Weed Control, Plant Physiology and Biochemistry

RECENT RESULTS OF RESEARCH ON PHOTOSYNTHESISIN SUGARCANE

D. Gross, C. W. Baldry, J. Coombs, C. Bucke and A. W. GordonTate & Lyle Ltd., Research Centre

Keston, Kent, England

ABSTRACT

Ever since Kortschak et al. in 1965 and Hatch and Slack in 1966 re­ported that in sugarcane the first stable products of photosynthetic CO2-fixation

are malate and aspartate instead of the usual 3-phosphoglycerate (PGA) , theproposition of the 4-carbon dicarboxylic acid, or fi-carboxylation, pathway as anew alternative scheme to the now classic Calvin cycle has aroused greatscientific interest. The question is whether in sugarcane fi-carboxylation of PEPsubstitutes for RuDP carboxylation to a significant degree, representing thusa primary process in CO2-fixation. Satisfactory separation of mesophylJ (grana­type) chloroplasts from bundle sheath (non-grana type) chloroplasts has beenachieved, but, due to release of large quantities of phenolic compounds andtheir interaction with enzymes during the isolation procedure, the biologicalactivity was greatly impaired. Attempts to reverse the inhibitory effects byvarious chemical additives were only partly successful. It could, however, bedemonstrated that isolated chloroplasts carried out a light-dependent car­boxylation of phospho-enol pyruvate, giving rise to malate and oxaloacetateas the first stable products.

INTRODUCTION

The work of Kortschak et aI. (10) and Hatch and Slack (9) has shownthat, when sugarcane leaves are exposed to 14C0 2 in short term photosynthesisexperiments, the pathway by which CO 2 is incorporated into sugar differs fromthat postulated by Bassham and Calvin (4) for algae and temperate plants.Great interest was aroused as to the significance of the new pathway. Accordingto the Calvin scheme, CO 2 is combined enzymically with a 5~carbon acceptor mole­cule, ribulose-I, 5-diphosphate (RuDP), to yield two 3-carbon molecules ofphosphoglyceric acid (PGA); which is the first measurable product of photo­synthesis. According to the scheme proposed by Hatch and Slack, and Kortschaket aI. (the HSK scheme), the primary acceptor molecule is the 3-carbon com­pound phospho-enol pyruvic acid, which combines with CO 2 to yield the 4-Cdicarboxylic acid, oxaloacetate; the reaction is catalyzed by the enzyme phospho­enol pyruvate carboxylase (PEP carboxylase) as follows:1. Phospho-enol pyruvate + CO 2 + H 20 ~ oxaloacetate + orthophosphate2. Aspartate ee oxaloacetate ee malate

Reduction of oxaloacetate in the presence of malic dehydrogenase wouldyield malate and transamination; in the presence of glutamate-aspartate trans­aminase it would give rise to aspartate as the first identifiable stable photo­synthetic product.

1147

Sucrose, starch

AMP+

PPi--........2Pi

\\,Pi+ ATP

c~

Fig. 1.

C',"The H~K, or j3-carb<.>xylation, pathway ~s reported to be operatin ii~.of tropical grasses, such as sugarcane, maize, sorghum, and in Certai~ d~ a nUtnqesuch as Amaranthus edulis and A triplex. These plants are said ICotyledO:ilssynthetically more efficient ~han temperate plants operating ona~heto be. Pho.tO~.alone because they show higher rates of apparent photosynth . Cal~lll cYcle}sistance to photosaturation and a CO 2 compensation point lesIs, a hIgher re.·affinity of PEP carboxylase for CO 2 is much higher than that

Co;e to zero. The

ylase, and the still somewhat hypothetical transcarboxylation 0 RUl?P carhox_an aid to overall efficiency in CO 2 assimilation. step nlIght act as

