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. 2018 Feb 9;176(4):2737–2749. doi: 10.1104/pp.17.01771

MYB52 Negatively Regulates Pectin Demethylesterification in Seed Coat Mucilage1

Dachuan Shi a,2, Angyan Ren a,2, Xianfeng Tang b, Guang Qi b, Zongchang Xu a, Guohua Chai b, Ruibo Hu b, Gongke Zhou b, Yingzhen Kong a,3
PMCID: PMC5884589  PMID: 29440562

MYB52 is a transcriptional activator that represses pectin demethylesterification during seed coat mucilage maturation.

Abstract

Pectin, which is a major component of the plant primary cell walls, is synthesized and methyl-esterified in the Golgi apparatus and then demethylesterified by pectin methylesterases (PMEs) located in the cell wall. The degree of methylesterification affects the functional properties of pectin, and thereby influences plant growth, development and defense. However, little is known about the mechanisms that regulate pectin demethylesterification. Here, we show that in Arabidopsis (Arabidopsis thaliana) seed coat mucilage, the absence of the MYB52 transcription factor is correlated with an increase in PME activity and a decrease in the degree of pectin methylesterification. Decreased methylesterification in the myb52 mutant is also correlated with an increase in the calcium content of the seed mucilage. Chromatin immunoprecipitation analysis and molecular genetic studies suggest that MYB52 transcriptionally activates PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), PMEI14, and SUBTILISIN-LIKE SER PROTEASE1.7 (SBT1.7) by binding to their promoters. PMEI6 and SBT1.7 have previously been shown to be involved in seed coat mucilage demethylesterification. Our characterization of two PMEI14 mutants suggests that PMEI14 has a role in seed coat mucilage demethylesterification, although its activity may be confined to the seed coat in contrast to PMEI6, which functions in the whole seed. Our demonstration that MYB52 negatively regulates pectin demethylesterification in seed coat mucilage, and the identification of components of the molecular network involved, provides new insight into the regulatory mechanism controlling pectin demethylesterification and increases our understanding of the transcriptional regulation network involved in seed coat mucilage formation.

Pectin is a group of galacturonate -rich polymers that is abundant in the primary cell walls and the middle lamella of dicots and nongraminaceous monocots (Mohnen, 2008). It comprises the matrix in which the cellulose are embedded. Three major types of pectic polysaccharides, homogalacturonan (HG), rhamnogalacturonan I (RG-I), and substituted galacturonans have been identified (Caffall and Mohnen, 2009). HG has a backbone composed of 1,4-linked α-galacturonic acid (GalA) residues and accounts for approximately 65% of the pectin in cell walls (Zablackis et al., 1995; Mohnen, 2008). RG-I has a backbone composed of alternating 1,4-linked α-GalA and 1,2-linked α-rhamnose (Rha) residues and accounts for between 20% and 35% of wall pectin.

HG is synthesized in the Golgi apparatus, where its GalA residues are also methylesterified (Pelloux et al., 2007; Wolf et al., 2009; Driouich et al., 2012). After secretion into the wall, the methyl-esterified HG is de-esterified by pectin methylesterases (PMEs; Goubet and Mohnen, 1999a, 1999b). Many PMEs catalyze blockwise desterification, which results in the formation of contiguous GalAs that, in the presence of Ca2+, may form “egg-box” structures that increase wall stiffness and influence wall porosity (Micheli, 2001). Other PMEs catalyze random de-esterification, which generates low-methylesterified HG that is a substrate for polygalacturonases and pectate lyases (Micheli, 2001). Such activities may weaken the cell wall (Daas et al., 2001; Wakabayashi et al., 2003; Pelloux et al., 2007; Jolie et al., 2010). The patterns and degrees of methylesterification are critical for the mechanical and physiological properties of the pectin network and thus affect the elasticity, extensibility, and porosity of the cell wall (Willats et al., 2001b; Peaucelle et al., 2012).

There is increasing evidence that PMEs have a role in many biological processes in both vegetative and reproductive development, as well as in plant responses to biotic and abiotic stress (Lionetti et al., 2007; Raiola et al., 2011). PME activity is affected by pH, ionic strength, and by PME inhibitors (PMEIs), which reversibly bind to the enzyme (Micheli, 2001; Di Matteo et al., 2005). In Arabidopsis (Arabidopsis thaliana), there are 66 different genes that encode PME proteins and 71 genes that encode PMEI proteins (Wang et al., 2013). Since PMEs and PMEIs are involved in diverse biological processes, it is believed that a complex regulatory network has evolved to orchestrate their expression.

Arabidopsis seed coat mucilage is a useful model system to study the properties of plant cell walls due to its nonessentiality and easy extractability compared to cell walls in other tissues and organs. The seed coat mucilage is rapidly extruded when the seed coat epidermal cells are wetted with water. The mucilage itself is composed predominantly of RG-I (Macquet et al., 2007) together with small amounts of HG (Rautengarten et al., 2008), hemicelluloses (Yu et al., 2014), and cellulose (Harpaz-Saad et al., 2011, 2012; Sullivan et al., 2011; Ben-Tov et al., 2015). For decades, Arabidopsis seed coat epidermal cell development has been a model system to investigate cell wall polysaccharide biosynthesis and the relevant gene regulatory networks. More than 50 genes, including transcription factors and functional enzymes, have been identified and shown to be necessary for mucilage synthesis, maturation, and release in Arabidopsis (Francoz et al., 2015).

Studies of several PME-related genes involved in pectin maturation in seed coat mucilage have provided strong evidence that HG demethylesterification has a role in determining mucilage properties. For example, PMEI6 prevents HG demethylesterification in seed coat epidermal cells, and the pmei6 mutant has a mucilage extrusion defect (Saez-Aguayo et al., 2013). The subtilisin-like Ser protease, SBT1.7, is believed to activate PMEI or repress PME during mucilage modification. The sbt1.7 mutant also has a mucilage extrusion defect (Rautengarten et al., 2008). PECTIN METHYLESTERASE58 (PME58) mediates the molecular interactions between HG and other mucilage components by demethylesterifying mucilage HG (Turbant et al., 2016). FLYING SAUCER1 (FLY1) has been proposed to regulate the degree of methylesterification (DM) of seed coat mucilage by recycling PME enzymes (Voiniciuc et al., 2013).

Despite recent advances in identifying the seed coat mucilage enzymes involved in pectin demethylesterification, little is known about the transcriptional regulatory mechanisms involved. To date, only two transcriptional regulators, LEUNIG_HOMOLOG/MUCILAGE MODIFIED1 (LUH/MUM1) and SEEDSTICK (STK), have been shown to regulate pectin demethylesterification in seed coat mucilage (Bui et al., 2011; Walker et al., 2011; Saez-Aguayo et al., 2013; Ezquer et al., 2016). LUH/MUM1 represses the transcription of PMEI6, SBT1.7, and FLY1 (Bui et al., 2011; Walker et al., 2011; Saez-Aguayo et al., 2013). STK positively regulates the transcription of PMEI6 and negatively regulates SBT1.7, while STK and LUH/MUM1 may repress each other (Ezquer et al., 2016). Together, these results suggest that a complex regulatory network exists to coordinate seed coat mucilage demethylesterification.

