THESIS Iii-*fin'“?! O . ‘ in .9 ". - 3" lief. 3} ($-1- I'i'vvfim-‘U‘rl err-~— O 3.x 0'- .0 I '- ° ... _°¢ ' . lie.» 1-- ‘7’ l “-i‘ J bub-r '- l P L_ This is to certify that the thesis entitled Ph/SID/Dfl; 3/ gray/l: and qpra/,'o~ I'M Rice presented by 16/5, Raff/N has been accepted towards fulfillment of the requirements for PA-D degreein BO/‘aN/Y l Major professor Hans Kende Date (.411,th 0-7639 MSU is an Affirmative Action/Equal Opportunity Institution )V1ESI_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from .A-IlrIIIIL. your record. FINES will B be charged if book is returned after the date stamped below. PHYSIOLOGY OF GROWTH AND AERATION IN RICE By llya Raskin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1984 3:321 "a U \ ABSTRACT PHYSIOLOGY OF GROWTH AND AERATION IN RICE By Ilya Raskin Isolated stem sections were used to study the rapid acceleration of internodal elongation in deep-water rice (Oryza sativa L. cv. Habiganj Aman II) caused by submer- gence. The effect of submergence on rice was, at least in part, mediated by C2H4, which accumulated in the air spaces of submerged sections. This accumulation resulted from slower diffusion of C2H4 from the tissue into the water and from increased CZHA synthesis in the submerged internodes, triggered by reduced concentrations of 02. Increased CZHA levels accelerated internodal elongation and inhibited the growth of leaves. The enhancement of internodal elongation by C2H4 was particularly pro- nounced in an atmosphere rich in C02 and low in 02. The effect of submergence on the growth of stem sections of deep-water rice could be mimicked by eXposing non-submerged sections to a gas mixture which is similar to the gaseous atmosphere in the internodal lacunae of submerged sections, namely 3% 02, 6% COZ, 91% N2 (by vol.) and 1 ul 1"1 C2H4° Ethylene probably causes internodal elongation in rice by increasing the activity of endogenous gibberellins. Stem sections excised from plants that had been watered with a solution of 10'6 M tetcyclacis, an inhibitor of gibberellin biosynthesis, did not elongate when submerged in the same solution nor when exposed to 1 ul 1'1 C2H4 in air. Applied gibberellic acid (GA3) restored the rapid internodal elonga- tion in submerged and C2H4-treated sections to the levels observed in control sections that had not been treated with tetcyclacis. Submergence led to the rapid mobilization of starch from the older regions of rice internodes, which had been formed prior to submergence. Starch disappearance was accompanied by a 70-fold increase in a-amylase activity. A similar increase in a-amylase activity was detected in response to C2H4 and GA3. Submergence also caused a 28-fold increase in the translocation of photosynthetic assimilates from the leaves to the internodes and younger regions of culms. As in the adult rice plants, submergence altered the growth of different organs in rice seedlings. It markedly enhanced coleoptile and mesocotyl growth and inhibited leaf growth. Morphological changes similar to those observed in submerged seedlings were observed in seedlings grown in sealed containers in darkness. Ethylene, high levels of C02 and reduced levels of 02 found in sealed containers contributed equally to the increase in coleOptile and carbon fixation by enlarging the surface of the gas-liquid interface available for C02 uptake from the water. Deep- water rice plants without air layers did not grow in response to submergence and the submerged parts of the plant deteriorated as evidenced by a rapid loss of chlorOphyll and protein. ACKNOWLEDGEMENTS I sincerely thank my major professor, Dr. Hans Kende, for providing me with intelligent, sensitive and patient guidance and for teaching me not only how to receive a Ph.D. but how to be one. I extend my gratitude to the members of _ my guidance committee, Dr. Dave Dilley, Dr. Norman Good, Dr. Andrew Hanson and Dr. Jan Zeevaart for their constructive advice and encouragement. Last, but not least, I thank all other members of the faculty, post-docs, and graduate students of the D.O.E. Plant Research Laboratory for the wonderful time which I had in Michigan. ii TABLE OF CONTENTS Page. LIST OF FIGURES . . . . . . . . . . . . . . . . . . . v LIST OF TABLES . . . . . . . . . . . . . . . . . . . . viii CHAPTER GENERAL INTRODUCTION 0 O O I O O O O O O O O O C O O O 1 . CHAPTER 1 - REGULATION OF GROWTH IN STEM SECTIONS OF DEEP WATER RICE . . . . . . . . . . . . . 1. Abstract . . . . . . . II. Introduction . . . . III. Materials and Methods IV. Results . . . . . . . . V. Discussion . . . . . . VI. References . . . . . . Q C _.|—i ‘CVC‘LJIKJ'I .9 CHAPTER 2 - THE ROLE OF GIBBERELLIN IN THE GROWTH OF SUBMERGED DEEP-VJATER RICE o o o o o o o o 12 1. Abstract . . . . . . . . . . . . . . 13 II. Materials and Methods . . . . . . . . 15 III. Results . . . . . . . . . . . . . . . 17 IV. Discussion . . . . . . . . . . . . . 19 V. Literature Cited . . . . . . . . . . 21 CHAPTER 3 - EFFECT OF SUBMERGENCE ON TRANSLOCATION, STARCH CONTENT AND a-AMYLASE ACTIVITY IN DEEP WATER RICE . . . . . . . . . . . . . 28 I. Abstract . . . . . . . . . . . . . . 29 II. Introduction . . . . . . . . . . . 30 III. Materials and Methods . . . . . . . . 31 IV. Results and Discussion . . . . . . . 34 V. References . . . . . . . . . . . . . 36 CHAPTER 4 - REGULATION OF GROWTH IN RICE SEEDLINGS . . 41 I. Abstract . . . . . . . . . . . . . . 42 II. Materials and Methods . . . . . . . . 43 III. Results . . . . . . . . . . . . . . . 45 IV. Discussion . . . . . . . . . . . . . 49 V. References . . . . . . . . . . . . . 52 iii CHAPTER 5 - CHAPTER 6 - _ CHAPTER 7 - CONCLUSIONS THE ROLE OF AIR LAYERS IN THE DEEP-WATER RICE . . . . . . . 1. Abstract . . . . . . . . II. Materials and Method . III. Results . . . . . . . . IV. Discussion . . . . . . . V. Literature Cited . . . MECHANISM OF AERATION IF RICE I. Mass Flow of Gases II. Role of Air Layers III. Discussion . . . . . VI. References . . . . . AERATION OF A METHOD FOR MEASURING LEAF VOLUME, DENSITY, THICKNESS AND INTERNAL GAS VOLUME . . . . . iv Page Figure 1.1 1.2 1.3 2.2 2.3 3.1 3.2 3.3 4.2 LIST OF FIGURES Page The morphology of stem sections of deep-water rice incubated in air and submerged in water 7 Ethylene evolution from internodes excised from rice stem sections that had been submerged in H20 or incubated in air . . . . . . . . . . . 9 Ethylene production by internodes and leaves excised from rice stem sections incubated in air or in different gas mixtures. . . . . . 10 Effect of GA3 on the growth of internodes and leaves of rice stem sections submerged in 10‘6M TCY solution or distilled water . . . . . . . 24 Effect of GA3 on the growth of internodes and leaves of rice stem sections incubated in a stream of air or 3% 02, 6% 002, 91% N2 (by vol.) and 1 p1-1 czn4 . . . . . . . . . . 25 Effect of low GA3 concentrations on the growth of internodes and leaves of rice stem sections treated with 10'6M TCY and incubated in the stream of air or air containing 1 ul 1‘ C2H4 27 Starch content of different regions of internodes excised from submerged or air-incubated rice stem sections . . . . . . . . . . . . . . . . 38 a-Amylase activity in different regions of inter- nodes excised from submerged or air-incubated rice stem sections . . . . . . . . . . . . . . 39 Effect of submergence and incubation in air on the partitioning of 4C-labeled assimilates in modified rice stem sections . . . . . . . . 40 The time course of growth of etiolated rice seedlings (cv. M-9) in sealed and aerated containers . . . . . . . . . . . . . . . . . . 46 Growth of etiolated seedlings of different rice varieties in sealed and aerated containers . . 47 V Figure Page 4.3 Time course of C2H4 evolution from etiolated rice seedlings (cv. M-9) after 6 days of incubation in air or in different gas mix- tures . . . . . . . . . . . . . . . . . . . . 47 4.4 The effect of C2H4 on the growth of rice seedlings (cv. M-9) under a 16-h photOperiod . 49 4.5 Time course of elongation of completely submerged and air-grown rice seedlings . . . . . . . . . 56 5.1 Mass flow of air along a submerged rice leaf blade 0 O O O O O O O O O O I O O O O O O O O 57 5.2 Diffusion of ethane through the air layers around submerged rice leaves . . . . . . . . . 58 5.3 02 movement through the air layers in darkness determined by the oxidation of reduced methYlene blue 0 O O O O I O O O O O O O O O O 58 5.4 Mass flow of air into the air layers in darkness 59 5.5 Mass flow of gases into the air layers in dark- ness measured as the rate of volume change in the head space containing air, N2, or 02 . . . 59 5.6 Mass flow of gases in and out of air layers . . 59 5.7 Photosynthetic carbon fixation in submerged leaf blades of rice with intact air layers and following elimination of air layers by Triton X-100 treatment . . . . . . . . . . . . 60 5.8 Physiological importance of air layers for the elongation response and for the maintenance of Chl and protein levels . . . . . . . . . . 60 5.9 Model for the mass flow of gases through air layers 0 O O O O I O O O O C I O O O O O O O O 61 6.1 Mass flow of gases into the underwater parts of a partially submerged rice plant . . . . . . . 72 6.2 The role of roots in the mass flow of gases into a rice plant . . . . . . . . . . . . . . . . . 74 6.3 The effect of the 602 concentration in the solution around the plant on the rate and direction of the gas flow . . . . . . . . . . 75 vi Figure Page 6.4 Reversibility of the effect of C02 on mass flow of gasses . . . . . . . . . . . . . . . . 76 6.5 The role of air layers in the conductance of gases 0 O O O O O O I O O C O O C O O I O O O 78 7.1 Analytical balance adapted for weighing leaves under water . . . . . . . . . . . . . . . . . 80 vii LIST OF TABLES Table Page 1.1 Concentrations of 02, C02 and C2H4 in the internodal lacuane of rice stem sections . . . 7 1.2 Effects of 02, C02, C2H4 and submergence on the elongation of rice stem sections incubated under a 13- h photOperiod . . . . . . . . . . . 8 1.3 Effects of 02, C02, C2H4 and submergence on the elongation of rice stem sections incubated in darkness . . . . . . . . . . . . . . . . . 8 1.4 Effects of 02, C02, C2H4, submergence and anoxia on the elongation of rice stem sections incubated under continuous light . . . . . . . 8 1.5 Effects of AVG and ACC on growth of submerged rice stem sections . . . . . . . . . . . . . . 9 1.6 Effects of sumbergence and different gas mixtures on the levels of free and conjugated ACC in the internodes of rice stem sections . . . . . . . 10 2.1 Number of subepidermal cells in the growing region of rice internodes . . . . . . . . . . 22 2.2 Effect of leaf sheath removal on internodal elongation in rice stem sections . . . . . . . 23 3.1 Effect of C H4 and GA3 on internodal elongation and a-amyIase activ1ty in the older regions of the internodes formed before isolation of rice stem sections . . . . . . . . . . . . . . 37 4.1 Effect of different gas mixtures on the growth of etiolated rice seedlings . . . . . . . . . 48 4.2 Effect of different concentrations of C2H4 on the growth of etiolated rice seedlings . . . . 48 viii Table 4.3 7. 2 Effect of darkness and light on the growth of rice seedlings submerged at different depths Of water 0 C O O O O O O O O O O O O O O O 0 Length of coleOptiles of selected cereals as a function of depth of planting in vermiculite Comparison of leaf parameters in different cereals O O O O O O O O O O O I O O O O O 0 Average volume, weight density, volume of the internal air spaces, and density of the non- gaseous leaf content of leaves of Phaseoulus vulgaris and Oryza sativa . . . . . . . . . Average thickness of Phaseolus vulgaris trifoliates and midlaminar regions of Oryza sativa leaves . . . . . . . . . . . . . . . ix Page 50 50 57 81 GENERAL INTRODUCTION Rice occupies 11 percent of the world's arable land and is grown almost exclusively for human consumption. It constitutes half of the diet of 1.6 billion people living in many underdeveloped areas of the world where no other crop _ can be grown. Rice thrives in a wide variety of habitats ranging from the humid tropics of India, Thailand, Vietnam and the Philippines, to the hot and arid lands of Pakistan, Iran and Egypt, and the cool climates high in the mountains of Nepal and Northern China. Rice is grown either on dry land, in shallow paddies or in the flooded plains covered with several meters of water. This dissertation is primarily concerned with deep- water or floating rice, grown predominantly in low-lying areas of Southeast Asia which are flooded every year during the rainy season. Seeds of deep-water rice are planted on flood plains or dry river beds at least a month before the rainy season starts. By the onset of the monsoon rains, deep-water rice has gained the ability to elongate as fast as 20 to 25 cm a day in order to keep some of its foliage above the rising water. Deep-water rice plants grow in water 1 to 6 m deep, usually forming a mat on the water surface. All deep-water rice varieties are photoperiodic and are naturally selected in such a way that panicle emergence coincides with the end of the rainy season. Since no more internodes can be formed after the initiation of flowers, the end of vegetative development coincides with the loss of elongation ability. Harvesting is done after the flood waters have receded, although early maturing varieties are harvested from boats. Deep-water rice has deveIOped special adaptive mechan- isms in order to survive the extreme conditions of its habi— tat. First, it possesses a regulatory mechanism enabling it to increase the growth rate in order to keep at least part of the foliage above the surface of rapidly rising waters during the first part of the monsoon season. Total submer- gence for more than 1 week results in plant death. Second— ly, deep-water rice has an efficient aeration system which can transport 02 all the way to the roots in order to maintain aerobic metabolism in the submerged organs. Roots and shoots of rice rapidly deteriorate without adequate supply of 02. Of all rice varieties, deep-water rice has been studied least. No work has been done on the mechanism by which 02 and C02 is supplied to the submerged organs of the plant. Practically nothing has been known about the physiological basis of internodal elongation induced by partial flooding, in spite of the possibility of increasing grain yields in flooded areas by increasing the elongation ability of high-yielding rice varieties which could not otherwise survive rapid flooding. There is also a great potential for using deep-water rice as a model system in the study of regulation of plant growth. This dissertation focusses on the physiological mechanisms involved in aeration and regulation of growth in partially flooded deep-water rice. The similarities of growth regulation in rice seedlings and deep—water rice plants are also discussed. Chapter 1 Regulation of Growth in Stem Sections of Deep-Water Rice Planla (1984) 160:66—72 Planta, (C; Springer-Verlag 1984 Regulation of growth in stem sections of deep-water rice Ilya Raskin and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing. Ml 48824. USA Abstract. Submergence in water greatly stimulates internodal elongation in excised stem sections of deep-water rice (Oryza sativa L. cv. “Habiganj Aman 11“) and inhibits growth of leaf blades and leafsheaths. The highest rates ofinternodal growth have been observed in continuous light. Very little growth occurs in submerged sections kept in dark- ness or incubated under N2 in the light. The effect of submergence on the growth of deep-water rice is, at least in part, mediated by C2114, which accu- mulates in the air spaces of submerged sections. This accumulation results from increased C2H4 synthesis in the internodes of submerged sections and reduced diffusion of C2H4 from the tissue into the water. Increased Czl-I4 levels accelerate inter- nodal elongation and inhibit the growth of leaves. Compounds capable of changing the rate of C2H4 synthesis, namely aminoethoxyvinylglycine, an in- hibitor of C2H4 synthesis, and l-aminocyclopro- pane-l-carboxylic acid, the immediate precursor of C2H4, have opposite effects on growth of inter- nodes and leaves. The enhancement of internodal elongation by C2H4 is particularly pronounced in an atmosphere of high CO2 and low 02. The in- crease in C2114 synthesis in internodes of sub- merged sections is primarily triggered by reduced atmospheric concentrations of 02- The rate of CZH4 evolution by internodes isolated from stem sections and incubated in an atmosphere of low 02 is up to four times greater than that of isolated internodes incubated in air. In contrast. CZH4 evo- lution from the leaves is reduced under hypoxic conditions. The effect of submergence on growth of stem sections of deep-water rice can be mim- icked by exposing non-submerged sections to a gas mixture which is similar to the gaseous atmosphere in the internodal lacunae of submerged sections, .-i/i/irt-riminmx ACC = 1-aminocyclopropane~l-carhoxylic acid; A \'(i = aminoethoxyvinylglycinc namely 3% Oz, 6% C02. 91% N2 (by vol.) and lul l“1 C2H4. Our results indicate that growth responses obtained with isolated rice stem sections are similar to those of intact deep-water rice plants. Key words: Carbon dioxide (ethylene. rice) — Deep- water rice — Ethylene (rice) — 0ry:a (growth regula- tion) — Oxygen (ethylene synthesis). Introduction Floating or deep-water rice is mainly grown in the flood plains of Southeast Asia. Deep-water rice plants are capable of rapid elongation during the rainy season when the plants become partially sub- merged (see review in De Datta 1981). Growth rates of 20-25 cm a day have been recorded, with most of the elongation taking place in the sub- merged internodes (Vergara et al. 1976). In con- trast to internodes. the lower leaves of deep-water rice stop elongating and eventually die when they become totally submerged. Ethylene stimulates the growth of the inter- nodes of non-submerged deep-water rice plants. and endogenous C2H4, which accumulates in the submerged internodes. is, at least in part, responsi- ble for the stimulation ofgrowth under water (Me- traux and Kende 1983a). lnternodal elongation in response to submergence is based on activation of cell division in the intercalary meristem and subse~ quent elongation of the newly formed cells (Me- traux and Kende 1983 b). The use of isolated stem sections. described in this report, has provided us with a system of re- duced complexity and increased versatility to study the submergence response in deep-water rice. We have evaluated the roles of C2114, 0: and CO2 in controlling the growth ofdeep-water rice plants. l. Raskin and H. Kende: Growth in rice stem sections The Opposite effects of these gases on the growth of internodes and leaves have been examined and related to the growth habit of deep-water rice culti- vars. Material and methods Chemicals. l-Aminocyclopropane-l-carboxylic acid (ACC) was purchased from Calbiochem-Behring Corp.. La Jolla. Cal., USA; aminoethoxyvinylglycine (AVG) was a gift from Dr. M. Lieberman (US. Department of Agriculture, Beltsville. Md, USA). Plant material. Seeds of deep-water rice (0r_r:a sativa L. cv. “ Habiganj Aman II “) were obtained from the Bangladesh Rice Research Institute (Dacca, Bangladesh). Rice was germinated and grown as described in Métraux and Kende (1983a). Stem sections. 