Phytol.

(19SX), 109, 265 277

C4 plants as valuable model experimental systems for the study of photosynthesis

BY ROBERT T. FURBANK' AND CHRISTINE H. EOYER^ Division of Plant Industry, CSIRO, Canberra, Australia ^Research Institute for Photosynthesis, University of Sheffield, Sheffield SIO 2TN, UK {Received 11 December 1987; accepted 15 February 1988)
CONTENTS Summary 265

I. II. III. IV.

Introduction Anatomical and biochemical differentiation Regulation of carbon partitioning Experimental .systems and applications 1. Isolated mesophyll tissue 2. Isolated mesophyll protoplasts

265 267 267 269 269 272

3. Isolated mesophyll cells 4. Isolated bundle-sheath tissue V. Whole leaf measurements VI. Possibilities of future development Acknowledgements References

273 273 274 27.S 275 275

SUMMARY T w o decades have passed since the C4 cycle was first described. In this time, an increasingly large body of information has shown that the complexities of structure and metabolism associated with C4 photosynthesis may be successfully exploited to understand further the molecular biology, biochemistry and structure-function relationships of photosynthetie cells. Key words: Photophosphorylation, carbon assimilation, chlorophyll a fluorescence, sucrose, protein phosphorylation.

I.

I N T R O I) U C T 1 O N

Photosynthesis in vascular plants is a light-depend e n t process which fixes CO., itito carbohydrate u s i n g the enzyme RuBP* carboxylase. The involvem e n t of this enzyme in photosytithesis is universal throughout the plant kingdom and the source of CO2 is ultimately from the atmosphere. The route through which CO,^ reaches RuBP does, h()we\er, vary between plant types. Vascular plants are divided into 3 gt-oups according to their mechanisms of CO.2 fixation; C3, C4 and CAM. C3 plants 'trap'
* Abbrcviatiotis: 9AA, 9-amin()acridinc; C3, .^ carhon comp o u n d ; C4, 4 carbon compound; CAM, crassulacean acid metabolism; DHAP, diliydroxyacetone phosphate; F6P, fructose 6-phosphatu; FBP, fructose f ,6-bisphosphatL-; ( i l P , RIUCOSC 1-phosphate; G6P, glucose 6-phosphate; M.A,!^, malate; NADM E , NAD malic enzyme; NADP-ME, NADP malic enzyme; O A A , o.xaloacetate; PEP, pliosphocnolpyruvate; PEP-CK, phosphocnolpyruvate carfioxykinase; PG.\, phosphoglycerate; Pi, orthophosphate; PPi, pyrophosphate, PYR, pyru\ate; R.SP, ribose-5-phosphatc; RuBP, ribulose-f .5-bispliosphatc; sucrosoP, sucrose-6-phosphate; TP, triose phosphate.

CO, from the intercellular spaces solely hy the action of RuBP carboxylase and are limited by the CO. content ofthe atmosphere (r. 340 ppm). Both C4 and CAM plants use the enzyme PEP carboxylase to fix atmospheric CO., into C4 acids which are then decarboxylated tJ supply CO, to RuBP carboxylase (Hatch, 1977, 1978). PEP-carboxylase which catalyses the reactioti of HCO,- with PEP to form OAA initiates the primary route of carbon assimilation for the photosynthetic pathway in C4 and CAM plants, whereas in C3 plants and algae it plays an auxiliary role. This allosteric enzyme that is regulated by a^ number of metabolites (Doncaster & Leegood,' 1987), catalyses a highly exergonic reaction and the reverse reaction cannot be demonstrated. The C4 cycle is essentially an additional step in photosynthesis; an ATP-dependent CO., 'pump'. C.-\M plants use this mechanism with a temporal separation of the two events in CO, fixation : PEP carboxylase fixes atmospheric CO., into MAL at night while RuBP carboxylase fixes the CO., released by decarboxylation C4 acid in the cells during tbe day. This

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R. T. Furbank and Christine H. Foyer

Malate

OxQloQcetate (C-A-)

ATP+NADPH

ADP+ NADP

Starch

Figure 1. Diagrammatic representation ofthe C4 cycle showing the carhoxylation and decarboxylation phases of the pathway with the \ariations in decarboxylation types as follows: NADP-ME (NNNSH ), NAIJ-ME ( ) . r'EF-CK (I I). Decarboxylation of C4 acids releases CO, which is fixed in the bundle sheath chloroplast by the Calvin cycle (C3). There is some doubt about the fate of PEP produced by decarboxylation of oxaloacetate in PEP-CK species. PEP may return directly to the mesophyll or be converted to PYR via PYR kinnse in the bundle sheath. allows the stomata to remain closed during the dav, producing high CO.j concentrations in the leaf and conserving water. Unlike CAM plants, the CO. 'pump' of C4 photosynthesis (Hatch, 1978) relies upon the spatial separation of metabolic pathways so that the carboxylation of RuBP occurs in the bundle sheath compartment, which is not in free gaseous communication with the atmosphere. The CO., fixed in the Calvin cycle is derived from C4 acids, produced by the incorporation of atmospheric CO,, into PRP in the mesophyll cells (Fig. I). The advantage conferred by this CO.^ 'pump' is that COo can be concentrated in the bundle sheath at up to lO-fold atmospheric levels (Hatch & Osmond, 1976). This results in CO.^ saturation of RuBP carboxylasc and suppression of photorospiration, a process which considerably reduces photosynthetic efficiency in C3 plants. The specilized Krantz anatomy (Hatch & Osmond, 1976) of C4 plants also results in improved water use cHiciency in arid climates. Consequently, the economic importance of C4 plants is considerable, ranging from high yield crops such as sugarcane and maize to the most virulent weeds (Edwards & Huber, 1981). For many years C3 species such as pea and spinach have been used as 'typical' plants for biochemical studies. It is erroneous to assume that all plants behave identically either in an adaptive sense or mechanistically. With this qualification in mind, however, C4 plants show great promise as 'biochemical tools' for probing the fundamental processes of pbotosynthesis. In this work we will show that photosynthetic tissues from C4 species ha\'e been successfully used to study problems associated with topography, function and regulation of photosynthesis. The C4 paradigm provides an opportunity to observe and exploit the totally different expression of the same genome in adjacent cell types in such a manner as to segregate and optimize the functioning of the carboxylation and decarboxylation phases of C4 photosynthesis (Fig. 1). CAM plants (which may bc regarded as a variation on the C4 theme), in which the carboxylation and decarboxylation phases of the pathway arc separated not in space but in time, provide an additional tool to study regulation of protein synthesis and function on a diurnal basis. Undoubtedly C4 monoctoylcdonous species provide tbe ideal system in which to study regulation of gene expression, as cell differentiation into bundle sheath and mesophyll occurs longitudinally throughout the leaf with a gradient in leaf age from base to tip.

