ABA Levels and Sensitivity in Developing Wheat Embryos of Sprouting Resistant and Susceptible Cultivars

A sprouting-resistant and a sprouting-susceptible wheat cultivar were utilized to examine the role of ABA levels and sensitivity responses in wheat embryonic germination. Endogenous embryonic ABA levels were measured in both cultivars throughout grain maturation utilizing a new and sensitive ABA immunoassay. Embryonic ABA levels of each cultivar were similar with the sprouting-susceptible cultivar having about a 25% lower ABA level than that of the sprouting-resistant cultivar. Larger differences between the cultivars were noted in sensitivity to ABA, as measured by capability of ABA to block embryonic germination. ABA inhibited embryonic germination much more effectively in the sprouting-resistant cultivar.     

Metabolic Changes in Partially Dormant Wheat Seeds during Storage

Changes in germination, seedling growth, respiration, response to applied gibberellic acid, and glucose-U-¹⁴C utilization were investigated in partially dormant wheat (Triticum aestivum L., Pa 151 × 107) seeds which were stored under various conditions for periods up to 1 year. Only seeds stored at −20 C and 12.4% moisture maintained partial dormancy, which was overcome by germinating in 10⁻³m gibberellic acid. Germination and seedling growth of seeds stored at 25 C and 15.1% moisture declined within 12 weeks and the percentage of seeds infected with storage fungi increased. Gibberellic acid produced faster growing seedlings, particularly from those seeds with partial dormancy, but did not overcome growth reduction which was caused by deterioration. Seeds kept under laboratory conditions (B), 25 C and 12.1% moisture (C), and 25 C and 15.1% moisture (D) for 12 weeks utilized 35, 55, and 80% less glucose, respectively, than those stored at −20 C and 12.4% moisture (A). Seeds stored under B and C consistently had higher germination, growth, and respiratory rates than seeds from A and D. The respiratory rate declined as deterioration advanced under D. Respiratory quotients ranged from 1.0 for seeds stored under A to 1.6 for seeds stored under D.     

小麦种子成熟和萌发过程中的假萌发素活性

用SDS-PAGE方法研究了假萌发素(ψG)在小麦种子成熟和萌发过程中活性的变化.结果表明:在种子成熟过程中只有ψG表达,扬花后10 d,在颖壳、内外桴、种皮和果皮中皆可检测到ψG的草酸氧化酶活性,随着发育进程的推进,ψG的活性增大.在种子萌发过程中,在小麦品种中育5号的维管束过渡区中除了萌发素G和G'外,还可检测到ψG的草酸氧化酶活性.由于ψG在种子成熟过程中主要存在于颖壳、内外桴、果皮及种皮这些保护组织中,且开始大量表达的时间正是生长接近停止时,于是推测ψG很可能通过降解草酸产生H2O2而推动这些组织细胞壁的木质化.     

A Natural Fluorescent Protein That Changes Its Fluorescence Color during Maturation

The gene of a new red fluorescent protein zoan2RFP from coral polyp Zoanthus sp., a homologue of the known green fluorescent protein from the jellyfish Aequorea victoria, was cloned. At early stages of maturation, zoan2RFP exhibits green fluorescence, which then turns to the red one. A similar phenomenon was recently reported for the E5 mutant of the red fluorescent coral protein DsRed. Zoan2RFP differs from E5 by faster maturation kinetics and the complete disappearance of green fluorescence in the mature protein. Naturally occurring proteins of this type can be considered as intermediate forms between the green and red fluorescent proteins, which are formed during the microevolution of fluorescent proteins.     

Postinfectional Changes of Lipid Metabolism in Potato Tuber Resistant and Susceptible to Phytophthora infestans

The effect of Phytophthora infestans on the lipid content and composition as well as lipid acyl hydrolase activity in the potato tuber of two cultivars, Nysa – horizontal resistant and Bintje –susceptible were investigated. In the susceptible cultivar the infection resulted in: a qualitative changes in aminolipids, an increase in steroid content, an inconsiderable decrease in amount of glycolipids and a slight drop in FFA, no significant changes in composition of total, FA and slight loss in acyl hydrolase activity. The following changes were observed in the resistant cultivar: a considerable enhancement of steroid content (reached in about 400 %), a slight increase in amount of glycolipid, a rapid drop of acyl hydrolase activity and FFA content and a considerable increase in oleic acid level in total FA. In the response to both infection and aging in both cultivars no significant quantitative changes in lipid-phosphorus and sulfolipids were noted.     

