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Rab GTPases play an important role in regulating intracellular vesicular trafficking in eukaryotic cells. Previously, we found that Oryza sativa rice Rab11 (OsRab11) is required for the regulation of vesicular trafficking from the trans- Golgi network (TGN) to the plasma membrane (PM) and/or vacuoles. To further elucidate the relationship between vesicular trafficking and abiotic and biotic stresses, we determined OsRab11 expression levels under several environmental stress conditions. OsRab11 expression was induced by pathogens, jasmonic acid (JA), and high salt treatment. Under high salt conditions, dominant negative OsRab11(S28N) mutant plants exhibited a hypersensitive phenotype similar to that of sos1-1, whereas overexpressed-OsRab11 plants showed resistance to high salt stress. When the expression of vacuolar and PM Na+/H+ antiporter genes such as AtNHX1, AtNHX2, and AtSOS1 was examined, there was no significant difference between the wild-type and OsRab11(S28N) mutant plants. However, PM trafficking of AtSOS1-green fluorescent protein (GFP) in 35S::AtSOS1-GFP sos1-1 plants was severely impaired by T7-OsRab11(S28N) expression. Similarly, vacuolar trafficking of AtNHX2-GFP was inhibited by T7-OsRab11 (S28N) expression. These results indicate that trafficking of PM and vacuolar antiporter proteins by OsRab11 is important for high salt stress resistance.

The use of fossil fuels now induces two major issues. First, fossil fuel burning is increasing atmospheric carbon dioxide (CO2) concentrations and, in turn, global warming. Second, fossil fuel resources are limited and will thus decrease in the long run. As a potential solution, there is a need for ecological manufacturing processes that convert raw plant materials into chemical products. For instance, raw plants can be directly converted into hydroxymethylfurfural, which is a versatile intermediate for the synthesis of valuable biofuels such as dimethylfuran and 5-ethoxymethyl-2-furfural. This technology has two benefits for chemical sustainability. First, the pretreatment step is eliminated, thus contributing to reduction of CO2 emissions. Second, plants are sustainable resources versus fossil fuels, which are limited. Here, we review current sustainable technologies for the production of biobased products and hydroxymethylfurfural from plants, using in particular ionic liquids. Plant sources include poplar, switchgrass, miscanthus, weed plants, and agave species.

A simple conversion process of inulin polymer into hydroxymethylfurfural (HMF) was developed using Bronsted acid catalyst (HCl) in the presence of an ionic liquid, 1-octyl-3-methylimidazolium chloride ([OMIM]Cl). In addition, the physicochemical properties of inulin particle and its depolymerixation products were scrutinized. FESEM and XRD diffraction frequency showed that inulin particles are clustered in a granulated formation and their molecular structure is highly amorphous. FT-IR analysis identified five characteristic frequency regions: Region 1; 700–900, Region 2; 900–1,200, Region 3; 1,200–1,350; Region 4; 1,350–1,500, and Region 5; 1,530–1,800 cm−1. HPLC analysis confirmed that the major composition of inulin consists of fructose and glucose. The synthesis of HMF was significantly affected by the Bronsted catalyst and its concentration. Its highest yield (63.1±5.1 dwt%) was achieved at 0.3M HCl in the presence of [OMIM]Cl. No presence of the Bronsted catalyst exhibited negligible HMF yield (2.3±1.1 dwt%). Our results demonstrate that the Bronsted catalyst plays a pivotal role in the catalytic process of HMF synthesis from inulin polymer.

Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

A new green technology was developed using citrus peel waste to produce hydroxymethylfurfual (HMF). FT-IR analysis of the waste showed 4 characteristic vibration modes (C H, C O, C OH, and C O/COO−), contributing to sugars. XRD and FESEM elucidated that the waste and its hydrolysate consist of highly amorphous clusters. HCl increased HMF yield by 1.4-fold. CrF3 increased its yield by 1.7-fold. At 0.2 of the stoichiometric ratio value, HMF yield was highest. The highest HMF yield was achieved in the reaction mixture of 4 g [OMIM]Cl, 1 mL ethyl acetate, 0.1 g CrF3, 5 mL 0.3 M HCl, and 0.5 g biomass.