20 cm long and containing the highest two nodes and the top-most internode. were excised from the main stems and tillers of six- to ten-week-old plants with a sharp razor blade (Fig. 1A). The sections were cut in such a fashion that the lower node was 2 cm above the basal cut. We used only sections in which the expanding. youngest internode was 1 to 7 cm long. All leaf sheaths originating from nodes not included in the section were peeled from the latter. The initial length of each internode was measured by holding the section in front of a strong light which allowed localization of the two nodes in- cluded in each section. Following 3d of experimental treat- ments. each section was cut open longitudinally to determine the final length of the internode or internodes if any new ones had developed during the incubation. Growth of the leaves was determined by subtracting the internodal growth from the total increase in section length. The term “leaf“, as we use it, includes the leaf sheaths and the bases of the leaf blades that grow out of the original 20-cm-long stem section during the course of the experiment (Fig. 1 B). Growth of submerged sections. Ten to 15 sections were placed in an upright position in a 100-ml glass beaker which was then filled with glass beads to prevent the submerged sections from floating up. Each beaker containing the sections was lowered to the bottom of a H volumetric cylinder, 42 cm deep. filled to the rim with distilled H20 or experimental solution. All experiments were performed at 27° C. either in darkness. or under a 13-h photoperiod, or in continuous light (cool-white ...7 fluorescent tubes; 70 pmol m ' s“ 1). Growth in different gas mixtures. Ten to 15 excised stem sections were placed upright in a 100-ml glass beaker containing 30 ml of distilled 1120. Each beaker containing the sections was placed in a 2.5-1 plastic cylinder. 60 cm deep, which was fitted with a gas-tight lid and with inlet and outlet tubings. The flow rate of air or gas mixtures through the cylinders was maintained at 80 ml min". Experiments were carried out at 27° C either in continuous light (70 timol m ‘2 s‘ ‘). or under a 13-h photo- period (same photon flux as above). or in darkness. Nitrogen, O2 and CO2 were supplied from high-pressure gas cylinders. Gas mixtures were prepared with gas-pressure regulators and rotameters containing three calibrated flow-meter tubes equipped with high-accuracy valves and a mixing tube (Mathe- son Gas Products. Joliet, 111.. USA). Compressed laboratory air was used for all flow-through air treatments. Gas mixtures and air were humidified to 100% relative humidity by bubbling them through water. They were divided and dispersed to the incubation cylinders with flow meter boards (Pratt ct al. 1960). Ethylene was added to the gas stream with a Cyliy-dil'fusion apparatus (Saltveit 1978). When stem sections were to be treated with C2H,-free air or gas mixtures. the gases were passed through a 25-cm-long column (7cm inner diameter) packed with Purafil (Purafil, Atlanta, 021., USA) to remove contaminating traces of C211,. Analysis of internodal gases. Each section was completely sub- merged in water in a large sink, and both ends of the internode were severed with a sharp razor blade. Gases from the interno- dal lacunae were allowed to escape into an inverted test tube filled with water. lnternodal gases of sections used for the same treatment were collected in one test tube which was then stop- pered with a serumw'ial cap before being taken out ofthe water. A 2-ml sample was withdrawn from each test tube with a gas- tight syringe to determine the concentration of Oz and CO2 using a gas chromatograph equipped with a thermal conductivi- ty detector (Model GC8700; Carle Instruments, Anaheim, CaL. USA). Ethylene was determined by gas chromatography of l-ml gas samples (Kende and Hanson 1976). The gas volumes withdrawn for analyses were replaced with equivalent volumes of water. Measurement of C 2H 4 production. The internodes were sepa- rated from most of the leaf tissue with a transverse cut across the upper node. Only the leaf tissues above the upper node were used for the determination of CZH4 synthesis. The single leaf sheath around the internode was carefully peeled away and discarded. A second transverse cut across the bottom node severed the internode from the 2-cm-long basal portion of the section. Excised internodes were weighed and placed in 30-ml test tubes (three internodes per test tube) containing 2 ml of distilled water or a solution of 10" M AVG. The leaf tissue from three sections was weighed and placed in one 60-ml test tube containing 3 ml ofdistilled water. All test tubes were stop~ pered with rubber serum-vial caps, each of which was fitted with a 3.5-inch, 16-gauge (9 cm long. 1.17 mm inner diameter) and a 1.5-inch, 16-gauge (4 cm long, 1.17 mm inner diameter) hypodermic needle to provide an inlet and an outlet for the gas stream. Each test tube was flushed for 1 h with the same gas mixture (40 ml min" ‘) in which the stem sections had been incubated before separation of internodes and leaf sheaths. ln- ternodes from sections that had been submerged in H20 were placed in test tubes and flushed with a gas mixture of 3% 02. 6% CO2 and 91% N2 (by ml). After 1 h, inlet and outlet needles were tightly stoppered, and the test tubes were placed on an orbital shaker operating at 20 cycles min” ‘. They were kept in the light (photon flux 60 umol m" s‘ ‘) at 24° C. One- ml gas samples were withdrawn every hour with a tuberculine syringe inserted through the serum-vial cap and were replaced with air; CzH4 was determined by gas chromatography (Kende and Hanson 1976). Determination of AC C and A CC-con/ugate. Exeised internodes were ground in liquid N2 in a mortar with a pestle. The resulting powder was extracted with 2 vols. of 70% (v/v) ethanol and centrifuged at 12.000 g for 15 min using a Sorvall RC-2B centri- fuge and a 58-34 rotor (DuPont lnstruments-Sorvall, Wilm- ington, Dcl.. USA). The level of ACC-conjugate (presumed to be malonyl ACC) was determined by adding HCl to the supernatant to give a final concentration of 2 M HCl followed by hydrolysis at 100° C for 4 h. The hydrolyzatc was neutralized with 2 M NaOH. The level of ACC was determined according to Lizada and Yang (1979). The amount of conjugated ACC was determined by subtracting the amount of ACC in the non- hydronch sample from the amount of ACC in the hydrolyzed sample. Internal ACC standards were used to correct for losses during extraction and hydrolysis. Fig. IA. B. The morphology of stem sections of deep-water rice incubated in air and submerged in water. A Longitudinal median section through a 20-cm-long stem section. The second highest node of the stem (N2) was 2 cm above the basal cut (C2). This lower node was separated from the highest node (.\'I) by the youngest internode (I). The stem section between the highest node and the upper cut (Cl) consisted of leaf sheaths and the developing youngest leaves (L). B Sections that had been incubated in a 2.5-1 cylinder through which air was passed (80 ml min’ ‘) or were submerged in water under continuous light. After 3d. the sections were cut open. and the length of the internode was measured. The position of the highest node is indicated by the arrows. The site of the original upper excision is also indicated (--CUT--) Results Stem sections isolated from deep-water rice plants were incubated in a stream of air or were sub- merged in water under continuous light for 3d (Fig. 1). The final length of the sections following both treatments was similar. However, when the stems were slit open. it became evident that the internodes of the non-submerged sections had elongated very little and that growth of the sections was based mainly on elongation of the leaf sheaths and leaf blades. In contrast. the internodes of the submerged sections had increased several fold in length while leaf growth was inhibited. The levels of CO2 and CzH,1 increased up to 20— and lOO-fold. respectively. and the level of O2 declined by as much as tenfold in the internodal lacunae of submerged sections compared to see- 7 I. Raskin and H. Kende: Growth in rice stern sections Table 1. Concentrations 01°02. CO2 and CiH4 in the internodal lacunae of rice stem sections incubated in a stream of air (80 ml min' 1) or submerged in H20. Each value is the pooled average of 10 sections Gas Submerged Air control End Middle End Middle of 2nd of 3rd of 2nd of 3rd dark light dark light period period period period 02 (%, v/v) 2.1 7.4 19.7 22.1 CO2 (‘11,, V/V) 6.7 1.1 1.6 0.06 C,H_l (pl 1’ l) 1.0 0.9 0.01 0.01 tions incubated in air (Table 1). In submerged sec- tions, the air spaces of leaf sheaths also contained about 1pll‘l CZH4 as determined by vacuum evacuation of leaf sheaths under water (data not shown). Air and six different mixtures of 02. CO2 and C2H4 in N2 were used to evaluate the effect of high concentrations of CO2 and CZH4 and low concentrations of 02 on growth of internodes and leaves. The gas mixtures, which were passed through the chambers in which sections were incu- bated under a 13-h photoperiod. contained either 21% 02, 0.03% CO2 (both v/v) and no CZI-I4 or concentrations of these gases that were close to those in the internodal lacunae of submerged sec- tions at the end of the dark period, namely 3% 02, 6% CO2 (both v/v) and 1 pl 1’1 C2H4. Ethyl- ene at 1 pl 1'1 in air and 3% O2 enhanced interno- dal growth by 6.9 and 4.4 times. respectively (Ta- ble 2). When 1 pl 1‘1 C2H4 and 3% (v/v) 02 were supplied in the same gas mixture, internodal growth was enhanced about tenfold. High concen- trations of C02 (6%, v/v), in a gas mixture con- taining 21% (v/v) O2 and no CZH4 had very little effect on internodal elongation in non-submerged sections. However, when 1pll’1 of C2H4 was added to the above gas mixture, the growth of internodes was increased more than ninefold. While internodal elongation was strongly pro- moted by C2H4, leaf growth was inhibited when C2H4 was added to air or the other gas mixtures. For example, 1 p11" CZI-I4 added to the gas mix- ture containing 3% (v/v) O2 inhibited growth of deep-water rice leaves by about 70% (Table 2). The effect of submergence on growth of internodes and leaves in deep-water rice stem sections was closely mimicked by passing a gas mixture of 3% (v/v) Oz. 6% (v/v) CO2 and 1 pl 1'1 C3H4 in N2 through the chambers containing rice stern sec- tions. I. Raskin and H. Kende: Growth in rice stem sections Table 2. Effects of 02. C02, CIH‘ and submergence on the elongation of rice stem sections incubated under a 13-h photoperiod for 3 d. Air and gas mixtures (all v/v) were passed through the incubation cylinders at 80 ml min' ‘. Each value is the average of 11 sections : SE Treatment Length increase (mm) , - C 2H4 concentration in internode Intemode Leaf Total section (pl 1‘ 1) Air 9.5: 1.2 105.5: 9.9 115.0:10.4 0.02 Air + 1 pl 1“ CIH. 65.2: 7.8 62.9:11.9 128.1 :17.8 0.9 21% O, + 6% C0, + 73% N2 13.3: 2.8 118.3: 6.4 131.6: 6.8 0.02 21% O, + 6% C0, + 73% N, + 1 pl 1'1 CZH‘ 88.1 :11.1 90.6:12.5 178.7:21.4 1.0 3% 02 + 0.03% CO2 + 97% N2 41.4: 5.1 76.2: 7.7 117.6: 7.2 0.01 3% 02 + 003% C02 + 97% N2 + 1 pl 1'1 CZH4 95.9:11.7 35.1: 3.5 141.0:12.9 0.8 3% 02 + 6% CO2 + 91% N2 54.7: 4.7 111.9: 6.9 166.6: 10.4 0.02 3% 02 + 6% CO2 + 91% N2 + 1 pl 1'1 CZH‘ 95.3: 9.6 52.8: 4.2 148.1 : 8.1 1.1 Submerged 93.4:10.1 61.8: 7.8 155.2:14.3 1.0 Table 3. Effects of 02, C02. C2H4 and submergence on the elongation of rice stem sections incubated in darkness for 3 d. Air and the gas mixtures (all vtv) were passed through the incubation cylinders at 80 ml min“. Each value is the average of“ sections : SE Treatment Length increase (mm) CzH‘ concentration in internode Internode Leaf Total section (pl 1‘ ‘) Air 5.1: 1.3 83.6: 7.4 88.7: 7.9 0.01 Air + 1pll"C2H4 - 25.4: 5.9 25.9: 2.7 51.3: 6.1 0.66 21% 02 + 6% CO2 + 73% N2 16.8: 4.5 128.4:14.2 145.2:16.0 0.02 21% 02 + 6% CO2 + 73% N2 + 1 pl“l CzH,t 62.2: 12.9 58.3: 16.6 120.5:25.3 0.70 % 02 + 0.03% CD; + 97% N2 17.1 : 3.5 72.0: 2.9 89.1 : 3.5 0.01 3% O, + 0.03% CO2 + 97% N2 + 1pl“C2H‘ 61.8: 11.8 33.4: 10.4 95.2: 17.1 0.80 % O, + 6".) CO2 + 91% N2 27.1: 4.1 84.0: 8.8 111.1:10.6 0.02 3% 02 + 6% CO2 + 91% N2 + 1 p11" C2114 60.1 :12.5 30.1: 3.4 90.2:12.6 0.64 Submerged in H20 7.7: 0.9 22.0: 2.7 29.7: 3.0 0.10 Submerged in 0.1 M sucrose 9.4: 1.1 18.9: 2.0 28.3: 2.0 0.12 Table 4. Effects of 02, C02, C2144, submergence and anoxia on the elongation of rice stem sections incubated under continuous light for 3 d. The gases (v/v) were passed through the incubation cylinders at 80 ml min“. Each value is the average of 15 sections : SE Treatment Length increase (mm) C2H4 concentration in internode Intemode Leaf Total section (pl 1' ‘) Air 10.9: 0.9 121.4:106 132.3:11.7 0.02 3% O, + 6% CO2 + 91% i\',_ + 1 p11“ C2H4 123.4:11.5 38.7: 4.0 162.1:12.9 0.8 Submerged 118.0: 7.7 61.9: 7.6 179.9: 11.8 1.1 100% N2 5.1 : 0.9 10.3: 1.2 15.4: 1.5 <0.01 In darkness, the response of internodes and leaves to different concentrations of 02, CO2 and C2H4 was qualitatively similar to that obtained under a 13-h photoperiod (Table 3). However, growth in all gas mixtures was significantly smaller in the dark than under a 13-h photOperiod (com- pare with Table 2). Very little growth of internodes and leaves occurred when sections were submerged in distilled water or 0.1 M sucrose in continuous darkness. The highest rates of internodal growth in response to a mixture of 3% (v/v) Oz, 6% (v/v) C02 and 1 pl 1‘1 of C2H4 or submergence were observed in stem sections of deep-water rice incu- bated under continuous light (Table 4). Very little growth was observed under continuous light if N2 was passed through the incubation chambers. An attempt was made to evaluate the role of endogenous CZH4 in the promotion of growth of I. Raskin and H. Kende: Growth in rice stem sections Table 5. Effects of AVG and ACC on the growth of submerged rice stem sections kept under a 13-h photoperiod for 2.5 (1. Each value is the average of 12 sections : SE Treatment Length increase (mm) C2“; concentration in internode Internode Leaf Total section (pl 1‘ 1) Air control 6.5 : 0.9 69.5 : 4.6 76.0 : 4.9 0.01 Submerged in H20 66.4:7.3 51.5:6.1 117.9: 14.7 1.0 Submerged in 10'5 M AVG 31.7:5.6 78.8:8.3 110.5: 12.2 0.08 Submerged in 10’5 M ACC 72.8:7.6 36.5:7.0 109.3: 9.8 2.1 Submerged in 10"5 M AVG + 10" M ACC 52.6:3.6 41.7:5.3 94.3: 7.9 1.5 submerged stern sections using AVG, an inhibitor of C2H4 biosynthesis, and ACC, the immediate precursor of C2H4. Aminoethoxyvinylglycine (10‘5 M) inhibited internodal elongation in sub- merged sections by 52% and promoted leaf growth by 53% compared with sections submerged in dis- tilled water (Table 5). 1-Aminocyclopropane-1- carboxylic acid (10‘5 M) increased internodal elongation by 10% and decreased leaf growth by 29% compared with sections submerged in water; it partially reversed the action of AVG on the sub- merged sections. However. even at concentrations above 10-5 M, ACC did not completely counter- act the action of AVG (results not shown), probab- ly because AVG inhibited some other important processes in addition to C2H4 synthesis. The rise in the concentration of C2H4 in sub- merged rice stem sections may be caused by re- duced diffusion of CZH4 from the tissue into the water while the rate of C2H4 synthesis remains unchanged. Alternatively, C2H4 accumulation in the tissues may be the combined result of enhanced C2H4 synthesis in and reduced CZH4 diffusion from submerged stem sections. Since it is not possi- ble to compare CZH4 evolution in submerged and nonsubmerged sections directly, we transferred the internodal tissue from submerged stem sections into test tubes containing 3% O2 and 6% CO2 (both v/v), i.e. concentrations that were similar to those found in the internodal lacunae of sub- merged sections, and determined C2H4 evolution under these conditions. lnternodal tissues from sections that had been incubated in air were trans- ferred into test tubes containing air (Fig. 2). Inter- nodal tissues from submerged sections evolved over five times more C2114 than internodal tissue from air-incubated sections, and AVG (10‘5 M) inhibited C2114 evolution by 80—90% in both in- stances. We compared the effeet of low 02 and high C02 concentrations on C 2H 4 synthesis in interno- dal and leaf tissue from stem sections that had Sum-.335 0,. satco2 Air-OM! SUM. *37002. 69oCO2+AVG Air *AinAVG 00123456 Time (h) Fig. 2. Ethylene evolution from internodes excised from rice stem sections that had been submerged in H20 or incubated in a stream of air (80 ml min‘ ‘) under continuous light. lnter- nodes excised from submerged sections were sealed in 30-ml test tubes containing 3% Oz, 6% CO2 and 91% N2 (by vol.) and 2 ml of distilled H20 (0) or 10'5 M AVG solution (0). Internodes excised from the sections incubated in air were sealed in 30-ml test tubes containing air and 2ml H20 (I) or 10" M AVG solution (0). Each point is the average of three replicate test tubes containing three internodes each. Ver- tical bars denote : SE. When no bars are given. the SE is smaller than the symbol used. The lower or upper part of the bar is omitted when it would interfere with another SE bar been incubated previously in different gas mixtures (Fig.3). Ethylene evolution was measured in the same gas mixtures in which the sections had been incubated before the separation of the internodes from the leaf tissue. lnternodal tissue excised from sections and incubated in a stream of gas contain- ing 3% O2 evolved up to four times more C2H4 than did internodes excised from sections incu- bated in a stream of air or 21% Oz. 6% CO2 and 73% N2 (by vol.) (Fig.3). The reverse was true for leaves. These evolved the largest amounts of C2H4 in air or in a gas mixture of 21% 02, 6% C02, 73% N2 (by vol.). In contrast to interno- dal tissue, leaf tissue evolved the least CZI-I4 in l. Raskin and H. Kende: Growth in rice stem sections 1 0 157.02 .emo2 200_ A. lnternodes 3x0,.o.os-/.co, G O r 5 O 1 Air 0| 0 1 217-02 ,67-C0: 1 l l l l l _ 8. Leaves 0‘ O 2|%0,.6'/.C01 C2H4 Evolutlon (pmol 9" FW) 6‘- o l 3° ' no. ears-mo. '5 ' mover/mo, A 1 l I l l l "o l 2 3 4 5 6 Ti me (h) Fig. 3A. B. Ethylene production by internodes (A) and leaves (B) excised from rice stem sections that had been incubated in a stream of air (I). 3% 01.0.03% C02. 97% N2 (0), 21% Oz. 6% C01. 73% N2 (A) or 3% 02, 6% C02, 91% N1 (0) (all by vol.) under continuous light. Air and the respective gas mixtures were passed through the incubation cylinders at 80 ml min ‘ ‘. For ethylene determinations. internodal and leaf tissues were isolated and incubated in 30- or 60-ml test tubes. respectively. containing 2 or 3 ml H20 and the same gas mix- tures with which the whole stem sections had been treated ear- lier. Each point is the average of three replicate test tubes, each containing internodes or leaf tissue from three sections. The SE is given by the vertical bars (see legend Fig. 2) Table 6. Effects of submergence and different gas mixtures on the levels of free and conjugated ACC in internodes of rice stem sections incubated under continuous light. Gases (v/v) were passed through the incubation cylinders at 80 ml min". Each value is the pooled average from 10 internodes Treatment ACC Conjugated (nmol ACC 3“ FW) (nmol 8" FW) Air 2.1 24.3 3% 0:. 003% C02. 97% N2 2.5 28.5 21% 0:. 6% C01. 73% N2 2.1 34.4 3% 0:. ”"u C02. 