INTERACTION OF GIBBERELLIC ACID AND INDOLE- ACETIC ACID IN THE DIFFERENTIATION OF WOUND VESSEL MEMBERS

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1 INTERACTION OF GIBBERELLIC ACID AND INDOLE- ACETIC ACID IN THE DIFFERENTIATION OF WOUND VESSEL MEMBERS BY L. W. ROBERTS AND D. E. FOSKET Department of Biological Sciences, University of Idaho, Moscow, Idaho, and Biology Department, Brookhaven National Laboratory, Upton, New York, U.S.A. {Received 15 January 1965) SUMMARY Sucrose-agar media supplemented with indole-acetic acid (0.05 ppm) and gibberellic acid (0.05 or 0.5 ppm) increased the formation of wound vessel members in transverse tissue slices excised from the second internode of Coletis blumeni (Bentb.) stems. Tbe treated tissue slices showed the initiation of cambial activity, whorls of wound vessel members in the cortex, and adventitious root formation after prolonged incubation. Also, incompletely differentiated secondary xylem cells and wound vessel members were observed which exhibited relatively thin secondary wall striations, and stained weakly with phloroglucinol-hcl and Safranin O. INTRODUCTION Several lines of investigation have indicated an interaction between GA (gibberellic acid) and IAA (indole-acetic acid) in xylem differentiation. Bradley and Crane (1957) showed that GA treatment stimulated cambial activity in stems of apricot spur shoots and resulted in greatly increased numbers of secondary xylem elements. Wareing (19^8) postulated an interaction between GA and IAA in xylem formation resulting from cambial activity. Treatment of Lycospersicon escitlentuni L. with GA increased the length of annular-spiral elements and pitted scalariform cells, and increased the percentage of pitted xylem cells (Davis and Holmes, 1962). Soybeans treated with GA produced relatively thin-walled vessel elements with greater numbers of xylem parenchyma cells (Bostrack and Struckmeyer, 1964). Odhnoff (1963) noted that xylem differentiation was initiated closer to the apex in GA-treated bean roots than in controls. Gautheret (1961) indicated the xylogenic effect of GA in tissue cultures of Jerusalem artichoke, and Netien (1957) reported the morphogenic nature of GA in cultivated xylem fragments of Jerusalem artichoke. Recently Digby and Wareing (1964) have shown that the ratio of exogenous GA to IAA is important in regulating cambial activity and xylem differentiation. Previous research has demonstrated the feasibility of studying the differentiation of wound vessel members in isolated transverse slices of Coleus blumeni (Benth.) stem internode tissues incubated on sucrose-agar media supplemented with various growth factors (Fosket and Roberts, 1964). The present study was undertaken in order to determine whether the effect of GA was primarily on cambial activation or on xylem differentiation, and to determine whether or not GA acts through an auxin-mediated mechanism.

2 6 L. W. ROBERTS AND D. E. FOSKET M.^TERIALS AND METHODS In order to remove endogenous auxin sources, the apical bud, axillary buds, and leaves were removed from vegetative shoots of Coleiis blumeni (Benth.). The second internode was excised 48 hours later, surface sterilized with a saturated calcium hypochlorite solution, and rinsed in four changes of sterile distilled water. A transverse tissue slice, approximately 2 mm thick, was removed from the basal end of each internodal segment and rinsed in three changes of sterile distilled water. The tissue slices were incubated in Petri dishes on semi-solid media containing 2% sucrose, 1% agar, and supplemented with GA, IAA, or mixtures of the two. Three replicates of each treatment were made; each replicate consisting of nine tissue slices with three slices per plate. The slices were incubated 7 days under continuous fluorescent light at 20" C and subsequently cleared by the sodium hydroxide-chloral hydrate method (Roberts and Fosket, 1962) and stained with an aqueous solution of Safranin O. Temporary water mounts were made of the stained slices by squashing them between a cover glass and a slide. Counts were made of the completely differentiated wound vessel members only and no attempt was made to count those incompletely differentiated. RESULTS In a previous publication it was reported that media supplemented with IAA (5 or 0.5 ppm) inhibited the formation of wound vessel members whereas IAA at 0.05 ppm was highly stimulatory to the differentiation of them (Fosket and Roberts, 1964). In the present experiment the addition of GA had no significant effect on the number of regenerated members at o.^ or 0.05 ppm, but the addition of 5.0 ppm GA was slightly inhibitor}' (Table i). There was a great increase in the numbers of wound vessel members Table i. The mean number of completely differentiated zvoiind vessel members regenerated in 2 mm thick tissue slices incubated on sucrose-agar media containing gibbei-ellic acid and mixtures of gibber el lie acid and IAA Treatment 0.05 ppm GA ppm IAA 0.5 ppm GA ppm IAA 0.05 ppm GA Control (sucrose agar alone) 0.5 ppm GA 5.0 ppm GA 5.0 ppm GA PPni IAA No. cells regenerated* 2100" 1616" SS4'' 483'''' 428" 267' 265'= % inhibition or stimulation stimulation 234.6" 0 stimulation 14.7% stimulation % inhibition 44.7'''o inhibition 45.1% inhibition * The mean number of wound vessel memhers regenerated is given. Logarithmic transformations of these data showed that means followed hy diflferent letters are significantly different at the 5",, level. Means followed by the same letters are not significantly different. differentiated in the presence of combinations of GA and IAA in low concentrations. The synergistic effect of the combination of 0.5 ppm GA with 0.05 ppm IAA is clearly shown. In addition to this effect on the numbers of completely differentiated members, tissue slices incubated on media containing GA (0.5 or 0.05 ppm) and IAA (0.05 ppm exhibited the following characteristics: (a) initiation of cambial activity, (b) formation of incompletely differentiated secondary xylem elements and wound vessel members, (c) spherical masses of wound vessel members in the parenchyma of the cortex, and (d) initiation of adventitious roots after 14 days incubation. The wound vessel members

