Mechanism of resistance to quinclorac in smooth crabgrass (Digitaria ischaemum)

The mechanism of resistance to quinclorac was investigated in a smooth crabgrass biotype [Digitaria ischaemum (Schreb. ex Schweig) Schreb. ex Muhl] from Tulare County, California. Quinclorac (8.96 kg a.i. ha⁻¹) had no effect (P = 0.18) on the resistant (R) biotype, but reduced fresh weight of a susceptible (S) biotype by 93%. After treatment with 4.48 kg a.i. quinclorac ha⁻¹, the S biotype produced about three times more ethylene than the R biotype and accumulated cyanide in tissues. Similar amounts of endogenous cyanide resulting from treatment with KCN reproduced quinclorac phytotoxicity. Pre-treatment with the ACC synthase inhibitor AVG reduced quinclorac phytotoxicity by 37% and ethylene production by 89%. These data suggest a target site-based mechanism of resistance involving stimulation of ACC synthesis and accumulation of cyanide. Also, the R biotype had four times more β-cyanoalanine synthase activity than the S biotype, suggesting a higher ability to detoxify cyanide.     

Multiple effects of a naturally occurring proline to threonine substitution within acetolactate synthase in two herbicide-resistant populations of Lactuca serriola

Two populations of Lactuca serriola L. with resistance to acetolactate synthase (ALS)-inhibiting herbicides were discovered in wheat fields at two locations more than 25 km apart in South Australia. Both resistant populations carried a single base change within a highly conserved coding region of the ALS gene that coded for a single amino acid modification within ALS. The modification of proline 197 to threonine resulted in an enzyme that was highly resistant (>200-fold) to inhibition by sulfonylurea herbicides and moderately resistant to triazolopyrimidine and imidazolinone herbicides. The herbicide-resistant ALS was also less sensitive to inhibition by the branched-chain amino acids valine and leucine. In addition, the resistant enzyme had a lower K_m for pyruvate. However, extractable ALS activity was similar between resistant and susceptible plants. The substitution of threonine for proline 197 within ALS has multiple impacts on ALS enzyme activity in L. serriola that may influence the frequency of this resistant allele in the environment.     

Enhanced mycotoxin production of a lipase-deficient <Emphasis Type="Italic">Fusarium graminearum</Emphasis> mutant correlates to toxin-related gene expression

Fusarium graminearum causes important diseases of small-grain cereals and maize and produces several mycotoxins. Among them, deoxynivalenol (DON) and zearalenone (ZEA) can accumulate in feedstuffs and foods to health-threatening levels. Although DON is important for fungal virulence in wheat, disease severity in the field does not correlate with mycotoxin concentrations. We compared gene expression and mycotoxin production of lipase-deficient mutants (Δfgl1), strongly reduced in virulence, and the respective wild-type isolate. Δfgl1 mutants exhibited up-regulated DON production during wheat head infection. On isolated wheat kernels, DON was only produced in low quantities, but higher in wild-type than in Δfgl1 mutants. In contrast, neither wild-type nor Δfgl1 mutants produced ZEA during wheat head infection. However, ZEA was clearly detectable on wheat kernels. Here, Δfgl1 mutants revealed a dramatically enhanced ZEA production. We could correlate the altered amounts of DON and ZEA directly with the expression of the toxin-related genes Tri5 for DON and PKS4 and PKS13 for ZEA. Based on Tri5 expression and the infection pattern of the wild-type and Δfgl1 mutants, we suggest that the transition zone of rachilla and rachis is important in the induction of DON synthesis. Gene expression studies indicate an involvement of the lipase FGL1 in regulation of 8 PKS genes and ZEA production.     

除草剂与乙酰乳酸合成酶相互作用的分子机理研究进展

综述了酵母乙酰乳酸合成酶(ScALS)与磺酰脲类除草剂氯嘧磺隆(chlorimuron-ethyl,CE)形成的复合物在0.28 nm分辨率下的晶体结构及拟南芥乙酰乳酸合成酶(AtALS)与磺酰脲类和咪唑啉酮类除草剂复合物的三维结构。除草剂的分子结构与酶、底物并不相似,但它们与酶形成的复合物可阻断底物进入酶活性位点通路而起抑制作用。连接磺酰脲的10个氨基酸残基同样连接咪唑喹啉酸,另有6个残基只与磺酰脲而不与咪唑喹啉酸相连,有2个残基只与咪唑喹啉酸而不与磺酰脲相连,即两种抑制剂占据了特别的重叠位点,但以不同方式连接。抗性杂草的产生是因为突变株ALS的残基位点变异,从而引起除草剂与ALS结合方式的变化。这些研究对进一步理解除草剂与靶分子的作用方式及除草剂的分子药物设计具有重要的指导作用。     

Molecular basis of diverse responses to acetolactate synthase-inhibiting herbicides in sulfonylurea-resistant biotypes of Schoenoplectus juncoides

