Puccinia graminis f. sp. tritici Population Causing Recent Wheat Stem Rust Epidemics in Kazakhstan Is Highly Diverse and Includes Novel Virulence Pathotypes.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a reemerging disease that caused severe epidemics in northern Kazakhstan and western Siberia in the period of 2015 to 2019. We analyzed 51 stem rust samples collected between 2015 and 2017 in five provinces in Kazakhstan. A total of 112 Pgt races were identified from 208 single-pustule isolates. These races are phenotypically and genotypically diverse, and most of them are likely of sexual origin. No differentiation of phenotypes and single-nucleotide polymorphism genotypes was observed between isolates from Akmola and North Kazakhstan provinces, supporting the idea of a wide dispersal of inoculum in the northern regions of the country. Similarities in virulence profiles with Pgt races previously reported in Siberia, Russia, suggest that northern Kazakhstan and western Siberia constitute a single stem rust epidemiological region. In addition to the races of sexual origin, six races reported in Europe, the Caucasus, and East Africa were detected in Kazakhstan, indicating that this epidemiological region is not isolated, and spore inflow from the west occurs. Virulence alone or in combination to several genes effective against the Ug99 race group was detected, including novel virulence on Sr32 + Sr40 and Sr47. The occurrence of a highly diverse Pgt population with virulence to an important group of Sr genes demonstrated the importance of the pathogen's sexual cycle in generating new and potentially damaging virulence combinations.

Evidence for Systemic Infection by Puccinia horiana, Causal Agent of Chrysanthemum White Rust, in Chrysanthemum.

Puccinia horiana, causal agent of the disease commonly known as chrysanthemum white rust (CWR), is a quarantine-significant fungal pathogen of chrysanthemum in the United States and indigenous to Asia. The pathogen was believed to have been eradicated in the United States but recently reappeared on several occasions in northeastern United States. The objective of the study presented here was to determine whether P. horiana could systemically infect chrysanthemum plants, thus providing a means of survival through winters. Scanning and transmission electron microscopy revealed the development of P. horiana on the surface and within leaves, stems, or crowns of inoculated chrysanthemum plants artificially exposed to northeastern U.S. winter temperatures. P. horiana penetrated leaves directly through the cuticle and then colonized the mesophyll tissue both inter- and intracellularly. An electron-dense material formed at the interface between fungal and host mesophyll cells, suggesting that the pathogen adhered to the plant cells. P. horiana appeared to penetrate mesophyll cell walls by enzymatic digestion, as indicated by the absence of deformation lines in host cell walls at penetration sites. The fungus was common in vascular tissue within the infected crown, often nearly replacing the entire contents of tracheid cell walls. P. horiana frequently passed from one tracheid cell to an adjacent tracheid cell by penetration either through pit pairs or nonpitted areas of the cell walls. Individual, presumed, fungal cells in mature tracheid cells of the crown and stems arising from infected crowns suggested that the pathogen might have been moving at least partially by means of the transpiration stream. The demonstration that chrysanthemum plants can be systemically infected by P. horiana suggests that additional disease control measures are required to effectively control CWR.

First Report of Leaf Spot Caused by Cladosporium herbarum on Centaurea solstitialis in Greece.

Centaurea solstitialis L. (yellow starthistle), family Asteraceae, an invasive weed in California and the western United States, is targeted for biological control. In the summer of 2003, an epidemic of unknown etiology on dying C. solstitialis plants was observed near Kozani, Greece (40°22'07″N, 21°52'35″E, elevation, 634 m). Plants had necrotic light brown leaf spots on the lower leaves and the decurrent leaf bases along the stems. Often, necrotic lesions extended along the stems to the capitula. Virtually all plants in a solid stand of C. solstitialis (approximately 0.5 ha) showed disease symptoms. Diseased plants were collected, air dried, and sent to the quarantine facility of the Foreign Disease-Weed Science Research Unit (FDWSRU), USDA/ARS, Fort Detrick, MD. On the basis of culture growth (45-cm diameter after 2 weeks at 25°C on malt extract agar), fungal morphology (1), and comparison with 21 internal transcribed spacer sequences in GenBank, the putative causal organism was identified as Cladosporium herbarum (Pers.:Fr.) Link. (teleomorph = Davidiella tassiana (De Not.) Crous & U. Braun). Sixteen C. solstitialis plants in the rosette stage and 16 plants in the bolted stage were inoculated with an aqueous suspension of spores (106 conidia ml-1) and placed in an environmentally controlled chamber at 25°C with 8 h of dew and 12 h of light daily. Plants in the rosette stage were resistant, but the fungus was very aggressive on bolted plants. Within 4 to 6 days of inoculation, necrosis developed on leaves and stems and then spread up the stems to the capitula, often resulting in plant death. The fungus also infected developing flowers. Cladosporium herbarum was reisolated from each of the 16 bolted C. solstitialis plants in two separate tests at the FDWSRU and from all bolted inoculated plants at the European Biological Control Laboratory (EBCL) in Greece. In the greenhouse at the EBCL, the pathogen readily spread to (and was isolated from) another 10 noninoculated C. solstitialis plants in close vicinity to the inoculated C. solstitialis plants. Results of host range tests will establish if this isolate of Cladosporium herbarum has the potential as a biological control agent of C. solstitialis in the United States and does not pose a threat to other Centaurea spp. used in horticulture. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 863446). Live cultures are being maintained at the FDWSRU and EBCL, Greece. To our knowledge, this is the first report of a disease caused by Cladosporium herbarum on C. solstitialis. Reference: (1) M. H. M. Ho et al. Mycotaxon 72:115, 1999.

