Progress 09/01/04 to 08/31/09
Outputs
OUTPUTS: 1044 homozygous T-DNA insertion lines from the SALK collection were obtained through ABRC (UNR-Suppl Table 1). Of these 1008 lines have been bulked by the Mittler lab (UNR-Suppl Table 2). 1008 bulked up knockout lines were already stress tested for cold, heat and oxidative stress by the Mittler lab (results are shown in UNR Suppl Table 2). These results are being uploaded to the Bioassay and Phenotype Database (BAP DB, http://bioweb.ucr.edu/bapdb/; please see UCR - Thomas Girke part for further description) and a manuscript describing the database is in preparation. Approximately 20-25% of the lines show a phenotype (increased or decreased tolerance to a specific abiotic stress). The Mittler lab has also screened 383 lines for tolerance to salinity and osmotic stress and this data was already uploaded to the BAP DB database. Interestingly, in many of the lines we observe an inverse correlation between tolerance/ sensitivity to salinity and tolerance/sensitivity to oxidative stress. To begin the functional annotation of proteins with unknown function, which are involved in the oxidative stress response of Arabidopsis (Arabidopsis thaliana), we generated transgenic Arabidopsis plants that constitutively expressed 23 different POFs (four of which were specific to Arabidopsis) and 18 different proteins with defined features. All were 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 were subjected to osmotic, salinity, cold, and heat stresses. More than 70% of all expressed proteins conferred tolerance to oxidative stress. In contrast, >90% of the expressed proteins did not confer enhanced tolerance to the other abiotic stresses tested, and approximately 50% rendered plants more susceptible to osmotic or salinity stress. Two Arabidopsis-specific POFs, and an Arabidopsis and Brassica-specific protein of unknown function, conferred enhanced tolerance to oxidative stress. Our findings suggest that tolerance to oxidative stress involves mechanisms and pathways that are unknown at present, including some that are specific to Arabidopsis or the Brassicaceae. Over a 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 which 2045 have no obvious homologs in rice or poplar and are likely to be involved in species- or phylogenetic-specific functions in Arabidopsis. The study of POFs is an important endeavor that will shed much needed light on the genetic properties that make any given plant species unique. Furthermore, with respect to many species-specific features, such studies show that we seem to be limited in what we can expect to learn from a model plant such as Arabidopsis. PARTICIPANTS: PIs: Ron Mittler, John C. Cushman, Jeff Harper, Marty Gollery. Post-docs/Research Associates: Mustafa Morsy, Gouthu Satyanarayana, Sahi Koussevitzky, Yong Hwa Lee, Luhua Song, Gad Miller. Graduate Students: Abu Yobi, Steve McDowell, Christine Kuhns (visiting from Germany) Undergraduate Students: Mitch Hegedus, Diana Cenariu, Katy Gilmore, Layne Jarrett, Leigh Armijo, Hiroe Sejima, Jesse Coutu, Krista Murray, Alicia Hegie, Alicia Kishpaugh, Katie Dean, Leticia Rodriguez, Frederick Villaluz, Serena Huntington, Virginia Smith, Katy Gilmore. High School Students: Sydney Smith, Alexis Thrower, Rachel Tam, Stephanie Chen, Ashley Moore (upward bound high school students and now undergraduate student). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The methods for abiotic stress screens we have optimized are being used by many other investigators. Detailed protocols are available from our website. We have contributed to the general field of biology by showing that proteins with obscured features are species-specific: 1. 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. 2. Gollery M, Harper JF, Cushman JC, Mittler T, Mittler R. (2007) What we don't know can hurt us. Trends in Plant Science. 12:492-496 Results from this project are on several websites connected by our Systomics Network (http://www.faculty.ucr.edu/~tgirke/) Genome Cluster Database (GCD) http://bioweb.ucr.edu/databaseWeb/index.jsp Plant Unknown-eome DB (POND) http://bioinfo.ucr.edu/projects/Unknowns/external/index.html Plant Gene Expression Database (PED) http://bioinfo.ucr.edu/projects/Unknowns/external/express.html Bioassay and Phenotype Database (BAP DB) http://bioweb.ucr.edu/bapdb/
Publications
- Suzuki N, Mittler R (2006) Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol. Plant. 126, 45-51.
- Shulaev V, Cortes D, Miller G, Mittler R (2008) Metabolomics for plant stress response. Physiol Plant. 132, 199-208.
- Morsy M, Gouthu S, Orchard S, Thorneycroft D, Harper JF, Mittler R, Cushman JC. (2008) Charting plant interactomes: possibilities and challenges. Trends Plant Sci. 13, 183-191.
- Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and abiotic stress. Physiol Plant. 133, 481-489.
- Horan K, Jang C, Bailey-Serres J, Mittler R, Shelton C, Harper JF, Zhu JK, Cushman JC, Gollery M, Girke T (2008) Annotating genes of known and unknown function by large-scale coexpression analysis. Plant Physiol. 147, 41-57.
