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Feb 28, 2011 - In Arabidopsis, the outer integument cell differentiation is a highly dynamic process and can be divided into five stages (Western et al. 2000 ...
Journal of Integrative Plant Biology 2011, 53 (5): 399–408

Research Article

LEUNIG_HOMOLOG and LEUNIG Regulate Seed Mucilage Extrusion in Arabidopsis F

Minh Bui, Nathan Lim, Paja Sijacic and Zhongchi Liu



Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA ∗ Corresponding author Tel: +1 301 405 1586; Fax: +1 301 314 1284; E-mail: [email protected] F Articles can be viewed online without a subscription. Available online on 28 February 2011 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipb doi: 10.1111/j.1744-7909.2011.01036.x

Abstract LEUNIG (LUG) and LEUNIG_HOMOLOG (LUH) encode two closely related Arabidopsis proteins, belonging to the Gro/TLE family of transcriptional co-repressors. These two genes were previously shown to exhibit partially overlapping functions in embryo and flower development. In this report, the role of both LUH and LUG on seed mucilage extrusion was examined. Seed mucilage extrusion occurs after the seeds are imbibed, serving as functional aid in seed hydration, germination, and dispersal. While luh-1 mutants exhibited strong defects in seed mucilage extrusion, lug-3 mutants exhibited a minor phenotype in mucilage extrusion. Further characterization indicates that luh-1 does not exhibit any obvious defect in seed epidermal cell differentiation, mucilage synthesis, or mucilage deposition, suggesting a specific role of LUH in mucilage extrusion. This seed mucilage phenotype of luh-1 is identical to that of mucilage modified 2 (mum2) mutants. MUM2 encodes a β-galactosidase required for the modification of the mucilage. Quantitative reverse transcription polymerase chain reaction of RNA extracted from siliques detected a slight decrease of MUM2 mRNA in the luh-1 mutant compared to the wild type. Together, LUH and possibly LUG may specifically regulate mucilage extrusion by promoting the expression of genes required for mucilage maturation. Bui M, Lim N, Sijacic P, Liu Z (2011) LEUNIG_HOMOLOG and LEUNIG regulate seed mucilage extrusion in Arabidopsis. J. Integr. Plant Biol. 53(5), 399–408.

Introduction Seed mucilage is a functional aid in seed hydration, germination, and dispersal (Fahn 1982; Penfield et al. 2001). The primary component of mucilage is carbohydrate-based pectin. The three main types of pectins are homogalacturonan (HG), rhamnogalacturonan I (RG I), and rhamnogalacturonan II (Arsovski et al. 2010), which are synthesized, secreted, and deposited by the seed outer integument, which consists of two cell layers, the outermost epidermal cell layer and a second subtending cell layer. In Arabidopsis, the outer integument cell differentiation is a highly dynamic process and can be divided into five stages (Western et al. 2000, 2001; Windsor et al. 2000). Fertilization of ovules triggers outer integument cell expansion (stage 1), followed by amyloplast accumulation (stage 2), and mucilage synthesis and secretion into the apoplastic

space (stage 3). The mucilage secretion into the apoplastic space forces cytoplasmic rearrangement, leading to a volcanoshaped cytoplasmic column in the center of the epidermal cells in each seed. At stage 4, starch granule degradation is accompanied by secondary wall deposition. Epidermal cells are marked by a central volcano-like secondary cell wall known as the columella (Western et al. 2000; Windsor et al. 2000). Stage 5 is the desiccation of seeds. When seeds are planted, they absorb water (imbibition). The mucilage previously trapped in the apoplastic space expands and erupts through the primary cell wall, generating a gelatin-like ring encapsulating the seeds. Previous studies have identified a large number of genes required for seed coat differentiation and mucilage release. They include TRANSPARENT TESTA GLABRA1 (TTG1), TTG2, TRANSPARENT TESTA2 (TT2), TT8, GLABRA2 (GL2), ENHANCER OF GLABRA3 (EGL3), MYB5, MYB61 and  C

