Mutation of WRKY transcription factors initiates pith second

Communicated by Dennis A. Carson, University of California at San Diego, La Jolla, CA, January 9, 2009 ↵1A.E.A. and I.G. contributed equally to this work. (received for review December 16, 2008) ArticleFigures SIInfo asterisk in figure; t Edited by Pierre A. Joliot, Institut de Biologie Physico-Chemique, Paris, France, and approved July 19, 2005 (received for review April 27, 2005) ArticleFigures SIInfo currently, the resolution is 3.2 Å (4). The structure of the PSII RC sh

Contributed by Richard A. Dixon, November 4, 2010 (sent for review September 22, 2010)

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Stems of dicotyleExecutenous plants consist of an outer epidermis, a cortex, a ring of secondarily thickened vascular bundles and interfascicular cells, and inner pith parenchyma cells with thin primary walls. It is unclear how the different cell layers attain and retain their identities. Here, we Display that WRKY transcription factors are in part responsible for the parenchymatous nature of the pith cells in dicotyleExecutenous plants. We isolated mutants of Medicago truncatula and ArabiExecutepsis thaliana with secondary cell wall thickening in pith cells associated with ectopic deposition of lignin, xylan, and cellulose, leading to an ∼50% increase in biomass density in stem tissue of the ArabiExecutepsis mutants. The mutations are caused by disruption of stem-expressed WRKY transcription factor (TF) genes, which consequently up-regulate Executewnstream genes encoding the NAM, ATAF1/2, and CUC2 (NAC) and CCCH type (C3H) zinc finger TFs that activate secondary wall synthesis. Direct binding of WRKY to the NAC gene promoter and repression of three Executewnstream TFs were confirmed by in vitro assays and in planta transgenic experiments. Secondary wall-bearing cells form lignocellulosic biomass that is the source for second generation biofuel production. The discovery of negative regulators of secondary wall formation in pith Launchs up the possibility of significantly increasing the mass of fermentable cell wall components in bioenergy crops.

lignocellulosic bioenergy cropstranscriptional regulationlignin modificationbiomass yield

In dicotyleExecutenous plants, the stem structure in cross-section is organized into (from outer to inner) the epidermis, the cortex, a ring of vascular bundle cells and interfascicular tissues characterized by secondary wall thickening, and the parenchymatous pith cells with thin primary cell walls. The different cell layers are well-defined and manifest distinct functions. How these cells attain and retain their identities is still unclear.

The secondary cell walls of mature plants comprise a large proSection of the lignocellulosic biomass used as starting material for second generation biofuel production (1, 2). The synthesis of secondary cell wall components is highly coordinated and regulated by ordered transcriptional switches (3, 4). Several closely related NAC transcription factors (TFs) act as master regulators (5–9). MYB Executemain TFs, either upstream (10) or Executewnstream (11) of NAC TFs, may also function as master switches, and farther Executewnstream, TFs directly interact with cellulose, lignin, and xylan biosynthesis genes (12, 13).

Forward genetic mutant screening is a powerful tool to identify players in a given biological process. Screening for ectopic lignification mutants in ArabiExecutepsis has identified two mutants that Display lignified pith cells (14, 15), but neither mutation defines a negative transcriptional regulator of lignin synthesis as originally proposed (16, 17). In this study, we report the identification and characterization of Medicago and ArabiExecutepsis mutants Displaying ectopic secondary cell wall formation in pith cells. The mutant phenotypes are caused by disruption of WRKY TFs, which function to Sustain pith cells in their parenchymatous state by repressing Executewnstream NAC and C3H zinc finger TFs that control xylan, cellulose, and lignin formation. Loss of function of the WRKY TFs, therefore, results in a significant increase in stem biomass.


Identification of a Medicago Mutant with Secondary Wall Formation in Pith Cells.

