Seed Coat Mucilage Research Articles

Unlocking the in vitro and in vivo antioxidant and anti-inflammatory activities of polysaccharide fractions from Lepidium sativum seed-coat mucilage

Inflammation coupled with oxidative stress contribute to the pathogenicity of various clinical disorders. Oxidative stress arises from an imbalance between production of reactive oxygen species (ROS) and antioxidant defense system, leading to cellular damages. The study investigated the antioxidant and anti-inflammatory effects of polysaccharides isolated from Lepidium sativum seed-coat mucilage. The water-soluble polysaccharides were extracted from mucilage and fractionated using gel permeation chromatography. The radical scavenging potential of various fractions was determined using DPPH, H2O2, and lipid peroxidation assays. The most effective EC50 was recorded for F53 (57.41 ± 1.34 μg/mL), followed by F20 (69.19 ± 0.61 μg/mL) and F52 (75.06 ± 0.45 μg/mL). In vitro anti-inflammatory effect was determined through human membrane stabilization assay while the in vivo effect was evaluated using a carrageenan-induced paw edema in mouse model where F53 demonstrated significant (P = 0.05) anti-inflammatory potential (92.60 % compared to diclofenac sodium 91.46 %). GC-MS analysis revealed the presence of galacturonic acid and glucuronic acid as main acidic monosaccharides along with varying quantities of rhamnose, arabinose, and maltose as prominent neutral monosaccharides. The study concludes that cress seed mucilage contains potent antioxidant and anti-inflammatory polysaccharides. Further studies on the mode of action of these polysaccharides could provide deeper insights into their potential use as antioxidant and anti-inflammatory agents.

The transcription factors ZAT5 and BLH2/4 regulate homogalacturonan demethylesterification in Arabidopsis seed coat mucilage.

The level of methylesterification alters the functional properties of pectin, which is believed to influence plant growth and development. However, the mechanisms that regulate demethylesterification remain largely unexplored. Pectin with a high degree of methylesterification is produced in the Golgi apparatus and then transferred to the primary cell wall where it is partially demethylesterified by pectin methylesterases (PMEs). Here, we show that in Arabidopsis (Arabidopsis thaliana) seed mucilage, pectin demethylesterification is negatively regulated by the transcription factor ZINC FINGER FAMILY PROTEIN5 (ZAT5). Plants carrying null mutations in ZAT5 had increased PME activity, decreased pectin methylesterification, and produced seeds with a thinner mucilage layer. We provide evidence that ZAT5 binds to a TGATCA motif and thereby negatively regulates methylesterification by reducing the expression of PME5, HIGHLY METHYL ESTERIFIED SEEDS (HMS)/PME6, PME12, and PME16. We also demonstrate that ZAT5 physically interacts with BEL1-LIKE HOMEODOMAIN2 (BLH2) and BLH4 transcription factors. BLH2 and BLH4 are known to modulate pectin demethylesterification by directly regulating PME58 expression. The ZAT5-BLH2/4 interaction provides a mechanism to control the degree of pectin methylesterification in seed coat mucilage by modifying each transcription factor's ability to regulate the expression of target genes encoding PMEs. Taken together, these findings reveal a transcriptional regulatory module comprising ZAT5, BLH2, and BLH4, that functions in modulating the demethylesterification of homogalacturonan in seed coat mucilage.

Creating yellow seed Camelina sativa with enhanced oil accumulation by CRISPR‐mediated disruption of Transparent Testa 8

SummaryCamelina (Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well‐organized seed coat mucilage layer. Transcriptomic analysis of CsTT8‐edited seeds revealed significantly increased expression of the lipid‐related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis‐related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced‐oil germplasm in camelina. The resulting lines may directly contribute to future net‐zero carbon energy production or be combined with other traits to produce desired lipid‐derived bioproducts at high yields.

Brassica napus BnaA09.MYB52 enhances seed coat mucilage accumulation and tolerance to osmotic stress during seed germination in Arabidopsis thaliana.

