Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum)
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Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.
KeywordsArabinogalactan proteins Extensins Prolyl 4-hydroxylase Solanum lycopersicum Tobacco rattle virus Virus induced gene silencing
We would like to thank Prof. Dr. Constantinos Fasseas (Electron Microscopy Laboratory, Agricultural University of Athens, Greece), Prof. Dr. Polydefkis Hatzopoulos and Dr. Christos Michaelides (Department of Agricultural Biotechnology, Agricultural University of Athens, Greece) for their help in Scanning Electron Microscopy analysis, Dr. Panagiotis Kefalas (Mediterranean Agronomic Institute of Chania, Greece) for synthesizing β-Yariv reagent and Prof. Dr. Paul J. Knox (Centre for Plant Sciences, University of Leeds, UK) for providing the extensin and AGP monoclonal antibodies. This work benefited from the networking activities within the European funded COST ACTION FA1106 “Qualityfruit”.
Conflict of interest
The authors declare that they have no conflict of interest.
- Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72: 248–254Google Scholar
- Daras G, Rigas S, Penning B, Milioni D, McCann MC, Carpita NC, Fasseas C, Hatzopoulos P (2009) The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 (AtCesA3) has a dominant-negative effect on cellulose synthesis and plant growth. New Phytol 184(1):114–126. doi: 10.1111/j.1469-8137.2009.02960.x PubMedCrossRefGoogle Scholar
- Dauplais M, Lecoq A, Song J, Cotton J, Jamin N, Gilquin B, Roumestand C, Vita C, de Medeiros CL, Rowan EG, Harvey AL, Menez A (1997) On the convergent evolution of animal toxins Conservation of a diad of functional residues in potassium channel-blocking toxins with unrelated structures. J Biol Chem 272(7):4302–4309PubMedCrossRefGoogle Scholar
- De Cnodder T, Vissenberg K, Van Der Straeten D, Verbelen JP (2005) Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid: a matter of apoplastic reactions. New Phytol 168(3):541–550. doi: 10.1111/j.1469-8137.2005.01540.x PubMedCrossRefGoogle Scholar
- De Tullio MC, Paciolla C, Dalla Veechia F, Rascio N, D’Emerico S, De Gara L, Liso R, Arrigoni O (1999) Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation. Planta 209(4):424–434. doi: 10.1007/s004250050745 PubMedCrossRefGoogle Scholar
- Kivirikko KI, MR, Pihlajaniemi T (1991) Hydroxylation of proline and lysine residues in collagens and other animal and plant proteins. In: Harding JJ, Crabbe MJC (eds) Focus on post-translational modifications of proteins. CRC, Boca Raton, FL, pp 1–51Google Scholar
- Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer EL, Eddy SR, Bateman A, Finn RD (2012) The Pfam protein families database. Nucleic Acids Res 40(Database issue):D290–D301. doi: 10.1093/nar/gkr1065 PubMedCentralPubMedCrossRefGoogle Scholar
- Sambrook JFE, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- The Tomato Genome Consortium (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485(7400):635–641. doi: 10.1038/Nature11119
- Velasquez SM, Ricardi MM, Dorosz JG, Fernandez PV, Nadra AD, Pol-Fachin L, Egelund J, Gille S, Harholt J, Ciancia M, Verli H, Pauly M, Bacic A, Olsen CE, Ulvskov P, Petersen BL, Somerville C, Iusem ND, Estevez JM (2011) O-glycosylated cell wall proteins are essential in root hair growth. Science 332(6036):1401–1403. doi: 10.1126/science.1206657 PubMedCrossRefGoogle Scholar
- Vigani G, Morandini P, Murgia I (2013) Searching iron sensors in plants by exploring the link among 2′-OG-dependent dioxygenases, the iron deficiency response and metabolic adjustments occurring under iron deficiency. Front Plant Sci 4:169Google Scholar