Progress 03/04/04 to 02/28/09
Outputs
OUTPUTS: 1. Cytosolic Apx1 is essential for protecting the chloroplastic ROS scavenging system. In the absence of cytosolic Apx1 the entire hydrogen peroxide scavenging system of Arabidopsis collapses and major oxidative damage occurs. Based on early work showing that intact chloroplasts are very sensitive to the external application of hydrogen peroxide, we hypothesize that cytosolic Apx1 is important for protecting the chloroplast from hydrogen peroxide that is generated in the cytosol or leaks from the mitochondria or peroxisomes. In the absence of cytosolic Apx1, stromal Apx is unable to protect the chloroplast from externally produced hydrogen peroxide. However, in the absence of stromal Apx, cytosolic Apx1 is sufficient to protect the chloroplast. 2. The zinc-finger protein Zat12 is required for Apx1 expression during oxidative stress. In the absence of Zat12 (knockout-Zat12), Apx1 expression is not elevated in plants in response to oxidative stress. Zat12 is also required for Zat7 and WRKY25 expression. Overexpression of Zat12 enhances the tolerance of plants to oxidative stress. We hypothesize that Zat12 is a key component of the hydrogen peroxide signal transduction pathway of Arabidopsis functioning upstream of Zat7, WRKY25 and Apx1. 3. The zinc-finger protein Zat12 plays a key role in the response of plants to oxidative stress, osmotic stress and salinity. It controls a defense regulon of 42 transcripts that include different light and osmotic stress response transcripts. 4. The heat shock transcription factor 21 (HSF21) is required for Apx1 expression during oxidative stress. In plants expressing a dominant-negative construct for HSF21, Apx1 expression is not elevated in response to oxidative stress. HSF21 is also required for Zat12 expression. Based on biochemical characterization of HSFs in fly and mammalian cells and based on our genetic analysis, we hypothesize that HSF21 functions as an early component of the hydrogen peroxide signal transduction pathway acting as a redox-response transcription factor that may be a direct sensor of oxidative stress in plants. 5. RbohD is essential to maintain the ROS-response signal and may link calcium signaling with ROS signaling. 6 .The transcriptional co-activator MBF1c is a master regulator of basal thermotolerance in Arabidopsis. 7. The EAR-motif of the zinc finger protein Zat7 is required for abiotic stress tolerance in Arabidopsis. 8. The zinc-finger protein Zat10 is a key regulator of abiotic stress tolerance in Arabidopsis and has a modulating function as a positive/negative regulator. PARTICIPANTS: PI: Ron Mittler Minority high school students: Rachel Tam Stephanie Kao Undergraduate Students: Adrienne Goetz Jessie Coutu Alicia Hegie (previously Coutu) Barrett Abel Nicholas James Lyle Serena Huntington (Minority) Leigh Armijo (Minority) Ahmet Sodek Mitch Hegedus (Minority) Graduate Stdents: Nobuhiro Suzuki (Ph.D.) Sultan Ciftci (Ph.D.). Jessie Coutu (Ph.D.). Postdoc: Sholpan Davletova Ph.D. Gad Miller Ph.D. Shai Koussevitzky Ph.D. Visiting Scholars: A.N.MISRA, Professor&Head, Biosci.&Biotechnol. F.M.University, India TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our main contributions to the field of ROS metabolism in plants (now interlinked with almost all fields of plant biology) are: 1.Annotation of all major ROS-scavenging genes in Arabidopsis (Reported in our 2004 Trends in Plant Sci paper). 2.Microarray data for a time course experiment in which hydrogen peroxide stress was imposed on plants internally. This is the first complete time course analysis in which oxidative stress was internal and resulted from the absence of a key hydrogen peroxide enzyme. It is, in our view, superior to experiments in which chemicals or hydrogen peroxide are applied to plants externally. The data is reported in our Plant Cell paper and is available from Tair and our website. 3.Assigning a function to cytosolic Apx1 in protecting the chloroplast from 'external' hydrogen peroxide stress (i.e., Hydrogen peroxide that
Publications
- Koussevitzky S, Nott A, Mockler T.C, Hong F, Sachetto-Martins G, Surpin M, Lim J,Mittler R, and Chory J, "Multiple signals from damaged chloroplasts converge on a common pathway to regulate nuclear gene expression", Science, p. 715, vol. 316, (2007).
