Comparison of corticocancellous bone graft from the anterolateral metaphysis of the distal radius versus iliac crest for the treatment of unstable scaphoid nonunion with humpback deformity.

BACKGROUND: Corticocancellous bone grafting from the iliac crest is acceptable treatment for unstable scaphoid nonunion with a viable proximal pole. However, harvesting graft from the iliac crest is associated with donor site morbidity and the requirement of general anesthesia. Thus, bone grafting from the anterolateral metaphysis of the distal radius (DR) can be a treatment option. However, no study has compared the clinical effect between the two grafting techniques. METHODS: From 2014 to 2019, patients with unstable scaphoid nonunion with humpback deformity underwent corticocancellous bone grafting from the anterolateral metaphysis of the DR (group DR) or iliac crest (group IC). Humpback deformity was determined by evaluating the scapholunate angle (SLA) ≥ 60°, intrascaphoid angle (ISA) ≥ 45°, and radiolunate angle (RLA) ≥ 15° from preoperative radiographs and computed tomography scans. The SLA, ISA, and RLA served to gauge carpal alignment. The operative time, grip strength, active range of motion (ROM), the Modified Mayo Wrist score (MMWS), and Disabilities of Arm, Shoulder, and Hand (DASH) score were assessed postoperatively. RESULTS: Thirty-eight patients qualified for the study (group DR, 15; group IC, 23). Union rates did not differ by patient subset (group DR, 100%; group IC, 95.7%; P = .827), and grip strength, ROM, MWS, and DASH score were similar between groups at the last follow-up. The operative time (minutes) was significantly shorter in group DR (median, 98; quartiles, 80, 114) than in group IC (median, 125; quartiles, 105, 150, P < .001). The ISA, RLA, and SLA improved postoperatively in both groups (P < 0.001). The degree of restoring carpal alignment, as evaluated by SLA, showed superior correction capability in group DR (median, 25.3% quartiles, 21.1, 35.3, P < 0.05). Donor site complications were not significantly different between the groups. CONCLUSIONS: Corticocancellous bone graft from the anterolateral metaphysis of the DR for unstable scaphoid nonunion is associated with a shorter operation time and comparable results with that from the iliac crest in regard to union, restoration of carpal alignment, and wrist function. LEVEL OF EVIDENCE: Level III.

The Ve-resistance locus, a plant signaling intercept.

MAIN CONCLUSION: The Ve-resistance locus in tomato and potato affects both stress/defense and growth, consistent with a signaling intercept and a competitive regulatory mechanism. Acting in an antagonistic fashion, the two genes comprising the tomato Ve-resistance locus have been shown to influence both the defense/stress cascade, which causes wilt symptoms, and plant growth (Nazar et al. in Planta 247:1339-1350, 2018c); in contrast, both have been reported to elevate wilt resistance in potato or Arabidopsis. In a further examination of this influence in potato transformed with the Ve1 gene, effects are again demonstrated with respect to both disease resistance and crop productivity consistent with the Ve locus being a signaling intercept and the antagonistic effects, previously observed in tomato. The results support a competitive model in which the tomato Ve1 and Ve2 proteins act to reduce the detrimental effects of the defense/stress cascade and energy transfers to the developing potato tubers.

Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production.

Potato (Solanum tuberosum L.) is generally considered to be sensitive to drought stress. Even short periods of water shortage can result in reduced tuber production and quality. We previously reported that transgenic potato plants expressing the sweet potato orange gene (IbOr) under the control of the stress-inducible SWPA2 promoter (referred to as SOR plants) showed increased tolerance to methyl viologen-mediated oxidative stress and high salinity, along with increased carotenoid contents. In this study, in an effort to improve the productivity and environmental stress tolerance of potato, we subjected transgenic potato plants expressing IbOr to water-deficient conditions in the greenhouse. The SOR plants exhibited increased tolerance to drought stress under greenhouse conditions. IbOr expression was associated with slightly negative phenotypes, including reduced tuber production. Controlling IbOr expression imparted the same degree of drought tolerance while ameliorating these negative phenotypic effects, leading to levels of tuber production similar to or better than those of wild-type plants under drought stress conditions. In particular, under drought stress, drought tolerance and the production of marketable tubers (over 80g) were improved in transgenic plants compared with non-transgenic plants. These results suggest that expressing the IbOr transgene can lead to significant gains in drought tolerance and tuber production in potato, thereby improving these agronomically important traits.

