Vitamins and their derivatives synergistically promote hair shaft elongation ex vivo via PlGF/VEGFR-1 signalling activation.

BACKGROUND: Although vitamins or their derivatives (Vits), such as panthenyl ethyl ether, tocopherol acetate, and pyridoxine, have been widely used in topical hair care products, their efficacy and mode of action have been insufficiently studied. OBJECTIVE: To elucidate the biological influence of Vits on hair follicles and determine the underlying mechanisms. METHODS: A mouse vibrissa hair follicle organ culture model was utilized to evaluate the effects of Vits on hair shaft elongation. Gene and protein expression analyses and histological investigations were conducted to elucidate the responsible mechanisms. A human hair follicle cell culture was used to assess the clinical relevance. RESULTS: In organ culture models, the combination of panthenyl ethyl ether, tocopherol acetate, and pyridoxine (namely, PPT) supplementation significantly promoted hair shaft elongation. PPT treatment enhanced hair matrix cell proliferation by 1.9-fold compared to controls, as demonstrated by Ki67-positive immunoreactivity. PPT-treated mouse dermal papillae exhibited upregulated Placental growth factor (Plgf) by 1.6-fold compared to controls. Importantly, the addition of PlGF neutralizing antibodies to the ex vivo culture diminished the promotive effect on hair growth and increase in VEGFR-1 phosphorylation achieved by PPT. A VEGFR-1 inhibitor also inhibited the promotion of hair growth. Microarray analysis suggested synergistic summation of individual Vits' bioactivity, putatively explaining the effect of PPT. Moreover, PPT increased PlGF secretion in cultured human dermal papilla cells. CONCLUSION: Our findings suggested that PPT promoted hair shaft elongation by activating PlGF/VEGFR-1 signalling. The current study can shed light on the previously underrepresented advantage of utilizing Vits in hair care products.

[A Rapid Determination Method of Metals in Foods for Emergency Response to Health Crisis].

A rapid determination method for emergency response to health crisis caused by metals in foods, was developed using microwave decomposition equipment and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The method was assessed for 18 elements (Al, As, B, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sb, Se, Sn, Tl and Zn) in 5 kinds of beverages and 7 kinds of foods. A single-laboratory method validation study was performed using food samples added with 20 mg/kg of each metal. Trueness was 88-108% and intralaboratory reproducibility was 0.2-11.3%. Time required for analysis was less than 3 hr. Thus, the presented method could be useful for rapid analysis of metals involved food poisoning cases.

Anti-inflammatory effects of differential molecular weight Hyaluronic acids on UVB-induced calprotectin-mediated keratinocyte inflammation.

BACKGROUND: The biological functions of Hyaluronic acid are related to its molecular weight and binding to its receptor, Toll-like receptor4 (TLR4) or CD44. Recent studies have shown that low-molecular-weight Hyaluronic acid (LMW-HA) exhibits proinflammatory effects, while high-molecular-weight Hyaluronic acid (HMW-HA) functions as an anti-inflammatory factor. UVB-induced epidermal inflammation is mainly mediated by endogenous molecules, such as damage-associated molecular patterns (DAMPs), that cause severe skin damage by activating TLR signaling pathways. OBJECTIVE: Since both LMW- and HMW-HA have inhibitory functions on TLR-mediated macrophage inflammation, HA is assumed to suppress UVB-induced DAMP-mediated inflammation in the skin. In this study, both Ultra- low-molecular-weight Hyaluronic acid (uLMW-HA) and HMW-HA were found to inhibit UVB-induced keratinocyte inflammation. METHODS: HaCaT cells were treated with medium containing Hyaluronic acid at the appropriate concentration after 15 mJ/cm2 irradiation. Secreted protein levels were determined with ELISA kits. Expression levels of proteins downstream of TLR4 were detected by Simple Western system. RESULTS: By competitively binding to TLR4, uLMW-HA downregulated Calprotectin-induced TRAF6 expression, which might be the direct process by which uLMW-HA decreased UVB-induced IL-6 secretion. Reduced　CD44 variant (CD44v) expression in keratinocytes attenuated the inhibitory effect of both uLMW-HA and HMW-HA on UVB-induced inflammation, which indicated the involvement of CD44v in HA-regulated anti-inflammatory activity. CONCLUSION: Overall, this research indicates that Hyaluronic acid is more than a moisturizer; it is also a biologically effective material that can prevent the excessive skin inflammation caused in daily life, especially in the late stages after sunburn.

