Functional GNA expressed in Escherichia coli with high efficiency and its effect on Ceratovacuna lanigera Zehntner

The mannose-specific GNA (Galanthus nivalis agglutinin, snowdrop lectin) are the resistant proteins with many bioactivities. Snowdrop lectin is different with plant organs and development periods in lectin species, content, and bioactivities. It is an effective and cheap way to obtain much active GNA through overexpression of GNA gene in Escherichia coli. Constructs encoding mature GNA fused with an N-terminal pelB signal sequence protein (PelB) were expressed in E. coli with high efficiency. Recombinant protein productivity was higher than values published before. The insecticidal activity of purified recombinant proteins was assayed on feeding sugarcane wooly aphid (Ceratovacuna lanigera Zehntner), as well as spraying on sugarcane plants infected by aphids. The insecticidal activity was found to be comparable to native GNA. Oral delivery has obvious positive implications for crop protection against insect pests since peptides can be present in, or sprayed on, plant tissues susceptible to damage. A highly efficient expression of functional recombinant GNA would decrease the cost of GNA and promote its wide use, especially to give crop protection in the field.     

A novel lipid transfer protein from the dill Anethum graveolens L.: isolation,structure, heterologous expression,and functional characteristics

A novel lipid transfer protein, designated as Ag‐LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag‐LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag‐LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag‐LTP spatial structure contains four α ‐helices, forming the internal hydrophobic cavity, and a long C‐terminal tail. The measured volume of the Ag‐LTP hydrophobic cavity is equal to ~800 A³, which is much larger than those of other plant LTP1s. Ag‐LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A new pathogenesis-related protein,LrPR4, from <Emphasis Type="Italic">Lycoris radiata</Emphasis>, and its antifungal activity against <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A new Lycoris radiata pathogenesis-related (PR)-4 gene, LrPR4 was isolated. LrPR4 encodes a 142 amino acid protein with a predicted molecular mass of 15.43 kDa and pI of 7.56. The putative LrPR4 shows high similarity to PR4 type proteins from various plant species and belongs to the Barwin family. Like other PR4s from monocot plants, LrPR4 protein contains a conserved Barwin domain and has a signal peptide at its N-terminus. The recombinant LrPR4 protein expressed in Escherichia coli showed activity towards hydrolysing RNA from L. radiata bulbs and antifungal activity. The results of this study suggest that LrPR4 may play a role in the disease resistance responses of plant against pathogen attacks though its antifungal activity.     

OsSEND-1: a new RAD2 nuclease family member in higher plants

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H₂O₂, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.     

Nitrate Reductase and Glucose-6-phosphate Dehydrogenase Activities as Influenced by Leaf Age and Addition of Protein to Extraction Media

Lea fage influences the level of activity and the decay rate of the enzymes nitrate reductase (NR: E.C. 1.6.6.1) and glucose-6-phosphate dehydrogenase (G6PD; E.C. 1.1.1.49) extracted with and without protein in the extraction media. Such influence was determined in three plant species: corn (Zea mays L.), oats (Arena sativa L.), and tobacco (Nicotiana tabacum L.). Leaves of different ages were obtained from plants of various ages, or by removal of leaf blades from different positions on a single shoot. NR activity (per g fresh weight) declined as leaves of these plant species became older, especially when extraction was effected with conventional media (i.e. without added protein). The instability of NR in these extracts increased as leaves became older. Decay of NR in vitro was exponential with time. Addition of protein [3 %(w/v) casein or bovine serum albumin] to extraction media markedly increased the level of NR activity and its stability, especially in older leaves of all three plant species. Addition of protein did not affect the level of G6PD extracted from corn leaf blades, but slightly enhanced its activity in extracts from the oldest leaf blade of oats. G6PD activity also declined as leaf blades of corn and oats became older.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

The contribution of leaching to the rapid release of nutrients and carbon in the early decay of wetland vegetation

