regulation of photosynthesis byend-product … · storer (up to 160 milligrams of sucrose per...

Plant Physiol. (1992) 99, 1443-14480032-0889/92/99/1443/06/$01.00/0

Received for publication January 2, 1992Accepted February 24, 1992

Regulation of Photosynthesis by End-Product Accumulation inLeaves of Plants Storing Starch, Sucrose, and Hexose Sugars'

Eliezer E. Goldschmidt and Steven C. Huber*Department of Horticulture, The Hebrew University of Jerusalem, Rehovot, Israel 76100 (E.E.G.);

and U.S. Department of Agriculture, Agricultural Research Service, and Departments of Crop Science and Botany,North Carolina State University, Raleigh, North Carolina 27695-7631 (S.C.H.)

ABSTRACT

In the present study, leaves of different plant species weregirdled by the hot wax collar method to prevent export of assimi-lates. Photosynthetic activity of girdled and control leaves wasevaluated 3 to 7 days later by two methods: (a) carbon exchangerate (CER) of attached leaves was determined under ambient CO2concentrations using a closed gas system, and (b) maximum pho-tosynthetic capacity (Amax) was determined under 3% CO2 with aleaf disc 02 electrode. Starch, hexoses, and sucrose were deter-mined enzymically. Typical starch storers like soybean (Glycinemax L.) (up to 87.5 milligrams of starch per square decimeter ingirdled leaves), cotton (Gossypium hirsutum L.), and cucumber(Cucumis sativus L.) responded to 7 days of girdling by increased(80-100%) stomatal resistance (r.) and decreased A.... (>50%). Onthe other hand, spinach (Spinacia oleracea L.), a typical sucrosestorer (up to 160 milligrams of sucrose per square decimeter ingirdled leaves), showed only a slight reduction in CER and almostno change in Amax. Intermediate plants like tomato (Lycopersiconesculentum Mill.), sunflower (Helianthus annuus L.), broad bean(Vicia faba L.), bean (Phaseolus vulgaris L.), and pea (Pisum sativumL.), which upon girdling store both starch and sucrose, respondedto the girdle by a considerable reduction in CER but only moderateinhibition of A..., indicating that the observed reduction in CERwas primarily a stomatal response. Both the wild-type tobacco(Nicotiana sylvestris) (which upon girdling stored starch and hex-oses) and the starchless mutant (which stored only hexoses, up to90 milligrams per square decimeter) showed 90 to 100% inhibitionof CER and approximately 50% inhibition of Amax. In general,excised leaves (6 days) behaved like girdled leaves of the respectivespecies, showing 50% reduction of Am... in wild-type and starchlessN. sylvestris but only slight decline of A., in spinach. The resultsof the present study demonstrate the possibility of the occurrenceof end-product inhibition of photosynthesis in a large number ofcrop plants. The long-term inhibition of photosynthesis in girdledleaves is not confined to stomatal responses since the A.,,a declinedup to 50%. The inhibition of Am.., by girdling was strongest in starchstorers, but starch itself cannot be directly responsible, because thestarchless mutant of N. sylvestris was also strongly inhibited. Sim-ilarly, the inhibition cannot be attributed to hexose sugars either,because soybean, cotton, and cucumber are among the plants moststrongly inhibited although they do not maintain a large hexosepool. Spinach, a sucrose storer, showed the least inhibition in bothgirdled and excised leaf systems, which indicates that sucrose is

' The research reported in this publication was funded by the U.S.Department of Agriculture, Agricultural Research Service, and theNorth Carolina Agricultural Service. E.E.G. was the North Carolina-Israel Exchange Scholar for 1990 at the Department of Crop Science,North Carolina State University, Raleigh, NC.

1443

probably not directly responsible for the end-product inhibition ofphotosynthesis. The occurrence of strong end-product inhibitionappears to be correlated with high acid-invertase activity in fullyexpanded leaves. The inhibition may be related to the nature ofsoluble sugar metabolism in the extrachloroplastic compartmentand may be caused by a metabolite that has different rates ofaccumulation and turnover in sucrose storers and other plants.