Here, we provide evidence that AtMYB52, a R2R3-MYB transcription factor, has a role in pectin maturation in the seed coat mucilage. We show that MYB52 negatively regulates pectin demethylesterification in the seed coat mucilage. Using electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR), we demonstrate that MYB52 directly binds to the promoters of PMEI6, SBT1.7, and PMEI14 both in vivo and in vitro, which is also confirmed by the genetic evidence provided in this study.

RESULTS

The Expression Pattern of MYB52 Is Correlated with Seed Coat Mucilage Production

MYB52 is expressed in all tissues during plant growth, especially during silique development, according to the Arabidopsis AtGenExpress database (Supplemental Fig. S1; Schimid et al., 2005). We further investigated MYB52 expression in developing siliques at 4 days post anthesis (DPA), 8 DPA, 10 DPA, and 13 DPA (Fig. 1A). MYB52 expression increased rapidly after 4 DPA and peaked at 8 DPA, the stage where seed coat mucilage accumulates. From 8 DPA to 10 DPA, MYB52 expression was maintained at its maximum level and then decreased at 13 DPA. We next performed in situ hybridization at different developmental stages to more precisely determine expression levels and locate MYB52 transcripts during seed coat differentiation (Fig. 1B). We used a MYB52 antisense probe to show that MYB52 is mainly expressed throughout the initial and middle stages of seed coat differentiation (Fig. 1B). MYB52 transcripts appeared at the heart stage (4 DPA) and peaked at 8 and 10 DPA (Fig. 1B). Moreover, the epidermal cell layer demonstrated a clear hybridization signal at 8 DPA, with intense expression in the basal epidermal cells where starch granules accumulate (Fig. 1B). At 10 DPA, transcripts were localized in the cytoplasm surrounding the starch granules (Fig. 1B). Virtually no signal was detected at 13 DPA, which is consistent with our RT-qPCR results (Fig. 1, A and B). In control experiments, no hybridization of the sense probe was detected at any developmental stage. Taken together, our results provide evidence that during seed coat development MYB52 is expressed when mucilage polysaccharides are synthesized (Fig. 1, A and B).

Figure 1.

Figure 1.

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Expression pattern of MYB52 and seed coat mucilage phenotypes of two MYB52 T-DNA insertion mutants. A, MYB52 transcript levels in the developing seed coat. The transcript level at 4 DPA was set as 1. Values indicate the means, and error bars represent the sd values of three biological replicates. Statistical significance was tested compared to 4 DPA using the unpaired t test (***P < 0.001). B, In situ hybridization of the seed coat of developing seeds at 4, 8, 10, and 13 DPA using MYB52-specific sense and antisense probes. Red arrow indicates where the MYB52 transcript is located. CO, Columella; MU, mucilage; SG, starch granules; CP, cytoplasm; OI, two cell-layered outer integument region; II, three cell-layered inner integument region. Scale bars = 50 μm. C, Schematic representation of the T-DNA insertion sites (green triangles) in the myb52 mutants. Gray boxes represent untranslated regions, black boxes represent exons, and black lines represent introns. D, MYB52 expression in the myb52 mutants determined using RT-PCR. Primers used are listed in Supplemental Table S1. E, Staining of wild- type (WT), myb52-1, and myb52-2 seeds by vigorous shaking for 1 h at 200 rpm in water containing 0.01% ruthenium red. Scale bar = 100 μm.

MYB52 Defect Results in a Mucilage Phenotype under Vigorous Shaking

Two independent lines (SALK_138624 and SALK_118938), each carrying a T-DNA insertion in MYB52 (Fig. 1C), were obtained from the Arabidopsis Biological Resource Center. SALK_138624 has a T-DNA inserted in the third exon of MYB52, whereas the SALK_118938 line carries a T-DNA insertion in the second intron. Homozygous plants for the two insertion lines were named myb52-1 and myb52-2, respectively. No MYB52 transcripts were discernible in the myb52-1 or myb52-2 mutants using the primers shown in Supplemental Table S1, suggesting that they are both null mutants (Fig. 1D).

To determine if seed coat mucilage formation is affected in myb52-1 and myb52-2, their mucilage was examined visually using ruthenium red staining (RRS). There were no discernible differences in the appearance of the mucilage of wild-type and myb52-1 seeds when the seeds were gently shaken by hand (data not shown). However, after shaking the seeds vigorously for 1 h, the mucilage layer remaining on the myb52-1 mutant seeds was substantially thinner than the corresponding layer of wild-type seeds (Fig. 1E). The seed coat mucilage phenotype of myb52-2 was similar to that of myb52-1, suggesting that the mucilage defect is a consequence of the T-DNA insertions in the MYB52 gene (Fig. 1E). Unless otherwise specified, myb52-2 was used in all subsequent experiments.

Seed Coat Morphology Is Unaltered in myb52

Sections of developing seeds at stages 10 and 13 DPA were examined to determine if the MYB52 mutation affected seed coat morphology (Supplemental Fig. S2). The shape of the myb52-2 epidermal cells was not affected. Scanning electron microscopy of the surface of the mature seeds showed that the columella and radial cell wall of wild type and myb52-2 were similar, suggesting that mutating MYB52 had no discernible effect on seed coat morphology (Supplemental Fig. S2).

The myb52 Mutant Seeds Have Less Nonadherent Mucilage and More Adherent Mucilage Than Wild-Type Seeds

Nonadherent mucilage was extracted by using water or ethylenediaminetetraacetic acid (EDTA) and the sugar contents of the mucilage were determined using HPLC (Fig. 2A; Supplemental Table S2; Supplemental Table S3). There was a 30% increase in the amount of sugar solubilized from myb52 mutant seeds by EDTA compared to water (Fig. 2A). No differences were observed in the amount of sugar solubilized from wild-type seeds by water and by EDTA as previously reported (Turbant et al., 2016; Rautengarten et al., 2008).

Figure 2.

Figure 2.

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Biochemical and immunolabeling analysis of seed coat mucilage polysaccharides and pectin methylesterification status in wild-type and myb52 seed coat mucilage. A, Comparison of total sugar contents in nonadherent seed coat mucilage of the wild-type (WT), myb52-1, and myb52-2 extracted by shaking for 1 h at 200 rpm in water or EDTA. B, Methanol released from the whole mucilage of the WT and the mutants. Whole mucilage was extracted by shaking seeds in 50 mm EDTA on the TissueLyser 11 (Qiagen) at 25 Hz. C, Immunolabeling of WT and myb52-2 seeds with CCRC-M38. Scale bar = 50 μm. D, Immunolabeling of WT and myb52-2 seeds with JIM5. Scale bar = 50 μm. E, Immunolabeling of WT and myb52-2 seeds with JIM7. Scale bar = 50 μm. F, ELISA assays of the adherent mucilage from the WT and myb52-2 using the CCRC-M38, JIM5, and JIM7 antibodies. Nonadherent mucilage was removed by shaking seeds in water at 200 rpm. The adherent mucilage was then extracted by shaking the water-extracted seeds in 50 mm EDTA on the TissueLyser 11 (Qiagen) at 25 Hz. Values indicate the means, and error bars represent the sd values of three biological replicates. Statistical significance was tested compared to the wild type using the unpaired t test (***P < 0.001).