The existence of a second carboxylation route in temping malate as a primary product has been known for many erate plants yield_role in overall photosynthesis was considered to be a mino years (5). But itsgested t~at the le~el of PEP carbo~ylase ~ctivity in sugarcane\~~e. It Was sug_grasses IS much higher than that m Calvm-type plants and th other tropical

at for RuDp.car-

Oxaloacetate .is assumed to donate. CO 2 to the CJcarboxylation to RuDP in the presence of a transcarboxa.lsugar phosphates, and eventually sucrose: y a3. RuDP + oxaloacetate + H 20 ~ 2 PGA + pyruvate.is then regenerated from pyruvate. .4. Pyruvate + orthophosphate + ATP ~ phospho-enol . ,.,phate + AMP. DVl'l1'.,-;

The main reactions may be incorporated in the .LUllO •WinO'

1148

D. GROSS, C. W. BALDRY, J. COOMBS, C. BUCKE, A. W. GORDON 1149

boxylase the converse is the case (14). Higher levels of RuDP carboxylase inHSK plants have been reportedsince (7,6) and confirmed by us.

The question is whether this is a sure indication that in, for instance,sugarcane p-carboxylation of phospho-enol pyruvate substitutes for RuDP car­boxylation to a significant degree, representing a primary event in the pathwayof CO 2 incorporation.

It has been established by Laetsch et al. (11,12) that sugarc,!-ne, in com­mon with certain tropical grasses, possesses 2 types of chloroplasts, i.e., one typecontaining stacked thylakoid membranes or grana and usually found in themesophyll tissue, and the other type without appreciable amounts of grana, thelamellar type, present in the adjacent bundle sheath tissue. The bundle sheathchloroplasts normally contain a great amount of starch grains. Most of the RuDPcarboxylase and malic enzyme can be found in the bundle sheath chloroplasts.HSK plants have a characteristic concentric arrangement of bundle sheath cellsand palisade type mesophyll cells around the vascular bundles, i.e. the Kranztype anatomy.

The question arises whether different functions in the photosyntheticprocess may be ascribed to each type of chloroplast, and, if so, what is the roleof the dimorphic chloroplasts in the HSK-pathway. To answer this question, thesugarcane chloroplasts would not only have to be isolated intact and biologicallyfully active but also separated from one another as effectively as possible.

METHODS AND RESULTS

Separation of Chloroplasts

Both types of chloroplasts may be obtained by disruption of the leaf.Since the non-grana type contains much more starch, the average density ofthe 2 types differs. On this basis, the chloroplasts should be separable by densitygradient centrifugation. It was found that by grinding cane leaves in pyrophos­phate-buffered sucrose solutions in a cooled Waring Blendor for 8 sec, filteringthe brei through several layers of muslin, and centrifuging the filtrate at 4000 gfor 30 sec in a refrigerated centrifuge, a pellet of mixed chloroplasts could beobtained, the ratio of grana-type to non-grana type being approximately 6: 1(Fig. 2). When layering 10 ml of a suspension of mixed chloroplasts in an in­cubation medium, 0.35 M with respect to sucrose, over 10 ml of 50% sucrosesolution in a 50-ml glass tube in a swing-out bucket head and centrifuging atabout 900 g for 15 min, a dark green band consisting predominantly of grana­type chloroplasts was obtained at the interface between the 2 solutions. A palegreen pellet was recovered from the bottom of the tube. The pellet containedmainly non-grana-type chloroplasts (about 50%), starch grains, cell debris, aswell as some grana-type chloroplasts. Tg~ efficiency of the separation dependedon the starch content of the chloroplasts, The chloroplasts separated by thismethod reduced DCPIP (phenol-indo-2:6 dichloro-pheno) in the light at abouthalf the rate of preparations not subjected to the separation procedure. Bothtypes showed light-dependent reduction of ferricyanide. A subsequent refinementof the separation system, introducing a 3rd layer of 73% (w jw) sucrose, enablesthe non-grana type to be separated from the debris. (Fig. 3 and 4) .

