The drop plate method as an alternative for Azospirillum spp viable cell enumeration within the consensus protocol of the REDCAI network

Abstract Quality evaluation of commercial inoculants is essential to warrant an adequate cropresponse to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cellsas an alternative to the spread plate technique, which is currently proposed in the consensusprotocol of the REDCAI network. Between 14 and 25 private and public laboratories partici-pated in three independent trials. We obtained consistent and robust results that allowed toconfirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensusprotocol.

Resumen La evaluación de la calidad de los inoculantes comerciales es fundamental para garantizar una adecuada respuesta de los cultivos a la inoculación dentro de un marco de bioseguridad. En este sentido, el objetivo de este trabajo fue la estandarización y validación de la técnica de la microgota para la cuantificación de Azospirillum como metodología alternativa a la técnica de siembra en superficie, propuesta actualmente en el protocolo consenso de la Red de Calidad de Inoculantes, REDCAI. Entre 14 y 25 laboratorios, tanto privados como públicos, participaron de tres ensayos independientes. A partir de ellos se obtuvieron resultados reproducibles y robustos que permiten confirmar que ambas técnicas son equivalentes y concluir que la técnica de recuento por la microgota es una alternativa adecuada para ser incluida dentro del mencionado protocolo consenso.

Metabolic changes associated with chilling injury tolerance in tomato fruit with hot water pretreatment.

Hot water treatment (HWT) of tomato (Solanum lycopersicum L.) fruit reduces the symptoms of chilling injury (CI). The aim of this study was to identify metabolites associated with HWT-induced CI tolerance in tomato fruit cv. Imperial. Mature green tomatoes with HWT (42°C/5 min) and control were stored under chilling conditions (5°C/20 days) and then ripened (21°C/7 days). Methanol extracts from pericarp were analyzed for total phenolics (TP), antioxidant activity (AoxA), and metabolic profiling by UPLC-DAD-MS and GC-MS. After cold storage and ripening, HWT fruit showed less CI, higher TP, and AoxA than control. It also showed an increased accumulation of phenolics, sugars, and some alkaloids that may be mediated by azelaic acid, glutamine, and tryptophan. The levels of N-feruloyl putrescine, esculeoside AII, and hydroxy-α-tomatine II were reduced. The better metabolic performance of HWT fruit under cold storage was associated with a higher accumulation of several metabolites (e.g., antioxidants and osmolytes) in ripening fruit. PRACTICAL APPLICATION: The identification of metabolites associated with the reduction of chilling injury (CI) symptoms in HWT tomato fruit extends the understanding of the mechanisms involved in CI tolerance. This information provides targets that could be used to develop strategies for preventing CI (e.g., genetic improvement of tomato, direct application of key metabolites). The application of such strategies will increase the economic value and decrease postharvest losses.

The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network.

Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.

Characterization of peptides with antioxidant activity and antidiabetic potential obtained from chickpea (Cicer arietinum L.) protein hydrolyzates.

Alcalase hydrolyzates were prepared from the albumin (AH) and globulin (GH) fractions of eight chickpea (Cicer arietinum L.) genotypes from Mexico and 10 from other countries. Protein content, antioxidant activity (AA) (ABTS, DPPH), and degree of hydrolysis were evaluated and the best genotype was selected by principal component analysis. The hydrolyzates of the chosen genotype were analyzed for its antidiabetic potential measured as inhibition of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP4). Peptide profiles were obtained by liquid chromatography-mass spectrometry (UPLC-DAD-MS), and the most active peptides were analyzed by molecular docking. The average antioxidant activity of albumin hydrolyzates was higher than that of globulin hydrolyzates. ICC3761 was the selected genotype and peptides purified from the albumin hydrolyzate showed the best antioxidant activity and antidiabetic potential (FEI, FEL, FIE, FKN, FGKG, and MEE). FEI, FEL, and FIE were in the same chromatographic peak and this mixture showed the best ABTS scavenging (78.25%) and DPP4 inhibition (IC50  = 4.20 µg/ml). MEE showed the best DPPH scavenging (47%). FGKG showed the best inhibition of α-amylase (54%) and α-glucosidase (56%) and may be a competitive inhibitor based on in silico-predicted interactions with catalytic amino acids in the active site of both enzymes. These peptides could be used as nutraceutical supplements against diseases related to oxidative stress and diabetes. PRACTICAL APPLICATION: This study showed that chickpea protein hydrolyzates are good sources of peptides with antidiabetic potential, showing high antioxidant activity and inhibition of enzymes related to carbohydrate metabolism and type 2 diabetes. These hydrolyzates could be formulated in functional foods for diabetes.

