A naturally occurring mutation in an Arabidopsis accession affects a beta-D-galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed mucilage

The Arabidopsis thaliana accession Shahdara was identified as a rare naturally occurring mutant that does not liberate seed mucilage on imbibition. The defective locus was found to be allelic to the mum2-1 and mum2-2 mutants. Map-based cloning showed that MUCILAGE-MODIFIED2 (MUM2) encodes the putative beta-D-galactosidase BGAL6. Activity assays demonstrated that one of four major beta-D-galactosidase activities present in developing siliques is absent in mum2 mutants. No difference was observed in seed coat epidermal cell structure between wild-type and mutant seed; however, weakening of the outer tangential cell wall by chemical treatment resulted in the release of mucilage from mum2 seed coat epidermal cells, and the mum2 mucilage only increased slightly in volume, relative to the wild type. Consistent with the absence of beta-D-galactosidase activity in the mutant, the inner layer of mucilage contained more Gal. The allocation of polysaccharides between the inner and outer mucilage layers was also modified in mum2. Mass spectrometry showed that rhamnogalacturonan I in mutant mucilage had more branching between rhamnose and hexose residues relative to the wild type. We conclude that the MUM2/BGAL6 beta-D-galactosidase is required for maturation of rhamnogalacturonan I in seed mucilage by the removal of galactose/galactan branches, resulting in increased swelling and extrusion of the mucilage on seed hydration.     

The Arabidopsis MUM2 gene encodes a beta-galactosidase required for the production of seed coat mucilage with correct hydration properties

Seed coat development in Arabidopsis thaliana involves a complex pathway where cells of the outer integument differentiate into a highly specialized cell type after fertilization. One aspect of this developmental process involves the secretion of a large amount of pectinaceous mucilage into the apoplast. When the mature seed coat is exposed to water, this mucilage expands to break the primary cell wall and encapsulate the seed. The mucilage-modified2 (mum2) mutant is characterized by a failure to extrude mucilage on hydration, although mucilage is produced as normal during development. The defect in mum2 appears to reside in the mucilage itself, as mucilage fails to expand even when the barrier of the primary cell wall is removed. We have cloned the MUM2 gene and expressed recombinant MUM2 protein, which has beta-galactosidase activity. Biochemical analysis of the mum2 mucilage reveals alterations in pectins that are consistent with a defect in beta-galactosidase activity, and we have demonstrated that MUM2 is localized to the cell wall. We propose that MUM2 is involved in modifying mucilage to allow it to expand upon hydration, establishing a link between the galactosyl side-chain structure of pectin and its physical properties.     

Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis

In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1–5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion.     

抱茎独行菜MUM4基因的克隆及分析

抱茎独行菜(Lepidium perfoliatum L.)为十字花科具典型粘液繁殖体植物,为探究该植物中种皮粘液质基因（MUCILAGE-MODIFIED4,MUM4,该基因在拟南芥中编码NDP-L-鼠李糖合成酶)的功能,通过生物信息学分析设计引物克隆得到抱茎独行菜MUM4基因,命名为LpMUM4。同源比对分析结果表明,LpMUM4与拟南芥AtMUM4基因具有很高的一致性。qRT-PCR结果表明,该基因在抱茎独行菜各组织中均有表达,在角果和根中的表达量最高,且其表达量随角果的发育表现出渐强的趋势。免疫组织化学定位分析表明,LpMUM4基因于角果发育的早期阶段在内珠被和外珠被都有表达,而在外珠被的表皮和亚表皮中表达量更高,至角果发育的最后阶段,其表达集中于表皮和亚表皮层,这可能与抱茎独行菜的外珠被发育成种皮及粘液质的生成有关。将LpMUM4基因转化拟南芥,该基因的过表达对位于粘液质合成途径中的上游基因AtTTG1具有显著的抑制作用。表型比对观察显示,转基因拟南芥与其野生型植株形态无显著差异,这可能是因为抱茎独行菜种皮的发育和粘液质的形成是一个多基因调控的复杂过程,某一基因的过表达或许不会引起明显的表型变化。     

