Effect of Flooding on Starch Accumulation in Chloroplasts of Sunflower ( Helianthus annuus L.)

Abstract

ABSTRACI Chloroplasts in leaves of sunflower (Helianthus aNums L. cv hybrid 894) whose roots were flooded for 4 days showed an increase in the level of starch in chloroplasts when examined with the electron microscope. Starch determination showed significantly higher levels in leaves of flooded plants. Chloroplast and mitochondrial strcture seemed otherwise normal. Research has shown that plant water potential often is not adversely affected by flooding (4, 10) while leaf conductance is frequently reduced (2, 4, 13). Reduced leaf conductance is often cited as the cause ofreduced photosynthesis ofwaterlogged plants (2, 17). However, Moldau (13) examined the effect of flooding on bean plants (Phaseolus vulgaris L.) and concluded that an insufficient supply of metabolites from the roots in addition to stomatal 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 maintaining photosynthesis. Cytokinins decline in the bleeding sap from flooded roots (5) and application of cytokinins has been partially successful in maintaining photosynthesis rates in plants with waterlogged or partially excised roots (3, 7). Cytokinins maintain ribulose bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) synthesis (7). However, the lack of full recovery with cytokinin application implies still other factors are involved. In our examination of the effect of flooding on sunflowers, we found a reduction in photosynthesis after 4 d without a major reduction in leaf conductance (19) further suggesting factors other than stomatal conductance were influencing photosyn-thesis. It seemed possible that loss of chloroplast integrity was responsible and the present study was undertaken. Electron microscope studies of plant tissues grown under anaerobic conditions have been reported in roots of tomato (14), coleoptiles of rice (22), and seedlings of Echinochloa (21). We are not aware of any published reports which describe the effect of root system flooding on the chloroplast structure and starch accumulation in leaves. 'Partial support for this research was supplied by a Medical and Biological Research Grant from Washington State University to R. L. W. Scientific Paper No. SP 6533, College of Agriculture, Washington State University. MATERIALS AND METHODS Sunflower plants (Helianthus annuus L. cv Hybrid 894) were grown in a greenhouse from seed in 10-cm square pots, one plant per pot, containing a peat, pumice, and sand mixture (55:30:15). A complete fertilizer (Peters 20:20:20) was supplied to plants daily for 3 weeks, until the start ofan experiment. A photon flux at the top of the plants of 450 to 550 ,E m-2-s' during a 15-h photoperiod was provided by 1000-w high pressure sodium lamps. Temperature was maintained at 25C during the day and 15°C at night with a RH of 40 to 60%. Flooding was accomplished by immersing plant roots in water and maintaining the water level 1 to 2 cm above the soil surface throughout the experiment. 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 smaller strips while immersed in a few drops of 3% glutaraldehyde in 6 mM (pH 7) phosphate buffer. The leaf strips were put into vials of the same fixative and aspirated. After 2 to 6 h, the samples were rinsed in four 15-min changes of6 mm buffer. Post-fixation was with 1% O0s4 in the same buffer for 2 h. Dehydration was by a standard ethanol series and the samples were embedded in Epon 812. Silver-gold sections were stained with Reynold's lead citrate and uranyl acetate. The stained sections were examined on a Zeiss 10 transmission electron microscope. Fixative mixtures made using phosphate buffer solutions that were stronger than 6 mm caused an obvious loss of turgor in the leafstrips of both flooded and control plants. The need for 6 mm phosphate buffer was empirically determined and measured with a Wescor 5130A (Wescor, Inc., Logan, UT) vapor pressure osmometer. The osmolarity of the buffer alone was approximately 100 mOsm while the osmolarity of the complete fixative mixture was 290 mOsm. Starch determinations were made following a procedure similar to that of Loescher and Nevins (11). The third leaf pairs from 10 plants of each treatment were collected at approximately midway in the light cycle, frozen in liquid N2, lyophilized, and ground to a fine powder. Samples (50 mg) from each leaf pair were extracted twice with hot 80% ethanol:water (v/v) solution and once with water to remove soluble carbohydrates. The tissue residue was then suspended in 2 ml of 20 mm sodium phosphate (pH 6.9) and 6 mm NaCl. Test tubes were placed in a boiling water bath for 15 min to gelatinize the starch. The samples were cooled 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 and terminated by adding 10 ml of distilled H20. One-ml aliquots 195