BackgroundPolyphenol oxidase (PPO) activity in plants is a trait with potential economic, agricultural and environmental impact. In relation to the food industry, PPO-induced browning causes unacceptable discolouration in fruit and vegetables: from an agriculture perspective, PPO can protect plants against pathogens and environmental stress, improve ruminant growth by increasing nitrogen absorption and decreasing nitrogen loss to the environment through the animal's urine. The high PPO legume, red clover, has a significant economic and environmental role in sustaining low-input organic and conventional farms. Molecular markers for a range of important agricultural traits are being developed for red clover and improved knowledge of PPO genes and their structure will facilitate molecular breeding.ResultsA bacterial artificial chromosome (BAC) library comprising 26,016 BAC clones with an average 135 Kb insert size, was constructed from Trifolium pratense L. (red clover), a diploid legume with a haploid genome size of 440–637 Mb. Library coverage of 6–8 genome equivalents ensured good representation of genes: the library was screened for polyphenol oxidase (PPO) genes.Two single copy PPO genes, PPO4 and PPO5, were identified to add to a family of three, previously reported, paralogous genes (PPO1–PPO3). Multiple PPO1 copies were identified and characterised revealing a subfamily comprising three variants PPO1/2, PPO1/4 and PPO1/5. Six PPO genes clustered within the genome: four separate BAC clones could be assembled onto a predicted 190–510 Kb single BAC contig.ConclusionA PPO gene family in red clover resides as a cluster of at least 6 genes. Three of these genes have high homology, suggesting a more recent evolutionary event. This PPO cluster covers a longer region of the genome than clusters detected in rice or previously reported in tomato. Full-length coding sequences from PPO4, PPO5, PPO1/5 and PPO1/4 will facilitate functional studies and provide genetic markers for plant breeding.
SUM M ARYRoot system senescence and nitrogen (N) release from red clover (Trifolium pratense L.) plants, grown under semi-sterile conditions and a controlled environment, were studied for 28 days following temporary or prolonged abiotic stress. Plants stressed temporarily, to simulate grazing, recovered with no additional N lost in leachate. In contrast, plants subjected to prolonged stress that simulated overwintering conditions and inhibited shoot re-growth survived stress lasting 7 days, but plant viability was reduced to 50% by 14 days and 0% at 21 days. There were no significant differences in root protein, catalase activity, root death index or total N loss in leachate over 21 days, but by 28 days total N loss in leachate increased to 214% above control levels, with a 433% increase in total oxidized N. This increase in N loss between 21 and 28 days indicated the start of cellular breakdown of the root system, coinciding with the failure of plants to recover.Key enzyme activities and protein concentrations in nodules decreased rapidly over 10 days' prolonged stress. cDNA-amplified fragment length polymorphism (AFLP) analysis identified contaminating bacterial and fungal genes, along with plant gene sequences with consistent or altered expression profiles. Four plant sequences, glyceraldehyde-3-phosphate dehydrogenase (Tp-gapdh1), nodule senescence reduced (Tp-nsr1), nodule senescence enhanced (Tp-nse1) and a cysteine protease gene (Tp-cp8) were differentially expressed throughout the plant: Tp-nsr1 and Tp-nse1 have potential as molecular markers for nodule senescence.Root and nodule death in agricultural legumes, such as red clover, are implicated in N release into watercourses and the wider environment. Differences in the ability of these plants to survive prolonged stress lasting 14 days, and the delayed release of root N into leachate until 28 days after the stress, highlight the potential for the development of new red clover varieties with different rates of root system senescence.
Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRIDMseI digestion. Four fragments generated by EcoAGTDMseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. \ canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10-1 to 10-5 ᴍ) incubated for 21 days on aseptic tissue culture media WPM containing 1 μᴍ Cu. Zn2+ caused phytotoxicity only at high concentrations (10-1 to 10-2 ᴍ). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.
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