- 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.
- Koussevitzky S, Suzuki N, Huntington S, Armijo L, Sha W, Cortes D, Shulaev V, Mittler R (2008) Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination. J Biol Chem. 283, 34197-34203.
- Chaudhary B, Hovav R, Flagel L, Mittler R, Wendel JF (2009) Parallel expression evolution of oxidative stress-related genes in fiber from wild and domesticated diploid and polyploid cotton (Gossypium). BMC Evolutionary Biology, 10:378.
- Tunc-Ozdemir M, Miller G, Kim J, Sodek A, Koussevitzky S, Misra AM, Luhua S, Mittler R, Shintani D (2009) Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis. Plant Physiol. 151, 421-32.
- Chang F-I, Curran A, Woolsey R, Quilici D, Cushman J, Mittler R, Harmon A, Harper J, (2009) Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana. Proteomics 9, 2967-85
- Miller G, Schlauch K, Tam R, Cortes D, Torres MA,, Shulaev V, Dangl JL, Mittler R (2009) The plant NADPH oxidase RbohD mediates rapid, systemic signaling in response to diverse stimuli. Science Signaling. 18;2(84):ra45.
- Mittler R, Blumwald E (2010) Genetic engineering for modern agriculture: Challenges and perspectives. Ann. Rev. Plant Biol. Invited Review.
- Miller G, Honig A, Stein H, Suzuki N, Mittler R, Zilberstein A (2009) Unraveling delta1-pyrroline-5-carboxylate (P5C)/proline cycle in plants by uncoupled expression of proline oxidation enzymes. J Biol Chem. 284, 26482-92.
- Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R. (2009) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. 2009 Aug 27. [Epub ahead of print]
- Mittler. R (2006) Abiotic Stress, the Field Environment and Stress Combination. Trends Plant Sci. 11, 15-19.
- Miller G, Mittler R (2006) Could plant HSFs function as hydrogen peroxide sensors Ann. Bot. 98, 279-288.
- Bailey-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.
- 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.
- 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.
- 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.
- Suzuki N, Bajad S, Shuman J, Shulaev V, Mittler R (2008) The transcriptional co-activator MBF1c is a key regulator of thermotolerance in Arabidopsis thaliana. J Biol Chem. 283, 9269-9275.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Project Website: http://bioinfo.ucr.edu/projects/Unknowns/external/index.html Over a 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 which 2045 have no obvious homologs in rice or poplar and are likely to be involved in species- or phylogenetic-specific functions in Arabidopsis. The study of POFs is an important endeavor that will shed much needed light on the genetic properties that make any given plant species unique. Furthermore, with respect to many species-specific features, such studies show that we seem to be limited in what we can expect to learn from a model plant such as Arabidopsis. 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
1000 mutants were screened for tolerance to abiotic stress. 42 different POFs were expressed in transgenic plants and analyzed. It was found that genes of unknown function encode species-specific featues.
Publications
- 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].
- Gollery M, Harper J, Cushman J, Mittler T, Mittler R (2007) POFs: what we do not know can hurt us. Trends Plant Sci. 12, 492-496.
- Horan K, Jang C, Bailey-Serres J, Mittler R, Shelton C, Harper JF, Zhu JK, Cushman JC, Gollery M, Girke T (2008) Annotating genes of known and unknown function by large-scale coexpression analysis. Plant Physiol. 147, 41-57.
- Morsy M, Gouthu S, Orchard S, Thorneycroft D, Harper JF, Mittler R, Cushman JC. (2008)Charting plant interactomes: possibilities and challenges. Trends Plant Sci. 13, 183-191.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Activities: Experiments were conducted on mutants of unknown function. Events:: A post doc (Shai Koussevitzky Ph.D.) was trained. Resaults were presented in the following conferences: 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. Dissemination: http://www.ag.unr.edu/ROS/
PARTICIPANTS: Shai Koussevitzky Ph.D.
TARGET AUDIENCES: Plant Science Community
PROJECT MODIFICATIONS: None
Impacts
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. 4. 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 and it functions upstream to Zat7, WRKY25 and Apx1. 5. The heat shock transcription factor 21 (HSF21) is required for Apx1 expression during oxidative stress. 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. 6. The transcriptional co-activator MBF1c enhances the expression of a number of oxidative stress response genes (including Zat12, Zat7 and Apx2). Expression of MBF1c in plants can enhance their tolerance to a combination of heat and osmotic stress (a major cause of abiotic stress damage in plants). 7. The EAR domain of Zat7 is involved in the response of plants to salinity stress. 8. RbohD is essential to maintain the ROS-response signal and may
link calcium signaling with ROS signaling.
Publications
- 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.
- 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.
- Shulaev V, Cortes DF, Miller G, Mittler R (2007) Metabolomics for plant stress response. Physiol. Plant In press.
- Miller G, Shulaev V, Mittler R (2007) Reactive oxygen species and abiotic stress. Physiol. Plant In press.
