Development of anAgrobacterium-mediated transformation system for regenerating garland chrysanthemum (Chrysanthemum coronarium L.)

Although efficient shoot regeneration and selection are essential for genetic transformation mediated byAgrobacterium, success has been limited with the garland chrysanthemum (Chrysanthemum coronarium L.). In this study, we developed a useful protocol for shoot regeneration with leaf disk explants. The optimal concentrations of NAA and BA were 0.2 mg L⁻¹ and 0.5 mg L⁻¹, respectively. To optimize the selection system for regenerating plants from genetically transformed tissues, we tested the effects of four antibiotics (kanamycin, hygromycin, carbenicillin, and cefotaxime). Among them, 5 mg L^-1 hygromycin proved adequate as a selectable marker, whereas 500 mg L^-1 carbenicillin was effective in eliminating excessiveAgrobacterium after co-cultivation. Transgenic plants were obtained by first co-culturing garland chrysanthemum leaf disks withA. tumefaciens strain EHA105, which harbors plasmid pRCVII containing the hygromycin resistance (hpt) and β-glucuronidase (GUS) genes. After the transgenic plants were confirmed via Southern analysis, they were rooted in soil and appeared phenotypically normal. Our report is the first to describe the optimum conditions for producing transgenic plants of this species.     

A Correlation between Antioxidant Activity and Metabolite Release during the Blanching of Chrysanthemum coronarium L.

Liquid chromatography tandem mass spectrometry (LCMS/MS)-based metabolite profiling was applied to elucidate the correlation between metabolite release and antioxidant activity during water blanching of Chrysanthemum coronarium L. (CC). Some major metabolites showing differences between fresh CC and blanched CC (BCC) were selected by principal component analysis (PCA) and partial least-square discriminate analysis (PLS-DA) loading plots, and were identified as dicaffeoylquinic acid (DCQA), succinoyl-DCQA, and acetylmycosinol. By PLS regression analysis of the correlation between antioxidant components and effects, candidate antioxidative metabolites were predicted due to strong positive correlations with DCQA and succinoyl-DCQA, and by a relatively weak positive correlation with acetylmycosinol.     

Crystal structures of two homologous pathogenesis-related proteins from yellow lupine

Pathogenesis-related class 10 (PR10) proteins are restricted to the plant kingdom where they are coded by multigene families and occur at high levels. In spite of their abundance, their physiological role is obscure although members of a distantly related subclass (cytokinin-specific binding proteins) are known to bind plant hormones. PR10 proteins are of special significance in legume plants where their expression patterns are related to infection by the symbiotic, nitrogen-fixing bacteria. Here we present the first crystal structures of classic PR10 proteins representing two homologues from one subclass in yellow lupine. The general fold is similar and, as in a birch pollen allergen, consists of a seven-stranded beta-sheet wrapped around a long C-terminal helix. The mouth of a large pocket formed between the beta-sheet and the helix seems a likely site for ligand binding. The shape of the pocket varies because, in variance with the rigid beta-sheet, the helix shows unusual conformational variability consisting in bending, disorder, and axial shifting. A surface loop, proximal to the entrance to the internal cavity, shows an unusual structural conservation and rigidity in contrast to the high glycine content in its sequence. The loop is different from the so-called glycine-rich P-loops that bind phosphate groups of nucleotides, but it is very likely that it does play a role in ligand binding in PR10 proteins.     

Sequential and structural homology between intracellular pathogenesis-related proteins and a group of latex proteins

The intracellular pathogenesis-related proteins have been identified in a broad range of flowering plants. Some display quite different patterns of expression, in many cases unrelated to the pathogenic response. Nevertheless, these proteins are all very similar and in most cases share more than 35% sequence identity. In this report we investigate the significance of a rather weak similarity between the intracellular pathogenesis-related (IPR or PR-10) proteins and a group of proteins identified in the latex of opium poppy and in Arabidopsis, among others. A sequence analysis held together with the recently published three-dimensional structure of Bet v 1, an IPR protein from birch pollen, strongly suggests sequential and structural homology between the two protein families.     

Identification,purification, and characterization of pathogenesis-related proteins from virus-infected Samsun NN tobacco leaves

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).     

