Growth defence balance in grass biomass production: the role of jasmonates

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1 Journal of Experimental Botany Advance Access published February 22, 2015 Journal of Experimental Botany doi: /jxb/erv011 Review Paper Growth defence balance in grass biomass production: the role of jasmonates Christine Shyu and Thomas P. Brutnell* Donald Danforth Plant Science Center, St. Louis, MO 63132, USA * To whom correspondence should be addressed. TBrutnell@danforthcenter.org Received 11 November 2014; Revised 11 December 2014; Accepted 23 December 2014 Abstract Growth defence balance is the selective partitioning of resources between biomass accumulation and defence responses. Although it is generally postulated that reallocation of limited carbon pools drives the antagonism between growth and defence, little is known about the mechanisms underlying this regulation. Jasmonates (JAs) are a group of oxylipins that are required for a broad range of responses from defence against insects to reproductive growth. Application of JAs to seedlings also leads to inhibited growth and repression of photosynthesis, suggesting a role for JAs in regulating growth defence balance. The majority of JA research uses dicot models such as Arabidopsis and tomato, while understanding of JA biology in monocot grasses, which comprise most bioenergy feedstocks, food for human consumption, and animal feed, is limited. Interestingly, JA mutants of grasses exhibit unique phenotypes compared with well-studied dicot models. Gene expression analyses in bioenergy grasses also suggest roles for JA in rhizome development, which has not been demonstrated in Arabidopsis. In this review we summarize current knowledge of JA biology in panicoid grasses the group that consists of the world s emerging bioenergy grasses such as switchgrass, sugarcane, Miscanthus, and sorghum. We discuss outstanding questions regarding the role of JAs in panicoid grasses, and highlight the importance of utilizing emerging grass models for molecular studies to provide a basis for engineering bioenergy grasses that can maximize biomass accumulation while efficiently defending against stress. Key words: Bioenergy grasses, biotic stress, herbivory, jasmonate, maize, trade-off, Setaria viridis. Introduction A major challenge faced by plants is to respond efficiently to environmental stresses while successfully producing offspring. Several ecological and physiological studies have shown that environmental stresses often compromise growth (Coley et al., 1985; Herms and Mattson, 1992). The consensus that has emerged is that this trade-off between growth and defence is due to energy and resource reallocation (McKey, 1974; Coley et al., 1985; Herms and Mattson, 1992; Meldau et al., 2012). While growth and defence trade-off has been demonstrated from the cellular level to the evolutionary level, the mechanisms of resource partitioning have not yet been determined. It is suggested that the relationship between resources, growth, and defence is non-linear and that there is a critical threshold where defence responses are activated and growth rates maintained (Herms and Mattson, 1992). Therefore, achieving this balancing point between activation of defence and maintenance of growth is critical for breeding crops. A better balance between the two will allow crops to utilize resources efficiently to maximize productivity while still being responsive to environmental threats. Past breeding efforts have largely focused on breeding for higher grain yield, often at the expense of biomass (e.g. the Green Revolution). Given the rapid development toward lignocellulosic biofuels, biomass quantity and quality have emerged as traits of interest. Over the past decade, multiple research projects have been funded to develop a lignocellulosic bioenergy sector using multiple grass feedstocks including sugarcane, switchgrass, Miscanthus, and sorghum The Author Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please journals.permissions@oup.com

2 Page 2 of 12 Shyu and Brutnell (Vermerris, 2011). A deeper understanding of the control points and genes that influence that balance between growth and defence should enable the identification of gene networks, thus providing breeders with an opportunity to exploit existing or engineered variation to select for novel varieties through marker-assisted breeding or transgenics. Although energy and resource reallocation has been shown to be important for balancing plant growth and defence, understanding of the mechanisms underlying resource reallocation has just started to emerge. Recent studies point to the role of plant hormones, particularly brassinosteroids, in balancing growth and immune responses (Lozano-Duran et al., 2013; Fan et al., 2014; Lozano-Duran and Zipfel, 2015). These regulatory nodes are largely at the transcriptional level (Lozano-Duran et al., 2013; Fan et al., 2014). However, as sessile organisms, plants have evolved complex hormonal regulatory networks that underpin developmental control and defence responses, and multiple hormones fine-tune these signalling outputs (Pieterse et al., 2009, 2012; De Vleesschauwer et al., 2013). While some hormones have demonstrated roles in growth [e.g. auxin, cytokinin, and gibberellin (GA)] and others appear primarily to regulate defence [e.g. abscisic acid, ethylene, and salicylic acid (SA)], jasmonates (JAs) have a unique role in regulating both growth and defence responses. Commonly referred to as the wound hormone (Koo and Howe, 2009), JAs also play important roles in plant growth and development (Browse, 