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Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred Functional Trichotomy

Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred... Topical Review Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred 1[OPEN] Functional Trichotomy a,2,3 b Matthias Erb, and Daniel J. Kliebenstein Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland Department of Plant Sciences, University of California, Davis, California 95616 ORCID IDs: 0000-0002-4446-9834 (M.E.); 0000-0001-5759-3175 (D.J.K.) The plant kingdom produces hundreds of thousands of low molecular weight organic compounds. Based on the assumed functions of these compounds, the research community has classified them into three overarching groups: primary metabolites, which are directly required for plant growth; secondary (or specialized) metabolites, which mediate plant–environment interactions; and hormones, which regulate organismal processes and metabolism. For decades, this functional trichotomy of plant metabolism has shaped theory and experimentation in plant biology. However, exact biochemical boundaries between these different metabolite classes were never fully established. A new wave of genetic and chemical studies now further blurs these boundaries by demonstrating that secondary metabolites are multifunctional; they can function as potent regulators of plant growth and defense as well as primary metabolites sensu lato. Several adaptive scenarios may have favored this functional diversity for secondary metabolites, including signaling robustness and cost-effective storage and recycling. Secondary metabolite multifunctionality can provide new explanations for ontogenetic patterns of defense production and can refine our understanding of plant–herbivore interactions, in particular by accounting for the discovery that adapted herbivores misuse plant secondary metabolites for multiple purposes, some of which mirror their functions in plants. In conclusion, recent work unveils the limits of our current functional classification system for plant metabolites. Viewing secondary metabolites as integrated components of metabolic networks that are dynamically shaped by environmental selection pressures and transcend multiple trophic levels can improve our understanding of plant metabolism and plant–environment interactions. Plants can use simple, inorganic precursors to syn- required for the growth and development of plants thesize a large diversity of low M organic compounds. (Fernie and Pichersky, 2015). Secondary metabolites, This synthetic capacity helps plants to colonize diverse including major groups such as phenolics, terpenes, and challenging environments. Low M organic com- pounds are commonly separated by perspective func- tion into primary metabolites, secondary metabolites (also called specialized metabolites or natural pro- ducts), and plant hormones (Fig. 1; Taiz et al., 2015). Primary metabolites are highly conserved and directly This work was supported by the University of Bern, Swiss National Science Foundation (grant no. 155781 to M.E.), the European Research Council under the European Union’s Horizon 2020 Research and In- novation Program (grant no. ERC–2016–STG 714239 to M.E.), the National Science Foundation Division of Integrative Organismal Sys- tems (award no. 1655810), theNational Science Foundation Division of Molecular and Cellular Biosciences (grant no. 1906486 to D.J.K.); the National Institute of Food and Agriculture (hatch project no. CA–D–PLS–7033–H to D.J.K.), and the Danish National Research Foundation (grant no. DNRF99 to D.J.K.). Senior author. Author for contact: matthias.erb@ips.unibe.ch. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Matthias Erb (matthias.erb@ips.unibe.ch). M.E. and D.J.K. developed and wrote the paper. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.20.00433 Plant Physiology , September 2020, Vol. 184, pp. 39–52, www.plantphysiol.org  2020 American Society of Plant Biologists. All Rights Reserved. 39 Erb and Kliebenstein and nitrogen-containing compounds, are often lineage that plant secondary metabolites evolve in response to specific and aid plants to interact with the biotic and herbivore pressure, resulting in the evolution of re- abiotic environment (Hartmann, 2007). Finally, plant sistance mechanisms in herbivores. The resulting arms hormones are defined as small compounds that regu- race is thought to drive the diversity of plant second- late organismal processes, including the production ary metabolites and insect herbivores (Futuyma and of the other metabolites, by interacting with receptor Agrawal, 2009). proteins (Davies, 2004). Over the last decades, the distinction between pri- Despite the fact that definitions of secondary metab- mary metabolites, secondary metabolites, and plant olites are inherently diffuse (Hartmann, 2007; Pichersky hormones has proven a useful approximation. How- and Lewinsohn, 2011; Davies, 2013), the distinction be- ever, the emergence of a more detailed understanding tween primary metabolites, secondary metabolites, and of plant metabolism may require us to revisit this plant hormones has found its way into textbooks and functional partitioning (Neilson et al., 2013; Maag et al., shapes our thinking in plant biology to this day. An 2015; Kliebenstein, 2018; Pichersky and Raguso, 2018; illustrative example is the field of plant–herbivore in- Zhou et al., 2018). In particular, an increasing number teractions, where major efforts have gone into disen- of genetic and functional studies on plant secondary tangling how plants protect their primary metabolites metabolites are blurring the functional trichotomy by (serving as nutrients for herbivores) using secondary showing that plant secondary metabolites can have metabolites (serving as defenses for plants), and how regulatory functions and serve as precursors for pri- adapted herbivores manage to extract primary metab- mary metabolites. In this review, we discuss this evi- olites while avoiding the negative effects of secondary dence, mostly focusing on examples that rely on the metabolites (Awmack and Leather, 2002; Howe and use of natural knockout variants, mutants, and trans- Jander, 2008; Zhou et al., 2015; Erb and Reymond, genic plants altered in their capacity to produce certain 2019). In this context, plant hormones are investigated secondary metabolites in combination with chemical as regulators of primary and secondary metabolism, complementation assays to demonstrate activity of the defense, and resistance that may be manipulated by metabolites. We illustrate that for an increasing number adapted herbivores (Howe and Jander, 2008; Schuman of plant secondary metabolites, a strict functional sep- and Baldwin, 2016; Stahl et al., 2018), similar to patho- aration from regulators and primary metabolites may gens (Kazan and Lyons, 2014). The biochemical co- not do them justice and possibly hinders our progress in evolutionary arms–race theory (Ehrlich and Raven, understanding their roles for plant survival in hostile 1964), a key concept in plant–herbivore interactions environments. (Berenbaum and Zangerl, 2008; Jander, 2018), postulates INTEGRATION OF PLANT SECONDARY METABOLITES INTO REGULATION AND METABOLISM Early Evidence for Metabolic Integration of Secondary Metabolites In 1977, David Rhoades studied the properties of creosotebush (Larrea spp.) leaf resin. He found that the resin, which contained high levels of phenylpropanoid derivatives (lignans), absorbed ultraviolet radiation, reduced evaporative water loss across cellulose mem- branes, and had the capacity to form complexes with proteins, thus possibly reducing the digestibility of plant materials for herbivores (Rhoades, 1977, p. 281). Rhoades (1977) thus postulated that “.any chemical system possessed by a plant must necessarily be inte- grated into the total metabolic scheme and multiple functions are to be expected.” In other words, Rhoades (1977) proposed that secondary metabolites are not end Figure 1. Low molecular weight compounds in plants are functionally points, but integrated components of plant metabolism, classified as primary metabolites, secondary metabolites, or hormones. and may, by consequence, take on any number of Present work on plant secondary metabolites demonstrates that many of functions, similar to other plant metabolites. Indeed, them also have regulatory roles, and some are demonstrated precursors evidence was emerging at that time that secondary of primary metabolites. Note that primary metabolites and hormones metabolites may regulate growth and defense, as ex- also show functional overlap with the other metabolite classes (not discussed here). These findings blur the functional trichotomy of plant ogenously applied flavonoids could modulate polar metabolism and call for a reassessment of ecological and evolutionary auxin transport and catabolism (Stenlid, 1963; Stenlid, frameworks that are based on this model. 1976), glucosinolate breakdown products could replace 40 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites auxins in inducing hypocotyl bending (Hasegawa et al., sinapoylmalate (Kim et al., 2015). The phenylpropanoid 1986), and induced volatiles promoted resistance and phenotype is rescued in mutants that no longer produce defense regulation in neighboring trees (Baldwin and the substrate of CYP83B1, indole‐3‐acetaldoxime (Kim Schultz, 1983; Rhoades, 1983). et al., 2015), suggesting that it may be the aldoxime overaccumulation rather than the lack of downstream glucosinolates that suppresses sinapoylmalate. Sup- pressor screens showed that the phenylpropanoid Secondary Metabolites as Regulators of Plant Defense phenotype is also absent in plants that have mutated Following early preliminary evidence of secondary MEDa/b genes, which encode key components of a large multisubunit transcriptional complex that regulates metabolites regulating defenses, genetic evidence fol- lowed in 2009, when it was reported that Arabidopsis phenylpropanoid biosynthetic genes (Kim et al., 2015; (Arabidopsis thaliana) mutants defective in indole glu- Dolan et al., 2017). A recent study demonstrates that cosinolate biosynthesis no longer mount a callose de- a group of Kelch Domain F‐Box (KFB) genes that are fense response following Flg22 treatment. Callose involved in PAL inactivation (Zhang et al., 2013) are up- formation is rescued by adding 4-methoxy-indol-3- regulated in indole glucosinolate mutants in a MED5- ylmethylglucosinolate (Clay et al., 2009). The myrosinase dependent manner, whereas PAL-activity is suppressed PEN2 is required for this phenomenon, implicating (Kim et al., 2020). PAL-activity and sinapoylmalate ac- glucosinolate breakdown in callose regulation (Clay cumulation are (partially) rescued in glucosinolate- et al., 2009). Shortly thereafter, it was discovered that deficient KBF mutants (Kim et al., 2020). The model indole-derived benzoxazinoid secondary metabolites emerging from these studies is that aldoximes, which have a comparable callose regulatory function in ce- accumulate in CYP83B1 mutants, increase KFB-mediated reals. Benzoxazinoid-deficient bx1 maize (Zea mays) PAL degradation through MED5 transcriptional regula- mutants are defective in aphid- and chitosan-induced tion as well as other, yet unknown, mechanisms (Kim callose deposition, and callose induction is rescued by et al., 2015; Kim et al., 2020). As aldoximes are produced the addition of DIMBOA or DIMBOA-Glc (Ahmad by many different species, this form of defense regula- et al., 2011; Meihls et al., 2013). In both cases, the ca- tion may also occur beyond glucosinolate-producing pacity to regulate callose is structurally specific and plants (Kim et al., 2020). Interestingly, wheat lines over- depends on the modification of the indole-derived ring. expressing a maize benzoxazinoid O-methyl transferase In Arabidopsis, indol-3-ylmethylglucosinolate, which and thus accumulate more HDMBOA-Glc and less lacks a methylated hydroxy-group on the aromatic DIMBOA-Glc also show higher levels of the phenyl- ring, is inactive, whereas the methylated form is active propanoid ferulic acid, despite unaltered pool sizes of (Clay et al., 2009). In maize, DIMBOA-Glc, which lacks amino acid precursors (Li et al., 2018a), suggesting that a methylated hydroxy-group at the nitrogen, is active, phenolic compounds may also be regulated by other secondary metabolite pathways. whereas the methylated form (HDMBOA-Glc) is inac- tive (Li et al., 2018a). Whereas the callose response to Apart from glucosinolates and benzoxazinoids, benzoxazinoids is conserved between wheat (Triti- volatile secondary metabolites such as terpenoids, cum aestivum) and maize, they do not elicit callose in green-leaf volatiles, and aromatic