C 4 Mesophyll and Bundle Sheath

7/27/2019 C 4 Mesophyll and Bundle Sheath

1/14

Corresponding author: E-mail, [email protected]; Fax, +81-52-789-4063.

Plant Cell Physiol.50(10): 17361749 (2009) doi:10.1093/pcp/pcp116, available online at www.pcp.oxfordjournals.org The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

In C4 plants, mesophyll (M) chloroplasts are randomlydistributed along the cell walls, while bundle sheath (BS)chloroplasts are typically located in either a centripetal or

centrifugal position. We investigated whether theseintracellular positions are affected by environmentalstresses. When mature leaves of finger millet (Eleusinecoracana) were exposed to extremely high intensitylight, most M chloroplasts aggregatively re-distributed tothe BS side, whereas the intracellular arrangement ofBS chloroplasts was unaffected. Compared with thehomologous light-avoidance movement of M chloroplastsin C3 plants, it requires extremely high light (3,0004,000 mol m2s1) and responds more slowly (distinctivemovement observed in 1 h). The high light-inducedmovement of M chloroplasts was also observed in maize(Zea mays), another C4species, but with a distinct pattern

of redistribution along the sides of anticlinal walls,analogous to C3plants. The aggregative movement of Mchloroplasts occurred at normal light intensities (250500 mol m2s1) in response to environmental stresses,such as drought, salinity and hyperosmosis. Moreover, there-arrangement of M chloroplasts was observed in field-grown C4plants when exposed to mid-day sunlight, butalso under midsummer drought conditions. The migrationof M chloroplasts was controlled by actin filaments andalso induced in a light-dependent fashion upon incubationwith ABA, which may be the physiological signaltransducer. Together these results suggest that M and BScells of C4plants have different mechanisms controlling

intracellular chloroplast positioning, and that theaggregative movement of C4M chloroplasts is thought to

be a protective response under environmental stressconditions.

Keywords: C4 photosynthesis Chloroplast Eleusinecoracana Environmental stress Photo-relocationmovement Zea mays.

Abbreviations: BS, bundle sheath; DAPI, 4,6-diamidino-2-phenylindole; DMSO, dimethylsulfoxide; M, mesophyll; ME,malic enzyme.

Introduction

Chloroplasts can change their intracellular positions to opti-mize photosynthetic activity and/or to reduce photodam-age in response to light irradiation (Takagi 2003, Wada et al.2003, Sato and Kadota 2007). Thus, under high intensity

light irradiation, chloroplasts move away from light to mini-mize photodamage, while under low intensity irradiationthey move toward the light to maximize photosynthesis.The motility and positioning of chloroplasts appear to bemediated by actin filaments and/or microtubules (Wadaet al. 2003, Sato and Kadota 2007). A spatial reorganizationof actin filaments occurs during light-dependent redistribu-tion of chloroplasts. Actin filaments not only provide tracksfor chloroplast movement but also anchor the chloroplastsafter photo-orientation (Takagi 2003). These chloroplastphoto-relocation movements are widely observed in a vari-ety of plant species, from green algae to seed plants, althoughlittle attention has been given to C4 plants. There is one

report that some monocotyledonous C4 plants show thechloroplast photo-relocation movement in response to blue

Differential Positioning of C4Mesophyll and Bundle SheathChloroplasts: Aggregative Movement of C4Mesophyll

Chloroplasts in Response to Environmental StressesMasahiro Yamada1, Michio Kawasaki2, Tatsuo Sugiyama3, Hiroshi Miyake1and Mitsutaka Taniguchi1,1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi, 464-8601 Japan2Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, 036-8561 Japan3Research Institute of Life and Health Sciences, Chubu University, Kasugai, Aichi, 487-8501 Japan

1736 Plant Cell Physiol.50(10): 17361749 (2009) doi:10.1093/pcp/pcp116 The Author 2009.

RegularPaper


2/14

light (Inoue and Shibata 1974). However, particular behaviorof the chloroplasts was not described.

C4plants such as maize and finger millet have two typesof photosynthetic cells, mesophyll (M) and bundle sheath

(BS). Both cell types are arranged into a specialized Kranz-type leaf anatomy: BS cells surround the vascular tissueswhile M cells encircle the cylinders of the BS cells. The C4dicarboxylate cycle of photosynthetic carbon assimilation isdistributed between the two cell types and acts as a CO2pump to concentrate CO2 in the BS chloroplasts (Hatch1999, Kanai and Edwards 1999). The site of decarboxylationto feed CO2to BS chloroplasts is different between C4sub-types; in NADP-malic enzyme (ME) type species donation ofCO2from C4acids occurs in BS chloroplasts while in NAD-MEtype species it occurs in mitochondria in BS cells. M and BScells have well developed and numerous chloroplasts. Just asthe M and BS chloroplasts are structurally and functionally

differentiated, so their intracellular orientation is also differ-ent: M chloroplasts of all C4species are randomly distributedalong the cell walls, while BS chloroplasts are typically locatedin the centripetal position (close to the vascular tissue, as infinger millet) or the centrifugal position (close to M cells, asin maize). The intracellular orientation of BS chloroplasts isthought to have physiological significance. The centrifugalposition of BS chloroplasts is advantageous to metaboliteexchange between M cells and BS chloroplasts. In contrast,the centripetal position of BS chloroplasts maximizes thelength of the CO2diffusion pathway between BS and M cells,and minimizes CO2leakage from BS cells to M cells (Hattersleyand Browning 1981, von Caemmerer and Furbank 2003).The intracellular arrangement of BS chloroplasts is acquiredduring cell maturation (Miyake and Yamamoto 1987). Amechanism for keeping chloroplasts in the home positionoperates after establishment of the intracellular dispositionof chloroplasts, since the original arrangement of chloro-plasts can be re-established 12 h after disturbance bycentrifugation (Kobayashi et al. 2009). The intracellular posi-tioning of M and BS chloroplasts is dependent on the acto-myosin system and cytosolic protein synthesis, but nottubulin or light (Miyake and Nakamura 1993, Kobayashiet al. 2009). These findings suggest that M and BS cells in C4plants have different systems for chloroplast positioning; anM cell-specific system for dispersing chloroplasts and a BScell-specific system for holding chloroplasts in the centripe-tal or centrifugal disposition. These unique arrangements ofC4chloroplasts are thought to be caused by the cytoskeletalnetwork and vacuolar pressure (Kobayashi et al. 2009), butthe molecular mechanism is obscure at present.

