Coproduction of lipids and carotenoids by the novel green alga Coelastrella sp. depending on cultivation conditions.

A novel green alga Coelastrella sp. D3-1 was isolated, and its unique and significant lipid and carotenoid coproduction capability was characterised depending on cultivation conditions. The main component of produced lipids was triacylglycerol under nutrient depletion conditions, in which fatty-methyl-esters made up 20-44% of the dry cell weight (DCW) and consisted of abundant C16:0 and C18:1 fatty acids. The red (orange)-stage cells also produced a large portion of carotenoids (38.5% of the DCW) involving echinenone, canthaxanthin, and astaxanthin as major components accumulated over only 5-6 days under optimal conditions. Stress tests revealed resistance of the cells to pH 2-11, high temperatures (40-60 °C), ultraviolet irradiation, drought, and H2O2 treatment, thereby showing a robust nature. Both green- and red (orange)-stage cell extracts also showed antioxidant and anti-inflammatory abilities, implying that they have significant functions as useful biorefinery materials.

Molecular characterization of a novel soybean gene encoding a neutral PR-5 protein induced by high-salt stress.

In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four alpha-helixes and 16 beta-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2h after initiation of the stress and was highly induced between 18-72h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean.

Crucial roles of MZF1 and Sp1 in the transcriptional regulation of the peptidylarginine deiminase type I gene (PADI1) in human keratinocytes.

Peptidylarginine deiminases (PADs) catalyze the conversion of protein-bound arginine residues into citrulline residues in a calcium-dependent manner. The PAD1 gene (PADI1) is expressed in a few tissues, including the epidermis, where the protein is detected with a higher level in the more differentiated keratinocytes. Using quantitative reverse transcription-PCR experiments, we show that PADI1 mRNAs are more abundant in keratinocytes cultured with 1.2 than 0.15 mM calcium. We cloned and characterized the promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI1 coupled to the luciferase gene. We found that as few as 195 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Mutations of MZF1- or Sp1-binding sites markedly reduced PADI1 promoter activity. Chromatin immunoprecipitation assays revealed that MZF1 and Sp1/Sp3 bind to this region in vivo. Furthermore, MZF1 or Sp1 small interfering RNAs (siRNAs) effectively diminished PADI1 expression in keratinocytes cultured in both low- and high-calcium-containing medium. In addition, the expression of MZF1 and PAD1 increased in parallel when normal human epidermal keratinocytes underwent differentiation. These data indicate that MZF1 and Sp1/Sp3 binding to the promoter region drive the PADI1 expression.

Molecular characterization of a novel salt-inducible gene for an OSBP (oxysterol-binding protein)-homologue from soybean.

Oxysterol-binding protein (OSBP) and its homologues constitute a protein family in many eukaryotes from yeast to humans, which are involved in cellular lipid metabolism, vesicle transport and signal transduction. In this study, we characterized a novel salt-inducible gene for an OSBP-homologue from soybean (Glycine max [L.] Merr.). The soybean OSBP-homologous gene, denoted as G. max OSBP (GmOSBP), encoded a 789 aa putative protein with two characteristic domains; the pleckstrin homology (PH) domain and the ligand-binding (LB) domain, in the N- and C-terminus, respectively. The GmOSBP-PH domain showed localization into/around the nucleus in a transient subcellular localization assay. The phylogenetic relationship of the GmOSBP-LB domain to those in other OSBP-homologues suggested that GmOSBP might bind a lipid molecule(s) different from the ligand-candidates found for the human/yeast OSBP-homologues. The GmOSBP gene was constitutively transcribed in all of the soybean organs examined--root, stem and trifoliate leaf--at low levels and was highly induced in all these organs by high-salt stress (300 mM NaCl). Interestingly, gene expression of GmOSBP was also markedly induced in the senesced soybean cotyledon, which contains high levels of a variety of cellular lipids utilized for energy for germination and as membrane components. Therefore, we suggest that GmOSBP may be involved in some physiological reactions for stress-response and cotyledon senescence in the soybean.

Purification and characterization of three neutral extracellular isoperoxidases from rye leaves.

Rye (Secale cereale L.) seedlings; contain two major flavone glucuronides, luteolin 7-O-diglucuronyl-4'-O-glucuronide (L3GlcUA) (1) and luteolin 7-O-diglucuronide (L2GlcUA) (2) in abundance in the apoplast of primary leaves; express a large number of peroxidase isoenzymes; and release H(2)O(2) into the apoplast during primary leaf development. We purified and characterized three neutral extracellular peroxidase isoenzymes (rPOXs N1, N2, and N3) that can oxidize L2GlcUA as a natural substrate. The isoelectric points and molecular weights of rPOXs N1, N2, and N3 were 6.1, 7.2, and 6.3, and 42, 37, and 51 kDa, respectively. The optimum pH of the rPOXs N1, N2, and N3 were 5.5, 5.5, and 8.5, respectively, and their optimum temperatures ranged from 45 to 50 degrees C for all isoenzymes. rPOXs N1, N2, and N3 recognized flavonoids with 3', 4'-OH groups as potential substrates, but not flavonoids with a glycosylated 4'-OH group or those without a 3'-OH group. The activities on phenol-type substrates were high in the order of guaiacol>catechol>o-cresol for all isoenzymes. rPOXs N1, N2, and N3 exhibited broad reactivity with endogenous hydrogen donors including luteolin glucuronides derived from the apoplast of rye primary leaves.

NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes.

Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues into citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) bind to this region in vitro and in vivo. Moreover, mutation of the Sp1- or NF-Y-binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region.

