Confirmation and characterization of the first case of acetolactate synthase (ALS)-inhibitor resistance in Japanese brome (Bromus japonicus) in the US.

BACKGROUND: Japanese brome (Bromus japonicus Thumb.) is one of the problematic annual weeds in winter wheat (Triticum aestivum L.) and is generally controlled by acetolactate synthase (ALS) inhibitors. Repeated use of the ALS inhibitor propoxycarbazone-Na resulted in the evolution of resistance to this herbicide in three B. japonicus populations, i.e., R1, R2, and R3 in Kansas (KS). However, the level of resistance and mechanism conferring resistance in these populations is unknown. The objectives of this research were to (i) evaluate the level of resistance to propoxycarbazone-Na in R1, R2, and R3 in comparison with a known susceptible population (S1), (ii) investigate the mechanism of resistance involved in conferring ALS-inhibitor resistance, and (iii) investigate the cross-resistance to other ALS inhibitors. RESULTS: Dose-response (0 to 16x; x = 44 g ai ha-1 of propoxycarbazone-Na) assay indicated 167, 125, and 667-fold resistance in R1, R2 and R3 populations, respectively, compared to S1 population. ALS gene sequencing confirmed the mutations resulting in amino acid substitutions, i.e., Pro-197-Thr (R3, R1)/Ser (R2, R1) bestowing resistance to these ALS inhibitors. Such amino acid substitutions also showed differential cross-resistance to sulfosulfuron, mesosulfuron-methyl, pyroxsulam, and imazamox among resistant populations. Pretreatment with malathion (a cytochrome P450 enzyme-inhibitor) followed by imazamox treatment suggested cross-resistance to this herbicide possibly via metabolism only in R3 population. CONCLUSION: Overall, these results confirm the first case of target-site based resistance to ALS inhibitors in B. japonicus in the US, highlighting the need for exploring herbicides with alternative modes of action to enhance weed control in winter wheat. © 2024 Society of Chemical Industry.

Molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of the cold-resistant Patagonian grass Bromus pictus associated with fructan accumulation under low temperatures.

Fructans are fructose polymers synthesized from sucrose in the plant vacuole. They represent short- and long-term carbohydrate reserves and have been associated with abiotic stress tolerance in graminean species. We report the isolation and characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) gene from a Patagonian grass species, Bromus pictus, tolerant to drought and cold temperatures. Structural and functional analyses of this gene were performed by Southern and Northern blot. Sugar content, quality and fructosyltransferase activity were studied using HPAEC-PAD (high-pH anion-exchange chromatography with pulsed amperometric detection), enzymatic and colorimetric assays. The putative 6-SFT gene had all the conserved motifs of fructosyl-transferase and showed 90% identity at the amino acid level with other 6-SFTs from winter cereals. Expression studies, and determination of sugar content and fructosyl-transferase activity were performed on five sections of the leaf. Bp6-SFT was expressed predominantly in leaf bases, where fructosyltransferase activity and fructan content are higher. Bp6-SFT expression and accumulation of fructans showed different patterns in the evaluated leaf sections during a 7 d time course experiment under chilling treatment. The transcriptional pattern suggests that the B. pictus 6-SFT gene is highly expressed in basal leaf sections even under control temperate conditions, in contrast to previous reports in other graminean species. Low temperatures caused an increase in Bp6-SFT expression and fructan accumulation in leaf bases. This is the first study of the isolation and molecular characterization of a fructosyltransferase in a native species from the Patagonian region. Expression in heterologous systems will confirm the functionality, allowing future developments in generation of functional markers for assisted breeding or biotechnological applications.

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species Bromus pictus.

Fructans are fructose polymers synthesized in a wide range of species such as bacteria, fungi and plants. Fructans are synthesized by fructosyltransferases (FTs) and depolymerized by fructan exohydrolases (FEHs). Bromus pictus is a graminean decaploid species from the Patagonian region of Argentina, which accumulates large amounts of fructans even at temperate temperatures. The first gene isolated from B. pictus fructan metabolism was a putative sucrose:fructan 6-fructosyltransferase (6-SFT). Here, a complete cDNA of the first fructan exohydrolase (FEH) from B. pictus (Bp1-FEHa) was isolated using RT-PCR strategies. The Bp1-FEHa encoding gene is present as a single copy in B. pictus genome. Functional characterization in Pichia pastoris confirmed Bp1-FEHa is a fructan exohydrolase with predominant activity towards beta-(2-1) linkages. Its expression was analyzed in different leaf sections, showing the highest expression levels in the second section of the sheath and the tip of the blade. Bp1-FEHa expression was studied along with FEH and FT activities and fructan accumulation profile in response to chilling conditions during a 7-day time course experiment. Bp1-FEHa expression and FEH activity followed a similar pattern in response to low temperatures, especially in basal sections of the sheaths. In these sections the FEH and FT activities were particularly high and they were significantly correlated to fructan accumulation profile, along with cold treatment.