A new pink gerbera (Gerbera hybrida Hort.) cultivar 'Party Time' was developed from a cross between 'Hope' and 'Sunnyeo', followed by seedling and line selections at the Flower Research Institute of Gyeongnam Agricultural Research and Extension Services (GARES) in 2007. Detailed characteristics of the new cultivar were evaluated from 2005 to 2007. 'Party Time' has semi-double type large flowers in a good harmony with pink (RHS, 65-C) ray floret and brown center. It has a good, stable flower shape and strong peduncles, and its vase life was 12.7 days. The average flower yield of 'Party Time' was about 47.3 stems per plant a year in greenhouse yield trails carried out 2005 to 2007. 'Party Time' (Grant No. 2691) was registered to the Korea Seed and Variety Service (KSVS) for commercialization in 2009.

An eco-beneficial strategy for the production of hydroxymethylfurfural (HMF) was developed using kudzu root extract as a sustainable biomass material and the physicochemical properties of the extract characterized using FESEM, XRD, and FT-IR. The visual image of the kudzu extract and the XRD analysis revealed that the molecular structure of the extract is amorphous and its major composition would be starch. The FT-IR analysis showed 6 characteristic vibration modes contributing to starch and other sugars. The halide ligands of different types significantly affected the synthesis of HMF. The exceptional promotion effect on HMF synthesis was achieved by fluorine ligand of the chromium-based metal catalyst. The most effective stoichiometry ratio of catalyst-to-feed biomass for the synthesis of HMF from kudzu root extract was found at its ratio value of 0.2. The most optimal extraction condition for HMF synthesis from the kudzu extract occurred at 0.3 M HCl. The highest HMF production (34.8 ± 4.8 dwt%) was obtained in the reaction mixture containing 40% (w/v) ionic liquid, 10% (v/v) ethyl acetate, 1% (w/v) halide catalyst, and 50% (v/v) of 0.3 M HCl extract by undergoing the reaction at 130 °C for 12 h.

A simple production process of hydroxymethylfurfural (HMF) was developed by examining its reaction conditions in an ionic liquid, 3-methylimidazolium chloride ([OMIM]Cl). For this, effects of five parameters; extraction time, reaction time, HCl concentration, chromium dichloride (CrCl 2 ) catalyst, and raw plant biomass sources (girasol as a source of inulin and potato tubers as a source of starch); on the yield of HMF were scrutinized. The results showed that HMF yield was significantly affected by each parameter or their combination. The best extraction time and reaction time were identified at 2 h and 60 min, respectively. HCl concentration optima were determined at 0.3 M for girasol biomass and 0.5 M for potato tuber. The presence of CrCl 2 in the reaction mixture prepared from potato tuber biomass exhibited approximately a 2-fold increase in the yield of HMF (54.4% ± 5.1% dwt). In contrast, in girasol reaction mixture, its catalytic effect was not detected. The highest HMF yield (58.2 ± 5.8% dwt) was achieved in the reaction mixture of 4g [OMIM]Cl, 1 ml ethyl acetate, and 5 ml girasol extract (prepared at 2 h extraction in 0.3 M HCl) without CrCl 2 catalyst by undergoing the reaction at 120 °C for 60 min.

A simple two-step process was conducted to scrutinize new catalytic action of chromium fluoride (CrF 3 ) by evaluating its effect on the yield of hydroxylmethylfurfural (HMF) in ionic liquid (1-octyl-3-methylimidazolium chloride) using raw tapioca root biomass with high starch, and hydrolysis efficiency of the biomass extract was characterized by FT-IR spectrometry and FESEM image analysis. The FT-IR absorbance bands confirmed the presence of starch and other soluble sugars in the acidic biomass extract by presenting its frequency signals in the 950–1200 cm −1 regions. FESEM image showed that the efficiency of the biomass hydrolysis is time-dependent. The concentration of CrF 3 significantly affected the yield of HMF. Its highest yield (52.6 ± 4.8 dwt%) was achieved at 1% CrF 3 , but over 1% CrF 3 its yields began to decline. The combined addition of CrF 3 and CrBr 3 showed ca. 1.3-fold higher yield than CrF 3 alone. The reaction rates of HMF synthesis were considerably influenced by the stoichiometry ratio of CrF 3 to biomass. The highest reaction rate (0.41 g/g biomass/h) occurred at 0.2 ratio value.