- Mittler. R., Vanderauwera, S., Gollery, M., Van Breusegem, F., "The reactive oxygen gene network of plants.", Trends Plant Sci, p. 490, vol. 9,(2004).
- Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, Shulaev V, Schlauch K, Mittler R, "Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis.", Plant Cell, p. 268, vol. 17, (2005). Miller G, Suzuki N, Rizhsky L, Hegie A, Koussevitzky S, Mittler R, "Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development and response to abiotic stresses.", Plant Physiol., p. 1777, vol. 144, (2007).
- Miller G, Mittler R, "Could plant HSFs function as hydrogen peroxide sensors", Ann Bot., p. 279, vol. 98, (2005).
- Mittler. R, "Abiotic Stress, the Field Environment and Stress Combination.", Trends Plant Sci., p. 15, vol. 11, (2006).
- Gadjev, I., Vanderauwera, S., Gechev, T.S., Laloi, C., Minkov, I.N., Shulaev, V., Apel, K., Inze, D., Mittler, R., Van Breusegem, F., "Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis", Plant Physiol., p. 436, vol. 141, (2006).
- Suzuki N, Rizhsky L, Liang H, Shuman J, Shulaev V, Mittler R, "Enhanced tolerance to environmental stresses in transgenic plants expressing the transcriptional co-activator MBF1.", Plant Physiol., p. 1313, vol. 139, (2005).
- Suzuki N, Mittler R, "Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction", Physiol. Plant., p. 45, vol. 126, (2005). Davletova S, Schlauch K, Coutu J, Mittler R, "The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis", Plant Physiol., p. 847, vol. 139, (2005).
- Gollery M, Harper J, Cushman J, Mittler T, Girke T, Zhu J-K, Bailey-Serres J, Mittler R, "What Makes Species Unique The Contribution of Proteins with Obscure", Genome Biol., p. R57, vol. 7, (2006).
- Mittler R, Song L, Coutu J, Coutu A, Ciftci S, Kim YS, Lee H, Stevenson B, Zhu, J-K, "Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress.", FEBS Lett., p. 6537, vol. 580, (2006).
- Bailey-Serres, J., Mittler, R., "The roles of reactive oxygen species in plant cells.", Plant Physiol., p. 311, vol. 141, (2006). Ciftci-Yilmaz S, Morsy MR, Song L, Coutu A, Krizek BA, Lewis MW, Warren D, Cushman J, Connolly EL, Mittler R, "The EAR-motif of the C2H2 zinc-finger protein ZAT7 plays a key role in the defense response of Arabidopsis to salinity stress", J. Biol. Chem., p. 9260, vol. 282, (2007).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Project Website: http://www.ag.unr.edu/ROS/ Reactive oxygen species (ROS) play a dual role in plant biology acting on the one hand as important signal transduction molecules and on the other as toxic by-products of aerobic metabolism that accumulate in cells during different stress conditions. Because of their toxicity as well as their important signaling role, the level of ROS in cells is tightly controlled by a vast network of genes termed the 'ROS gene network'. Using mutants deficient in key ROS-scavenging enzymes, we have defined a signaling pathway that is activated in cells in response to ROS accumulation. Interestingly, many of the key players in this pathway, including different zinc finger proteins and WRKY transcription factors, are also central regulators of abiotic stress responses involved in temperature, salinity and osmotic stresses. Here, we describe our recent findings and discuss how ROS integrate different signals originating from different cellular compartments during abiotic stress. PARTICIPANTS: Principle Investigator: Dr. Ron Mittler Department of Biochemistry and Molecular Biology University of Nevada Mail Stop 200 Reno NV 89557 Office phone: 775-784-1384 (6031) Lab phone: 775-784-4071 Cell phone: 775-530-9823 Fax: 775-784-1650 e-mail: ronm@unr.edu Minority high school students: Rachel Tam Stephanie Kao Ashley Ho Undergraduate Students: Leigh Armijo (Minority) Hiroe Sejima Oktay Ince Meryem Betul Gunay Takehiro Aoyama Graduate Stdents: Nobuhiro Suzuki (Ph.D.) Diana Cenariu (Visiting Graduate Student from Romania) Postdoc: Gad Miller Ph.D. Shai Koussevitzky Ph.D. TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The ROS network was defined. Mutants were isolated with altered ROS tolerance. Key genes in the network were identified. Transgenic plants with enhanced tolerance to abiotic stress were developed.