In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage

A comprehensive analysis was carried out of the composition of seed coat mucilage from Arabidopsis thaliana using the Columbia-0 accession. Pectinaceous mucilage is released from myxospermous seeds upon imbibition, and in Arabidopsis consists of a water-soluble, outer layer and an adherent, inner layer. Analysis of monosaccharide composition in conjunction with digestion with pectolytic enzymes conclusively demonstrated that the principal pectic domain of both layers was rhamnogalacturonan I, and that in the outer layer this was unbranched. The macromolecular characteristics of the water-soluble mucilage indicated that the rhamnogalacturonan molecules in the outer layer were in a slightly expanded random-coil conformation. The inner, adherent layer remained attached to the seed, even after extraction with acid and alkali, suggesting that its integrity was maintained by covalent bonds. Confocal microscopy and monosaccharide composition analyses showed that the inner layer can be separated into two domains. The internal domain contained cellulose microfibrils, which could form a matrix with RGI and bind it to the seed. In effect, in the mum5-1 mutant where most of the inner and outer mucilage layers were water soluble, cellulose remained attached to the seed coat. Immunolabeling with anti-pectin antibodies indicated the presence of galactan and arabinan in the inner layer, with the latter only present in the non-cellulose-containing external domain. In addition, JIM5 and JIM7 antibodies labeled different domains of the inner layer, suggesting the presence of stretches of homogalacturonan with different levels of methyl esterification.     

The role of COBRA‐LIKE 2 function,as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization

The production of hydrophilic mucilage along the course of seed coat epidermal cell differentiation is a common adaptation in angiosperms. Previous studies have identified COBRA‐LIKE 2 (COBL2), a member of the COBRA‐LIKE gene family, as a novel component required for crystalline cellulose deposition in seed coat epidermal cells. In recent years, Arabidopsis seed coat epidermal cells (SCEs), also called mucilage secretory cells, have emerged as a powerful model system for the study of plant cell wall components biosynthesis, secretion, assembly and de muro modification. Despite accumulating data, the molecular mechanism of COBL function remains largely unknown. In the current research, we utilized genetic interactions to study the role of COBL2 as part of the protein network required for seed mucilage production. Using correlative phenotyping of structural and biochemical characteristics, unique features of the cobl2 extruded mucilage are revealed, including: ‘unraveled’ ray morphology, loss of primary cell wall ‘pyramidal’ organization, reduced Ruthenium red staining intensity of the adherent mucilage layer, and increased levels of the monosaccharides arabinose and galactose. Examination of the cobl2cesa5 double mutant provides insight into the interface between COBL function and cellulose deposition. Additionally, genetic interactions between cobl2 and fei1fei2 as well as between each of these mutants to mucilage‐modified 2 (mum2) suggest that COBL2 functions independently of the FEI‐SOS pathway. Altogether, the presented data place COBL2 within the complex protein network required for cell wall deposition in the context of seed mucilage and introduce new methodology expending the seed mucilage phenotyping toolbox.     

Differentiation of mucilage secretory cells of the Arabidopsis seed coat

In some plant species, including Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type with a unique morphology and containing large quantities of polysaccharide mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under normal laboratory conditions. Thus, the Arabidopsis seed coat offers a unique system with which to use genetics to identify genes controlling cell morphogenesis and complex polysaccharide biosynthesis and secretion. As a first step in the application of this system, we have used microscopy to investigate the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis and the synthesis, secretion, and extrusion of mucilage. During seed coat development in Arabidopsis, the epidermal cells of the outer ovule integument grow and differentiate into cells that produce large quantities of mucilage between the primary cell wall and plasma membrane. Concurrent with mucilage production, the cytoplasm is shaped into a column in the center of the cell. Following mucilage secretion the cytoplasmic column is surrounded by a secondary cell wall to form a structure known as the columella. Thus, differentiation of the seed coat mucilage cells involves a highly regulated series of events including growth, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis.     

