Regulation of C/N-metabolism in Source and Sink Tissues
Major focus in the Huber laboratory is on regulatory mechanisms controlling carbon-nitrogen metabolism in plant source and sink tissues, as these constitute important pathways that impact growth and development and crop yield. Processes of interest include sucrose synthesis and utilization; nitrate assimilation; and storage product formation in developing soybean seeds. Studies include several levels of control but most work is focused on posttranslational protein modifications, including protein phosphorylation, O-glycosylation, and S-nitrosylation. We are using Arabidopsis and field-grown maize and soybean to identify modified proteins that may be manipulated to impact productivity. Major current projects in the lab include the following:
Proteomics of 14-3-3 proteins.
The 14-3-3 proteins are phosphoserine-binding proteins that interact broadly with a wide range of cellular proteins involved in metabolism, signal transduction and gene expression. Early studies in our lab focused on the role of 14-3-3s in the regulation of the enzyme nitrate reductase (NR), which catalyzes the first step in the important process of nitrate assimilation. The activity of NR is regulated at the posttranslational level by phosphorylation of a serine residue (Ser543 in the hinge 1 region) followed by binding of a 14-3-3 protein to form an inactive complex. It was more recently shown that 14-3-3 proteins can also affect NR activity indirectly by affecting the proteolytic degradation of the NR protein. Currently we are using transgenic Arabidopsis plants expressing 14-3-3 directed mutants to explore mechanisms that regulate binding to targets in vivo, and to explore how 14-3-3 isoforms interact globally with cellular proteins. These fundamental studies may have practical application if, as suspected, transgenic manipulation of 14-3-3s can control the steady-state level of multiple proteins in crop plants.
Intracellular Localization of Sucrose synthase.
Sucrose synthase (SUS) is recognized as an important enzyme of sucrose metabolism in sink organs active in biosynthetic processes such as cell wall synthesis or accumulation of storage products (e.g., starch, protein, and oil in developing seeds). In source leaves, SUS is a low abundance protein associated with vascular tissue where it is thought to energize phloem transport. SUS is a soluble, globular phosphoprotein that has been assumed to be freely diffusing in the cytoplasm. However, recent results suggest that the enzyme may be compartmented within the cell as a result of association with the plasma membrane and the actin cytoskeleton. Interestingly, a synthetic peptide based on a region of the protein that may function in actin binding causes F-actin in animal cells to ‘bundle,’ thereby inhibiting cell division and cell movement. The peptide may have application as a cancer therapeutic (Winter et al. patent pending), and thus, studies of a plant metabolic enzyme may have unexpected application in the field of human health. We are also studying the association of SUS with plant membranes in vitro in order to identify potential membrane- and actin-binding domains. We are speculating that SUS at different locations within the cell may channel carbon into different metabolic pathways. If this is true, then controlling the localization of the enzyme may provide a new strategy to influence how growing cells use sucrose.
Regulation of soybean seed composition.
Developing soybean seeds receive sucrose and amino acids via the translocation stream and use these assimilates to form protein and oil (major storage products). Cultivars differ in protein:oil formation and in general, the two products are inversely related. The mechanisms that control seed composition are largely unknown, but are extremely important agronomically. We are speculating that the metabolic priority for a developing seed is to utilize available amino acids for protein synthesis, and that surplus C-skeletons (in excess of that required for protein synthesis), are then available for oil production. We are postulating that this coordination is achieved at the transcriptional level, where N-metabolites regulate expression of genes encoding enzymes of starch and lipid biosynthesis; and at the post-translational level, perhaps involving protein phosphorylation of key metabolic enzymes. We are currently testing these postulates.
Protein kinase specificity and oxidative stress.
We are interested in the factors that control protein kinase specificity in plants, with particular interest in the calcium-dependent protein kinases (CDPKs) and sucrose-nonfermenting (SNF)1-related protein kinases (SnRK1s). We have identified canonical and noncanonical phosphorylation motifs targeted by these kinases in vitro that may be useful in predicting new phosphorylation sites in proteins, and in application of molecular genetic approaches to modify phosphorylation-dependent processes. Fundamental studies of the biological mechanisms that control important plant processes may ultimately produce new approaches to increase the capacity of crop plants to produce and utilize nutrients that support growth of seeds, tubers and fruits. Furthermore, exciting new work suggests that methionine (Met) oxidation, which is a reversible and abundant modification of plant proteins even under normal conditions, may prevent phosphorylation when it occurs at critical positions in phosphorylation motifs. This can be clearly observed in vitro using synthetic peptides and MALDI-ToF MS analysis. We are exploring whether this effect also occurs in vivo, and whether it may play a previously unrecognized role in oxidative stress sensing/signaling.
