Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots

Abstract

Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resolution. However, the methodology for this type of research has not been well-developed. This paper reports developing a workflow of laser capture microdissection (LCM) followed by gel-liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)-based proteomics analysis for the identification of proteomes contained in individual cell layers of tomato roots. Thin-sections (~10-μm thick, 10 sections per root tip) were prepared for root tips of tomato germinating seedlings. Epidermal and cortical cells (5000–7000 cells per tissue type) were isolated under a LCM microscope. Proteins were isolated and then separated by SDS–polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion. The MS and MS/MS spectra generated using nanoLC-MS/MS analysis of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample. Based on the biological functions, proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells. Several of these proteins were found in Al-treated roots only. The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots. Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers, in both vertical and cross directions when cells are isolated from slides prepared using intact root tips, are the major challenges using the technology in proteomics analysis of plant roots.

References

[1] Ron M, Dorrity MW, de Lucas M, Toal T, Hernandez RI, Little SA et al. Identification of novel loci regulating interspecific variation in root morphology and cellular development in tomato. Plant Physiol 2013; 162: 755-768.

[2] Glover BJ . Differentiation in plant epidermal cells. J Exp Bot 2000; 51: 497-505.

[3] Blancaflor EB, Jones DL, Gilroy S . Alterations in the cytoskeleton accompany aluminum-induced growth inhibition and morphological changes in primary roots of maize. Plant Physiol 1998; 118: 159-172.

[4] Wu D, Shen H, Yokawa K, Baluška F . Alleviation of aluminium-induced cell rigidity by overexpression of OsPIN2 in rice roots. J Exp Bot 2014; 65: 5305-5315.

[5] Bizet F, Hummel I, Bogeat-Triboulot MB . Length and activity of the root apical meristem revealed in vivo by infrared imaging. J Exp Bot 2015; 66: 1387-1395.

[6] Opitz N, Marcon C, Paschold A, Malik WA, Lithio A, Brandt R et al. Extensive tissue-specific transcriptomic plasticity in maize primary roots upon water deficit. J Exp Bot 2015; 67: 1095-1107.

[7] Libault M, Chen S . Editorial: plant single cell type systems biology. Front Plant Sci 2016; 7: 35.

[8] Petricka JJ, Winter CM, Benfey PN . Control of Arabidopsis root development. Annu Rev Plant Biol 2012; 63: 563-590.

[9] Schmid MW, Schmidt A, Grossniklaus U . The female gametophyte: an emerging model for cell type-specific systems biology in plant development. Front Plant Sci 2015; 6: 907.

[10] Zhao X, Yang N, Wang T . Comparative proteomic analysis of generative and sperm cells reveal molecular characteristics associated with sperm development and function specialization. J Proteome Res 2013; 12: 5058-5071.

[11] Svozil J, Gruissem W, Baerenfaller K . Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues. Front Plant Sci 2015; 6: 376.

[12] Kerk NM, Ceserani T, Tausta SL, Sussex IM, Nelson TM . Laser captures microdissection of cells from plant tissues. Plant Physiol 2003; 132: 27-35.

[13] Petricka JJ, Schauer MA, Megraw M, Breakfield NW, Thompson JW, Georgiev S et al. The protein expression landscape of the Arabidopsis root. Proc Natl Acad Sci USA 2012; 109: 6811-6818.

[14] Matas AJ, Yeats TH, Buda GJ, Zheng Y, Chatterjee S, Tohge T et al. Tissue- and cell-type specific transcriptome profiling of expanding tomato fruit provides insights into metabolic and regulatory specialization and cuticle formation. Plant Cell 2011; 23: 3893-3910.

[15] Dembinsky D, Woll K, Saleem M, Liu Y, Fu Y, Borsuk LA et al. Transcriptomic and proteomic analyses of pericycle cells of the maize primary root. Plant Physiol 2007; 145: 575-588.

[16] Magnavaca R, Gardner CO, Clark RB . Evaluation of inbred maize lines for aluminum tolerance in nutrient solution. In: Gabelman HW, Louhman BC (eds). Genetic Aspects of Plant Mineral Nutrition. Martinus Nijhoff: Dordrecht, The Netherlands, 1987, pp 255-265.

[17] Shaff JE, Schultz BA, Craft EJ, Clark RT, Kochian LV . GEOCHEM-EZ: a chemical speciation program with greater power and flexibility. Plant Soil 2010; 330: 207-214.

[18] Okekeogbu I, Ye Z, Sangireddy SR, Li H, Bhatti S, Hui D et al. Effect of aluminum treatment on proteomes of radicles of seeds derived from Al treated tomato plants. Proteomes 2014; 2: 169-190.