A change in the intracellular disposition of C4chloroplastsin response to environmental stresses other than light wasinitially reported by Lal and Edwards (1996). They describedthat the chloroplasts and cytosol in M cells of drought-stressed maize, a monocot NADP-ME type C4 plant,

collapsed inwardly and BS chloroplasts lost their centrifugalposition. The effect of drought stress on chloroplast positionin Amaranthus cruentus, a dicot NAD-ME type C4plant, isnot as pronounced as for maize. However, the detailed

behavior of chloroplasts and its molecular mechanism werenot mentioned in the report. To gain a better understandingof chloroplast relocation movement in C4plants, we closelyinvestigated the intracellular disposition of chloroplasts inresponse to various environmental stresses and plant hor-mones in this study. When mature leaves of finger millet andmaize were exposed to high intensity light, M chloroplastsshowed aggregative movement but BS chloroplasts did not.The orientation movement of M chloroplasts was alsoobserved under natural growing conditions with high sun-light, salinity or drought stress. These findings suggest thatM and BS cells are also differentiated regarding the controlof intracellular chloroplast positioning in response to

environmental changes.

Results

Effect of light on the intracellular positions of M andBS chloroplasts in finger millet

M cells in leaf blades of finger millet, an NAD-ME type C 4plant, have a great number of chloroplasts dispersedrandomly along the cell walls, while BS cells have larger chlo-roplasts that are located in the centripetal position. A fiberilluminator with white light was used to illuminate leafblades attached to plants, at light intensities of 250, 2,000,3,000 or 4,000 mol quanta m2s1for 2 h (Fig. 1, Supple-mentary Fig. S1). Most of the M chloroplasts were dispro-portionately re-distributed to the BS side in response to thelight, and the centripetal positioning of M chloroplasts wasmore distinct at intensities >3,000 mol m2s1. Although afraction of the M chloroplasts did not migrate to the BS side,there is a possibility that the M chloroplasts gather to cir-cumvent high intensity light. In contrast, the centripetalarrangement of BS chloroplasts was unchanged, even thoughthey appeared to swell slightly after strong light illuminationand the degree of chloroplast association was marginallyreduced.

We also investigated the time course of M chloroplastmovement in response to strong light irradiation. Slightaggregative movement of M chloroplasts was observed 0.5 hafter strong light irradiation, and the one-sided distributionof M chloroplasts became more remarkable in a time-dependent manner (Fig. 2, Supplementary Fig. S2).

Effect of high intensity light on the intracellularpositions of maize chloroplasts

Maize is an NADP-ME type C4 plant. Compared with theNAD-ME type finger millet, maize has more numerous Mchloroplasts, while its BS chloroplasts are smaller and located

1737

Stress-responsive movement of C4chloroplasts

Plant Cell Physiol.50(10): 17361749 (2009) doi:10.1093/pcp/pcp116 The Author 2009.


3/14

in the centrifugal position (Fig. 3A, B). Given these differences,we examined whether maize also shows re-arrangement ofchloroplasts in response to high intensity light. After 2 h ofstrong light irradiation, maize M chloroplasts redistributed,but were found along the sides of anticlinal walls parallel tothe direction of irradiation (Fig. 3C, D). This re-arrangementpattern was obviously different from the pattern observed infinger millet in which M chloroplasts mainly migratedtowards the BS side and formed a partial ring around a cylin-der of BS cells. The centrifugal positioning of chloroplasts inmaize BS cells was not changed under the high intensitylight.

Effects of environmental stresses on the intracellularpositioning of chloroplasts

The extremely strong light intensity (>3,000 mol m2s1)that caused the obvious aggregative movements of M chlo-roplasts described above is far greater than plants encounterunder normal growing conditions. This strong light irradi-ance induced strong photoinhibition, because the PSII maxi-mum quantum yield (Fv/Fm) of finger millet leaf bladesdeclined from 0.8 to near 0.5 after 2 h of the strong light(4,000 mol m2s1) treatment. Therefore, there is a possibilitythat some stress provoked by photoinhibition acts as a trig-ger for the aggregative movement of M chloroplasts, in addi-tion to the possibility that strong light itself functions as asignal. We examined the impact of other environmentalstresses on the intracellular disposition of chloroplasts undernormal intensity light. To induce drought stress, watersupply was withheld from finger millet plants growing undernormal intensity light (500 mol m2s1). When leaf bladesclosed and began to fade after 57 d of water shortage, weobserved transverse leaf sections (Fig. 4A, B). Almost all of

the M chloroplasts were distributed towards the BS cells(Fig. 4B). We investigated the relationship between M chlo-roplast movement and water potential in leaf blades of fingermillet after disruption of the water supply (Fig. 4C). Whenthe water potential was between 0.53 and 0.15 MPa, chlo-roplast movement was observed in some sections but not inothers. When water potential was below 0.7 MPa, all of theM chloroplasts showed aggregative movement. The M chlo-roplast movement in response to drought stress was alsoobserved in maize (Supplementary Fig. S3).

Next, we observed the intracellular arrangement of chlo-roplasts in response to salinity or high osmotic stress (Fig. 5).In finger millet exposed to 3%NaCl (1 osmol kg1) in normalintensity light, most of the M chloroplasts migrated towardsthe BS cells but the centripetal arrangement of BS chloro-plasts was unchanged (Fig. 5B). As no significant differencewas observed in the Fv/Fmvalues of leaf blades between thecontrol and NaCl-treated plants (0.750.01, n= 4), it sug-gests that the M chloroplasts in the salinity-treated leavesresponded before the occurrence of photoinhibition. Theaggregative movement of M chloroplasts in salinity-stressedplants was also observed in semi-thin sections prepared fromresin-embedded leaves (Supplementary Fig. S4). M chloro-plasts were distributed toward BS cells but not along the cellwalls directly attached to BS cells. High salinity causes a com-bined stress due to an imbalance of ions and osmotic homeo-stasis. We also investigated the effect of osmotic stress onthe intracellular arrangement of chloroplasts in finger milletby supplying 20%polyethylene glycol (0.52 osmol kg1) as anexternal osmolyte (Fig. 5C). Only the M chloroplasts showeda change in intracellular positioning in response to highosmotic stress, similarly to the salinity stress. Therefore,strong osmotic stress clearly induces aggregative movementof M chloroplasts. Under these stress conditions, no obvious

Fig. 1 Effect of light intensity on the intracellular positions of M and BS chloroplasts in finger millet. Leaf blades of finger millet were continuously

irradiated with white light of intensity 250 (A and B), 2,000 (C and D), 3,000 (E and F) and 4,000 mol m2s1(G and H), respectively, at the adaxial

side for 2 h. B, D, F and H are magnified images. In each panel, the upper side of the leaf sections is the adaxial side. B, bundle sheath cell; M,

mesophyll cell; V, vascular bundle. Scale bars = 50 m.

