The Relationship between Growth and Indole-3-Acetic Acid Content of Roots of Pisum sativum L. Author(s): William L. Pengelly and John G. Torrey Reviewed work(s): Source: Botanical Gazette, Vol. 143, No. 2 (Jun., 1982), pp. 195-200 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/2474706 . Accessed: 03/04/2012 15:52 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. The University of Chicago Press is collaborating with JSTOR to digitize, preserve and extend access to Botanical Gazette. http://www.jstor.org BOT. GAZ. 143(2):195-200. 1982. ? 1982 by The Universityof Chicago. All rightsreserved. 0006-8071/82/4302-0004$02.00 THE RELATIONSHIP BETWEEN GROWTH AND INDOLE-3-ACETIC ACID CONTENT OF ROOTS OF PISUM SATIVUM L. WILLIAM L. PENGELLY1 AND JOHN G. TORREY Cabot Foundation,Harvard University,Petersham, Massachusetts 01366 The indole-3-aceticacid (IAA) contentof roots and shoots of light-grownpea seedlings(Pisizmi sali,zim L. 'Little Marvel') growingat differentrates was studied by radioimmunoassayduring the firstweek of germination.Different growth rates were obtained by daily irrigationwith either deionized water or a dilute Hoagland's mineralnutrient solution. Mineral nutrient-grownroots grew morerapidly, had more lateral roots,and initiatedlateral roots at a greaterdistance fromthe apex than did water-grownroots. Growth kineticswith both treatmentswere biphasic. There was an initial phase of rapid cell expansion lasting about 3 days duringwhich growth was insensitiveto externalmineral nutrient supply. This was followedby a slowergrowth phase consistingof a balance betweencell divisionand cell expansion.Withholding nutri- ents resultedin the progressiveinhibition of cell division duringthis second phase. At no time did the amount of IAA per gram freshweight or the numberof cells per gram freshweight differ significantly in roots treatedwith water or mineralnutrients, whereas the total amount of IAA per organ and the total numberor cells per organ were greaterin roots providedwith nutrients.The amount of IAA per gram freshweight changed dramaticallyin roots duringthe firstweek of germination,but this was correlated withchanges in the relativecontributions of cell divisionand cell expansionto freshweight growth rather than to growthrate. By contrast,the amount of IAA per gram freshweight in 3-mmroot tips showed a directrelationship with growthrate. The relationshipbetween IAA contentand growthin whole shoots was qualitativelyand quantitativelysimilar to that foundin whole roots. Introduction specific radioimmunoassay (RIA) (PENGELLY and The role ofindole-3-acetic acid (IAA) in the growth MEINS 1977). To relate IAA content to root growth, and development of roots has been the subject of w e studied the distribution of IAA in the root, considerable controversy.Early bioassays of tissue- changes in IAA content during growthand develop- extracted auxin showed that the amount and distri- ment, and IAA levels in plant material of the same bution of auxin in the root and shoot were similar age growing at differentrates, the latter experi- (THIMANN 1934). More recently, IAA has been mental condition being achieved by varying the determinedin roots by combined gas chromatogra- external mineral nutrient supply. For comparison, phy-mass spectrometry(GC-MS) (BRIDGES, HILL- similar studies with pea shoots were also carried out. MAN, and WILKINS 1973; ELLIOT and GREENWOOD 1974; RIVIER and PILET 1974). A comparison of Material and methods results using GC-MS and bioassay (BRIDGES et al. PLANTS.-Pea seeds (Pisum satizvumL. 'Little 1973; GREENWOODet al. 1973) indicated that, in Marvel,' Asgrow Seed Co., New Haven, Conn.) Zea, amounts of IAA in roots were similar to those were rinsed brieflyin tap water and then surface- in coleoptiles. sterilized for 10 min with a 10%' (wt/vol) filtered THIMANN (1937, 1977) emphasized the greater calcium hypochloritesolution containingeither a few sensitivityof roots than shoots to externallyapplied drops of Tween 80 (Polyoxyethylene [20] sorbitan auxin, the formerresponding to 100-1,000 times monooleate, Fisher Scientific,Springfield, N.J.) or a lower concentrations of IAA applied in solution. small amount of commercial detergent as a surfac- These observations led to the view that auxin rela- tant. The seeds were rinsed four times, soaked for tions are fundamentally differentin the root and 8 h in distilled water, sown in fine-particlewashed shoot, namely, that shoots normally contain sub- river sand (special grade #OON, Kesseli and Morse optimal amounts of IAA and hence are limited in Co., Worcester, Mass.) that had been previously their growth by auxin while roots contain supra- washed with deionized water, and watered thor- optimal amounts of IAA and are actively inhibited oughly wvithdeionized water. The seeds were grown by auxin. in a chamber (Sherer Model CEL 