Publications
- Miller G, Mittler R (2006) Could plant HSFs function as hydrogen peroxide sensors? Ann. Bot. 98, 279-288.
- ailey-Serres, J., Mittler, R. (2006) The Roles of Reactive Oxygen Species in Plant Cells. Plant Physiology Special Issue on Reactive Oxygen Species. Plant Physiol. 141, 311.
- Rizhsky L., Davletova S., Liang H. and Mittler R. (2004) The zinc-finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J. Biol. Chem. 279, 11736-11743.
- Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, Shulaev V, Schlauch K, Mittler R (2005) Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 17, 268-281.
- Davletova S, Schlauch K, Coutu J, Mittler R (2005) The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiol. 139, 847-856.
- Suzuki N, Mittler R (2006) Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol. Plant. 126, 45-51.
- Gadjev, I., Vanderauwera, S., Gechev, T.S., Laloi, C., Minkov, I.N., Shulaev, V., Apel, K., Inze, D., Mittler, R., Van Breusegem, F. (2006) Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis. Plant Physiol. 141, 436-445.
- Mittler R, Song L, Coutu J, Coutu A, Ciftci S, Kim YS, Lee H, Stevenson B, Zhu, J-K (2006) Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett. 580, 6537-6542.
- Ciftci-Yilmaz S, Morsy MR, Song L, Coutu A, Krizek BA, Lewis MW, Warren D, Cushman J, Connolly EL, Mittler R (2007) The ear-motif of the C2H2 zinc-finger protein ZAT7 plays a key role in the defense response of Arabidopsis to salinity stress. J Biol Chem. 282, 9260-9268.
- Koussevitzky S, Nott A, Mockler T.C, Hong F, Sachetto-Martins G, Surpin M, Lim J, Mittler R, and Chory J (2007) Multiple signals from damaged chloroplasts converge on a common pathway to regulate nuclear gene expression. Science 316, 715-719.
- Miller G, Suzuki N, Rizhsky L, Hegie A, Koussevitzky S, Mittler R (2007) Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development and response to abiotic stresses. Plant Physiol. 144, 1777-1785.
- Ciftci-Yilmaz S, Mittler R (2008) The zinc finger network of plants. Cell Mol Life Sci. 65, 1150-1160.
- Shulaev V, Cortes D, Miller G, Mittler R (2008) Metabolomics for plant stress response. Physiol Plant. 132, 199-208.
- Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and abiotic stress. Physiol Plant. 133, 481-489.
- Van Breusegem F, Bailey-Serres J, Mittler R (2008) Unraveling the tapestry of networks involving reactive oxygen species in plants. Plant Physiol. 147, 978-984.