抱茎独行菜种皮粘液质相关基因TTG1的克隆、表达分析及功能鉴定

抱茎独行菜（Lepidium perfoliatum L.）为十字花科具典型粘液质繁殖体植物,而TTG1基因（Transpa-rent testa glabra 1）所编码的蛋白是调控种皮细胞分化并影响粘液质释放的转录因子。目前关于TTG1基因在粘液质繁殖体植物中的研究报道较少,为探究TTG1基因在抱茎独行菜粘液质发育中的作用,本研究利用同源克隆技术获得抱茎独行菜TTG1基因cDNA开放阅读框（ORF）序列,命名为LpTTG1。序列分析表明,该基因ORF全长为1032 bp,编码343个氨基酸,含有WD40基序;qRT-PCR分析结果显示,该基因在抱茎独行菜各组织中均有表达,反映了该基因功能的多样性;免疫组织化学定位结果表明,LpTTG1在种子发育过程中内珠被和外珠被的表达水平变化与外珠被粘液质的合成过程相一致,推测该基因可能参与调控抱茎独行菜种皮的发育及粘液质的形成。将LpTTG1基因转化拟南芥,该基因的过量表达显著促进了粘液质合成途径下游基因AtMUM4在角果中的表达,表明该基因有可能参与粘液质合成途径调控,并促进下游产物MUM4的产生。然而,对LpTTG1转基因拟南芥与野生型植株表型的比较发现,两者种子形态及粘液质分泌与释放方式均无显著差异,这可能是因为抱茎独行菜种皮发育和粘液质形成是一个多基因调控的复杂过程,某一基因的过量表达也许不会引起明显的表型变化。     

The FEI2-SOS5 pathway and CELLULOSE SYNTHASE 5 are required for cellulose biosynthesis in the Arabidopsis seed coat and affect pectin mucilage structure

A common adaptation in angiosperms is the deposition of hydrophilic mucilage into the apoplast of seed coat epidermal cells during the course of their differentiation. Upon imbibition, seed mucilage, composed mainly of carbohydrates (i.e. pectins, hemicelluloses and glycans) expands rapidly, encapsulating the seed and aiding in seed dispersal and germination. The FEI1/FEI2 receptor-like kinases and the SOS5 extracellular GPI-anchored protein were previously shown to act on a pathway regulating cellulose biosynthesis during Arabidopsis root elongation. In the highlighted study, we demonstrated that FEI2 and SOS5 regulate the production of the cellulosic rays deposited across the inner adherent-layer of seed mucilage. Mutations in either fei2 or sos5 disrupted the formation of rays, which was associated with an increase in the soluble, outer layer of pectin mucilage and accompanied by a reduction in the inner adherent-layer. Mutations in CELLULOSE SYNTHASE 5 also led to reduced rays and mal-partitioning of the pectic component of seed mucilage, further establishing a structural role for cellulose in seed mucilage. Here, we show that FEI2 expressed from a CaMV 35S promoter complemented both root and seed mucilage defects of the fei1 fei2 double mutant. In contrast, expression of FEI1 from a 35S promoter complemented the root, but not the seed phenotype of the fei1 fei2 double mutant, suggesting that unlike in the root, FEI2 plays a unique and non-redundant role in the regulation of cellulose synthesis in seed mucilage. Altogether, these data suggest a novel role for cellulose in anchoring the pectic component of seed mucilage to the seed surface and indicate that the FEI2 protein has a function distinct from that of FEI1, despite the high sequence similarity of these RLKs.     

Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment

Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes. Here, we investigated the role of cellulose synthase (CESA) subunits CESA2, CESA5, and CESA9 in the seed coat epidermis. We characterized the roles of these CESA proteins in the seed coat by analyzing cell wall composition and morphology in cesa mutant lines. Mutations in any one of these three genes resulted in lower cellulose content, a loss of cell shape uniformity, and reduced radial wall integrity. In addition, we found that attachment of the mucilage halo to the parent seed following extrusion is maintained by cellulose-based connections requiring CESA5. Hence, we show that cellulose fulfills an adhesion role between the extracellular mucilage matrix and the parent cell in seed coat epidermal cells. We propose that mucilage remains attached to the seed coat through interactions between components in the seed mucilage and cellulose. Our data suggest that CESA2 and CESA9 serve in radial wall reinforcement, as does CESA5, but CESA5 also functions in mucilage biosynthesis. These data suggest unique roles for different CESA subunits in one cell type and illustrate a complex role for cellulose biosynthesis in plant developmental biology.     

Local Evolution of Seed Flotation in Arabidopsis

Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 β-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed.