Citral dehydrogenase involved in geraniol oxidation pathway: purification,characterization and kinetic studies from <Emphasis Type="Italic">Persicaria minor</Emphasis> (<Emphasis Type="Italic">Polygonum minus</Emphasis> Huds.)

Plants emit semiochemicals as alarm signals upon attack by herbivores or insect pests. Complex insect-plant interaction through alarm pheromones can be manipulated to improve crop protection. Geraniol, citral and geranic acid are monoterpenoid compounds from plants and they play a role as semiochemical alarm pheromones. In plants, the oxidation of geraniol into geranic acid is catalyzed by two oxidoreductases, geraniol dehydrogenase and citral dehydrogenase. In this study, citral dehydrogenase isoenzymes from Persicaria minor (Polygonum minus) leaves were purified to homogeneity and characterized. Enzyme purification through Toyopearl GigaCap Q-650 M column chromatography at pH 7.5 produced two activity peaks, suggesting the existence of two citral dehydrogenase isoenzymes. Both isoenzymes were different in isoelectric point and kinetic parameters but similar in pH and optimal temperature as well as in substrate specificity. Findings from this study will provide a basic understanding for the development of recombinant production of these particular enzymes. Further studies on molecular structure involved could be exploited in transgenic plant as an integrated pest management strategy.     

Toxicity of terpenes to spores and mycelium of Penicillium digitatum

Spores, although often considered metabolically inert, catalyze a variety of reactions. The use of spores instead of mycelium for bioconversions has several advantages. In this paper, we describe the difference in susceptibility of mycelium and spores against toxic substrates and products. A higher resistance of spores toward the toxic effects of bioconversion substrates and products is an advantage that has not been studied in detail until now. This paper shows that spores of Penicillium digitatum ATCC 201167 are on average over 2.5 times more resistant than mycelium toward the toxicity of substrates, intermediates, and products of the geraniol bioconversion pathway. Furthermore, the higher resistance of spores to citral was shown as an advantage in its biotransformation by P. digitatum. Using three different approaches the toxicity of the compounds were tested. The order of toxicity toward P. digitatum was, starting with the most toxic, citral > nerol/geraniol > geranic acid > methylheptenone > acetaldehyde.     

Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: Similar activity but difference in subcellular localization

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with K_m values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol. The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.     

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

Geraniol biotransformation-pathway in spores of Penicillium digitatum

Spores of Penicillium digitatum ATCC 201167 transform geraniol, nerol, citral, and geranic acid into methylheptenone. Spore extracts of P. digitatum convert geraniol and nerol NAD+-dependently into citral. Spore extract also converts citral NAD+-dependently into geranic acid. Furthermore, a novel enzymatic activity, citral lyase, which cofactor-independently converts citral into methylheptenone and acetaldehyde, was detected. These result show that spores of P. digitatum convert geraniol via a novel biotransformation pathway. This is the first time a biotransformation pathway in fungal spores has been substantiated by biochemical studies. Geraniol and nerol are converted into citral by citrol dehydrogenase activity. The citral formed is subsequently deacetylated by citral lyase activity, forming methylheptenone. Moreover, citral is converted reversibly into geranic acid by citral dehydrogenase activity.     

Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k(cat)/K(m) = 2.02 × 10(6) M(-1) s(-1)), followed by geraniol (k(cat)/K(m) = 1.57 × 10(6) M(-1) s(-1)). Apparent K(m) values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.     

Comparison of plant-based expression platforms for the heterologous production of geraniol

We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 μg/g fresh weight, fw), followed by the transient expression system (27 μg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 μg/g fw), and finally hairy root cultures (9 μg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 μg/g fresh biomass per day, whereas transient expression produced 5.9 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol.     

Rose-scented geranium (<Emphasis Type="Italic">Pelargonium</Emphasis> sp.) generated by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A₄ and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A₄ strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A₄ and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A₄ and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5 months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.     

转基因玉米双抗12-5-21的抗虫性及对草甘膦的耐受性

[目的]评价转cry1Ab/cry2Aj和G10evo-epsps基因玉米双抗12-5的杂交后代双抗12-5-21对亚洲玉米螟、黏虫、棉铃虫的抗性及对目标除草剂草甘膦的耐受性.[方法]分别在6叶期和花丝期、6叶期、花丝期对亚洲玉米螟、黏虫、棉铃虫进行田间人工接虫;自然条件下,收获期剖秆调查双抗12-5-21对鳞翅目害虫...     

Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance.