Development of molecular markers, based on chloroplast and ribosomal DNA regions, to discriminate three popular medicinal plant species, Cynanchum wilfordii, Cynanchum auriculatum, and Polygonum multiflorum.

Identification of plant species is important for standardizing herbal medicine. Cynanchum wilfordii (Baekshuoh in Korean) and Polygonum multiflorum (Hashuoh in Korean) are important oriental medicinal herbs in Korea, Japan, and China. Cynanchum auriculatum is a faster growing and more productive plant than C. wilfordii; and, it is not recognized as a medicinal plant in the Korean Pharmacopoeia. C. wilfordii, P. multiflorum, and C. auriculatum are often misidentified in the Korean herbal medicine marketplace due to their morphological similarities and similar names. In this study, we investigated molecular authentication of these three medicinal plants using DNA sequences in the TrnL-F chloroplast intergenic region. Specific species identification was achieved by detecting allelic variations of single nucleotide polymorphisms (SNPs) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and high resolution melting curve analysis. Our results demonstrate that the intraspecific genetic distance between C. wilfordii and C. auriculatum is relatively low. We also developed a quantitative PCR assay using species-specific TrnL-F primers, which allowed us to estimate the ratio of C. wilfordii and C. auriculatum using varying ratios of mixed genomic DNA template from the two species. Additionally, to identify species in hybrid plants produced by cross-fertilization, we analyzed nuclear ribosomal DNA internal transcribed spacer regions in C. wilfordii and C. auriculatum by ARMS-PCR. Our results indicate that SNP-based molecular markers, usable to barcode tools could provide efficient and rapid authentication of these closely related medicinal plant species, and will be useful for preventing the distribution of products contaminated with adulterants.

Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato.

In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1 µg g(-1)DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and ß-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

Accumulation of PrLeg, a Perilla legumin protein in potato tuber results in enhanced level of sulphur-containing amino acids.

Potato is the fourth staple food in the world, following rice, wheat, and maize, whereas tubers contain high quality of starch, relatively high amounts of vitamin C and many other important substances. It also contains relatively good quality of protein (about 3 to 6% of the dried weight) and patatin, and 11S globulin is a major storage protein with high level of lysine. However, tuber protein contains relatively low amounts of sulphur-containing amino acids, which may result in low nutritional value. Recently, we cloned a gene encoding PrLeg polypeptide, a seed storage protein from Perilla, which contains relatively higher levels of sulphur-containing amino acids. We transformed PrLeg cDNA into a potato plant to over-express under the direction of the tuber-specific promoter, patatin. Most of the transgenic lines identified through PCR and RT-PCR analyses were able to accumulate high amount of prLeg transcript in their tuber tissue, while very little or no transcript that were detected in their leaf tissues. The level of methionine content was elevated up to three-fold compared to non-transgenic parental line, without any significant changes in other amino acids, suggesting that further research is required to get a deeper insight into their nutritional value.

Enhanced antioxidant and protective activities on retinal ganglion cells of carotenoids-overexpressing transgenic carrot.

Carotenoids are considered to act as antioxidants and protect humans from serious disorders such as skin degeneration and ageing, cardiovascular disease, certain types of cancer, and age-related diseases of the eye. In this study, these chemopreventive activities of a carotenoids-overexpressing transgenic carrot were evaluated. The results of DPPH, hydroxyl, and superoxide radical scavenging tests demonstrate that the acetone extract obtained from the taproots of the carrot plants exhibits significant antioxidant activity. A higher activity was detected in the transgenic carrot extract compared with the wild-type extract. A chemopreventive activity test for degenerative diseases of the eye revealed that pretreatment with the carrot extract reduced cell death in a retinal ganglion cell line, RGC-5 cells exposed to 1-buthionine- (R,S)-sulfoximine and L-glutamic acid.

Overexpression of Arabidopsis YUCCA6 in potato results in high-auxin developmental phenotypes and enhanced resistance to water deficit.