1738

M. Yamada et al.



4/14

Fig. 2 Changes in the intracellular arrangement of chloroplasts in response to high intensity light. Leaf blades of finger millet were continuously

irradiated with the high intensity light (4,000 mol m2s1). Transverse sections were observed with the light microscope before (A and B) and

after 0.5 (C and D), 1 (E and F), 2 (G and H) and 3 h (I and J) of illumination. Scale bars = 50 m.

1739




5/14

plasmolysis was observed. Furthermore, when leaf segmentsof finger millet were deaerated in 1 M sorbitol (1 osmol kg1)and incubated with the same solution for 4 h in the light,plasmolysis of M cells was observed but the centripetalaggregation of M chloroplasts did not occur (data notshown). Therefore, we conclude that the chloroplast

movement in response to environmental stresses is notcaused directly by plasmolysis, which hardly occurs in plantsgrowing under atmospheric conditions.

To examine whether light irradiation is necessary for thechloroplast movement in response to environmentalstresses, finger millet was subjected to drought or salinity

Fig. 3 Change in the intracellular positions of maize chloroplasts in response to light irradiation. Leaf blades of maize were irradiated for 2 h with

normal intensity (250 mol m2s1) (A and B) or high intensity (4,000 mol m2s1) light (C and D), and transverse sections were examined. B and

D are magnified images. B, bundle sheath cell; M, mesophyll cell; V, vascular bundle. Scale bars = 50 m.

Fig. 4 Change in the intracellular position of chloroplasts in response to drought stress. Finger millet growing under the normal light condition

(500 mol m2s1during the light period) was exposed to drought stress by withholding the water supply. When leaf blades began to fade after

57 d, leaf sections were examined with a light microscope. (A) Control; (B) drought stress. In each panel, the upper side of the leaf sections is the

adaxial side. Scale bars = 50 m. (C) Relationship between M chloroplast movement and water potential in leaf blades. Water potential wasmeasured for 12 d after disruption of the water supply. At the same time, we checked whether M chloroplast movement occurred and the results

were plotted on a graph. The water potentials of non-stressed plants were 0.58 to 0.15 MPa.

1740

M. Yamada et al.



6/14

stress under dark conditions. Although the water potentialof leaf blades exposed to drought or salinity stress for 9 d wasdecreased to 1.830.18 MPa or 0.800.17 MPa (n= 4),respectively, the aggregative movement of M chloroplastswas not observed (data not shown). Therefore, it was con-cluded that light is required for the chloroplast movementin response to environmental stresses.

We also examined the intracellular arrangement of nucleiin response to salinity stress (Fig. 6). Although BS nucleiwere located close to M cells, M nuclei were distributedperipherally at the mid position, a little towards BS cells. Theintracellular positions of both types of nuclei were notchanged regardless of salinity stress. We further observedthe intracellular arrangement of mitochondria (Fig. 7). AllBS mitochondria were dominantly located close to vascularbundles but M mitochondria were randomly distributedin the cells. The intracellular positions of neither type ofmitochondria were changed regardless of salinity stress.

Effects of natural sunlight on the intracellularpositioning of chloroplasts

To investigate whether the chloroplast aggregative move-

ments occur in C4plants under natural conditions, we har-vested leaf blades of finger millet and maize exposed todirect mid-day sunlight in midsummer (1,800 mol m2s1)and a dry environment, and observed the transverse sections(Fig. 8). Only the M chloroplasts in finger millet and maizeshowed the aggregative movement. In finger millet, M chlo-roplast movement was more significant on the adaxial side(upper side of the leaf section) compared with the abaxialside (Fig. 8A). Maize chloroplasts in M cells that were locatedat the adaxial or abaxial side migrated towards the BS cells,similarly to drought stress (Fig. 8B). The Fv/Fmvalues of leafblades from finger millet and maize were 0.370.03 and0.410.03, respectively and, therefore, these plants had

experienced severe photoinhibition. At night-time, M chlo-roplasts of both plants returned to comparatively randompositions along the plasma membranes (Fig. 8C, D). TheFv/Fmvalues were recovered to normal values (0.810.01 forfinger millet and 0.750.01 for maize) after the end of thenight. These findings suggest that change in the intracellulararrangement of M chloroplasts is a general phenomenon infield-growing C4plants that are exposed to multiple envi-ronmental stresses, which cause severe photoinhibition.

Involvement of actin filaments in the intracellulararrangement of chloroplasts in response to stronglight irradiation

We investigated whether actin filaments participate in Mchloroplast movement in response to light irradiation.Cytochalasin B is a potent inhibitor of actin polymerization,and we had previously confirmed by immunodetection thatour pre-treatment of leaf segments with cytochalasin Bdisrupted actin networks (Kobayashi et al. 2009). Treatmentof finger millet with cytochalasin B showed a prominentinhibitory effect on the strong light-dependent movementof M chloroplasts, in contrast to treatment with dimethyl-sulfoxide (DMSO) as a control (Fig. 9A, B). Cytochalasin Bdid not affect the disposition of M chloroplasts under normalintensity light (Figs. 9C, D). The centripetal position of BSchloroplasts was unchanged irrespective of cytochalasin Btreatment.

Effect of plant hormones on the intracellularpositioning of chloroplasts

ABA accumulates and functions as a signal transducer inresponse to environmental stresses such as drought and soilsalinity (Zhang et al. 2006). To investigate the possibility ofthe involvement of ABA in the chloroplast movement inresponse to environmental stresses, we allowed leaf

Fig. 5 Change in the intracellular arrangement of chloroplasts in

response to salinity or high osmotic stress. Finger millet was supplied

with 3%NaCl or 20%polyethylene glycol solution to produce salinity

and high osmotic stress, respectively, for 5 d in normal intensity light

(500 mol m2s1during the light period), and transverse sections ofleaf blades were examined. (A) Control; (B) salinity stress; (C) high

osmotic stress. Scale bars = 50 m.