In plants, heteroblasty reflects the morphological adaptation during leaf development according to the external environmental condition and affects the final shape and size of organ. Among parameters displaying heteroblasty, leaf index is an important and typical one to represent the shape and size of simple leaves. Leaf index factor is eventually determined by cell proliferation and cell expansion in leaf blades. Although several regulators and their mechanisms controlling the cell division and cell expansion in leaf development have been studied, it does not fully provide a blueprint of organ formation and morphogenesis during environmental changes. To investigate genes and their mechanisms controlling leaf index during leaf development, we carried out molecular-genetic and physiological experiments using an Arabidopsis mutant. In this study, we identified macrophylla (mac) which had enlarged leaves. In detail, the mac mutant showed alteration in leaf index and cell expansion in direction of width and length, resulting in not only modification of leaf shape but also disruption of heteroblasty. Molecular-genetic studies indicated that mac mutant had point mutation in ROTUDJFOLIA3 (ROT3) gene involved in brassinosteroid biosynthesis and was an allele of rod-I mutant. We named it rnac/rot3-5 mutant. The expression of ROT3 gene was controlled by negative feedback inhibition by the treatment of brassinosteroid hormone, suggesting that ROT3 gene was involved in brassinosteroid biosynthesis. in dark condition, in addition, the expression of ROT3 gene was up-regulated and mac/rot3-5 mutant showed lower response, compare to wild type in petiole elongation. This study suggests that ROT3 gene has an important role in control of leaf index during leaf expansion process for proper environmental adaptation, such as shade avoidance syndrome, via the control of brassinosteroid biosynthesis.

The CCR4-CAF1-NOT complex-mediated degradation of mRNA is a fundamental aspect of gene regulation in eukaryotes. We herein examined the role of AtCAF1 in the innate immune and wound responses of plants. Our results showed that overexpression of AtCAF1 significantly down-regulated the transcript level of EFR but not FLS2 and BRI1, as well as abolished up-regulated expression pattern of EFR in response to wounding. Consistently, Agrobacterium-mediated transient expression of GUS was highly enhanced in the transgenic plants overexpressing AtCAF. Furthermore, JA responsive genes were down-regulated by overexpression of AtCAF, causing the transgenic plants overexpressing AtCAF more susceptible to necrotrophic fungal pathogen, Botrytis cinerea. These results suggest that The CCR4- CAF1-NOT complex-mediated degradation of mRNA negatively regulates wounding-mediated disease resistance in Arabidopsis thaliana.

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41-79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P(i)). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P(i) signaling pathway through the ubiquitin-26S proteasome system.

We report for the first time the direct conversion of raw grape berry biomass to hydroxymethylfurfural (HMF) using ionic liquid solvents with metal chloride catalysts. Exploiting raw plant biomass as a biorefinery feedstock is innovative for sustainable chemical industry. The use of the raw biomass to synthesize compounds can indeed lead to less energy consumption and less CO2 emissions. Using raw plant biomass skips pretreatment steps that are required to produce biomaterials such as carbohydrates or cellulosic biomass. Here, grape berry biomass was used as a raw chemical feedstock for the production of hydroxymethylfurfural, a key platform intermediate for syntheses of future renewable biofuels. We examined 3 ionic liquid solvents, 3 reaction temperatures, 5 chloride catalysts, and 5 concentrations of HCl. We found an increasing HMF yields depending on reaction conditions. 1-octyl-3-methylimidazolium chloride was most effective for HMF synthesis. Addition of HCl or metal chlorides alone showed little improvement. The highest HMF yield of about 100 mg HMF per mL of grape biomass extract was obtained using 0.3 M HCl, [OMIM]Cl, and CrCl2 at 100°C for 3 h. Our study provides a model system of sustainable production of valuable compounds from raw plant biomass.