1150 WEED CONTROL, ETC.

Fig. 2. a. Transverse section of fresh sugarcane leaf showing vascular bundle (V) surroundedby bundle sheath (BS) and parenchymatous tissue (P); b. Similar section cleared in boiling70% ethanol {or 30 min, washed in distilled water and stained' with iodine; showing localiza­tion of starch in the bundle sheath (S); c. grana-type chloroplasts (G); d. mixture of grana­type (G) and non-grana type chloroplasts (NG) containing starch (St) , contaminated withfree starch grains (FS) and cell debris (CD).

II p

a b

1-23 1·18 1-13

g/ml

c

40.0.

d

>=---:BO 40

%

Fig. 3. a. Separation of sugarcane chloroplasts into 2 factions on a continuous sucrose densitygradient; layer of gTana-type chloroplasts (g) and pellet (p) containing mainly non-granatype chloroplasts (n.g) , gnna-type chloroplasts and debris; b. change in density along thegradient, measured by refractometer on fractions collected dropwise; c. optical density at 652nm (chlorophyll absorbance) of fractions collected dropwise; d. percentage of 0-0 grana- andA-A non-grana-type chloroplasts, determined by haemocytometer counts on fractions from thegradient.

D. GROSS, C. W. BALDRY,. J. COOMBS, C. BUCKE, A. W. GORDON ll5l

a b c d e f

p

mg rate perIOOugChl.

1'4 1·2 1·0 2 I 0 6 3 0 2 1 0 2 I 0g/m

Fig. 4. a. separation of sugarcane chloroplasts on a discontinuous sucrose density gradient;b. change in density along the gradient; c. mg total chlorophyll recovered from each layer;d. mg protein recovered from each layer; e. relative rates of reduction of ferricyanide/per 100ttg chlorophyll/hour by chloroplasts; f. relative rates of photoreduction of dichlorophenol indo­phenol by chloroplasts.

Electron microscopy of fine sections of chloroplasts confirmed that chloro­plasts derived from the upper layer were of the grana-type, whereas chloroplastsfrom the lower layer were mainly of the starch-containing non-grana or lamellartype (Fig. 5).

Fig. 5. Electrn micrograph (X40,000) of sugarcane chloroplasts. a. Part of a chloroplastderived from the upper layer showing typical grana structure (gr); b. part of a chloroplastfrom the pellet showing starch grains (st) andahse~ce of grana. .

1152 WEED CONTROL, ETC.

Chloroplast Activity

It had been expected that separation of the 2 types of chloroplasts withgood yields would permit the investigation of their respective functions in the,process ofCO 2-fixation and sucrose biosynthesis. Intact chloroplasts with a fullcomplement of enzymes should be capable of reducing CO 2 to carbohydrates withthe concomitant evolution of oxygen. Chloroplasts which have lost the carboxy­lation activity may evolve oxygen when 3-phosphoglyceric acid is added, and thosewhich have lost ferredoxin will need an addition of ferredoxin and ferredoxin/NADP reductase. Finally, chloroplasts retaining only electron transfer abilitywill need the addition of a nonphysiological electron acceptor such as ferricya­nide. By determining the additives required to produce light-dependent oxygenevolution, the degree of damage to the photosynthetic pathways during the prep­aration of chloroplasts may be estimated.

Measurements using oxygen electrodes demonstrated that cane chloro­plasts evolved oxygen only on addition of ferricyanide, while spinach andsorghum chloroplasts, isolated by a similar technique, responded differently(Fig. 6).

Spinach

IO"P~I'

.····I

! No additions

i -I: PGA

~ /~~02

<:a

~88<:

"~~~~

Time

Fig. 6. Light-dependent oxygen evolution by isolated chloroplasts, with and without specificadditives, measured by means of oxygen electrodes. F = I p. mole ferricyanide, Fd = ferre­doxin; Fp = flavoprotein; NADP = nicotinamide adenine dinucleotide phosphate; PGA =3·phosphoglyceric acid. .