2009, Creelman and Mullet, 1997). Studies on hormone signalling in plants have largely focused on the dicot model Arabidopsis, providing an outstanding foundation for core biosynthesis and signalling pathways in response to JAs. However, most of the world s food and bioenergy feedstock sources are monocots (FAO Statistical Yearbook, 2013) a group that diverged from the eudicots ~200 million years ago (Wolfe et al., 1989). This evolutionary distance has resulted in profound variations in morphological, ecological, and developmental networks. Thus, perhaps it is not surprising that JA signalling in grasses reveals unique roles for JAs in sex determination, shoot morphology, and rhizomatous growth. In this review, we discuss the role of JAs in grasses, and highlight key questions in this field that remain to be explored. Overview of JA biosynthesis and signalling JAs are a group of oxylipins that are highly induced upon wounding and herbivore feeding (Koo et al., 2009). JA biosynthetic genes are also expressed in specific tissues during reproductive development, presumably leading to JA accumulation in specific cell types (Meyer et al., 1984; Hause et al., 2000; Ito et al., 2007). The JA biosynthetic pathway has been largely elucidated in Arabidopsis and tomato, and extensively reviewed (Gfeller et al., 2010; Lyons et al., 2013; Wasternack and Hause, 2013). The first step of JA biosynthesis starts in the chloroplast membrane, where lipids are cleaved by lipases to generate α-linolenic acid. Oxygenation of α-linolenic acid by 13-lipoxygenase (LOX) forms (13-S)- hydroperoxyoctadecatrienoic acid (13-HPOT). Allene oxide synthase (AOS) and allene oxide cyclase (AOC) then catalyse sequential reactions to generate the JA precursor oxophytodienoic acid (OPDA). OPDA is transferred to the peroxisome through a peroxisomal membrane transporter COMATOSE/ PEROXISOMAL ABC TRANSPORTER 1 (CTS/PXA1), and reduced to 12-oxophytoenoic acid (OPC-8) by OPDA reductase (OPR). OPC-8:0 CoA ligase (OPCL) then catalyses conversion of OPC-8 into OPC-8-CoA. After three rounds of β-oxidation by acyl-coenzyme A oxidase (ACX), JA is synthesized and secreted into the cytosol. Finally, JA is further conjugated with different amino acids, most importantly isoleucine (Ile) to form JA-Ile, the bioactive form of JA (Thines et al., 2007; Fonseca et al., 2009). Perception of JA-Ile occurs in the nucleus, where the F-box protein CORONATINE INSENSITIVE1 (COI1) and transcriptional repressor JASMONATE ZIM-DOMAIN (JAZ) protein function as co-receptors to perceive JA-Ile (Katsir et al., 2008; Sheard et al., 2010). JA-Ile functions as molecular glue that binds COI1 to JAZ, which then leads to ubiquitination of JAZ proteins and targeted degradation via the 26S proteasome (Chini et al., 2007; Tan et al., 2007; Thines et al., 2007). Degradation of JAZ proteins allows activation of downstream gene expression through JA-responsive transcription factors such as MYC2 (Lorenzo et al., 2004; Chini et al., 2007). Studies have shown that diverse JAZ proteins have different binding affinities and stabilities in the presence of bioactive JAs, leading to various strengths of signalling outputs (Chung and Howe, 2009; Chung et al., 2010; Shyu et al., 2012). Moreover, different JAZ proteins interact with various transcription factors, leading to a myriad of JA responses such as defence against insects, trichome development, and reproductive growth (Fernandez- Calvo et al., 2011; Qi et al., 2011; Song et al., 2013). Recently, JASMONATE-ASSOCIATED VQ MOTIF GENE 1 (JAV1) was identified as another layer of regulation to control JA signalling outputs. JAV1 was identified from a genetic screen for RNA interference (RNAi) lines that were resistant to the necrotrophic fungus Botrytis cinerea (Hu et al., 2013). The JAV1 RNAi line was also more resistant to herbivore feeding, and exhibited induced transcripts that were classified as defence or stress related. JAV1 was degraded in the presence of methyl jasmonate (MeJA) and wounding in a coi1-dependent manner, but did not interact with COI1, suggesting that another F-box protein downstream of COI1 might be involved in controlling JA signalling outputs. Interestingly, JAV1 RNAi lines did not have any developmental phenotypes that were previously shown to be involved in JA signalling. This suggests that JA-mediated defence responses and development responses can be uncoupled. Homologues that share the conserved VQ motif are present in rice, maize, and other plant species. Further comparative studies of JAV homologues in the grasses should reveal if JAV1 is a conserved component of JA signalling in the angiosperms. In maize, little is known about JA biosynthesis and perception. The most characterized enzyme family is the LOX family, in which loss-of-function mutants were shown to have striking phenotypes such as feminized tassel structures (Acosta et al., 2009) and altered responses to fungal pathogens (Christensen et al., 2013, 2014). Mutants in OPR7 and OPR8 have also been characterized, exhibiting phenotypes similar