Our goal was to quantify the coupled process of litter turnover and leaching as a source of nutrients and fixed carbon in oligotrophic, nutrient-limited wetlands. We conducted poisoned and non-poisoned incubations of leaf material from four different perennial wetland plants (Eleocharis spp., Cladium jamaicense, Rhizophora mangle and Spartina alterniflora) collected from different oligotrophic freshwater and estuarine wetland settings. Total phosphorus (TP) release from the P-limited Everglades plant species (Eleocharis spp., C. jamaicense and R. mangle) was much lower than TP release by the salt marsh plant S. alterniflora from N-limited North Inlet (SC). For most species and sampling times, total organic carbon (TOC) and TP leaching losses were much greater in poisoned than non-poisoned treatments, likely as a result of epiphytic microbial activity. Therefore, a substantial portion of the C and P leached from these wetland plant species was bio-available to microbial communities. Even the microbes associated with S. alterniflora from N-limited North Inlet showed indications of P-limitation early in the leaching process, as P was removed from the water column. Leaves of R. mangle released much more TOC per gram of litter than the other species, likely contributing to the greater waterborne [DOC] observed by others in the mangrove ecotone of Everglades National Park. Between the two freshwater Everglades plants, C. jamaicense leached nearly twice as much P than Eleocharis spp. In scaling this to the landscape level, our observed leaching losses combined with higher litter production of C. jamaicense compared to Eleocharis spp. resulted in a substantially greater P leaching from plant litter to the water column and epiphytic microbes. In conclusion, leaching of fresh plant litter can be an important autochthonous source of nutrients in freshwater and estuarine wetland ecosystems.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in theplasma membrane from rice leaf and root cells

Presence of a high-affinity binding protein for N-acetylchitooligosaccharide (fragments of chitin) elicitor in the plasma membrane from rice leaf and root cells was shown by affinity labeling experiments with an ¹²⁵I-labeled N-acetylchitooligosaccharide derivative. Binding studies also showed that binding site in the leaf cells has a high affinity to highly elicitor-active, larger chitin fragments but much lower or no affinity to less elicitor-active or elicitor-inactive oligosaccharides. The amount of the binding protein in the leaf cells was slightly smaller than that in the suspension-cultured cells but much larger compared to that in the root cells. These results indicate the possible- involvement of the elicitor binding protein in the perception of the elicitor signal in intact rice plant.     

Antifungal activity of the ribosome‐inactivating protein BE27 from sugar beet (Beta vulgaris L.) against the green mould Penicillium digitatum

The ribosome‐inactivating protein BE27 from sugar beet (Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter‐ and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA (rRNA) N‐glycosylase activity on the sarcin–ricin loop of the major rRNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C‐terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α‐helix and a β‐hairpin, similar to the γ‐core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell.     

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. Brian Reavy and Svetlana Bagirova contributed equally to this work.     

Perinuclear and nuclear envelope localizations of <Emphasis Type="Italic">Arabidopsis</Emphasis> Ran proteins

Using phragmoplastin-interacting protein 1 (PhrIP1) as bait, we isolated an Arabidopsis cDNA encoding Ran2, a small Ras-like GTP-binding protein. The interaction between PhrIP1 and Ran2 was confirmed by an in vitro protein–protein interaction assay with purified Ran2 and PhrIP1. The plant Ran2 shares high sequence homology, 78 and 86% at the amino acid level, with human Ran/TC4 and C. elegans Ran, respectively. Our results obtained from enzyme assays and Western blot analysis show that Ran2 has intrinsic GTPase activity and is present in the soluble fraction of Arabidopsis seedling extract. Fluorescent microscopy using anti-Ran2 antibody revealed that the Ran protein is localized in the perinuclear region with the highest concentration at the nuclear envelope. In contrast to its animal counterparts that are present in the nucleoplasm, the Ran protein is absent inside the nucleus. These results suggest that plant Ran proteins may be involved in mediation of nucleocytoplasmic transport and assembly of the nuclear envelope after karyokinesis in plant cells.     

