Plant Physiol. (1983) 73, 195-1980032-0889/83/73/0195/04/$00.50/0

Short Communication

Effect of Flooding on Starch Accumulation in Chloroplasts ofSunflower (Helianthus annuus L. '

Received for publication May 2, 1983 and in revised form July 11, 1983

ROBERT L. WAMPLE AND RONALD W. DAVISDepartment ofHorticulture and Landscape Architecture, Washington State University, Pullman,Washington 99164-6414 (R. L. W.); Electron Microscope Center, University ofIdaho, Moscow, Idaho83843 (R. W. D)

ABSTRACI

Chloroplasts in leaves of sunflower (Helianthus aNums L. cv hybrid894) whose roots were flooded for 4 days showed an increase in the levelof starch in chloroplasts when examined with the electron microscope.Starch determination showed significantly higher levels in leaves offlooded plants. Chloroplast and mitochondrial strcture seemed otherwisenormal.

Research has shown that plant water potential often is notadversely affected by flooding (4, 10) while leaf conductance isfrequently reduced (2, 4, 13). Reduced leaf conductance is oftencited as the cause ofreduced photosynthesis ofwaterlogged plants(2, 17). However, Moldau (13) examined the effect of floodingon bean plants (Phaseolus vulgaris L.) and concluded that aninsufficient supply of metabolites from the roots in addition tostomatal closure were responsible for reduced photosynthesis.Root excision, which may be similar to the effect ofwaterlogging,reduces photosynthesis but is not related to stomatal closure (6)and further suggests a broader role of the roots in maintainingphotosynthesis. Cytokinins decline in the bleeding sap fromflooded roots (5) and application of cytokinins has been partiallysuccessful in maintaining photosynthesis rates in plants withwaterlogged or partially excised roots (3, 7). Cytokinins maintainribulose bisphosphate (RuBP) carboxylase/oxygenase (EC4.1.1.39) synthesis (7). However, the lack of full recovery withcytokinin application implies still other factors are involved.

In our examination of the effect of flooding on sunflowers, wefound a reduction in photosynthesis after 4 d without a majorreduction in leaf conductance (19) further suggesting factorsother than stomatal conductance were influencing photosyn-thesis. It seemed possible that loss of chloroplast integrity wasresponsible and the present study was undertaken.

Electron microscope studies of plant tissues grown underanaerobic conditions have been reported in roots oftomato (14),coleoptiles of rice (22), and seedlings of Echinochloa (21). Weare not aware of any published reports which describe the effectof root system flooding on the chloroplast structure and starchaccumulation in leaves.

'Partial support for this research was supplied by a Medical andBiological Research Grant from Washington State University to R. L.W. Scientific Paper No. SP 6533, College of Agriculture, WashingtonState University.

MATERIALS AND METHODS

Sunflower plants (Helianthus annuus L. cv Hybrid 894) weregrown in a greenhouse from seed in 10-cm square pots, one plantper pot, containing a peat, pumice, and sand mixture (55:30:15).A complete fertilizer (Peters 20:20:20) was supplied to plantsdaily for 3 weeks, until the start ofan experiment. A photon fluxat the top of the plants of 450 to 550 ,E m-2 -s' during a 15-hphotoperiod was provided by 1000-w high pressure sodiumlamps. Temperature was maintained at 25C during the day and15°C at night with a RH of 40 to 60%. Flooding was accom-plished by immersing plant roots in water and maintaining thewater level 1 to 2 cm above the soil surface throughout theexperiment.Segments of the third leaf pair above the cotyledons of 3.5-

week-old plants were obtained from control and flooded plants.The segments were immediately cut into 1 mm x 1 cm or smallerstrips while immersed in a few drops of 3% glutaraldehyde in 6mM (pH 7) phosphate buffer. The leaf strips were put into vialsof the same fixative and aspirated. After 2 to 6 h, the sampleswere rinsed in four 15-min changes of6 mm buffer. Post-fixationwas with 1% O0s4 in the same buffer for 2 h. Dehydration wasby a standard ethanol series and the samples were embedded inEpon 812. Silver-gold sections were stained with Reynold's leadcitrate and uranyl acetate. The stained sections were examinedon a Zeiss 10 transmission electron microscope.

Fixative mixtures made using phosphate buffer solutions thatwere stronger than 6 mm caused an obvious loss of turgor in theleaf strips ofboth flooded and control plants. The need for 6 mmphosphate buffer was empirically determined and measured witha Wescor 5130A (Wescor, Inc., Logan, UT) vapor pressureosmometer. The osmolarity of the buffer alone was approxi-mately 100 mOsm while the osmolarity of the complete fixativemixture was 290 mOsm.

