OsIDD2, a zinc finger and INDETERMINATE DOMAIN protein, regulates secondary cell wall formation.

Previously, we found 123 transcription factors (TFs) as candidate regulators of secondary cell wall (SCW) formation in rice by using phylogenetic and co-expression network analyses. Among them, we examined in this work the role of OsIDD2, a zinc finger and indeterminate domain (IDD) family TF. Its overexpressors showed dwarfism, fragile leaves, and decreased lignin content, which are typical phenotypes of plants defective in SCW formation, whereas its knockout plants showed slightly increased lignin content. The RNA-seq and quantitative reverse transcription polymerase chain reaction analyses confirmed that some lignin biosynthetic genes were downregulated in the OsIDD2-overexpressing plants, and revealed the same case for other genes involved in cellulose synthesis and sucrose metabolism. The transient expression assay using rice protoplasts revealed that OsIDD2 negatively regulates the transcription of genes involved in lignin biosynthesis, cinnamyl alcohol dehydrogenase 2 and 3 (CAD2 and 3), and sucrose metabolism, sucrose synthase 5 (SUS5), whereas an AlphaScreen assay, which can detect the interaction between TFs and their target DNA sequences, directly confirmed the interaction between OsIDD2 and the target sequences located in the promoter regions of CAD2 and CAD3. Based on these observations, we conclude that OsIDD2 is negatively involved in SCW formation and other biological events by downregulating its target genes.

Identification of transcription factors involved in rice secondary cell wall formation.

Using co-expression network analysis, we identified 123 transcription factors (TFs) as candidate secondary cell wall regulators in rice. To validate whether these TFs are associated with secondary cell wall formation, six TF genes belonging to the MYB, NAC or homeodomain-containing TF families were overexpressed or downregulated in rice. With the exception of OsMYB58/63-RNAi plants, all transgenic plants showed phenotypes possibly related to secondary cell wall alteration, such as dwarfism, narrow and dark green leaves, and also altered rice cinnamyl alcohol dehydrogenase 2 (OsCAD2) gene expression and lignin content. These results suggest that many of the 123 candidate secondary cell wall-regulating TFs are likely to function in secondary cell wall formation in rice. Further analyses were performed for the OsMYB55/61 and OsBLH6 TFs, the former being a TF in which the Arabidopsis ortholog is known to participate in lignin biosynthesis (AtMYB61) and the latter being one for which no previous involvement in cell wall formation has been reported even in Arabidopsis (BLH6). OsMYB55/61 and OsBLH6-GFP fusion proteins localized to the nucleus of onion epidermal cells. Moreover, expression of a reporter gene driven by the OsCAD2 promoter was enhanced in rice calli when OsMYB55/61 or OsBLH6 was transiently expressed, demonstrating that they function in secondary cell wall formation. These results show the validity of identifying potential secondary cell wall TFs in rice by the use of rice co-expression network analysis.

Mutation in the gene encoding ubiquitin ligase LRSAM1 in patients with Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) represents a family of related sensorimotor neuropathies. We studied a large family from a rural eastern Canadian community, with multiple individuals suffering from a condition clinically most similar to autosomal recessive axonal CMT, or AR-CMT2. Homozygosity mapping with high-density SNP genotyping of six affected individuals from the family excluded 23 known genes for various subtypes of CMT and instead identified a single homozygous region on chromosome 9, at 122,423,730-129,841,977 Mbp, shared identical by state in all six affected individuals. A homozygous pathogenic variant was identified in the gene encoding leucine rich repeat and sterile alpha motif 1 (LRSAM1) by direct DNA sequencing of genes within the region in affected DNA samples. The single nucleotide change mutates an intronic consensus acceptor splicing site from AG to AA. Direct analysis of RNA from patient blood demonstrated aberrant splicing of the affected exon, causing an obligatory frameshift and premature truncation of the protein. Western blotting of immortalized cells from a homozygous patient showed complete absence of detectable protein, consistent with the splice site defect. LRSAM1 plays a role in membrane vesicle fusion during viral maturation and for proper adhesion of neuronal cells in culture. Other ubiquitin ligases play documented roles in neurodegenerative diseases. LRSAM1 is a strong candidate for the causal gene for the genetic disorder in our kindred.

