[3H]Zeatin was supplied through the transpiration stream to radish (Raphanus sativus L.) seedlings with roots excised. Formation of dihydrozeatin was not detected but numerous other metabolites were formed, including adenine, adenosine, AMP, zeatin riboside and zeatin riboside-5′-monophosphate. However, in labelled seedlings which had been left in water for 15 h, an unknown compound (raphanatin) was the dominant metabolite and accounted for about 25% of the total radioactivity extracted. A procedure for the isolation of this metabolite was devised and yielded 70 μg from 1600 seedlings. Raphanatin was characterized by mass and ultraviolet spectra and has been identified as 7-glucosylzeatin. It is an active and very stable metabolite which was located mainly in the cotyledon laminae and may be a storage form of the hormone. In contrast, labelled nucleotides, the other major metabolites of zeatin, were largely confined to the hypocotyls and petioles. Zeatin riboside-5′-monophosphate was the dominant metabolite in hypocotyls of de-rooted seedlings supplied with zeatin for 0.5–2 h. The majority of the radioactivity in the xylem sap was due to zeatin, but about 10% was present as zeatin riboside; nucleotides accounted for less than 10% of the radioactivity and labelled raphanatin was not detected.

Regulators of Cell Division in Plant Tissues

The chemical changes that occur in leaves as they grow old have been well characterized for many species. The endogenous factors which control and regulate these changes in plant cells remain, to a great extent, obscure, and the problem of why the cell eventually dies is yet unsolved. A normal feature of the ageing leaf blade is a continuous decline in protein level (1). The most rapid fall occurs during senescence and is associated with irreversible yellowing, loss of chlorophyll, and the eventual death of the organ. When a mature leaf is excised from the plant and the petiole kept in water, these same symptoms of senescence occur and, provided the petiole does not form roots, the protein content of the blade may fall to less than half the original value within a few days. If, however, the petiole forms roots, the behavior of the excised leaf is quite different. It will remain green and photosynthetically active, increasing in dry weight and total protein for periods extending, in some cases, to several years (2), much longer, in fact, than the leaf would survive in situ. The factors responsible for senescence and the decline of total protein in the leaf are not fully understood. Experiments with excised leaves have shown that the decrease in protein content of the blade is not necessarily due to a lack of carbohydrate, nitrogen, or other nutrients (14, 4) or to an inability of the cells themselves to synthesize amino acids (13) but is due, rather, to a failing ability to incorporate these amino acids into protein (11). Provided the leaf has a growing root system, the incorporation of amino acids proceeds normally, and it seems logical to assume that the roots metabolize and supply the blade with certain factors necessary for the continued synthesis of protein. During the last 4 years, several chemical substances have been shown to retard senescence of leaf blades, and in this respect the compounds would atppear to substitute either directly or indirectly for the unknown so-called root factors. In 1957, Person, Samborski, and Forsyth (10) demonstrated that both chlorophyll degradation and protein loss in detached wheat leaves are retarded if the blades are floated on solutions of benzimidazole at 50 mg/liter. In the same year, Richmond and Lang (12) showed that similar effects could be obtained if excised leaves of Xanthium are kept with their petioles dipping into solutions of kinetin at 5 mg/liter. Mothes and Engelbrecht (5) sprayed solutions of kinetin directly onto leaves of Nicotiana and reported (1959) that the retention of chlorophyll is localized to the areas of the blade to which kinetin is supplied. They found that labelled amino acids migrate to, and accumulate in, the treated parts of tobacco leaves, and they suggest that kinetin retards leaf senescence by causing the treated areas to act as loci for the accumulation of metabolites (5, 6). Extensive investigations have shown that both protein synthesis and ribonucleic acid synthesis is stimulated in kinetin-treated parts of tobacco leaves. (15). Mothes and Engelbrecht (7) conclude, however, that "accumulation, . . . is not the consequence of synthesis, but mass synthesis of protein, for example, is the consequence of an accumulation of amino acids." In 1959, (8) it was demonstrated that a number of auxins are markedly effective in retarding the senescence of attached or detached autumn leaves of Prunus serrulata-senriko. Where ethanolic solutions of esters of 2,4-dichlorophenoxyacetic acid are applied to the blades, the cells retain their green color for some 10 to 20 days longer than those in the surrounding untreated parts of the blade, or in the control leaves. The retention of green color in the treated areas is associated with an actively photosynthetic chlorophyll and, a maintenance of the initial protein level (9). Attempts to retard senescence by various treatments with kinetin proved unsuccessful in this species. Precise information is still meagre concerning the part played by either auxins or kinins in the retardation of the biochemical changes associated with senescence but it is clear that they must exert a control through some fundamental cellular processes. In this paper are described some effects of one kinin, kinetin, upon the metabolism of protein and nucleic acids during the senescence of excised leaves of Xanthium pennsylvanicum. The results provide evidence that kinetin may control senescence in these 1 Received Jan. 15, 1962. 2 Present address: Agricultural Research Council Unit of Experimental Agronomy, Department of Agriculture, Oxford.

