Source: UNIVERSITY OF NEVADA submitted to
THE RESPONSE OF PLANTS TO A COMBINATION OF DROUGHT STRESS AND HEAT SHOCK.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0204384
Grant No.
(N/A)
Project No.
NEV00338
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2005
Project End Date
Jun 30, 2008
Grant Year
(N/A)
Project Director
Mittler, R.
Recipient Organization
UNIVERSITY OF NEVADA
(N/A)
RENO,NV 89557
Performing Department
BIOCHEMISTRY
Non Technical Summary
The study of abiotic stress in plants has advanced considerably in recent years. However, the majority of studies testing the response of plants to changes in environmental conditions have focused on a single stress treatment applied to plants under controlled conditions. In contrast, in the field, a number of different stresses can occur simultaneously. These may include conditions such as drought, extreme temperature or high salinity and may alter plant metabolism in a novel manner that may be different from that caused by each of the different stresses applied individually1,2. Drought and heat shock represent an excellent example of two different stresses that occur in the field simultaneously, especially in semi-arid or drought-stricken areas1-4. Although drought stress and heat shock have been extensively studied5-7, relatively little is known about how their combination impact plants. We identified sucrose accumulation as a possible defense mechanism of plants against this stress combination. Our long-term objective is to develop different plants and crops with enhanced tolerance to a combination of drought stress and heat shock. A combination of drought stress and heat shock is common to many semi-arid or drought-stricken regions of Nevada. Developing plants and crops with enhanced tolerance to this stress combination will contribute significantly to Nevada agriculture and economy and directly address one of the major NAES research priorities.
Animal Health Component
(N/A)
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20324201040100%
Goals / Objectives
Our long-term objective is to enhance the tolerance of different plants and crops to a combination of drought stress and heat shock. Our specific objectives are: 1. Study the regulation of sucrose and proline biosynthesis during a combination of drought stress and heat shock in Arabidopsis. 2. Develop a screen to test the resistance of Arabidopsis plants to a combination of drought stress and heat shock and use the screen to test mutants altered in sucrose and proline biosynthesis. 3. Attempt to enhance the tolerance of Arabidopsis plants to a combination of drought stress and heat shock by expressing specific regulatory genes in transgenic plants.
Project Methods
We will perform biochemical, physiological and molecular measurements, as well as use different mutants and transgenic lines to test our hypothesis. In addition, we will attempt to enhance the tolerance of transgenic Arabidopsis plants to a combination of drought stress and heat shock using different regulatory genes previously identified by our gene-expression studies as specifically expressed in cells during the stress combination.

Progress 07/01/05 to 06/30/08

Outputs
OUTPUTS: Project Website: http://www.ag.unr.edu/Stress_Combination/ Within their natural habitat plants are subjected to a combination of different abiotic stresses, each with the potential to exacerbate the damage caused by the others. One of the most devastating stress combinations for crop productivity, which frequently occurs in the field, is drought and heat stress. In this study we conducted proteomic and metabolic analysis of Arabidopsis thaliana plants subjected to a combination of drought and heat stress. We identified 45 different proteins that specifically accumulated in Arabidopsis in response to the stress combination. These included enzymes involved in reactive oxygen detoxification, malate metabolism, and the Calvin cycle. The accumulation of malic enzyme during the combined stress corresponded with enhanced malic enzyme activity, a decrease in malic acid, and lower amounts of oxaloacetate, suggesting that malate metabolism plays an important role in the response of Arabidopsis to the stress combination. Cytosolic ascorbate peroxidase 1 (APX1) protein and mRNA accumulated during the stress combination. When exposed to heat stress combined with drought, an APX1-deficient mutant (apx1) accumulated more hydrogen peroxide and was significantly more sensitive to the stress combination than wild type. In contrast, mutants deficient in thylakoid or stromal/mitochondrial APXs were not more sensitive to the stress combination than apx1 or wild type. Our findings suggest that cytosolic APX1 plays a key role in the acclimation of plants to a combination of drought and heat stress. PARTICIPANTS: Principle Investigator: Dr. Ron Mittler Department of Biochemistry and Molecular Biology University of Nevada Mail Stop 200 Reno NV 89557 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. Visiting Scholars: A.N.MISRA, Professor&Head, Biosci.&Biotechnol. F.M.University, India TARGET AUDIENCES: Scientific community PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New mechanisms for controlling the response of plants to a combination of drought and heat were discovered. Transgenic soybean plants with enhanced tolerance to stress combination were developed.