Molecular cloning and characterization of a novel soybean gene encoding a leucine-zipper-like protein induced to salt stress.

To understand molecular responses to salt stress in soybean (Glycine max [L.] Merr.), we identified 106 salt-inducible soybean genes that expressed differentially at 72 h after 100 mM NaCl treatment using the cDNA-amplified fragment length polymorphism (AFLP) method. The genes were designated as G. max Transcript-Derived Fragments (GmTDFs). Among these genes, we characterized a soybean gene GmTDF-5 that encoded an unknown protein of 367 amino acids. The GmTDF-5 protein was a putative cytosolic protein with two leucine-zipper motifs at the N-terminal and was calculated as 40.7 kDa. Southern blot analysis indicated that GmTDF-5 presents as an intron-less single gene on soybean genome and possibly distributes narrowly throughout the higher plants. By 100 mM NaCl treatment, the gene expression of GmTDF-5 was induced in the stem and lower-expanded leaf, and the amount of mRNA increased 5.1- and 2.0-fold up to 72 h, respectively. Interestingly, GmTDF-5 expression in the upper-leaf appeared dramatically with 10.0-fold increase at 72 h after the salt stress, but not until 48 h. Hyperosmotic pressure (mannitol treatment) and dehydration also caused the increases similar to NaCl treatment in the levels of GmTDF-5 expression. These results suggest that GmTDF-5 might be a novel cytosolic leucine-zipper-like protein functioning in mature organs of soybean shoot against water-potential changes.

Regulation of the expression of peptidylarginine deiminase type II gene (PADI2) in human keratinocytes involves Sp1 and Sp3 transcription factors.

Peptidylarginine deiminases (PAD) convert protein-bound arginine residues into citrulline residues in a Ca(2+) ion-dependent manner. Among the five isoforms (PAD1, 2, 3, 4, and 6) existing in rodents and humans, PAD2 is the most widely expressed in both species, tissues, and organs. In order to study the mechanisms regulating the expression of the human PAD2 gene, PADI2, we characterized its promoter region using transfected human keratinocytes. A series of reporter gene constructions derived from the 2 kb region upstream of the transcription initiation site defined a minimal promoter sequence from nucleotides -132 to -41. This PADI2 region is GC-rich and lacks canonical TATA and CAAT boxes. Investigation of cis-acting elements in the region, further deletion analyses and electrophoretic mobility shift assays using specific antibodies revealed four Sp1-binding sites and identified Sp1 and Sp3 as binding factors important for the promoter activity. These results suggest that Sp1/Sp3 cooperation may provide a mechanism to control the transcription of PADI2.

Cloning of two cysteine proteinase genes, CysP1 and CysP2, from soybean cotyledons by cDNA representational difference analysis.

By cDNA representational difference analysis (cDNA RDA) and rapid amplification of cDNA ends (RACE), we isolated two cDNAs, CysP1 and CysP2, from the cotyledons of growing soybean (Glycine max (L.) Merr.) seedlings. CysP1 cDNA is 1265 bp in size with a 1089-bp open reading frame (ORF), and CysP2 cDNA is 1270 bp in size with a 1089-bp ORF. Either CysP1 or CysP2 encodes a cysteine proteinase (CPR) with a C-terminal KDEL motif. The similarities between CysP1 and CysP2 are 93.5% in nucleotide sequences and 93.6% in deduced amino acid sequences. Furthermore, we determined the nucleotide sequences of CysP1 genomic DNA (1846 bp) and CysP2 genomic DNA (1831 bp). Both consisted of four exons and three introns. RNA-blot analysis revealed that both CysP1 and CysP2 were expressed from 6 days after germination (DAG) to 13 or 14 DAG in the cotyledons of growing seedlings and did so in a short period (9-12 DAG) in rejuvenated cotyledons. The transcripts of CysP1 and CysP2 were also detected in the root, flower and pod of soybean plants. Their physiological roles in the cotyledons of growing seedlings are discussed.

UDP-glucuronic acid:soyasapogenol glucuronosyltransferase involved in saponin biosynthesis in germinating soybean seeds.

We detected UDP-glucuronic acid:soyasapogenol glucuronosyltransferase (UGASGT) activity in the microsomal fraction from germinating soybean (Glycine max [L.] Merr.) seed. A microsomal fraction was isolated from germinating soybean seed and treated with various detergents to solubilize the enzyme. UGASGT activity was monitored throughout purification using UDP-[U-(14)C]glucuronic acid and soyasapogenol B as substrates. Purification of UGASGT was achieved by HiTrap Q, Superdex 200, and HiTrap Blue chromatography procedures. This resulted in >205-fold enrichment relative to the starting homogenate. UGASGT was found to require divalent cations for activity. Studies on the substrate specificity of UGASGT demonstrated that the specificity for the sugar residue transferred was very high, as activity was scarcely found when UDP-glucuronic acid was replaced by other UDP sugars: UDP-glucose and UDP-galactose. Soyasapogenols, which are the aglycons of soybean saponin, are usable acceptors, but glycyrrhetinic acid, sophoradiol, beta-amyrin, and flavonoids are not. These findings suggest that this UGASGT was a specific enzyme for UDP-glucuronic acid as a donor and soyasapogenols as acceptors, and that it was related to the biosynthesis of the sugar chain in soybean saponin. This study provides a basis for the molecular characterization of a key enzyme in saponin biosynthesis in soybean. The isolation of the gene may enable its use in the elucidation of the biosynthesis and physiological role of saponins in soybean.