Detoxification without Intoxication: Herbicide Safeners Activate Plant Defense Gene Expression

Plants are frequently exposed to natural and synthetic toxins such as heavy metals, allelochemicals, organic pollutants, and pesticides. Consequently, plants must mount specific and coordinated defense mechanisms for survival under adverse growing conditions (Zhang et al., 2007). One such mechanism is the capability for metabolizing organic compounds from abiotic origin (xenobiotics). Extensive biotransformation is part of a strategy for coping with the potentially negative impacts of xenobiotics on plant growth and development. This is particularly evident in the capability for metabolic detoxification of herbicides; plants are able to detoxify herbicides by complex multistep processes that exhibit extraordinary diversity among species (Kreuz et al., 1996). The differential ability of plant species to metabolize a particular herbicide is widely exploited in modern agriculture by the use of selective herbicides that are safe to the crop but effectively control associated weeds. Another interesting feature of the herbicide detoxification system of some monocotyledonous crop species is inducibility by certain synthetic compounds collectively named “herbicide safeners.” Safeners are a group of chemically diverse compounds with the unique ability to protect grass crops from herbicide injury without reducing herbicide activity in target weed species (Davies and Caseley, 1999; Hatzios and Burgos, 2004). Safener protection from herbicide injury is accomplished by increasing the expression of genes encoding herbicide-metabolizing enzymes, such as the glutathione S-transferases (GSTs), cytochrome P450 monooxygenases (P450s), and several others (Hatzios, 1989; Farago et al., 1994; Riechers et al., 2005). Various safener chemical classes have been developed to enhance herbicide tolerance in maize (Zea mays), grain sorghum (Sorghum bicolor), rice (Oryza sativa), and small-grain cereals (Fig. 1). However, despite the widespread agronomic use of safeners for decades and ample information about their effects on increasing the activity of detoxification enzymes, there is virtually no knowledge of the molecular mechanisms for safener induction of their corresponding genes or signaling pathways being recruited. An exception is a safener-binding protein that was identified in maize, where its binding activity was characterized in maize seedlings (Walton and Casida, 1995) along with its gene expression patterns (Scott-Craig et al., 1998), although the precise role of this protein in the safener-mediated signaling pathway has yet to be established. Chemical structures of several herbicide safeners and their uses in arable crops. A, Flurazole (seed applied for grain sorghum). B, Cloquintocet-mexyl (foliar applied in small-grain cereals). C, Dichlormid (soil applied for maize). D, Benoxacor (soil applied for maize). E, Fenclorim (soil applied for rice). Safeners induce the expression of genes involved in plant defense and detoxification, such as GSTs and P450s, yet they are not toxic to the target plant and confer protection from herbicide injury. This suggests that safeners are tapping into an unidentified, preexisting signaling pathway for detoxification of endogenous toxins or xenobiotics (Riechers et al., 2005). A new hypothesis resulting from our most recent research is that safeners may be utilizing an oxidized lipid-mediated (oxylipins; Mosblech et al., 2009) or cyclopentenone-mediated signaling pathway, which subsequently leads to the expression of GSTs and other proteins involved in detoxification and plant defense (Weber, 2002; Loeffler et al., 2005; Mueller et al., 2008; Mueller and Berger, 2009). The goal of this Update on detoxification is to briefly review herbicide safener mechanism of action in light of the many recent findings related to oxylipins and their roles in signaling, induction of defense genes, and activation of detoxification responses in plants (Mueller et al., 2008; Mueller and Berger, 2009). The metabolism of herbicides in plants generally requires the sequential action of several enzymes (Kreuz et al., 1996; Bounds and Hutson, 2000). Metabolic reactions and enzymes most commonly involved are oxidations by P450s and hydrolyses by carboxylesterases (phase I reactions); conjugations to endogenous molecules such as glutathione (GSH) or Glc, catalyzed by GSTs or UDP-dependent glycosyltransferases (UGTs), respectively (phase II); transport of conjugates into the vacuole by carriers of the multidrug resistance-associated protein (MRP) family or by other transport mechanisms (phase III); and processing of conjugates involving reactions such as partial degradation, secondary conjugations, and incorporation as “bound residue” into cell wall constituents (phase IV). Among the less frequently observed biotransformations of herbicides are, to name but a few, oxidations at nitrogen or sulfur atoms; reductions of nitro groups, aldehydes, ketones, and α,β-unsaturated carbonyl compounds; and isomerizations (Bounds and Hutson, 2000). The enzymes involved in these less common reactions in plants have not always been identified. The initial step in the metabolism of a particular herbicide may not