D. Mark Hodges

Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

Abstract. The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, is considered a useful index of general lipid peroxidation. A common method for measuring MDA, referred to as the thiobarbituric acid-reactive-substances (TBARS) assay, is to react it with thiobarbituric acid (TBA) and record the absorbance at 532 nm. However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values. Extracts of plant tissues including purple eggplant (Solanum melongena L.) fruit, carrot (Daucuscarota L.) roots, and spinach (Spinacia oleracea L.) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm. A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA. The reliability and efficiency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L.) leaves containing interfering compounds and then measuring MDA recovery. Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques. Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol. If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 96.5%. Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L.) and spinach which had low or negligible concentrations of anthocyanidin derivatives. Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modified method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering compounds. This modified protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering compounds.

Lipophilic components of the brown seaweed, Ascophyllum nodosum, enhance freezing tolerance in Arabidopsis thaliana

Extracts of the brown seaweed Ascophyllum nodosum enhance plant tolerance against environmental stresses such as drought, salinity, and frost. However, the molecular mechanisms underlying this improved stress tolerance and the nature of the bioactive compounds present in the seaweed extracts that elicits stress tolerance remain largely unknown. We investigated the effect of A. nodosum extracts and its organic sub-fractions on freezing tolerance of Arabidopsis thaliana. Ascophyllum nodosum extracts and its lipophilic fraction significantly increased tolerance to freezing temperatures in in vitro and in vivo assays. Untreated plants exhibited severe chlorosis, tissue damage, and failed to recover from freezing treatments while the extract-treated plants recovered from freezing temperature of −7.5°C in in vitro and −5.5°C in in vivo assays. Electrolyte leakage measurements revealed that the LT50 value was lowered by 3°C while cell viability staining demonstrated a 30–40% reduction in area of damaged tissue in extract treated plants as compared to water controls. Moreover, histological observations of leaf sections revealed that extracts have a significant effect on maintaining membrane integrity during freezing stress. Treated plants exhibited 70% less chlorophyll damage during freezing recovery as compared to the controls, and this correlated with reduced expression of the chlorphyllase genes AtCHL1 and AtCHL2. Further, the A. nodosum extract treatment modulated the expression of the cold response genes, COR15A, RD29A, and CBF3, resulting in enhanced tolerance to freezing temperatures. More than 2.6-fold increase in expression of RD29A, 1.8-fold increase of CBF3 and two-fold increase in the transcript level of COR15A was observed in plants treated with lipophilic fraction of A. nodosum at −2°C. Taken together, the results suggest that chemical components in A. nodosum extracts protect membrane integrity and affect the expression of stress response genes leading to freezing stress tolerance in A. thaliana.

A Commercial Extract of Brown Macroalga (Ascophyllum nodosum) Affects Yield and the Nutritional Quality of Spinach In Vitro

The effects of extracts of the brown marine alga (Ascophyllum nodosum, ANE) on growth and biochemical and molecular changes in spinach were studied. Overall increases in biomass, chlorophyll, and antioxidant activity were observed at an application rate of 0.1 g L –1 ANE. Shoot fresh weight, dry-matter content, and total soluble protein showed 1.6-, 1.2-, and 1.5-fold increases, respectively. Total chlorophyll increased by 30% and total antioxidant capacity, phenolics, and flavonoid content increased by at least 33%. A 1.4-fold increase in chalcone isomerase activity was observed, whereas the activity of phenylalanine ammonia lyase was not affected. The ANE affected the transcript abundance of genes that affect sucrose and glycine betaine metabolism. The transcript abundance of cytosolic glutamine synthetase (GS1), betaine aldehyde dehydrogenase (BADH), choline monooxygenase (CMO), and glutathione reductase (GR) increased in plants treated with 0.1 g L –1 ANE.

Abiotic Stress in Harvested Fruits and Vegetables

Harvested fruits and vegetables can be potentially exposed to numerous abiotic stresses during production, handling, storage and distribution (Hodges, 2003). Some of these stresses can be minor in nature, resulting in no quality loss or, in some cases, in quality improvement (Hodges et al., 2005) during distribution. However, when the abiotic stress is moderate or severe, quality losses almost always are incurred at market (Toivonen, 2003ab Toivonen, 2005). One of the challenges facing fruit and vegetable production globally is that regional climate regimes are becoming more unpredictable from year to year. Hence understanding of effects of field abiotic stresses (e.g. drought, extreme temperatures, light and salinity) on postharvest stress susceptibility will become more important since postharvest stresses limit the storage and shelf life potential of fruits and vegetables (Toivonen, 2005). It is the intent of this chapter to first describe the nature of preand post-harvest abiotic stress events, delve into their importance for product quality and marketing and then explore the technologies available to begin managing the sensitivity of fruits and vegetables to stresses they encounter in the handling and distribution chain.