First report of Ser653Asn mutation endowing high-level resistance to imazamox in downy brome (Bromus tectorum L.).

BACKGROUND: Bromus tectorum L. is one of the most troublesome grass weed species in cropland and non-cropland areas of the northwestern USA. In summer 2016, a B. tectroum accession (R) that survived imazamox at the field-use rate (44 g ha-1 ) in an imidazolinone-tolerant (IMI-tolerant or Clearfield™) winter wheat field was collected from a wheat field in Carter County, MT, USA. The aim of this study was to determine the resistance profile of the B. tectroum R accession to imazamox and other ALS inhibitors, and investigate the mechanism of resistance to imazamox. RESULTS: The R B. tectorum accession had a high-level resistance (110.1-fold) to imazamox (IMI) and low to moderate-levels cross-resistance to pyroxsulam (TP) (4.6-fold) and propoxycarbazone (SCT) (13.9-fold). The R accession was susceptible to sulfosulfuron (SU) and quizalofop and clethodim (ACCase inhibitors), paraquat (PS I inhibitor), glyphosate (EPSPS inhibitor) and glufosinate (GS inhibitor). Sequence analysis of the ALS gene revealed a single, target-site Ser653Asn mutation in R plants. Pretreatment of malathion followed by imazamox at 44 or 88 g ha-1 did not reverse the resistance phenotype. CONCLUSION: This is the first report of evolution of cross-resistance to ALS-inhibiting herbicides in B. tectorum. A single-point mutation, Ser653Asn, was identified, conferring the high-level resistance to imazamox. © 2017 Society of Chemical Industry.

Non-target-site-based resistance to ALS-inhibiting herbicides in six Bromus rigidus populations from Western Australian cropping fields.

BACKGROUND: Bromus rigidus is a common weed species that has increased in cropping fields owing to limited control options. During a random field survey in Western Australia, six B. rigidus populations that had survived in-crop weed control programmes were collected. The study aimed to determine the resistance profile of these six populations. RESULTS: Based on dose-response studies, all six B. rigidus populations had a low-level resistance to sulfosulfuron and sulfometuron (both sulfonylurea herbicides) while remaining susceptible to herbicides with other modes of action. ALS in vitro activity assays revealed no differences in enzyme sensitivity between susceptible and resistant populations, while the use of malathion (a cytochrome P450 inhibitor) in combination with sulfosulfuron caused the resistant populations to behave like the susceptible population. CONCLUSION: This study established that these six B. rigidus populations have a low-level resistance to the ALS-inhibiting sulfonylurea herbicides, but are able to be controlled by other herbicide modes of action. The low-level, malathion-reversible resistance, together with a sensitive ALS, strongly suggest that a non-target-site enhanced metabolism is the mechanism of resistance.

Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance.

We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.

Characterization of cold-responsive extracellular chitinase in bromegrass cell cultures and its relationship to antifreeze activity.

A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.

Bromus fasciculatus presl--a third diploid progenitor of Bromus section Genea allopolyploids (Poaceae).

Allozyme variation of ten heterozymes of seven enzymes among five accessions of a rare diploid Bromus fasciculatus was analysed with the use of PAGE and compared with that for six other species of section Genea of the genus Bromus. Allozymes charasteristic for diploids B. tectorum and B. fasciculatus are combined in fixed heterozygous phenotypes of tetraploid B. rubens. Fixed heterozygous phenotypes of tetraploid B. madritensis combine one allozyme of B. fasciculatus with another of diploid B. sterilis at each of the loci studied. Of the three diploids studied, only B. sterilis fits well for a role of a genome donor for the polyploid B. diandrus-rigidus complex. Bromus fasciculatus thus appears to be a diploid ancestor for the two tetraploids of section Genea, B. rubens and B. madritensis.