IDENTIFICATION OF ULTRASTRUCTURAL AND BIOCHEMICAL MARKERS OF FROST AVOIDANCE IN THE CUTICULAR LAYER OF CORN

Abstract

Abiotic stresses are a critical factor in the reduction of yield. Corn has been identified as a highly economically important yet, frost sensitive crop. Climate change trends are show increased frost damage. The global need for food production is increasing and current production will not meet demand. Corn is killed at the moment of freezing and therefore, developing frost avoidance is essential. The primary obstacle limiting production of new more cold sensitive crops in the Canadian prairies is the cooler climate and early frost events in both spring and fall which are preventing widespread expansion. While many studies have examined corn chilling and frost sensitivity, the impact of simulated autumn temperatures (termed chilling pre-treatment) preceding a frost has not been reported. The effect of chilling pre-treatment, on subsequent freezing avoidance was studied in mature hybrid grain corn of four contrasting genotypes (256 and 675 [chilling sensitive]; 884 and 959 [chilling resistant]). Chilling pre-treatment (18°C/6°C, 10 days) induced physical and biochemical changes in the cuticular wax layer in all four genotypes. These changes were measured using a suite of complementary techniques including: thermal imaging, hydrophobicity, Confocal Laser Scanning Microscopy (CLSM), Attenuated Total internal Reflectance (ATR-FTIR), and Gas Chromatography Mass Spectrometry (GC-MS). In all corn genotypes studied, chilling pre-treatment induced a warmer freezing temperature than non-chilled. No significant genotypic differences were observed, however, genotypes 675 and 959 were least responsive to the stressor which resulted in the smallest change in freezing temperature induced by chilling pre-treatment. Hydrophobicity was reduced following chilling pre-treatment in all genotypes with the most significant effect observed in genotype 675. Cuticular thickness (μ=3.25 μm) remained unchanged over the ten-day chilling pre-treatment under controlled environment conditions. By contrast, over the five-week field conditions, cuticle thickness increased in all genotypes. Genotype 256 had a significantly thinner cuticle (-0.25 μm) than the other genotypes indicating genotypic variation is accentuated under field conditions and sensitive lines may have a thinner cuticle. In the growth chamber, chilling treatment induced increasing cutan, cutin, & cuticular wax only in Region 1 (CH3 functional group) according to ATR-FTIR within 2 μm of the adaxial surface layer. By contrast, field treatment induced a reduction in cutan, cutin, and cuticular wax in all regions (1, 2, & 3) (CH3, Asymmetrical CH2, Symmetrical CH2) to the same 2 μm depth of ATR sampling. A primary challenge of proofing cuticle based studies in the field is the extremely strong environmental influence (high light intensity, wind abrasion, insects, temperature fluctuations) which induce modifications on the cuticle. Using GC-MS analysis, 142 known compounds were identified in both controlled environment (chilling treatment) and field samples from the adaxial cuticular wax extraction of mature grain hybrid grain corn. Of those identified compounds, 28 were found to represent significant (P<0.05) variation between chilling treated and non-chilled treatments under both growth chamber and field conditions. This variation represented 5 Classes of key compounds (Alkane, Alcohol, Fatty Acid, Triterpenes and other). It is clear that chilling treatment modifies both physical and biochemical properties of the cuticular layer. The degree and rate of detectable chemical changes induced by chilling treatment indicate physical cuticular modifications likely are contingent on biochemical changes. This may be due to the great number of chemical modifications and signals needed to induce a physical modification. ATR applications are more reflective of the cuticular composition in cases where the entire cuticular thickness is within the depth of sampling (2 μm). The investigation of the dynamic process of cuticular wax modification following chilling treatment using complementary techniques in Zea mays appears to be a useful system with practical applications for evaluating the correlation between the cuticle as a barrier to abiotic stress and chilling treatment in a whole plant system.