compounds can also Arabidopsis, and intact glucosinolates do not elicit regulate plant defenses (Baldwin et al., 2006; Godard callose in maize (Li et al., 2018a). These studies show et al., 2008; Erb, 2018; Bouwmeester et al., 2019). Many that callose regulation by secondary metabolites is of these volatiles are released upon herbivore- or highly specific, tightly controlled, and likely evolved pathogen attack and are capable of directly inducing or repeatedly. The mechanism underlying secondary priming hormonal defense signaling pathways and metabolite–induced callose formation awaits to be resistance. In maize, for instance, mutants that are de- elucidated. Glucosinolates and benzoxazinoids may, fective in their capacity to produce volatile indole are for instance, promote callose production by regulat- unable to prime their systemic tissues to rapidly release ing hormonal pathways (Burow et al., 2015; Katz terpenes upon herbivore attack (Erb et al., 2015). Add- et al., 2015), through transcriptional regulation (Kim ing indole to the headspace of maize plants restores this et al., 2015), or by directly initiating callose formation priming phenotype (Erb et al., 2015). Rice (Oryza sativa) posttranslationally. plants also respond to indole through priming of early Interestingly, glucosinolates and benzoxazinoids defense signaling elements such as the map kinase also seem to regulate the accumulation of other sec- OsMPK3 (Ye et al., 2019). Transgenic plants that are ondary metabolites (Hemm et al., 2003; Kim et al., 2015; deficient in OsMPK3 expression are no longer respon- Li et al., 2018a). In Arabidopsis, mutants that are de- sive to indole, suggesting that indole acts via the fective in the atypical myrosinase PEN2 release lower priming of early defense signaling (Ye et al., 2019). In amounts of Trp-derived metabolites such as camalexin Arabidopsis, geranylgeranyl reductase1 mutants are de- upon flg22 treatment (Frerigmann et al., 2016) and in- fective in systemic acquired resistance against P. syringae fection by Pseudomonas syringae (Stahl et al., 2016). (Riedlmeier et al., 2017). Adding the pathogen-induced Furthermore, mutants defective in the CYP83B1 en- volatiles a-and b-pinene to the headspace of the mu- tant restores resistance, with the response depending zyme required for indole glucosinolate production also show lower accumulation of the phenylpropanoid on intact salicylic acid signaling and the AZELAIC ACID Plant Physiol. Vol. 184, 2020 41 Erb and Kliebenstein INDUCED (AZI1) gene (Riedlmeier et al., 2017). The unknown. Studies on the indole glucosinolate break- precise role of other volatile secondary metabolites that down product indole-3-carbinol have identified an can regulate defenses at physiological concentrations, unexpected target protein. Indole-3-carbinol accumu- including homoterpenes (Arimura et al., 2000) and green- lates upon wounding in Arabidopsis and rapidly re- leaf volatiles (Ameye et al., 2018), has not yet been ex- duces root growth upon exogenous application. In plored using genetic approaches, but their activity has vitro, indole-3-carbinol interferes with the interaction been demonstrated clearly through chemical comple- between auxin and its receptor TIR1 by binding at an mentation (Arimura et al., 2000; Engelberth et al., 2004; allosteric site (Katz et al., 2015). As the indolic glucosi- Frost et al., 2008; Meents et al., 2019). Further support for nolate catabolite likely binds directly to TIR1 (Katz the potential regulatory role of defense volatiles comes et al., 2015), one may argue that TIR1 acts as an from LOX2-silenced Nicotiana attenuata plants, which are indolic glucosinolate receptor that mediates the regu- deficient in the production of herbivory-induced, green- lation of growth by a plant secondary metabolite. leaf volatiles. In contrast with the other systems where Another link to auxin signaling was found with a volatiles induce defense, the LOX2 mutation leads to structurally unrelated aliphatic glucosinolate. This was stronger expression of defense-related genes in neighbors found by the initial observation that the auxin-sensitive than wild-type plants, suggesting that volatiles can also repressors IAA5, IAA6, and IAA19 strongly regulate suppress defenses (Paschold et al., 2006). 4-methylsulfinylbutyl glucosinolate (4-MSOB) levels in In summary, at least five classes of secondary me- dehydrated Arabidopsis plants (Salehin et al., 2019). tabolites (glucosinolates, benzoxazinoids, terpenes, ar- Iaa5,6,19 mutants fail to close their stomata upon drought omatics, and green-leaf volatiles) are now confirmed to stress, a phenotype that can be reverted by adding act as potential regulators of in planta defense. It is 4-MSOB (Salehin et al., 2019). Together with the finding exciting to speculate that there are many other sec- that glucosinolate biosynthesis and activation mutants ondary metabolites that play similar regulatory roles. are less tolerant to drought (Salehin et al., 2019), and that An important gap of knowledge is the mechanism glucosinolate breakdown products can trigger stomatal by which secondary metabolites regulate defenses. As closure in Arabidopsis and Vicia faba (Khokon et al., many of the secondary metabolites are chemically re- 2011; Hossain et al., 2013), these results provide evi- active (Farmer and Davoine, 2007; Hadacek et al., 2010), dence that aliphatic glucosinolates are involved in sto- it is possible that they act indirectly by depleting de- matal regulation. Interestingly, glucosinolate-mediated toxification enzymes, thus triggering the accumulation stomatal regulation requires a functional ROS receptor of known signaling molecules such as reactive oxygen kinase (GHR1; Salehin et al., 2019). Given that the my- species (ROS; Khokon et al., 2011). However, as dis- rosinase TGG1 accumulates in guard cells and is re- cussed below, secondary metabolites may also have quired for stomatal regulation (Zhao et al., 2008), and hormone-like properties by binding to specific receptor that glucosinolate breakdown products can regulate proteins (Katz et al., 2015). More work on the targets of stomatal closure through ROS production (Khokon secondary metabolites in planta is clearly warranted et al., 2011), it is conceivable that ROS link endoge- and would help to clarify the ecological and evolu- nous glucosinolates to stomatal regulation (Khokon tionary context of their capacity to regulate defenses. et al., 2011). Apart from growth and stomatal opening, glucosi- nolates may also regulate the circadian clock and flowering time. Natural presence/absence variation Secondary Metabolites as Regulators of Growth in the 2-oxoglutarate-dependent dioxygenase AOP2, and Development which converts methylsulfinylalkyl glucosinolates into Plants regulate their growth dynamically and often alkenyl glucosinolates, is linked to variation in the ex- reduce their investment into growth and development pression of the major flowering gene FLC and to vari- upon herbivore- or pathogen attack. This reduction in ation in flowering time (Kliebenstein et al., 2001; Atwell growth is thought to be largely due to the reconfigu- et al., 2010). Introducing a functional AOP2 into Ara- ration of a plant’s signaling network rather than a lack bidopsis Col-0 (a natural AOP2 knockout) confirmed of resources (Kliebenstein, 2016; Machado et al., 2017; the flowering time effect, identified a shift in the ex- Guo et al., 2018). Strikingly, plant secondary metabo- pression of circadian genes, and showed a 1-h decrease lites and their breakdown products are being (re)-dis- in clock periodicity (Kerwin et al., 2011). Abolishing covered as plant growth modulators, thus adding glucosinolate production using Myb transcription fac- another layer of regulation to growth-defense patterns. tor mutants led to the same periodicity shift, suggesting Again, glucosinolates provide a mechanistic example of that the effect may be linked to the presence of the how secondary metabolites can modulate growth. When 4-MSO glucosinolate in wild-type Col-0 (Kerwin et al., applied to the roots of Arabidopsis and many other plant 2011). The effect of the AOP locus on flowering time species, the aliphatic 3-hydroxypropylglucosinolate in- depends on the genetic background (Jensen et al., 2015), hibits root meristematic growth at physiological con- supporting the hypothesis that secondary metabolites centrations via an intact Target of Rapamycin pathway are integrated into a complex and variable regulatory network. How aliphatic glucosinolates directly regu- (Malinovsky et al., 2017). The exact molecular interac- tion partner of 3-hydroxypropylglucosinolate remains late gene expression networks and developmental 42 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites phenotypes such as flowering time remains to be possible that flavonols also function as signals and tested (Burow and Halkier, 2017). further work is needed to differentiate between these The present data suggest that glucosinolates can hypotheses. influence growth by multiple different mechanisms, Other secondary metabolites may also regulate plant including Target of Rapamycin regulation, auxin development. Diploid oat sad2 mutants that overpro- regulation, auxin-independent transcriptional regula- duce the triterpene b-amyrin produce shorter roots and tion, and auxin-mediated ROS accumulation (Katz significantly more root hairs than wild-type plants, et al., 2015; Kim et al., 2015; Malinovsky et al., 2017; phenotypes which are absent in other mutants of the Salehin et al., 2019). This diversity, paired with the pathway that do not overproduce b-amyrin (Kemen substantial variation in glucosinolate biosynthesis et al., 2014). However, this phenotype cannot be phe- within species, creates a wealth of metabolic networks nocopied by adding b-amyrin to roots, possibly because and phenotypes, which can be acted upon by natural its activity requires specific spatiotemporal accumula- selection. It is tempting to speculate that this diversity tion patterns (Kemen et al., 2014). In N. attenuata, is a reflection of the highly diverse habitats and envi- silencing a malonyltransferase that malonylates 17- ronments that a single species can inhabit and may Hydroxygeranylinalool diterpene glycosides reduces provide adaptive potential beyond conserved hor- floral style cell size and length (Li et al., 2018b). monal pathways. Knocking down diterpene glycoside production by si- In addition to glucosinolates, flavonoids are impli- lencing a geranylgeranyl diphosphate synthase abol- cated in regulating plant growth, development, and ishes the effect of the malonyltransferase, suggesting environmental responses. Exogenously applied flavo- that specific diterpene hexose decoration patterns are noids have long been known to modulate auxin trans- responsible for the flower phenotype (Li et al., 2018b). port (Stenlid, 1976). Evidence that flavonoids may also Furthermore, a labeling experiment in poplar recently act as endogenous growth regulators came from an uncovered that herbivore-attacked leaves can convert Arabidopsis chalcone synthase mutant, transparent testa benzyl cyanide, a herbivore-induced volatile, to the (tt4). tt4 plants show growth alterations that are char- auxin phenylacetic acid (Günther et al., 2018), thus acteristic of disturbed auxin localization, including re- providing a potential link between the catabolism of duced root growth and gravitropism (Brown et al., volatile secondary metabolites and the regulation of 2001). The tt4 mutant also displays increased auxin plant growth and development. transport (Murphy et al., 2000), which can be reversed The examples above show how secondary metabo- by adding the flavonoid precursor naringenin (Brown lites can modulate growth and development through a et al., 2001). Further mechanistic studies suggest that variety of mechanisms, some of which are barely dis- flavonoids modulate auxin transport through several tinguishable from mechanisms normally assigned to mechanisms, including interactions with auxin trans- plant hormones (Fig. 2). Whereas some of these sec- porters and transport-regulating proteins (Peer and ondary metabolite regulators are ancient and highly Murphy, 2007; Santelia et al., 2008). Arabidopsis roots conserved (e.g. flavonoids, terpenes), others evolved grow away from light and flavonoids accumulate in more recently (e.g. glucosinolates and benzoxazinoids) their light-exposed sides (Silva-Navas et al., 2016). The and are restricted to specific plant families. Plants thus tt4 mutant also shows reduced light avoidance, which have both a conserved and a unique, variable, and was linked to reduced auxin polar transport and re- flexible repertoire of regulators at their disposition to duced ROS