Nonadherent mucilage was obtained by treating the water-extracted seeds of wild type and myb52 mutants with 2 m NaOH. Although there was less sugar content in water-extracted nonadherent mucilage and more sugar content in alkaline-extracted adherent mucilage in the myb52 mutants than the wild type, the total sugar content was similar (Fig. 2A; Supplemental Table S2).

Taken together, these results suggest that in the myb52 mutant seeds, a portion of the outer layer of the mucilage is less soluble in water and remains associated with the inner mucilage layer, while a portion of the inner layer of mucilage is more readily solubilized by EDTA. EDTA is a Ca2+ chelator, which removes Ca2+ ions from the calcium bridges formed between GalA residues in low-methylesterified HG. The increased amounts of mucilage extracted by EDTA in myb52-1 and myb52-2 suggest that the relocation of mucilage is related to changes in the methylesterification status of HG, as it has been reported that decreased HG methylesterification leads to increased formation of calcium bridges (Micheli, 2001).

Pectin Demethylesterification in the Mucilage Is Modified in myb52

To ascertain if the DM of the mucilage is altered by the absence of MYB52, the amount of methanol released by alkaline de-esterification of the whole mucilage was determined (Ralet et al., 2012). The amounts of methanol released was reduced by more than 40% in the myb52-1 and myb52-2 seed coat mucilage compared to wild type (Fig. 2B), suggesting that the DM of HG is significantly decreased in the myb52 mutants.

To obtain additional evidence that MYB52 affects the DM of HG in seed coat mucilage, we compared labeling of wild-type and myb52-2 seed mucilage with a monoclonal antibody (mAb) that recognizes highly methylesterified HG (JIM7) and two mAbs that recognize sparsely methylesterified HG (CCRC-M38 and JIM5; Pattathil et al., 2010). In comparison to wild type, there was an increase in labeling intensity of the myb52-2 seeds with CCRC-M38 and JIM5 and a decrease in labeling intensity with JIM7 (Fig. 2, C–E). We also used the same mAbs to analyze the released adherent mucilage using enzyme linked immunosorbent assay (ELISA). Stronger binding of CCRC-M38 and JIM5 and weaker binding of JIM7 were again observed with myb52-2 mucilage relative to wild type (Fig. 2F). These results, together with the methanol release data, provide evidence that the DM of HG is decreased in both myb52 mutants.

PME Activity Is Increased in myb52

In Arabidopsis seed coat mucilage, HGs are de-methylesterified by PMEs, and therefore the DM is determined by PME activity. To investigate whether the decreased DM in myb52 is caused by increased PME activity, a PME activity assay was performed using total protein extracts from the whole seed coat mucilage of wild type and myb52. PME activity increased by 43% and 56% in myb52-1 and myb52-2, respectively, compared to wild type (Fig. 3A). Comparable results were obtained when PME activity was determined using proteins extracted from demucilaged seeds (Fig. 3B).

Figure 3.

Figure 3.

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PME activity analysis in mutants and fold-change differences in the expression levels of PME-related genes in myb52-2. A, PME activity in the whole seed coat mucilage of wild type (WT), myb52-1, myb52-2, pmei6, sbt1.7, pmei14, MYB52 OX, and luh-3. Whole mucilage was extracted by shaking seeds in 50 mm EDTA on the TissueLyser 11 (Qiagen) at 25 Hz. B, PME activity in the demucilaged seeds of WT, myb52-1, myb52-2, pmei6, sbt1.7, pmei14 MYB52 OX, and luh-3. Demucilaged seeds were obtained by shaking seeds in 50 mm EDTA as indicated in A to remove the whole mucilage. C, Fold-change differences in the expression levels of PMEI13, PME58, PMEI14, HMS, PMEI6, SBT1.7, and LUH in myb52-2 relative to the wild type. The fold change is shown in Log2 scale. D, PME activity in the whole seed coat mucilage of WT, myb52-2, and myb52-2 that overexpressed PMEI6, SBT1.7, or PMEI14, respectively. Error bars represent sem of three biological replicates. Statistical significance was tested using the unpaired t test (***P < 0.001) in A, B, and D.

MYB52 Regulates the Expression Level of Several PME-related Genes

To identify genes regulated by MYB52 during seed coat differentiation, we used Genevestigator (https://www.genevestigator.com) to perform a coexpression analysis. One hundred ninety-seven candidate genes that coexpress with MYB52 were identified with a cutoff correlation score of 0.842 (Supplemental Table S4). Notably, a group of pectin methylesterification-related genes, including two PMEI genes, PMEI13 and PMEI14, and two PME genes, PME58 and HIGHLY METHYL ESTERIFIED SEEDS (HMS), are coexpressed with MYB52 (Supplemental Table S4). To determine if these genes are regulated by MYB52, we performed RT-qPCR to determine their expression patterns in wild type and myb52-2 mutant seeds at 8 DPA (Fig. 3C). We also included the previously characterized gene, PMEI6, which encodes an inhibitor that inactivates PME by binding to the enzyme and the gene SBT1.7, which encodes a protease that degrades the PME protein (Saez-Aguayo et al., 2013; Rautengarten et al., 2008). The transcript level of HMS was up-regulated dramatically in the myb52 mutant, whereas the expression of PMEI14 and PMEI6 were reduced dramatically. The expression level of SBT1.7 also decreased substantially in myb52-2, whereas the expression levels of PME58 and PMEI13 only decreased slightly. These results suggest that MYB52 regulates PME- and PMEI-related genes to control PME activity.

MYB52 Directly Binds to the PMEI14, PMEI6, and SBT1.7 Promoters In Vitro and In Vivo

Promoter sequence analysis showed that the promoter regions of PMEI13, PMEI14, PMEI6, and SBT1.7 contain a MYB binding site (MBS), the AA(A/C)AAAC motif that is a well-known cis-acting element bound by MYB52 (Franco-Zorrilla et al., 2014). For these four genes, we used the promoter subfragments containing the MBS as probes in EMSA assays to assess MYB52 binding in vitro. We found that the GST-MYB52 fusion protein bound the MBS subfragments of PMEI14, PMEI6, and SBT1.7 (Fig. 4, A, C, and E) but not PMEI13 (data not shown). No mobility shift was observed upon reaction with GST alone, indicating that MYB52 bound specifically to the MBS in the promoters of these three genes in vitro.

Figure 4.

Figure 4.