3 JA biology in panicoid grasses Page 3 of 12 to tasselseed1 (ts1; Yan et al., 2012). Although few genetic studies have been reported, maize contains homologues of all known enzymes in the JA biosynthesis pathway, suggesting high homology for JA biosynthesis between monocots and eudicots (Table 1; Lyons et al., 2013). Despite the importance of JA signalling in orchestrating responses in growth and defence, it is striking that very little is known about JA signalling in maize and other panicoid grasses. MYC7, the maize homologue of Arabidopsis MYC2, is induced in mature maize leaves upon mechanical wounding and insect elicitor treatments (Engelberth et al., 2012). Studies in rice indicate that OsJAZ1 β-glucuronidase (GUS) is degraded upon JA treatment in a 26S proteasome-dependent manner (Cai et al., 2014). OsJAZ proteins also interact with OsbHLH148 to regulate drought tolerance (Seo et al., 2011). Thus, based on these limited studies in maize and rice, it appears that core mechanisms underlying JA signalling may be fairly conserved between monocots and eudicots. However, it is also likely that there are novel components to the signalling pathway present in monocot lineages that are absent in dicots. For instance, maize, rice, Brachypodium distachyon, and Setaria viridis all have three or more copies of COI1 (Table 1; Lee et al., 2013; Lyons et al., 2013). All three OsCOIs interact with OsJAZ proteins in the presence of coronatine, a molecular mimic of JA-Ile, in diverse combinations and strengths (Yamada et al., 2012; Lee et al., 2013; Cai et al., 2014), but only two of the three OsCOI genes are able to complement the Arabidopsis coi1 mutant (Lee et al., 2013). These results collectively suggest that although the core mechanism in JA perception appears to be conserved in monocots and eudicots, additional COI JAZ receptor complexes are present in grasses, presumably to generate a more complex network for JA signalling outputs (Fig. 1). JA-mediated defence against herbivores and pathogens Plants have evolved complex mechanisms to monitor and respond to herbivore and pathogen attack, including Table 1. The number of grass homologues of genes in JA biosynthesis and signalling the reallocation of mechanisms to enhance tolerance, prevent attack, or kill the attacker (Howe and Jander, 2008). Jasmonic acid exerts several layers of control in both the recognition and the magnitude of the response (Campos et al., 2014). In maize and sorghum, JA levels are strongly induced upon herbivory (Zhu-Salzman et al., 2004; Christensen et al., 2013; Kollner et al., 2013). In maize, transcripts that encode JA biosynthetic enzymes are also strongly up-regulated in response to herbivory or mechanical wounding, correlating with high levels of JA accumulation (Engelberth et al., 2007, 2012). High JA levels also correlate with increased terpene accumulation and inhibition of larval growth (Zhu-Salzman et al., 2004; Christensen et al., 2013; Kollner et al., 2013). In addition, higher levels of JA appear to have been selected in traditional breeding programmes of sorghum and maize for increased resistance to leaf-feeding fall armyworm and southwestern corn borer, possibly through the induction of sesquiterpines (Williams et al., 1990; Shivaji et al., 2010; Cheng et al., 2013). In maize, several Mutator (Mu) transposon-insertion lines that disrupt genes encoding JA biosynthetic enzymes are more susceptible to fungal pathogens and herbivore feeding (Yan et al., 2012; Christensen et al., 2013, 2014). Mutants in ZmLOX10, encoding a 13-LOX, an enzyme in the early steps of JA biosynthesis, produce lower JA levels upon wounding, and accumulate fewer secondary metabolites upon beet armyworm (Spodoptera exigua) treatment (Christensen et al., 2013). As expected, lox10 mutants are more susceptible to S. exigua in both laboratory and field conditions (Christensen et al., 2013). Mutants in lox12-1, a monocot-specific LOX12, are more susceptible to the fungal pathogen Fusarium verticillioides, and have reduced levels of JA, OPDA, and JA-Ile (Christensen et al., 2014). Double mutants in JA biosynthetic genes opr7 and opr8 also display increased susceptibility to F. verticillioides (Christensen et al., 2014) and increased susceptibility to S. exigua feeding (Yan et al., 2012), and are highly susceptible to the oomycete Pythium sp. (Yan et al., 2012). Taken together, these findings reveal that JA is required for Homologues were identified on Phytozome ( based on sequence similarity >50%, except for COI1 homologues which were identified with sequence similarities >60%. Arabidopsis gene IDs used for Phytozome query were: LOX2 (At3g45140), AOS (At5g42650), AOC2 (At3g25770), OPR3 (At2g06050), ACX1 (At4g16760), and COI1 (At2g39940). JAZ homologues were identified based on the presence of TIFY (Pfam ID: PF06200) and Jas domains (Pfam ID: PF09425). Arabidopsis thaliana Brachypodium distachyon Oryza sativa Setaria italica Sorghum bicolor Zea mays Panicum virgatum LOX AOS AOC OPR ACX COI JAZ No. of possible COI1 JAZ combinations

4 Page 4 of 12 Shyu and Brutnell Fig. 1. Hypothetical model for JA signalling in grasses. Grass systems have three copies of COI1 and multiple copies of JAZ repressors, allowing a greater number of COI JAZ and JAZ transcription factor (TF) combinatorial interactions that will facilitate a broad range of signalling outputs. Shown are three possible modes of JA responses where three different TFs control diverse JA signalling outputs through various JAZ TF promoter interactions. COI1, CORONATINE INSENSITIVE1; JAZ, JASMONATE ZIM-DOMAIN proteins. effective defence against both insect and fungal pathogens in maize. Although JA is a positive regulator of herbivore and fungal defence in maize and sorghum, the roles of JA in defence against nematodes are more complex (Oka et al., 1997; Cooper et al., 2005; Gao et al., 2008, 2009). Maize mutants with a disruption in the gene encoding 9-LOX accumulate higher levels of JA and thus might be expected to show enhanced defence response (Gao et al., 2008, 2009). Surprisingly this mutant line is more susceptible to root-knot nematodes and seed-infecting fungi Aspergillus flavus and Aspergillus nidulans (Gao