OBSERVATIONS ON THE CHEMICAL NATURE AND FORMATION OF AUXIN IN THE AVENA COLEOPTILE

ETIOLATED AVENA coleoptiles are among the plant tissues which have been thought to contain auxin a or b rather than indole acetic acid (IAA) as the natural growth hormone. The reasons for this belief have rested upon diffusion measurements which indicate a molecular weight for the natural growth hormone of twice that expected for IAA (Went, 1928; Heyn, 1935), as well as indications that the auxin of the coleoptile (Zea) is stable towards hot acid, a treatment considered to destroy pure IAA (K6gl et al., 1934). Attempts to directly isolate auxin from the Avena coleoptile have not as yet been successful, however. It is now clear that IAA must be considered as a natural plant auxin, particularly since the substance has been isolated in pure condition from higher plants (Berger and Avery, 1944; HaagenSmit et al., 1942; Dandliker, 1945). Auxins a and b have not, on the other hand, been isolated from higher plants since the initial work of Kogl et al. (1934) which dealt with exceptional samples of malt and corn oil of unusually high auxin content. Inactive pseudoauxin a is however reported to have been isolated in small amounts from corn in one instance (Haagen-Smit et al., 1942). This paper will present evidence obtained along lines not pursued by the earlier investigators which indicates that the bulk of the auxin in Avena coleoptiles is IAA. At least three different forms of auxin may be distinguished in plant tissues, the differentiation being based on the method used for extraction. These forms are: (1) The diffusible or free-moving auxin which is produced when living plant tissues with a freshly-cut surface are allowed to stand in contact with agar for short periods of time. (2) The auxin which can be extracted from non-living tissues by non-polar solvents such as ether. (3) The bound auxin, or that auxin which is only detectable in the Avena bioassay after rather drastic treatment such as alkaline or enzymatic hydrolysis. It has been recently shown that auxin is not produced during short ether extractions when the auxin is first immobilized by freezing, lyophilizing the tissue, and then extracting with ether at 0?C. (Wildman and Muir, 1948). For reasons which will later become evident, the auxin determined in this manner is believed to be the uncombined auxin present in the tissue at the irnmediate moment of freezing. Hence, the cold ether-extractable auxin might be referred to as "free" auxin, a designation which has been used by others to describe auxin extracted by ether (van Overbeek et al., 1945), 1Received for publication July 1, 1948. Supported in part by a grant from the American Cancer Society. We are greatly indebted to Miss Martha Kent for her expert assistance with the Avena tests. although the previous methods of extraction do not unambiguously eliminate the possibility of producing auxin during the extraction process. The lyophilization and 0?C. ether extraction procedure appears to prevent the enzymatic formation of IAA from tryptophan in other plant tissues and it is of interest to see whether this mechanism of auxin formation is also operative in the Avena coleoptile. METHODS AND MATERIALS.-Plant material .-All of the plant tissue used in this investigation was obtained from etiolated Avena coleoptiles, variety Siegeshafer. The plants were grown in the standard manner of Went and Thimann (1937) for Avena assay work. All work preliminary to extraction was carried out in phototropically-inactive light. Avena assay.-The Avena test was performed exactly as earlier described (Wildman et al., 1947). Results compared on the basis of curvatures were obtained from the same Avena test. EXPERIMENTAL RESULTS. Correlation between diffusible auxin and cold ether extractable auxin.From the beginning of modern auxin physiology, it has been known that auxin production is confined to a very small region of the tip of the Avena coleoptile. When 20-30 mm. coleoptiles are divided into 5 mm. sections and the basal part of each segment allowed to stand on agar only the tip segment yields auxin into the agar (Went, 1928; van Overbeek, 1941; Went and Thimann, 1937). Furthermore, there is a close relation between growth and auxin production in that the region of most active elongation is located immediately beneath the coleoptile tip where active auxin production takes place. It should be noted that the region of elongation itself is practically incapable of diffusing auxin into agar. Studies with Avena and corn coleoptiles, as well as other tissues, have shown that the diffusible auxin of a tissue is greater in amount than the auxin which is extracted with ether (van Overbeek, 1941). It follows therefore that during the diffusion period auxin is generated in the tip but not in other more basal sections. Since earlier experiments on determinations of extractable auxin (Thimann, 1934; Went, 1942) have not rigorously excluded the possibility of auxin formation during ether or chloroform extraction, the following experiments were carried out. Standard Avena coleoptiles 20-30 mm. in length were cut off at the tops of the holders, 5 mm. tips removed by decapitation scissors and the primary leaf removed. The decapitated coleoptile was then cut into two sections of 5 mm. in length, and a third section 10 mm. in length. All of the samples were immediately frozen in liquid air, dried by lyophil, ground to 40 mesh in a Wiley mill and stored over P205 in vacuo in darkness until used