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. 2023 Aug 17;64(8):906-919.
doi: 10.1093/pcp/pcad065.

MYB-bHLH-TTG1 in a Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required for Homogalacturonan Demethylesterification

Patrick J Allen  1 , Ross S Napoli  1 , Roger W Parish  1 , Song Feng Li  1
Affiliations

MYB-bHLH-TTG1 in a Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required for Homogalacturonan Demethylesterification

Patrick J Allen et al. Plant Cell Physiol. .

Abstract

MYB-bHLH-TTG1 (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat biosynthesis pathways via a multi-tiered regulatory mechanism. The MYB genes include MYB5, MYB23 and TRANSPARENT TESTA2 (TT2), which regulate GLABRA2 (GL2), HOMEODOMAIN GLABROUS2 (HDG2) and TRANSPARENT TESTA GLABRA2 (TTG2). Here, we examine the role of PECTIN METHYLESTERASE INHIBITOR14 (PMEI14) in seed coat mucilage pectin methylesterification and provide evidence in support of multi-tiered regulation of seed coat mucilage biosynthesis genes including PMEI14. The PMEI14 promoter was active in the seed coat and developing embryo. A pmei14 mutant exhibited stronger attachment of the outer layer of seed coat mucilage, increased mucilage homogalacturonan demethylesterification and reduced seed coat radial cell wall thickness, results consistent with decreased PMEI activity giving rise to increased PME activity. Reduced mucilage release from the seeds of myb5, myb23, tt2 and gl2, hdg2, ttg2 triple mutants indicated that HDG2 and MYB23 play minor roles in seed coat mucilage deposition. Chromatin immunoprecipitation analysis found that MYB5, TT8 and seven mucilage pathway structural genes are directly regulated by MYB5. Expression levels of GL2, HDG2, TTG2 and nine mucilage biosynthesis genes including PMEI14 in the combinatorial mutant seeds indicated that these genes are positively regulated by at least two of those six TFs and that TTG1 and TTG2 are major regulators of PMEI14 expression. Our results show that MYB-bHLH-TTG1 complexes regulate mucilage biosynthesis genes, including PMEI14, both directly and indirectly via a three-tiered mechanism involving GL2, HDG2 and TTG2.

Keywords: Arabidopsis thaliana; Cell walls; MBW; Mucilage; PMEI; Pectin; Seed coat; TTG1; Transcription factors.

