PONCIANO, D.C., J.S. SURFUS, C. SAMMONS, J.A. GAMON, and R.R. NAKAMURA.

California State University Los Angeles, Los Angeles, CA 90032, USA.

Using Arabidopsis thaliana as a model system, we are exploring the role of plant pigments as indicators of the ozone response in plants. However, since conventional methods are destructive and require considerable leaf tissue, they are not well suited to multi-temporal studies in this small-leaved species. The purpose of this study was to explore leaf spectral reflectance as a means of sampling pigment changes with leaf development and exposure to varying light levels. During leaf development, reflectance-based pigment indices revealed increased chlorophyll content and decreased xanthophyll cycle pigment content. As growth light increased towards full sun, both chlorophyll and xanthophyll levels decreased, and anthocyanin levels increased, indicating these plants were severely stressed in high irradiance, reflecting the intolerance of these shade-bred plants for bright light. These results demonstrate the utility of spectral reflectance as a non-invasive tool for quantifying plant pigments. Because these pigments are indirect indicators of physiological function, reflectance provides a useful method for assessing physiological changes with age and light exposure. Future work will apply this method to ozone-exposed plants.

The purpose of this experiment was to explore leaf spectral reflectance as a tool for following pigment changes of Arabidopsis thaliana with development and exposure to different light levels. Arabidopsis thaliana is an ideal plant for temporal studies because it is a small plant that has a short life cycle, plus it is widely adopted by plant physiologists as a model organism (Figure 1).

Conventional methods for quantifying pigments such as paper, gas, and liquid chromatography are not only expensive and tedious but require the sample to be destroyed, making it impossible to follow physiological responses through time. A solution to this problem is to spectral reflectance. Leaf spectral reflectance can be sampled using a leaf reflectometer (Figure 2); which allows rapid and repeatable sampling, even on tiny leaf regions less than one millimeter in diameter. By measuring light intensity as a function of wavelength this instrument can quantify color (Figure 3 & Figure 4).

Color can be assay using reflectance indices, which are numeric values calculated from leaf spectral reflectance. These indices can be used to assay pigment levels and provide indicators of integrated physiological state. At the leaf scale, a modified normalized vegetation index (MNDVI) can be used to analyze chlorophyll content (Gitelson and Merzlyak 1994, Gamon & Surfus in press). The photochemical reflectance index (PRI) is an index of photosynthetic radiation use efficiency that can be used to study the xanthophyll cycle pigments (Gamon et al. 1997). The ratio of red to green reflectance (Red/Green) can be used to estimate anthocyanin levels (Gamon & Surfus in press).

• Chlorophyll levels will increase during plant’s growth.

To examine the effect of growth light intensity, four trays of Arabidopsis thaliana were planted every other week. Plants were grown under four different light levels: high light (1500m molm^-2s^-1), medium light (1000m molm^-2s^-1), low light (500m molm^-2s^-1), and very low light (300m molm^-2s^-1).

Leaf spectral reflectance measurements for the four oldest cohorts were collected and vegetation indices were calculated (Table 1). The instrument used to collect spectral reflectance, a leaf reflectometer, is composed of a fiber optic leaf probe attached to a narrow-band spectral detector that contains a halogen lamp. The spectral detector is controlled by special software through a computer (Figure2).

To estimate relative pigment levels, we applied the reflectance indices indicated in table 1. Indices of chlorophyll and anthocyanin levels were measured using single reflectance scans, each taking approximately five seconds. To estimate pool sizes of xanthophyll cycle pigments, we used a kinetic sampling approach that took ten minutes per sample (Figure 5 & 6). These indices provided relative pigments levels; further work will establish absolute levels by calibrating reflectance indices against extracted pigments using HPLC (Gamon & Surfus in press).

Note: see introduction for Figure 2.

We want to thank Dr. Robert Vellanoweth, Dr. Scott Grover, and their labs for letting us use their facilities. We also thank David Fuentes for all his help with the computers, all theVCSARS lab members, our families, and friends for their help and support.