The milk protein, lactoferrin, is known to have antibacterial, antiviral, and antifungal activities. To explore the possibility of conferring disease resistance in plants by expressing this protein, the gene for the full-length human lactoferrin (HLF), as well as the N-lobe, the N-terminal half molecule (HLFN), was introduced into rice plants and expressed constitutively under the control of the cauliflower mosaic virus 35S promotor. Western blot analysis of leaves from HLF-transgenic rice plants showed an 80 kDa-band, which was about 1-2 kDa less than human milk lactoferrin. HLFN was expressed as a 45-kDa protein and retained its heparin-binding property. Deglycosylation experiments suggested that both proteins produced by the plants had plant-type oligosaccharide chains. The transgenic rice plants were assessed for resistance against disease-causing bacteria, virus, and fungi. Of the pathogens tested, significant resistance against Burkholderia (Pseudomonas) plantarii, the causative agent of bacterial seedling blight disease, was observed in the transgenic plants expressing HLF or HLFN.     

转基因玉米2HVB5的性状鉴定及遗传稳定性分析

为了明确转基因玉米2HVB5的目标性状及遗传稳定性,对回交转育郑58的BC₅S₁、BC₅S₂代转基因玉米2HVB5分别进行了Southern blot、ELISA、室内和田间生物活性测定、靶标除草剂草铵膦耐受性分析及农艺性状调查。结果显示,2HVB5中目的基因cry2Ah-vp和bar都是以单拷贝的形式整合到玉米基因组并稳定遗传,Cry2Ah-vp和PAT蛋白在2HVB5植株的不同时期、不同组织部位均有表达,其中在叶片中的表达量相对较高,分别达到2-3.5 μg/g FW（鲜重）和8-17 μg/g FW（鲜重）。室内生物活性检测结果表明,2HVB5转基因玉米对东方粘虫和棉铃虫有很高的抗性,接虫后4-5 d幼虫死亡率达100%,对草地贪夜蛾有明显的体重抑制。田间抗虫性鉴定结果也表明,2HVB5转基因玉米对东方粘虫和棉铃虫均达到高抗水平,平均抗性级别分别为1.19-1.29和0.60-0.73。2HVB5转基因玉米可耐受田间使用中剂量4倍量的草铵膦,农艺性状与对照郑58相比无显著差异。转基因玉米2HVB5遗传稳定,高抗虫耐除草剂,可用于玉米害虫尤其是夜蛾科害虫的防治,具有产业化应用前景。     

Sucrose-mediated priming of plant defense responses and broad-spectrum disease resistance by overexpression of the maize pathogenesis-related PRms protein in rice plants

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.     

Distribution of hydroxamic acids in zea mays tissues

The hydroxamic acid content of leaves of cereals correlates well with resistance to aphids. In maize these compounds were absent from xylem exudates and guttation drops. Lateral veins of leaves of 7-day-old maize plants contained 8 mmol/kg fr. wt. while the entire leaf contained only 4.2 mmol/kg fr. wt. In leaves of 20-day-old plants, these amounts decreased by ca one-third. In mesocotyls, the cortex and central vascular cylinder contained 1.3 and 2.2 mmol/kg fr. wt, respectively. In 12-day-old wheat plants, the complete leaves and their veins contained 2.4 and 6.4 mmol/kg fr. wt respectively. Thus, the concentration of hydroxamic acid was always higher in the vascular bundles.     

Constitutive expression of a fusion gene comprising Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) confers enhanced disease and insect resistance in transgenic tobacco

Plant defensins are small, basic cysteine-rich peptides that can inhibit the growth of a broad range of fungi or bacteria at micro-molar concentrations. They have been introduced as transgenes into different species to enhance host resistance to pathogens. In this study, a fusion gene of two defensins, Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) fused by a linker peptide of a polyprotein precursor from Impatiens balsamina was introduced into tobacco (Nicotiana tabacum var. Xanthi) via Agrobacterium-mediated leaf section transformation. Putative transgenic plants were confirmed by PCR analysis and integration of the fusion gene was confirmed by Southern blotting. RT-PCR analysis showed that the fusion gene was expressed in several confirmed transgenic plants. Western blotting analysis of crude protein extracts from leaves of the transgenic plants with anti-Tfgd2 and anti-RsAFP2 antibodies exhibited an 8 and 9 kDa bands corresponding to size of the fusion gene and confirmed the expression of fusion protein. When the leaves of transgenic plants were challenged with Rhizoctonia solani and Phytophthora parasitica var. nicotianae pathogens, they showed enhanced levels of disease resistance along with resistance to the generalist herbivore, Spodoptera litura larvae compared to control. Our results demonstrate that Tfgd2–RsAFP2 fusion protein is effective in protecting the transgenic plants against fungal and insect pathogens.     

