In plants, low temperature and dehydration activate a set of genes containing C-repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plant's response to cold and drought evolved from a common CBF-like transcription factor, first through gene duplication and then through promoter evolution.Many plants increase their tolerance to freezing after exposure to low nonfreezing temperatures-a phenomenon known as cold acclimation (Hughes and Dunn, 1996;Thomashow, 1998). The major component of this acquired freezing tolerance is the tolerance to dehydration stress caused by extracellular ice formation during the freezing process. The presence of ice lowers the water potential extracellularly and causes water to flow out of cells (Pearce, 1999). Thus, a major cause of freezing damage is the freeze-induced dehydration (Steponkus and Webb, 1992;Thomashow, 1998). Because a plant's ability to survive freezeinduced dehydration is related to its adaptation to drought, it is not surprising that plants respond to low temperature and drought very similarly at the molecular level . Many genes, such as RD (responsive to dehydration), ERD (early responsive to dehydration), COR (cold regulated), LTI (low-temperature induced), and KIN (cold inducible), are induced by both low temperature and drought stress (Ingram and Bartels, 1996;Pearce, 1999; Thomashow, 1999; Shinozaki and YamaguchiShinozaki, 2000). The similarity of cold and drought stresses is further demonstrated by experiments showing that mild drought stress can result in increased freezing tolerance in plants (Clavitier and Siminovitch, 1982;Siminovitch and Cloutier, 1983; Guy et al., 1992).Recently, a major transcriptional regulatory system that controls abscisic acid (ABA) independent gene expression in response to low temperature has been identified (Stockinger et al., 1997;Liu et al., 1998). The system is based on the C-repeat (CRT)/ dehydration-responsive element (DRE) cis-acting element and the trans-acting DNA-binding protein CBF/DREB1 (CRT-binding factor or DRE-binding protein). There are three CB...

The nitrogen concentration usually decreases in elevated [CO 2 ] (Wong 1979;Hocking & Meyer 1985;Hocking & Meyer 1991a, 1991bColeman et al. 1993;Pettersson, MacDonald & Stadenburg 1993;Rogers et al. 1993;McKee & Woodward 1994;Jacob, Greitner & Drake 1995;Nie et al. 1995;Poorter et al. 1997), indicating that nitrogen uptake lags behind carbohydrate synthesis and growth in elevated [CO 2 ]. The effect of elevated [CO 2 ] on the nitrate uptake rates per unit root weight is rather variable and apparently depends on the nitrogen concentration supplied (Larigauderie, Reynolds & Strain 1994) and the species. Whereas elevated [CO 2 ] increased the rate of nitrate uptake per unit root weight in loblolly pines (Bassirirad et al. 1996) and Prosopis glandulosa (Bassirirad et al. 1997), it did not alter nitrate uptake in Nardus agrostis (Bassirirad et al. 1997), and it decreased nitrate uptake in a mixed field community (Jackson & Reynolds 1996). Although nitrate uptake might be improved in elevated [CO 2 ] because plants possess more roots and exploit a larger soil volume (see, e.g. Stulen & den Hertog 1993;Pettersson et al. 1993;Jackson & Reynolds 1996), the decreased water flow in elevated [CO 2 ] will tend to decrease the root surface concentrations of nitrate (Van Vuuren et al. 1997).The organic nitrogen concentration decreases in elevated [CO 2 ] (Wong 1979;Curtis, Drake & Whigham 1989;Garbutt, Williams & Bazzaz 1990;Coleman et al. 1991;Hocking & Meyer 1991a, 1991bColeman & Bazzaz 1992;Gries, Kimball & Idso 1993;Pettersson et al. 1993;Körner & Miglietta 1994;Pettersson & MacDonald 1994; FerrarioMery et al. 1997;Poorter et al. 1997), indicating that nitrate assimilation fails to keep pace with growth. There is conflicting evidence with respect to the effect of elevated [CO 2 ] on nitrate reductase (NR) activity. Although elevated [CO 2 ] led to a small increase of NR activity in mustard (Maeskaya et al. 1990) and Vigna radiata (Sharma & Sen Gupta 1990), it produced a two-fold decrease of NR activity in wheat (Hocking & Meyer 1991a), maize (Purvis, Peters & Hageman 1974), and a 15-25% decrease in Nicotiana plumbaginifolia (Ferrario-Mery et al. 1997). It also led to a decrease of nitrite reductase activity in lettuce (Besford & Hand 1989). In Plantago major, elevated [CO 2 ] led to a transient increase in NR activity, that was reversed after a few days (Fonseca, Bowsher & Stulen 1997). These reports of a decrease of NR activity in elevated [CO 2 ] are rather surprising, because exogenous sugars lead to increased expression of Nia (Cheng et al. 1992;Vincentz et al. 1993;Krapp et al. 1993;Krapp & Stitt 1995;Morcuende et al. 1998) and post-translational activation of NR (Kaiser & Huber 1994; Huber, Bachman & Huber 1996) in detached leaves. Recently Geiger et al. (1998) showed that although elevated [CO 2 ] does not markedly increase the maximum NR activity in tobacco the diurnal regulation of NR is modified, allowing higher activity in the later part of the light period and during the night.Even less is kno...

