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Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Evolution of resistance to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we established the molecular basis of target site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, 1-3, and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than that of EiGS2. A novel point mutation resulting in a Ser59Gly substitution in EiGS1-1 was identified in glufosinate-resistant plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified mutant EiGS1-1 expressed in yeast was more tolerant to glufosinate than the wild-type variant. These transgenic results correlate with a more glufosinate-resistant GS in the crude tissue extract of resistant versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that Ser59 is not directly involved in glufosinate binding but is in contact with some important binding residues (e.g. Glu297) and especially with Asp56 that forms an intratoroidal contact interface. Importantly, the same Ser59Gly mutation was also found in geographically isolated glufosinate-resistant populations from Malaysia and China, suggesting parallel evolution of this resistance mutation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background and Aims: Resistance to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in wild radish (Raphanus raphanistrum) appears to be due to a complex, multifaceted mechanism possibly involving enhanced constitutive plant defence and alterations in auxin signalling. Based on a previous gene expression analysis highlighting the plasma membrane as being important for 2,4-D resistance, this study aimed to identify the components of the leaf plasma membrane proteome that contribute to resistance., Methods: Isobaric tagging of peptides was used to compare the plasma membrane proteomes of a 2,4-D-susceptible and a 2,4-D-resistant wild radish population under control and 2,4-D-treated conditions. Eight differentially abundant proteins were then targeted for quantification in the plasma membranes of 13 wild radish populations (two susceptible, 11 resistant) using multiple reaction monitoring., Key Results: Two receptor-like kinases of unknown function (L-type lectin domain-containing receptor kinase IV.1-like and At1g51820-like) and the ATP-binding cassette transporter ABCB19, an auxin efflux transporter, were identified as being associated with auxinic herbicide resistance. The variability between wild radish populations suggests that the relative contributions of these candidates are different in the different populations., Conclusions: To date, no receptor-like kinases have been reported to play a role in 2,4-D resistance. The lectin-domain-containing kinase may be involved in perception of 2,4-D at the plasma membrane, but its ability to bind 2,4-D and the identity of its signalling partner(s) need to be confirmed experimentally. ABCB19 is known to export auxinic compounds, but its role in 2,4-D resistance in wild radish appears to be relatively minor., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Aims: Resistance to synthetic auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) is increasing in weed populations worldwide, which is of concern given the recent introduction of synthetic auxin-resistant transgenic crops. Due to the complex mode of action of the auxinic herbicides, the mechanisms of evolved resistance remain largely uncharacterized. The aims of this study were to assess the level of diversity in resistance mechanisms in 11 populations of the problem weed Raphanus raphanistrum, and to use a high-throughput, whole-genome transcriptomic analysis on one resistant and one susceptible population to identify important changes in gene expression in response to 2,4-D., Methods: Levels of 2,4-D and dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance were quantified in a dose-response study and the populations were further screened for auxin selectivity, 2,4-D translocation and metabolism, expression of key 2,4-D-responsive genes and activation of the mitogen-activated proein kinase (MAPK) pathway. Potential links between resistance levels and mechanisms were assessed using correlation analysis., Key Results: The transcriptomic study revealed early deployment of the plant defence response in the 2,4-D-treated resistant population, and there was a corresponding positive relationship between auxinic herbicide resistance and constitutive MAPK phosphorylation across all populations. Populations with shoot-wide translocation of 2,4-D had similar resistance levels to those with restricted translocation, suggesting that reduced translocation may not be as strong a resistance mechanism as originally thought. Differences in auxin selectivity between populations point to the likelihood of different resistance-conferring alterations in auxin signalling and/or perception in the different populations., Conclusions: 2,4-D resistance in wild radish appears to result from subtly different auxin signalling alterations in different populations, supplemented by an enhanced defence response and, in some cases, reduced 2,4-D translocation. This study highlights the dangers of applying knowledge generated from a few populations of a weed species to the species as a whole.

