Project description:Jasmonoyl-isoleucine regulates defence, growth and developmental responses in eudicots. Bryophyte genomes have conserved sequences for all JA-Ile signalling pathway components, but in contrast to higher plants, the bioactive hormone has not been identified. We show that the JA-Ile receptor COI1 is functionally conserved in the bryophyte Marchantia polymorpha, but responds to a different ligand. Although Marchantia plants neither synthesize nor respond to JA-Ile, loss-of-function Mpcoi1 mutants have phenotypic defects reminiscent of COI1-dependent functions in Arabidopsis. AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness on M. polymorpha, as does a single amino acid substitution in MpCOI1 that switches ligand specificity. Mass spectrometry quantification of cyclopentenone derivatives, bioactivity analysis and COI1-ligand interaction assays pinpointed two isomers of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA) as the natural MpCOI1 ligands. Our results identify the ancestral jasmonate, confirm the functional conservation of its signalling pathway, and show that JA-Ile and COI1 emergence in higher plants from their ancestral counterparts required co-evolution of hormone biosynthetic complexity and receptor specificity.
Project description:The phytohormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) is a major regulator of developmental and stress responses in plants. The perception of JA-Ile involves the formation of a ternary complex with the F-box protein COI1 and a member of the JAZ family of co-repressors, which leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the co-receptor COI1-JAZ complex with higher affinity than JA-Ile. Based on the crystal structure of the co-receptor, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists of COR or JA-Ile. One derivative, Coronatine O-methyloxime (COR-MO), shows a strong activity preventing COI-JAZ interaction and the subsequent JAZ degradation. COR-MO efficiently reverts the effects of JA-Ile or COR treatments on JA-mediated responses, such as anthocyanin accumulation, root-growth inhibition and gene expression in Arabidopsis plants. Moreover, it potentiates plant resistance preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for Plant Biology research, our results underscore its biotechnological and agronomical potential for a safer and sustainable agriculture.
Project description:JA-signaling in plants is manipulated by COI1 that recruits the protein complex perceiving JA signal . To determine JA correlated with the function of tobacco COI1, NtCOI1-silenced and control before and after MeJA treatment were analyzed using microarray chip.
Project description:The phytohormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) is a major regulator of developmental and stress responses in plants. The perception of JA-Ile involves the formation of a ternary complex with the F-box protein COI1 and a member of the JAZ family of co-repressors, which leads to JAZ degradation. Coronatine (COR) is a bacterial phytotoxin that functionally mimics JA-Ile and interacts with the co-receptor COI1-JAZ complex with higher affinity than JA-Ile. Based on the crystal structure of the co-receptor, we designed ligand derivatives that spatially impede the interaction of the co-receptor proteins and, therefore, should act as competitive antagonists of COR or JA-Ile. One derivative, Coronatine O-methyloxime (COR-MO), shows a strong activity preventing COI-JAZ interaction and the subsequent JAZ degradation. COR-MO efficiently reverts the effects of JA-Ile or COR treatments on JA-mediated responses, such as anthocyanin accumulation, root-growth inhibition and gene expression in Arabidopsis plants. Moreover, it potentiates plant resistance preventing the effect of bacterially produced COR during Pseudomonas syringae infections in different plant species. In addition to the utility of COR-MO for Plant Biology research, our results underscore its biotechnological and agronomical potential for a safer and sustainable agriculture. Two-condition experiment, Col-0 vs Col-0 plants treated 2h with Coronatine 0-methyloxime (COR-MO) and Col-0 plants treated 2h with Coronatine vs Col-0 plants treated 2h with Coronatine plus COR-MO. Biological replicates: 4 control and 4 treated replicates
Project description:DICER-like 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 four distinct DCL genes and stably silenced their expression by RNAi in Nicotiana attenuata, a model system for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal to the plants. Manduca sexta larvae performed significantly better on ir-dcl3and 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. Gene expression in leaves of Nicotiana attenuata wild type, irDCL3 and irDCL4 plants was measured at 1 hour after elicitation with oral secretions of Manduca sexta larvae. Three independent experiments were performed with wild type plants and three independent experiments were performed with irDCL3 and irDCL4 plants. A total of 811 genes were identified as differentially regulated in irDCL3 and irDCL4 compared to wild type plants.
Project description:DICER-like 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 four distinct DCL genes and stably silenced their expression by RNAi in Nicotiana attenuata, a model system for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal to the plants. Manduca sexta larvae performed significantly better on ir-dcl3and 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.