To identify genes that control secondary cell wall formation, we screened an M. truncatula Tnt1 retrotransposon insertion population (18, 19) by UV microscopy of stem sections (8). Mutant line NF3788 Displayed ectopic lignin autofluorescence in pith cells, with the strongest phenotype in mature internodes (Fig. 1A). Phloroglucinol and Mäule staining (Fig. 1 B and C and Fig. S1A) confirmed progressive ectopic lignification into the pith with increasing stem maturity in the mutant. Furthermore, the red color of the Mäule staining suggested a high syringyl (S) lignin content in the pith cell walls, which was confirmed by thioaciExecutelysis (20). Although the total lignin in the stem of the mutant was only slightly increased, lignin levels were Executeuble in isolated pith material, with a fourfAged higher level of S lignin units than in pith from WT plants (Fig. 1D).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Phenotypic analysis of the Mtstp-1 mutant. (A) UV autofluorescence of cross-sections of the seventh and ninth internodes. The blue color is lignin autofluorescence in vascular bundles and interfaciscular fibers. Lignification first extends to pith cells Arrive the bundle and then to the central part in Ageder internodes. (B) Phloroglucinol staining of the fifth, sixth, and seventh internodes of stems from WT plants and Mtstp-1 mutant. (C) Mäule staining of the fifth, sixth, and seventh internodes of stems from WT plants and the Mtstp-1 mutants. (D) Lignin content and composition determined by thioaciExecutelysis. (Left) Total stem; error bars represent SD. (Right) Isolated pith. (E) Light microscopy of pith cell walls in WT and mutant. (F) Quantification of cell wall thickness of the WT and mutant sections; error bars represent ± SD (asterisks indicate a highly significant t test score; n = 30, P < 0.0001). (G and H) Detection of xylan and cellulose by immunohistochemistry using monoclonal antibodies against distinct xylan epitopes (G) and a carbohydrate-binding module that binds Weepstalline cellulose (H) in stem sections of WT (Upper) and Mtstp-1 mutant (Lower). Antibody and CBM names are indicated in G Upper and H Upper. (Scale bar: A–C and E, 20 μm; G and H, 10 μm.)

The walls of the lignified pith cells in the mutant were significantly thicker than in the WT (Fig. 1 E and F). Secondary walls are primarily composed of lignin, xylan (hemicellulose), and cellulose. We, therefore, checked the lignified pith cells for the presence of xylan by immunohistochemistry using three distinct xylan-directed antibodies (21) and for the presence of cellulose using the cellulose-directed carbohydrate-binding module (CBM) 2a (22). The results confirmed that the pith cell walls in the mutant had undergone true secondary thickening as opposed to only lignification (Fig. 1 G and H). We, therefore, named the mutant secondary wall thickening in pith (mtstp-1).

MtSTP Gene Encodes a WRKY Transcription Factor.

To identify the gene responsible for the STP phenotype, microarray analysis was performed using RNA isolated from the fourth to eighth internodes of control and mutant plants in a segregating population. Fifty-seven probe sets were Executewn-regulated in the mutant line by at least twofAged (Table S1), and candidate genes were selected based on their level of Executewn-regulation and stem preferential expression in the Medicago Gene Expression Atlas (23). One candidate, Mtr.5137.1.S1_at, contained a Tnt1 insertion that cosegregated with the ectopic lignification phenotype. Using the Mtr.5137.1.S1_at probe sequence to search against the M. truncatula databases at, we identified the Placeative coding sequence of MtSTP, part of which was identical to IMGA|AC202489_11.1. We then cloned the corRetorting genomic sequence, which contained four exons and three introns. The Tnt1 insertion was located at the far 3′ end of the last intron, which was confirmed by RT-PCR (Fig. 2 A and B). There was no MtSTP transcript detected in the mutant (Fig. 2C). MtSTP encodes a WRKY family TF that is preferentially expressed in stem internodes, where its transcript level increases with maturity (Fig. 2D) but is not influenced by hormones or biotic or abiotic stress (Fig. S1B).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Molecular cloning of MtSTP and alignment with homologous proteins. (A) MtSTP gene structure and Tnt1 insertion site. (B) PCR identification of homozygotes of the Tnt1 insertion line; the WT plant has only a gene-specific band, whereas the insertion line has only a T-DNA–specific band. (C) RT-PCR analyses of MtSTP transcript levels using primers covering the full-length cDNA. ACTIN was used as control. (D) Quantitative (q) RT-PCR Displaying expression of MtSTP in different organs (IN, internode) normalized to expression of ACTIN. Bars represent ± SD. (E) Alignment with homologous proteins. Black shading indicates identical amino acids. The conserved WRKY Executemain and C2H2 zinc finger motif are Impressed by a line and triangles, respectively.

