ERF4 and MYB52 transcription factors play antagonistic roles in regulating homogalacturonan de-methylesterification in Arabidopsis seed coat mucilage.

Homogalacturonan (HG), a component of pectin, is synthesized in the Golgi apparatus in its fully methylesterified form. It is then secreted into the apoplast where it is typically de-methylesterified by pectin methylesterases (PME). Secretion and de-esterification are critical for normal pectin function, yet the underlying transcriptional regulation mechanisms remain largely unknown. Here, we uncovered a mechanism that fine-tunes the degree of HG de-methylesterification (DM) in the mucilage that surrounds Arabidopsis thaliana seeds. We demonstrate that the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor (TF) ERF4 is a transcriptional repressor that positively regulates HG DM. ERF4 expression is confined to epidermal cells in the early stages of seed coat development. The adhesiveness of the erf4 mutant mucilage was decreased as a result of an increased DM caused by a decrease in PME activity. Molecular and genetic analyses revealed that ERF4 positively regulates HG DM by suppressing the expression of three PME INHIBITOR genes (PMEIs) and SUBTILISIN-LIKE SERINE PROTEASE 1.7 (SBT1.7). ERF4 shares common targets with the TF MYB52, which also regulates pectin DM. Nevertheless, the erf4-2 myb52 double mutant seeds have a wild-type mucilage phenotype. We provide evidence that ERF4 and MYB52 regulate downstream gene expression in an opposite manner by antagonizing each other's DNA-binding ability through a physical interaction. Together, our findings reveal that pectin DM in the seed coat is fine-tuned by an ERF4-MYB52 transcriptional complex.

MYB-bHLH-TTG1 Regulates Arabidopsis Seed Coat Biosynthesis Pathways Directly and Indirectly via Multiple Tiers of Transcription Factors.

MYB-bHLH-WDR (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat development including mucilage and tannin biosynthesis. The R2R3 MYBs MYB5, MYB23 and TRANSPARENT TESTA2 (TT2) participate in the MBW complexes with the WD-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). These complexes regulate GLABRA2 (GL2) and TTG2 expression in developing seeds. Microarray transcriptome analysis of ttg1-1- and wild-type (Ler) developing seeds identified 246 TTG1-regulated genes, which include all known metabolic genes of the tannin biosynthetic pathway. The first detailed TTG1-dependent metabolic pathways could be proposed for the biosynthesis of mucilage, jasmonic acid (JA) and cuticle including wax ester in developing seeds. We also assigned many known and previously uncharacterized genes to the activation/inactivation of hormones, plant immunity and nutrient transport. The promoters of six cuticle pathway genes were active in developing seeds. Expression of 11 genes was determined in the developing seeds of the combinatorial mutants of MYB5, MYB23 and TT2, and in the combinatorial mutants of GL2, HOMEODOMAIN GLABROUS2 (HDG2) and TTG2. These six TFs positively co-regulated the expression of four repressor genes while three of the six TFs repressed the wax biosynthesis genes examined, suggesting that the three TFs upregulate the expression of these repressor genes, which, in turn, repress the wax biosynthesis genes. Chromatin immunoprecipitation analysis identified 21 genes directly regulated by MYB5 including GL2, HDG2, TTG2, four repressor genes and various metabolic genes. We propose a multi-tiered regulatory mechanism by which MBWs regulate tannin, mucilage, JA and cuticle biosynthetic pathways.

HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy.

Developmental and Reproductive Effects of Iron Oxide Nanoparticles in Arabidopsis thaliana.

Increasing use of iron oxide nanoparticles in medicine and environmental remediation has led to concerns regarding exposure of these nanoparticles to the public. However, limited studies are available to evaluate their effects on the environment, in particular on plants and food crops. Here, we investigated the effects of positive (PC) and negative (NC) charged iron oxide (Fe2O3) nanoparticles (IONPs) on the physiology and reproductive capacity of Arabidopsis thaliana at concentrations of 3 and 25 mg/L. The 3 mg/L treated plants did not show evident effects on seeding and root length. However, the 25 mg/L treatment resulted in reduced seedling (positive-20% and negative-3.6%) and root (positive-48% and negative-negligible) length. Interestingly, treatment with polyethylenimine (PEI; IONP-PC coating) also resulted in reduced root length (39%) but no change was observed with polyacrylic acid (PAA; IONP-NC coating) treatment alone. However, treatment with IONPs at 3 mg/L did lead to an almost 5% increase in aborted pollen, a 2%-6% reduction in pollen viability and up to an 11% reduction in seed yield depending on the number of treatments. Interestingly, the treated plants did not show any observable phenotypic changes in overall size or general plant structure, indicating that environmental nanoparticle contamination could go dangerously unnoticed.