Changes in Level and Activity of Phospholipid Transfer Protein during Maturation and Germination of Maize Seeds

The variations of the amounts of phospholipid transfer proteins (PLTP), determined by ELISA and immunoblotting methods, were followed during the maturation and germination of maize (Zea mays L.) seeds. Changes of the amounts of PLTP occur during seed maturation. The levels of PLTP, low in the first 3 weeks after fecondation, strongly raised 3 to 5 weeks after, then reached and maintained a high value (10% of total soluble proteins) during the last steps of maturation. These variations, determined by ELISA, are in accordance with the observations made by immunoblotting. Changes in phospholipid transfer activity were also found when protein extracts prepared from seeds at different stages of maturation were assayed for transfer activity. The levels of PLTP were also determined during the germination of maize seeds and the early growth of the plantlets, both in the endosperm and the aerial parts. While no major change was observed in the endosperm, a high increase in PLTP level was found in the aerial part of the plantlet, both by ELISA and immunoblotting. An enhancement of the phospholipid transfer activity was parallely observed in the protein extracts of plantlets at various stages of germination. These results are consistent with an in vivo correlation between the synthesis of phospholipid transfer protein, observed during the maturation and germination of maize seeds, and the biogenesis of membranes which involves intracellular movements of phospholipids.     

Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrP^C was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrP^d) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrP^d originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.     

Changes in the Contents of Free and Protein Amino Acids in Tobacco Seeds and Placentae during Maturation

Changes in the levels of protein and free amino acids in theseeds and placentae of Nicotiana tabacum were studied duringseed development. Seed maturation was completed 24 days afteranthesis. During maturation, protein rapidly accumulated inthe seeds between the 6th and 18th day, along with an appreciablecompositional change in the protein amino acids as the proportionsof glutamic acid and arginine increased. The amount of freeamino acids in the seeds gradually decreased throughout maturation.The major free amino acid on the 6th day after anthesis wasglutamine, which then drastically decreased between the 6thand 12th day with increases of glutamic acid, proline, arginineand alanine. The latter amino acids decreased thereafter untilthe 24th day. On the other hand, the amount and composition of the proteinsin the placentae did not change significantly throughout seedmaturation. In the early stage of development, the major freeamino acids in the placentae were glutamine, asparagine andglutamic acid, while in the later stage asparagine was mostabundant. (Received March 12, 1982; Accepted August 16, 1982)     

Sunlight-Exposed Biofilm Microbial Communities Are Naturally Resistant to Chernobyl Ionizing-Radiation Levels

Background

The Chernobyl accident represents a long-term experiment on the effects of exposure to ionizing radiation at the ecosystem level. Though studies of these effects on plants and animals are abundant, the study of how Chernobyl radiation levels affect prokaryotic and eukaryotic microbial communities is practically non-existent, except for a few reports on human pathogens or soil microorganisms. Environments enduring extreme desiccation and UV radiation, such as sunlight exposed biofilms could in principle select for organisms highly resistant to ionizing radiation as well.

Methodology/Principal Findings

To test this hypothesis, we explored the diversity of microorganisms belonging to the three domains of life by cultivation-independent approaches in biofilms developing on concrete walls or pillars in the Chernobyl area exposed to different levels of radiation, and we compared them with a similar biofilm from a non-irradiated site in Northern Ireland. Actinobacteria, Alphaproteobacteria, Bacteroidetes, Acidobacteria and Deinococcales were the most consistently detected bacterial groups, whereas green algae (Chlorophyta) and ascomycete fungi (Ascomycota) dominated within the eukaryotes. Close relatives to the most radio-resistant organisms known, including Rubrobacter species, Deinococcales and melanized ascomycete fungi were always detected. The diversity of bacteria and eukaryotes found in the most highly irradiated samples was comparable to that of less irradiated Chernobyl sites and Northern Ireland. However, the study of mutation frequencies in non-coding ITS regions versus SSU rRNA genes in members of a same actinobacterial operational taxonomic unit (OTU) present in Chernobyl samples and Northern Ireland showed a positive correlation between increased radiation and mutation rates.

Conclusions/Significance

Our results show that biofilm microbial communities in the most irradiated samples are comparable to non-irradiated samples in terms of general diversity patterns, despite increased mutation levels at the single-OTU level. Therefore, biofilm communities growing in sunlight exposed substrates are capable of coping with increased mutation rates and appear pre-adapted to levels of ionizing radiation in Chernobyl due to their natural adaptation to periodical desiccation and ambient UV radiation.     

Effects of Helminthosporium victoriae Toxin on Germination and Aleurone Secretion by Resistant and Susceptible Seeds

Oat seeds of cultivars susceptible and resistant to Helminthosporium victoriae were held for various times in pathogen-produced, host-specific toxin solutions; control seeds were in water. Seeds were then washed thoroughly and incubated on moist paper, or dried and stored for 2-3 weeks before germination was attempted. In both cases, germination of susceptible seeds was prevented by previous exposure to toxin for 1 hour or more. Control seeds and treated resistant seeds grew normally. Toxin did not affect O₂ uptake or loss of carbohydrates from seeds for the first 12 hours of imbibition. After 12 hours, toxin-treated susceptible seeds had higher respiration and lost more carbohydrates than did control seeds. Experiments with embryoless seeds showed that toxin blocked synthesis and secretion of α-amylase by susceptible but not by resistant aleurone cells. Resting aleurone cells were exposed briefly to toxin, then dried and stored until all toxin was gone. Susceptible aleurone cells treated in this way failed to produce α-amylase following exposure to gibberellic acid, while controls and resistant treated aleurone tissues produced amylase. Susceptibility or resistance to toxin appears to be expressed in resting and metabolically active tissues.     