Starch determinations were made following a procedure simi-lar to that of Loescher and Nevins (11). The third leaf pairs from10 plants of each treatment were collected at approximatelymidway in the light cycle, frozen in liquid N2, lyophilized, andground to a fine powder. Samples (50 mg) from each leaf pairwere extracted twice with hot 80% ethanol:water (v/v) solutionand once with water to remove soluble carbohydrates. The tissueresidue was then suspended in 2 ml of 20 mm sodium phosphate(pH 6.9) and 6 mm NaCl. Test tubes were placed in a boilingwater bath for 15 min to gelatinize the starch. The samples werecooled and 0.2 ml of pancreatic a-amylase (Sigma, Type I-A)solution (0.1 ml/100 ml buffer) was added to each test tube.Samples were incubated in a 37°C water bath for 16 h andterminated by adding 10 ml of distilled H20. One-ml aliquots

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196 WAMPLE AND DAVIS Plant Physiol. Vol. 73, 1983*

§g3= _a;9il_ 44 _ ~~~~.r.j'3i.t__- s ,, Lz }'' i'

P.5v|*lI~il. _ w_ 1 _.; _

FIG. 1. Electron micrograph of palisade parenchyma cells from leaves of control (a) and flooded (b) sunflower plants after 96 h. Representativestarch grains are labeled S.

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FLOODING AND STARCH ACCUMULATION IN CHLOROPLASTS

were taken and reducing sugar analyses were made using adinitrosalicylate method (12). The experiment was repeated fourtimes.

RESULTS AND DISCUSSION

Sections of flooded and control plant leaves examined withthe electron microscope revealed a comparatively large amountof starch in chloroplasts of the flooded specimens (Fig. 1, a andb). The most striking difference between control and floodedspecimens is the amount of starch present in the chloroplasts. Itappears that larger starch granule size in chloroplasts of floodedspecimens is a primary cause of this. What we had expected tofind was a degeneration of chloroplast membranes on a largeenough scale to account for the previously observed decrease inphotosynthesis ( 19). In this initial study, that did not seem to be

Table 1. Effect ofFlooding (4 Days) on Starch Accumulation inSunflowers

Fifty-mg samples of the third leaf pair of control and flooded plantswere used for enzymic starch determination following soluble carbohy-drate extraction. Mean ± SD.

Maltose Equivalents

Control Flooded

Mmol/50 mg dry wt tissueTissueonly 1.4 ±0.4 2.5 ±0.6Enzyme 0.8 0.8Boiled enzyme + tissue 1.4 ± 0.5 2.5 ± 0.6Unboiled enzyme + tissue 16.2 ± 3.3 31.7 ± 4.6~~- sr

FIG. 2. Electron micrograph of an area of a parenchyma cell fromthe leaf of a flooded plant. Starch grains (S), a portion of a nucleus (N),mitochondria (M), and intact grana (unlabeled arrows) are visible.

the case.Chemical analysis after extraction of soluble carbohydrates

verified the starch accumulation seen in the electron micrographs(Table I). Hooded leaves had almost twice as much starch(measured as maltose equivalents) as control plants.

Reports by Rackam (16) and Wildman (25) suggest that highlevels of starch accumulation may influence the rate of CO2fixation directly through distortion of thylakoid membranes orby reduction in the light reaching thylakoid membranes. Ourobservations suggest that, although mitochondria appear normal,chloroplast thylakoids in flooded sunflowers are displaced due tostarch accumulation but are probably still intact (Fig. 2). A morethorough examination of the substructure in chloroplasts offlooded sunflower plants will be the subject of a subsequentinvestigation.

Starch accumulation in flooded sunflowers may reflect reducedphloem transport as a consequence of a decline in root metabo-lism caused by low 02 levels. Root growth and metabolism isundoubtedly a major sink in young sunflowers. With this reducedsink strength and phloem transport, there would be an accumu-lation of cytoplasmic sucrose in the leaves and, as suggested byHerold (9), could cause a build-up of triose and hexose phos-phates as a consequence of feedback inhibition of sucrose phos-phate synthetase (EC 2.4.1.14) (18) by sucrose. This woulddeplete the pool of free phosphate (Pi) in the cytoplasm andinhibit export of triose phosphates from the chloroplast since Piis required for the exchange process (8). Inhibition of Pi uptakeby flooded plants as a consequence of increased endogenousethylene as demonstrated for Zea mays L. by Jackson et al. (10)could also contribute to reduced cytoplasmic Pi levels. Theresultant accumulation of triose phosphates and 3-P-glyceratewould lead to a stimulation of chloroplastic starch synthesis (15)and may account for the observations reported here. The accu-mulation of triose phosphates in the chloroplast may directlycause photosynthetic rates to decline (23). In addition or alter-natively, a decrease in osmotic potential caused by soluble car-bohydrate accumulation could cause photosynthesis to declineas a result of intrathylakoid acidification as suggested by Berkow-itz and Gibbs (1).Other reports of starch accumulation in flooded plants are

inconsistent. For example, Wiedenroth and Poskuta (24) foundno increase in 14C incorporation into leaf starch of flooded wheatplants while Trought and Drew (20) attributed an increase inshoot dry weight of flooded wheat plants during the first 8 d tostarch accumulation. The mechanism responsible for starch ac-cumulation in flooded sunflowers remains unclear at this time.Studies designed to examine the hypotheses presented above andto explore the possible role that starch accumulation plays inother physiological responses to flooding are planned.

LITERATURE CITED

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198 WAMPLE AND DAVIS

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Plant Physiol. Vol. 73, 1983

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