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Drought at flowering and grain filling greatly reduces maize (Zea mays) yield. Climate change is causing earlier and longer-lasting periods of drought, which affect the growth of multiple maize organs throughout development. To study how long periods of water deficit impact the dynamic nature of growth, and to determine how these relate to reproductive drought, we employed a high-throughput phenotyping platform featuring precise irrigation, imaging systems, and image-based biomass estimations. Prolonged drought resulted in a reduction of growth rate of individual organs-though an extension of growth duration partially compensated for this-culminating in lower biomass and delayed flowering. However, long periods of drought did not affect the highly organized succession of maximal growth rates of the distinct organs, i.e. leaves, stems, and ears. Two drought treatments negatively affected distinct seed yield components: Prolonged drought mainly reduced the number of spikelets, and drought during the reproductive period increased the anthesis-silking interval. The identification of these divergent biomass and yield components, which were affected by the shift in duration and intensity of drought, will facilitate trait-specific breeding toward future climate-resilient crops. When affected by drought, the plant responds by adjusting the growth of individual organs but not the coordination between the organs.

Hyperspectral imaging is a promising tool for non-destructive phenotyping of plant physiological traits, which has been transferred from remote to proximal sensing applications, and from manual laboratory setups to automated plant phenotyping platforms. Due to the higher resolution in proximal sensing, illumination variation and plant geometry result in increased non-biological variation in plant spectra that may mask subtle biological differences. Here, a better understanding of spectral measurements for proximal sensing and their application to study drought, developmental and diurnal responses was acquired in a drought case study of maize grown in a greenhouse phenotyping platform with a hyperspectral imaging setup. The use of brightness classification to reduce the illumination-induced non-biological variation is demonstrated, and allowed the detection of diurnal, developmental and early drought-induced changes in maize reflectance and physiology. Diurnal changes in transpiration rate and vapor pressure deficit were significantly correlated with red and red-edge reflectance. Drought-induced changes in effective quantum yield and water potential were accurately predicted using partial least squares regression and the newly developed Water Potential Index 2, respectively. The prediction accuracy of hyperspectral indices and partial least squares regression were similar, as long as a strong relationship between the physiological trait and reflectance was present. This demonstrates that current hyperspectral processing approaches can be used in automated plant phenotyping platforms to monitor physiological traits with a high temporal resolution.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

The recognition of phytophagous insects by plants induces a set of very specific responses aimed at deterring tissue consumption and reprogramming metabolism and development of the plant to tolerate the herbivore. The recognition of insects by plants requires the plant's ability to perceive chemical cues generated by the insects and to distinguish a particular pattern of tissue disruption. Relatively little is known about the molecular basis of insect perception by plants and the signalling mechanisms directly associated with this perception. Importantly, the insect feeding behaviour (piercing-sucking versus chewing) is a decisive determinant of the plant's defence response, and the mechanisms used to perceive insects from different feeding guilds may be distinct. During insect feeding, components of the saliva of chewing or piercing-sucking insects come into contact with plant cells, and elicitors or effectors present in this insect-derived fluid are perceived by plant cells to initiate the activation of specific signalling cascades. Although receptor-ligand interactions controlling insect perception have yet not been molecularly described, a significant number of regulatory components acting downstream of receptors and involved in the activation of defence responses against insects has been reported. Some of these regulators mediate changes in the phytohormone network, while others directly control gene expression or the redox state of the cell. These processes are central in the orchestration of plant defence responses against insects.

