RuBPCase activase mediates growth-defense tradeoffs: Silencing RCA redirects JA flux from JA-Ile to MeJA to attenuate induced defense responses in Nicotiana attenuata

RuBPCase activase (RCA), an abundant photosynthetic protein is strongly down-regulated in response to Manduca sexta’s oral secretion (OS) in Nicotiana attenuata. RCA-silenced plants are impaired not only in photosynthetic capacity and growth, but also in jasmonic acid (JA)-isoleucine (Ile) signaling, and herbivore resistance mediated by JA-Ile dependent defense traits. These responses are consistent with a resource-based growth-defense trade-off. Since JA+Ile-supplementation of OS restored WT levels of JA-Ile, defenses and resistance to M. sexta, but OS supplemented individually with JA- or Ile did not, the JA-Ile deficiency of RCA-silenced plants could not be attributed to lower JA or Ile pools or JAR4/6 conjugating activity. Similar levels of JA-Ile derivatives after OS elicitation indicated unaltered JA-Ile turnover and lower levels of other JA-conjugates ruled out competition from other conjugation reactions. RCA-silenced plants accumulated more methyl jasmonate (MeJA) after OS elicitation, which corresponded with increased jasmonate methyltransferase (JMT) activity. RCA-silencing phenocopies JMT over-expression, wherein elevated JMT activity redirects OS-elicited JA flux towards inactive MeJA, creating a JA sink which depletes JA-Ile and its associated defense responses. Hence RCA plays an additional non-photosynthetic role in attenuating JA-mediated defenses and their associated costs potentially allowing plants to anticipate resource-based constraints on growth before they actually occur. Keywords: Herbivory, jasmonate methyl transferase, jasmonate signaling, Manduca sexta, methyl jasmonate, Nicotiana attenuata, plant defense, RuBPCase activase Introduction Plants are attacked by a variety of herbivores and in response, plants activate defenses which can directly or indirectly affect the attacking herbivores (Kessler & Baldwin, 2001; Steppuhn et al., 2004; Zavala et al., 2004). Plants are thought to deploy two alternative strategies against herbivores: (1) resistance and (2) tolerance. These strategies are well studied and are explained by different theories. Among these, the optimal defense theory (OD) (Mckey, 1974; Mckey, 1979; Rhoades, 1979) enjoys the most empirical support. This theory proposes that the distribution of defenses within a plant reflects the fitness value of the tissue for the plant, with higher value tissues being better defended than the less valuable tissues. Moreover, this theory assumes that defenses are costly and a trade-off exists between defense and growth, which in turn explains the prevalence of inducible defenses (Coley et al., 1985; Heil & Baldwin, 2002). Hence during herbivore attack, plants re-adjust their resource investment strategies to reoptimize their allocation of resources to resistance and tolerance mechanisms, growth and reproduction. Under these circumstances, a rapid reallocation of resources to tolerance rather than defense response could maximally reduce the negative fitness consequences of herbivore attack (Schwachtje & Baldwin, 2008). However, very little is known about the molecular mechanisms that plants use to optimize their resource allocation after herbivore attack. For example, while tolerating herbivory, plants allocate newly assimilated carbon to their roots to be used for post-herbivory re-growth, rather than transporting it to the young leaves (Schwachtje et al., 2006). When Nicotiana attenuata is attacked by its specialist lepidopteran herbivore, Manduca sexta, fatty acid amino acid conjugates (FACs), present in larval oral secretions (OS) activate early defense responses by activating the jasmonic acid (JA) signaling network (Halitschke et al., 2001). JA, a linolenic acid-derived compound, is rapidly and transiently accumulated after herbivory (Creelman et al., 1992; Farmer & Ryan, 1992; Baldwin et al., 1994). JA biosynthesis begins in chloroplasts after lipase activation, which release fatty acids from the membrane lipids. Free linolenic acid is converted to 13S hydroperoxyoctadecatrienoic acid (HPOT) by a specific lipoxygenase which is subsequently converted to 12-oxo-phytodienoic acid (OPDA) by allene oxide synthase (AOS) and allene oxide cyclase (AOC). OPDA is transported to the peroxisome and after reduction and three cycles of β-oxidation by the acyl CoA oxidase 1 enzymes, multifunctional protein, and L-3-ketoacyl CoA-thiolase, is transformed to JA (Schaller & Stintzi, 2009). JA is then exported to the cytosol through the peroxisomal membrane by membrane proteins (Arai et al., 2008), where it is further metabolized. The accumulation of JA is regulated not only by JA biosynthetic genes and their associated transcription factors but also by the availability of fatty acid precursor (Howe & Schilmiller, 2002; Chung et al., 2008; Paschold et al., 2008; Skibbe et al., 2008; Kallenbach et al., 2010). A portion of JA is conjugated to different amino acids of which the isoleucine conjugate (JA-Ile) associates with Coronatine insensitive 1 (COI1) to promote the degradation of Jasmonate ZIM domain (JAZ) repressors by the 26S proteasome (Thines et al., 2007). The degradation of the JAZ repressor releases the MYC 2 transcription factor from repression and activates JA-responsive genes involved in plant defense (Fonseca et al., 2009; Memelink, 2009). JA-Ile is the active molecule triggering downstream defense responses and therefore, the magnitude of JA-Ile is directly correlated with the magnitude of a plant’s defense response. In N. attenuata, silencing the expression of JAR4/6, the enzyme conjugating JA and Ile, attenuates JA-Ile production and resistance against attack from M. sexta larvae (Wang et al., 2007). A comparative proteomic-transcriptomic study revealed that while defense-related genes are up-regulated after herbivory, photosynthesis-related genes are down-regulated (Giri et al., 2006; Bilgin et al., 2010). Remarkably, herbivore attack causes a greater reduction in a plant’s photosynthetic capacity than would be predicted based on the canopy area removed by the herbivore (Zangerl et al., 2002). RuBPCase activase (RCA), an abundant photosynthetic protein is strongly down-regulated after herbivore attack or simulated herbivory (Giri et al., 2006). RCA modulates the activity of RuBPCase, the major photosynthetic protein involved in carbon fixation, by removing inhibitory sugar phosphates from the active site of enzyme (Portis, 2003). RCA’s role in photosynthesis and growth is well studied and RCA-deficient plants have reduced photosynthetic rates, growth, and accumulate less biomass (He et al., 1997; Ilyin et al., 2005). Reduced growth limits the food available for herbivores; therefore, decreasing growth could be a part of plant’s defense strategy (Hermsmeier et al., 2001; Hahlbrock et al., 2003). In addition, a decrease in carbon (C) supply could alter the expression of genes of enzymes involved in C-utilization and storage (Koch, 1996). Previously, we observed that in addition to impaired photosynthetic capacity and growth, RCA-silenced N. attenuata plants were impaired in JA-Ile signaling, herbivore resistance and many defense traits that mediate resistance (Mitra & Baldwin, 2008) (Fig. 1). The reduction in photosynthesis and growth associated with RCA-silencing was congruent with RCA’s biochemical function as revealed from work with Arabidopsis, tobacco, and rice (He et al., 1997; Ilyin et al., 2005). However, the decrease in JA-Ile accumulation after RCA-silencing was novel. Prior experimentation had ruled out limitations in the Ile pool at the wound site or activity of the conjugating enzyme as being responsible for the attenuated JA-Ile levels of RCA-silenced plants (Mitra & Baldwin, 2008). As a member of the AAA+ (for ATPases associated with a variety of cellular activities) protein family, RCA may also be involved in other cellular processes (Ogura & Wilkinson, 2001). In different plant systems, the regulation of RCA in response to UV-B light, ozone, drought, and heat stress (Pelloux et al., 2001; Liu et al., 2002; Bota et al., 2004; Demirevska-Kepova et al., 2005) suggests that RCA is involved in diverse stress-related functions. Recently, RCA in Arabidopsis was found to be down-regulated at both transcript and protein levels in a COI1-dependent manner, after elicitation with JA (Shan et al., 2011). The increased susceptibility of RCA-deficient N. attenuata plants to herbivore attack (Mitra & Baldwin, 2008) suggested an additional, defense-related role for RCA other than in RuBPCase activation. Figure 1 An overview of the consequences of herbivory and RCA-silencing in N. attenuata plants Previously, we characterized two independently transformed RCA-silenced lines (line 1 and line 2) with similar degrees of reductions in photosynthetic rate, JA-Ile levels, and resistance against M. sexta larvae (Mitra & Baldwin, 2008). Here we used a single RCA-silenced line (line 2) to elucidate the mechanisms responsible for its attenuated JA-Ile accumulation and herbivore resistance. We examine its JA metabolism and JA-signaling after simulated herbivory with JA or Ile or both (JA+Ile) supplementations. Since adenylation of JA initiates its conjugation to amino acids and adenylation is an energy-demanding process (Staswick et al., 2002), a decrease in photosynthetic capacity may reduce the ATP supply required for JA adenylation. By extending the dark period in wild type (WT) plants, we examined the effect of reduced net carbon gain on JA-adenylation and consequently on JA-Ile accumulation. Lastly, we examine the growth of RCA-silenced plants after simulated herbivory and methyl jasmonate (MeJA) treatment.