Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis.

KEY MESSAGE: We investigated the functions of two cyanobacterial HemY protoporphyrinogen IX oxidase (PPO) genes with in vitro and in vivo assays and evaluated their applicability as resistance traits to PPO-inhibiting herbicides. We isolated HemY-type protoporphyrinogen IX oxidase (PPO) genes from cyanobacteria, OnPPO gene from Oscillatoria nigro-viridis PCC7112 and HaPPO gene from Halothece sp. PCC7418. The alignment of amino acid sequences as well as phylogenetic analyses conducted showed that OnPPO and HaPPO are classified as HemY-type PPO and are more closely related to plastidic PPOs than to mitochondrial PPOs. The PPO-deficient Escherichia coli BT3 strain, which requires heme supplementation, could obtain normal growth in the absence of heme supplementation when complemented with OnPPO and HaPPO. The enzyme assays of OnPPO, HaPPO, and Arabidopsis thaliana PPO1 (AtPPO1) proteins each revealed different kinetic properties in terms of catalytic efficiency, substrate affinity, and the degree of inhibition by PPO inhibitors. In particular, the catalytic efficiencies (kcat/Km) of OnPPO and HaPPO were approximately twofold higher than that of AtPPO1. The elution profiles of all three PPOs, acquired by size-exclusion chromatography, showed only a single peak with a molecular weight of approximately 52-54 kDa, which corresponds to a monomeric form. Moreover, functional complementation with OnPPO and HaPPO in AtPPO1-silenced Arabidopsis resulted in restored growth, whereas AtPPO1-silenced wild type Arabidopsis suffered necrotic death. In addition, we observed that overexpression of OnPPO and HaPPO in Arabidopsis conferred resistance to the PPO-inhibiting herbicides tiafenacil and saflufenacil. These results suggest that two HemY-type PPOs of cyanobacteria can functionally substitute for plastidic PPO activity in Arabidopsis and can enhance resistance to tiafenacil and saflufenacil.

Biochemical and physiological mode of action of tiafenacil, a new protoporphyrinogen IX oxidase-inhibiting herbicide.

We conducted biochemical and physiological experiments to investigate the mode of action of tiafenacil (Terrad'or™), a new protoporphyrinogen IX oxidase (PPO)-inhibiting pyrimidinedione herbicide. Analysis of the half-maximal inhibitory concentration (IC50) against recombinant PPO enzymes from various plant species, including amaranth (Amaranthus tuberculatus), soybean (Glycine max), arabidopsis (Arabidopsis thaliana), and rapeseed (Brassica napus), showed that tiafenacil had an IC50 of 22 to 28 nM, similar to the pyrimidinedione herbicides butafenacil and saflufenacil and the N-phenylphthalimide herbicide flumioxazin. By contrast, tiafenacil exhibited 3- to 134-fold lower IC50 values than the diphenyl ether herbicides fomesafen, oxyfluorfen, and acifluorfen. Tiafenacil is non-selective and is herbicidal to both dicots and monocots, such as the weeds velvetleaf (Abutilon theophrasti), amaranth, and barnyardgrass (Echinochloa crus-galli) as well as the crops soybean, rapeseed, rice (Oryza sativa), and maize (Zea mays) at concentrations ranging from 1 to 50 µM. Treatment of plant tissue with tiafenacil in darkness resulted in the accumulation of protoporphyrin IX. Subsequent exposure to light increased the content of malondialdehyde and significantly decreased the Fv/Fm values of chlorophyll fluorescence. The results suggest that tiafenacil is a new PPO-inhibiting pyrimidinedione herbicide.

Differential responses of three sweetpotato metallothionein genes to abiotic stress and heavy metals.

Metallothioneins (MTs) are cysteine-rich, low molecular weight, metal-binding proteins that are widely distributed in living organisms. Plants produce metal-chelating proteins such as MTs to overcome the toxic effects of heavy metals. We cloned three MT genes from sweetpotato leaves [Ipomoea batatas (L.) Lam.]. The three IbMT genes were classified according to their cysteine residue alignment into type 1 (IbMT1), type 2 (IbMT2), and type 3 (IbMT3). IbMT1 was the most abundantly transcribed MT. It was predominantly expressed in leaves, roots, and callus. IbMT2 transcript was detected only in stems and fibrous roots, whereas IbMT3 was strongly expressed in leaves and stems. The IbMT expression profiles were investigated in plants exposed to heavy metals and abiotic stresses. The levels of IbMT1 expression were strongly elevated in response to Cd and Fe, and moderately higher in response to Cu. The IbMT3 expression pattern in response to heavy metals was similar to that of IbMT1. Exposure to abiotic stresses such as methyl viologen (MV; paraquat), NaCl, polyethylene glycol (PEG), and H2O2 up-regulated IbMT expression; IbMT1 responded strongly to MV and NaCl, whereas IbMT3 was induced by low temperature and PEG. Transgenic Escherichia coli overexpressing IbMT1 protein exhibited results suggest that IbMT could be a useful tool for engineering plants with enhanced tolerance to environmental stresses and heavy metals.

Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures.

The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of ß-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

Downregulation of the lycopene ε-cyclase gene increases carotenoid synthesis via the ß-branch-specific pathway and enhances salt-stress tolerance in sweetpotato transgenic calli.

Lycopene ε-cyclase (LCY-ε) is involved in the first step of the α-branch synthesis pathway of carotenoids from lycopene in plants. In this study, to enhance carotenoid synthesis via the ß-branch-specific pathway [which yields ß-carotene and abscisic acid (ABA)] in sweet potato, the expression of IbLCY-ε was downregulated by RNAi (RNA interference) technology. The RNAi-IbLCY-ε vector was constructed using a partial cDNA of sweet potato LCY-ε isolated from the storage root and introduced into cultured sweet potato cells by Agrobacterium-mediated transformation. Both semi-quantitative Reverse transcription polymerase chain reaction (RT-PCR) of carotenoid biosynthesis genes and high-performance liquid chromatography (HPLC) analysis of the metabolites in transgenic calli, in which the LCY- εgene was silenced, showed the activation of ß-branch carotenoids and its related genes. In the transgenic calli, the ß-carotene content was approximately 21-fold higher than in control calli, whereas the lutein content of the transgenic calli was reduced to levels undetectable by HPLC. Similarly, expression of the RNAi-IbLCY-ε transgene resulted in a twofold increase in ABA content compared to control calli. The transgenic calli showed significant tolerance of 200 mM NaCl. Furthermore, both the ß-branch carotenoids content and the expression levels of various branch-specific genes were higher under salt stress than in control calli. These results suggest that, in sweet potato, downregulation of the ε-cyclization of lycopene increases carotenoid synthesis via the ß-branch-specific pathway and may positively regulate cellular defenses against salt-mediated oxidative stress.

Expression of the sweetpotato R2R3-type IbMYB1a gene induces anthocyanin accumulation in Arabidopsis.

R2R3-type MYB transcription factors (TFs) play important roles in transcriptional regulation of anthocyanins. The R2R3-type IbMYB1 is known to be a key regulator of anthocyanin biosynthesis in the storage roots of sweetpotato. We previously showed that transient expression of IbMYB1a led to anthocyanin pigmentation in tobacco leaves. In this article, we generated transgenic Arabidopsis plants expressing the IbMYB1a gene under the control of CaMV 35S promoter, and the sweetpotato SPO and SWPA2 promoters. Overexpression of IbMYBa in transgenic Arabidopsis produced strong anthocyanin pigmentation in seedlings and generated a deep purple color in leaves, stems and seeds. Reverse transcription-polymerase chain reaction analysis showed that IbMYB1a expression induced upregulation of several structural genes in the anthocyanin biosynthetic pathway, including 4CL, CHI, F3'H, DFR, AGT, AAT and GST. Furthermore, overexpression of IbMYB1a led to enhanced expression of the AtTT8 (bHLH) and PAP1/AtMYB75 genes. high-performance liquid chromatography analysis revealed that IbMYB1a expression led to the production of cyanidin as a major core molecule of anthocyanidins in Arabidopsis, as occurs in the purple leaves of sweetpotato (cv. Sinzami). This result shows that the IbMYB1a TF is sufficient to induce anthocyanin accumulation in seedlings, leaves, stems and seeds of Arabidopsis plants.

Three Brassica rapa metallothionein genes are differentially regulated under various stress conditions.