91% NJ 2.3 28.6 Submerged 1.9 21.4 gas mixtures containing low levels of 02. We veri- tied the fact that the differences in C2H4 produc— tion in internodes and leaves isolated from stem sections were not the result of differences in the wound response following excision. When inter- nodes and leaves from submerged and non-sub- merged sections incubated in different gas mixtures were cut longitudinally into two or four portions, Czl-l4 production was not further stimulated (re- sults not shown). Submergence or incubation of stem sections in different gas mixtures did not significantly affect the endogenous levels of free ACC or conjugated ACC. presumed to be malonyl ACC (Table 6). All experiments described in this paper were repeated at least three times with similar results. Discussion The growth response of submerged deep-water-rice stem sections is very similar to that of partially submerged, intact deep-water rice plants (Métraux and Kende 1983a, b). Elongation of internodes is greatly stimulated while leaf growth is inhibited. Our data indicate that stimulation of internodal growth and inhibition of leaf growth in submerged deep-water rice can be explained both by increased C2H4 synthesis in the internodal tissue and by ac- cumulation of C2H4 in the internal air spaces of the submerged organs. The enhancement of CZH4 synthesis in the internodes and inhibition of C2114 synthesis in the leaves of deep-water rice is brought about by low levels of 02 (Fig. 3A) as found in the lacunae of submerged stem sections (Table 1). In this respect. internodes of deep-water rice differ from most other plant tissues where ethylene syn- thesis is inhibited at low 02 levels (e.g. Raskin and Kende 1983). Stimulation of C2H4 synthesis under hypoxic conditions (5% 02) has previously been reported for maize and barley roots (Jackson 1982). The opposite effects of AVG, an inhibitor, and ACC, a promoter of C2H4 synthesis on the growth of leaves and internodes agree well with the changes in the growth rates of internodes and leaves under water (Tables 2, 5). Accumulation of Czl-I4 in the submerged plant parts results from the almost 10“-fold slower rate of CZH4 diffusion through water when compared with C2H4 diffu- sion through air (Musgrave et al. 1972). Although the internodal tissue of non-submerged sections incubated under hypoxic conditions produces as much CZH4 as do internodes from sections that have been submerged (Figs. 2, 3A). no C2114 accu- mulation in the internodal lacunae of non-sub- merged sections has been observed (Table 2) be- cause of the high rate of (32H4 diffusion from the non-submerged tissue into the air. Carbon dioxide at the high levels found in the lacunae of submerged sections promotes interno— dal elongation to a minor extent and does not sti- mulate C2H4 production (Table 2. Fig. 3A). This is in contrast to etiolated rice seedlings in which CO2 at high concentrations does promote growth of the coleoptiles and mesocotyls (Raskin and Kende 1983). However, CO2 at high levels en- hances the stimulatory effect of C2114 on interno- dal growth in rice stem sections and decreases the inhibitory effect of C3H4 on leaf growth (Table 2). Therefore. the full effect of submergence on growth of stem sections of deep-water rice can be mim- icked most closely by incubating non-submerged sections in a gas stream whose composition is simi- lar to that of the internodal lacunae, namely 3% Oz, 6% C02, and 1 ul l‘l CZH4 (Table 2). Internodes and leaves do not elongate under anaerobic conditions (Table 4), and very little growth occurs in submerged sections kept in con- tinuous darkness (Table 3). The CZH4 concentra- tion in the internodal lacunae of submerged sec- tions was about ten times lower in the dark, i.e. in the absence of photosynthetic 02, than in the light (Tables 3, 4). This agrees with the previous observation that CZI-I4 synthesis is inhibited under anaerobic conditions (Hansen 1942). In conclusion. 02 at concentrations found in submerged internodes (approx. 3%) enhances C2H4 synthesis in internodes of deep-water rice. Ethylene. which accumulates in submerged stems, promotes growth ofinternodes and inhibits growth of leaves. The stimulatory effect of C2H4 on inter- nodal growth is enhanced at the elevated levels of CO2 which occur in submerged internodes (ap- prox. 6%). Deep-water rice is adapted to grow very rapidly when partially submerged in water. This adaptation is based on the ability of deep-water rice to react to reduced 02 and elevated CO2 and C2H4 levels within the submerged stems. We thank R. deZacks for help in preparing the plant material. We also acknowledge gratefully the help of Dr. S.M.H. Zaman. Director of the Bangladesh Rice Research Institute. who pro- ll I. Raskin and H. Kende: Growth in rice stem sections vided seeds of deep-water rice. This research was supported by the National Science Foundation through grant No. PCM 81-09764 and by the US. Department of Energy under Con- tract No. DE-ACOZ-76ER01338. References De Datta, SK. (1981) Principles and practices of rice produc- tion. Wiley. New York Hansen. E. (1942) Quantitative study of ethylene production in relation to respiration of pears. Bot. Gaz. (Chicago) 103. 543-558 Jackson. MB. (1982) Ethylene as a growth promoting hormone under flooded conditions. In: Plant growth substances 1982. pp. 291—301, Wareing, P.F., ed. Academic Press, London New York Kende, H., Hanson. AD. (1976) Relationship between ethylene and senescence in morning-glory flower tissue. Plant Phys- iol. 57. 523-527 Lizada. M.C.C., Yang, SF. (1979) A simple and sensitive assay for 1-aminocyclopropane-1ocarboxylic acid. Anal. Biochem. 100, 140—145 Metraux, J.-P., Kende. H. (1983a) The role of ethylene in the growth response of submerged deep water rice. Plant Phys- iol. 72. 441—446 Metraux. J.-P., Kende, H. (1983b) The cellular basis of the elongation response in submerged deep-water rice. Planta 160, 73—77 Musgrave, A., Jackson, M.B.. Ling E. (1972) Callitriche stem elongation is controlled by ethylene and gibberellin. Nature (London) New Biol. 238. 93-96 Pratt. H.K., Workman, M., Martin, F.W., Lyons, J.M. (1960) Simple method for continuous treatment of plant material with metered traces ofcthylene or other gases. Plant Physiol. 35, 609—611 Raskin, I., Kende. H. (1983) Regulation of growth in rice seed- lings. J. Plant Growth Regul.. in press Saltveit, M.E., Jr. (1978) Simple apparatus for diluting and dispensing trace concentrations of ethylene in air. Hort- Science 13. 249-151 Vergara. B.S.. Jackson. 8., De Datta. SK. (1976) Deep-water rice and its response to deep-water stress. In: Climate and rice, pp. 301—319. International Rice Research Institute. Los Banos, Philippines Received 27 July; accepted 12 September 1983 Chapter 2 The Role of Gibberellin in the Growth Response of Submerged Deep-Water Rice 13 ABSTRACT We have shown previously that C2H4, which accumulates in the air spaces of submerged stem sections of rice (Oryza sativa L. cv. "Habiganj Aman II") is involved in regulating the growth reSponse caused by submergence. The role of gibberellins in the submergence response was studied using tetcyclacis (TCY), a new plant growth retardant, which inhibits gibberellin biosynthesis. Stem sections excised from plants that had been watered with a solution of 10‘6 M TCY for 7-lO d did not elongate when submerged in the same solution or when exposed to l ul 1" C2H4 in air. Gibberellic acid (6A3) at 3 x 10'7 M overcame the effect of TCY and restored the rapid internodal elongation in submerged and ethylene-treated sections to the levels observed in control sections that had not been treated with TCY. The effect of lO'8 to 3 x l0"7 M GA3 on internodal elongation was enhanced two- to eight-fold when l ul l"1 C2H4 was added to the air passing through the chamber in which the sections were incubated. 6A3 and C2H4 caused a similar increase in cell divisions in the intercalary meristem of rice internodes. Thus, ethylene may cause internodal elongation in rice by increasing the activity of endogenous GAS. 14 When deep-water rice plants become partially submerged, internodal elongation is greatly enhanced (l,8). This submergence response is, at least in part, mediated by increased levels of C2H4, the synthesis of which is stimulated in submerged internodes (3). The increase in C2H4 synthesis in submerged internodes is triggered by reduced concentrations of 02 (7). This mechanism of growth regulation enables rice plants to adjust their height to the depth of the surrounding water. This paper examines the involvement of 6A3 and light in the submergence response of rice using isolated stem sections. Tetcyclacis (TCY), a new growth retardant, which blocks GA biosynthesis at the oxidative reactions between ent-kaurene and ent-kaurenoic acid (6), has been used to lower the levels of endogenous GAs in the rice plant. Involvement of GAS has been suspected because they are known to promote elongation of filflfli internodes (2). Gibberellins have also been shown to mediate the stimulatory effect of C2H4 on stem elongation in the semi-aquatic plant Callitriche platycarpa (5). l5 MATERIAL AND METHODS Chemicals. Gibberellic acid (6A3) was a gift from Merck and Co., Inc. (Rahway, N.J., USA), TCY [S-(4-chlorophenyl)-3,4,5,9,l0-pentaazatetracyclo- 5,4,1,02’6,08’11-dodeca-3,9-diene] was a gift of BASF (Limburgerhof, F.R.G.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). Plant material. Seeds of deep-water rice (Oryza sativa L. cv. "Habiganj Aman II") were obtained from the Bangladesh Rice Research Institute (Dacca, Bangladesh). Rice was germinated and grown as described by Métraux and Kende (3). 'Twenty-cm-long stem sections containing the top-most internode were excised, subjected to submergence or gas treatments and measured as described by Raskin and Kende (7). All experiments were performed at 270 C either in continuous light (cool-white fluorescent tubes; 70 umole m'zs‘1) or in darkness. Stem sections used for TCY treatments were excised from rice plants which had been watered daily for 7-l0 d with l0"6 M TCY in half-strength Hoagland solution. The term “leaf", as we use it, includes the leaf sheath and the bases of the leaf blades that grow out of the original 20-cm-long stem section during the course of the experiment. Microscopy. For the determination of cell numbers in the elongation zones of rice internodes, stem sections were incubated for 2 d in air or in a gas mixture consisting of 3% 02, 6% coz, 91% N2 (by vol.) and l pl 1-1 C2H4. Stem sections kept in this gas mixture were standing in 40 ml of distilled water while the stem sections incubated in air were either standing in 40 ml of distilled water or 5 x l0“6 M GA3 solution. The regions of the internodes which grew during the last 20 h of treatment were excised with a razor blade. Thin, free-hand longitudinal sections were cut from the surface of each internode l6 to cover the whole length of the elongated region. The sections were stained with methylene violet, and the number of subepidermal cells in files of l.l mn length was counted in 6 different regions distributed evenly along the newly elongated region of each internode. The total number of cells was calculated from the average number of cells in each region examined. .- n O . c 2 rt 3 3.. ..vi a\a 5 via .a_ .. ,>r‘A:J . .1; .4 . s . ~i név , p . ... r v #3 ..i .... ..-. .3 fl m; ,1. .... n c ... n i . . Q n—» ... l7 RESULTS Stem sections excised from TCY-treated and control plants were submerged in lO’6 M TCY solution or distilled water, respectively, in the light for 3 d. Submergence stimulated internodal elongation in control sections but not in TCY-treated sections (Fig. lA). Gibberellic acid added to the solution in which the sections were submerged promoted internodal growth, expecially in TCY-treated sections. At about 3 x l0"7 M, 6A3 restored the submergence reSponse of TCY- treated internodes to the level of control (-TCY) internodes. Saturating concentrations of GA3 (10'4 M) increased internodal growth of submerged control sections by 54% while the internodal growth of the sections submerged in TCY was promoted 22-fold at the same concentration of GA3. In contrast to internodes, rice leaves were less inhibited in growth by TCY (Fig. lB). Addition of 0A3 increased leaf length in control and TCY-treated sections to a similar extent. We showed earlier that the submergence response could be mimicked by exposing non-submerged sections to a gas mixture which was similar to the gaseous atmosphere in the internodal lacunae of submerged sections, namely 3% 02, 6% C02, 9l% N2 (by vol.) and l ul l‘1 C2H4 (7). Internodes of stem sections incubated in this gas mixture for 3 d responded similarly to GA3 as did submerged sections (Fig. 2A) while internodes of stem sections kept in air showed a much greater response. lnternodal elongation of sections incubated in 3% 02, 6% C02, 91% N2 and l ul 1‘1 C2H4 and treated with 10-4 M 0A3 was promoted by 3% while the internodal growth of sections incubated in air was enhanced l8-fold by the same concentration of 0A3. Leaf growth was two- to threefold inhibited by 3% 02, 6% C02, 9l% N2 and l pl l‘1 C2H4 when compared to leaf growth in air (Fig. 28). The amount of GA3-induced leaf growth was similar in both filo in qr‘r‘f‘r .v i'uivyx A d I.’ ‘«H a ‘ 18 treatments. Ethylene did not enhance internodal elongation in sections standing in 40 ml of 10‘5 M TCY solution for 3 d (Fig. 3A). When low concentrations of GA3 were added to the TCY solution, the increase in internodal elongation was 2.4 to 8 times larger with ethylene in the atmosphere than without. Again, C2H4 inhibited leaf growth at all 6A3 concentrations, and only a small enhancement of leaf growth by GA3 was observed in both treatments (Fig. 38). Cell divisions in the internodes of stem sections that had been submerged or incubated in 3% 02, 6% C02, 9i% N2 and i pi i-l C2H4 for 3 d was greatly enhanced (4). We found that both 5 x l0“6 M GA3 and a gas mixture of 3% 02, 6% C02, 9l% N2 and l pl 1'1 C2H4 increased the number of cells in the growing region of rice internodes l7 fold with very little if any difference in the average cell length (Table I). Also, comparable amounts of [3H]thymidine were incorporated into the DNA of the newly elongated region of internodes in sections treated with 5 x l0“6 M GA3 in air or with the above gas mixture (data not shown). The internode within the rice stem sections used in our experiments is covered by a single leaf sheath which originates at the lower of the two nodes of the section. When these leaf sheaths were removed and the exposed internodes illuminated, growth of the internodes in response to submergence, 10'5 M 6A3 and 3% 02, 6% C02, 9i% N2 and i pi i-l C2H4 was severly inhibited (Table II). In darkness, the inhibition of growth of exposed internodes was much less pronounced or, in the case of GA3-treated internodes, no inhibition was evident at all. Wrapping the lower l0 cm of submerged stem sections with aluminum foil to darken the exposed internode also increased growth of the exposed internodes <:ompared to those not wrapped in foil (Table II). Thus, exposing internodes to l ight strongly inhibited the effects of submergence, GA3 and C2H4 on internodal elongation. rii"f‘-' " ,TE '33 I : 19 DISCUSSION Figures l-3 indicate that C2H4 is likely to cause internodal elongaton in rice by increasing the activity of endogenous GAS. Ethylene may either increase the sensitivity of internodal tissue to endogenous GAS or increase the concentration of physiologically active GAS in the rice internode. This hypothesis is supported by the following results: (i) Stem sections excised from plants that had been watered with 10'6 M TCY did not elongate when submerged in the same solution or when exposed to l pl 1'1 C2H4 in air (Figs. 1,3). Addition of GA3 at concentrations above l0"7 M restored rapid internodal growth in submerged and C2H4-treated internodes. (ii) Low GA3 concentrations (lO‘8 to leO‘7 M) were much more effective in promoting internodal elongation when l pl l"1 C2H4 was added to the air (Fig. 3). (iii) Saturating concentrations of GA3 (lO'5 to lO'4 M) enhanced internodal elongation in the sections incubated in air to the levels observed in the gas mixture containing 3% 02, 6% C02, 9l% N2 and l pl l"1 C2H4 (Fig. 2). (iv) GA3 stimulated cell divisions in the intercalary meristems of rice internodes to the same extent as did the gas ‘ mixture of 3% 02, % C02, 9l% N2 and l pl C2H4 (Table I). The following chain of events appears to take place following submersion of rice stem sections. The level of 02 in the tissue is greatly reduced as a result of submergence, and lowered 02 concentrations stimulate ethylene synthesis (7). Ethylene accumulates in the submerged internode because its diffusion in water is l0,000 times slower than in air. Ethylene promotes rapid internode elongation by enhancing the activity of endogenous GAS. In this respect, the Situation in rice may be similar to that in Callitriche platycarpa ivhere promotion of stem elongation by ethylene is also dependent on the presence of GAS (5). 20 Acknowledgements. We thank R. deZacks for help in preparing the plant material. 2l LITERATURE CITED DE DATTA SK l98l Principles and practices of rice production. Wiley, New York KAUFMAN PB, P DAYANANDAN l983 Gibberellin-induced growth in_Avgna internodes. In_A Crozier, ed, The biochemistry and physiology of gibberellins, Vol 2, Praeger Publishers, New York, pp l29-l57 ‘ METRAUX J-P, H KENDE l983 The role of ethylene in the growth response of submerged deep water rice. Plant Physiol. 72:44l-446 METRAUX J-P, H KENDE l984 The cellular basis of the elongation response in submerged deep-water rice. Planta l60:73-77 MUSGRAVE A, MB JACKSON, E LING 1972 Callitriche stem elongation is controlled by ethylene and gibberellin. Nature New Biol. 238:93-96 RADEMACHER W, J JUNG, E HILDEBRANDT, JE GRAEBE 1983 Influence of the bioregulator tetcyclacis (BAS l06..W) on gibberellin biosynthesis and the hormonal status of plants. _Ln AR Cooke, ed, Proceedings, lOth Annual Meeting, Plant Growth Regulator Society of America, Plant Growth Regulator Soc. of America, Lake Alfred, Florida, pp 36-4l RASKIN I, H KENDE l984 Regulation of growth in stem sections of deep-water rice. Planta l60:66-72 VERGARA BS, B JACKSON, SK DE DATTA l976 Deep water rice and its response to deep water stress. _In Climate and rice, International Rice Research Institute, Los Bafios, Philippines, pp 30l—3l9 22 Table I. Number of Subepidermal Cells in the Growing Regions of Rice Internodes Rice stem sections were incubated for 2 d in flow-through chambers (2.5 l) through which air or a mixture of 3% 02, % C02, 9l% N2 (by vol.) and l pl 1'1 C2H4 were passed at 80 ml min'l. The sections kept in air had their cut ends 2 cm deep in 40 ml of water or 5 x lO'6 M GA3 solution while sections kept in the above gas mixture were standing in 40 ml of water. Cells were counted in the regions that had elongated during last 20 h of incubation. Increase in Total number Average cell Treatment internodal length of cells in one length (mm) file of the newly (pm) elongated region Air + H20a l.l :_O.4 l8.7 :_ 5.7 57.3 :_2.6 Air + 5xlO'6 M GA3b 4l.3 : 2.9 308.4 : 25.l l35.0 : 3.8 3% 02, 6% C02, 9l% N2 39.0 :_l.8 3l6.5 :_ll.4 l23.3 :_3.7 + 7 (ll l-l C2H4b an ll OJ + U) [‘1 b”- l ON + U) m 23 Table II. Effect of Leaf Sheath Removal on Internodal Elongation in Rice Stem Sections The air and gas mixtures (all by vol.) were passed through the 2.5-l incubation cylinders at 80 ml min‘l. Treatment Leaf sheath Conditions Increase in internodal length (mill) Submerged + light 95.4 i 5.8 " " - light 2.9 i 0.5 Submerged, wrappeda + light 95.l :_6.3 " " " " - light 48.3 i 4.2 10'5 M GA3, air + darkness 8l.5 _+_ 9.5 " " - darkness 86.6 i 9.4 " " ' + light 121.8 1 6.5 " " - light l7.5 : 5.0 3%03 +6%602 +9i%N2 H M H C2H4 + darkness 66.6 i 5.7 " " — darkness 46.0 :_5.9 " " + light 99.8 : 9.l " " - light l2.0 + 2.l aThe lower lO cm of the stem sections were wrapped in a lO x 4 cm piece of aluminum foil. 24 160 ... A.hnemnodes _Tcy I 140 «— +TCY 120 -— 100 -— MK) 80—— h 60 -— E v 40 «- 3: l.