Trypsin Proteinase Inhibitor Activity Research Articles

Solar UV-B radiation and ethylene play a key role in modulating effective defenses against Anticarsia gemmatalis larvae in field-grown soybean.

Solar UV-B radiation has been reported to enhance plant defenses against herbivore insects in many species. However, the mechanism and traits involved in the UV-B mediated increment of plant resistance are unknown in crops species, such as soybean. Here, we studied defense-related responses in undamaged and Anticarsia gemmatalis larvae-damaged leaves of two soybean cultivars grown under attenuated or full solar UV-B radiation. We determined changes in jasmonates, ethylene (ET), salicylic acid, trypsin protease inhibitor activity, flavonoids, and mRNA expression of genes related with defenses. ET emission induced by Anticarsia gemmatalis damage was synergistically increased in plants grown under solar UV-B radiation and was positively correlated with malonyl genistin concentration, trypsin proteinase inhibitor activity and expression of IFS2, and the pathogenesis protein PR2, while was negatively correlated with leaf consumption. The precursor of ET, aminocyclopropane-carboxylic acid, applied exogenously to soybean was sufficient to strongly induce leaf isoflavonoids. Our results showed that in field-grown soybean isoflavonoids were regulated by both herbivory and solar UV-B inducible ET, whereas flavonols were regulated by solar UV-B radiation only and not by herbivory or ET. Our study suggests that, although ET can modulate UV-B-mediated priming of inducible plant defenses, some plant defenses, such as isoflavonoids, are regulated by ET alone.

Solar UV-B radiation modulates chemical defenses against Anticarsia gemmatalis larvae in leaves of field-grown soybean

Although it is well known that solar ultraviolet B (UV-B) radiation enhances plant defenses, there is less knowledge about traits that define insect resistance in field-grown soybean. Here we study the effects of solar UV-B radiation on: a) the induction of phenolic compounds and trypsin proteinase inhibitors (TPI) in soybean undamaged leaves or damaged by Anticarsia gemmatalis neonates during six days, and b) the survival and mass gain of A. gemmatalis larvae that fed on soybean foliage. Two soybean cultivars (cv.), Charata and Williams, were grown under plastic with different transmittance to solar UV-B radiation, which generated two treatments: ambient UV-B (UVB+) and reduced UV-B (UVB−) radiation. Solar UV-B radiation decreased survivorship by 30% and mass gain by 45% of larvae that fed on cv. Charata, but no effect was found in those larvae that fed on cv. Williams. TPI activity and malonyl genistin were induced by A. gemmatalis damage in both cultivars, but solar UV-B radiation and damage only synergistically increased the induction of these compounds in cv. Williams. Although TPI activity and genistein derivatives were induced by herbivory, these results did not explain the differences found in survivorship and mass gain of larvae that fed on cv. Charata. However, we found a positive association between lower larval performance and the presence of two quercetin triglycosides and a kaempferol triglycoside in foliage of cv. Charata, which were identified by HPLC-DAD/MS2. We conclude that exclusion of solar UV-B radiation reduce resistance to A. gemmatalis, due to a reduction in flavonol concentration in a cultivar that has low levels of genistein derivatives like cv. Charata.

Intraspecific variation in defense against a generalist lepidopteran herbivore in populations of Eruca sativa (Mill.)

Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ-induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense-regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase-associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.

DICER‐like Proteins and Their Role in Plant‐herbivore Interactions in Nicotiana attenuata

DICER-like (DCL) proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified and stably silenced four distinct DCL genes by RNAi in Nicotiana attenuata (Torrey ex. Watson), a model for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal. Manduca sexta larvae performed significantly better on ir-dcl3 and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2 ir-dcl3 plants were impaired in JA accumulation, while JA-Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.

