chloroplasts from the leaves of C4 plants. The agranal bundle sheath chloro- plasts are inactive in the Hill reaction, whereas granal bundle sheath and granal.
of the National Academy of Sience8 Proceedings Vol. 67, No. 1, pp. 18-25, September 1970
Deficient Photosystem II in Agranal Bundle Sheath Chloroplasts of C4 Plants K. C. Woo,* Jan M. Anderson,f N. K. Boardmantt W. J. S. Downton,4 C. B. Osmond,* and S. W. Thornet AUSTRALIAN NATIONAL1 UNIVBR8ZTY AND COMMONWEALTTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANIZATION, CANBERRA, AUSTRALIA
Communicated by R. N. Robertson, June 4, 1970
Abstract. A method is described for separating mesophyll and bundle sheath chloroplasts from the leaves of C4 plants. The agranal bundle sheath chloroplasts are inactive in the Hill reaction, whereas granal bundle sheath and granal mesophyll chloroplasts exhibit normal photosystem II activity. The agranal bundle sheath chloroplasts are deficient in photosystem II; they lack cytochrome b-559 and the fluorescence bands associated with photosystem II. All the chloroplasts exhibit photosystem I activity. Leaves of plants with the C4-dicarboxylic acid pathway of photosynthesis1 contain two distinctive layers of chlorophyll-containing cells: the outer mesophyll layer and the inner bundle sheath layer surrounding the vascular bundles.2 Chloroplasts of the mesophyll cells contain grana, but those of the bundle sheath exhibit varying degrees of grana development depending on the species. Earlier studies' in which leaf sections were treated with the Hill oxidant, tetranitro blue tetrazolium chloride (TNBT), showed that noncyclic electron flow from water was restricted to chloroplasts contaiuing grana. The agranal bundle sheath chloroplasts of Sorghum were incapable of TNBT reduction unless artificial electron donors were provided to photosystem I. This paper describes a technique for separating mesophyll and bundle sheath chloroplasts from the leaves of (X plants. The method is based on the differential resistance of the bundle sheath and mesophyll cells to breakage.4 The species used in this study were selected to provide a suitable range of grana development in the bundle sheath chioroplasts. We have examined the photochemical and fluorescence properties and cytochrome contents of the isolated mesophyll and bundle sheath chloroplasts. The results indicate that the bundle sheath chloroplasts lacking grana are deficient in photosystem II but they contain an active photosystem I. Granacontaining chloroplasts, both mesophyll and bundle sheath, have a functional photosystem II. Materials and Methods. Seedlings of Zea mays L. (var. NES 1002), Sorghum bicolor L. (var. Texas 610), and Atriplex spongiosa F. v. M. were grown in a glass house for 2-3 weeks. Leaves (10-15 g samples) were cut into strips (2-3 mm wide) and blended in a Sorvall Omnimixer for 5 sec at 50% of the line voltage in 100 ml of isolation medium (0.33 M sorbitol, 30 mM TES buffer, pH 7.2, 1 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, 5 mAI 18
VOL. 67, 1970 PHOTOSYSTEM BUNDLE SHEATH CHLOROPLASTS
19
2-mercaptoethanol, 0.5% bovine serum albumin, and 2% polyclar AT). This grinding procedure is effective in breaking mesophyll cells, but very few of the more resistant bundle sheath, cells. Unbroken cells and some cell debris were removed by filtration through two layers of Miracloth. The filtrate was centrifuged for 2 min at 300 X g and the pellet discarded. Mesophyll chloroplasts were obtained by centrifuging the supernatant for 10 min at 1000 X g and resuspending the pellet in the suspension medium (0.33 M sorbitol, 10 mM phosphate buffer, pH 7.4, 1 mM MgCl2, and 0.5% bovine serum albumin). The residue from the Miracloth was resuspended in 100 ml of isolation medium and blended for 3-5 min in the Omnimixer at 100% of line voltage. This procedure disrupts the remaining mesophyll cells and leaves the intact bundle sheath cells attached to lengths of vascular tissue. The blending time was varied for each species to give good yields of bundle sheath cells. The brei was filtered through Miracloth, and the residue washed and suspended in 50 ml of isolation medium. Examination in the light microscope showed that the bundle sheath cells were devoid of mesophyll cells. Fragments of bundle sheath chloroplasts were obtained by blending the resuspended residue in a Ten Broeck homogenizer or a Janke Kunkle mill with glass beads. The homogenate was filtered through Miracloth and the filtrate centrifuged for 2-3 min at 1000 X g to remove starch grains and cell debris. Bundle sheath chloroplast fragments were then sedimented by centrifugation for 20 min at 10,000 X g, and resuspended in a minimum volume of suspension medium. All operations were performed at 0-4CC. Hill reaction activities were measured by an oxygen electrode (Rank), with NADP as electron acceptor. Samples were illuminated for 2 min with red light (Wratten 29 filter; X >600 nm) of intensity 4 X 105 erg cm-2 sec-'. Photoreduction of NADP was measured at 340 nm with ascorbate-dichlorophenolindophenol (DCIP) as electron donor and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). Light-induced absorbance changes were measured with a Chance-Aminco dual wavelength spectrophotometer (American Instrument Co.). Actinic monochromatic light was provided by a 650 watt tungsten iodide lamp and Balzer interference filters. Light intensities were measured with an YSI-Kettering model 65 radiometer. Cytochromes were identified from reduced minus oxidized difference spectra, measured at 200C and 77°K in a Cary model 14R recording spectrometer, as described previously.5 Measurements at 77°K were performed in 60% glycerol by the single freezing procedure. Fluorescence emission spectra at 77°K were recorded in 60% glycerol on a fluorescence spectrometer incorporating automatic correction for photomultiplier and monochromator responses, and variation in output of light source.6'7 Fluorescence quantum efficiencies were calculated as described previously.7
Results and Discussion. The mesophyll chloroplasts of Sorghum bicolor contain well-developed grana but the bundle sheath chloroplasts are transversed by single unappressed lamellae (agranal). In contrast, grana are well developed in the bundle sheath chloroplasts of Atriplex spongiosa and somewhat less developed in the mesophyll cells.8 In Zea mays, the bundle sheath chloroplasts are essentially similar to those of Sorghum bicolor except for an occasional region of appressed lamellae; the mesophyll chloroplasts have good grana. The degree of grana formation in these species is reflected by the chlorophyll a/b ratios.9 Photochemical activities: Hill reaction activities of isolated chloroplasts with NADP as electron acceptor are shown in Table 1. M\Iesophyll chloroplasts from the three species gave good rates of oxygen evolution and there were no significant differences between the species. In contrast, the bundle sheath chloroplasts of Sorghum were inactive in the Hill reaction and oxygen evolution was not detectable. Bundle sheath chloroplasts of Zea mays gave traces of oxygen, but the rates of evolution were very low. The bundle sheath chloroplasts of Atriplex gave rates of oxygen production comparable to those of its
BOTANY: WOO ET AL.
20
PROC. N. A. S.
TABLE 1. Photochemical activities of isolated chloroplasts. Chloroplast type
Sorghum bicolor mesophyll Sorghum bicolor bundle sheath Zea may8 mesophyll Zea mays bundle sheath
Atriplex spongiosa mesophyll Atriplex spongiosa bundle sheath
Hill reaction* (,uatoms[01/mg
NADP reductiont
chlorophyll/hr)
chlorophyll/hr)
159 0
38 43
168
41
traces (