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APETALA2 (AP2) (Jofuku et al. 1994; Rerie et al. 1994; Penfield et al. 2001; Western et al. 2001; Johnson et al. 2002; Zhang et al. 2003; Li et al. 2009; Arsovski et al. 2010). These genes, which encode transcription factors, not only regulate seed outer integument cell differentiation but also other developmental processes such as root hair initiation, trichome differentiation, and flower organ identity determination. Most of these mutants’ seed epidermis exhibit a reduced or absent columella. Another type of mucilage mutant exhibits defects that are specific to seed mucilage biosynthesis or maturation, and may define target genes of transcription factors described above. Mucilage Modified 4 (MUM4) encodes an NDP (nucleoside diphosphate)-rhamnose synthase necessary for rhamnogalacturonan I (RGI) synthesis (Usadel et al. 2004; Western et al. 2004; Oka et al. 2007). mum4 mutants have reduced seed coat columella size and reduced mucilage extrusion (Western et al. 2001). MUM4 is upregulated by GL2, that is downstream of TTG1 and AP2 (Western et al. 2004). On the other hand, MUM2 encodes a member of glycosyl hydrolase Family 35 βgalactosidase (BGAL6) required for mucilage maturation postsynthesis and post-secretion into the apoplastic space (Dean et al. 2007; Macquet et al. 2007). mum2 seed outer integument synthesizes normal amounts of mucilage but fails to extrude the mucilage upon imbibition. MUM2 acts to remove the galactose/galactan branches to increase the hydrophilic properties of the mucilage, which is needed for normal hydration and expansion of the mucilage. Other mutants with similar phenotypes to mum2 include mutants of PATCHY (AtBXL1) which codes for a bifunctional βD-xylosidase/a-L-arabinofuranosidase. patchy mutants exhibited patchy mucilage release and delayed seed germination. PATCHY (AtBXL1) was suggested to trim off rhamnogalacturonan I arabinan side-chains of the mucilage (Arsovski et al. 2009). A subtilisin-like serine protease coded by AtSBT1.7 affects mucilage extrusion by either directly degrading a pectin methylesterase (PME), or proteolytically cleaving and thus activating a PME inhibitor. Thus, AtSBT1.7 prevents excessive demethylesterification of the mucilage and/or of the primary cell wall (Rautengarten et al. 2008). More recently, mutants of the Arabidopsis GALACTURONOSYLTRANSFERASE (GAUT)11 was shown to cause reduced mucilage release and lower mucilage galacturonic acid levels. gaut11 may affect cell wall modification or biosynthesis of galacturonic acid in mucilage (Caffall et al. 2009). Nevertheless, it is not known what regulators may control the stage- and integument tissue-specific expression of mucilage modifiers like MUM2, PATCHY (AtBXL1), AtSBT1.7, and GAUT11. Previously, our lab has been characterizing two homologous transcriptional co-repressors, LUH and LUG, both belonging to the Groucho/TUP1 family of transcription co-repressors, which included TOPLESS1 in Arabidopsis (Conner and Liu

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2000; Long et al. 2006; Liu and Karmarkar 2008; Sitaraman et al. 2008). Both LUH and LUG were shown to regulate floral, leaf, and embryo development, and luh; lug double mutants are embryo lethal (Sitaraman et al. 2008; Stahle et al. 2009). LUG and LUH directly interact with a co-factor SEUSS (SEU) that bridges the interaction between LUG/LUH co-repressors and target-specific DNA-binding transcription factors such as APETALA1 (AP1) (Sridhar et al. 2004, 2006; Sitaraman et al. 2008). In this study we showed that mutants of LUH exhibit a mucilage extrusion defect similar to that of mum2 mutants. This similarity in phenotype led us to investigate the potential regulatory relationship between LUH and MUM2 and the involvement of LUG and SEU in this process.

Results luh-1 mutant seeds exhibit mucilage extrusion defects Previously we isolated a putative null allele of LUH, luh-1, in the Col-er background via the Arabidopsis TILLING project (Sitaraman et al. 2008). We reported that luh-1 seeds exhibited reduced germination rate (Sitaraman et al. 2008). In addition, luh-1 seeds tended to clump to each other when they were soaked in water. To understand the basis of these defects, we tested mucilage release in luh-1. Dry seeds of luh-1 and the wild type (both Col-er and L-er accessions) were soaked in liquid containing 0.01% toluidine blue and then observed under a dissecting microscope. A halo of gelatinous mucilage was observed that encapsulated each wild-type seed (Figure 1A, B). In contrast, there was a complete absence of mucilage surrounding the luh-1 seeds (Figure 1C). This mucilage defect is specifically caused by a loss of LUH as 35S::LUH fully rescued the mucilage defect of luh-1 (Figure 1D). The released mucilage could be separated into two distinct layers, the loose and water-soluble outer layer that can be easily shaken off and the denser inner layer that is more tightly associated with the seed (Western et al. 2001). luh-1 lacks both mucilage layers, while the wild type as well as luh-1; 35S::LUH possess both mucilage layers (Figure 1A-H). The effect of luh1 mutation in seed mucilage release is highly penetrant, with 95% luh-1 seeds lacking mucilage compared to 1.6% wild-type (Col-er) seeds (Figure 1I–L; Figure 2). luh-1; 35S::LUH is similar to the wild type with only 4.2% seeds lacking mucilage upon imbibition. An elevated LUH expression level caused by the strong 35S promoter in luh-1; 35S::LUH plants did not increase the amount of seed mucilage. Thus, LUH expression level is not proportional to the amount of mucilage produced.