C4 plants as model experimental systnns

267
thetically active bundle sheath and mesophyll tissue (Hatch & Osmond, 1976). The isolated cell types often provide less complex conditions for the study of chloroplast reactions than in C3 plants and an opportunity to manipulate metabolic pathways isolated in individual compartments.

II.

.^^ N A T O M U ' A L A N D B I O C H F. M I C A L

D I F F E U 1; N T I A T I O N

T h e C4 piitliwiiy has evolved independenth' in a large number of plant families, hoth monocotyledons and dicotyledons. The presence of enzymes of the C4 cycle in C3 plants and the polyphyletic origin of the C4 pathway lead to the conclusion that only small genetic alterations involving changes in gene expression would be necessary for the transition from C3 to C4 photosynthesis. The formation of Kranz-like anatomy would he one of the first of several necessary mutational stages (Moore, 1982). All C3-C4 intermediate species have a leaf anatomy somewhere between that of C3 and C4 plants combined with a photosynthetic CO., compensation point that also falls between the two (> 40//I 1"' for C3 and < 5 /i\ 1 ' for C4 plants). C4 plants show a large degree of interspecific variation in biochemistry and anatomy and ha\e been sub-divided into 3 groups according to the enzyme used for decarboxylation of C4 acids in the bundle sheath: NADP-MR type, NAD-MK type and PEP-CK type. The latter two types produce aspartate in the mesophyll as the C4 metabolite for translocation to the bundle sheath whereas NADP-ME types produce MAL. ln C4 plants the photorespiratory pathway is not directly linked to the Calvin cycle as it is in C3 plants where the whole process occurs within each ceil. In C3 plants, the enzyme glycerate kinase links the pathways of photosynthesis and photorespiration by returning carbon to the CaKin cycle. The cellular differentiation in C4 plants has resulted in the majority of the enzymes of the photorespiratory cycle being localized largely or even exclusively in the bundle-sheath cells but glycerate kinase, the final enzyme of the pathway for cycling carbon, is localized exclusively in the mesophyll cell chloroplasts (Usuda & Edwards, 1980). T h u s , the con\-ersion of glycerate to PCJA is confined exclusively to mesophyll chloroplasts. The mesophyll and bundle-sheath cell types are remarkably different in enzyme function and soluble and membrane protein composition. Eor example, the mesophyll cell contains abundant amounts of the enzymes PEP carboxylase and PYR Pi-dikinase while the Calvin cycle enzymes, RuBP carboxylase, ribulose 5-phosphate kinase, EBP and sedoheptulose 1,7-bisphosphatase, are localized in the bundlesheath chloroplasts (Hatch & Osmond, 1976). The difference 1 1 1 enzyme activities is accounted tor largely by differential protein distribution between the cell types rather than the presence of inactive enzymes or cell-type specific inhibitors, ft is this morphological and biochemical differentiation of the C4 cell types which provides plant biochemists with a unique experimental system to study photosynthetic processes, particularly now that techniques are availafile wfiicfi allow isolation of photosyn-

III.

RI-(UM.ATION

OF CARBON

PARTITIONING

Principal features of C4 photosynthesis are high rates of photosynthesis, sucrose formation, assimilate export and prodvicti\ ity compared with C3 plants. C4 photosynthesis requires high irradiances. Photos\'nthetic carfion assimilation is not completely light-saturated even at full sunlight and changes in irradianee result in large fluctuations in carf:)on assimilation (Moss, Musgrave & Lemon, 196f). Phere is considerable scope in the C"4 system for profjing the regulation of carbon assimilation, carbon partitioning and source-sink interactions. Pronotinced diurnal \ariations in enzyme activities, photosynthesis, sucrose synthesis and export have been found in maize leaves (Kalt-Torres et al. 1987; Kalt-Torres & lluber, f987; Usuda et al., 1987). Kalt-Torres et al. (1987) have suggested that, because maize exports comparati\ ely more carbon during the day than the C3 species soybean and spinach, the partitioning of carbon may be different in C3 and C4 species, resulting in different patterns of metaholism and diurnal growth. The site of sucrose synthesis is determined by the distribution of sucrose-P synthase between the bundle sheath and mesophyll cells and varies considerably between species. Recently it has been shown that young leaves of Zea mays (F"urbank, Stitt & Eoyer, 1985) and other C4 species (Usuda & P^dwards, 1980) synthesize sucrose almost exclusivelv in the mesophyll compartment (fig. 2) and that

Mesophyli

Time (min}

Figure 2. 'f'hc time-course of laf^clling of ["C]-sucrose in the mesopfiyll and bundle-sheath fractions of maize leaves during a pulse-chase experiment as described by Furbank, Stitt & Foyer (1985) demonstrating that values approximating to fOO"',, of total leaf sucrose synthesis are obtained for the mesophyll cells of maize.

