutant, such complete loss of adaxial domain
or appearance of abaxial characters in the adaxial surface has not
been observed, and domain specification is intact. Unlike most
polarity factors that exhibit domain-specific expression in leaf
primordia, such as HD-ZIP III and KAN family genes (Kerstetter
et al., 2001; McConnell et al., 2001; Emery et al., 2003), STF
expression is not axially confined (Figure 4B). The expression
domains of STF and the presence of intact dorsoventral polarity in
the stfmutant are consistent with recent observations reported for
maw (Vandenbussche et al., 2009). Our work on STF/LAM1, along
with the studies on MAW, suggests that polarity governs only the
initial phase where founders are recruited at the adaxial–abaxial
boundary. Subsequent assembly of specialized cell layers and
outgrowth of flattened lamina is dependent on WOX function. We
propose that STF function at the adaxial–abaxial boundary is
required for cell proliferation, cellular differentiation, and expansion
controlling lamina elaboration in themediolateral axis. In addition to
STF, LAM1, MAW, NS1, and NS2, which are WOX genes, class I
HD-ZIP genes have been reported to control tendril and bract
morphogenesis without necessarily disrupting polarity. For exam-
ple, the tendril-less (tl ) mutation in pea revealed that tendrils are
modified leaves where lamina outgrowth is inhibited by repressive
function ofTL (Hofer et al., 2009), and a related gene inArabidopsis,
LATEMERISTEM IDENTITY1 (LMI1), is required for suppression of
bract formation (Saddicetal., 2006). It remains tobeshown if class I
HD-ZIP genes target or interact with WOX genes in leaf primordia.
STF Controls Blade Outgrowth and Vein Patterning
Auxin has been described as the key factor that modulates
growth and pattern formation during vascular morphogenesis
Figure 7. Metabolic Profiling in 4-Week-Old Leaves of Wild-Type and lam1 Mutant N. sylvestris.
Representative common metabolites are shown. Colors indicate downregulated (green) and upregulated (red) metabolites in lam1 mutant compared
with the wild type (WT). The black color shows metabolites that are unchanged. The numbers 1 through 6 at the top indicate replicates of lam1 and wild-
type samples each from six individual plants. Statistical significance was calculated using Student’s t test (***P < 0.001, **P < 0.01, and *P < 0.05).
2134 The Plant Cell
(Scarpella et al., 2006; Cano-Delgado et al., 2010; Scarpella et al.,
2010). Recently, Scarpella et al. (2010) have suggested that auxin
internalization from PIN1 convergence points in the epidermal
layer induces formation of veins and that auxin maxima generated
by PIN1 polarity is the major factor that orchestrates vascular
patterning and blade outgrowth. However, auxin response factors
that specifically and differentially regulate auxin signaling in the
outer epidermal layer have not been identified, nor is there a
mechanism for local auxin biosynthesis that could account for
continued generation of PIN1 polarity as the leaf expands. The
pattern of veindisruptionand lamina growth arrest in the stfmutant
fits the mechanism of blade outgrowth and venation coupling
described by Scarpella et al. (2010) and suggests that the STF
regulation of vascular patterning and lamina outgrowth may be
connected to auxin signaling at the leaf margins.
STFMayRegulateBladeOutgrowthandVascularPatterning
by Modulating Auxin and Cytokinin Homeostasis
Our microarray analysis identified a strong connection between
the stfmutant and transcriptional changes in hormone-responsive
genes, especially genes associated with auxin (Table 1). Consis-
tent with this, stf and lam1 mutants accumulate less free IAA in
their leaves (Figures 6C to 6E). The reduced auxin cannot be
simply a consequence of the narrow leaf mutant phenotype
because ectopic expression of STF in N. sylvestris results in a
smaller leaf, yet there is a twofold increase in free IAA compared
with controls (Figure 8A). Auxin is a multifunctional phytohormone
required for cell division and cell expansion and has been
Figure 8. Ectopic Expression of Mt STF and Nb STF1 in N. sylvestris.
(A) Free IAA content in mature leaves of lam1 mutant, Wild type, STF:
GUS transgenic control, and 35S:STF transgenic plant. Values are the
mean and SE of five replicates (***P < 0.001). FW, fresh weight.
(B) 35S:STF transgenic plant showing upward curling leaf phenotype.
(C) 35S:Nb-STF1 transgenic leaf showing upward curling phenotype.
(D) Wild-type leaf.
(E) Left, 4-week-old wild-type (WT) N. sylvestris; right, 17-month-old
transgenic plant with highest STF overexpression showing shoot and
root deformation.
(F) Close-up of the transgenic plant in (E) showing two large tumors.
Scale bars = 5 cm.
Figure 9. Exogenous Application of Auxin and Cytokinin Partially Res-
cues the lam1 Lamina.
(A) and (B)Wild-type N. sylvestris (A) and lam1 (B) shoots treated with 10
mM BAP. Inset shows leaf branching.
(C) and (D)Wild-typeN. sylvestris (C) and lam1 (D) shoots treated with 10
mMBAP plus 10mM IAA. Inset shows partially formed petiole and blade.
(E) and (F)Wild-type N. sylvestris (E) and lam1 (F) shoots treated with 10
mM BAP plus 1 mM IAA. Inset shows partially formed petiole and blade.
Note that lam1 leaves are uniformly thin and cannot be distinguished into
petiole and lamina. Scale bars = 1 cm.
[See online article for color version of this figure.]
Regulation of Lamina by STENOFOLIA 2135
associated with a plethora of developmental programs, including
leaf margin serration, reproductive development, leaf vascular
patterning, and blade outgrowth (Scarpella et al., 2006, 2010;
Koenig et al., 2009; Pagnussat et al., 2009; Perrot-Rechenmann,
2010; Bilsborough et al., 2011), indicating that the stf phenotypes
are subsets of auxin-mediated developmental programs. It has
been proposed that intra- and intercellular auxin gradients are
critical in conveying positional information during auxin signal
transduction (Benkova et al., 2003; Vanneste and Friml, 2009). It is
conceivable that a twofold reduction in total free auxin content in
the mutant could disrupt auxin gradients that convey develop-
mental signals. Under the model of Scarpella et al. (2010), STF/
LAM1 could function by controlling the spatial and temporal
availability of auxin at the leaf margin. Normal auxin response
and transport, but reduced free IAA associatedwith lowmetabolic
flux in lam1 mutant, suggests that STF/LAM1 may be involved in
the modulation of auxin homeostasis.
However, auxin is not the only hormone altered in stf and lam1.
Abscisic acid is also lower in lam1 mutants and higher than wild
type in STF-overexpressing N. sylvestris (see Supplemental
Figure 8B online). Moreover, the stem elongation defect in the
lam1 mutant can be rescued by GA application (McHale, 1992),
which together with the downregulation of GA biosynthetic
enzymes in stf microarray, suggests that STF has a pleiotropic
effect on GA biosynthesis. The microarray analysis also revealed
that 1-aminocyclopropane-1-carboxylic acid oxidase (a key
enzyme in ethylene biosynthesis), and His-containing phospho-
transmitter protein1 (a key element in two-component cytokinin
signal transduction phosphorelay) are strongly downregulated
(Table 1). More importantly, the highest STF overexpressor
transgenic plants developed typical auxin and cytokinin over-
production phenotypes, including tumors (Figure 8). lam1 is
sensitive to cytokinin application and responded by forming leaf
branches similar to auxin treatment, but the application of auxin
and cytokinin together partially rescued the lam1 lamina (Figure
9). Together, these data suggest the integration of multiple
hormones (Jaillais and Chory, 2010) during leaf morphogenesis
coordinated by auxin:cytokinin ratio in leaf primordia.