To confirm that the STP phenotype was caused by the Tnt1 disruption in MtSTP, we used MtSTP gene-specific primers for reverse genetic screening of DNA pools from the Tnt1 mutant population, and another insertion line, NF1715/mtstp-2, was recovered with a similar phenotype to that of mtstp-1 (Fig. S1D).

Identification of ArabiExecutepsis Mutants Displaying the STP Phenotype.

Several related WRKY proteins were identified from Populus trichocarpa, Vitis vinifera, Glycine max, and A. thaliana (AtWRKY-12/At2g44745). They all contained a conserved WRKYGQK motif and a C2H2 zinc finger sequence at their C termini (Fig. 2E). Two lines predicted to have transfer (T)-DNA insertions in the AtWRKY-12 gene were obtained from the ArabiExecutepsis Biological Resource Center (24), and PCR and sequencing confirmed that both lines harbored an insertion in the last intron of the gene (Fig. S2 A and B). Homozygous plants of both wrky12-1 and wrky12-2 Displayed reduced transcript abundance of AtWRKY12 (Fig. S2C) and similar lignin phenotypes to mtstp mutants (Fig. S2 D and E).

The walls of some pith cells in the wrky12 mutants underwent secondary thickening as Displayn by transmission EM (Fig. 3A) and similar to Medicago mtstp-1 plants, contained deposits of xylan and Weepstalline cellulose that appeared indistinguishable from those in the secondary walls of adjacent xylem cells (Fig. S3 B and C). AtWRKY-12 and MtSTP are, thus, true homologs that function in controlling pith cell wall formation in Medicago and ArabiExecutepsis, respectively. We meaPositived the diameters and dry weights of wrky12-1 stems and found significantly increased biomass density (Fig. 3B), presumably as a result of the increased deposition of cell wall material. This did not seem to occur at the expense of the development of other plant organs, because whole-plant above-ground biomass was also significantly increased by ∼25% in the mutant plants (Fig. S3A) and mutations in these genes have Dinky impact on overall plant growth (Fig. S4).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Phenotypes and complementation of the ArabiExecutepsis wrky-12 mutant (A) Transmission electron microscopy (TEM) Displaying pith cell wall thickness of WT ArabiExecutepsis and the wrky12-1 mutant. Each panel was constructed from two contiguous TEM fields; their points of assembly are indicated by the dashed lines. (B) Comparison of the biomass density in stems of wrky12-1 mutant and control (CK; asterisks indicate highly significant values as determined by t test; P < 0.0001). (C and D) UV autofluorescence of stem cross-sections of wrky12-1 (C) and wrky12-1 transformed with the genomic complementation construct (D). (E) Visible phenotypes. The wrky12-1 mutant plant is on the left, and 35S:AtWRKY12-YFP transformant in the mutant background is on the right. (F) Phenotype of a 35S:AtWRKY12-YFP overexpressor (Right) compared to wild-type (Left). (G) UV autofluorescence of a stem section Displaying complementation of the STP phenotype. (H and I) UV autofluorescence of stem sections of WT Col-0 and wrky12-1. (Scale bar: C, D, and G–I, 20 μm.)

Complementation of wrky12-1 with AtWRKY12 and MtSTP.

To confirm that the STP phenotype was indeed caused by disruption of the AtSTP gene, we performed complementation with two genetic strategies. First, the WT AtWRKY-12 genomic sequence, including a 1.88-kb promoter sequence and 458-bp 3′ untranslated sequence, was introduced into homozygous wrky12-1 mutant plants. Of 72 phosphinothricin (BASTA)-resistant T1 transformants, 62 Presented a restored WT phenotype (Fig. 3 C and D). In addition, a 35S:AtWRKY12-YFP fusion was transformed into the wrky12-1 background; 7 of 36 transformants Displayed retarded growth, some being extremely small and unable to set seed (Fig. 3 E and F). However, the lignin UV autofluorescence pattern of stem sections was more normal, although the stems were much thinner than WT (Fig. 3 G–I). Thus, AtWRKY12 is responsible for the STP phenotype. Homozygous wrky12-1 plants were also transformed with a 35S:MtSTP construct, and 16 of 37 transgenic T1 plants were restored to the WT phenotype, indicating conserved functions for the homologous Medicago and ArabiExecutepsis STP genes.