Jasmonate signalling plays a central role in activating the plethora of responses that are elicited by herbivory. Solanum nigrum plants silenced in the expression of genes involved in jasmonic acid biosynthesis (irlox3),conjugation (irjar4) and perception (ircoi1) were used to study the function of these genes in the field and in the regulation of transcriptional and metabolic responses. In the field, damage from Noctuidea larvae was four- to fivefold higher on irlox3 and ircoi1 than on wild-type (WT) plants, whereas damage to irjar4 plants was similar to WT levels. Damage rates reflected plant survival rates; fewer irlox3 (78%) and ircoi1 (22%) plants survived compared with irjar4 and WT plants of which all plants survived. Gene expression profiling in leaves 3 h after simulated herbivory revealed differential regulation of ~700 genes in irlox3 and ircoi1 plants but of only six genes in irjar4 compared with WT plants. Surprisingly, transcriptional responses were not reflected in metabolomic responses; 48 h after simulated herbivory, irjar4 plants showed a 50% overlap in their metabolic profile with ircoi1 plants. Together, these results reveal that SnJAR4 does not play a direct role in herbivore defence, but suggests that SnJAR4 is involved in responses other than those to herbivory.

The activation of enzymatic oxylipin biosynthesis upon wounding, herbivory and pathogen attack depends on the biochemical activation of lipases that make polyunsaturated fatty acids (PUFAs) available to lipoxygenases (LOXs). The identity and number of the lipases involved in this process remain controversial and they probably differ among plant species. Analysis of transgenic Nicotiana attenuata plants (ir-gla1) stably reduced in the expression of the NaGLA1 gene showed that this plastidial glycerolipase is a major supplier of trienoic fatty acids for jasmonic acid (JA) biosynthesis in leaves and roots after wounding and simulated herbivory, but not during infection with the oomycete Phytophthora parasitica (var. nicotianae). NaGLA1 was not essential for the developmental control of JA biosynthesis in flowers and for the biosynthesis of C(6) volatiles by the hydroperoxide lyase (HPL) pathway; however, it affected the metabolism of divinyl ethers (DVEs) early during infection with P. parasitica (var. nicotianae) and the accumulation of NaDES1 and NaLOX1 mRNAs. Profiling of lysolipids by LC-MS/MS was consistent with a rapid activation of NaGLA1 and indicated that this lipase utilizes different lipid classes as substrates. The results revealed the complexity and specificity of the regulation of lipase-mediated oxylipin biosynthesis, highlighting the existence of pathway- and stimulus-specific lipases.

The recognition of insect and pathogen attack requires the plant's ability to perceive chemical cues generated by the attacker. In contrast to the recognition of microbe-associated molecular patterns and effectors, little is known about the molecular recognition of herbivore-associated elicitors (HAEs) and the signaling mechanisms operating in plants after their perception. HAE perception depends strongly on the natural history of both plants and insects and it is therefore expected that many of the responses induced by different HAEs are specific to the species involved in the interaction. The interaction between Nicotiana attenuata and the specialist lepidopteran Manduca sexta presents a relevant biological system to understand HAE perception and signal transduction systems in plants.

Homeodomain-leucine zipper type I (HD-Zip I) proteins are plant-specific transcription factors associated with the regulation of growth and development in response to changes in the environment. Nicotiana attenuata NaHD20 was identified as an HD-Zip I-coding gene whose expression was induced by multiple stress-associated stimuli including drought and wounding. To study the role of NaHD20 in the integration of stress responses with changes in growth and development, its expression was silenced by virus-induced gene silencing (VIGS), and control and silenced plants were metabolically and developmentally characterized. Phytohormone profiling showed that NaHD20 plays a positive role in abscisic acid (ABA) accumulation in leaves during water stress and in the expression of some dehydration-responsive genes including ABA biosynthetic genes. Moreover, consistent with the high levels of NaHD20 expression in corollas, the emission of benzylacetone from flowers was reduced in NaHD20-silenced plants. Additionally, bolting time and the opening of the inflorescence buds was decelerated in these plants in a specific developmental stage without affecting the total number of flowers produced. Water stress potentiated these effects; however, after plants recovered from this condition, the opening of the inflorescence buds was accelerated in NaHD20-silenced plants. In summary, NaHD20 plays multiple roles in N. attenuata and among these are the coordination of responses to dehydration and its integration with changes in flower transitions. Fil: Ré, Delfina Adela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Instituto Max Planck Institut für Chemische Okologie; Alemania Fil: Dezar, Carlos Alberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Baldwin, Ian T.. Instituto Max Planck Institut für Chemische Okologie; Alemania Fil: Bonaventure, Gustavo. Instituto Max Planck Institut für Chemische Okologie; Alemania

