Proton nuclear magnetic resonance (1H NMR) spectra of leaves from 50 plant species were obtained at a spectrometer frequency of 470 MHz. Water present in leaf samples gives rise to characteristic spectral patterns. Most species show only one broad 1H NMR peak; however, the leaves of some plants display complex, orientation-dependent spectra in which a common three-line pattern is discerned. The pattern varies with the angle between the leaf surface and the external magnetic field. Proton relaxation measurements show the presence of at least two water compartments in the leaves. The compartments are responsible for different components of the spectral pattern. EPR spectra, obtained at 35 GHz and at a temperature of -180TC, of plant leaf sections are dominated by the strong signals of manganous ions. We find that most plant leaves have isotropic Mn2+ EPR spectra. However, in some species (including ones that exhibit orientation-dependent 'H NMR spectra) we detect orientation-dependent intensities in the forbidden lines; the spectra indicate that Mn2+ ions occupy binding sites with axial or lower symmetry on nonrandomly oriented membranes. Both the NMR and the EPR results suggest that the chloroplasts of some plants are preferentially aligned with respect to the leaf surface.Photosynthesis takes place in a highly ordered membrane system (1). The membranes (thylakoids) occur in parallel stacks (grana) inside the chloroplasts, and most chloroplasts are found within layers of specialized cells. The existence of such a highly structured system suggests the possibility that photosynthetic membranes may be preferentially aligned with respect to the leaf surface. Indeed, some plant cells actively control the orientation of their chloroplasts in response to light stimuli (2).It is difficult to measure the net alignment of thylakoids in a leaf. Grana are easily viewed by electron microscopy, but to do so it is necessary to focus on individual chloroplasts or on very small regions of the leaf. Net thylakoid alignment (as a statistical property) might be more readily measured if the membranes provided an orientation-dependent spectroscopic signal. We have found such signals in both NMR and EPR spectra.Plant leaves are mostly water. Therefore, one might expect the 1H NMR spectrum of a leaf to be essentially the spectrum of water-a single line. In previous 1H NMR studies of plant material a single broad line usually has been observed. One well documented exception is in the spectrum of dogwood stems, which have orientation-dependent patterns caused by geometrical effects of the sample shape (3). Previous 1H NMR studies of plant tissue have concentrated on the effects of freezing (4). Spin-lattice (tj) and spin-spin (t0) relaxation times have also been measured; for example, exchange times between water compartments were determined in ivy bark (5). Leaf samples seldom have been studied.The EPR spectrum of a plant leaf always shows the presence of manganous ions (6, 7); most leaf manganese is concentrated in the chloropla...

Everyone who has seen the flash of a firefly has observed the phenomenon of bioluminescence. Biological light production in various animals, plants, and bacteria has only recently been understood in molecular terms. Light production is dependent upon ATP in firefly tails (1) and reduced coenzymes such as NADH or FMNH2 in some bacteria (2). These findings form the basis for using bioluminescence as a sensitive analytical tool for detecting extremely small quantities of ATP and NADH down to 10"15 and 10"14 moles, respectively (3,4). Bioluminescent methods have wide-ranging utility for analysis; they have been used in detection of bacterial contamination, in determination of cell viability, in developing sensitive immunochemical assays, and in research areas where sensitive enzyme assays are required (3,5).Described below is a bioluminescent assay for ATP and NADH that meets the needs of an undergraduate biochemistry laboratory course. This experiment provides students with experience in both bioluminescence and analytical biochemistry yet requires limited instrumentation, preparation, and expense. The experiment as described can be run in one 3-hr time period and has potential for being used in conjunction with the measurement of metabolic levels of nucleotides and coenzymes.