The 'end-product inhibition of photosynthesis' hypothesis,as proposed over 100 years ago by Boussingault (3), statesthat the accumulation of assimilates in an illuminated leafmay be responsible for a reduction in the net photosynthesisrate of that leaf. In their 1968 review, Neales and Incoll (17)concluded that satisfactory proof of the hypothesis mustdepend upon two levels of investigation: (a) demonstrationof a negative correlation between photosynthesis rate andassimilate level in the leaf; and (b) elucidation of the bio-chemical mechanism involved.Review of the literature shows that the 'negative correla-

tion' between photosynthesis and carbohydrate accumula-tion is still not readily observable with all plants tested. Whenseveral plant species were compared, plants responded dif-ferently to conditions favoring photosynthate accumulation(7, 14, 18).Another point already raised by Neales and Incoll (17)

concerns the possibility that depression of CER2 may resultfrom increased r5 and not necessarily from inhibition ofphotosynthesis per se. Attempts to establish the nonstomatalnature of the feedback inhibition of photosynthesis havebeen made in several recent studies, utilizing various experi-mental approaches (1, 15). According to Mayoral et al. (15),short-term responses of girdled leaves (1-3 d) are likely toresult from stress-induced stomatal closure, whereas long-term inhibition (4-7 d) is predominantly nonstomatal.The physiological-biochemical mechanism underlying the

feedback inhibition, when observed, is still a matter of con-jecture. Among the major ideas proposed is one that suggeststhat the accumulation of starch interferes in some way withphotosynthesis (e.g. ref. 16) and an opposite view, whichclaims that the inhibition must be caused by the accumulation

2 Abbreviations: CER, carbon exchange rate; rs, stomatal resistance;Amax, maximum photosynthetic capacity.

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GOLDSCHMIDT AND HUBER

of soluble sugars (e.g. sucrose). Critical examination of thesehypotheses has proven difficult so far. Several recent studiesexpressed the view that some photosynthetic metabolites,rather than the end products themselves, must be responsiblefor the feedback inhibition (2, 6, 8, 10, 18).The approach adopted in the present study was to look for

end-product inhibition of photosynthesis in a range of plantspecies representing different leaf carbohydrate storage hab-its. In addition to typical starch and sucrose storers, we alsoexamined a starchless tobacco mutant that accumulates nei-ther starch nor sucrose (9, 12). Photosynthesis was deter-mined as CER under ambient CO2 as well as by measurementof Amnax under 3% CO2, which circumvents the stomatalinfluence (3). We wanted to determine whether there was arelationship between the photosynthetic response to girdlingand the carbohydrate storage pattem, which, if observed,might shed some light also on the biochemical mechanismsinvolved.

MATERIALS AND METHODS

Plant Material

Soybean (Glycine max L. Merr. cv Ransom), cotton (Gos-sypium hirsutum L.), cucumber (Cucumis sativus L.), tomato(Lycopersicon esculentum Mill.), broad bean (Vicia faba L.),sunflower (Helianthus annuus L.), and bean (Phaseolus vul-garis L.) were grown in a soil mixture under greenhouseconditions. Pea (Pisum sativum L. cv Progress 9'), spinach(Spinacia oleracea L. cv Dark Green Bloomsdale), and tobacco(Nicotiana sylvestris L., wild type and starchless mutant NS458) were grown initially in growth chambers (12 h light,24°C/12 h dark, 180C) and transferred to the greenhouseseveral days before the start of experiments.

Girdling Experiments

Mature, fully expanded leaves were used. Girdling was

performed by the wax collar method (17). Balsam woodcollars were sealed to petioles with modeling clay such thata 1.5- to 3.0-cm section of petiole was enclosed. Hot wax

(80-850C) was poured around the petiole. In Nicotiana leaves,which have no petioles, the girdle was applied to the midrib.In broad bean, which has short petioles, the girdle was

applied to side branches with several leaves. Girdles were

inspected at the end of the experiments. In successful girdles,the part of the petiole exposed to the wax was severelydesiccated. Data obtained from leaves with incomplete girdleswere discarded. Several experiments were conducted witheach plant species. Usually one girdled leaf and one compa-rable control leaf were present on each plant; experimentsconsisted of three to five replicate plants.

Detached Leaves

Leaves of N. sylvestris and spinach were excised and im-mediately placed in beakers filled with distilled water, wherethe petioles were recut under water. Leaves were held in thegrowth chamber for periods up to 6 d.