Temporal and spatial expression of mRNAs encoding pathogenesis-related proteins during ethylene-promoted leaflet abscission in Sambucus nigra*

Differential screening of a cDNA library generated from RNA extracted from ethylene-treated leaflet abscission zones of Sambucus nigra resulted in the isolation of 20 abscission-related clones. These clones could be grouped into seven families. Sequencing of members of these families revealed that the majority encoded pathogenesis-related (PR) proteins, and these could be identified by sequence homology as a polyphenol oxidase (PPO), a PR-1 type protein, a Chial type chitinase, a PR-4 type protein similar to the potato win peptides, a PR-6 type proteinase inhibitor, a Chia4 type chitinase and a metallothionein-like protein (Coupe, Taylor & Roberts 1995, Planta 197, 442–447). Northern analysis revealed that these mRNAs were not expressed in freshly excised material but accumulated primarily in the abscission zone tissue after 18 h of exposure to ethylene at a time when abscission of the leaflet explants had reached 70%. Expression of the PPO and the Chia4-type chitinase was ethylene-dependent while that of the PR-4 type was up-regulated in the abscission zone tissue in the absence of the gas. The characterization of these mRNAs and their encoded proteins is presented and their possible roles during abscission are discussed.     

Molecular modeling of pathogenesis-related proteins of family 5

The family of pathogenesis-related (PR) 5 proteins have diverse functions, and some of them are classified as thaumatins, osmotins, and inhibitors of α-amylase or trypsin. Although the specific function of many PR5 in plants is unknown, they are involved in the acquired systemic resistance and response to biotic stress, causing the inhibition of hyphal growth and reduction of spore germination, probably by a membrane permeabilization mechanism or by interaction with pathogen receptors. We have constructed three-dimensional models of four proteins belonging to the Rosaceae and Fagaceae botanical families by using the technique of comparative molecular modelling by homology. There are four main structural differences between all the PR5, corresponding to regions with replacements of amino acids. Folding and the secondary structures are very similar for all of them. However, the isoelectric point and charge distributions differ for earch protein.     

Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro

The Bet v. 1 gene family of birch encodes the major pollen allergens as well as pathogenesis-related (PR) proteins that are induced by microbes in somatic tissues. These PR proteins belong to a group of conserved intracellular defense-related proteins that have been termed 'ribonuclease-like' PR proteins, on the basis of the partial sequence homology observed between PR1, a Bet v 1-homologue from parsley, and a recently characterized ginseng ribonuclease. However, this enzymatic activity has not yet been demonstrated, not for any of the members of this family of PR proteins, nor for the related pollen allergens. We have investigated the possible nuclease activity of Bet v 1 using apparently homogeneous preparations of natural Bet v 1 purified from birch pollen, and a recombinant non-fusion protein purified from E. coli extracts. We report here that Bet v 1 proteins indeed possess an intrinsic ribonucleolytic activity as they can digest different RNA substrates in vitro, but show no activity on single or double-stranded DNA.     

蕾后期和花前期切花菊品种'神马'不同部位叶片光合作用和叶绿素荧光特性的比较

对蕾后期和花前期切花菊(Chrysanthemum morifolium Ramat.)品种'神马'('Jinba')不同部位叶片光合作用参数日变化、叶绿素荧光参数、光响应曲线及参数进行了研究.结果表明:蕾后期和花前期品种'神马'叶片蒸腾速率(Tr)、气孔导度(Gs)和净光合速率(Pn)的日变化均为单峰曲线,峰值出现在10:00或12:00;胞间CO2浓度(Ci)的日变化则先降低后升高,谷值出现在12:00.蕾后期和花前期品种'神马'叶片Tr、Ci和Gs值的平均值总体上随叶片位置降低而逐渐升高;蕾后期不同部位叶片Pn值的平均值差异较小,花前期叶片Pn值的平均值则随叶片位置降低而逐渐降低.随着叶片位置降低,蕾后期和花前期品种'神马'叶片的初始荧光(Fo)、最大荧光(Fm)、可变荧光(Fv)、表观量子效率(AQE)和最大净光合速率(Pmax)以及蕾后期的暗呼吸速率(Rd)均逐渐降低,而花前期的Rd值以及蕾后期和花前期的光补偿点(LCP)均逐渐升高.随着光合有效辐射(PAR)升高,蕾后期不同部位叶片以及花前期中部叶和下部叶的Pn值呈先急剧升高后趋于平稳的变化趋势,而花前期上部叶的Pn值则呈先急剧升高后逐渐下降的变化趋势.研究结果显示:在切花菊设施栽培过程中适当补充光照可提高切花菊品质.     

