stenofolia regulates blade outgrowth and leaf vascular … · stenofolia regulates blade outgrowth...

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 [email protected] authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Million Tadege([email protected]) and Kirankumar S. Mysore ([email protected]).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.]


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.


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.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.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.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.


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.



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.


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


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.

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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

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