Photosynthesis and Respiration Measurements

Net CER was measured as CO2 depletion in a closed systemusing a Li-Cor model 6000 Portable Photosynthesis System.3Amax was determined by the Hansatech leaf disc oxygenelectrode in 3% CO2 (4). Respiration measurements were alsoconducted with the leaf disc electrode. Plants were kept inthe dark for at least 30 min and usually for several hoursprior to respiration measurements.

Leaf Carbohydrate Analysis

Leaf tissue (three to five 11-mm diameter discs per leaf)was extracted with 80% ethanol. Sucrose and hexose sugarsin the supernatant were measured enzymatically and starchwas determined after digestion with amyloglucosidase asdescribed previously (20). The ethanolic extracts were alsoused for soluble sugar component analysis on an HPLCsystem, as described by Robbins and Pharr (19).

Acid Invertase Assays

Leaves were harvested in the morning and frozen in liquidN2 prior to storage at -800C. Soluble enzyme extraction, anddesalting and assay were as previously described (12).

RESULTS

The long-term effects of girdling (6-7 d) on certain pho-tosynthetic parameters of different species are summarizedin Tables I and II. Plants are arranged according to thecarbohydrates that accumulated in leaves after export isrestricted by phloem girdling. At the top of Table I are starchaccumulators (soybean, cotton, cucumber), followed byplants that accumulate various proportions of starch andsoluble sugars (N. sylvestris wild type, tomato, sunflower,broad bean, bean, pea) and at the bottom is spinach, whichaccumulates predominantly sucrose. Leaves were harvestedat the beginning of the photoperiod to assess the forms ofcarbohydrate that accumulated in girdled leaves and werenot remobilized or metabolized at night.

Photosynthetic activity was assessed by two methods. First,we measured photosynthesis (as CO2 uptake) and stomatalresistance under ambient conditions in the greenhouse. Sec-ond, we measured Amax (as 02 evolution) using the leaf discelectrode to circumvent any stomatal limitations. As shownin Table II, the typical starch storers showed strong inhibitionof CER accompanied by an upsurge in r5. The Amax of theseplants was also inhibited by more than 50%. Both the N.sylvestris wild type, which stores starch as well as hexoses,and the starchless mutant, which stores only hexoses (seeFig. 3), showed strong depression of CER along with in-creased r,. The Amax of the wild type was inhibited 74% bygirdling and only 43% in the starchless mutant. The differ-ence between wild type and the starchless mutant in inhibi-

3Mention of a trademark or proprietary product does not consti-tute a guarantee or warranty of the product by the U.S. Departmentof Agriculture or the North Carolina Agricultural Research Serviceand does not imply its approval to the exclusion of other productsthat may also be suitable.

Plant Physiol. Vol. 99, 19921 444



END-PRODUCT INHIBITION OF PHOTOSYNTHESIS

Table I. Carbohydrate Accumulation in Control and Girdled Leaves of Various SpeciesLeaf samples were taken at the start of the photoperiod, 7 d after girdling. Values are means of

four to six determinations. For clarity, SES are shown only for total carbohydrates.Carbohydrates

Plant Leafa TotalStarch Hexose Sucrose

mg.dm-2Soybean C 33 3.3 2.2 39 ±4

G 80 5.9 9.3 95 ± 8Cotton C 28 1.2 0 30 ± 6

G 49 0.6 0 49 ± 10Cucumber C 137 0 0 137 ± 19

G 119 0.9 0 120± 13N. sylvestris wild C 71 16 10 98 ± 12

type G 75 91 5.9 172 ± 11N. sylvestris mutant C 0 20 15 35 ± 10

G 0 59 5.9 65 ± 8Tomato C 58 27 1.1 86 ± 9

G 41 62 2.4 105 ± 6Sunflower C 19 10 3.1 32 ± 9

G 48 37 29 114±45Broad bean C 1.4 0 4.6 6± 2

G 26 20 78 123 ± 15Bean C 33 12 4.2 49±11

G 39 21 15 75±22Pea C 13 1.0 7.0 21 ± 5

G 50 23 28 100 ± 5Spinach C 9.5 0 0.5 10 ± 4

G 9.7 0 124 134 ±16a C = control; G = girdled.