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R. T. Furbank and Christine H. Foyer

BUNDLE SHEATH
glucose - gkicose
(n) (n + 1)

MESOPHYLL CYTOSOL
PGA

STARCH

ADPglucose ATP

^ -

PGA

PYR

TP

TP

MAL

CYTOSOL

FBP

Figure 3. Diagiammatic representation of mctabolitf Huxcs betwcLMi the mesophyll and bundle-sheatli cells and accompanying pathways of sucrose and starch synthesis in NA1)P-MI<' C4 species such as Zea mays. (Although MAL and OAA are shown to share the same the chloroplast envelope translocator, it worthy of note that they arc most prohably transported on separate carriers).

starch synthesis is confined to the bundle sbeath under normal growth regimes. Accordingly the enzymes specific for suerose synthesis are localized in the mesophyll cells and absent from those of the bundle sheath (Eurbank, Stitt & Eoyer, 1985; Mbaku, Eritz, & Bowes, 1978). This strict compartmentation may not hold true for other NADP-MK species (Chen el al., 1974; Mbaku et al., 1978; Ohsugi & lluber, 1987). In Zea mays starch is found mainly in tbe bundle-sbeath cells of plants grown under normal illumination (Downton & Hawker, 1973) but with plants grown under continuous illumination starch was found in both cell types. Similarly, induction of sucrose-P synthase in the bundle sheath requires high irradiance (Eurbank et nl., 1985; Ohsugi & Huber, 1987). The level of irradiance therefore plays a fundamental role in determining assimilate partitioning and distribution between tbe bundle-sheath and mesophyll cells. Consequently the system shows considerable potential for the study of gene expression related to carbon metabolism. In C4 plants a large proportion of tbe carbon fixed in photosynthesis travels between

the two cell types (Eig. 3). Large gradients of several transport metabolites between the mesophyll and bundle-sbeath cells of maize bave been described (Stitt & Heldt, 1985; Leegood 1985). This transport involves not only C4 acids but also PCiA and TP. In NADP-ME species such as maize, tbe C4 cycle is closely coupled to the activity of the C3 cycle since a significant proportion of the P(iA formed in the bundle-sheath cells is reduced in the mesophyll cells and returned to the bundle sheath as TP. NAD-ME and PEP-CK species generate sufficient NADPH in the bundle-sheath chloroplasts to allow reduction to proceed and no reducing power is exported from tbe mesophyll to the bundle-sheatb. However, since tbe mesophyll chloroplasts of all the sub-types contain high capacities for PGA reduction (Usuda & Edwards, 1980) and the PCA-TP shuttle between bundle sheath and mesophyll operates in all the C4 sub-groups, it appears that transport to tbe mesophyll is universal in C4 photosynthesis. Consequently tbe TP formed may be used for the synthesis of suerose in NAD-ME and PEP-CK C4 types as well as the NADP-ME species. Ohsugi &

C4 plants as model experimental systems Huber (1987) have shown that the relative content of extractable acti\ity of sucrose-P synthase in the cell types betweeen light and darkness (and also the degree ol light-activation of this enzyme) varied considerably between the C4 sub-groups. In NADF-ME species such as Zea mays the discrete localization of sucrose phosphate synthase in the mesophyll cytosol means that the relationship between carbon assimilation and sucrose synthesis is complicated such that the rate ot sucrose synthesis a n d Pi recycling back to the chloroplast cannot be coordinated in the simple manner which facilitates co-regulation in C3 species (Walker, 1976; Furbank, Foyer & Walker, 1987). However, the Hux of carbon into sucrose in maize closely follows the rate of photosynthesis (Muber et al., 1987) with the availability of assimilated carbon (reflected in the levels of n^etabolites) being an important regulating factor. C 4 species therefore have considerable potential for studying the intimate association between carbon assimilation and assimilate partitioning (Fig. 3). In this case, providing a suitable mesophyll preparation could be devised, the regulation of sucrose synthesis could be examined in a system incapable of directing carbon into starch.
IV. EXPERl M I'NTAI. SYSTKMS A . N APPLICATIONS

269 because it is fast growing and robust. It does tiot require highly specialized growth conditions and the individual tissues can be easily separated and manipulated. Howe\er, mesophyll cells, protoplasts and chloroplasts can be isolated from a variety of C4 monocotyledons and also some dicotyledons, to provide tissues from representatives of each of the decarboxylation types. A useful general procedure for isolation of intact mesophyll chloroplasts from a wide range of C4 species has been devised by Jenkins & Russ (1984), enabling them to be separated from leaf homogenates by centrifugation through a percoll cushion. Intact chloroplasts penetrate and pass through the layer of percoll while the broken chloroplasts and other cell debris do not, providing a good separation between the two fractions. This procedure has been shown to yield 80-95 " intact mesophyll chloroplasts from species such as Atriplex spongiosa, Zea mays. Sorghum bicolor, Panicum miliaceiim and Panicum maximum. With some modifications (for example, the density of percoll cushion) this procedure should be generally applicable to most C4 species but, when it is unsuccessful, chloroplasts can be prepared by the controlled rupture of isolated mesophyll protoplasts [see section IV. (1), part. 2]. A major virtue of the C4 mesophyll chloroplast as an experimental system is the absence of a complete Benson-Calvin cycle (Figs 1 and 3). In the NADPME species (such as Zea mays), isolated chloroplasts will reduce OAA to MAL and convert PYR to PEP (Day & Hatch, 1981). The latter reaction is catalysed bv the enzvme PYR Pi-dikinase the activity of which