- Ciftci-Yilmaz S, Mittler R, (2007) The zinc finger network of plants. CMLS In press.
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Progress 01/01/06 to 12/31/06
Outputs
We are awaiting the homozygoused lines generated in A and by the Ecker project. We have established protocols for all screens and conducted the different stress screens on known mutants and transgenic plants to obtain a baseline for our stress assays. The following screens were established: Heat (38oC; 1, 6, 24 and 48 hour), Cold (4 or 10oC; 1, 2 and 3 days), Osmotic stress (50, 100, 200 and 300 mM Sorbitol), Salinity (50, 100, 150 and 200 mM NaCl), Ferric-citrate (0.01, 0.1, 0.5 and 1 mM), High light (for 24, 48 and 72 hour), and Paraquat ( 0.01, 0.1, 0.5, 1 and 10 μM). The following known lines were so far tested as references: Over expressor-At5g59820, Knockout-At5g59820, Over expressor-At3g24500, Knockout-At1g07890, Antisense-At1g20630, Knockout-At4g08390, Double Antisense-At1g20630 X Knockout-At1g07890; Knockout-At5g47910; Knockout-At3g30775. The following unknown knockout lines were screened so far: At2g41090; At4g31100; At5g52310, At5g52300, At4g25580. We
are in the process of cloning and expressing the following 61 unknown cDNA clones in Arabidopsis: At2g41650, At4g17070, At1g32220, At5g19875, At5g27830, At5g59080, At5g50350, At5g43750, At5g19120, At5g18040, At3g60980, At3g51610, At3g43520, At4g12000, At4g08940, At4g05590, At1g72060, At3g30300, At3g23910, At3g14430, At3g20340, At3g19515, At3g16670, At3g08670, At3g10020, At3g02170, At1g13340, At1g19020, At1g64360, At1g52200, At1g78410, At1g21520, At1g80130, At1g73120, At1g50170, At1g50290, At1g11210, At1g14870, At2g22080, At2g04795, At2g21195, At1g27330, At2g15560, At2g32210, At2g24150, At1g63980, At2g19310, At2g44240, At2g40000, At5g52310, At5g52300, At4g25580, At5g04930, At3g27870, At1g68710, At3g25610, At1g13210, At1g26130, At1g17500, At1g72700, At1g54280, At3g13900, At1g59820, At5g44240. Once we complete this first round we will test the lines for tolerance/sensitivity to stress. Each of the vectors we are making contains a C-terminal GFP fusion for localization and a tag for
pull-down assays and complex analysis.
Impacts
We have found that most of the differences in gene context between species could be attributed to genes with unknown functions. Genes of unknown function might therefore represent a group of novel young genes that have independently evolved to determine the unique properties of different species (manuscript in preparation). We have tentatively identified function in 186 heretofore unidentified genes through the PANTHER program.
Publications
- 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.
- 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.
- 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
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Progress 01/01/05 to 12/31/05
Outputs
We are currently generating over 100 transgenic lines expressing genes of unknowns function. We are screening any knockout lines for genes with unknown function available in the ABRC stock, as well as the lines we are generating. We have established a project website. We performed bioinformatics analysis of genes with unknown function present in all published eukaryotic genomes.
Impacts
Assign a stress-related function to genes with unknown function in Arabidopsis. Determine the importance of genes with unknown function to species diversity. Identify genes that could be used in different crops to enahnce the tolerance of plants to abiotic stress.
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 stress in transgenic plants expressing the transcriptional co-activator MBF1. Plant Physiol. 139, 1313-1322.
- Gollery M, Harper J, Cushman J, Mittler T, Girke T, Zhu J-K, Bailey-Serres J, Mittler R (2005) What makes species unique? A role for genes with unknown function. PNAS submitted.
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Progress 01/01/04 to 12/31/04
Outputs
The long-term goal of this research project is to facilitate the assignment of function to every gene in Arabidopsis thaliana. To date, the function of the proteins encoded by more than 20% of plant genes is completely unknown, with the function of up to 43% of these proteins poorly characterized. The specific focus of this research is genes of unknown function and the networks that respond to environmental stress, such as chilling, drought, salt, flooding, high light, and oxidative environments. Environmental stress is the primary cause of crop loss world-wide. A plants survival and growth is dependent on its ability to cope with environmental stresses. It is clear that a plants stress response results from a complex set of changes in gene networks and metabolism. While 1000s of genes have been linked to stress networks, in only a few cases have their biochemical and genetic functions been elucidated. In fact, most of the stress-regulated genes identified to date
have completely unknown function(s). Major areas of progress: 1. Construction of transgenic plants expressing genes with unknown function is underway. 2. Bioinformatic analysis of gene with unknown function (family analysis, expression...), is underway.
Impacts
Identification of genes with unknown function that could be used to enahnce the tolerance of crop plants to abiotic stresses.
Publications
- No publications reported this period
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