A maize insulin-like growth factor signals to a transduction pathway that regulates protein synthesis in maize

Abstract

Insulin and insulin-like growth factors (IGFs) are well-characterized regulators in higher eukaryotic cells that control biological processes such as cell growth and survival, and selective translation of mRNAs. This research presents the purification of a 20 kDa protein, isolated from maize tissue, with IGF activity. The protein was purified from 48 h-germinated maize embryonic axes by G-50 Sephadex fractionation followed by affinity chromatography through a bovine insulin antibody-Sepharose column. This protein proved to significantly speed up maize germination and seedling growth. At the molecular level, Zea mays IGF (ZmIGF) enhanced phosphorylation of S6 ribosomal protein (rp) on the 40 S ribosomal subunit, in a similar way as observed when bovine insulin is applied to maize axes during germination. Rapamycin, a specific inhibitor of the insulin-stimulated signal transduction pathway, prevented S6 rp phosphorylation in maize axes. Moreover, ZmIGF stimulated [(35)S]methionine incorporation into rps, above the level of overall cytoplasmic proteins. Either incubation with anti-insulin antibody, heat treatment (60 degrees C) or trypsin digestion abolished this ZmIGF effect. It is proposed that ZmIGF is an endogenous maize growth factor that regulates the synthesis of specific proteins through a pathway similar to that of insulin or IGFs in animal tissues.

Coordination of cell growth and cell division in maize (Zea mays L.) relevance of the conserved TOR signal transduction pathway

“…ZmIGF (maize growth factor) was purified from maize axes after 48 h of seed germination by sephadex G-5O and DEAE sepharose column chromatographies. The factor was further purified through affinity chromatography using bovine insulin antibody (SigmaAldrich) as ligand (García-Flores et al 2001). The ZmlGF peptide was identified using dot-blot analysis-tested insulin antibody.…”

“…The tissues were homogenized and soluble proteins extracted and measured by the Bradford method (Bradford 1976). Proliferating cell nuclear antigen (PCNA) or cyclin D (CycD) proteins were immuno-precipitated from the samples using the correspondent Arabidopsis thaliana antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and adsorbed by protein A-Sepharose beads (Zymed Lab, San Francisco, CA; García-Flores et al 2001). The immuno-precipitates were resolved by 12% SDS-polyacrylamide gel electrophoresis (Laemmli 1970) and transferred to a PVDF membrane (BioRad, Hercules, CA) for western blot analysis.…”

“…Two biological systems stimulated either with insulin or ZmIGF were set to measure DNA synthesis. Specific incubation and labeling periods were set for each system to meet the active cell division period (García-Flores et al 2001). System A: Maize embryonic axes, excised from 15-h germinated seeds, were incubated for 15 more h at 25°C in Murashige and Skoog (MS;1962) medium to reach the period of first cell division observed (Baiza and Sánchez-de Jiménez 1989).…”

“…The ZmlGF peptide was identified using dot-blot analysis-tested insulin antibody. ZmIGF units were determined from a dot-blot standard curve made with known amounts of insulin (García-Flores et al 2001). Insulin and rapamycin, the TOR inhibitor, were purchased from Sigma-Aldrich.…”

“…Nonetheless, orthologs of TOR and S6K proteins have been described for Arabidopsis (Menand et al 2002) and maize (Reyes-de la Cruz et al 2004;Agredano-Moreno et al 2007), showing that these kinases are fundamental in plant development. Interestingly, insulin is capable of stimulating PI3K-TOR pathway in maize tissues, and it significantly speeds up maize germination and seedling growth, similar to a maize peptide (ZmIGF), recognized by insulin antibody present in embryonic axes or maize calluses (García-Flores et al 2001). In both cases, these effects were inhibited by rapamycin (Dinkova et al 2007).…”