The expression profiles of three Brassica rapa metallothionein genes (BrMT 1-3) were determined in 7-day-old seedlings exposed to various exogenous factors including plant hormones, heavy metals and abiotic stresses. BrMT1, BrMT2, and BrMT3 were representatives of MT gene type 1, type 2, and type 3, respectively, according to their cysteine alignment. BrMT2 showed a relatively higher basal expression level compared to BrMT1 and BrMT3 under normal conditions. The BrMT1 transcript was markedly increased by various factors including ethephon, polyethylene glycol and hydrogen peroxide, with no down-regulation evident. On the contrary, BrMT2 expression was down-regulated by abscisic acid, salicylic acid, and methyl jasmonate. Heavy metals did not increase BrMT2 expression. BrMT3 expression was only marginally and non-significantly up- and down-regulated by the stress conditions tested. Promoter regions of BrMT1 and BrMT2 display different cis-acting elements supporting the different responses of both genes against various stresses. The results demonstrate the differential regulation of BrMT1-3 by various plant exogenous factors, and indicate the utility of the BrMT1 promoter as a multiple stress inducible promoter.

Down-regulation of ß-carotene hydroxylase increases ß-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato.

Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. ß-Carotene hydroxylase (CHY-ß) is a key regulatory enzyme in the beta-beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both ß-carotene to ß-cryptoxanthin and ß-cryptoxanthin to zeaxanthin. To increase the ß-carotene content of sweetpotato through the inhibition of further hydroxylation of ß-carotene, the effects of silencing CHY-ß in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-ß was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-ß construct. This construct was introduced into cultured cells of white-fleshed sweetpotato (cv. Yulmi). Reverse transcription-polymerase chain reaction analysis confirmed the successful suppression of IbCHY-ß gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene ß-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-ß gene expression changed the composition and levels of carotenoids between non-transgenic (NT) and transgenic cells. In transgenic line #7, the total carotenoid content reached a maximum of 117 µg/g dry weight, of which ß-carotene measured 34.43 µg/g dry weight. In addition, IbCHY-ß-silenced calli showed elevated ß-cryptoxanthin and zeaxanthin contents as well as high transcript level P450 gene. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) in transgenic cells was more than twice that in NT cells. RNA-IbCHY-ß calli increased abscisic acid (ABA) content, which was accompanied by enhanced tolerance to salt stress. In addition, the production of reactive oxygen species measured by 3,3'-diaminobenzidine (DAB) staining was significantly decreased in transgenic cultured cells under salt stress. Taken together, the present results indicate that down-regulation of IbCHY-ß increased ß-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity.

The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system.

MYB transcription factors play important roles in transcriptional regulation of many secondary metabolites including anthocyanins. We cloned the R2R3-MYB type IbMYB1 complementary DNAs from the purple-fleshed sweet potato (Ipomoea batatas L. cv Sinzami) and investigated the expression patterns of IbMYB1 gene with IbMYB1a and IbMYB1b splice variants in leaf and root tissues of various sweet potato cultivars by reverse transcription-polymerase chain reaction. The transcripts of IbMYB1 were predominantly expressed in the purple-fleshed storage roots and they were also detectable in the leaf tissues accumulating anthocyanin pigments. In addition, transcript levels of IbMYB1 gene were up-regulated by treatment with methyl jasmonate or salicylic acid in leaf and root tissues of cv. White Star. To set up the intragenic vector system in sweet potato, we first evaluated the utilization of the IbMYB1 gene as a visible selectable marker. The IbMYB1a was transiently expressed in tobacco leaves under the control of a constitutive cauliflower mosaic virus 35S promoter, a root-specific and sucrose-inducible sporamin promoter, and an oxidative stress-inducible sweet potato anionic peroxidase2 promoter. We also showed that overexpression of IbMYB1a induced massive anthocyanin pigmentation in tobacco leaves and up-regulated the transcript levels of the structural genes in anthocyanin biosynthetic pathway. Furthermore, high-performance liquid chromatography analysis revealed that the expression of IbMYB1a led to production of cyanidin as a major core molecule of anthocyanidins in tobacco leaves. These results suggest that the IbMYB1 gene can be applicable to a visible marker for sweet potato transformation with intragenic vectors, as well as the production of anthocyanin as important nutritive value in other plant species.

Scopolin-hydrolyzing beta-glucosidases in roots of Arabidopsis.