Biotransformation of the Mycotoxin Deoxynivalenol in Fusarium Resistant and Susceptible Near Isogenic Wheat Lines

In this study, a total of nine different biotransformation products of the Fusarium mycotoxin deoxynivalenol (DON) formed in wheat during detoxification of the toxin are characterized by liquid chromatography—high resolution mass spectrometry (LC-HRMS). The detected metabolites suggest that DON is conjugated to endogenous metabolites via two major metabolism routes, namely 1) glucosylation (DON-3-glucoside, DON-di-hexoside, 15-acetyl-DON-3-glucoside, DON-malonylglucoside) and 2) glutathione conjugation (DON-S-glutathione, “DON-2H”-S-glutathione, DON-S-cysteinyl-glycine and DON-S-cysteine). Furthermore, conjugation of DON to a putative sugar alcohol (hexitol) was found. A molar mass balance for the cultivar ‘Remus’ treated with 1 mg DON revealed that under the test conditions approximately 15% of the added DON were transformed into DON-3-glucoside and another 19% were transformed to the remaining eight biotransformation products or irreversibly bound to the plant matrix. Additionally, metabolite abundance was monitored as a function of time for each DON derivative and was established for six DON treated wheat lines (1 mg/ear) differing in resistance quantitative trait loci (QTL) Fhb1 and/or Qfhs.ifa-5A. All cultivars carrying QTL Fhb1 showed similar metabolism kinetics: Formation of DON-Glc was faster, while DON-GSH production was less efficient compared to cultivars which lacked the resistance QTL Fhb1. Moreover, all wheat lines harboring Fhb1 showed significantly elevated D3G/DON abundance ratios.     

Histological and Ultrastructural Changes in Leaves and Stems of Resistant and Susceptible Chickpea Cultivars to Ascochyta rabiei

The histo- and cytopathological effects in resistant (ILC-195) and susceptible (Canitez-87) chickpea cultivars were examined by light, transmission and scanning electron microscopy 3, 5 and 7 days after inoculation (d.a.i) of seedlings with Ascochyta rabiei. The fungus produced typical appressoria that penetrated both cuticle and stomata. The resistant plants had physical barriers and a cuticle layer against fungal penetration 3 d.a.i. The fungus spread intercellularly and subepidermally in the leaves and stems of susceptible plants 3 d.a.i., and was followed 5 d.a.i. by cell plasmolysis, degeneration of organelles and of cellulose, but not lignified, walls. Pycnidia formation occurred between 5 and 7 d.a.i. 7 d.a.i., organelle degeneration, pycnidia formation and symptom severity increased. Tracheidal elements, including lignified elements, were almost intact in both resistant and susceptible cultivars. In the susceptible plants, lignin cell walls were slightly degraded after 7 days. There was less cell degeneration and pycnidia formation in resistant plants. Some electron-dense large bodies and lipid granules were observed within intracellular fungal hyphae in infected cells of resistant plants 7 d.a.i.     

Identification of Embryoid-Abundant Genes That Are Temporally Expressed during Pollen Embryogenesis in Wheat Anther Cultures

Uninucleate microspores in anther cultures of bread wheat (Triticum aestivum cv Pavon) are capable of producing haploid pollen embryoids and plants. To gain an understanding of this alternate pathway of pollen development, we constructed a cDNA library to young pollen embryoids, isolated embryoid-specific genes, and analyzed their expression patterns during morphogenesis. Two embryoid-abundant clones, pEMB4 and 94, were expressed very early during culture, suggesting that these genes are associated with development and are not simply expressed as a consequence of differentiation. The accumulation patterns of five cloned mRNAs may indicate the activation of specific genes associated with the major morphological and physiological activities connected with the differentiation of embryoids in vitro. These results suggest that embryoid-abundant gene expression is causally related to this pathway because gene expression is spatially and temporally specific and is not observed when microspores are cultured under noninductive conditions.     

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.

Methodology/Principal Findings

Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.

Conclusions/Significance

These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.     

Phenols in Cotton Seedlings Resistant and Susceptible to Alternaria macrospora

In healthy cotton seedlings, stems have a lower phenol content than leaves, but resistant plants have an altogether relatively higher phenol content than susceptible plants. Phenols extracted from infected plants can inhibit the growth of A. macrospora in vitro. In cotton plants infected with A. macrospora, phenols are oxidized by polyphenoloxidase rather than peroxidase and catalase. The main oxidative activity was around the developing necrotic area but activity was detected far from, necrosis as well. Though pre–inoculation mechanical injuries operated the phenol oxidation mechanism in the plant, they neither prevented nor encouraged the increase in disease severity. Isozyme pattern showed that contribution of all participants in the pathological interaction to the oxidative mechanism occurred in the diseased plant. A negative linear correlation was found between polyphenoloxidase activity, phenol accumulation and resistance. This study suggests that the phenol oxidative mechanism, participates in cotton plant resistance to A. macrospora.