Plant Morphology 21 (1) : 87-91, 2009. REGULAR PAPER Effects of chloroplast dysfunction in a subpopulation of leaf mesophyll cells on photosynthetic and respiratory activities of a whole leaf: A study using variegated leaves of Hedera helix L. Naoko Yoshioka, Yuki Imanishi, Keiko Yasuda, and Atsushi Sakai Department of Biological Sciences, Faculty of Science, Nara Women’s University, Kitauoya-nishi-machi, Nara 630-8506, Japan Author for correspondence: A. Sakai, [email protected] Summary: A variegated leaf of Hedera helix is composed of green and white mesophyll cells with and without photosynthetic capacity, respectively. We measured the photosynthesis and dark respiration rates, as well as CO2 compensation points, of H. helix leaves with various extents of variegation. The photosynthetic rate (on an area basis) of the variegated leaves increased almost linearly according to the increase in the proportion of green area to total leaf area. In contrast, dark respiration rate was nearly constant irrespective of the extent of leaf variegation. These results suggest that chloroplast dysfunction in white mesophyll cells did not drastically affect photosynthetic activity of green mesophyll cells, or respiratory rates of both green and white mesophyll cells. CO2 compensation point was elevated when the proportion of green area became extremely low, indicating that the proportion of non-photosynthetic cells within a photosynthetic organ could affect its CO2 compensation point. Key words: CO2 compensation point, photosynthesis, respiration, variegation. INTRODUCTION Leaf variegation is characterized by the presence of green mesophyll cells of normal appearance present in white or yellow (or pale green) sectors in normally green the same leaves. In an Arabidopsis variegation mutant im, leaves (Aluru et al. 2006). The various causes of this the green leaf sectors exhibited increased photosynthetic variegation include plastid ribosome deficiency (Han et oxygen evolution rates under CO2-saturating conditions, al. 1992, Hess et al. 1993), alteration of plastid genome compared with the wild-type, all-green leaves (Aluru et (Stoike and Sears 1998), mitochondrial dysfunction al. 2001). This increase in photosynthesis may be a (Martínez-Zapater et al. 1992, Sakamoto et al. 1996), and means of compensating for a lack of photosynthesis in deficiencies in carotenoid biosynthesis (Wetzel et al. white sectors. Thus chloroplast dysfunction in a 1994). Variegated plants have played prominent roles in subpopulation of mesophyll cells in variegated leaves research on genetics (e.g., discovery of non-Mendelian can affect respiratory/photosynthetic characteristics of a inheritance) and morphology (e.g., analysis of cell whole leaf in several ways. lineage in leaf development). Moreover, they provide us In the present study, we tried to examine whether with excellent opportunities to study inter-organellar and chloroplast dysfunction in white sector cells indeed inter-cellular interactions. affects photosynthetic capacities of the green sectors and White sectors of variegated leaves are, at least in respiratory activity within the same cells, by analyzing some cases, formed by viable cells containing the relationship between the extent of leaf variegation undifferentiated, defective plastids (e.g., Kato et al. and photosynthetic/respiratory rates, using variegated 2007). In an Arabidopsis variegation mutant var2, leaves of ivy (Hedera helix L.). We also investigated the expression of nuclear-encoded genes for chloroplast impact of leaf variegation on CO2 compensation point, photosynthetic proteins was strongly repressed in white which is used as a convenient and useful parameter to sectors (Kato et al. 2007), suggesting that retrograde distinguish C3 from C4 photosynthesis (Edwards and signaling (Susek et al. 1993) from plastids to nucleus is Walker 1983). The results suggest that chloroplast involved in the formation of these sectors (Nott et al. dysfunction in a subpopulation of leaf cells had no 2006). Moreover, Yoshida et al. (2008) reported that the apparent effects on respiratory rates of white sector cells amount of mitochondrial protein, porin, was larger in and photosynthetic capacities of green sector cells. The white than in green sectors of var2. Hedtke et al. (1999) results also demonstrated that an extreme increase in reported that the levels for mitochondrial DNA and white sectors (non-photosynthetic tissues) within a leaf mitochondrial transcripts were elevated in white leaves significantly elevated CO2 compensation point. of albostrians barley mutants that have deficiency in plastid ribosome. These reports suggest that chloroplast MATERIALS AND METHODS dysfunction in white sectors may elevate respiratory activities of the white cells by affecting the amount Plant material and a parameter to evaluate extent of and/or function of mitochondria. In addition to these leaf variegation intra-cellular (inter-organellar) interactions, inter-cellular The plants of H. helix L. used in this study were in the interactions may also exist; i.e., the