Both cane and sorghum chloroplasts showed light-independent. oxygenconsumption. This was stimulated by addition of catechol or chlorogenic acid,both o-diphenol compounds. It was found that the cane leaf contains high con­centrations of phenolic compounds and o-diphenol oxidase. Cane leaf extract

rD. GROSS, C. W. BALDRY, J. COOMBS, C. BUCKE, A. W. GORDON 1'153

could be shown to inhibit both oxygen evolution and CO 2-fixation by otherwisephotochemically active spinach chloroplasts. This seemed -to establish a plausibleconnection between phenolic inhibitors and deficient photosynthetic activity ofisolated cane chloroplasts.

Mechanism of Inhibition

The action of cane extracts, phenolic compounds isolated from cane, andauthentic phenols on CO 2-fixation, oxygen evolution, photophosphorylation, elec­tron transport, and enzymes involved in CO2-assimilation was investigated in de­tail, using spinach or Chenopodium chloroplasts, or crude enzyme preparationsas models. CO 2-assimilation and particularly the enzymic fixation of CO 2 in thepresence of ribose-a-phosphate and ATP were more sensitive to inhibition thanphotophosphorylation, electron transfer or the enzyme phospho-enol pyruvatecarboxylase.

It was found that o-diphenolrO, oxidoreductase activity (phenol oxidase)in cane leaf supernatant or chloroplast preparations was about 30 times that insimilar spinach preparations. It was assumed that large amounts of polyphenoliccompounds liberated by macerating the leaves may be converted to quinones bypolyphenol oxidase.

Our results (2) clearly demonstrated that chlorogenic acid and caffeicacid were the major o-diphenols in extracts of cane leaves. These compoundsinhibited reactions associated with CO 2-fixation by the PCR cycle, but CO 2­

assimilation due to PEP carboxylase activity was less sensitive to this kind of in­hibition (Fig. 7 and 8). The high level of PEP carboxylase observed would beconsistent with this assumption.

control

5minutesI--l

catechol

caffeic acidchlorogenic acid

LIGHTON

1iO'1pmole o-dohenol

Fig. 7. Inhibitory effects of o-diphenols on light-dependent oxygen evolution by spinach chloro­plasts (with N'aHCOg as snbstrate).

Pb.

WEED CONTROL, F.TC.

( ~D

~?

/fa.

//

//

/./

//

//

//

//

/I

//

//

//

//

//

I/

II

. 0·5

oUJ

~

52UJ

C\l00'25

~o:2;:::1..

1154

o 1 2IJMOLES FERRICYANIDE ADDED

Fig. 8. Effect of caffeic acid on stoichiometry of ferricyanide by sugarcane chloroplasts (100 JLgchlorophyll in 3 ml HEPES buffer) . a. theoretical stoichiometry; b. no added o-diphenol; c. plus0.3 JL moles caffeic acid; d. plus 0.6 JL moles'blffeic acid.

Reversal of Inhibition

Inhibition of enzymes by o-diphenols may be prevented or reduced byremoving the substrates, such as oxygen and phenolic compounds, inhibiting thephenoloxidase, or reducing the end products (quinones) back to phenols. Agreat number of additives, such as sulphydryl (SH) reagents, polymers, reducingcompounds and copper chelators, were examined for their effects on caneo-diphenoloxidase activity (3). Oxygen evolution and o-diphenoloxidase-catalysedO 2 consumption were measured polarographically, using Clark-type electrodes.Photosynthetic CO 2-fixation by chloroplasts and dark enzymic carboxylation re­actions with ribose-5-phosphate and ATP or with phospho-enol pyruvate as sub­strates were determined by measuring incorporation of 14C0 2 into acid-stableproducts.

Of the sulphydryl reagents tested, viz. thioglycollate, f3-mercaptoethanol,dithioerythritol, glutathione and cysteine, thioglycollate proved the most effectiveinhibitor of phenoloxidase (o-diphenol.Ojoxidoreductasej activity in cane leaves.A mxr solution caused almost immediate inhibition of enzyme activity. A similardegree of inhibition was observed after a pre-incubation period of 5 minuteswith f3-mercaptoethanol (Fig. 9).

