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

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. 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.

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress.

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Seasonal expression of a dehydrin gene in sibling deciduous and evergreen genotypes of peach (Prunus persica [L.] Batsch).

A cDNA library was created from cold-acclimated bark tissue of peach and selectively probed using an antibody directed against the lysine-rich consensus region of dehydrin proteins. Several clones were thus obtained which had a high degree of sequence similarity to other dehydrin genes. Northern analysis, using clone 5a, indicated that a 1.8 kb transcript was seasonally expressed in sibling deciduous and evergreen genotypes of peach, and also inducible by water deficit in cv. Rio Oso Gem. The evergreen and deciduous genotypes differ significantly in both their ability to cold-acclimate and in the seasonal expression of the dehydrin transcript and protein. In both genotypes, the transcript was maximally expressed during winter and undetectable in May-July. The evergreen genotype (less cold-tolerant), however, displayed transcript accumulation which lagged behind and declined sooner than in the deciduous genotype. Protein expression was similar to transcript expression, however, protein expression in the evergreen genotype lagged considerably behind transcript accumulation in the fall. This indicates that several levels of regulation of dehydrin proteins may exist during cold acclimation. A genomic clone (G10a) was isolated which contained the full-length dehydrin gene, designated ppdhn1. The peach dehydrin gene encodes 472 amino acids with a predicted size of 50,020 Da. The encoded protein (PCA60) contains nine of the lysine-rich repeats characteristic of dehydrins and two DEYGNP motifs at the amino acid terminus. A genomic blot, probed with clone 5a under stringent conditions, indicated that one or two highly homologous genes are present in peach, whereas an additional member was detected under low-stringency conditions. It is suggested that several members of the dehydrin gene family may exist in peach that vary in their relation to ppdhn1.