1. Isolated mesophvll /issue 7.ea mays is the C4 species most frequently used in studies of photosynthesis, presumably because of the particular significance of corn as a crop and also

ATP

2ADP AMP+PPi

Pi PyruvatePyruvate

ATP

Phosphoenol pyruvate Pi

NADPH NADP t

Phosphoenol pyruvate

Oxaloacetate -

-^Oxaloacetate ATP
ADP

^ Malate

- Malate

NADPH

NADP Glyceraldehyde phosphate

G lycerate 3-phosphate Dihydroxy acetone phosphate

Glycerate 3-phosphate

1 3 Diphospho glycerate

1
Dihydroxyacetone phosphate

Figure 4. Diagrammatic illustration of the metabolism of PVR, OAA and (;A3P showing the energy requirements in the reactions catalysed by (1) PYR Pi-dikinase; (2) adenylate kinase; (3) pyrophosphatase; (4) NADP-MAL dehydroKenase; (5) PC-X kinase; (6) NADP-glyceraldehyde-3-phosphate dehydrogenase; (7) TP isomerase.

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R. T. Furhank and Christine H. Foyer

Table 1. Pliotosynlhctic oxygen evolution in isolated mesophyll chloroplasts^

Net Oj evolution (/Miiol mg ' chl h Zea mays Paniciim miliacemn Panicmn maximum (PKP-CK type) 137
-21 n.t.

Substrate
3-PGA PYR OAA PYR + OAA

(NADP-MK type)
235 -2 60 84

(NAD-ME type)
111 -28 n.t. 14

A'rPiNAOPH (per mol substrate)

1:1 2:0 0:1 2:1

22

* Chloroplasts were prepared and assayed using stable isotope mass-spcctromotry as described by I<\irbanl<, Badger & Osmond (192). n.t., not tested.

has been shown to be regulated by protein phosphorylation (Hurncll & Hatch, 1985, Foyer, 1984). Because this stromal enzyme utilizes ATP and produces AMP, this has necessitated an important modification in the metabolism in C4 mesophyll chloroplasts, where the activity of the enzyme adenylate kinase is directly coupled to C4 photosynthesis (Fig. 4) and the removal of AMP generated by the PYR Pi-dikinase reaction (Hatch, 1982). The concentration of adenylate kinase in C4 species is 10 times that in C3 plants. There is also a large increase in the activity of this enzyme when etiolated seedlings are greened under illumination (Hatch, Slack & Osmond, 1969), as with other photosynthetic enzymes. In addition to metabolizing intermediates of tbe C4 pathway, mesopbyll chloroplasts will also reduce PGA to T l \ Thus, the metabolism of I^YR, OAA and PCiA involves individual reaction sequences, using the light reactions of the chloroplast thylakoid as a common source of ATP and N A DPI I. Since each of these reactions has a difTerent recjuirement for ATP and NADPII (iMg. 4), the energy demand on the isolated chloroplast can be easily controlled and manipulated according to the substrate added. The rates of these partial reactions in mesophyli chloroplasts from 3 species are .shown in Table 1. This system has recently been appHed to the study of the interaction between stromal and thylakoid reactions, l^he addition of PYR as an ATP sink, stimulates a high rate of cyclic electron How in mesophyll cbloroplasts of Zea mays (Crowther et al., 1983) and drastically reduces the stromal A T P / A D P ratio (Fernyhough, Foyer & Horton, 1983), permitting a demonstration of the capacity for cyclic photophosphorylation to produce ATP without NADPH oxidation. Such studies are more complex when C3 chloroplasts are used because of the cycling of photosynthetic intermediates. Similarly, the response of pseudocyclic photophosphorylation to ATP demand and NADP levels has been studied using stable isotope mass-spectrometry and isolated mesophyll chloroplasts (Furbank, Badger & Osmond, 1983); OAA was here used as a metabolic sink

for NADPH and PYR as a sink for ATP (Fig. 4). Also, the absence of RuBP carboxylase activity in the mesopbyll chloroplast allowed the elucidation of the kinetic behaviour of an in vivo Mehler reaction without the complication of photorespiratory oxygen consumption, a problem wbich has plagued such studies in C3 systetns (Furbank, Badger and Osmond, 1982). Chlorophyll a Huorescetice at room termperature has become a widely used non-intrusn'e probe ot photosyntbetic function and regulation ni vivo (Schrcibcr, 1983, Sivak & Walker, 198.S). However, chlorophyll fluorescence quenching is a cotnplex and often ambiguous parameter since it contains two components. These are photochemical and nonphotochemical quenching (Krause, Vernotte & Briantais, 1982). Photochemical quenching ((/g) results from oxidation of the primary electron acceptor oi photosystem II, Q^, and is generally thought to reflect the rate of electron transport and hence photosyntbetic flux (Schreiber & Bilgcr, 1987). Nonphotochemical quenching of chlorophyll a Huorescencc (f/^,.) includes a number of processes not principally involving non-cyclic electron transport. These include bigb energy-state quencbing (Krause et al., 1982) due to the formation of a ApH {q^,) and energization of tbe chloroplast thylakoid membrane system, photoinbibitory quenching of Huorescence ((/,), and quenching of fluorescence via changes in distribution of energy between the photosystems (known as state transitions) termed (/.,. (see Horton & Hague, 1988). The major component of nonphotochemical quenching under normal conditions appears to be high energy-state quenching (Krause, et al., 1982; Krause & Laasch, 1987), which is sensitive to the rate of metabolic ATP utilization and provides a valuable probe of photosynthetic carbon metabolism. The relationship between these two mechanisms of fluorescence quenching and photosynthesis is most clearly demonstrated with C4 mesopbyll cbloroplasts wbere the metabolic requirements for NADPH and ATP are easily manipulated by the addition of diflerent substrates (Fig. 4).