- Luhua S, Ciftci-Yilmaz S, Harper J, Cushman J, Mittler R (2008) Enhanced tolerance to oxidative stress in transgenic Arabidopsis plants expressing proteins of unknown function. Plant Physiol. 148, 280-292.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: Experiments were conducted on mutants lacking key reactive oxygen scavenging enzymes. These included protein RNA and metabolite analysis and isolation of signal transduction mutants. Events: A postdoc (Shai Koussevitzky) and a graduate student (Sultan Ciftci) were trained. Results were presented in the following symposia: 2007 Gordon Research Conference, Temperature Stress, CA. 2007 North Carolina Plant Molecular Biology Consortium Seminar Series. 2007 Departmental Seminar, Reed College, Oregon. 2007 "ROS in PLANTS", Ghent, Belgium. 2007 Workshop on Redox Signal Integration. Bielefeld University, Germany. Services: Two undergraduate students were trained (Leigh Armijo and Mitch Hegedus). Products:http://bioinfo.ucr.edu/projects/Unknowns/external/index.html
PARTICIPANTS: Sultan Ciftci (Ph.D.). Shai Koussevitzky Ph.D.
TARGET AUDIENCES: Plant Science community
PROJECT MODIFICATIONS: None
Impacts
Over one quarter of all eukaryotic genes encode proteins with obscure features that lack currently defined motifs or domains (POFs). Interestingly, most of the differences in gene repertoire among species were recently found to be attributed to POFs. A comparison of the Arabidopsis, rice and poplar genomes reveals that Arabidopsis contains 5069 POFs. Of these 2045 have no obvious homologs in rice or poplar and are likely to be involved in species- or phylogenetic-specific functions in Arabidopsis. We believe that the study of POFs is an important endeavor that will shed much needed light on the genetic properties that makes any given plant species unique. To begin the functional annotation of POFs that are involved in the oxidative stress response of Arabidopsis thaliana we generated transgenic Arabidopsis plants that constitutively express 27 different POFs (of which 4 were unique to Arabidopsis), and 13 other proteins of unknown function that contained a previously
defined domain(s) or motif(s). All previously found to be expressed in response to oxidative stress in Arabidopsis. Transgenic plants were tested for their tolerance to oxidative stress imposed by paraquat or t-butyl hydroperoxide, or subjected to osmotic, salinity, cold and heat stresses. More than 70% of all expressed proteins conferred tolerance to oxidative stress. In contrast, over 90% of the expressed proteins did not confer enhanced tolerance to the other abiotic stresses tested, and about 50% rendered plants more susceptible to osmotic or salinity stress. Interestingly, 2 Arabidopsis specific POFs, and one Arabidopsis-specific protein of unknown function that contained a zinc finger domain, conferred enhanced tolerance to oxidative stress. Our findings suggest that tolerance to oxidative stress in Arabidopsis could involve mechanisms and pathways that are unknown at present, including some that may even be specific to Arabidopsis or the Brassica family.
Publications
- Song L, Ciftci-Yilmaz S, Harper J, Cushman J, Mittler R (2008) Enhanced tolerance to oxidative stress in transgenic Arabidopsis thaliana plants expressing proteins of unknown function. Submitted.
- Gollery M, Harper J, Cushman J, Mittler T, Mittler R (2007) POFs: what we dont know can hurt us. Trends Plant Sci. 12, 492-496
- Horan K, Jang C, Gollery M, Harper J, Cushman J, Mittler R, Zhu J-K, Shelton C, Bailey-Serres J, Girke T (2008) Annotating Genes of Known and Unknown Functions by Large-Scale Co-Expression Analysis. Plant Physiol. Submitted.