always lead to a complete detoxification or can even constitute an activation step (e.g. hydrolysis of an esterified “proherbicide” precursor). However, the formation of herbicide conjugates with GSH or Glc fundamentally alters their physicochemical properties, rendering the compounds more polar and unable to freely diffuse across cellular compartments or translocate throughout the symplast. Furthermore, such conjugates are usually amenable to further metabolism and can be accepted by energized carriers for their transport out of the cytosol. Plants thus possess an integrated multistep system for the detoxification of potentially harmful compounds such as herbicides (Kreuz et al., 1996) in addition to systems that enable plants to cope with the consequences of xenobiotic-induced stresses, such as antioxidative and radical-scavenging components. A unique and intriguing feature of safeners is their apparent ability to coordinately induce a whole array of critical components that constitute herbicide detoxification pathways in plants (Zhang et al., 2007). These aspects of safener action have attracted considerable interest from both industry and academic researchers since the discovery of the first herbicide safeners approximately 50 years ago (Hatzios, 1989; Davies and Caseley, 1999). One of the most important reactions for herbicide metabolism is catalyzed by P450 enzymes, which typically introduce a reactive functional group suitable for further metabolism and detoxification. The most common P450-mediated reactions are hydroxylations of aromatic rings or alkyl groups and dealkylations resulting from the formal hydroxylation of a carbon adjacent to an oxygen or nitrogen atom (Kreuz et al., 1996; Barrett, 2000). Plant P450 proteins are encoded by very large and diverse multigene families consisting of approximately 350 genes in rice and 250 genes in Arabidopsis (Arabidopsis thaliana), whose protein are involved in processes such as secondary and et al., 2004). The metabolism of herbicides from several important chemical classes is by P450 to crop et al., and the the and roles of P450 and herbicide in rice and maize are more et al., et al., et al., is ample from in metabolism and with that P450 are by However, for expression of specific P450s at the is et al., and information is at the or protein The of I or P450-mediated reactions to the corresponding or or Glc The for molecules are encoded in plant by large multigene with in Arabidopsis et al., et al., 2009). of this family roles in plant secondary and detoxification of toxins by are some that safeners enhance the of herbicides in plants (Kreuz et al., Hatzios and Burgos, 2004). the safener (Fig. which the of in the metabolism of the herbicide (Kreuz et al., has been to the metabolic of endogenous in by increasing the activity of a and an et al., The group of plant enzymes involved in herbicide metabolism is the which the of the cellular to an of The mechanism an addition of the GSH to the with or of a group (e.g. a or from the Plant GSTs a large and diverse with genes encoded by the Arabidopsis and have been on and into several et al., with the and classes being most in plants et al., et al., GSTs are proteins that are in the under conditions but have diverse as in et al., 2009). proteins have been to the of to safeners in (Riechers et al., addition to their activity as involved in several diverse endogenous roles for plant GSTs have been as in and as in GSH and transport of reactive and as GSH et al., and et al., The activity is of particular for the of certain GSTs the of to the corresponding their to GSTs of the and classes GSH activity et al., 2009). Plant GSTs to a of heavy metals, in which of reactive oxygen species is a common et al., et al., 2009). recent have GSTs as components of cell signaling pathways and as of et al., The first for safener of expression at the was in a with the safener on the induction of a in maize et al., addition to safeners enhance the activity of enzymes involved in and or GSH an of GSH et al., which is in with the for GSH as a for detoxification of and gene expression is on in Arabidopsis et al., 2005; et al., 2005; Mueller et al., safener of gene expression is on by the induction of activity in several plant species (Riechers et al., et al., et al., and et al., 2005; et al., 2007). genes or proteins are for A, protein and activity of and with was by Mueller et of and by the safeners and in maize seedlings et al., activity of and and activity of was by Mueller et GSTs GSH activity et al., of and gene expression is on in Arabidopsis et al., 2005; et al., 2005; Mueller et al., safener of gene expression is on by the induction of activity in several plant species (Riechers et al., et al., et al., and et al., 2005; et al., 2007). genes or proteins are for A, protein and activity of and with was by Mueller et of and by the safeners and in maize seedlings et al., activity of and and activity of was by Mueller et GSTs GSH activity et al., have that and conjugates are into the vacuole of plant by and that safeners enhance this transport The first for the of activity in plants was the that the GSH of the herbicide and other compounds was into by in the et al., genes to the a of the have been identified