Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco

Abstract

Stress perception and signal transduction leading to tolerance involve a complex interplay of different gene products. We describe here the isolation and characterization of an intronless gene (OSISAP1) from rice encoding a zinc-finger protein that is induced after different types of stresses, namely cold, desiccation, salt, submergence, and heavy metals as well as injury. The gene is also induced by stress hormone abscisic acid. Overexpression of the gene in transgenic tobacco conferred tolerance to cold, dehydration, and salt stress at the seed-germination/seedling stage as reflected by the percentage of germination/green seedlings, the fresh weight of seedlings, and their developmental pattern. Thus, OSISAP1 seems to be an important determinant of stress response in plants.

Fig. 1.

Comparison of deduced amino acid sequence from OSISAP1 and other zinc-finger proteins. Conserved cysteine and histidine residues are indicated in bold type. Conservation of amino acids at the C-terminal AN1-type zinc-finger (a) and the N-terminal A20 type zinc-finger (b) of OSISAP1 vis-à-vis PVPR3 (P. vulgaris pathogenesis-related 3 protein; ref. 23), AWP1 (Homo sapiens protein associated with PRK1; ref. 22), mZNF216 (Mus musculus zinc-finger protein 216; ref. 24), hZNF216 (H. sapiens zinc-finger protein 216; ref. 24), PEM6 (Ciona savigyni posterior end mark 6 protein; ref. 26), and XLULFP (Xenopus laevis ubiquitin-like fusion protein; ref. 25).

Fig. 2.

Expression pattern of OSISAP1 after different stresses to rice seedlings: cold (a), chemical modulators of membrane fluidity (b), salt and desiccation (c), submergence (d), heavy metals (e), mechanical wounding (f), and ABA (g). C, WT rice control without exposure to stress. OSISAP1 cDNA was used as a radiolabeled probe for Northern hybridization in all cases. The lower panels in all cases show ethidium bromide-stained rRNA for equivalent loading and RNA quality.

Fig. 3.

Expression of OSISAP1 in leaves of untransformed (UT) and transformed (SAPcL8, SAPcL9, SAPcL11, SAPcL22, and SAPcL43) tobacco. Other details are similar to those described for Fig. 2.

Fig. 4.

Effect of cold stress on tobacco seedlings from WT and OSISAP1-overexpressing transgenic lines (SAPcL8, SAPcL9, SAPcL11, SAPcL22, and SAPcL43). (a) Twenty-one-day-old seedlings were grown at 8 ± 1°C for 15 days and transferred to culture-room conditions. Fresh weight was recorded for cold-stressed seedlings after 15 days of cold-stress recovery. Fresh weights of unstressed seedlings of the same age also were recorded and designated as control. Absolute variation of two experiments is shown at the top of each bar. (b) Twenty-one-day-old seedlings of untransformed and OSISAP1-overexpressing lines were cold-stressed at 8 ± 1°C for 15 days and then transferred back to MSH for recovery. Photographs of representative seedlings of WT and five transgenic lines were taken after 15 days of recovery.

Fig. 5.

Effect of dehydration stress on tobacco seedlings from WT and OSISAP1-overexpressing transgenic lines (SAPcL8, SAPcL9, SAPcL11, SAPcL22, and SAPcL43). Seeds were germinated on 0.3 M (a) and 0.4 M (b) mannitol. (c) Relative fresh weight of 8-day-old seedlings germinated on 0.3 or 0.4 M mannitol. The fresh weight is shown relative to the fresh weight of unstressed seedlings. Absolute variation of two experiments is shown in a–c. (d) Representative seedlings of WT and five transgenic lines taken after 8 days of germination on 0.3 M (Upper) and 0.4 M (Lower) mannitol.

Fig. 6.

Effect of salt stress on tobacco seedlings from WT and T1 progenies of transgenic lines (SAPcL8, SAPcL9, SAPcL11, SAPcL22, and SAPcL43) overexpressing OSISAP1. (a) Twenty-one-day-old seedlings were salt-stressed in 250 mM NaCl for 4 days and then transferred back to MSH for recovery. After 8 days of recovery, seedlings of WT and transgenic lines that showed no apparent signs of chlorosis were counted. Absolute variation in two experiments is shown at the top of each bar. (b) Twenty-one-day-old seedlings were stressed with 250 mM NaCl for 4 days and then transferred back to MSH for recovery. Photographs of representative seedlings of WT and five transgenic lines were taken after 8 days of recovery.