Factors contributing to ice nucleation and sequential freezing of leaves in wheat

Abstract

Main conclusion Anatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest leaves and potentially meristematic regions. Abstract Infrared thermography (IR) has been used to observe wheat leaves freezing independently and in an age-related sequence with older leaves freezing first. To determine mechanisms that might explain this sequence of freezing several analytical approaches were used: (1) The size of xylem vessels, in proximity to where freezing initiated, was measured to see if capillary freezing point depression explained sequential freezing. The sequence of freezing in the four youngest leaves was correlated, with the largest vessels freezing first. (2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes explained the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves suggesting a prominent role for this sugar as compared to other simple sugars and fructans. Among individual free amino acids proline and serine were correlated to the freezing sequence, with younger leaves having the highest concentrations. (3) Microflora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of bacteria and fungi were correlated to the freezing sequence for all leaves, and species or genera associated with high ice nucleation activity were absent in younger leaves. Moisture content and transcript expression of ice binding proteins were also measured. As expected, our results show that no single mechanism explains the freezing sequence observed via infrared analyses. While these multiple mechanisms are operative at different levels according to the leaf age, they seem to converge when it comes to the protection of vital meristematic tissues. This provides potential phenotypic characters that could be used by breeders to develop more winter-hardy genotypes.

Visualising the effect of freezing on the vascular system of wheat in three dimensions by in‐block imaging of dye‐infiltrated plants

“…What appeared to be age‐dependent infiltration of dye solution could confirm the suggestion 24 that leaf position in the crown may at least partially explain age dependent freezing in wheat. Ice formation was initiated in roots and progressed acropetally from the crown into leaves with the youngest leaves attached to the top of the reservoir freezing last 24 …”

“…Also note that despite the expanded reservoir, the vascular bundles above and to the side of the reservoir did not appear to be altered by the freeze treatment. The bar in 'A' is 1 mm and also applies to 'B' and 'C' dye solution could confirm the suggestion 24 that leaf position in the crown may at least partially explain age dependent freezing in wheat. Ice formation was initiated in roots and progressed acropetally from the crown into leaves with the youngest leaves attached to the top of the reservoir freezing last.…”

“…Ice formation was initiated in roots and progressed acropetally from the crown into leaves with the youngest leaves attached to the top of the reservoir freezing last. 24 In leaves, dye solution was not confined to xylem vessels but as stated above, was contained within the mestome sheath of the vascular bundle (Figure 3). The mestome sheath in roots as well as leaves apparently prevented movement of dye solution outside the vascular bundle.…”

The effect of cold acclimation, deacclimation and reacclimation on metabolite profiles and freezing tolerance in winter wheat

“…The actively dividing shoot apical meristem (SAM) cells within the crown region are vital to cold perception and ensured vernalization of overwintering plants, as well as spring re-growth after the above-ground tissues are lost to freezing damage ( Wellensiek, 1964 ; Tanino and McKersie, 1985 ). Amino acids and soluble sugars can lower the freezing point and act as cryoprotectants during CA ( Bocian et al, 2015 ; Chai et al, 2019 ; Livingston et al, 2021 ). The increased accumulation of hexose phosphates in crowns, as compared to the leaves, indicates the active breakdown of starch or sucrose into glucose and fructose ( Kaplan et al, 2007 ).…”

“…Moreover, dehydrinsproteins, which act as protectants against abiotic stresshave been shown to accumulate in winter wheat leaves and crowns during CA, and to positively correlate with FT (Vítámvás et al, 2019(Vítámvás et al, , 2021. Elevated amino acid concentrations are likely to contribute to increased FT by acting as osmoregulators (Livingston et al, 2021), which is not the case with starch. These differences between carbon and nitrogen metabolism in the two treatment groups may be the reason behind significantly decreased FT of winter wheat, grown under higher low-temperature CA conditions (Figure 6).…”