Temperature Impact On Secondary Metabolite Development in Turfgrasses

1. Temperature Impact On Secondary Metabolite Development in Turfgrasses Nangle E.J.1*, D.S. Gardner, J.M. Metzger, T.K. Danneberger, L.E. Rodriguez-Saona2 1 Dept Of Horticulture and Crop Science, The Ohio State University, Columbus, Ohio 2Dept of Food Science and Technology, The Ohio State University, Columbus, Ohio Abstract Secondary metabolites are compounds which influence plant response to stressful environments. There are numerous compounds that are broadly classified as carotenoids o ranthocyanins. These compounds are considered important in plant response to stresses such as high light intensity, increased UV-B incidence and possibly to drought tolerance. Their role in turfgrasses is not well understood and further work into their production in response to specific stresses is warranted.. Creeping bentgrass (Agrostis stolonifera) cultivars ‘L-93’ and ‘Penncross’, Tall fescue (Festuca arundinicea) ‘Barvado’ and Bermudagrass (Cynodon dactylon) ‘Transcontinental’ were planted in 11cm diameter pots with 80/20 w/v sand/peat mix and propagated with sufficient irrigation and fertilization to prevent nutrient or water stress. These commonly used C3 and C4 turfgrasses were subjected to three temperature levels. Standard greenhouse temperature ~20 °C and light intensities of ~800 μmol m-2 s-1, 10 °C and 2 °C in light intensities of ~600 μmol m-2 s-1. The development of anthocyanins and carotenoids was measured over time scales of 0, 6, 12, 24 and 48 hrs. Samples grown at room temperature and 10 °C showed greater variability in anthocyanin content while samples treated in 2 °C produced a rapid response to the temperature change. In warmer temperatures without light, all grasses produced higher levels of anthocyanins while when light intensity increased and temperature decreased there was a significant drop in anthocyanin quantities in turfgrasses grown 2 °C. Bermudagrass produced the lowest levels of anthocyanins but showed the greatest response to temperature. Total carotenoids were highest in Tall fescue but the response to changes in temperature varied. In cooler temperatures with light there was a greater decrease in carotenoid content in C3 turfgrass plants. The results indicate that turfgrasses respond differently to temperature changes and also indicates potential for understanding damage in higher light conditions and cooler temperatures to turfgrass plants. Fig 3. Anthocyanin production over a 48hr time scale at a temperature of 10 °C. Error bars indicate standard error of the mean. Fig 2. Anthocyanin production over a 48hr time period at a temperature of 2°C. Error bars indicate standard error of the mean. Introduction • Light is predominantly used by plants in the 400 nm – 700 nm range known as PAR (photosynthetically active radiation). The energy levels related to these wavelengths are beneficial to plant growth and development. Light outside of this range contains different energy levels. • Predicted changes in climate may lead to alteration of spectral composition with increased levels of UV-B light in particular of concern (1) • Turfgrasses contain phenolic and terpenoid type compounds which have been related to protection from damaging light sources. These pigments respond to a range of conditions in a variety of plants including seasonal change, nutrient availability (1). • The impact of temperature on production of these compounds may affect the grasses ability to tolerate photoinhibition and damage to photosynthetic apparatus. There may already be evidence of this with chilling stress seen in the southern part of the United States. Fig 4. Carotenoid production over a 48hr time period at a temperature 2°C. Error bars indicate standard error from the mean. Fig 5. Carotenoid production at 10 °C over a 48 hr time period. Error bars indicate standard error from the mean. Objectives • To test three temperatures for indication of optimal temperature for production of carotenoids and anthocyanins in four commonly used turfgrasses. • To measure the difference in anthocyanins and carotenoids across a range of turfgrasses both C4 and C3 and over time. Materials and Methods Light environments • Two light environments were used. Conviron growth chambers were used to create a light intensity of ~600 μmol m-2 s-1 while greenhouse lighting averaged ~800 μmol m-2 s-1. • Three 18 x 12 cm pots of ‘L-93’ and ‘Penncross’ (Agrostis stolonifera L.), Tall fescue (Festuca arundinicea) ‘Barvado’ and Bermudagrass (Cynodon dactylon) ‘Transcontinental’ were placed in a randomized