lug but not seu exhibited weak mucilage defects LUG encodes a protein with a high level of sequence similarity to LUH (Sridhar et al. 2004; Sitaraman et al. 2008). We

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Figure 1. Mucilage extrusion after phenotype of wild type (WT) and luh-1. Toluidine blue staining of unshaken (A–D) and shaken seeds (E–H), and lower magnification photos of shaken seeds (I–L). Bars indicate 200 µm in (A–H) and 500 µm in (L), (I–L) have the same magnification. (A, E, I) Wild type (Col-er). (B, F, J) Wild type (L-er). (C, G, K) luh-1. (D, H, L) luh-1; 35S::LUH. (M) A wild-type (Col-er) seed pre-treated with ethylene diamine tetra acetate (EDTA). (N) A luh-1 seed pre-treated with EDTA. Shown is a thin layer of mucilage that encapsulates the luh-1 seed.

tested if lug-3, a strong loss-of-function mutant, also exhibited seed mucilage defects. Most lug-3 mutant seeds still release mucilages (Figure 2). However, lug-3 mutant seeds had a significant reduction of the outer mucilage layer (Figure 3B). The inner mucilage layer of lug-3 was thin and often torn (Figure 3F, J). This weakness became more apparent when the seeds were shaken, as the water was often littered with mucilage fragments. This contrasts with the inner mucilage layer of the wild type seeds that mostly remained intact (Figure 1E). Thus, LUG does have a role, albeit a relatively minor one compared to LUH, in seed mucilage synthesis or maturation.

Both LUH and LUG directly but independently interact with SEU (Sridhar et al. 2004; Sitaraman et al. 2008). We thus tested seu-1 seeds for any seed mucilage defects. seu-1 mutants secreted mucilage normally (Figure 3C, G, K) but had a slightly higher percentage of seeds that lacked mucilage (11%) as compared to 6.4% in the wild type (L-er) (Figure 2). This difference however is not statistically significant. Double seu-1; luh-1 mutants exhibit defects identical to luh-1 (Figure 3D, H, L), suggesting either that SEU acts in the same pathway as LUH but its function is masked by a redundant gene such as SEUSS_LIKE (SLK) (Bao et al. 2010), or that

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Figure 2. Percentage of seeds that failed to release mucilage upon imbibition. Error bar stands for SD based on three biological replicates (except for ap2–2 with only two biological replicates). “luh-1 (EDTA)” indicates luh-1 seeds treated with ethylene diamine tetra acetate before they were stained with toluidine blue.

SEU does not play a role in mucilage extrusion. The ap2 mutant seeds, which served as a control here, were previously reported to exhibit seed mucilage defect due to a failure in outer integument cell differentiation (Jofuku et al. 1994; Western et al. 2001; Ohto et al. 2009). The ap2 mutant seed epidermis lacks columella (Figure 4I) and the underlying palisade cells. As a result, ap2 seeds make little to no mucilage (Figure 2; Figure 3A, E, I).

luh-1 seeds do not exhibit significant changes in seed coat morphology or integument cell differentiation The absence of seed mucilage release upon imbibition can be attributed to defects in several dependent or independent processes. First, the outer integument cells could develop or differentiate improperly, leading to an absence of seed coat layer and columella, as in ap2 mutants (Figure 4I) (Jofuku et al. 1994; Western et al. 2001). Second, the outer integument cells could fail to synthesize or secrete mucilage to apoplastic space, resulting in a reduced or abnormal columella, as in the case of mum4 (Western et al. 2001, 2004). Third, the outer integument cell layer could synthesize and secrete mucilage properly into the apoplastic space but fail to extrude mucilage post-imbibition due to improper modification of mucilage, which is similar to mum2 mutants (Western et al. 2001; Dean et al. 2007). Heavy metal chelating agents such as ethylene diamine tetra acetate (EDTA) can force the release of trapped mucilage in mucilage extrusion mutants such as mum2