Effect of Adjuvant on Glyphosate Effectiveness, Retention, Absorption and Translocation in Lolium rigidum and Conyza canadensis.

Glyphosate retention, absorption and translocation with and without adjuvant were examined in Lolium rigidum and Conyza canadensis in greenhouse and laboratory settings to develop an understanding of the influence of the selected adjuvant on glyphosate activity. Tests on whole plants show that the dose of herbicide needed to reduce dry weight by 50% (GR50) or plant survival (LD50) decreases by mixing glyphosate and adjuvant to 22%-24% and 42%-44% for both populations of L. rigidum and C. canadensis, respectively. This improvement in efficacy could be attributed to the higher herbicide retention and lower contact angle of the glyphosate + adjuvant drops on the leaf surface compared to the glyphosate solution alone. Plants of both species treated with 14C-glyphosate + adjuvant absorbed more glyphosate compared to non-adjuvant addition. Furthermore, the movement of the herbicide through the plant was faster and greater with the adjuvant. Our results reveal that the use of adjuvants improves the effectiveness of glyphosate in two of the most important weeds in agricultural crops in Mediterranean countries.

ADP-Glucose Pyrophosphorylase Is Located in the Plastid and Cytosol in the Pulp of Tropical Banana Fruit (Musa acuminata).

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch synthesis in seeds, tubers and fruits. UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of sucrose metabolism in the cytosol while alkaline phosphatase (ALP) is a marker enzyme of the amyloplast that keeps the production of ADPG by removing PPi. Unripe banana accumulates starch in the pulp during development, while ripe fruits are characterized by the accumulation of soluble sugars. The aim of the study was to compare starch granule structure, carbohydrate levels, subcellular location and activities of three enzymes: AGPase, UGPase and ALP. Protein extracts from the cytosolic and amyloplastidial fractions were obtained from the pulp of banana fruit at three developmental stages (11, 16 and 21 weeks after flowering) and analyzed by electrophoresis and immunodetection. Protein profiles were similar during ripening, showing a main electrophoretic band at 50-55 kDa. Higher protein content was found in the cytosolic than in the amyloplastidial fraction. Starch granules and ALP activity were enriched in the amyloplast, whereas AGPase showed a subcellular distribution similar to UGPase. Immunoblot analysis also confirmed the presence of AGPase in both cytosol and amyloplast. AGPase activity was higher in the cytosol than in the amyloplast. Both AGPase activity and western blot band intensity were highest at 16 weeks. UGPase activity was highest at 21 weeks. We conclude that cytosolic production of ADP-glucose is not an exclusive feature of cereal endosperms due to plant breeding, but it also occurs in fruits of non-domesticated plants such as tropical banana (Musa acuminata). This work increases our understanding about pyrophosphorylase activities in the pulp of banana fruit.

Anthocyanins of Pithecellobium dulce (Roxb.) Benth. Fruit Associated with High Antioxidant and α-Glucosidase Inhibitory Activities.

Red arils of Pithecellobium dulce fruit, commonly known as guamuchil, show high antioxidant (AOx) and α-glucosidase inhibitory (IαG) activities, which have been mainly associated with the content of unknown anthocyanins. In this study, the AOx (i.e., DPPH and ABTS as Trolox equivalents, µmol TE/g) and IαG (as half-maximal inhibitory concentration, IC50, mg/mL) activities of the anthocyanin-rich fraction (ARF) obtained from red arils were contrasted with those of the methanol extract (ME), and the main ARF anthocyanins were characterized by HPLC-DAD-ESI-MS, GC-MS and 1H-NMR. The AOx and IαG values of the ARF (DPPH = 597.8; ABTS = 884.01; IαG = 0.06) were better than those of the ME (DPPH = 41.5; ABTS = 142.3; IαG = 17.5); remarkably, the ARF IαG value was about 42 times lower than that of acarbose. The main anthocyanins in ARF were pelargonidin 3-O-glucoside and cyanidin 3-O-glucoside. Thus, the consumption of red P. dulce arils could provide health benefits for prevention/treatment of chronic degenerative diseases such as diabetes.

Multiple Resistance Evolution in Bipyridylium-Resistant Epilobium ciliatum After Recurrent Selection.