SummaryAntisense expression of a full length cDNA encoding plastid aldolase led to decreased expression of aldolase at the transcript and protein level in several 'antisense' potato transformants. To quantify the inhibition, activity was compared in corresponding leaves down a plant and in plants of different ages. Aldolase activity was decreased by 32-43%, 56-71%, 79-83% and 91-97% in A-70, A-3, A-51 and A-2. Separation on a Q-Sepharose-FF column showed the decrease was due to inhibition of plastid aldolase. The transformants showed a small increase of Rubisco activity, a small decrease of phosphoribulokinase activity, and larger but subproportional decreases of sedoheptulose-1,7-biphosphatase and plastid fructose-1,6-bisphosphatase activity. Ambient photosynthesis was inhibited by 10%, 40%, 66% and 85% in A-70, A-3, A-51 and A-2. The transformants contained increased triose phosphates, and very low ribulose-1,5-bisphosphate and glycerate-3-phosphate. Chlorophyll fluorescence indicated that photosystem II was more reduced and thylakoid energization was increased. Starch synthesis was decreased by 16% and 36% in A-70 and A-3, whereas sucrose synthesis was less strongly inhibited. Plant growth was not significantly altered in A-70, was decreased by 41% in A-3, and was severely inhibited in plants with under 20% of wild-type aldolase activity. Although plastid aldolase catalyses a readily reversible reaction, possesses no known regulatory properties, and would appear irrelevant for the control of metabolism and growth, small changes in its activity have marked consequences for photosynthesis, carbon partitioning and growth.

Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydrationresponsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show that B. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulated Bn115 gene, an ortholog of the Arabidopsis CBF-targeted COR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivum L. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate.Plants vary greatly in their abilities to survive freezing temperatures (Sakai and Larcher, 1987). Whereas plants from tropical regions have essentially no capacity to withstand freezing, herbaceous plants from temperate regions can survive freezing at temperatures ranging from Ϫ5 to Ϫ30°C, depending on the species. It is significant that the maximum freezing tolerance of plants is not constitutive, but is induced in response to low temperatures (below approximately 10°C), a phenomenon known as "cold acclimation" (Hughes and Dunn, 1996;Thomashow, 1999). Nonacclimated wheat (Triticum aestivum L. cv Norstar) plants, for instance, are killed at freezing temperatures of about Ϫ5°C, but after cold acclimation, can survive temperatures down to about Ϫ20°C. Determining what accounts for the differences in freezing tolerance between plant species and the molecular basis of cold acclimation is of basic scientific interest and has the potential to provide new approaches to improve the freezing tolerance of plants, an important agronomic trait.A recent advance in understanding cold acclimation in Arabidopsis was the discovery of the C-repeat/dehydration-responsive element binding factor (CBF) cold-response pathway (see Thomashow, 2001). Arabidopsis encodes a small fami...