OsCAD2 is the major CAD gene responsible for monolignol biosynthesis in rice culm.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step of monolignol biosynthesis. The rice genome contains 12 CAD-like genes, and whereas the proteins encoded by OsCAD2 and OsCAD7 are known to function in monolignol biosynthesis, the degree to which these enzymes contribute to this process and the involvement of the enzymes encoded by the remaining ten genes is unclear. This paper investigates the role of OsCAD2 and the nine other OsCAD-like proteins in monolignol biosynthesis. Among the OsCAD genes analyzed, OsCAD2, an enzyme belonging to the bona fide CAD phylogenetic group, was the most abundantly expressed gene in the uppermost internode, and was expressed at levels that were more than seven times greater than those of the second most abundantly expressed gene, OsCAD1. Promoter-GUS analysis of OsCAD2 (pCAD::GUS) in the internode, sheath, and roots revealed that GUS expression was strong in tissues that accumulated high levels of lignin. Furthermore, expression always preceded lignin accumulation, showing the tight correlation between OsCAD2 expression and monolignol biosynthesis. Additionally, expression of pCAD::GUS was well synchronized with that of rice caffeic acid 3-O-methyltransferase (OsCOMT::GUS), suggesting that the two enzymes function cooperatively during monolignol biosynthesis. Co-expression network analysis of eight OsCAD genes further revealed that, among the OsCAD genes, expression of OsCAD2 was most tightly associated with the transcription of lignin biosynthesis-related genes. These results suggest that OsCAD2 is largely responsible for monolignol biosynthesis in rice, which is similar to that indicated for the predominant role of other plant bona fide CAD protein to monolignol biosynthesis.

Genotypic variations in non-structural carbohydrate and cell-wall components of the stem in rice, sorghum, and sugar vane.

We evaluated genetic variations in the non-structural carbohydrate (NSC) and the cell-wall components of stem in rice, sorghum, and sugar cane to assess the potential suitability of these gramineous crops for bioethanol production. For NSC, the maximum soluble sugar concentration was highest in sugar cane, followed by sorghum with sucrose. The major NSC in rice was starch, but there were wide variations in the starch to soluble sugar ratios among the cultivars. The total concentration of cell-wall components was negatively correlated with the NSC concentration, indicating competition for carbon sources. Among the cell-wall components, lignin was relatively stable within each group. The major sugar species composing hemicellulose was xylose in all crop groups, but there were differences in composition, with a higher fraction of arabinose and glucose in rice as compared to the other crops. In rice, there was less lignin than in sorghum or sugar cane; this might be advantageous for the efficient saccharification of cellulose.

Improvement in cell proliferation on silicone rubber by carbon nanotube coating.

Silicone rubbers are widely used as tissue implants because of their flexibility and chemical stability. However, they have limited cellular adhesiveness and may cause problems in the long term. In this study, a coating of carbon nanotubes (CNTs) was applied to silicone rubber to improve its cellular adhesiveness. Scanning electron micrograph of this coating revealed that CNTs had formed a densely packed meshwork; the Ra values and protein adsorption capacity were enhanced. Although the contact angle did not change after coating, it decreased after immersion into a culture medium. After cultivation for 6 d, while Saos-2 cells were hardly observed on untreated silicone, the cells proliferated on CNT-coated silicone. Thus, CNT coating might be a simple and effective solution to problems associated with silicone implants.

Adhesion of human osteoblast-like cells (Saos-2) to carbon nanotube sheets.

Carbon nanotubes (CNTs) exhibit excellent cell proliferation properties, which can serve as a scaffold for cell culturing. However, there are only a few reports on adhesion of osteoblast-like cells to a CNT sheet. In this study, we investigated adhesion of osteoblast-like cells to single-walled carbon nanotube (SWNT) and multi-walled carbon nanotube (MWNT) sheets and compared these adhesions with that on a cell culture polystyrene dish by using a cell adhesion test and a scanning electron microscope. The MWNT sheets exhibited faster adhesion of cells at an initial stage than SWNT sheets and cell culture polystyrene dish. The number of attached cells on the MWNT sheets seemed to be greater than on SWNT sheets and cell culture polystyrene. Moreover, the MWNT sheets exhibited both high speed and good capacity for cell adhesion. However, the surface of the MWNT sheets was such that it facilitated cell adherence but hindered the spreading of the attached cells. Interestingly, cell adhesion to CNT sheets was significantly influenced by pre-coating with serum. These results indicate that CNT sheets would play an important role in adsorption of serum proteins, which would consequently facilitate cell adhesion, and that the MWNT sheets have a high cell adhesiveness.

