We further investigated the role of the Arabidopsis CBF regulatory genes in cold acclimation, the process whereby certain plants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced in response to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/ dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 or CBF3 (also known as DREB1b and DREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expression of target COR (cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate that overexpression of CBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we show that it results in multiple biochemical changes associated with cold acclimation: CBF3-expressing plants had elevated levels of proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 also had elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increased expression of the key Pro biosynthetic enzyme ⌬ 1 -pyrroline-5-carboxylate synthase. These results lead us to propose that CBF3 integrates the activation of multiple components of the cold acclimation response.

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens 1.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like SUC, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCI), intermediate (100 mM NaCI), and high (300 mM NaCI) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO, assimilation response to changing internal CO, partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased SUC and starch pools in leaf tissues, e.g. the ratio of mannitol to SUC increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by "C labeling) from SUC into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (MCPR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in MCPR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.The polyols were the first class of compounds to be termed compatible solutes (Brown and Simpson, 1972), and many of these compounds (acyclic polyols, e.g. sorbitol, mannitol, and glycerol, and substituted cyclic polyols, e.g. pinitol) and several related derivatives (e.g. glycerol glucoside) play roles in stress protection in almost all classes of living organisms,

Kinetically improved diacylglycerol acyltransferase (DGAT) variants were created to favorably alter carbon partitioning in soybean (Glycine max) seeds. Initially, variants of a type 1 DGAT from a high-oil, high-oleic acid plant seed, Corylus americana, were screened for high oil content in Saccharomyces cerevisiae. Nearly all DGAT variants examined from high-oil strains had increased affinity for oleoyl-CoA, with S 0.5 values decreased as much as 4.7-fold compared with the wild-type value of 0.94 mM. Improved soybean DGAT variants were then designed to include amino acid substitutions observed in promising C. americana DGAT variants. The expression of soybean and C. americana DGAT variants in soybean somatic embryos resulted in oil contents as high as 10% and 12%, respectively, compared with only 5% and 7.6% oil achieved by overexpressing the corresponding wildtype DGATs. The affinity for oleoyl-CoA correlated strongly with oil content. The soybean DGAT variant that gave the greatest oil increase contained 14 amino acid substitutions out of a total of 504 (97% sequence identity with native). Seed-preferred expression of this soybean DGAT1 variant increased oil content of soybean seeds by an average of 3% (16% relative increase) in highly replicated, single-location field trials. The DGAT transgenes significantly reduced the soluble carbohydrate content of mature seeds and increased the seed protein content of some events. This study demonstrated that engineering of the native DGAT enzyme is an effective strategy to improve the oil content and value of soybeans.

Olive (Olea europaea L. cv. Frantoio) plants grown hydroponically in a glasshouse were supplied with half‐strength Hoagland solutions containing 0, 50, 100, and 200 mM NaCl for 4 weeks and subsequently supplied with the standard solution without NaCl to relieve salinity stress. Two complete stress‐relief cycles were repeated on the same plant material during one growing season. Growth was inhibited at all salt levels, but most growth parameters of plants treated with 50 or 100 mM NaCl returned to control levels after 4 weeks of relief. More severely stressed plants (200 mM NaCl) recovered to only 60% of the growth of the controls after 4 weeks. During relief, plants treated with 50 and 100 mM NaCl had net photosynthetic rates and stomatal conductances higher than the controls. Increasing the NaCl concentration of the external solution from 0 to 200 mM decreased both leaf pre‐dawn water potential (from ‐0.3 to ‐1.0 MPa) and osmotic potential (from ‐2.1 to ‐2.7 MPa). The sodium concentration in the leaves of plants treated with 200 mM NaCl reached maximum levels of 211 and 388 mM (expressed on a tissue water basis) at the end of the first salinity and relief periods, respectively. Leaf chloride concentrations were 359 and 223 mM at the same sampling dates. These data indicate that the inhibitory effects of salinization on growth and gas exchange of the salt‐tolerant olive cv. Frantoio can be readily reversed when salinity is relieved, despite the marked accumulation of potentially toxic ions (Na+. Cl) in the leaf.

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase 14C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent Km (M6P) was 15.8 millimolar, but the apparent Km (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg2+, Ca2+, Zn2+, and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPHdependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.Sugar alcohols (acyclic polyols or alditols) are obtained when the aldo or keto group of a sugar is reduced to a hydroxyl. Mannitol, the most frequently occurring sugar alcohol in plants, is particularly abundant in algae and has been detected in at least 70 higher plant families. It is a major carbohydrate in many members of some dicot families, e.g. the Scrophulariaceae, Oleaceae, Rubiaceae, and Apiaceae (2). Until recently, however, little information has been available on mannitol's role in higher plants (16,17 that it is an early photosynthetic product (27, 29) and present in phloem tissue or phloem exudates of celery (family Apiaceae) (9) and species in many other families, e.g. the Oleaceae (30). Other physiological roles have been proposed, including osmoregulation, storage and recycling of reducing power, and service as a compatible solute (16,17), but very little is known of mannitol metabolism in higher plants. A M6PR2 has been reported as being located in the cytosol of mesophyll protoplasts from celery (27). Preliminary labeling data derived from celery and privet were responsible for the initial assays for reductase activity with M6P and mannitol 1-P as substrates.Here we demonstrate the formation of M6P and mannitol 1-P as early photosynthetic products in celery and privet (family Oleaceae). We also report evidence for the role and importance ofM6PR and its characteristics in mannitol biosynthesis in celery. MATERIALS AND METHODS Plant Mate...