New aspects of rubber biosynthesis. Following a review of the site of rubber biosynthesis in Hevea brasiliensis and Parthenium argentalum, evidence is given for the initiation of polyisoprene molecules from (ranMerpenoid precursors including geranylgeranyl pyrophosphate. All franj‐14C‐geranylgeraniol has been isolated from incubations of H. brasiliensis latex serum with 14C‐isopentenyl pyrophosphate. Gel‐filtration chromatography of the serum yields very small rubber particles of high biosynthetic activity, and two proteinaceous fractions. One of these increases the biosynthesis of rubber and may contain the enzyme, isopentenyldiphosphate δ‐isomerase, whilst the other appears to inhibit rubber formation. The nature and molecular weight of the rubber formed in vitro is discussed and a mechanism for the de novo formation of rubber particles is suggested.

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Fresh rubber latex obtained from Hevea bra8iliensis consists of a suspension of several types of particles in an aqueous serum, and can be separated into fractions by high-speed centrifuging (Cook & Sekhar, 1953). The top layer in the centrifuge tube consists mainly of rubber particles, below which is a layer of almost clear serum. At the bottom of the tube is a layer referred to in this paper as the 'bottom fraction', which contains sedimentable bodies including the lutoid particles described by Homans & van Gils (1948). In preliminary work an aqueous extract of the bottom fraction was found to contain several electrophoretically distinct protein components. This observation has been confirmed and elaborated by Moir & Tata (1960). This paper describes the isolation and characterization of a major component which is anionic at pH 8-6 and for which the name hevein is proposed. EXPERIMENTAL Materials. Freeze-dried samples of the bottom fraction of freshly tapped latex were used throughout. Collection of the latex and centrifugal separation and freeze-drying of the bottom fraction were carried out by the staff of the Rubber Research Institute of Malaya, as described by McMullen (1959). All freeze-dried fractions were sealed under high vacuum in Malaya, and on arrival at our Laboratory were stored at-150 until required. Elementary analyses. Total N was determined by the micro-Kjeldahl method and C, H and ash by Pregl microcombustion. The S content of the ash and gaseous combustion products was measured turbidimetrically as BaSO4 after complete dissolution of the ash in dilute HC1. Where the amount of material available for analysis was limited, and the ash content was known to be low (as in purified hevein), C, H and S were determined on the same sample with a microcombustion tube containing a roll of silver and PbO2 only. P was determined colorimetrically with ammonium molybdate after wet oxidation. Soluble proteins were extracted from the freeze-dried solids with 0*05M-phosphate buffer, pH 6-8, or with 0-05 m-Na2B407, pH 9-3. The protein-N content of the extract was then determined by tannic acid precipitation and Kjeldahl analysis (Archer & Sekhar, 1955). Purified protein samples were dried to constant weight in vacuo at 800 before analysis.

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Ethephon (I) is used commercially to prolong the flow of latex from the rubber tree after tapping (Yield stimulation). The compound is applied to the bark in the region of the tapping cut and the effect on latex flow is due to the ethylene released by chemical decomposition, since gaseous ethylene itself is also a very effective stimulant. When 14C-I is applied to the bark of a young Hevea seedling, it is absorbed into the plant by processes which appear to be largely non-metabolic. Ethylene formation commences immediately at the site of application, and the gas is quickly translocated throughout the plant. Translocation of I to all parts of the plant also occurs and the accumulation of 14C in the bark above the zone of application is greater than that below. Chromatographic analysis has shown that compounds other than I remain in the plant tissue. Experiments using 14C-I have shown that detached leaves are able to convert a considerable proportion of the compound to at least twelve non-volatile acid products. One of these is a conjugate of I with an unidentified material. A major component of the products is 2-hydroxyethylphosphonic acid (II), which is itself converted to a number of compounds in leaves. The application of I to bark from mature Hevea, results in the formation of a single substance which is also a conjugate of I. Neither I nor II is effective in inducing the formation of ethylene from endogenous precursors in vegetative Hevea tissue. Ethylene is poorly metabolized by Hevea leaves and the evidence available indicates that it is unlikely that any of the compounds produced from I are metabolites of ethylene.