Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant

Abstract

Temperate plants develop a greater ability to withstand freezing in response to a period of low but nonfreezing temperatures through a complex, adaptive process of cold acclimation. Very little is known about the signaling processes by which plants perceive the low temperature stimulus and transduce it into the nucleus to activate genes needed for increased freezing tolerance. To help understand the signaling processes, we have isolated mutants of Arabidopsis that are constitutively freezing-tolerant in the absence of cold acclimation. Freezing tolerance of wild-type Arabidopsis was increased from -5.5 degreesC to -12.6 degreesC by cold acclimation whereas the freezing tolerance of 26 mutant lines ranged from -6.8 degreesC to -10.6 degreesC in the absence of acclimation. Plants with mutations at the eskimo1 (esk1) locus accumulated high levels of proline, a compatible osmolyte, but did not exhibit constitutively increased expression of several cold-regulated genes involved in freezing tolerance. RNA gel blot analysis suggested that proline accumulation in esk1 plants was mediated by regulation of transcript levels of genes involved in proline synthesis and degradation. The characterization of esk1 mutants and results from other mutants suggest that distinct signaling pathways activate different aspects of cold acclimation and that activation of one pathway can result in considerable freezing tolerance without activation of other pathways.

Figure 1

Freezing survival of wild-type and esk1 plants. (A) Nonacclimated wild-type (Left) and esk1 plants were frozen at −8°C under the protocol described under Methods. (B) Percent survival of nonacclimated and acclimated plants after freezing to different temperatures. Nonacclimated wild-type (•) and esk1 (○) were frozen in a temperature-controlled chamber as described under Methods. At the temperatures shown, samples of plants were removed from the chamber, allowed to recover, and scored for survival. Alternatively, wild-type (▪) and esk1 (□) plants were cold acclimated at 4°C for 2 days before being subjected to the same freezing test. The data are means ± SE for three separate experiments.

Figure 2

Growth habits of wild-type (Left) and esk1 plants. (A) Plants grown for 30 days at 22°C under 150 μmol quanta m⁻²⋅s⁻¹ continuous light. (B) Plants grown for 7 days at 22°C and then for 60 days at 4°C under 90 μmol quanta m⁻²⋅s⁻¹ continuous light.

Figure 3

Gel-blot analysis of transcripts from five cold-regulated genes in wild-type (WT) and esk1 Arabidopsis. Plants were grown at 22°C under 150 μmol quanta m⁻²⋅s⁻¹. Total RNA (10 μg) isolated from leaf tissue of nonacclimated plants (NA) or from plants cold acclimated at 4°C for 2 days (CA) was separated on a 1.2% agarose-formaldehyde gel and probed successively with cDNAs corresponding to the cold-regulated genes indicated. The ribosomal 25S rRNA was visualized on the nylon membrane by using ethidium bromide to demonstrate equal loading. Quantitative comparisons reported in the text are based on PhosphorImager analyses of the blots. The experiment was repeated four times with similar results.

Figure 4

Proline accumulation and expression of proline metabolism genes in wild-type (WT) and esk1 Arabidopsis. (A) Proline levels in nonacclimated plants (open bars), in plants cold acclimated at 4°C for 2 days (shaded bars), and in plants 10 hr after application of 100 mM proline (solid bars). Harvested leaf tissue was frozen in liquid nitrogen and lyophilized. Free amino acids were extracted and quantified by using an amino acid analyzer. (B) Gel-blot analysis of transcript levels for genes encoding Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline oxidase (AtPOX). Methods are those described in Fig. 3.