accumulation, both of which can regulate adjust growth and development, which likely contrib- cell division and elongation (Gayomba et al., 2010; utes to their potential to colonize variable and chal- Silva-Navas et al., 2016). tt4 also displays lower accu- lenging habitats. mulation of flavonols and increased ROS levels in guard cells, phenotypes associated with more rapid absisic acid–induced stomatal closure (Watkins et al., Secondary Metabolites as Primary Metabolites 2014). An additional link between flavonoid biosyn- thesis, ROS accumulation, and plant development was If secondary metabolites can regulate growth, uncovered recently in tomato (Solanum lycopersicum; development, and defense, can they also function Muhlemann et al., 2018). The are mutant is defective in a as primary metabolites? Whereas primary metabo- flavonol 3-hydroxylase (F3H), displays reduced flavo- lites are highly conserved, secondary metabolites nol and increased ROS accumulation in pollen grains, evolve dynamically and are inherently variable in and suffers from reduced pollen tube growth and in- structure and production (Wink, 2008). This rapid tegrity. The pollen tube phenotype can be rescued by evolution would seem to complicate their integra- the addition of antioxidants (Muhlemann et al., 2018). tion into the most fundamental workings of plant Flavonols are thus thought to act as antioxidants that metabolism because it would require a rapid evolu- reduce ROS accumulation and thereby regulate plant tion of enzymes to connect these new structures into development (Hernández et al., 2009; Muhlemann the more conserved metabolic pathways. However, et al., 2018). However, the oxidation state of a cell can evidence for secondary metabolites that are not directly influence signaling by altering disulfide bridge strictly essential, but nevertheless contribute to pri- formation or other protein modifications. Thus, it is mary metabolism, is emerging. In Arabidopsis, plants Plant Physiol. Vol. 184, 2020 43 Erb and Kliebenstein Figure 2. Glucosinolates and benzoxazinoids as examples of secondary metabolites that blurr the functional trichotomy of plant metabolism. Dif- ferent functions of glucosinolates in Arabidopsis and benzoxazinoids in maize and wheat are depicted. Genes that are known to be involved in the different functions are indicated. Note that a direct role of benzoxazinoids and glucosinolates as plant primary metabolites (for instance, in the context of nitrogen/sulfur and/or energy storage) has not been clearly demonstrated so far. *MEDs and KFBs are likely regulated by aldoxime pre- cursors of glucosinolates. For references, see the article. with mutations in the flavonoid pathway upstream are proposed to take the deglycosylated cyanogen of the FLAVANONE-3-HYDROXYLASE (F3H) show and directly release ammonia and the corresponding a reduction in the respiratory cofactor ubiquinone acetate (Jenrich et al., 2007). Further support for the (coenzyme Q; Soubeyrand et al., 2018). Ubiquinone potential of cyanogenic glucosides as a primary me- levels can be restored by adding dihydrokaempferol or tabolite store came from overexpression of a hydrox- kaempferol to the mutants. Labeling experiments dem- ynitrile lyase, which is involved HCN formation in onstrate that the aromatic ring of kaempferol is integrated cassava (Manihot esculenta). Theseplantshavede- into ubiquinone, and that heme-dependent peroxidases creased concentrations of cyanogenic glycosides and likely use kaempferol to produce 4-hydroxybenzoate as increased concentrations of total amino acids, sug- a substrate for ubiquinone (Soubeyrand et al., 2018). gesting that cyanogenic glycosides may be degraded The integration of flavonoids into primary metabo- and reintegrated into primary metabolism (Narayanan lism is perhaps not surpising, because they represent et al., 2011). The potential integration of other secondary one of the oldest and most conserved classes of sec- metabolites such as glucosinolates is currently under in- ondary metabolites (albeit with substantial interspecific vestigation. In Arabidopsis, sulfur deficiency induces the variation in glycosylation patterns). Flavonoid evolu- expression of the myrosinases BGLU28 and BGLU30 tion precedes the emergence of many innovations in (Maruyama-Nakashita et al., 2003). Under sulfur-limiting plant primary metabolism, such as C photosynthesis. conditions, bglu28/30 double mutants accumulate higher Whether younger, more specialized secondary metab- levels of intact aliphatic glucosinolates, contain lower olites can act as primary metabolites is not well un- amounts of Cys and protein sulfur content, and grow derstood. This lack of knowledge is closely related to a less than wild-type plants, suggesting that glucosino- limited understanding of secondary metabolite catab- lates may serve as sulfur-storage molecules (Zhang olism. Where do these compounds go when they are no et al., 2020). longer needed? One would assume that reintegrating Detailed biochemical characterization and metabolic secondary metabolites into primary metabolism is profiling of secondary-metabolite mutants, combined beneficial for plants (Neilson et al., 2013). Such a rein- with complementation and labeling experiments, are tegration pathway has been proposed for cyanogenic required to further corroborate the potential roles of glycosides (Selmar et al., 1988). Upon deglycosylation, secondary metabolites in primary metabolism and to HCN may be assimilated into Asn via the formation of identify additional secondary metabolites that serve as b-cyano-Ala (Selmar et al., 1988). Indeed, two sorghum primary-metabolite precursors. Degradation of many (Sorghum bicolor) nitrilases are capable of producing different secondary metabolites has been observed Asn from b-cyano-Ala (Jenrich et al., 2007). An al- under specific environmental conditions (Negi et al., ternative pathway not involving the release of HCN 2014; Zipor et al., 2015). Furthermore, alterations in was suggested in sorghum. In this system, nitrilases primary metabolites are observed in various plants 44 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites with altered secondary metabolism (Mayer et al., 2001; between secondary metabolites and hormones, for Narayanan et al., 2011; Huber et al., 2016; Machado instance (Malinovsky et al., 2017; Sun et al., 2019b). et al., 2017; Zhang et al., 2020), and the accumulation Overall, the functional integration of secondary me- of specific secondary metabolites has been associated tabolites at a given point in evolution is a likely with storage and growth in microevolutionary studies consequence of the interaction between complex (Heath et al., 2014). Keeping an open mind about the environments with highly connected plant metabolic capacity of plants to evolve integrated metabolic networks. Below, we discuss the potential benefits of networks is warranted to gain a better comprehen- plant secondary metabolite metabolic integration that sion of the prevalence and importance of secondary may have favored their use as regulators and primary metabolites as precursors of primary metabolites. metabolites. Plant Secondary Metabolites as Reliable Readouts of Secondary Metabolites as Facilitators of Defense Activation Micronutrient Uptake Plants control defense activation to save metabolic An additional example that further blurs the dis- energy and avoid self-damage. Defense investment tinction between primary and secondary metabolism is is typically titrated through feedback regulation, plant micronutrient uptake. Grasses excrete low M including both positive and negative feedback loops compounds into the rhizosphere to chelate micronu- that are built into early defense signaling (Hu et al., trients such as iron and thus make them biologically 2015; Li et al., 2015) and hormonal networks (Gilardoni available (Curie and Briat, 2003). Recent work suggests et al., 2011; Liu et al., 2019). A limitation of these feedback that secondary metabolites are likely important for iron loops is that they do not provide direct information about uptake in both herbs and grasses. Chemical removal of the final level of defense activation (i.e. the production excreted phenolic acids from the nutrient solution of of defense metabolites per se). Because herbivores and red clover (Trifolium pretense) was found to result in iron pathogens may interfere with the production of defense deficiency in red clover (Jin et al., 2007). Subsequently, compounds at many levels, including in the final steps an Arabidopsis mutant, which is deficient in the 2- of biosynthesis (Jones et al., 2019), integrating them oxoglutarate-dependent dioxygenase Feruloyl-CoA directly into regulatory feedback loops may allow 69-Hydroxylase 1 and thus no longer able to pro- plants to more accurately monitor and adjust defense duce coumarins, was found to suffer from iron defi- accumulation. Using secondary metabolites as de- ciency under alkaline conditions (Schmid et al., 2014). fense activation readouts may also help plants to Similarly, young maize benzoxazinoid mutants that do no longer produce and excrete benzoxazinoids optimize synergies between different defenses and to were found to suffer from iron deficiency when compensate for accidental failures of specificde- fense pathways. The increasing number of examples growing in the presence of iron salts (Hu et al., 2018). showing that plant secondary metabolites regulate Both benzoxazinoids and coumarins are able to che- defenses (see section “Secondary Metabolites as late iron in vitro (Bigler et al., 1996; Mladenka et al., Regulators of Plant Defense”) hint at the existence of 2010). Because these complexes are essential for plant such systems. growth and development by providing essential As many secondary metabolites are compartmental- micronutrients, they should, according to definition, ized and/or stored in inactive forms, their decom- be classified as primary metabolites, thus provid- partmentalization and/or activation likely also helps ing another illustration of how secondary metabo- plants to recognize tissue damage and other forms of lites can turn into primary metabolites under given environmental stress. In this case, the metabolites conditions. wouldbeusedasdamage–associated molecular patterns (DAMPs). Green-leaf volatiles are an example of second- ary metabolites that are also DAMPs (Tanaka et al., 2014; ADAPTIVE EXPLANATIONS FOR METABOLIC Quintana-Rodriguez et al., 2018). Another potential INTEGRATION OF SECONDARY METABOLITES example of secondary metabolites as DAMPs is the There isnow ampleevidencefor secondaryme- previously discussed links between indolic glucosino- tabolites that are regulators and precursors of pri- lates and DIMBOA regulation of callose upon pathogen mary metabolites. But why would plants evolve attack. Interestingly in this case, the secondary metab- an integrated metabolism in which the same metab- olite/DAMPs are linked to endogenous responses to olite class has multiple functions that incorporate pathogen-associated molecular patterns (e.g. FLS2; growth, development, defense, and regulation? Plants Clay et al., 2009) and stomatal closure upon drought have large, interconnected metabolic networks at their stress (Salehin et al., 2019). disposition. Natural selection acts on these metabolic Given these considerations, secondary metabolites networks, resulting in the evolution of network topol- may be common readouts of defense activation and ogies that maximize fitness. Over evolutionary time, damage may have favored their evolution as defense these topologies likely include dynamic transitions regulators. Plant Physiol. Vol. 184, 2020 45 Erb and Kliebenstein Figure 3. Functional integration of plant secondary metabolites shapes plant–herbivore and tritrophic interactions. Schematic representation of how different functions of secondary metabolites are used by plants, herbivores, and natural enemies of her- bivores is shown. Plants use secondary metabolites for multiple purposes, including resistance, regulation, and primary me- tabolism (see Fig. 2). Recent work suggests that this multifunctionality is mirrored in adapted herbivores, which also use secondary metabolites for multiple purposes, including similar and new functions. Little is known about how adapted natural enemies use secondary metabolites, but multifunctional integration across three trophic levels is likely (Box 2). Circles represent hypothetical individual secondary metabolites (for color code, refer to Figs. 1 and 2). Solid lines indicate metabolic connections within an organism. Dashed lines indicate similar functions of the same compounds in different organisms. Metabolic Network Specialization as a Potential Means to Multifunctionality as a Cost-Saving