From lab to nature: assessing injury in xylem parenchyma cells

The maximum northern survival limit of several woody plant species was thought to be defined through the ability of various organs to deep supercool including flower buds, bark and xylem parenchyma cells (XPCs) (Tumanov and Krasavtsev 1959, Sakai and Weiser 1973, Quamme et al. 1973, George et al. 1974, Burke et al. 1976, Rajashekar and Burke 1978, Ashworth et al. 1988). The phenomenon of deep supercooling to avoid intracellular freezing damage has long been considered an important area of study. Historically, trees were categorized into those which did and did not deep supercool (Burke et al. 1976). Karlson et al. (2004) revealed XPC-related phylogenetic inheritance of dehydrins which appeared to delineate supercooling and non-supercooling types of Cornus. The absence of a deep supercooling strategy was thought to enable expansion of those tree types beyond the deep supercooling tree line limit of −40 °C (Burke et al. 1976). However, the fundamental significance of XPCs becomes apparent with additional reports that: (i) the deep supercooling temperature limit can approach −70 °C (Gusta et al. 1983, Fujikawa and Kuroda 2000, Kuroda et al. 2003) and (ii) deep supercooling is a widespread strategy of freezing avoidance from tropical (Kuroda et al. 1997), to temperate (Fujikawa and Kuroda 2000) and boreal tree species (Kuroda et al. 2003, Kasuga et al. 2007). The apple (Malus domestica L. Borkh.) is one of the most recognizable and ubiquitous fruits in the temperate world. In the USA alone, production of apples increased by 2% from 2010 to 2011 reaching 9.51 billion pounds with a net value to the grower of over US$2.2 billion (Perez et al. 2011). Globally, apple production increased by 10% between 2003 and 2007 to 64 million metric tonnes with China representing 43% of world output share (Lynch 2010). Of all the risk factors influencing annual apple production, temperature is the most significant. Unlike most other temperate fruit crops in which photoperiod plays a major role, dormancy induction in apple and pear are directly influenced by temperature (Heide and Pestrud 2005). This in turn will influence the timing of subsequent freezing stress resistance and winter survival. A common misconception is that global warming will reduce the incidence of injury due to low-temperature stress. In reality, earlier springs have placed more deciduous crops at risk due to earlier bloom dates and subsequent frost kill (Storey and Tanino 2011, Hänninen and Tanino 2011). In 2007, over US$2 billion damage to horticulture crops resulted from a massive freeze from Texas to New York State over one weekend in April (Gu et al. 2008). Recently, an estimated 80% of the apple flower buds in Ontario, Canada were damaged and this occurred in late April, 2012, due to a combined effect of an early warm spring and late spring frosts (Kuitenbrouwer 2012). As devastating as this may seem, flower buds will still be produced for next year's crop, but more serious would be the loss of the tree itself. It is said that a system is only as good as its weakest link. In the case of apple tree survival, the XPCs of the trunk are the most critical factors to freezing stress resistance and therefore the weakest link to long-term survival of the whole tree (Quamme et al. 1972, Quamme and Hampson 2004). The resultant injury to the XPC is observed as a darkened area within the central trunk, commonly known as ‘blackheart’ (Figure. 1). XPC resists injury through freezing avoidance (deep supercooling) to very low, mid-winter temperatures. However, if ice nucleation and freezing do occur in the intracellular space of the XPC, cells are immediately killed. The Russian scientists, Tumanov and Krasavtsev (1959), were likely the first to suggest that northern trees are injured by freezing of supercooled water. Blackheart in (a) M. domestica ‘Amur Red’ apple; (b) 3-year-old M. domestica ‘Ottawa 3′ dwarfing apple rootstock; and (c) Pyrus ­communis L. ‘John’ pear. Again, global warming may not necessarily result in expanded production areas of apple. Using 78 years of weather and apple production data, Caprio and Quamme (1999) ­provided compelling evidence that minimum temperatures associated with Arctic outflows in the Okanagan Valley of British Columbia, Canada are responsible for winter injury. The November to mid-January period was the key time point in apple tree survival/productivity. The authors concluded that the northern range of apple production due to global warming was not anticipated to expand as initially predicted. Although Arctic outflows are increasingly less frequent with higher minimum temperatures, they are a limiting factor due to their negative impact on XPC intracellular freezing. In spite of the importance of deep supercooling and XPCs, there are surprisingly few recent papers on this phenomenon. In this issue, Pramsohler et al. (2012) provide several important new findings contributing to our knowledge of the location of the specific freezing events within the XPC, and transferability of information based on detached stems to reflect whole plant responses. I shall expand on two of