C4 plants as model experimeiital systems

A

271

iticreases electron tratisport and (/g. ^Pbis is a classical example of 'photosynthetic control' of electrotT 0-8 transport by bigb thylakoid ApH which is difficult to Pyruvate mimic iti any other system. This simple experiment Oxaloacetate also clearly demonstrates a close relationship beoOoo<3oa tween (/g and tbe rate of electron transport, ln this 0-6 OOCb system where RuBP carboxylase/oxygenase is o ^300000000 I^E absent (and hence O, uptake by the photorespiratory pathway), net O., evolution reflects electroti transport much more accurately tbatT iti C3 cbloroplasts, cells 0-4 or leaves. Experiments of tbe type sbown in Figure 5 bave recently been used to demonstrate a linear relationship between tbe rate of electron tratTsport 0-2 and tbe redox state of Q^ under a variety of conditions. Tbylakoid protein pbospborylation, an important mecbanism contributing to the efficiency atnd regu1 1 1 1 lation of photosynthesis, can be readily manipB 70 ulated in a C4 system. The thylakoid membranes contain protein kinase enzymes which phosphorylate specific thylakoid polypeptides (Bennett 1977, 60 J Coughlan & Hind, 1986). Of particular importance 1 -" 9AA is the phosphorylation of the chlorophyll cz/A-bindI '/' 1 50 ing protein, an event wbich causes an alteration in \ / the distribution of absorbed photons in such a way as \ to decrease the rate of excitation of photosystem II. \ o 40 The phosphorylation of this protein is controlled by E' the redox state of the plastoquinone pool and is "o believed to provide the molecular basis for tbe state 30 1 to state 2 adaptis'e transition (Horton et al., 1981, 3 Bennett, Steinback & Arntzen, 1980). Reversible 20 phosphorylation/dephosphorylation changes may thus contribute to the regulation of relative rates of cyclic and non-cyclic electron How by facilitating a 10 cbange in energy distribution and redox poise in response to botb redox state and demand for .ATP. The flexible manipulation of the energy and redox 0 states of the mesophyll chloroplast stroma has \ provided an ideal system for studying rcgulatioti of 1 1 phosphorylation of thylakoid membrane proteins 1 2 3 4 5 6 (Fernybough, Foyer & Horton, 1983, 1984). Work Time (min) witb intact maize mesopbyll cbloroplasts has conFigure 5. The eflect of PYR (20 mM) on q^^ and (/,., {.\), Hrtned the involvement of the redox state as a major ApH (measured by 9AAfluorescence)and O., evolution (B) in maize mesophyll chloroplasts metabolizing ().'\A (1 factor controlling the regulation of the phosmM) in the presence ot Pi (.S niM). Chlorophyll fluorescence phorylation of the light-har\-esting chlorophyll ajhand Oj evolution were measured as described in Furbank, binding protein but has not produced any evidence Foyer & Walker (1987) using the chloroplast isolation for the presence of an effect of the A T P / A D P ratio procedures and assay medium according to lenkins & Kuss upon kinase acti\ity (Fernyhough et al., 1983). The (1984). addition of pyru\'ate to isolated maize mesophyll chloroplasts causes the A T P / A D P ratio in the Figure 5 sbows the effect of PYR addition on f/(j, (/,.. stroma to fall. In Figure 6, for example, the A T P / and ApH (9AA in Fig. 5B) in isolated mesophyll ADP ratio fell from 1-6 to O-l after 10 min irradiance chloroplasts of maize that were mctabolizitijj; OAA. and yet phosphorylation of the light-har\ esting With OAA as substrate, etiergy-dependent quench- chlorophyll (-///;-binding protein (tbe pbospborylated ing and ApH are high because there is no con- polypeptide species at 26000 kDa in Fig. 6) was sumption of ATP to dissipate tbis energy. The stitnulated compared witb that in the absence of addition of PYR (which via the enzyme PYR, Pi- substate. 1 lowe\ er, tbe addition of OAA which dikinase consumes 2 mol All-" per mol PYR) col- causes utilization of reducing equnalents without lapses both ApH atnd 7,,. (demonstrating tbe elose ATP consumption increased the measured .ATP/ relatiotisbip between tbese two paratneters) and
Chlorophyll fluonBscence quenching

'

evolution

VJ

272

R. T. Furbank and Christine H. Foyer

100,000

32,000 26000

9000
B

mm

Figure 6. Autoradiogram to illustrate the eomparati\ e phosphorylation of thyiakoid polypeptides from maize mesophyll chloroplasts metabolizing different substrates. Intact chloroplasts were prepared from mesophyll protoplast and phosphorylated with [''-P]-Pi as described by Fernyhough, Foyer & Morton (1983, 1984) Thyiakoid membrane polypeptide phosphoryhition in chloroplasts incubated in the presence of (A) 2 mM OAA; (B) in the absence of substrate; (C) 10 mM PYK; (D) 3 niM glycerate-3-ph()sphate; (I'^) 2 mM OAA plus 10 mM PYR is shown. Molecular weights (kDa) of the major pliosphorylated species are given.