Chromium halides were introduced for the sustainable production of hydroxymethylfurfural (HMF) from raw acorn biomass using an acidic ionic liquid. The free sugars (glucose and maltose) released by the acidic hydrolysis of the biomass were confirmed by the FT-IR absorption bands around 995-1014cm(-1) and HPLC. FESEM analysis showed that the acorn biomass contains various sizes of starch granules and their structures were severely changed by the acidic hydrolysis. An optimal concentration of HCl for the HMF yields was 0.3M. The highest HMF yield (58.7+1.3dwt%) was achieved in the reaction mixture of 40% [OMIM]Cl+10% ethyl acetate+50% 0.3M HCl extract containing a mix of CrBr(3)/CrF(3). The combined addition of two halide catalysts was more effective in the synthesis of HMF (1.2-fold higher on average) than their single addition. The best productivity of HMF was found at 15% concentration of the biomass and at 50%, its relative productivity declined down to ca. 0.4-fold.

2-Aminoindan-2-phosphonic acid (AIP), a specific competitive phenylalanine ammonia lyase (PAL) inhibitor was applied to a suspension cell culture of Cistanche deserticola. The effects of AIP treatment on cell growth, PAL activity, contents and yields of total phenolic compound, salidroside and four phenylethanoid glycosides (PheGs) are investigated. The results demonstrated that, 0.5 and 2.0 μM AIP treatments had similar effects on the measurements investigated in this study. AIP treatment resulted in significant decreases in PAL activity, total phenolic compounds content, and PheGs content. Linear regression analysis showed that PAL activity had a high correlation coefficient with the total phenolic compound content and the four PheGs contents. Total PAL activity-time area under curve (AUC) had a high correlation coefficient with the total phenolic compound yield and the yields of five tested compounds in untreated cell samples. In AIP-treated cells, total PAL activity-time AUC retained a high correlation with the total phenolic compound yield and the yields of three tested compounds, echinacoside, acteoside, and tubuloside A, but not salidroside and cistanoside A. The difference could be caused by the different biosynthetic origins of each of the tested compounds. These results demonstrate the important role of PAL in the biosynthesis of PheGs in the suspension cell culture of C. deserticola.

Chicory roots were introduced for the direct synthesis of hydroxymethylfurfural (HMF), a platform chemical using HCl and the ionic liquid, 1-octyl-3-methylimidazolium chloride ([OMIM]Cl). HCl played an important role in the hydrolysis of chicory sugar polymers. The highest inulin conversion (60.6 ± 4.4% dwt) occurred with 0.3 M HCl. Increasing HCl concentration up to 1.5 M did not improve inulin conversion. Negligible inulin hydrolysis was found in the absence of HCl. The highest HMF yield (50.9 ± 5.4% dwt) was obtained in a reaction mixture containing 50% of the reaction solvent ([OMIM]Cl + ethyl acetate) and 50% of the root extract prepared in 0.3 M HCl solution. Increasing the HCl concentration resulted in a decrease in HMF yield. The addition of CrCl 2 catalyst decreased HMF yields by an average of 25%. The highest productivity of HMF was achieved in the reaction mixture containing 50% reaction solvent (an admix of 80% [OMIM]Cl + 20% ethyl acetate) and 50% chicory root extract prepared from 20% (w/v) root biomass concentration.

Starch can be exploited as an abundant resource for sustainable production of diverse chemical intermediates such as hydroxymethylfurfural (HMF). We present a simple process of producing HMF from starches using an ionic liquid, 1-octyl-3-methylimidazolium chloride ([OMIM]CI), and CrCl 2 catalyst. The addition of HCl and CrCl 2 significantly affected the yields of HMF on the whole. CrCl 2 increased the yields of HMF by approximately two-folds on average. The synthesis of HMF was most effective between 60 and 90 min reaction time at both concentrations of 0.3 and 0.5 M HCl. Starch concentration had influence on the productivity of HMF. At 20% starch concentration with 0.3 and 0.5 M HCl, its productivity was greatest (2.8 folds high). Eight starch sources (corn, wheat, rice, potato tuber, sweet potato, tapioca, acorn, and kudzu starch) were tested for the synthesis of HMF. The highest yields of HMF (73.0 ± 3.8 wt%) were obtained in tapioca starch dissolved in 0.5 M HCl.