Of the polymers tried, such as bovine serum albumin (BSA) , polyvinyl­pyrrolidone (PVP) and polyethylene glycol 4000 (carbowax), the first 2 sub­stances proved effective. Effects of SH reagents and polymers on CO 2-fixation inthe dark by cane leaf preparations were only observed if the additive was in­cluded in the grinding medium. Most SH reagents when used as concentrationsbetween .10':'3 and 10-4 M stimulated CO 2-assimilation, with ribose-5-phosphateand ATP as substrates (Fig. 10).

D. GROSS, C. W. BALDRY, J. COOMBS, C. BUCKE, A. W. GORDON 1155

o 10-6 10-4 10-2 1 2 3M CONCENTRATION GRAMS PER100ml

Fig. 9. Inhibition of sugarcane leaf o-diphenoloxidase, measured by means of oxygen electrodes,after 5 min per-incubation with SH reagents and polymers: 0 thioglycollate; .... ,B-mercapto.ethanol; • glutathione; L. dithioerythritol; 0 cysteine; • BSA; \l PVP; 'f carbowax.

d.

a.

o,~-+---+---l+---+----+---lc.

500 I,) b. ;<7~~ 1/'1/·0::100 L~_g-o.-o _v_v_v_v

g§

500 •

~ P':~, V·300 ;/ ,

Y !," '! a \ e.,.."' \ .---.

100 ... ,;..... .. ''0 /.,.-.-.-.

10 6 104 102 1 2

M CONCENTRATION GRAMS PER 100 ml

O,L--,J-,;~_'::':-;;__:=*__-+__-!;-..J

Fig. 10. Effects of SoH reagents (a and c) and polymers (b and d) on dark enzymic C02fixation by sugarcane leaf brei, with ribose-5-phosphate and ATP (a and b) or phospho-enolpyruvate (c and .d) as substrates. Symbols as in Fig. 9.

By addition of suitable protective compounds to the grinding medium, wesucceeded in isolating chloroplasts from cane leaves which were capable of carry­ing out an important intermediate stage in the HSK cycle.

Light-phospho-enol Pyruvate-dependent CO2-fixation

An addition of 10-3 M thioglycollate and I% carbowax to the grindingmedium made it possible for cane chloroplasts to be isolated which could be

1156 WEED CONTROL, ETC.

shown to carry out a light-dependent carboxylation of phospho-enol pyruvate(I) .

We found in our experiments (I) that no significant C()2-fixation byisolated sugarcane chloroplasts could be observed in the. absence of added sub­strate or with ribose-5-phosphate, indicating that the photosynthetic carbon re­duction cycle was inactive. However, when phospho-enol pyruvate was addedand the preparation illuminated, 14C0 2 incorporation into stable products waslinear over periods of up to 45 min, with maximum rates of the order .of 2,umoles CO 2 fixed/rng chlorophyll/hour. An examination of the products of thereaction by chromatography and autoradiography showed that asignificantfrac­tion of the original radioactivity was located in malate (90%) and oxaloacetate(10%) .

Addition of PCA or oxaloacetate (OAA) inhibited the. reaction, as didNADP, ferredoxin and ferredoxin/Na.D'P reductase. Differential centrifugationof ground cane J~aves showed that .much of the carboxylase activity was linkedwith chloroplast membrane fragments. The results are consistent with the fixa­tion of CO 2 by a chloroplast membrane-bound PEP carboxylase, followed bya rapid photoreduction, possibly mediated by ferredoxin, of the oxaloacetate tomalate.

The rapid removal of the first product of photosynthesis could accountfor the known high resistance of sug(lrcane to photosaturation, as the possibilityof end product accumulation and irlhibition would be minimised. Apart fromthe high rates of photosynthesis, f3-carboxylation could also account for the abil­ity of sugarcane to reduce CO 2-concentration of the atmosphere to near zero(low compensation point) .