Strategy Resist Manipulation Producing secondary metabolites has energetic and Herbivores, pathogens, and viruses can interfere with metabolic costs (Gershenzon, 1994). These costs are not defense hormone signaling and thereby manipulate always evident (Züst et al., 2011; Machado et al., 2017), plants for their own benefit (Kazan and Lyons, 2014; Stahl and may mostly occur under specific environmental et al., 2018). The high degree of conservation in defense conditions such as strong competition and nutrient hormone signaling may in fact favor the evolution of bi- limitation (Cipollini et al., 2018). Plants likely manage otic manipulation of plant signaling (Berens et al., 2017). costs of secondary metabolite production through the For example, if an attacking organism evolves the ability regulation of biosynthesis, but controlled recycling of to alter jasmonate signaling, this may provide it a fitness the resulting compounds would enhance the plants benefitonawide varietyofhost plantsand mayreduce ability to recoup costs in challenging environments the advantage for plants to evolve new inducible resis- (Neilson et al., 2013). Secondary metabolites that are tance mechanisms regulated by these hormones. One induced upon environmental stress could for instance possibility to solve this problem would be to use less- be recycled back into primary metabolism once the conserved metabolites as defense regulators. If a plant stress subsides. One way of testing this hypothesis is to had the ability to use these metabolites, it would be manipulate secondary metabolite recycling by target- less likelytofallpreytohostswitching by hormone- ing enzymes involved in their degradation, such as manipulating enemies. The evolution of (specialized) glucosidases (Morant et al., 2008) or nitrilases (Jenrich secondary metabolites into regulatory networks may thus et al., 2007). With use of this approach, a link between be promoted through the evolution of manipulation the degradation of cyanogenic glycosides and plant strategies in plant enemies. Clear examples supporting protein supply was uncovered (Narayanan et al., 2011), this hypothesis are currently lacking. As the biosynthesis supporting the hypothesis that reintegration of sec- of defense-regulating secondary metabolites such as glu- ondary compounds into primary metabolism may be cosinolates is at least partially controlled by conserved advantageous for the plant. A caveat of this approach is phytohormonal pathways (Schweizer et al., 2013), plant that it remains difficult to disentangle a direct contri- enemies that are capable of overcoming these conserved bution of the generated catabolites to primary metab- pathways may also suppress more specificregulators. olism from their potential regulatory roles. A more Interestingly, an opposite pattern has also been found for detailed understanding of secondary metabolite sig- the tomato leaf spot fungus, which uses a hydrolase to naling and catabolism would help to explore the role of detoxify steroidal glycoalkaloids and benefits from the secondary metabolite reintegration as a cost-saving defense-suppressing properties of the resulting break- strategy. down products (Bouarab et al., 2002). This illustrates that Another way to minimize costs is to use the same specialized plant enemies may also misuse the regulatory secondary compound for multiple purposes (Neilson properties of secondary metabolites of their host plants. et al., 2013). As many secondary compounds are 46 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites Box 1. Case study of secondary metabolite multifunctionality. Cited articles: Glauser et al., 2011; Robert et al., 2012, 2017; Maag et al., 2016. Box 2. Multifunctionality of plant secondary metabolites in tri- trophic interactions. Cited articles: Fink and Brower, 1981; Hunter, 2003; Sarfraz et al., 2009; Sloggett and Davis, 2010; Aartsma et al., chemically reactive, they need to be managed by the 2017; Rafter et al., 2017; Robert et al., 2017; Turlings and Erb, 2018; plant through (potentially costly) storage, inactivation, Sun et al., 2019a; Ugine et al., 2019; Zhang et al., 2019. and/or resistance mechanisms, including specialized cells, ducts, and glands (Sirikantaramas et al., 2008). By employing the same compound class for multiple ECOLOGICAL CONSEQUENCES OF THE purposes, plants may spread these fixed costs across METABOLIC INTEGRATION OF more fitness components and increase their competi- SECONDARY METABOLITES tiveness. Metabolic costs may also be lowered by using the same biosynthetic machinery to produce different The separation of low M compounds into primary compounds for different purposes. Whereas the cost- metabolites, secondary metabolites, and hormones has saving aspects of multifunctionality are difficult to shaped our ecological and evolutionary thinking of quantify, multifunctionality seems to be a widespread plant–environment interactions. If we abolish this view property of secondary metabolites, as discussed above, in favor of a more integrated perspective (i.e. where and it is difficult for this multifunctionality to evolve secondary metabolites can have regulatory roles and without benefit. can provide precursors for primary metabolites), we Plant Physiol. Vol. 184, 2020 47 Erb and Kliebenstein can derive new hypotheses on plant defense patterns herbivores to metabolize these compounds. Recent ex- and plant–herbivore interactions. These hypotheses are amples also hint at the possibility that plant secondary likely to improve our understanding of the ecological metabolites may have hormonal functions in herbi- roles of plant secondary metabolites in the future. vores. In rice, knocking down CYP71A1, a gene re- sponsible for the production of serotonin, a monoamine neurotransmitter, reduces the performance of the rice Ontogenetic Patterns of Secondary Metabolite Production brown planthopper (Nilaparvata lugens). Adding sero- tonin to an artificial diet enhances its performance (Lu Many secondary metabolites show distinct onto- et al., 2018), suggesting that the herbivore may benefit genetic accumulation patterns, with concentrations from the hormonal properties of this plant metabolite. varying over time and between tissues. Ecological the- Plants may also benefit from producing secondary ory explains this within-plant variation using resource metabolites that act as (de)-regulators of herbivore constraints, allocation costs, and variation in herbivore physiology. Spinach (Spinacia oleracea), for instance, pressure (McKey, 1974; van Dam, 2009; Meldau et al., produces the molting hormone 20-hydroxyecdysone 2012; Schuman and Baldwin, 2016; Barton and Boege, (Bakrim et al., 2008), which can interfere with caterpil- 2017). The above theories are all based on costs and lar development (Kubo et al., 1983). benefit relationship, with the benefit typically being In general terms, a plant’s metabolism is shaped by a limited to herbivore resistance. Given the blurred tri- dynamic landscape of environmental selection pres- chotomy of plant secondary metabolism, the ecological sure; conversely, the metabolic network of herbivores balance sheet may be improved by taking into account is shaped by the functional and chemical potential of multifunctionality (Barton and Boege, 2017). A drop in plant metabolites within the herbivore’s own selection secondary metabolite levels, as is often observed a few landscape. One can thus expect that, similar to what weeks after germination or at the onset of flowering, for Rhoades postulated for plants (Rhoades, 1977), any instance (Meldau et al., 2012; Barton and Boege, 2017), chemical system taken up by a herbivore must neces- may reflect an increased need of primary metabolites sarily be integrated into its total metabolic scheme, and and nutrients rather than a drop in herbivore pressure. multiple functions of plant secondary metabolites are to Similarly, strong expression of secondary metabolites in roots may not only be the result of high tissue value and a high risk of root herbivore attack, but may simply reflect additional functions of the compounds such as micronutrient uptake and microbial conditioning (Hu et al., 2018; Stringlis et al., 2018). Our understanding of ontogenetic allocation patterns of secondary metabo- lites may thus improve if we take their full metabolic integration and potential multifunctionality into ac- count and do not limit their considered benefits to herbivore resistance. Defense Metabolites in Plant–Herbivore Interactions The functional trichotomy used to define plant me- tabolites has also shaped our understanding of how these metabolites influence plant–herbivores interac- tions. Herbivores are assumed to forage for primary metabolites while trying to avoid the negative effects of secondary metabolites through behavioral and meta- bolic adaptations (Behmer, 2009; Stahl et al., 2018). If we accept that secondary metabolites can also be regula- tors and precursors of primary metabolites, then it be- comes conceivable that they may have similar roles in herbivores. The root-feeding larvae of the western corn rootworm for instance forage for iron-benzoxazinoid complexes to acquire iron and improve their growth, thus effectively using a plant secondary metabolite as a primary metabolite (Hu et al., 2018). Several other herbivores also gain more weight in the presence of plant secondary metabolites (Meldau et al., 2009; Richards et al., 2012; Marti et al., 2013; Veyrat et al., 2016; Wetzel et al., 2016), and it is conceivable that some of these effects may be due to the capacity of the 48 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites be expected, some of which likely mirror their multiple secondary metabolites are highly integrated into plant functions in plants (Fig. 3). Specialist herbivores are metabolism and can serve as both regulators and pri- known to use secondary metabolites as infochemicals mary metabolites. Thus, it is likely that most secondary (e.g. foraging cues), and some also sequester defenses to metabolites have additional functions for plants. Tak- protect themselves against herbivore natural enemies ing into account these additional functions (see Out- (Nishida, 2002; Opitz and Müller, 2009), in analogy to standing Questions), we can refine key concepts in the use of these chemicals as defense regulators and plant-environment interactions and improve our un- resistance factors in plants (Fig. 3). Cabbage aphids derstanding of the chemical ecology of plants and their (Brevicoryne brassicae) are an illustrative example in enemies. this context, as they can activate glucosinolates by producing their own myrosinases (Bridges et al., 2002; ACKNOWLEDGMENTS Kazana et al., 2007). This allows them to use glucosi- We would like to thank Mike Blatt for the invitation to write this Inaugural nolates as two-component defense system against Topical Review, Pierre Mateo for drawing chemical structures, and Christelle predators (Kazana et al., 2007). As glucosinolate A.M. Robert, Clint Chapple, Jonathan Gershenzon, and two anonymous reviewers as well as the Twitter community for helpful comments on an earlier breakdown products (isothiocyanates) also increase version of this manuscript. aphid responses to alarm pheromones (Dawson et al., Received April 7, 2020; accepted June 15, 2020; published July 7, 2020. 1987), it was proposed that aphid-released isothiocya- nates may also act as danger signals (Bridges et al., 2002). Another example where herbivores use second- LITERATURE CITED ary metabolites for several purposes that mirror their Aartsma Y, Bianchi FJJA, van der Werf W, Poelman EH, Dicke M (2017) multiple uses by plants are again benzoxazinoids, Herbivore-induced plant volatiles and tritrophic interactions across which are used as defense metabolites and sidero- spatial scales. New Phytol 216: 1054–1063 phores by a specialist root herbivore in maize (Box 1). Ahmad S, Veyrat N, Gordon-Weeks R, Zhang Y, Martin J, Smart L, Apart from mirroring plant functions, adapted herbi- GlauserG,Erb M, FlorsV,FreyM,etal (2011) Benzoxazinoid me- vores can also use plant secondary metabolites for tabolites regulate innate immunity against aphids and fungi in maize. herbivore-specific functions. Cyanogenic glycosides, Plant Physiol 157: 317–327 for instance, can be used by specialized lepidoptera as Ameye M, Allmann S, Verwaeren J, Smagghe G, Haesaert G, Schuurink RC, Audenaert K (2018) Green leaf volatile production by plants: A defenses and nuptial gifts (Zagrobelny et al., 2018), and meta-analysis. New Phytol 220: 666–683 glucosinolates are part of the pheromone blend of flea Arimura G, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J beetles (Phyllotreta striolata; Beran et al., 2016). (2000) Herbivory-induced volatiles elicit defence genes in lima bean These examples illustrate that, as in plants, sec- leaves. Nature 406: 512–515 ondary metabolites can act as defenses, regulators, Atwell S, Huang YS, Vilhjálmsson BJ, Willems G, Horton M, Li Y, Meng and precursors of primary metabolites in herbivores. 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Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred Functional Trichotomy