Sulfonylurea-resistant biotypes of Schoenoplectus juncoides were collected from Nakafurano, Shiwa, Matsuyama, and Yurihonjyo in Japan. All of the four biotypes showed resistance to bensulfuron-methyl and thifensulfuron-methyl in whole-plant experiments. The growth of the Nakafurano, Shiwa, and Matsuyama biotypes was inhibited by imazaquin-ammonium and bispyribac-sodium, whereas the Yurihonjyo biotype grew normally after treatment with these herbicides. The herbicide concentration required to inhibit the acetolactate synthase (ALS) enzyme by 50% (I₅₀), obtained using in vivo ALS assays, indicated that the four biotypes were > 10-fold more resistant to thifensulfuron-methyl than a susceptible biotype. The Nakafurano, Shiwa, and Matsuyama biotypes exhibited no or little resistance to imazaquin-ammonium, whereas the Yurihonjyo biotype exhibited 6700-fold resistance to the herbicide. The Nakafurano and Shiwa biotypes exhibited no resistance to bispyribac-sodium, but the Matsuyama biotype exhibited 21-fold resistance and the Yurihonjyo biotype exhibited 260-fold resistance to the herbicide. Two S. juncoides ALS genes (ALS1 and ALS2) were isolated and each was found to contain one intron and to encode an ALS protein of 645 amino acids. Sequencing of the ALS genes revealed an amino acid substitution at Pro₁₉₇ in either encoded protein (ALS1 or ALS2) in the biotypes from Nakafurano (Pro₁₉₇ → Ser₁₉₇), Shiwa (Pro₁₉₇ → His₁₉₇), and Matsuyama (Pro₁₉₇ → Leu₁₉₇). The ALS2 of the biotype from Yurihonjyo was found to contain a Trp₅₇₄ → Leu₅₇₄ substitution. The relationships between the responses to ALS-inhibiting herbicides and the amino acid substitutions, which are consistent with previous reports in other plants, indicate that the substitutions at Pro₁₉₇ and Trp₅₇₄ are the basis of the resistance to sulfonylureas in these S. juncoides biotypes.     

Transient expression of an active human interferon-beta in lettuce

Using Agrobacterium mediated transient expression method, plant bivalent expression vector pBI121 containing GUS as a report gene was transformed into lettuce (Lactuca sativa). Through designed orthogonal analysis, intact lettuce leaves infiltrated with 200 μM acetosyringone and 0.8 OD₆₀₀ bacterial suspensions under vacuum for 30 min, then co-cultured at 24 °C for 6 ds produced a maximum GUS protein of 2.5% TSP with 21.39 nmol mg⁻¹ min⁻¹ MU activity, which was 19 times of the control (1.31 nmol mg⁻¹ min⁻¹ MU). Employed these optimized conditions HuIFN-beta was expressed in lettuce leaves. Western blot and antivirus bioactivity analyses confirmed the HuIFN-beta achieved by agrobacterium infiltration had a high biological activity (3.1 × 10⁴ IU/mL). To our knowledge, it is the first detailed orthogonal optimizing study of Agrobacterium mediated transient expression and the first report on the production of the biologically active therapeutic proteins produced by Agrobacterium mediated transient expression in lettuce. In summary, transient expression by Agrobacterium vacuum infiltration can be adopted as an efficient, inexpensive and small-scaled plant expression system for therapeutic protein production.     

Sucrolytic activities in cherry tomato fruits in relation to temperature and solar radiation

A study has been made concerning the influence of two environmental factors (temperature and solar radiation) on sugar content and sucrolytic activity in cherry tomato fruits during the crop cycle. For this, Solanum lycopersicum cv. Naomi plants were grown in an experimental greenhouse. Three fruit samples were taken over the entire production period: the first sampling at the beginning of harvest [85 days after transplanting (dat)], the second at mid-harvest (160 dat), and the third at the end of harvest (229 dat). The values for temperature and solar radiation peaked in the third sampling, coinciding with an increase in lipid peroxidation, without lowering yield with respect to previous samplings. Regarding the sugar content in the cherry tomatoes, our results showed that the increase in temperature and solar radiation diminished the sucrose content at 229 dat and raised the hexoses (glucose and fructose) as well as starch content, produced primarily by the enzyme sucrose synthase. On the contrary, the rest of the enzymes responsible for sucrose degradation, acid and neutral invertases, showed no notable changes in their activity at the end of the crop cycle. In short, our results suggest that the increase in sucrolytic activity, induced mainly by sucrose synthase under these conditions, contributes to the mechanisms of antioxidant defense by supplying precursors (glucose and fructose) of antioxidant compounds in order to restrict the massive accumulation of ROS and thereby avoid the appearance of cell necrosis and reduce yield losses.     

Effects of carvedilol on metabolic mechanism of endogenous NOS inhibitor in human umbilical vein endothelial cells treated with oxidized free radical

AIM: To observe the effects of oxidized free radical (OFR) on dimethylarginine dimethylaminohydrolase (DDAH) activity and concentration in human umbilical vein endothelial cells (HUVECs), and to investigate the metabolic mechanism of endogenous NOS inhibitor and the role of carvedilol. METHODS: HUVECs of 3-6th passage, cultured with modified Jaffes method, were divided into three groups: (1) cells cultured with equivalent DMEM medium as control; (2) OFR intervention groups, 0.01 mmol/L, or 0.1 mmol/L OFR was added respectively; (3) drug intervention groups: 0.1 mmol/L OFR plus 10 μmol/L carvedilol. ADMA, nitric oxide (NO), endothim (ET), L-citrulline concentrations and the activity of NOS in conditioned medium were measured after 24 h exposure. ADMA concentration in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. RESULTS: Compared with control group, ADMA and ET concentrations were increased, while the level of NO and the activity of NOS decreased and relevant to the concentrations of OFR. We assayed DDAH activity by determining L-citrulline formation from ADMA. The concentration of L-citrulline was decreased, while the DDAH expression had no obvious change. With the role of carvedilol, ADMA, ET concentrations were decreased, while the level of NO, L-citrulline and the activity of NOS increased. CONCLUSION: Endothelial dysfunction induced by OFR is associated with the increase in ADMA concentration and reduction of DDAH activity, but not DDAH expression. Carvedilol promotes the degradation of ADMA through increasing activity of DDAH and improving endothelium function.