The use of herbicides with different modes of action is the primary strategy used to control weeds possessing resistance to a single mechanism of action (MOA). However, this practice can lead to selection for generalist resistance mechanisms and may cause resistance to all MOAs. In this research, we characterized the resistance to diquat/paraquat (bipyridiliums) in an Epilobium ciliatum biotype (R1) collected in an olive orchard from Chile, where alternatives herbicides (2,4-D, glyphosate, glufosinate, flazasulfuron and pyraflufen-ethyl) with different MOAs were used, but they have also showed failure in controlling this species. Because the resistance/susceptibility patterns of the R1 biotype to glufosinate, 2,4-D and pyraflufen-ethyl were not clear, a recurrent resistance selection was carried out in field and greenhouse using these herbicides on R1 plants for three generations (R2 biotype). One biotype that was never treated with herbicides (S) was included as control. Results indicated that the S biotype was controlled at the field dose of all herbicides tested. The biotype R1 exhibited resistance to diquat, paraquat and flazasulfuron and natural tolerance to glyphosate. The R2 biotype displayed resistance to glufosinate, 2,4-D and pyraflufen-ethyl with LD50 (herbicide dose to kill 50% of plants) values higher than field doses in all assays. Physiological and biochemical studies determined the resistance to diquat of the R1 biotype, which was due to impaired translocation. The resistance to flazasulfuron in the R1 and R2 biotypes was confirmed by the low sensitivity of the acetolactate synthase (ALS) activity compared to the S biotype. The similar accumulation of shikimate in treated S, R1, and R2 plants with glyphosate supported the existence of innate tolerance to this herbicide in E. ciliatum. Resistance to glufosinate, 2,4-D and pyraflufen-ethyl in the R2 biotype, acquired after recurrent selection, was determined by low sensitivity of the glutamine synthetase, low accumulation of ethylene and protoporphyrinogen IX oxidase, respectively, in comparison to the S biotype. Epilobium ciliatum from Chilean olive orchards had resistance to only two MAOs (photosystem I and ALS inhibitors), but resistance to five MOAs could occur in the next cropping seasons, if alternatives to weed management, other than herbicides, are not included.

Phenolic profiles and their contribution to the antioxidant activity of selected chickpea genotypes from Mexico and ICRISAT collections.

Chickpea (Cicer arietinum L.) genotypes, nine kabuli from Mexico and 9 desi from other countries, were investigated for their phenolic profiles and antioxidant activity (AA). Phenolics in methanol extracts (ME) were analyzed by ultra-performance liquid chromatography coupled to diode array detection and mass spectrometry (UPLC-DAD-MS), whereas the AA was measured as Trolox equivalents (TE) by ABTS, DPPH and FRAP methods. Twenty phenolic compounds were identified in the ME and their levels showed a great variability among the chickpea genotypes. Phenolic acids and flavonoids were the most abundant compounds in kabuli and desi genotypes, respectively. The AA values (µmol TE/ 100 g dw) by ABTS (278-2417), DPPH (52-1650), and FRAP (41-1181) were mainly associated with the content of sinapic acid hexoside, gallic acid, myricetin, quercetin, catechin, and isorhamnetin, suggesting they are the main compounds responsible for the AA. The sum of the AA obtained for standards of these compounds evaluated at the concentration found in the extracts accounted for 34.3, 69.8, and 47.0% of the AA in the extract by ABTS, DPPH, and FRAP, respectively. In the AA by DPPH, most of the mixtures of these compounds resulted in synergistic interactions. Three desi genotypes with black seeds (ICC 4418, ICC 6306, and ICC 3761) showed the highest AA and flavonoids content, whereas the most promising kabuli genotypes were Surutato 77, Bco. Sin. 92, and Blanoro that showed the highest values of phenolic acids. These genotypes represent good sources of antioxidants for the improvement of nutraceutical properties in chickpea.

Pro-106-Ser mutation and EPSPS overexpression acting together simultaneously in glyphosate-resistant goosegrass (Eleusine indica).

