Progress 10/01/04 to 09/30/08
Outputs
OUTPUTS: During the past year, our work resulted in the publication of (1) one peer-reviewed research article; (2) one workshop report; (3) one comprehensive review that summarizes progress in the redox biology field we launched forty years ago; (4) an analysis of the application of proteomics, a rapidly developing technology for analyzing wheat; (5) an historical article describing the emergence of some of our work on the Berkeley campus; and (6) a special journal issue I helped edit to honor a luminary in our field, Andrew A. Benson, on the occasion of his 90th birthday. I was also on sabbatical leave in Germany, supported by a Humboldt Research Award, where I performed research at the University of Munich and the Max Plant Institute of Molecular Plant Physiology in Golm. During my stay, I participated in three symposia and conferences and gave 16 seminars and lectures in Germany and other parts of Europe. Articles are listed below. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Although a principal source of energy and protein for millions of the world's poorest people, the nutritional value of sorghum (Sorghum bicolor L Moench) is diminished because of low digestibility of grain protein and starch. To address this problem, we analyzed the properties of two sorghum lines that have a common pedigree but differ in digestibility. Consistent with results based on a ruminal fluid assay, the protein and starch of one line (KS48) was more thoroughly digested than that of the other (KS51) using in vitro assays based on pepsin and alpha-amylase. The indigestibility of KS51 relative to KS48 was shown to be due to (i) a greater abundance of disulfide-bonded proteins; (ii) presence in KS51 of non-waxy starch and the accompanying granule-bound starch synthase; and (iii) the differing nature of the protein matrix and its interaction with starch. The current findings suggest that each of these factors should be considered in efforts to enhance the nutritional value of sorghum grain. During the year, we also continued work on our wheat allergy project. We strengthened evidence that thioredoxin overexpressed in the endosperm lower the allergenicity of wheat proteins.
Publications
- 1. Buchanan, B.B., Wong, J.H., Cho, M.-J., Kim, Y.-B., Jung, H.R., Kim, H.K., Sorigsaki, S., Lemaux, P.G., Moss, R.B., Teuber, S.S. and Frick, O.L. 2007 The dog as a model for assessing food allergens in wheat. In: Gluten Proteins 2006. pp. 338-382. Edited by G. L. Lookhart and P.K.W. Ng. American Association of Cereal Chemists, St. Paul, MN. 2. Schuermann P. and Buchanan B. B. (2008) The Ferredoxin/Thioredoxin System of Oxygenic Photosynthesis. Antioxid. Redox Signal. 10, 1235-1274. 3.Hurkman, W. J., Vensel, W. H., DuPont, F. M., Altenbach, S. B. and Buchanan, B. B. 2008 Endosperm and Amyloplast Proteomes of Wheat Grain. In In: Plant Proteomics: Technologies, Strategies, and Applications (G. Kumar and R. Rakway, eds.), John Wiley & Sons, Inc., NY. pp. 207-222. 4. Wong, J. H., Lau, T., Cai, N., Singh, J., Pedersen, J. F., Vensel, W. H., Hurkman, W. J., Lemaux, P. G. and Buchanan, B. B. (2008) Digestibility of protein and starch from sorghum (Sorghum bicolor) is linked to biochemical and structural features of grain endosperm. J. Cereal Sci. 49, 73-82. 5. Buchanan, B. B. 2007 Thioredoxin: An unexpected meeting place. Photosyn. Res. 92, 145-148. 6. Buchanan, B. B., Douce, R. and Lichtenthaler, H. K. 2007 Andrew A. Benson. Photosyn. Res. 92, 143-144. 7. Buchanan, B. B., Douce, R. and H. K. Lichtenthaler (2007) Editors, A Tribute to Andrew A. Benson, Special Issue of Photosynthesis Research, Vol. 92, No. 2, pp. 142-271.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Our research during the past year is based on the application of proteomics to address two major agricultural problems: drought tolerance (using a cereal, wheat) and germination (with a legume, Medicago truncatula). With wheat, we studied protein changes in grain in response to drought. Three wheat genotypes differing in genetic background were cultivated in field under well-watered and drought conditions by following a randomized complete block design with four replications. The overall effect of drought was highly significant as determined by grain yield and total dry matter. About 650 spots were reproducibly detected and analyzed on 2-DE gels. Of these, 121 proteins showed significant change under drought condition in at least one of the genotypes. Mass spectrometry analysis using MALDI-TOF/TOF led to the identification of 57 proteins. Two-thirds of identified proteins were thioredoxin (Trx) targets, in accordance with the link between drought and oxidative stress.
Further, because of contrasting changes in the tolerant and susceptible genotypes studied, several proteins emerge as key participants in the drought response. In addition to providing new information on the response to water deprivation, the present study offers opportunities to pursue the breeding of wheat with enhanced drought tolerance using identified candidate genetic markers. The 2-DE database of wheat seed proteins is available for public access at http://www.proteome.ir. The Medicago work built on our earlier experience with cereals which we showed that germination is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Trx appears to play a role in this transition in cereals. It is not known, however, whether Trx-linked redox changes are restricted to cereals or whether they take place more broadly in germinating seeds. To gain information on this point, we
have investigated a model legume, Medicago truncatula. Two complementary gel-based proteomic approaches were followed to identify Trx targets in seeds: Proteins were (1) labeled with a thiol-specific probe, monobromobimane (mBBr), following in vitro reduction by an NADP/Trx system, or (2) isolated on a mutant Trx affinity column. Altogether, 111 Trx-linked proteins were identified with few differences between axes and cotyledons. Fifty nine were new, 34 found previously in cereal or peanut seeds, and 18 in other plants or photosynthetic organisms. In parallel, the redox state of proteins assessed in germinating seeds using mBBr revealed that a substantial number of proteins that are oxidized or partly reduced in dry seeds became more reduced upon germination. The patterns were similar for proteins reduced in vivo during germination or in vitro by Trx. In contrast, glutathione and glutaredoxin were less effective as reductants in vitro. Overall, more than half of the potential targets
identified with the mBBr labeling procedure were reduced during germination. The results provide evidence that Trx functions in the germination of seeds of dicotyledons as well as monocotyledons.