Seed coat mucilage plays an important role in promoting seed germination under adversity. Previous studies have shown that Arabidopsis thaliana MYB52 (AtMYB52) can positively regulate seed coat mucilage accumulation. However, the role of Brassica napus MYB52 (BnaMYB52) in accumulation of seed coat mucilage and tolerance to osmotic stress during seed germination remains largely unknown. We cloned the BnaA09.MYB52 coding domain sequence from B. napus cv ZS11, identified its conserved protein domains and elucidated its relationship with homologues from a range of plant species. Transgenic plants overexpressing BnaA09.MYB52 in the A. thaliana myb52-1 mutant were generated through Agrobacterium-mediated transformation and used to assess the possible roles of BnaA09.MYB52 in accumulation of seed coat mucilage and tolerance to osmotic stress during seed germination. Subcellular localization and transcriptional activity assays demonstrated that BnaA09.MYB52 functions as a transcription factor. RT-qPCR results indicate that BnaA09.MYB52 is predominantly expressed in roots and developing seeds of B. napus cv ZS11. Introduction of BnaA09.MYB52 into myb52-1 restored thinner seed coat mucilage in this mutant to levels in the wild type. Consistently, expression levels of three key genes participating in mucilage formation in developing seeds of myb52-1 were also restored to wild type levels by overexpressing BnaA09.MYB52. Furthermore, BnaA09.MYB52 was induced by osmotic stress during seed germination in B. napus, and ectopic expression of BnaA09.MYB52 successfully corrected sensitivity of the myb52-1 mutant to osmotic stress during seed germination. These findings enhance our understanding of the functions of BnaA09.MYB52 and provide a novel strategy for future B. napus breeding.

Disruption of the Arabidopsis Acyl-Activating Enzyme 3 Impairs Seed Coat Mucilage Accumulation and Seed Germination.

The Acyl-activating enzyme (AAE) 3 gene encodes an oxalyl-CoA synthetase that catalyzes the conversion of oxalate to oxalyl-CoA as the first step in the CoA-dependent pathway of oxalate catabolism. Although the role of this enzyme in oxalate catabolism has been established, its biological roles in plant growth and development are less understood. As a step toward gaining a better understanding of these biological roles, we report here a characterization of the Arabidopsis thaliana aae3 (Ataae3) seed mucilage phenotype. Ruthidium red (RR) staining of Ataae3 and wild type (WT) seeds suggested that the observed reduction in Ataae3 germination may be attributable, at least in part, to a decrease in seed mucilage accumulation. Quantitative RT-PCR analysis revealed that the expression of selected mucilage regulatory transcription factors, as well as of biosynthetic and extrusion genes, was significantly down-regulated in the Ataae3 seeds. Mucilage accumulation in seeds from an engineered oxalate-accumulating Arabidopsis and Atoxc mutant, blocked in the second step of the CoA-dependent pathway of oxalate catabolism, were found to be similar to WT. These findings suggest that elevated tissue oxalate concentrations and loss of the oxalate catabolism pathway downstream of AAE3 were not responsible for the reduced Ataae3 seed germination and mucilage phenotypes. Overall, our findings unveil the presence of regulatory interplay between AAE3 and transcriptional control of mucilage gene expression.

Influence of nanoscale spatial architecture of seed and root mucilage on its wettability

Mucilage secreted by root-tips may alter hydraulic conditions at the root-soil interface and seed coat mucilage increases water availability for seeds. Both significantly affect the water content dynamics of the rhizosphere. Therefore, we examined the nanostructure of different types of dried mucilage (root or seed mucilage and mucilage from different species) and how it affects their wettability. We hypothesized that with increasing roughness wettability of dried mucilage decreases based on the Cassie−Baxter relation due to entrapped air in holes formed in the mucilage layer during drying. In order to visualize the nanostructure of different mucilage species, we dried root mucilage from aeroponically grown seedlings (maize, cress, flax, wheat, barley) and seed mucilage (cress and flax) as thin layers on flat mica surfaces. Spatial structures of the samples were visualized by atomic force microscopy. Variogram analysis was used to analyze the spatially resolved topography data, and the coverage of mucilage was quantified using GeoDict® software. Moreover, by the pore size distribution, the effect of hole-size dispersity on mucilage's wettability was investigated. Our results demonstrate that mucilage of different species have different nanostructures. In contrast to expectations, the wettability of dried mucilage does not follow the Cassie−Baxter relation and does not decrease with increasing roughness. We conclude that wettability of surfaces decreases with increasing mucilage coverage. The lower the coverage the more hydrophilic area of the underlying mineral surface contributes to the overall wettability.

MYB-bHLH-TTG1 ina Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required forHomogalacturonan Demethylesterification.

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.

Analysis of Pectin-derived Monosaccharides from Arabidopsis Using GC-MS.

Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography-mass spectrometry (GC-MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC-MS. Key features Comparative analysis of monosaccharide content in Arabidopsis-derived pectin between different genotypes or different treatments. Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue. Allows quick analyses of neutral and acidic monosaccharides simultaneously. Graphical overview.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.).