Glyphosate has been used for more than 15 years for weed management in citrus groves in the Gulf of Mexico, at up to 3-4 applications per year. Goosegrass (Eleusine indica (L.) Gaertn.) control has sometimes failed. In this research, the mechanisms governing three goosegrass biotypes (Ein-Or from an orange grove, and Ein-Pl1 and Ein-Pl2 from Persian lime groves) with suspected resistance to glyphosate were characterized and compared to a susceptible biotype (Ein-S). Dose-response and shikimate accumulation assays confirmed resistance of the resistant (R) biotypes. There were no differences in glyphosate absorption, but the R biotypes retained up to 62-78% of the herbicide in the treated leaf at 96 h after treatment (HAT), in comparison to the Ein-S biotype (36%). The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity in the Ein-Or and Ein-S biotypes was over 100-fold lower than the Ein-Pl1 and Ein-Pl2 ones. The latter showed a high EPSPS-basal activity, a mutation at Pro-106-Ser position in the EPSPS gene, and EPSPS overexpression. The EPSPS basal and EPSPS overexpression were positively correlated. The R goosegrass biotypes displayed poor glyphosate translocation. Furthermore, this grassweed showed, for the first time, two mechanisms at the target-site level (Pro-106-Ser mutation + EPSPS overexpression) acting together simultaneously against glyphosate.

First Resistance Mechanisms Characterization in Glyphosate-Resistant Leptochloa virgata.

Leptochloa virgata (L.) P. Beauv. is an annual weed common in citrus groves in the states of Puebla and Veracruz, Mexico limiting their production. Since 2010, several L. virgata populations were identified as being resistant to glyphosate, but studies of their resistance mechanisms developed by this species have been conducted. In this work, three glyphosate-resistant populations (R8, R14, and R15) collected in citrus orchards from Mexico, were used to study their resistance mechanisms comparing them to one susceptible population (S). Dose-response and shikimic acid accumulation assays confirmed the glyphosate resistance of the three resistant populations. Higher doses of up to 720 g ae ha-1 (field dose) were needed to control by 50% plants of resistant populations. The S population absorbed between 7 and 13% more 14C-glyphosate than resistant ones, and translocated up to 32.2% of 14C-glyphosate to the roots at 96 h after treatment (HAT). The R8, R14, and R15 populations translocated only 24.5, 26.5, and 21.9%, respectively. The enzyme activity of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was not different in the S, R8 and R14 populations. The R15 Population exhibited 165.9 times greater EPSPS activity. Additionally, this population showed a higher EPSPS basal activity and a substitution in the codon 106 from Proline to Serine in the EPSPS protein sequence. EPSPS gene expression in the R15 population was similar to that of S population. In conclusion, the three resistant L. virgata populations show reduced absorption and translocation of 14C-glyphosate. Moreover, a mutation and an enhanced EPSPS basal activity at target-site level confers higher resistance to glyphosate. These results describe for the first time the glyphosate resistance mechanisms developed by resistant L. virgata populations of citrus orchards from Mexico.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico.

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world.

Healthy Ready-to-Eat Expanded Snack with High Nutritional and Antioxidant Value Produced from Whole Amarantin Transgenic Maize and Black Common Bean.

The snack foods market is currently demanding healthier products. A ready-to-eat expanded snack with high nutritional and antioxidant value was developed from a mixture (70:30) of whole amarantin transgenic maize (Zea mays L.) and black common bean (Phaseolus vulgaris L.) by optimizing the extrusion process. Extruder operation conditions were: feed moisture content (FMC, 15-25 %, wet basis), barrel temperature (BT, 120-170 °C), and screw speed (SS, 50-240). The desirability numeric method of the response surface methodology (RSM) was applied as the optimization technique over four response variables [expansion ratio (ER), bulk density (BD), hardness (H), antioxidant activity (AoxA)] to obtain maximum ER and AoxA, and minimum BD, and H values. The best combination of extrusion process variables for producing an optimized expanded snack (OES, healthy snack) were: FMC = 15 %/BT = 157 °C/SS = 238 rpm. The OES had ER = 2.86, BD = 0.119 g/cm (3) , H = 1.818 N, and AoxA = 13,681 µmol Trolox equivalent (TE)/100 g, dry weight. The extrusion conditions used to produce the OES increased the AoxA (ORAC: +18 %, ABTS:+20 %) respect to the unprocessed whole grains mixture. A 50 g portion of OES had higher protein content (7.23 vs 2.32 g), total dietary fiber (7.50 vs 1.97 g), total phenolic content (122 vs 47 mg GAE), and AoxA (6626 vs 763 µmol TE), and lower energy (169 vs 264 kcal) than an expanded commercial snack (ECS = Cheetos™). Because of its high content of quality protein, dietary fiber and phenolics, as well as high AoxA and low energy density, the OES could be used for health promotion and chronic disease prevention and as an alternative to the widely available commercial snacks with high caloric content and low nutritional/nutraceutical value.

Effectiveness of hydrothermal-calcium chloride treatment and chitosan on quality retention and microbial growth during storage of fresh-cut papaya.