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Figures

Fig. 1
Fig. 1
Analysis of GUS expression driven by PMEI14 and PAE1 promoters and subcellular localization of PMEI14. (A–J) GUS expression driven by PMEI14 (At1g56100) promoter in developing seeds at (A) 4 DAP, at (B) 7 DAP and (C) no GUS, (D) in ovules, (E) in developing roots, (F) in leaf petioles in 2-week-old seedlings and (G) in leaf tips. (H) and (I) A semi-thin section (6 µm) of a PMEI14 promoter::GUS seed at ∼5 DAP stained with safranin (dark-field microscopy with GUS activity in seed coat, embryo and endosperm). (J) Semi-thin sections (3–4 µm) of PMEI141.2::GFP::GUS seeds (line 47) at ∼5 DAP counterstained with safranin (arrows indicating GUS activity). (K) Schematic diagram showing the annotated structure of the Arabidopsis PMEI14 gene based on Expressed Sequence Tag data The Arabidopsis Information Resource (TAIR). The full-length annotation shows five exons separated by four introns [bottom arrows, locations of primers for (L)]. (L) qRT-PCR expression analysis of PMEI14 mRNA transcriptional splice variants in developing (globular to walking stick stages) wild-type (Col-0) seeds and 2-week-old whole seedlings relative to the UBIQUITIN10 (UBQ10) gene (At4g05320). (M) GUS expression driven by PAE1 (At1g09550) promoter in wild-type (Col-0), gl2 and ttg2 seeds. Only one silique was chosen from each different PCR confirmed transgenic line (∼10 transgenic lines per mutant background). (N) Cross-sections of PAE1 promoter::GUS expression in developing wild-type (Col-0), gl2 and ttg2 seeds. Bars: (A–C) 500 µm, (D, E and M) 1 mm, (F) 5 mm, (G) 2 mm, (H and I) 100 µm, (J) 5 µm and (N) 25 µm. Abbreviations: AM, amyloplasts; CZSC, chalazal seed coat region; CZE: chalazal endosperm, En: endosperm, Em: embryo, MCE: micropylar endosperm, Pa: palisade.
Fig. 2
Fig. 2
Radial cell wall development and mucilage release in pmei14-3 mutant seed coats. (A) Scanning electron micrographs of seed coat MSCs of mature wild-type (Col-0), pmei14-3 (SM_3_38019), tt2 and ttg2 mutants and PMEI14 complementation lines. Bracketed lines represent regions across radial cell walls that were measured. (B) and (C) Mean radial cell wall thickness values in wild-type (Col-0) and mutant seeds. The values were averaged over 10 biological replicates with at least over 100 cell wall measurements per replicate. Statistical analysis was performed using one-way ANOVA and the Tukey post hoc test. Bars with different letters are significantly different at P < 0.05. Data are shown as mean ± SD. (D) Mucilage release of wild-type (Col-0) and pmei14-3 mutant seeds was observed following treatment with 250 mM KOH. Arrows indicate the released outer water-soluble mucilage layer of wild-type seeds. Scale bars: (A) 10 µm and (D) 200 µm.
Fig. 3
Fig. 3
HG pectin methylesterification in wild-type and pmei14-3 mutant seeds. (A) Immunofluorescence labeling of methylesterfied HG in seed mucilage and columellae of wild-type (Col-0), pmei14-3 and PMEI14.2 and PMEI14.1 complementation lines. Confocal fluoresence microscopy wasperformed and optical sections were obtained showing adherent mucilage released from whole seeds following imbibition. Low or unesterfied HG was labeled with LM19 and highly esterified HG with LM20 in mucilage and columellae. Control replicate experiments were performed without LM19 or LM20. Laser gain values were fixed for each antibody treatment to allow for image comparison and fluorescence quantification relative to the scale of fluorescence intensity. Scale bars: 50 µm. (B) and (C) Quantification of fluorescence intensity following mucilage and columellae immunolabeling of methylesterfied HG in seeds of wild-type (Col-0), pmei14-3 and proPMEI14::PMEI14 complementation lines. Fluorescence intensity was determined using maximum fluorescence values with background fluorescence subtracted from each individual image. Immunolabeling was performed on three biological replicates of 20–50 seeds per replicate. Statistical analysis was performed using one-way ANOVA and the Tukey post hoc test. Bars with different letters are significantly different at P < 0.05. Data are shown as mean ± SD. Abbreviation: col, columellae.
Fig. 4
Fig. 4
ChIP-qPCR analysis of enriched MYB5-binding regions of TTG1-dependent mucilage pathway genes. (A) ChIP-qPCR analysis. The values represent mean fold enrichment (+AB/−AB, n = 3 biological replicates) following normalization using a control sequence from the Arabidopsis ACTIN7 gene (At5g09810) (Supplementary Figure 9). Chromatin immunoprecipitation enrichment values above the threshold of 2-fold were considered as ‘positively enriched’ post-normalization. A sequence from the GAPC2 gene (At1g13440) was used as a non-enriched negative control. Statistical analysis was performed using one-way ANOVA and the Tukey post hoc test. Bars with different letters are significantly different at P < 0.05. Data are shown as mean ± SD. (B) A subset of 11 promoters are presented. Underlines and ‘+’ represent enriched regions, while underlines and “−” represent regions tested that were not enriched. Q: Quantified amplicon. (C) Enrichment levels of the first intron of the MUM2 gene where MYB5 binds to the intron region in at least two locations. Bars represent ±SD. (D) Logos of sequences enriched in promoter and intergenic regions in MYB5 ChIP analysis. Logo bars: ±SE. Abbreviation: AB, anti-MYC antibody; Q, quantified amplicon; SE, standard error.
Fig. 5
Fig. 5
Expression of TTG1-dependent mucilage pathway genes in myb5, myb23, tt2 and gl2, hdg2, ttg2 combinatorial mutant seeds. qRT-PCR expression analysis of MYB5 target genes in developing seeds (globular to walking stick stages) of single, double and triple mutants. The transcript levels were presented as transcript abundance. Values shown in wild-type (Col-0) and mutant seeds were averaged over three biological replicates. The UBIQUITIN10 (UBQ10) gene (At4g05320) was used as an internal reference gene for all experiments. Statistical analysis was performed using one-way ANOVA and the Tukey post hoc test. Bars with different letters are significantly different at P < 0.05. Data are shown as mean ± SD.
Fig. 6
Fig. 6
Mucilage release and seed color phenotypes of gl2, ttg2 and hdg2 mutant combinations. (A) Levels of mucilage release following staining with 0.05% ruthenium red solution. Dry seeds of wild-type (Col-0) and mutants were shaken in ddH2O or 50 mM EDTA treatments for 30 min before staining. (B) Quantification of mucilage release levels in three classes, namely, full, partial or no mucilage release. The data were calculated from three independent experiments and are shown as percentages ± SD. The total values show the total number of seeds examined. n.d., not detected. (C) Seed color phenotypes of wild-type (Col-0) and the mutant combinations in three biological replicates. Scale bars: (A) 100 µm and (C) 500 µm. Abbreviation: n.d., not detected.
Fig. 7
Fig. 7
Model of the MYB-bHLH-TTG1-regulated seed coat mucilage pathway. MYB5-bHLH-TTG1 complexes regulate the expression of mucilage biosynthesis genes directly and indirectly via a multi-tiered regulatory pathway comprising transcriptional activators and repressors. The MBW complexes directly regulate mucilage biosynthesis genes and the tier 2 regulator genes: GL2, HDG2 and TTG2. The three tier 2 genes themselves also regulate mucilage biosynthesis genes. Solid arrows indicate modes of gene regulation supported by a combination of transcriptomic, qRT-PCR and ChIP analyses (Li et al. 2020 and this study). Dotted arrows (middle) indicate modes of gene regulation supported by qRT-PCR analysis only. HDG2 was shown to directly regulate CESA5, which plays a role in seed coat cellulose biosynthesis (Kong et al. 2021). The dashed arrow (top) indicates direct MYB5 gene regulation supported by ChIP analysis. The thick compound arrows (bottom) from metabolic genes towards mucilage biosynthesis represent indirect regulation. TTG2 directly regulates GL2 (Xu et al. 2022).
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