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages. Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

Profiling and characterization of Camelina sativa (L.) Crantz meal proteins

AbstractProtein from camelina seed is a valuable co‐product that can be derived from the meal remaining after oil extraction. The current study describes the types and physicochemical properties of the major proteins present in camelina meal. Seed coat mucilage, which interferes with protein extraction, was removed from whole seeds by digestion with Viscozyme® and lipids were removed with hexane to obtain demucilaged/defatted meal. Protein comprised 51.3% of meal dry matter and the eight essential amino acids comprised 40.8% of total amino acids. The meal polypeptide profile showed bands originating from cruciferin (~44.1 and 51.7 kDa), napin (~14 kDa) and oil body proteins (OBP; ~15–20 kDa) resembling that of other crucifers. Cruciferins (11 isoforms) were the predominant proteins, while vicilins (6 isoforms) also were identified among the proteins soluble at pH 8.5. Among the proteins soluble at pH 3, napins (5 isoforms) comprised the majority, though late embryogenesis abundant proteins also were found. Camelina cruciferin and napin were confirmed to possess predominantly β‐sheet and α‐helix secondary structures, respectively. Camelina cruciferin structure was highly sensitive to changes in pH of the medium and underwent acid‐induced denaturation at pH 3, but exhibited high thermal stability (>80°C) at neutral and alkaline pHs. The structure of camelina napins was less sensitive to pH. The major proteins associated with oil bodies were oleosins (6 isoforms). Identification and characterization of the properties of camelina meal proteins will enable strategic paths for co‐product valorization such as developing plant protein ingredients.

Pectin methyltransferase QUASIMODO2 functions in the formation of seed coat mucilage in Arabidopsis

Pectin, cellulose, and hemicelluloses are major components of primary cell walls in plants. In addition to cell adhesion and expansion, pectin plays a central role in seed mucilage. Seed mucilage contains abundant pectic rhamnogalacturonan-I (RG-I) and lower amounts of homogalacturonan (HG), cellulose, and hemicelluloses. Previously, accumulated evidence has addressed the role of pectin RG-I in mucilage production and adherence. However, less is known about the function of pectin HG in seed coat mucilage formation. In this study, we analyzed a novel mutant, designated things fall apart2 (tfa2), which contains a mutation in HG methyltransferase QUASIMODO2 (QUA2). Etiolated tfa2 seedlings display short hypocotyls and adhesion defects similar to qua2 and tumorous shoot development2 (tsd2) alleles, and show seed mucilage defects. The diminished uronic acid content and methylesterification degree of HG in mutant seed mucilage indicate the role of HG in the formation of seed mucilage. Cellulosic rays in mutant mucilage are collapsed. The epidermal cells of seed coat in tfa2 and tsd2 display deformed columellae and reduced radial wall thickness. Under polyethylene glycol treatment, seeds from these three mutant alleles exhibit reduced germination rates. Together, these data emphasize the requirement of pectic HG biosynthesis for the synthesis of seed mucilage, and the functions of different pectin domains together with cellulose in regulating its formation, expansion, and release.

Diversification of SEC15a and SEC15b isoforms of an exocyst subunit in seed plants is manifested in their specific roles in Arabidopsis sporophyte and male gametophyte.

The exocyst complex is an octameric evolutionarily conserved tethering complex engaged in the regulation of polarized secretion in eukaryotic cells. Here, we focus on the systematic comparison of two isoforms of the SEC15 exocyst subunit, SEC15a and SEC15b. We infer that SEC15 gene duplication and diversification occurred in the common ancestor of seed plants (Spermatophytes). In Arabidopsis, SEC15a represents the main SEC15 isoform in the male gametophyte, and localizes to the pollen tube tip at the plasma membrane. Although pollen tubes of sec15a mutants are impaired, sporophytes show no phenotypic deviations. Conversely, SEC15b is the dominant isoform in the sporophyte and localizes to the plasma membrane in root and leaf cells. Loss-of-function sec15b mutants exhibit retarded elongation of hypocotyls and root hairs, a loss of apical dominance, dwarfed plant stature and reduced seed coat mucilage formation. Surprisingly, the sec15b mutants also exhibit compromised pollen tube elongation in vitro, despite its very low expression in pollen, suggesting a non-redundant role for the SEC15b isoform there. In pollen tubes, SEC15b localizes to distinct cytoplasmic structures. Reciprocally to this, SEC15a also functions in the sporophyte, where it accumulates at plasmodesmata. Importantly, although overexpressed SEC15a could fully complement the sec15b phenotypic deviations in the sporophyte, the pollen-specific overexpression of SEC15b was unable to fully compensate for the loss of SEC15a function in pollen. We conclude that the SEC15a and SEC15b isoforms evolved in seed plants, with SEC15a functioning mostly in pollen and SEC15b functioning mostly in the sporophyte.

