Mutation of the core pentamer, CCGAC, of two putative low temperature responsive elements (LTREs) in the 5'-proximal region for the winter Brassica napus cold-induced gene BN115 was carried out. Analyses of transient expression of the resultant mutated BN115 promoter-GUS fusions revealed the loss of low-temperature regulation by the promoter. This indicates that the CCGAC sequence is critical to the low-temperature response in the BN115 gene. In contrast, mutation of two G-boxes, CACGTG, staggered between the LTREs in the same region of the promoter did not alter cold-inducible gene expression. Replacement of a possible enhancer region of the BN115 promoter with the enhancer from the CaMV 35S promoter resulted in a several-fold increase in low temperature-induced GUS activity.

~A cDNA clone, pBNll5, encoding a low-temperature-regulated transcript in winter Brassica napus has been isolated. Northern blot analyses show that levels of transcripts hybridizing to p B N l l 5 increase within 24 h of exposure of B. napus to low temperature, peak at 3 d, and then remain at an elevated level for the duration of the cold treatment (up t o 10 weeks). Transferring plants from 2°C to room temperature results i n the loss of detectable transcripts hybridizing to p B N l l 5 within 1 d. The transcript was not detected in RNA isolated from roots of cold-acclimated B. napus. Results of in vivo labeling of nascent RNA in leaf discs of B. napus with thiouridine suggest that regulation of expression may be transcriptional, at least at the onset of cold temperature. Although drought stress leads t o a slight increase in transcript level at room temperature, neither a brief exposure to elevated temperatures nor exogenous application of abscisic acid resulted in the appearance of the transcript represented by pBNll5. Furthermore, transcripts hybridizing to p B N l l 5 were present at the same levels whether the plants were acclimated in the light or dark. Hybridization experiments show that p B N l l 5 hybridizes strongly to cold-regu- Low temperature is a major trigger for the acquisition of freezing tolerance in plants capable of cold acclimation (Levitt, 1980). Although it has been shown that biochemical, morphological, and physiological changes occur in plant cells during cold acclimation (Sakai and Larcher, 1987; JohnsonFlanagan and Singh, 1988;Singh and Laroche, 1988;Guy, 1990)' direct evidence was obtained only recently to show that low temperature regulated the accumulation of specific mRNAs during cold acclimation (Thomashow, 1990). The appearance of nove1 transcripts during cold acclimation has been observed in alfalfa (Mohapatra et al., 1989), wheat (Lin et al., 1990; Houde et al., 1991), barley (Cattivelli and Bartels, 1990;Dunn et al., 1990), Arabidopsis (Hajela et al., 1990; ' Publication No. 1414 171 Kurkela and Franck, 1990;Nordin et al., 1991; Gilmour et al., 1992), and Brassica (Orr et al., 1992). Furthermore, DNA sequences corresponding to these cold-specific or cold-regulated transcripts have also been isolated and characterized by differential screening of cDNA libraries constructed from these species. These mRNAs appear rapidly upon exposure of the plant to low temperatures, and deduced amino acid sequences of the products of some of these cold-regulated genes have been determined. A number of cold-induced transcripts have been shown to hybridize to transcripts of rab (responsive to ABA) genes (Hahn and Walbot, 1989) or to encode polypeptides containing amino acid sequence motifs (Guo et al., 1991; Gilmour et al., 1992;Houde et al., 1992) found in rab proteins (Skriver and Mundy, 1990), suggesting that they may play a role in the toleration of the cellular desiccation stress that accompanies extracellular freezing. Other cold-induced transcripts encode polypeptides containi...

Low temperature triggers the acquisition of frost tolerance in plant species that are capable of developing cold hardiness. Recently, the transcription of specific genes was observed to be induced, at least in part, by low temperature in alfalfa (9), Arabidopsis (5, 7, 10), barley (2, 3), and wheat (6, 8) during cold acclimation. In wheat and Arabidopsis, the cold-induced

A cDNA clone, pBN59, was isolated by differential screening of a cDNA library of winter Brassica napus during cold acclimation. Nucleotide sequence of BN59 was found to be homologous to that encoding the 70 kDa subunit of the vacuolar H(+)-ATPase in plants. Transcripts hybridizing to BN59 accumulated during exposure to low temperatures and to the exogenous application of abscisic acid (ABA). Western blot analyses also indicated an increase in the 70 kDa subunit during cold acclimation. The accumulation of an endomembrane H(+)-ATPase is consistent with the observation of osmotic adjustment, increases in endogenous ABA and the proliferation of endomembranes during cold acclimation.

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