Variations in radioactive cesium accumulation in wheat germplasm from fields affected by the 2011 Fukushima nuclear power plant accident.

Decreasing the transfer of radioactive cesium (RCs) from soil to crops has been important since the deposition of RCs in agricultural soil owing to the Fukushima nuclear power plant accident of 2011. We investigated the genotypic variation in RCs accumulation in 234 and 198 hexaploid wheat (Triticum spp.) varieties in an affected field in 2012 and 2013, respectively. The effects of soil exchangeable potassium (ExK) content to RCs accumulation in wheat varieties were also evaluated. A test field showed fourfold differences in soil ExK contents based on location, and the wheat varieties grown in areas with lower soil ExK contents tended to have higher grain RCs concentrations. RCs concentrations of shoots, when corrected by the soil ExK content, were positively significantly correlated between years, and RCs concentrations of shoots were significantly correlated with the grain RCs concentration corrected by the soil ExK content. These results indicated that there were genotypic variations in RCs accumulation. The grain to shoot ratio of RCs also showed significant genotypic variation. Wheat varieties with low RCs accumulations were identified. They could contribute to the research and breeding of low RCs accumulating wheat and to agricultural production in the area affected by RCs deposition.

Evaluation of the resistance effect of QTLs derived from wild soybean (Glycine soja) to common cutworm (Spodoptera litura Fabricius).

Common cutworm (CCW) is a serious herbivorous insect pest of soybean. Previously, we conducted an antixenosis bioassay (measuring feeding preference) with CCW using recombinant inbred lines (RILs) derived from a cross between a wild soybean (Glycine soja) collected in Hiroshima prefecture (JP110755) and the leading cultivar, Fukuyutaka. The analysis revealed quantitative trait loci (QTLs) for antixenosis resistance, qRslx3 and qRslx4. In the present study we developed another RIL population using Fukuyutaka and a different G. soja, collected in Kumamoto prefecture (G406). An analysis revealed an antixenosis resistance QTL on chromosome 7, and the resistant allele of the QTL was derived from G406. The chromosomal position of the QTL was almost the same as that of CCW-2, a previously-reported antibiosis resistance QTL for CCW, detected in a F2 population derived from a cross between Fukuyutaka and a resistant cultivar Himeshirazu. These QTLs could be the same locus; however, G406 and Himeshirazu are likely to possess different alleles, because Himeshirazu allele exhibits no antixenosis effect. We expect that pyramiding of the resistance QTLs derived from G. soja will contribute to the development of CCW resistant cultivars.

Isolation and characterization of induced mutants in the gene associated with seed cadmium accumulation in soybean.

Food contamination by cadmium (Cd) is a serious threat to human health. Thus, it is imperative to prevent Cd accumulation in staple crops like soybean. The development of low Cd accumulating cultivars is an effective solution. To this end, it is essential to identify the gene(s) controlling seed Cd accumulation. Although Glyma.09G055600 (GmHMA3) seems to be associated with Cd accumulation in soybean, it has not been established if it is responsible for seed Cd accumulation. In the present study, the effect of GmHMA3 on seed Cd accumulation in soybean was validated using three independent GmHMA3 mutants isolated from an ethyl methanesulfonate-induced soybean mutant library. Each of mutant had an amino acid substitution in GmHMA3 and segregating progenies were developed by crossing the original cultivar with each of the three mutants. The relationship between these three mutations and seed Cd accumulation was investigated. While two of them significantly increased seed Cd accumulation corresponding to previous reports of a natural missense mutation in GmHMA3, the other slightly decreased seed Cd accumulation. Overall, these results indicate that GmHMA3 is responsible for seed Cd accumulation in soybean.

Rapid Quantification of Radioactive Strontium-90 in Fresh Foods via Online Solid-Phase Extraction-Inductively Coupled Plasma-Dynamic Reaction Cell-Mass Spectrometry and Its Comparative Evaluation with Conventional Radiometry.