Possible Mechanisms for STF Function
We envision three scenarios for how STF may function in mod-
ulating hormone homeostasis and integration. In the first sce-
nario, STF regulates tissue-specific hormone homeostasis by
controlling one or more hormone-conjugating enzymes. In sup-
port of this hypothesis, jasmonic acid carboxyl methyltransfer-
ase and putative auxin and cytokinin glucosyltransferases are
upregulated in the stfmutant (Table 1). It is known that activation
of auxin-conjugating enzymes leads to auxin depletion pheno-
types and impairs auxin-mediated plant growth and environ-
mental responses (Jackson et al., 2002; Qin et al., 2005; Park
et al., 2007; Tognetti et al., 2010). In this scenario, failure ofSTF to
control IAA/cytokinin glucosyltransferase in the stf mutant could
lead to the buildup of IAA/cytokinin-glucoside conjugate, which
signals excess IAA/cytokinin and this feeds back to sugar
metabolism to slow down biosynthesis by reducing or rechan-
neling hexoses (see Supplemental Figure 9 online). Decreased
Glc and Fru, but increased Suc have been shown to be associ-
ated with reduced auxin biosynthesis in the maize cell wall
invertase mutant mn1 (LeClere et al., 2010). This decrease in
hexoses, in turn, could downregulate the entire shikimate path-
way, leading to reduced auxin biosynthesis, and could also affect
other hormones (see Supplemental Figure 9 online).
In the second scenario, STFmodulates the auxin:cytokinin ratio
by regulating cytokinin signaling through activation of a histidine-
containing phosphotransmitter protein, which is strongly down-
regulated in stf or by repressing A-type two-component response
regulators, RR9 and RR15, which are modestly upregulated in stf,
analogous to repression of ARR7 and ARR15 by WUS in Arabi-
dopsis (Leibfried et al., 2005). Complementation of lam1 with At
WUS expression (Figure 10) favors this possibility. Cytokinin
Figure 10. Arabidopsis WUSComplements the lam1Mutant Phenotype.
(A)Untransformed wild-type (WT)N. sylvestris grown in tissue culture MS
media. Right panel shows wild-type leaf cleared with lactic acid for
looking at the venation pattern.
(B) lam1 mutant transformed with STF:GUS construct as negative
control. Right panel shows lam1 leaf cleared with lactic acid.
(C) lam1 mutant complemented with STF:WUS construct. Right panel
shows complemented leaf cleared with lactic acid. Note that the lamina
and venation phenotypes are complemented.
[See online article for color version of this figure.]
2136 The Plant Cell
signaling is known to affect auxin biosynthesis, and although the
interaction could be antagonistic in roots (Dello Ioio et al., 2008;
Muller and Sheen, 2008; Moubayidin et al., 2009), auxin-cytokinin
crosstalk in shoots is thought to be synergistic (Pernisova et al.,
2009; Zhao et al., 2010). In this hypothesis, cytokinin response is
the primary target of STF, whereas auxin and other hormones are
altered as a result of hormone crosstalk or a change in the auxin:
cytokinin ratio.
In the third scenario, STF regulates other transcription factors
that may be directly or indirectly connected to auxin/cytokinin
activity. Two SHORT ROOT/SCR-related GRAS family transcrip-
tion factors and one D-type cyclin (CYCD6;1 homolog) are
downregulated in stf. SHORT ROOT and SCR are regulators of
cell proliferation in leaves (Dhondt et al., 2010) and directly target
D-type cyclins to control cell cycle progression (Sozzani et al.,
2010). The combined downregulation of GRAS genes and
CYCD6 could lead to premature exit from the cell cycle in stf.
Other possible targets includeHOMEOBOXPROTEIN 1, which is
abscisic acid responsive and a repressor of auxin-responsive
lateral organ boundary domain protein in M. truncatula roots
(Ariel et al., 2010), BOP1/2, LOBD38, TCP3, and KNAT2/KNAT6-
relatedKNOX genes, which all have the potential to coordinate or
interact with the auxin/cytokinin signaling machinery.
However, the above three scenarios are not mutually exclusive,
and it remains to be shown if they act in concert. For example,STF
may repress auxin/cytokinin-glucosyltransferaseandalso activate
cytokinin by activating a His-containing phosphotransmitter pro-
tein at the same time tomaintain the right auxin:cytokinin ratio and
homeostasis at the leaf margins. The synergistic interaction of
auxin and cytokinin could then presumably deliver the instructive
spatiotemporal signal to TCPs, GRAS genes, and the cell cycle to
execute morphogenic functions. In this context, STIMPY/WOX9-
mediated coordination of sugar and cytokinin signaling to the cell
cycle has been proposed for shoot meristem establishment in
Arabidopsis (Skylar et al., 2010, 2011). Identifying the direct target
(s) ofSTFwill enlighten our understanding of themechanismof this
fundamental process.
METHODS
Mutant Screening and Cloning of STF
Insertional mutagenesis in Medicago truncatula genotype R108 using
Tnt1 retrotransposon and screening conditions in the greenhouse have
been previously described (Tadege et al., 2008, 2009). Forward genetics
screening of 5600 Tnt1-tagged lines under standard conditions (16 h/8 h
and 248C/198C day/night cycles) in the greenhouse for leaf blademutants
have identified sevenmutants with identical phenotypes of stf designated
stf1-1 to stf1-7. The cloning of STF by genotyping of FST was performed
as previously described (Tadege et al., 2008).
The stf mutant phenotype segregates as a recessive mutant (95 wild
types to 30mutants in stf1-2 heterozygous) in the seven independent lines.
Forty-one FST were recovered from stf1-1 by TAIL-PCR using a combina-
tion of Tnt1-specific primers (Tnt1-F1, Tntail 1, 2, 3, and LTR3 or LTR5) and
arbitrary primers AD1 or AD2 (see Supplemental Table 1 online). Of 22 FST
genotyped in a segregatingpopulationof stf1-1mutants using FST-specific
primers, two FST were identified with homozygous Tnt1 insertions that
cosegregated with the narrow leaf mutant phenotype. Only one of the two
FST (75insert1) was also found to be tagged in the other six stfmutant lines
and cosegregated with the mutant phenotype. The gene corresponding to
this FST was designated STF and the full-length sequence, including its
promoter, was amplified from genotype R108 using primers STF1F and
STF1R from the sequenced M. truncatula A17 genome. All primers are
listed in Supplemental Table 1 online.