tion of Amax reflects the fact that Amax of the mutant is alwayslow relative to that of the wild type (Table II). Plants of the'intermediate' group (tomato, sunflower, broad bean, bean)also showed strong inhibition of CER, but increases in r, were

more moderate. The Amax was inhibited to a lesser extent,ranging from 35% in tomato to 13% in bean. Pea probablyalso belongs in this group; results from another experiment(not shown) indicate that pea leaf CER is only slightly inhib-ited by girdling. Spinach showed the least inhibition ofphotosynthesis after girdling: 16% inhibition of CER and 7%

inhibition of Am,,. This behavior of spinach was consistentlyconfirmed in at least five separate girdling experiments. Thephotosynthetic and carbohydrate storage pattems typical foreach plant, although somewhat less intense, were obtainedalso after 4 d of girdling (data not shown).Some experiments were also conducted with detached

leaves (6 d), which are analogous in several respects to girdledleaves (17). As expected, both genotypes of N. sylvestris showstrong depression of Amax compared with only 20% depres-sion in spinach (Table III).

Table II. Photosynthetic Parameters of Control and Girdled Leaves of Various PlantsCER was determined 2 to 4 h into the photoperiod, and Amax was determined in the afternoon (9-12 h into the photoperiod). Measurements

were taken 7 d after girdling, except those for cotton, which were taken after 6 d. Values are means of four to six determinations.CER rs Amax

PlantControl Girdled Inhibition Control Girdled Increase Control Girdled Inhibitionmg CO2-dm-2-h-h % s-cm'i % Umo 02/dm 2-h-1 %

Soybean 27.6 5.2 81 0.9 6.2 589 12.5 5.1 59Cotton 29.4 4.3 85 0.7 4.1 486 12.6 6.1 51Cucumber 15.7 5.2 67 1.0 2.9 190 14.6 6.0 59N. sylvestris wild 22.0 1.5 93 0.6 5.5 817 18.5 4.7 74

typeN. sylvestris 20.1 -1.3 100 0.7 7.1 914 7.0 4.0 43

starchlessmutant

Tomato 10.8 1.7 84 2.6 10.3 296 11.0 7.2 35Sunflower 43.3 14.0 68 0.7 2.3 229 22.4 17.2 23Broad bean 25.6 9.7 62 0.9 1.9 111 18.5 12.7 31Bean 8.7 1.7 80 6.8 12.8 88 7.8 6.8 13Pea 20.0 14.0 30Spinach 41.3 34.6 16 0.5 0.9 80 45.3 42.1 7

1445




When the percentage inhibition ofAmax in the girdled leaves(Table II) is compared with the starch levels (Table I) of theseplants, a striking negative correlation is evident (Fig. 1). Theonly point that clearly diverges from the correlation is thatof the starchless N. sylvestris (symbol 5 in Fig. 1), which hadstrong Amrax inhibition without accumulating any starch.

Inhibition of photosynthetic capacity by girdling was alsocorrelated with activity of the enzyme acid invertase. Asshown in Figure 2, high-invertase-type species (closed sym-bols) tended to show strong inhibition of Amax in girdledleaves, whereas low-invertase-type species (open symbols)showed less inhibition of Amax. This is consistent with thegeneralization that high-invertase-type species tend to bestarch accumulators, and suggests that extrachloroplasticsugar metabolism may be somehow related to the inhibitionof Am,ax by girdling.The kinetics of carbohydrate accumulation in leaves fol-

lowing petiole girdling was monitored in soybean (Fig. 3A),spinach (Fig. 3B), and N. sylvestris wild type (Fig. 3C) andstarchless mutant (Fig. 3D). As in previous experiments,leaves were sampled for carbohydrate determination at thebeginning of the photoperiod to maximize the distinctionbetween control and girdled leaves. In soybean (Fig. 3A),starch was the only carbohydrate accumulated. Accumulationcontinued throughout the experiment, although the rate ofaccumulation gradually diminished. Some increase in basalstarch level also occurred in the nongirdled control leaveswith the increase in leaf age. In contrast, spinach accumulatedonly sucrose (Fig. 3B). Sucrose level was already high by day2, peaked at day 4, and declined after that. In N. sylvestris,the wild type accumulated starch until day 4 and hexosesthroughout the experiment. In the nongirdled controls, thebasal starch level was rather high throughout the experiment,but hexoses did not accumulate (Fig. 3C). Hexoses were theonly major carbohydrate accumulated in the starchless mu-tant of N. sylvestris (Fig. 3D). Hexose levels peaked by day 4,after which there was some decline. HPLC-sugar analyses ofethanolic extracts from soybean, both Nicotiana genotypes,and spinach confirmed the presence of glucose, fructose, andsucrose and did not indicate the appearance of other solublesugars in either girdled or control leaves.The capacity of leaves to accumulate sucrose may be related

Table I1. Amax of Detached (6 d) Leaves and Attached ControlLeaves from Wild Type and Starchless Mutant (NS458) ofN. sylvestris and SpinachThe cut petioles of the detached leaves were kept in degassed

water in the growth chamber under the same conditions as thecontrol plants.