Autophosphorylation of receptor-like protein kinases.
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require two receptor-like protein kinases, known as BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), for hormone perception and signal transduction. These receptor-like kinases are embedded in the plasma membrane with an extracellular portion that binds the ligand (in this case brassinosteroid) and a cytoplasmic domain that has potential protein kinase activity. We are collaborating on a multi-investigator NSF2010 project to determine whether plant receptor-like kinases function similarly to many animal receptor kinases, which exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. Identifying phosphorylation sites that are required for recognition and/or phosphorylation of downstream substrates may provide an approach to manipulate the hormonal control of plant growth and development.
Protein carbonylation and global change.
Plants are continually producing reactive oxygen species (ROS) that can irreversibly modify cellular components including proteins, resulting in dinitrophenylhydrazine (DNPH)-reactive carbonyl formation (a recognized marker for irreversible protein oxidative damage). In both growth chamber grown Arabidopsis and field-grown soybean leaves numerous proteins [including Rubisco (large and small subunits), Rubisco activase, and chlorophyll a/b binding protein (LhcII)] were found to contain DNPH-reactive carbonyls. Interestingly, protein carbonyl content seems to be increased by exposure of plants to elevated CO2. This is unexpected, because inhibition of photorespiration at elevated CO2 would be expected to decrease production of ROS in vivo. Although the basis remains unclear, our working model is that growth at elevated CO2 results in an initial oxidation of proteins and that, with time, plants adjust by increasing antioxidant capacity. This may explain why plants at elevated CO2 are more resistant to O3 injury, and why seed yield of some soybean cultivars is not significantly increased when plants are grown at elevated CO2. We are currently using proteomic approached and MALDI-TOF mass spectrometry to identify proteins differing in abundance and/or level of oxidation from leaves grown at elevated CO2 compared to ambient CO2.
Man-Ho Oh, Research Associate
Clayton Larue, Research Associate
Jisen Zhang, Visiting Scientist
Xia Wu, Graduate Student
Sean Park, Undergraduate Student
1. Winter, H. and Huber, S.C. 2000. Regulation of sucrose metabolism in higher plants. Localization and regulation of activity of key enzymes. Crit. Rev. Plant Sci. 19: 31-67. (also published as Crit. Rev.Biochem.Mol.Biol.35:253-289)
2. Foyer, D.H., Ferrario-Méry, S. and Huber, S.C. 2000. Regulation of carbon fluxes in the cytosol. Co-ordination of sucrose synthesis, nitrate reduction, and organic acid and amino acid biosynthesis. In: Photosynthesis: Physiology and Metabolism (Leegood, R.C., Sharkey, T.D. and von Caemmerer, S., eds), Chapter 8, Kluwer Academic Publishers, pp. 177-203.
3. Toroser, D. and Huber, S.C. 2000. Carbon and nitrogen metabolism and reversible protein phosphorylation. In: Plant Protein Kinases (M Kreis and JC Walker, eds), Advances in Botanical Research incl. Advances in Plant Pathology, Academic Press, pp. 435-458.
4. Winter, H. and Huber, S.C. 2000. Sucrose synthase:actin interaction. Sucrose metabolism and the actin cytoskeleton. In: Actin: A Dynamic Framework for Multiple Plant Cell Functions (CJ Staiger, F Baluska, D Volkmann and P Barlow, eds), Chapter 7, Kluwer Academic Publishers, The Netherlands, pp. 119-128.
5. Toroser, D., Plaut, Z. and Huber, S.C. 2000. Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate. Plant Physiol., 123: 403-411.
6. Athwal, G.S., Lombardo, C.R., Huber, J.L., Masters, S.C., Fu, H. and Huber, S.C. 2000. Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. Plant Cell Physiol. 41: 523-533.
7. Oh, M.-H., Ray, R.K., Huber S.C., Asara, J.M., Gage, D.A. and Clouse, S.D. 2000. Recombinant brassinosteroid-insensitive1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. Plant Physiol. 124: 751-765.