PLANT PHYSIOLOGY , Volume 184 (1): 14 – Jul 7, 2020

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Abstract

Topical Review Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred 1[OPEN] Functional Trichotomy a,2,3 b Matthias Erb, and Daniel J. Kliebenstein Institute of Plant Sciences, University of Bern, 3013 Bern, Switzerland Department of Plant Sciences, University of California, Davis, California 95616 ORCID IDs: 0000-0002-4446-9834 (M.E.); 0000-0001-5759-3175 (D.J.K.) The plant kingdom produces hundreds of thousands of low molecular weight organic compounds. Based on the assumed functions of these compounds, the research community has classified them into three overarching groups: primary metabolites, which are directly required for plant growth; secondary (or specialized) metabolites, which mediate plant–environment interactions; and hormones, which regulate organismal processes and metabolism. For decades, this functional trichotomy of plant metabolism has shaped theory and experimentation in plant biology. However, exact biochemical boundaries between these different metabolite classes were never fully established. A new wave of genetic and chemical studies now further blurs these boundaries by demonstrating that secondary metabolites are multifunctional; they can function as potent regulators of plant growth and defense as well as primary metabolites sensu lato. Several adaptive scenarios may have favored this functional diversity for secondary metabolites, including signaling robustness and cost-effective storage and recycling. Secondary metabolite multifunctionality can provide new explanations for ontogenetic patterns of defense production and can refine our understanding of plant–herbivore interactions, in particular by accounting for the discovery that adapted herbivores misuse plant secondary metabolites for multiple purposes, some of which mirror their functions in plants. In conclusion, recent work unveils the limits of our current functional classification system for plant metabolites. Viewing secondary metabolites as integrated components of metabolic networks that are dynamically shaped by environmental selection pressures and transcend multiple trophic levels can improve our understanding of plant metabolism and plant–environment interactions. Plants can use simple, inorganic precursors to syn- required for the growth and development of plants thesize a large diversity of low M organic compounds. (Fernie and Pichersky, 2015). Secondary metabolites, This synthetic capacity helps plants to colonize diverse including major groups such as phenolics, terpenes, and challenging environments. Low M organic com- pounds are commonly separated by perspective func- tion into primary metabolites, secondary metabolites (also called specialized metabolites or natural pro- ducts), and plant hormones (Fig. 1; Taiz et al., 2015). Primary metabolites are highly conserved and directly This work was supported by the University of Bern, Swiss National Science Foundation (grant no. 155781 to M.E.), the European Research Council under the European Union’s Horizon 2020 Research and In- novation Program (grant no. ERC–2016–STG 714239 to M.E.), the National Science Foundation Division of Integrative Organismal Sys- tems (award no. 1655810), theNational Science Foundation Division of Molecular and Cellular Biosciences (grant no. 1906486 to D.J.K.); the National Institute of Food and Agriculture (hatch project no. CA–D–PLS–7033–H to D.J.K.), and the Danish National Research Foundation (grant no. DNRF99 to D.J.K.). Senior author. Author for contact: matthias.erb@ips.unibe.ch. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Matthias Erb (matthias.erb@ips.unibe.ch). M.E. and D.J.K. developed and wrote the paper. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.20.00433 Plant Physiology , September 2020, Vol. 184, pp. 39–52, www.plantphysiol.org  2020 American Society of Plant Biologists. All Rights Reserved. 39 Erb and Kliebenstein and nitrogen-containing compounds, are often lineage that plant secondary metabolites evolve in response to specific and aid plants to interact with the biotic and herbivore pressure, resulting in the evolution of re- abiotic environment (Hartmann, 2007). Finally, plant sistance mechanisms in herbivores. The resulting arms hormones are defined as small compounds that regu- race is thought to drive the diversity of plant second- late organismal processes, including the production ary metabolites and insect herbivores (Futuyma and of the other metabolites, by interacting with receptor Agrawal, 2009). proteins (Davies, 2004). Over the last decades, the distinction between pri- Despite the fact that definitions of secondary metab- mary metabolites, secondary metabolites, and plant olites are inherently diffuse (Hartmann, 2007; Pichersky hormones has proven a useful approximation. How- and Lewinsohn, 2011; Davies, 2013), the distinction be- ever, the emergence of a more detailed understanding tween primary metabolites, secondary metabolites, and of plant metabolism may require us to revisit this plant hormones has found its way into textbooks and functional partitioning (Neilson et al., 2013; Maag et al., shapes our thinking in plant biology to this day. An 2015; Kliebenstein, 2018; Pichersky and Raguso, 2018; illustrative example is the field of plant–herbivore in- Zhou et al., 2018). In particular, an increasing number teractions, where major efforts have gone into disen- of genetic and functional studies on plant secondary tangling how plants protect their primary metabolites metabolites are blurring the functional trichotomy by (serving as nutrients for herbivores) using secondary showing that plant secondary metabolites can have metabolites (serving as defenses for plants), and how regulatory functions and serve as precursors for pri- adapted herbivores manage to extract primary metab- mary metabolites. In this review, we discuss this evi- olites while avoiding the negative effects of secondary dence, mostly focusing on examples that rely on the metabolites (Awmack and Leather, 2002; Howe and use of natural knockout variants, mutants, and trans- Jander, 2008; Zhou et al., 2015; Erb and Reymond, genic plants altered in their capacity to produce certain 2019). In this context, plant hormones are investigated secondary metabolites in combination with chemical as regulators of primary and secondary metabolism, complementation assays to demonstrate activity of the defense, and resistance that may be manipulated by metabolites. We illustrate that for an increasing number adapted herbivores (Howe and Jander, 2008; Schuman of plant secondary metabolites, a strict functional sep- and Baldwin, 2016; Stahl et al., 2018), similar to patho- aration from regulators and primary metabolites may gens (Kazan and Lyons, 2014). The biochemical co- not do them justice and possibly hinders our progress in evolutionary arms–race theory (Ehrlich and Raven, understanding their roles for plant survival in hostile 1964), a key concept in plant–herbivore interactions environments. (Berenbaum and Zangerl, 2008; Jander, 2018), postulates INTEGRATION OF PLANT SECONDARY METABOLITES INTO REGULATION AND METABOLISM Early Evidence for Metabolic Integration of Secondary Metabolites In 1977, David Rhoades studied the properties of creosotebush (Larrea spp.) leaf resin. He found that the resin, which contained high levels of phenylpropanoid derivatives (lignans), absorbed ultraviolet radiation, reduced evaporative water loss across cellulose mem- branes, and had the capacity to form complexes with proteins, thus possibly reducing the digestibility of plant materials for herbivores (Rhoades, 1977, p. 281). Rhoades (1977) thus postulated that “.any chemical system possessed by a plant must necessarily be inte- grated into the total metabolic scheme and multiple functions are to be expected.” In other words, Rhoades (1977) proposed that secondary metabolites are not end Figure 1. Low molecular weight compounds in plants are functionally points, but integrated components of plant metabolism, classified as primary metabolites, secondary metabolites, or hormones. and may, by consequence, take on any number of Present work on plant secondary metabolites demonstrates that many of functions, similar to other plant metabolites. Indeed, them also have regulatory roles, and some are demonstrated precursors evidence was emerging at that time that secondary of primary metabolites. Note that primary metabolites and hormones metabolites may regulate growth and defense, as ex- also show functional overlap with the other metabolite classes (not discussed here). These findings blur the functional trichotomy of plant ogenously applied flavonoids could modulate polar metabolism and call for a reassessment of ecological and evolutionary auxin transport and catabolism (Stenlid, 1963; Stenlid, frameworks that are based on this model. 1976), glucosinolate breakdown products could replace 40 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites auxins in inducing hypocotyl bending (Hasegawa et al., sinapoylmalate (Kim et al., 2015). The phenylpropanoid 1986), and induced volatiles promoted resistance and phenotype is rescued in mutants that no longer produce defense regulation in neighboring trees (Baldwin and the substrate of CYP83B1, indole‐3‐acetaldoxime (Kim Schultz, 1983; Rhoades, 1983). et al., 2015), suggesting that it may be the aldoxime overaccumulation rather than the lack of downstream glucosinolates that suppresses sinapoylmalate. Sup- pressor screens showed that the phenylpropanoid Secondary Metabolites as Regulators of Plant Defense phenotype is also absent in plants that have mutated Following early preliminary evidence of secondary MEDa/b genes, which encode key components of a large multisubunit transcriptional complex that regulates metabolites regulating defenses, genetic evidence fol- lowed in 2009, when it was reported that Arabidopsis phenylpropanoid biosynthetic genes (Kim et al., 2015; (Arabidopsis thaliana) mutants defective in indole glu- Dolan et al., 2017). A recent study demonstrates that cosinolate biosynthesis no longer mount a callose de- a group of Kelch Domain F‐Box (KFB) genes that are fense response following Flg22 treatment. Callose involved in PAL inactivation (Zhang et al., 2013) are up- formation is rescued by adding 4-methoxy-indol-3- regulated in indole glucosinolate mutants in a MED5- ylmethylglucosinolate (Clay et al., 2009). The myrosinase dependent manner, whereas PAL-activity is suppressed PEN2 is required for this phenomenon, implicating (Kim et al., 2020). PAL-activity and sinapoylmalate ac- glucosinolate breakdown in callose regulation (Clay cumulation are (partially) rescued in glucosinolate- et al., 2009). Shortly thereafter, it was discovered that deficient KBF mutants (Kim et al., 2020). The model indole-derived benzoxazinoid secondary metabolites emerging from these studies is that aldoximes, which have a comparable callose regulatory function in ce- accumulate in CYP83B1 mutants, increase KFB-mediated reals. Benzoxazinoid-deficient bx1 maize (Zea mays) PAL degradation through MED5 transcriptional regula- mutants are defective in aphid- and chitosan-induced tion as well as other, yet unknown, mechanisms (Kim callose deposition, and callose induction is rescued by et al., 2015; Kim et al., 2020). As aldoximes are produced the addition of DIMBOA or DIMBOA-Glc (Ahmad by many different species, this form of defense regula- et al., 2011; Meihls et al., 2013). In both cases, the ca- tion may also occur beyond glucosinolate-producing pacity to regulate callose is structurally specific and plants (Kim et al., 2020). Interestingly, wheat lines over- depends on the modification of the indole-derived ring. expressing a maize benzoxazinoid O-methyl transferase In Arabidopsis, indol-3-ylmethylglucosinolate, which and thus accumulate more HDMBOA-Glc and less lacks a methylated hydroxy-group on the aromatic DIMBOA-Glc also show higher levels of the phenyl- ring, is inactive, whereas the methylated form is active propanoid ferulic acid, despite unaltered pool sizes of (Clay et al., 2009). In maize, DIMBOA-Glc, which lacks amino acid precursors (Li et al., 2018a), suggesting that a methylated hydroxy-group at the nitrogen, is active, phenolic compounds may also be regulated by other secondary metabolite