Three beta-glucosidases (At1g66270-BGLU21, At1g66280-BGLU22, and At3g09260-BGLU23) were purified from the roots of Arabidopsis and their cDNAs were expressed in insect cells. In addition, two beta-glucosidase binding protein cDNAs (At3g16420; PBPI and At3g16430; PBPII) were expressed in Escherichia coli and their protein products purified. These binding proteins interact with beta-glucosidases and activate them. BGLU21, 22 and 23 hydrolyzed the natural substrate scopolin specifically and also hydrolyzed to some extent substrates whose aglycone moiety is similar to scopolin (e.g. esculin and 4-MU-glucoside). In contrast, they hydrolyzed poorly DIMBOA-glucoside and did not hydrolyze pNP- and oNP-glucosides. We determined the physicochemical properties of native and recombinant BGLUs, and found no differences between them. They were stable in a narrow pH range (5-7.5) and had temperature and pH optima for activity at 35 degrees C and pH 5.5, respectively. As for thermostability, >95% of their activity was retained at 40 degrees C but dramatically decreased at >50 degrees C. The apparent K(m) of native and recombinant enzymes for scopolin was 0.73 and 0.81 mM, respectively, and it was 5.8 and 9.7 mM, respectively, for esculin. Western blot analysis showed that all three enzymes were exclusively expressed in roots of seedlings but not in any other plant part or organ under normal conditions. Furthermore, spatial expression patterns of all eight genes belonging to subfamily 3 were investigated at the transcription level by RT-PCR.

Exogenous sucrose utilization and starch biosynthesis among sweet potato cultivars.

Three sweetpotato cultivars were investigated for their starch content and amylose/amylopectin ratio. Ym starch contains 87.2% amylopectin and 12.8% amylose, when total starch was calculated as 100%. The Zm cultivar contains 33.6% amylopectin and 18.2% amylose, and its total starch was calculated as 51.8% of that of Ym. The Hm cultivar contains 39.1% amylopectin and 30.5% amylose, and its total starch was 69.6%. We analyzed the expression levels of starch and sucrose biosynthesis-related genes including AGPases a, b, and c; sucrose synthases I and II; starch synthase I; GBSS I; and SBEs I and II. All genes tested in this experiment were detected only in Ym, while several genes showed very faint or no expression in Zm and Hm. We also measured tissue-specific expression of these genes in whole plants of Ym. Most of the genes are expressed in the stem and roots of the plants. Expression profiles of starch synthesis-related genes of the sweetpotato leaves were investigated after supplementing the different concentrations of sucrose solution. All genes in Ym were clearly induced by sucrose, but the expression levels of some of these genes did not change in Zm and Hm. The total starch content of Ym, Zm, and Hm gradually increased over time on addition of 3%, 6%, and 9% sucrose concentrations. The greatest accumulation was observed in Ym at 48h, and it was almost 2.24 times higher than that of the (0%) control, while Zm and Hm showed 1.76 and 1.91 times higher levels of starch, respectively. These results indicate that cooperative expression of all related genes is essential for starch biosynthesis from sucrose. This is the first report on different sucrose contents and the efficiency with which exogenous sucrose switches on gene expression of starch biosynthesis-related genes among cultivars.

Comparative characterization of the Arabidopsis subfamily a1 beta-galactosidases.

The Arabidopsis genome contains 17 predicted beta-galactosidase genes, all of which belong to glycosyl hydrolase (GH) Family 35. These genes have been further grouped into seven subfamilies based on sequence similarity. The largest of these, subfamily a1, consists of six genes, Gal-1 (At3g13750), Gal-2 (At3g52840), Gal-3 (At4g36360), Gal-4 (At5g56870), Gal-5 (At1g45130), and Gal-12 (At4g26140), some of which were characterized in previous studies. We report here the purification and biochemical characterization of recombinant Gal-1, Gal-3, Gal-4 and Gal-12 from Pichiapastoris, completing the analysis of all six recombinant proteins, as well as the isolation and characterization of the native Gal-2 protein from Arabidopsis leaves. Comparison of the relative expression levels of the subfamily a1 beta-galactosidases at the mRNA and protein levels uncovered evidence of differential regulation, which may involve post-transcriptional and post-translational processes. In addition, this study provides further support for the proposed function of the subfamily a1 beta-galactosidases in cell wall modification based on analysis of the organ-specific expression and subcellular localization of Gal-1 and Gal-12. Our study suggests that, despite some differences in individual biochemical characteristics and expression patterns, each member of the family has the potential to contribute to the dynamics of the Arabidopsis plant cell wall.

Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during As stress.