AmaxPlant Inhibition

Intact Detached

mg CO2, dmrn2 *h-' %

N. sylvestris wild 19.2 5.1 73type

N. sylvestris 9.7 5.6 42starchlessmutant

Spinach 28.4 22.7 20

0

*9

20 k

aEC

4

0z0-

m

z

zw

wa.

40

*7

\ "I06

60 _

80

SOYBEAN2 COTTON3 CUCUMBER4 NICOTIANA - WT5 NICOTIANA - MUT6 TOMATO7 SUNFLOWER8 BROADBEAN9 BEAN

10 PEASPINACH

*.3

4

40 80LEAF STARCH (mg-dm52)

120

Figure 1. Relation between starch levels accumulated and per-centage inhibition of Amax in girdled leaves of various plants. Amaxdeterminations were made in the afternoon. The starch data aretaken from Table I.

00-

x

0

0ILz0

m

x

z

0 25 50 75 100 125 150 175

ACID INVERTASE (/.Lmol*gj'FW-h ')

Figure 2. Relation between inhibition of Amax in girdled leaves andactivity of soluble acid invertase. See inset of Figure 1 for identifi-cation of species.

1446 Plant Physiol. Vol. 99, 1992

*8

*2



END-PRODUCT INHIBITION OF PHOTOSYNTHESIS

80

60

40

IE 20

3

I

ir 120

w 0

160

1201

80

40

®W-- SPINACH

33'V~~~~~As

lL &

8 0

DAYS AFTER GIRDLING

4

Figure 3. Time course of carbohydrate levels in control (oplsymbols) and girdled (closed symbols) leaves of soybean (A); spiach (B); N. sylvestris wild type (C); and N. sylvestris starchless muta(NS458) (D). Leaves were sampled in the morning. Mean valufrom three to five replicates are shown.

to the activity of acid invertase (12). We have comparsucrose concentration late in the photoperiod with acidvertase activity in leaves of control and girdled plantsspinach, sunflower, bean, and tomato. As shown in Figuresucrose accumulated in girdled leaves only when the ac

invertase activity was below an apparent 'threshold' activiof 10 ,umol of sucrose g-' fresh weight h-1. It is interestirthat sunflower leaves do not normally accumulate sucros

but do so when girdled (Table I). The accumulation of sucro

in girdled sunflower leaves was associated with a reducticin acid invertase activity from above to below the 'thresholactivity (Fig. 4).

DISCUSSION

The purpose of the present study was to examine tirelationship between the feedback inhibition of photosy:thesis and the carbohydrate accumulation patterns in a wicrange of plant species. We identified a long-term (4-7inhibition of photosynthesis in girdled leaves (Tables II ar

III) of some but not all species. Although this inhibiticgenerally seemed to have a stomatal component, the A,data suggested that the photosynthetic process per se w,also inhibited. This long-term inhibition appeared to besome relationship to the carbohydrate accumulation patter

The correlation between Amax inhibition and starch accu-

mulation (Fig. 1) would seem to support the 'starch inhibitionhypothesis,' were it not for the starchless mutant of N.sylvestris, which was also rather strongly inhibited in theabsence of starch accumulation (Tables II and III). Hexosescannot be responsible for the inhibition either, because thestrongly inhibited starch storers (soybean, cotton, cucumber)did not maintain high levels of hexose sugars in the longterm (Table I, Fig. 3). Sucrose is not a likely candidate for theinhibitor, of course, because the 'intermediate' sucrose storers(sunflower, broad bean, bean, pea) showed only weak inhi-bition (Table I) and the prominent sucrose storer, spinach,showed the least inhibition (Table I, Fig. 3B). Thus, we againreach the conclusion that none of the principal photosyn-thetic end products was directly responsible for the apparentfeedback inhibition.