The broad‐leaf herbicide 2,4‐dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp

Synthetic chemical elicitors of plant defense have been touted as a powerful means for sustainable crop protection. Yet, they have never been successfully applied to control insect pests in the field. We developed a high-throughput chemical genetics screening system based on a herbivore-induced linalool synthase promoter fused to a β-glucuronidase (GUS) reporter construct to test synthetic compounds for their potential to induce rice defenses. We identified 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin homolog and widely used herbicide in monocotyledonous crops, as a potent elicitor of rice defenses. Low doses of 2,4-D induced a strong defensive reaction upstream of the jasmonic acid and ethylene pathways, resulting in a marked increase in trypsin proteinase inhibitor activity and volatile production. Induced plants were more resistant to the striped stem borer Chilo suppressalis, but became highly attractive to the brown planthopper Nilaparvata lugens and its main egg parasitoid Anagrus nilaparvatae. In a field experiment, 2,4-D application turned rice plants into living traps for N. lugens by attracting parasitoids. Our findings demonstrate the potential of auxin homologs as defensive signals and show the potential of the herbicide to turn rice into a selective catch crop for an economically important pest.

Three MAPK Kinases, MEK1, SIPKK, and NPK2, are not Involved in Activation of SIPK after Wounding and Herbivore Feeding but Important for Accumulation of Trypsin Proteinase Inhibitors

We identified three Nicotiana attenuata mitogen-activated protein kinase (MAPK) kinases (MAPKKs), NaMEK1, NaSIPKK, and NaNPK2, whose transcript levels were up-regulated in a wild tobacco plant, N. attenuata, after wounding and simulated herbivore attack. A virus-induced gene silencing approach was used to analyze the functions of these three MAPKKs in plant defense against the tobacco hornworm Manduca sexta. NaMEK1 and NaSIPKK influenced the accumulation of the precursor of jasmonic acid (JA), 12-oxo-phytodienoic acid, and silencing NaSIPKK enhanced the levels of wounding- and herbivory-induced JA. In-gel kinase assays indicated that all three MAPKKs were not required for the activation of NaSIPK, an important MAPK in plant responses to wounding and herbivory. However, NaMEK1, NaSIPKK, and NaNPK2 appeared to regulate the levels of trypsin proteinase inhibitor activity. Bioassays revealed that M. sexta larval growth was not impaired on N. attenuata plants silenced in NaMEK1, NaSIPKK, or NaNPK2 expression. Our findings suggest that NaSIPKK is involved in JA biosynthesis after herbivore attack without activating NaSIPK, suggesting that the network of MAPK signaling in N. attenuata's defense responses against herbivore attack is more complicated than previously thought.

BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata's responses to herbivory

BAK1 is a co-receptor of brassinosteroid (BR) receptor BRI1, and plays a well-characterized role in BR signalling. BAK1 also physically interacts with the flagellin receptor FLS2 and regulates pathogen resistance. The role of BAK1 in mediating Nicotiana attenuata's resistance responses to its specialist herbivore, Manduca sexta, was examined here. A virus-induced gene-silencing system was used to generate empty vector (EV) and NaBAK1-silenced plants. The wounding- and herbivory-induced responses were examined on EV and NaBAK1-silenced plants by wounding plants or simulating herbivory by treating wounds with larval oral secretions (OS). After wounding or OS elicitation, NaBAK1-silenced plants showed attenuated jasmonic acid (JA) and JA-isoleucine bursts, phytohormone responses important in mediating plant defences against herbivores. However, these decreased JA and JA-Ile levels did not result from compromised MAPK activity or elevated SA levels. After simulated herbivory, NaBAK1-silenced plants had EV levels of defensive secondary metabolites, namely, trypsin proteinase inhibitors (TPIs), and similar levels of resistance to Manduca sexta larvae. Additional experiments demonstrated that decreased JA levels in NaBAK1-VIGS plants, rather than the enzymatic activity of JAR proteins or Ile levels, were responsible for the reduced JA-Ile levels observed in these plants. Methyl jasmonate application elicited higher levels of TPI activity in NaBAK1-silenced plants than in EV plants, suggesting that silencing NaBAK1 enhances the accumulation of TPIs induced by a given level of JA. Thus NaBAK1 is involved in modulating herbivory-induced JA accumulation and how JA levels are transduced into TPI levels in N. attenuata.