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

An assessment model for rating high-threat crop pathogens.

ABSTRACT Natural, accidental, and deliberate introductions of nonindigenous crop pathogens have become increasingly recognized as threats to the U.S. economy. Given the large number of pathogens that could be introduced, development of rapid detection methods and control strategies for every potential agent would be extremely difficult and costly. Thus, to ensure the most effective direction of resources a list of high-threat pathogens is needed. We address development of a pathogen threat assessment model based on the analytic hierarchy process (AHP) that can be applied world-wide, using the United States as an illustrative example. Previously, the AHP has been shown to work well for strategic planning and risk assessment. Using the collective knowledge of subject matter expert panels incorporated into commercial decision-making software, 17 biological and economic criteria were determined and given weights for assessing the threat of accidental or deliberately introduced pathogens. The rating model can be applied by experts on particular crops to develop threat lists, especially those of high priority, based on the current knowledge of individual diseases.

First Report of Puccinia jaceae var. solstitialis Pycnia on Yellow Starthistle in the United States.

The rust fungus Puccinia jaceae (Otth) var. solstitialis field isolate FDWSRU 84-71 (formerly TR 84-96) was first released in California for the biological control of yellow starthistle (YST; Centaurea solstitialis) in July 2003. This isolate was collected by S. S. Rosenthal in 1984, east of Yarhisar and Hafik (SIVAS), Turkey. It is macrocyclic and autoecious (1), completing its entire life cycle on YST. Observations were made in field plots west of Woodland, CA that had been inoculated on a monthly regimen between January and June of 2005. On February 22 and March 2, 2006, pycnia were observed in and around one of the plots (38°42.767'N, 121°53.732'W at an altitude of 57 m). Pycnia were yellow, flask shaped, and small (less than 100 µm in diameter), occurring in clusters on abaxial leaf surfaces or on petioles. DNA was isolated from pycnial and uredinial samples collected from the site and used for polymerase chain reaction amplification of the ITS2 region with P. jaceae specific primers. Sequences of amplicons from both samples were identical to FDWSRU 84-71 (GenBank Accession No. AF047728). Further amplification of pycnial DNA with PCR primers specific to the released P. jaceae isolate (2) produced the expected 851-bp amplicon. To our knowledge, this is the first report of pycnia from Puccinia jaceae var. solstitialis in the United States, suggesting that the YST rust is fully functional and completes its life cycle in California. References: (1) D. B. O. Savile. Can. J. Bot. 48:1553, 1970. (2) L. F. Yourman and D. G. Luster. Biol. Control 29:73, 2004.

Aphid (Hemiptera: Aphididae) species composition and potential aphid vectors of plum pox virus in Pennsylvania peach orchards.