This paper describes a rapid quantification method for radioactive strontium (90Sr) in fresh foods (perishable foods) and has been comparatively evaluated with the common classical radiometric quantification method. Inductively coupled plasma-dynamic reaction cell-mass spectrometry with online solid-phase extraction (cascade-ICP-MS) rapidly determines 90Sr in a pure water-based sample. Despite its advantages, its application to fresh foods (perishable foods) has not yet been reported; however, the analytical potential of this method for fresh foods must be evaluated. In this study, 90Sr was determined in 12 fresh foods via improved cascade-ICP-MS (Icas-ICP-MS). Addition and recovery tests were demonstrated using real samples of grape, apple, peach, Japanese pear, rice, buckwheat, soybean, spinach, shiitake mushroom, grass, sea squirt, and flounder. With a decomposed solution of Japanese pear, the measurement value coincided with the amount of spiked 90Sr. The reproducibility of the measurements was represented by relative standard deviations of 14.2 and 5.0% for spiked amounts of 20 and 200 Bq/kg, respectively (n = 10), and the recovery rates were 93.7 ± 7.1%. In this case, the limit of detection (LOD) was 2.2 Bq/kg (=0.43 pg/kg). These results were compared with the data obtained using a common classical radiometric quantification method (nitrate precipitation-low background gas flow counter (LBC) method) in the same samples. Both the methods showed equivalent performances with regard to reproducibility, precision, and LODs but different analysis times. Icas-ICP-MS required ∼22 min for analysis, whereas the nitrate precipitation-LBC method required 20 days, confirming that Icas-ICP-MS is the suitable method for analyzing 90Sr in fresh foods.

Genetic and functional characterization of Sg-4 glycosyltransferase involved in the formation of sugar chain structure at the C-3 position of soybean saponins.

Triterpenoid saponins are specialized metabolites, which are abundant in soybean seeds. They have a wide variety of effects on human health and physiology. The composition of sugar chain attached to the aglycone moiety of saponins can be controlled by genetic loci, such as Sg-1, 3, and 4. Among these, the homozygous recessive sg-4 impairs the accumulation of saponins that have an arabinose moiety at the second position of the C-3 sugar chain (i.e., saponins Ad and ßa) in the hypocotyls. In this study, we found that sg-4 cultivars are disabled in Glyma.01G046300 expression in hypocotyls. This gene encodes a putative glycosyltransferase (UGT73P10) and is a homolog of GmSGT2 (UGT73P2) whose recombinant protein has been previously shown, in vitro, to conjugate the second galactose moiety at the C-3 position of soyasapogenol B monoglucuronide (SBMG). The sg-4 phenotype (absence of saponins Ad and ßa in hypocotyls) was restored by introducing the Glyma.01G046300 genomic DNA fragment that was obtained from the Sg-4 cultivar 'Ibarakimame 7'. Although Glyma.01G046300 is expressed in the cotyledons even in the sg-4 cultivars such as 'Enrei', the induced premature stop codon mutation (W244*) resulted in impaired accumulation of saponin ßa in this tissue also in the 'Enrei' genetic background. Furthermore, the recombinant Glyma.01G046300 protein was shown to conjugate the second Ara moiety at the C-3 position of SBMG using UDP-Ara as a sugar donor. These results demonstrate that Sg-4 is responsible for conjugation of the second Ara moiety at the C-3 position of soybean saponins.

Identification and dissection of single seed weight QTLs by analysis of seed yield components in soybean.

Single seed weight (SSW), or seed size, is a seed yield components (SYC) in soybean, and it is suggested that the genetic factors regulating SSW are involved in the control of other SYCs. The quantitative trait loci (QTLs) for SSW and their effects on the other SYCs were investigated using a recombinant inbred line population derived from typical small- and large-seeded cultivars that were cultivated in two different environments. QTL analysis detected four environmentally stable QTLs for SSW, two of which coincided with the defined loci, qSw17-1 and Ln. The effects of the other loci, qSw12-1 and qSw13-1, were confirmed by analyzing residual heterozygous line progenies derived from the recombinant population. These four QTL regions were also involved in the control of an additional SYC, namely the large-seeded allele at each locus that reduced either the number of pods per plant or the number of ovules per pod. These results suggest the presence of at least two different regulatory mechanisms for SSW. Isolation of genes responsible for these QTLs provides an important tool in the understanding and utilization of SSW diversity for soybean breeding.

