Source: UNIVERSITY OF KENTUCKY submitted to
GLYCEROLIPID METABOLISM AND ITS ROLE IN PLANT DEFENSE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0202371
Grant No.
2005-35319-15347
Project No.
KY012021
Proposal No.
2004-03287
Multistate No.
(N/A)
Program Code
51.8
Project Start Date
Dec 15, 2004
Project End Date
Dec 14, 2008
Grant Year
2005
Project Director
Kachroo, P.
Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Presently, control of pathogens relies primarily on chemical applications and in the long run this strategy is likely to be untenable. Host resistance provides the grower a cost effective and environmentally sound method to combat plant diseases. The outcome of the interaction of plants with a given pathogen is governed by several factors including specific interaction between various defense signaling pathways. Among various signaling molecules proposed to modulate defense responses, salicylic acid and jasmonic acid elicit distinct responses and undergo extensive cross talk. We have recently shown that fatty acids and glycerolipid metabolism play an important role in plant defense. In the proposed work we will use several different molecular and biochemical approaches to elucidate the mechanisms through which glycerolipid metabolism participates in defense signaling. The purpose of this study is to investigate role of glycerolipid metabolism in plant defense.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21224991040100%
Goals / Objectives
Isolation and characterization of Arabidopsis mutants that are tolerant to glycerol. Isolation, identification and characterization of proteins that bind to oleic acid.
Project Methods
In the proposed work, we aim to isolate mutants that render plants tolerant to glycerol and study their role in plant defense. Mutants will be sreened on glycerol containing medium. In addition, we will attempt to identify oleic acid binding proteins and study their role in plant defense. Affinitiy purification method will be developed to isolate oleic acid binding proteins. The sequence obtained from these proteins will be used to isolate the gene and characterize its function.

Progress 12/15/04 to 12/14/08

Outputs
OUTPUTS: Study of fatty acid and glycerolipid metabolism has resulted in novel findings that have improved our understanding of Arabidopsis and soybean defense signaling pathways. These research findings were presented in three oral and three poster presentations at annual society (American Phytopathological Society, American Society of Plant Biology) and two presentation at an international conference (International Group Working on Legume and Vegetable Viruses). Work related to this project has also resulted in the training of two undergraduate students, one graduate student, and one postdoctoral researcher. PARTICIPANTS: Pradeep Kachroo (PI;) Mihir Mandal (graduate student); Tom Muse (undergradute student); Anna Rush (undergraduate student); Ella Konnova (postdoctoral researcher) TARGET AUDIENCES: Plant science research community via publications and conference presentations, undergraduate students via research opportunities related to the project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Stearoyl-acyl carrier protein-desaturase (SACPD)-catalyzed synthesis of oleic acid (18:1) is an essential step in fatty acid biosynthesis. Arabidopsis mutants (ssi2) with reduced SACPD activity accumulate salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens. We have now shown that reduced levels of 18:1 induce similar defense-related phenotypes in soybean. The SACPD silenced plants contained reduced 18:1, which led to increased SA levels, and increased expression of resistance genes. These results suggest that soybean and Arabidopsis respond similarly to 18:1-derived cues by inducing a novel broad-spectrum resistance-conferring pathway, even though they differ significantly in their lipid biosynthetic pathways. We have also established a novel link between glycerol-3-phosphate metabolism and plant defense. The G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. We have also identified a novel fatty acid component, acyl carrier protein (acp) 4, which regulates 18:1 levels in the plant plastids. Acyl carrier protein 4 also participates in cuticle formation by regulating fatty acid flux. We also showed that a defect in cuticle, which forms the outermost structure of the leaf, impairs the plants ability to perceive the mobile systemic acquired resistance (SAR) signal in distal tissues. The defective SAR in acp4 plants was not due to impairment in salicylic acid (SA)-, methyl SA-, or jasmonic acid-mediated pathways but was associated with the impaired cuticle of acp4 leaves. These findings emphasize the fact that the plant cuticle also participates in signaling mechanisms besides serving as a physical barrier against environmental stress.

