A Dof Transcription Factor, SCAP1, Is Essential for the ... · Figure 2. SCAP1 Encodes a Dof-Type Transcription Factor Whose Expression Starts at a Late Stage of Guard Cell Differentiation

Please cite this article in press as: Negi et al., A Dof Transcription Factor, SCAP1, Is Essential for the Development of FunctionalStomata in Arabidopsis, Current Biology (2013), http://dx.doi.org/10.1016/j.cub.2013.02.001

A Dof Transcription Factor, S

Current Biology 23, 1–6, March 18, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2013.02.001

ReportCAP1,

Is Essential for the Developmentof Functional Stomata in Arabidopsis

Juntaro Negi,1 Kosuke Moriwaki,1 Mineko Konishi,2

Ryusuke Yokoyama,3 Toshiaki Nakano,1 Kensuke Kusumi,1

Mimi Hashimoto-Sugimoto,1 Julian I. Schroeder,4

Kazuhiko Nishitani,3 Shuichi Yanagisawa,2 and Koh Iba1,*1Department of Biology, Faculty of Sciences, KyushuUniversity, Fukuoka 812-8581, Japan2Biotechnology Research Center, The University of Tokyo,Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan3Department of Developmental Biology and Neurosciences,Graduate School of Life Sciences, Tohoku University, Sendai980-8578, Japan4Cell and Developmental Biology Section, Division ofBiological Sciences, University of California, San Diego,La Jolla, CA 92093, USA

Summary

Stomata are highly specialized organs that consist of pairs

of guard cells and regulate gas and water vapor exchangein plants [1–3]. Although early stages of guard cell differen-

tiation have been described [4–10] and were interpreted inanalogy to processes of cell type differentiation in animals

[11], the downstream development of functional stomatalguard cells remains poorly understood. We have isolated

an Arabidopsis mutant, stomatal carpenter 1 (scap1), thatdevelops irregularly shaped guard cells and lacks the ability

to control stomatal aperture, including CO2-induced

stomatal closing and light-induced stomatal opening.SCAP1 was identified as a plant-specific Dof-type transcrip-

tion factor expressed in maturing guard cells, but not inguard mother cells. SCAP1 regulates the expression of

genes encoding key elements of stomatal functioning andmorphogenesis, such as K+ channel protein, MYB60 tran-

scription factor, and pectin methylesterase. Consequently,ion homeostasis was disturbed in scap1 guard cells, and

esterification of extracellular pectins was impaired so thatthe cell walls lining the pores did not mature normally. We

conclude that SCAP1 regulates essential processes ofstomatal guard cell maturation and functions as a key tran-

scription factor regulating the final stages of guard celldifferentiation.

Results and Discussion

We isolated stomatal carpenter 1 (scap1) as a mutant impairedin CO2-dependent leaf temperature change from an M2 popu-lation of ethyl methanesulfonate-mutagenized Arabidopsisplants, using thermography [12]. In this mutant, the typicalchanges of stomatal conductance that occur in wild-type(WT) plants in response to CO2 (Figure 1A) and light (Figure 1B)were inhibited. Themutant was defective also in the regulationof transpiration in response to drought stress (Figure 1C). Asubset of stomata in this mutant appeared morphologicallyabnormal (Figure 1D), indicating a disruption in pore

*Correspondence: [email protected]

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morphogenesis. In particular, the ventral cell walls, whichform the inner surface of the pore, appeared floppy andseemed to remain adhered in mature stomata. This phenom-enon was observed in approximately 50% of the stomataexamined. Although the remaining 50% of total stomataappeared normal morphologically, all stomata of the scap1mutant probably lack the ability to control stomatal aperture,because the scap1 mutant was completely insensitive tochanges in CO2 concentration and light intensity (Figures 1Aand 1B). To clarify the timing of morphological defects occur-ring during stomatal development, we investigated themorphology of stomatal lineage cells from meristemoids tomature guard cells. The morphological defects occurred afterguard mother cells were divided to form young stomata (Fig-ure 1E), suggesting that SCAP1 is a late-acting gene in guardcell differentiation.By map-based cloning, we identified the SCAP1 gene as