Effect of Kinetin on Protein & Nucleic Acid Metabolism in Xanthium Leaves During Senescence.

Present evidence indicates that only the root systems of plants have been shown conclusively to synthesize cytokinins. Although most of these compounds are apparently exported to the shoot via the xylem, there are indications that more attention should be given to the possibility of translocation through the phloem. Within mature leaves the cytokinins derived from the roots are converted to inactive or storage forms by means of glucosylation. While it would appear that glucosylation could occur in all living plant cells whenever the cytokinins are no longer required for active growth, and could provide the plant with a potential reservoir of cytokinins, very little is known with regard to the transport and reutilisation of these compounds.

The synthesis, transport and metabolism of endogenous cytokinins

After the discovery of kinetin (Miller et al. 1956, J. Am. Chem. Soc. 78: 1345–1350) there was a flurry of syntheses that led to the finding of 6-benzylaminopurine (BA), an active and easily obtainable cytokinin. Much research into cytokinin physiology was subsequently done with this substance. Further, the isolation and unequivocal identification of natural BA and the high biological activity of its meta-hydroxylated analogues stimulated the search for other natural aromatic cytokinins. Screening was accomplished by ELISA of HPLC fractions using antisera against ortho- and meta-hydroxybenzyladenosine. Subsequent isolation and decisive identification by mass spectrometry led to discovery of a broad spectrum of endogenous plant growth substances structurally similar to a highly active compound, meta-topolin (6-[3-hydroxybenzyl-amino]purine), and to its less active analogue, ortho-topolin (6-[2-hydroxybenzyl-amino]purine). The structures of such aromatic cytokinins suggest considerably different biosynthetic pathways from that of zeatin and related isoprenoid cytokinins. From a physiological viewpoint, aromatic cytokinin metabolism can be classified under four main headings analogous to isoprenoid cytokinins: interconversion, hydroxylation, conjugation, and oxidative degradation. This review attempts to put into context what is known about 9-alkyl-BAs and compares their metabolism in regard to the practical use of cytokinins in agriculture and biotechnology. The recently discovered unusual specificity of additionally C2,N9-disubstituted aromatic cytokinins toward cell cycle kinases, suggests that these cytokinin-derived growth regulators may selectively inhibit certain steps of the cell cycle. The functional overlap of the aromatic cytokinins with those of their isoprenoid counterparts and cytokinin inhibitors, in relation to growth and developmental processes in plants, has yet to be determined.

The aromatic cytokinins

Biosynthesis and Metabolism of Plant Hormones

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Dennis R. McCalla

Papers

Rapid bioassay for kinetin & kinins using senescing leaf tissue

The metabolism of a kinin, benzyladenine

On the effects of auxins on membrane permeability and pectic substances in bean endocarp.