[19] Zhou S, Palmer M, Zhou J, Bhatti S, Howe KJ, Fish T et al. Differential root proteome expression in tomato genotypes with contrasting drought tolerance exposed to dehydration. J Amer Soc Hort Sci 2013; 138: 131-141.

[20] Zhou S, Sauvé R, Thannhauser TW . Proteome changes induced by aluminum stress in tomato roots. J Exp Bot 2009; 60: 1849-1857.

[21] Fernandez-Pozo N, Menda N, Edwards JD, Saha S, Tecle IY, Strickler SR et al. The Sol Genomics Network (SGN)-from genotype to phenotype to breeding. Nucl Acids Res 2014; 43: D1036-D1041.

[22] Yang Y, Qiang X, Owsiany K, Zhang S, Thannhauser TW, Li L . Evaluation of different multidimensional LC-MS/MS pipelines for isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of potato tubers in response to cold storage. J Proteome Res 2011; 10: 4647-4660.

[23] Thimm O, Bläsing O, Gibon Y, Nagel A, Meyer S, Krüger P et al. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J 2004; 37: 914-939.

[24] Usadel B, Nagel A, Thimm O, Redestig H, Blaesing OE, Palacios-Rojas N et al. Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of coresponding genes, and comparison with known responses. Plant Physiol 2005; 138: 1195-1204.

[25] Kwasniewski M, Nowakowska U, Szumera J, Chwialkowska K, Szarejko I . iRootHair: a comprehensive root hair genomics database. Plant Physiol 2013; 161: 28-35.

[26] Feist P, Hummon AB . Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int J Mol Sci 2015; 16: 3537-3563.

[27] Von Hagen J . Proteomics Sample Preparation. John Wiley & Sons Inc: Hoboken, NJ, USA, 2011.

[28] Wu X, Xiong E, Wang W, Scali M, Cresti M . Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis. Nat Protoc 2014; 9: 362-374.

[29] Dzieciatkowska M, Hill R, Hansen KC . GeLC-MS/MS analysis of complex protein mixtures. Methods Mol Biol 2014; 1156: 53-66.

[30] Sherman MY, Qian QB . Less is more: improving proteostasis by translation slow down. Trends Biochem Sci 2013; 38: 585-591.

[31] Nelissen H, Clarke JH, De Block M, De Block S, Vanderhaeghen R, Zielinski RE et al. DRL1, a homolog of the yeast TOT4/KTI12 protein, has a function in meristem activity and organ growth in plants. Plant Cell 2003; 15: 639-654.

[32] Schneider K, Mathur J, Boudonck K, Wells B, Dolan L, Roberts K . The ROOT HAIRLESS 1 gene encodes a nuclear protein required for root hair initiation in Arabidopsis. Genes Dev 1998; 12: 2013-2021.

[33] Wang H, Lockwood SK, Hoeltzel MF, Schiefelbein JW . The ROOT HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis. Genes Dev 1997; 11: 799-811.

[34] Yuen CY, Sedbrook JC, Perrin RM, Carroll KL, Masson PH . Loss-of-function mutations of ROOT HAIR DEFECTIVE3 suppress root waving, skewing, and epidermal cell file rotation in Arabidopsis. Plant Physiol 2005; 138: 701-714.

[35] Marzec M, Szarejko I, Melzer M . Arabinogalactan proteins are involved in root hair development in barley. J Exp Bot 2015; 66: 1245-1257.

[36] Song XF, Yang CY, Liu J, Yang WC . RPA, a class II ARFGAP protein, activates ARF1 and U5 and plays a role in root hair development in Arabidopsis. Plant Physiol 2006; 141: 966-976.

[37] Tominaga M, Yokota E, Vidali L, Sonobe S, Hepler K, Shimmen T . The role of plant villin in the organization of the actin cytoskeleton, cytoplasmic streaming and the architecture of the transvacuolar strand in root hair cells of Hydrocharis. Planta 2000; 210: 836-843.

[38] Baluška F, Salaj J, Mathur J, Braun M, Jasper F, Šamaj J et al. Root hair formation: F-actin-dependent tip growth is initiated by local assembly of profilin-supported F-actin meshworks accumulated within expansin-enriched bulges. Dev Biol 2000; 227: 618-632.

[39] Richards KD, Snowden KC, Gardner RC . Wali6 and wali7. Genes induced by aluminum in wheat (Triticum aestivum L.) roots. Plant Physiol 1994; 105: 1455-1456.

[40] Olea F, Pérez-García A, Cantón FR, Rivera ME, Cañas R, Avila C et al. Up-regulation and localization of asparagine synthetase in tomato leaves infected by the bacterial pathogen Pseudomonas syringae. Plant Cell Physiol 2004; 45: 770-780.