Silencing NaTPI Expression Increases Nectar Germin, Nectarins, and Hydrogen Peroxide Levels and Inhibits Nectar Removal from Plants in Nature

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellent, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.

Pectin methylesterase NaPME1 contributes to the emission of methanol during insect herbivory and to the elicitation of defence responses in Nicotiana attenuata

Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60–120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.

Jasmonates and its mimics differentially elicit systemic defence responses in Nicotiana attenuata

Coronalon (6-ethyl indanoyl isoleucine), a synthetic jasmonate mimic, is known to regulate levels of transcripts and secondary metabolites that are commonly elicited by methyl jasmonate (MeJA) in a variety of plants. The ability of coronalon and its derivative (In-L-Ile-Me) to elicit MeJA-activated transcriptional and defence responses [nicotine and trypsin proteinase inhibitors (TPIs)] was compared in treated and systemic untreated tissues of wild-type (WT) and NaLOX3-silenced Nicotiana attenuata plants which are unable to activate either local or systemic defence responses. Coronalon and its derivative significantly regulated 71% and 86% of genes up-regulated by MeJA and 53% and 66% of the genes down-regulated by MeJA in the treated leaves, but only 3% and 7% of all regulated genes in untreated, but phylotactically connected, leaves of WT plants. Consistent with their ability to elicit transcriptional responses in treated tissues, coronalon and In-L-Ile-Me increased nicotine and TPIs when applied to the tissues in which these metabolites are produced, namely roots and leaves. However, treating roots elicited TPI activity in leaves in both WT and NaLOX3-silenced plants, suggesting that mimics can be transported apoplastically from roots to leaves in the xylem. This response was lower in NaLOX3-silenced plants, suggesting that the ability of coronalon and In-L-Ile-Me to elicit TPI responses in leaves after root treatments requires intact jasmonic acid (JA) signalling. Treating leaves did not elicit detectable changes in endogenous JA levels but did decrease free salicylic acid contents. It is concluded that coronalon and In-L-Ile-Me elicit jasmonate responses in treated tissues and could be valuable tools for dissecting local and systemic jasmonate signalling networks in plants.

Co(i)‐ordinating defenses: NaCOI1 mediates herbivore‐ induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses

Arabidopsis and tomato plants mutated in the F-box protein COI1 mediating jasmonate (JA) responses are more susceptible to herbivores in laboratory trials, but the exact mechanisms of COI1-mediated resistance are not known. We silenced COI1 by transformation with an inverted repeat construct (ir-coi1) in Nicotiana attenuata, a plant the direct and indirect defenses of which against various herbivores have been well studied. ir-coi1 plants are male sterile and impaired in JA-elicited direct [nicotine, caffeoylputrescine and trypsin proteinase inhibitor (TPI) activity] and indirect (cis-alpha-bergamotene emission) defense responses; responses not elicited by JA treatment (ethylene production and flower TPI activity) were unaffected. Larvae of Manduca sexta, a common herbivore of N. attenuata, gained three times more mass feeding on ir-coi1 than on wild-type (WT) plants in glasshouse experiments. By regularly moving caterpillars to unattacked leaves of the same plant, we demonstrate that larvae on WT plants can grow and consume leaves as fast as those on ir-coi1 plants, a result that underscores the role of COI1 in mediating locally induced resistance in attacked leaves, and the importance of herbivore movement in avoiding the induced defenses of a plant. When transplanted into native habitats in the Great Basin Desert, ir-coi1 plants suffer greatly from damage by the local herbivore community, which includes herbivores not commonly found on N. attenuata WT plants. Choice assays with field-grown plants confirmed the increased attractiveness of ir-coi1 plants for both common and unusual herbivores. We conclude that NaCOI1 is essential for induced resistance in N. attenuata, and that ir-coi1 plants highlight the benefits of herbivore movement for avoiding induced defenses.

