Abstract
Protein microarray technology has emerged as a powerful new approach for the study of thousands of proteins simultaneously. Protein microarrays have been used for a wide variety of applications for the human and yeast systems. In a recent study, we demonstrated that Arabidopsis functional protein microarrays can be generated and employed to characterize the function of plant proteins. The arrayed proteins were produced using an optimized large-scale plant-based expression system. In a proof-of concept study, 173 known and novel potential substrates of calmodulin (CaM) and calmodulin-like proteins (CML) were identified in an unbiased and high-throughput manner. The information documented here on novel potential CaM targets provides new testable hypotheses in the area of CaM/Ca2+-regulated processes and represents a resource of functional information for the scientific community.
Key Words: protein microarray, calmodulin, protein-protein interactions, large scale protein production
At the foundation of the remarkable complexity of all living organisms, lays an intricate network of interactions between their cellular components—proteins, nucleic acids, sugars and other molecules. Organisms' functions such as growth and development, reproduction, and responses to pathogens and environmental stresses are ultimately the result of constant assembly and disassembly of macromolecular complexes within cells. Therefore, revealing and cataloging these associations will lead to a comprehensive understanding of cellular signaling pathways and regulatory networks of the organism.
Availability of DNA microarray-based high throughput technology has aided in large-scale analyses of organisms' genome as evident by their application in transcript mapping, gene expression profiling, detecting mutations and deletions, and mapping transcription factor binding sites. Although DNA microarray experiments are invaluable, little can be inferred from these studies about the functions of encoded gene products. Recently, protein microarray technology has emerged as a powerful approach for the study of hundreds or thousands of proteins simultaneously in a controlled experimental setup. Yeast and human protein microarrays have been used for a wide variety of applications including the study of protein-protein interactions, protein-nucleic acid interactions, targets of small molecule, antibody specificity, protein kinase substrates, as well as posttranslational modifications.1 An important advantage offered by protein microarrays is the capacity to study, in an unbiased manner, the in vitro behavior of a large number of macromolecules in various types of biochemical reactions. Therefore, novel substrates and subsequently biological functions of proteins can be predicted and then validated through additional methods.
In a recent study, we demonstrated that Arabidopsis functional protein microarrays can be constructed and employed to investigate protein function.2 Specifically, we used high-density Arabidopsis protein microarrays containing 1,133 proteins to investigate targets of calmodulin (CaM) and calmodulin-like proteins (CML).
An Optimized Homologous Protein Production System for Constructing Functional Arabidopsis Protein Microarrays
A critical step in the generation of functional protein microarrays is the construction of a high quality expression library from which a large number of distinct protein samples can be produced. For this, we built a high quality expression library of tagged ORFs, ATEC (Arabidopsis thaliana expression collection) in a versatile plant expression vector. ATEC collection is represented by multiple gene families including protein kinases, transcription factors, protein with unknown functions, heat-shock proteins, P450 cytochromes, protein degradation-related proteins, CaMs/CMLs and CaM/CML-binding proteins, and others.
Another important requirement to construct functional protein microarrays is that the purified proteins have to be functional, that is, they should retain their cellular activity and act similarly to their cellular counter-parts, when on the protein chip. Therefore, proteins must be produced and purified in a biological system that allows native folding and post-translational modifications to occur, and provides necessary cofactors for protein activity. In this respect, we tested the performance of two eukaryotic systems for protein expression, Saccharomyces cerevisiae and Nicotiana benthamiana. Our experiments clearly demonstrated that although proteins were expressed at comparable levels in both systems, the plant-produced proteins retained their enzymatic function after purification compared to yeast expression system.2
Based on these results, a large-scale plant-based protein expression and production system was optimized and employed to generate 1133 Arabidopsis proteins. These purified recombinant proteins were used to generate first high density Arabidopsis protein microarrays. To demonstrate the utility, these protein microarrays were probed with several CaM and CML proteins to identify their binding partners.