Hydroxymethylfurfural (HMF) is an important chemical intermediate, but it has not been widely used be- cause of low yields and high production costs. Sucrose is available at lower costs than other sugars and thus could be a biomass-derived abundant source for HMF production. In this study, a catalytic process for efficiently producing HMF from sucrose was scrutinized using 1-methyl-3-octylimidazolium chloride ((MOIM)Cl) as a reaction solvent, and HCl and metal chlorides (CrCl2 and Zncl2) as a catalyst. The rate of sucrose hydrolysis was relatively much faster in the reactions with HCl than without it. The hydrolysis of sucrose to fructose and glucose was affected by its reaction time. The mixed solvent of 50% (MOIM)Cl and 50% sucrose solution with HCl was more effective in HMF synthesis than single solvent alone. The addition of ZnCl2 and CrCl2 increased HMF yields by approximately 1.2-1.8-fold and its higher yield was found in the latter. The highest yield (82.0±3.9 wt%) in HMF production was achieved in the reaction mixture containing 5 g (MOIM)Cl and 5 mL of 20% sucrose solution with 0.5 M HCl plus CrCl2 at 30 min reaction time. How- ever, 0.3 M HCl was more effective for the HMF productivity than 0.5 M HCl.

Recently, hydroxymethylfurfrual (HMF) has been highlighted as a key intermediate for the production of liquid biofuels and other valuable compounds. We used sesame roots as a biocatalyst to synthesize HMF using flask cultures. The synthesis of HMF was identified by GC-mass analysis. The highest root growth was observed in cultures with 1.0 mg/l NAA at , while root growth was not found in those without NAA treatment. When silver nitrate () was added, the root growth was greatest in those treated with 0.5 mg/l and cultured at . In the case of HMF synthesis, its highest yield was obtained in those treated with 0.5 mg/l NAA at , but low HMF was detected in those treated without naphthaleneacetic acid (NAA). The addition of to the culture medium showed a 8-10% reduction in HMF yield compared to that of the control, indicating its inhibitory effect on the synthesis of HMF. On the whole, an optimal culture temperature for HMF synthesis seemed to be between .

【A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl- $\beta$ -D-N,N'-diacetylchitobioside and 4-methylumbelliferyl- $\beta$ -D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.】

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.

Phosphate, a favorable phosphorous form for plant, is one of major nutrient elements for growth and development in plants. Plants exhibit various physiological and biochemical responses in reaction to phosphate starvation in order to maintain phosphate homeostasis. Of them, expression of high affinity phosphate transporter gene family and efficient uptake of phosphate via them is a major physiological process for adaption to phosphate deficient environment. Although the various genetic resources of high affinity phosphate transporter are identified recently, little is known about their functions in plant that is prerequisite information before applying to crop plants to generate valuable transgenic plants. We demonstrated that Arabidopsis transgenic plants over-expressing two different high affinity phosphate transporter gens, OsPT1 and OsPT7, derived from rice, exhibit better growth responses compared with wild-type under phosphate starvation condition. Specially, OsPT7 gene has proven to be more effective to generate Arabidopsis transgenic plant tolerant to phosphate deficiency than OsPT1. Furthermore, the expression level of AtPT1 gene that is one of reporter genes specifically induced by phosphate starvation was significantly low compared with wild-type during phosphate starvation. Taken together, these results collectively suggest that over expression of OsPTl and OsPT7 genes derived from monocotyledonous plant function efficiently in the dicotyledonous plant, relieving stress response caused by phosphate starvation and leading to better growth rate.

The regeneration and growth of bulblets from the bulb scale segments of Hyacinthus orientalis L. cv. Pink Pearl were more efficient in IBA than IAA at the same concentrations (1.0 and 3.0 ㎎/l).. The normal (base-down) orientation of explants was more effective for bulblet regeneration and root growth than the inverted (base-up) orientation. The growth of bulblets and roots was increased higher in the perlite than the agar medium. These results suggested that the alternate culture system, first cultured in the agar medium for bulblet regeneration, and then in the perlite medium for bulblet growth, may be more useful for efficient in vitro culture of hyacinth (H. orientalils) cv. Pink Pearl.