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MYB52 directly activates PMEI6, SBT1.7, and PMEI14 expression. A, C, and E, EMSA analysis of the binding of MYB52 protein to the promoters of PMEI6 (A), PMEI14 (C), and SBT1.7 (E), respectively. GST was used as a negative control. B, D, and F, ChIP analysis showing that MYB52-MYC interacts with the MBS-containing regions within the PMEI6, SBT1.7, and PMEI14 promoters. The top shows the schematic diagrams of the promoter regions of PMEI6 (B), SBT1.7 (D), and PMEI14 (E). The green boxes indicate the MBS-containing sequences detected by ChIP assays. The underlined regions indicate promoter fragment without MYB binding site that was used as a negative control. Siliques at 8–10 DPA stages harvested from WT and 35S:MYB52-MYC transgenic plants were used for ChIP experiments. RT-qPCR was used to quantify the enriched DNA fragments in the PMEI6, SBT1.7, and PMEI14 promoters. G and H, Transcription activity analysis showing that MYB52 activated PMEI6, PMEI14, and SBT1.7 expression in Arabidopsis leaf protoplasts. The effector construct contained the MYB52 cDNA driven by the 35S promoter. The reporter construct consisted of the GUS reporter gene driven by the PMEI6, PMEI14, or SBT1.7 promoters individually. The GUS expression in the protoplasts transfected with no effector was used as a control and was set to 1. Error bars represent ±sd of three biological replicates. Statistical significance was tested by the unpaired t test (*P < 0.05).

To determine whether the PMEI14, PMEI6, and SBT1.7 promoters are targets of MYB52 in vivo, we performed ChIP assays using 8 to 10 DPA Arabidopsis siliques expressing recombinant MYB52-MYC driven by the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 4, B, D, and F). Our RT-qPCR results show that MYB52 specifically binds to the PMEI14, PMEI6, and SBT1.7 promoters in planta. There was no discernible differential amplification of the negative control containing non-MBS sites in the MYB52-MYC line (Fig. 4, B, D, and F). These results indicate that MYB52 likely activates the expression of PMEI14, PMEI6, and SBT1.7 through direct binding to their promoters.

Transactivation assays were also performed in Arabidopsis protoplasts to determine if MYB52 directly regulates the expression of PMEI14, PMEI6, and SBT1.7. After transformation with the effector construct (p35S-MYB52 or p35S), the reporter construct containing the LUCIFERASE (LUC) gene, and the internal control construct containing the GUS gene were cotransformed into Arabidopsis leaf protoplasts, and relative GUS activity in the transfected protoplasts was calculated and normalized against LUC activity (Fig. 4G). GUS activity in protoplasts transformed with GUS under the control of the PMEI6, SBT1.7, or PMEI14 gene promoters increased 4- to 10-fold relative to the control (Fig. 4H).

To further confirm that MYB52 activates PME16, SBT1.7, and PMEI14, we examined the transcription level of these three genes in a MYB52 overexpression line (Fig. 5A). The expression levels of PMEI14, PMEI6, and SBT1.7 were upregulated in MYB52 overexpression lines, suggesting that MYB52 transcriptionally activates PMEI14, PMEI6, and SBT1.7 in vivo.

Figure 5.

Figure 5.

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Fold-change differences in the expression levels of PMEI6, SBT1.7, PMEI14, and MYB52 in MYB52 overexpression line and the luh-3 mutant. A, Fold-change (Log2) differences in the expression levels of PMEI6, SBT1.7, and PMEI14 in the MYB52 overexpression line (MYB52 OX) relative to wild type. B, Fold-change (Log2) differences in the expression levels of MYB52, PMEI6, SBT1.7, and PMEI14 in luh-3 relative to wild type. Error bars represent the sd of three biological replicates. Statistical significance was tested using the unpaired t test (*P < 0.05)

Genetic Evidence that MYB52 Regulates PMEI14, PMEI6, and SBT1.7

We have provided evidence that PMEI14, PMEI6, and SBT1.7 are positively regulated by MYB52. PMEI6 and SBT1.7 have been reported to inhibit PME activity in seed coat mucilage (Saez-Aguayo et al., 2013). Thus, to determine if PMEI14 has a similar function in seed coat mucilage, we obtained two homozygous pmei14 lines (SALK_206157 and SALK_020742). RT-PCR analysis confirmed that they were null mutants for PMEI14 (Supplemental Fig. S3). RRS showed that both pmei14-1 and pmei14-2 have similar mucilage phenotypes as myb52 when seeds were vigorously shaken for 1 h in water containing 0.01% ruthenium red at 200 rpm (Supplemental Fig. S4). Moreover, we could rescue the myb52 mucilage phenotype by individually overexpressing PMEI14, PMEI6, and SBT1.7 in myb52-2 (Supplemental Fig. S4). Overexpressing MYB52 driven by the CaMV 35S promoter (MYB52-OX) in wild-type plants had no substantial effect on seed coat mucilage phenotype (Supplemental Fig. S4).

To determine whether the PME-related genes affect mucilage HG methylesterification, the amounts of methanol released from the whole seed coat mucilage of pmei6, sbt1.7, pmei14, and MYB52 OX lines were measured (Fig. 2B). Compared to wild type, methanol release decreased significantly in pmei6, sbt1.7, and pmei14, while it increased significantly in MYB52 OX. Consistent with the decreased methanol content, the seed coat mucilage PME activities of pmei6, sbt1.7, and pmei14 were increased compared to wild type, while PME activity decreased in MYB52 OX (Fig. 3A), indicating that MYB52 and PMEI14, as well as PMEI6 and SBT1.7, repress PME activity.

We also performed PME activity assays on seeds that had their mucilage removed (Fig. 3B). PME activity in demucilaged pmei6 and sbt1.7 seeds increased significantly compared with wild type, which is consistent with previous studies (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). As expected, the MYB52 OX seed demonstrated a discernible decrease in PME activity compared with the wild type. However, no difference in PME activity was observed for pmei14 and wild-type demucilaged seeds, suggesting that PMEI14 activity might be confined to the seed coat mucilage.

Taken together, these data indicate that PMEI14, PMEI6, and SBT1.7 are direct targets of MYB52, supporting a model in which MYB52 modulates HG demethylesterification by activating the expression of PMEI14, PMEI6, and SBT1.7.

LUH/MUM1 Transcriptionally Activates MYB52 and PMEI14

LUH/MUM1 is a transcriptional regulator known to be involved in mucilage pectin maturation (Bui et al., 2011; Huang et al., 2011; Walker et al., 2011). Consistent with a previous study, the methanol content increased and PME activity decreased in luh compared with the wild type (Figs. 2B and 3A; Saez-Aguayo et al., 2013). To determine whether the expression level of MYB52 is regulated by LUH/MUM1, we used RT-qPCR to compare the expression levels of MYB52 and its three direct targets in 8 DPA wild-type and luh-3 seeds (Fig. 5B). The transcript levels of MYB52 were downregulated in the luh-3 mutant compared to the wild type, indicating that LUH/MUM1 activates the expression of MYB52. The three target genes of MYB52 were all regulated in the same way in luh-3 as they were in myb52, suggesting that LUH/MUM1 transcriptionally activates PMEI14, PMEI6, and SBT1.7, which is consistent with previous reports on the regulation role of LUH/MUM1 on PMEI6 and SBT1.7 (Saez-Aguayo et al., 2013). The expression level of LUH in myb52-2 was similar to that in the wild type, suggesting that MYB52 acts downstream of the LUH regulatory pathway (Fig. 3C).

