Legumes, and a very few non-legume plant species, are known to possess functioning haemoglobin genes. We describe here the characterization of a haemoglobin cDNA isolated from barley. The deduced amino acid sequence shows 71% amino acid identity with a non-legume haemoglobin gene, a further 16% of the residues being conservative replacements. The barley cDNA also hybridizes to genomic sequences in rye, maize and wheat. The demonstration of a gene from a monocotyledon with close sequence homology to the known non-legume plant haemoglobins fills a major gap in the known distribution of haemoglobin genes in the plant kingdom. The expression of the gene is induced in isolated barley aleurone layers exposed to anaerobic conditions, and the roots of flooding-stressed barley plants. The expression of the RNA under anoxic conditions is similar to that of a known anaerobic response gene, alcohol dehydrogenase. Our results suggest that the increased expression of haemoglobin RNA is an integral part of the normal anaerobic response in barley. The findings are discussed in the light of current theories of haemoglobin function and evolution.

A reverse transcription loop-mediated isothermal amplification of DNA (RT-LAMP) for detection of Potato virus Y (PVY) was developed. In this procedure, a set of four primers matching a total of six sequences of the coat protein (CP) gene of PVY were designed in such a way that a loop could be formed and elongated during DNA amplification. Using PVY CP complementary DNA clones as templates, the LAMP reaction was optimized by adjusting the concentrations of MgSO4, dNTPs, and Bst DNA polymerase. The effects of fragment length of target DNA on LAMP also were investigated. Two-step and one-step RT-LAMPs were performed using RNA extracts of various PVY cultures, and the results were correlated with two-step reverse transcription polymerase chain reaction (RT-PCR) for detection of PVY. Further, the turbidity caused by precipitation of magnesium pyrophosphate formed in positive RT-LAMP reactions was used to measure the amplification by utilizing a time-saving spectrophotometric method. The one-step RT-LAMP-turbidity method gave results comparable with the two-step RT-PCR method for detection of PVY from potato leaf and tuber samples. Of the total 240 samples, 234 were diagnosed similarly by both methods.

Previous studies have shown that plant hemoglobin (Hb) mRNA is expressed in barley (Hordeum vdgare 1.) aleurone layers during hypoxia. We have examined the effect of a number of respiratory inhibitors on barley aleurone layers to determine the factors that influence Hb gene expression. Respiratory inhibitors that reduce O, consumption, such as CO, cyanide, and antimycin A, strongly enhanced Hb mRNA levels. Treatment with the oxidative phosphorylation uncoupler 2,4-dinitrophenol markedly increased O, consumption and had a similar positive effect on Hb gene expression. Hb transcript levels were also stimulated by the ATP synthase inhibitor oligomycin. l h e results suggest that the expression of Hb is not directly influenced by O, usage or availability but is influenced by the availability of ATP in the tissue.Hb exists widely in organisms ranging from prokaryotes to eukaryotes (Appleby et al., 1988; Wittenberg and Wittenberg, 1990). It has been found in a number of plant species (Appleby, 1992), most notably dicots engaged in symbiotic relationships with bacteria. The expression of a plant Hb gene in the seed tissue of a monocot (Taylor et al., 1994) has raised the possibility that the molecule may be more universally present in plants and contribute to plant growth and development in a manner beyond its role in the N,-fixation process in dicots.Hb is characterized by its conserved structure, high O, affinity, and reversible combination with O, in the ferrous state. Its function is normally associated with the facilitation of O, diffusion, O, storage, and O, utilization in organisms. Legume Hb has been well studied in legume symbiosis, where it is believed to act as an O, carrier to the symbiosomes of root nodules, supplying O, to the bacterial respiratory chain while preserving a low free-O, concentration (Appleby, 1992). However, the significance of the presence of Hb in nonnodulating plants has not been resolved. Studies have shown that in nonnodulating dicots Hb occurs mainly in the roots a t a concentration of approximately 100 nM, which is probably lower than the concentration of free-dissolved O, in the cells (Appleby et al., 1988 MATERIALS A N D METHODSSeeds of barley (Hordeum vulgare L. cv Harrington, provided by the Canadian Grain Commission, Winnipeg, Manitoba) were de-embryonated and a small portion of the dista1 end was cut off. The resulting half-seeds were surface-sterilized for 30 min in 1% (w/v) NaOCl and rinsed thoroughly in distilled water. After 2 d of imbibition at 22°C in darkness, 25 aleurone layers were separated from the starchy endosperm, placed in a sterile 50-mL conical flask containing 1.5 mL of an incubation medium (water or 0.1 M phosphate buffer, pH 7.2), and incubated with slow agitation (65 cycleslmin) at room temperature. Treatment with Respiratory lnhibitorsUnless otherwise stated, freshly prepared barley aleurone layers were incubated in 0.1 M phosphate buffer (pH 7.0) containing various additions. The Cyt c oxidase inhibitor KCN was used at a final concentration of 0.8 mM,...

Potato field isolates (Solanum tuberosum) of Potato virus Y (PVY) collected from the midwestern and western United States were characterized using serological, molecular, and biological assays. PVY field isolates were grouped into the previously defined categories: PVY(O), European PVY(NTN), North American PVY(NTN), and PVY(N:O) recombinant and four previously undefined groups. Studies reported here agree with published reports from Europe and elsewhere in North America as PVY isolates capable of causing veinal necrosis in tobacco indicator plants appear in high frequency. In contrast to European experiences, PVY tuber necrosis isolates have a PVY(O) coat protein rather than that of PVY(N). Several PVY(N:O) recombinant isolates induced potato tuber necrotic ringspot disease (PTNRD) in the highly susceptible potato cv. Yukon Gold. The PTNRD symptoms produced by these PVY(N:O) recombinants were atypical compared with lesions found on the same cultivar infected with either the European or North American PVY(NTN) isolates. These PVY(N:O) isolates produced a roughly circular, sunken necrotic lesion on the surface of the tuber instead of the typical external sunken ring pattern displayed by PVY(NTN) isolates. This study establishes the complex nature of PVY populations within the U.S. potato industry and clearly demonstrates the diverse nature of PVY in the United States.

Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99 %. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90 % sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60 % and the Terminal Right domain less than 90 % sequence similarity, while the remainder of the molecule has greater than 97 % similarity with PSTVd. Because of its less-than 90 % sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid ; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid.

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