Comparison of long-term up-regulated genes during induction of freezing tolerance by cold and ABA in bromegrass cell cultures revealed by microarray analyses

A suspension culture of bromegrass (Bromus inermis Leyss cv. Manchar) provides a unique system for studying cold hardiness mechanism, where freezing tolerance can be independently induced by two different stimuli, cold and exogenous abscisic acid (ABA). Freezing tolerance was induced in bromegrass cells by exposure to low temperatures for 7 days (LT50: -8.1 A degrees C) and also by incubation with ABA for 7 days at 25 A degrees C (LT50: -12.3 A degrees C) compared to the control cells grown without ABA at 25 A degrees C (LT50: -3.9 A degrees C). To characterize freezing tolerance mechanisms involved in both systems, bromegrass genes up-regulated after 7 days of low temperature or ABA treatment were analyzed using rice cDNA microarrays. In total, 300 and 479 clones were identified as cold- and ABA-inducible genes, respectively. Among them, 149 clones were induced both by cold and ABA treatments. Several genes were newly found to be cold-inducible from our microarray results. The increased expression of 8 selected genes in the microarray results was confirmed by Northern blot analyses. Northern blot analyses of three of these genes during 13 days of exposure to 4 A degrees C confirmed a gradually increased and long-term up-regulated expression during cold acclimation. In agreement with physiological and protein studies, these two cold hardiness induction systems exhibited fairly different transcriptome profiles: cold stress was characterized by triggering numerous genes involved in protein degradation/synthesis and RNA maintenance in addition to cold stress-related genes whilst ABA treatment was characterized by inducing numerous genes involved in seed formation and functional genes related to biotic and abiotic stresses.