Plasma-membrane dynamics in live protoplasts from maize (Zea mays L.) roots were characterized and examined for relationships as to the ability of the protoplasts to synthesize new cell walls and develop to cells capable of division. The lateral diffusion-coefficients and mobile fractions of fluorescence-labeled plasma-membrane proteins and lipids were measured by fluorescence photobleaching recovery. Small but significant effects on the diffusion of membrane proteins were observed after treatments with oryzalin or amiprophosmethyl, microtubule-disrupting drugs that increased the mobile fraction, and after treatments with cytochalasins B or D, microfilament-disrupting drugs that decreased the diffusion coefficient. A number of parameters were tested for correlative effects on membrane dynamics and protoplast performance in culture. Protoplasts isolated with a cellulase preparation from Trichoderma viride showed faster membrane-protein diffusion and a lower frequency of development to cells capable of division than did protoplasts isolated with a cellulase preparation from T. reesei. Membrane proteins in maize A632, a line less capable of plant regeneration from callus, diffused with a smaller diffusion coefficient but a greater mobile fraction than did membrane proteins in maize A634, a line with greater regeneration capacity. The plasma membranes of A632 and A634 protoplasts also differed with regard to lateral-diffusion characteristics of phospholipid and sterol probes, although the presence of both rapidly and slowly diffusing lipid components indicated the apparent existence of lipid domains in both A632 and A634. The protoplasts of the two lines did not differ significantly, however, in either wall regeneration or frequency of development to cells capable of division.

The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St.Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both A wide variety of methods were used to prepare specimens of S. secundatum for fluorescence measurements. Both epidermal peels and whole leaf tissues were examined. Epidermal strips were peeled from the leaves through use of a razor blade. Upon separation from the leaves, the epidermal peels were placed on a glass plate and rubbed gently with a pencil eraser to remove adherent mesophyll cells. Ice-cold distilled water was added during the rubbing operation. The epidermal peels were then washed twice and held in either distilled water or Tris buffer (0.37 M sucrose, 25 mM EDTA, 10 mM NaCl, 10 mm Tris, pH 7.6) during a 30 min to 1 h period of dark adaptation before fluorescence measurement. The absence of adherent mesophyll chloroplasts was verified by examination under the fluorescence microscope. Viability ofthe guard cells was confirmed by staining with fluorescein diacetate.Whole leaf tissue to be used for fluorescence measurements was dark-adapted either as detached leaves or as whole plants. Detached leaves were held on wet filter paper to prevent dehydration during dark adaptation. Dark adaptation was carried out at 25°C for times ranging from 30 min to 4 h.Fluorescence induction curves were measured on two different instruments. Most of the measurements were performed with a modified fluorescence microscope as described in Figure 1. Although conceptually similar to the microscope systems used by others (9, 10, 25) to measure guard cell fluorescence, this system had several special features which contributed to improved selectivity and sensitivity. In addition to standard mercury arc illumination, the microscope was equipped with two laser light sources. The argon ion laser was operated in the all-lines mode and emitted a light beam that was filtered at the microscope to provide either blue excitation (several wavelengths in the range 457.9-488.0 nm) or green excitation (514.5 nm). Alternatively, the light beam from a He-Ne laser could be selected to provide 429 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

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A modified fluorescence microscope system was used to measure chlorophyll fluorescence and delayed light emission from mesophyll and bundle sheath cells in situ in fresh-cut sections from leaves of Panicum miliaceum L. The fluorescence rise in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated leaves and the slow fluorescence kinetics in untreated leaves show that mesophyll chloroplasts have larger photosystem 11 unit sizes than do bundle sheath chloroplasts. The larger photosystem 11 units imply more efficient noncyclic electron transport in mesophyll chloroplasts. Quenching of slow fluorescence also differs between the cell types with mesophyll chloroplasts showing complex kinetics and bundle sheath chloroplasts showing a relatively simple decline. Properties of the photosynthetic system were also investigated in leaves from plants grown in soil containing elevated NaCI levels. As judged by changes in both fluorescence kinetics in DCMU-treated leaves and delayed light emission in leaves not exposed to of the growth of the area over this fluorescence induction curve (22, 23) have revealed two distinct components: a fast, nonexponential component (a) and a slower, exponential component (j3). These two components have sometimes been attributed to two different forms of PSII, termed PSIIa and PSIIO, which are considered to be distinct physical entities (22). Alternatively, it has been suggested that the biphasic nature ofthe fluorescence rise simply reflects different degrees of connectivity between PSII units (5). Other explanations are that the heterogeneity of the induction curve is due to the ability of DCMU to inhibit linear electron flow away from PSII (14, 15), or that it is a consequence of different degrees of connectivity between the light-harvesting complex and the PSII reaction center (26). Although no general agreement has yet been reached for the mechanism of this biphasicity, it is widely accepted that analysis of the induction curve provides a useful probe of PSII organization and function.Decay of the slow fluorescence kinetics is largely the result of two quenching mechanisms: quenching by the reoxidation of Q, which reflects the redox state of the electron transfer system, and quenching by other factors which are largely dependent upon the magnitude of the pH gradient across the thylakoid membrane (17). Slow fluorescence kinetics from leaves are intimately related to changes in the rates of lightdependent oxygen evolution and carbon metabolism (12,30).Delayed light emission is a result of the recombination of positive charges on the donor side of PSII with negative charges on the acceptor side. This charge recombination is sometimes considered to be a true reversal of the primary photoreaction of photosynthesis (18). Delayed light emission from PSI is thought to be much less than that from PSII (4, 27). Treatments such as heat, salt, electron transport inhibitors, etc., which alter the photosynthetic properties of PSII, will alter delayed light emission as well as Chl fluorescence...