Enzyme Studies

Attempts have been made to obtain new information on the properties,distribution and metabolic control reactions of the major enzymes associatedwith sucrose metabolism and biosynthesis. This included a detailed investigationof alkaline pyrophosphatase. A key enzyme in the HSK-pathway is pyruvate:phosphate dikinase, producing phospho-enol pyruvate to maintain CO 2,fixation

by means of PEP carboxylase. Sufficient PEP can only be formed if the otherproducts of the reaction, such as pyrophosphate and AMP, are removed rapidlyenough. .

It has been suggested recently that an inorganic pyrophosphate (PPase)is present in high activities only in plants exhibiting the HSK-pa.thway. We havefound that the activity is highest in the tropical grasses, but a temperate plantsuch as spinach has a specific activity inferior only to sugarcane and maize, butsuperior to sorghum and Panicum fasciculatum.The same applies to Cheno­podium album. There seems to be no clear-cut difference in the PPase activitybetween plants having the HSK-pathway and other plants.

An examination of the intracellular distribution of PPase in sugarcane,spinach and Chenopodium album showed the PPase activity to be concentratedin the chloroplasts and associated with photophosphorylase particles which arereadily released from the chloroplasts.

The presence of starch grains in the non-grana type chloroplasts prompteda study of the localization of amylase activity in sugarcane chloroplasts. It has

-"j :r.'..'.~ ~'. I,'

.- . .

D. GROSS, C. W. BALDRY, J. COOMBS, C. BUCKE, A. W. GORDON 1157

been established by Bourne et al. (8) that most of the activity in cane leavesis a-amylase and that most of it is associated with the chloroplasts and partic­ularly with those of the non-grana type. The non-grana type showed an activitytwice as high as that of the grana-type, which may be interpreted as havinga more efficient method for starch degradation than the grana-type. The starchmetabolism has been further elucidated by the discovery that starch phosphory­lase, with a pH optimum of 6.5, is mainly localised in the non-grana type.

Release of Enzymes by Differential Grinding

The progressive release of carboxylating enzymes has been correlated withthe breakdown of specific tissues during the mechanical disruption of sugarcaneleaves, using a differential grinding technique. A correlation between the releaseof enzymes and that of protein, chlorophyll, phenol oxidase and phenolic com­pounds was studied. By use of gentle grinding followed by more vigorous macer­ation with abrasives, the contents of mesophyll cells and bundle sheath cellscan be released separately. The gentle grinding to release the most fragile cellswas accomplished by stirring the tissue with acid-washed sand in a mortar, usinga pestle. After washing to remove the contents of the mesophyll cells, the con­tents of the tougher cells were released by grinding vigorously with anti-bumpinggranules or glass balls.

The release of photosynthetic CRC activity from sugarcane leaves wassimilar to that of chlorophyll. In contrast, the release of ,B-carboxylation activityfollowed that of total protein. Evidence was found for the release of malicenzyme at the same stage of progressive grinding I as of ribulose diphosphatecarboxylase. Malic enzyme is the final component of the enzyme pathway,shuttling CO

2from the atmosphere to the site of CO2-fixation by ribulose dis­

phosphate carboxylase. PEP carboxylase and other enzymes concerned with themechanism were released earlier in the grinding process.

Fig. 11 shows the distribution of total and specific activities of the car-

a 8..

240

I--

>.~:t=

.~

U I--« ~

ci.(/)

b

1 2 34 1 2":·34110 ma Protein I

Fig. 11. Progressive release of a. PEP carboxylase and b. ribulose diphosphate carboxylase

activity from sugarcane leaf tissue.

boxylating enzymes in the 4 fractions from sugarcane leaf tissue. RuDP car­boxylase activity appears to be released during the rupture of the mesophyll

1158 WEED CONTROL, ETC.

cells, when the grana-type chloroplasts are also released. PEP carboxylase is re­leased earlier in the grinding process, suggesting it to be associated with thecytoplasm of the mesophyll cells. This would seem to indicate the cytoplasm asthe site of primary carboxylation.