Preponderance of Oxygenated Sesquiterpenes and Diterpenes in the Volatile Oil Constituents of Lactuca serriola L. Revealed Antioxidant and Allelopathic Activity

Using synthetic chemicals in industry and agriculture has led to several environmental problems. Thus, plant products derived from volatile oils (VOs) could be a potential green source for bioherbicides. Little is known about the VOs of Lactuca serriola. Hence, the present study aimed to characterize the VOs chemical composition from the aerial parts of L. serriola, assessment of antioxidant activity, and evaluate allelopathic potential against the noxious weed Bidens pilosa. The VOs were extracted by hydrodistillation and analyzed by GC/MS. The VOs from the Egyptian ecospecies of L. serriola were found to comprise 34 compounds mainly oxygenated sesquiterpenes and diterpenes. The major compounds were isoshyobunone (64.22 %), isocembrol (17.35 %), and alloaromadendrene oxide‐1 (7.32 %). So, L. serriola can be considered as a good source for isoshyobunone, considering that it has a much higher concentration than any other plants. Also, this plant has a high content of the oxygenated diterpene compound, isocembrol, which is rarely found in the VOs of most plants. The VOs expressed strong antioxidant activity. Also, for the first time, our results showed a strong allelopathic activity of VOs from L. serriola on germination and seedling growth of the noxious weed, B. pilosa. We suppose that the activity of the VOs from L. serriola could be attributed to these previously mentioned major compounds, as they represent about 89 % of the total identified oil constituents. Nevertheless, to evaluate these compounds as new allelochemicals, further study is needed to test the allelopathic activity of authenticated standard of these compounds either singular or in combination on several weeds as well as evaluate the safety, and improve the efficacy and stability at the field scale.     

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA‐binding protein ODB1 was identified from a mitochondrial extract after DNA‐affinity purification. ODB1 (organellar DNA‐binding protein 1) co‐purified with WHY2, a mitochondrial member of the WHIRLY family of plant‐specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52‐1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo‐protein complexes. Both proteins co‐immunoprecipitated in a DNA‐dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single‐stranded than for double‐stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co‐immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52‐like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild‐type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology‐mediated recombination. These observations identify ODB1 as a further component of homologous recombination‐dependent DNA repair in plant mitochondria.     

Ginkgo biloba Expresses Calreticulin,the Major Calcium-Binding Reticuloplasmin in Eukaryotic Cells

Calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was characterized in Ginkgo biloba L. pollen and seeds. Electrophoretic analysis of the partly purified extracts showed the presence of two protein bands of 57 and 50kDa apparent molecular masses, which strongly cross-reacted with antibodies against plant calreticulins. Amino acid sequence comparison with other plant and animal calreticulins revealed a much higher similarity of the N-terminus of Ginkgo calreticulins with the homologue from angiosperms rather than with that from mammals. The finding of calreticulin in Ginkgo is indicative of the conservation also in gymnosperms of Ca²⁺ homeostatic mechanisms, which seem to rely on the same molecular components as all eukaryotic cells.     

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA as a lacZ fusion protein was selected in a dapA ^- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.     

Expression of recombinant staphylokinase,a fibrin-specific plasminogen activator of bacterial origin,in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) plants

One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the β-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.     

茶树CsPLK基因的鉴定与表达分析

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Host-parasite Interrelationship in Phosphatase Activity

Infection by Cuscuta and Orobanche causes significant losses in dry solids and protein content in host plant or plant part. Changes occur in phosphatase activity towards fructose-1,6-diphosphate at alkaline pH and β-glycerophosphate at acid pH, expressed per mg protein or g fresh tissue. The leaves of all hosts infected by Orobanche show an increase in the alkaline fructosediphosphatase activity, whereas as far as the infection by Cuscuta is concerned the general response is a decrease in the enzyme in the shoots. The alterations in the phosphatase activity towards β-glycerophosphate at acid pH in the shoots are not consistent. However, there is a marked increase in the acid phosphatase activity against β-glycerophosphatase in the roots of the infected hosts. The significance of these findings has been discussed in the light of host-parasite interrelationship.