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(Dean et al. 2007) and patchy/atbxl1 (Arsovski et al. 2009) as absence of Ca++ is believed to reduce the binding of pectin chains through calcium bridges, causing more extensive hydration of the mucilage and/or weakening the primary wall. We thus tested if EDTA treatment of luh-1 seeds could help release the trapped mucilage. Indeed, approximately 51% of EDTA-treated luh-1 seeds could now release mucilage, as opposed to only 6% of EDTAuntreated luh-1 seeds (Figure 2). However, the secreted mucilage layer is significantly thinner than the wild-type control (Figure 1M, N) but resembles EDTA-treated mum2 mutants (Dean et al. 2007). This suggests that either luh-1 synthesizes mucilage at a reduced level or that luh-1 mucilage has limited ability to expand as mum2. Scanning electron microscopy (SEM) was used to examine epidermal morphology of dry seeds. Wild-type seeds have hexagonal shaped epidermal cells and a protruding central columella in each epidermal cell (Figure 4A, B, E, F). luh-1 seed epidermal cells appeared normal with columella in the center of each epidermal cell (Figure 4C, G). However, the columella appeared more rectangular and had thicker radial walls compared to the wild type. luh-1; 35S::LUH seeds were wild type-like (Figure 4D, H). lug-3 and seu-1 single (Figure 4J, K), and seu-1; luh-1 double (Figure 4L) mutant seed epidermis also appeared normal. Thus, in contrast to ap2 seeds (Figure 4I), luh-1 does not appear to cause defects in integument cell differentiation or mucilage synthesis/secretion. Normal amounts of mucilage secretion into the apoplastic space are necessary for cytoplasmic rearrangement leading to volcano-shaped columella (Western et al. 2001). The normal epidermal morphology and the presence of mucilage released by EDTA treatment suggest that the mucilage defect of luh-1 likely occurs after mucilage synthesis and secretion and may reside in mucilage modification/maturation similarly to mum2. To further confirm that mucilage is properly synthesized and deposited, plastic sections and histological staining were performed on the wild-type and luh-1 seeds. luh-1 mutants produced and accumulated mucilage in apoplastic space similarly to wild-type seeds (Figure 5). Each outer integument cell has a centrally raised columella. Amyloplasts were visible in the cytoplasm of both wild-type and luh-1 cells together with mucilage accumulation in the apoplastic space flanking the columella. Therefore, luh-1 is capable of synthesizing and depositing mucilage but fails to extrude it upon imbibition.

Examination of MUM2 and other cell wall modification enzymes in luh-1 Because luh-1 and mum2-1 mutants exhibited highly similar seed mucilage phenotypes, which include normal epidermal

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Figure 3. Mucilage extrusion phenotypes of ap2, seu, and lug. Toluidine blue staining of unshaken (A–D) and shaken seeds (E–H), and lower magnification photos of imbibed seeds (I–L). Bars indicate 200 µm in (A–H) and 500 µm in (I) and (L), (J, K) have similar magnifications as L. (A, E, I) ap2–2. (B, F, J) lug-3. (C, G, K) seu-1. (D, H, L) seu-1; luh-1.

morphology and normal mucilage release upon EDTA treatment (Western et al. 2001), LUH and MUM2 may both act in mucilage maturation. While MUM2 encodes a β-galactosidase involved in cleaving the pectin branches, LUH encodes a putative transcriptional co-repressor. LUH may positively regulate MUM2 expression by repressing the expression of a negative regulator of MUM2. We tested if MUM2 expression is reduced in luh-1 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RNA isolated from luh-1 siliques at 7 d post-anthesis (7 DPA), when mucilage synthesis and modification occurs (Western et al. 2000, 2001). We detected a very slight reduction (12%) of MUM2 in luh-1 (Figure 6B). One possible explanation is that the effect of LUH on MUM2 expression is specific in the outer integument tissue and thus difficult to detect when we extracted RNA from the whole silique tissue. Alternatively, other BGAL genes in Arabidopsis may be regulated by LUH, and a reduction of these other BGAL genes in luh-1 is responsible for the mum2-like phenotype in luh-1.