Cloning ofMedicago sativa, Nicotiana benthamiana, and Nicotiana
sylvestris STL Sequences
Ms STL1, Ms STL2, and partial Nb STL1 sequences were isolated by RT-
PCR from shoot apex-enriched tissue of seedlings using primers
STFcd1F and STFcd1R from conserved STF regions. A partial Ns STL1
sequence was amplified by RT-PCR fromwild-typeN. sylvestris using Nb
STL1-derived primersNbSTLfd andNbSTLrs, and a full-length sequence,
including its promoter and 39 untranslated region (UTR), was isolated by
TAIL-PCR (Liu et al., 1995) using primers NsSTL391F, NsSTL392F, and
NsSTL591R, NsSTL592R in combination with arbitrary primers AD1 and
AD2. Full-length Nb STL1 was amplified using Ns STL1-derived primers
NsSTLfrd and NsSTLrvs. All other STLs were identified by BLAST search
from NCBI or the respective genome databases. After functional confir-
mation, Nb STL1 and Ns STL1 were renamed Nb STF1 and Ns STF1,
respectively. Deletion of Ns STF1 in the lam1mutant was identified using
Ns STF1-specific primers; F1 and R1 for the middle of the genomic
sequence, F1 andR2 for coding sequence (CDS) extending to the 39UTR,
and F2 and R3 for the promoter region and 59 UTR (see Supplemental
Table 1 online for primer sequences).
Transgene Construction and Plant Transformation
A 5.3-kb genomic fragment of the M. truncatula STF gene was amplified
using primersSTFgattB1 andSTFgattB2 andwas cloned in gateway vector
pMDC99 for plant transformation (see Supplemental Table 2 online). A
genomic DNA fragment of STF containing a 2663-bp region immediately
upstream of the translation start was cloned into pMDC162 vector for the
STF:GUS construct using primerspSTF1F and pSTF1R. TheDR5promoter
was cloned into pMDC162 vector for theDR5:GUS construct using primers
DR5gusattB1p and DR5gusattB2p. For overexpression constructs, the Mt
STF CDS and Nb STF1 CDS were cloned into pMDC32 using primers
STFcdattB1 and STFcdattB2 for STF and NbSTF1attB1 and NbSTF1attB2
for Nb STF1. For the STF:WUS construct, the AtWUS1CDSwas amplified
from PRS:WUS plasmid using WUSgattB1 and WUSgattB2 primers and
was cloned in front of the STF promoter in pMDC162 vector (see Supple-
mental Table 2 online). Constructs were introduced into Agrobacterium
tumefaciens by electroporation. A. tumefaciens strain AGL1 was used for
M. truncatula transformation as described (Cosson et al., 2006) and strain
GV2260 was used for N. sylvestris transformation.
Phylogenetic Analysis
Phylogenetic analysis was performed using full-length and homeodomain
region amino acid sequences (see Supplemental Data Set 1 online).
Sequences were aligned using Clustal W, and a neighbor-joining phylo-
genetic tree was constructed using MEGA 4 software. The most parsi-
monious trees with bootstrap values from 1000 trials were shown.
Tissue Fixation and Embedding
Leaf samples from 4-week-old seedlings of M. truncatula were cut into
small pieces and collected directly into 4% formaldehyde made in PHEM
buffer (60 mM Pipes, 25 mM HEPES, 2 mM MgCl, and 10 mM EGTA, pH
6.9). Samples were vacuum infiltrated for 30min and then left in the fixative
solution for an additional 2 h. After fixation, samples were washed three
times in PHEMbuffer and dehydrated by passing through a graded ethanol
Regulation of Lamina by STENOFOLIA 2137
series of 25%, 50%, 75%, 95%, and 100%ETOH, each step lasting for 1 h
at room temperature and repeating three times for the 100% ETOH step.
Ethanolwas then replacedwith a graded series of low-melting-temperature
Steedman’s wax in ethanol (WAX:ETOH). The Steedman’s wax was pre-
pared by melting 900 g of polyethylene glycol 400 distearate (Sigma-
Aldrich) and 100 g of 1-hexadecanol (Sigma-Aldrich) and stirring at 658C,
aliquoted in 50-mL volumes and stored at2208C. The wax was melted at
388C before use. Samples were treated with 1ETOH:1WAX once for 3 h,
1ETOH:3WAX once for 3 h, and 100% WAX three times for 3 h. Tissues
were then embedded in plastic molds and after hardening at room
temperature, blocks were stored at 48C prior to sectioning. Embedded
tissues were sectioned at 15 to 20 mm using a Leica RM 2235 rotary
microtome (Leica Microsystems). The wax was removed by treating
sections with absolute ethanol. After air drying, sections were stained
with Toluidine Blue (Sigma-Aldrich) for visibility and were viewed under an
Olympus BX-51 compound microscope (Hitschfel Instruments).
Tissue Preparation for Light Microscopy
Equivalent leaves from 4-week-old R108 and stfmutant plants were fixed
in 6:1 ethanol:glacial acetic acid overnight at room temperature and were
washed with 95% ethanol twice for 10 min. Samples were then cleared
with 85% lactic acid over 24 h until the tissue became transparent.
Cleared tissues were mounted with 30% glycerol on glass slide and were
viewed under anOlympus BX-51 compound microscope equipped with a
digital camera.
SEM
Leaf tissue from 4-week-old R108 and stf1-2 plants were vacuum
infiltrated in fixative solution (3% glutaraldehyde in 25 mM phosphate
buffer, pH 7.0) for 1 h. Samples were further fixed with 1.0% osmium
tetroxide overnight, dehydrated in graded ethanol series, critical point
dried, coated with Gold as previously described (Wang et al., 2008), and
viewed using a ZEISS DSM-960A scanning electron microscope (Carl
Zeiss MicroImaging).
RNA in Situ Hybridization
RNA in situ hybridization with digoxigenin-labeled STF-specific probe
was performed on shoot apices of M. truncatula plants grown for 12 d
after germination or on inflorescence apices as described (Ferrandiz
et al., 2000). RNA antisense and sense probeswere generatedwith the T7
and SP6 polymerases, respectively, using a 780-bp STF cDNA template,
which included the last 431 bp of CDS plus 355 bp of the 39 UTR.
Histochemical GUS Staining
GUS staining assay was performed as described (Zhao et al., 2001) and
images of GUS staining patterns of tissues were collected with a digital
cameramounted on anOlympusBX-51 compoundmicroscope (Hitschfel
Instruments) or an Olympus SZX-16 Stereoscope (Hitschfel Instruments).