Publications

  • Xia Y, Gao Q-M, Yu Keshun, Navarre D, Hildebrand D, Kachroo A, Kachroo P (2009) Acyl carrier protein regulates oleate levels and systemic acquired resistance in Arabidopsis. Cell Host and Microbe (In press).
  • Chanda B, Venugopal, SC, Kulshrestha S, Navarre DA, Downie B, Vaillancourt L, Kachroo A, Kachroo P. (2008). Glycerol-3-phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis. Plant Physiology 147:2017-2029.
  • Kachroo A, Fu D-Q, Havens W, Navarre DA, Kachroo P, Ghabrial SA. (2008). An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean. Molecular Plant-Microbe Interactions 21:564-575. (Cover feature).


Progress 12/15/06 to 12/14/07

Outputs
Our studies with ssi2 and its suppressors indicate that reduced 18:1 levels in the ssi2 plants are responsible for altered defense signaling. Furthermore, replenishing 18:1 levels results in restoration of wild-type-like signaling in the mutant. This suggests that 18:1 or an 18:1-derived molecule(s) acts as the signal for restoring altered defense signaling in ssi2 plants. In plants, changes in the levels of oleic acid (18:1), a major monounsaturated fatty acid (FA), results in the alteration of salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. In addition to SSI2, the Arabidopsis genome encodes six S-ACP-DES-like enzymes, the native expression levels of which are unable to compensate for a loss-of-function mutation in ssi2. The presence of low levels of 18:1 in the fab2 null mutant indicates that one or more S-ACP-DES isozymes contribute to the 18:1 pool. Biochemical assays show that in addition to SSI2, four other isozymes are capable of desaturating 18:0-ACP but with greatly reduced specific activities, which likely explains the inability of these SSI2 isozymes to substitute for a defective ssi2. However, overexpression of the S-ACP-DES1 isoform in ssi2 plants results in restoration of 18:1 levels and thereby rescues all ssi2-associated phenotypes. Transcript level of S-ACP-DES isoforms is reduced in high 18:1-containing plants. Enzyme activities of the desaturase isoforms in a 5-fold excess of 18:1-ACP show product inhibition of up to 73%. Together these data indicate that 18:1 levels are regulated at both transcriptional and post-translational levels. Interestingly, reduction of 18:1 can also induce HRT gene expression and confer resistance to Turnip Crinkle Virus (TCV). Resistance to TCV is dependent upon the resistance (R) gene, HRT, and a recessive locus rrt. Resistance is also dependent on salicylic acid (SA), EDS1 and PAD4. Exogenous application of SA confers resistance in RRT-containing plants by increasing HRT transcript levels in a PAD4-dependent manner. However, the 18:1-regulated pathway is independent of SA, rrt, EDS1 and PAD4. Reducing the levels of 18:1, via a mutation in the SSI2-encoded S-ACP-DES, or by exogenous application of glycerol, increased transcript levels of HRT as well as several other R genes. Second site mutations in the ACT1-encoded glycerol-3-phosphate (G3P) acyltransferase or GLY1-encoded G3P dehydrogenase restored 18:1 levels in HRT ssi2 plants and reestablished a dependence on rrt. Resistance to TCV and HRT gene expression in HRT act1 plants was inducible by SA but not by glycerol, while that in HRT pad4 plants was inducible by glycerol but not SA. The low 18:1-mediated induction of R gene expression was also dependent on ACT1 but independent of EDS1, PAD4 and RAR1. Intriguingly, TCV inoculation did not activate this 18:1-regulated pathway in HRT plants, but instead resulted in the induction of several genes that encode 18:1-synthesizing isozymes. These results suggest that the 18:1-regulated pathway may be specifically targeted during pathogen infection and that altering 18:1 levels may serve as a novel strategy for promoting disease resistance.

Impacts
Presently, control of pathogens relies primarily on chemical applications but in the long run, this strategy is likely to be untenable. Host resistance provides the grower a cost-effective and environmentally-sound method to combat plant diseases. The outcome of the interaction of plants with a given pathogen is governed by several factors including specific interactions between various defense signaling pathways. Among several signaling molecules proposed to modulate defense responses, salicylic acid and jasmonic acid elicit distinct responses and undergo extensive cross-talk. This project will allow us to decipher molecular mechanisms regulating plant defense responses to pathogens. By elucidating the signaling mechanisms through which plant defense responses are activated, new strategies for developing environmentally-sound and cost effective disease resistant crops can be developed.