Indole-3-acetic acid (IAA), a major plant auxin, is produced in both tryptophan-dependent and tryptophan-independent pathways. A major pathway in Arabidopsis thaliana generates IAA in two reactions from tryptophan. Step one converts tryptophan to indole-3-pyruvic acid (IPA) by tryptophan aminotransferases followed by a rate-limiting step converting IPA to IAA catalyzed by YUCCA proteins. We identified eight putative StYUC (Solanum tuberosum YUCCA) genes whose deduced amino acid sequences share 50%-70% identity with those of Arabidopsis YUCCA proteins. All include canonical, conserved YUCCA sequences: FATGY motif, FMO signature sequence, and FAD-binding and NADP-binding sequences. In addition, five genes were found with ~50% amino acid sequence identity to Arabidopsis tryptophan aminotransferases. Transgenic potato (Solanum tuberosum cv. Jowon) constitutively overexpressing Arabidopsis AtYUC6 displayed high-auxin phenotypes such as narrow downward-curled leaves, increased height, erect stature, and longevity. Transgenic potato plants overexpressing AtYUC6 showed enhanced drought tolerance based on reduced water loss. The phenotype was correlated with reduced levels of reactive oxygen species in leaves. The results suggest a functional YUCCA pathway of auxin biosynthesis in potato that may be exploited to alter plant responses to the environment.

Identification of a C2H2-type zinc finger transcription factor (ZAT10) from Arabidopsis as a substrate of MAP kinase.

Mitogen-activated protein kinases (MAPKs or MPKs) are one of the most important and conserved signaling molecules in plants. MPKs can directly modulate gene expression by the phosphorylation of transcription factors. However, only a few target substrates of MPKs have been isolated. Here, we identified a C(2)H(2)-type zinc finger transcription factor from Arabidopsis, ZAT10, as a substrate of MPKs. Using in vitro and in vivo protein-protein interaction analyses, we demonstrated that ZAT10 directly interacted with MPK3 and MPK6. ZAT10 was phosphorylated by recombinant Arabidopsis MPK3 and MPK6 in a kinase assay. Furthermore, ZAT10 was also phosphorylated by native MPK3 and MPK6 prepared from Arabidopsis plants in an in-gel kinase assay. Mass spectrometry analysis of phosphopeptides was used to determine two MPK phosphorylation sites in ZAT10. These sites were verified by site-directed mutagenesis and in vitro kinase assays.

YUCCA6 over-expression demonstrates auxin function in delaying leaf senescence in Arabidopsis thaliana.

The Arabidopsis thaliana YUCCA family of flavin monooxygenase proteins catalyses a rate-limiting step in de novo auxin biosynthesis. A YUCCA6 activation mutant, yuc6-1D, has been shown to contain an elevated free IAA level and to display typical high-auxin phenotypes. It is reported here that Arabidopsis plants over-expressing YUCCA6, such as the yuc6-1D activation mutant and 35S:YUC6 transgenic plants, displayed dramatic longevity. In addition, plants over-expressing YUCCA6 exhibited classical, delayed dark-induced and hormone-induced senescence in assays using detached rosette leaves. However, plants over-expressing an allele of YUCCA6, that carries mutations in the NADPH cofactor binding site, exhibited neither delayed leaf senescence phenotypes nor phenotypes typical of auxin overproduction. When the level of free IAA was reduced in yuc6-1D by conjugation to lysine, yuc6-1D leaves senesced at a rate similar to the wild-type leaves. Dark-induced senescence in detached leaves was accompanied by a decrease in their free IAA content, by the reduced expression of auxin biosynthesis enzymes such as YUCCA1 and YUCCA6 that increase cellular free IAA levels, and by the increased expression of auxin-conjugating enzymes encoded by the GH3 genes that reduce the cellular free auxin levels. Reduced transcript abundances of SAG12, NAC1, and NAC6 during senescence in yuc6-1D compared with the wild type suggested that auxin delays senescence by directly or indirectly regulating the expression of senescence-associated genes.

Production of serotonin by dual expression of tryptophan decarboxylase and tryptamine 5-hydroxylase in Escherichia coli.

A plant-specific biogenic amine, serotonin, was produced by heterologous expression of two key biosynthetic genes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), in Escherichia coli. The native T5H, a cytochrome P450 enzyme, was unable to be functionally expressed in E. coli. Through a series of N-terminal deletions or additions of tagging proteins, we generated a functional T5H enzyme construct (GST∆37T5H) in which glutathione S transferase (GST) was translationally fused with the N-terminal 37 amino acid deleted T5H. Dual expression of GST∆37T5H and TDC using a pCOLADuet-1 E. coli vector produced serotonin at concentrations of approximately 24 mg l⁻¹ in the culture medium and 4 mg l⁻¹ in the cells. An optimum temperature of approximately 20 °C was required to achieve peak serotonin production in E. coli because the low induction temperature gave rise to the highest soluble expression of GST∆37T5H.

The sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter confers high-level expression of the GUS reporter gene in the potato tuber.

Molecular farming refers to the process of creating bioengineered plants with the capability of producing potentially valuable products, such as drugs, vaccines, and chemicals. We have investigated the potential of the sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter and its transit peptide (TP) as an expression system for the mass production of foreign proteins in potato. The ibAGP1 promoter and its TP sequence were transformed into potato along with beta-glucuronidase (GUS) as a reporter gene, and GUS activity was subsequently analyzed in the transgenic potato plants. In tuber tissues, GUS activity in transgenic plants carrying only the ibAGP1 promoter (ibAGP1::GUS) increased up to 15.6-fold compared with that of transgenic plants carrying only the CaMV35S promoter (CaMV35S::GUS). GUS activity in transgenic plants was further enhanced by the addition of the sweetpotato TP to the recombinant vector (ibAGP1::TP::GUS), with tuber tissues showing a 26-fold increase in activity compared with that in the CaMV35S::GUS-transgenic lines. In leaf tissues, the levels of GUS activity found in ibAGP1::GUS-transgenic lines were similar to those in CaMV35S::GUS-lines, but they were significantly enhanced in ibAGP1::TP::GUS-lines. GUS activity gradually increased with increasing tuber diameter in ibAGP1::GUS-transgenic plants, reaching a maximum level when the tuber was 35 mm in diameter. In contrast, extremely elevated levels of GUS activity - up to about 10-fold higher than that found in CaMV35S::GUS-lines - were found in ibAGP1::TP::GUS-transgenic lines at a much earlier stage of tuber development (diameter 4 mm), and these higher levels were maintained throughout the entire tuber developmental stage. These results suggest that the sweetpotato ibAGP1 promoter and its TP are a potentially strong foreign gene expression system that can be used for molecular farming in potato plants.

Tomato phenylalanine ammonia-lyase gene family, highly redundant but strongly underutilized.

Phenylalanine ammonia-lyase (PAL) is an important enzyme in both plant development and pathogen defense. In all plants it is encoded by a multi-gene family, ranging in copy number from four in Arabidopsis to a dozen or more copies in some higher plants. Many studies indicate that alternate genes are differentially regulated in response to environmental stimuli. In this study, Southern blot and dot blot analyses in tomato indicate a surprisingly large family of related sequences with approximately 26 copies in the diploid genome, some easily distinguished by restriction enzyme digestion. Analyses of a BAC genome library suggest that the genes are generally not clustered. A more detailed comparison of the gene sequences using PCR to isolate the individual copies and reverse transcription-PCR to study the transcripts that they encode indicates a significant diversity in the gene sequences themselves, but surprisingly only one mRNA transcript can be detected even when additional expression is induced by pathogen growth or wounding. Consistent with previous reports in other plants, a parallel study with a closely related plant, the potato, indicates a much broader utilization of the PAL genes, highlighting the unusual nature of this family in tomato and of the mechanism(s) that silences so many members. Plant transformation analyses further demonstrate the presence of very active silencing, suggesting aggressive competition between PAL gene duplication and copy inactivation during PAL gene evolution.

Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes.

The plant-specific serotonin derivatives feruloylserotonin (FS) and 4-coumaroylserotonin (CS) are synthesized by the enzymes 4-coumarate:coenzyme A ligase (4CL) and serotonin N-hydroxycinnamoyltransferase (SHT). To express these genes coordinately, SHT was fused in-frame with the self-processing FDMV 2A sequence followed by 4CL in a single open reading frame and introduced into Escherichia coli or Saccharomyces cerevisiae. The transgenes were abundantly expressed in both recombinant microbes, but functional expression was achieved only in yeast, with cleavage at the 2A sequence yielding monomeric SHT-2A and 4CL as judged by immunoblot and product analyses. In the presence of an exogenous supply of precursors such as serotonin and ferulic acid, the recombinant yeast synthesized 4.5 mg l(-1) FS in the medium while 0.02 mg l(-1) FS was produced in the cells. Time-course analysis indicated peak accumulation of FS at 24 h after induction, and this level was maintained until 96 h. The optimum precursor concentration was 2 mM. A series of serotonin derivatives was produced by adding various cinnamate derivative precursors with serotonin; 2.5 mg l(-1) caffeoylserotonin (CaS) and 1.4 mg l(-1) CS were produced, whereas no sinapoylserotonin or cinnamoylserotonin was yielded.

Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity.

Seed germination is a complex physiological process in plants that can be affected severely by heavy metals. The interference of germination by cadmium stress has not been well documented at the proteomic level. In the present study, in order to investigate the protein profile alternations during the germination stage following exposure to cadmium, a proteomic approach has been adopted in combination with morphological and physiological parameters. Seeds were exposed with a wide range of cadmium between 0.2 and 1.0 mM. Increases of cadmium concentration in the medium resulted in increased cadmium accumulation in seeds and TBARS content, whereas germination rate, shoot elongation, biomass, and water content were decreased significantly. Temporal changes of the total proteins were investigated by two-dimensional electrophoresis (2-DE). Twenty-one proteins were identified using MALDI-TOF mass spectrometry, which were upregulated at least 1.5-fold in response to cadmium stress. The identified proteins are involved in several processes, including defense and detoxification, antioxidant, protein biosynthesis, and germination processes. The identification of these proteins in the cadmium stress response provides new insight that can lead to a better understanding of the molecular basis of heavy metal responses of seeds at the germination stage.

A comparison of the carotenoid accumulation in Capsicum varieties that show different ripening colours: deletion of the capsanthin-capsorubin synthase gene is not a prerequisite for the formation of a yellow pepper.

Ripe pepper (Capsicum sp.) fruits can display a range of colours from white to deep red. To understand better the regulatory mechanisms of the carotenoid biosynthetic pathways that underlie these ripening colours, Capsicum varieties that show seven different fully ripe colour types were analysed. The levels and composition of the carotenoid accumulation in these samples at different stages of ripening were measured, and the resulting data were analysed in conjunction with the expression patterns of the carotenoid biosynthetic genes. It was found that red peppers accumulate increasing levels of total carotenoids during ripening, whereas non-red peppers accumulate lower levels of total carotenoids of varying composition. The expression levels of the phytoene synthase, phytoene desaturase, and capsanthin-capsorubin synthase (Ccs) genes are high in peppers with high levels of total carotenoid, whereas one or two of these genes are not expressed in peppers with lower levels of total carotenoid. Surprisingly, it was found that the Ccs gene is present in two Capsicum varieties whose ripe colour is yellow. This gene has never previously been shown to be present in yellow peppers. Sequence analyses of the Ccs gene further revealed two structural mutations in yellow peppers that may result in either a premature stop-codon or a frame-shift. Taken together with the fact that the Ccs transcript is not detectable in yellow peppers, our current results suggest that nonsense-mediated transcriptional gene silencing of Ccs and not the deletion of this gene is responsible for yellow ripening in Capsicum.

Molecular and biochemical characterizations of dehydroascorbate reductase from sesame (Sesamum indicum L.) hairy root cultures.

Dehydroascorbate reductase (DHAR) is a biotechnologically or physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction for most higher plants. A DHAR cDNA was isolated from sesame (Sesamum indicum L.) hairy roots, and its structure and biochemical properties were characterized to provide some information about its expressional and biochemical profiles in the hairy root cultures. The cDNA contained a catalytic motif CXXS, which may be indicative of a thiol-dependent redox function. A fusion DHAR expressed in an Escherichia coli expression system was purified with four purification steps until a homogeneous single band signal was seen in an acrylamide gel, and its antibody was prepared for Western blot analyses. The biochemical results showed that the purified recombinant DHAR had an optimal pH of around 6.0, which was different from those (pH 7.8-8.2) of other plant species. The temperature optimal for the DHAR activity was in a relatively wide range of 30-60 degrees C. It was proved by a real-time RT-PCR technique that the transcription activity of the DHAR was about 2-5-fold higher during the first 3 week cultures than during the latter 3 week ones. The highest activity of the sesame DHAR was detected in the 4 week cultures of the hairy roots, after which its activity was rapidly decreased to approximately 80%, suggesting that the most active DHAR occurred in this culture period. Western blot analyses confirmed that the presence of DHAR enzyme was identified in both cultures of the fused E. coli and the sesame hairy roots.