— g 20 -— 8 e// O O l Illfi l l l 1 1 100 '7 B. Leaves 80 -— 60 -— 4o _— 20 -- i I; i i l i l o ' 0.01 0.1 1 10 100 GA3 (MM) Fig. 'l. A,B Effect of GA3 on the growth of internodes (A) and leaves (B) of rice stem sections submerged in lO"6 M TCY solution (0) or distilled water (a) for 3 d in continous light. Each point is the average of l4 sections. Vertical bars denote :_SE. The lower or upper part of the bar is omitted when it would interfere with another SE bar. 25 Fig. 2. A,B Effect of GA3 on the growth of internodes (A) and leaves (8) of rice stem sections incubated in a stream of air (a) or 3% 02’ 6% C02, 9l% N2 (by vol.) and l pl l‘1 C2H4 (+02,+C02 + C2H4) (0) under continuous light for 3 d. Sections standing upright in lOO-ml glass beakers containing 40 ml of different GA3 concentrations in distilled water were placed in 2.5-l plastic cylinders through which air or the above gas mixture was passed at 80 ml min’]. Each point is the average of 25 sections :_SE (see legend Fig. l). When no bars 'are given, the SE is smaller than the symbol used. GROWTH (mm) 26 140-- lco,, io2 + 02H4 A. Internodes 120-- 100 —- H 80-- 60—t 40*- 20-. 7I/ , 0 IL ‘7f l l i i i B. Leaves Ah 160-- 140*- 120-* W 80“ lCOzi loz T C2H4 O l j]; l i i ii 0 O 01 0.1 1 10 100 GROWTH (mm) 27 1204 100~~ 60-~ 40 -- 2()-- A. Internodes 100-~ 8C) -1 40-- B. Leaves GA3, (nM) Ah'+ C2H4 Adr Ah'+ C2H4 Fig. 3. A,B Effect of low GA3 concentrations on the growth of internodes (A) and leaves (B) of rice stem sections treated with lO'6 M TCY and incubated in a stream of air (a) or air containing l pl l'1 C2H4 (o). The sections, standing upright in lOO-ml glass beakers containing 40 ml of TO"6 M TCY solution with different GA3 concentrations, were placed in 2.5-l plastic cylinders through which air or air with C2H4 was passed at 80 ml min‘l in continuous light for 3 d. Each point is the average of l4 sections :_SE (see legend Fig. 2). Chapter 3 Effect of Submergence on Translocation, Starch Content and a-Amylase Activity in Deep-Water Rice 3.),iuhv' ’.——- ”fir-y" i. _ i‘ ”.2 (:0 Jr"... ___—— . . » .r‘MHOlOS c'vv [iv-\- U. ‘ ..J 4r :A (i. *‘a-vr J 5m- ; A'- i‘., ’2 l J“: InUDL ' a) L IV 0 0‘ CD _4.‘ a. .4. ‘7) (”N 29 Abstract. Submergence induces rapid internodal elongation in deep—water rice (Oryza sativa L. cv. ”Habiganj Aman II"). We investigated the metabolic activities which help to support such fast growth. Three days of submergence in water under continuous light led to the mobilization of 65% of the starch from those regions of rice internodes which had been formed prior to submergence. Disappearance of starch was accompanied by a 70-fold enhancement of a-amylase activity. Similar increases inta-amylase were detected in response to ethylene and gibberellic acid (GA3). Submergence also caused a 26-fold increase in the translocation of photosynthetic assimilates from the leaves to the internodes and younger regions of the culms. These physiological processes are likely to provide the metabolic energy required for internodal elongation in response to submergence. Key words: a-Amylase - Deep-water rice - Oryza (growth regulation) - Partitioning - Starch. 30 Introduction Internodes of deep-water rice elongate rapidly, up to 25 cm a day, in response to submergence (Vergara et al. 1976; Métraux and Kende l983). Internodal elongation is primarily caused by C2H4 which accumulates in the submerged culms (Métraux and Kende 1983). Growth of internodes in response to submergence or C2H4 can be reproduced in excised stem sections of deep water rice (Raskin and Kende 1984a). Ethylene appears to promote growth by increasing the activity of endogenous gibberellins (GAS) (Raskin and Kende 1984b). The submergence response in whole deep-water rice plants and stem sections is based on the activation of cell division in the intercalary meristem and subsequent elongaton of the newly formed cells (Métraux and Kende 1984). It has been shown earlier that the "tops" of air-grown rice contain Significantly larger amounts of starch than those of submerged rice plants (Yamaguchi 1973) and that amylase activity in rice leaves and internodes increases during submergence (Yamaguchi and Sato 1963). We have examined in greater detail the sources of energy which help sustain the large increase in cell division activity and elongation in the internodes of submerged stem sections of deep-water rice. We have also investigated the effects of GA3 and ethylene, both of which enhance growth of stem sections, on a-amylase activity. nil) u! Ci'l ... . Fl 3 31 Materials and methods Plant material. Seeds of deep-water rice (Oryza sativa L. cv. "Habiganj Aman II") were obtained from the Bangladesh Rice Research Institute (Dacca, Bangladesh). Rice was germinated and grown as described by Metraux and Kende (1983). Twenty-cm-long stem sections containing the top-most internode were excised, submerged, treated with air or C2H4 and measured as described by Raskin and Kende (1984a). All experiments were performed at 270 C in continuous light (cool-white fluorescent tubes; 70 pmole m'zs'l). Chemicals. NaHl4CO3 Solution (2.1 GBq mmol'l) was purchased from ICN, Chemical and Radioisotype Div. (Irvine, Cal., USA). Gibberellic acid (GA3) was a gift from Merck and Co. (Rahway, N.J., USA). All other chemicals and enzymatic reagents were purchased from Sigma Chemical Co. (St. Louis, Mo., USA). a-Amylase determination. Internodes of 5 or 6 stem sections were measured to determine their length increase during the experimental treatment, excised with a razor blade and separated into the old regions formed before the experimental treatment and the new regions formed during the experimental treatment. The internodal tissue was sliced into small pieces, weighed, and l g was ground at 1-30 C in a mortar with a pestle in 3 ml of 25 mM sodium acetate buffer (pH 5.4) containing 10 mM CaClz, 0.1% (v/v) Triton X-100 and 20 mg polyvinylpolypyrrolidone (PVP). The slurry was centrifuged at 12,000 g_for 15 min using a Sorvall RC-ZB centrifuge and a 55—34 rotor (DuPont Instruments- Sorvall, Wilmington, Del., USA). a-Amylase in the supernatant was assayed according to Jones and Varner (1967). The pellet was saved for starch determination. One enzyme unit (E.U.) of a-amylase activity is the amount of enzyme that causes a change in absorbance of l at 620 nm during 10 min. 34 P\v ..-- do. 'e .. div 3. P... a J. c .. . . s . a 1.. w a r L.» . 3 8‘ a o .4. . u ..i. .o if IiNI ‘i‘i. ; .‘ -\- § I A r O I 32 Starch determination. The pellet left after a-amylase extraction was resuspended in 10 ml hot 80% ethanol and centrifuged at 12,000 g_for 15 min. Extraction with hot 80% ethanol and centrifugation was repeated once more to remove residual chlor0phyll and soluble sugars. The resulting pellet was extracted with 10 m1 of boiling 0.02 M KOH for 15 min, cooled to room temperature and centrifuged at 8,000 g for 15 min. One ml aliquots of the supernatant were added to 2 ml of 50 mM sodium acetate buffer (pH 4.5) in which 50 mg of amyloglucosidase (1,4-a-D-glucan glucohydrolase from Rhizopus mold) were dissolved. The reaction mixture was incubated at 460 C for 90 min, boiled for 2 min and centrifuged at 13,000 g for 15 min in a Micro-Centrifuge Model 2358 (Fisher Scientific, Pittsburg, Pa., USA) for 15 min. Glucose in the supernatant was analyzed enzymatically using the hexokinase and glucose-6-phosphate dehydrogenase diagnostic kit. Carbon translocation. For the translocation experiments, so-called modified stem sections were used. They consisted of stem sections as described by Raskin and Kende (1984a) except that the outer leaf originating from the lower of the two nodes remained attached. It was trhnned to a size of 70 cm above, the basal end of the section (see Fig. 3). Twelve such modified stem sections were placed in each of two 2.5-1 cylinders, 60 cm deep, so that 10 cm of the leaf blade protruded above the cylinder rim. One of the cylinders was filled to the rim with distilled water while the other, containing only 50 ml of distilled water in the bottom, was continously flushed with air at the rate of 80 m1 min'l. Glass beads were placed on the bottom of the submergence cylinder to prevent sections from floating up. After 3 d, six sections were selected from each treatment. The top 7 cm of each leaf were introduced into a 1.1-l transparent plastic chamber through which air containing 14C02 (2.4 MBq 14C l‘l) was passed for 15 min with a circulating pump. 14C02 was obtained by injecting 50 pi of a Na214co3 solution into a 250-ml giass flask containing 33 100 m1 of 30% (w/v) H3P04. The flask was connected with Tygon tubing to the pump and the leaf chamber. Supplementary illumination was provided with 150 W incandescent bulbs during exposure to 14C02 to give a total light intensity 2 S-i in the leaf chamber of 200 pmol m’ . Following 14C02 exposure, the stem sections remained in the incubation cylinders for 210 min under the same conditions as those preceding labeling. Thereafter, the modified stem sections were dissected into 6 different regions: the 7 cm tip of the leaf blade which had been exposed to 14C02, the remainder of the leaf blade, the leaf Sheath, the lower half of the internode containing the intercalary meristem, the upper half of the internode, the remainder of the section containing the apical meristem and younger leaf sheaths. Each region was weighed and ground in liquid N2 in a mortar with a pestle. The resulting powder was extracted with 4 volumes of 75% ethanol and centrifuged at 12,000 g for 20 min. One ml aliquots of the supernatant were dried overnight at 70° C, resuspended in 200 pl of 75% ethanol and combusted in a Tri-Carb Sample Oxidizer (Model B 306, Packard Instrument Co., Downers Grove, 111., USA). The pellets were resuspended in 10 ml 75% ethanol and centrifuged at 12,000 g for 15 min. Resuspension and centrifugation were repeated once more. The pellets were dried overnight at 70° C and combusted in the sample oxidizer. Radioactivity was determined in a Tri-Carb Liquid Scintillation Spectrometer (Model 3255). Measurement of photosynthetic rates. The net rate of photosynthetic carbon fixation was measured with a Type 225-MK II Infra-Red Gas Analyzer (Analytical Development Co., Hertfordshire, U.K.). .1 Iii 34 Results and discussion Internodes of air-grown rice plants contain large amounts of starch (about 10% of their dry weight). Submergence under continuous light led to rapid disappearance of starch from the older regions of the internodes, which were already present before the start of submergence (Fig. 1). Those portions of the internodes which were formed during submergence, contained 18 to 41 times less starch than the internodes of control sections incubated in air. Decrease of starch content in the older part of the internodes coincided with a marked increaSe in a-amwlase activity. (Fig. 2). No increase in a-amylase activity was detected in the young internodal tissue formed during submergence of rice stem sections and in the sections incubated in air. We showed earlier that acceleration of internodal growth in response to submergence was mediated by ethylene and that accumulation of ethylene in the lacunae of submerged internodes increased the activity of endogenous gibberellins (Raskin and Kende l984b). An increase in a-amylase activity comparable to that of submerged sections was also detected in the older regions of internodes of non-submerged sections treated with 1 pl 1‘1 C2H4 or 5x10‘7 M GA3 (Table l). Submergence, C2H4 and GA3 also caused a 6- to lZ-fold increase in internodal elongation compared to sections incubated in air. The submergence response was associated with a 26-fold increase in the translocation of photosynthetic assimilates from the above-water parts of the leaves to the rapidly growing internodes and younger regions of the culms (Fig. 3). When rice stem sections, to which a 70-cm—long leaf remained attached at the lower of two nodes, were submerged with the tip of the leaf protruding above the water, 18.1% of the total 14C fixed in the uppermost 7 cm of the leaf were transported to the internode during 210 min following labeling. In con- trol sections incubated in air, only 0.7% of the total labeled photosynthetic na— '1 fl .4 .f as.- ('2 vi. ..x .\¢ 35 assimilates (ethanol soluble and insoluble) were translocated to the internode. The total amount of 14C incorporated into submerged sections was 3.2 times greater than that incorporated into sections kept in air. Gas exchange studies showed that under Similar light conditions, the net rate of photosynthetic carbon fixation in the above-water leaf tips of submerged sections was 14.8 mg C02 dm‘2 h‘l while in the tips of air-incubated sections it was 4.8 mg C02 dm'2 h'l. At least part of this difference appeared to be caused by wider opening of the stomata on the above-water parts of the leaves of submerged sections when compared to the stomata on the leaves of sections kept in air (data-not shown). Our results indicate that two physiological processes help to satisfy the increased demand for energy, substrates and osmotica in rapidly dividing and elongating cells of the sumberged internodes of deep-water rice. First, flooding causes a marked increase in a-amylase activity in rice internodes. This is accompanied by hydrolysis of internodal starch. Second, submergence greatly enhances the rate of carbon export from the leaves to the stem, indicating an increased Sink strength in the growing internodes. We gratefully acknowledge the technical assistance of R. deZacks. This work was supported by the National Science Foundation through Grant No. PCM 81-09764 and by the U.S. Department of Energy under Contract No. DE-ACOZ-76ER01338. Ht . I: 36 References Jones, R.L., Varner, J.E. (1967) The bioassay of gibberellins. Planta 12, 53-59 Métraux, J.-P., Kende, H. (1983) The role of ethylene in the growth response of submerged deep water rice. Plant Physiol. 12, 441-446 Métraux, J.-P., Kende, H. (1984) The cellular basis of the elongation response in submerged deep-water rice. Planta 160, 73-77 Raskin, 1., Kende, H. (l984a) Regulation of growth in stem sections of deep-water rice. Planta 160, 66-72 Raskin, I., Kende, H. (l984b) The role of gibberellin in the growth response of submerged deep-water rice. Manuscript in preparation. Vergara, B.S., Jackson, 8., De Datta, S.K. (1976) Deep-water rice and its response to deep-water stress. In: Climate and rice. pp. 301-319, International Rice Research Institute, Los Bafios, Philippines Yamaguchi, T., Sato, T. (1963) Studies on floating rice. (2) The effect of water level treatment upon carbohydrate content and the amylase and invertase activities. Proc. Cr0p Sci. Soc. Japan 31, 357-361 Yamaguchi, T. (1973) Studies on the floating rice III. Effects of raising water level on the growth and carbohydrate contents of the tops. Proc. Crop Sci. Soc. Japan 42, 29-34 Received ; accepted 37 Table 1. Effect of C2H4 and GA3 on internodal elongation (: SE) and a-amwlase activity in the older regions of the internodes formed before isolation of rice stem sections. Air and air containing 1 pl 1"1 C2H4 were passed at 80 ml min"1 through the incubation cylinders in which sections were standing upright in 100 m1 glass beakers containing 40 m1 of distilled water or 5x10'7 M GA3 solution. Each number is the average of 5 or 6 sections incubated for 3 d in continuous light. Treatment Increase in internodal a-Amylase activity length (mm) (E.U.) Air 8.6 1 1.9 1.3 Submerged 98.4 i 13.1 19.2 Air +1 pl i-l C2H4 53.9: 6.6 i5.2 5x10‘7MGA3 99.8 i 9.5 14.6 38 24.“. Old, Air 2o-i A l 3 Pp. lo) 16" C» E 12“ J: (J L— GS 4—0 8"“- U? ° 0 Old, Subm. 4—r- New,Subm. h l 0 i i i i i r i i 0 10 2O 30 4O 50 60 70 80 90 ' Time (h) _[ig;_l. Starch content of different regions of internodes excised from submerged or air—incubated rice stem sections kept in continuous light. A-——A Older internodal region of stem sections formed prior to isolation of the sections from the plant and incubated in air. o-——o Older internodal region of submerged sections formed prior to isolation of the section. o-—-o Younger internodal region formed after the stem sections had been submerged. Each point is the pooled average of 5 or 6 sections. 39 O A ‘3 0 Old, Subm. DJ 20 -— o ' V O O .3; 16 -.. o ..>. o '8 <1: 12 -— ° 0) 8 '3'; 8 ‘— o E l 4 -- ° :5 New,Subm. , E e /, Old, Air 0 i . ‘= — 0 10 2o 30 4'0 5'0 6'0 7'0 ‘8'0 9'0 Time (h) _Eig;_2. a—Amylase activity in different regions of internodes excised from submerged or air-incubated rice stem sections kept in continuous light. A—-—A Older internodal region of stem sections incubated in air that had been formed prior to isolation of these sections. o——-o Older internodal region of submerged sections formed prior to isolation of the sections. o-——o Younger internodal region formed after the stem sections had been submerged. Each point is the pooled average of 5 or 6 sections. Submerged (II 4:. :P‘ on jigng3. Effect of submergence and incubation in air on the partitioning of 14C-labeled assimilates in modified rice stem sections kept in continuous light (% incorporation, ethanol soluble/ethanol insoluble fractions). 14C02 was supplied to 7-cm-long tips of the leaf blades. Sections were separated into different regions 210 min after the end of the exposure to 14C02. Total 140 incorporation in submerged stem sections was 37.6 KBq per section while in air— incubated sections total incorporation was 11.7 KBq per section. Chapter 4 Regulation of Growth in Rice Seedlings 42 J Plant Growth Regul (1983) 2:193-203 Joann-lo! ‘Plant Growth Regulation © 1983 Springer-Verlag Regulation of Growth in Rice Seedlings Ilya Raskin and Hans Kende MSU-DOE Plant Research Laboratory. Michigan State University, East Lansing, Michigan 48824 USA Received June 23. 1983; accepted August 10. 