PARTICIPANTS: In this work I collaborated with a number of individuals both within and without UC Berkeley. Outside colleagues included Dr. Francois Montrichard and her graduate student, Ms. Fatima Alkhalfioui of Angers, France. In our wheat work, we initiated a collaboration with Dr. Ghasem Hosseini Hajheidari and his group in Karaj, Iran. This proved to be a very successful collaboration, one that I hope will continue in the future. Long term collaborations were continued with my Berkeley colleagues, Dr. Peggy Lemaux and Dr. Joshua Wong, a senior research associate in our laboratory. Those experiments are ongoing.
TARGET AUDIENCES: Our research addresses problems that have the potential for societal application. We try to reach that community.
PROJECT MODIFICATIONS: None.
Impacts
Our wheat work provided experimental evidence linking drought tolerance to oxidative stress via the regulatory protein, Trx. The finding that Trx and several target proteins were enriched in a drought-tolerant genotype relative to its susceptible counterparts has set the stage for plant breeders to select for genes responsible for drought tolerance. These trials have been initiated by our collaborators in Iran. With respect to germination, our evidence that redox changes of the type established for cereals occur in a legume indicates that these changes take place in a broad spectrum of monocot and diccot crops. In view of these findings, it is now possible to determine whether Trx or Trx-related proteins are associated with dormancy or precocious germination, processes that are detrimental to agriculture worldwide.
Publications
- Hajheidari M, Eivazi A, Buchanan BB, Wong JH, Majidi I, Salekdeh GH. 2007 Proteomics uncovers a role for redox in drought tolerance in wheat. J Proteome Res. 2007 :1451-60.
- Alkhalfioui F, Renard M, Vensel WH, Wong J, Tanaka CK, Hurkman WJ, Buchanan BB, Montrichard F. 2007 Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds. Plant Physiol. 144:1559-79
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Progress 01/01/06 to 12/31/06
Outputs
During the past year, we undertook a long neglected problem that is key to our understanding of the biology of cereal endosperm. In contrast to chloroplasts, our knowledge of amyloplasts, organelles that synthesize and store copious amounts of starch in heterotrophic plant tissues, is in a formative stage. To help fill this gap, a proteomic analysis of amyloplasts isolated from the starchy endosperm of wheat seeds (10 days post-anthesis) was conducted. The study has led to the identification of 289 proteins that function in a range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen and sulphur metabolism, nucleic acid-related reactions, synthesis of various building blocks, protein-related reactions, transport, signalling, stress, and a variety of other activities grouped under 'miscellaneous'. The function of 12% of the proteins was unknown. The results highlight the role of the amyloplast as a starch-storing
organelle that fulfills a spectrum of biosynthetic needs of the parent tissue. When compared with a recent proteomic analysis of whole endosperm, our studies demonstrated the advantage of using isolated organelles in proteomic studies. The second phase of our work this year was to investigate the role of redox in regulating enzymes in amyloplasts. Using proteomics and immunological methods, we identified the components of the ferredoxin/thioredoxin system (ferredoxin, ferredoxin/thioredoxin reductase and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase (FNR). However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxin-thioredoxin reductase (FTR) and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the
identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism, whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.
Impacts
The impact of the work accomplished is as expressed in the original application, namely, it is relevant to the yield of grain harvested from cereals and other plants. In this case, we have characterized the amyloplast of wheat using proteomics. We have determined the protein (enzyme) complement of the organelle and demonstrated the presence of the ferredoxin/thioredoxin system, a regulatory mechanism known to play a central role in chloroplasts. The data suggest that the system could be equally important in grain amyloplasts. Increased understanding of the organelle will most likely prove useful as we continue to improve grain as a food and, in the future, as a biofuel.
Publications
- Balmer, Y., Vensel, W.H., Dupont, F.M., Buchanan, B.B., Hurkman, W.J. (2006) Proteome of amyloplasts isolated from developing wheat endosperm presents evidence of broad metabolic capability. J Exp Bot. 57, 1591-1602.
- Balmer, Y., Vensel, W.H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W.J., Buchanan, B.B. (2006) A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts. Proc Natl Acad Sci USA 103, 2988-2993.
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Progress 01/01/05 to 12/31/05
Outputs
A primary goal of this project is to understand the factors that determine the quality and performance of seeds. Through the development of an understanding of seed development, it is possible to manipulate seeds, potentially resulting in new mechanisms for controlling germination, important to the malting industry, and strategies to prevent premature sprouting, a notable problem in wheat and barley. Toward this goal, one approach to the problem is to understand the central features of embryogenesis a process critical to the development and ultimately the properties of the seed. The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65 to 94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos
at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing
2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.
Impacts
The yield of grain harvested from cereals and other plants is determined by a number of factors, including those that affect seed development. The hormone auxin is of prime importance in this connection. The present findings add to our understanding of mode of auxin action a longstanding problem in plant biology and agriculture. Knowledge of how auxin acts and how it accumulates is one of the goals of this project.
Publications
- Malgorzata D. G., Zhang, S., Harada, J.J. and Lemaux, P.G. 2005 Leafy cotyledon genes are essential for induction of somatic embryogenesis of Arabidopsis Planta 222, 977-988.
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