The Helianthus annuus (sunflower) HAHB4 transcription factor belongs to the HD-Zip family and its transcript levels are strongly induced when sunflower plants are attacked by herbivores, mechanically damaged or treated with methyl-jasmonic acid (MeJA) or ethylene (ET). Promoter fusion analysis, in Arabidopsis and in sunflower, demonstrated that induction of HAHB4 expression by these treatments is regulated at the transcriptional level. In transiently transformed sunflower plants HAHB4 expression upregulates the transcript levels of several genes involved in JA biosynthesis and defense-related processes such as the production of green leaf volatiles and trypsin protease inhibitors (TPI). In HAHB4 sunflower overexpressing tissue, increased activities of lipoxygenase, hydroperoxide lyase and TPI are detected whereas in HAHB4-silenced tissue these activities are reduced. Transgenic Arabidopsis thaliana and Zea mays plants ecotopically expressing HAHB4 also exhibit higher transcript levels of defense-related genes and when Spodoptera littoralis or Spodoptera frugiperda larvae are placed on each species, respectively, larvae consumed less and gain less mass compared with larvae feeding on control plants. Arabidopsis plants ectopically expressing HAHB4 had higher amounts of JA, JA-isoleucine and ET compared with control plants both before and after wounding, but reduced levels of salicylic acid (SA) after wounding and bacterial infection. We conclude that HAHB4 coordinates the production of phytohormones during biotic stress responses and mechanical damage, specifically by positively regulating JA and ET production and negatively regulating ET sensitivity and SA accumulation. Fil: Manavella, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Dezar, Carlos Alberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Bonaventure, Gustavo. Instituto Max Planck Institut für Chemische Okologie; Alemania Fil: Baldwin Ian Thomas. Instituto Max Planck Institut für Chemische Okologie; Alemania Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

Background: Arabidopsis AtHB7 and AtHB12 transcription factors (TFs) belong to the homeodomain-leucine zipper subfamily I (HD-Zip I) and present 62% amino acid identity. These TFs have been associated with the control of plant development and abiotic stress responses; however, at present it is not completely understood how AtHB7 and AtHB12 regulate these processes. Results: By using different expression analysis approaches, we found that AtHB12 is expressed at higher levels during early Arabidopsis thaliana development whereas AtHB7 during later developmental stages. Moreover, by analysing gene expression in single and double Arabidopsis mutants and in transgenic plants ectopically expressing these TFs, we discovered a complex mechanism dependent on the plant developmental stage and in which AtHB7 and AtHB12 affect the expression of each other. Phenotypic analysis of transgenic plants revealed that AtHB12 induces root elongation and leaf development in young plants under standard growth conditions, and seed production in water-stressed plants. In contrast, AtHB7 promotes leaf development, chlorophyll levels and photosynthesis and reduces stomatal conductance in mature plants. Moreover AtHB7 delays senescence processes in standard growth conditions. Conclusions: We demonstrate that AtHB7 and AtHB12 have overlapping yet specific roles in several processes related to development and water stress responses. The analysis of mutant and transgenic plants indicated that the expression of AtHB7 and AtHB12 is regulated in a coordinated manner, depending on the plant developmental stage and the environmental conditions. The results suggested that AtHB7 and AtHB12 evolved divergently to fine tune processes associated with development and responses to mild water stress. Fil: Ré, Delfina Adela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina Fil: Capella, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina Fil: Bonaventure, Gustavo. Max Planck Institut Jena; Bélgica Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina

Nicotiana attenuata plants silenced in the expression of GLYCEROLIPASE A1 (ir-gla1 plants) are compromised in the herbivore- and wound-induced accumulation of jasmonic acid (JA). However, these plants accumulate wild-type (WT) levels of JA and divinyl-ethers during Phytophthora parasitica infection. By profiling oxylipin-enriched fractions with targeted and untargeted liquid chromatography-tandem time-of-flight mass spectrometry approaches, we demonstrate that the accumulation of 9-hydroxy-10E,12Z-octadecadienoic acid (9-OH-18:2) and additional C18 and C19 oxylipins is reduced by ca. 20-fold in P. parasitica-infected ir-gla1 leaves compared with WT. This reduced accumulation of oxylipins was accompanied by a reduced accumulation of unsaturated free fatty acids and specific lysolipid species. Untargeted metabolic profiling of total leaf extracts showed that 87 metabolites accumulated differentially in leaves of P. parasitica-infected ir-gla1 plants with glycerolipids, hydroxylated-diterpene glycosides and phenylpropanoid derivatives accounting together for ca. 20% of these 87 metabolites. Thus, P. parasitica-induced oxylipins may participate in the regulation of metabolic changes during infection. Together, the results demonstrate that GLA1 plays a distinct role in the production of oxylipins during biotic stress responses, supplying substrates for 9-OH-18:2 and additional C18 and C19 oxylipin formation during P. parasitica infection, whereas supplying substrates for the biogenesis of JA during herbivory and mechanical wounding.

Nicotiana attenuata HSPRO (NaHSPRO) is a negative regulator of seedling growth promoted by the fungus Piriformospora indica. Homologs of NaHSPRO in Arabidopsis thaliana (i.e., AtHSPRO1 and AtHSPRO2) are known to physically interact with the AKINβγ subunit of the SnRK1 complex. To investigate whether NaHSPRO is associated with SnRK1 function during the stimulation of seedling growth by P. indica, we studied N. attenuata plants silenced in the expression of NaGAL83 (as-gal83 plants)--a gene that encodes for the regulatory β-subunit of SnRK1--and plants silenced in the expression of both NaHSPRO and NaGAL83 (ir-hspro/as-gal83 plants). The results showed that P. indica differentially stimulated the growth of both as-gal83 and ir-hspro/as-gal83 seedlings compared with control seedlings, with a magnitude similar to that observed in ir-hspro seedlings. Thus, we showed that, similar to NaHSPRO, NaGAL83 is a negative regulator of seedling growth stimulated by P. indica. We propose that the effect of NaHSPRO on seedling growth is associated with SnRK1 signaling.

Plants employ a large variety of defense strategies to resist herbivores, which require transcriptional reprogramming of cells and profound changes in plant metabolism. Due to the large number of genes involved in defense processes, rapid screening strategies are essential for elucidating the contributions of individual genes in the responses of plants to herbivory. However, databases and seed banks of mutant plants which allow rapid retrieval of mutant genotypes are limited to a few model plant species, namely, Arabidopsis thaliana and Oryza sativa (rice). In other plants, virus-induced gene silencing (VIGS) offers an efficient alternative for screening the functions of individual genes in order to prioritize the allocations of the large time investments required to establish stably transformed RNAi-silenced lines. With VIGS, it is usually possible to achieve strong, specific silencing of target genes in the ecological models Nicotiana attenuata and Solanum nigrum, allowing the rapid assessment of gene silencing effects on phytohormone accumulation, signal transduction and accumulation of defense metabolites. VIGS plants are also useful in bioassays with specialist and generalist herbivores, allowing direct verification of gene function in plant resistance to herbivores.