et al., 2008, 2009). These results indicate a negative role for JA in regulating plant defence against nematodes. These findings are contradictory to studies in tomato and oat, which show increased resistance to nematodes upon exogenous application of JAs (Oka et al., 1997; Cooper et al., 2005). Therefore, the role of JAs in defending against nematodes may involve a more complex regulation compared with defence against herbivores that consume shoot tissues. The role of JAs in responding to bacterial pathogens appears to be specific for each host pathogen pair. The well-characterized interaction of Arabidopsis thaliana with Pseuodomonas syringae pv. tomato DC3000 (reviewed in Xin and He, 2014) has shown that the secreted phytotoxin effector coronatine, structurally similar to JA-Ile, binds to the COI JAZ receptor complex with a higher affinity relative to JA-Ile (Katsir et al., 2008; Fonseca et al., 2009). This results in strong induction of JA-induced gene expression that antagonizes SA-mediated defence responses (Laurie-Berry et al., 2006). In rice, JAs appear to promote defence against Xanthomonas oryzae pv. oryzae (Xoo), as pre-treatment with JA results in stronger resistance to pathogen attack (Yamada et al., 2012). JAZ and LOX expression also increases in Xoo-infected rice leaves (Grewal et al., 2012; Yamada et al., 2012). The role of JA in bacterial pathogen resistance in panicoid grasses is unclear, but in recent years bacterial pathogens such as Goss s bacterial wilt (Clavibacter michiganensis subsp. nebraskensis) have become increasingly threatening for corn production (Malvik et al., 2010; Friskop et al., 2014). Thus, studies of bacterial pathogen responses in panicoid grasses will provide resources for comparative studies and also potential for engineering crops with targeted resistance. JA responses are also subject to developmental control in maize (Bosak et al., 2013; Yan et al., 2014). In artificial herbivory and caterpillar feeding experiments, JA levels were induced at similar levels in both young and juvenile tissues, but volatiles accumulated to higher levels in juvenile plants. Conversely, cystatin-like protease inhibitors (PIs) were more highly induced in young seedlings than in juvenile plants upon herbivore treatment, correlating with reduced performance of S. exigua larval growth reared on young seedlings compared with juvenile plants (Bosak et al., 2013). These results are consistent with recent characterization of opr7 opr8 mutants where longer coleoptiles, sheath, and leaf blades were observed in first and second leaves but not in leaves initiated later in development (Yan et al., 2014). These studies suggest that younger seedlings respond to herbivory and JA-mediated growth inhibition in a more direct and constitutive manner, while juvenile plants use less direct defence mechanisms (Bosak et al., 2013). Not only do JA responses dynamically change between developmental stages, but responses drastically differ between different segments of the same leaf (Kollner et al., 2013). When herbivory occurs in the middle or base of a leaf, sesquiterpenes, JAs, and JA-Ile accumulate distal but not proximal to the wound site. A similar effect is observed when middle leaf segments are treated with volicitin, crude regurgitant elicitors, and mechanical wounding, where JAs accumulate preferentially at the tip of the leaf (Engelberth et al., 2007). In an independent study, transcripts of the JA biosynthesis gene AOS also accumulate in tip segments of maize leaves when middle segments are treated with insect elicitors, aligning with JA accumulation patterns in previous studies (Engelberth et al., 2012). Taken together, these results suggest that JA signals are transmitted towards the tip of the leaf, presumably to maximize the range of signal transduction through volatiles to attract natural enemies that prey upon herbivores (Kollner et al., 2013). The roles of JAs in regulating growth and development Perhaps the most surprising effect of a disruption in JA biosynthesis in grasses is the maize tasselseed1 (ts1) mutant phenotype, which has female flower structures developed on the male tassel (Fig. 2; Acosta et al., 2009). Studies have demonstrated roles for JAs in reproductive growth, including male fertility in Arabidopsis, female fertility in tomato, and, more recently, spikelet development in rice (Li et al., 2004; Browse, 2009; Creelman and Mullet., 1997; Cai et al., 2014). Due to the unique morphology of separate male and female inflorescences in maize, a role for JAs in sex determination was also revealed (Acosta et al., 2009; Yan et al., 2012). The ts1 locus was positionally cloned, identifying the TS1 protein as

5 Fig. 2. Examples of JA-related phenotypes that are unique to panicoid grasses. Upper panel: ear shoot elongation phenotype in maize opr7 opr8 (as described in Yan et al., 2012): the two plants on the left are wild-type maize, while the two plants on the right are opr7 opr8 (image courtesy of Yuanxin Yan and Mike Kolomiets). Bottom left: feminized tassel in tasselseed1 (Acosta et al., Reprinted with permission from the AAAS.). Bottom right: Miscanthus spring rhizomes (image courtesy of Kankshita Swaminathan). a 13-LOX (LOX8) involved in JA biosynthesis. The ts1 tassels had significantly lower JA levels, and the feminized tassel phenotype could be rescued with exogenous application of JAs (Acosta et al., 2009). Although no herbivore infection assays have been reported for ts1 thus far, ts1 mutant seedling leaves have lower endogenous JA levels compared with the wild type in both control and wounded conditions (Christensen et