Microarray Analysis
Microarray was performed on RNA extracted from 0.5- to 0.8-mm shoot
apices of 4-week-old seedlings. Three independent stf alleles (stf1-1,
stf1-2, and stf1-3) and their corresponding wild type in segregating F2
populations were used. The three lines were treated as three biological
replicates, and for each replicate, a pooled tissue collected from20 plants
was used to make RNA preparation. Total RNA was extracted using
RNeasy Plant Mini Kit (Qiagen). The microarray analysis was performed
using Medicago Affymetrix GeneChip. Probe labeling, hybridization, and
scanning were conducted according to the manufacturer’s instructions
(Affymetrix). For eachmicroarray sample, the .CEL file was exported from
GeneChip Operating System software (Affymetrix) and imported into
robust multi-chip average for normalization. The presence/absence call
for each probe set was obtained from dCHIP. Differentially expressed
genes between the stf mutant and the wild type were selected based on
“associative analysis” (Dozmorov and Centola, 2003) using Matlab
(MathWorks). In this method, the background noise presented between
replicates and technical noise during microarray experiments was mea-
sured by the residual presented among a group of genes whose resid-
uals are homoscedastic. Genes whose residuals between the compared
sample pairs that are significantly higher than the measured background
noise level were considered to be differentially expressed. A selec-
tion threshold of 2 for transcript ratios and a Bonferroni-corrected P value
threshold of 8.15954E-07 were used. The Bonferroni-corrected P value
threshold was derived from 0.05/N in these analyses, where N is the
number of probe sets (61,278) on the chip.
Quantitative RT-PCR
Total RNA from 0.5- to 0.8-mm shoot apices of 4-week-old plants was
extracted using RNeasy Plant Mini Kit (Qiagen) and the RNA was treated
with Turbo DNase (Ambion) to remove contaminating genomic DNA. Two
micrograms of total RNA was used for cDNA synthesis using the Omni-
script Kit (Qiagen). Primers were designed to anneal near the 39 end or at
the 39 UTR (see Supplemental Table 4 online). Three stf mutants (stf1-1,
stf1-2, and stf1-3) and their segregating wild type were used as biological
replicates for the analysis and three technical replicates were run for
each. The qRT-PCR analysis was performed as described in Pfaffl (2001)
using Power SYBRGreen PCRmastermix (Applied Biosystems) in an ABI
Prism 7900 HT sequence detection system (Applied Biosystems). Gene
expression was normalized using the expression of the EF1a and with
UBQ5 used as housekeeping genes. Relative gene expression for each
gene in the mutant plants was compared with that obtained for wild type,
which was arbitrarily set to 1.0.
Quantification of Auxin
Quantification of free IAAwasperformedusingGC–selective ionmonitoring–
MS essentially as described (Chen et al., 1988) using fresh tissue from
equivalent leaves of 4-week-old wild-type, mutant, and transgenic plants
with the following modifications. 0.5 g of fresh tissue was ground and
extractedwith 2mLof65% isopropanol in 200mMimidazolebuffer, pH7.0.
d7-IAA (100 pM) (CDN) was added as an internal standard and was
equilibrated in the extract for 1 h at 48C.The extractwas centrifuged and the
supernatant was diluted to 12.5 mL using water. The diluted extract was
then applied to a conditioned amino anion exchange column (BAKER-10
SPE 3 mL). After the diluted extract passed through the column, aspiration
was continued for 30 s and the column was washed sequentially with 2.0
mL of each of hexane, ethyl acetate, acetonitrile, and methanol. The IAA
was eluted from the amino column using 3.0 mL of 2% acetic acid in
methanol. The acidic methanol eluent was evaporated to near dryness and
the residue was redissolved in 10% aqueous methanol and applied to a
C18 SPE column. The column was washed with 10% aqueous methanol
containing 1% acetic acid, and IAA was eluted from the column with 2%
acetic acid in methanol. The acidic methanol was evaporated to dryness,
and the residue was redissolved in 50 mL of methanol. IAA was methylated
by the addition of 2mL of 2.0M trimethylsilyldiazomethane (Sigma-Aldrich),
the reaction was allowed to go for 30 min at room temperature and excess
trimethylsilyldiazomethane was quenched by the addition of 2 mL of 2.0 M
acetic acid in hexane. One microliter of methylated IAA solution was
injected inanAgilent 6890GCconnected to 5973MSdetectorwith electron
ionization source (Agilent Technologies). The injector was at 2808C and in
splitlessmode.Theoven temperaturewas initially at 708C for 2min and then
ramped to 3158C at 58C/min. The monitored ions were mass-to-charge
2138 The Plant Cell
ratio 130 and 137, which correspond to the quinolinium ions from IAA and
d7-IAA, respectively, as well as the mass-to-charge ratio 189 and 196 for
the corresponding molecular ions. Dwell times were 100 ms for each ion.
Abscisic acid was quantified by reanalyzing the same samples using
abscisic acid internal standard.
Metabolite Profiling
Leaves from equivalent positions of lam1 and wild-type plants were
collected from 4-week-old plants grown in growth cabinets. Metabolite
analysis by GC-MSwas performed as described (Broeckling et al., 2005).
The GC system used was an Agilent 6890 GC coupled to a 5973 mass
spectrometry detector. One microliter of samples was injected at a 15:1
split ratio and the injector was held at 2808C. Separation was achieved on
DB-5MS column (J & W Scientific; 60 m, 0.25 mm i.d., and 0.25-mm film).
Helium was used as carrier gas at constant flow of 1 mL/min. The
temperature program was 2 min at 808C followed by a 58C/min ramp to
3158C and this was held at 3158C for 12 min. Mass spectra were scanned
fromm/z 50 to 650with the acquisition rate of 2 spectra/s. Acquiredmass
spectra were deconvoluted using AMDIS software, and metabolite iden-
tifications were achieved by mass spectral matching to the Noble
Foundation’s in-house EIMS spectral library of authentic compounds,
the publicly available GOLM library (http://csbdb.mpimpgolm. mpg.de/
csbdb/dbma/msri.html), and the NIST08 library. Peak selection and
alignment were performed using MET-IDEA software (Broeckling et al.,
2006). The area of each peak was normalized against the area of the
internal standard, and absolute quantification for selected metabolites
was achieved using authentic standard calibration curves.
Application of Auxin and Cytokinin to Shoot Apex
For foliar spray of auxin, 10 mL of 1, 10, 50, or 100 mM NAA was sprayed
per plant to shoots of 2-week-old seedlings daily for 10 d. For local
treatments of apices with hormones, 10mM IAA, 10mMNAA, 10mM IAA
plus 10 mM BAP, or 1 mM IAA plus 10 mM BAP were dissolved in a
prewarmed (508C) lanolin paste. The paste was manually administered to
shoot apices of 2-week-old N. sylvestris and lam1 seedlings or 6-week-
old lam1 plants with pipette tips.
Root Elongation Assay
Tomeasure root elongation in the presence of auxin, auxin polar transport
inhibitor, or auxin biosynthetic intermediates, seeds were germinated on
0.5 MS plates and 9-d-old seedlings were transferred to 0.5 MS plates
containing different concentrations of IAA, NAA, 2,4-D, Trp, or NPA. Root
length was measured 4 or 5 d after transfer. Auxins and all other chem-
icals used in this assays were obtained from Sigma-Aldrich.
Accession Numbers
Sequence data from this article can be found in the EMBL/GenBank data
libraries under accession numbers STF, JF276252; Ns STF1, JF276252;
Ms STL1, JF276252 and Ms STL2, JF276252 (see also Supplemental
Table 3 online). Microarray data from this manuscript can be found in
ArrayExpress under accession number E-MEXP-3187.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Scanning Electron Micrograph of R108 and
stf Leaf Surfaces and Measurement of Leaf Size.