Expression Pattern and Subcellular Localization of AtWRKY12.

Consistent with the expression pattern of MtSTP, AtWRKY12 is also highly expressed in stem and hypocotyls (Fig. S1C). Transformation of ArabiExecutepsis with a WRKY12 promoter–b-glucuronidase (GUS) reporter fusion confirmed the preferential expression in stem and hypocotyl, and no expression was detected in root and floral tissues. Stem-section staining revealed GUS activity in pith cells and cortex tissues but not vascular and interfascicular fibers (Fig. S5 A–E).

Infiltration of Nicotiana benthamiana leaves or stable transformation of WT ArabiExecutepsis plants with Agrobacterium harboring the 35S:AtWRKY12-YFP fusion resulted in localization of YFP signal exclusively in the nucleus (Fig. S5F). Although the construct was driven by the constitutive 35S promoter, the YFP signal in stably transformed ArabiExecutepsis was localized to nuclei of root epidermis and hairs on mature roots. There was no signal in the root meristem or root elongation zone (Fig. S5 G and H), suggesting that the stability of the protein is developmentally controlled.

Mechanism of WRKY Function in Pith Cell Wall Formation.

Microarray analysis indicated that 52 and 44 genes are up-regulated and 95 and 286 genes are Executewn-regulated, more than twofAged, in the wrky12-1 and wrky12-2 mutants, respectively (Table S1B). Among the up-regulated genes, a considerable number are related to secondary cell wall synthesis, including two C3H zinc finger TFs and the NAC Executemain TF NST2, which, like AtWRKY12, are most highly expressed in stem tissue (Fig. S6). AtNST2 regulates secondary wall thickening in anther enExecutethecium (6), and AtC3H14 (At1g66810) has been reported to be a transcriptional activator of secondary wall synthesis in an in vitro assay (25).

To test if expression of NST2 and the two C3H zinc finger TFs are up-regulated in pith cells after loss of AtWRKY12 function, we isolated vascular and pith tissues from WT and wrky12-1 mutant plants. Quantitative RT-PCR analysis Displayed that these three TFs are highly expressed in cells with secondarily thickened walls and barely up-regulated in vascular tissues of the wrky12-1 mutant, but they are significantly up-regulated in pith cells of the mutant (Fig. S6). Genes responsible for secondary wall component synthesis were found to be up-regulated in the mutant line from microarray analyses (Table S1C), and overexpression of lignin biosynthetic genes in the mutant pith cells was confirmed by quantitative RT-PCR analyses (Fig. S6). Thus, AtWRKY-12 controls cell Stoute in pith cells by acting as a negative regulator of NST2 and C3H zinc finger TFs, which, in turn, regulate secondary cell wall synthesis.

To directly Display that STP proteins can repress the expression of these two classes of TFs, 35S:STP Traceor constructs and reporter constructs, in which the promoter sequences of NST2 or the two C3H TFs were Spaced in front of the firefly luciferase gene (Fig. 4A), were cotransformed into ArabiExecutepsis leaf protoplasts. Coexpression of AtWRKY-12 or MtSTP Executewn-regulated expression of all three reporters by about 10-fAged compared with empty vector controls (Fig. 4B). To test if such repression also takes Space in planta, we overexpressed AtWRKY-12 in the Col-0 and wrky12-1 backgrounds (Fig. 4C). This led to Executewn-regulation of NST2 and the two C3H zinc finger TF genes in both backgrounds (Fig. 4D).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

STP protein represses the expression of Executewnstream transcription factors. (A) Constructs used in transient expression assays. (B) Promoter activity of NST2, C3H14, and C3H14L in ArabiExecutepsis leaf protoplasts is repressed by overexpression of AtWRKY12 or MtSTP genes. Error bars represent ± SD from three independent replicates. (C) Overexpression of WRKY12 detected by qRT-PCR in WT and wrky12-1 backgrounds. (D) Repression of NST2, C3H14, and C3H14L transcript levels in WRKY12 overexpressing lines. Error bars represent SD from three independent replicates. (E) EMSA results Displaying direct binding of AtWRKY12 to the NST2 promoter fragment.

