SummaryThe expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 298C, is strongly induced by treatments at 48C. At sublethal temperatures of 10 and 158C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a coldinducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a signi®cant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.

Constitutive expression of the rice cold-inducible Osmyb4 gene in transgenic Arabidopsis (Arabidopsis thaliana) plants improves adaptive responses to cold and drought stress, most likely due to the constitutive activation of several stress-inducible pathways and to the accumulation of several compatible solutes (e.g., glucose, fructose, sucrose, proline, glycine betaine and some aromatic compounds). Although the Osmyb4 gene seems able to activate stress responsive pathways in different species, we previously reported that its specific effect on stress tolerance depends on the transformed species. In the present work, we report the effects of the Osmyb4 expression for improving the stress response in apple (Malus pumila Mill.) plants. Namely, we found that the ectopic expression of the Myb4 transcription factor improved physiological and biochemical adaptation to cold and drought stress and modified metabolite accumulation. Based on these results it may be of interest to use Osmyb4 as a tool for improving the productivity of woody perennials under environmental stress conditions.

ABSTRACT31P nuclear magnetic resonance spectroscopy was used to measure intracellular pH in living tissues. Oxygen deprivation caused fast cytoplasmic acidification from pH 7.4 to 7.0 in shoots of rice, Oryza sativa L. var arborio, a species highly resistant to anoxia. Acidification was complete after 10 minutes of anoxia. Alkalinization of both cytosplasm and vacuole followed thereafter. In the anoxia intolerant wheat shoots, Triticum aestivum L. var MEK, the same treatment caused a sharper cytoplasmic acidification, from pH 7.4 to 6.6, which occurred during a period of 2 hours. Cytoplasmic acidification continued with progress of anoxia and there was no vacuolar alkalinization comparable to the one observed in rice. In wheat oxygen, withdrawal also caused the reduction of both glucose-6-phosphate level and of metabolic rate. It also induced heavy losses of inorganic phosphate from tissues. Conversely, in rice, glucose-6-phosphate level and metabolic rate were increased and inorganic phosphate leakage from tissues was completely absent. These results are discussed in relation to the mechanisms of plant resistance to anoxia.According to studies on maize root tips, resistance to anoxia resides in the ability of some plants in exploiting the initial, lactate-based cytoplasmic acidification, to stop further lactate production and to activate at the same time alcoholic fermentation (22). The the synthesis of new isozymes and lasted for days, suggesting a long-term function for LDH in plants subjected to ROA. In the highly anoxia-tolerant rice shoots, alcoholic fermentation started in the absence of (21) or with limited lactate production (14). Instead, a prolonged though low succinate production was found (16).The response of plants to ROA has been shown to be accompanied by a revolution in the expression of genetic information. In particular, it results in the increase in several glycolytic and fermentative enzyme activities (24). Apparently, the success of resisting to anoxia is mediated by an increase in carbon flux through glycolytic and fermentative chains. Such an increase may be enhanced by acclimatization of plants through pretreatment in hypoxia (10).Anoxia caused acidification of cell sap in shoot tissues of sensitive plants like wheat and barley. In contrast, cell sap alkalinization has been observed in resistant species like rice and Echinochloa crus-galli (14). Metabolic proton consumption has been indicated as an important device used by plants to counteract or prevent cell sap acidosis (14). In the present study we evaluated whether and to what extent cytoplasmic acidification was still induced by ROA in a resistant plant like rice in spite of the previously reported alkalinization of cell sap in this species (14,15 RESPONSE TO ANOXIA IN RICE AND WHEAT SEEDLINGSand circulation of incubation media (6). The central glass tube of the airlift system for the upward flow of gas and medium had a diameter of 5 mm. Gases were delivered inside this tube, just above the signal detecting zone at the rate of 5 ...

In this paper, we report the metabolic and molecular changes in response to cold and drought induced in Osmyb4 transgenic Arabidopsis thaliana compared with the wildtype (WT). The rice Osmyb4 gene codes for a transcription factor (Myb4) induced by cold treatment and, in Arabidopsis transgenic plants, improves cold and freezing tolerance [Vannini C, Locatelli F, Bracale M, Magnani E, Marsoni M, Osnato M, Mattana M, Baldoni E, Coraggio I (2004) Plant J 37: 115-127].Here, we report the ability of Myb4 to induce also drought tolerance in Arabidopsis transgenic plants. By the use of nuclear magnetic resonance (NMR) and enzymatic assays, we showed that several compatible solutes (glucose, fructose, sucrose, proline, glycine betaine and sinapoyl malate) accumulate in higher amount in Osmyb4-overexpressing plants with respect to the WT, both under normal and stress conditions. Considering proline, we also found that in transgenic plants the levels of the mRNAs coding for D 1 -pyrroline-5-carboxylate synthase (EC not assigned) and for D 1 -pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) were higher and lower, respectively. The constitutive activation of several stress-inducible pathways and different kinetics in the accumulation of several metabolites, in Myb4 transgenic plants, may represent an advantage to prepare plants to face the stress condition. Moreover, these results taken together suggest that Myb4 integrates the activation of multiple components of stress response.Abbreviations -LC-NMR, liquid chromatography-NMR; NMR, nuclear magnetic resonance; PAL, phenylalanine ammonia lyase; P5CDH, D 1 -pyrroline-5-carboxylate dehydrogenase; P5CS, D 1 -pyrroline-5-carboxylate synthase.

Accumulation of amino acids was studied in rice roots of 3-day-old seedlings subjected for 48h to anaerobic conditions. Alanine and Gaba were the main amino acids accumulated under anoxia. Their synthesis was strongly inhibited by MSX and AZA, inhibitors of glutamine synthetase and glutamate synthase. These activities increased after 8h of anaerobic treatment and, by immunoprecipitation of 35S-labeled proteins, it was shown that glutamine synthetase and ferredoxin-dependent glutamate synthase were synthesized during the treatment. These findings indicate that the glutamine synthetase/glutamate synthase cycle play an important role in anaerobic amino acid accumulation.