Supplemental Figure 2. Phylogenetic Analysis of Closely Related
WOX Proteins using Full-Length and Homeodomain Regions.
Supplemental Figure 3. Gene Structure and Multiple Amino Acid
Sequence Alignment of STF and STL Proteins.
Supplemental Figure 4. Multiple Amino Acid Sequence Alignment of
the Homeodomain Region of WUS-, PRS-, and STF-Related Proteins.
Supplemental Figure 5. Multiple Amino Acid Sequence Alignment of
the C-terminal Region of WUS-, PRS-, and STF-Related Proteins.
Supplemental Figure 6. STF:GUS Expression in M. truncatula.
Supplemental Figure 7. Treatment of lam1 Shoots and Roots with
Auxins, Trp, and NPA.
Supplemental Figure 8. Extreme Phenotypes of Nb STF1 Over-
expression in N. sylvestris and Measurements of Abscisic Acid in lam1
and Transgenic Lines.
Supplemental Figure 9. Putative Model for the Mechanism of STF
Function.
Supplemental Table 1. Primers Used for Recovering FST, Genotyp-
ing FST, and Cloning of STLs.
Supplemental Table 2. Primers Used for Plasmid Construction.
Supplemental Table 3. Primers Used for qRT-PCR.
Supplemental Table 4. Accession Numbers or Gene Identifiers of
Sequences Used for Multiple Sequence Alignment and Phylogenetic
Tree Construction.
Supplemental Data Set 1. Text File of Alignment Corresponding to
Supplemental Figure 2 Online.
ACKNOWLEDGMENTS
We thank David Meinke for critical reading of the manuscript; Stacy
Allen, Keri Wang, Preston Larson, Tamding Wangdi, Vagner Benedito,
and Elison Blancaflor for technical assistance; Jiri Friml for providing the
DR5:GUS plasmid; Michael Scanlon for PRS:WUS plasmid; and Hee-
Kyung Lee and Janie Gallaway for taking care of tissue culture and
greenhouse plants. This material is based on work supported by the
National Science Foundation under Grant EPS-0814361 and DBI
0703285, and in part by the Samuel Roberts Noble Foundation. Work
by A.B. and F.M. was supported by the Spanish Ministerio de Ciencia e
Innovacion (Grant BIO2009-10876) and the Generalitat Valenciana.
AUTHOR CONTRIBUTIONS
M.T. designed the research, performed research, analyzed data, andwrote
the paper;H.L.,M.B., Y.T, and F.M. performed researchand analyzeddata;
A.B., J.W., C.M.R., and L.N. performed research; P.R. and L.S. contributed
analytical tools; N.A.M. analyzed data and edited the paper; and K.S.M.
designed the research, analyzed data, and edited the paper.
ReceivedMarch 17, 2011; revised June 6, 2011; accepted June 14, 2011;
published June 30, 2011.
REFERENCES
Aida, M., Ishida, T., Fukaki, H., Fujisawa, H., and Tasaka, M. (1997).
Genes involved in organ separation in Arabidopsis: An analysis of the
cup-shaped cotyledon mutant. Plant Cell 9: 841–857.
Ariel, F., Diet, A., Verdenaud, M., Gruber, V., Frugier, F., Chan,
R., and Crespi, M. (2010). Environmental regulation of lateral root
Regulation of Lamina by STENOFOLIA 2139
emergence in Medicago truncatula requires the HD-Zip I transcription
factor HB1. Plant Cell 22: 2171–2183.
Barkoulas, M., Hay, A., Kougioumoutzi, E., and Tsiantis, M. (2008). A
developmental framework for dissected leaf formation in the Arabi-
dopsis relative Cardamine hirsuta. Nat. Genet. 40: 1136–1141.
Benkova, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertova,
D., Jurgens, G., and Friml, J. (2003). Local, efflux-dependent auxin
gradients as a common module for plant organ formation. Cell 115:
591–602.
Bilsborough, G.D., Runions, A., Barkoulas, M., Jenkins, H.W.,
Hasson, A., Galinha, C., Laufs, P., Hay, A., Prusinkiewicz, P.,
and Tsiantis, M. (2011). Model for the regulation of Arabidopsis
thaliana leaf margin development. Proc. Natl. Acad. Sci. USA 108:
3424–3429.
Bolduc, N., and Hake, S. (2009). The maize transcription factor
KNOTTED1 directly regulates the gibberellin catabolism gene
ga2ox1. Plant Cell 21: 1647–1658.
Braybrook, S.A., and Kuhlemeier, C. (2010). How a plant builds leaves.
Plant Cell 22: 1006–1018.
Broeckling, C.D., Huhman, D.V., Farag, M.A., Smith, J.T., May, G.D.,
Mendes, P., Dixon, R.A., and Sumner, L.W. (2005). Metabolic
profiling of Medicago truncatula cell cultures reveals the effects of
biotic and abiotic elicitors on metabolism. J. Exp. Bot. 56: 323–336.
Broeckling, C.D., Reddy, I.R., Duran, A.L., Zhao, X., and Sumner,
L.W. (2006). MET-IDEA: Data extraction tool for mass spectrometry-
based metabolomics. Anal. Chem. 78: 4334–4341.
Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M.,
Hudson, A., and Martienssen, R.A. (2000). Asymmetric leaves1
mediates leaf patterning and stem cell function in Arabidopsis. Nature
408: 967–971.
Cano-Delgado, A., Lee, J.Y., and Demura, T. (2010). Regulatory
mechanisms for specification and patterning of plant vascular tissues.
Annu. Rev. Cell Dev. Biol. 26: 605–637.
Chen, K.H., Miller, A.N., Patterson, G.W., and Cohen, J.D. (1988). A
rapid and simple procedure for purification of indole-3-acetic acid
prior to GC-SIM-MS analysis. Plant Physiol. 86: 822–825.
Cosson, V., Durand, P., d’Erfurth, I., Kondorosi, A., and Ratet, P.
(2006). Medicago truncatula transformation using leaf explants.
Methods Mol. Biol. 343: 115–127.
David-Schwartz, R., Koenig, D., and Sinha, N.R. (2009). LYRATE is a
key regulator of leaflet initiation and lamina outgrowth in tomato. Plant
Cell 21: 3093–3104.
Dello Ioio, R., Nakamura, K., Moubayidin, L., Perilli, S., Taniguchi,
M., Morita, M.T., Aoyama, T., Costantino, P., and Sabatini, S.
(2008). A genetic framework for the control of cell division and
differentiation in the root meristem. Science 322: 1380–1384.
Dhondt, S., Coppens, F., De Winter, F., Swarup, K., Merks, R.M.H.,
Inze, D., Bennett, M.J., and Beemster, G.T.S. (2010). SHORT-ROOT
and SCARECROW regulate leaf growth in Arabidopsis by stimulating
S-phase progression of the cell cycle. Plant Physiol. 154: 1183–1195.
Dinneny, J.R., Yadegari, R., Fischer, R.L., Yanofsky, M.F., and
Weigel, D. (2004). The role of JAGGED in shaping lateral organs.