Publications

  • Kachroo A., Kachroo P. (2007) Salicylic acid-, jasmonic acid- and ethylene-mediated regulation of plant defense signaling. In: Genetic Engineering, Principles and Methods. Ed. J. Setlow. 28: 55-83.
  • Kachroo, A., Shanklin, J., Lapchyk, L., Whittle, E., Hildebrand, D., Kachroo, P. (2007) The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis. Plant Molecular Biology 63: 257-271.
  • Chandra-Shekara, A.C, Venugopal, S.C., Barman, S.R., Kachroo, A., Kachroo, P. (2007) Plastidial fatty acid levels regulate resistance gene-dependent defense signaling in Arabidopsis. Proceedings of the National Academy of Sciences USA, 104:7277-7282.


Progress 12/15/05 to 12/14/06

Outputs
Stearoyl-ACP-desaturase (S-ACP-DES)-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is the key step regulating levels of unsaturated fatty acids (FAs) in the cell. A mutation in the ssi2-encoded S-ACP-DES results in constitutive expression of the salicylic acid (SA) pathway and repression of certain jasmonic acid (JA)-mediated responses. Our studies with ssi2 and its suppressors indicate that reduced 18:1 levels in these plants are responsible for altered defense signaling. Furthermore, replenishing 18:1 levels results in restoration of wild-type-like signaling in the mutant. This suggests that 18:1 or an 18:1-derived molecule(s) participates in the SA- and JA-mediated defense signaling pathways of Arabidopsis. We have identified four genes which participate in the prokaryotic FA pathway and loss-of-function of which restores various SA- and JA-regulated phenotypes in ssi2 plants. Mutations in the genes encoding for glycerol-3-phosphate (G3P) acyltransferase (ACT1) and G3P dehydrogenase (GLY1) restore wild-type-like phenotypes in the ssi2 plants. A loss-of-function mutation in oleate- or linolate-desaturase also confers partial restoration of the morphological phenotype but does not restore altered defense signaling. Interestingly, the 18:1 content in wild-type plants can be lowered by exogenous glycerol application, converting these plants to ssi2 mimics. Analyses of mutants impaired in SA signaling or the FA pathway revealed that glycerol induces SA-signaling via the isochorishmate synthase (SID2)-dependent pathway. Furthermore, mutants impaired in glycerol catabolism or the generation of G3P do not induce the SA pathway or show any reduction in 18:1 upon glycerol application. Since a mutation in ssi2-encoded S-ACP-DES interferes with the normal functioning of the plants, we examined the role of other Arabidopsis S-ACP-DES isoforms in defense signaling. In addition to SSI2, the Arabidopsis genome encodes six S-ACP-DES-like enzymes, the native expression levels of which are unable to compensate for a loss-of-function mutation in ssi2. The presence of low levels of 18:1 in the fab2 null mutant indicates that one or more S-ACP-DES isozymes contribute to the 18:1 pool. Biochemical assays show that in addition to SSI2, four other isozymes are capable of desaturating 18:0-ACP but with greatly reduced specific activities, which likely explains the inability of these SSI2 isozymes to substitute for a defective ssi2. Lines containing T-DNA insertions in S-ACP-DES1 and S-ACP-DES4 show that they are altered in their lipid profile but contain normal 18:1 levels. However, overexpression of the S-ACP-DES1 isoform in ssi2 plants results in restoration of 18:1 levels and thereby rescues all ssi2-associated phenotypes. Thus, high expression of a low specific activity S-ACP-DES is required to compensate for a mutation in ssi2. Transcript levels of S-ACP-DES isoforms is reduced in high 18:1-containing plants. Enzyme activities of the desaturase isoforms in a five-fold excess of 18:1-ACP show product inhibition of up to 73%. Together, these data indicate that 18:1 levels are regulated at both transcriptional and post-translational levels.

Impacts
Presently, control of pathogens relies heavily on chemical applications but, in the long run, this strategy is likely to be untenable. Host resistance provides the grower a cost-effective and environmentally-sound method to combat plant diseases. The outcome of the interaction of plants with a given pathogen is governed by several factors, including specific interactions between various defense signaling pathways. Among several signaling molecules proposed to modulate defense responses, salicylic acid and jasmonic acid elicit distinct responses and undergo extensive "cross-talk". By elucidating the signaling mechanisms through which plant defense responses are activated, new strategies for developing environmentally-sound and cost-effective disease resistant crops can be developed.

