Development of a New Cold-Tolerant Maize (<i>Zea mays</i> L.) Germplasm Using the <i>ICE1</i> Gene from <i>Arabidopsis thaliana</i>

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION (ICE1) expression in response to cold stress, RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar. Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations. From the regenerated plantlets, three T1 lines were screened and identified by PCR. A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize (Zea mays L.) genomes of the three T1 generations. Under low temperature-stress conditions (4°C), the relative conductivity levels decreased by 27.51%–31.44%, the proline concentrations increased by 12.50%–17.50%, the malondialdehyde concentrations decreased by 16.78%–18.37%, and the peroxidase activities increased by 19.60%–22.89% in the T1 lines compared with those of the control. A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots, stems, and leaves of the T1 lines. ICE1 positively regulates the expression of the CBF genes in response to cold stress. Thus, this study showed the successful transformation of maize with the ICE1 gene, resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.     

OST1‐mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses

Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3‐like), β‐subunits of a nascent polypeptide‐associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo, and facilitates their interaction with C‐repeat‐binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1‐BTF3‐CBF module regulates plant response to cold stress.     

Mapping of barley homologs to genes that regulate low temperature tolerance in Arabidopsis

We investigated the allelic nature and map locations of Hordeum vulgare (barley) homologs to three classes of Arabidopsis low temperature (LT) regulatory genes—CBFs, ICE1, and ZAT12—to determine if there were any candidates for winterhardiness-related quantitative trait loci (QTL). We phenotyped the Dicktoo × Morex (D×M) mapping population under controlled freezing conditions and in addition to the previously reported 5H-L Fr-H1 QTL, observed three additional LT tolerance QTLs on 1H-L, 4H-S, and 4H-L. We identified and assigned either linkage map or chromosome locations to 1 ICE1 homolog, 2 ZAT12 homologs, and 17 of 20 CBF homologs. Twelve of the CBF genes were located on 5H-L and the 11 with assigned linkage map positions formed 2 tandem clusters on 5H-L. A subset of these CBF genes was confirmed to be physically linked, validating the map position clustering. The tandem CBF clusters are not candidates for the D×M LT tolerance Fr-H1 QTL, as they are ~30 cM distal to the QTL peak. No LT tolerance QTL was detected in conjunction with the CBF gene clusters in Dicktoo × Morex. However, comparative mapping using common markers and BIN positions established the CBF clusters are coincident with reported Triticeae LT tolerance and COR gene accumulation QTLs and suggest one or more of the CBF genes may be candidates for Fr-H2 in some germplasm combinations. These results suggest members of the CBF gene family may function as components of winterhardiness in the Triticeae and underscore both the importance of extending results from model systems to economically important crop species and in viewing QTL mapping results in the context of multiple germplasm combinations. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Climate‐dependent variation in cold tolerance of weedy rice and rice mediated by OsICE1 promoter methylation

The mechanisms by which weedy rice (Oryza sativa f. spontanea) has adapted to endure low‐temperature stress in northern latitudes remain unresolved. In this study, we assessed cold tolerance of 100 rice varieties and 100 co‐occurring weedy rice populations, which were sampled across a broad range of climates in China. A parallel pattern of latitude‐dependent variation in cold tolerance was detected in cultivated rice and weedy rice. At the molecular level, differential cold tolerance was strongly correlated with relative expression levels of CBF cold response pathway genes and with methylation levels in the promoter region of OsICE1, a regulator of this pathway. Among all methylated cytosine sites of the OsICE1 promoter, levels of CHG and CHH methylation were found to be significantly correlated with cold tolerance among accessions. Furthermore, within many of the collection locales, weedy rice shared identical or near‐identical OsICE1 methylation patterns with co‐occurring cultivated rice. These findings provide new insights on the possible roles that methylation variation in the OsICE1 promoter may play in cold tolerance, and they suggest that weedy rice can rapidly acquire cold tolerance via methylation patterns that are shared with co‐occurring rice cultivars.     

INDUCER OF CBF EXPRESSION 1 integrates cold signals into FLOWERING LOCUS C‐mediated flowering pathways in Arabidopsis

Plants constantly monitor changes in photoperiod and temperature throughout the year to synchronize flowering with optimal environmental conditions. In the temperate zones, both photoperiod and temperature fluctuate in a somewhat predictable manner through the seasons, although a transient shift to low temperature is also encountered during changing seasons, such as early spring. Although low temperatures are known to delay flowering by inducing the floral repressor FLOWERING LOCUS C (FLC), it is not fully understood how temperature signals are coordinated with photoperiodic signals in the timing of seasonal flowering. Here, we show that the cold signaling activator INDUCER OF CBF EXPRESSION 1 (ICE1), FLC and the floral promoter SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) constitute an elaborate signaling network that integrates cold signals into flowering pathways. The cold‐activated ICE1 directly induces the gene encoding FLC, which represses SOC1 expression, resulting in delayed flowering. In contrast, under floral promotive conditions, SOC1 inhibits the binding of ICE1 to the promoters of the FLC gene, inducing flowering with a reduction of freezing tolerance. These observations indicate that the ICE1‐FLC‐SOC1 signaling network contributes to the fine‐tuning of flowering during changing seasons.