1741




7/14

segments from non-stressed finger millet to absorb ABAduring incubation for 16 h under low intensity light. ThisABA treatment induced the centripetal assembly of M chlo-roplasts (Fig. 10). We confirmed that treatment with ABAabove 3 M was effective in causing this arrangement ofchloroplasts. When the incubation with ABA was conducted

in the dark, the chloroplast movement did not occur(data not shown). Incubation with other plant hormones(IAA, 2,4-D, GA3 and kinetin) in the light had no effect onthe intracellular positioning of chloroplasts (data notshown). Various concentrations of NaCl (0.33%) and H2O2(1100 mM) also had no effect (data not shown).

Fig. 6 Effect of salinity stress on the intracellular positions of nuclei. Transverse sections of leaf blades from control (A and B) or salinity-stressed

(C and D) finger millet were stained with DAPI and observed under a bright-field (A and C) or fluorescence (B and D) microscope. B and D are

merged images of the bright-field and fluorescence images. Nuclei were detected as white particles in cells. Scale bars = 50 m.

Fig. 7 Effect of salinity stress on the intracellular positions of mitochondria. Transverse sections of leaf blades from control (AC) or salinity-

stressed (DF) plants were stained with rhodamine 123. Mitochondria (yellow) and chloroplasts (red) were imaged using confocal laser scanning

microscopy. C and F are enlarged images of M cells, and the right side in the two panels is the BS side. Scale bars = 50 m.

1742

M. Yamada et al.



8/14

Fig. 8 Aggregative movement of M chloroplasts in field-grown finger millet and maize in midsummer. Leaf blades of finger millet (A and C) and

maize (B and D) growing under natural midsummer conditions with high radiation and a dry environment were sampled in the middle of the day

(14:00 h; atmosphere temperature, 35C; light intensity, 1,800 mol m2s1; A and B) or during the night (3:00 h; atmosphere temperature, 26C;

C and D) of a fair day, and transverse sections were examined. In each panel, the upper side of the leaf sections is the adaxial side. Scale

bars = 50 m.

Fig. 9 Effect of cytochalasin B on the intracellular arrangement of chloroplasts in response to light irradiation. Leaf segments excised from leaf

blades of finger millet were deaerated in 0.5%(v/v) dimethyl sulfoxide (DMSO) with or without 50 M cytochalasin B, and floated on the

solution for 2 h under room light (


9/14

Discussion

Stronger light and longer exposure times arerequired for aggregative movement of C4Mchloroplasts compared with C3M chloroplasts

Photo-relocation movement of chloroplasts is widelyobserved in a variety of plant species. In this study, we found

that M chloroplasts of C4plants showed aggregative move-ment in response to strong light. Extremely high light inten-sities >3,000 mol m2s1were needed to induce an obviousmovement of M chloroplasts in normally growing C4plants(Fig. 1, Supplementary Fig. S1). Inoue and Shibata (1974)reported that absorbance of leaves from five graminaceousC4species decreased in response to blue light (about 86 molquanta m2s1), but the light intensity was much lower thanthat necessary for the obvious aggregative movementinduced by white light in our experiment. They used leavesincubated in darkness for 1 d before measurement and,therefore, the leaves might become more susceptible tolight. We also confirmed that blue light could induce thecentripetal positioning of M chloroplasts but the extent oflocalization was not prominent (data not shown). Inoue andShibata did not report the precise migration pattern of chlo-roplasts, and the wavelength dependency of the aggregativemovement remains to be investigated.

The aggregative movement of M chloroplasts in C4and C3plants differs in light intensity and time required. C3M chloro-plasts respond to much lower light intensities than C4M chlo-roplasts. For example, the apparent light avoidance movementof chloroplasts in dark-adapted Arabidopsis thaliana leaf

occurs upon illumination with blue light at 5 W m2(about19 mol m2s1) (Trojan and Gabrys 1996). The extent ofchloroplast avoidance movement inA. thalianaincreases inresponse to the intensity of white light and reaches a maxi-

mum at about 500 mol m2s1(Kasahara et al. 2002). Simi-larly, the maximum chloroplast movement in redwood sorreloccurs upon illumination with blue light at 250 mol m2s1(780 mol m2s1of daylight) (Brugnoli and Bjrkman 1992).The time required for obvious observation of chloroplastmovement is also shorter in C3plants. For example, chloro-plasts of redwood sorrel, sunflower and Arabidopsis start tomove after only a few minutes of light irradiation (Brugnoliand Bjrkman 1992, Trojan and Gabrys 1996). In leaf epider-mal cells of the aquatic angiosperm Vallisneria gigantea,about half the chloroplasts move out of the area irradiatedwith high intensity blue light within the first 15 min of irra-diation, and the percentage increases to 80%after 30 min

(Sakurai et al. 2005). In contrast, the extent of chloroplastmovement was low after 30 min of high intensity light irra-diation (Fig. 2, Supplementary Fig. S2) and, therefore, theresponse of C4 M chloroplasts to strong light seems to beslow. C4plants generally adapt to high intensity light and,therefore, C4photosynthetic cells might not be as suscepti-ble to light-inducing stresses in comparison with C3M cells.Moreover, growth conditions might be another factor toyield the differential light responsiveness. C3plants are gen-erally grown under lower intensity light compared with C 4plants and, therefore, photoinhibition and chloroplastmovement for photoprotection in C3plants is more likely tooccur at relatively low light intensities.

Aggregative movement of C4M chloroplasts wasinduced in response to environmental stresses

The chloroplast movement in M cells of finger milletoccurred under normal intensity light (500 mol m2s1)under stress conditions such as drought, salinity or hyperos-mosis (Figs. 4, 5). These abiotic stresses are thought toreduce the threshold intensity of light at which aggregativemovement of M chloroplasts occurs. Ionic and osmoticstresses originating from salinity cause damage to metabolicprocesses and the ultrastructure of chloroplasts (Yamane et al.2003, Hasan et al. 2005, Morales et al. 2006, Omoto et al.2009). Although supplying plant roots with 20%polyethyl-ene glycol solution induced re-arrangement of M chloro-plasts (Fig. 5C), incubation of leaf segments with 1 M sorbitolor 3%NaCl solutions whose osmolality was twice as high asthat of the polyethylene glycol solution had no effect onchloroplast arrangement. Therefore, it is thought that somesignal associated with the osmotic stress is generated in adomain outside of leaf tissue and influences M chloroplastmovement. A decrease in water potential during watershortage is also important in M chloroplast re-arrangement(Fig. 4C). However, another factor may be involved in the

Fig. 10 Effect of ABA on the intracellular arrangement of chloroplasts.

Leaf segments excised from leaf blades of finger millet were deaerated

in 0.1% ethanol with or without 10 M ABA and floated on

the solution for 16 h under low intensity light (100 mol m2s1).