1983 Abstract. Etiolated rice seedlings (Oryza sativa L.) exhibited marked mor- phological differences when grown in sealed containers or in containers through which air was passed continuously. Enhancement of coleoptile and mesocotyl growth and inhibition of leaf and root growth in the sealed containers (“enclosure syndrome") were accompanied by accumulation of C02 and C2H4 in and depletion of 02 from the atmosphere. Ethylene (1 M I“), high levels of C02. and reduced levels of 02 contributed equally to the increase in coleoptile and mesocotyl growth. The effect of enclosure could be mimicked by passing a gas mixture of 3% Oz, 82% N2, 15% C03 (all v/v), and 1 pl 1’1 C2H4 through the vials containing the etiolated seed- lings. The effects of high CO; and low 02 concentrations were not mediated through increased C3H4 production. The enclosure syndrome was also ob- served in rice seedlings grown under water either in darkness or in light. The length of the rice coleoptile was positively correlated with the depth of planting in water-saturated vermiculite. The length of coleoptiles of wheat, barley, and oats was not affected by the depth of planting. In rice. the length of coleoptile was determined by the levels of 02, C02. and ethylene, rather than by light. This regulatory mechanism allows rice seed- lings to grow out of shallow water in which the concentration of 02 is limiting. The effect of different gases on the growth of rice seedlings is not well defined, in spite of the large number of publications on this topic. It has long been known that rice seeds have the unique ability to germinate at very low levels of O; or even in the absence of it (Taylor 1942, Vlamis and Davis 1943). It has 43 l. Raskin and H. Kende also been observed that coleoptile growth is stimulated and leaf and root growth inhibited in rice seedlings grown under water (Kordan 1977, Turner et al. 1981, Yamada 1954). These effects of submergence on the morphology of rice seedlings have been explained on the basis of reduced 0; supply under water. Indeed. elongation of the rice coleoptile is enhanced at reduced 0; tensions (Ohwaki 1967, Ranson and Parija 1955). Low concentrations of eth- ylene injected into sealed flasks containing rice seedlings also cause increased elongation of the rice mesocotyl and coleoptile (lmaseki et al. 1971, Ku et al. 1970, Miller and Miller 1974, Suge 1971). Indeed, Ku et al. (1970) have ascribed to ethylene the major role in the promotion of coleoptile growth. Recently, Atwell et a1. (1982) have suggested that ethylene is mainly responsible for the stimulation of rice coleoptile elongation in stagnant water and that C03, an antagonist of ethylene, inhibits coleoptile growth. According to these same authors, elongation of the coleoptile is relatively insensitive to 02 supply. With one exception (Ku et al. 1970, Table 2), all the above experiments on the effect of ethylene on growth of rice seedlings have been performed with plants in closed containers. Since enclosure leads to changes in the concentration of 02 and C02 in the atmosphere, the true contribution of ethylene to the regulation of growth is difficult to ascertain from the literature. In this paper, we evaluate the contributions of 03, C02. C2H4, and submer- gence in water to the stimulation of growth of etiolated and lightagrown rice seedlings. An attempt is also made to assign a physiological significance to the growth response induced by these gases in rice in comparison to other cereals. Finally, we have investigated whether the response of seedlings of different rice cultivars to altered gas atmospheres and to submergence is symptomatic for the later growth habit of these varieties. For example, deep-water rice plants that are at least 21 days old exhibit a dramatic growth response to ethylene (Métraux and Kende 1983). Do these same varieties at the seedling stage Show a greater response to ethylene and altered C02 and Oz atmOSpheres than do varieties not adapted to deep water? Materials and Methods Plant Material. The following rice (Oryza sativa L.) cultivars were used in this study: M-9 (seeds provided by Dr. J. N. Rutger, University of California, Davis, California): Labelle (seeds provided by Dr. T. Johnston, University of Arkansas, Stuttgart, Arkansas); IR-8 (seeds provided by Dr. R. S. Bandurski, Michigan State University, East Lansing. Michigan): Habiganj Aman III and VII (seeds provided by Dr. S. M. H. Zaman, Bangladesh Rice Research In- stitute, Dacca, Bangladesh); Pin Gaew 56 and Thavalu (seeds provided by Dr. B. S. Vergara, International Rice Research Institute, Los Bafios, Philippines). Most of the experiments were performed with the California semi-dwarf cul- tivar M-9. Seeds were sterilized in 2% (w/v) sodium hypochlorite solution, rinsed five times with sterile distilled water, and germinated in darkness at 30°C in sterile Petri dishes (9-cm diameter) containing 12 ml of sterile distilled water. After 2 days, seedlings with coleoptiles about 1 mm in length were selected for experimental treatments. 44 Growth of Rice Seedlings Growth in Closed and Flow-through Containers. Ten preselected 2-day-old seedlings were transferred under dim white light to one 40-ml shell vial con- taining 2 ml of distilled water. which formed a S-mm-deep layer in the bottom of the vial. The shell vials were tightly stoppered with serum vial caps in order to study growth of seedlings in sealed containers. Alternatively. serum vial caps were fitted with a 3‘/2-inch. 16—gauge hypodermic needle as an inlet and a l‘/2-inch. l6-gauge hypodermic needle as an outlet for experiments in which a continuous flow of air or a gas mixture was passed through the vial at 30 ml min“. Seedlings were handled under sterile conditions. all laboratory ware was sterilized, and the gases were passed through a Millipore filter (0.45 pm pore size. Millipore Corp., Bedford. Massachusetts). Unless mentioned oth- erwise. all experiments were carried out in complete darkness at 27°C. The mixtures of N2, 03. and CO; were either purchased from Matheson Gas Products (Joliet, Illinois) in high-pressure gas cylinders or prepared with gas regulators and flow meters (Matheson Gas Products). Compressed laboratory air was used for all flow-through air treatments. The gas mixtures and air were humidified to 100% relative humidity by being bubbled through water and were dispersed to the vials containing the seedlings with a flowmeter board (Pratt et al. 1960). C3114 was added to the gas stream with a C3114 diffusion apparatus (Saltveit 1978). To prepare C2H4-free gas mixtures or C3H4-free air, the gases were passed through a 25—cm-long column (7 cm l.D.) packed with Purafil (Purafil Inc.. Atlanta. Georgia). For 02 and C02 determinations. 2-ml gas samples were withdrawn with a gas-tight syringe and analyzed using a gas chromatograph equipped with a thermal conductivity detector (Model GC 8700, Carle Instru- ments Inc.. Anaheim, California). C2H4 was determined by gas chromatog- raphy of l-ml gas samples (Kende and Hanson 1976). C2114 Evolution in Different Gas Mixtures. Two-day-old etiolated rice seedlings were incubated in vials through which different gas mixtures were passed con- tinuously for 6 days. At the end of the incubation period, the vials were tightly stoppered. and l-ml gas samples were withdrawn through the serum vial caps with gas-tight syringes at hourly intervals for QB, determinations. A dim green flash light was turned on for less than 2 min during withdrawal of gas samples. Conditions of sterility were maintained as above. C 3H4 and Submergence Effects in Light. Two-day-old seedlings were individ- ually sown in 20-ml plastic pots filled with fine Vermiculite and placed in an environmental growth chamber under the following conditions: day tempera- ture 25°C. night temperature 22°C. relative humidity 60%, 16-h photoperiod with a light intensity of 350 uE m‘~ s“l at seedling level. Daily watering with '/4-Strength Hoagland‘s solution kept the Vermiculite saturated with water. Six 8-day-old seedlings. 7 to 8 cm long, were selected and placed into two cylin- drical glass containers (8 1 volume) equipped with inlet and outlet tubing. Air with and without C3114 was passed through the containers at a flow rate of 400 45 l. Raskin and H. Kende ml min”. C2114 at a concentration of 5 p1 1“l was added to the air stream as described above. Each day. seedlings were taken out of the container for 5 min for length measurements. Seedlings used in submergence experiments were grown in the same envi- ronmental chamber under the same conditions. Five 9-day-old seedlings were completely submerged in a 40-1 glass tank filled to the top with distilled H20. The tanks were kept in the same environmental chamber in which the seedlings had been grown. Seedlings remained submerged during growth measurements. Growth of Seedlings at Different Water Depths. Ten 2-day-old seedlings were incubated in 40-ml shell vials filled with distilled H30 to the depth of 0.5, 2. 4, 6. and 8 cm. Vials were incubated at 27°C in darkness or in continuous light (intensity 7O pE m ‘2 s‘ 1). Air was passed at a flow rate of 30 ml min“1 through the head space above the water of all vials except for those that were used to examine the effect of enclosure. Growth of Seedlings at Different Depths of Vermiculite. Sixteen 2-day-old seedlings of rice, cv. M-9, of wheat. cv. Ionia. of barley, cv. Lakeland. and of oats) cv. Korwood. were planted at depths of 1, 2, 4, 6, and 8 cm in fine Vermiculite in 800-m1, square plastic pots with holes in the bottom. The pots were kept in darkness at 27°C in plastic trays filled with water to keep the Vermiculite saturated with water for the duration of the experiment. Results Etiolated rice seedlings (cv. M-9) grown in aerated or sealed containers exhib- ited marked differences in morphology (Fig. lA-C). The altered composition of gases in the atmosphere caused by enclosure of the seedlings stimulated growth of the mesocotyl and the coleOptile and inhibited growth of the leaves. Enclosure also led to inhibition of root development (data not shown), which was in agreement with earlier observations (e.g. Kordan l976a). The levels of 02, C03, and Czl-I4 inside the sealed vials containing the seedlings were mea- sured daily (Fig. 1D). After 8 days of incubation. the sealed vials contained 3% 02, 21% C02, and 0.9 Mi 1.1 C2H4 (all V/V). The growth of etiolated rice seedlings of different varietal groups incubated for 7 days in sealed and aerated containers was compared to that of M-9 (Fig. 2). Enclosure caused a similar increase in coleOptile and mesocotyl growth and inhibition of leaf growth in all varieties tested. The rice cultivars used in this experiment included a Texas lowland variety Labelle. the semi-dwarf cultivar IR-8 from the Philippines. the Sri Lanka flood-tolerant variety Thavalu. the Bangladesh deep-water rice variety Habiganj Aman Vll, and the Thai deep- water rice variety Pin Gaew 56. Results similar to those shown in Fig. 2 were also observed with the deep-water cultivars Habiganj Aman III. Leb Mue Nahng, and Kalar Harsall (data not shown). Six different mixtures of N3, 03. C03, and C2H4, in comparison to air. were 46 Growth of Rice Seedlings A (Total he: ht) B (Coleopiile 8 Mesocot I) act 9 ao- y 70' 70" 60L ’6‘ sor 50- g 50- 407 g 40'- Coleopiile 30- g 30*- i a 201' 20 Mesocotyl E io- 10 ‘ V 1 1 1 1 L L 1 L a l f ' A 0 (Gas Com siiion) o 80*- C (Highest Leaf) 9? 32__ DO C2H4 ~08 A ‘ 11\" T 3 70'. E 28»- “0.7 2 2 1 60 '5 24' “0.6 E 50 g 20 C02 -o.5 E 40‘ :c)’ 15»- -0.4 E 30 ’71, 12- -o.3 ‘3’ 20 8 8- 802 U 02 f 10 “5 4- -o.1 N O L 1 1 1 1 1 1 1 6' O 1 1 1 L1 1 l_ 1 J 1 U 40 80 120 ISO 200 40 80 120 160 200 Time (h) Time (h) Fig. l. A-C: The time course of growth of etiolated rice seedlings (cv. M-9) in sealed (0) and aerated (O) containers. D: The time course of changes in 02. C03. and C3H4 concentrations inside the containers. Twenty-four 40-ml shell vials. each containing 10 2-day-old seedlings, were sealed. while 24 other vials were continuously flushed with air at a flow rate of 30 ml min". Every 24 h. seedlings from three randomly chosen sealed and aerated vials were measured with a ruler and discarded. Gas samples for C02. 02, and CZH. determinations were withdrawn from the sealed vials with gas-tight syringes before the vials were opened. A-C—each point is the average value for 30 plants. D—each point is the average value for three vials. Vertical bars denote S.E. When no error bar is given. the SE. is smaller than the symbol used. used to evaluate the contribution of high concentrations of C03 and C2H4 and low concentrations of 02 on the growth of etiolated rice seedlings. In the artificial gas mixtures, the concentrations of 02. C02, and ethylene were ad- justed singly or in combination to the concentrations found in sealed vials containing seedlings after several days of incubation, namely 3% Oz. 15% C02, and l ptl l“l ethylene (all v/v). The total length of the seedlings and the length of their coleoptiles, mesocotyls, and leaves were measured after 7 days of incubation (Table 1). The results demonstrated that enclosure increased co- leoptile length by 160%, mesocotyl length by 200%, and inhibited leaf growth by 44%. compared to seedlings incubated in containers that were continuously flushed with air. High C02 (15%), low 02 (3%), and C3H4 (1 pl 1") applied individually in the gas stream were responsible for about one-third of the total stimulation of coleoptile growth that was observed in the sealed containers. Combined treatment with any two of these gases elicited about two-thirds of the response caused by enclosure. The combination of C3H4, high C03, and low 0; closely mimicked the effect of enclosure on the growth of etiolated rice seedlings (Table l). Ethylene had some stimulatory effect on leaf growth in air and high CD; but inhibited leaf growth in low 03. While the relative length of 47 l. Raskin and H. Kende g3 R-B Labelle 80'- 7 s 70- ‘ 60- ‘ s 50" fl ‘ a 40- 8 ‘ s s 30" q - 201- ‘i ... (I) E L A "" g 10 :3 1: G rrcj , r eerie Tic u L, .- .HSRLQQDI Bin m Ihgltfllll 0’ ’ 21112911111 :5 c s 3 80" " g 14>- A1! 70-° « :12r L02 60+- - 6 IO- mZLOZ 50" s s ‘ V 3L 0 § 40l- ° 4 I; 6'- . S > o 301- ' 111' 4" 0 in 201- o -1 2" '0- ~ 0. l s a 2. a a O 1’ c 11 1. 1' c a, L 'r c 3,1» Time (h) Fig. 2 (left). Growth of etiolated seedlings of different rice varieties in sealed and aerated con- tainers. Twenty 2-day-old seedlings of each variety were incubated for 7 days in sealed (5) and aerated (a) 40-ml shell vials (10 seedlings per vial). The air flow was 30 ml min". Total length of seedlings (T) and length of coleoptile (C). mesocotyl (M). and longest leaf (L) were measured after 7 days of incubation. Each point is the average value for 20 seedlings. Vertical bars denote S.E. Fig. 3 (right). Time course of C2H4 evolution from etiolated rice seedlings (cv. M-9) after 6 days of incubation in 40-ml shell vials: in a continuous flow of air (0); 0.03% C02. 3% 02. 97% N3 ([1): 15% C03, 3% 02. 82% N2 (0), and 15% C02, 21% 03. 64% N: (A). Prior to CzH, measurements. the vials were sealed. and CIH, accumulation in the vials was monitored for 6 h. All gas concen- trations are given on a v/v basis; the gas flow was 30 ml min". Each point is the average value of triplicate samples. Vertical bars denote S.E. different organs of etiolated rice seedlings was markedly altered as a result of enclosure, the total length of the rice seedlings was only slightly affected. The dose response of etiolated rice seedlings (cv. M-9) to C3H4 was deter- mined by incubating seedlings for 7 days in containers through which air con- taining l, 5, and 10 pl 1'l of QR, was passed continuously. The ability of CZHJ to promote the elongation of seedlings was close to saturation at 1 pl 1 “ (Table 2). However, C2H4-induced acceleration of coleOptile and mesocotyl growth could account for only about 30% of the growth acceleration observed in sealed containers. Since the enhancement of coleOptile and mesocotyl growth in high CO: and low 02 could be caused by higher rates of C3114 production, the evolution of CZH.‘ from etiolated rice seedlings (cv. M-9) incubated in mixtures of 15% C03 48 Growth of Rice Seedlings Table 1. Effect of different gas mixtures on the growth of etiolated rice seedings. Length (mm) Longest Treatment Total Coleoptile Mesocotyl leaf Air(21% 02 + 0.03% C02) 61 t 2.9 23 :0.- 0.5 3 z 0.3 57 z 3.0 Air (21% 02 + 0.03% C02) + 1 p11“ C2114 71 z 2.7 31 z 0.3 4 z 0.3 66 1- 2.7 21% 02 + 15% C02 63 z 2.7 32 z 1.1 6 2 0.6 55 z 3.5 21% 02 + 15% C0; + l 11.11’l CZH. 72 z 2.0 44 z 1.5 9 z 0.7 61 z 2.3 3% 0; + 0.03% C02 77 z 4.1 33 1- l 2 5 z 0.3 71 ‘1: 4.4 3% 02 + 0.03% C02 + 1 pl l'l C211. 64 t 4.0 44 z 1.5 6 t 0.4 51 z 5.8 3% 02 + 15% C02 59 2: 4.0 40 1: 0.9 8 z 0.5 47 :0.- 5.0 3% 02 + 15% C0; + 1 p11" C211, 65 r 2.2 55 t 1.6 10 t 0.6 39 t 3.2 Sealed vial 69 t 2.3 59 t 1.4 9 .t 0.5 32 1 2.6 Rice seedlings (cv. M-9) were treated as indicated for 7 days. All gas mixtures were made up in N; and were passed through the 40-ml incubation flasks at 30 ml min". Concentrations of gases are given on a v/v basis. Each number is the average value for 30 seedlings incubated in three separate containers :0.- S.E. Table 2. Effect of different concentrations of C2H4 on the growth of etiolated rice seedlings. Length (mm) Treatment Total Coleoptile Mesocotyl Longest leaf Air 66 z 2.5 25 1- 0.8 2 1- 0.2 64 1- 2.5 Air + CZH, (1 pl 1") 77 I 2.4 32 z 0.7 4 r 0.2 73 z 2.6 Air + C214. (5 p11") 80 1- 3.6 35 z 1.2 4 t 0.3 75 1- 3.8 Air + C211. (10 p11") 82 .4.- 3.4 36 2.- 0.8 5 x 03 77 a: 3.5 Sealed vial 68 1- 2.1 56 1- 1.9 8 t l l 37 1- 3.7 Rice seedlings (cv. M-9) were treated as indicated for 7 days. Ethylene was continuously added to the air stream passing at 30 ml min‘I through the 40-rnl incubation vials. Each number is the average value for 30 seedlings incubated in three containers : S.E. and 3% 02 was measured (Fig. 3). The highest rates of QR, release were observed in seedlings grown in air rather than in high C02 or low 02. C2H4 at a concentration of 5 pl 1‘l supplied for 6 days in the air stream stimulated elongation of 8-day-old rice seedlings (cv. M-9) grown under a 16- h photoperiod (Fig. 4). Total height of the seedlings and length of the highest leaf sheath were increased by C2114. Similar results were obtained with the deep-water rice varieties Habiganj Aman III and VII (data not shown). Nine-day-old rice seedlings (cv. M-9) continued to grow even when com- pletely submerged in distilled water and kept under a 16-h photoperiod (Fig. 5). The slight initial increase in the growth rate caused by submergence was lost after 4 days of flooding. Similar results were obtained with the deep-water rice varieties Habiganj Aman III and VII (data not shown). To evaluate the relative contributions of continuous light (intensity 70 pE m'2 s") and re- stricted gas exchange on the growth of rice seedlings, 2-day-old seedlings were submerged under different depths of water in light and in darkness. 1n dark- 49 l. Raskin and H. Kende 04 0 Total H919.“ C2H4 30" Comrol Control 24 24 E ’e V 8 z 18 E |31~ ‘6 .9 5 Cth 5*; i "J '2 Control 12" l 6” 6" l 0 1 l 1 4 l 1 1 1 1 1 1 1 1 o 1 2 3 4 5 6 Co 1 2 3 4 5 6 7 Time (days) Tl rne (days) Fig. 4 (left). The effect of CIH, on the growth of rice seedlings (cv. M-9) under a 16-h photoperiod. CzH, (5 pl 1") was added to the stream of water-saturated air passing through the 8-1 incubation chamber at a flow rate of 400 ml min". The seedlings were 8 days old at the start of the Czl-l, treatment. Each point is the average value of total height (0) and longest leaf sheath (A) of 6 seedlings. Vertical bars denote S.E. Fig. 5 (right). Time course of elongation of completely sub- merged (O) and air-grown (A) rice seedlings (cv. M-9) under a 16-h photoperiod. The seedlings were 9 days old at the start of the experiment. Each point is the average value of five seedlings. Vertical bars denote S.E. ness, 2-day-old rice seedlings planted at water depths below 40 mm deve10ped coleoptiles and mesocotyls of similar lengths. as did etiolated, enclosed seed- lings (Table 3). Characteristically, leaf growth was inhibited in seedlings planted at depths below 40 mm. Even in continuous light, seedlings planted 40 mm below the water surface responded with a greater than 4-fold increase in co- 1e0ptile length as compared with air-grown seedlings exposed to light of the same intensity. Coleoptiles of rice seedlings submerged to depths below 40 mm in light were actually 70% larger than coleOptiles of aerated seedlings grown in darkness under 5 mm of water. The effect of restricted gas exchange on seedling growth could also be dem- onstrated when seedlings were planted at different depths of vermiculite. The length of etiolated rice coleOptiles showed a strong positive correlation to the depth of planting (Table 4). In complete darkness. the coleoptiles of rice seed- lings planted at a depth of 80 mm were about 3 times longer than the coleoptiles of seedlings planted at a depth of 10 mm. The depth of planting had no effect on the length of the coleoptiles of etiolated wheat. barley, and oat seedlings. Discussion Based on our results, it is difficult to single out any one gas (C02, 02. or C2H4) as being most important for the stimulation of growth of rice seedlings. Enclo- 50 Growth of Rice Seedlings Table 3. Effect of darkness and light on the growth of rice seedlings submerged at different depths ofwaier. - . Depth of Length (mm) submergence Treatment (mm) Total Coleoptile Mesocotyl Longest leaf Darkness 5 77 t 3.7 24 2: 1.0 2 t 0.3 75 t 3.8 Darkness 20 74 z 4.0 34 z 1.6 7 2°.