Identification of the ICE1 (inducer of CBF expression) gene in a Pinus sp.

Rolando Rojas


Freezing temperatures cause the destabilization of plant cell membranes caused by cytoplasmic dehydration, limiting a plantís natural range and causing major crop loss worldwide. Membrane systems are susceptible to severe water loss from within the cell to the intercellular space due to the formation of extracellular ice as temperatures drop below 00C. In temperate regions where temperatures can vary greatly between summer and winter seasons, plants can acquire a tolerance for freezing temperatures with exposure to low nonfreezing temperatures (0-150C). The mechanism, plant cold acclimation, has been studied in a model organism, Arabidopsis thaliana, along with other angiosperm species that are agriculturally significant, like Vitis vinifera and Solanum lycopersicum. The process of cold acclimation is dependent on the Cold Regulated (COR) proteins, induced by the cold signaling pathway, that stabilize membranes against injury resulting from freeze induced cellular dehydration. Understanding the nature of the genes and mechanisms regulating this pathway provides insight as to how temperature can affect the distribution of plant assemblages and the evolution of a plant population, and it can provide better strategies to improve the freezing tolerance of agronomically significant plants [1,2].

Our understanding of the cold signaling pathway which involves the ICE (inducer of CBF/DREB1 expression), CBF/DREB1 (C-repeat binding factor/dehydration responsive element binding) and COR genes continues to grow but much work needs to be done, in diverse species of angiosperms and gymnosperms, to grasp the full array of effects freezing temperatures have on plants. As presently understood, signal transduction begins as the temperature drops below 150C. An unknown sensor triggers cytoplasmic calcium levels to increase which signals the activation of kinases in the cytosol. The inactive ICE1 binding protein, constitutively expressed in the cytosol, gets phosphorylated allowing this transcription factor to bind a cis-element in the promoter of CBF3/DREB1A activating its expression. Induction of the CBF3/DREB1A gene produces a transcription factor that binds to cis-elements in the promoter of the COR genes that encode: chloroplast membrane phospholipids able to withstand freeze induced membrane trauma (COR15a), dehydrins important for membrane stabilization and the prevention of protein aggregation at low temperatures (COR47), and polypeptides that reduce membrane permeability (COR6.6). ICE1 is negatively regulated by HOS1, an E3 ubiquitin-protein ligase, and positively regulated by SIZ1, an E3 SUMO-protein ligase, via ubiquitylation and SUMOylation respectively [1,2,3,4].

My study aims to extend our knowledge of cold acclimation in seed plants by investigating a gene involved in the cold signaling pathway of gymnosperms. Pines are valued for their timber and wood pulp, and grow densely in temperate and tropical regions often outcompeting hardwood species [5]. The goal of this project is to amplify and characterize the ICE1 gene in Pinus nigra (black pine) making use of degenerate primers constructed from 12 angiosperm ICE1 sequences.

Materials and Methods
Degenerate primers were used to amplify a region of the ICE gene in Pinus nigra. Regions with the highest conservation, and suitable primer design requirements were used as locations for degenerate primer design. Pine needles were collected from Pinus nigra and DNA was harvested using a silicon-column protocol. PCR was performed with the degenerate primers on extracted black pine DNA. Electrophoresis was used to separate and visualize the PCR products. The expected size band was purified using a gel extraction protocol and sequenced. BLAST analysis was performed on the sequence.

Degenerate primers successfully amplified a ~500 base PCR product (Fig.1) using a 45 pmole concentration on 1 ng of Pinus nigra DNA. The primer concentration was increased, from 5 pmoles that is commonly used in 20 uL PCR reaction, to account for the decrease in potency of the degenerate primers (effective primer concentration decreases as the number of oligonucleotides that are coded by the degenerate primer increases). The band was excised, purified by gel extraction, sequenced and analyzed with BLAST.

The BLAST analysis on the sequence produced a top match to white spruce (Fig.2), a relative of black pine in the Pinaceae family, along with subsequent close matches to ICE1 sequences (Tab.1), belonging to angiosperms.

The utility of degenerate primers is apparent when attempting to characterize homologous genes in novel organisms. The mixture of oligonucleotides encoded by the a degenerate primer can reduce the specificity of the PCR amplification but it increases the likelihood of hybridization to the novel gene. This study demonstrated that the ICE1 gene is encoded in the genome of Pinus nigra, a cold hardy gymnosperm. This sequence information is a prelude to cold regulated gene expression studies in Pinus and can be used to explore contiguous regions of the black pine ICE1 gene. Logically, research into Pinus nigra HOS1 and SIZ1 genes should be done to understand the mechanism that regulates the ICE1 transcription factor in response to seasonal changes. Determining the nature of the genes and processes responsible for the activation of the cold-acclimation response will help our understanding of the natural world [1].


1.Thomashow MF. PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms. Annu Rev Plant Physiol Plant Mol Biol. 1999;50:571-599.
2.Miura K, Furumoto T. Cold signaling and cold response in plants. Int J Mol Sci. 2013;14(3):5312-37.
3.Chinnusamy V, Zhu J, Zhu JK. Cold stress regulation of gene expression in plants. Trends Plant Sci. 2007;12(10):444-51.
4.Chinnusamy V, Ohta M, Kanrar S, et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 2003;17(8):1043-54.
5.USDA Forest Service Staff. Pinus nigra. US Forest Service.
7.Nucleotide BLAST. National Center for Biotechnological Information.

Low temperatures are an environmental stress that influence plant growth and development, limits a plantís natural range, and causes major crop loss world wide. Plants have evolved a cold acclimation mechanism that enhances their tolerance to freezing temperatures. The accumulation of these defenses against freezing trauma is dependent on the activation of Cold Regulated (COR) gene expression. This study aims to amplify and characterize the ICE1 (inducer of CBF/DREB1 expression) gene in Pinus nigra (black pine) making use of degenerate primers constructed by using ICE1 sequences of 12 angiosperms. A region of the ICE1 gene in black pine was PCR amplified and sequenced. This study presents the first and only sequence information on the ICE1 gene in pine.


Figure 1-2%(v/w) agarose gel visualized with ethidium bromide under a UV lamp. The first lane shows an ~500 base PCR product (top band), amplified from Pinus nigra DNA using degenerate primers, separated by electrophoresis.

Figure 2-Closest BLAST result for the Pinus nigra PCR product that was sequenced. The sequence matched Picea glauca. commonly known as white spruce, a relative of black pine in the Pinaceae family.

Figure 3-List of closest matching BLAST results to amplified sequence.

Full Paper


Thank you to Catharina Grubaugh, Kate Reid, Dr. Berish Rubin, and Anthony Evans for their assistance and guidance during this project.

This document was last modified 05/07/2015.
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