Molecular Breeding of Sorghum bicolor, A Novel Energy Crop.

Currently, molecular breeding is regarded as an important tool for the improvement of many crop species. However, in sorghum, recently heralded as an important bioenergy crop, progress in this field has been relatively slow and limited. In this review, we present existing efforts targeted at genetic characterization of sorghum mutants. We also comprehensively review the different attempts made toward the isolation of genes involved in agronomically important traits, including the dissection of some sorghum quantitative trait loci (QTLs). We also explore the current status of the use of transgenic techniques in sorghum, which should be crucial for advancing sorghum molecular breeding. Through this report, we provide a useful benchmark to help assess how much more sorghum genomics and molecular breeding could be improved.

Increased lodging resistance in long-culm, low-lignin gh2 rice for improved feed and bioenergy production.

Lignin modification has been a breeding target for the improvements of forage digestibility and energy yields in forage and bioenergy crops, but decreased lignin levels are often accompanied by reduced lodging resistance. The rice mutant gold hull and internode2 (gh2) has been identified to be lignin deficient. GH2 has been mapped to the short arm of chromosome 2 and encodes cinnamyl-alcohol dehydrogenase (CAD). We developed a long-culm variety, 'Leaf Star', with superior lodging resistance and a gh phenotype similar to one of its parents, 'Chugoku 117'. The gh loci in Leaf Star and Chugoku 117 were localized to the same region of chromosome 2 as the gh2 mutant. Leaf Star had culms with low lignin concentrations due to a natural mutation in OsCAD2 that was not present in Chugoku 117. However, this variety had high culm strength due to its strong, thick culms. Additionally, this variety had a thick layer of cortical fiber tissue with well-developed secondary cell walls. Our results suggest that rice can be improved for forage and bioenergy production by combining superior lodging resistance, which can be obtained by introducing thick and stiff culm traits, with low lignin concentrations, which can be obtained using the gh2 variety.

Ontogenetic and tissue-specific expression of preproghrelin in the Atlantic halibut, Hippoglossus hippoglossus L.

Ghrelin is a conserved vertebrate hormone that affects both GH release and appetite. We have cloned and characterized Atlantic halibut preproghrelin cDNA and examined for the first time preproghrelin expression during fish larval development using quantitative real-time PCR. In addition, cellular sites of expression in larvae and tissue-specific expression in 3-year-old halibut were studied. A full-length cDNA for preproghrelin was isolated from halibut stomach tissue. The 899 bp cDNA encodes an open reading frame of 105 amino acids that is comprised of a signal peptide and two peptides with high similarity to ghrelin and obestatin. The deduced amino acid sequence of halibut ghrelin peptide (GSSFLSPSHKPPKGKPPRA) shows significant conservation relative to other teleostean sequences and is identical to human ghrelin for the first seven amino acids of the sequence. The putative obestatin peptide is well-conserved among fishes but shares limited similarity with its human counterpart. Expression of ghrelin was localized to two different cell types in the stomach of larval halibut by in situ hybridization. However, sensitive PCR assays on tissues collected from 3-year-old fish additionally identified ghrelin transcripts in pyloric caecae, intestine, and in immature ovary and testis. Ontogenetic studies detected ghrelin expression prior to exogenous feeding during larval development (hatching and mouth-opening stages) with increased expression occurring through metamorphosis. This increase was pronounced during climax metamorphosis and coincided with stomach differentiation. Patterns of preproghrelin expression suggest that ghrelin has important roles during and after larval development in halibut, and that ghrelin is associated with digestive and gonadal tissues in this teleost.