A DP ratio to 2-3 and yet suppressed protein which tbe epidermis has been removed, or wbicb bas pbospborylation (Fig. 6). Tbis indicates tbat modu- been finely sliced to aid penetration of a mixture of lation of thyiakoid protein kinase activity was exerted pectinase and cellulase enzymes. After digestion by redox state ratber tban tbe ATP/ADP ratio. (usually for 2-3 b at 25-30 C under weak illuminaHowever, bigb rates of ATP consumption which tion), protoplasts are released and wasbed in a cause reduction of ApH (and resultant low f?,,) favour suitable medium; tbe bundle-sbeatb tissue remains protein pbospborylation. Higb ApH and resulting undigested. Intact mespbyll protoplasts are then bigb (ly probibit protein pbosphorylation (Ferny- separated from tbe bundle-sbeatb strands and cell bough et al., 1984; Oxborougb & Horton, 1987; debris by gradient centrifugation. Mesopbyll protoOxborougb, Lee & Morton, 1987). In addition tbis plasts can be prepared from representative C4 species system provided corroborative evidence to support of all three decarboxylation types, but two species, tbe proposition tbat a basic function of protein Zea mays (Day, Jenkins & Hatcb, 1981) and Panicum pbospborylation was to modify excitation energy miliaceuni (Edwards et al., 1979), yield tbe bighest distribution in sucb a way as to re-route electron How pbotosyntbetic activities at present. to meet tbe requirements of tbe stroma for ATP and Intact mesopbyll protoplasts are incapable of NADPH. pbotosyntbetic CO.^ fixation or CO.^-dependent O., evolution, and are largely impermeable to all substrates other tban CO.^. Tbus mesopbyll protoplasts 2. Isolated mesophyll protoplasts are extremely limited as in vitro pbotosyntbetic Protoplasts (plant cells where tbe cell wall bas been systems. However, once tbe mesopbyll protoplast removed by enzymic digestion) may be prepared plasmalemma is ruptured to yield a mixture of intact from C4 plants by tbe digestion of leaf tissue from cbloroplasts and otber cytoplasmic organelles, bigh

C4 plajits as model experimental systems rates of metabolism can be promoted and measured by the addition of rele\ant substrates. Commonly, cbloroplasts and protoplast extracts are prepared by passing a suspension of protoplasts through a nylon mesh with pore size of 20 //m. l^his ruptures the plasmalemma and tonoplast membranes while allowing the chloroplasts to pass through, retaining complete integrity of structure and function (Edwards et al., 1979). Until improved methods of differential centrifugation through percoll became available this method was the only procedure for obtaining intact mesophyll chloroplasts. l'rotoplast extracts are rarely used but may prove valuable in studies where the interaction between cytosol and cbloroplast is of interest.
3. Isolated mesophyll cells

273 the chosen metabolites to the reaction medium in which photosynthesis and sucrose synthesis was assayed and, hence, the regulation of an entire pathway involving chloroplasts and cytosol could be easily examined. 4. Isolated bimdle-sheath tissue Isolated bundle-sheath tissue has not been as extensively exploited as an experimental system, despite the ease of isolating strands from a variety of C4 plants (Huber, Kanai & Edwards, 1973 ; Hatch & Kagawa, 1976; Chapman, Berry & Hatch, 1980) and also protoplasts from some species (Edwards et aL, 1979; Moore, Ku & Edwards, 1984). Bundle-sheath strands can be quickly prepared from many species (particularly Panicum miliaceum, Urochloa panieoides, Zea mays, Chloris gayana and some Atriplex and A)naranthus species) by differential grinding procedures. Bundle-sheath strands are readily removed from mesophyll tissue by vigorous blending and subsequent filtration and washing. In contrast, bundle-sheath protoplasts are much more difficult to prepare, and yields are often extremely low and highly variable (see Edwards et aL, 1979). A limitation to the bundle-sheath system is that the strands are extremely resistant to breakage and extraction. This means that the isolation of intact functional bundle-sheath chloroplasts or other subcellular compartments is virtually impossible using current homogenization procedures. The protoplast system overcomes this difficulty, but the impermeability of protoplasts and also their fragility can cause difficulties. The isolated bundle-sheath tissue contains the enzyme systems necessary for decarboxylating C4 acids and a fully functional photosynthetic carbon reduction cycle, identical to its C3 counterpart. Photosynthetically competent bundle-sheath strands from species in each of the 3 decarboxylation types display different properties and variable assimilation capabilities (Table 2). An interesting

When macerated in an appropriate medium most C4 lea\ es yield a mixture of mesophyll chloroplasts and other sub-cellular organelles together with tbylakoids, other membranous vesicles and cell fragments. However, the leaves of the some C4 plants, primarily from the genus Digitaiia can be ground in a mortar and pestle to produce functional mesophyll cells and bundle-sheath strands. The preparation of mesophyll cells in this way, described some 17 years ago by Edwards & Black (1971), is quick and simple. I'he isolated mesophyll cells are readily applicable to photosynthetic studies since, unlike protoplasts, they are permeable to substrates (Mbaku, et aL, 1978). This property may be due to the retention in cells of plasmodesmatal channels wbich become sealed in protoplasts. Isolated C4 mesophyll cells would provide a valuable system in which to examine the regulation of sucrose and starch synthesis. In this system the effects of indi\ idual metabolites (such as PGA, T P , Pi and most importantly fructose 2,6-bisphosphate) on the rate of sucrose synthesis could be examined in situ. In this system the concentrations of cytosolic metabolites could be varied simply by the addition of

Table 2. ^^CO.^ fixation by isolated bundle sheath strands*

CO.ifixation rate (//mol mg ' chl h Zea mays (NAOP-MK type) 0 98 112
130

Substrate CO., DHAP + CO,, RSP + DHAP + CO,,

MAL + R5P+DHA"P

Panicuyn miliaceum (NAD-ME type) 82 n.e. n.e. n.e.

