Detection and origin of abscissic acid and proline in honey

This paper attempts to establish the origin and amount of both abscissic acid (ABA) and proline in honey. Two isomers of the sesquiterpene phytohormone cis trans (ct)-and trans trans (tt)-ABA are found in nature, but only the cis trans isomer is physiologically active. It induces dormancy in plants or parts of plants and plays an important role in closing stomata, especially in water deficient conditions. Proline (an amino acid) has protective functions (eg in stabilizing membranes) if the plant is under stress (eg water deficiency). Both substances occur in relatively high amounts in pollen (fig 5). Honey contains ABA and proline originating from pollen. It is important to evaluate the origin of proline, since it has been suggested that this substance is added to honey solely by the bee. To identify the origin of ABA and proline systematically, their possible natural sources were analyzed : nectar, honeydew, pollen and bees. Further investigations were carried out on aphids, honey from sugar fed bees and natural honeys. For separation of ABA and proline solid phase extraction by C18-columns was used. Purification of ABA was effected by thin layer chromatography. ABA was quantified by HPLC and enzyme linked immunosorbent assay (ELISA), the proline was measured by a colorimetric test. The results obtained by HPLC and ELISA show good agreement (table III). Honey contains considerable amounts of ABA and proline (table II). Compared with natural honeys, the honey from sugar-fed bees (ZFH) has a significantly lower level of both ABA and proline (see fig 1). In contrast to bees which have visited nectar sources (rape field, BR 1,2 in fig 2), starved bees (BW 1-4, fig 2) contain less (endogenous) proline and no ABA at all (see fig 2). This corresponds to the observation that proline and ABA are present in nectar (see fig 3, SN = Sanseveria-nectar; CN : Cymbidium-nectar; RN : Rape-nectar; TN = Deadnettle-nectar). Aphids (honeydew) show very different amounts of these substances (see fig 4, AS = Aphis sp on Tilia vulgaris; MR 1,2 = Macrosiphum rosae on Rosa hybrida white (1), red (2); CR = Cryptomyzus ribis on Ribes rubrum L; SM = Sappaphis mali on Malus domestica Borkh; HP = Hyalopterus pruni on Prunus domestica L; HT = Honeydew of Myzus cerasi on Prunus cerasus L). The results show that ct-ABA is added to the honey mainly from nectar, honeydew and pollen, and not by the bee itself. Possibly the bee might be responsible for conversion of ct- into tt-ABA, since honey contains rather more tt-ABA than does the nectar. The proline content in honey originates in the nectar of some species investigated (Lamium album L, Brassica napus napus L, Sanseveria trifasciata Prain, Cymbidium sp), in honeydew (from Myzus cerasi on Prunus cerasus L), and in some pollen (fig 5) as well as in the honeybee itself. It isproposed that the ratio of ct- to tt-ABA content (instead of the proline content) of honey depends on the extent of manipulation by the bees in converting nectar into honey, which in turn reflects upon nectar water content and environmental conditions