Plum pox, an invasive disease recently identified in Pennsylvania stone fruit orchards, is caused by the aphid-transmitted Plum pox virus (genus Potyvirus, family Potyviridae, PPV). To identify potential vectors, we described the aphid species communities and the seasonal dynamics of the dominant aphid species within Pennsylvania peach orchards. Aphids were trapped weekly in 2002 and 2003 from mid-April through mid-November within two central Pennsylvania orchards by using yellow and green water pan traps. In total, 42 aphid species were identified from both orchards over 2 yr. Within orchards, actual species richness ranged from 24 to 30 species. The Abundance Based Coverage Estimator predicted species richness to range from 30 to 36 species, indicating that trap catches were identifying most aphid species expected to occur in the orchard. Three species, Rhopalosiphum maidis (Fitch), Aphis spiraecola Patch, and Myzus persicae (Sulzer), were consistently dominant across locations and years. Orchard-trapped populations of these three species peaked in a similar chronological sequence each year. As expected, trap color influenced the total number and distribution of the predominate species collected. However, the same dominant species occurred in both yellow and green traps. Based on the seasonal population dynamics reported here and on published vector efficacy studies, the most probable significant PPV vector was identified as A. spiraecola. If the PPV pathogen escapes current quarantine or if subsequent reintroductions of PPV occur, these data will be useful for developing plum pox management strategies.

Cercosporella acroptili and Cercosporella centaureicola sp. nov.--potential biological control agents of Russian knapweed and yellow starthistle, respectively.

Russian knapweed (Acroptilon repens [L.] DC.) and yellow starthistle (Centaurea solstitialis L.) are invasive weeds in the western United States, and both weeds are targeted for biological control. Cercosporella acroptili (Bremer) U. Braun was identified as a possible biological control agent for A. repens, and a morphologically similar Cercosporella sp. recently was found damaging to C. solstitialis in the field. Because both fungi are potentially important for biological control of the respective weeds, studies were undertaken to ascertain whether the isolates were identical based on morphology, pathogenicity, growth and spore production, and genetics (molecular characterization of the internal transcribed spacer regions of the ribosomal RNA genes). Differences in these variables between the two isolates were sufficient to indicate that the isolate from C. solstitialis was distinct and justified a new description at the species level: Cercosporella centaureicola sp. nov.

Improved Detection of Tilletia indica Teliospores in Seed or Soil by Elimination of Contaminating Microorganisms with Acidic Electrolyzed Water.

Acidic electrolyzed water (AEW) is a germicidal product of electrolysis of a dilute solution (e.g., 0.4% vol/vol) of sodium chloride. This solution can be used to disinfest wheat seed or soil samples being tested for teliospores of Tilletia indica, causal agent of Karnal bunt, without risk of damaging the teliospores. The AEW used in this study had a pH of 2.5 to 2.8 and oxidation-reduction potential of approximately 1,130 mV. In simulations of routine extractions of wheat seed to detect teliospores of T. indica, the effectiveness of a 30-min AEW treatment was compared with a 2-min 0.4% sodium hypochlorite (NaOCl) treatment to eradicate bacteria and nonsmut fungi. Each treatment reduced bacterial and fungal populations in wheat seed extracts by 6 to 7 log10 units when determined on 2% water agar with antibiotics. Reductions of 5 log10 units or more were observed on other media. NaOCl and AEW also were very effective at eliminating bacteria and fungi from soil extracts. In studies to detect and quantitate T. indica teliospores in soil, AEW was nearly 100% effective at eliminating all nonsmut organisms. Free chlorine levels in AEW were very low, suggesting that compounds other than those with chlorine play a significant role in sanitation by AEW. The low pH of AEW was shown to contribute substantially to the effectiveness of AEW to reduce microorganisms. A standardized protocol is described for a 30-min AEW treatment of wheat seed washes or soil extracts to eliminate contaminating microorganisms. A significant advantage of the use of AEW over NaOCl is that, with AEW, teliospore germination is not reduced and usually is stimulated, whereas teliospore germination declines after contact with NaOCl. The protocol facilitates detection and enumeration of viable teliospores of T. indica in wheat seed or soil and the isolation of pure cultures for identification by polymerase chain reaction. The germicidal effects of AEW, as demonstrated in this study, illustrate the potential of AEW as an alternative to presently used seed disinfestants.

First Report of Silybum marianum as a Host of Puccinia punctiformis.