MUM2/BGAL6 is a member of a gene family with 17 members. To identify other BGAL genes with a role in seed mucilage maturation in Arabidopsis, we used AtGEN microarray data (Schmid et al. 2005) to identify BGAL genes that are highly expressed during seed development (Figure 6A). Out of 16 BGAL genes present in the AtGEN data, approximately seven were expressed at relatively higher level during seed development: At4g36360 (BGAL3), At4g38590 (BGAL14), At2g04060, At2g32810 (BGAL9), At1g45130 (BGAL5), At5g56870 (BGAL4), and At5g63800 (BGAL6/MUM2) (Figure 6A). Several of these were selected for further analysis with semi-qRT-PCR (data not shown) and qRT-PCR (Figure 6B) using gene-specific primers. However, none showed any significant difference in mRNA expression level between wild type and luh-1 (Figure 6B). We also tested BXL1 and GAUT11 due to their specific defects in mucilage modification. luh-1 mutants showed a slight increase of BXL1 expression and no significant change in GAUT11 expression compared to the wild type (Figure 6B).

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Figure 4. Scanning electron microscopy photos of seed epidermal morphology. Bars indicate 100 µm in (A–D, H, I–L) and 10 µm in (E–G).

Figure 5. Mucilage is synthesized and deposited by the epidermal cells of luh-1 seeds. (A) A diagram illustrating the two cell layers of outer integument during active mucilage synthesis and deposition stage, when starch granules accumulate at the center column (grey) of the cell. Starch granules also accumulate in the inner cell layer. Mucilage deposited around the central column is colored purple. The outer primary wall is colored red. The thickening secondary wall is black. (B and C) Plastic section of seed outer integument. Starch granules gathered in the central column of the mucilage-secreting epidermal cell as well as the cell layer beneath. Mucilage accumulation between the outer primary wall and the protoplast is visible in both wild-type and luh-1 mutants.

Discussion LUG and LUH encode two highly homologous transcriptional co-repressors with 44% sequence identity. They are the most

similar pair among the 12 Gro/TLE type co-repressors in Arabidopsis (Liu and Karmarkar 2008). Previously, we showed that LUG plays a more prominent role than LUH in regulating floral homeotic gene expression in flower organ identity specification,

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Figure 6. Expression analysis of cell wall modification enzymes. (A) Heatmap of 16 Arabidopsis BGAL genes showing their normalized expression values during seed development. Seed stages are defined as stages 3 (mid-globular to early heart embryos), 4 (early to late heart embryos), and 5 (late heart to mid torpedo embryos) still encased inside the siliques; and stages 6 (mid to late torpedo embryos), 7 (late torpedo to early walking-stick embryos), 8 (walking-stick to early curled cotyledons embryos), 9 (curled to early green cotyledons embryos), and 10 (green cotyledons embryos). Stages 3, 4, and 5 are silique tissues containing seeds of corresponding stages. Mucilage synthesis and secretion reaches a peak around stage 7 (early walking stick), and the starch granules begin to degrade around stage 8 (curled cotyledon) (Western et al. 2000; Windsor et al. 2000). The mean normalized value from AtGEN (Schmid et al. 2005) was used in generating the clustergram with the Matlab RC13 (Mathworks) Bioinformatics Toolbox. Log 2 value is shown. (B) Quantitative reverse transcription polymerase chain reaction analysis of selective cell wall modification enzyme genes in luh-1 mutants.

and the two genes are functionally redundant in regulating embryo development (Sitaraman et al. 2008). Here, we report that luh-1 mutants exhibit stronger defects in seed mucilage extrusion than lug-3, suggesting a more prominent role of LUH in regulating seed mucilage release. Due to embryonic lethality

of luh-1; lug-3 double mutants we are unable to determine the genetic interaction between luh-1 and lug-3 in seed mucilage extrusion. Unlike luh-1 or lug-3 mutants, seu-1 did not appear to cause defects in mucilage extrusion nor did it enhance the defects of