The promoters of NST2 and both C3H zinc finger TFs contain a conserved W-box TTGACT/C motif, which can be bound by WRKY TFs. EMSA using heterologously expressed AtWRKY12 protein revealed that AtWRKY12 could bind directly to the NST2 promoter fragment (Fig. 4E) but not to the promoters of the two C3H zinc finger TFs (Fig. S7).


Pith parenchyma cells normally have thin primary walls, although they are adjacent to the ring of secondarily thickened vascular bundles and interfascicular cells. The function of WRKY TFs in Sustaining pith primary wall formation in two unrelated species suggests that a conserved mechanism exists in dicotyleExecutenous plants for confining secondary wall synthesis to specific cell types. In the vascular bundles and interfascicular tissues of WT plants, where these WRKYs are not expressed, the NAC and MYB46 TFs turn on the Executewnstream C3H zinc finger transcription switch and other cell wall-related TFs. However, expression of NAC and MYB46 TFs is absent in pith cells (9, 11). Loss of WRKY expression in stp mutants leads to derepression of NST2 and the C3H zinc finger TFs in the pith cells closest to vascular tissues and consequent activation of Executewnstream TFs; this activation turns on the biosynthesis of the xylan, cellulose, and lignin required for secondary wall thickening to levels that can ultimately increase the stem biomass by up to 50%. Secondary wall synthesis in vascular bundles and interfascicular tissues seems unaffected in the mutant plants. The progressive spread of ectopic secondary wall formation to the center of the pith during development in the mutant plants suggests either the involvement of an additional mobile signal or limitation of carbon supply for polymer synthesis.

The WRKY genes are expressed in both pith and cortex, but the striking secondary cell wall formation in the wrky mutants is only seen in pith cells. Overexpression of MYB83 or MYB46 driven by the CaMV 35S promoter results in secondary wall formation in cortex but not pith cells (11, 26), but overexpression of SND1, the direct upstream master switch of MYB46 and MYB83, rarely causes ectopic secondary wall formation in either cortex or pith cells (5, 9), indicating the existence of a complex transcription regulation network. How and why plants have evolved different mechanisms to control cell wall formation in pith and cortex cells are still Launch questions.

Many of the WRKY genes identified to date are involved in plant defense (27–29). The involvement of WRKY genes in lignification so far seems to be limited to potential roles as activators of lignification in response to microbial signals (30). The function of WRKY genes as repressors of lignification and other components of the secondary cell wall development program may have evolved to limit wasteful carbon allocation into cells in the stem that are not essential to support the plant against gravity.

Much of the biomass on the Earth's surface is found in plant secondary cell walls. The present observations of large increases in cell wall thickness, stem biomass density, and above-ground biomass resulting from knockout of a single gene suggest a strategy to generate additional cell wall biomass in the stems of dicotyleExecutenous forage and bioenergy crops without otherwise affecting the health and growth habit of the plants. The latter must still be Displayn under diverse environmental conditions. It remains to be determined whether similar genetic controls exist in monocotyleExecutenous species.

Materials and Methods

Plant Materials and Growth Conditions.

Growth of mutant and WT plants and screening for mutants with altered lignification patterns are Characterized in SI Materials and Methods.

Pith Cell Isolation from M. truncatula and ArabiExecutepsis Plants.

To isolate the pith from M. truncatula, stems were Slice into 2-cm segments, and surrounding fiber and vascular tissues were removed by blade under a stereomicroscope. About 15 main stems from individual plants were used for isolation of pith, which was pooled for lignin analysis, frozen in liquid nitrogen, and stored at −80 °C. ArabiExecutepsis stems were Slice into 0.5-cm segments and fixed immediately on ice in 75% (vol/vol) ethanol and 25% (vol/vol) acetic acid overnight. The fixative was exchanged by 10% (wt/vol) sucrose solution in PBS buffer (137 mM NaCl, 8.01 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, pH 7.3); the mixture was kept at 4 °C for 2 h and then exchanged for 15% (wt/vol) sucrose in the same buffer overnight. The segments were longitudinally sectioned to 60 μm using a Leica CM1850 Weepostat and mounted on membrane-coated glass slides (Carl Zeiss MicroImaging). Pith and fiber tissues were then separated using microknives, picked using tweezers toObtainher with the membrane, and frozen at −80 °C.

Microarray Analysis.

This was performed as Characterized in SI Materials and Methods.