Alpha-glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis.

Novel mutations in the RSW1 and KNOPF genes were identified in a large-scale screen for mutations that affect cell expansion in early Arabidopsis embryos. Embryos from both types of mutants were radially swollen with greatly reduced levels of crystalline cellulose, the principal structural component of the cell wall. Because RSW1 was previously shown to encode a catalytic subunit of cellulose synthase, the similar morphology of knf and rsw1-2 embryos suggests that the radially swollen phenotype of knf mutants is largely due to their cellulose deficiency. Map-based cloning of the KNF gene and enzyme assays of knf embryos demonstrated that KNF encodes alpha-glucosidase I, the enzyme that catalyzes the first step in N-linked glycan processing. The strongly reduced cellulose content of knf mutants indicates that N-linked glycans are required for cellulose biosynthesis. Because cellulose synthase catalytic subunits do not appear to be N glycosylated, the N-glycan requirement apparently resides in other component(s) of the cellulose synthase machinery. Remarkably, cellular processes other than extracellular matrix biosynthesis and the formation of protein storage vacuoles appear unaffected in knf embryos. Thus in Arabidopsis cells, like yeast, N-glycan trimming is apparently required for the function of only a small subset of N-glycoproteins.

The re-establishment of desiccation tolerance in germinated Arabidopsis thaliana seeds and its associated transcriptome.

The combination of robust physiological models with "omics" studies holds promise for the discovery of genes and pathways linked to how organisms deal with drying. Here we used a transcriptomics approach in combination with an in vivo physiological model of re-establishment of desiccation tolerance (DT) in Arabidopsis thaliana seeds. We show that the incubation of desiccation sensitive (DS) germinated Arabidopsis seeds in a polyethylene glycol (PEG) solution re-induces the mechanisms necessary for expression of DT. Based on a SNP-tile array gene expression profile, our data indicates that the re-establishment of DT, in this system, is related to a programmed reversion from a metabolic active to a quiescent state similar to prior to germination. Our findings show that transcripts of germinated seeds after the PEG-treatment are dominated by those encoding LEA, seed storage and dormancy related proteins. On the other hand, a massive repression of genes belonging to many other classes such as photosynthesis, cell wall modification and energy metabolism occurs in parallel. Furthermore, comparison with a similar system for Medicago truncatula reveals a significant overlap between the two transcriptomes. Such overlap may highlight core mechanisms and key regulators of the trait DT. Taking into account the availability of the many genetic and molecular resources for Arabidopsis, the described system may prove useful for unraveling DT in higher plants.

Mutations in UDP-Glucose:sterol glucosyltransferase in Arabidopsis cause transparent testa phenotype and suberization defect in seeds.

In higher plants, the most abundant sterol derivatives are steryl glycosides (SGs) and acyl SGs. Arabidopsis (Arabidopsis thaliana) contains two genes, UGT80A2 and UGT80B1, that encode UDP-Glc:sterol glycosyltransferases, enzymes that catalyze the synthesis of SGs. Lines having mutations in UGT80A2, UGT80B1, or both UGT80A2 and UGT8B1 were identified and characterized. The ugt80A2 lines were viable and exhibited relatively minor effects on plant growth. Conversely, ugt80B1 mutants displayed an array of phenotypes that were pronounced in the embryo and seed. Most notable was the finding that ugt80B1 was allelic to transparent testa15 and displayed a transparent testa phenotype and a reduction in seed size. In addition to the role of UGT80B1 in the deposition of flavanoids, a loss of suberization of the seed was apparent in ugt80B1 by the lack of autofluorescence at the hilum region. Moreover, in ugt80B1, scanning and transmission electron microscopy reveals that the outer integument of the seed coat lost the electron-dense cuticle layer at its surface and displayed altered cell morphology. Gas chromatography coupled with mass spectrometry of lipid polyester monomers confirmed a drastic decrease in aliphatic suberin and cutin-like polymers that was associated with an inability to limit tetrazolium salt uptake. The findings suggest a membrane function for SGs and acyl SGs in trafficking of lipid polyester precursors. An ancillary observation was that cellulose biosynthesis was unaffected in the double mutant, inconsistent with a predicted role for SGs in priming cellulose synthesis.