the key novel results below. First, freezing events (high- and low-temperature exotherms, HTE and LTE, respectively) are monitored through the latent heat of fusion released upon the change in state from liquid water to solid ice. HTE are the initial freezing events of the apoplast which in apple occur in the −1.9 to −4.7 °C range in the system of Pramsohler et al. (2012) and are generally non-injurious in cold-acclimated apple. However, the LTE represents the intracellular freezing of the XPC, which causes death in those cells. Using azalea buds as a model system, Graham (1971) was the first to observe that the LTE was associated with injury. In apple, Pramsohler et al. (2012) reported that LTE occurred around −12.7 °C in the summer, and by winter the process of acclimation reduced the LTE temperature to −36.9 °C. Although acclimational changes to enhance deep supercooling is beyond the scope of this article, both extracellular and intracellular alterations occur in XPCs (see Fujikawa et al. (2009) for a review). The HTE and LTE are traditionally captured through differential thermal analysis (DTA) using thermocouples to measure heat release upon freezing (Burke et al. 1976). However, potential artefacts due to the thermocouples themselves are avoided through the non-invasive technique of infrared video thermography (IRVT). Wisniewski et al. (1997) pioneered the use of IRVT to determine the location of ice nucleation and the pattern of ice propagation in intact plants on the whole organ level. Infrared DTA (IDTA) builds on both techniques, such that higher spatial resolution at the tissue level (200 ∝m) can be non-invasively observed for both nucleation and ice propagation (Hacker and Neuner 2007). Pramsohler et al. (2012) utilized IDTA to further define the location of the LTE in XPCs and showed discrete LTE in which freezing events were non-random. Exotherms were brightest in the primary xylem near the pith tissue, although other less distinct exotherms were also detected in the sapwood. This finding that the primary xylem is the most susceptibile tissue to freezing damage is consistent with Ketchie and Kammereck (1987) who used tetrazolium chloride to localize the site of injury in apple. Second, the ability to control environmental conditions, perform multiple cycles of experiments in a short period of time and take measurements under easier working conditions has resulted in a disproportionate number of papers based in the laboratory or greenhouse. Very few papers have recorded freezing events in whole trees in the field (Ashworth et al. 1985) and the disconnect between the laboratory and field results has often been an area of criticism (Gusta et al. 2009). Thus, the comparison by Pramsohler et al. (2012) of both HTE and LTE in four systems of detached stems and intact plants under controlled environments and field conditions is an important contribution that addresses the perennial question of the transferability of information across scales, i.e., whether data from detached stems reflect whole plant responses. Furthermore, a special in situ freezing ­chamber was constructed for the field that was clamped onto twigs attached to the tree in order to induce a freezing event. Intact trees in the field had a higher average HTE (−1.9 °C) than the other three methods of freezing (intact twig frozen in the freezing chamber in the field, detached twig frozen under field conditions, detached twig frozen under controlled freezing tests in the laboratory) which all expressed an equivalent HTE temperature of around −4.7 °C. LTE were more challenging since most LTE were not detectable in all four systems. Nevertheless, their results clearly showed intact field trees initiate apoplastic HTE freezing at a significantly higher temperature than methods using detached stems or freezing chambers on attached twig sections. Moreover, although damage to the XPC is generally considered irreversible, the authors show through lateral bud growth that full recovery is still possible even under 60% damage. It is a useful reminder of the necessity to examine regrowth responses for long-term survival and not simply to rely on detached twigs with isolated viability tests as a sole measure of survival in the field. Apple is one of the most significant world fruit crops and despite global warming, tree survival is at risk due to the limiting factor of its XPCs. Pramsohler et al. (2012) are one of a handful investigating this phenomenon and one of the few groups currently studying responses in apple. They provide critical information on the location of susceptible XPC tissue, provide new information through a comparison of field and controlled conditions in the intact tree, intact and detached twigs, and remind us of the importance of taking our experiments through to recovery before making conclusions. Considering the central importance of XPCs to the long-term survival of an increasing number of tree species, renewed research in avoidance and deep supercooling is warranted, particularly in the light of global warming issues. I am grateful to Danielle Way for her helpful comments.

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