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.

SummaryPrevious studies on cold-triggered events leading to Ca 2+ in¯ux during cold acclimatization have been conducted on either unicellular cyanobacterium Synechocystis or plant cell suspensions, and used transcript levels of cold-induced genes as end-point markers. Whether the results of these studies are valid for intact plants or their organs is not known. Here we examine cold signaling in transgenic Brassica napus seedlings carrying, in addition to the endogenous cold-inducible BN115 gene, the b-glucuronidase (GUS) gene placed under control of the BN115 promoter. The activity of BN115 promoter was monitored at the transcriptional and translational levels by determining accumulation of BN115 transcripts and by histochemical assay of GUS activity. Cold-activation of BN115 was strongly inhibited by the membrane¯uidizer benzyl alcohol, but mimicked at 25°C by the membrane rigidi®er dimethylsulfoxide (DMSO). The cold induction of BN115 was also inhibited by stabilizers of microtubules and actin micro®laments, taxol and jasplakinolide, respectively, but was mimicked at 25°C by microtubule destabilizer oryzalin or colchicine, or by micro®lament destabilizer latrunculin B. Gd 3+ or ruthenium red prevented the cold activation of BN115, but Ca 2+ ionophore A23187 or cyclic ADP-ribose activated it at 25°C. Inhibitors of tyrosine kinases, protein kinase C and phosphoinositide kinases prevented the cold activation of BN115, but inhibitors of protein phosphatases (PP) 1 and 2 A activated BN115 at 25°C. Constitutively expressed GUS activity in another transgenic line of the same cultivar of B. napus, was not affected by cold or any of the chemical treatments used in the experimentation. Activation of BN115 at 25°C by DMSO, Ca 2+ ionophore, cADPR, and by inhibitors of PP1 and 2A was accompanied by an increased freezing tolerance. It was concluded that the cold-activation of BN115 requires membrane rigidi®cation, cytoskeleton reorganization, Ca 2+ in¯ux and action of several types of protein kinases.

The effects of overexpression of two Brassica CBF/DREB1-like transcription factors (BNCBF5 and 17) in Brassica napus cv. Westar were studied. In addition to developing constitutive freezing tolerance and constitutively accumulating COR gene mRNAs, BNCBF5- and 17-overexpressing plants also accumulate moderate transcript levels of genes involved in photosynthesis and chloroplast development as identified by microarray and Northern analyses. These include GLK1- and GLK2-like transcription factors involved in chloroplast photosynthetic development, chloroplast stroma cyclophilin ROC4 (AtCYP20-3), beta-amylase and triose-P/Pi translocator. In parallel with these changes, increases in photosynthetic efficiency and capacity, pigment pool sizes, increased capacities of the Calvin cycle enzymes, and enzymes of starch and sucrose biosynthesis, as well as glycolysis and oxaloacetate/malate exchange are seen, suggesting that BNCBF overexpression has partially mimicked cold-induced photosynthetic acclimation constitutively. Taken together, these results suggest that BNCBF/DREB1 overexpression in Brassica not only resulted in increased constitutive freezing tolerance but also partially regulated chloroplast development to increase photochemical efficiency and photosynthetic capacity.

Cold acclimation of winter cereals and other winter hardy species is a prerequisite to increase subsequent freezing tolerance. Low temperatures upregulate the expression of C-repeat/dehydration-responsive element binding transcription factors (CBF/DREB1) which in turn induce the expression of COLD-REGULATED (COR) genes. We summarize evidence which indicates that the integration of these interactions is responsible for the dwarf phenotype and enhanced photosynthetic performance associated with cold-acclimated and CBF-overexpressing plants. Plants overexpressing CBFs but grown at warm temperatures mimic the cold-tolerant, dwarf, compact phenotype; increased photosynthetic performance; and biomass accumulation typically associated with cold-acclimated plants. In this review, we propose a model whereby the cold acclimation signal is perceived by plants through an integration of low temperature and changes in light intensity, as well as changes in light quality. Such integration leads to the activation of the CBF-regulon and subsequent upregulation of COR gene and GA 2-oxidase (GA2ox) expression which results in a dwarf phenotype coupled with increased freezing tolerance and enhanced photosynthetic performance. We conclude that, due to their photoautotrophic nature, plants do not rely on a single low temperature sensor, but integrate changes in light intensity, light quality, and membrane viscosity in order to establish the cold-acclimated state. CBFs appear to act as master regulators of these interconnecting sensing/signaling pathways.

~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...

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Arabidopsis thaliana GOLDEN2-LIKE (GLK1 and 2) transcription factors regulate chloroplast development in a redundant manner. Overexpression of AtGLK1 (35S:AtGLK1) in Arabidopsis also confers resistance to the cereal pathogen Fusarium graminearum. To further elucidate the role of GLK transcription factors in plant defence, the Arabidopsis glk1 glk2 double-mutant and 35S:AtGLK1 plants were challenged with the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2. Compared with Col-0, glk1 glk2 plants were highly resistant to Hpa Noco2, whereas 35S:AtGLK1 plants showed enhanced susceptibility to this pathogen. Genetic studies suggested that AtGLK-mediated plant defence to Hpa Noco2 was partially dependent on salicylic acid (SA) accumulation, but independent of the SA signalling protein NONEXPRESSOR OF PATHOGENESIS-RELATED 1 (NPR1). Pretreatment with jasmonic acid (JA) dramatically reversed Hpa Noco2 resistance in the glk1 glk2 double mutant, but only marginally affected the 35S:AtGLK1 plants. In addition, overexpression of AtGLK1 in the JA signalling mutant coi1-16 did not increase susceptibility to Hpa Noco2. Together, our GLK gain-of-function and loss-of-function experiments suggest that GLK acts upstream of JA signalling in disease susceptibility to Hpa Noco2. In contrast, glk1 glk2 plants were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, whereas 35S:AtGLK1 plants exhibited heightened resistance which could be maintained in the absence of JA signalling. Together, the data reveal that AtGLK1 is involved in JA-dependent susceptibility to the biotrophic pathogen Hpa Noco2 and in JA-independent resistance to the necrotrophic pathogen B. cinerea.