Publications

  • 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.
  • Mittler. R (2006) Abiotic Stress, the Field Environment and Stress Combination. Trends Plant Sci. 11, 15-19.
  • 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.
  • Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl Acad Sci U S A. 104, 19631-19636.
  • 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.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Activities: Experiments were conducted on gain and loss of function lines for MBF1c. Analysis included RNA protein and metabolite profiling. Events: A graduate student (Nobuhiro Suzuki) and a post doc (Gad Miller) were trained. Results 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. Services: Two undergraduate students were trained (Serena Huntington and Alicia Hegie). Products: It was discovered that MBF1c is a master regulator of thermotolerance. Dissemination: http://www.ag.unr.edu/Stress_Combination/ PARTICIPANTS: Gadi Miller (Postdoc) Nobuhiro Suzuki (Ph.D.) TARGET AUDIENCES: Plant Scientific community PROJECT MODIFICATIONS: None

Impacts
The ability of an organism to acclimate to its environment is a key determinant in its global distribution and capacity to compete with other organisms. The heat stress response, a highly conserved environmental and developmental program in eukaryotic and prokaryotic organisms, is an important component of the acclimation response of plants. Previous studies have shown that heat shock transcription factors (HSFs) play an important role in thermotolerance in plants and other organisms, controlling the expression of different heat shock proteins (HSPs) and detoxifying enzymes. In contrast, although several other pathways, involving ethylene, salicylic acid (SA) and trehalose were recently shown to play a central role in thermotolerance in plants, a key regulator of these responses was not identified. Here we report that the highly conserved transcriptional co-activator, multiprotein bridging factor 1c (MBF1c), is a key regulator of thermotolerance in Arabidopsis thaliana. MBF1c protein accumulates rapidly and is localized to nuclei during heat stress. MBF1c is required for thermotolerance and functions upstream to SA, trehalose, ethylene, and pathogenesis-related protein 1 during heat stress. In contrast, MBF1c is not required for the expression of transcripts encoding HSFA2 and different HSPs. Interestingly, MBF1c interacts with trehalose phosphate synthase 5 (TPS5), that is also heat inducible, and mutants deficient in TPS5 are thermosensitive. Our results provide evidence for the existence of a tightly-coordinated heat stress-response network, involving trehalose-, SA- and ethylene-signaling pathways, that is under the control of MBF1c.

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
  • Suzuki, N., Shuman, J., Shulaev, V, Mittler, R. (2007) Regulation of thermotolerance in Arabidopsis. J. Biol. Chem. In press


Progress 01/01/06 to 12/31/06

Outputs
Progress report: We made considerable progress this year in the following areas: MBF1c: We discovered that MBF1c is a master regulator of basal thermotolerance in Arabidopsis. MBF1c is required for the activation of all 3 pathways involved in basal thermotolerance (ethylene, salicylic acid and trehalose), and overexpression of MBF1c in transgenic plants makes plants more tolerant to heat stress, osmotic stress, heat stress combined with osmotic stress, and pathogen attack. MBF1c, therefore, is an excellent candidate to enhance the tolerance of plants and crops to a large set of biotic and abiotic stresses, especially stresses that prevail in dry regions such as Nevada. Transgenic soybean plants expressing MBF1c provided significantly higher yield (seed weight and number), compared to wild type plants, when grown under heat stress conditions. Companies such as Monsanto and Syngenta are interested in testing the gene in corn and soybean. A manuscript is in preparation. Proteomic analysis of drought and heat stress: We have previously identified over 700 transcripts and 7 metabolites that specifically accumulate in plants in response to a combination of drought and heat stress. We have now performed proteomics analysis of plants subjected to a combination of drought and heat stress. We identified 53 proteins that accumulate and 8 proteins that are suppressed in plants specifically in response to a combination of drought and heat stress. Ascorbate peroxidae 1 (Apx1) was identified as one protein that accumulates specifically in response to the stress combination. We tested a mutant deficient in this protein and found that apx- plants are highly sensitive to the stress combination. We have thus identified the first plant gene that is required for a combination of drought and heat stress. We have also identified a very interesting pattern of metabolic adjustment in plants subjected to a combination of drought and heat stress. If this pattern would hold true in subsequent experiments we will be able to report a very interesting finding in a top journal. We are conducting pathway analysis to test our results using mutants. Two manuscripts are in preparation. Testing new genes and mutants: We are testing the potential of different genes, we have identified by our GenChip analysis of drought and heat combination, to enhance the tolerance of plants to a combination of drought and heat stress. We have promising results, but need additional time and funds to confirm. Confirmation of our results could results in additional patent applications.

