Over a 24-h light-dark cycle, the level of mRNA coding for nitrate reductase (NR; EC 1.6.6.1) in the leaves of nitrate-fed Nicotiana tabacum L. plants increased throughout the night and then decreased until it was undetectable during the day. The amount of NR protein and NR activity were two-fold higher during the day than at night. When plants were transferred to continuous light conditions for 32 h, similar variations in NR gene expression, as judged by the above three parameters, still took place in leaf tissues. On the other hand, when plants were transferred to continuous dark conditions for 32 h, the NR-mRNA level continued to display the rhythmic fluctuations, while the amount of NR protein and NR activity decreased constantly, becoming very low, and showed no rhythmic variations. After 56 h of continuous darkness, the levels of NR mRNA, protein and activity in leaves all became negligible, and light reinduced them rapidly. These results indicate the circadian rhythmicity and light dependence of NR expression.

Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdategrown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed.In higher plants, the regulation of NR' has been studied extensively under various physiological conditions. A number of factors such as light, mineral nutrition, growth regulators and environmental stress have been shown to affect its activity (21). The precise mechanisms by which these factors modulate NR expression are, at present, not well understood. Molecular tools such as NR monoclonal antibodies (8) and a NR cDNA probe (3) have been obtained in our laboratory to study the regulation of the NR gene expression in tobacco.Higher plant NADH-NR (EC 1.6.6.1) is a nuclear geneencoded homodimer, having a subunit mol wt of 105,000 to 115,000 and containing flavin adenine dinucleotide, hemeFe, and molybdenum cofactor as prosthetic groups (4). Molybdenum is directly implicated in the electron transfer for nitrate reduction. Tungsten, a metal classified with Cr, Mo, and U in the Mendeleieff table, can compete with molybdenum for incorporation into the enzyme complex and results in enzyme inactivation (15,17). In this work, we show that ' Abbreviations: NR, nitrate reductase; rbcS, ribulose bisphosphate carboxylase small subunit. the substitution of tungstate for molybdate in the nutrient solution enhances strongly the expression ofthe NR structural gene. MATERIALS AND METHODS Plant Material and Experimental ConditionsTobacco seeds (Nicotiana tabacum cv Xanthi) were germinated and grown on a sand-peat mixture (1/1, v/v) in the greenhouse. A nitrate and ammonium-containing nutrient solution (10) was applied daily. After 1 month, the seedlings were transferred to a controlled culture room for experimentation under the following conditions: 85% RH, 16 h photoperiod (180 ,umol* m-2 s', fluorescent lam...

Heat stress occurring at reproductive stages can result in significant and permanent damage to crop yields. However, previous genetic studies in understanding heat stress response and signaling were performed mostly on seedling and plants at early vegetative stages. Here we identify, using a developmentally defined, gain-of-function genetic screen with approximately 18 000 Arabidopsis thaliana activation-tagged lines, a mutant that maintained productive seed set post-severe heat stress during flowering. Genome walking indicated this phenotype was caused by the insertion of 35S enhancers adjacent to a nuclear localized transcription factor AtMYB68. Subsequent overexpression analysis confirmed that AtMYB68 was responsible for the reproductive heat tolerance of the mutant. Furthermore, these transgenic Arabidopsis plants exhibited enhanced abscisic acid sensitivity at and post-germination, reduced transpirational water loss during a drought treatment, and enhanced seed yield under combined heat and drought stress during flowering. Ectopic expression of AtMYB68 in Brassica napus driven either by 35S or by heat-inducible promoter recapitulated the enhanced reproductive heat stress and drought tolerance phenotypes observed in the transgenic Arabidopsis. The improvement to heat stress is likely due to enhanced pollen viability observed in the transgenic plants. More importantly, the transgenic canola showed significant yield advantages over the non-transgenic controls in multiple locations, multiple season field trials under various drought and heat stress conditions. Together these results suggest that AtMYB68 regulate plant stress tolerance at the most important yield determining stage of plant development, and is an effective target for crop yield protection under current global climate volatility.

terize some physiological and biochemical aspects of these mutants and in particular to determine whether the nitrate reduction mutation (NR-) involves changes in the photosynthetic machinery. MATERIALS AND METHODSPlant Material. Nitrate reductase deficient clones of Nicotiana plumbaginifolia were isolated for protoplast cultures by selecting for chlorate-resistant colonies. Seedlings were regenerated from these colonies and grown in vitro on a medium containing 5 mm diammonium succinate as sole nitrogen source (5). These seedlings died when transferred on a medium containing 10 mM KNO3 as sole nitrogen source. Seedlings incubated for 1 week on this medium were assayed for in vitro nitrate reductase activity and found to express levels of nitrate reductase activity lower than 0.5% of the controls.Various attempts were made to grow these seedlings in the greenhouse, but no growth was observed when seedlings were watered with a nutrient solution containing NH4NO3. This was assumed to result from ammonium poisoning of plants unable to utilize nitrate. Addition oforganic acid to the nutrient solution resulted in the development of bacterial infestation. Conse

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