Silybum marianum (L.) Gaertn. (milk thistle) is a problematic invasive weed in the western United States. The rust fungus, Puccinia punctiformis (F. Strauss) Rohl., is found throughout the world as a pathogen of Cirsium arvense (L.) Scop. (Canadian thistle). Recently, plants of S. marianum grown from surface-disinfested seeds in our quarantine greenhouse were parasitized by a rust. Apparently, an isolate of P. punctiformis collected from C. arvense in Turkey that was present in the greenhouse had spread to adjacent S. marianum plants and caused infection without applying any artificial dew period. Ribosomal internal transcribed spacer region sequences from fungal spore DNA isolated from the two hosts were identical. Initial signs on S. marianum were abundant, fragrant spermogonia on large leaves. These signs occur on secondary shoots of C. arvense and are indicative of systemic fungal infection (1). As the fungus infection developed on S. marianum, uredinia and urediniospores were produced. Sori on older leaves also produced teliospores. Urediniospores from infected leaves were harvested and sprayed uniformly on eight 17-day-old plants of S. marianum grown in isolation from P. punctiformis. The spore suspension consisted of 4 mg urediniospores suspended in 40 ml distilled water. Inoculated plants were incubated for 18 h in a dew chamber at 20°C in the dark and transferred to a greenhouse (20 to 25°C, 30 to 50% relative humidity, and natural light). After 13 days, uredia with urediniospores developed on four of the plants. Using the same procedure, inoculations were repeated on plants of S. marianum and S. eburneum Coss. & Durieu (the only other species described in the genus) with urediniospores of a domestic isolate of the fungus from C. arvense in Maryland. Of 51 inoculated plants of S. marianum, 23 became infected and produced uredinia. None of the 12 inoculated plants of S. eburneum showed symptoms of infection. In nature, C. arvense and S. marianum occupy different ecological areas. C. arvense is found predominately in humid temperate habitats, while S. marianum is found in habitats with a dry Mediterranean climate. Life cycles of each host are also different. C. arvense is a perennial that emerges in spring and dies back in winter, while S. marianum is a winter annual that emerges in fall and dies in late spring. Because of the differences in life cycles combined with the different geographical distribution, P. punctiformis from C. arvense may rarely encounter susceptible S. marianum plants in the field. Since fungal spores can be produced routinely on artificially inoculated plants, there might be potential to use P. punctiformis for biological control of S. marianum. To our knowledge, this is the first report of S. marianum as a host for P. punctiformis. Reference: (1) A. H. R. Buller. Puccinia sauveolens and its sexual process. Page 345 in: Researches on Fungi. Vol VII. The Sexual Process in the Uredinales, Toronto, Canada, 1950.

First Report of Musk Thistle Rust (Puccinia carduorum) in California and Nevada.

Musk thistle, Carduus nutans L., is an introduced weed of pastures, rangelands, and natural areas in much of North America. Puccinia carduorum Jacky, an autoecious rust fungus from Turkey, has been evaluated for biological control of musk thistle since 1978, including a field study near Blacksburg, VA, from 1987 to 1990. After release of the fungus in Virginia, rusted musk thistle was found in eight eastern states by 1992, in Missouri by 1994 (1), and in Oklahoma by 1997 (2). A rust disease was discovered on musk thistle near Mt. Shasta, CA, on 22 September 1998, and near Mogul, NV, on 12 August 1999. The pathogen was identified as P. carduorum on the basis of pathogenicity on musk thistle and urediniospore morphology (ovate spores, 21 µm diameter, three germ pores equatorial in location, and echinulations over the upper two-thirds to three-quarters of urediniospores). Ribosomal RNA internal transcribed spacer DNA sequences (ITS1 and ITS2) were identical to those from the isolate obtained after the field release in Virginia, verifying that the California isolate is P. carduorum. The initial California infestation was observed on a few plants late in the season, and by September 2000, nearly 100% of plants were infected. The occurrence of P. carduorum in California is apparently the result of natural, unaided spread of the fungus on musk thistle from the East Coast of the United States. References: (1) A. B. A. M. Baudoin and W. L. Bruckart. Plant Dis. 80:1193, 1996. (2) L. J. Littlefield et al. Plant Dis. 82:832, 1998.

Identification, characterization, and relatedness of luteovirus isolates from forage legumes.

ABSTRACT Virus isolates from forage legumes collected from eight different states were identified as luteoviruses closely related to soybean dwarf luteovirus dwarfing (SbDV-D) and yellowing (SbDV-Y) described in Japan. All isolates produced reddened leaf margins in subterranean clover and were transmitted in a persistent manner by Acrythosiphon pisum, but not by Aulacorthum solani. Specific monoclonal antibodies raised against SbDV-Y were differentially reactive with endemic isolates. Immunoblots probed with a SbDV-D polyclonal antiserum showed single 26-kDa coat protein bands, confirming close serological relatedness to SbDV. Analyses of genomic and subgenomic double-stranded RNAs and northern blot analyses confirmed genomic relatedness to SbDV. Based on our results, we conclude that the U.S. luteovirus isolates studied comprise a strain or strains of the soybean dwarf virus that have clovers as common hosts and the pea aphid as a common vector.