al., 2013). Feminized tassels are also observed in the opr7 opr8 double mutant of maize, which is defective in JA biosynthesis, and the phenotype can be rescued by exogenous application of JAs (Yan et al., 2012). In addition to the strong sex determination phenotype, opr mutants display long ear shanks, have reduced anthocyanin accumulation (Fig. 2), and display a wide range of phenotypes that are related to defence (Yan et al., 2012), thus confirming the role of JAs in regulating a broad range of growth and defence responses. The roles JA biology in panicoid grasses Page 5 of 12 of JAs in reproductive growth and defence responses suggests the potential for fine-tuning growth and defence signalling outputs through regulation of JA signalling. In an RNA sequencing (RNA-seq) analysis of Miscanthus rhizomes at budding and senescing stages of the life cycle, expression of genes encoding JAZ proteins and JA biosynthetic enzymes and several JA biosynthesis transcripts were observed to be rapidly induced in budding spring rhizomes (Fig. 2; Barling et al., 2013). This suggests another potential role for JA in rhizome shoot development. Important bioenergy feedstocks, including Miscanthus and sugarcane, are all propagated through rhizomes. Thus, understanding the role of JAs in regulating rhizome shoot development will provide opportunities to engineer perennial crops that have more uniform emergence and potentially higher accumulation of shoot biomass. JA-induced growth inhibition JA was first isolated from the fungal pathogen Lasiodiplodia theobromae and shown to be an inhibitor of rice seedling growth (Aldridge et al., 1971; Yamane et al., 1981). Exogenous application of JA to inhibit root growth of seedlings then became widely utilized as a bioassay to screen for mutants that have altered JA responses (Feys et al., 1994; Lorenzo et al., 2004). In addition to seedlings, wounding of mature plants also results in reduced biomass (Yan et al., 2007; Zhang and Turner, 2008). Moreover, maize mutant lines that have higher endogenous JA levels exhibit reduced shoot and root growth in comparison with the wild type (Gao et al., 2008). Mechanisms that underpin JA-triggered growth inhibition have just started to be revealed. JA alters both cell proliferation and elongation in plants (Chen et al., 2011; Noir et al., 2013). In roots, Chen et al. (2011) demonstrated that JA-induced growth inhibition is regulated through MYC2-mediated repression of PLETHORA1 and PLETHORA2 expression to regulate root stem cell patterning. In leaves, application of MeJA or wound treatments delays cell cycle progression that is mediated through COI1and other core JA signalling components (Zhang and Turner, 2008; Noir et al., 2013). Findings from large-scale transcriptome analyses correlate with these results, showing that transcripts encoded by cell cycle genes are largely repressed in the presence of JAs (Pauwels et al., 2008; Noir et al., 2013). These results collectively suggest that JAs arrest growth partially by inhibiting the cell cycle and cell division through transcription factors such as MYC2 in roots. Detailed mechanisms of how JAs repress cell proliferation in leaves are yet to be determined. Studies in multiple plant species have shown that induction of JAs leads to suppression of photosynthesis and growthrelated gene expression (Reymond et al., 2004; Zhu-Salzman et al., 2004; Salzman et al., 2005; Zou et al., 2005; Giri et al., 2006; Park et al., 2006; Mitra and Baldwin, 2008; Bilgin et al., 2010). This phenomenon is also observed in panicoid grasses. In multiple cultivars of Sorghum bicolor, both exogenous application of MeJA and greenbug (a phloem-feeding aphid)

6 Page 6 of 12 Shyu and Brutnell treatment led to suppression of photosynthesis-related gene expression, while defence-related genes were strongly induced (Zhu-Salzman et al., 2004; Salzman et al., 2005). Surprisingly, understanding of how JAs suppress photosynthesis-related gene expression and whether or not this suppression leads to decreased photosynthetic efficiency is limited. A recent study in Arabidopsis showed that exogenous application of a JA-Ile mimic, coronatine, leads to inhibition of photosynthetic efficiency [steady-state quantum efficiency of photosystem II (PSII)] in a delayed and transient manner (Attaran et al., 2014). However, the delayed inhibition of photosynthesis by coronatine was not observed in a mutant with a defect in stomatal closure. This suggests that reduced photosynthetic efficiency upon application of coronatine was due to delayed stomatal opening that further hindered photosynthesis instead of a direct repression of photosynthesis. The authors concluded that JA signalling in Arabidopsis uncoupled growth inhibition from photosynthesis, and suggested a more prominent role for JAs in inhibiting growth (Attaran et al., 2014). Although repression of photosynthetic gene expression by JAs is observed in grass systems (Zhu-Salzman et al., 2004; Salzman et al., 2005), the mechanisms by which JAs lead to repression of photosynthetic gene expression in grass systems has yet to be determined. Plant growth requires selective remodelling of the primary cell wall (Benatti et al., 2012). To date, the majority of studies examining JA signalling and cell wall biosynthesis were conducted with Arabidopsis, which has a classical type I primary cell wall typical of most dicots and non-commelinid monocots (Carpita and Gibeaut, 1993). Microarray analysis of MeJA-treated Arabidopsis cell cultures showed repression of primary cell wall biosynthesis and remodelling genes such as cellulose synthase and pectate