While the phytotoxic responses of arsenic (As) on plants have been studied extensively, based on physiological and biochemical aspects, very little is known about As stress-elicited changes in plants at the proteome level. Hydroponically grown 2-wk-old rice seedlings were exposed to different doses of arsenate, and roots were collected after 4 days of treatment, as well as after a recovery period. To gain a comprehensive understanding of the precise mechanisms underlying As toxicity, metabolism, and the defense reactions in plants, a comparative proteomic analysis of rice roots has been conducted in combination with physiological and biochemical analyses. Arsenic treatment resulted in increases of As accumulation, lipid peroxidation, and in vivo H(2)O(2) contents in roots. A total of 23 As-regulated proteins including predicted and novel ones were identified using 2-DE coupled with MS analyses. The expression levels of S-adenosylmethionine synthetase (SAMS), GSTs, cysteine synthase (CS), GST-tau, and tyrosine-specific protein phosphatase proteins (TSPP) were markedly up-regulated in response to arsenate, whereas treatment by H(2)O(2) also regulated the levels of CS suggesting that its expression was certainly regulated by As or As-induced oxidative stress. In addition, an omega domain containing GST was induced only by arsenate. However, it was not altered by treatment of arsenite, copper, or aluminum, suggesting that it may play a particular role in arsenate stress. Analysis of the total glutathione (GSH) content and enzymatic activity of glutathione reductase (GR) in rice roots during As stress revealed that their activities respond in a dose-dependent manner of As. These results suggest that SAMS, CS, GSTs, and GR presumably work synchronously wherein GSH plays a central role in protecting cells against As stress.

Evaluation of the extracellular proteins in full-scale activated sludges.

The proteins present in the extracellular polymeric substances (EPS) of activated sludge flocs were investigated using three cation-associated extraction methods. The subproteomes generated from four full-scale activated sludges were subsequently fractionated by ammonium sulfate precipitation and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that each extraction method led to unique SDS-PAGE protein profiles, which provided strong evidence that the extracted proteins are uniquely associated with specific cations in activated sludge flocs. The comparison of protein profiles across sludges from different treatment plants revealed that extracts obtained using a cation-exchange resin exhibited similar protein banding patterns while sulfide- and base-extracted EPS led to more variable protein profiles. Analysis of several SDS-PAGE bands by liquid chromatography-tandem mass spectrometry of tryptic digests led to the identification of several bacterial proteins as well as sewage-derived polypeptides (human elastase IIIA and keratins). Their putative roles in activated sludges and their association with targeted cations are proposed.

Vicianin hydrolase is a novel cyanogenic beta-glycosidase specific to beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) in seeds of Vicia angustifolia.

The cyanogenic disaccharide glycoside, vicianin [mandelonitrile beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside)], is accumulated in seeds of Vicia angustifolia var. segetalis. Vicianin hydrolase (VH) catalyzes the hydrolysis of vicianin into mandelonitrile and a disaccharide vicianose. VH was purified from the seeds using DEAE-, CM- and Con A-Sepharose chromatography, and the molecular mass of the purified VH was estimated to be 56 kDa on SDS-PAGE. The N-terminal amino acid sequence of the purified VH was determined, and a cDNA encoding VH was obtained. The deduced VH protein consists of a 509 amino acid polypeptide containing a putative secretion signal peptide. It shares about 50% identity with various kinds of plant beta-glycosidases including tea leaf beta-primeverosidase and furcatin hydrolase, and is classified in family 1 of the glycosyl hydrolases. The VH transcript was detected abundantly in seeds and moderately in flowers, but only slightly in leaves, stems and roots, indicating that the organ distribution of VH expression is similar to that of the substrate vicianin. The recombinant VH was produced in insect cells with a baculovirus system, and was compared with the native VH in terms of substrate specificity. Both enzymes hydrolyzed vicianin to release vicianose, demonstrating that VH is a disaccharide-specific beta-glycosidase. VH also hydrolyzed the mandelonitrile beta-glucoside prunasin to some extent but did not hydrolyze the gentiobioside amygdalin, both of which contain the same aglycone as vicianin. Thus, VH is a unique cyanogenic glycosidase showing high glycone specificity for the disaccharide vicianoside.

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35.

Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.

Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1.

Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf beta-primeverosidase, which is another disaccharide glycosidase specific to beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant beta-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a Km value of 2.2 mm and specifically hydrolyzed the beta-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed beta-primeverosides and beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) but poorly hydrolyzed beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-d-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl beta-D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other beta-D-glucopyranosides. Stereochemical analysis using 1H NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant beta-glucosidases revealed that FH clusters with beta-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene.

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.