pathways. whereas the methylated form (HDMBOA-Glc) is inac- tive (Li et al., 2018a). Whereas the callose response to Apart from glucosinolates and benzoxazinoids, benzoxazinoids is conserved between wheat (Triti- volatile secondary metabolites such as terpenoids, cum aestivum) and maize, they do not elicit callose in green-leaf volatiles, and aromatic compounds can also Arabidopsis, and intact glucosinolates do not elicit regulate plant defenses (Baldwin et al., 2006; Godard callose in maize (Li et al., 2018a). These studies show et al., 2008; Erb, 2018; Bouwmeester et al., 2019). Many that callose regulation by secondary metabolites is of these volatiles are released upon herbivore- or highly specific, tightly controlled, and likely evolved pathogen attack and are capable of directly inducing or repeatedly. The mechanism underlying secondary priming hormonal defense signaling pathways and metabolite–induced callose formation awaits to be resistance. In maize, for instance, mutants that are de- elucidated. Glucosinolates and benzoxazinoids may, fective in their capacity to produce volatile indole are for instance, promote callose production by regulat- unable to prime their systemic tissues to rapidly release ing hormonal pathways (Burow et al., 2015; Katz terpenes upon herbivore attack (Erb et al., 2015). Add- et al., 2015), through transcriptional regulation (Kim ing indole to the headspace of maize plants restores this et al., 2015), or by directly initiating callose formation priming phenotype (Erb et al., 2015). Rice (Oryza sativa) posttranslationally. plants also respond to indole through priming of early Interestingly, glucosinolates and benzoxazinoids defense signaling elements such as the map kinase also seem to regulate the accumulation of other sec- OsMPK3 (Ye et al., 2019). Transgenic plants that are ondary metabolites (Hemm et al., 2003; Kim et al., 2015; deficient in OsMPK3 expression are no longer respon- Li et al., 2018a). In Arabidopsis, mutants that are de- sive to indole, suggesting that indole acts via the fective in the atypical myrosinase PEN2 release lower priming of early defense signaling (Ye et al., 2019). In amounts of Trp-derived metabolites such as camalexin Arabidopsis, geranylgeranyl reductase1 mutants are de- upon flg22 treatment (Frerigmann et al., 2016) and in- fective in systemic acquired resistance against P. syringae fection by Pseudomonas syringae (Stahl et al., 2016). (Riedlmeier et al., 2017). Adding the pathogen-induced Furthermore, mutants defective in the CYP83B1 en- volatiles a-and b-pinene to the headspace of the mu- tant restores resistance, with the response depending zyme required for indole glucosinolate production also show lower accumulation of the phenylpropanoid on intact salicylic acid signaling and the AZELAIC ACID Plant Physiol. Vol. 184, 2020 41 Erb and Kliebenstein INDUCED (AZI1) gene (Riedlmeier et al., 2017). The unknown. Studies on the indole glucosinolate break- precise role of other volatile secondary metabolites that down product indole-3-carbinol have identified an can regulate defenses at physiological concentrations, unexpected target protein. Indole-3-carbinol accumu- including homoterpenes (Arimura et al., 2000) and green- lates upon wounding in Arabidopsis and rapidly re- leaf volatiles (Ameye et al., 2018), has not yet been ex- duces root growth upon exogenous application. In plored using genetic approaches, but their activity has vitro, indole-3-carbinol interferes with the interaction been demonstrated clearly through chemical comple- between auxin and its receptor TIR1 by binding at an mentation (Arimura et al., 2000; Engelberth et al., 2004; allosteric site (Katz et al., 2015). As the indolic glucosi- Frost et al., 2008; Meents et al., 2019). Further support for nolate catabolite likely binds directly to TIR1 (Katz the potential regulatory role of defense volatiles comes et al., 2015), one may argue that TIR1 acts as an from LOX2-silenced Nicotiana attenuata plants, which are indolic glucosinolate receptor that mediates the regu- deficient in the production of herbivory-induced, green- lation of growth by a plant secondary metabolite. leaf volatiles. In contrast with the other systems where Another link to auxin signaling was found with a volatiles induce defense, the LOX2 mutation leads to structurally unrelated aliphatic glucosinolate. This was stronger expression of defense-related genes in neighbors found by the initial observation that the auxin-sensitive than wild-type plants, suggesting that volatiles can also repressors IAA5, IAA6, and IAA19 strongly regulate suppress defenses (Paschold et al., 2006). 4-methylsulfinylbutyl glucosinolate (4-MSOB) levels in In summary, at least five classes of secondary me- dehydrated Arabidopsis plants (Salehin et al., 2019). tabolites (glucosinolates, benzoxazinoids, terpenes, ar- Iaa5,6,19 mutants fail to close their stomata upon drought omatics, and green-leaf volatiles) are now confirmed to stress, a phenotype that can be reverted by adding act as potential regulators of in planta defense. It is 4-MSOB (Salehin et al., 2019). Together with the finding exciting to speculate that there are many other sec- that glucosinolate biosynthesis and activation mutants ondary metabolites that play similar regulatory roles. are less tolerant to drought (Salehin et al., 2019), and that An important gap of knowledge is the mechanism glucosinolate breakdown products can trigger stomatal by which secondary metabolites regulate defenses. As closure in Arabidopsis and Vicia faba (Khokon et al., many of the secondary metabolites are chemically re- 2011; Hossain et al., 2013), these results provide evi- active (Farmer and Davoine, 2007; Hadacek et al., 2010), dence that aliphatic glucosinolates are involved in sto- it is possible that they act indirectly by depleting de- matal regulation. Interestingly, glucosinolate-mediated toxification enzymes, thus triggering the accumulation stomatal regulation requires a functional ROS receptor of known signaling molecules such as reactive oxygen kinase (GHR1; Salehin et al., 2019). Given that the my- species (ROS; Khokon et al., 2011). However, as dis- rosinase TGG1 accumulates in guard cells and is re- cussed below, secondary metabolites may also have quired for stomatal regulation (Zhao et al., 2008), and hormone-like properties by binding to specific receptor that glucosinolate breakdown products can regulate proteins (Katz et al., 2015). More work on the targets of stomatal closure through ROS production (Khokon secondary metabolites in planta is clearly warranted et al., 2011), it is conceivable that ROS link endoge- and would help to clarify the ecological and evolu- nous glucosinolates to stomatal regulation (Khokon tionary context of their capacity to regulate defenses. et al., 2011). Apart from growth and stomatal opening, glucosi- nolates may also regulate the circadian clock and flowering time. Natural presence/absence variation Secondary Metabolites as Regulators of Growth in the 2-oxoglutarate-dependent dioxygenase AOP2, and Development which converts methylsulfinylalkyl glucosinolates into Plants regulate their growth dynamically and often alkenyl glucosinolates, is linked to variation in the ex- reduce their investment into growth and development pression of the major flowering gene FLC and to vari- upon herbivore- or pathogen attack. This reduction in ation in flowering time (Kliebenstein et al., 2001; Atwell growth is thought to be largely due to the reconfigu- et al., 2010). Introducing a functional AOP2 into Ara- ration of a plant’s signaling network rather than a lack bidopsis Col-0 (a natural AOP2 knockout) confirmed of resources (Kliebenstein, 2016; Machado et al., 2017; the flowering time effect, identified a shift in the ex- Guo et al., 2018). Strikingly, plant secondary metabo- pression of circadian genes, and showed a 1-h decrease lites and their breakdown products are being (re)-dis- in clock periodicity (Kerwin et al., 2011). Abolishing covered as plant growth modulators, thus adding glucosinolate production using Myb transcription fac- another layer of regulation to growth-defense patterns. tor mutants led to the same periodicity shift, suggesting Again, glucosinolates provide a mechanistic example of that the effect may be linked to the presence of the how secondary metabolites can modulate growth. When 4-MSO glucosinolate in wild-type Col-0 (Kerwin et al., applied to the roots of Arabidopsis and many other plant 2011). The effect of the AOP locus on flowering time species, the aliphatic 3-hydroxypropylglucosinolate in- depends on the genetic background (Jensen et al., 2015), hibits root meristematic growth at physiological con- supporting the hypothesis that secondary metabolites centrations via an intact Target of Rapamycin pathway are integrated into a complex and variable regulatory network. How aliphatic glucosinolates directly regu- (Malinovsky et al., 2017). The exact molecular interac- tion partner of 3-hydroxypropylglucosinolate remains late gene expression networks and developmental 42 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites phenotypes such as flowering time remains to be possible that flavonols also function as signals and tested (Burow and Halkier, 2017). further work is needed to differentiate between these The present data suggest that glucosinolates can hypotheses. influence growth by multiple different mechanisms, Other secondary metabolites may also regulate plant including Target of Rapamycin regulation, auxin development. Diploid oat sad2 mutants that overpro- regulation, auxin-independent transcriptional regula- duce the triterpene b-amyrin produce shorter roots and tion, and auxin-mediated ROS accumulation (Katz significantly more root hairs than wild-type plants, et al., 2015; Kim et al., 2015; Malinovsky et al., 2017; phenotypes which are absent in other mutants of the Salehin et al., 2019). This diversity, paired with the pathway that do not overproduce b-amyrin (Kemen substantial variation in glucosinolate biosynthesis et al., 2014). However, this phenotype cannot be phe- within species, creates a wealth of metabolic networks nocopied by adding b-amyrin to roots, possibly because and phenotypes, which can be acted upon by natural its activity requires specific spatiotemporal accumula- selection. It is tempting to speculate that this diversity tion patterns (Kemen et al., 2014). In N. attenuata, is a reflection of the highly diverse habitats and envi- silencing a malonyltransferase that malonylates 17- ronments that a single species can inhabit and may Hydroxygeranylinalool diterpene glycosides reduces provide adaptive potential beyond conserved hor- floral style cell size and length (Li et al., 2018b). monal pathways. Knocking down diterpene glycoside production by si- In addition to glucosinolates, flavonoids are impli- lencing a geranylgeranyl diphosphate synthase abol- cated in regulating plant growth, development, and ishes the effect of the malonyltransferase, suggesting environmental responses. Exogenously applied flavo- that specific diterpene hexose decoration patterns are noids have long been known to modulate auxin trans- responsible for the flower phenotype (Li et al., 2018b). port (Stenlid, 1976). Evidence that flavonoids may also Furthermore, a labeling experiment in poplar recently act as endogenous growth regulators came from an uncovered that herbivore-attacked leaves can convert Arabidopsis chalcone synthase mutant, transparent testa benzyl cyanide, a herbivore-induced volatile, to the (tt4). tt4 plants show growth alterations that are char- auxin phenylacetic acid (Günther et al., 2018), thus acteristic of disturbed auxin localization, including re- providing a potential link between the catabolism of duced root growth and gravitropism (Brown et al., volatile secondary metabolites and the regulation of 2001). The tt4 mutant also displays increased auxin plant growth and development. transport (Murphy et al., 2000), which can be reversed The examples above show how secondary metabo- by adding the flavonoid precursor naringenin (Brown lites can modulate growth and development through a et al., 2001). Further mechanistic studies suggest that variety of mechanisms, some of which are barely dis- flavonoids modulate auxin transport through several tinguishable from mechanisms normally assigned to mechanisms, including interactions with auxin trans- plant hormones (Fig. 2). Whereas some of these sec- porters and transport-regulating proteins (Peer and ondary metabolite regulators are ancient and highly Murphy, 2007; Santelia et al., 2008). Arabidopsis roots conserved (e.g. flavonoids, terpenes), others evolved grow away from light and flavonoids accumulate in more recently (e.g. glucosinolates and