In a previous study aimed at identifying regulators of Nicotiana attenuata responses against chewing insects, a 26-nucleotide tag matching the HSPRO (ORTHOLOG OF SUGAR BEET Hs1pro - 1) gene was found to be strongly induced after simulated herbivory (Gilardoni et al., 2010). Here we characterized the function of HSPRO during biotic interactions in transgenic N. attenuata plants silenced in its expression (ir-hspro). In wild-type plants, HSPRO expression was not only induced during simulated herbivory but also when leaves were inoculated with Pseudomonas syringae pv tomato DC3000 and roots with the growth-promoting fungus Piriformospora indica. Reduced HSPRO expression did not affect the regulation of direct defenses against Manduca sexta herbivory or P. syringae pv tomato DC3000 infection rates. However, reduced HSPRO expression positively influenced early seedling growth during interaction with P. indica; fungus-colonized ir-hspro seedlings increased their fresh biomass by 30% compared with the wild type. Grafting experiments demonstrated that reduced HSPRO expression in roots was sufficient to induce differential growth promotion in both roots and shoots. This effect was accompanied by changes in the expression of 417 genes in colonized roots, most of which were metabolic genes. The lack of major differences in the metabolic profiles of ir-hspro and wild-type colonized roots (as analyzed by liquid chromatography time-of-flight mass spectrometry) suggested that accelerated metabolic rates were involved. We conclude that HSPRO participates in a whole-plant change in growth physiology when seedlings interact with P. indica.

Choice of host plants by phytophagous insects is essential for their survival and reproduction. This choice involves complex behavioral responses to a variety of physical and chemical characteristics of potential plants for feeding. For insects of the order Hemiptera, these behavioral responses involve a series of steps including labial dabbing and probing using their piercing mouthparts. These initial probing and feeding attempts also elicit a rapid accumulation of phytohormones, such as jasmonic acid (JA), and the induced defense metabolites they mediate. When Nicotiana attenuata plants are rendered JA deficient by silencing the initial committed step of the JA biosynthesis pathway, they are severely attacked in nature by hemipteran leafhoppers of the genus Empoasca . By producing N. attenuata plants silenced in multiple steps of JA biosynthesis and perception and in the biosynthesis of the plant’s three major classes of JA-inducible insecticidal defenses, we demonstrate that the choice of plants for feeding by Empoasca leafhoppers in both nature and the glasshouse is independent of the accumulation of major insecticidal molecules. Moreover, this choice is independent of the presence of Candidatus Phytoplasma spp. and is not associated with detectable changes in plant volatiles but instead depends on the plant´s capacity to mediate JA signaling. We exploited this trait and used Empoasca leafhoppers to reveal genetic variation in JA accumulation and signaling hidden in N. attenuata natural populations.

The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 (LecRK1) has been recently identified as a component of the mechanism used by plants to suppress the Manduca sexta-triggered accumulation of salicylic acid (SA). The suppression of the SA burst by LecRK1 allows for the unfettered induction of jasmonic acid (JA)-mediated defense responses against M. sexta herbivory. LecRK1 contains a multi-domain extracellular region composed of a G-type Lectin domain and a PAN-AP domain separated by a variable sequence with low similarity to an EGF domain. The LecRK1 intracellular region is composed of a single domain structure with predicted Ser/Thr protein kinase activity. The multi-domain structure of the extracellular region of LecRK1 adds a level of complexity in terms of the potential ligands that this receptor protein could recognize.

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)–induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.