511-38) illumi- In view of the continuinguncertainty surrounding nated with warm-white fluorescent lamps supple- IAA and root growth,we examined in detail the IAA mented with incandescent lamps with an average of content of Pisum sativumroots using a sensitive and ca. 350 ,E m-2 s-. The chamber was operated on a 16-h light-cyclewith temperaturesof 25 C day and I Present address and address for correspondenceand re- prints:W. L. PENGELLY, Departmentof Chemistryand Bio- 19 C dark. On the beginning of day 2 (24-27 h of chemical Sciences, Oregon Graduate Center, 19600 N. W. germination) and daily thereafter,the plants were Walker Road, Beaverton,Oregon 97006. watered with eithera 0.25-strengthHoagland's solu- Manuscript receivedA ugutst1981; revisedmanutscript received tion (HOAGLAND and ARNON 1950) or deionized November1981. water. 195 196 BOTANICAL GAZETTE [JuNE TISSUE EXTRACTION.-The pea seedlings were (Amersham, ArlingtonHeights, Ill.) and 5.0 ml of harvested and rinsed in distilled water in the dark- toluene containing, per 1 liter, 6 g 2,5-diphenyl- ened laboratory.The cotyledons were removed with oxazole (PPO) and 2.5 mg 1, 4-bis-2-(5-phenyloxa- a razor blade at the base of their petioles, and the zolyl) benzene (POPOP). remainderof the seedling was bisected into root and Radioactive determinationswere made using the shoot by a single transversecut throughthe cotyle- liquid scintillationmethod in the Isotope Facilities donary node. Root or shoot pieces were blotted dry of the Biological Laboratories, Harvard University, with paper towels, weighed, and immediately im- Cambridge, Massachusetts, and samples were cor- mersed in aqueous methanol (80% vol/vol, ana- rected for quenching by the channels ratio method. lytical reagent grade) prechilled to - 15 C and held The amount of IAA in the extract was determined on ice. by comparing the fraction of [3H]IAA bound with For studies of the IAA content in the root apex, the external standard curve. Assays were validated 12-mm root tips of intact seedlings were sliced into either by comparing the values obtained using dif- 3-mm sections with a razor blade cutter, weighed ferent volumes of the plant extract or by adding rapidly in groups of five, and immersed in the cold internal standards of unlabeled IAA to assays of methanol. Typically, 0.07-2.0 g freshweight of tissue plant extracts (PENGELLY and MEINS 1977). were used for each IAA measurement. Plant tissues CELL COUNTS.-The total cell number per root in 25 volumes of cold methanol were ground for 15 was determined by counting acid-macerated tissue min on ice with a mortar and pestle together with in the microscope. Roots were soaked in a solution a small amount of w ashed and ignited sand of 5%,0(wt/vol) chromiumtrioxide and 5%O(wt/vol) and ca. 105 dpm (6 X 106 Bq) of freshly puri- hydrochloric acid (FoSKET and TORREY 1969) for fied [5-3H]IAA (27 Ci mmol-1, Schwarz-Mann, 48 h. The tissue-acid mixture was then passed Orangeburg, N.Y.) added for recovery estimates through a no. 18 (0.84 mm i.d.) hypodermic needle (PENGELLY and MEINS 1977). During grinding, a using a 10-mlglass syringeand diluted appropriately second 25 volumes of methanol were added. The with water to give about 50-100 cells per micro- homogenate wvasvacuum filteredin a Buichnerfun- scopic field. Each cell number determinationrepre- nel. The residue was washed twice with 25 volumes sents two or three replicate experiments and the of methanol, scraped fromthe filterpaper, and ho- counting of at least 4,000 cells. mogenized for a second 15 min, firstwith a small amount of methanol to insure complete homogeni- Results zation and then with 50 volumes of methanol. The GROWTH AND THE EFFECT OF MINERAL NUTRI- second homogenate was filteredand washed as the TION.-In preparation for measurements of IAA, first; filtrates and washes were pooled; and the we firststudied the growth of pea seedlings during methanol was removed by rotary evaporation at the firstweek of germination.To compare IAA con- 37 C. The aqueous residue (10-20 ml) was broughtto tent with growth, we wanted to examine plant pH 8.5 with the addition of 10 ml of 0.5 M K2HPO4 tissues of the same age growing at differentrates. and partially purified by repeated diethyl ether- We found that limitingmineral nutrients to seedlings bufferpartitioning (PENGELLY and MEINS 1977) to grown in sand had a marked effecton growth,and yield a final 1.0-ml extract in phosphate-buffered seedlings provided with only deionized water grew saline (PBS; 0.1 M K2HPO4, 0.14 M\1NaCl, pH 8.0). considerably more slowly than those provided with Recovery of radiolabeled tracer averaged ca. 60%'. mineral nutrients(table 1). Both the root and shoot RIA.-Plant extracts in PBS were analyzed for were affected in this way, but the effecton root IAA content by RIA, using a procedure modified growth was most striking.