Effect of seed coat mucilage, temperature, and photoperiod on germination of four Arabian desert species

In this study, we assessed the effects of mucilage presence, thermoperiod, and photoperiod on seed germination percentage and germination velocity (mean germination time) of four Arabian desert species. We hypothesized that mucilage presence, thermoperiod, and photoperiod and their interaction would influence seed germination. Seeds with and without mucilage were germinated under different alternating temperature regimes of 15/25, 20/30, and 25/35 °C night/day temperatures with a 12/0 h light/dark photoperiod. Results showed that the incubation temperature affected the germination of all the studied species. The photoperiod significantly affected the germination of Boerhavia elegans Choisy, Salvia aegyptiaca L., and Sporobolus ioclados (Trin.) Nees, while the mucilage presence influenced the germination of S. ioclados only. The interaction between mucilage presence, temperature, and photoperiod significantly influenced the germination percentage of B. elegans and S. aegyptiaca. Neither removal of the mucilage nor light conditions affected the germination percentages of Sporobolus spicatus (Vahl) Kunth. However, the temperature affected the germination of S. spicatus. The germination of all the studied species was faster for demucilaged seeds. Ecologically, the mucilaginous seed coat is considered an important adaptation for dispersal as it anchors seeds on the ground and holds water around the seed during the germination stage in the stressful arid desert habitats.

The Class II KNOX family members KNAT3 and KNAT7 redundantly participate in Arabidopsis seed coat mucilage biosynthesis.

The production of Arabidopsis seed mucilage involves complex polysaccharide biosynthetic pathways and developmental processes in seed epidermal cells. Although the polysaccharide components of Arabidopsis seed mucilage have been identified, their regulatory mechanism requires further investigation. Here, we show that Class II KNOX gene family members KNAT3 and KNAT7 play an essential role in regulating mucilage production in the early developmental stages of Arabidopsis seeds. Double mutant knat3knat7 resulted in defective seed mucilage production and columellae formation, whereas knat3 showed a normal phenotype compared with wild type, and the mucilage thickness in knat7 was slightly disturbed. Rhamnogalacturonan I (RG-I) and its biosynthetic substrates galacturonic acid and rhamnose were reduced in both the adherent and soluble mucilage of knat3knat7. Comparative transcriptome analysis on whole seeds suggested that polysaccharide, glucosinolate and anthocyanin biosynthetic pathways were specifically repressed in knat3knat7. Transient co-expression of KNAT3 and KNAT7 with promoter regions of candidate genes in Arabidopsis protoplasts revealed that both KNAT3 and KNAT7 act as positive regulators of the RG-I biosynthetic gene MUCILAGE-MODIFIED 4 (MUM4, AT1G53500). Collectively, our results demonstrate that KNAT3 and KNAT7 are multifunctional transcription factors in secondary cell wall development and redundantly modulate mucilage biosynthesis in Arabidopsis seeds.

Mutation of CESA1 phosphorylation site influences pectin synthesis and methylesterification with a role in seed development

Cell wall biogenesis is required for the production of seeds of higher plants. However, little is known about regulatory mechanisms underlying cell wall biogenesis during seed formation. Here we show a role for the phosphorylation of Arabidopsis cellulose synthase 1 (AtCESA1) in modulating pectin synthesis and methylesterification in seed coat mucilage. A phosphor-null mutant of AtCESA1 on T166 (AtCESA1T166A) was constructed and introduced into a null mutant of AtCESA1 (Atcesa1-1). The resulting transgenic lines showed a slight but significant decrease in cellulose contents in mature seeds. Defects in cellulosic ray architecture along with reduced levels of non-adherent and adherent mucilage were observed on the seeds of the AtCESA1T166A mutant. Reduced mucilage pectin synthesis was also reflected by a decrease in the level of uronic acid. Meanwhile, an increase in the degree of pectin methylesterification was also observed in the seed coat mucilage of AtCESA1T166A mutant. Change in seed development was further reflected by a delayed germination and about 50% increase in the accumulation of proanthocyanidins, which is known to bind pectin and inhibit seed germination as revealed by previous studies. Taken together, the results suggest a role of AtCESA1 phosphorylation on T166 in modulating mucilage pectin synthesis and methylesterification as well as cellulose synthesis with a role in seed development.