lyase (Pauwels et al., 2008). Likewise, RNA-seq analysis of coronatine-treated Arabidopsis during a time course also showed repression of EXPANSIN 8A, which is important for cell wall extension during growth (Li et al., 2003; Attaran et al., 2014). These results are consistent with the observation of JA-triggered inhibition of cell proliferation and elongation. Conversely, grasses have a type II primary wall, which differs from type I walls in its hemicellulose composition, relative abundance of pectin, and the presence of monolignol precursors as structural components (Carpita, 1996). As expected, multiple gene families encoding cell wall biosynthesis enzymes are differentially expressed between Arabidopsis and the grasses (Penning et al., 2009). Microarray analysis of MeJA-treated sorghum led to strong induction of putative primary and secondary cell wall-related cellulose synthases (Salzman et al., 2005). This suggests that JAs may have as yet unrevealed roles in regulating cell wall growth in grasses. To date, cell wall-related phenotypes in JA biosynthetic mutants have not been reported in either monocots or dicots. However, elevated JA accumulation is common to several cellulosedeficient mutants, and is frequently accompanied by ectopic lignification (Sánchez-Rodríguez et al., 2010). Expression of genes involved in monolignol biosynthesis upon JA treatment is more consistent between monocots and dicots. Genes known to be involved in monolignol biosynthesis were strongly induced in MeJA-treated Arabidopsis cell cultures, consistent with higher phenylpropanoid levels in the corresponding culture (Pauwels et al., 2008). Transcripts encoding caffeoyl CoA O-methyl-transferases enzymes involved in monolignol biosynthesis are expressed at higher levels upon MeJA treatment in sorghum seedlings (Salzman et al., 2005). These findings collectively support the phenomenon by which lignin levels can be induced upon biotic stresses to function as physical barriers against pathogens (Sattler and Funnell-Harris, 2013). Understanding how JA regulates cell wall biosynthesis in grasses would provide useful insights into engineering grasses with improved biomass quality for lignocellulosic ethanol production. Rapid growth at the expense of defence response JA signalling and the shade avoidance syndrome (SAS) To date, the best characterized JA-mediated growth and defence balance mechanism is in the study of the SAS. SAS is the phenomenon in which physiological and morphological changes occur when plants are subjected to vegetative shading (e.g. high density planting) (Smith and Whitelam, 1997). Changes such as elongation of stems and changes in leaf angle allow plants to outcompete neighbours for limiting light needed for photosynthesis (Smith, 1995; Dorn et al., 2000; Bell and Galloway, 2008). Although these morphological changes allow plants to grow taller and project out from the canopy, the SAS comes with a cost. Under vegetative shading, biomass and yields are reduced (Liu and Tollenaar, 2009; Leone et al., 2014) and defence responses including responses to herbivores, and bacterial and fungal pathogens are attenuated (Moreno et al., 2009; Ballare et al., 2012; Cerrudo et al., 2012; de Wit et al., 2013; Ballare, 2014; Moreno and Ballare, 2014). Shade attenuates defence responses by antagonizing JA signalling through increased levels of JAZ proteins and the transcription factor MYC2 (Moreno et al., 2009; Cerrudo et al., 2012; de Wit et al., 2013; Kegge et al., 2013; Cargnel et al., 2014; Chico et al., 2014; Leone et al., 2014). Repression of defence responses triggered by shade is reduced in JA mutants (Moreno et al., 2009). In other words, shade responses are enhanced in JA mutants (Robson et al., 2010), suggesting that JA is required for regulating the trade-off between growth in response to shade and defence against stresses. Mechanisms that underpin this balance have just started to be revealed, and multiple studies point to the JAZ repressors as central targets in rewiring growth and defence (Pieterse et al., 2014). JAZ10 expression is induced in shade-treated seedlings (Moreno et al., 2009). Shade-treated jaz10 mutant lines restore JA responses that are reduced in wild-type plants (Leone et al., 2014), and transgenic lines overexpressing JAZ1-GUS display enhanced responses to shade (Robson et al., 2010). Moreover, JAZ proteins are more stable in shade conditions and in phya and phyb mutants that continuously exhibit SAS (Robson et al., 2010; Chico et al., 2014; Leone et al., 2014). These results collectively suggest that JAZ proteins are required

7 for repressing JA-dependent defence responses under shade conditions. Chico et al. (2014) demonstrated that additional regulation through JAZ-interacting transcription factors such as MYC2 are also required for trade-off balance when plants are subjected to shade. MYC2 is stabilized in blue or red light, but destabilized in far-red light (shade) and dark conditions (Chico et al., 2014). phya and phyb are both required for MYC2 stability, and myc2 myc3 myc4 mutants are not responsive to shade (Chico et al., 2014). These results along with studies on JAZ-mediated trade-off responses have led to a model in which JAZ proteins are stabilized and MYC transcription factors are turned over to repress JA-dependent defence responses and accelerate growth under shade conditions (Chico et al., 2014). Surprisingly little is known about the role of JAs in regulating shade responses in maize and other panicoid grasses. The JA biosynthetic mutant opr7 opr8 was recently reported to have longer mesocotyls under darkness and red