At5g65590, which encodes an uncharacterized DNA bindingwith one finger (Dof) transcription factor (Figure 2A; see alsoFigure S1A available online). The scap1 mutation possessesa single C-to-T nucleotide substitution, causing an R65-to-Cexchange in the Dof domain (Figure 2A) that is required forDNA binding [13]. Thus, scap1 probably is a loss-of-functionallele. Introduction of the SCAP1 open reading frame with itsnative promoter into scap1 plants fully restored theWT pheno-type, confirming that At5g65590 is SCAP1 (Figure S1B). Wealso confirmed that SCAP1 RNAi plants exhibited similarphenotypes to the scap1 mutant (Figures S1C–S1F). Toexamine promoter activity and the localization of the geneproduct, we used the native SCAP1 promoter to drive expres-sion of the GUS reporter and the translational fusion of a full-length SCAP1 protein and GFP (SCAP1-GFP). The latterconstruct complemented the scap1 phenotype, indicatingthat the SCAP1-GFP fusion protein was functional (Fig-ure S1B). GUS expression driven by the SCAP1 promoterwas highest in guard cells (Figure 2B). The SCAP1-GFP fusionprotein was localized in the nuclei of guard cells (Figure 2C).Arabidopsis guard cells develop via three stages of asym-metric and symmetric cell divisions [9, 10]. Passage fromone stage to the next is promoted by SPCH (asymmetric entrydivision) [5], MUTE (meristemoid to guard mother cell) [6], andFAMA/FLP (guard mother cell to guard cells) [7, 8]. No GFPsignal was detected in meristemoids, guard mother cells, orrecently divided guard cells (Figure 2D; Figure S2), indicatingthat SCAP1 is not involved in the early stages of guard celldifferentiation. The timing of SCAP1 expression paralleledthat of SLAC1, an S-type anion channel that plays an essentialrole in the regulation of stomatal closure [12, 14]. These find-ings suggest that SCAP1 acts as a transcription factor thatcontrols guard cell maturation and the achievement of fullfunctionality.Dof factors are plant-specific transcription factorswith func-

tions in a variety of physiological contexts [13], and guard cell-specific expression of a K+ channel protein genewasmediatedby Dof-binding consensus sequences in its promoter region[15]. Consequently, an unidentified Dof factor, or factors,was proposed to be involved in guard cell-specific geneexpression [16–19]. We therefore investigated the role of

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mailto:[email protected]

A B C

D

E

Figure 1. A Mutation in SCAP1 Impairs Stomatal Movement and Morphogenesis

(A) Responses of stomatal conductance in scap1 and wild-type (WT) plants to changes in CO2 concentration.

(B) Time courses of stomatal responses to changing light intensity, monitored with an Arabidopsis whole-rosette gas-exchange system. Values shown are

means 6 SE (n = 4).

(C) Weight loss from detached leaves of WT and scap1, as a measure of drought stress tolerance. Values shown are means 6 SE (n = 4).

(D) Light micrographs of WT and scap1 stomata of mature leaves. In scap1, the ventral cell walls appear floppy and are often irregularly curved. Scale bars

represent 10 mm.

(E) Light micrographs of stomatal lineage cells at several stages of the stomatal development in WT and scap1. Morphological defects characteristic of the

scap1 mutant were seen only after young stomata formed. Scale bars represent 10 mm.