Chloris gayana (PEP-CK type)

65

ATP: NADPH requirement 3:2 1:0 1:0 1:0

n.e. n.e. n.e.

+ CO, * Bundle-sheath strands were prepared and assayed as described by FurbanU & Badger, 1983 and Woo, Geraud & Furhank (I9H3). n.e., negligible effect.

274

- T. Furbank and Christine II. Foyer examine the sensitu'ity of cyclic photophosphorylation to inhibitors and to determine tbe maximum capacity for ATP synthesis of cyclic photophosphorylation (Woo et al., 1983). .Also, this system has proved useful in interpreting several non-intrusive probes of tbylakoid processes, such as Pji^ electrochromic shifts and chlorophyll ii fluorescence (Leegood et al., 1983). Bundle-sheath thylakoids are particularly useful for demonstrating relationships between structure and function in thylakoid membranes. In freezeetcbing studies tbe face closest to tbe intratbylakoid lumen is desigtiated tbe E-face and in mesophyll chloroplasts it is studded with large particles (14-17 nm in diameter). In agranal bundle-sbeath chloroplasts from species such as Z. mays the tiumbers of these large particles are greatly reduced relative to the mesophyll chloroplasts (Miller, Miller & Mcltityre, 1977). From these and other studies it has been sbown tbat tbe large particles on tbe E-face are tbe PS II complexes and tbeir associated lightharvesting pigment protein complexes and that their presence is required for grana formation. Similarly comparisons of the proteins of the mesophyll and bundle sbeatb chloroplasts of maize have been used to identify PS II cbloropbyll-proteins arid polypeptides (Bassi et al., 1985).
V. WHOLI; LEAF ME.ASUREMENTS

characteristic of these strands is the proliferation of plasmodesmata found on the bundle sheath cell (Evert, Eschrich & Heyser, 1977; 1978) and the extreme permeability of isolated strands to a wide variety of compounds, despite high levels of photosyntbetic integrity (Matcb & Kagawa, 1976). It is probable that plasmodesmatal channels in bundlesheath strands remain open after isolation, wbich would account for the permeability of these cells. If this is the case, bundle-sheath strands would provide an excellent 'semi-/w vivo' system in which to examine plasmodesmatal functioti. There are many species-dependent characteristics of C4 plants which can be experimentally exploited. A particularly useful example can be found with tbe NADP-ME type C4 species which show enormous interspecific variation in amounts of PS II in the bundle sbeatb cbloroplasts. In Zea mays. Sorghum bicolor and many other NADP-ME type C4 species, the chloroplasts are essentially agranal, that is stacking of the thylakoid membranes is either greatly reduced or absent. Tbese bundle-sheath chloroplasts are also PS Il-deficient, exhibiting little or no net O,^ evolution wben illuminated (Ku et. al., 1974, Hatcb & Osmond, 1976). However, the absence of grana in C4 NADP-ME species may simply be related to babitat because the occurrence of welldefined grana may be an important adaptation to a low light environment. Thus the shade-adapted C4 grass Paspatum conjugatum contains well defined grana stacks and rates of non-cyclic electron transport in other species (such as some Digitaria species) approach those found in C3 chloroplasts (Broglie, Coruzzi & Chua, 1984). The agranal bundle sheath chloroplasts in species such as Sorghum bicolor and Z. mays clearly have reduced PS II and water-splitting functions but in addition tbey are deficient in ferredoxin-NADP reductase, tbe enzyme required for tbe photoreduction of NADP. Since in sucb chloroplasts tbis is reduced to 10-20",;, of that present in the mesophyll chloroplast (Leegood, 1985) much of the reducing power required to sustain PGA reduction in the Calvin cycle and other NADPH-requiring processes in the stroma must be derived from malate decarboxylation (a cbloroplast reaction in NADP-ME types) or via a shuttle of PGA and TP between the mesophyll and bundle-sbeatb cells (Hatcb & Osmond, 1976; Cbapman, et al.. Hatch, 1980; Woo, Geraud & Furbank, 1983). Tbis is sbown in Table 2 where bundle-sheath strands from Z. mays are seen to fix CO., only in the presence of a reduced carbon source sucb as TP or R5P, and even then CO, fixation is greatly stimulated by addition of MAL. Tbus, during pbotosyntbesis in vivo, tbe bundlesbeatb chloroplasts of Z. mays must rely almost completely on cyclic pbotopbospborylation for tbeir ATP supply to regenerate RuBP. Isolated bundlesbeatb strands from Z. mays bave been used to