light conditions, as well as shorter leaf bundles under red light conditions (Yan et al., 2014), implying similar roles for JAs in regulating growth and defence trade-offs under shade. A novel receptor kinase WOUND-RESPONSIVE AND PHYTOCHROME- REGULATED KINASE1 (WPK1) was identified in maize to be transcriptionally activated by wounding, JA treatment, and red light (He et al., 2005). Genetic characterization of WPK1 would provide critical information on how WPK1 is involved in integrating JA and light responses. Inspired by studies in Arabidopsis, characterization of JAZ proteins and MYC transcription factors in grasses will also shed light on mechanisms underlying growth and defence balance in shade conditions. Breeding and engineering for plants that can efficiently defend against stresses under shade will allow high density planting of bioenergy grasses and increased biomass yield per acre. JAZ transcription factor pairs as potential nodes for regulating growth and defence signalling outputs Several steps in the JA signalling pathway have been implicated for determining specific signalling outputs. JA-Ile can be conjugated into hydroxyl and dicarboxy forms that are less active (Koo et al., 2011, 2014; Heitz et al., 2012). JAZ proteins and splice variants have different affinities to interact with COI1 through diversified JAZ COI1 interaction motifs, resulting in a range of JAZ stabilities and different strengths of JA-induced responses (Chung and Howe, 2009; Chung et al., 2010; Shyu et al., 2012). JAZ proteins also interact with a broad range of transcription factors to specify JA signalling outputs (Fernandez-Calvo et al., 2011; Qi et al., 2011; Song et al., 2013). The mechanisms by which JAZ proteins recruit co-repressors and transcription factors also differ slightly (Pauwels et al., 2010; Shyu et al., 2012; Moreno et al., 2013). Moreover, members of the JAZ family have diverse expression patterns (Chung et al., 2008; Sehr et al., 2010; Demianski et al., 2012; Figueroa and Browse, 2012) that presumably lead to spatiotemporally specific JA responses. Interestingly, all of JA biology in panicoid grasses Page 7 of 12 these regulation points are centered on the JAZ family of proteins. Indeed, JAZ proteins are highly conserved across the plant kingdom (Chico et al., 2008; Bai et al., 2011). Moreover, panicoid grasses have a large number of JAZ proteins (Bai et al., 2011), suggesting greater diversity for JA-regulated signalling outputs that could potentially be manipulated (Fig. 3; Table 1). Another mechanism of regulation that is emerging is through the cross-talk of hormonal signalling pathways. Recently, several reports have suggested a direct interaction between GA and JA through JAZ DELLA interactions (Wild et al., 2012; Yang et al., 2012; Hou et al., 2013). DELLA proteins are repressors in the GA signalling pathway that are rapidly turned over in the presence of GA (Dill et al., 2001, 2004). Loss-of-function DELLA mutants exhibit decreased sensitivity to JAs, and mutations that lead to decreased JA signalling enhance GA responses (Hong et al., 2012; Wild et al., 2012; Yang et al., 2012; Hou et al., 2013). An OsCOI1 RNAi line demonstrated characteristic GA overexpression phenotypes including elongated cells, increased internode lengths, and increased plant height (Yang et al., 2012). Similar phenotypes were found in Arabidopsis, where coi1 had elongated petioles and flowered earlier than the wild type (Yang et al., 2012). Indeed, DELLA proteins interact with MYC2 to regulate JA signalling outputs, specifically the genes that encode sesquiterpene synthases (Wild et al., 2012). JAZ proteins and DELLA proteins physically interact with each other to modulate MYC2 signalling outputs (Wild et al., 2012; Yang et al., 2012; Hou et al., 2013). These studies collectively suggest a model where JAZs and DELLAs antagonize each other to promote JA or GA signalling outputs to balance growth and defence (Hou et al., 2013; Huot et al., 2014). It is interesting to note that mutations in a GA biosynthetic enzyme (Sasaki et al., 2002) or signalling component (Peng et al., 1999) underlie the phenotypes associated with the Green Revolution, suggesting that past breeding efforts have selected for reduced GA response to increase yield. However, the enhancement of JA responses through reduced GA response may have also been an indirect target of these breeding efforts. Molecular characterization of JAZ and DELLA function in panicoid grasses can provide potential to unravel the antagonistic relationship between JAs and GA, and develop plants that can generate significant biomass quantities and qualities while efficiently defending against biotic stresses. Conclusions and future directions JAs regulate a broad range of responses from herbivore and pathogen attack to growth and development. While the roles for JA in sex determination, ear shoot growth, rhizomatous development, and cell wall biosynthesis have been identified in the grasses, the biochemical and molecular details of regulation are less clear. Many of these responses are unique to grasses. As grasses are the primary source of food for human consumption and animal feed, and currently serve as the largest feedstock for the bioenergy industry, it is critical to