in many plant species et al., the of Arabidopsis and rice and genes, However, plant have been with to their and transport The mechanism for conjugates in the vacuole is not and may on the particular species and chemical of the et al., is a mechanism into the detoxification of the herbicide is into an transport mechanism et al., with the safener (Fig. the activity of a for or conjugates in from et al., in encoding GSTs to and a by et al., and in to in (Zhang et al., 2007). The functional for the of conjugates from the by may be to protect enzymes from et al., or to of of conjugates in the et al., into the processing of GSH conjugates is by the action of enzymes such as and to their corresponding conjugates et al., 1996; et al., 2007). in Arabidopsis the of processing pathways to in the vacuole and in the et al., the resulting conjugates are usually further by secondary to or by the action of and activity to the (Kreuz et al., 1996; Bounds and Hutson, 2000). However, the of safeners on these or the functional in herbicide tolerance in crops has been is a large of that safeners enhance the metabolic of P450s, and involved in the processes for herbicides has been that safeners are in plants by the systems as herbicides and induce their metabolism as as the metabolism of other many safeners as and GSH conjugates in plants et al., 1989; et al., 1996; et al., and conjugates of the safener identified in maize cell et al., and of GSH of the rice safener in Arabidopsis cell et al., that these safeners are with has been that the of is the GSH pathway in Arabidopsis and that this activity to rice and Arabidopsis cell et al., a to rice plants injury from the herbicide This activity for a safener was to be the of a of activity an step et al., with these effectively activity in seedlings (Riechers et al., but is not GSH in or in these on safener metabolism with plants or cell not to the or components of the signaling pathway involved in safener mechanism of although of GSH of the of of the detoxification pathway et al., 2008; The of safener effects on the expression and activity of diverse proteins involved in detoxification for integrated and in a in with safener and metabolism to the mechanism of action and signaling pathways by safener-mediated signaling pathways for the of defense genes and activation of detoxification pathways in Safeners may into a signaling pathway (e.g. and and Mueller and Berger, an signaling pathway et al., a signaling pathway and et al., to induce the expression of proteins involved in herbicide detoxification in is that safeners may an signaling pathway to plant defense gene but signaling and signaling pathways for the of plant gene that the that a of enzymes can metabolize herbicides or safeners as to detoxification and herbicide tolerance in crops. for of this The first safeners into developed for herbicides of the and classes for maize, grain and rice (Hatzios, These herbicides are in plants GSH et that (Fig. and related safeners of the the of both GSH and activity the herbicide in maize and are typically applied with their safeners to the at or in maize or grain The of herbicide is the the most are the and where of very are et al., 2004). Benoxacor (Fig. a safener developed for the herbicide in maize, metabolism of in maize (Kreuz et al., A was the safener to the tolerance of maize seedlings to the herbicide et al., and was at the and activity in the (Riechers et al., the chemically safeners and the of protein in the cell of the (Riechers et al., a safeners for and their on herbicide metabolism by GSTs in the that detoxify the herbicide and from the new as the from the (Kreuz et al., 1989; et al., Riechers et al., these on the role of GSTs in herbicide metabolism and crop tolerance to herbicides have that the expression of many and is or and that genes to However, is important to that many GSTs to a of in Arabidopsis et al., 2009) and and abiotic tolerance in and plants et al., without from herbicide injury in Arabidopsis to safener by induction of specific and and yet Arabidopsis and other are not from herbicide injury by safeners et al., 2002; and that some other mechanism or of the detoxification pathway must be in safener protection in crops. The pathway in which leads to the of in both and is a critical signaling pathway involved in plant responses to a of as or and abiotic as and (Fig. and related compounds defense responses to and and the of many other plant processes and and 2009). the expression of genes and plant and responses and signaling pathways and and is a of with the of from activity and to in reactions catalyzed by and and 2009). The of the pathway, is from by activity by of can be further to and conjugates that may have and has been as the of the pathway and as the in plant signaling However, has that activity may among the and conjugates as as its and 2009). other and and the as as and may possess and unique of cell as as the expression of plant defense and detoxification genes (Weber, 2002; Mueller and Berger, and 2009). has that oxidized and can to a diverse array of signaling compounds or et al., and 2009). of their diverse structures and these signaling molecules effects on gene expression patterns yet some in their of gene expression