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Progress 01/01/06 to 12/31/06
Outputs
Major findings for this period: 1. The transcriptional co-activator MBF1c is a master regulator of basal thermotolerance in Arabidopsis. 2. The EAR-motif of the zinc finger protein Zat7 is required for abiotic stress tolerance in Arabidopsis. 3. The zinc-finger protein Zat10 is a key regulator of abiotic stress tolerance in Arabidopsis and has a modulating function as a positive/negative regulator. 4. A cytosolic ROS-response pathway can suppress PCD induced by a peroxisomal ROS-response pathway in Arabidopsis and tobacco. Opportunities for training and development: Two postdocs, three graduate students, three undergraduates and two high school students were trained during the third year of the project (active hands-on training in the lab, lab meetings and scientific discussions, journal clubs and development of bioinformatics-computer skills). The students and postdocs were first and foremost trained in proper scientific methods and experimental design. The focus of
the training was on the analysis of knockout plants for key ROS-scavenging enzymes using a variety of methods including protein and RNA blots, GFP and luciferase imaging, microarray analysis and analysis of transgenic plants and knockouts. The PI presented results from the project at several meetings and seminars: 2005 Departmental seminar, Iowa State University, Iowa. 2005 Departmental seminar, Rutgers University, New Jersey. 2006 Institute Lecture, Institute of Life Sciences, Hebrew University, Jerusalem, Israel. 2006 Syngenta Corporation, Chapel Hill, NC 2006 Keystone meeting, Plant Responses to Abiotic Stress, Cooper Mountain, Colorado. 2006 Gordon Research Conference on Slat and Water Stress, Oxford University, UK. 2006 International Symposium on Dynamic Organelles in Plants, Okazaki, Japan. The PI is now serving as a Monitoring Editor in Plant Physiology.
Impacts
Our main contributions to the field of ROS metabolism in plants (now interlinked with almost all fields of plant biology) are: 1. Reconstructing the signal transduction pathway activated in plant in response to the accumulation of hydrogen peroxide in the cytosol. 2. Assigning a function to MBF1c as a master regulator of basal thermotolernace. 3. Assigning a function to Zat10 in the signal transduction pathway that senses hydrogen peroxide and activates Apx2 and FSD1 expression. 4. Assigning a function to the EAR-domain of zinc finger proteins. Our main contribution to other disciplines in science include a demonstration that NADPH oxidases might function as signal enhancers during oxidative stress and that ROS bridge between biotic and abiotic stresses. In addition, we provided genetic evidence that heat shock transcription factors could function as important sensors of ROS in plants. Our main contribution to agriculture and medicine is the demonstration that MBF1c
can enhance the tolerance of transgenic plants to heat stress, osmotic stress and their combination, and that Zat12 and Za10 can enhance the tolerance of transgenic plants to oxidative stress, osmotic stress and salinity stress. At least one biotech company (Syngenta) has shown interest in MBF1c and the PI was invited in February to present his results on this gene at the company.
Publications
- Gadjev, I., Vanderauwera, S., Gechev, T.S., Laloi, C., Minkov, I.N., Shulaev, V., Apel, K., Inze, D., Mittler, R., Van Breusegem, F. (2006) Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis. Plant Physiol. 141, 436-445.
- Gollery M, Harper J, Cushman J, Mittler T, Girke T, Zhu J-K, Bailey-Serres J, Mittler R (2006) What Makes Species Unique? The Contribution of Proteins with Obscure Features. Genome Biol. 7(7):R57 [Epub ahead of print].
- Mittler R, Song L, Coutu J, Coutu A, Ciftci S, Kim YS, Lee H, Stevenson B, Zhu, J-K (2006) Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett. In Press.
- Bailey-Serres, J., Mittler, R. (2008) The roles of reactive oxygen species in plant cells. Plant Physiol. 2006 Jun;141(2):311.
- Ciftci, S., Coutu, A., Song, L. And Mittler, R. (2006) The EAR-motif of the zinc finger protein Zat7 is required for salinity tolerance in Arabidopsis. J. Biol. Chem. Submitted.
- Koussevitzky, S., Mockler, T.C., Hong, F., Huang, Y., Mittler, R. and Chory, J. (2006) The transcription factor ABI4 regulates nuclear gene expression in response to multiple chloroplast signals. Science Submitted.
- Suzuki, N., Shuman, J., Shulaev, V. and Mittler, R. (2006) The transcriptional co-activator MBF1c is a key regulator of basal thermotolerance in Arabidopsis. Proc. Natl. Acad. Sci. USA. Submitted.