Resistance to auxinic herbicides is increasing in a range of dicotyledonous weed species, but in most cases the biochemical mechanism of resistance is unknown. Using (14)C-labelled herbicide, the mechanism of resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) in two wild radish (Raphanus raphanistrum L.) populations was identified as an inability to translocate 2,4-D out of the treated leaf. Although 2,4-D was metabolized in wild radish, and in a different manner to the well-characterized crop species wheat and bean, there was no difference in metabolism between the susceptible and resistant populations. Reduced translocation of 2,4-D in the latter was also not due to sequestration of the herbicide, or to reduced uptake by the leaf epidermis or mesophyll cells. Application of auxin efflux or ABCB transporter inhibitors to 2,4-D-susceptible plants caused a mimicking of the reduced-translocation resistance phenotype, suggesting that 2,4-D resistance in the populations under investigation could be due to an alteration in the activity of a plasma membrane ABCB-type auxin transporter responsible for facilitating long-distance transport of 2,4-D., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background and Aims: Dormancy in Lolium rigidum (annual ryegrass) seeds can be alleviated by warm stratification in the dark or by application of fluridone, an inhibitor of plant abscisic acid (ABA) biosynthesis via phytoene desaturase. However, germination and absolute ABA concentration are not particularly strongly correlated. The aim of this study was to determine if cytokinins of both plant and bacterial origin are involved in mediating dormancy status and in the response to fluridone., Methods: Seeds with normal or greatly decreased (by dry heat pre-treatment) bacterial populations were stratified in the light or dark and in the presence or absence of fluridone in order to modify their dormancy status. Germination was assessed and seed cytokinin concentration and composition were measured in embryo-containing or embryo-free seed portions., Key Results: Seeds lacking bacteria were no longer able to lose dormancy in the dark unless supplied with exogenous gibberellin or fluridone. Although these seeds showed a dramatic switch from active cytokinin free bases to O-glucosylated storage forms, the concentrations of individual cytokinin species were only weakly correlated to dormancy status. However, cytokinins of apparently bacterial origin were affected by fluridone and light treatment of the seeds., Conclusions: It is probable that resident microflora contribute to dormancy status in L. rigidum seeds via a complex interaction between hormones of both plant and bacterial origin. This interaction needs to be taken into account in studies on endogenous seed hormones or the response of seeds to plant growth regulators., (© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background and Aims: α-Amylase in grass caryopses (seeds) is usually expressed upon commencement of germination and is rarely seen in dry, mature seeds. A heat-stable α-amylase activity was unexpectedly selected for expression in dry annual ryegrass (Lolium rigidum) seeds during targeted selection for low primary dormancy. The aim of this study was to characterize this constitutive activity biochemically and determine if its presence conferred insensitivity to the germination inhibitors abscisic acid and benzoxazolinone., Methods: α-Amylase activity in developing, mature and germinating seeds from the selected (low-dormancy) and a field-collected (dormant) population was characterized by native activity PAGE. The response of seed germination and α-amylase activity to abscisic acid and benzoxazolinone was assessed. Using an alginate affinity matrix, α-amylase was purified from dry and germinating seeds for analysis of its enzymatic properties., Key Results: The constitutive α-amylase activity appeared late during seed development and was mainly localized in the aleurone; in germinating seeds, this activity was responsive to both glucose and gibberellin. It migrated differently on native PAGE compared with the major activities in germinating seeds of the dormant population, but the enzymatic properties of α-amylase purified from the low-dormancy and dormant seeds were largely indistinguishable. Seed imbibition on benzoxazolinone had little effect on the low-dormancy seeds but greatly inhibited germination and α-amylase activity in the dormant population., Conclusions: The constitutive α-amylase activity in annual ryegrass seeds selected for low dormancy is electrophoretically different from that in germinating seeds and its presence confers insensitivity to benzoxazolinone. The concurrent selection of low dormancy and constitutive α-amylase activity may help to enhance seedling establishment under competitive conditions.

Background and Aims: Karrikinolide (KAR(1)) is a smoke-derived chemical that can trigger seeds to germinate. A potential application for KAR(1) is for synchronizing the germination of weed seeds, thereby enhancing the efficiency of weed control efforts. Yet not all species germinate readily with KAR(1), and it is not known whether seemingly non-responsive species can be induced to respond. Here a major agronomic weed family, the Brassicaceae, is used to test the hypothesis that a stimulatory response to KAR(1) may be present in physiologically dormant seeds but may not be expressed under all circumstances., Methods: Seeds of eight Brassicaceae weed species (Brassica tournefortii, Raphanus raphanistrum, Sisymbrium orientale, S. erysimoides, Rapistrum rugosum, Lepidium africanum, Heliophila pusilla and Carrichtera annua) were tested for their response to 1 µm KAR(1) when freshly collected and following simulated and natural dormancy alleviation, which included wet-dry cycling, dry after-ripening, cold and warm stratification and a 2 year seed burial trial., Key Results: Seven of the eight Brassicaceae species tested were stimulated to germinate with KAR(1) when the seeds were fresh, and the remaining species became responsive to KAR(1) following wet-dry cycling and dry after-ripening. Light influenced the germination response of seeds to KAR(1), with the majority of species germinating better in darkness. Germination with and without KAR(1) fluctuated seasonally throughout the seed burial trial., Conclusions: KAR(1) responses are more complex than simply stating whether a species is responsive or non-responsive; light and temperature conditions, dormancy state and seed lot all influence the sensitivity of seeds to KAR(1), and a response to KAR(1) can be induced. Three response types for generalizing KAR(1) responses are proposed, namely inherent, inducible and undetected. Given that responses to KAR(1) were either inherent or inducible in all 15 seed lots included in this study, the Brassicaceae may be an ideal target for future application of KAR(1) in weed management.

Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0-42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development.

Twenty-two amino acid substitutions at seven conserved amino acid residues in the acetohydroxyacid synthase (AHAS) gene have been identified to date that confer target-site resistance to AHAS-inhibiting herbicides in biotypes of field-evolved resistant weed species. However, the effect of resistance mutations on AHAS functionality and plant growth has been investigated for only a very few mutations. This research investigates the effect of various AHAS resistance mutations in Lolium rigidum on AHAS functionality and plant growth. The enzyme kinetics of AHAS from five purified L. rigidum populations, each homozygous for the resistance mutations Pro-197-Ala, Pro-197-Arg, Pro-197-Gln, Pro-197-Ser or Trp-574-Leu, were characterized and the pleiotropic effect of three mutations on plant growth was assessed via relative growth rate analysis. All these resistance mutations endowed a herbicide-resistant AHAS and most resulted in higher extractable AHAS activity, with no-to-minor changes in AHAS kinetics. The Pro-197-Arg mutation slightly (but significantly) increased the K(m) for pyruvate and remarkably increased sensitivity to feedback inhibition by branched chain amino acids. Whereas the Pro-197-Ser and Trp-574-Leu mutations exhibited no significant effects on plant growth, the Pro-197-Arg mutation resulted in lower growth rates. It is clear that, at least in L. rigidum, these five AHAS resistance mutations have no major impact on AHAS functionality and hence probably no plant resistance costs. These results, in part, explain why so many Pro-197 AHAS resistance mutations in AHAS have evolved and why the Pro-197-Ser and the Trp-574-Leu AHAS resistance mutations are frequently found in many weed species.

Background and Aims: The smoke-derived compound karrikinolide (KAR(1)) shows significant potential as a trigger for the synchronous germination of seeds in a variety of plant-management contexts, from weed seeds in paddocks, to native seeds when restoring degraded lands. Understanding how KAR(1) interacts with seed physiology is a necessary precursor to the development of the compound as an efficient and effective management tool. This study tested the ability of KAR(1) to stimulate germination of seeds of the global agronomic weed Brassica tournefortii, at different hydration states, to gain insight into how the timing of KAR(1) applications in the field should be managed relative to rain events., Methods: Seeds of B. tournefortii were brought to five different hydration states [equilibrated at 15 % relative humidity (RH), 47 % RH, 96 % RH, fully imbibed, or re-dried to 15 % RH following maximum imbibition] then exposed to 1 nm or 1 microm KAR(1) for one of five durations (3 min, 1 h, 24 h, 14 d or no exposure)., Key Results: Dry seeds with no history of imbibition were the most sensitive to KAR(1); sensitivity was lower in seeds that were fully imbibed or fully imbibed then re-dried. In addition, reduced sensitivity to KAR(1) was associated with an increased sensitivity to exogenously applied abscisic acid (ABA)., Conclusions: Seed water content and history of imbibition were found to significantly influence whether seeds germinate in response to KAR(1). To optimize the germination response of seeds, KAR(1) should be applied to dry seeds, when sensitivity to ABA is minimized.

Dormancy release in imbibed annual ryegrass (Lolium rigidum Gaud.) seeds is promoted in the dark but inhibited in the light. The role of abscisic acid (ABA) in inhibition of dormancy release was found to be negligible, compared with its subsequent effect on germination of dormant and non-dormant seeds. Inhibitors of ABA metabolism had the expected effects on seed germination but did not influence ABA concentration, suggesting that they act upon other (unknown) factors regulating dormancy. Although gibberellin (GA) synthesis was required for germination, the influence of exogenous GA on both germination and dormancy release was minor or non-existent. Embryo ABA concentration was the same following treatments to promote (dark stratification) and inhibit (light stratification) dormancy release; exogenous ABA had no effect on this process. However, the sensitivity of dark-stratified seeds to ABA supplied during germination was lower than that of light-stratified seeds. Therefore, although ABA definitely plays a role in the germination of annual ryegrass seeds, it is not the major factor mediating inhibition of dormancy release in imbibed seeds.