Concerning the link between the photosynthetic inhibitioncaused by girdling and the type of carbohydrate accumulated,it seems that all of the strongly inhibited plants have one

property in common: they do not accumulate high levels ofsucrose. As shown in a previous study (12), the ability ofmature leaves to store sucrose is dependent upon the absenceof acid invertase. Indeed, soybean, cotton, cucumber, andboth the wild type and starchless mutant of N. sylvestrisretain high acid invertase in mature leaves (12), as doestomato (Table III), and all of these species showed significantinhibition of Amax by girdling. Spinach, on the other hand,had the lowest acid invertase activity of the species testedand was also least affected, in terms of Amax, by girdling. We

en propose that enhanced hydrolysis of sucrose occurs withinin- the vacuoles of high-invertase-type species when girdled,knt which leads to 'cycling' of and hexose When

ies sucrose hydrolysis exceeds the enzymatic capacity of thehexose kinases to rephosphorylate the released glucose andfructose, hexose sugars accumulate to a significant extent. In

ed some species, the hexose pool is maintained whereas inn-of4,idityng;e,ise

D)nd'

hen-ded),id:nnaxas

barn.

125

E

w

en0

U)en

w

-i

100 F

75F

50

25

0 10 20 30 40 50

ACID INVERTASE (y1ttoI*g'FWW')

Figure 4. Concentration of sucrose in control (open symbols) andgirdled (closed symbols) leaves of tomato, bean, sunflower, andspinach in relation to the activity of soluble acid invertase. Leaveswere sampled in the afternoon after 4 d of girdling.

I I I I IV SPINACH

_

"THRESHOLD". 1SUNFLOWER

v I

J3s BEAN TOMATOI i I I

A IA

1447

04

%J




others, such as soybean, the accumulation of hexose sugarsoccurs only transiently after phloem girdling (13). It may bethat appearance of free hexose sugars (even transiently) caninitiate down-regulation of the Calvin cycle and, hence,inhibition of Amax.A phloem girdle applied to high-invertase plants may be

functionally analogous to tobacco (22) and tomato (5) plantstransformed to express high levels of yeast invertase in thecell wall. In the transgenic plants, export is blocked as sucrosein the apoplast is hydrolyzed to hexose sugars, which cannotbe loaded into the phloem. Rather, the hexose sugars aretaken up into the mesophyll cells, thereby blocking export.As a result, there is accumulation of carbohydrate and re-duced growth.

In the study of Stitt et al. (22), it was established that theinhibition of photosynthesis occurred as the result of down-regulation of the Calvin cycle. Although the exact mechanisminvolved is unknown, it is relevant to note that Sheen (21)has recently demonstrated that sucrose and glucose inhibitedthe transcriptional activity of the promoters for seven pho-tosynthesis genes isolated from maize. Thus, buildup of(possibly) specific forms of carbohydrate (e.g. hexose sugars)may down-regulate the Calvin cycle as a result of effects ongene expression.From the sucrose storage aspect, the most interesting spe-

cies are those that do not normally accumulate sucrose butdo so when girdled. The best representative of such an'intermediate' species from the present study is sunflower.In girdled leaves, it is are able to store substantially higherthan usual amounts of sucrose (Table I), suggesting somegirdling-induced shift in carbohydrate metabolism. This fitsvery well with the relatively low acid invertase activity ofsunflower, which declines even further in girdled leaves (Fig.4). It is tempting to speculate that girdling drives the acidinvertase activity just below the critical 'threshold' level ofapproximately 10 ,mol of sucrose gg- h-', thereby enablingthe accumulation of sucrose.

Nevertheless, the results obtained with girdled spinachpresent some problems. It seems difficult to reconcile the lackof pronounced long-term inhibition of photosynthesis (TableII) with the early termination of the sucrose buildup (Fig. 3B).The possibility that girdled leaves have high rates of respi-ration that utilize the excess carbohydrates could not beconfirmed (data not shown). A solution might, perhaps, besuggested by the results of Housley and Pollock (11). Workingwith excised Lolium leaves, which maintained high photosyn-thetic rates for several days, they found that following theinitial accumulation of sucrose the leaves diverted their syn-thesis to high oligosaccharides (fructans). Although spinachis not a fructan accumulator, the possible occurrence ofanalogous metabolic changes in girdled spinach leaves shouldbe explored.We have shown that loss of Amax as a result of end-product

accumulation varies dramatically among species. It is mostpronounced in typical starch storers, but starch per se seemsnot to be involved. Rather, the loss of Amax may involvecycling of sucrose and hexose sugars in the cytosol thatultimately results in down-regulation of the Calvin cycle.Future studies will be needed to establish which of the Calvin

cycle enzymes have been affected, and what signal(s) isinvolved.