Sequence and expression in Escherichia coli of the coat protein gene of the dwarfing strain of soybean dwarf luteovirus.

The nucleotide sequence of the coat protein gene of the dwarfing (D) strain of soybean dwarf luteovirus (SbDV) was determined from cloned cDNA. The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated molecular mass of 22.2 kDa. A major portion of the coat protein open reading frame (ORF) was expressed in Escherichia coli as a pET fusion protein and the product was detected by western blot analysis using SbDV-D polyclonal antibodies. Comparison of the deduced coat protein amino acid sequence to that from the yellowing (Y) strain of SbDV demonstrated 88% identity.

First Report of Musk Thistle Rust (Puccinia carduorum) in Oklahoma.

Musk thistle, Carduus thoermeri (Carduus nutans subsp. leiophyllus), is an important, introduced pasture weed in central and northeastern Oklahoma. Puccinia carduorum was introduced into the United States from Turkey as a potential biological control for musk thistle. P. carduorum has not been reported previously in Oklahoma, thus precluding its field release for biological control research without APHIS approval. There is evidence the organism has moved westward since the initial field studies that began in 1987 in Virginia. In 1994 it was found in Missouri (1). In early November 1997, in Rogers County, Oklahoma, scattered populations of C. thoermeri were found that had moderate to heavy levels of infection with a rust fungus. The pustules contained mostly teliospores; based on teliospore and urediniospore morphology, the fungus was identified as P. carduorum. The morphology and dimensions of urediniospores (21 × 21 µm, avg.) and teliospores (35 × 21 µm, avg.), and the restriction of echinulations to the upper two-thirds to three-fourths of urediniospores, were consistent with P. carduorum. Infection studies with field inoculum were conducted at both Oklahoma State University and USDA-FDWSRU. Rust-infected leaves collected in Oklahoma were air dried and maintained at room temperature for 2 months prior to use as inoculum. Small, symptomless, first-year rosettes of musk thistle were transplanted from the field into a mixture of soil, sand, and peat moss in pots and placed into a growth chamber maintained at 20°C. Seeds of C. thoermeri planted into pots containing the same mixture were maintained in the same chambers. After approximately 6 to 8 weeks, when seedlings and transplants were growing vigorously, both groups of plants were dusted with urediniospores and teliospores from the dried leaves. Inoculated plants were placed either into a 20°C dew chamber for 24 h or were atomized with distilled water, placed into sealed, transparent, polyethylene bags and returned to the 20°C growth chamber for 24 h, after which time the bags were removed. Both sets of plants were then maintained at 20 to 25°C. Chlorotic flecks developed on inoculated leaves after 7 to 8 days; uredinia and urediniospores were present within 10 days after inoculation. Urediniospores from those leaves had the same dimensions and ornamentation pattern as those originally obtained from field collections. A DNA sequence analysis was conducted on the rRNA ITS2 region, which was polymerase chain reaction-amplified from genomic DNA (2) extracted from urediniospores of the Oklahoma isolate grown at FDWSRU. The sequence of the ITS2 region from those urediniospores was identical to the sequence (GenBank accession no. U57351) obtained from the isolate 7803 of P. carduorum from Turkey, used in the Virginia field studies. The confirmed presence of P. carduorum in Oklahoma will enable field research with this rust for management of musk thistle in the state. References: (1) A. B. A. M. Baudoin and W. L. Bruckart. Plant Dis. 80:1193, 1996. (2) Y. T. Berthier et al. Appl. Environ. Microbiol. 62:3037, 1996.

Polymorphic restriction patterns of ribosomal internal transcribed spacers in the biocontrol fungus Puccinia carduorum correlate with weed host origin.

The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified by PCR and used to develop genetic markers for isolates of Puccinia carduorum being evaluated for biological control of Carduus thoermeri (musk thistle). Unique patterns were produced upon restriction of ITS DNA amplified from four separate Puccinia spp. Restriction patterns of ITS DNA of isolates of P. carduorum from Carduus acanthoides and C. thoermeri were distinct from those of P. carduorum from Carduus tenuiflorus and Carduus pycnocephalus. By this technique, isolates of P. carduorum from four different weed hosts can be differentiated from other Puccinia spp. and separated into two host groups.