Recently, STK has been identified and shown to participate in the regulation of pectin demethylesterification in seed coat mucilage (Ezquer et al., 2016). Thus, we determined the expression level of STK in MYB52 OX, luh, and myb52-2. Compared to the wild type, the expression level of STK increased in MYB52 OX and luh, but decreased in myb52-2, suggesting that the expression of STK is induced by MYB52 (Supplemental Fig. S5).

Calcium Ion Content Is Increased in myb52 and pmei14 Adherent Mucilage

The demethylesterification of HG facilitates the formation of Ca2+ bridges between two HG molecules, leading to the enhancement of calcium-mediated cross links between neighboring polygalacturonans (Willats et al., 2001a). To investigate if increased Ca2+ cross linking occurs in myb52-2 and pmei14-1 seed coat mucilage, we first performed immunolabeling with the mAb 2F4, which specifically recognizes HG cross linked by calcium ions (Liners et al., 1989). The labeling intensity is much stronger in myb52-2 and pmei14-1 seeds than in wild type, suggesting that there is an increase in the amount of Ca2+ cross linked HG in the mutants (Fig. 6A). We then used inductively coupled plasma mass spectrometry to show that the amounts of calcium in myb52-2 (1.79 μm g−1) and pmei14 (1.85 μm g−1) adherent mucilage were almost double that of wild type (0.98 μm g−1; Fig. 6B).

Figure 6.

Figure 6.

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Immunolabeling and biochemical analysis of calcium in adherent mucilage. A, Detection of calcium cross linked HG in wild type, myb52, and pmei14 adherent mucilage using the 2F4 mAb. Scale bar = 50 μm. B, Calcium concentrations of adherent mucilage determined using ICP-MS. Calcium content is presented as the mean value (mg g−1 extract) of three biological replicates. Error bars represent sd values of three biological replicates. Statistical significance was tested by the unpaired t test (***P < 0.001).

DISCUSSION

Four genes, PMEI6, SBT1.7, FLY1, and PME58, are known to participate in HG demethylesterification (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013; Turbant et al., 2016). Characterization of the pmei6, sbt1.7, fly1, and pme58 mutants also suggest that the methylesterification status of HG has a role in mucilage maturation (Ezquer et al., 2016). However, the mechanisms underlying transcriptional regulation of HG demethylesterification have been largely unknown. Previous studies have demonstrated that LUH/MUM1 is a transcriptional activator of PMEI6 and SBT1.7 (Rautengarten et al., 2008; Saez-Aguayo et al., 2013), whereas STK represses SBT1.7 and LUH/MUM1 and activates PMEI6 (Ezquer et al., 2016), indicating the existence of a complex regulatory mechanism for HG demethylesterification during mucilage maturation (North et al., 2014). In our study, we have shown that seed coat mucilage maturation is altered and pectin demethylesterification increased in myb52 mutants. Thus, MYB52 is likely an important transcription factor in mucilage maturation.

MYB52 Negatively Regulates Pectin Demethylesterification by Acting as an Activator of PMEI6, PMEI14, and SBT1.7 in Seed Coat Mucilage

Our RT-qPCR and in situ hybridization results showed that the temporal and spatial expression patterns of MYB52 is correlated with mucilage production (Fig. 1, A and B), suggesting that MYB52 plays regulatory roles in mucilage formation. Further investigation revealed that the loss of MYB52 led to increased PME activity in the seed coat mucilage, along with decreased HG methylesterification. These data suggest that MYB52 may be involved in regulating pectin demethylesterification in the seed coat mucilage.

Through ChIP-seq and EMSA analysis, we have demonstrated that MYB52 directly binds to the promoters of PMEI6, PMEI14, and SBT1.7 both in vitro and in vivo (Fig. 4). The regulatory role of MYB52 was further assessed by transient transactivation assays in Arabidopsis protoplasts, which showed that MYB52 not only binds to the promoters of these genes but also up-regulates PMEI6, PMEI14, and SBT1.7 promoter activity (Fig. 4). Consistent with these results, 8 DPA seeds of plants overexpressing MYB52 had increased transcript levels of PMEI6, PMEI14, and SBT1.7 (Fig. 5A). Furthermore, the seed coat mucilage phenotype and the increased PME activity in myb52-2 mutants was suppressed when PMEI6, PMEI14, and SBT1.7 were overexpressed separately (Fig. 3D; Supplemental Fig. S4). Taken together, we conclude that MYB52 negatively regulates PME activity by acting as an activator of PMEI6, PMEI14, and SBT1.7 during seed coat mucilage pectin maturation. As PMEI6, PMEI14, and SBT1.7 inhibit PME activity, it is not surprising that the PME activity was increased in myb52 relative to wild type (Fig. 3, A and B), which in turn resulted in reduced methanol release in myb52 whole seed coat mucilage and demucilaged seed (Fig. 2B). That is, MYB52 negatively regulates the demethylesterification process during pectin maturation in the seed coat mucilage.

MYB52 has been proposed to regulate secondary cell wall (SCW) biosynthesis (Zhong et al., 2008; Cassan-Wang et al., 2013). However, the direct targets of MYB52 and the transcriptional role of MYB52 have not yet been identified. Zhong et al. (2008) concluded that MYB52 was an activator of SCW biosynthesis based on the phenotype of decreased SCW thickness in chimeric MYB52 repressor lines. In contrast, Cassan-Wang et al. (2013) proposed that MYB52 encoded a transcriptional repressor of SCW biosynthesis based on the enhanced lignification phenotype observed in the myb52 mutant. In our study, we have shown that MYB52 acts as a transcriptional activator of PMEI6, PMEI14, and SBT1.7 during seed coat mucilage demethylesterification. Moreover, an increased amount of unesterified HG cross linked by more Ca2+ ions was found in the myb52 mutant. Previous studies have shown that Ca2+-bridged HG may initiate the biosynthesis of lignin polymers during SCW deposition in xylem cells, which raises the prospect that PME may be involved in cell wall lignification (Dunand et al., 2002; Lairez et al., 2005; Wi et al., 2005). It is possible that the increased lignin deposition observed in myb52 by Cassan-Wang et al. (2013) may be at least partially due to elevated PME activity in myb52. However, further experiments are required to determine whether MYB52 affects SCW biosynthesis directly or indirectly.

PMEI14 Functions in Mucilage Pectin Maturation

Several loss-of-function phenotypes of PME genes have been described. For example, VANGUARD1, PROTEIN PHOSPHATASE METHYLESTERASE1, QUARTET, and PME48 influence pollen germination or pollen tube growth (Pina et al., 2005; Tian et al., 2006; Leroux et al., 2015), whereas HMS is involved in embryo cell wall loosening (Levesque-Tremblay et al., 2015). PME58 and PMEI6 have been reported to have roles in the maintenance of seed coat mucilage structure (Saez-Aguayo et al., 2013; Turbant et al., 2016). In our study, we have provided evidence that PMEI14, which functions in regulating seed coat mucilage pectin demethylesterification, is a direct target of MYB52. The increased PME activity in the pmei14 mutant suggests that PMEI14 is a PMEI. Although the DM of seed coat mucilage in pmei14, pmei6, and sbt1.7 decreased to a similar level, there were no discernible mucilage extrusion defects in the pmei14 mutant, which is distinct from the severe extrusion defects observed in pmei6 and sbt1.7 (Supplemental Fig. S4). DM and PME activity assays of whole and demucilaged seeds revealed that the decrease in DM and increase in PME activity in pmei14 relative to wild type were only observed in seed coat mucilage (Fig. 3A). However, mutations in PMEI6 and SBT1.7 affect DM and PME activity both in whole mucilage and demucilaged seeds (Figs. 2B and 3, A and B). Since it has been reported that the extrusion phenotype observed in both pmei6 and sbt1.7 is caused by the inability to fragment the outer primary cell wall (Rautengarten et al., 2008; Saez-Aguayo et al., 2013), we hypothesize that PMEI14 function is confined to seed coat mucilage, whereas PMEI6 and SBT1.7 affect the demethylesterification of pectin both in the seed coat mucilage and in the primary cell wall, leading to cell wall rigidification and delayed mucilage release in mutant seeds. PMEI6 and SBT1.7 are expressed in all tissues, whereas PMEI14 is predominantly expressed in the seed (Supplemental Fig. S1), which further supports the spatially restricted role of PMEI14. Consistent with our observation, Turbant et al. (2016) pointed out that PME58 tends to function specifically in mucilage-producing cells. The results of these studies suggest that a group of PME/PMEI genes may function exclusively in seed coat mucilage maturation, although further detailed analysis of these genes is required to substantiate this notion.

The Relocation of RG-I in myb52 Is Related to Increased Concentrations of Ca2+ and the DM of HG

It has been proposed that cellulose, xylan, and the DM of HG in seed coat mucilage may determine mucilage adherence (Saez-Aguayo et al., 2013; Griffiths et al., 2015, 2016; Ralet et al., 2016). We found that compared to wild type, the total sugar content of nonadherent mucilage decreased in myb52, whereas the total sugar content of the adherent mucilage increased (Fig. 2A; Supplemental Table S2). This indicates that decreasing HG methylesterification may also induce the relocation of RG-I from the nonadherent layer to the adherent layer, as has been observed in the pmei6, sbt1.7, and fly1 mutants (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013; Turbant et al., 2016). Demethylesterified HG can form Ca2+ ion cross links that may increase the strength of the pectin gel matrix. It is generally assumed that in these mutants reduced methylesterification in HG is accompanied by an increase in Ca2+ ion concentration, which contributes to mucilage adherence in the adherent layer (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013; Turbant et al., 2016), although no accurate Ca2+ ion quantification has been reported. Here, we have demonstrated that the concentration of Ca2+ ions increased in the adherent mucilage of myb52 and pmei14 (Fig. 6B; Liners et al., 1989). We propose that in myb52, the increased amount of Ca2+ ions promotes increased cross linking of the demethylesterified HG, which in turn attracts the nonadherent mucilage into the adherent layer. This is consistent with the result that more mucilage is extracted from the myb52 mutant by the divalent cation chelator EDTA than by water (Fig. 2A).

A Model for Transcriptional Regulation of Pectin Demethylesterification

Recent studies have revealed that the methylesterification status of HG plays a critical role in mucilage maturation (Ezquer et al., 2016). However, until now, only LUH/MUM1 (Bui et al., 2011; Huang et al., 2011; Walker et al., 2011) and STK (Ezquer et al., 2016) have been shown to be involved in regulating mucilage HG demethylesterification.

Here, a model is proposed to depict the role of MYB52 during seed coat mucilage pectin demethylesterification (Fig. 7). MYB52 positively regulates PMEI6, SBT1.7, and PMEI14 directly, which in turn negatively regulate the pectin demethylesterification process. MYB52 acts downstream of LUH. LUH could also positively regulate PMEI6, SBT1.7, and PMEI14, possibly through the positive regulation of MYB52.

Figure 7.

Figure 7.

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Proposed regulatory network for the role of MYB52 during the process of seed coat mucilage pectin demethylesterification. MYB52 negatively regulates pectin demethylesterification during pectin maturation by direct transcriptional activation of two genes (PMEI6, PMEI14) that encode PME inhibitors and the gene SBT1.7 that encodes a protease. MYB52 acts downstream of LUH and is positively regulated by LUH. MYB52 could also transcriptionally activate STK. LUH could positively regulate PMEI14, PMEI6, and PMEI14, possibly through the positive regulation of MYB52. Solid line indicates direct transcriptional regulation, and dashed line represents possible indirect transcriptional regulation.

The mutants pmei6, sbt1.7, and luh have similar defects in mucilage extrusion, whereas myb52 and pmei14 have no extrusion defects but similar seed coat mucilage phenotypes. Thus, we propose that although LUH/MUM1 and MYB52 may exhibit some overlapping regulatory roles, MYB52 may regulate HG demethylesterification more specifically during seed coat mucilage production. LUH/MUM1 is a master switch regulating several processes, including the demethylesterification of pectin during primary cell wall biosynthesis. We found that MYB52 positively regulates the expression of the recently identified gene STK, implying that the mechanisms controlling mucilage maturation are far more complex than previously thought.

In all, we have identified a transcription factor that negatively regulates the process of pectin demethylesterification by activating genes involved in mucilage maturation. The complex transcriptional regulatory roles of MYB52, LUH/MUM1, and STK identified here and in previous studies suggest that these regulators work cooperatively to maintain PME activity homeostasis and thus control the status of HG methylesterification. Knowledge of the role of MYB52 gained in this study provides a foundation for further examination of the regulatory networks that control pectin demethylesterification.

MATERIALS AND METHODS

Plant Material

Seeds were germinated on Murashige and Skoog medium, and the seedlings were transplanted into commercial soil. Plants were grown under long-day conditions (16-h light/8-h dark cycle at 22°C). Mutant lines for myb52 (SALK_118938 and SALK_138624), pmei14 (SALK_206157 and SALK_020742), luh-3 (SALK_107245), pmei6-2 (GK_790B12), and sbt1.7-1 (GK_140B02) were obtained from the Arabidopsis Biological Resource Center or The European Arabidopsis Stock Centre. Plants were genotyped by PCR using the primers listed in Supplemental Table S1 to identify homozygous insertion lines.

RRS and Scanning Electron Microscopy

For mucilage phenotype characterization, mature seeds were shaken for 1 h at 200 rpm in deionized water containing ruthenium red (0.01% w/v). The stained seeds were then observed using a bright-field microscope (SZX16; Olympus). Seed coat morphology was observed on a scanning electron microscope. Dry seeds were mounted on stubs using an adhesive disc and then coated with platinum using a Hitachi E1045 ion sputter. The surface of the seeds was observed using a Hitachi S4800 scanning electron microscope (Hitachi High-Technologies) at an accelerating voltage of 20 kV.

Mucilage Extraction

Nonadherent and adherent mucilage were prepared using a modified protocol (Yu et al., 2014). Dry seeds (50 mg) were accurately weighed and suspended in distilled water (3 mL) and shaken for 1 h at 200 rpm. The suspensions were centrifuged for 5 min × 300g, and the supernatants were collected. The seeds were washed twice with 1 mL distilled water. The supernatants and washes were then combined and lyophilized to give nonadherent mucilage. The water-extracted seeds were suspended in 2 m NaOH (3 mL) and shaken for 1 h at 200 rpm. The supernatants were obtained by centrifugation and the seeds were washed twice with distilled water (1 mL). The supernatants and washes were pooled and adjusted to pH 7 by adding acetic acid. The neutralized solution was then dialyzed (Mr cut off 3,500) for 24 h against deionized water, lyophilized, and weighed. Nonadherent mucilage was also solubilized with 50 mm EDTA, pH 8.0, using the same procedure described for water. The EDTA-extracted supernatants were dialyzed and lyophilized. The water-extracted nonadherent mucilage, the EDTA-extracted nonadherent mucilage, and the alkaline-extracted adherent mucilage were used for monosaccharide analysis. Pictures of ruthenium-red-stained seeds postextraction are shown in Supplemental Fig. S6.

RNA Isolation, RT-PCR, and RT-qPCR

Arabidopsis (Arabidopsis thaliana) flowers were marked and the siliques at 4, 8, 10, and 13 DPA were collected manually. Total RNA was extracted with an RNeasy Mini Kit (Qiagen). First-strand cDNA synthesis was performed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). RT-PCR was carried out using ACTIN2 as the reference (Huang et al., 2011). RT-qPCR was performed with the LightCycler 480 Real-Time PCR System (Roche) using GAPC as the internal control (Huang et al., 2011). The data were analyzed using the 2−ΔΔCt method (Livak and Schmittgen, 2001).

Immunocytochemical Analysis and ELISA

Whole-seed immunolabeling was performed as described (Yu et al., 2014). The 2F4 antibody was purchased from PlantProbes (www.plantprobes.net), and secondary antibodies were from Thermo Fisher Scientific (www.thermofisher.com). The CCRC and JIM mAbs were used with phosphate-buffered saline (PBS) buffer, whereas the 2F4 mAb required tris buffered calcium and saline (TCS) buffer (20 mm Tris-HCl, pH 8.2, 0.5 mm CaCl2, and 150 mm NaCl). Whole seeds were blocked for 1 h in 3% (w/v) milk protein in PBS/TCS and then washed with PBS/TCS. The blocked seeds were incubated for 1.5 h in primary antibody solution diluted 10-fold with 3% (w/v) skim milk powder protein in PBS/TCS. Seeds were then gently washed three times in PBS and incubated for 1 h in the absence of light with Alexa Fluor488-tagged donkey anti-rat immunoglobulin G (Thermo Fisher Scientific) for JIM5 and JIM7 mAbs or a donkey anti-mouse immunoglobulin G (Thermo Fisher Scientific) for CCRC-M38 and 2F4 mAbs that were diluted 200-fold with 3% (w/v) skim milk powder in PBS/TCS. After staining for 15 min with Calcofluor White (1:5 diluted in PBS/TCS, Sigma-Aldrich), images of the stained seeds were obtained using a FluoView FV1000 spectral confocal laser microscope using a 405- and 488-nm laser.

Adherent mucilage was extracted and ELISA was performed as previously described (Yu et al., 2014). For mucilage extraction, nonadherent mucilage was removed by shaking seeds (10 mg) in water at 200 rpm, then the adherent mucilage was extracted by shaking the water-extracted seeds in 50 mm EDTA (0.3 mL) with a TissueLyser 11 (Qiagen) at 25 Hz. The extracted adherent mucilage was used in the ELISA.

Resin Embedding for Bright-Field Microscopy

Arabidopsis flowers were marked and the siliques were collected manually at 4, 8, 10, and 13 DPA. Siliques were fixed in 0.1 m K phosphate, pH 7.0, containing glutaraldehyde (2% v/v). The fully permeated siliques were dehydrated using a series of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%). After embedding in Spurr’s resin, sections (1 µm) were cut and stained using toluidine blue O (0.1% w/v in water) and ruthenium red (0.01% w/v in water). Sections were examined with a bright-field microscopy (SZX16; Olympus).

Plasmid Construction and Plant Transformation

MYC was commercially synthesized and inserted into the Pcambia1300 CFP vector, which was then modified by digestion with KpnI-XbaI (Fast digest, Fermentas) followed by ligation using a commercial DNA Ligation Kit (D6022, TAKARA) to obtain 35S:MYC vector. For the MYC tagged MYB52 overexpression vector, the full-length coding DNA sequence (CDS) of MYB52 without the stop codon was amplified from wild-type cDNA and then inserted into the KpnI site of the 35S:MYC vector using the commercial DNA Ligation Kit to obtain pro35S:MYB52-MYC vector.

To generate MYB52-, PMEI6-, PMEI14-, and SBT1.7-overexpressing vectors, full-length CDSs of MYB52, PMEI6, PMEI14, and SBT1.7 were amplified and ligated into KpnI/XbaI-digested Pcambia35tlegfps2#4 vector using the commercial DNA Ligation Kit (D6022, TAKARA).

EMSAs

EMSAs were performed essentially as described by Chai et al. (2014). A GST-AtMYB52 gene fusion was generated in the pGEX4T-1 vector and expressed in Escherichia coli. The recombinant GST-AtMYB52 protein was purified using GST∙Bind (Merck) as recommended by the manufacturer. The candidate genes’ promoter subfragments amplified with 5′ biotin-labeled primers were used as the probes. The EMSA assay was conducted using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher) as recommended by the manufacturer.

ChIP and qPCR

ChIP assays were performed as described previously with minor modifications (Morohashi et al., 2009; Chen et al., 2013). In brief, 2 g of immature siliques at the 8-to-10-DPA stage were collected from wild-type and pro35S:MYB52-MYC transgenic plants and ground to a fine powder in liquid nitrogen. The chromatin complexes were isolated and sonicated to shear DNA into approximately 200 to 800 base pair fragments. The sonicated chromatin solutions were then immunoprecipitated with MYC-specific antibodies. As an input control for data normalization, a portion of prepared DNA that was not incubated with MYC antibody was also included. The precipitated DNA was recovered and quantified by PCR amplification using the primers listed in Supplemental Table S1. To quantify the specific promoters that contained the MBS motif recognized by MYB52, RT-qPCR analysis was performed as described above.