Development 131: 1101–1110.
Dozmorov, I., and Centola, M. (2003). An associative analysis of gene
expression array data. Bioinformatics 19: 204–211.
Efroni, I., Eshed, Y., and Lifschitz, E. (2010). Morphogenesis of simple
and compound leaves: A critical review. Plant Cell 22: 1019–1032.
Eklof, S., Astot, C., Sitbon, F., Moritz, T., Olsson, O., and Sandberg, G.
(2000). Transgenic tobacco plants co-expressing Agrobacterium IAA
and ipt genes have wild-type hormone levels but display both auxin-
and cytokinin-overproducing phenotypes. Plant J. 23: 279–284.
Emery, J.F., Floyd, S.K., Alvarez, J., Eshed, Y., Hawker, N.P., Izhaki,
A., Baum, S.F., and Bowman, J.L. (2003). Radial patterning of
Arabidopsis shoots by class III HD-ZIP and KANADI genes. Curr. Biol.
13: 1768–1774.
Ferrandiz, C., Gu, Q., Martienssen, R., and Yanofsky, M.F. (2000).
Redundant regulation of meristem identity and plant architecture by
FRUITFULL, APETALA1 and CAULIFLOWER. Development 127:
725–734.
Guo, M., Thomas, J., Collins, G., and Timmermans, M.C.P. (2008).
Direct repression of KNOX loci by the ASYMMETRIC LEAVES1
complex of Arabidopsis. Plant Cell 20: 48–58.
Ha, C.M., Jun, J.H., Nam, H.G., and Fletcher, J.C. (2007). BLADE-ON-
PETIOLE 1 and 2 control Arabidopsis lateral organ fate through
regulation of LOB domain and adaxial-abaxial polarity genes. Plant
Cell 19: 1809–1825.
Haecker, A., Gross-Hardt, R., Geiges, B., Sarkar, A., Breuninger, H.,
Herrmann, M., and Laux, T. (2004). Expression dynamics of WOX
genes mark cell fate decisions during early embryonic patterning in
Arabidopsis thaliana. Development 131: 657–668.
Hagemann, W., and Gleissberg, S. (1996). Organogenetic capacity of
leaves: The significance of marginal blastozones in angiosperms.
Plant Syst. Evol. 199: 121–152.
Hasson, A., Plessis, A., Blein, T., Adroher, B., Grigg, S., Tsiantis, M.,
Boudaoud, A., Damerval, C., and Laufs, P. (2011). Evolution and
diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis
leaf development. Plant Cell 23: 54–68.
Hay, A., and Tsiantis, M. (2010). KNOX genes: Versatile regulators of
plant development and diversity. Development 137: 3153–3165.
Hofer, J., Turner, L., Moreau, C., Ambrose, M., Isaac, P., Butcher, S.,
Weller, J., Dupin, A., Dalmais, M., Le Signor, C., Bendahmane, A.,
and Ellis, N. (2009). Tendril-less regulates tendril formation in pea
leaves. Plant Cell 21: 420–428.
Horiguchi, G., Kim, G.T., and Tsukaya, H. (2005). The transcription
factor AtGRF5 and the transcription coactivator AN3 regulate cell
proliferation in leaf primordia of Arabidopsis thaliana. Plant J. 43: 68–78.
Hu, Y., Xie, Q., and Chua, N.H. (2003). The Arabidopsis auxin-inducible
gene ARGOS controls lateral organ size. Plant Cell 15: 1951–1961.
Husbands, A.Y., Chitwood, D.H., Plavskin, Y., and Timmermans,
M.C. (2009). Signals and prepatterns: New insights into organ polarity
in plants. Genes Dev. 23: 1986–1997.
Ikeda, M., Mitsuda, N., and Ohme-Takagi, M. (2009). Arabidopsis
WUSCHEL is a bifunctional transcription factor that acts as a repres-
sor in stem cell regulation and as an activator in floral patterning. Plant
Cell 21: 3493–3505.
Jackson, R.G., Kowalczyk, M., Li, Y., Higgins, G., Ross, J., Sandberg,
G., and Bowles, D.J. (2002). Over-expression of an Arabidopsis gene
encoding a glucosyltransferase of indole-3-acetic acid: Phenotypic
characterisation of transgenic lines. Plant J. 32: 573–583.
Jaillais, Y., and Chory, J. (2010). Unraveling the paradoxes of plant
hormone signaling integration. Nat. Struct. Mol. Biol. 17: 642–645.
Jasinski, S., Piazza, P., Craft, J., Hay, A., Woolley, L., Rieu, I.,
Phillips, A., Hedden, P., and Tsiantis, M. (2005). KNOX action in
Arabidopsis is mediated by coordinate regulation of cytokinin and
gibberellin activities. Curr. Biol. 15: 1560–1565.
Jun, J.H., Ha, C.M., and Fletcher, J.C. (2010). BLADE-ON-PETIOLE1
coordinates organ determinacy and axial polarity in Arabidopsis by
directly activating ASYMMETRIC LEAVES2. Plant Cell 22: 62–76.
Kant, S., Bi, Y.M., Zhu, T., and Rothstein, S.J. (2009). SAUR39, a small
auxin-up RNA gene, acts as a negative regulator of auxin synthesis
and transport in rice. Plant Physiol. 151: 691–701.
Kerstetter, R.A., Bollman, K., Taylor, R.A., Bomblies, K., and
Poethig, R.S. (2001). KANADI regulates organ polarity in Arabidopsis.
Nature 411: 706–709.
Kim, G.T., Shoda, K., Tsuge, T., Cho, K.H., Uchimiya, H., Yokoyama,
R., Nishitani, K., and Tsukaya, H. (2002). The ANGUSTIFOLIA gene
2140 The Plant Cell
of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the
arrangement of cortical microtubules in leaf cells and expression of a
gene involved in cell-wall formation. EMBO J. 21: 1267–1279.
Kim, G.T., Tsukaya, H., and Uchimiya, H. (1998). The ROTUNDIFOLIA3
gene of Arabidopsis thaliana encodes a new member of the cytochrome
P-450 family that is required for the regulated polar elongation of leaf
cells. Genes Dev. 12: 2381–2391.
Koenig, D., Bayer, E., Kang, J., Kuhlemeier, C., and Sinha, N. (2009).
Auxin patterns Solanum lycopersicum leaf morphogenesis. Develop-
ment 136: 2997–3006.
Koyama, T., Furutani, M., Tasaka, M., and Ohme-Takagi, M. (2007).
TCP transcription factors control the morphology of shoot lateral
organs via negative regulation of the expression of boundary-specific
genes in Arabidopsis. Plant Cell 19: 473–484.
Koyama, T., Mitsuda, N., Seki, M., Shinozaki, K., and Ohme-Takagi,
M. (2010). TCP transcription factors regulate the activities of ASYM-
METRIC LEAVES1 and miR164, as well as the auxin response, during
differentiation of leaves in Arabidopsis. Plant Cell 22: 3574–3588.
Laux, T., Mayer, K.F., Berger, J., and Jurgens, G. (1996). The
WUSCHEL gene is required for shoot and floral meristem integrity in
Arabidopsis. Development 122: 87–96.