8. Kaiser, W.M. and Huber, S.C. 2001. Regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. J. Exp. Bot. 52: 1981-1989.
9. Huang, J.-Z., and Huber, S.C. 2001. Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Influence of proline and basic amino acid residues at selected positions. Plant Cell Physiol. 42: 1079-1087.
10. Huang, J.-Z., Hardin, S.C. and Huber, S.C. 2001. Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4. Arch. Biochem. Biophys. 393: 61-66.
11. Long, J.C., Zhao, W., Rashotte, A.M., Muday, G.K. and Huber, S.C. 2002. Gravity stimulated changes in auxin and invertase gene expression in Zea mays L. pulvinal cells. Plant Physiol. 128: 591-602.
12. Athwal, G.S. and Huber, S.C. 2002. Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase. Plant J. 29: 1-14.
13. Huber, S.C., MacKintosh, C. and Kaiser, W.M. 2002. Metabolic enzymes as targets for 14-3-3 proteins. Plant Mol. Biol. 50: 1053-1063.
14. Tang, G.-Q., Hardin, S.C., Dewey, R. and Huber, S.C. 2003. A novel C-terminal proteolytic processing of cytosolic pyruvate kinase, its phosphorylation and degradation by the proteosome in developing soybean seeds. Plant J. 34: 77-95 (featured on cover).
15. Shen, W., Clark, A.C. and Huber, S.C. 2003. The C-terminal tail of Arabidopsis 14-3-3 omega functions as an autoinhibitor and may contain a tenth alpha-helix. Plant J. 34: 473-484.
16. Hardin, S.C., Tang, G.-Q., Scholz, A., Holtgraewe, D., Winter, H. and Huber, S.C. 2003. Phosphorylation of sucrose synthase at serine-170: occurrence and possible role as a signal for proteolysis. Plant J. 35: 588-603.
17. Hardin, S.C. and Huber S.C. 2004. Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves. Plant Physiol. Biochem. 42: 197-208.
18. Hardin, S.C., Winter, H. and Huber, S.C. 2004. Phosphorylation of the amino-terminus of maize sucrose synthase in relation to membrane association and enzyme activity. Plant Physiol. 134: 1427-1438.
19. Huber, S.C. and Hardin, S.C. 2004. Numerous post-translational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels. Current Opinion Plant Biology 7: 318-322.
20. Sebastia, C.H., Hardin, S.C., Clouse, S.D., Kieber, J.J. and Huber, S.C. 2004. Identification of a new motif for CDPK phosphorylation in vitro that suggests ACC synthase may be a CDPK substrate. Arch. Biochem. Biophys. 428: 81-91.
21. Sebastia, C.H., Marsolais, F., Saravitz, C., Israel DW, Dewey R and Huber S.C. 2005. Metabolic profiles of free amino acids suggests a possible role for Asn in the control of storage product accumulation in developing soybean seeds. J. Exp. Bot. 56:1951-1963.
22. Wang, X.F., Goshe, M.B., Soderlom, E., Phinney, B.S., Kuchar, J., Li, J., Asami, T., Yoshida, S. Huber, S.C. and Clouse, S.D. 2005. Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase. Plant Cell 17:1685-1703.
23. Tang, G.-Q., Novitzky, B., Griffin, C., Huber, S.C. and Dewey, R. 2005. Functional characterization of two closely related soybean (Glycine max) oleate desaturase enzymes: evidence of regulation through differential stability and phosphorylation. Plant J. 44: 433-446.
24. Ehsan, H., Ray, W.K., Phinney, B., Wang, X., Huber, S.C., and Clouse, S.D. 2005. Interaction of Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase with a homolog of mammalian TGF-b receptor interacting protein. Plant J. 43: 251-261.
1. Brake, J., Wilson, R.F., Burton, J.W., Huber, S.C., Israel, D.W., Shannon, G., Anand, S.C.,Sleper, D.A., Pantalone, V.R., and Wilcox, J.R. 2002. Current status of “designer” soybeans – oil and protein traits. American Soybean Association MITA (P) No. 087/10/2002.
Provisional patent application filed Oct 17, 2003. D.N. 0239.99, “Synthetic peptides that cause F-actin bundling and block actin depolymerization.” Inventors: H. Winter, S.C. Huber and C. Larabell.
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