Transcription Activity Assay

To detect the regulatory activity of MYB52 on the three PME-related gene promoters, the PMEI6, PMEI14, and SBT1.7 promoters were individually ligated upstream of a GUS reporter of a modified pBI221 vector (35S promoter had been removed) to create the reporter constructs. The MYB52 coding region was ligated downstream of the 35S promoter of a modified pBI221 vector (GUS reporter had been removed) to create the effector construct. The LUC reporter carrying the LUCIFERASE gene driven by the CaMV 35S promoter was used for determination of the transfection efficiency. In each experiment, the GUS expression level in protoplasts transfected with no effector was used as a control and was set to one. All experiments were performed in three biological triplicates.

In Situ Hybridization

In situ hybridization was performed as described before with minor modifications (Yu et al., 2014). Developing siliques were fixed in a phosphate solution (4% paraformaldehyde in 0.1 m K phosphate, pH 7.0) and embedded with Paraplast Plus (Sigma) chips, sectioned 8 μm thick, and dewaxed. The MYB52 coding region was ligated to the pGM-T vector, and probes were generated in vitro using the SP6 or T7 RNA polymerase transcription kit (Roche). The in situ hybridization was performed according to Mayer et al. (1998).

Monosaccharide Compositional Analysis

The extracted mucilage was hydrolyzed for 2 h at 121°C with 0.5 mL of 2 m trifluoroacetic acid and then vacuum dried. The hydrolysates were then reacted for 30 min at 70°C with 1-phenyl-3-methyl-5-pyrazolone in 0.3 m NaOH. The monosaccharide derivatives were analyzed on a Waters HPLC System equipped with a Hypersil ODS-2 C18 column (4.6 × 250 mm; Thermo Scientific). An equal molar mixture of l-Fuc, l-Rha, l-Ara, d-Gal, d-Glc, d-Xyl, d-Man, and d-GalA was also derivatized and used as the standard.

Methanol Release Detection and PME Activity

Whole mucilage was used for methanol release assays. Whole mucilage was collected by vigorously shaking 10 mg seeds in 50 mm EDTA (200 μL) on the TissueLyser 11 (Qiagen) at 25 Hz. Ruthenium red staining was performed to ensure that both the nonadherent and adherent layers of mucilage were removed. The degree of mucilage methylesterification was determined as described by Voiniciuc et al. (2013). In brief, methanol was released by alkaline de-esterification (2 m NaOH for 1 h) of the mucilage. The solution was then neutralized with 2 m HCl. The released methanol was oxidized with alcohol oxidase (0.5 U, Sigma-Aldrich). A colorimetric method was used to quantify the released methanol at 412 nm. The amount of methanol released was calculated as described (Lionetti et al., 2007; Voiniciuc et al., 2013).

Whole mucilage and demucilaged seeds were used for PME activity analysis. Whole mucilage was extracted as stated above. To obtain the demucilaged seeds, mature seeds (20 mg) were shaken in 50 mm EDTA (200 μL) on the TissueLyser 11 (Qiagen) at 25 Hz to remove the whole mucilage. The seeds were then washed with distilled water and collected as demucilaged seeds. For PME activity analysis, total protein extracts were obtained using a One Step Plant Active Protein Extraction Kit (C510004, Sangon Biotech). Protein concentrations were determined according to the Bradford method (Bradford, 1976). Proteins (10 µg) were loaded into 6 mm-diameter wells in 1% agarose gels containing citrus fruit pectin (0.1% (w/v; ≥85% esterified, Sigma-Aldrich), citric acid (12.5 mM), and 50 mm Na2HPO4, pH 6.5. The gels were kept overnight at 28°C before being stained for 45 min with ruthenium red 0.01% (w/v) and washed for 4 h in water. The stained gels were photographed and the intensity of staining quantified with ImageJ 1.34S software (Saez-Aguayo et al., 2013). Measurements were performed in triplicate and data normalized with the wild-type area set to one.

Inductively Coupled Plasma Mass Spectrometry

Alkaline extracted adherent mucilage was prepared using the same protocol for monosaccharide analysis as described above (see “Mucilage Extraction” section). However, the dialysis procedure was omitted to preserve the calcium in the mucilage. ICP-MS was performed on an iCAP Qc ICP mass spectrometer (Thermo Fisher Scientific) equipped with a Superdex-75 HR10/30 column. The column was eluted at 0.6 mL min−1 with 10 mm ammonium formate, pH 5.0. Ca2+ was selectively detected as recommended by the manufacturer.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: At1g17950 (MYB52), At2g47670 (PMEI6), At5g67360 (SBT1.7), At1g56100 (PMEI14), At4g15750 (PMEI13), At2g32700 (LUH/MUM1), and At4g09960 (STK).

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. MYB52 and PMEI14 are predominantly expressed during seed development stages (the boxed area), while LUH, SBT1.7, and PMEI6 are expressed ubiquitously in all organs and different development stages.

  • Supplemental Figure S2. Sections and seed morphology of developing wild-type (WT) and myb52-2 seeds.

  • Supplemental Figure S3. RT-PCR analysis of gene transcripts in the pmei14 mutant lines.

  • Supplemental Figure S4. Analysis of mucilage phenotypes in the wild-type, mutant, and transgenic lines using ruthenium red staining.

  • Supplemental Figure S5. Fold-change differences in the expression levels of STK in MYB52 OX, luh-3, and myb52-2 mutants compared with that in the wild type.

  • Supplemental Figure S6. Ruthenium red (RR) staining of wild type seeds after using different seed coat mucilage extraction methods.

  • Supplemental Table S1. Primers used in this study.

  • Supplemental Table S2. Glycosyl residue compositions of mucilage extracted sequentially with water (nonadherent mucilage) and NaOH (adherent mucilage) from Arabidopsis wild-type, myb52-1, and myb52-2 seeds (μg/mg intact seeds).

  • Supplemental Table S3. Glycosyl residue compositions of EDTA-extracted nonadherent mucilage from Arabidopsis wild-type, myb52-1, and myb52-2 seeds (μg/mg intact seeds).

  • Supplemental Table S4. List of 197 candidate genes that are coexpressed with MYB52 identified based on Genevestigator data sets.

Acknowledgments

The authors thank Malcolm O’Neill (Complex Carbohydrate Research Center, University of Georgia) for his critical suggestions and helpful revision of the manuscript, Michael G. Hahn (Complex Carbohydrate Research Center, University of Georgia) for providing the CCRC antibody, and Paul Knox (University of Leeds, UK) for providing the JIM5 and JIM7 antibodies.

Footnotes

1

This work was supported by the National Natural Science Foundation of China (31670302 and 31470291), the National Key Technology R&D Program (2015BAD15B03-05), the Taishan Scholar Program of Shandong (to G.Z.), the Elite Youth Program of CAAS (to Y.K.), and the China Postdoctoral Science Foundation (2015M571173).

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