Winter hardiness is an important trait for grapevine breeders and producers, so identification of the regulatory mechanisms involved in cold acclimation is of great potential value. The work presented here involves the identification of two grapevine ICE gene homologs, VaICE1 and VaICE2, from an extremely cold-tolerant accession of Chinese wild-growing Vitis amurnensis, which are phylogenetically related to other plant ICE1 genes. These two structurally different ICE proteins contain previously reported ICE-specific amino acid motifs, the bHLH-ZIP domain and the S-rich motif. Expression analysis revealed that VaICE1 is constitutively expressed but affected by cold stress, unlike VaICE2 that shows not such changed expression as a consequence of cold treatment. Both genes serve as transcription factors, potentiating the transactivation activities in yeasts and the corresponding proteins localized to the nucleus following transient expression in onion epidermal cells. Overexpression of either VaICE1 or VaICE2 in Arabidopsis increase freezing tolerance in nonacclimated plants. Moreover, we show that they result in multiple biochemical changes that were associated with cold acclimation: VaICE1/2-overexpressing plants had evaluated levels of proline, reduced contents of malondialdehyde (MDA) and decreased levels of electrolyte leakage. The expression of downstream cold responsive genes of CBF1, COR15A, and COR47 were significantly induced in Arabidopsis transgenically overexpressing VaICE1 or VaICE2 upon cold stress. VaICE2, but not VaICE1 overexpression induced KIN1 expression under cold-acclimation conditions. Our results suggest that VaICE1 and VaICE2 act as key regulators at an early step in the transcriptional cascade controlling freezing tolerance, and modulate the expression levels of various low-temperature associated genes involved in the C-repeat binding factor (CBF) pathway.

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Chinese Wild-Growing Vitis amurensis ICE1 and ICE2 Encode MYC-Type bHLH Transcription Activators that Regulate Cold Tolerance in Arabidopsis

The microRNA156 (miR156) family ‘has’ been well demonstrated to regulate plant growth and development. However, no reports focused on the roles of miR156s in environmental stress responses. In previous studies, we identified 18 cold stress responsive microRNAs in rice by using microarray analysis. Here, in the present study, we focused on the biological function of one of these cold responsive microRNAs, OsmiR156k. We generated the transgenic rice overexpressing OsmiR156k under the control of CaMV35S promoter, and verified the presence of OsmiR156k by using Southern blot analysis. We found that overexpression of OsmiR156k inhibited the seedling growth at the very early seedling stage under cold stress. Furthermore, OsmiR156k overexpression decreased plant cold tolerance at the young seedling growth stage, as evidenced by lower survival rates, chlorophyll contents and proline contents. As expected, we also suggested the down-regulated expression of the cold stress responsive genes, 01g22249 and OsP5CS, and OsmiR156k-tagated SPL genes, SPL3, SPL14 and SPL17, in the OsmiR156k transgenic lines. Taken together, our findings suggest that overexpression of OsmiR156k decreased the tolerance to cold stress in rice.

Overexpression of OsmiR156k leads to reduced tolerance to cold stress in rice (Oryza Sativa)

A novel Zea mays ssp. mexicana L. MYC-type ICE-like transcription factor gene ZmmICE1, enhances freezing tolerance in transgenic Arabidopsis thaliana

Zoysia japonica MYC type transcription factor ZjICE1 regulates cold tolerance in transgenic Arabidopsis

Cold stress is very detrimental to crop production. However, only a few genes in rice have been identified with known functions related to cold tolerance. To meet this agronomic challenge more effectively, researchers must take global approaches to select useful candidate genes and find the major regulatory factors. We used five Gene expression omnibus series (GSE) data series of Affymetrix array data, produced with cold stress-treated samples from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), and identified 502 cold-inducible genes common to both japonica and indica rice cultivars. From them, we confirmed that the expression of two randomly chosen genes was increased by cold stress in planta. In addition, overexpression of OsWRKY71 enhanced cold tolerance in ‘Dongjin’, the tested japonica cultivar. Comparisons between japonica and indica rice, based on calculations of plant survival rates and chlorophyll fluorescence, confirmed that the japonica rice was more cold-tolerant. Gene Ontology enrichment analysis indicate that the ‘L-phenylalanine catabolic process’, within the Biological Process category, was the most highly overrepresented under cold-stress conditions, implying its significance in that response in rice. MapMan analysis classified ‘Major Metabolic’ processes and ‘Regulatory Gene Modules’ as two other major determinants of the cold-stress response and suggested several key cis-regulatory elements. Based on these results, we proposed a model that includes a pathway for cold stress-responsive signaling. Results from our functional analysis of the main signal transduction and transcription regulation factors identified in that pathway will provide insight into novel regulatory metabolism(s), as well as a foundation by which we can develop crop plants with enhanced cold tolerance.

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Genome-Wide Identification and Analysis of Genes, Conserved between japonica and indica Rice Cultivars, that Respond to Low-Temperature Stress at the Vegetative Growth Stage.

ItICE1, a ICE1-like gene, was isolated from a cDNA library from cold-treated woad (Isatis tinctoria L.) tissues. Expression analysis revealed that the ItICE1 gene was expressed constitutively and was predominant in the leaves of woad seedlings and that its mRNA accumulation was altered by salt stress and abscisic acid application, but not by dehydration and cold stresses. The transgenic rice lines overexpressing ItICE1 showed no growth retardation under normal growth conditions as well as enhanced tolerance to cold stress. Physiological assays showed that ItICE1 not only increased the accumulation of free proline and chlorophyll in transgenic rice lines under cold stress, but also reduced malondialdehyde content and electrolyte leakage. The analysis of gene expression in transgenic rice lines indicated that the maize ubiquitin promoter could respond to cold stress and upregulate ItICE1 gene expression level under its control. Under cold stress conditions, transgenic lines had a remarkably increased expression of OsDREB1A, J013078A14, 001-125-G03, 001-023-B08 and J023042N13 compared to wild-type plants (P < 0.05), implying that ItICE1 functions in the CBF/DREB1 cold-response pathway. These results demonstrate that ItICE1 plays an important regulatory role in the improvement of tolerance to cold stress in rice and is potentially useful for improving the cold tolerance of other plants.

Minghui Zhao

Papers

Overexpression of a ItICE1 gene from Isatis tinctoria enhances cold tolerance in rice