(A) Control; (B) ABA treatment. Scale bars = 50 m.

1744

M. Yamada et al.



10/14

induction of chloroplast movement, because M chloroplastmovement was occasionally observed in leaves showing highwater potential above 0.53 MPa.

Previously it was reported that water stress induced cen-

tripetal re-arrangement of M chloroplasts in leaves of the C4plant, maize (Lal and Edwards 1996) and the C4Crassulaceanacid metabolism (CAM) cycling plant, Portulaca grandiflora(Guralnick et al. 2002). Their intracellular localization is simi-lar to the typical aggregative arrangement of M chloroplastswhich we observed in finger millet, but not in maize, duringhigh light stress. The chloroplast rearrangement of C4M cellsis thought to be induced by a combination of light and envi-ronmental stresses. In our experiments, most M chloroplastsin finger millet leaves moved towards the BS, but some Mchloroplasts remained scattered around the opposite side.In maize leaves irradiated by high intensity light, M chloro-plasts were distributed along the sides of the anticlinal walls

(Fig. 3C, D), but the direction of chloroplast movement infield-growing water-stressed maize was rather towards theBS (Fig. 8B), similar to the observation of Lal and Edwards(1996) under drought stress. Therefore, C4M chloroplastsmight show light avoidance movement similar to C3M chlo-roplasts, but prominent aggregation of M chloroplastsoccurs in C4plants that receive severe stresses for long peri-ods of time.

C4plants attain higher rates of photosynthesis in full sun-light and are also more efficient in water use compared withC3plants (Hatch 1992). As a result, C4plants are said to bemore tolerant to environmental stresses. We found aggrega-tive movement of M chloroplasts of finger millet and maizegrowing in a field in midsummer (Fig. 8). The leaf surface atthat time was exposed to a light intensity of about 1,800 molm2s1, which was not high enough to induce chloroplastmovement in the laboratory. The field-grown plants can besubject to other stresses in addition to high intensity light.Under the mid-day field condition, plants were exposed tostrong light and high temperature for several hours. Althoughplants were well watered, a high transpiration rate maynonetheless cause low leaf water potential (Hirasawa andHsiao 1999). Indeed, the field-growing plants that we mea-sured showed severe photoinhibition at mid-day. Thus, acombination of stresses may induce chloroplast movementin C4plants in the field. The intracellular disposition of Mchloroplasts changes diurnally as the aggregative arrange-ment is partially eliminated at night-time when plantsrecover from photoinhibition.

Possible physiological roles of the aggregativeM chloroplast movement

A study with Arabidopsis mutants revealed that chloroplastavoidance movement decreases the amount of light absorp-tion by chloroplasts, and therefore protects plants fromphotodamage under high light (Kasahara et al. 2002). C4

plants growing under environmental stresses are exposed toan excess of light energy and are subjected to photoinhibi-tion (Lal and Edwards 1996, Jia and Lu 2003, Xu et al. 2008).Under these conditions, the assemblage of M chloroplasts is

thought to provide photoprotection through mutual shad-ing of the chloroplasts, similarly to C3chloroplasts. Actually,we observed an increase in light transmittance through leafblades in response to high intensity light (SupplementaryFigs. S1, S2). Although the degree of PSII photoinhibition byhigh intensity light is similar between M and BS thylakoids ofmaize (Pokorska and Romanowska 2007), M chloroplasts aremore sensitive to the damaging effect of salinity than are BSchloroplasts (Hasan et al. 2005, Omoto et al. 2009). Previ-ously, we found that salinity-induced damage in M chloro-plasts of maize and rice is light dependent, and not due todirect effects of excessive accumulation of sodium in the leaftissues (Mitsuya et al. 2003, Hasan et al. 2005). We therefore

assumed that reactive oxygen species are involved in thechloroplast damage induced by salinity (Mitsuya et al. 2003,Yamane et al. 2004a, Yamane et al. 2004b, Hasan et al. 2005).Moreover, the distribution of antioxidant enzymes isreported to be different between M and BS cells in maize(Doulis et al. 1997, Foyer et al. 2002). It is presumed that anti-oxidant status could be different between the photosyn-thetic cell types under stress conditions. Under the salinitystress that caused aggregative movement of M chloroplasts(Fig. 5B), symptoms of photoinhibition were not observed.The C4M chloroplast movement may be one means of pho-toprotection which occurs prior to photoinhibition.

Another possible role of C4 M chloroplast movement ismaintenance of photosynthetic activity under stress condi-tions. Most M chloroplasts in finger millet moved towardthe BS, unlike C3chloroplasts that migrate to the cell wallsparallel to strong light. The centripetal aggregation of C4Mchloroplasts might be to enable communication with BScells. The centripetal position of M chloroplasts shortens thediffusion pathway of metabolites between M and BS cells,and may contribute to keeping C4 photosynthesis active.Moreover, leakiness of CO2 from BS cells is increased instressed C4 plants (Ghannoum 2009). M chloroplasts andcytosol might move towards the BS to refix CO2 releasedfrom BS cells more efficiently. However, the centripetalaggregation of M chloroplasts towards the BS side couldincrease the diffusion distance between the intercellular airspace and the primary carboxylation step (cytosolic phos-phoenolpyruvate carboxylase and M chloroplast) and, there-fore, decrease the production of C4dicarboxylates (Lal andEdwards 1996, Tholen et al. 2008). Indeed, the chloroplastavoidance response inA. thalianaleaves results in a smallerchloroplast surface area adjacent to intercellular airspacesand decreases internal conductance to CO2diffusion (Tholenet al. 2008). Attempts to characterize the relationship betweenchloroplast disposition and photosynthetic parameters are

1745




11/14

currently in progress to determine whether C4plants adaptto refix released CO2under environmental stress conditionswhich lead to stomatal closure.

Molecular mechanism of M chloroplast movementA potent inhibitor of actin polymerization, cytochalasin B,inhibited the aggregative movement of M chloroplasts inresponse to high intensity light (Fig. 9). Therefore, actin fila-ments are considered to participate in the M chloroplastmovement. Actin filaments encircle M and BS chloroplastsof finger millet and maize, and seem to be involved in theirpositioning and anchorage (Kobayashi et al. 2009). The acto-myosin system is necessary for arrangement of both chloro-plasts during cell maturation and rearrangement ofchloroplasts after disturbance by centrifugal force (Miyakeand Nakamura 1993, Kobayashi et al. 2009). The involve-ment of actin filaments as a track in chloroplast photo-

relocation movement has been confirmed in several C3plantspecies by pharmacological studies (Wada et al. 2003). Abasket structure of microfilaments surrounding ArabidopsisM chloroplasts was observed with immunofluorescent label-ing (Kandasamy and Meagher 1999). Actin filaments changetheir organization before and after chloroplast movement,and also function in anchoring chloroplasts to the site(Takagi 2003). As C4 M chloroplasts move toward the BSside, the M cells might possess a system for determining cellpolarity and machinery for polarized motility. Whether asimilar motility system for chloroplast movement works inboth C3and C4plant cells remains to be investigated.