benzoxazinoids) their light-exposed sides (Silva-Navas et al., 2016). The and are restricted to specific plant families. Plants thus tt4 mutant also shows reduced light avoidance, which have both a conserved and a unique, variable, and was linked to reduced auxin polar transport and re- flexible repertoire of regulators at their disposition to duced ROS accumulation, both of which can regulate adjust growth and development, which likely contrib- cell division and elongation (Gayomba et al., 2010; utes to their potential to colonize variable and chal- Silva-Navas et al., 2016). tt4 also displays lower accu- lenging habitats. mulation of flavonols and increased ROS levels in guard cells, phenotypes associated with more rapid absisic acid–induced stomatal closure (Watkins et al., Secondary Metabolites as Primary Metabolites 2014). An additional link between flavonoid biosyn- thesis, ROS accumulation, and plant development was If secondary metabolites can regulate growth, uncovered recently in tomato (Solanum lycopersicum; development, and defense, can they also function Muhlemann et al., 2018). The are mutant is defective in a as primary metabolites? Whereas primary metabo- flavonol 3-hydroxylase (F3H), displays reduced flavo- lites are highly conserved, secondary metabolites nol and increased ROS accumulation in pollen grains, evolve dynamically and are inherently variable in and suffers from reduced pollen tube growth and in- structure and production (Wink, 2008). This rapid tegrity. The pollen tube phenotype can be rescued by evolution would seem to complicate their integra- the addition of antioxidants (Muhlemann et al., 2018). tion into the most fundamental workings of plant Flavonols are thus thought to act as antioxidants that metabolism because it would require a rapid evolu- reduce ROS accumulation and thereby regulate plant tion of enzymes to connect these new structures into development (Hernández et al., 2009; Muhlemann the more conserved metabolic pathways. However, et al., 2018). However, the oxidation state of a cell can evidence for secondary metabolites that are not directly influence signaling by altering disulfide bridge strictly essential, but nevertheless contribute to pri- formation or other protein modifications. Thus, it is mary metabolism, is emerging. In Arabidopsis, plants Plant Physiol. Vol. 184, 2020 43 Erb and Kliebenstein Figure 2. Glucosinolates and benzoxazinoids as examples of secondary metabolites that blurr the functional trichotomy of plant metabolism. Dif- ferent functions of glucosinolates in Arabidopsis and benzoxazinoids in maize and wheat are depicted. Genes that are known to be involved in the different functions are indicated. Note that a direct role of benzoxazinoids and glucosinolates as plant primary metabolites (for instance, in the context of nitrogen/sulfur and/or energy storage) has not been clearly demonstrated so far. *MEDs and KFBs are likely regulated by aldoxime pre- cursors of glucosinolates. For references, see the article. with mutations in the flavonoid pathway upstream are proposed to take the deglycosylated cyanogen of the FLAVANONE-3-HYDROXYLASE (F3H) show and directly release ammonia and the corresponding a reduction in the respiratory cofactor ubiquinone acetate (Jenrich et al., 2007). Further support for the (coenzyme Q; Soubeyrand et al., 2018). Ubiquinone potential of cyanogenic glucosides as a primary me- levels can be restored by adding dihydrokaempferol or tabolite store came from overexpression of a hydrox- kaempferol to the mutants. Labeling experiments dem- ynitrile lyase, which is involved HCN formation in onstrate that the aromatic ring of kaempferol is integrated cassava (Manihot esculenta). Theseplantshavede- into ubiquinone, and that heme-dependent peroxidases creased concentrations of cyanogenic glycosides and likely use kaempferol to produce 4-hydroxybenzoate as increased concentrations of total amino acids, sug- a substrate for ubiquinone (Soubeyrand et al., 2018). gesting that cyanogenic glycosides may be degraded The integration of flavonoids into primary metabo- and reintegrated into primary metabolism (Narayanan lism is perhaps not surpising, because they represent et al., 2011). The potential integration of other secondary one of the oldest and most conserved classes of sec- metabolites such as glucosinolates is currently under in- ondary metabolites (albeit with substantial interspecific vestigation. In Arabidopsis, sulfur deficiency induces the variation in glycosylation patterns). Flavonoid evolu- expression of the myrosinases BGLU28 and BGLU30 tion precedes the emergence of many innovations in (Maruyama-Nakashita et al., 2003). Under sulfur-limiting plant primary metabolism, such as C photosynthesis. conditions, bglu28/30 double mutants accumulate higher Whether younger, more specialized secondary metab- levels of intact aliphatic glucosinolates, contain lower olites can act as primary metabolites is not well un- amounts of Cys and protein sulfur content, and grow derstood. This lack of knowledge is closely related to a less than wild-type plants, suggesting that glucosino- limited understanding of secondary metabolite catab- lates may serve as sulfur-storage molecules (Zhang olism. Where do these compounds go when they are no et al., 2020). longer needed? One would assume that reintegrating Detailed biochemical characterization and metabolic secondary metabolites into primary metabolism is profiling of secondary-metabolite mutants, combined beneficial for plants (Neilson et al., 2013). Such a rein- with complementation and labeling experiments, are tegration pathway has been proposed for cyanogenic required to further corroborate the potential roles of glycosides (Selmar et al., 1988). Upon deglycosylation, secondary metabolites in primary metabolism and to HCN may be assimilated into Asn via the formation of identify additional secondary metabolites that serve as b-cyano-Ala (Selmar et al., 1988). Indeed, two sorghum primary-metabolite precursors. Degradation of many (Sorghum bicolor) nitrilases are capable of producing different secondary metabolites has been observed Asn from b-cyano-Ala (Jenrich et al., 2007). An al- under specific environmental conditions (Negi et al., ternative pathway not involving the release of HCN 2014; Zipor et al., 2015). Furthermore, alterations in was suggested in sorghum. In this system, nitrilases primary metabolites are observed in various plants 44 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites with altered secondary metabolism (Mayer et al., 2001; between secondary metabolites and hormones, for Narayanan et al., 2011; Huber et al., 2016; Machado instance (Malinovsky et al., 2017; Sun et al., 2019b). et al., 2017; Zhang et al., 2020), and the accumulation Overall, the functional integration of secondary me- of specific secondary metabolites has been associated tabolites at a given point in evolution is a likely with storage and growth in microevolutionary studies consequence of the interaction between complex (Heath et al., 2014). Keeping an open mind about the environments with highly connected plant metabolic capacity of plants to evolve integrated metabolic networks. Below, we discuss the potential benefits of networks is warranted to gain a better comprehen- plant secondary metabolite metabolic integration that sion of the prevalence and importance of secondary may have favored their use as regulators and primary metabolites as precursors of primary metabolites. metabolites. Plant Secondary Metabolites as Reliable Readouts of Secondary Metabolites as Facilitators of Defense Activation Micronutrient Uptake Plants control defense activation to save metabolic An additional example that further blurs the dis- energy and avoid self-damage. Defense investment tinction between primary and secondary metabolism is is typically titrated through feedback regulation, plant micronutrient uptake. Grasses excrete low M including both positive and negative feedback loops compounds into the rhizosphere to chelate micronu- that are built into early defense signaling (Hu et al., trients such as iron and thus make them biologically 2015; Li et al., 2015) and hormonal networks (Gilardoni available (Curie and Briat, 2003). Recent work suggests et al., 2011; Liu et al., 2019). A limitation of these feedback that secondary metabolites are likely important for iron loops is that they do not provide direct information about uptake in both herbs and grasses. Chemical removal of the final level of defense activation (i.e. the production excreted phenolic acids from the nutrient solution of of defense metabolites per se). Because herbivores and red clover (Trifolium pretense) was found to result in iron pathogens may interfere with the production of defense deficiency in red clover (Jin et al., 2007). Subsequently, compounds at many levels, including in the final steps an Arabidopsis mutant, which is deficient in the 2- of biosynthesis (Jones et al., 2019), integrating them oxoglutarate-dependent dioxygenase Feruloyl-CoA directly into regulatory feedback loops may allow 69-Hydroxylase 1 and thus no longer able to pro- plants to more accurately monitor and adjust defense duce coumarins, was found to suffer from iron defi- accumulation. Using secondary metabolites as de- ciency under alkaline conditions (Schmid et al., 2014). fense activation readouts may also help plants to Similarly, young maize benzoxazinoid mutants that do no longer produce and excrete benzoxazinoids optimize synergies between different defenses and to were found to suffer from iron deficiency when compensate for accidental failures of specificde- fense pathways. The increasing number of examples growing in the presence of iron salts (Hu et al., 2018). showing that plant secondary metabolites regulate Both benzoxazinoids and coumarins are able to che- defenses (see section “Secondary Metabolites as late iron in vitro (Bigler et al., 1996; Mladenka et al., Regulators of Plant Defense”) hint at the existence of 2010). Because these complexes are essential for plant such systems. growth and development by providing essential As many secondary metabolites are compartmental- micronutrients, they should, according to definition, ized and/or stored in inactive forms, their decom- be classified as primary metabolites, thus provid- partmentalization and/or activation likely also helps ing another illustration of how secondary metabo- plants to recognize tissue damage and other forms of lites can turn into primary metabolites under given environmental stress. In this case, the metabolites conditions. wouldbeusedasdamage–associated molecular patterns (DAMPs). Green-leaf volatiles are an example of second- ary metabolites that are also DAMPs (Tanaka et al., 2014; ADAPTIVE EXPLANATIONS FOR METABOLIC Quintana-Rodriguez et al., 2018). Another potential INTEGRATION OF SECONDARY METABOLITES example of secondary metabolites as DAMPs is the There isnow ampleevidencefor secondaryme- previously discussed links between indolic glucosino- tabolites that are regulators and precursors of pri- lates and DIMBOA regulation of callose upon pathogen mary metabolites. But why would plants evolve attack. Interestingly in this case, the secondary metab- an integrated metabolism in which the same metab- olite/DAMPs are linked to endogenous responses to olite class has multiple functions that incorporate pathogen-associated molecular patterns (e.g. FLS2; growth, development, defense, and regulation? Plants Clay et al., 2009) and stomatal closure upon drought have large, interconnected metabolic networks at their stress (Salehin et al., 2019). disposition. Natural selection acts on these metabolic Given these considerations, secondary metabolites networks, resulting in the evolution of network topol- may be common readouts of defense activation and ogies that maximize fitness. Over evolutionary time, damage may have favored their evolution as defense these topologies likely include dynamic transitions regulators. Plant Physiol. Vol. 184, 2020 45 Erb and Kliebenstein Figure 3. Functional integration of plant secondary metabolites shapes plant–herbivore and tritrophic interactions. Schematic representation of how different functions of secondary metabolites are used by plants, herbivores, and natural enemies of her- bivores is shown. Plants use secondary metabolites for multiple purposes, including resistance, regulation, and primary me- tabolism (see Fig. 2). Recent work suggests that this multifunctionality is mirrored in adapted herbivores, which also use secondary metabolites for multiple purposes, including similar and new functions. Little is known about how adapted natural enemies use secondary metabolites, but multifunctional integration across three trophic levels is likely (Box 