complete block design in both light environments. • The ambient temperature in the greenhouse was controlled at ~20 c, while growth chambers were set to have14 hrs of either 10 or 2 ° C with a return to 20° C and dark conditions for 10 hours. All plants in light environments were dark acclimated for 24hrs prior to initiation of trials. • Pots received sufficient nutrients and irrigation to prevent onset of deficiency or drought stress. Treatments • Turfgrasses were subjected to the temperature changes over a time scale of 0, 6, 12, 24, and 48 hrs. At 0, 6, 12, 24 and 48hrs samples were subject to light intensity and temperature (Fig 1a-c). • The turfgrass canopy was maintained at ~4.5 cm with sufficient irrigation to prevent drought stress during the experiments. • Samples were taken using liquid nitrogen and frozen immediately at -80 ° C and stored in the laboratory prior to analysis. Laboratory Analysis • Clippings were freeze dried and then anthocyanins were extracted using an acetone separation method of the water soluble compounds (2). A spectrophotometer (Shimadzu Inc.) was used to measure absorbance's at 530 and 660 nm. • Turfgrass carotenoids were extracted using a similar acetone extraction method and then absorbance readings were used to calculate total carotenoid content μg ml (3). • Chlorophylls A, B and totals (Moran, 1982) (4) were measured using spectrophotometry. The ratio of Chl A to B was also worked out with the absorbance's taken. Proc GLM was used to analyze the data with repeated measures for the time over all treatments. Fig 6. Chlorophyll levels in four turfgrasses at 2°C over a 48hr time scale. Error bars indicate standard error from the mean. Fig 7. Chlorophyll levels in four turfgrasses grown over a 48hr time period at 10 °C. Error bars indicate standard error from the mean. Results Conclusions Anthocyanins are affected by temperatures and to ensure that there is no spikes due to decreased temperatures or vice versa it is recommended that temperatures be maintained at ~20°C for future turfgrass research. Further work into mRNA may give a clearer analysis of the responses of both precursors to anthocyanins and carotenoids, chalcone synthase and phytoene synthase. Carotenoids are also impacted by temperature and maintaining plants at ~20 °C compared to cooler temperatures may provide more consistent results in turfgrass research into these secondary metabolites. • The reduction in temperature led to a spike in anthocyanin production early in the trial (Fig 2.) while overall there was more variability at warmer temperatures (Fig 3.). There was on average slightly higher absorbance's in plants that were treated with cooler temperatures. • Bermuda had the lowest quantity of carotenoids at the cooler temperatures and this may be an indication as to why chilling stress occurs in the southern states. An inability to reduce the negative effect of photoinhibition may lead to the discoloration seen in winter. (Fig 4 and 5.). There was also more variability at the warmer temperatures indicating that there is a possible enzymatic effect with temperature variations. • Across all turfgrasses when there was an initial decrease (6hrs) of carotenoids and anthocyanins there was a resulting decrease in the chlorophyll content (Fig 6 and 7). After this there was some variability within the levels. • The absorbance measurement of anthocyanins which was taken at 530nm may not be the most accurate absorbance for anthocyanins within turfgrass. A scan of the absorbance spectra indicated peak absorbance was ~505 nm and this indicates that further work into specific anthocyanin development in certain conditions is warranted. References 1 Barker, N.R., S. Nogues, and D.J. Allen, 1997. Phtosynthesis and photoinhibition, p 95-112. In P.J. Lumsden (ed.) Plant and UV-B: Responses to environmental changes. Cambridge Univ Press, Cambridge UK. 2 Rabino, I. and A.L. Mancinelli, 1986. Light, temperature and anthocyanin production. Plant Physiol. 81, 922-924. 3 Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: Pigments of photosynthetic membranes. Methods Enzymol. 148, 350-382. 4 Moran, R. 1982. Formulae for Determination of Chlorophyllous Pigments Extracted with N,N- Dimethylformamide. Plant Physiol. 69, 1376 – 1381. Acknowledgments Figure 1a. Growth Chamber with UV lights currently installed as project progresses. Figure 1b. Growth chamber with fluorescent bulbs and UV Lamps running. Figure 1c. Turfgrasses in greenhouse prior to initiation of trials • The authors would like to thank Christiaan Arends of Barenbrug USA for seed supply.