Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

MYB4: A RICE TRANSCRIPTIONAL FACTOR INVOLVED IN COLD TOLERANCE

OSNATO M.*, BRACALE M.**, LOCATELLI F.*, VANNINI C.**, MAGNANI E.*, CORAGGIO I.*

* Istituto Biosintesi Vegetali, CNR, via Bassini 15, 20133, Milano

** DBSF, Università dell’Insubria, via J. H. Dunant 3, 21100 Varese, Italy

cold tolerance, myb factors, rice, arabidopsis, transgenic plants

In our laboratory, several rice myb genes induced by different types of stress have been cloned. In particular Osmyb4 codes for a transcription factor 257 aminoacids long, expressed at low levels in rice coleoptiles grown for three days at 29°C and it is strongly induced by treatments at 4°C of only two hours. We carried out characterisation studies of expression of Osmyb4 in different rice cultivars agronomically known to be tolerant or sensitive evaluating both the time and the temperature of induction. Three days seedling were treated for 2 or 4 hours at 4 °C or for 6 hours at several temperatures (15 °C, 10 °C and 4 °C). In any cases, seedling keeped at 29 °C were used as control. In all the assayed cultivars (Arborio, Loto, S. Andrea e Saturno) the expression of myb4 is already induced after 2 hours of 4 °C treatment, its increase is detectable at 15°C and reaches its maximum at 10°C. Both of these temperatures are sublethal for rice, and genes induced by these conditions are considered important for protection from a more extreme stress.

Furthermore, we demonstrated the trascriptional activity of Myb4 on the bean pal2 (phenylalanine ammonio lyase) promoter in tobacco protoplast transient assays. The pal2 promoter is known to be induced by several stresses and to be trascriptionally activated in vivo by the coordinated action of Myb and bZip transcriptional factors.

We also tested in two transient expression systems (rice calli and tobacco protoplasts) the Myb4 transcriptional activity on the promoters of several cold induced genes: paf93 and pt59 of barley and des9 of potato. On their sequences putative binding sites for Myb factors are present. paf93 and pt59 code for two chloroplastic proteins and their expression is regulated also by drought and light respectively. In our conditions, they were not activated neither by cold, nor by Myb4. D9-desaturase (encoded by des9) catalysing the formation of double bonds in the fatty acids of the membrane lipids, have a key function in cold acclimation. Its promoter was induced in both systems by Myb4 over-expression and/or by cold.

Finally, to evaluate the Osmyb4 function in vivo, its cDNA was constitutively expressed in Arabidopsis thaliana plants (cv. Wassilewskija), under the control of the constitutive CaMV35S promoter. Transformed Arabidopsis thaliana plants, have been assayed for the myb4 expression and investigated several physiological parameters in control and in cold conditions. In particular, we evaluated the photooxidative damnage at 2 °C (measured as Fv/Fm) and the cellular membrane damnage in chilling and freezing conditions (ion leakage).

The Myb4 overexpressing plants show a improuved cold tolerance, both as general phenotipic aspect that as membrane integrity and efficiency of photosynthesis.

Analysis of homozigote plants obtained through self-fertilisation of transformed individuals are in progress.