Phytochemistry, Vol. 29, No. 3, pp. 765 768, 1990. 003 I 9422/90 $3.00 + 0.00 Printed in Great Britain. 0 1990 Pergamon Press plc EFFECTS OF PHYSIOLOGICAL AND ONTOGENETICAL AGEING ON STEROL LEVELS AND COMPOSITION IN PEA LEAVES MARIE E. OLSSON and CONNY LILJENBERG Department of Plant Physiology, Botanical Institute, University of GGteborg, S-413 19 Giiteborg, Sweden zyxwvutsrqponmlkj (Received in revised form 7 August 1989) Key Word Index-Pisum sativum; Fabaceae (Leguminosae); pea; ageing; maturity; senescence; sterols; campesterol; stigmasterol; sitosterol. Abstract-Free, esterified and glycosylated sterols of pea leaves were investigated during different stages of ageing. Samples for sterol analysis were collected both from leaves of different maturity of plants with 12 leaves and from newly unfolded leaves of plants at juvenile and mature stage. Free sterols were the most abundant form. The total level of sterols decreased with increasing physiological age, mainly due to a decline in the levels of stigmasterol and an unidentified sterol, while sitosterol remained constant. The total content of sterols was highest in the newly unfolded leaves from the juvenile plants. The unidentified sterol decreased from juvenile to mature stage, while stigmasterol and sitosterol remained constant. The results indicate that the membrane sterol composition reflects the developmental level that is prevailing. Stigmasterol might have a regulatory function in physiological ageing. INTRODUCTION Changes in cell membranes have often been pointed out to be important early events in the process of ageing [l-3]. Ageing in plants can be regarded from two differ- ent aspects: physiological ageing which is connected with the term senescence and ontogenetical ageing which refers to the process of passing through different phases, e.g. the juvenile and the mature phase. Most research within this area has been devoted to physiological ageing Phospholipid levels has been found to decline during senescense [l, 4, 51. Membranes of senescing cells have been reported to exhibit changes in levels and composi- tion of sterols [6-S]. However, in general, the biochemical mechanisms underlying the process of ontogenetical age- ing are far from known. The sterol levels during one phase of increasing maturity, the floral differentiation, has been studied. Sterol levels were found to decrease at or just prior to floral development [S, 91. However, there are few reports which compare the changes in membrane composition during physiological ageing and during ontogenetical ageing. In the present work the alterations in levels and composition of free, esterified and glycosylated membrane sterols in pea leaves at different ages are investigated. For some of the experiments, newly unfolded top leaves from juvenile (12- days-old) and adult (32-days-old, flowering) plants were used. Thereby the physiological maturity was regarded as being maintained constant while two different ontogen- etical phases were investigated. In other experiments, the analyses were performed with leaves at two different stages of physiological maturity by using samples of newly unfolded leaves and of the sixth leaf (counted from the base of the plant). RESULTS Free sterols were the most abundant form in a11stages of maturity investigated. Steryl glycosides and acylated steryl glycosides were present at low levels, and therefore, of those only the identity of the major one, sitosterol, was confirmed by mass spectrometry. Steryl esters were not present. According to the preliminary GLC analysis, choles- terol appeared to be present in large quantities. However, GC-MS failed to confirm the component as cholesterol, but revealed another unidentified sterol. The identifica- tion of the latter as a sterol was based on the following: (i) it co-chromatographed with the other sterols on TLC, (ii) two different derivatives, (trimethylsilyl ethers and acet- ates) displayed similar behaviour of reference sterols and the unknown component upon GLC analysis and (iii) the mass spectrum gave a fragmentation pattern similar to other sterols. The [M] + of the trimethylsilyl ether was at m/z 482. Prominent peaks were [M - Me] +, [M -(Me),- SiOH]+, [M- 129]+, [M-(Me),SiOH-side chain]+, [M-211]+, [M-(Me),SiOH-side chain-C,H,]+. The ratio dry weight/fresh weight of leaves at various stages of physiological maturity was rather constant (data not shown). As the pea plants grew older, visible senes- tense started in the oldest leaves at the stem base while the rest of the plant looked fully green and healthy. The leaf used, representing a physiologically older stage, was the lowest leaf that still did not show any visible sign of chlorosis (the sixth leaf). Sterol analysis revealed a signifi- cant decrease in levels of free sterols of the older leaves (Table 1). The sitosterol/stigmasteroI ratio of the older leaves increased, mainly due to a decline in the stigmas- terol content. The level of the unknown sterol, as we11 as campesterol, was highest in the younger leaves. Sitosterol was the major sterol of the glycosides and acylated glycosides in both young and old leaves (Tables 2 and 3). As with the free sterols, the ratio sitosterol/stigmasteroI of the glycosides and acylated glycosides increased with age. In contrast to the results of leaves from the two stages of physiological maturity, the newly unfolded leaves from plants of different ages showed a slightly increased ratio 765