Resistance management in a native plant: nicotine prevents herbivores from compensating for plant protease inhibitors

Plants deploy chemical defenses in complex mixtures, which are thought to be adaptive, but experimental tests have used artificial diets rather than plants. Herbivore attack on Nicotiana attenuata rapidly increases the production and accumulation of trypsin proteinase inhibitors (TPI) and the toxic alkaloid nicotine. By transgenically silencing their respective biosynthetic genes, we were able to abolish TPI activity and reduce inducible nicotine by 85%. Nicotine production was not affected by silencing pi or vice versa, and transformation did not alter levels of other metabolites examined. Spodoptera exigua, a native generalist herbivore that can compensate for heterologous TPI expression, performed better on TPI- or nicotine-deficient plants compared with the wild-type. Because of a compensatory feeding response to TPI when nicotine is absent, larvae performed better on nicotine-deficient plants than they did on plants silenced in both defenses. The antifeedant toxin, nicotine, prevents this compensatory response. We conclude that N. attenuata counters an insect adaptation with a defensive synergism.

Independently silencing two JAR family members impairs levels of trypsin proteinase inhibitors but not nicotine

Jasmonic acid (JA)-amino acid conjugates are important JA metabolites that activate JA responses. However, our understanding of their involvement in herbivore defenses is limited. We identified a new Arabidopsis jasmonate resistant 1 (JAR1) homologue in Nicotiana attenuata (N. attenuata) and named it jasmonate resistant 6 (JAR6). JAR6 clustered closely with Arabidopsis JAR1 and the recently reported jasmonate resistant 4 (JAR4), another JAR1 homologue in N. attenuata, in a phylogenic analysis. The strong elicitation of JAR6 transcripts by wounding and treatment with Manduca sexta (M. sexta) oral secretions (OS), which mimics herbivore attack, suggests it plays a role in herbivore defense. Independently silencing JAR4 or JAR6 by transforming N. attenuata with inverted repeat JAR4 or JAR6 constructs significantly reduced levels of not only JA-Ile plus JA-Leu but also JA-Val in OS-elicited leaves, suggesting JAR4 and JAR6 are functionally redundant and their amino acid substrates are not highly specific to individual amino acids. A new JA conjugate, JA-Gln, whose levels are much higher than those of the other JA conjugates in WT plants, was not affected in JAR4- or JAR6-silenced lines, implying that another JA-conjugating enzyme exists in N. attenuata. Neither JA-ACC, the second most abundant JA conjugate in Arabidopsis seedlings, nor JA-Met or JA-Trp, was detectable in N. attenuata. Levels of trypsin proteinase inhibitors (TPIs) in JAR4- and JAR6-silenced plants were significantly reduced, but nicotine levels were normal. We conclude that both JAR4 and JAR6 conjugate JA to Ile, Val, and Leu, and that both positively regulate TPI activity.

Systemin in Solanum nigrum. The Tomato-Homologous Polypeptide Does Not Mediate Direct Defense Responses

We extend Ryan's seminal work on the 18-amino acid polypeptide systemin in tomato's (Solanum lycopersicum) systemic wound response to the closely related solanaceous species Solanum nigrum. We compared wild-type plants to plants transformed with an inverted repeat prosystemin construct (IRSys) to silence the expression of the endogenous S. nigrum prosystemin gene. In wild-type plants elicited with wounding + oral secretions from Manduca sexta larvae, trypsin-proteinase inhibitors (TPIs) accumulated even though prosystemin transcripts were down-regulated. Neither reducing the endogenous systemin levels by RNAi nor complementing the plants with systemin by exogenously supplying the polypeptide through excised stems significantly increased TPI activity, indicating that systemin and TPIs are not correlated in S. nigrum. The performance of two herbivore species from two feeding guilds, M. sexta larvae and Myzus persicae nicotianae, did not differ between wild-type and IRSys plants, demonstrating that varying endogenous systemin levels do not alter the direct defenses of S. nigrum. Field experiments with wild-type and IRSys plants and the flea beetle Epitrix pubescens supported these glasshouse data. That levels of oral secretion-elicited jasmonic acid did not differ between wild-type and IRSys plants suggests that systemin is unlikely to mediate jasmonate signaling in S. nigrum as it does in tomato. We conclude that the tomato-homologous polypeptide does not mediate direct defense responses in S. nigrum.