A phase I trial of B7-transfected or parental lethally irradiated allogeneic melanoma cell lines to induce cell-mediated immunity against tumor-associated antigen presented by HLA-A2 or HLA-A1 in patients with stage IV melanoma. NCI protocol T93-0161. BRMP protocol 9401.

T cell lines generated by primary in vitro stimulation with B7-expressing HLA-A2+ melanoma cells lyse HLA-A2+ melanomas, but not non-melanomas that are HLA-A2+. Other data have demonstrated lack of response of these T cell lines against non-HLA-A2 melanomas. These concepts are verified by data from MALME MEL, which is killed, and MALME FIB, which is not. In no case was lysis directed at targets expressing potential allo-antigens (except for HLA-A2+ melanomas). A19 and Aw33 have not been excluded as possible allo-targets (but no data suggests they are). In total, it appears that much of the lytic activity observed in the two T cell lines is directed against HLA-A2-restricted, melanoma-specific antigens.

Fe-Chelate Reductase Activity of Plasma Membranes Isolated from Tomato (Lycopersicon esculentum Mill.) Roots : Comparison of Enzymes from Fe-Deficient and Fe-Sufficient Roots.

Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (-Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3-))(2). NADH and Fe(3+)(citrate(3-))(2)K(m) values for reductase in PM from +Fe and -Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from -Fe tomatoes. The pH optimum for Fe-chelate reductase was 6.5. Fe-chelate reductases from -Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n-octyl beta-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and -Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from -Fe PM. We conclude that PM from roots of +Fe and -Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM.

Multiple forms of plant cytochromes p-450.

Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases are implicated in the metabolism of sterols, terpenes, gibberellins, isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 are involved in the detoxification of herbicides (chlorotoluron, primsulfuron, and diclofop) includes photoreversible CO inhibition of the reactions, and a requirement for O(2) and NADPH. Several cytochromes P-450, M(r) 45,000 to 65,000, have been isolated, including hydroxylases of cinnamic acid, 3,9-dihydroxypterocarpan, and digitoxin. In some cases the purified cytochrome P-450 has been successfully reconstituted with NADPH:cytochrome P-450 reductase (M(r) 72,000-84,000 protein). This reductase appears to be a nonspecific electron donor to different forms of cytochrome P-450. Immunological techniques and specific inhibitors (triazoles, imidazole derivatives) are being used to characterize plant cytochromes P-450 and the NADPH:cytochrome P-450 reductase. Specific cytochromes P-450 are induced by wounding or pathogens, others are expressed in specific cell types. Plant cytochromes P-450 are found in various subcellular locations, including endoplasmic reticulum, plasma membranes, glyoxysomes, and perhaps mitochondria. A cytochrome P-450 demethylase from avocado has recently been sequenced and found to have a hydrophobic N terminus similar to the membrane anchor of cytochromes P-450 from other organisms. The existence of cytochromes P-450 in different subcellular locations suggests that there are many genes for cytochromes P-450 in plants which have yet to be identified and classified.

Development of Endoplasmic Reticulum and Glyoxysomal Membrane Redox Activities during Castor Bean Germination.

Redox activities, NADH:ferricyanide reductase, NAD(P)H:cytochrome reductases, and NADH:ascorbate free-radical reductase, are present in endoplasmic reticulum (ER) and glyoxysomal membranes from the endosperm of germinating castor bean (Ricinus comminus L. var Hale). The development of these functions was followed in glyoxysomes and ER isolated on sucrose gradients from castor bean endosperm daily from 0 through 6 days of germination. On a per seed basis, glyoxysomal and ER protein, glyoxysomal and ER membrane redox enzyme activities, and glyoxylate cycle activities peaked at day 4 as did the ER membrane content of cytochrome P-450. NADH:ferricyanide reductase was present in glyoxysomes and ER isolated from dry seed. This activity increased only about twofold in glyoxysomes and threefold in ER during germination relative to the amount of protein in the respective fractions. The other reductases, NADH:cytochrome reductase and NADH:ascorbate free-radical reductase, increased about 10-fold in the ER relative to protein up to 4 to 5 days, then declined. NADPH:cytochrome reductase reached maximum activity relative to protein at day 2 in both organelles. The increases in redox activities during germination indicate that the membranes of the ER and glyoxysome are being enriched with redox proteins during their development. The development of redox functions in glyoxysomes was found to be coordinated with development of the glyoxylate cycle.

Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots.

Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity.