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

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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

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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. The equation to calculate GAI, a parameter that represents the extent of leaf variegation, was defined based on these observations. For example, GAIs of completely green, pale green, and white leaves should be 1.0, 0.5, and 0, respectively.

Effects of leaf variegation on the photosynthetic capacity and dark respiration rate Rates of photosynthesis and dark respiration of H. helix leaves with various extents of variegation are shown in Figs. 2A and 2B, respectively. Gross photosynthesis rate increased almost linearly according to the increase of Fig. 2. Effects of leaf variegation on photosynthetic/respiratory GAI (r = 0.90, d. f. = 28, P < 0.001), with no apparent characteristics. (A), Relationship between gross photosynthesis convexity. Leaf discs with zero GAI still had a potential rate under CO2-saturating conditions and GAI. Results from 29 to generate oxygen under illumination, suggesting that leaf discs are shown. (B), Relationship between dark chloroplasts should still be present somewhere in the respiration rate measured in ambient air (i.e., under low CO2 apparently all-white sectors. concentration) and GAI. Results from 27 leaf discs are shown. Dark respiration rate did not rise or fall according to (C), Relationship between CO2 compensation point and GAI. the increase in GAI (r = 0.24, d. f. = 26, P > 0.1). These Results from 21 leaves are shown. Inset in (C) represents light results suggest that chloroplast dysfunction in white response curves of leaves with high GAIs (filled circles, GAI = 0.75) and low GAIs (open circles, GAI = 0.05) measured under sector cells did not affect the photosynthetic capacity of neighboring green sector cells, or the respiratory function ambient CO2 concentration (ca. 400 ppm). of mitochondria in the same cells. system, the air blown out of the leaf chamber was introduced into the IRGA and returned back to the leaf CO2 compensation points were inversely related to GAI chamber by continuous pumping with a peristaltic pump. CO2 compensation points of H. helix leaves with various The CO2 concentration in the air circulating in the closed extents of variegation are shown in Fig. 2C. Inset shows system should either rise (as a result of net release of light-response curves of representative leaves with high CO2 by respiration) or fall (as a result of net absorption and low GAI values (0.75 and 0.05, respectively), of CO2 by photosynthesis) until CO2 compensation point measured in ambient air (i.e., under low CO2 is reached. Thus, the CO2 concentration in the system concentration). The result confirmed higher was monitored continuously, and the plateau value photosynthetic rate and almost equal respiratory rate of (accomplished within 2 h) was considered as CO2 high-GAI compared with low-GAI leaves. CO2 compensation point of the sample leaf. CO2 compensation points of leaves with high GAI values compensation point was measured at 27˚C and PPFD at ranged from 50 to 80 ppm, which fell within -2 -1 500 μmol quanta m s . representative values for C3 plants. However, CO2

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compensation point increased gradually as GAI declined; constant, there was an almost linear correlation between it exceeded 100 ppm when GAI fell below 0.3, and GAI and rate of gross photosynthesis (Fig. 2A). This reached 500 ppm when GAI became close to zero. This linear proportionality indicates that the GAI is an result clearly demonstrated that CO2 compensation point appropriate parameter to represent the extent of leaf of a variegated leaf changed in response to variegation. The linearity also suggests that the non-photosynthetic cells as a proportion of total photosynthetic capacities of green sectors in H. helix mesophyll cells. variegated leaves are, on a leaf area basis, nearly constant. DISCUSSION This conclusion may contradict the opinion that green sectors in variegated leaves have a higher Effects of chloroplast dysfunction on the respiratory photosynthetic capacity than wild-type green leaves to activity of white mesophyll cells fulfill the large sink demand by neighboring white Several lines of evidence suggest that chloroplast sectors that have lost photosynthetic function (Aluru et dysfunction up-regulates the function of mitochondria al., 2001, 2006). If photosynthetic capacity of green within the same cells. Hedtke et al. (1999) reported an sectors is indeed affected by a sink demand by increase in the amounts of mitochondrial DNA and neighboring white sectors, a small green sector near a mitochondrial transcripts in the chloroplast-deficient large white sector should exhibit higher photosynthetic leaves of albostrians barley. However, they did not capacity than a large green sector near a small white investigate the level of mitochondrial protein or the sector, because sink demand for the green sector should respiratory activity of mitochondria. Yoshida et al. be larger in the former than in the latter. Nonetheless, our (2008) reported higher accumulation of a mitochondrial result did not fit this prediction, suggesting that protein, porin, in white sectors of Arabidopsis var2 photosynthetic rates of green sectors were nearly mutant leaves. However, they also reported that the total constant, irrespective of the sizes of neighboring white (i.e., cyanide-resistant plus cyanide-sensitive) respiratory sectors. Thus, we tentatively conclude that the presence rate of variegated leaves did not differ from that of of non-photosynthetic white sectors in H. helix wild-type, all-green leaves. Thus, the possibility of variegated leaves exerts little effect on the photosynthetic up-regulation of mitochondrial respiratory function in capacity of the neighboring green sectors. plastid-deficient cells is still uncertain. Whether green sectors in variegated leaves actually In the present study, we tried to investigate the had increased photosynthetic capacity should be differences in respiratory rates between green mesophyll examined carefully. Aluru et al. (2001, 2006) measured cells and white mesophyll cells by measuring respiratory photosynthesis rates on a chlorophyll basis, and found rates of leaf discs with various extents of variegation and increased photosynthesis in green sectors of variegated then analyzing the relationship between the two leaves of Arabidopsis im mutant. However, comparison parameters, respiratory rates and GAIs. If white cells on a chlorophyll basis may not be appropriate in this case, indeed had higher respiratory rates, there should be a because they also reported that im green sectors exhibit a negative correlation between GAI and respiratory rate. higher chlorophyll a/b ratio than wild-type leaves. High However, respiratory rate was nearly constant, chlorophyll a/b ratio indicates a small antennae size of irrespective of the differences in GAI (Fig. 2B). Thus, we photosystems. Therefore, when normalized on a tentatively conclude that chloroplast dysfunction in white chlorophyll basis, a specimen with high chlorophyll a/b sector cells in H. helix variegated leaves had little impact ratio (i.e., green sectors of im leaves) should include a on respiratory activity of white cells. larger number of photosystems. Estimation of It is apparent that dissection of green and white photosynthetic rates on an area or weight basis is sectors and direct comparison of their respiratory rates preferable. On the other hand, our method may not be should give more direct and conclusive results. However, sensitive enough to detect slight differences in the such an analysis is difficult for such leaves with a photosynthetic capacity between green sectors. Moreover, marble-like pattern of variegation as in H. helix; it is in the present study, variegated leaves with different quite difficult to cut out enough mono-colored sectors of extents of variegation were compared. With our a size suitable for measuring O2 consumption rates in the methodology, it is difficult to detect increased system with a gas-phase O2 electrode. To examine the photosynthesis in green sectors if the photosynthetic validity and generality of our conclusion, further capacity of green sectors within a whole leaf is affected investigation on various plant species with variegated simultaneously. Extensive comparison between all-green leaves is necessary. Our methodology will provide a leaves and green sectors of variegated leaves is necessary convenient and useful means for analyzing the effects of to examine this possibility. The final conclusion awaits chloroplast dysfunction on respiratory and/or further investigations of various variegated plant species. photosynthetic activity, especially in leaves with a marble-like variegation pattern. Effects of the presence of non-photosynthetic white mesophyll cells on CO2 compensation point Effects of chloroplast dysfunction on the photosynthetic CO2 compensation point (the CO2 level where gross capacity of the neighboring green mesophyll cells photosynthesis equals respiration) is an important and In contrast to the respiratory rates that were nearly useful parameter to distinguish C4 from C3 plants

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(Edwards and Walker 1983); in general, CO2 chloroplast development affects mitochondrial gene compensation point is nearly zero in C4 plants, but is 50 and transcript levels. Plant J. 19: 635-643. to 70 ppm in C3 plants. H. helix leaves with high GAI Hess, W. R., Prombona, A., Fieder, B., Subramanian, A. exhibited a CO2 compensation point of representative C3 R., Börner, T. (1993) Chloroplst rps 15 and the rpo plants. However, our results also demonstrated that B/C1/C2 gene cluster are strongly transcribed in leaves with low GAI exhibited extraordinarily high CO2 ribosome-deficient plastids: evidence for a compensation points. This result demonstrated that CO2 functioning non-chloroplast-encoded RNA compensation point could be elevated when an extremely polymerase. EMBO J. 12: 563-571. large amount of non-photosynthetic cells were included Kato, Y., Miura, E., Matsushima, R., Sakamoto, W. in the focal photosynthetic organ. Although the (2007) White leaf sectors in yellow variegated2 are magnitude of elevation was striking, our result is quite formed by viable cells with undifferentiated plastids. reasonable because CO2 compensation point is Plant Physiol. 144: 952-960. determined by the balance between gross photosynthetic Martínez-Zapater, J. M., Gil, P., Capel, J., Somerville, rate and respiratory rate. C.R. (1992) Mutations at the Arabidopsis CHM locus promote rearrangements of the mitochondrial genome. ACKNOWLEDGEMENTS Plant Cell 4: 889-899. The authors would like to thank Dr. A. Furukawa, who Nott, A., Jung, H.-S., Koussevitzky, S., Chory, J. (2006) died in July 2008, for his technical advice at the start of Plastid-to-nucleus retrograde signaling. Annu Rev. this study. The authors also thank Dr. T. Mimura and Dr. Plant Biol. 57: 739-759. S. Sakaguchi for their useful discussion, and Miss M. Sakamoto, W., Kondo, H., Murata, M., Motoyoshi, F. Takusagawa and Miss Y. Sawai for their critical reading (1996) Altered mitochondrial gene expression in a of the manuscript. This work was partly supported by maternal distorted leaf mutant of Arabidopsis induced Grants-in-Aid for Scientific Research on Priority Areas by chloroplast mutator. Plant Cell 8: 1377-1390. (No. 16085208 to A.S.) from the Japan Society for the Stoike, L. L., Sears, B. B. (1998) Plastome promotion of Science. mutator-induced alterations arise in Oenothera chloroplast DNA through template slippage. Genetics REFERENCES 149: 347-353. Aluru, M. R., Bae, H., Wu, D., Rodermel, S. R. (2001) Susek, R. E., Ausubel, F. M., Chory, J. (1993) Signal The Arabidopsis immutans mutation affects plastid transduction mutants of Arabidopsis uncouple nuclear differentiation and the morphogenesis of white and CAB and RBCS gene expression from chloroplast green sectors in variegated plants. Plant Physiol. 127: development. Cell 74: 787-799. 67-77. Wetzel, C. M., Jiang, C.-Z., Meehan, L. J., Voytas, D. F., Aluru, M. R., Yu, F., Fu, A., Rodermel, S. (2006) Rodermel, S. R. (1994) Nuclear-organelle Arabidopsis variegation mutants: new insights into interactions: The immutans variegation mutant of chloroplast biogenesis. J. Exp. Bot. 57: 1871-1881. Arabidopsis is plastid autonomous and impaired in Edwards, G. E., Walker, D. A. (1983) C3, C4: Mechanism, carotenoid biosynthesis. Plant J. 6: 161-175. and cellular and environmental regulation, of Yoshida, K., Watanabe, C., Kato, Y., Sakamoto, W., photosynthesis. Blackwell Scientific Publications, Noguchi, K. (2008) Influence of chloroplastic Oxford. photo-oxidative stress on mitochondrial alternative Han, C.-D., Coe, E. H. Jr, Martienssen, R. A. (1992) oxidase capacity and respiratory properties: a case Molecular cloning and characterization of iojap (ij), a study with Arabidopsis yellow variegated 2. Plant pattern striping gene of maize. EMBO J. 11: Cell Physiol. 49: 592-603. 4037-4046. Hedtke, B., Wagner, I., Börner, T., Hess, W. R. (1999) Received: 24 January 2009 / Accepted: 23 February 2009 Inter-organellar crosstalk in higher plants: impaired

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