Cold-induced freezing tolerance in Arabidopsis

Abstract

Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance.

Figure 1

Freezing survival of Arabidopsis after different acclimation (acclim) treatments 1 week after freezing. A, Three-week-old plants were acclimated at 1°C with a 16-h photoperiod for 0 (UN), 1, 2, or 3 d or acclimated for 3 d followed by deacclimation at 21°C for 1 d (+1d de) or 2 d (+2d de). The plants shown were frozen to −4°C (left), −7°C (middle), or −10°C (right). B, Three-week-old plants were not cold acclimated (UN) or kept for 3 d at 1°C in darkness (3d 1°D) or a 16-h photoperiod (3d 1°L) before freezing to −6°C, −8°C, or −12°C. C, Three-week-old plants were acclimated under 12-h light periods with low temperature (1°C) during the light and high temperature (21°C) during the dark (1°L/21°D), with high temperature during the light and low temperature during the dark (21°L/1°D), or at 1°C for both light and dark (1°L/1°D) for 2, 3, or 5 d before freezing to −8°C. D, Three-week-old plants were acclimated at 1°C and 16 h of light for 2 or 3 d in the presence or absence of 50 μm DCMU before freezing to −8°C.

Figure 2

Accumulation of sugar and Pro and dry weight changes during cold acclimation of Arabidopsis. At different times after transfer to 1°C, total soluble sugar (A) or Pro (B) was measured in leaves. Data points in A and B are averages from three to five independent sets of plants acclimated at 1°C with a 16-h photoperiod (▪) or deacclimated at 21°C (⋄) and from two independent sets of plants acclimated at 1°C with a 16-h photoperiod and treated with 50 μm DCMU (▵) or at 1°C in darkness (•). C, Dry weight changes during cold acclimation: Fresh weight (FW) and dry weight were measured in whole plants harvested at different times after transfer to 1°C. Data presented are averages of dry weights as percentages of fresh weights of independent sets of plants acclimated at 1°C with a 16-h photoperiod (▪), 3 d of acclimation followed by deacclimation at 21°C (⋄) (n = 3), or 1°C in darkness (•) (n = 3). Error bars, which are covered by the data points in some cases, indicate sd.

Figure 3

Comparison of freezing survival, sugar accumulation, and Pro accumulation in Arabidopsis plants cold acclimated under different light periods. A, Average lowest freezing temperature survived by plants cold acclimated under different light periods. Sets of plants were grown for 3 weeks at 21°C and acclimated at 1°C for 1, 2, 3, or 5 d. Light periods used were: constant light (⋄; n = 3), 18 h (□; n = 3), 12 h (▵; n = 6), 6 h (×; n = 3), or 3 h (○; n = 3) of light or darkness (•; n = 3). Sets of plants were then frozen to a series of temperatures indicated on the y axis. After thawing, plants were allowed to recover at 21°C. The lowest temperature survived by most of the plant leaves after a 1-week recovery period is shown. B, Soluble sugar content of Arabidopsis leaves acclimated under different light periods as in A. Total soluble sugar content is expressed as micrograms of soluble sugar per milligram leaf fresh weight (FW). C, Pro accumulation in plants acclimated under different light periods as in A. Pro content is expressed in micrograms per milligram leaf fresh weight. Data are averages from two to six independent sets of plants, with each set representing pools from 8 to 15 plants. Error bars, which are covered by the data points in some cases, indicate sd.

Figure 4

Accumulation of mRNAs at low temperature. Shown here are northern blots of total RNA prepared from leaves harvested at the indicated times after transfer to 1°C under a 16-h light period, in darkness, or under a 16-h photoperiod in the presence (+) or absence (−) of DCMU. Blots in A were hybridized with AC1–6, a probe for a cold-induced transiently expressed mRNA isolated by differential display PCR. Blots in B, C, and D were hybridized to COR15a, COR47, and COR78, respectively, all of which are abundant cold-induced mRNAs (Hajela et al., 1990; Thomashow et al., 1993).