Impacts
We have demonstrated elevated yield in MBF1c-transgenic soybean and Arabidopsis plants subjected to drought, heat and their combination, as well as identified many unique genes, proteins and metabolites that are elevated or suppressed in plants in response to a combination of drought and heat stress. Because drought and heat stress combination is a major cause of yield loss in the US, causing billions of dollars in damages, and because we are the first to perform these molecular experiments, and have generated a large body of data, many people in industry, research institutes and universities all around the world are interested in our findings. We are making a significant impact in publications, invitations to industry and international seminars/conferences and patent application. We have just submitted a new NSF grant on this research subject, utilizing the new data we obtained. Our research on MBF1c revealed that this gene is a master regulator of basal thermotolerance in plants (paper was submitted to PNAS). This is a major finding in the field of plant biology, because a master regulator of basal thermotolerance has been sought after for many years now. Our work in the coming year would enable us to determine the mode of function of MBF1c and to extend our analysis of MBF1c in other model or crop plants. This is very important because for successful patenting of MBF1c we would need to demonstrate a broad function of this gene in different plants.

Publications

  • New Website (2006): www.ag.unr.edu/Stress_Combination
  • Work on the project was presented in the following international conferences and industry seminars (2006-2007):
  • 2006 Syngenta Corporation, Chapel Hill, NC 2006 Keystone meeting, Plant Responses to Abiotic Stress, Colorado. 2006 Gordon Research Conference Water Stress in Plants, UK. 2006 International Symposium on Dynamic Organelles, Okazaki, Japan. 2007 Gordon Research Conference, Temperature Stress, CA. 2007 North Carolina Plant Molecular Biology Consortium Seminar Series (5 companies and 3 universities).
  • Papers acknowledging the grant (2006-2007):
  • 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.
  • 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.
  • 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. In press.
  • 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.
  • Koussevitzky, S., Suzuki, N., Shulaev, V. and Mittler, R. (2007) Proteomic analysis of drought and heat stress combination in Arabidopsis reveals a key role for APX1. In preparation.
  • Patent applications (2006): U.S. Patent No. entitled: Enhanced tolerance to environmental stresses in transgenic plants expressing the transcriptional co-activator MBF1 (In preparation).


Progress 01/01/05 to 12/31/05

Outputs
Climate changes over the past decades subjected large areas of our planet to desertification and drought. Crop plants cultivated in affected areas frequently encounter extreme environmental conditions that include a combination of drought and other stresses, such as heat or salinity. Tolerance to a combination of drought and heat is a well-known breeding target in maize, grasses and other crops. However, a transgenic approach that enhances the tolerance of plants to this stress combination is not yet available. Here, we report that constitutive expression of the eukaryotic transcriptional co-activator, multiprotein bridging factor 1 (MBF1c), in Arabidopsis thaliana enhances the tolerance of transgenic plants to osmotic stress, heat stress and their combination. We further show that MBF1 expression enhances the tolerance of transgenic plants to these stresses by partially activating the ethylene-response signal transduction pathway. MBF1 proteins could be used to enhance the tolerance of plants to a combination of abiotic stresses. Preliminary results with transgenic soybean show enhanced growth and productivity in transgenic plants expressing MBF1c.

Impacts
We are the first to identify, test and confirm a transgene (MBF1c) that can enahnce the tolerance of plants to a combination of drought and heat stress. This stress combination caused an estimated 120 billion dollars of damages to US agriculture between 1984 and 2004.

Publications

  • 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.
  • Mittler. R (2006) Abiotic Stress, the Field Environment and Stress Combination. Trends Plant Sci. 11, 15-19 (plus cover of journal)
  • Suzuki N, Mittler R (2006) Reactive oxygen species and temperature stresses: A delicate balance between signaling and destruction. Physiol. Plant. 126, 45-51.