Even though M chloroplasts show aggregative movement

in response to salinity stress, nuclei and mitochondria didnot change their positions (Figs. 6, 7). In contrast, light-dependent nuclear positioning was reported in leaf cells ofA. thalianaand prothallial cells ofAdiantum capillus-veneris(Iwabuchi et al. 2007, Tsuboi et al. 2007). While both thenuclear and chloroplast photo-relocation movements sharephotoreceptors and cytoskeletons, some componentsinvolved in the moving machinery are thought to be specificto each organelle (Iwabuchi et al. 2007). Recently, blue light-induced co-localization of mitochondria with chloroplastswas shown in Arabidopsis palisade M cells (Islam et al. 2009).The authors presumed a relationship of the co-localizationwith their mutual metabolic interactions. The nuclear andmitochondrial movement in C3leaves is speculated to be anadaptive response for light as well as chloroplast photo-relocation movement, while the aggregative movement ofC4M chloroplasts independent of nuclei and mitochondriamay be induced for a special physiological requirement asso-ciation with C4photosynthesis.

Treatment of finger millet leaf segments with ABAinduced the centripetal assembly of M chloroplasts in alight-dependent manner (Fig. 10). Because ABA was vacuuminfiltrated into the leaf segments, M chloroplast movement

is thought to be caused by a direct effect of ABA on M cellsand not by secondary effects such as stomatal closure. Par-ticipation of ABA in chloroplast movement has also beenreported in succulent plants (Kondo et al. 2004). Clumping

of chloroplasts in response to water stress was first found incortical cells of P. grandiflorastems (Guralnick et al. 2002).After that, Kondo et al. (2004) showed that chloroplasts in avariety of succulent CAM plants become densely clumpedunder combined light and water stress. The chloroplastclumping induced by ABA is dependent on light. ABA, whichis a signal transducer in response to environmental stresses,is proposed to function as a trigger for the chloroplast move-ments in C4and CAM plants. Because M chloroplast move-ment occurred in the leaf segments irradiated with highintensity light (Fig. 9A), ABA may be synthesized in theleaves and initiate chloroplast movement, as well as ABAwhich is synthesized in roots and transported to leaves. Light

is essential to chloroplast movement induced by ABA, and itis also required for the aggregative movement of C4M chlo-roplasts in response to drought or salinity stress. Under envi-ronmental stress conditions, a decrease in consumption ofreducing equivalents can result in accumulation of electronsin the photosynthetic electron transport chain, that pro-duces harmful reactive oxygen species. Thus, reactive oxygenspecies are another potential trigger for chloroplast move-ment. Indeed, it was reported that hydrogen peroxide is gen-erated by high fluence blue light in Arabidopsis M cells andwas suggested to promote chloroplast avoidance movementin the presence of blue light (Wen et al. 2008). However, theincubation of leaf segments of finger millet with various con-centrations of hydrogen peroxide had no effect on the intra-cellular arrangement of chloroplasts. This indicates thathydrogen peroxide itself cannot induce chloroplast move-ment in C4plants, but further work is required to determinewhether other reactive oxygen species affect chloroplastmovement in C4M cells.

In summary, the present study has shown the aggregativemovement of C4M chloroplasts in response to environmen-tal stresses. The movement is light dependent, and evidenceis provided that it is mediated by ABA. At present, thephysiological significance and molecular mechanism of thechloroplast response are unknown and need further study.

Materials and Methods

Plant materials and growth conditions

Finger millet (Eleusine coracana L. Gaertn. cv. Yukijirushi)and maize (Zea maysL. cv. Golden Cross Bantam T51) weregrown in vermiculite in a growth chamber with 14 h of illu-mination (500 mol m2s1) at 28C and 10 h of darkness at20C per day. Plants were fertilized regularly with Arnon andHoagland solution (Arnon and Hoagland 1940) duringgrowth. The middle regions of the fourth leaf blades from

1746

M. Yamada et al.



12/14

plants of about 4 weeks old were normally used forexperiments.

The experiments with field-growing plants were con-ducted in August 2008 at the University Farm of Nagoya

University. Plants were grown in well-watered and periodi-cally fertilized soil for 10 weeks, and fully-matured leaveswere used for experiments.

High-light treatment

A fiber illuminator illuminated the middle regions of thefourth leaf blades with a halogen lamp (MHF-150L, Moritex,Tokyo, Japan or PICL-NEX, Nippon P-I Co. Ltd., Tokyo, Japan)at a distance of 2.5 cm. The photosynthetic photon flux den-sity at the leaf surface was checked with a quantum meter(LI-250, LI-COR, Lincoln, NE, USA). Small segments (55 mmsquare) were excised from the treated leaf blades and vacuuminfiltrated for 10 min with fixation buffer [50 mM PIPES-

NaOH, pH 6.9, 4 mM MgSO4, 10 mM EGTA, 0.1%(w/v) TritonX-100, 200 M phenylmethylsulfonyl fluoride, 5%(v/v) form-aldehyde and 1% (v/v) glutaraldehyde]. After incubation at4C overnight, the fixed segments were embedded in 5%(w/v) agar and sectioned at 7080 m with a micro-slicer(DTK-3000W, Dosaka EM, Kyoto, Japan). Transverse sectionswere observed with a light microscope (BX51, Olympus,Tokyo, Japan) equipped with a CCD camera (DP70, Olympus).Chlorophyll fluorescence was measured with a portable chlo-rophyll fluorometer PAM-2100 (Walz, Effeltrich, Germany).

Stress treatment

Three- to four-week-old plants were exposed to drought

stress by withholding water supply until the appearance ofthe first sign of wilting. Leaf segments were then excisedfrom the upper developed leaf blades and fixed as describedabove. Transverse sections were observed with the lightmicroscope. Water potential in leaves was measured with aWP4 Dewpoint Meter (Decagon Devices, Pullman, WA,USA).