Current Biology Vol 23 No 62


SCAP1 in guard cell-specific gene expression by microarrayexperiments. We selected 1,540 genes that are expressed inguard cells, but not in mesophyll cells [16], and compared theirexpression levels in scap1 and WT guard cells (Figure 3A;Table S1). The scap1 mutation resulted in decreased expres-sion of a number of genes, including genes for several factorsdirectly involved in stomatal opening and closure: GORK, anoutward K+ channel protein [20]; PYL2, a regulatory compo-nent of ABA receptor 2 [21, 22]; and MYB60, an essential

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transcriptional regulator for guard cell movements [23]. Thus,SCAP1 is not amere transcription factor for guard cell-specificexpression of a single gene but probably a key factor for guardcell function.The results of a dual-luciferase transient reporter assay

revealed that, in guard cell protoplasts (GCPs), SCAP1 acti-vates the GORK and MYB60 promoters (Figure 3B), whichhave several potential Dof-binding sites (T/A-AAAG) (Fig-ure 3C). Furthermore, in a chromatin immunoprecipitation

B

DStomatal fuctioning

scap1mutationCys

At5g65590(AtDof5.7)

A

Coding region

UTR

C SCAP1-GFPHoechst

33342

SLAC1-GFP

SCAP1-GFP

Mature stomataYoung stomataGMCMeristemoid

Figure 2. SCAP1 Encodes a Dof-Type Transcription Factor Whose Expression Starts at a Late Stage of Guard Cell Differentiation

(A) SCAP1 gene structure and the protein structure of the Dof domain. Cysteine residues conserved in Dof domain proteins are shown in red. The R65-to-C

substitution caused by the scap1 mutation is indicated in blue.

(B) GUS staining of pSCAP1::GUS transformants shows preferential SCAP1 expression in guard cells.

(C) Subcellular localization of SCAP1-GFP protein in guard cells. Nuclei were stained by Hoechst 33342 (blue). Scale bars represent 10 mm.

(D) The SCAP1-GFP accumulates in nuclei of young guard cells and mature guard cells, but not in meristemoids or guard mother cells. The timing of SCAP1

expression resembles that of SLAC1 (an S-type anion channel protein). Scale bars represent 10 mm.

Dof Factor Essential for Functional Stomata3


(ChIP) assay using a functional SCAP1-FLAG fusion proteinexpressed from a genomic fragment (Figure S1B), weobserved robust enrichment of GORK and MYB60 promoterfragments, including Dof-binding sites (Figure 3D). Theseresults indicated that SCAP1 directly binds and then activatestheGORK andMYB60 promoters.We also showed that SCAP1activated the GORK and MYB60 promoters not only in GCPsbut also in mesophyll protoplasts (Figure S3A), suggestingthat expression of SCAP1 alone may be sufficient to induceexpression of its target genes during stomatal maturation.Consistent with the phenotype of the scap1 mutant, theseresults suggest that SCAP1 is a direct regulator for the genesessential for guard cell function.

Interestingly, the expression of genes controlling cell wallarchitecture was also altered by the scap1 mutation (Fig-ure 3A). In scap1 guard cells, the expression of PME6, which

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encodes a pectinmethylesterase (PME), was repressed partic-ularly strongly, whereas expression of the pectin methylester-ase inhibitor gene was enhanced. Cells secrete pectin as afully methylesterified polymer that is demethylesterified extra-cellularly by PME (EC 3.1.1.11) [24]. The demethylesterifiedpolymer can form Ca2+ bridges between individual pectinmolecules that tend to stiffen the wall [25, 26]. We investigateddifferential demethylesterification of pectins in the scap1mutation using two monoclonal antibodies, JIM5 and JIM7,for the differential detection of methylesterified pectins. JIM5binds preferentially to less methylesterified pectins, whereasJIM7 recognizes a highly methylesterified pectin epitope [26,27]. JIM7 binding was detected in ventral walls of scap1 guardcells, but not in the WT (Figure 4A). By contrast, JIM5 stainingwas similar in mutant and WT and was not restricted to theventral walls (Figure 4A). Thus, the demethylesterification of

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A

B

C

D

Figure 3. SCAP1 Is a Transcription Factor that Regulates Guard Cell-Specific Genes

(A) Relative expression levels of 1,540 stomatal genes that are induced or repressed by the scap1mutation based onmicroarray data. By qRT-PCR analysis

(Table S2), we confirmed that expression of GORK, MYB60, and PME6 was repressed strongly in scap1. Expression levels were normalized against the

UBQ10 expression as an internal control. Values shown are means 6 SE (n = 4).

