Molecular genotyping of herbicide resistance in P. minor: ACCase resistance.

Little seed canary grass (Phalaris minor Retz.) populations resistant to herbicides that inhibit acetyl-CoA carboxylase (ACCase) represent an increasingly important weed control problem in northern India. The objective of this study was to develop DNA-based markers to differentiate herbicide-resistant and herbicide-susceptible population of P. minor. Primers were designed to amplify the conserved region carrying two reported mutations Trp2027 to Cys and Ile2041 to Asn conferring ACCase inhibitor resistance in several grass weeds and subjected to single-strand conformational polymorphism (SSCP) to detect the mutations. Five distinctive electrophoretic patterns on non-denaturing PAGE were observed, and four patterns were found to be associated with ACCase herbicide resistance in P. minor. The PCR-SSCP test developed in this study confirmed 17 resistant populations to contain mutations in CT domain of ACCase gene. This is the first report of rapid and easy molecular diagnosis of ACCase herbicide-resistant and herbicide-sensitive population of P. minor through PCR-SSCP analysis.

Isolation of the P5CS gene from reed canary grass and its expression under salt stress.

Reed canary grass (RCG) is a perennial grass traditionally cultivated for forage. It is also used as fuel to produce energy in Finland and Sweden, and other countries have expressed interest in the cultivation of RCG. In China, arable land is limited. Salinity is considered to be a major factor limiting plant crop development and productivity. To boost biofuel production of RCG and extend its range in saline soil, we seek to improve its salt tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS plays a crucial role in proline biosynthesis. We isolated a P5CS gene from RCG, designated B231P5CS (GenBank accession No. JQ622685). B231P5CS is a fragment (971 bp) that encodes a 323-amino acid polypeptide. We also cloned an actin gene fragment from RCG as a reference gene in expression analysis of B231P5CS gene. Expression analysis revealed that B231P5CS transcripts were upregulated in leaves after treatment with salt (200 mM NaCl) and that transcript levels of B231P5CS reached a maximum 12 h after exposure, which was 14.69 times the level in control plants. The trends of expression were exactly opposite in roots; transcripts were downregulated after salt treatment. Proline concentration increased in leaves after stress. In contrast, proline content of roots decreased up to 3.6-fold relative to controls. Changes in proline concentration after stress were correlated with B231P5CS expression. Our results suggest that B231P5CS is a stress-inducible gene and plays a non-redundant role in plant development. This gene may be used to improve stress tolerance of RGC and other bioenergy feedstock.

ß-Pinene inhibited germination and early growth involves membrane peroxidation.

ß-Pinene, an oxygenated monoterpene, is abundantly found in the environment and widely occurring in plants as a constituent of essential oils. We investigated the phytotoxicity of ß-pinene against two grassy (Phalaris minor, Echinochloa crus-galli) and one broad-leaved (Cassia occidentalis) weeds in terms of germination and root and shoot growth. ß-Pinene (0.02-0.80 mg/ml) inhibited the germination, root length, and shoot length of test weeds in a dose-response manner. The inhibitory effect of ß-pinene was greater in grassy weeds and on root growth than on shoot growth. ß-Pinene (0.04-0.80 mg/ml) reduced the root length in P. minor, E. crus-galli, and C. occidentalis over that in the control by 58-60, 44-92, and 26-85 %, respectively. In contrast, shoot length was reduced over the control by 45-97 % in P. minor, 48-78 % in E. crus-galli, and 11-75 % in C. occidentalis at similar concentrations. Further, we examined the impact of ß-pinene on membrane integrity in P. minor as one of the possible mechanisms of action. Membrane integrity was evaluated in terms of lipid peroxidation, conjugated diene content, electrolyte leakage, and the activity of lipoxygenases (LOX). ß-Pinene (≥0.04 mg/ml) enhanced electrolyte leakage by 23-80 %, malondialdehyde content by 15-67 %, hydrogen peroxide content by 9-39 %, and lipoxygenases activity by 38-383 % over that in the control. It indicated membrane peroxidation and loss of membrane integrity that could be the primary target of ß-pinene. Even the enhanced (9-62 %) activity of protecting enzymes, peroxidases (POX), was not able to protect the membranes from ß-pinene (0.04-0.20 mg/ml)-induced toxicity. In conclusion, our results show that ß-pinene inhibits root growth of the tested weed species through disruption of membrane integrity as indicated by enhanced peroxidation, electrolyte leakage, and LOX activity despite the upregulation of POX activity.

How nitrogen and sulphur addition, and a single drought event affect root phosphatase activity in Phalaris arundinacea.

Conservation and restoration of fens and fen meadows often aim to reduce soil nutrients, mainly nitrogen (N) and phosphorus (P). The biogeochemistry of P has received much attention as P-enrichment is expected to negatively impact on species diversity in wetlands. It is known that N, sulphur (S) and hydrological conditions affect the biogeochemistry of P, yet their interactive effects on P-dynamics are largely unknown. Additionally, in Europe, climate change has been predicted to lead to increases in summer drought. We performed a greenhouse experiment to elucidate the interactive effects of N, S and a single drought event on the P-availability for Phalaris arundinacea. Additionally, the response of plant phosphatase activity to these factors was measured over the two year experimental period. In contrast to results from earlier experiments, our treatments hardly affected soil P-availability. This may be explained by the higher pH in our soils, hampering the formation of Fe-P or Fe-Al complexes. Addition of S, however, decreased the plants N:P ratio, indicating an effect of S on the N:P stoichiometry and an effect on the plant's P-demand. Phosphatase activity increased significantly after addition of S, but was not affected by the addition of N or a single drought event. Root phosphatase activity was also positively related to plant tissue N and P concentrations, plant N and P uptake, and plant aboveground biomass, suggesting that the phosphatase enzyme influences P-biogeochemistry. Our results demonstrated that it is difficult to predict the effects of wetland restoration, since the involved mechanisms are not fully understood. Short-term and long-term effects on root phosphatase activity may differ considerably. Additionally, the addition of S can lead to unexpected effects on the biogeochemistry of P. Our results showed that natural resource managers should be careful when restoring degraded fens or preventing desiccation of fen ecosystems.

Selection of plants for roles in phytoremediation: the importance of glucosylation.

Over-expression and transposon mutagenesis in root cultures of Arabidopsis thaliana demonstrated the importance of the family 1 glycosyltransferase UGT72B1 in catalysing the N-glucosylation of the persistent pollutant 3,4-dichloroaniline (DCA). In phytotoxicity studies with DCA in seedlings, over-expression of UGT72B1 enhanced sensitivity, whereas the knockouts were more resistant than the controls. In contrast, manipulating the expression of UGT72B1 had no effect on the O-glucosylation, or toxicity, of chlorophenols. When N-glucosylation was disrupted in plants, radioactivity derived from [14C]-DCA became covalently bound into high molecular weight insoluble material, principally associated with the lignin fraction. This suggested that insolubilization into stable cell wall components represented a more effective mechanism of DCA detoxification than the formation of N-glycosidic conjugates. A screen of plants used in remediation, identified low levels of N-glucosyltransferase activity in switchgrass and high activities in reed canary grass. When incubated with [14C]-DCA, reed canary grass plants accumulated soluble N-glycosides of DCA, whereas switchgrass formed insoluble residues. Consistent with the results obtained in studies with Arabidopsis, phytotoxicity trials with DCA demonstrated that switchgrass was more tolerant than reed canary grass. Our studies provide a new biochemical basis for selecting plants for useful remediating traits towards specific classes of pollutants.