TRM4 is essential for cellulose deposition in Arabidopsis seed mucilage by maintaining cortical microtubule organization and interacting with CESA3.

The differentiation of the seed coat epidermal (SCE) cells in Arabidopsis thaliana leads to the production of a large amount of pectin-rich mucilage and a thick cellulosic secondary cell wall. The mechanisms by which cortical microtubules are involved in the formation of these pectinaceous and cellulosic cell walls are still largely unknown. Using a reverse genetic approach, we found that TONNEAU1 (TON1) recruiting motif 4 (TRM4) is implicated in cortical microtubule organization in SCE cells, and functions as a novel player in the establishment of mucilage structure. TRM4 is preferentially accumulated in the SCE cells at the stage of mucilage biosynthesis. The loss of TRM4 results in compact seed mucilage capsules, aberrant mucilage cellulosic structure, short cellulosic rays and disorganized cellulose microfibrils in mucilage. The defects could be rescued by transgene complementation of trm4 alleles. Probably, this is a consequence of a disrupted organization of cortical microtubules, observed using fluorescently tagged tubulin proteins in trm4 SCE cells. Furthermore, TRM4 proteins co-aligned with microtubules and interacted directly with CELLULOSE SYNTHASE 3 in two independent assays. Together, the results indicate that TRM4 is essential for microtubule array organization and therefore correct cellulose orientation in the SCE cells, as well as the establishment of the subsequent mucilage architecture.

Extensive Natural Variation in Arabidopsis Seed Mucilage Structure.

Hydrated Arabidopsis thaliana seeds are coated by a gelatinous layer called mucilage, which is mainly composed of cell wall polysaccharides. Since mucilage is rich in pectin, its architecture can be visualized with the ruthenium red (RR) dye. We screened the seeds of around 280 Arabidopsis natural accessions for variation in mucilage structure, and identified a large number of novel variants that differed from the Col-0 wild-type. Most of the accessions released smaller RR-stained capsules compared to the Col-0 reference. By biochemically characterizing the phenotypes of 25 of these accessions in greater detail, we discovered that distinct changes in polysaccharide structure resulted in gelatinous coatings with a deceptively similar appearance. Monosaccharide composition analysis of total mucilage extracts revealed a remarkable variation (from 50 to 200% of Col-0 levels) in the content of galactose and mannose, which are important subunits of heteromannan. In addition, most of the natural variants had altered Pontamine Fast Scarlet 4B staining of cellulose and significantly reduced birefringence of crystalline structures. This indicates that the production or organization of cellulose may be affected by the presence of different amounts of hemicellulose. Although, the accessions described in this study were primarily collected from Western Europe, they form five different phenotypic classes based on the combined results of our experiments. This suggests that polymorphisms at multiple loci are likely responsible for the observed mucilage structure. The transcription of MUCILAGE-RELATED10 (MUCI10), which encodes a key enzyme for galactoglucomannan synthesis, was severely reduced in multiple variants that phenocopied the muci10-1 insertion mutant. Although, we could not pinpoint any causal polymorphisms in this gene, constitutive expression of fluorescently-tagged MUCI10 proteins complemented the mucilage defects of a muci10-like accession. This leads us to hypothesize that some accessions might disrupt a transcriptional regulator of MUCI10. Therefore, this collection of publicly-available variants should provide insight into plant cell wall organization and facilitate the discovery of genes that regulate polysaccharide biosynthesis.

[Biological characteristics of defensin and its disease-resistance genetic engineering].

Defensin is a kind of cysteine-rich small peptide, which has a broad spectrum of resistance to bacteria with a special resistance mechanism. So far, a large number of studies on defensins have been reported, and the different types of defensins have been isolated from various organisms. A broad prospect of application on defensins has been displayed both in genetic engineering and medicine field. This article reviewed the classification and the biological characteristics of defensins, including mammalian α-, ß-, θ-defensins, insect defensins, and plant defensins. The molecular structures, antibacterial activities, and antibacterial mechanisms of these definsins were summarized. The two mechanisms of de-fensin, including independent membrane mechanism and targeting of intracellular compounds by defensins, are ex-pounded. This paper also summarized the researches on isolation and expression of defensin genes and disease resistance genetic engineering of mammal and plant defensins. A prospect of the future applications of defensin both in biophar-maceutical sciences and plant disease resistance genetic engineering was discussed.