presence of white mesophyll cells may affect photosynthetic capacities of -87- The areas of green, pale green, and white sectors and total leaf area were determined using NIH image 1.62 software (National Institute of Health, Bethesda, Maryland, USA) following image scanning of the leaves or leaf discs. Microscopic observations Cross-sections of leaf tissues were made by hand-sectioning with razors and examined under a microscope (BX-60, Olympus, Tokyo, Japan) without fixation and staining. Measurement of photosynthetic and respiratory activities of leaf discs with oxygen electrode Leaf discs (3.6 cm2) were cut out from the leaf blades, and their gross photosynthesis rates and dark respiration rates were measured with a closed chamber system equipped with a gas-phase O2 electrode (LD2/2, Hansatech, Norfolk, UK) according to the manufacturer's instructions. The gross photosynthesis rate was estimated by measuring the rates of both O2 generation under -2 -1 illumination (500 μmol quanta m s) and O2 consumption in the dark, at 27˚C and 5% CO2. The dark respiration rate was estimated by measuring the rate of O2 consumption in the dark at 27˚C in ambient air (approximately 400 ppm CO2). Light response curve of photosynthesis under low CO2 concentration Light response curves of photosynthesis by detached leaves of H. helix were drawn using a preliminary open-flow system equipped with an infra-red gas analyzer (IRGA). A sample leaf, whose leaf stalk was inserted into a small water-tight tube filled with water, was sealed in a plastic bag (Hybri-bag soft, Cosmobio, Tokyo, Japan). The bag (functioning as a leaf chamber) was equipped with two tubing adapters (working as an Fig. 1. Morphological observations of variegated leaves of H. air inlet and air outlet, respectively) connected to tubing helix. (A), H. helix leaves with various extents of variegation. (B), Cross section of green sector. (C), Cross section of pale through which air was blown with a high-speed green sector. (D), Cross section of white sector. Bars in (A) and peristaltic pump. The flow rate of the air was measured (D) represent 5 cm and 200 μm, respectively. with a flow-meter. The leaf chamber was immersed in a water-bath maintained at 27˚C, and the leaf within the juvenile phase. Expanded, mature leaves were collected bag was irradiated with a reflector lamp (PR-500WB, from plants that were planted at Nara Women’s National, Osaka, Japan) through a water jacket. The University (Nara City, Japan), and used for all the photosynthetically active photon flux density (PPFD) experiments. Leaf stalks of collected leaves were was controlled in a step-wise manner with appropriate immediately cut again in water to prevent cavitation and shading filters at 500, 300, 150, 75, 30, 6, and 0 μmol -2 -1 individually placed in a water-tight tube filled with water. quanta m s , and the CO2 concentrations of the air at The detached leaves were immediately transported to the the inlet and outlet of the leaf chamber were measured, in laboratory, and used for experiments. turn, with an IRGA (LI-800, LI-COR). After As a variegated leaf of H. helix was composed of measurement, the sample leaf was dried for 3 days at three areas, namely, green sectors, pale green sectors, 80˚C and weighed. Net photosynthesis and dark and white sectors (see Fig. 1), the extent of leaf respiration rates were calculated from the CO2 variegation for individual leaves or leaf discs was concentrations measured at inlet and outlet of the leaf evaluated by using a “green area index (GAI)” defined chamber, flow rate of the air, and sample leaf dry weight. by the following formula: CO2 compensation point GAI = (green sector area + 0.5 * pale green sector The CO2 compensation point was measured with a area)/(green sector area + pale green sector area + white closed circulation system equipped with a leaf chamber sector area) (described in the previous section) and an IRGA. In this -88- RESULTS A variegated leaf of Hedera helix is composed of green, pale green, and white sectors Images of some H. helix leaves with various extents of variegation are shown in Fig. 1. A variegated leaf of H. helix was composed of green sectors, pale green sectors, and white sectors. In green sectors, mesophyll cells contained chloroplasts and were green. In white sectors, all mesophyll cells lacked chloroplasts and appeared white. In pale green sectors, several layers of mesophyll cells were green, while the rest of the layers were white. The white cells contained non-green plastids, as revealed by 4’-6-diamidino-2-phenylindole (DAPI)- fluorescence microscopic observations of protoplasts (data not shown). Pale green sectors could be further classified into several color levels, as the number of green cell layers differed between different pale green sectors. However, on average, the number of green cell layers in pale green sectors appeared to be roughly half that in the green sectors. No apparent differences in leaf anatomy (e.g., number of cell layers and leaf thickness) were observed among the green, pale green, and white sectors.