Distribution of Enzymes

Previous experiments had shown that the majority of enzymes of canechloroplasts were lost during the isolation process, Consequently, enzyme' ac­tivities in chloroplast preparations cannot be equated with the activities in vivo.

The sucrose gradient method for separating the two types of chloroplastwas modified by the addition of a-Layer of 25% sucrose above that of 50%sucrose. This served to separate the upper layer of chloroplasts (grana-type)from the supernatant which contains high enzyme activities. None of the chloro­plast preparations separated on sucrose gradients showed R5P carboxylation sys­tems or pyruvate:Pi dikinase activity. However, a number of other enzymes couldbe detected and their levels of activity in each type determined, ". \'

These results, in particular those on malic enzyme, tend to agree withthose obtained by progressive grinding. The presence of PEP carboxylase in bothtypes is interesting, especially as it is stimulated by light in each case. The ap­parent absence of PPasefrom the non-grana type is surprising, as this enzymeis believed to be essential for the activiry of pyruvate-Pi dikinase, which is ap­parently associated with bundle sheath cells.

The overall picture obtained would seem to confirm the results of otherworkers that PEP carboxylase is associated with mesophyll cells and that malicenzyme, pyruvate: Pi dikinase and the R5P carboxylation system are associatedwith less easily ruptured cells, plausibly the bundle sheath cells. However, it wassubsequently found that the differential release of inhibitory phenols and phenoloxidase may complicate the interpretation of enzymic data.

DISCUSSION

The 2 most important discoveries made in the last few years with re­gard to sugarcane were that a) it contained 2 types of chloroplasts, located withinspecific tissues (11,12) and b) that the primary products of short term carbonfixation experiments, using 14C0 2, were oxaloacetate, malate and aspartate, Theseobservations have led to the proposal of the Hatch-Slack-Kortschak (HSK)scheme of 4-carbon dicarboxylic acid photosynthesis in sugarcane and relatedtropical grasses.

Fixation of CO 2 into malic acid was well known as a dark reaction, sothat at first it did not look like a normal photosynthetic reaction to Kortschaket al. (10); but later experiments confirmed the light-dependent nature of thereaction, with PGA appearing after malate and aspartate and before the hexosephosphates.

There is a possibility of 2 interconnected pathways proceeding simultane­ously in sugarcane and related plants, i.e., CO 2 being partly fixed by the nor­mal Calvin cycle and partly by /i-carboxylation. The key feature in the pro­posed HSK-pathway is that the primary carboxylation is catalyzed by PEPcarboxylase, giving rise first to oxaloacetate and then by reduction to malate

D. GROSS, C. W. BALDRY, J. COOMBS, C. BUCKE, A. W. GORDON 1159

and aspartate. Released CO 2 , in the presence of malic enzyme, is subsequentlytransferred to RuDP of the Calvin pathway by a transcarboxylatiorr step whichis still purely hypothetical. It is very difficult to prove experimentally whethertranscarboxylation occurs or; more likely, decarboxylation and re-fixation ofCO 2 by RuDP carboxylase. Alternatively, oxaloacetate might serve as the sub­strate for a speculative transcarboxylase, donating enzyme-bound CO 2 to asuitable acceptor molecule such as RuDP.

Much of the evidence put forward is based on enzyme studies. Thus,HSK plants are said to have low levels of RuDP carboxylase and high levels ofPEP carboxylase. They also have higher levels of pyruvate:phosphate dikinase,required for the regeneration of PEP from pyruvate, than regular Calvin plants.Higher levels of RuDP carboxylase in HSK plants have been reported sinceand confirmed by us.