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Progress 01/01/05 to 12/31/05
Outputs
This year we focused our research activity on 4 different genes (Zat12, Zat7, MBF1c, HSF21) identified by our microarray analysis of knockout Apx1 plants: Zat12 (At5g59820; Putative zinc-finger protein) We generated Zat12 promoter::luc fusions and tested the spatial and temporal expression of this gene in response to different stresses. This reporter system is now used for the identification of EMS mutants that are deficient in reactive oxygen signaling. We tested knockouts for Zat12 and transgenic plants expressing Zat12 for abiotic stress tolerance and found that Zat12 plays a key role in plants in the response to oxidative stress, osmotic stress and salinity stress. Microarray analysis of transgenic plants expressing Zat12 identified 42 transcripts that are elevated in their expression in response to Zat12 expression. Twenty three of these are also elevated in wild type plants subjected to hydrogen peroxide stress. Microarray results for Zat12 plants, Zat12 plants
subjected to hydrogen peroxide stress, wild type plants and wild type plants subjected to hydrogen peroxide stress were deposited in online databases and can be accessed via genevestigator and other resources. Zat7 (At3g46070/080/090; Putative zinc-finger protein) We generated transgenic plants that express Zat7. This protein contains a transcription suppression domain (EAR-domain) at its C-terminal and two zinc finger domains. To study the role of the EAR domain we obtained mutants that contain a deletion in the region or a mutated domain. We also obtained RNAi lines for Zat7 (the gene appears as a triple tandem repeat in the Arabidopsis genome and we are not pursuing knockout mutagenesis). MBF1c (At3g24500; Transcriptional co-activator) We generated transgenic plants that express MBF1c and obtained a knockout for it. Stress screens determined that MBF1c can enhance the tolerance of transgenic plants to heat, osmotic stress, heat combined with osmotic stress and pathogen infection.
Knockout plants for MBF1c are more sensitive to heat stress. MBF1c appears to work by activating a defense pathway that is dependent on ethylene signaling. MBF1c augments the reactive oxygen defense response of plants, but not the heat or drought responses. We performed microarray analysis and RNA blots to show enhanced expression of several ROS-response genes in MBF1c-expressing plants during heat stress. Microarray results for MBF1c plants were deposited in online databases and can be accessed via genevestigator and other resources. GFP-fusions of MBF1c show heat-induced nuclear localization of this protein. HSF21 (At4g18880; Heat shock transcription factor) We generated transgenic plants that express HSF21 and obtained knockouts for HSF21. We also generated dominant-negative lines for HSF21. RNA gel blot analysis suggests that HSF21 functions upstream to Zat12 and Apx1. We are currently performing stress screens as well as biochemical assays to determine if HSF21 is a
redox-response transcription factor. Our working hypothesis is that HSF21 is a key sensor of hydrogen peroxide. In its absence Zat12 ans Apx1 are not induced in response to light stress.
Impacts
Our main contributions to the field of ROS metabolism in plants (now interlinked with almost all fields of plant biology) are: 1. Assigning a function to HSF21 in the signal transduction pathway that senses hydrogen peroxide and activates Apx1 expression. 2. Assigning a function to Zat12 in the signal transduction pathway that senses hydrogen peroxide and activates Apx1 expression. 3. Assigning a function to RbohD in the signal transduction pathway that senses hydrogen peroxide and activates Apx1 expression. 4. Assigning a function to MBF1c in the signal transduction pathway that senses hydrogen peroxide and activates Apx1 expression. 5. Determining the function of the EAR-domain of Zat7 during abiotic stress. Our main contribution to other disciplines in science include a demonstration that NADPH oxidases might function as signal enhancers during oxidative stress and that ROS bridge between biotic and abiotic stresses. In addition, we provided genetic evidence that
heat shock transcription factors could function as important sensors of ROS in plants. Our main contribution to agriculture and medicine is the demonstration that MBF1c can enhance the tolerance of transgenic plants to heat stress, osmotic stress and their combination, and that Zat12 can enhance the tolerance of transgenic plants to oxidative stress and osmotic stress. At least one biotech company (Syngenta) has shown interest in MBF1c and the PI was invited in February to present his results on this gene at the company.
Publications
- Davletova S, Schlauch K, Coutu J, Mittler R (2005) The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiol. 139, 847-856.