The transcription factor HAHB10 belongs to the sunflower (Helianthus annuus) HD-Zip II subfamily and it has been previously associated with the induction of flowering. In this study it is shown that HAHB10 is expressed in sunflower leaves throughout the vegetative stage and in stamens during the reproductive stage. In short-day inductive conditions the expression of this gene is induced in shoot apexes together with the expression of the flowering genes HAFT and HAAP1. Transgenic Arabidopsis plants expressing HAHB10 cDNA under regulation either by its own promoter or by cauliflower mosaic virus (CaMV) 35S exhibited an early flowering phenotype. This phenotype was completely reverted in a non-inductive light regime, indicating a photoperiod-dependent action for this transcription factor. Gene expression profiling of Arabidopsis plants constitutively expressing HAHB10 indicated that specific flowering transition genes such as FT, FUL, and SEP3 were induced several fold, whereas genes related to biotic stress responses, such as PR1, PR2, ICS1, AOC1, EDS5, and PDF1-2a, were repressed. The expression of HAHB10 and of the flowering genes HASEP3 and HAFT was up-regulated by both salicylic acid (SA) treatment and infection with a virulent strain of Pseudomonas syringae. Basal SA and jasmonic acid (JA) levels in Arabidopsis plants ectopically expressing HAHB10 were similar to those of control plants; however, SA levels differentially increased in the transgenic plants after wounding and infection with P. syringae while JA levels differentially decreased. Taken together, the results indicated that HAHB10 participates in two different processes in plants: the transition from the vegetative to the flowering stage via the induction of specific flowering transition genes and the accumulation of phytohormones upon biotic stresses.

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glutamate [corrected] (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.

Plastids are the central orchestrators of the early and late responses to wounding and herbivory in plants. This organelle houses some of the most important enzymes involved in the biogenesis of intra and extracellular signals that mediate defense responses against these stresses. Among these enzymes are the ones initiating the biosynthesis of oxylipins [e.g., jasmonic acid (JA) and C(6) volatiles], terpenoid volatiles and phenolic compounds, including both volatile [e.g., methylsalicylate (MeSA)] and non-volatile compounds [e.g., salicylic acid (SA)]. Plastids also play a major role in orchestrating changes in primary metabolism during herbivory and thereby in the reallocation of carbon and nitrogen to different functions in response to herbivory. How the primary stress signals generated by mechanical damage and herbivory reach the plastid to activate the rapid synthesis of these signal molecules is at present largely unknown.

Plants can distinguish mechanical damage from larval folivory through the recognition of specific constituents of larval oral secretions (OS) which are deposited on the surface of leaf wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of the OS of Lepidopteran larvae and they are strong elicitors of herbivore-induced defense responses in several plant species, including the wild tobacco Nicotiana attenuata. When OS from Manduca sexta larvae is deposited on N. attenuata wounded leaves, the major FAC N-linolenoyl-glutamic acid (18:3-Glu) is modified within seconds by a heat labile process. Some of the major modified forms are oxygenated products derived from 13-lipoxygenase activity and one of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of enhanced JA biosynthesis and differential monoterpene emission in N. attenuata leaves.

CORONATINE INSENSITIVE 1 (COI1) is a well-known key player in processes downstream of jasmonic acid (JA) biosynthesis: silencing COI1 in Nicotiana attenuata (ir-coi1) makes plants insensitive to JA, prevents the up-regulation of JA-mediated defenses and decreases the plant's resistance to herbivores and pathogens. In agreement with previous studies, we observed that regulation of several JA biosynthesis genes elicited by Manduca sexta oral secretions (OS) is COI1 dependent. In response to wounding and application of OS ir-coi1 plants accumulate 75% less JA compared with wild-type plants (WT), resembling JA levels found in plants silenced in the key enzyme in JA biosynthesis LIPOXYGENASE 3 (as-lox). However, while OS-elicited as-lox plants also accumulated lower levels of the JA-conjugate JA-isoleucine (JA-Ile) than did WT plants, JA-Ile accumulation in ir-coi1 was higher, prolonged and peaked with a delay of 30 min. In vivo substrate feeding experiments of N. attenuata demonstrate that the increased and prolonged JA-Ile accumulation pattern in ir-coi1 is not the result of altered substrate availability, i.e. of JA and/or Ile, but is due to an approximately 6-fold decrease in JA-Ile turnover. These results provide the first evidence for a second, novel regulatory feedback function of COI1 in enhancing JA-Ile turnover. Hence, in addition to its control over JA biosynthesis, COI1 might fine-tune the dynamics of the jasmonate response after induction by herbivore elicitors.