MUD1, a RING-v E3 ubiquitin ligase, has an important role in the regulation of pectin methylesterification in Arabidopsis seed coat mucilage

Pectin is one of the major components of plant primary cell wall polysaccharides. The degree of pectin methylesterification (DM) plays an important role in the process of plant growth. However, little is known about the underlying regulatory mechanisms during the process of pectin demethylesterification. Here, we characterized mucilage defect 1 (mud1), a novel Arabidopsis thaliana mutant, which displays increased mucilage adherence resulting from increased activities of pectin methylesterases (PMEs) and decreased degree of pectin methylesterification (DM). MUD1 encodes a nuclear protein with a Really Interesting New Gene (RING)-v domain and is highly expressed in developing seed coat when seed coat mucilage starts to accumulate. We have demonstrated that MUD1 has E3 ubiquitin ligase activity in vitro. The expression of PME-related genes, including MYB52, LUH, SBT1.7, PMEI6, and PMEI14 decreased considerably in mud1. We propose that MUD1 acts as an ubiquitin ligase potentially regulating the DM of pectin by post-transcriptionally removing proteins that normally negatively regulate the level or activity of PMEs in the seed coat mucilage.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

The Carboxyl-Terminus of TRANSPARENT TESTA GLABRA1 Is Critical for Its Functions in Arabidopsis.

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1′s functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Multiple strategies of plant colonization by beneficial endophytic Enterobacter sp. SA187.

Although many endophytic plant growth-promoting rhizobacteria have been identified, relatively little is still known about the mechanisms by which they enter plants and promote plant growth. The beneficial endophyte Enterobacter sp. SA187 was shown to maintain the productivity of crops in extreme agricultural conditions. Here we present that roots of its natural host (Indigofera argentea), alfalfa, tomato, wheat, barley and Arabidopsis are all efficiently colonized by SA187. Detailed analysis of the colonization process in Arabidopsis showed that colonization already starts during seed germination, where seed-coat mucilage supports SA187 proliferation. The meristematic zone of growing roots attracts SA187, allowing epiphytic colonization in the elongation zone. Unlike primary roots, lateral roots are significantly less epiphytically colonized by SA187. Root endophytic colonization was found to occur by passive entry of SA187 at lateral-root bases. However, SA187 also actively penetrates the root epidermis by enzymatic disruption of plant cell wall material. In contrast to roots, endophytic colonization of shoots occurs via stomata, whereby SA187 can actively re-open stomata similarly to pathogenic bacteria. In summary, several entry strategies were identified that allow SA187 to establish itself as a beneficial endophyte in several plant species, supporting its use as a plant growth-promoting bacterium in agriculture systems.

Galactoglucomannan structure of Arabidopsis seed-coat mucilage in GDP-mannose synthesis impaired mutants.

Cell-wall polysaccharides are synthesized from nucleotide sugars by glycosyltransferases. However, in what way the level of nucleotide sugars affects the structure of the polysaccharides is not entirely clear. guanosine diphosphate (GDP)-mannose (GDP-Man) is one of the major nucleotide sugars in plants and serves as a substrate in the synthesis of mannan polysaccharides. GDP-Man is synthesized from mannose 1-phosphate and GTP by a GDP-Man pyrophosphorylase, VITAMIN C DEFECTIVE1 (VTC1), which is positively regulated by the interacting protein KONJAC1 (KJC1) in Arabidopsis. Since seed-coat mucilage can serve as a model of the plant cell wall, we examined the influence of vtc1 and kjc1 mutations on the synthesis of mucilage galactoglucomannan. Sugar composition analysis showed that mannose content in adherent mucilage of kjc1 and vtc1 mutants was only 42% and 11% of the wild-type, respectively, indicating a drastic decrease of galactoglucomannan. On the other hand, structural analysis based on specific oligosaccharides released by endo-β-1,4-mannanase indicated that galactoglucomannan had a patterned glucomannan backbone consisting of alternating residues of glucose and mannose and the frequency of α-galactosyl branches was also similar to the wild type structure. These results suggest that the structure of mucilage galactoglucomannan is mainly determined by properties of glycosyltransferases rather than the availability of nucleotide sugars.