LeClere, S., Schmelz, E.A., and Chourey, P.S. (2010). Sugar levels
regulate tryptophan-dependent auxin biosynthesis in developing
maize kernels. Plant Physiol. 153: 306–318.
Leibfried, A., To, J.P., Busch, W., Stehling, S., Kehle, A., Demar, M.,
Kieber, J.J., and Lohmann, J.U. (2005). WUSCHEL controls meri-
stem function by direct regulation of cytokinin-inducible response
regulators. Nature 438: 1172–1175.
Lin, W.C., Shuai, B., and Springer, P.S. (2003). The Arabidopsis
LATERAL ORGAN BOUNDARIES-domain gene ASYMMETRIC
LEAVES2 functions in the repression of KNOX gene expression and
in adaxial-abaxial patterning. Plant Cell 15: 2241–2252.
Liu, Y.G., Mitsukawa, N., Oosumi, T., and Whittier, R.F. (1995).
Efficient isolation and mapping of Arabidopsis thaliana T-DNA in-
sert junctions by thermal asymmetric interlaced PCR. Plant J. 8:
457–463.
Long, J.A., Moan, E.I., Medford, J.I., and Barton, M.K. (1996). A
member of the KNOTTED class of homeodomain proteins encoded by
the STM gene of Arabidopsis. Nature 379: 66–69.
Martın-Trillo, M., and Cubas, P. (2010). TCP genes: A family snapshot
ten years later. Trends Plant Sci. 15: 31–39.
Matsumoto, N., and Okada, K. (2001). A homeobox gene, PRESSED
FLOWER, regulates lateral axis-dependent development of Arabidop-
sis flowers. Genes Dev. 15: 3355–3364.
Mayer, K.F., Schoof, H., Haecker, A., Lenhard, M., Jurgens, G., and
Laux, T. (1998). Role of WUSCHEL in regulating stem cell fate in the
Arabidopsis shoot meristem. Cell 95: 805–815.
McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and
Barton, M.K. (2001). Role of PHABULOSA and PHAVOLUTA in
determining radial patterning in shoots. Nature 411: 709–713.
McHale, N.A. (1992). A nuclear mutation blocking initiation of the lamina
in leaves of Nicotiana sylvestris. Planta 186: 355–360.
McHale, N.A. (1993). LAM-1 and FAT genes control development of the
leaf blade in Nicotiana sylvestris. Plant Cell 5: 1029–1038.
McHale, N.A., and Marcotrigiano, M. (1998). LAM1 is required for
dorsoventrality and lateral growth of the leaf blade in Nicotiana.
Development 125: 4235–4243.
Mizukami, Y., and Fischer, R.L. (2000). Plant organ size control:
AINTEGUMENTA regulates growth and cell numbers during organo-
genesis. Proc. Natl. Acad. Sci. USA 97: 942–947.
Moubayidin, L., Di Mambro, R., and Sabatini, S. (2009). Cytokinin-
auxin crosstalk. Trends Plant Sci. 14: 557–562.
Muller, B., and Sheen, J. (2008). Cytokinin and auxin interaction in root
stem-cell specification during early embryogenesis. Nature 453:
1094–1097.
Nardmann, J., Ji, J., Werr, W., and Scanlon, M.J. (2004). The maize
duplicate genes narrow sheath1 and narrow sheath2 encode a
conserved homeobox gene function in a lateral domain of shoot
apical meristems. Development 131: 2827–2839.
Nath, U., Crawford, B.C., Carpenter, R., and Coen, E. (2003). Genetic
control of surface curvature. Science 299: 1404–1407.
Nikovics, K., Blein, T., Peaucelle, A., Ishida, T., Morin, H., Aida, M.,
and Laufs, P. (2006). The balance between the MIR164A and CUC2
genes controls leaf margin serration in Arabidopsis. Plant Cell 18:
2929–2945.
Ori, N., et al. (2007). Regulation of LANCEOLATE by miR319 is required
for compound-leaf development in tomato. Nat. Genet. 39: 787–791.
Pagnussat, G.C., Alandete-Saez, M., Bowman, J.L., and Sundaresan,
V. (2009). Auxin-dependent patterning and gamete specification in the
Arabidopsis female gametophyte. Science 324: 1684–1689.
Palatnik, J.F., Allen, E., Wu, X., Schommer, C., Schwab, R.,
Carrington, J.C., and Weigel, D. (2003). Control of leaf morphogen-
esis by microRNAs. Nature 425: 257–263.
Park, J.-E., Park, J.-Y., Kim, Y.-S., Staswick, P.E., Jeon, J., Yun, J.,
Kim, S.-Y., Kim, J., Lee, Y.-H., and Park, C.-M. (2007). GH3-mediated
auxin homeostasis links growth regulation with stress adaptation re-
sponse in Arabidopsis. J. Biol. Chem. 282: 10036–10046.
Pernisova, M., Klıma, P., Horak, J., Valkova, M., Malbeck, J.,
Soucek, P., Reichman, P., Hoyerova, K., Dubova, J., Friml, J.,
Zazımalova, E., and Hejatko, J. (2009). Cytokinins modulate auxin-
induced organogenesis in plants via regulation of the auxin efflux.
Proc. Natl. Acad. Sci. USA 106: 3609–3614.
Perrot-Rechenmann, C. (2010). Cellular responses to auxin: Division
versus expansion. Cold Spring Harb. Perspect. Biol. 2: a001446.
Pfaffl, M.W. (2001). A new mathematical model for relative quantifica-
tion in real-time RT-PCR. Nucleic Acids Res. 29: e45.
Poethig, R.S. (1997). Leaf morphogenesis in flowering plants. Plant Cell
9: 1077–1087.
Qin, G., Gu, H., Zhao, Y., Ma, Z., Shi, G., Yang, Y., Pichersky, E.,
Chen, H., Liu, M., Chen, Z., and Qu, L.J. (2005). An indole-3-acetic
acid carboxyl methyltransferase regulates Arabidopsis leaf develop-
ment. Plant Cell 17: 2693–2704.
Reinhardt, D., Pesce, E.R., Stieger, P., Mandel, T., Baltensperger, K.,
Bennett, M., Traas, J., Friml, J., and Kuhlemeier, C. (2003). Regu-
lation of phyllotaxis by polar auxin transport. Nature 426: 255–260.
Saddic, L.A., Huvermann, B., Bezhani, S., Su, Y., Winter, C.M., Kwon,
C.S., Collum, R.P., and Wagner, D. (2006). The LEAFY target LMI1 is a
meristem identity regulator and acts together with LEAFY to regulate
expression of CAULIFLOWER. Development 133: 1673–1682.
Scanlon, M.J., Schneeberger, R.G., and Freeling, M. (1996). The
maize mutant narrow sheath fails to establish leaf margin identity in a
meristematic domain. Development 122: 1683–1691.
Scarpella, E., Barkoulas, M., and Tsiantis, M. (2010). Control of leaf and
vein development by auxin. Cold Spring Harb. Perspect. Biol. 2: a001511.