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luh-1 in seed mucilage extrusion. These results suggest that either SEU acts in the same pathway as LUH but its function is masked by a redundant gene such as SEUSS_LIKE (SLK) (Bao et al. 2010), or suggest SEU does not play a role in mucilage extrusion. Our data strongly support a defect of luh-1 mutants in mucilage extrusion rather than mucilage synthesis or deposition. These defects are highly similar to those of mum2, mum1 and, to some extent, patchy/atblx1 and gaut11 (Arsovski et al. 2009; Caffall et al. 2009). Because LUH encodes a transcription corepressor rather than a cell wall modification enzyme, one possibility is that LUH regulates the expression of MUM2 or other cell wall modification enzymes. In the absence of LUH, these cell wall modification enzymes are either expressed at a lower level or not expressed at all. However, qRT-PCR failed to reveal a significant reduction of MUM2 mRNA in luh-1 silique collected at 7 DPA. One possibility is that LUH is required to positively regulate other BGAL genes with a similar role as MUM2 or LUH may regulate other enzymes involved in mucilage modification or primary wall modification. Nevertheless qRT-PCR failed to detect any expression differences in BGAL3, BGAL9, AtBXL1, and GAUT11 (Figure 6B). Expression of additional BGALs, At4g38590 (BGAL14), At2g04060, was not detectable using the same RNA samples (data not shown). The interpretation of these results could be twofold: either we have not yet examined the correct target gene, or we failed to discover a reduction of MUM2 or other BGAL genes due to the averaging effect of isolating the entire silique tissue. Perhaps, regulation of MUM2 by LUH only occurs in the outer integument cells and could only be detected in those cell types. Further work such as in situ hybridization or isolation of outer integument tissues for qRT-PCR will be necessary to test these alternative possibilities. Finally, LUH could regulate expression of genes that regulate MUM2 post-transcriptionally such as modification of enzyme activities through phosphorylation. The seed mucilage secretory cells of Arabidopsis provide an ideal model for the discovery of novel genes as well as novel regulatory networks for directing proper synthesis, secretion, and modification of cell wall components. The dramatic and highly penetrant phenotype of luh mutants in seed mucilage extrusion identified LUH as a major player in cell wall biology and a regulator, rather than an enzyme, in this highly specific stage of seed coat maturation. Considering that plant cell walls are important for all aspects of plant cell growth, development, its interaction with the environment, and the formation of specialized structures such as cotton fibers and seed mucilage, understanding how LUH regulates seed mucilage maturation may open doors for new discoveries in plant cell wall biology.

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Materials and Methods Plant growth and materials Arabidopsis thaliana wild-type and mutant plants were grown under long day conditions (16 h light, 8 h dark) at 20 ◦ C and 65% humidity. luh-1, seu-1, lug-3 mutants were previously described (Liu and Meyerowitz 1995; Franks et al. 2002; Sitaraman et al. 2008). luh-1 is isolated in a Columbia erecta (Col-er) background; seu-1 and lug-3 are in a Landsberg erecta (L-er) background.

Morphological characterization For plastic section, siliques were fixed overnight in 4% paraformaldehyde in 0.1 M sodium phosphate buffer (1× PBS), pH 7.0. Next day, samples were washed twice with 1× PBS and dehydrated at 1 h intervals with ethanol series at 30%, 50%, 70%, 95%, and twice with 100%. Infiltration and embedding steps were done using JB-4 Embedding Kit according to the manufacturer’s protocol (Polysciences; (www.polysciences.com/SiteData/docs/123/ 204a24a728c8ffee6471ef33ede1e5d9/123.pdf). Sections (1– 2 µm thick) were made using a manual Sorvall Porter-Blum JB-4 Microtome placed in water on superfrost/plus slides (Fisher Scientific), and allowed to dry on a slide warmer at 42 ◦ C overnight. Sections were stained with 0.01% toluidine blue O in 0.01% sodium borate for 5 min, de-stained, and then photographed under a compound scope. To view seed mucilage extrusion defects, wild-type and mutant seeds of approximately similar harvest and storage time were stained with 0.01% toluidine blue O in 0.01% sodium borate for 20 min. They were gently transferred with a pipette to plates filled with water, visualized, and then photographed under a Zeiss Stemi 2000C stereoscope. For EDTA pretreatment, seeds were shaken for 90 min in 0.05 M EDTA, then rinsed, and stained with 0.01% toluidine blue O (in 0.01% sodium borate) before viewing. For SEM, seeds were dry mounted on aluminum stubs (Ted Pella), coated with gold-palladium in a Denton DV 503 Vacuum Evaporator, and photographed with an Amray 1820D SEM at 5.0 kV.