- O.6 67 z 4.4 Darkness 4O 86 z 4.4 49 1- 1.2 9 z 0.5 78 z 4.7 Darkness 6O 76 z 2.8 62 z: 1.1 10 t 0.7 50 z 5.7 Darkness 80 79 3.- 2.1 70 1- 2.1 10 t 0.7 19 z 2.1 Darkness, sealed 5 78 t 2.3 64 t 2.7 12 2: 1.6 35 t 3.2 Light 5 50 z 0.9 9 z 0.7 0 50 z 0.9 Light 20 55 t 2.2 23 t 0.7 0 55 r 2.2 Light 40 50 1- 2.6 40 t 1.2 0 44 z 4.1 Light ' 6O 50 z 2.0 40 1- 1.3 1 z: 0.1 46 z 3.0 Light 80 47 t 3.3 39 z 1.3 2 r. 0.2 42 z 4.1 Light. sealed 5 53 z 2.3 14 z 0.7 0 53 z 2.3 Rice seedlings (cv. M-9) were treated as indicated for 7 days. Each number is the average value for 20 seedlings from duplicate treatments :- S.E. Table 4. Length of coleoptiles of selected cereals as a function of the depth of planting in Ver- miculite. Depth of planting (mm) Length of incubation Plant 10 20 4O 6O 80 (days) Rice (cv. M-9) 16 t 0.6' 25 t 0.8 35 z 1.4 43 I 2.4 49 2: 1.3 6 Oats (cv. Korwood) 62 1- 0.9 58 z 1.5 60 1- 1.0 59 1- 1.8 59 2: 1.2 5 Wheat (cv. Ionia) 69 t 1.7 73 2: 1.7 69 2: 1.9 70 z 1.8 65 z 2.4 5 Barley (cv. Lakeland) 46 t 1.7 43 t 1.2 44 z 1.3 43 z 2.4 46 :t 1.3 5 ‘ Average coleOptile length (mm) of 16 plants : S.E. sure of 2-day-old rice seedlings in sealed containers produced what we call the “enclosure syndrome.” The development of the enclosure syndrome could be mimicked by passing a mixture of 3% Oz, 82% N2, 15% C02. and 1 pl 1“ C2H4 through the vials containing rice seedlings. Therefore, the morphological changes observed during enclosure are caused by the combined effects of in- creased C02 and C2H4 and decreased 02 levels in the ambient atmosphere. The effect of each of these gases appeared to be additive for the stimulation of coleoptile and mesocotyl growth in sealed containers (Table l). The use of the flow-through system with eight different gas mixtures allowed us to differentiate among the effects of C02. 03. and C3H4 on the growth of rice seedlings. Because of the involvement of respiratory gases (CO; and 03) in the regulation of the morphogenesis of rice seedlings. an independent eval- uation of the role of C3H4 can only be accomplished when the atmosphere around the plant is continuously renewed. The use of sealed containers in 51 I. Raskin and H. Kende which C2H4 was injected (Ku et al. 1970. Miller and Miller 1974) led to an overestimation of the QR, effect on growth. Similarly, the marked enhance- ment of coleoptile elongation under water may not be ascribed only to low 0; supply. as suggested by Kordan (l976b). Our results contradict those of Atwell et al. (1982), who found coleOptile elongation to be largely unaffected by low 0; supply and who suggest that growth of the coleoptile is inhibited by C02. CO; was found to stimulate C2H4 synthesis and release in the leaves of corn. Xanthium, and rice (Grodzinski et al. 1982, Kao and Yang 1982). Simi- larly, 5% O; was found to enhance C2H4 production in apical segments of maize roots (Jackson 1982). However, in etiolated rice seedlings, C3H4 evolution was inhibited by 15% CO; or 3% 02 (Fig. 3). Thus, it is unlikely that high CO; and/ or low 02 levels stimulate the growth of rice coleoptiles and mesocotyls through the enhancement of C2114 production. However. high CO; and/or low 02 concentrations may sensitize seedlings to C2H4. Alternatively. these three gases may stimulate growth independently of each other. For example, high concentrations of C02 could enhance growth of etiolated rice coleoptiles through cell-wall acidification. as has been described for oat coleoptiles (Evans et al. 1971). The stimulatory effect of low 02 on coleoptile and mesocotyl growth may be based on either reduced accumulation of hydroxyproline-rich protein in the cell walls (Hoson and Wada 1980), or on acidification of the cell wall as a result of acid production during fermentation (Hochachka and Momm- sen 1983), or on inhibition of IAA-oxidase activity (Schneider and Wightman I974. Yamada 1954). An enclosure syndrome of comparable magnitude was observed in traditional lowland, semi-dwarf, flood-tolerant, and deep-water rices. Thus. the magnitude of the enclosure syndrome cannot be used to distinguish between different varietal groups, e.g. between deep-water and regular rice cultivars. The enclosure syndrome could also be observed when the gas exchange between the seedling and the environment was restricted, either in seedlings submerged in water or in seedlings planted in water—saturated vermiculite. Under both conditions, the length of the rice coleOptile was positively corre- lated to the depth of planting (Tables 3 and 4). In the case of submerged seed- lings. this correlation has already been observed by Kefford (1962) and Yamada (1954). We have shown that restricted gas exchange overrode the photoinhi- bition of coleOptile elongation in rice. Therefore. elevated CO; and ethylene levels and reduced 03 tensions are more important factors in regulating growth of rice coleoptiles than is light. In contrast. the growth of wheat, barley. and oat coleOptiles was not affected by the depth of planting. In rice. the stimulation of coleoptile and mesocotyl growth by increased concentrations of CO; and ethylene and decreased levels of 02 is a unique adaptive feature that permits rice seedlings to grow rapidly in shallow waters and waterlogged soils. While coleoptile and mesocotyl growth are promoted under hypoxic conditions. leaf growth is strongly inhibited. Once the tip ofthe coleoptile emerges into the air, rapid growth of the leaf commences. Acknowledgments. We thank R. deZacks for help in the preparation of plant material. This re- search was supported by the National Science Foundation through Grant No. PCM 81-09764 and by the Department of Energy under Contract No. AC02—76ERO-l338. 52 Growth of Rice Seedlings References Atwell BJ. Waters 1. Greenway H (1982) The effect of oxygen and turbulence on elongation of coleoptiles of submergence-tolerant and -intolerant rice cultivars. J Exp Bot 33: 1030—1044 Evans ML. Ray PM. Reinhold L (1971) Induction of coleoptile elongation by carbon dioxide. Plant Physiol 47:335-341 Grodzinski B. Boesel I. Horton RF (1982) Ethylene release from leaves of Xumhr'um strumarium L. and Zea mays L. J Exp Bot 332344-354 Hochachka PW. Mommsen TP (1983) Protons and anaerobiosis. Science 219:1391-1397 Hoson T. Wada S (1980) Role of hydroxyproline-rich cell wall protein in growth regulation of rice coleoptiles grown on or under water. Plant Cell Physiol 21:511-524 Imaseki H. Pjon CJ. Furuya M (1971) Phytochrome action in Oryza sativa L. IV. Red and far red reversible effect on the production of ethylene in excised coleoptiles. Plant Physiol 48:241- 244 Jackson MB (1982) Ethylene as a growth promoting hormone under flooded conditions. In: Wareing PF (ed) Plant growth substances 1982. Academic Press. London. New York. pp 291- 301 Kao CH. Yang SF (1982) Light inhibition of the conversion of l-aminocyclopropane-1-carboxy1ic acid to ethylene in leaves is mediated through carbon dioxide. Planta 155:261—266 Kefford NP ( 1962) Auxin-gibberellin interaction in rice coleoptile elongation. Plant Physiol 37:380- 386 Kende H. Hanson AD (1976) Relationship between ethylene evolution and senescence in morning- glory flower tissue. Plant Physiol 572523—527 Kordan HA (1976a) Adventitious root initiation and growth in relation to oxygen supply in ger- minating rice seedlings. New Phytol 76:81-86 Kordan HA (1976b) Oxygen as an environmental factor influencing normal morphogenetic devel- opment in germinating rice seedlings. J Exp Bot 27:947—952 Kordan HA (1977) ColeOptile emergence in rice seedlings in different oxygen environments. Ann Bot 41:1205-1209 Ku HS. Suge H. Rappaport L. Pratt HK (1970) Stimulation of rice coleoptile growth by ethylene. Planta 902333-339 Métraux J—P. Kende H (1983) The role of ethylene in the growth response of submerged deep water rice. Plant Physiol 722441-446 Miller JH. Miller PM (1974) Ethylene and the responses to light of rice seedlings. Physiol Plant 30:206-21 1 Ohwaki Y ( 1967) Growth of rice coleoptiles in relation to oxygen concentrations. Sci Rep Tohoku Univ Ser IV 33:1-5 Pratt HK, Workman M. Martin FW, Lyons JM (1960) Simple method for continuous treatment of plant material with metered traces of ethylene or other gases. Plant Physiol 35:609-611 Ranson SL, Parija B (1955) Experiments on growth in length of plant organs. 11. Some effects of depressed oxygen concentrations. J Exp Bot 6:80-93 Saltveit ME. Jr ( 1978) Simple apparatus for diluting and dispensing trace concentrations of ethylene in air. HortSci 13:249-251 Schneider EA. Wightman F (1974) Metabolism of auxin in higher plants. Ann Rev Plant Physiol 25:487—5 13 Suge H (1971) Stimulation ofoat and rice mesocotyl growth by ethylene. Plant Cell Physiol 12:831— 837 Taylor DL t 1942) Influence of oxygen tension on respiration, fermentation. and growth in wheat and rice. AmJ Bot 291721-738 Turner FT. Chen C-C. McCauley GN (1981) Morphological development of rice seedlings in water of controlled oxygen levels. Agronomy J 73:566-570 Vlamis J. Davis AR (1943) Germination. growth. and respiration of rice and barley seedlings at low oxygen pressures. Plant Physiol 18:685-692 Yamada N ( 1954) Auxin relationships of the rice coleoptile. Plant Physiol 29:92—96 Chapter 5 The Role of Air Layers in the Aeration of Deep-Water Rice Plant Physiol. (1983) 72. 447-454 54 0032-0859/33," "ill-”0447 .‘08/50050/0 How Does Deep Water Rice Solve Its Aeration Problem1 'N Received for publication November 16. 1982 and in revised form February 8, 1983 lva RASKIN xso HANS KENDE MS U-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 ABSTRACT In partially flooded deep water rice (0:: ':a sativa L. cv Habiganj Aman ll). continuous air layers trapped between the hydrophobic. corrugated surface of the leaf blades and the surrounding water constitute the major path of aeration. The conduction of gases through the internal air spaces of the leaf is negligible compared to the conduction of gases through the external air layers. The total volume of the air layers on both sides of a leaf blade is about 45‘} of the volume of the leaf blade itself. The size of the air layers around submerged leaf blades of cereals not adapted to conditions of partial flooding. tag. of oats. barley. and wheat. is considerably smaller than that of rice. Gases move through the air layers not only by diffusion but also by mass flow. In darkness. air is drawn down from the atmosphere through the air layers along a pressure gradient created by solubilization of respiratory CO: in the surrounding water. In light. pho- tosynthetic 0 3 is expelled through the air layers to the atmosphere because the solubility of 02 in water is much lower than that of C02. Air layers greatly increase the rate of photosynthetic carbon fixation by enlarging the surface of the gas-liquid interface available for CO; uptake from the water. Air layers are vital for the survival of the partially submerged rice plant. When leaves are washed with a dilute solution of a surfactant (Triton X400). no air layers are formed under water. Plants without air layers do not grow in response to submergence. and the submerged parts of the plant deteriorate as evident by rapid loss of chlorophyll and protein. Air layers provide a significant survival advantage even to completely submerged rice plants. Floating or deep water rice is mainly grown in the floodplains of Southeast Asia where the water can rise up to 6 m during the rainy season 16). Floating rice has great agronomic importance because it is the subsistence crop in many densely populated areas where no other crop can be grown. The distinguishing character- istic of this rice is its ability to elongate with rising waters. Growth rates of 20 to 25 cm/d have been recorded in response to sub- mergence. with the total plant height reaching up to 7 m (15). Survival of deep water rice depends on its ability to keep part of its foliage above the water surface. Completely submerged plants cease to elongate and eventually die (14). Flooding imposes a severe stress on rice plants as O: and CO; supplies become limiting under water. The slow diffusion of gases in water (10.000 times slower than in air) greatly curtails the gas exchange between the plant and the surrounding water. This problem would be further aggravated if the gas exchange were limited primarily to the relatively small area of stomata on the rice leaf. It is commonly believed that the aeration requirements of the submerged organs of rice and other plants tolerant to partial flooding are met by O; entering the above-water parts of the ' Supported by National Science Foundation Grant l’(‘.\l 81-09764 and the U. S. Department of Energy Contract DE-ACUZ-76ERO-l338. leaves through the stomata and diffusing to the submerged organs via the internal air spaces loosely termed aerenchyma (1. 2. 4, 7. 9). This view fails to explain the aeration mechanism in deep water rice. In rice, the aerenchyma occupies a significant volume of the roots, internodes. and leaf sheaths. However. aerenchyma is poorly developed in the lower half of the leaf blade and is completely absent in the upper half of the blade (10). Thus. the aerenchyma cannot provide the necessary air connection between the atmosphere and the underwater organs of deep water rice plants which can survive prolonged flooding with only their leaf tips above the water. This paper presents evidence for the existence of air layers between the corrugated. hydrophobic surface of a submerged rice leaf and the surrounding water. These air layers provide an aeration path which is vital for the partially flooded plant. The mechanism of gas movement through the air layers and the function of air layers in the gas exchange between plant and water are also described. MATERIALS AND METHODS Plant Material and Growth Conditions. Seeds of 3 Bangladesh floating rice variety (Oryza sativa L. cv Habiganj Aman 11) were obtained from the Bangladesh Rice Research Institute (Dacca. Bangladesh). Rice plants were grown as described previously ( 12). Seeds of wheat (cv Ionia). barley (cv Lakeland). and oats (cv Korwood) were sown singly in one-quart plastic pots in potting soil of the same composition as described before (12). These plants were grown in an environmental chamber under the following conditions: day temperature. 24°C; night temperature. 21°C. RH. 60%; 16-h photoperiod with a light intensity of 350 pE m“2 s'1 at soil level. Plants were watered twice daily with half-strength Hoagland solution. All experiments were performed with 33- to 55-d-old plants. In the case of rice. this age corresponds to the time of internode elongation. Only fully expanded. healthy look- ing blades from the upper leaves. 9 to 12 mm in width. were used in our experiments. Leaf sections were excised from the midpor- tions of the leaf blades with a sharp razor blade. Chemicals. Ethane was purchased from Matheson Gas Products (Joliet. IL). NaH”CO;. (7.9 mCi/mmol) was from New England Nuclear. and platinum black was from Fisher Scientific Co. All other chemicals were purchased from Sigma Chemical Co. Triton X-100 Treatment. Formation of air layers on the sub- merged parts of leaves was prevented by wetting the leaves or leaf sections with a 0.05% (v/v) Triton X-100 solution, followed by a thorough rinse with distilled H30. Ethane Diffusion Measurements. The experimental set-up for ethane diffusion measurements is shown in Figure 2. The diffusion experiments were performed in light (100 [IE m”2 s") at 24°C. The blade of an attached leaf was introduced into a U-shaped tube which contained water in the bottom. The tip and the lower part of the leaf protruded into the head spaces in the right and left arm of the U-tube. respectively. while 15 cm ofthe midportion of the blade became submerged in the water. Ethane was injected into the closed head space in the right arm of the tube to yield a final concentration of4.5% (v/v). The atmospheric pressure in the right arm of the U-tube was maintained by periodic insertion of a hypodermic needle through the ethane injection port. Ethane was assayed in the sampling compartment of the left arm through which air was passed at a rate of 2 ml min“. One-ml gas samples were withdrawn through the sampling port with a gas-tight sy- ringe, and the ethane content was determined by GC using the conditions of Kende and Hanson (l 1). Samples were withdrawn slowly to keep constant pressure in the sampling compartment. The open outlet tubing was sufficiently long to prevent drawing of air from the outside into the sampling compartment. Application of Archimedes" Principle for the Determination of the Volumes of Air Layers and Leaves. The fresh weight of three 7-cm-long leaf sections (P1). excised from the midportion of the leaf blades. was measured. and the sections were attached to a metal clamp of sufficient weight to keep the sections submerged in H;O. The clamp with the leaf sections was suspended with a wire from the pan hook of an analytical balance and submerged in distilled H20. The weight of the submerged clamp with the sections (P2) was measured. Thereafter. the leaf sections were treated with Triton X-100 to eliminate the air layers. and the weight of the submerged clamp with sections (P3) was measured again. Elimination ofair layers led to a decrease in buoyant force (8). whereby B = P3 — P2. According to Archimedes’ principle. the buoyant force exerted on the air layers is equal to the weight of water displaced by the air layers. The volume of air layers ( V...) can then be calculated from: B =— (I) PH20 V." where pup is the density of water. The volume of air layers on each side of a leaf section was determined after wetting each side of the section separately with Triton X400 solution. The volume of the leaf sections was also calculated by applying Archimedes’ principle: Bleaf Puzo (2) Vleaf = where 8a.; is the buoyant force acting on the submerged sections without air layers and equals Bleaf=Pl-'(P3_P4) (3) where P. is the weight of the submerged clamp. The density ofthe leaf section (p;..,.{) was determined as: P) leaf .01er = (4) The area of the leaf sections was measured with a portable area meter. model LI-3000 (Lambda Instruments Co., Lincoln. NE). equipped with a transparent belt conveyer accessory. Oxygen Movement in the Air Layers. A glass cylinder (40 cm deep. 4.2 cm i.d.) was filled with 450 ml ofa 15 mg L"1 methylene blue solution. leaving a head space of 65 ml. Forty mg of Pt-black powder was added to the solution as a catalyst. Reduction of methylene blue was accomplished by bubbling gases through the solution in the following order: N2 for 4 min. H2 until the solution became colorless. and N2 for 10 min to remove H: from the solution and fill the head space with N2. After the reduction was completed. the cylinder was tightly stoppered. All experiments were performed at 28°C in darkness with a green safe light turned on only during measurements. Plants were kept in darkness for l h before the experiment. The Opposite ends of an excised leaf blade were clamped. adaxial surface outward. to a thin glass rod. At time zero. the stopper was removed from the cylinder. and the leaf blade fixed to the glass rod was lowered into the solution through the head space filled with N2. One cm of the leaf blade 55 RASKIN AND KENDE Plant Physiol. Vol. 72. 