ACKNOWLEDGMENTS

The assistance of Mr. Mitchell Hayes with growing of the plantsand of Mr. Mark Bickett with experimental techniques is gratefullyacknowledged. Thanks are due also to Professor D.M. Pharr forproviding the HPLC sugar analyses.

LITERATURE CITED

1. Azcon-Bieto J (1983) Inhibition of photosynthesis by carbohy-drates in wheat leaves. Plant Physiol 73: 681-686

2. Bagnall DJ, King RW, Farquhar GD (1988) Temperature-de-pendent feedback inhibition of photosynthesis in peanut.Planta 175: 348-354

3. Boussingault JB (1868) Agronomie, Chimie Agricole et Physiol-ogie, Ed 2. Mallet Bachelier, Paris, pp 236-312

4. Delieu T, Walker DA (1981) Polarographic measurement ofphotosynthetic oxygen evolution by leaf discs. New Phytol 89:165-178

5. Dickenson CD, Altabella T, Chrispeels MJ (1991) Slow-growthphenotype of transgenic tomato expressing apoplastic inver-tase. Plant Physiol 95: 420-425

6. Foyer CH (1987) The basis for source-sink interaction in leaves.Plant Physiol Biochem 25: 649-657

7. Geiger DR (1976) Effects of translocation and assimilate demandon photosynthesis. Can J Bot 54: 2337-2345

8. Geiger DR (1987) Understanding interactions of source and sinkregions in plants. Plant Physiol Biochem 25: 659-666

9. Hanson KR, McHale NA (1988) A starchless mutant of Nicotianasylvestris containing a modified plastid phosphoglucomutase.Plant Physiol 88: 838-844

10. Herold A (1980) Regulation of photosynthesis by sink activity:the missing link. New Phytol 86: 131-144

11. Housley TL, Pollock CJ (1985) Photosynthesis and carbohydratemetabolism in detached leaves of Lolium temulentum. NewPhytol 99: 499-507

12. Huber SC (1989) Biochemical mechanism for regulation of su-crose accumulation in leaves during photosynthesis. PlantPhysiol 91: 656-662

13. Huber SC, Huber JA, Hanson DR (1990) Regulation of thepartitioning of products of photosynthesis. In I Zelitch, ed,Perspectives in Biochemical and Genetic Regulation of Pho-tosynthesis. Alan R Liss, New York, pp 85-101

14. Mauney JR, Guinn G, Fry KE, Hesketh JD (1979) Correlationof photosynthetic carbon dioxide uptake and carbohydrateaccumulation in cotton, soybean, sunflower and sorghum.Photosynthetica 13: 260-266

15. Mayoral ML, Plaut Z, Reinhold L (1985) Effect of translocation-hindering procedures on source leaf photosynthesis in cuc-umber. Plant Physiol 77: 712-717

16. Nafziger ED, Koller HR (1976) Influence of leaf starch concen-tration on CO2 assimilation in soybean. Plant Physiol 57:560-563

17. Neales TF, Incoll LD (1968) The control of leaf photosynthesisrate by the level of assimilate concentration in the leaf: areview of the hypothesis. Bot Rev 34: 107-125

18. Plaut Z, Mayoral ML, Reinhold L (1987) Effect of alteredsink:source ratio on photosynthetic metabolism of sourceleaves. Plant Physiol 85: 786-791

19. Robbins NS, Pharr DM (1988) Effects of restricted root growthon carbohydrate metabolism and whole plant growth of Cuc-umis sativus L. Plant Physiol 87: 409-413

20. Rufty TW Jr, Huber SC (1983) Changes in starch formation andactivities of sucrose phosphate synthase and cytoplasmic fruc-tose-1,6-bisphosphatase in response to source-sink alterations.Plant Physiol 72: 474-480

21. Sheen J (1990) Metabolic repression of transcription in higherplants. Plant Cell 2: 1027-1038

22. Stitt M, von Schaewen A, Willmitzer L (1990) 'Sink regulationof photosynthetic metabolism in transgenic tobacco plantsexpressing yeast invertase in their cell wall involves a down-regulation of the Calvin cycle and up-regulation of glycolysis.Planta 183: 40-50

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