Scarpella, E., Marcos, D., Friml, J., and Berleth, T. (2006). Control of
leaf vascular patterning by polar auxin transport. Genes Dev. 20:
1015–1027.
Shimizu, R., Ji, J., Kelsey, E., Ohtsu, K., Schnable, P.S., and
Scanlon, M.J. (2009). Tissue specificity and evolution of meristematic
WOX3 function. Plant Physiol. 149: 841–850.
Shleizer-Burko, S., Burko, Y., Ben-Herzel, O., and Ori, N. (2011).
Dynamic growth program regulated by LANCEOLATE enables flexible
leaf patterning. Development 138: 695–704.
Skylar, A., Hong, F., Chory, J., Weigel, D., and Wu, X. (2010). STIMPY
mediates cytokinin signaling during shoot meristem establishment in
Arabidopsis seedlings. Development 137: 541–549.
Regulation of Lamina by STENOFOLIA 2141
Skylar, A., Sung, F., Hong, F., Chory, J., and Wu, X. (2011). Metabolic
sugar signal promotes Arabidopsis meristematic proliferation via G2.
Dev. Biol. 351: 82–89.
Sozzani, R., Cui, H., Moreno-Risueno, M.A., Busch, W., Van Norman,
J.M., Vernoux, T., Brady, S.M., Dewitte, W., Murray, J.A., and
Benfey, P.N. (2010). Spatiotemporal regulation of cell-cycle genes by
SHORTROOT links patterning and growth. Nature 466: 128–132.
Sussex, I.M. (1955). Morphogenesis in Solanum tuberosum L.: Exper-
imental investigation of leaf dorsoventrality and orientation in the
juvenile shoot. Phytomorphology 5: 286–300.
Tadege, M., Wang, T.L., Wen, J., Ratet, P., and Mysore, K.S. (2009).
Mutagenesis and beyond! Tools for understanding legume biology.
Plant Physiol. 151: 978–984.
Tadege, M., Wen, J., He, J., Tu, H., Kwak, Y., Eschstruth, A., Cayrel,
A., Endre, G., Zhao, P.X., Chabaud, M., Ratet, P., and Mysore, K.S.
(2008). Large-scale insertional mutagenesis using the Tnt1 retro-
transposon in the model legume Medicago truncatula. Plant J. 54:
335–347.
Tattersall, A.D., Turner, L., Knox, M.R., Ambrose, M.J., Ellis, T.H.,
and Hofer, J.M. (2005). The mutant crispa reveals multiple roles for
PHANTASTICA in pea compound leaf development. Plant Cell 17:
1046–1060.
Timmermans, M.C., Hudson, A., Becraft, P.W., and Nelson, T. (1999).
ROUGH SHEATH2: A Myb protein that represses knox homeobox
genes in maize lateral organ primordia. Science 284: 151–153.
Tognetti, V.B., et al. (2010). Perturbation of indole-3-butyric acid
homeostasis by the UDP-glucosyltransferase UGT74E2 modulates
Arabidopsis architecture and water stress tolerance. Plant Cell 22:
2660–2679.
Tsukaya, H. (2006). Mechanism of leaf-shape determination. Annu. Rev.
Plant Biol. 57: 477–496.
Uchida, N., Townsley, B., Chung, K.H., and Sinha, N. (2007). Regu-
lation of SHOOT MERISTEMLESS genes via an upstream-conserved
noncoding sequence coordinates leaf development. Proc. Natl. Acad.
Sci. USA 104: 15953–15958.
Vandenbussche, M., Horstman, A., Zethof, J., Koes, R., Rijpkema,
A.S., and Gerats, T. (2009). Differential recruitment of WOX tran-
scription factors for lateral development and organ fusion in Petunia
and Arabidopsis. Plant Cell 21: 2269–2283.
Vanneste, S., and Friml, J. (2009). Auxin: A trigger for change in plant
development. Cell 136: 1005–1016.
Waites, R., and Hudson, A. (1995). Phantastica: A gene required for
dorsoventrality of leaves in Antirrhinum majus. Development 121:
2143–2154.
Waites, R., Selvadurai, H.R.N., Oliver, I.R., and Hudson, A. (1998).
The PHANTASTICA gene encodes a MYB transcription factor in-
volved in growth and dorsoventrality of lateral organs in Antirrhinum.
Cell 93: 779–789.
Wang, H., Chen, J., Wen, J., Tadege, M., Li, G., Liu, Y., Mysore, K.S.,
Ratet, P., and Chen, R. (2008). Control of compound leaf develop-
ment by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in Medi-
cago truncatula. Plant Physiol. 146: 1759–1772.
White, D.W.R. (2006). PEAPOD regulates lamina size and curvature in
Arabidopsis. Proc. Natl. Acad. Sci. USA 103: 13238–13243.
Xu, L., Xu, Y., Dong, A., Sun, Y., Pi, L., Xu, Y., and Huang, H. (2003).
Novel as1 and as2 defects in leaf adaxial-abaxial polarity reveal the
requirement for ASYMMETRIC LEAVES1 and 2 and ERECTA func-
tions in specifying leaf adaxial identity. Development 130: 4097–4107.
Yamada, M., Greenham, K., Prigge, M.J., Jensen, P.J., and Estelle,
M. (2009). The TRANSPORT INHIBITOR RESPONSE2 gene is re-
quired for auxin synthesis and diverse aspects of plant development.
Plant Physiol. 151: 168–179.
Zambryski, P., Tempe, J., and Schell, J. (1989). Transfer and function
of T-DNA genes from agrobacterium Ti and Ri plasmids in plants. Cell
56: 193–201.
Zhang, X., Zong, J., Liu, J., Yin, J., and Zhang, D. (2010). Genome-
wide analysis of WOX gene family in rice, sorghum, maize, Arabidop-
sis and poplar. J. Integr. Plant Biol. 52: 1016–1026.
Zhao, Y., Christensen, S.K., Fankhauser, C., Cashman, J.R., Cohen,
J.D., Weigel, D., and Chory, J. (2001). A role for flavin monooxy-
genase-like enzymes in auxin biosynthesis. Science 291: 306–309.
Zhao, Z., Andersen, S.U., Ljung, K., Dolezal, K., Miotk, A., Schultheiss,
S.J., and Lohmann, J.U. (2010). Hormonal control of the shoot stem-
cell niche. Nature 465: 1089–1092.
2142 The Plant Cell
DOI 10.1105/tpc.111.085340; originally published online June 30, 2011; 2011;23;2125-2142Plant Cell
MysoreYuhong Tang, Lloyd Sumner, Pascal Ratet, Neil A. McHale, Francisco Madueño and Kirankumar S.
Million Tadege, Hao Lin, Mohamed Bedair, Ana Berbel, Jiangqi Wen, Clemencia M. Rojas, Lifang Niu,Nicotiana sylvestrisand
Medicago truncatula Regulates Blade Outgrowth and Leaf Vascular Patterning in STENOFOLIA
This information is current as of September 15, 2020
Supplemental Data /content/suppl/2011/06/15/tpc.111.085340.DC1.html
References /content/23/6/2125.full.html#ref-list-1
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