1983 was left above the surface of the solution. and the cylinder remained open so that air could diffuse into the head space. The O: movement along the adaxial surface was followed by measuring the distance between the color front and the surface of the solution every 5 min. Manometric Measurement of Gas Flow into and out of Air Layers. The manometric set-up depicted on the inset of Figure 4 was used to measure the mass flow of gases from the atmosphere into the air layers and vice versa. The set-up consisted of a 60-cm- long glass tube. containing 70 ml of distilled H30. A leaf blade was placed into the tube such that the leaf tip protruded into the small head space. The tube was closed at both ends with rubber stoppers. Two air-exchange needles and a 50-ttl glass micropipet (manometric tube). which connected the atmosphere to the head space. were fitted through the top rubber stopper. When the air exchange needles were closed. the movement ofa water bubble in the manometric tube reflected the volume change in the head Space. Eleven manometric set-ups. 10 with excised leaf blades (5 control. 5 Triton X-100 treated) and one without leaf blade (thermobarometer) were immersed in a constant temperature bath at 30°C. Volume changes shown by the thermobérometer were subtracted from the volume changes in the other manometric set- ups to correct for fluctuations in the atmospheric pressure or slight changes of temperature in the water bath. Measurement of 1‘C02 Fixation. Twelve randomly selected 6- cm-long leaf sections (six Triton X-100 treated and six control) were preincubated in 6 mM NaHCO; in 0.15 not Tricine buffer. pH 7.86. in the light (250 tiE m‘2 5"). After 15 min. the sections were quickly transferred to the same medium containing NaH”CO.i (0.5 pCi/ml) under the same light conditions. After 5. 10. and 15 min. two Triton X-lOO-treated and two control sections were removed from the incubation medium and frozen in liquid N2. Frozen sections were ground in liquid N; in a mortar with a pestle. The resulting powder was transferred to small test tubes. and 2 ml of l M HCl in 80% ethanol was added to the powder to liberate ”CO; from residual. unmetabolized NaH”CO;.. The test tubes were dried overnight at 70°C. and their contents were combusted in a Tri-Carb sample oxidizer. model B 306 (Packard Instrument Co.. Downers Grove. IL). Radioactivity was deter- mined in a Tri-Carb liquid scintillation spectrometer. model 3255. Submergence Tests. Plants were randomly divided into three groups. and half of the plants in each group was treated with Triton X-100 (treatments 3. 4. and 6. Fig. 8) while the other half was left as nontreated control (treatments I. 2. and 5). Plants of the first group. referred to as ‘partially submerged' (treatments 1 and 3) were lowered with plastic lines into a BOO-L Nalgene tank (Nalge. Rochester. NY) filled to the rim with deionized H;O so that only 15 cm of the foliage remained above the water. The tanks were located in the same environmental chamber where the plants had been grown. Plants of the second group. referred to as ‘completely submerged‘ (treatments 2 and 4) were lowered to the bottom of the tank and left completely submerged for the whole duration of the experiment. Plants of the third group (treatments 5 and 6). referred to as ‘not-submerged.’ remained under the same growing conditions as before. Plant elongation and protein and Chl contents were measured after 90 h ofexperimental treatments. Protein and Chl Determinations. Leaf discs. 7 mm in diameter. were cut with a cork borer from the midportions of the leafblades. In partially submerged plants. protein and Chl contents were determined only in the underwater midportions of those leaf blades which had tips above the water. The leaf discs from each treatment were randomized and floated. adaxial surface up. on water in a Petri dish. Seven randomly selected leaf discs were homogenized in a glass homogenizer with a motor-driven glass plunger in 3 ml of ISO mM Tris-HCl buffer. pH 7.9. containing 0.1% (v/v) Triton X-100. The homogenate was centrifuged at 120003 for 15 min using a Sorvall RC-ZB centrifuge and an SS- 56 AERATION 1N DEEP WATER RICE 20cm (I9ml) TREATMENT WITH TRITON X'IOO FLOW ABAXIAL SIDE ADAXIAL SIDE (ml-min") -— — 0.2410023 + — 0.20:0.017 ' 80% — + 0.05: 0008 20% + + 0.00 1 000 FIG. I. Mass flow of air along a submerged rice leaf blade. To eliminate air layers. either the adaxial. abaxial. or both sides of the blade were washed with a 0.05% (v/v) solution of Triton X—lOO. Each value is the mean of measurements using 20 randomly selected leaf blades :55. 34 rotor (DuPont Instruments-Sorvall. Wilmington. DE). Protein in the supernatant was determined according to Bradford (5) using the Bio-Rad protein assay mixture (Bio—Rad Laboratories). For C hl determinations. seven randomly selected leaf discs were ho- mogenized in a glass homogenizer with motor-driven glass plunger in It) ml 80’} acetone (v/v). The homogenate was centrifuged at 6.0002 for IO min. Total Chl was determined according to Ar- non (3). RESULTS Conduction of Gases through Air Layers along Leaves. Con- ductance of air through the air layers on both sides of the leaf was demonstrated using the experimental set-up shown in Figure I. A detached leaf blade of rice. with the tip cut off, was introduced through water into an inverted cylinder which had a small head space of air. The slight reduction of pressure in the head space created by the weight of the water column caused a mass llow of J”. 0.24 ml min". along the leaf from the outside into the head space. When both sides ofthe leaf had been washed with a Triton X-IUU solution. no detectable air flow occurred (Fig. I). Similar results (not shown) were obtained when a lS-mm wide ring of clear nail polish had been applied around the basal portion ofthe suhnlh’fged leal' blade to disrupt the continuity of the air layers. These results indicate that the mass flow of gases through the internal air spaces of the submerged leaf or through the water is negligible compared to the mass tlow through the external air layers. Treating either side of the leaf Wllh Triton X-IOO demon- strated that about 80% of the air flow was conducted through the air layer on the adaxial and 20% through the air layer on the abaxial surface of the blade (Fig. l). While the results shown in Figure l demonstrate the possibility of a mass flow of gases through the air layers along a pressure gradient. Figure 2 shows the diffusion of gases through the air layers along a concentration gradient. Ethane diffused from the right side ofthe U-tube through the air layers along the submerged part of the leaf to the sampling compartment on the left side of the tube. After 50 min. the ethane concentration in the air at the exit port ofthe sampling companment reached a constant average level of S3 [l.l L". Only traces of ethane appeared in the sampling compartment when the air layers were eliminated by treatment with Triton X- 100 or interrupted by applying a l-cm wide ring of paraffin oil around the leaf. Ethane has been chosen as gas for this experiment because its diffusion coefficient and solubility in water are close to those of O: and because its concentration can be easily determined by gas chromatography. The data from Figure 2 can be used to calculate the combined cross-sectional area A (cm!) of the air layers on both sides of a leaf blade provided that the lateral loss of ethane from the air layers is small. The steady state equation of one-dimensional diffusion according to Armstrong (I) is: _ DA (Co "’ Cl) L J (5) where .l is the rate ofdiffusion of ethane through the air layers (g s“), D the diffusion coefficient of ethane (0.128 cmJ 5". see Ref. 13). G, (g cm“) the concentration of the ethane in the right arm. Ci the concentration of ethane in the sampling compartment (g cm‘”). and L (15 cm) the length of the diffusion path which is equal to the length of the submerged portion of the leaf blade. J can be calculated from: J = C1!" (6) where C. is the steady state concentration of ethane in the sam- pling companment after about 50 min (83 id L"| or 0.” pg cm‘”) and F is the flow rate of air in the sampling companment (cm3 s“). The value ofA can be calculated after substitution of Equa- tion 6 for J in Equation 5. From the above equations, the cross- sectional area A of the air layers around an l.l-cm wide leaf was calculated to be 0.0070 cm". A theoretical curve for the diffusion of ethane into the sampling companment as a function oftime (Fig. 2) could be derived from the equation for one-dimensional diffusion modified from Ja- cobs (8), n’w’Dl ADC "" : 0(1+2 2 (—l)"e' L) F n-l where I is the time (s) from the start of the experiment. The combined cross-sectional area of the air layers on both sides of a l.l-cm wide leaf blade A used to solve Equation 7 was determined by applying Archimedes' principle (see below). Based on these measurements, A was found to be 0.0006 cm‘ (Table l), which was very close to the value of 0.0070 cm: obtained in the difl'usion experiments. The time needed to attain a constant concentration of ethane in the sampling compartment was longer than predicted on the basis of Equation 7 (Fig. 2). This discrepancy very likely arose because of initial losses of ethane to the water and the leaf. Such lateral losses are not taken into account in Equation 7. Measurement of the Volumes of Air Layers and Leaves. The volumes of air layers and leaves as well as leaf density and thickness were determined by applying Archimedes‘ principle (Table l). The ability to trap air along a submerged leaf was also present in cereals other than rice. 9.3. in oats, barley. and wheat Cl (7) 57 RASKIN AND KENDE Plant Physiol. Vol. 72, I983 90 .0 o ... 0 ("Calls E '5. V Sampling w po \, Leaf 2 <1 I y. . . u Distilled water (50ml) 1 - A A . Ln " ‘ ' : : n ’ . v .. U 20 4O 60 80 IOO |20 I40 |60 I& 200 220 540 TIME (min) Ft( 2. 2. Diffusion of ethane through the air layers around submerged rice leaves. The diffusion of ethane into the sampling companment was determined using l.l-cm wide leaf blades with intact air layers (0). following elimination of air layers with Triton X-IOO (I). or interruption of the continuity of air layers by a l-cm wide ring of paraffin oil (A). The diffusion of ethane through water without a leaf(><) was also determined. The computer-generated. theoretical curve for the diffusion of ethane into the sampling companment is given by the broken line. The concentration of ethane in the right compartment ofthe U tube was 45% was pertormed at 24°C and at a light intensity of l00 iiE m"2 s". t’v/v) and the flow rate of air in the sampling compartment was 2 ml min' '. The experiment Table I. Comparison of Leaf Parameters in Different Cereals All measurements were made by applying Archimedes' cross- -sectional (597cm X10 X l.lcm principle (see‘ Materials and Methods" ). The combined area of the air layers on both sides of a 1.1-cm wide rice leaf was 0.0066 cm‘ l cm" Parameter Rice Wheat' Barley" Oats' Leaf density (g cm”) 0.79 :I: 0.02” 0.83 :t 0.004 0.80 t 0.005 0.79 1 0.007 Average leaf thickness (mm) 0.14 1- 0.003‘ 0.2l .t 0.004 0.27 : 0.0l 0.28 1- 0.004 Volume of air layers over I 5.97 :t 0.3c LSS : 0.15 2.27 :t 0.22 l.l9 a: 0.1] cm‘ of the leaftcm" X 10’ Volume of air/volume leaf 0.44 :t 0.02c 0.09 3: 0.007 0.08 :I: 0.009 0.04 :5: 0.004 Volume of air layers over abaxial side (”2) 24.8‘ Volume of air layers over adaxial side ((7) 75.2‘1 ’n= 10:55. "21:17:55. =27tse. dn= l0. (Table l). However, the ratio of the volume of the air layers to the volume of the leaf in rice was five times larger than that in wheat, close to six times larger than that in barley. and II times larger than that in oats. It can be calculated that the leaves of a completely submerged 65- d-old rice plant of I45 cm height with a total le ll blade area of 34 dm trap about 20 cm of air in their air layer s. The rice leaf is about half as thick as the leaves of the other cereals (Table I). In thinner leaves, the diffusion of gases to the mesophyll cells is more rapid because of the shorter diffusion path. Although the density of rice leaves is not significantly different from that of wheat. barley. and cat leaves. the density of the submerged rice leaves is lower because of the larger air layers. The size of air layers of nonfloating rice varieties. such as M 9. is similar to that of Habiganj Aman II (data not shown). Movement of Oxygen in the Air Layers. ln darkness. air layers 58 AERATION IN DEEP WATER RICE 36- U N I —~ 'TR ITON __ N in N 03 O b m l I I I DEPTH OF COLOR FRONT (cm) i?) l +TRITON n-J—H 0 5 io IS 20 25 30 TIME (min) FIG. 3. O_. movement through the air layers in darkness determined by the oxidation of reduced methylene blue (l5 mg L"). 02 movement from the atmosphere through the air layers along the adaxial surface of a partially submerged rice leaf was followed by measuring the position of the blue color front relative to the surface of the solution at 28°C. Every 5 min. a green safe light was turned on for about 5 s to take each measurement. (0), Leaves with intact air layers; (I), Triton X-lOO-treated leaves without air layers. Each point represents the mean of measurements usmg five leaf blades; the vertical bars denote SE. conducted atmospheric 0; to the lower portions of the submerged leaf blades (Fig. 3). The downward movement of 02 through the air layers was much faster than liquid phase diffusion of 0: down from the water surface. The lateral loss of 02 from the air layers due to the respiratory demand of the leaf and solubility of O; in water did not seem to prevent longitudinal movement of 02 along the air layers. Mass Flow of Gases through Air Layers: the ‘Snorkel' Function. Figures 4 to 6 demonstrate that there is directional mass flow of gases in and out of the air layers. In darkness. flow of air into the air layers caused the volume of the head space to decrease continuously. The rate ofthis decrease diminished monotonically with time (Fig. 4). Even after I2 h of dark incubation. the untreated blades withdrew air from the head space at the rate of .2 _Ill min". Elimination of air layers with Triton X-IOO greatly diminished the initial rate of volume reduction and brought it to zero within 3 h. Mass flow of gases from the head space into the air layers was also inhibited when the air in the head space was exchanged with N; (Fig. 5). The withdrawal of gas into the air layers totally ceased within 2 ll after the introduction of N2. while it continued for the duration ofthe experiment in the air controls. When the air in the head space was exchanged with 0,). the high rate of O; uptake from the head space was maintained for the duration of the experiment (Fig. 5). Figure 6 shows the changes in the volume of the head space during the following schedule of light-dark treatments: 5 h dark- ness _. 75 min light (200 iiE m" s") —’ 5 h darkness. In the case C .. +Trilon S E Water Bubble Manometric Tube Air Exchange Needle: . Head Space -éfl (lml) "L ~31! . . V;._Dl5lllled if. Water (70ml) {Ll—J“ Leaf ”el' .fl RATE OF VOLUME CHANGE IN HEAD SPACE (Ml-min") n'i gl 0 2 4 6 8 IO l2 TIME (h) FIG. 4. Mass llow ofair into the air layers in darkness measured as the rate of volume change in the head space of the manometric set-up containing an excised leaf blade with intact air layers (0). and following elimination of air layers by Triton X-IOO treatment (I). The experiment was performed at 30° C. After each measurement of volume change. the head space was flushed with 10 ml of air through the air exchange needles which were then tightly closed again. Each point represents the mean of measurements using five leaf blades and is adjusted for the fluctuation in the atmospheric pressure recorded by the thermobarometer. of untreated leaf blades. illumination led to immediate reversal in the direction of gas movement. Cases were expelled from the air layers into the head space at an average rate of 6.7 id min". When the light was turned off. the direction of gas movement was immediately reversed again. Air from the head space was drawn back into the air layers. causing a continuous decrease in the head space volume. The similar rates of volume changes at the begin- ning of the first and second dark period indicated that light fully restored the high rate of gas withdrawal from the head space. During the second dark period. the high initial rate of mass flow decreased again with kinetics similar to those observed during the first dark period. Partially or completely submerged. illuminated leaf blades withour air layers slowly formed small bubbles of photosynthetic 0; along the leaf surface. The formation of these bubbles increased the volume inside the manometric set-up (Fig. 6). CO: Uptake through Air Layers: the ‘Gill‘ Fttnction. Submerged plants use CO; dissolved in water for photosynthesis. ln herba— ceous plants. CO; is primarily taken up through the stomata. Therefore. the surface area through which CO; is absorbed by submerged leaves without air layers is largely limited to the combined area of the stomatal pores. Air layers greatly increase ... . n .n m In“ 1 r I c .h C .. ..w r .\. n .I. I r H V ' ,H....._1,p.-m:.:,.- .. p... .- T .. .. K . ‘ vs , r C 4U n. a. z E i ‘ ~ K“ ... a rl VI 5.. ix ... .n; .. u 3. . . n ,. p. i. a n. ‘w on nix .~‘ I I ; a. w. . T .-. V .. 3 .; ... n . u .v. x . 4 . RATE OF VOLUME CHANGE IN HEAD SPACE (,ul- min") l 6 8 TlMElh) FlG. 5. Mass flow of gases into the air layers in darkness measured as the rate of volume change in the head space containing air (9). N; (A). or O.» in). Nitrogen or O) was bubbled through the water of the manometric set-ups for at least 2 h. Thereafter. the leaf blades were introduced into the cylinders (time 0). the manometers were stoppered. and the head spaces of the manometric set-ups were flushed with N2 or 02 through the air exchange needles for 20 min at a flow rate of 30 ml min". After flushing, the air exchange needles were tightly stoppered again. The thermobaro- meters were prepared like the manometric set—ups. except that no leaf was inserted into them. In the case of the 02 atmosphere. the head space was flushed with It) ml 0,» after every fourth measurement of volume change. The experiment was performed at 30°C. Each point is the mean of measurements using five leaf blades and is adjusted for the fluctuation in the atmospheric pressure recorded by the thermobarometer. the surface area for CO.» absorption from the surrounding water. thus increasing the amount of C02 available for photosynthesis. The results shown in Figure 7 demonstrate this gill function of air layers. Leaf sections submerged in a solution of Nal-l”C03 incor- porated HC into photosynthetic products at a rate that was 9- to lO-fold higher with air layers than without. Physiological Significance of Air Layers. Elimination of air layers led to loss of Chi and protein in the underwater parts of partially submerged leaf blades (compare treatments 1 and 3. Fig. 8. A and B) and severely reduced the elongation response to flooding (compare treatments l and 3, Fig. 8. C and D). Cont- pletely submerged plants also lost protein and Chi. and no elon- gation response to flooding was observed. The most severe mani- festation of these symptoms was seen in completely submerged plants without air layers (compare treatments 2 and 4, Fig. 8, A- D). Therefore. even under conditions of total submergence. air layers conferred some adaptive advantage to plants. Treatments 5 and 6 (Fig. 8. A-D) showed that Triton X-lOO by itself did not cause loss of pr0tein and Chl and did not inhibit growth. This experiment also confirmed that partial submergence stimulated growth and internode elongation of plants with intact air layers (compare treatments l and 5. Fig. 8. C and D). 