Isolation and Characterization of the Soybean Sg-3 Gene that is Involved in Genetic Variation in Sugar Chain Composition at the C-3 Position in Soyasaponins.

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.

A vacuolar sorting receptor-independent sorting mechanism for storage vacuoles in soybean seeds.

The seed storage proteins of soybean (Glycine max) are composed mainly of glycinin (11S globulin) and ß-conglycinin (7S globulin). The subunits of glycinin (A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3) are synthesized as a single polypeptide precursor. These precursors are assembled into trimers with a random combination of subunits in the endoplasmic reticulum, and are sorted to the protein storage vacuoles. Proteins destined for transport to protein storage vacuoles possess a vacuolar sorting determinant, and in this regard, the A1aB1b subunit contains a C-terminal peptide that is sufficient for its sorting to protein storage vacuoles. The A3B4 subunit, however, lacks a corresponding C-terminal sorting determinant. In this study, we found that, unlike the A1aB1b subunit, the A3B4 subunit does not bind to previously reported vacuolar sorting receptors. Despite this difference, we observed that the A3B4 subunit is sorted to protein storage vacuoles in a transgenic soybean line expressing the A3B4 subunit of glycinin. These results indicate that a protein storage vacuolar sorting mechanism that functions independently of the known vacuolar sorting receptors in seeds might be present in soybean seeds.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo).

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism (mog) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf (rl) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD (PPD) that regulates arrest of cell divisions in meristematic cells. We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes.

Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69.

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized ß-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BACKGROUND: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. RESULTS: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. CONCLUSIONS: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.

Diversity of seed cesium accumulation in soybean mini-core collections.

Radiocesium is an extremely harmful radionuclide because of its long half-life; it is important to reduce its transfer from contaminated soil into crops. Here we surveyed genetic variation for seed cesium (Cs) concentration in soybean mini-core collections representing large genetic diversity. The collections grown over 3 years in rotational paddy fields exhibited varying seed Cs concentrations with significant year-to-year correlations, although the phenotypic stability of Cs concentration was lower than that of the congeners potassium (K) and rubidium (Rb). Although Cs is supposedly accumulated in plants via the K transport system, there was no apparent relationship between Cs and K concentrations, whereas a clear positive correlation was observed between Cs and Rb concentrations. Cs and K concentrations in seed showed slightly positive and negative correlations, respectively, with days to flowering. We selected several high or low Cs accumulator candidates on the basis of the 3 years of seed concentration data. These two groups showed significantly different seed Cs concentrations in another field. The differences could not be explained by flowering time alone. These results suggest that genetic variation for seed Cs concentration is present in soybean germplasm and would be useful for breeding low Cs-accumulating varieties.

Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)-related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio-temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3-MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale-colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.

A mutable albino allele in rice reveals that formation of thylakoid membranes requires the SNOW-WHITE LEAF1 gene.

Active DNA transposons are important tools for gene functional analysis. The endogenous non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate various transposon-insertion mutants because nDart1-0 elements tend to insert into genic regions under natural growth conditions. We have developed a specific method (nDart1-0-iPCR) for efficient detection of nDart1-0 insertions and successfully identified the SNOW-WHITE LEAF1 (SWL1) gene in a variegated albino (swl1-v) mutant obtained from the nDart1-promoted rice tagging line. The variegated albino phenotype was caused by insertion and excision of nDart1-0 in the 5'-untranslated region of the SWL1 gene predicted to encode an unknown protein with the N-terminal chloroplast transit peptide. SWL1 expression was detected in various rice tissues at different developmental stages. However, immunoblot analysis indicated that SWL1 protein accumulation was strictly regulated in a tissue-specific manner. In the swl1 mutant, formations of grana and stroma thylakoids and prolamellar bodies were inhibited. This study revealed that SWL1 is essential for the beginning of thylakoid membrane organization during chloroplast development. Furthermore, we provide a developmental perspective on the nDart1-promoted tagging line to characterize unidentified gene functions in rice.

Seed coat pigmentation in transgenic soybean expressing the silencing suppressor 2b gene of Cucumber mosaic virus.

KEY MESSAGE: Soybean expressing the Cucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing. RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor (I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation.

Latent TGF-ß binding protein 4 promotes elastic fiber assembly by interacting with fibulin-5.

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-ß binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-ß because TGF-ß-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-ß-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.