Three plants per pot were grown in a 300 ml plastic potfilled with vermiculite in the growth chamber. High salinitytreatment was achieved by supplying 30 ml per day of Arnonand Hoagland solution containing 3%(w/v) NaCl for 5 d. Forhigh osmotic stress, 15 ml d1 of 20% (w/v) polyethyleneglycol 6,000 solution was supplied for 5 d. Transversesections of the fixed leaf segments were observed withthe light microscope. The osmolality values of the solutionswere determined by the freezing point method in anOsmotoron-5 (Orion Riken Inc., Tokyo, Japan).

For microscopic observation of semi-thin sections, leafsegments were fixed as previously reported (Omoto et al.2009). Semi-thin sections (1 m thickness) were cut withglass knives on an ultramicrotome. Then, the sections werestained with toluidine blue O and observed with the lightmicroscope.

Nuclear and mitochondrial staining

For nuclear staining, leaf segments from the salinity-stressedplants were fixed as described above and transverse sections

were stained with 1 mg ml1

4,6-diamidino-2-phenylindole(DAPI) for 1 h. After washing with distilled water for 10 min

twice, the sections were imaged with a light microscope(BX51, Olympus) equipped with an epifluorescence system(U-LH100HG, Olympus).

For mitochondrial staining, non-fixed leaf segments fromthe salinity-stressed plants were tucked into carrot blocksand sectioned with a microslicer. Transverse sections werestained in PME buffer (50 mM PIPES-NaOH, pH 6.9, 5 mMMgSO4, 5 mM EGTA and 0.15 M NaCl) containing 1 M rho-damine 123 for 4 min. After washing with PME buffer for10 min twice, the sections were imaged with a confocal laserscanning microscope (LSM5 PASCAL, Carl Zeiss, Germany).

Rhodamine 123 was excited with the 488 nm wavelength ofan ArKr laser and the images were collected using a BP505530 bandpass filter. Autofluorescence of chloroplasts wasexcited with the 543 nm wavelength of a HeNe laser andimaged using an LP560 longpass filter. Serial confocal opticalimages at 0.50 m intervals were collected, and projectionsof 2040 m thickness were created with LSM ImagingBrowser software.

Chemical treatment

For cytochalasin treatment, small leaf segments (5 5 mmsquare) were excised and vacuum infiltrated for 10 min with0.5%(v/v) DMSO with or without 50 M cytochalasin B (MP

Biomedicals, Irvine, CA, USA). After floating on the samesolution for 2 h, the leaf segments were exposed to normal(250 mol m2s1) or high light (4,000 mol m2s1) for 2 h.Then, the leaf segments were fixed and transverse sectionswere observed with a light microscope.

For ABA treatment, small leaf segments were excised andvacuum infiltrated for 10 min with 0.1%(v/v) ethanol withor without 10 M ABA. After floating on the same solutionfor 16 h under low light (100 mol m2s1), the leaf segmentswere fixed and transverse sections were observed with a lightmicroscope.

For other chemical treatments, small leaf segments wereexcised and vacuum infiltrated for 10 min with 10 mM MES-

KOH (pH 6.9) containing IAA (0.3, 1 or 3 M), 2,4-D (3, 10 or30 M), GA3(15, 50 or 150 M), kinetin (30, 100 or 300 M),ABA (1, 3, 10 or 30 M), NaCl [0.3, 1 or 3%(w/v)] or H2O2(1, 5, 10, 20 or 100 mM). After floating on the same solutionfor 16 h under low light (100 mol m2s1), the leaf segmentswere hand-sectioned with a razor blade and transversesections were observed with a light microscope.

Supplementary data

Supplementary data are available at PCP online.

1747




13/14

Funding

The Ministry of Education, Culture, Sports, Science andTechnology (Grants-in-Aid for Scientific Research No.

21380014).

Acknowledgments

We thank Mr. Yasuki Tahara, University Farm of NagoyaUniversity, for growing the plants.

References

Arnon, D.I. and Hoagland, D.R. (1940) Crop production in

artificial solutions and soils with special reference to factors

influencing yield and absorption of inorganic nutrients. Soil Sci.50:

463471.

Brugnoli, E. and Bjrkman, O. (1992) Chloroplast movements in leaves:

influence on chlorophyll fluorescence and measurements of light-induced absorbance changes related to pH and zeaxanthin

formation. Photosynth. Res.32: 2335.

Doulis, A.G., Debian, N., KingstonSmith, A.H. and Foyer, C.H. (1997)

Differential localization of antioxidants in maize leaves. Plant Physiol.

114: 10311037.

Foyer, C.H., Vanacker, H., Gomez, L.D. and Harbinson, J. (2002)

Regulation of photosynthesis and antioxidant metabolism in maize

leaves at optimal and chilling temperatures: review. Plant Physiol.

Biochem.40: 659668.

Ghannoum, O. (2009) C4photosynthesis and water stress. Ann. Bot.

103: 635644.

Guralnick, L.J., Edwards, G., Ku, M.S.B., Hockema, B. and Franceschi, V.R.

(2002) Photosynthetic and anatomical characteristics in the C4-

crassulacean acid metabolism-cycling plant, Portulaca grandiflora.

Funct. Plant. Biol.29: 763773.

Hasan, R., Ohnuki, Y., Kawasaki, M., Taniguchi, M. and Miyake, H.

(2005) Differential sensitivity of chloroplasts in mesophyll and

bundle sheath cells in maize, an NADP-malic enzyme-type C4plant,

to salinity stress. Plant Prod. Sci.8: 567577.

Hatch, M.D. (1992) C4 photosynthesis: an unlikely process full of

surprises. Plant Cell Physiol.33: 333342.

Hatch, M.D. (1999) C4photosynthesis: a historical overview. InC4Plant

Biology. Edited by Sage, R.F. and Monson, R.K. pp. 1746. Academic

Press, San Diego.

Hattersley, P.W. and Browning, A.J. (1981) Occurrence of the suberized

lamella in leaves of grasses of different photosynthetic types. I. In

parenchymatous bundle sheaths and PCR (Kranz) sheaths.

Protoplasma109: 371401.

Inoue, Y. and Shibata, K. (1974) Comparative examination of terrestrial

plant leaves in terms of light-induced absorption changes due to

chloroplast rearrangements. Plant Cell Physiol.15: 717721.

Islam, M.S., Niwa, Y. and Takagi, S. (2009) Light-dependent intracellular

positioning of mitochondria inArabidopsis thalianamesophyll cells.

Plant Cell Physiol.50: 10321040.