(B) SCAP1 regulates GORK and MYB60 promoter activity in a transient assay. The pGORK::LUC or pMYB60::LUC reporter plasmid and the

35S::SCAP1 or 35S::SCAP1(m) effector plasmid were cotransfected into guard cell protoplasts. SCAP1(m) represents mutated SCAP1 that has a scap1

mutation (R65S, Figure 2A). The empty vector (pBI221) served as a control. Firefly luciferase (luc) activity was normalized against the activity of Renilla

(legend continued on next page)


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A

B C

Figure 4. SCAP1 Is Required for Dimethylesterifi-

cation of Pectin in Guard Cell Walls and Ion

Homeostasis in Guard Cells

(A) In scap1, ventral cell walls appear floppy and

are often irregularly curved (light micrographs).

Epidermal strips were probed with monoclonal

antibodies that bind to methylesterified pectin

(JIM7) or unesterified pectin (JIM5). Labeling

was detected with an Alexa Fluor 488-labeled

secondary antibody and visualized by fluores-

cence microscopy.

(B and C) Guard cell protoplasts (GCPs) were iso-

lated from leaves of WT and scap1 plants. GCP

volumes (B) and organic and inorganic ion levels

(C) were quantified after incubation with or

without white light (80 mmol m22 s21) for 1 hr.

Values shown are means 6 SE (n = 4). Asterisks

indicate significant differences between values

of GCPs incubated with or without light (p < 0.05).

Dof Factor Essential for Functional Stomata5


pectins seemed to be suppressed in the ventral walls of scap1guard cells, suggesting that SCAP1 is involved in the controlof guard cell wall mechanical properties. Increased highlymethylesterified content in the ventral walls suggested a lowerabundance of intermolecular crosslinking in the pectin fractionof the wall, possibly resulting in a floppier, less sturdy wall.This interpretation, which is in line with the aberrant appear-ance of the unusual stomata in the scap1 mutant (Figure 4A),could provide an explanation for the reduced efficiency ofstomatal function observed in the mutant. In WT guard cells,ventral walls are less extensible than other cell wall portions,which forces them to bend outward when the cell expandsreversibly under high turgor and results in the opening of thestomatal pore. An increased elastic extensibility of the scap1ventral walls, which may be induced by an increased fractionof noncrosslinked pectins, could render this biomechanicalmachinery ineffective. Because we did not detect transactiva-tion of the 2 kb PME6 promoter by SCAP1 in our transientreporter assays, we conclude that SCAP1 might regulate

luciferase derived from an internal control plasmid. Values shown are means6 SE (n = 4). Asterisks indica

at p < 0.05.

(C) Putative Dof-binding sites on the plus (top) and minus (bottom) strand of the upstream regions of t

positions of fragments amplified in (D).

(D) ChIP-qPCR. Guard cell protoplasts of SCAP1-FLAG plants were harvested for a chromatin immunopre

(2Ab) anti-FLAG antibody. qRT-PCR was used to quantify enrichment of theGORK andMYB60 promoter

ing to the promoter region of a gene (At4g23150; CRK7) that was not expressed in guard cells were used

indicate significant differences compared to the control values (2Ab) at p < 0.05.