Functional Analysis of Several Arabidopsis CaMS/CMLs Targets Using High-Density Protein Microarrays
In contrast to the yeast and vertebrates, plants contain a large family of CaM and CaM-like proteins.3 The diversity of plant CaM/CMLs structures translates in variability in their target preference. Plant CaM/CML bind a wide variety of substrates.4 Some of these targets are homologous to animal CaM targets, but some of the targets are unique to plants. To identify other targets of plant CaMs/CMLs, protein microarray containing 1133 proteins were probed with 3 CaMs (CaM1, CaM6 and CaM7) and 4 CMLs (CML8, CML9, CML10 and CML12). Our analysis identified 173 putative CaM/CML substrates, belonging to various protein families. Interestingly, we found that the majority of the targets were specific to one or a small subset of CaMs/CMLs.2
Analysis of Gene Ontology (GO) categories in our set of predicted CaM/CML targets identified several over-represented biological processes (Fig. 1 and Table 1). There is a large repertoire of previously identified CaM-binding proteins that are linked to plant response to various environmental stresses. Also, genes encoding specific plant CaM and CaM-related proteins are induced by abiotic factors such as cold, and biotic stressors like fungal proteins, viruses, and pathogenic bacteria.5
Figure 1.
Targets of CaMs/CMLs identified from the protein microarrays belongs to different biological processes based on the Gene ontology (Go) analyses.
Table 1.
Analysis of gene ontology (GO) categories over-represented in the CaM/CML target list
| GO-ID | P-Value | Genes in Test Set | Accession # | Gene Description |
| 48409 | 5.67E-03 | Flower development | AT5G23260 | | TT16 | MADS-BOX PROTEIN, TRANSPARENT TESTA16 |
| AT3G58780 | | SHP1 | AGAMOUS-LIKE MADS BOX AGL1 / SHATTERPROOF 1 | |||
| AT5G60910 | | AGL8 | AGAMOUS-LIKE MADS BOX AGL8 / FRUITFULL | |||
| 10154 | 7.29E-04 | Fruit development | AT3G61880 | | CYP78A9 | CYTOCHROME P450, PUTATIVE |
| AT5G60910 | | AGL8 | AGAMOUS-LIKE MADS BOX AGL8 / FRUITFULL (AGL8) | |||
| 48481 | 4.02E-03 | Ovule development | AT5G23260 | | TT16 | MADS-BOX PROTEIN, TRANSPARENT TESTA16 |
| AT3G58780 | | SHP1 | AGAMOUS-LIKE MADS BOX AGL1 / SHATTERPROOF 1 | |||
| 48440 | 7.56E-03 | Carpel development | AT5G23260 | | TT16 | MADS-BOX PROTEIN, TRANSPARENT TESTA16 |
| AT3G58780 | | SHP1 | AGAMOUS-LIKE MADS BOX AGL1 / SHATTERPROOF 1 | |||
| 48467 | 8.25E-03 | Gynoecium development | AT5G23260 | | TT16 | MADS-BOX PROTEIN, TRANSPARENT TESTA16 |
| AT3G58780 | | SHP1 | AGAMOUS-LIKE MADS BOX AGL1 / SHATTERPROOF 1 | |||
| 48413 | 1.75E-02 | Floral whorl development | AT5G23260 | | TT16 | MADS-BOX PROTEIN, TRANSPARENT TESTA16 |
| AT3G58780 | | SHP1 | AGAMOUS-LIKE MADS BOX AGL1 / SHATTERPROOF 1 | |||
| 9651 | 3.77E-03 | Response to salt stress | AT3G19290 | | ABF4 | ABA-RESPONSIVE ELEMENT-BINDING PROTEIN 2 |
| AT2G31180 | | MYB FAMILY TRANSCRIPTION FACTOR (MYB14) | |||
| AT2G23290 | | MYB FAMILY TRANSCRIPTION FACTOR | |||
| AT5G35410 | | SOS2 | CBL-INTERACTING PROTEIN KINASE 24 (CIPK24) | |||
| 6970 | 6.55E-03 | Response to osmotic stress | AT3G19290 | | ABF4 | ABA-RESPONSIVE ELEMENT-BINDING PROTEIN 2 |
| AT2G31180 | | MYB FAMILY TRANSCRIPTION FACTOR (MYB14) | |||
| AT2G23290 | | MYB FAMILY TRANSCRIPTION FACTOR | |||
| AT5G35410 | | SOS2 | CBL-INTERACTING PROTEIN KINASE 24 (CIPK24) | |||
| 42829 | 5.84E-03 | Physiological defense response | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| AT4G26070 | | MEK1 | MITOGEN-ACTIVATED PROTEIN KINASE KINASE | |||