SummaryEven though plastid aldolase catalyses a reversible reaction, does not possess properties allowing it to contribute to 'fine' regulation, and would therefore be considered unimportant for the control of metabolism and growth, antisense transformants with a 50-70% decrease in aldolase activity showed an inhibition of photosynthesis and growth. We now show that acclimation of photosynthesis to growth conditions includes and requires changes in plastid aldolase activity. Wild-type potato plants and transformants were grown at low irradiance (70 µmol m -2 sec -1 ), and at high irradiance (390 µmol m -2 sec -1 ) at 400 or 800 p.p.m. carbon dioxide. (i) Ambient photosynthesis was always inhibited by a 30-40% decrease of aldolase activity, the strongest inhibition being observed when plants were growing in high irradiance and elevated carbon dioxide. (ii) The inhibition was due to a low rate of ribulose-1,5-bisphosphate regeneration in low light, exacerbated by an inadequate rate of starch synthesis in high light and elevated carbon dioxide. Decreased expression of aldolase in antisense transformants was also accompanied by a Received 14 August 1998; revised 14 December 1998; accepted 17 December 1998. *For correspondence (fax ϩ49 6221 54 5859; e-mail mstitt@botanik1.bot.uni-heidelberg.de). † Volker Haake and Michael Geiger contributed equally to this paper. Abbreviations: A, photosynthesis; c i , intracellular CO 2 concentration; FBPase, plastid fructose-1,6-bisphosphatase; Fru6P, fructose-6-phosphate; FW, fresh weight; Glc6P, glucose-6-phosphate; g H2O , stomatal conductance for water; NADP-GAPDH, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; PGK, glycerate-3-phosphate kinase (3-phosphoglycerate kinase); PRK, ribulose-5-phosphate kinase (5-phosphoribulokinase); SBPase, sedoheptulose-1,7-bisphosphatase; WUE, water use efficiency; pAld, transcript for plastidic aldolase; pFbp, transcript for plastidic FBPase; RbcS, transcript for the small subunit of Rubisco; Tkt, transcript for transketolase; 3PGA, glycerate-3-phosphate; Ru1,5bisP, ribulose-1,5-bisphosphate.

SummaryTobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon-and nitrogen-rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen-replete wild-type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wildtype plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen de®ciency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.

Antibodies raised against a peptide fragment (residues 60-456) of potato sucrose phosphate synthase (SPS) were used to investigate whether potato plants contain multiple forms of SPS. When a partially purified preparation of SPS from cold-stored potato tubers was separated on 5 % polyacrylamide gel electrophoresis (PAGE), four immunopositive bands were found with estimated molecular weights of 125, 127, 135 and 145 kDa. These bands were also found in rapidly prepared extracts and were termed SPS-la, SPS-lb, SPS-2 and SPS-3, respectively. Direct evidence that SPS-la and SPS-lb represent active SPS was provided by the finding that both are greatly reduced in plants expressing an antisense sequence derived from the potato leaf SPS gene. SPS-2 was not decreased in the antisense plants, indicating that it has a significantly different sequence. Evidence that SPS-2 represents active SPS was obtained by showing that the amount of SPS-la and SPS-lb protein remaining in the leaves and tubers of antisense potato plants was too low to account for the remaining SPS activity. The four immunopositive SPS forms had different tissue distributions. SPS-la was the major form in all tissues except petals, sepals and stamens. SPS-lb and SPS-2 were absent in very young growing tissues hut were present as minor forms in source leaves and sprouting tubers. The SPS-lh level was especially high in petals and sepals, and the SPS-

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