Rapid degradation of fresh-cut papaya limits its marketability. Hydrothermal treatments in combination with a calcium dip, applied to whole fruit before slicing, and also the application of chitosan as a coating film, have been found to have very good results in maintaining the quality of fresh-cut fruits. Based on these considerations, the aim of this study was to evaluate the effect of hydrothermal treatment (HT; 49 °C, 25 min) containing calcium chloride (Ca; 1%, w/v) followed by dipping in chitosan (Chit; 1%, w/v, 3 min) on the physical, chemical, and microbial qualities of papaya slices stored at 5 °C for 10 d. Pulp color, firmness, ascorbic acid, total phenolics, ß-carotene, and lycopene were evaluated every 2 d while the microbial quality (mesophilics, psychrophilics, molds, and yeasts) was evaluated every 5 d. Fruit treated with HT-Ca and HT-Ca + Chit showed better color and firmness retention than Control and Chit. Papaya slices treated with HT-Ca + Chit had higher nutritional content and lower microbial growth at the end of storage. The application of the HT-Ca + Chit could be used to reduce deterioration processes, maintaining physical, chemical, and microbial qualities and increasing the shelf life of fresh-cut papaya stored at 5 °C.

Microsatellite-based genetic diversity among accessions of maize landraces from Sinaloa in México.

In the state of Sinaloa México, traditional farmers still cultivate maize accessions with a wide diversity of morphological characteristics, but the gene reservoir maintained in these populations has been poorly studied and it is being lost due to changes in land use and the adoption of hybrid commercial varieties. The aim of this study was to evaluate the genetic diversity of some of these maize populations to contribute to their preservation. Twenty eight accessions were used for the analysis. DNA was extracted from 396 individuals and probed with 20 microsatellites distributed across the maize genome. A total of 121 alleles were obtained (average of 6.1 alleles per locus) and a total genetic diversity of 0.72. The UPGMA-cluster analysis, model-based population structure and principal component analysis revealed three major groups, one formed mainly by accessions of races typical of the Northwestern lowlands (Chapalote, Dulcillo del Noroeste, Tabloncillo Perla, Blando de Sonora and Elotero de Sinaloa) and the other two with accessions mainly from Tabloncillo and Tuxpeño. The high number of alleles per locus and total genetic diversity found in this study demonstrate a broad genetic basis of the accessions of maize landraces from Sinaloa, representing a gene reservoir useful in breeding programs.

Technological properties, antioxidant activity and total phenolic and flavonoid content of pigmented chickpea (Cicer arietinum L.) cultivars.

Chickpeas are rich sources of highly nutritious protein and dietary fibre; the health benefits of consuming legumes such as antioxidant activity (AoxA) could be effective for the expansion of their food uses. The technological properties and antioxidant potential of five pigmented chickpea cultivars were evaluated. Protein content of the grains varied from 24.9 to 27.4 g/100 g sample (dw). The cooking time (CT) of the whole grains ranged from 90.5 to 218.5 min; the lowest CT corresponded to Black ICC3761 cultivar. The total phenolic content (TPC) and AoxA [oxygen radical absorbance capacity (ORAC) value] varied from 1.23 to 1.51 mg GAE/g sample (dw) and from 5011 to 5756 µmol TE/100 g sample (dw), respectively; Red ICC13124 showed the highest ORAC value. The differences in technological properties and AoxA among cultivars could be used in chickpea breeding programmes. Chickpea cultivars could contribute significantly to the management and/or prevention of degenerative diseases associated with free radical damage.

High antioxidant activity mixture of extruded whole quality protein maize and common bean flours for production of a nutraceutical beverage elaborated with a traditional Mexican formulation.

The objective of this study was to determine the best combination of extrusion process variables for the production of whole quality protein maize (EQPMF) and common bean (ECBF) flours to prepare a high antioxidant activity mixture (EQPMF + ECBF) suitable to produce a nutraceutical beverage with high acceptability elaborated with a traditional Mexican formulation. Processing conditions were obtained from a factorial combination of barrel temperature (BT = 120-170 °C) and screw speed (SS = 120-200 rpm). Response surface methodology was applied to obtain maximum values for antioxidant activity (A ( ox ) A) of the flour mixture (EQPMF + ECBF) and acceptability (A) of the nutraceutical beverage. The best combinations of extrusion process variables for EQPMF and ECBF to prepare an optimized mixture (60%EQPMF + 40%ECBF) were BT = 98 °C/SS = 218 rpm and BT = 105 °C/SS = 83 rpm, respectively. The optimized mixture had A ( ox ) A = 14,320 µmol Trolox equivalent (TE)/100 g sample dry weight (dw) and a calculated protein efficiency ratio (C-PER) of 2.17. A 200 ml portion of a beverage prepared with 25 g of the optimized flour mixture had A ( ox ) A = 3,222 µmol TE, and A = 89 (level of satisfaction "I like it extremely"). This nutraceutical beverage could be used as an alternative to beverages with low nutritional/nutraceutical value, such as those prepared with water, simple sugars, artificial flavoring and colorants, which are widely offered in the market.