59 RASKIN AND KENDE Plant Physiol. Vol. 72. 1983 DARK LlG.HT DARK 7r 0 fl ‘ol’ 6.— '5 E 5... 3 Lu L) 4’- i <1 0. m ‘l o 3" < LtJ I z 2* m (.9 Z l" < I U +TRITON m - . 2 3 _J O > u. 0 UJ ’— 5 I Cl 6'1“ i I I + r . i 1 2 3 4 5- s Time(h)' Fig. 3 A_ The effect of the C02 concentration in the solution around the plant on the rate and direction of the gas flow. A rice plant was placed in a 71-cm-tall plastic tank filled with 14 l of 90 mM phosphate buffer (pH 6.9l). Mass flow of gases was monitored as described for Fig. l except that 4 leaves were used for the measurements, and the experiment was performed in light of lOO uE m"25'1 intensity at 24 0C. After the initial rate of gas intake was determined for 30 min, C02 from a high-pressure gas cylinder was bubbled through the solution using an air stone placed at the bottom of the submergence tank (first arrow). After 5 h 10 min, the leaves were removed from the leaf chamber (second arrow), and changes in the head space volume were monitored for another 50 min. The recorder tracing on the left side of the graph represents loss of gas from the head space of the leaf chamber, i.e. flow of gas into the plant. The tracing on the right side represents gain of head space volume, i.e. release of gas into the head space. _B The pH change in the submergence tank was monitored with a pH meter (Model PHM 26, Radiometer, Copenhagen, Denmark) equipped with a combination electrode anAn1 CV 0791C\ anH nnnnnn+nA +n a nh3n+ nnnAnAnw 76 fig;_g_ Reversibility of the effect of C02 on mass flow of gases. Two rice plants were placed in two 52-cm-tall plastic tanks filled either with l0 l of 90 mM phosphate buffer (pH 7), or 0.23 M KOH solution saturated with C02 (pH 7). Saturation with C02 was achieved by bubbling C02 through the KOH solution until pH 7 was reached. The five largest culms were cut with a razor blade 2 cm above the water surface. The other culms and leaves were cut below the water surface. The above-water end of a single culm was introduced into a 7-cm-long glass chamber of 2.5 ml volume, which was then partially immersed in water so that the head Space volume was l.5 ml. A chamber was connected with thin Tygon tubing to one end of a horizontally placed glass tube, i.d. 2 mm, into which a bubble of 40% ethanol was introduced from the other end. The glass tube was calibrated so that the rate of the liquid bubble movement, recorded every l0 min, represented the rate of volume change in the head space caused by the flow of gases into and out of the culm. All measurements were performed in light (lOO uE m'zs‘l) at 240 C. Since mass flow of gases in only one out of five culms was measured, the total flow for each plant was about 5 times larger than shown here. After 95 min, the plants were interchanged between the two solutions and equilbrated for 30 min before measurements of gas flow were resumed. ge Rate of Volume Chan (All min“) 77 --5 N (O h 0 O O O I I i I l Plant 1 Apia-'11:?“ +002 +co2 I O) C i ii) I. O O O i I l 0 O m w VP-Lantfi1/ ' -co2 90 120 m . o o: 0 Time (min) 150 180 210 f—E \ (\ -c+‘ - (Cl Q CHI Gas Intake (ml) 78 20 15 1O o n: A c» G>1O 120 150 1 O 210 240 2 0 3 0 Time (min) 60 90 Fig. 5 The role of air layers in the conductance of gases. The experimental procedure was the same as outlined for Fig. l except that only one leaf was kept above the water for the gas intake measurements. Prior to the beginning of the experiment, the top 6 cm of the leaf tip were cut off, and the cut surface was sealed with transparent nail polish. 0f the remaining leaf, 6 cm protruded from the water into the leaf chamber. The shaded areas represent the regions of the leaf that were covered with nail polish. Chapter 7 A Method for Measuring Leaf Volume, Density, Thickness and Internal Gas Volume HurtSt'ir'm't’ 18( 5):698—699. l 983 . 80 A Method for Measuring Leaf Volume, Density, Thickness, and Internal Gas Volume Ilya Raskin MS U -DOE Plant Research Laboratory. Michigan State University. East Lansing. MI 48824 Additional index words. A bstract. Phaseolus vulgaris. Oryza sativa A fast. simple, and accurate method is presented for the determination of volume. thickness. and density of leaves and the volume of internal air spaces of leaves and other plant organs. The method is based on the application of Archimedes’ principle and involves the determination of the buoyant force acting on plant organs submerged in water. There is a noticeable lack of reliable meth- ods for measuring volume. thickness. and density of leaves and size of the internal air spaces of leaves despite the importance of these parameters in understanding leaf mor- phogenesis and photosynthetic productivity (l. 10). Microscopes (4). micrometers (l I). and linear variable transducers (8) have been used to determine leaf thickness. Leaf vol- ume has been measured volumetrically (3). and superficial leaf density (mg cm‘3) has been measured with a B-gaugc (2). The vol- ume of intercellular air spaces has been de- tcrmincd by direct microscopic measurements of sectioned tissue (7) or by weighing the tissue in air before and after infiltration with water or isoosmotic solutions (6). This paper describes a fast and accurate method for determining many physical pa- rameters of the leaf by applying Archimedes' principle. the derivation of which can be found in any standard physics textbook. The method requires simple equipment which should be available to every experimenter: it is easily performed and can be used on leaves of any size and shape. This method can be applied equally well to the determination of physical parameters of other plant organs and tissues: its accuracy is limited only by the precision of the analytical balance used in weighing the plant material. Archimcdcs’ principle states that the buoy- ant force acting upon a body immersed in a fluid is equal to the weight of the fluid dis- placed by the body: 8 = W H] where B is the buoyant force. p is the density of the Ilmd (weighttunit volumct. and V is RCL’L‘HL‘U for publication March 25. I983. Sup- ported by the LS. Department of Energy under contract no. l)E-:\C03~76l§RO- 1338‘ and by grant no. l'(‘ S' l “0764 from the National Science Foun- dation to Hans Kcndc. in whose laboratory this work “as performed. The author thanks H. Kcndc and] -l’, Slctraux for hclpt'ul discmsions. The cost of PUI‘IMIIIRL' this paper was defrayed in part by the payment of page charges. Lindcr postal regu- lations. llll\ paper thcrctorc must be hereby markcd tltllt'fllu'fllt‘lll solely to indicate lhh fact the volume of the immersed body. Thus. a close approximation of B acting on the body completely submerged in water can be ob- tained from the difference in the weight of the body in air (Wm) and its weight in water (W water): B = \V'Lllf - WKJICT [2] Combining equations l and 2: watt _ \Vwater = pv [3] Determination of leaf volume. It is clear from equation 3. that only 2 values are re- quircd for the determination of leaf volume-— namely. the weight of the leaf in air (WM) and its weight in water (“7“,“). A simple assembly can be used to measure the weight of the leaf in water (Fig. l). A holder. con- sisting ofa Hoffman clamp of sufficient weight to keep the plant material submerged in water. is clipped to 2 hypodermic needles, which pierce the leaf. Larger leaves may be cut into several sections if necessary. The holder with the whole leaf or leaf sections is then sus- pended from the pan hook of an analytical balance and submerged in distilled water. The weight of the leaf in water can be calculated by subtracting the weight of the submerged holder with the leaf (Wm, ., Md“) from the weight of the submerged holder (thw). The weight of the leaf in water is usually negative became of the positive buoyancy of leaves. Thus. the volume of the leaf (VW) from equation 3 is: w _ (\Vboldcr _ V _ air H lcal ‘ holilcr) lcaf " H () P 2 . [4| where pH30 is the density of water. If the weight is measured in milligrams. the volume of the leaf. in cubic millimeters. is numerically equal to Wm — (Wham-a Wm, , hum“) because pH3() at room temper- attire can be approximated as I mg mm~ ‘. An cxpcrimcntal error may be introduced in the determination of V“ by the presence of small air bubbles trapped on the surface of the submerged leaf. Air bubbles increase the buoyancy of the sample and lead to an mer- cstimation of leaf volume. This problem can be overcome by wetting the leaf with a sur- factant solution: c.g.. a 0.05% (v/v) Solution of Triton X-l00. prior to determining WW - Md“. Surfactant trcatment effectively eliminated all air bubbles from the leaf sur- face. Column 3 in Table I gives the average volumes of l2 fully developed trifoliate leaves of Plzusmlus vulgaris L. cv. Sacramento and fully expanded leaf blades of Oryza sativa L. . cv. Habiganj Aman ll collected randomly from 12 eight-week-old plants grown in the greenhouse. Columns l and 2 in Table 1 contain the average fresh weights of bean and rice leaves (WW) and their weight in water (wholder ' wlcaf + holder)~ TCSPCC‘IVCIY- Determination of leaf density. Leaf density (pr) can be calculated from the equation: pleaf = wlcaf/Vleaf l5] Column 4 in Table l contains the average densities : SD of l2 bean and rice leaves calculated from equation 5. Determination of the volume of the internal air spaces of leaves. Cases in the internal spaces in the leaf can be replaced by vacuum infiltration with a 0.05% Triton X-100 so- lution. The plant material fixed to the holder is placed in the bottom of the desiccator or vacuum flask partially filled with surfactant solution so that the tissue is submerged to- tally. Agitation of the desiccator and sec- tioning of the leaves aSSist the infiltration process. The vacuum. about 500 mm Hg. should be applied and released at least 4 times. with every evacuation lasting about 40 sec. The infiltrated tissue attached to the holder is then weighed in water as described above. The buoyant force exerted on the intercellular air spaces (B,,,,pm,) is: B air spaces = [6] where Wm m, , mm“ is the weight of the infiltrated leaf with the holder in water and WI“, , Wk, is the weight of the same leaf and a holder in water before infiltration. For all practical purposes. the density of the 0.05% Triton X-l00 solution inside the lcaf cart be considered equal to the density of water. Therefore. the internal gas volume of the leaf winf. leaf + holder — wlcaf v holder 0 [311130 [:1 l O _Anolyticol O ____l 0 Balance if Pen Hook Distilled Woter Clamp . Leof Sections Fig. l. Analytical balance adapted for weighing lcaycs underwater. tntttrlitA 'iniptittxolv ‘muntng putiilmition to; (throng tiiiniinttiv at" to uotiienilqttd V ' t )f\ Ittg‘lnti” tiioit pniiitttlnu 'l“,\ I‘Htil J-“l“l‘() '15le 81 Table 1. Average volume. weight density. volume of the internal air spaces. and density of the nongascous leaf content of leaves of Phaseoulus vu/earis’ and Uri-:0 sutiva’ collected from 8-week-old plants. 1 2 3 4 5 6 7 8 \vau’ vair \pace) Vnungas pmgn Leaf WW," V...“ pk.“- V01 V01 Density of fresh Underwater Leaf Leaf internal (7e leaf vol nongaseous nongaseous wt wt vol density air spaces occupied by leaf content leaf content Plant (mg) (mg) (mm‘) (mg mm") (mm‘) air spaces (mm’) (mg mm") Bean 2501 r 605 -714 z 234 3215 :2 827 0.78 z 0.02 754 z 234 23.5 r 1.9 2461 I 602 1.02 z 0.01 Rice 850 t 138 -—634 z 104 1483 r 239 0.57 1- 0.01 585 1' 96 39.5 t 1.0 898 z 145 0.95 t 0.01 ‘Each leaflet of the trifoliate was cut into 2 pieces. and the resulting 6 sections were used for all measurements. Each number is the average obtained from 12 leaves : SD. >Each leaf blade was cut transversely into 6 or 7 pieces. which were used for all measurements. Each number is the average obtained from 12 leaves : SD. (Vair spaces) is given by _ B _iir \EJv L“ vair pH30 : \tht' lcal . holder — W’lcaf ‘ holder [7] szO No differences in electrical conductivity of Triton X-lOt) solution before and after infil- tration of 4 rice leaves were detected. indi- cating that infiltration does not cause any detectable leakage of ionic solutes from the cells. The possible swelling of tissue infiltrated with hypotonic Triton X- 100 solution will not affect the value of the leaf thickness calcu- lated from Archimedes' principle. Swelling. however. would pose a serious problem if the infiltrated tissue is blotted and weighed in air according to other methods of deter- mination of the intercellular air volume (6). Columns 5 and 6 in Table 1 contain the average volume of the internal air spaces of 12 bean and rice leaves and the average per- centage of the total leaf volume occupied by the air spaces : 50. Determination of the density of the non- gaseous leaf content. The volume of the liq- uid and solid components ofthe leaf(megas), which comprise the nongascous leaf content. can be calculated by subtracting the volume of the internal air spaces (Var mm) from the volume of the leaf (me). The density of the nongaseous leaf content. pump“... is: = wleat/v [8] Column 8 in Table 1 contains the average density of the nongaseous leaf content of 12 priongas nongas Table 2. leaves collected from 8-weekoold plants. bean and rice leaves 1' so. Determination of average leaf thickness. The average leaf thickness (TM) can be de- termined from the volume of a leaf section of known area (AW): Tleaf = vleaf/Alcaf [9] The surface area of cut-out or punched-out leaf sections (using. for example. a large cork borer) with square. rectangular. or circular shapes can be determined easily from the linear dimensions. The most accurate values of the leaf thickness can be obtained from large sections. the average density of which equals the density of the whole leaf. For ex- ample. using small sections containing dis- proportionately large or small amounts of vascular tissue can cause errors in determi- nation of average leaf thickness. Sectioning can be avoided by determining the area of the whole leaf using a variety of methods (5. 9). Column 6 in Table 2 contains the average thickness 2 so of8 randomly selected. fully developed bean trifoliates and mid-laminar regions of rice leaves collected from 8-week- old plants grown in the greenhouse. The thickness of 20 randomly selected bean leaflets from the same bean plants was mea- sured with a micrometer for comparison with the results of Table 2. Care has been taken not to position the micrometer on ridges on the leaf surface. The average thickness ob- tained with the micrometer was 0.29 mm x 0.06 SD. The average thickness calculated from Table 2 was 0.26 mm : 0.01. The thickness of the mid-laminar region of 20 randomly selected rice leaves measured with the micrometer was 0.25 mm I 0.03. coni- Average thickness of Phasen/us vulgaris" trifoliates and mid-laminar region of Oryza sativa" l 2 3 4 5 6 Alcat w.‘lf winter VIC-ll Tlcaf Combined Fresh Underwater Combined Avg thick- area of wt of wt of vol of ness of No. sections sections sections sections leaf : 50 Plant sections 1mm“) (mg) (mg) lmm") (mm) Bean 6 3435 727 1- 38 ~180 : I7 907 z 46 0.26 : 0.01 Rice 2or3 2825 t 546 315 z 51 - 122 t 25 437 t 72 0.16 z 0.01 "Two leaf discs. 27 mm iii diameter. were punched out from each leaflet of the trifoliate with a cork borer. The average thickness of the resulting 6 discs was measured as described in the text. Each number is the average obtained from 8 leaves. '.-\ inid-laniinar region of a rice leaf was cut transversely itito 2 rectangular pieces (about 7 cm long). The area of the sections was calculated as a product of length and width. Each number is the average obtained from 8 leaves. pared to 0.16 mm 1 0.01 as calculated from equation 9 (Table 2). Larger values of leaf thickness are obtained with the micrometer because the micrometer measures the maxi- mum distance between the leaf surfaces which are not even (1 l ). 1n highly corrugated leaves (e.g.. those of rice). the average leaf thick- ness determined with the buoyancy method and maximum leaf thickness determined with the micrometer are significantly different. The values obtained by the buoyancy method seem to represent the best approximation for av- erage thickness of the leaf. Literature Cited 1. Beakbane. AB. 1967. A relationship be tween leaf structure and growth potential in apple. Ann. Appl. Biol. 60:67—76. Jones. HQ. 1973. Estimation of plant water status with the beta-gauge. Agr. Mcteorol. 11:540—546. . 3. Huxley. PA. 1971. Leaf volume: a simple method for measurement and some notes on its use in studies of leaf growth. J. Appl. Ecol. 8:147—153. 4. Maksymowych. R. 1973. Thickness of the lamina. p. 25—26. In: M. Abercrombie. DR. Newth. and 1.0. Torrey (eds). Analysis of leaf development. Cambridge Univ. Press. Cambridge. UK. 5. Obraztsov. A.S. and V.M. Kovalev. l976. Volumetric method for determining area of the leaf surface in plants and plantings. So- viet Plant Physiol. 23:911-914. 6. Smith. .l.A.C. and S. Heuer. 1981. Deter- mination of volume of intercellular spaces in leaves and some values for CAM plants. Ann. Bot. 48:915-917. 7. Turrel. F.M. 1936. The area of the internal exposed surface of dicotyledon leaves. Amer. J. Bot. 23:255—264. 8. Tyree. M.T. and 5.1. Cameron. 1977. A new technique for measuring oscillatory and diur- nal changes in leaf thickness. Can. J. For. Res. 7:540—546. 9. Watson. DJ. 1952. The physiological basis of variation in yield. Adv. Agroii. 4:101— 145. 10. Wilson. D. and .l.P. Cooper. 1967. Assim- ilation of Loliimi in relation to leaf meso- phyll. Nature 214:989—992. 11. Woodward. El. 1979. The differential tem- perature responses of the growth of certain plant species from different altitudes: 11. Analysis of the control and morphology of leaf extension and specific leaf area of l’lllt'llnl bertolmiii DC. and I’. ulpintmi 1.. New Phy- tol. 82:397—405. Es) 82 CONCLUSIONS The growth responses of deep-water and non-deep-water rice varieties to flooding can be reproduced in excised stem sections. Submergence reduces oxygen levels in the internodal lacunae. Lower oxygen concentrations, in turn, stimu- late ethylene synthesis in the internodal tissue. Ethylene accumulation in the underwater parts of rice causes rapid internodal elongation and inhibition of leaf growth. Ethylene action is probably mediated by an increase in the activity of endogenous gibberellins. drAmylase activity in submerged and ethylene-treated in- ternodes increases greatly. Simultaneously, submergence increases the translocation of photosynthetic assimi- lates from the leaves to the internodal region. Submerged parts of rice leaves are surrounded by continuous air layers which provide the major aeration path in the rice plant. Gases move in the rice plant not only by diffusion but, primarily, by mass flow down a pressure gradient created by the solubilization of respiratory C02 in water.