et al., 2005; et al., 2005; Mueller et al., 2008; Mueller and Berger, 2009). are diverse from and can be or oxylipins are reactions in or cell where as for their and of and and et al., 2009). These reactive oxylipins have been as for and in plant and systems (Mueller and Berger, 2009). oxylipins its and which are part of the pathway in this pathway and its enzymes have been to the activity of and defense gene activation in to et al., and 2009). not in their and structures but in their reactive species the and and the and and 2007). of their origin or for the of oxylipins possess as signaling molecules in to (Mueller and Berger, 2009). oxylipins several common properties, a that in binding to or of to an and the ability to proteins and induce genes and enzymes involved in detoxification (Mueller and Berger, 2009). Safeners are typically may with cellular such as and are of detoxification enzymes in plants (Zhang et al., 2007). However, several chemical classes of safeners have been developed for a across these classes is not apparent (Fig. 1). Furthermore, information on for safener action in plants is and that under conditions with GSH of et al., 1989; et al., 1996; et al., safeners be to a reactive by a metabolic step in the the formation of and in to is the of oxidized or oxylipins (Mueller and Berger, 2009). oxylipins and a induction of genes involved in secondary metabolism as as detoxification and responses and defense genes (Mueller and Berger, of secondary is not specific to oxylipins (Mueller and Berger, since and oxylipins the of secondary and defense or This in a reactive in to the of and cellular However, safeners and oxylipins have a and unique on the coordinated induction of detoxification genes and enzymes genes and enzymes P450s, (e.g. GSH and of which are to in the sequential for cellular detoxification of xenobiotics (Kreuz et al., 1996; et al., 2005). addition to the proteins safeners the expression of an involved in and GSH et al., 2007). enzymes that the (Mueller et al., and GSTs that GSH to the of (Mueller et al., 2008; and Safeners coordinately induce and several proteins (Zhang et al., which GSH to many herbicide (Kreuz et al., 1996) and to safeners as et al., 1989; et al., 1996; et al., These findings a induction and plant defense and detoxification mechanisms (Fig. However, many herbicides are for GSTs but are not of GSTs or other detoxification enzymes (Zhang et al., they their effects on herbicides have a specific target or a protein that to or with the to the binding of the protein in and to the of and 2000). with safeners are not to with a specific target that leads to cellular or yet they are by the defense signaling system and effectively genes and enzymes involved in detoxification (Zhang et al., 2007). gene expression are observed or with or in yet some of genes are observed as Mueller and Berger, 2009). One interesting to further is or abiotic can be with these unique gene expression may be that or effects on defense gene expression and the resulting plant tolerance or of are observed in to of diverse oxylipins most or to have on a specific or of signaling in to a has been that the diversity and of oxylipins have been in and that a array of oxylipins with may to defense gene expression and the resulting responses (Mueller and Berger, 2009). Safeners and oxylipins have a common activity in that they both induce the expression of defense genes and detoxification responses in plants (Zhang et al., Mueller and Berger, 2009). is that safeners induce the expression of enzymes involved in an detoxification pathway et al., 1996; et al., et al., 2007). with this a that the from the expression of genes involved in plant defense and detoxification several P450s, and et al., 2005; These that and other oxylipins as may plant defense and detoxification responses in a to of with the from to et al., to the safener in crops from herbicide injury (Zhang et al., 2007). with this has been in the that induction of detoxification genes to be most by oxylipins (Mueller and Berger, 2009) and that this coordinated detoxification in Arabidopsis the sequential and pathway for metabolism in plants (Kreuz et al., 1996; et al., et al., 2009). these findings the hypothesis that safeners may be tapping into a signaling pathway, which subsequently the expression of proteins involved in a detoxification pathway in plants addition to the and of to be involved in the signaling for activation of detoxification responses in plants et al., 2008; Mueller et al., 2008; Mueller and Berger, with xenobiotics or oxylipins in the activation of and genes that activation in their et al., 1996; et al., These are by of the protein family et al., coordinated induction of detoxification and plant defense genes in to several diverse or may be a common in the et al., 1996; et al., 2008; Mueller et al., 2008; et al., The coordinated induction of genes involved in plant defense and secondary in particular the P450 gene family in has been in to diverse abiotic and et al., et al., 2004). A mechanism for the coordinated induction of detoxification enzymes, which the pathway, has been and characterized in systems et al., 2009). the is to and in the by a to of that with the in the several on and in the is