Iwabuchi, K., Sakai, T. and Takagi, S. (2007) Blue light-dependent

nuclear positioning in Arabidopsis thaliana leaf cells. Plant Cell

Physiol.48: 12911298.

Jia, H. and Lu, C. (2003) Effects of abscisic acid on photoinhibition in

maize plants. Plant Sci.165: 14031410.

Kanai, R. and Edwards, G.E. (1999) The biochemistry of C4 photo-

synthesis. InC4Plant Biology. Edited by Sage, R.F. and Monson, R.K.

pp. 4987. Academic Press, San Diego.

Kandasamy, M.K. and Meagher, R.B. (1999) Actinorganelle interaction:

association with chloroplast in Arabidopsis leaf mesophyll cells.Cell Motil. Cytoskel.44: 110118.

Kasahara, M., Kagawa, T., Oikawa, K., Suetsugu, N., Miyao, M. and Wada, M.

(2002) Chloroplast avoidance movement reduces photodamage in

plants. Nature420: 829832.

Kobayashi, H., Yamada, M., Taniguchi, M., Kawasaki, M., Sugiyama, T.

and Miyake, H. (2009) Differential positioning of C4mesophyll and

bundle sheath chloroplasts: recovery of chloroplast positioning

requires the actomyosin system. Plant Cell Physiol.50: 129140.

Kondo, A., Kaikawa, J., Funaguma, T. and Ueno, O. (2004) Clumping

and dispersal of chloroplasts in succulent plants. Planta 219:

500506.

Lal, A. and Edwards, G.E. (1996) Analysis of inhibition of photosynthesis

under water stress in the C4 speciesAmaranthus cruentusand Zea

mays: electron transport, CO2fixation and carboxylation capacity.Aust. J. Bot.23: 403412.

Mitsuya, S., Kawasaki, M., Taniguchi, M. and Miyake, H. (2003) Light

dependency of salinity-induced chloroplast degradation. Plant Prod.

Sci.6: 219223.

Miyake, H. and Nakamura, M. (1993) Some factors concerning the

centripetal disposition of bundle sheath chloroplasts during the leaf

development of Eleusine coracana.Ann. Bot.72: 205211.

Miyake, H. and Yamamoto, Y. (1987) Centripetal disposition of bundle

sheath chloroplasts during the leaf development of Eleusine

coracana.Ann. Bot.60: 641647.

Morales, F., Abadia, A. and Abadia, J. (2006) Photoinhibition and

photoprotection under nutrient deficiencies, drought and salinity.

In Photoprotection, Photoinhibition, Gene Regulation, and

Environment. Edited by Demmig-Adams, B., Adams, W.W.I. andMattoo, A.K. pp. 6585. Springer, The Netherlands.

Omoto, E., Kawasaki, M., Taniguchi, M. and Miyake, H. (2009)

Salinity induces granal development in bundle sheath chloroplasts

of NADP-malic enzyme type C4 plants. Plant Prod. Sci. 12:

199207.

Pokorska, B. and Romanowska, E. (2007) Photoinhibition and D1

protein degradation in mesophyll and agranal bundle sheath

thylakoids of maize. Funct. Plant Biol.34: 844852.

Sakurai, N., Domoto, K. and Takagi, S. (2005) Blue-light-induced

reorganization of the actin cytoskeleton and the avoidance response

of chloroplasts in epidermal cells of Vallisneria gigantea. Planta221:

6674.

Sato, Y. and Kadota, A. (2007) Chloroplast movements in response to

environmental signals. InThe Structure and Function of Plastids.

Edited by Wise, R.R. and Hoober, J.K. pp. 527537. Springer,

New York.

Takagi, S. (2003) Actin-based photo-orientation movement of

chloroplasts in plant cells.J. Exp. Biol.206: 19631969.

Tholen, D., Boom, C., Noguchi, K., Ueda, S., Katase, T. and Terashima, I.

(2008) The chloroplast avoidance response decreases internal

conductance to CO2diffusion inArabidopsis thalianaleaves. Plant

Cell Environ.31: 16881700.

Trojan, A. and Gabrys, H. (1996) Chloroplast distribution inArabidopsis

thaliana (L) depends on light conditions during growth. Plant

Physiol.111: 419425.

1748

M. Yamada et al.



14/14

Tsuboi, H., Suetsugu, N., Kawai-Toyooka, H. and Wada, M. (2007)

Phototropins and neochrome1 mediate nuclear movement in the

fernAdiantum capillus-veneris. Plant Cell Physiol.48: 892896.

von Caemmerer, S. and Furbank, R.T. (2003) The C4 pathway: an

efficient CO2pump. Photosyn. Res.77: 191207.Wada, M., Kagawa, T. and Sato, Y. (2003) Chloroplast movement.

Annu. Rev. Plant Biol.24: 455468.

Wen, F., Xing, D. and Zhang, L. (2008) Hydrogen peroxide is involved in

high blue light-induced chloroplast avoidance movements in

Arabidopsis.J. Exp. Bot.59: 28912901.

Xu, Z.Z., Zhou, G.S., Wang, Y.L., Han, G.X. and Li, Y.J. (2008) Changes in

chlorophyll fluorescence in maize plants with imposed rapid

dehydration at different leaf age.J. Plant Growth Regul.27: 8392.

Yamane, K., Kawasaki, M., Taniguchi, M. and Miyake, H. (2003)

Differential effect of NaCl and polyethylene glycol on the

ultrastructure of chloroplasts in rice seedlings.J. Plant Physiol.160:

573575.

Yamane, K., Rahman, S., Kawasaki, M., Taniguchi, M. and Miyake, H.

(2004a) Pretreatment with antioxidants decreases the effects of salt

stress on chloroplast ultrastructure in rice leaf segments (OryzasativaL.). Plant Prod. Sci.7: 292300.

Yamane, K., Rahman, M.S., Kawasaki, M., Taniguchi, M. and Miyake, H.

(2004b) Pretreatment with a low concentration of methyl viologen

decreases the effects of salt stress on chloroplast ultrastructure in

rice leaves (Oryza sativaL.). Plant Prod. Sci.7: 435441.

Zhang, J.H., Jia, W.S., Yang, J.C. and Ismail, A.M. (2006) Role of ABA in

integrating plant responses to drought and salt stresses. Field Crops

Res.97: 111119.

(Received June 6, 2009; Accepted August 2, 2009)

1749



C 4 Mesophyll and Bundle Sheath

Documents

Transcript of C 4 Mesophyll and Bundle Sheath