Publications

  • Kachroo, A., Venugopal, S.C., Navarre, D.A., Lapchyk, L., Kachroo, P*. (2006) Metabolite signaling; role of fatty acids in plant defense. In "Biology of Molecular Plant-Microbe Interactions", Eds, F. Sanchez, C. Quinto, I. M. Lopez-Lara, O. Geiger. Volume 5: 195-201.
  • Kachroo, P., Chandra-Shekara, A.C., Klessig, D. (2006) Plant signal transduction and defense against viral pathogens. Advances in Viral Research 66: 161-191.


Progress 12/15/04 to 12/14/05

Outputs
Stearoyl-ACP-desaturase (S-ACP-DES)-mediated conversion of stearic acid (18:0) to oleic acid (18:1) is the key step regulating levels of unsaturated fatty acids (FAs) in the cell. A mutation in the ssi2-encoded S-ACP-DES results in constitutive expression of the salicylic acid (SA) pathway and repression of certain jasmonic acid (JA)-mediated responses. Our studies with ssi2 and its suppressors indicate that reduced 18:1 levels in these plants are responsible for altered defense signaling. Furthermore, replenishing 18:1 levels results in restoration of wild-type-like signaling in the mutant. This suggests that 18:1 or an 18:1-derived molecule(s) participates in the SA- and JA-mediated defense signaling pathways of Arabidopsis. We have identified four genes which participate in the prokaryotic FA pathway and loss-of-function of which restores various SA- and JA-regulated phenotypes in ssi2 plants. Mutations in the genes encoding for glycerol-3-phosphate (G3P) acyltransferase (ACT1) and G3P dehydrogenase (GLY1) restore wild-type-like phenotypes in the ssi2 plants. A loss-of-function mutation in oleate- and linolate-desaturase also confers partial restoration of the morphological phenotype but does not restore altered defense signaling (Kachroo et al., 2005). Interestingly, the 18:1 content in wild-type plants can be lowered by exogenous glycerol application, converting these plants to ssi2 mimics. Analyses of mutants impaired in SA signaling or the FA pathway revealed that glycerol induces SA-signaling via the isochorishmate synthase (SID2)-dependent pathway. Furthermore, mutants impaired in glycerol catabolism or the generation of G3P do not induce the SA pathway or show any reduction in 18:1 upon glycerol application. The act1 mutant also does not show any reduction in 18:1 levels due to its inability to acylate G3P with 18:1. Consequently, plants overexpressing the ACT1 gene are hypersensitive to exogenous glycerol application. Thus, we have demonstrated that 18:1 levels in plastids are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of SA- and JA-mediated signaling pathways. Since a mutation in ssi2-encoded S-ACP-DES interferes with the normal functioning of the plants, we examined the role of other Arabidopsis S-ACP-DES isoforms in defense signaling. The Arabidopsis genome is annotated to carry six other S-ACP-DES encoding genes. We have studied their expression in various mutant backgrounds, examined the effect of overexpression of some of these in the wild-type as well as ssi2/fab2 background, and determined specific activities of all isoforms expressed in leaf tissue. Overexpression of SSI2 isoforms is able to rescue all ssi2-related phenotypes and restore wild type-like morphology by upregulating 18:1 levels. Knockout lines were also obtained for two of these desaturases, but these did not have any effect on defense phenotypes or 18:1 levels. However, the plants containing knockout mutations did show reduced total lipid content. Taken together, these results further confirm our assertion that 18:1 levels are important for normal defense signaling in plants.

Impacts
Presently, control of pathogens relies primarily on chemical applications but in the long run, this strategy is likely to be untenable. Host resistance provides the grower a cost-effective and environmentally-sound method to combat plant diseases. The outcome of the interaction of plants with a given pathogen is governed by several factors including specific interactions between various defense signaling pathways. Among several signaling molecules proposed to modulate defense responses, salicylic acid and jasmonic acid elicit distinct responses and undergo extensive cross-talk. This project will allow us to decipher molecular mechanisms regulating plant defense responses to pathogens. By elucidating the signaling mechanisms through which plant defense responses are activated, new strategies for developing environmentally-sound and cost effective disease resistant crops can be developed.

Publications

  • Kachroo, P., Srivathsa C. V., Navarre, D.A., Lapchyk, L., Kachroo, A. (2005) Role of salicylic acid and fatty acid desaturation pathways in ssi2-mediated signaling. Plant Physiology 139, 1717-1735.