A strong constitutive gene expression system derived from ibAGP1 promoter and its transit peptide.

To develop a strong constitutive gene expression system, the activities of ibAGP1 promoter and its transit peptide were investigated using transgenic Arabidopsis and a GUS reporter gene. The ibAGP1 promoter directed GUS expression in almost entire tissues including rosette leaf, inflorescence stem, inflorescence, cauline leaf and root, suggesting that the ibAGP1 promoter is a constitutive promoter. GUS expression mediated by ibAGP1 promoter was weaker than that by CaMV35S promoter in all tissue types, but when GUS protein was targeted to plastids with the aid of the ibAGP1 transit peptide, GUS levels increased to higher levels in lamina, petiole and cauline leaf compared to those produced by CaMV35S promoter. The enhancing effect of ibAGP1 transit peptide on the accumulation of foreign protein was tissue-specific; accumulation was high in lamina and inflorescence, but low in root and primary inflorescence stem. The transit peptide effect in the leaves was maintained highly regardless of developmental stages of plants. The ibAGP1 promoter and its transit peptide also directed strong GUS gene expression in transiently expressed tobacco leaves. These results suggest that the ibAGP1 promoter and its transit peptide are a strong constitutive foreign gene expression system for transgenesis of dicot plants.

Two sweetpotato ADP-glucose pyrophosphorylase isoforms are regulated antagonistically in response to sucrose content in storage roots.

The transcriptional regulation of ADP-glucose pyrophosphorylase (AGPase) genes in detached leaves in response to exogenous sucrose has been investigated earlier; however the effects of endogenous sucrose on AGPase gene transcription in leaves or starch-accumulating tissues have not yet been determined. We therefore have investigated the relationship between endogenous sucrose content in the storage tissues of sweetpotato (Ipomoea batatas cv. Yulmi) and the rate of transcription of the two sweetpotato AGPase isoforms, ibAGP1 and ibAGP2, by means of transient expression analysis of their promoters. Sequence analysis of the two promoters identified putative sucrose-responsive elements on the ibAGP1 promoter and, conversely, putative sucrose-starvation elements on the ibAGP2 promoter. Transient expression analyses on transverse storage root sections revealed that the ibAGP1 and ibAGP2 promoters directed strong expression in the sweetpotato storage roots (diameter: 1.5 cm). Sucrose contents of the sweetpotato storage roots were positively correlated with growth of the storage root. In the storage roots, ibAGP1 promoter activity became stronger with increasing endogenous sucrose levels, while ibAGP2 promoter activity became markedly weaker. Consequently, ibAGP2 was expressed primarily during the early stages of storage root development, whereas ibAGP1 was abundantly expressed in the later stages, during which a profound level of starch accumulation occurs. The antagonistic regulation of the two promoters in response to endogenous sucrose levels was also confirmed in carrot (Daucus carota L. cv. Hapa-ochon) taproots.

Expression and characterization of extracellular fungal phytase in transformed sesame hairy root cultures.

A recombinant fungal phytase was produced by cultures of sesame hairy roots transformed with Agrobacterium rhizogenes, purified and its molecular properties were characterized. Its transcription level and the phytase production were rapidly increased after 4 weeks of the cultures, suggesting that its transcription and protein synthesis might concur. Western blot analysis provided evidence that the recombinant fungal phytase was secreted into the liquid culture medium of the hairy roots. The phytase enzyme secreted was purified by three steps of ultrafiltration, DEAE-Sepharose ion exchange chromatography, and Sephadex G-100 size-exclusion chromatography. As a result, one single band signal was observed with SDS-PAGE, indicating that the purification step was reasonable. The positive signs of both the zymogram and the PAS staining on SDS-PAGE suggested that the activity of the final product phytase was active and glycosylated. The optimal reaction temperature of the phytase was between 50 and 60 degrees C and at over 60 degrees C its activity was reduced by 30-90%, depending on the temperatures applied. Pre-incubation at temperatures of 20-50 degrees C showed stable catalytic activity, while at over 50 degrees C the phytase activity was gradually decreased by 90%. The optimal pH was between 4 and 5 pH values for the recombinant fungal phytase, while for native phytase it was at pH 5.0. Addition of iron ion inhibited the phytase activity but treatments of some cations, EDTA, and PMSF showed no effect on the activity or slightly stimulated it positively.