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PME6 expression through interactionswith motifs in the region outside of thepromoter, such as the far upstreamsequence and introns. Alternatively,SCAP1 might affect PME6 expressionthrough regulating expression of anadditional transcription factor or factors.In the pme6 mutant, we did not detectany notable stomatal morphologicaldefects, but stomatal CO2 sensitivitywas lower by 18% and light sensitivitywas lower by 34% compared to WT(Figures S4C and S4D, p < 0.05). Thesephenotypes were weaker than thescap1 mutant. A possible explanationfor this result is that SCAP1 affects theexpression of multiple factors involved

in stomatal functioning (Figure 3A; Figure S3B), so that scap1mutant phenotype cannot be explained by a defect in a singlecomponent.We examined whether the scap1 mutation also affects ion

balance in guard cells. To avoid any possible effects ofabnormal scap1 cell walls, we prepared GCPs. The WTGCPs showed the well-known swelling response when illumi-nated, but scap1 GCPs did not (Figure 4B). In accordancewith this finding, the usual light-induced accumulation ofinorganic and organic ions was not observed in scap1 GCPs(Figure 4C). These results indicated that SCAP1 is requiredfor ion homeostasis in guard cells, a result consistent withthe decreased expression of genes directly involved instomatal opening and closure in the scap1 mutant (Figure 3A;Figure S3B).To investigate the effects of ectopically overexpressed

SCAP1, we made the CaMV35S::SCAP1 construct and trans-formed plants; however, these plants were bleached duringgrowth (data not shown), so a restricted expression pattern

te significant differences compared to the control

he GORK and MYB60 genes. Thick lines indicate

cipitation (ChIP) experiment with (+Ab) or without

(Table S2). As a negative control, primers anneal-

. Values shown are means 6 SE (n > 3). Asterisks



for SCAP1 (Figures 2B–2D) may be important for properSCAP1 functioning.

In summary, SCAP1 is a Dof-type transcription factorexpressed during the late stage of guard cell differentiation(Figure 2). A mutation in SCAP1 impairs stomatal openingand closing (Figures 1A–1C) and represses the expressionof genes involved in stomatal movement (Figure 3A; Fig-ure S3B). SCAP1 also functions as a transcriptional activatorthat directly induces GORK and MYB60 expression (Figures3B–3D). Furthermore, SCAP1 influences essential biome-chanical parameters, as demonstrated by the modified cellwall structure (Figure 4A) and the disturbed ion homeostasisin scap1 guard cells (Figure 4C). Thus, our findings suggestthat SCAP1 is a key transcription factor that controls thefinal stage of guard cell differentiation by regulating theexpression of multiple genes responsible for stomatal matura-tion and function. Further study of SCAP1 will pave the way toa better understanding of processes essential for stomatalmaturation and provide an opportunity to engineer stomatalfunction.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data

libraries under the accession numbers in Table S1 or as follows: SCAP1,

At5g65590; GORK, At5g37500; MYB60, At1g08810; PME6, At1g23200;

SLAC1, At1g12480. The microarray data set is deposited in the Gene

Expression Omnibus (GEO) with accession number GSE43964.

Supplemental Information

Supplemental Information includes four figures, Supplemental Experi-

mental Procedures, and two tables and can be found with this article online

at http://dx.doi.org/10.1016/j.cub.2013.02.001.

Acknowledgments

We thank L.G. Smith for critical reading of the manuscript. We also thank N.

Kawahara and Y. Johno for technical assistance. This research was sup-

ported by Grants-in-Aid for Scientific Research on Innovative Areas

21114002 (K.I.), 21114004 (S.Y.), and 22380043 (S.Y.) from the Ministry of

Education, Culture, Sports, Science and Technology of Japan; by the

Program for Promotion of Basic and Applied Research for Innovations in

Bio-oriented Industry (K.I.); by the CREST program from the Japan Science

and Technology Agency (S.Y.); by National Institutes of Health grant

R01GM060396; by National Science Foundation grant MCB0918220

(J.I.S); and by the Mitsubishi Foundation (K.I.).

Received: September 19, 2012

Revised: January 7, 2013

Accepted: February 1, 2013

Published: February 28, 2013

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A Dof Transcription Factor, SCAP1, Is Essential for the ... · Figure 2. SCAP1 Encodes a Dof-Type Transcription Factor Whose Expression Starts at a Late Stage of Guard Cell Differentiation