Evolution of proteinase inhibitor defenses in North American allopolyploid species of Nicotiana

We studied the jasmonate (JA)-elicited trypsin-proteinase inhibitor (TPI) anti-herbivore defense system in North American Nicotiana to understand how complex polygenetic traits evolve after allopolyploidy speciation. N. quadrivalvis (Nq) and N. clevelandii (Nc) are allotetraploid descendant species of the ancestors of the diploid species N. attenuata (Na) and N. obtusifolia (No). From cDNA, intron and promoter sequence analyses, and Southern blotting, we deduced that only the maternally derived No TPI genes were retained in the tetraploid genomes (Nq, Nc), whereas the sequences of the paternal Na ancestor were deleted. The number of TPI repeats in different Nicotiana taxa was independent of phylogenetic associations. In Na, TPI activity and mRNA transcript accumulation as well as JA levels increased dramatically above wound-induced levels when the oral secretions (OS) from Manduca sexta larvae were introduced into wounds. This OS-mediated amplification of defense signaling and downstream response was also found in the tetraploid genomes but was absent from No; in No, OS treatment suppresses TPI mRNA accumulation and activity and does not increase JA accumulation. Hence, the tetraploids retained components of Na's signaling system, but lost Na's TPI genes and used No's TPI genes to retain a functional TPI defense system, underscoring the genomic flexibility that enables complex polygenic traits to be retained in allopolyploid species.

Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.

Constitutive and inducible trypsin proteinase inhibitor production incurs large fitness costs in Nicotiana attenuata.

Plant trypsin proteinase inhibitors (TPIs) are potent herbivore- and jasmonate (JA)-induced defenses, but support for the commonly invoked explanation for their inducible expression, namely their associated fitness costs, has been elusive. To determine whether the expression of TPIs incurs fitness costs, we expressed 175 bp of the seven-domain pi from Nicotiana attenuata in an antisense orientation in a TPI-producing genotype (WT) of N. attenuata to reduce TPI expression. Moreover, we expressed the full-length seven-domain pi in a sense orientation under control of a constitutive promoter to restore TPI activity in a natural genotype unable to produce TPIs because of a mutation in its endogenous pi gene. Lifetime reproductive output was determined from high and low TPI-producing plants of the same genetic background with and without JA elicitation and grown in the same pot to simulate natural competitive and nutrient regimes. Transformants with either low or no TPI activity grew faster and taller, flowered earlier, and produced more seed capsules (25-53%) than did neighboring TPI-producing genotypes, and JA elicitation increased TPI production and decreased seed capsule production further. Growth under high light levels only marginally reduced these fitness costs. Results were similar regardless of whether TPI activity was suppressed or restored by transformation: the larger the difference in TPI activity between neighbors, the larger the difference in seed capsule production (R(2) = 0.57). TPI production is costly for a plant's components of fitness when grown under realistic competitive regimes and is consistent with the hypothesis that inducibility evolved as a cost-saving mechanism.

Fitness benefits of trypsin proteinase inhibitor expression in Nicotiana attenuata are greater than their costs when plants are attacked.

BackgroundThe commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores.ResultsIn order to determine whether the putative benefits of TPI production outweigh its cost, we transformed N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R2 = 0.56) with seed capsule production per plant.ConclusionsOur results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in M. sexta performance translates into a fitness benefit for the plant.