thus protein by from et al., 2009). The proteins with of I and detoxification genes such as GSTs or to their resulting in protection et al., 2009). is that safeners may a signaling mechanism to the activation system in (Fig. signaling may in or in with signaling pathways and Mueller and Berger, 2009) to coordinately the expression of enzymes involved in herbicide metabolism and detoxification (Zhang et al., on in their expression and since most safeners (Fig. 1). of this the of the gene an et al., which may to the of genes in systems et al., 2009). Another interesting is that proteins to the group of that constitute the plant that are involved in signaling of GSTs and in Arabidopsis (Mueller et al., 2008; Mueller and Berger, 2009). is the and molecular that initial safener and the (e.g. activity and herbicide A mechanisms for signaling have been in the Safeners may the activity or of that with in the of plant defense genes and the expression of genes encoding enzymes involved in herbicide detoxification (Hatzios, 1989; Davies and Caseley, 1999; Hatzios and Burgos, 2004). research and to the molecular components of the herbicide detoxification pathway in (Zhang et al., 2007). A was that by safener and in of to a group of as part of a multigene family common to plants et al., they are for their in and responses to and abiotic and 2009). plant genes are by a of and et al., et al., 2002; et al., for and 2009). proteins (Zhang et al., and and are by safener in Consequently, is that safeners may be tapping into a signaling pathway et al., 2005; et al., as by the induction of expression (Fig. which subsequently leads to the expression of GSTs and other proteins involved in detoxification and plant defense in a (Zhang et al., 2007). the to that was by which was further by et al., 2004). Arabidopsis is interesting that many of the genes in I that are by oxylipins and safeners are by with the the gene encoding et al., 2005). as and in to in a et al., and in their et al., et al., the of the may be particularly important for of the pathway, as the of these classes of signaling molecules and 2009). of in to may have from activity on esterified or from et al., by in this safeners expression as et al., et al., are safeners the of to the induction of and proteins that and of the (Mueller et al., this a be in maize, or rice seedlings in to safener to of and et al., Safeners are an intriguing group of that possess an molecular mechanism for crops from herbicide injury. Safeners induce the expression of plant defense and detoxification genes, such as P450s, and yet they are not toxic to is that safeners into preexisting signaling pathways for detoxification, a signaling pathway (Mueller and Berger, which subsequently leads to the expression of GSTs and other proteins involved in herbicide detoxification and defense responses (Fig. unique of safeners crop and plant are along with several of research that further and information safener mechanism of safeners not herbicide tolerance in plants such as Arabidopsis et al., 2002; and or 1989; Davies and Caseley, may be to to the safener from to crops. not to safeners at the despite in and expression et al., 2002; and for the of to safeners herbicide of herbicide-metabolizing with of the expression of enzymes, a in some other of the herbicide detoxification pathway, or some of these components. the safener be into this new weed by the of herbicide crops and target weed species as as new chemical control for weeds. knowledge of critical in the or of genes encoding detoxification enzymes, or a of gene expression is by lead to the precise of expression in gene be to introduce into important crops such as maize, grain and et al., or for in the such as or This new be from the of new gene expression systems for plant the of genes by into plants and chemically gene expression safener to the of a plant researchers to the resulting at or growth and One of interest and to safener-mediated signaling is to safeners an in of specific oxylipins et al., which be accomplished Safeners plant secondary metabolism in and the grass weed et al., as a in addition to into signaling is important to specific or precise the use of safeners be for expression in plants et al., Another important of safener research that is is to the metabolic of safeners in crops. is there a common among diverse safeners (Fig. that is important in activity in other being a and or formation important et al., as by the with et al., further P450 metabolism or other I for activation and further and induction by safeners (Fig. with a specific P450 such as the induction of GSTs by and molecular aspects of safener mechanism of action to the of cellular detoxification is important to and patterns of gene expression et al., 2002; Riechers et al., et al., the of the encoded detoxification proteins and to diverse (Riechers et al., and further of detoxification proteins a role in herbicide metabolism (Zhang and et al., patterns of gene expression with herbicides by et al., in to safeners or oxylipins be from the of the of plant responses to a of detoxification, in with endogenous signaling a molecular be of interest to of gene expression in to at the or a the cellular and metabolism of and conjugates further in