8 Page 8 of 12 Shyu and Brutnell Fig. 3. Phylogenetic tree of JAZ proteins from maize, sorghum, switchgrass, Setaria, rice, and Arabidopsis. A Neighbor Joining phylogenetic tree was generated using the conserved TIFY and Jas motif amino acid sequences with MEGA 5 (Tamura et al., 2011). Setaria italica JAZ genes are highlighted in magenta, and Arabidopsis thaliana JAZ genes are highlighted in blue, demonstrating that diversification of JAZ genes occurred after monocot and eudicot divergence. conduct molecular studies in grasses to understand and eventually manipulate these responses to increase productivity. JAV1 and JAZs are both potential candidates for manipulating growth defence balance in panicoid grasses. As JAV1 appears to regulate JA-mediated defence but not growth responses (Hu et al., 2013), it may be possible to alter the expression of JAV1 or downstream partners to enhance defence responses without compromising growth. Manipulation of JAZ proteins under cell type- or tissue-specific controls may be another mechanism to fine-tune JA responses to pathogen attack while limiting negative consequences on plant growth. For instance, by expressing JAZ genes strictly in leaf tissues, negative consequence on flower development may be obviated as biochemical pathways would not be impacted in floral or stem tissues. A unique feature in grass systems is the continuous development of leaves along a basipetal axis (Nelson, 2011), and this gradient has been exploited to examine responses to wounding and herbivore treatments, as discussed above (Engelberth et al., 2007, 2012; Kollner et al., 2013). Although the signals that propagate through the leaf are unknown, recent studies have highlighted the dynamic gene expression and metabolite changes that occur along the grass leaf developmental gradient (Li et al., 2010; Majeran et al., 2010; Facette et al., 2013). Transcripts expressed at the base of the maize leaf encode proteins required for hormone biosynthesis, cell division, and cell wall biosynthesis, while transcripts accumulating in the middle and tip of the leaf primarily encode proteins required for photosynthesis. Therefore, an outstanding question is how leaves respond to herbivore responses during this dynamic

9 developmental process. In sorghum, herbivore feeding causes down-regulation of photosynthesis-related gene expression (Zhu-Salzman et al., 2004; Salzman et al., 2005). Given that photosynthesis-related transcripts are highly expressed at the tip, where JA levels also accumulate to increased levels upon wounding, measuring photosynthetic efficiency and transcriptome profiles of mature leaf tip segments upon wounding will yield a deeper understanding of the effect of JA on photosynthesis and repression of growth. Additional cell type-specific and developmental stage-specific analyses of JA responses will also open up many avenues to understand how JA orchestrates such a myriad of responses (Ballare, 2014; Huot et al., 2014). Research in the well-established eudicot model Arabidopsis has provided the molecular foundation to our understanding of JA signalling and its role in regulating growth and defence trade-offs. However, due to profound differences in anatomy, physiology, reproductive biology, and evolutionary pressures, it is expected that regulatory networks between monocots and eudicots will also be divergent (Shiu et al., 2004; Greenup et al., 2009; De Vleesschauwer et al., 2013). Emerging grass models including Brachypodium distachyon and Setaria viridis have started to gain momentum as genetic and genomic resources are being developed for these communities (Brutnell et al., 2010; Bragg et al., 2015). Setaria viridis is of particular interest for bioenergy grasses because it is a C 4 panicoid grass that is closely related to Mischanthus, switchgrass, sugarcane, and sorghum (Brutnell et al., 2010). The genome of its domesticated relative Setaria italica is sequenced, and crossing and transgenic tools are available for molecular and genetic studies (Bennetzen et al., 2012; Jiang et al., 2013). Setaria viridis has a rapid growth rate (6 8 weeks generation time) and small stature that makes it ideal for laboratory conditions (Brutnell et al., 2010). Utilization of newly characterized chemicals that function as JA inhibitors also broadens the range of experiments that can be performed to understand JA biology (Meesters et al., 2014; Monte et al., 2014). We are entering an exciting stage of JA biology where tools of comparative genomics can be applied to gain a deeper understanding of JA signalling and diversification in the grasses. Our understanding of JA biology in panicoid grasses and its role in balancing growth and defence trade-offs is just beginning. Utilizing advanced techniques such as high-throughput sequencing and emerging genetic resources to characterize and compare JA signalling mechanisms can provide insights into how plants can orchestrate complex signalling pathways to balance growth and defence and thrive under both natural and artificial environments. Outcomes from these studies will push the field another step forward to achieve the long-term goal of engineering bioenergy crops that can accumulate biomass of higher quantity and quality while efficiently defending against environmental challenges. Acknowledgements We would like to thank Dr Sankalpi Warnasooriya, Dr Carla Coelho, Ms Rachel Mertz, and Ms Kim Maxson-Stein for critically reading of and commenting on the manuscript. We also acknowledge Drs Mike Kolomiets, JA biology in panicoid grasses Page 9 of 12 Yuanxin Yan, and Kankshita Swaminathan for providing images for Figure 2 of this article. and Dr James Schnable for identifying JAZ homologues listed in Table 1 and Figure 3. This work was supported by USDA- AFRI NIFA Postdoctoral Fellowship to CS, and DOE grant DE-SC to TB. We apologize to those whose work could not be cited in this review due to space limitations. 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