2). Circles represent hypothetical individual secondary metabolites (for color code, refer to Figs. 1 and 2). Solid lines indicate metabolic connections within an organism. Dashed lines indicate similar functions of the same compounds in different organisms. Metabolic Network Specialization as a Potential Means to Multifunctionality as a Cost-Saving Strategy Resist Manipulation Producing secondary metabolites has energetic and Herbivores, pathogens, and viruses can interfere with metabolic costs (Gershenzon, 1994). These costs are not defense hormone signaling and thereby manipulate always evident (Züst et al., 2011; Machado et al., 2017), plants for their own benefit (Kazan and Lyons, 2014; Stahl and may mostly occur under specific environmental et al., 2018). The high degree of conservation in defense conditions such as strong competition and nutrient hormone signaling may in fact favor the evolution of bi- limitation (Cipollini et al., 2018). Plants likely manage otic manipulation of plant signaling (Berens et al., 2017). costs of secondary metabolite production through the For example, if an attacking organism evolves the ability regulation of biosynthesis, but controlled recycling of to alter jasmonate signaling, this may provide it a fitness the resulting compounds would enhance the plants benefitonawide varietyofhost plantsand mayreduce ability to recoup costs in challenging environments the advantage for plants to evolve new inducible resis- (Neilson et al., 2013). Secondary metabolites that are tance mechanisms regulated by these hormones. One induced upon environmental stress could for instance possibility to solve this problem would be to use less- be recycled back into primary metabolism once the conserved metabolites as defense regulators. If a plant stress subsides. One way of testing this hypothesis is to had the ability to use these metabolites, it would be manipulate secondary metabolite recycling by target- less likelytofallpreytohostswitching by hormone- ing enzymes involved in their degradation, such as manipulating enemies. The evolution of (specialized) glucosidases (Morant et al., 2008) or nitrilases (Jenrich secondary metabolites into regulatory networks may thus et al., 2007). With use of this approach, a link between be promoted through the evolution of manipulation the degradation of cyanogenic glycosides and plant strategies in plant enemies. Clear examples supporting protein supply was uncovered (Narayanan et al., 2011), this hypothesis are currently lacking. As the biosynthesis supporting the hypothesis that reintegration of sec- of defense-regulating secondary metabolites such as glu- ondary compounds into primary metabolism may be cosinolates is at least partially controlled by conserved advantageous for the plant. A caveat of this approach is phytohormonal pathways (Schweizer et al., 2013), plant that it remains difficult to disentangle a direct contri- enemies that are capable of overcoming these conserved bution of the generated catabolites to primary metab- pathways may also suppress more specificregulators. olism from their potential regulatory roles. A more Interestingly, an opposite pattern has also been found for detailed understanding of secondary metabolite sig- the tomato leaf spot fungus, which uses a hydrolase to naling and catabolism would help to explore the role of detoxify steroidal glycoalkaloids and benefits from the secondary metabolite reintegration as a cost-saving defense-suppressing properties of the resulting break- strategy. down products (Bouarab et al., 2002). This illustrates that Another way to minimize costs is to use the same specialized plant enemies may also misuse the regulatory secondary compound for multiple purposes (Neilson properties of secondary metabolites of their host plants. et al., 2013). As many secondary compounds are 46 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites Box 1. Case study of secondary metabolite multifunctionality. Cited articles: Glauser et al., 2011; Robert et al., 2012, 2017; Maag et al., 2016. Box 2. Multifunctionality of plant secondary metabolites in tri- trophic interactions. Cited articles: Fink and Brower, 1981; Hunter, 2003; Sarfraz et al., 2009; Sloggett and Davis, 2010; Aartsma et al., chemically reactive, they need to be managed by the 2017; Rafter et al., 2017; Robert et al., 2017; Turlings and Erb, 2018; plant through (potentially costly) storage, inactivation, Sun et al., 2019a; Ugine et al., 2019; Zhang et al., 2019. and/or resistance mechanisms, including specialized cells, ducts, and glands (Sirikantaramas et al., 2008). By employing the same compound class for multiple ECOLOGICAL CONSEQUENCES OF THE purposes, plants may spread these fixed costs across METABOLIC INTEGRATION OF more fitness components and increase their competi- SECONDARY METABOLITES tiveness. Metabolic costs may also be lowered by using the same biosynthetic machinery to produce different The separation of low M compounds into primary compounds for different purposes. Whereas the cost- metabolites, secondary metabolites, and hormones has saving aspects of multifunctionality are difficult to shaped our ecological and evolutionary thinking of quantify, multifunctionality seems to be a widespread plant–environment interactions. If we abolish this view property of secondary metabolites, as discussed above, in favor of a more integrated perspective (i.e. where and it is difficult for this multifunctionality to evolve secondary metabolites can have regulatory roles and without benefit. can provide precursors for primary metabolites), we Plant Physiol. Vol. 184, 2020 47 Erb and Kliebenstein can derive new hypotheses on plant defense patterns herbivores to metabolize these compounds. Recent ex- and plant–herbivore interactions. These hypotheses are amples also hint at the possibility that plant secondary likely to improve our understanding of the ecological metabolites may have hormonal functions in herbi- roles of plant secondary metabolites in the future. vores. In rice, knocking down CYP71A1, a gene re- sponsible for the production of serotonin, a monoamine neurotransmitter, reduces the performance of the rice Ontogenetic Patterns of Secondary Metabolite Production brown planthopper (Nilaparvata lugens). Adding sero- tonin to an artificial diet enhances its performance (Lu Many secondary metabolites show distinct onto- et al., 2018), suggesting that the herbivore may benefit genetic accumulation patterns, with concentrations from the hormonal properties of this plant metabolite. varying over time and between tissues. Ecological the- Plants may also benefit from producing secondary ory explains this within-plant variation using resource metabolites that act as (de)-regulators of herbivore constraints, allocation costs, and variation in herbivore physiology. Spinach (Spinacia oleracea), for instance, pressure (McKey, 1974; van Dam, 2009; Meldau et al., produces the molting hormone 20-hydroxyecdysone 2012; Schuman and Baldwin, 2016; Barton and Boege, (Bakrim et al., 2008), which can interfere with caterpil- 2017). The above theories are all based on costs and lar development (Kubo et al., 1983). benefit relationship, with the benefit typically being In general terms, a plant’s metabolism is shaped by a limited to herbivore resistance. Given the blurred tri- dynamic landscape of environmental selection pres- chotomy of plant secondary metabolism, the ecological sure; conversely, the metabolic network of herbivores balance sheet may be improved by taking into account is shaped by the functional and chemical potential of multifunctionality (Barton and Boege, 2017). A drop in plant metabolites within the herbivore’s own selection secondary metabolite levels, as is often observed a few landscape. One can thus expect that, similar to what weeks after germination or at the onset of flowering, for Rhoades postulated for plants (Rhoades, 1977), any instance (Meldau et al., 2012; Barton and Boege, 2017), chemical system taken up by a herbivore must neces- may reflect an increased need of primary metabolites sarily be integrated into its total metabolic scheme, and and nutrients rather than a drop in herbivore pressure. multiple functions of plant secondary metabolites are to Similarly, strong expression of secondary metabolites in roots may not only be the result of high tissue value and a high risk of root herbivore attack, but may simply reflect additional functions of the compounds such as micronutrient uptake and microbial conditioning (Hu et al., 2018; Stringlis et al., 2018). Our understanding of ontogenetic allocation patterns of secondary metabo- lites may thus improve if we take their full metabolic integration and potential multifunctionality into ac- count and do not limit their considered benefits to herbivore resistance. Defense Metabolites in Plant–Herbivore Interactions The functional trichotomy used to define plant me- tabolites has also shaped our understanding of how these metabolites influence plant–herbivores interac- tions. Herbivores are assumed to forage for primary metabolites while trying to avoid the negative effects of secondary metabolites through behavioral and meta- bolic adaptations (Behmer, 2009; Stahl et al., 2018). If we accept that secondary metabolites can also be regula- tors and precursors of primary metabolites, then it be- comes conceivable that they may have similar roles in herbivores. The root-feeding larvae of the western corn rootworm for instance forage for iron-benzoxazinoid complexes to acquire iron and improve their growth, thus effectively using a plant secondary metabolite as a primary metabolite (Hu et al., 2018). Several other herbivores also gain more weight in the presence of plant secondary metabolites (Meldau et al., 2009; Richards et al., 2012; Marti et al., 2013; Veyrat et al., 2016; Wetzel et al., 2016), and it is conceivable that some of these effects may be due to the capacity of the 48 Plant Physiol. Vol. 184, 2020 Blurred Functional Boundaries of Plant Metabolites be expected, some of which likely mirror their multiple secondary metabolites are highly integrated into plant functions in plants (Fig. 3). Specialist herbivores are metabolism and can serve as both regulators and pri- known to use secondary metabolites as infochemicals mary metabolites. Thus, it is likely that most secondary (e.g. foraging cues), and some also sequester defenses to metabolites have additional functions for plants. Tak- protect themselves against herbivore natural enemies ing into account these additional functions (see Out- (Nishida, 2002; Opitz and Müller, 2009), in analogy to standing Questions), we can refine key concepts in the use of these chemicals as defense regulators and plant-environment interactions and improve our un- resistance factors in plants (Fig. 3). Cabbage aphids derstanding of the chemical ecology of plants and their (Brevicoryne brassicae) are an illustrative example in enemies. this context, as they can activate glucosinolates by producing their own myrosinases (Bridges et al., 2002; ACKNOWLEDGMENTS Kazana et al., 2007). This allows them to use glucosi- We would like to thank Mike Blatt for the invitation to write this Inaugural nolates as two-component defense system against Topical Review, Pierre Mateo for drawing chemical structures, and Christelle predators (Kazana et al., 2007). As glucosinolate A.M. Robert, Clint Chapple, Jonathan Gershenzon, and two anonymous reviewers as well as the Twitter community for helpful comments on an earlier breakdown products (isothiocyanates) also increase version of this manuscript. aphid responses to alarm pheromones (Dawson et al., Received April 7, 2020; accepted June 15, 2020; published July 7, 2020. 1987), it was proposed that aphid-released isothiocya- nates may also act as danger signals (Bridges et al., 2002). Another example where herbivores use second- LITERATURE CITED ary metabolites for several purposes that mirror their Aartsma Y, Bianchi FJJA, van der Werf W, Poelman EH, Dicke M (2017) multiple uses by plants are again benzoxazinoids, Herbivore-induced plant volatiles and tritrophic interactions across which are used as defense metabolites and sidero- spatial scales. New Phytol 216: 1054–1063 phores by a specialist root herbivore in maize (Box 1). Ahmad S, Veyrat N, Gordon-Weeks R, Zhang Y, Martin J, Smart L, Apart from mirroring plant functions, adapted herbi- GlauserG,Erb M, FlorsV,FreyM,etal (2011) Benzoxazinoid me- vores can also use plant secondary metabolites for tabolites regulate innate immunity against aphids and fungi in maize. herbivore-specific functions. 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PLANT PHYSIOLOGYOxford University Press

Published: Jul 7, 2020

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