Leaf shape is controlled early on by initiation at the shoot apical meristem (SAM), as well as by changes in the rates and planes of cell division and the polarity-dependent differentiation of leaf cells. To elucidate the regulation of this differentiation by signal(s) from the SAM, we screened for mutations in genes that might be involved in these early processes. A novel recessive mutant, 356-2 [identified as a new allele of thedeformed root and leaf1 (drl1) mutant], was isolated from a collection ofDs transposon insertion lines. The356- 2/drl1- 101 mutant produces narrow, filamentous leaves and defective mer-istems. Its palisade cells have a spongy cell-like structure and are fewer in number, indicating that the leaves are abaxialized. Interestingly, some of those filament-like leaves have no vascular tissues inside their blades.DRL1 encodes a protein similar to the yeast elongator-associated protein (EAP) KTI12. The amino acid sequence of DRL1 is universally conserved in prokaryotes and eukaryotes. These facts suggest that DRL1 might positively regulate leaf polarity and SAM activity by controlling cell proliferation and differentiation.

The effects of various abiotic factors, including polyethylene glycol (PEG), silver nitrate (AgNO3) and potassium phosphate (KH2PO4) on biomass growth, extracellular production of recombinant fungal phytase and its transcription activity by realtime RT-PCR were examined with transformed sesame hairy roots. The PEG treatments decreased both biomass growth (80.5≈82.3%) and phytase production (82.1≈96.4%) at all concentrations tested, except 1.0 g/L PEG, which increased biomass growth to 112.4% of that of the control. The AgNO3 treatments also resulted in reduced biomass growth (77≈92%) and phytase production (84.4≈96.3%) at all concentrations applied except 1.5 mg/L AgNO3, at which biomass was increased to 109.6% of that of untreated roots. The potassium phosphate treatments increased biomass growth, production of recombinant fungal phytase and its transcription until a concentration of 340 mg/L was attained, and at 510 mg/L a rapid decrease was observed in all of the aforementioned parameters. Combined treatments of PEG (1 g/L) and AgNO3 (1.5 mg/L) exhibited both positive and negative influences on biomass growth and production and transcription of the recombinant fungal phytase when medium containing 170 and 340 mg/L of potassium phosphate were used, respectively. The addition of PEG alone to culture medium containing potassium phosphate at both of the aforementioned concentrations decreased biomass while increasing production and transcription of recombinant fungal phytase. Conversely, the addition of AgNO3 alone to culture medium increased biomass but decreased production and transcription of fungal phytase. Throughout these experiments, the most effective treatment for enhanced biomass growth, and enhanced production and transcription of recombinant fungal phytase was attained when the 1 g/L PEG and 1.5 mg/L AgNO3 were combined in medium containing 340 mg/L potassium phosphate.

AvrRpt2 protein triggers hypersensitive response (HR) and strong disease resistance when it is translocated from a bacterial pathogen Pseudomonas sp. to host plant cells containing a cognate RPS2 resistance protein through Type III Secretion System (TTSS). However, AvrRpt2 protein can function as the effector that suppresses a basal defense and enhances the disease symptom when functional RPS2 resistance protein is absent in the infected plant cells. Using Affymetrix Arabidopsis DNA chip, we found that many genes were specifically regulated by AvrRpt2 protein in the rps2 Arabidopsis mutant. Here, we showed that expression of AtERF11 that is known as a member of B1a subcluster of AP2/ERF transcription factor family was down regulated specifically by AvrRpt2. To determine its function in plant resistance, we also generated the Arabidopsis thaliana transgenic plants constitutively overexpressing AtERF11 under CaMV 355 promoter, which conferred an enhanced resistance against a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. Thus, these results collectively suggest that AtERF11 plays a role as a positive regulator for disease resistance against biotrophic bacterial pathogen in plant.