RNA extraction and qRT-PCR analysis Total RNA was isolated from 7DPA siliques of Col-er and luh1 plants using an RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was synthesized from 1 µg total RNA using a QuantiSure First-strand cDNA kit (Accugen Biosciences). Tenfold diluted cDNA (1 µL) was used as a template in real-time PCR analysis. qRT-PCR reactions used iQ SYBR Green Supermix (Bio-Rad

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Table 1. Gene-specific primers used in quantitative reverse transcription polymerase chain reaction Forward primer 5 →3

Gene

Reverse primer 5 →3

Efficiency (%)

MUM2

TTCTCTTCTCCGGTTCCATCCACT

TCCAAGCTTAGGCTCATGGAGGTT

BGAL3

ACATACCAGGTGGGACTGAAAGG

AGGCTGAGGCTTTTGTACAGTTAAGG

BGAL9

GGACGTATCAGGTGGGATTGAAGG

GAAGGTGAAGCATCGGTCTCCAAA

BXL1

TTCAACGCTAAGGTCACCCAACAAG

TCAGCACATGTGGGCTTTCCATT

96

GAUT11

CTCTGTCGCTGGTTTAGTTCTC

CTTGTGACCTCTTCCGTGAAGT

97

GAPC1

CCAGTCACTGTTTTCGGCATCA

AGCTGCAGCCTTGTCTTTGTCA

98

Laboratories), and were run and analyzed on CFX96 RealTime System (Bio-Rad Laboratories). Annealed temperatures for all primers were at 60 ◦ C, except for GAUT11, which was annealed at 57 ◦ C. Gene-specific primers used in qRT-PCR are listed in Table 1. The housekeeping gene GLYCERALDEHYDE3-PHOSPHATE DEHYDROGENASE C SUBUNIT 1 (GAPC1, At3g04120) was used as a reference gene. The Ct for each gene was subtracted from the Ct for GAPC1 to yield the CtWT and Ctluh−1 . Formula 2−Ct , where -Ct equals CtWT minus Ctluh−1 , gave the fold expression difference. The error bar represents SD based on three technical repeats.

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biosynthesis of pectin and xylan in cell walls and seed testa. Mol. Plant 2, 1000–1014. Conner J, Liu Z (2000) LEUNIG, a putative transcriptional corepressor that regulates AGAMOUS expression during flower development. Proc. Natl. Acad. Sci. USA 97, 12902–12907. Dean GH, Zheng H, Tewari J, Huang J, Young DS, Hwang YT, Western TL Carpita NC, McCann MC, Mansfield SD, Haughn GW (2007) The Arabidopsis MUM2 gene encodes a β-galactosidase required for the production of seed coat mucilage with correct hydration properties. Plant Cell 19, 4007–4021. Fahn A (1982) Plant Anatomy, Ed 3. Pergamon Press. Franks RG, Wang C, Levin JZ, Liu Z (2002) SEUSS, a member of a novel family of plant regulatory proteins, represses floral

Acknowledgements

homeotic gene expression with LEUNIG. Development 129, 253– 263.

We would like to thank Parsa Hosseini for gene expression analysis (Figure 6A), and co-advisor Dr William Higgins for his support and guidance to M. B. The authors wish to thank Courtney Hollender for the cover photo and Tim Maugel for scanning electron microscopy assistance (contribution #97 of the Laboratory for Biological Ultrastructure, University of Maryland, College Park). The work was supported by agrant from the National Science Foundation (IOB0616096) to Z. L.

Jofuku KD, den Boer BG, Van Montagu M, Okamuro JK (1994) Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6, 1211–1225. Johnson CS, Kolevski B, Smyth DR (2002) TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14, 1359– 1375. Li SF, Milliken ON, Pham H, Seyit R, Napoli R, Preston J, Koltunow, AM, Parish RW (2009) The Arabidopsis MYB5 transcription factor

Received 6 Feb. 2011

Accepted 14 Feb. 2011

regulates mucilage synthesis, seed coat development, and trichome morphogenesis. Plant Cell 21, 72–89. Liu Z, Karmarkar V (2008) Groucho/Tup1 family co-repressors in plant

References

development. Trends Plant Sci. 13, 137–144. Liu Z, Meyerowitz EM (1995) LEUNIG regulates AGAMOUS expres-

Arsovski AA, Haughn GW, Western TL (2010) Seed coat mucilage cells of Arabidopsis thaliana as a model for plant cell wall research. Plant Signal Behav. 5, 796–801. Arsovski AA, Popma TM, Haughn GW, Carpita NC, McCann

sion in Arabidopsis flowers. Development 121, 975–991. Long JA, Ohno C, Smith ZR, Meyerowitz EM (2006) TOPLESS regulates apical embryonic fate in Arabidopsis. Science 312, 1520– 1523.

MC. Western TL (2009) AtBXL1 encodes a bifunctional β-D-

Macquet A, Ralet MC, Loudet O, Kronenberger J, Mouille G,

xylosidase/alpha-L-arabinofuranosidase required for pectic arabi-

Marion-Poll A, North HM (2007) A naturally occurring mutation

nan modification in Arabidopsis mucilage secretory cells. Plant

in an Arabidopsis accession affects a β-D-galactosidase that in-

Physiol.150, 1219–1234.

creases the hydrophilic potential of rhamnogalacturonan I in seed

Bao F, Azhakanandam S, Franks RG (2010) SEUSS and SEUSSLIKE transcriptional adaptors regulate floral and embryonic development in Arabidopsis. Plant Physiol. 152, 821–836. Caffall KH, Pattathil S, Phillips SE, Hahn MG, Mohnen D (2009) Arabidopsis thaliana T-DNA mutants implicate GAUT genes in the

mucilage. Plant Cell 19, 3990–4006. Ohto MA, Floyd SK, Fischer RL, Goldberg RB, Harada JJ (2009) Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in Arabidopsis. Sex Plant Reprod. 22, 277–289.

408

Journal of Integrative Plant Biology

Vol. 53

No. 5

2011

Oka T, Nemoto T, Jigami Y (2007). Functional analysis of Ara-

Stahle MI, Kuehlich J, Staron L, von Arnim AG, Golz JF (2009)

bidopsis thaliana RHM2/MUM4, a multidomain protein involved in

YABBYs and the transcriptional corepressors LEUNIG and LE-

UDP-D-glucose to UDP-L-rhamnose conversion. J. Biol. Chem.

UNIG_HOMOLOG maintain leaf polarity and meristem activity in

282, 5389–5403.

Arabidopsis. Plant Cell 21, 3105–3118.

Penfield S, Meissner RC, Shoue DA, Carpita NC, Bevan MW (2001)

Usadel B, Kuschinsky AM, Rosso MG, Eckermann N, Pauly M

MYB61 is required for mucilage deposition and extrusion in the

(2004) RHM2 is involved in mucilage pectin synthesis and is

Arabidopsis seed coat. Plant Cell 13, 2777–2791.

required for the development of the seed coat in Arabidopsis. Plant

Rautengarten C, Usadel B, Neumetzler L, Hartmann J, Bussis D,

Physiol. 134, 286–295.

Altmann T (2008) A subtilisin-like serine protease essential for

Western TL, Burn J, Tan WL, Skinner DJ, Martin-McCaffrey L,

mucilage release from Arabidopsis seed coats. Plant J. 54, 466–

Moffatt BA, Haughn GW (2001) Isolation and characterization of

480.

mutants defective in seed coat mucilage secretory cell development

Rerie WG, Feldmann KA, Marks MD (1994) The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis. Genes Dev. 8, 1388–1399. Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M,

in Arabidopsis. Plant Physiol. 127, 998–1011. Western TL, Skinner DJ, Haughn GW (2000) Differentiation of mucilage secretory cells of the Arabidopsis seed coat. Plant Physiol. 122, 345–356.

Scholkopf B, Weigel D, Lohmann JU (2005) A gene expression

Western TL, Young DS, Dean GH, Tan WL, Samuels AL, Haughn

map of Arabidopsis thaliana development. Nat. Genet. 37, 501–

GW (2004) MUCILAGE-MODIFIED4 encodes a putative pectin

506. Sitaraman J, Bui M, Liu Z (2008) LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development. Plant Physiol. 147, 672–681. Sridhar VV, Surendrarao A, Gonzalez D, Conlan RS, Liu Z (2004) Transcriptional repression of target genes by LEUNIG and SEUSS, two interacting regulatory proteins for Arabidopsis flower development. Proc. Natl. Acad. Sci. USA 101, 11494–11499. Sridhar VV, Surendrarao A, Liu Z (2006) APETALA1 and SEPA-

biosynthetic enzyme developmentally regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis seed coat. Plant Physiol. 134, 296–306. Windsor JB, Symonds VV, Mendenhall J, Lloyd AM (2000) Arabidopsis seed coat development: Morphological differentiation of the outer integument. Plant J. 22, 483–493. Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A (2003) A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development 130, 4859–4869.

LLATA3 interact with SEUSS to mediate transcription repression during flower development. Development 133, 3159–3166.

(Co-Editor: Chun-Ming Liu)