Laetsch (13) has exterisively discussed the functional significance of chloro­plast dimorphism. He thinks that the chloroplasts of the HSK plants have sep­arated the functions of fixing CO 2 and making starch, so that most of the CO 2

is fixed in the mesophyll layer and end products (probably sugars) are rapidlytransported to the bundle sheath chloroplasts where they are stored as starch.The starch is removed during the night. An effective transport· system is pro­vided by the close proximity of the leaf cells from the vascular tissue in theseplants, the distance being seldom more than one cell. .Should the bundle sheathcells be directly involved in transport, then precursors and products may bemoved about with even greater efficiency.

The specialized structure of the chloroplasts could also account for theresistance of the HSK plants to photosaturation and their apparent lack ofphotorespiration. The former could be explained by the rapid removal of inter­mediate compounds from the mesophyll chloroplasts, preventing end productinhibition of primary carbon fixation, the latter by complete absence of a photo­respiration system or by re-assimilation of CO 2 produced by photorespirationbefore it becomes detectable. Either way, these characteristics could readilyaccount for the high photosynthetic efficiency of sugarcane and kindred plants.By contrast, Calvin plants lose a significant amount of the fixed CO 2 by photo­respiration. The low CO 2 compensation point characteristic of sugarcane could'be correlated to leaf structure and carbon fixation pattern. Walker and Crofts(15) have recently subjected the HSK pathway to a critical examination and con­

cluded that the existence of a second carboxylation system giving rise to malatecan generally be accepted. The question that has to be unequivocally answeredis whether or not p-carboxylation is a primary reaction and substitutes forRuDP-carboxylation in HSK plants.

Several of the outstanding questions of the carbon cycle could be resolvedif it were possible to isolate the 2 types of chloroplast in a fully functional state.But so far, the difficulties involved have proved formidable. Sugarcane leavesare hardened by silica and contain highi;'concentrations of phenolic compounds.These tend to be released during the isolation procedure so as to inhibit the nat­ural enzyme systems of the chloroplasts. Some progress on reversing the inhibi­tory action has been made and evidence for part of the HSK pathway thus pro­duced. Alternatively, a great deal of relevant information may be obtained fromplants related to sugarcane or with similar metabolic pathways and lesser prob­lems of chloroplast isolation. The vconclusions drawn from such experiments

1I60 WEED CONTROL, ETC.II

could be tested by following the metabolism of specifically labelled substratesincorporated by intact sugarcane tissue.

REFERENCES

1. Baldry, C. W., C. Bucke, and J: Coombs. 1969. Lightjphosphoenol pyruvate-dependentcarbon dioxide fixation by isolated sugar cane chloroplasts. Biochem. Biophys. Res.Commun, 37:828-832.

2. Baldry, C. W., C. Bucke, J. Coombs, and D. Gross. 1970. Phenols, phenoloxidase andphotosynthetic activity of chloroplasts isolated from sugar, cane and spinach. Planta(Berlin) 94:107-123.

3. Baldry, C. W., C. Bucke, and J. Coombs. 1970. Effects of some phenoloxidase inhibitorson chloroplasts and carboxylating enzymes of sugar cane and spinach. .Planta (Berlin)94:124-133. .

4. Bassham, J. A., and M.. Calvin. 1957. The Path of Carbon in Photosynthesis. Prentice-HaiL.Inc. Englewood Cliffs, New Jersey.. " .

5. Bassham, J. A., and M. Kirk. 1960. Dynamics of photosynthesis of carbon compounds.1. Carboxylation reactions. Biochim.- Biophys. Acta. 43:447-464.

6. Berry, J. A.,W. J. S. Downton, and E. B. Tregunna, 1970. The photosynthetic carbonmetabolism of Zea mays and Gomphrena globose. The location of the C02 fixation andthe carboxyl transfer reactions. Can. J. Bot., 48:777·786.

7. Bjorkman, 0., and E. GauhL 1969. Carboxydismutaseactivity in plants with and without.a-carboxylation photosynthesis. Planta (Berlin), 88:197-203.

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