Oxalic acid-induced resistance to Rhizoctonia solani in rice is associated with induction of phenolics,peroxidase and pathogenesis-related proteins

Abstract

Oxalic acid (1 mM) when applied as a foliar spray to rice plants induced resistance to challenge infection with Rhizoctonia solani, the rice sheath blight pathogen. Maximum reduction in sheath blight incidence was observed when the plants were sprayed with oxalic acid three days before inoculation with the fungus. The biochemical alterations in rice plants treated with oxalic acid was also investigated. When rice plants were treated with oxalic acid, a two-fold increase in phenolic content in leaf sheaths was recorded three days after treatment. Phenylalanine ammonia-lyase and peroxidase activities increased significantly starting from two days after treatment. Peroxidase (PO) isozyme analysis indicated that PO-3 and PO-4 were induced two days after treatment with oxalic acid. Western blot analysis revealed that two chitinases (28 and 35 kDa) and two β-1,3-glucanases (30 and 32 kDa) were strongly induced in rice sheaths four to six days after treatment with oxalic acid. Immunoblot analysis of protein extracts from oxalic acid-treated plants demonstrated the induction of a 23 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antibody. These results suggest that the enhanced activities of defense enzymes and defense-related compounds in oxalic acid-treated rice plants may contribute to resistance against R. solani.     

Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Summary Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.     

Induction of pathogen resistance and pathogenesis-related genes in tobacco by a heat-stable Trichoderma mycelial extract and plant signal messengers

Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica . The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.     

Molecular investigations of pathogenesis-related Bet v 1 homologues in Passiflora (Passifloraceae)

The major birch pollen allergen, Bet v 1, responsible for allergic reactions in many areas of the world, is homologous to a large number of pathogenesis-related proteins (PRs), identified as PR10. As part of a long-range investigation of these types of proteins and of evolution in Passiflora,DNA sequences from eight Bet v 1 homologue isoforms were obtained from five species of this genus in Brazil, and their sequences compared among themselves and with 30 others from 8 different species, classified in different taxonomic units. The objective was a first characterization of these PRs in wild passionflowers, and their use for evolutionary and applied investigations. High interspecific, but low intraspecific variability was observed, as expected from multigenic families subjected to concerted evolution. The relationships obtained both within Passiflora and between it and seven other genera probably best reflect functional similarities than evolutionary history.     

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20–30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential expression of pathogenesis-related proteins and defense enzymes in banana: interaction between endophytic bacteria,Banana bunchy top virus and Pentalonia nigronervosa

Banana bunchy top disease caused by Banana bunchy top virus is the most serious viral disease of banana and plantain worldwide. The virus is transmitted by the aphid vector Pentalonia nigronervosa in a persistent manner. This paper deals with the effect of the interaction between plant growth promoting endophytic bacteria, Banana bunchy top virus, and the banana aphid Pentalonia nigronervosa in the expression of Pathogenesis-related proteins (PR-proteins) and defense enzymes in banana. The existence of virus in the aphids was confirmed by ELISA, DIBA and PCR. PCR could amplify 1100-bp replicase gene of BBTV from viruliferous aphids. A significant increase in the enzymatic activity of all measured PR proteins and defense enzymes, as compared to control plants, was seen in the plants inoculated with endophytic bacteria and challenged with viruliferous aphids. Native gel electrophoresis revealed expression of more isoforms of PR proteins viz., peroxidase and chitinase in the banana plants challenged with mixtures of plant growth promoting endophytic bacteria and viruliferous aphids. Enhanced activity of a PR-2 protein viz., β-1,3-glucanase was also noticed in the viruliferous aphids infested plants. Some of the defense-related enzymes viz., Polyphenol oxidase and Phenylalanine ammonia lyase and phenolic compounds were also upregulated, up to 5 days after aphid infestation and thereafter there was a reduction in the enzymatic activity. Thus, there exist a differential accumulation of PR proteins and defense-related enzymes, when there is tri-tropic interaction between endophytic bacteria, virus, and insect and the role of the endophytic bacteria in the defense mechanisms against insect pests needs to be elucidated.