$THE NEW PHYl^OLOGIST, PLATE 3 Photomicrographs of incompletely dirterentiatcd \\oiind \cssel members tormc-d in Coteiis bhimeni (Benth.$

3 THE NEW PHYl^OLOGIST, PLATE 3 Photomicrographs of incompletely dirterentiatcd \\oiind \cssel members tormc-d in Coteiis bhimeni (Benth.) lnternodal tissue slices incubated 7 days on a sucriise-akar medium supplemented with GA (0.05 ppm) and IAA (0.05 ppml. No. I. Incompletely differentiated ^^'^'^I (a) showing partial formation of secondary wall striations. x S40. Xo. 2. Strands of w ound \ essels SIIOWIHL; lrreuailar staining " ith Satranin (). i 1 o. I,. W. ROBERTS AND P. E. FOSKET G Sr'BSTAXrES ( facing 6)

Growth substances and wound vessel members 7 resulting from treatment with mixtures of GA and IAA were observed in plates of cells contiguous with the primary vascular bundles and in spherical masses

4 Growth substances and wound vessel members 7 resulting from treatment with mixtures of GA and IAA were observed in plates of cells contiguous with the primary vascular bundles and in spherical masses which occurred in the cortex at the corners of the stem slices, adjacent to the large vascular bundles. Tissue slices in which the vascular tissue had been excised, i.e. solely parenchyma ofthe pith, failed to form wound vessel members under the same conditions. The term wound vessel member has been defined as a lignified cell with unique wall striations and simple perforation plates arising from a wound response (Roberts and Fosket, 1962). Those observed in the cortical regions were typical, but the sheets of members contiguous with the primary vascular tissue exhibited forms ranging from the typical wound vessel members to cell types indistinguishable from the scalariform-reticulate xylem elements that result from cambial activity in the normal Coleus stem. It is significant that treatment with GA and IAA together resulted in the differentiation of wound vessel members similar in appearance to normal xylem elements derived from cambial activity in Coleus. All reticulated cells were included in the cell counts as wound vessel members provided that differentiation was complete at the time of counting. Initiation of adventitious roots occurred from the meristematic mantle of cells encircling the cortical wound vessel member masses in a manner similar to the phenomenon observed by Steward et al. (1958) in secondary phloem cultures of carrot tap-root. The concentration of GA and IAA which stimulated maximum wound vessel member differentiation also resulted in incomplete differentiation of both tracheary elements formed from cambial activity and wound vessel members themselves (Plate 3, No. i). Such incompletely differentiated cells possessed thin secondary wall striations. Although critical cytochemical tests for lignin were not performed, it appears that these cells are poorly lignified on the basis of weak staining with the phloroglucinol-hcl and with Safranin O reagents, Plate 3, No. 2). It is possible that the media containing both GA and IAA delayed some stage in the differentiation sequence. It is of interest that Digby and Wareing (1964) also reported the appearance of incompletely differentiated tracheary elements in debudded shoots of Popuhis robusta and Vitis vinifera following the application of GA (500 ppm) and IAA (100 ppm) in lanolin. Dissection and maceration of tissues from the interfascicular region showed large numbers of tracheids and fibre-tracheids, scalariform-reticulate vessels with simple and scalariform perforation plates, and heavily-pitted xylem parenchyma cells. Many of these secondary xylem cell types can be observed from interfascicular cambial activity in the fourth internode of the intact (untreated) plant. Because of the diversity of intergrading secondary xylem cell types found in both cultured second internodes and in mature stems of untreated plants, it is impossible to state whether or not treatment with GA and IAA mixtures resulted in more than precocious cambial activity. The cambial activity in such treated material was expressed largely as secondary xylem with the differentiation of relatively little secondary phloem. The precocious stimulation of cambial activity in second internode tissues was not unexpected in view of the previous results obtained with woody tissues (Bradley and Crane, 1957; Wareing, 1958; Digby and Wareing, 1964). DISCUSSION It is impossible to determine accurately the ratio of GA to IAA which affects any differentiation process since the exogenous growth substances may trigger metabolic processes that, in turn, produce either GA or IAA or some other growth factor. For example, substances released by the initiated cambial activity may interact with the exogenous