Project description:Jasmonates are fatty acid derived hormones that regulate multiple aspects of plant development, growth and stress responses. Bioactive jasmonates differ between vascular plants and bryophytes (using jasmonoyl-L-isoleucine; JA-Ile and dinor-12-oxo-10,15(Z)-phytodienoic acid; dn-OPDA, respectively), but bind an evolutionarily conserved COI1 receptor. Whilst the biosynthetic pathways of JA-Ile in the model vascular plant Arabidopsis thaliana have been elucidated, the details of dn-OPDA biosynthesis in bryophytes are still unclear. Here, we identify an ortholog of Arabidopsis Fatty Acid Desaturase 5 (AtFAD5) in the model liverwort Marchantia polymorpha and show that FAD5 function is ancient and conserved between species separated by more than 450 million years of independent evolution. Similar to AtFAD5, MpFAD5 is required for the synthesis of 7Z-hexadecenoic acid. Consequently, in Mpfad5 mutants the hexadecanoic pathway is blocked, the dn-OPDA levels almost completely depleted and normal chloroplast development is impaired. Our results demonstrate that the main source of dn-OPDA in Marchantia is the hexadecanoic pathway and the contribution of the octadecanoid pathway, i.e. from OPDA, is minimal. Remarkably, despite extremely low levels of the bioactive hormone (dn-OPDA), MpCOI1-mediated responses to wounding and insect feeding can still be activated in Mpfad5 mutants, suggesting that dn-OPDA is not the only bioactive jasmonate and COI1 ligand in Marchantia.
Project description:Floral Nectaries Many plants secrete a rich floral nectar to entice visitation by insect and avian pollinators. In turn, these pollinators transfer pollen between flowers increasing plant fecundity. The nectary is the floral organ that secretes nectar into the base of the flower. The size and abundance of the ornamental tobacco nectaries (Nicotiana sp.) will permit us to isolate up to several grams of nectaries at each stage to obtain the necessary amounts of RNA for probe preparation. Our primary goals to understand the biochemistry the nectary, so that we can manipulate nectary function to increase pollinator visitation. We have previously conducted an EST study and have identified 13596 cDNAs from three different stages of nectary development (Stage 6, immature, presecretory nectaries; Stage 12, mature nectaries at floral anthesis; and nectaries, 44 hours after fertilization. In our efforts to evaluate the transcriptional program for the Nicotiana nectary we are proposing to evaluate nectary mRNAs by hybridization with the potato microarrays. We have preliminary evidence that wholesale transcriptional reprogramming (60% of the transcriptome) occurs during nectary maturation and again following fertilization. Our goal is to understand these processes at a biochemical level so that we can begin manipulating nectary function to improve nectar quality and quantity thereby increasing the attractiveness of flowers to insect pollinators. Such improvements have the potential to result in increases in insect visitation, seedset, and ultimately yield for insect pollinated crops. We are also making significant efforts to understand the restructuring of the nectary during its lifecycle. Many changes occur during nectary development and the observed transcriptional reprogramming makes sense the when these many changes are accounted for. Keywords: Loop design
Project description:Floral Nectaries Many plants secrete a rich floral nectar to entice visitation by insect and avian pollinators. In turn, these pollinators transfer pollen between flowers increasing plant fecundity. The nectary is the floral organ that secretes nectar into the base of the flower. The size and abundance of the ornamental tobacco nectaries (Nicotiana sp.) will permit us to isolate up to several grams of nectaries at each stage to obtain the necessary amounts of RNA for probe preparation. Our primary goals to understand the biochemistry the nectary, so that we can manipulate nectary function to increase pollinator visitation. We have previously conducted an EST study and have identified 13596 cDNAs from three different stages of nectary development (Stage 6, immature, presecretory nectaries; Stage 12, mature nectaries at floral anthesis; and nectaries, 44 hours after fertilization. In our efforts to evaluate the transcriptional program for the Nicotiana nectary we are proposing to evaluate nectary mRNAs by hybridization with the potato microarrays. We have preliminary evidence that wholesale transcriptional reprogramming (60% of the transcriptome) occurs during nectary maturation and again following fertilization. Our goal is to understand these processes at a biochemical level so that we can begin manipulating nectary function to improve nectar quality and quantity thereby increasing the attractiveness of flowers to insect pollinators. Such improvements have the potential to result in increases in insect visitation, seedset, and ultimately yield for insect pollinated crops. We are also making significant efforts to understand the restructuring of the nectary during its lifecycle. Many changes occur during nectary development and the observed transcriptional reprogramming makes sense the when these many changes are accounted for. Keywords: Loop design 30 hybs total