Intact C4 leaves themselves can also be used to great advantage in the study of thylakoid and carbon metabolism reactiotis. Leaves of Z. mays are particularly useful for determitiing the relationship between carboti metabolism and cblorophyll fluorescence. The deficiency of PS II m the bundle sheath chloroplasts meatis that whole leaf variable fluorescence emanates almost exclusively from the mesophyll thylakoids, considerably simplifying interpretation of the signal (Furbank & Walker, 1985). Also, concentrations of pbotosynthetic metabolites are bigber tban in C3 plants and consequently easier to measure, facilitating correlation of key metabolites witb cbaracteristics of tbe fluorescence signal. Althougb tbis tecbnique bas been applied to tbe study of induction in C4 plants (Ireland, Long & Baker, 1984; Leegood & Furbank, 1984) and to the effects of cbilling (Baker, East & Long, 1983) in parallel with measurements of carbon assitTiilation (Long, East & Baker, 1983), it bas not yet beeti extensively used to expand our knowledge of chlorophyll fluoresence quenching mechanisms /// vitH> (Ireland, Baker & Long, 1985). In addition to studies on metabolism, Z. mays has been used effectively to study the effects of photooxidati ve damage oti photosynthetic membrane structure (Bacbmann et al., 1973) and gene expression (Harpster, Mayfield & Taylor, 1984; Mayfield & Taylor, 1984). Studies using carotenoid-deHcient

C4 plants as model expertmental systems

275

mutants have shown that photo-oxidative damage to strands have a molecular exclusion limit of 800-900 Da. In addition the difl'usion constants for a variety the chloroplast specifically aftects the accumulation of a number cytosolic mRNAs encoding chloroplast of compounds ha\e been calculated using assays proteins. Photo-oxidation appears to block exclu- coupled to cytosolic reactions within the cells (M. Hatch, personal communication). The bundle sively the accumulation of a specific group oi mRNAs such that certain proteins like some PS II sheath cell system therefore has considerable potenpolypeptides, the light-harvesting chlorophyll ajb- tial for the study of plasmodesmatal function and binding protein, the Rieske iron-sulphur protein anci intercellular transport. Such \iable functioning plastocyanin are severely decreased in treated plants systems from vascular plants in which to study (Burgess & Taylor, 1987). This has led to the plasmodesmatal metabolism and action ? zntro ha\ e suggestion that the developmental status of the been una\ailable. chloroplast is communicated to the nucleus and that C3 C4 intermediate species have been \aluable in tbe effect of chloroplast photo-oxidation is to destroy the study of the evolution of C4 photosynthesis tbe 'signal' originating in the chloroplast that (Peisker, 1986). In addition, several of the characpromotes optimal expression of nuclear genes en- teristics of C3-C4 intermediate species such as the coding chloroplast proteins (Burgess & Taylor, processes that afford a lower photosynthetic CO.^ 1987). compensation point and reduced rates ol photorespiration relatix-e to C3 plants may provide indications of mechanisms by which photorespiration VI. POSSI Bl L1TI1-;S FOR i'VIU.OPMHNT could be reduced in C3 plants (Monson. Edwards & A major area of expansion in studies of photo- Ku, 1984). Isolated bundle-sheath strands may also be of synthesis is the application of knowledge of molecular biology to C4 plant systems, in order to \alue in the study of the regulation of cellular exploit the vast opportunities afforded by the metabolism and intercellular transport. The cytosol manipulation of transposable genetic elements. It sugar-phosphate and Pi contents of bundle-sheath has already been shown that enzymes of the C3 and cells can be easily manipulated allowing the study of interactions between chloroplast and cytosol in a C 4 pathways are regulated by a combination of system capable of starch and, in some cases, sucrose m R N A accumulation and post-transcriptional consynthesis. Similar opportunities for exploitation trol (Martineau & Taylor, 198S). This work also exist in the C4 mesophyll cell system. T o date, suggests a strong developmental link between studies have been aimed at optimising conditions for morphological difierentiation of C4 tissue and gene CO,, fixation in isolated bundle sheath strands. expression of C4 enzymes. Such a system pro\ ides There has been little consideration of the regulatory the potential for examination of links between genes implications such as Pi-cycling w ithin the chloroplast with widely diverse products but functionally assostroma as a result of starch synthesis, its relation to ciated roles in photosynthesis. T h e technique of Pi-cycling between the cytosol and chloroplast as a m R N A in situ hybridization has been de\eloped in result of sucrose synthesis, and fiuxes across the Zea mays by virtue of the cell-specific expression of chloroplast phosphate translocator. In an analogous genes for RuBP carboxylase and PEP carboxylase fashion, regulation of mitochondrial respiration providing a \ aluable tool in plant molecular biology could be examined in a system closely approximating (Martineau & Taylor, 1986). In addition, since light the in vitro condition and the relationships between evidently dictates the presence oi enzymes associated mitochondrial and chloroplast energy transduction with the light reactions and also carbon metabolism explored. in mesophyll tissue (Downton & Hawker, 1973, Bedbrook et aL, 1978, Ohsugi & Huber, 1987), the further application of molecular genetic analysis to A ( K N O W L E I) C I-: M E N T S the photoregulated expression of genes for proteins We wish to thank Professors M. D. Hatch, G. E. I'^dwards associated with photosynthesis will have a profound and W. C. Taylor for constructive discussion. This work impact on our understanding of the initiation, was funded by the Agricultural and I<'ood Researcli production and assembly of basic metabolic proCouncil, UK. cesses and their regulation. The extreme permeability of bundle-sheath strands to low-molecular-vveight compounds provides a unique system for manipulating the cytosolic composition of an 'intact' cell. It appears that in isolated bundle-sheath cells the plasmodesmata remain intact and functional (as Judged by electron microscopy and transport studies: J.Burnell and S. Craig, personal communication). Recently, it has been shown that the plasmodesmata of these isolated
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