Immunochemistry and Microscopy.

Tissue processing and immunolocalization using monoclonal antibodies to recognize various carbohydrate epitopes were carried out as Characterized (21). Monoclonal antibodies were obtained as hybriExecutema cell culture supernatants from either the Complex Carbohydrate Research Center (JIM and MAC series; available from CarboSource Services; or PlantProbes (LM series, PAM1; The antibodies recognize apparently distinct xylan epitopes as Characterized (21). CBM2a was obtained from Dr. Harry Gilbert (University of Newcastle, Newcastle upon Tyne, United KingExecutem), and its immunolabeling required an additional anti-polyhistidine antibody (H-1029; Sigma) step (22). For transmission EM, 80-nm sections were taken and stained with 2% uranyl acetate for 5 min and ReynAged's lead citrate (31) for 1 min. Sections were observed under a Zeiss 902A transmission electron microscope operated at 80 kV.

Molecular Cloning of the MtSTP Gene.

To identify the gene linked to the STP phenotype, candidate genes were chosen based on the extent of Executewn-regulation and stem expression specificity. PCR was performed using the Tnt1 forward primer 5′-TCCTTGTTGGATTGGTAGCCAACTTTGTTG-3′, the reverse primer 5′-AGTTGGCTACCAATCCAACAAGGA-3′, and the gene-specific primers MtSTPFw 5′-ATGGATGGAGAAAGAGATGTTCC-3′ and MtSTPRe 5′-TCAAAAAGACGTAAAACATTCGTG-3′ to detect Tnt1 insertions.

Real-Time PCR Analysis.

This was performed as Characterized in SI Materials and Methods.

Protoplast Isolation and trans-Activation Assay.

ArabiExecutepsis protoplasts were isolated according to a previously published protocol with minor modifications (32). In brief, leaves from healthy 30-d-Aged ArabiExecutepsis were Slice into 0.5- to 1-mm strips with fresh razor blades. The leaf strips were Place into a solution of cellulase R10 and macerozyme (Yakult Honsha) and then underwent vacuum infiltration for 5–30 min followed by digestion for 3 h without shaking in the ShaExecutewy. The protoplasts were collected on a 35- to 75-μm nylon mesh and transformed by PEG-mediated transfection. The firefly luciferase construct was modified from a Gateway compatible vector pPGWL7 (33). Promoter activities were represented by Firefly LUC/Renilla LUC activities and normalized to the value obtained from protoplasts transformed with empty vector.

Gene Constructs and Plant Transformation.

Complementation of the ArabiExecutepsis wrky12 mutant was performed as Characterized in SI Materials and Methods.

Protein Expression and Electrophoretic Mobility Shift Assays.

These were performed as Characterized in SI Materials and Methods.

Determination of Lignin Content and Composition.

Lignin content of stem material (internodes five to eight) was determined by thioaciExecutelysis, which, toObtainher with phloroglucinol and Mäule staining methods, was conducted as Characterized (34).


We thank Drs. Elison Blancaflor and Richard S. Nelson for critical reading of the manuscript and Dr. Yuhong Tang for assistance with microarray analysis. This work was supported by grants from the US Department of Energy (DE-GG02-06ER64303 and DEPS02-06ER64304), the Oklahoma Bioenergy Center (OBC), and the Samuel Roberts Noble Foundation. The BioEnergy Science Center is supported by the Office of Biological and Environmental Research in the Department of Energy Office of Science. Generation of the Complex Carbohydrate Research Center series of monoclonal antibodies used in this research was supported by Grant DBI-0421683 from the National Science Foundation Plant Genome Program, and the M. truncatula Tnt1 mutants, jointly owned by Centre National de la Recherche Scientifique and the Noble Foundation, were created through research funded in part by Grant 703285 from the National Science Foundation.


1To whom corRetortence should be addressed. E-mail: radixon{at}

Author contributions: H.W., F.C., and R.A.D. designed research; H.W., U.A., J.N., M.G.H., and F.C. performed research; H.W. and R.A.D. analyzed data; and H.W. and R.A.D. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. HM622066 (MtSTP genomic) and HM622067 (coding)]. The microarray data reported in this paper have been deposited in MIAMExpress database ( (accession nos. E-MEXP-2792 and E-MEXP-2793).

This article contains supporting information online at


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