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Article,

The Effect of Water, Sugars, and Proteins on the Pattern of Ice Nucleation and Propagation in Acclimated and Nonacclimated Canola Leaves

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Plant Physiology, (2004)

Abstract

Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2C h21) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.
BibTeX key
Gusta2004
entry type
article
year
2004
journal
Plant Physiology
pages
1642--1653
volume
135
citeulike-article-id
1523734
priority
2
posted-at
2007-07-31 06:20:30
pdf
\\biostore\desktop\$\u2531135\Desktopideas and theory\papers\Gustaetal\_04\_supercooling\_exotherms\_sugars\_hydration\_canola.pdf
comment
(private-note)Supercooling: (bacteria)(gusta et al. 2004) Gusta et al. 2004 Acclimated plants could survive supercooling better. They were able to get the water to the apoplast quicker. Hardened plants can tolerate more of their water to be frozen. (which is quite the opposite, right? OK I'm confised) 23 in Single and Marcellos 1981 Gusta et al. 2004 drier leaves have a greater capacity to supercool. (acclimated leaves were drier) At 26.9C, no further exothermic events were observed in the nonacclimated leaves; instead they remained isothermal with the chamber temperature indicating the absence of any additional water freezing (Fig. 1E, top). However, a second exothermic event was observed in the acclimated leaves, indicating the freezing of a significant quantity of additional water which was not observed in nonacclimated leaves The time needed to reach freezing equilibrium was much longer for acclimated versus nonacclimated leaves In general, average water content decreased and dry weight increased with acclimation, although the distribution was not homogeneous. When freezing was observed at 23.3C, a slow mild exothermic event occurred across the entire leaf in both the nonfrozen plants (Fig. 2, A?C, top) and thawed plants previously frozen to 26C, However, in freeze-killed leaves, a large exothermic event immediately occurred as freezing progressed from the point of nucleation across the leaves. When the temperature was lowered to 24.7C, an additional fraction of water froze in leaves of both the nonfrozen plants (Fig. 2E, top) and thawed plants previously frozen to 26C Leaves obtained from nonfrozen plants became isothermal after 15 to 20 min, while leaves obtained from freeze-killed plants required 40 min indicating water was still available for freezing Cell sap removed from acclimated and non-acclimated leaves froze at different rates (slower in acclimated leaves) Nonwetted leaves obtained from fully acclimated plants readily supercooled to temperatures as low as 214C, whereas nonacclimated leaves supercooled only to 29C or 210C. Leaves wetted with a fine spray of water supercooled from 26C to 29C irrespective if they were acclimated or not. Fully acclimated nonwetted canola (cv Express) leaves were supercooled to 23C, 26C, 29C, 212C, or 215C prior to initiating freezing with the use of ice crystals. The leaves were held isothermal at one of these selected temperatures for 0, 0.5, 2, or 24 h and afterward evaluated for electrolyte leakage. Surprisingly, supercooled, cold acclimated canola leaves when frozen at 29C sustained little injury as determined by the LT50 results (Table IV). Leaves supercooled to 212C suffered little injury for up to 2 h, whereas after 24 h ion leakage increased. Freezing of leaves supercooled to 215C was devastating, resulting in over 40\% ion leakage in samples collected 0.5 h after the initiation of freezing. In addition, Zimmerman (1982) reported that during freezing, transient pores can form in the plasma membrane allowing for the bulk flow of solutes from the symplast to the apoplast. Olien (1967) as well as Livingston and Henderson (1998) also demonstrated solutes were released from the symplast during a mild freeze and were concentrated in the apoplastic water. Reaney and Gusta (1999) postulated these solutes would reduce the rate of ice growth through the cell wall. These solutes form a fluid zone that surrounds cells as freezing progresses and prevents ice adhesion to the plasma membrane which otherwise can result in lysis (Olien and Smith, 1981). The release of solutes from the symplast may account for the increase in freezing tolerance following a mild nonlethal frost (Trunova, 1965). The initial exotherm would have been either water vapour or free liquid water ;-) relationship between dry weight, wet weight, acclimation and freezing nonacclimated leaves had high water content. freeze-killed leaves lost compartmentalisation and therefore more water froze at -3.5 Wisniewski et al. (2001), using IRVT, demonstrated that nonwetted herbaceous plants always supercooled to a lower temperature than wetted plants.

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