Costs of induced volatile production in maize

Herbivore‐induced plant volatiles have been shown to serve as indirect defence signals that attract natural enemies of herbivores. Parasitoids and predators exploit these plant‐provided cues to locate their victims and several herbivores are repelled by the volatiles. Recently, benefits, in terms of plant fitness, from the action of the parasitoids were shown for a few systems. However, the cost of production of herbivore‐induced volatiles for the plant remains unknown. Here, we estimate the fitness cost of the production of induced volatiles in maize, Zea mays. Plants were treated with regurgitant of Spodoptera littoralis or with the elicitor volicitin and we measured dry weight of plant parts at specific times after treatments. After a two‐week treatment period, the dry‐weight of leaves of induced plants was lower than that of un‐induced plants, suggesting a metabolic cost for induced defence. However, maize plants seem to compensate for this loss during subsequent growth, since seed production at maturity was not different for unharmed plants and plants treated with caterpillar regurgitant. For volicitin treated plants a small but significant reduction in seed production was found. It is likely that the treatments also induced the production of other defence compounds, which will contribute to the cost. Yet, a comparison of six maize inbred lines with distinct differences in volatile emissions showed a strong correlation between the intensity of induced emissions and reduction in plant performance. An analysis of the terpenoids that accumulated in the leaves of the inbred lines revealed non‐volatilised compounds are constitutively present in maize and only the volatilised compounds are induced. Interestingly, the lines that released the largest amounts of induced volatiles also contained more of the non‐volatile terpenoids. Based on these results and results from a previous study on the benefits of attracting parasitoids, we conclude that costs of induced volatile production in plants are counterbalanced by the benefits as long as natural enemies of the herbivores are present in the environment.     

Genome-wide analysis of terpene synthases in soybean: functional characterization of GmTPS3

Terpenes (terpenoids or isoprenoids) constitute a large class of plant natural products and play numerous functional roles in primary and secondary metabolism as well as inecological interactions. This study presents a genomic analysis of 23 putative soybean (Glycine max) terpene synthase genes (GmTPSs) distributed over 10 of 20 chromosomes. The GmTPSs are grouped into six types based on gene architecture and sequence identity. Sequence alignment indicates that most GmTPSs contain the conserved aspartate-rich DDX₂D motif, and two clades encoded by TPS-a and TPS-b contain variations of an arginine-rich RRX₈W motif. Quantitative real-time PCR analysis demonstrated that GmTPSs were predominantly expressed in reproductive organs. Heterologous expression followed by enzymatic assay suggested that GmTPS3 functions as a geraniol synthase. We also generated transgenic tobacco plants ectopically expressing GmTPS3. In dual-choice feeding-preference and force-feeding assays, the transgenic tobacco lines expressing GmTPS3 exhibited enhanced resistance to cotton leafworms and an increased level of geraniol. Taken together, these data provide a comprehensive understanding of the TPS family in soybeans and suggest a promising approach to engineering transgenic plants with enhanced insect resistance.     

Changes in photosynthetic carbon flow in transgenic rice plants that express C4-type phosphoenolpyruvate carboxykinase from Urochloa panicoides

A cDNA encoding phosphoenolpyruvate carboxykinase (PCK) of Urochloa panicoides (a PCK-type C4 plant) was expressed in rice (Oryza sativa cv Tsukinohikari) plants under the control of the promoter of a maize (Zea mays) gene for phosphoenolpyruvate carboxylase or pyruvate, orthophosphate dikinase with the transit peptide of the small subunit of Rubisco. Crude extracts prepared from the green leaves of transgenic plants had high PCK activity and the newly expressed PCK was localized in chloroplasts. In labeling experiments with (14)CO(2) up to 20% of the radioactivity was incorporated into 4C compounds (malate, oxaloacetate, and aspartate) in excised leaves of transgenic plants, as compared with about 1% in excised leaves of control plants. There was a positive correlation between PCK activity and the extent of labeling of 4C compounds. When L-[4-(14)C]malate was fed to excised leaves the extent of incorporation of radioactivity into sucrose was 3-fold greater in transgenic plants than in control plants and the level of radiolabeled aspartate was significantly lower in transgenic plants. These results indicate that the ectopic expression of PCK in rice chloroplasts was able partially to change the carbon flow in mesophyll cells into a C4-like photosynthetic pathway. Such a strategy appears to provide a possible method for enhancing the photosynthetic capacity of C3 plants.     

Role of hydroxycinnamic acids in the infection of maize silks by Fusarium graminearum Schwabe

In the current study, the hydroxycinnamic acids in silks of diverse maize inbred lines differing in Fusarium resistance were determined at several times after inoculation with Fusarium graminearum or sterile water as control. The main objective was to determine the possible relationship between the hydroxycinnamic acid changes in silks and ear rot resistance. Several changes in the cell-wall-bound hydroxycinnamic acid concentrations were observed after inoculation with F. graminearum, although these changes were not directly correlated with genotypic resistance to this fungus. Ester-bound ferulic acid decreased, probably due to degradation of hemicellulose by hydrolytic enzymes produced by Fusarium spp., while p-coumaric acid and diferulates showed slight increases that, in conjunction, did not result in delayed F. graminearum progression through the silks. It is important to note that the decrease of ferulic acid in the F. graminearum treatment was faster in susceptible than in resistant genotypes, suggesting a differential hemicellulose degradation in silk tissues. Therefore, the ability of the maize genotypes to slow down that process through hemicellulose structural features or xylanase inhibitors needs to be addressed in future studies.