The processes for leaf development in dicotyledonous plants are surprisingly complex, while the mechanism of controlling and coordinating them is poorly understood. To characterize the fundamental features of the leaf development of Arabidopsis, we first attempted to isolate mutants that alter leaf morphology. Here, leaf morphological mutant of Arabidopsis, lanceolata1 (lan1) which has small and narrow leaves have isolated and characterized. To clarify the function of LAN1 in organ development, we characterized lan1-7 mutant using an anatomical and genetic approach. The lan1-7 mutant had reduced size of foliage leaves and reduced dimensions of stems. A reduction both in cell size and in cell number was evident at the cellular level in the lan1 mutant, revealing that LAN1 gene appears to affect cell division at an earlier stage and cell elongation throughout the development of leaf primordia. From the analysis of heterogeneous plant with lan1 mutation and 35S-AG transgenic plant, AG gene is revealed to regulate leaf morphology under the control of 35S promoter. Thus, MADS-box gene was revealed to have some relationship to that of LAN1 gene at certain stage in leaf development processes.

The differential transcription activity of dehydroascorbate reductase (DHAR) was scrutinized in the transformed hairy roots, leaves, stems, roots, and developing seeds of sesame (Sesamum indicum L.). Its relative levels of expression were compared via the threshold cycle (CT) method, using real-time RT-PCR. Ubiquitous expression of DHAR in all organs was confirmed with both reai-time and conventional RT-PCR. With the former, DHAR transcript levels were, unexpectedly, 4.7-fold higher in the stem tissue than in the hairy roots; the lowest levels were detected in the seeds. It was possible to determine the transcription activity of hairy root DHAR, with a low amount of total RNA (0.5 ng), using real-time RT-PCR but not with conventional RT-PCR gel analysis. This indicated that the former is more sensitive and efficient than the latter for the detection of gene expression. We also characterized DHAR cDNA cloned from transformed hairy roots, and found that sequence identity for the deduced amino acids of the DHAR enzyme was shared at 60 to 83% among plant species. The algorithm prediction and phylogenetic analysis suggested that the cloned cDNA polypeptide is cytosolic DHAR. Another feature of the cloned cDNA polypeptide was the presence of a CXXS instead of CXXC motif in the active center of the DHAR enzyme.

The scale segments of the bulblets of Hyacinthus orientalis L. cv. Anna Marie were examined to improve their growth and development with cold-pretreatment and sucrose. The cold-pretreated (4 °C for 4 months) segments showed higher growth and better development of the bulblets on medium without sucrose than ones stored at 20 °C. A rapid decrease in starch content of bulb pieces was found during the first 2 weeks in all cultures and thereafter the content decreased gradually. A scanning electron microscopic observation during the bulblet growth and development showed a gradual decreasing trend of the starch granules from 2 to 16 weeks of the cultures. SDS-PAGE electrophoresis revealed the presence of a characteristic polypeptide of approximately 45 kD, which is assumed to be a major storage protein in the bulblets.

Sesame (Sesamum indicum L.) hairy roots were transformed with a fungal (Aspergillus) phytase and their culture conditions were surveyed for the extra-cellular production of the recombinant phytase protein in shake flasks. Kanamycin resistance of sesame hairy roots was observed at 50 μg ml−1 kanamycin sulfate and southern hybridization analysis confirmed the existence of the phytase gene in the hairy root genomic DNA. The continuous dark condition was more effective for both the root growth and phytase production than light. Slightly higher root growth was determined at 30 °C than 26 °C in Murashige & Skoog (MS) medium supplemented with 3% sucrose, while the final phytase production was greatest in MS medium with 5 or 3% sucrose at both temperatures of 26 and at 30 °C. Among the culture media used, full-strength MS medium was exclusively efficient for production of the recombinant phytase. Most rapid increase rates in both the root growth and phytase production were detected at the 4th week of the culture periods and thereafter their rates began to decrease. Our results indicated that 5–6-week culture periods may be necessary for the maximal phytase production. Western analysis revealed that even though the phytase proteins expressed were measured with greater activities in the liquid medium than in the root tissues, they were still retained in the tissues.