- Suzuki N, Rizhsky L, Liang H, Shuman J, Shulaev V, Mittler R (2005) Enhanced tolerance to environmental stresses in transgenic plants expressing the transcriptional co-activator MBF1. Plant Physiol. 139, 1313-1322.
- Suzuki N, Mittler R (2005) Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol. Plant. in press.
- Miller G, Mittler R (2005) Could plant HSFs function as hydrogen peroxide sensors? Ann. Bot. in press.
- Mittler. R (2006) Abiotic Stress, the Field Environment and Stress Combination. Trends Plant Sci. in press.
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Progress 01/01/04 to 12/31/04
Outputs
Research and Education Activities: Major research activity: We have developed a knockout model for one of the key hydrogen peroxide-removal enzymes of plants (cytosolic ascorbate peroxidase; Apx1; At1g07890) and used this model to induce an internal state of oxidative stress in Arabidopsis, using a time course design. Major findings: 1. Cytosolic Apx1 is essential for protecting the chloroplastic ROS scavenging system. In the absence of cytosolic Apx1 the entire hydrogen peroxide scavenging system of Arabidopsis collapses and major oxidative damage occurs. Based on early work showing that intact chloroplasts are very sensitive to the external application of hydrogen peroxide, we hypothesize that cytosolic Apx1 is important for protecting the chloroplast from hydrogen peroxide that is generated in the cytosol or leaks from the mitochondria or peroxisomes. In the absence of cytosolic Apx1, stromal Apx is unable to protect the chloroplast from externally produced
hydrogen peroxide. However, in the absence of stromal Apx, cytosolic Apx1 is sufficient to protect the chloroplast. 2. The zinc-finger protein Zat12 is required for Apx1 expression during oxidative stress. In the absence of Zat12 (knockout-Zat12), Apx1 expression is not elevated in plants in response to oxidative stress. Zat12 is also required for Zat7 and WRKY25 expression. Overexpression of Zat12 enhances the tolerance of plants to oxidative stress. We hypothesize that Zat12 is a component of the hydrogen peroxide signal transduction pathway of Arabidopsis functioning upstream of Zat7, WRKY25 and Apx1. 3. The heat shock transcription factor 21 (HSF21) is required for Apx1 expression during oxidative stress. In plants expressing a dominant-negative construct for HSF21, Apx1 expression is not elevated in response to oxidative stress. HSF21 is also required for Zat12 expression. Based on biochemical characterization of HSFs in fly and mammalian cells and based on our genetic analysis,
we hypothesize that HSF21 functions as an early component of the hydrogen peroxide signal transduction pathway acting as a redox-response transcription factor that may be a direct sensor of oxidative stress in plants. Training and Development: Opportunities for training and development: One postdoc and four undergraduates were trained during the first year of the project (active hands-on training in the lab, lab meetings and scientific discussions, journal clubs and development of bioinformatics-computer skills). The students and postdoc were first and foremost trained in proper scientific methods and experimental design. The focus of the training was on the analysis of knockout plants for key ROS-scavenging enzymes using a variety of methods including protein and RNA blots, microarray analysis and analysis of transgenic plants and knockouts on agar plates
Impacts
Zat12 may be used to produce transgenic crop plants resistant to oxidative stress.
Publications
- Davletova S. Rizhsky L., Liang H., Shuman J., Coutu, J., Shulaev V., Oliver, D. and Mittler, R. (2005) "Cytosolic Ascorbate Peroxidase 1 is a Central Component of the Reactive Oxygen Gene Network of Arabidopsis.", The Plant Cell 17, 268-281.
- Mittler. R., Vanderauwera, S., Gollery, M., Van Breusegem, F. (2004) "The reactive oxygen gene network of plants.", Trends Plant Sci ., p. 490, vol. 9. Published
- Rizhsky L., Davletova S., Liang H. and Mittler R. (2004) "The zinc-finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J. Biol. Chem., p. 1173, vol. 279. Published
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