Photosynthesis Research 57: 203–213, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. 203 Regular paper Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (10-d-old) leaves and young (2-d-old) leaves Benoît Schoefs 1 , Martine Bertrand 2 & Yves Lemoine 3 1 Laboratory of Biomembranes, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic; 2 National Institute for Marine Sciences and Techniques, Conservatoire National des Arts et M´ etiers, BP 324, 50103 Cherbourg Cedex, France; 3 Laboratory of Plant Cytophysiology and Phycology (SN2), University of Lille 1, 59655 Villeneuve d’Ascq, France Received 4 November 1997; accepted in revised form 23 June 1998 Key words: carotenoid, chlorophyll b formation, chlorophyllide a esterification, accumulation of photosynthetic pigments, HPLC, protochlorophyllide a Abstract Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumu- lation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages. Abbreviations: Chl – chlorophyll; Chlide – chlorophyllide, C X-Y – Chlide a having absorbance and fluorescence maxima at X and Y nm at 77 K; DHGG – dihydrogeranylgeraniol; GG – geranylgeraniol; HPLC – High Per- formance Liquid Chromatography; Pchl – protochlorophyll; Pchlide – protochlorophyllide, Ph – phytol; THGG – tetrahydrogeranylgeraniol Introduction Dark-grown angiosperm leaves synthesize carotenoids but are devoid of Chl (Morren 1858). In the absence of light, they accumulate a Chl precursor, namely Pchlide a, a modified collection of xanthophylls and little or no carotene. Usually no carotenoid precursor may be ob- served, except if leaves remain for a very long period in the dark (e.g. Schoefs et al. 1995). When dark-grown plants are illuminated, carotenoid accumulation in leaves is triggered (Goodwin and Phagpolngarm 1960). In the same time photoactive Pchlide a is transformed to Chlide a which is then esterified by GG pyrophosphate which after three suc- cessive dehydrogenations yielding Chlide a Ph, is integrated into pigment–protein complexes (i.e. Chl a; reviewed by Rüdiger 1992; Franck and Schoefs 1996). Chl a accumulation during greening exhibits a lag phase of several hours whose length appears to depend upon the age: the younger the leaf, the shorter the lag phase (Akoyunoglou and Argyroudi-Akoyunoglou 1966, Sisler and Klein 1963). Chl b formation starts after that of Chl a. It is generally accepted that it is synthesized at the expense of Chl(ide) a (reviewed by Schoefs and Bertrand 1997). 10-d-old dark-grown leaves are not encountered in nature since prolonged darkness does not occur usu- ally. In natural conditions, proplastids of young leaves