5 8 L. W. ROBERTS AND D. E. FOSKET GA or IAA to produce some of the described effects. The initiated cambial activity may release some substance to the primary vascular system that is highly stimulatory to the differentiation of wound vessel members. If such a substance is formed, it is not likely to be diffusible IAA. Previous experiments (Eosket and Roberts, 1964) have shown that the addition of exogenous IAA (0.5 ppm and higher) is inhibitory to the formation of wound vessel members. The xylogenic effect of exogenous GA may be due to either increased diffusible auxin (Kuraishi and Muir, 1964), decreased IAA oxidase (Pilet, 1957), or decreased peroxidase activity (McCune and Galston, 1959). There is some justification for assuming that GA lowers the peroxidase activity associated with lignification since the incompletely differentiated wound vessel members appear to be weakly lignified. Ng (1963) reported increased numbers of wound vessel members were formed in Coleus stem slices incubated in the presence of the hydrazide of ^-hydroxybenzoic acid (2 X 10"«M). The latter, a lignification inhibitor, strongly blocked the cytochemicallydemonstrable peroxidase activity localized in the developing wound vessel members and also increased their numbers. On the other hand, similar slices incubated in the presence of the lignin precursor eugenol (0.0^ nim) exhibited smaller numbers of wound vessel members with enhanced lignification and extremely high peroxidase activity as compared to control slices. Ng (1963) suggested that the peroxidase level, as affected by eugenol and hydrazide substrates, respectively, was associated with both lignification and IAA oxidase activity. ACKNOWLEDGMENTS This research was supported by funds from the National Science Foundation (G-17741). The authors express their thanks to Mr. Dale Hansen for his technical assistance. REFERENCES BosTR-\CK, J. M. & STRUCKMEYER, B. E. (1964). Effects of gibberellic acid on the anatomy of soybeans {Glycine max). Am. J. Bot., 51, 6ti. BRADLEY, M. \'. & CR.^NE, J. C. (1957). Gibberellin-stimulated cambial activity in stems of apricot spur shoots. Science, 126, 972. DAVIS, E. L. & HOLMES, P. J. (1962). Morphogenetic effect of gibberellic acid on the xylem oi Lycopersicon esculentuni L. Phyton, 19, 31. DiGBY, J. & WAREING, P. E. (1964). The role of growth hormones in cambial activity. Proc. loth int. Bot. Congr., Edinburgh, p. 357 (abstract). EOSKET, D. E. & ROBERTS, L. W. (1964). Induction of wound-vessel differentiation in isolated Coleus stem segments in vitro. Am. J. Bot., 51, 19. G.^LTHERET, R. J. (1961). Action conjuguee de l'acide gibberellique, de la cinetine et de l'acide indoleacetique sur Ies tissus cultives m vitro, particulierement sur ceux de Topmainbour. C. r. hebd. Seanc. Acad. Sci., Paris, 253, KuR.'MSHi, S. & AIuiR, R. M. (1964). The relationship of gibberellin and auxin in plant growth. PL Cell PhyswL, 5, 61. McCuNE, D. C. & GALSTON, A. W. (1959). Inverse effects of gibberellin on peroxidase activity and growth in dwarf strains of peas and corn. PL PhysioL, Lancaster, 34, 416. NETIEN, G. (1957). Action des gibberellines sur la culture des tissus vegetaux cultives in vitro. C. r. hebd. Seanc. Acad. Sci., Paris, 244, NG, K. C. {1963). A histochemical study of the role of lignification and peroxidase activity in zvound-vessel differentiation. M.S. tbesis. University of Idaho. ODHNOEF, C. (1963). The effect of gibberellin and phenylboric acid on xylem differentiation and epidermal cell elongation in bean roots. Physiologia PL, 16, 474. PILET, P. E. (1957). Action des gibberellines sur l'activite auxines-oxydasique de tissus cultives in vitro. C. r. hebd. Seanc. Acad. Sci., Paris, 245, ROBERTS, L. W. & FOSKET, D. E. (1962). Further experiments on wound-vessel formation in stem wounds of Coleus. Bot. Gaz., 123, 247. STEWARD, F. C, MAPES, M. O. & MEARS, K. (1958). Growth and organized development of cultured cells. II. Organization in cultures grown from freely susperided cells. Am. J. Bot., 45, 705. WAREING, P. F. (1958). Interaction between indoleacetic acid and gibberellic acid in cambial activity. Nature, Lond., 181, 1744.

Topic 15. The Shoot System

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