SDS–PAGE analysis of scale extracts of Hyacinthus orientalis L. cv. Carnegie exhibited various polypeptide bands in electrophoresis gels. As the culture period of the hyacinth bulb scales increased, some polypeptide bands disappeared and some new bands appeared. The most outstanding feature was that a very strong 29 kD polypeptide band was observed during the early stages of scale culture, but after 8 weeks of culture, the polypeptide band was nearly undetectable in the gel, indicating that this polypeptide may be one of a family of major storage proteins in the hyacinth scales. Treatment with IBA at concentrations of 1.5 and 3.0 mg/l showed higher regeneration and growth of the bulblets formed from the scale segments of bulbs than those treated with IAA at the same concentrations. No recognizable difference was observed between cultures on either perlite or agar media. The normal (base-down) orientation of the explants had greater influence on the root growth than their inverted (base-up) orientation. The above data suggested that a two-step culture system (first cultured in the agar medium, then in the perlite medium) may be more useful for efficient in vitro culture of this hyacinth cultivar.

A cDNA (SeFAD2) encoding a microsomal ω-6 desaturase was characterized in association with its temporal expression and changes in linoleic acid levels in sesame (Sesamum indicum L.) oilseeds. Much higher sequence identities of the SeFAD2 protein were found with microsomal ω-6 (71–81%) than with plastidial ω-6 desaturases (22–24%) and the sequences within the conserved histidine boxes were some different from those of other plant histidine boxes examined. Phylogenetic analysis revealed that the SeFAD2 gene might have independently diverged as a different subfamily or family member. At least 1–2 copies of the SeFAD2 gene existed in sesame genome and its expression was seed-specific. The SeFAD2 gene vigorously expressed between 18 and 27 days after flowering (DAF). Changes in linoleic acid levels occurred coincidently with changes in the SeFAD2 transcript levels according to the development of sesame oilseeds. It was assumed that the SeFAD2 gene is responsible for production of storage linoleic acid in sesame seeds.

R2R3 MYB transcription factors play regulatory roles in plant responses to various environmental stresses and nutrient deficiency. In this study, we isolated and designated OsMYB4P, an R2R3 MYB transcription factor, from rice (Oryza sativa L. 'Dongjin') under phosphate-deficient conditions. OsMYB4P was localized in the nucleus and acted as a transcriptional activator. Transcriptional levels of OsMYB4P in cell suspension, shoots, and roots of rice increased under phosphate-deficient conditions. Shoots and roots of OsMYB4P-overexpressing plants grew well in high- and phosphate-deficient conditions. In addition, root system architecture was altered considerably as a result of OsMYB4P overexpression. Under both phosphate-sufficient and -deficient conditions, more Pi accumulated in shoots and roots of OsMYB4P-overexpressing plants than in the wild type. Overexpression of OsMYB4P led to greater expression of Pi transporter-family proteins OsPT1, OsPT2, OsPT4, OsPT7, and OsPT8 in shoots, and to decreased or unchanged expression of these proteins in roots, with the exception of OsPT8. These results demonstrate that OsMYB4P may be associated with efficient utilization of Pi in rice through transcriptional activation of Pi homeostasis-related genes.

GDP dissociation inhibitor (GDI) plays an essential role in regulating the state of bound nucleotides and subcellular localizations of Rab proteins. In our previous study, we showed that OsGDI3 facilitates the recycling of OsRab11 with a help of OsGAP1. In this study, we show that OsGDI3 complement the yeast sec19-1 mutant, a temperature-sensitive allele of the yeast GDI gene, suggesting that OsGDI3 is a functional ortholog of yeast GDI. To obtain further knowledge on the function of OsGDI3, candidate OsGDI3-interacting proteins were identified by yeast two-hybrid screens. OsMAPK2 is one of OsGDI3 interacting proteins from yeast two-hybrid screens and subject to further analysis. A kinase assay showed that the autophosphorylation activity of OsMAPK2 is inhibited by OsGDI3 in vitro. In addition, ectopic expressions of OsGDI3-in Arabidopsis cause reductions at the level of phosphorylated AtMPK in phosphorylation activity. Taken together, OsGDI3 functions as a negative regulator of OsMAPK2 through modulating its kinase activity.

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 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.