| AT3G12250 | | TGA6 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| AT1G22070 | | TGA3 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| 51869 | 6.55E-03 | Physiological response to stimulus | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| AT4G26070 | | MEK1 | MITOGEN-ACTIVATED PROTEIN KINASE KINASE | |||
| AT3G12250 | | TGA6 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| AT1G22070 | | TGA3 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| 6951 | 9.60E-04 | Response to heat | AT5G37670 | | 15.7 KDA CLASS I-RELATED SMALL HEAT SHOCK PROTEIN-LIKE |
| AT5G56030 | | HSP81 2 | HEAT SHOCK PROTEIN 81-2 (HSP81-2) | |||
| AT5G02500 | | HSC70 1 | HEAT SHOCK COGNATE 70 KDA PROTEIN 1 | |||
| AT1G11270 | | F-BOX FAMILY PROTEIN | |||
| 9755 | 1.18E-02 | Hormone-mediated signaling | AT3G19290 | | ABF4 | ABA-RESPONSIVE ELEMENT-BINDING PROTEIN 2 |
| AT1G69270 | | RPK1 | RECEPTOR LIKE PROTEIN KINASE 1 | |||
| AT1G10210 | | ATMPK1 | MITOGEN-ACTIVATED PROTEIN KINASE | |||
| 9814 | 8.84E-03 | Defense response, incompatible interaction | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| AT3G12250 | | TGA6 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| AT1G22070 | | TGA3 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| 45087 | 1.53E-02 | Innate immune response | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| AT3G12250 | | TGA6 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| AT1G22070 | | TGA3 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| 9627 | 5.08E-03 | Systemic acquired resistance | AT3G12250 | | TGA6 | BZIP FAMILY TRANSCRIPTION FACTOR |
| AT1G22070 | | TGA3 | BZIP FAMILY TRANSCRIPTION FACTOR | |||
| 9738 | 1.05E-02 | Abscisic acid mediated signaling | AT3G19290 | | ABF4 | ABA-RESPONSIVE ELEMENT-BINDING PROTEIN 2 |
| AT1G69270 | | RPK1 | RECEPTOR LIKE PROTEIN KINASE 1 | |||
| 42436 | 1.19E-02 | Indole derivative catabolism | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 6569 | 1.19E-02 | Tryptophan catabolism | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 46218 | 1.19E-02 | Indolalkylamine catabolism | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 42402 | 1.19E-02 | Biogenic amine catabolism | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 42219 | 1.78E-02 | Amino acid derivative catabolism | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 9074 | 1.78E-02 | Aromatic amino acid family catabolism | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 10120 | 1.78E-02 | Camalexin biosynthesis | AT4G39950 | | CYP79B2 | CYTOCHROME P450 79B2, PUTATIVE (CYP79B2) |
| 6006 | 9.11E-04 | Glucose metabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| AT4G37870 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| 6092 | 1.62E-03 | Main pathways of carbohydrate metabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| AT4G37870 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| 19318 | 2.74E-03 | Hexose metabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| AT4G37870 | | PHOSPHOENOLPYRUVATE CARBOXYKINASE (ATP) | |||
| 15980 | 3.52E-03 | Energy derivation by oxidation of organic compounds | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| AT4G37870 | | PHOSPHOENOLPYRUVATE CARBOXYKINASE (ATP) | |||
| 6096 | 2.01E-03 | Glycolysis | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 6007 | 8.18E-03 | Glucose catabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 46365 | 8.51E-03 | Monosaccharide catabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 19320 | 8.51E-03 | Hexose catabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 46164 | 9.19E-03 | Alcohol catabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 16052 | 1.35E-02 | Carbohydrate catabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 44275 | 1.35E-02 | Cellular carbohydrate catabolism | AT5G47810 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN |
| AT1G20950 | | PYROPHOSPHATE—FRUCTOSE-6-PHOSPHOTRANSFERASE | |||
| AT4G26270 | | PHOSPHOFRUCTOKINASE FAMILY PROTEIN | |||
| 45788 | 5.97E-03 | Regulation of cell shape | AT5G23260 | | TT16 | MADS-BOX PROTEIN, TRANSPARENT TESTA16 |
Protein array screening predicted CaM/CML to interact to a group of proteins with recognized roles in processes related to plant response to biotic stresses such as innate immune response, systemic acquired resistance (SAR) and camalexin biosynthesis. Two of the bZIP transcription factors, TGA3 and TGA6 that bind CaMs are critical components of salicylic acid (SA)-mediated signal transduction pathway.6,7 CaM is known to enhance the ability of TGA3 to bind a C/G-box promoter element.8 Another interesting putative target is represented by CYP79B2, a Cytochrome P450 enzyme that functions in the conversion of tryptophan to indole alkaloids (phytoalexins). Phytoalexins such as camalexins are secondary metabolites that accumulate in response to pathogen attack. It has been hypothesized that CaM/Ca2+ regulate the synthesis of phytoalexins through at least one component, the CaM-binding IQD1 transcription factor.9 Another putative CaM-binding protein, MAPKK1, is known to be activated by MEKK1 in response to wounding,10 and participates in defense responses to bacterial elicitor flagellin.11
Heat-shock proteins (Hsp), mediators of cellular responses to elevated temperatures and other environmental factors, are well-known CaM targets. Mammalian CaM binds Hsp proteins.12 CaM also interacts with the cytosolic maize Hsp70 and inhibits its intrinsic ATPase activity.13 In our assays, several Arabidopsis heat-shock proteins interacted with CaM/CMLs. Two members of the Hsp90 family, the Heat shock 70 kDa protein 1 (Hsp70-1) and Hsp81-2 interacted with all CaM isoforms.
A group of seven receptor and receptor-like kinases (RLK) putative targets identified on protein arrays are known to function in cell-surface receptor-linked signal transduction (Table 1). LRR-type RLKs are involved in mediating developmental and defense-related cellular responses via binding extra cellular ligands and activating downstream signaling pathways. Moreover, interactions with CaM, phosphorylation or dephosphorylation are processes shown to activate animal and plant RLKs.14–16
Conclusions
Previous research has discovered a large and varied repertoire of CaM/CMLs substrates in plants, thus has led to understanding CaM/CML regulation of many biological processes. Here we demonstrated that use of protein arrays for dissecting CaM/CML-regulated signaling in Arabidopsis. Known and novel potential CaM/CML substrates were identified in an unbiased and high-throughput manner. The information documented here on novel potential CaM targets provides new testable hypotheses in the area of CaM/Ca2+-regulated processes and represents a resource of functional information for the scientific community.
Acknowledgements
This work is supported by Yale University Genomics and Proteomics Center pilot grant and the NSF Arabidopsis 2010 grant (MCB-0313514 and DBI-0519853).
Footnotes
Previously published online as a Plant Signaling & Behavior E-publication: http://www.landesbioscience.com/journals/psb/article/4416
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