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Chilling stress can have severe impacts on the growth, development and productivity of maize worldwide. In the present study, cDNA amplified fragment length polymorphism (cDNA-AFLP) analysis was used to evaluate gene expression in maize during chilling treatments (6°C) over four time periods (0, 2, 6 and 12 h). A total of 441 transcript-derived fragments (TDFs) induced by lowtemperature treatment were detected. Based on the sequence analysis, the 58 TDFs of known functions were involved in metabolism, photosynthesis, signal transduction and defence responses etc., suggesting that maize undergoes a complex adaptive process in response to low temperatures. Three full-length cDNA, encoding MAP-KKK (mitogen-activated protein kinase kinase kinase), CLC-D (chloride channel D) and RLK (receptor-like protein kinases) homologues, were isolated from maize through in silico cloning and named as ZmMAPKKK, ZmCLC-D and ZmRLK, respectively. Finally, the expression patterns of the three genes showed a significant increase of differential expression after chilling stress as analysed by semi-quantitative RT-PCR and real-time qRT-PCR. This study provides important clues to understanding low-temperature regulation mechanisms in maize and the three candidate genes involved in chilling responses need further research to determine their usefulness in breeding new resistance cultivars.

A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.