Mechanism of resistance to ACCase-inhibiting herbicides in wild oat (Avena fatua ) from Latin America.

Whole-plant response of two suspected resistant Avena fatua biotypes from Chile and Mexico to ACCase-inhibiting herbicides [aryloxyphenoxypropionate (APP), cyclohexanedione (CHD), and pinoxaden (PPZ)] and the mechanism behind their resistance were studied. Both dose-response and ACCase enzyme activity assays revealed cross-resistance to the three herbicide families in the biotype from Chile. On the other hand, the wild oat biotype from Mexico exhibited resistance to the APP herbicides and cross-resistance to the CHD herbicides, but no resistance to PPZ. Differences in susceptibility between the two biotypes were unrelated to absorption, translocation, and metabolism of the herbicides. PCR generated fragments of the ACCase CT domain spanning the potential mutations sited in the resistant and susceptible biotypes were sequenced and compared. A point mutation was detected in the aspartic acid triplet at the amino acid position 2078 in the Chilean biotype and in isoleucine at the amino acid position 2041 in the Mexican wild oat biotype, which resulted in a glycine triplet and an asparagine triplet, respectively. On the basis of in vitro assays, the target enzyme (ACCase) in these resistant biotypes contains a herbicide-insensitive form. This is the first reported evidence of resistance to pinoxaden in A. fatua.

Characterization of monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the endosperm.

A quantitative trait locus has previously been identified in maize (Zea mays L.) that influences the level of free amino acids in the endosperm, especially those from the aspartate pathway: lysine, threonine, methionine, leucine, and isoleucine. Because this locus occurs in a region of the genome containing ask2, a monofunctional aspartate kinase, the nature of the monofunctional aspartate kinase genes in the parental inbreds, Oh545o2 and Oh51Ao2, was investigated. Two genes, Ask1 and Ask2 were isolated, and Ask2 was mapped to the ask2 locus. Nucleotide sequence analysis of the Ask2 alleles from Oh545o2 and Oh51Ao2 showed they differ by one amino acid. Both alleles complemented a yeast aspartate kinase mutant, hom3, and based on the growth of the yeast mutant it appeared that Ask2-Oh545o2 produces an enzyme with greater total activity than that encoded by the Oh51Ao2 allele. The results suggest that the higher level of free amino acids derived from the aspartate pathway in Oh545o2 endosperm results from a single amino acid change in the ASK2 enzyme that has pleiotropic effects on its activity.

Cytoskeletal proteins are coordinately increased in maize genotypes with high levels of eEF1A.

The opaque2 (o2) mutation increases the Lys content of maize (Zea mays) endosperm by reducing the synthesis of zein storage proteins and increasing the accumulation of other types of cellular proteins. Elongation factor 1A (eEF1A) is one of these proteins, and its concentration is highly correlated with the amount of other Lys-containing proteins in the endosperm. We investigated the basis for this relationship by comparing patterns of protein accumulation and gene expression between a high (Oh51Ao2) and a low (Oh545o2) eEF1A inbred, as well as between high and low eEF1A recombinant inbred lines obtained from their cross. The content of alpha-zein and several cytoskeletal proteins was measured in high and low eEF1A inbred lines, and the levels of these proteins were found to correlate with that of eEF1A. To extend this analysis, we used an endosperm expressed sequence tag microarray to examine steady-state levels of RNA transcripts in developing endosperm of these genotypes. We identified about 120 genes coordinately regulated in association with eEF1A content. These genes encode proteins involved in several biological structures and processes, including the actin cytoskeleton, the endoplasmic reticulum, and the protein synthesis apparatus. Thus, higher levels of eEF1A in o2 mutants may be related to a more extensive cytoskeletal network surrounding the rough endoplasmic reticulum and increased synthesis of cytoskeleton-associated proteins, all of which contribute significantly to the Lys content of the endosperm.