than 40%. In these conditions, C684–696is preferentially formed. This suggests that C684–696formation (see
Secs. III.B and III.C) is essential for further assembly of PSII. This is confirmed by the observation that
in young leaves C684–696is formed only in low quantities (Figure 4A) even when the percentage of pho-
toreduction is 100% and consequently the development of the photosynthetic apparatus is very slow
[27,29,67] (see also later).
The results of these experiments emphasize the central role of the Pchlide-Chlide cycle in the bio-
genesis of the photosynthetic apparatus. The cycle not only is used to produce Chl but also acts as the pri-
mary regulator of the synthesis of polypeptides. It also explains why the reaction centers of PSI and PSII
are synthesized before the antennae, which slowly accumulate thereafter [97].
In gymnosperms, the Chlide produced in the dark continuously activates the transcription of the
polypeptides required for the assembly of the photosynthetic apparatus. Therefore, the 77 K fluorescence
spectra of dark-grown gymnosperm cotyledons [36,88] presented the typical bands of PSI and PSII. A
similar observation was made with dark-grown primary needles [24], a tissue noted for its inability to syn-
thesize Chl in the dark (e.g., Ref. 93).
It can be deduced from several experiments that the expression of several nuclear genes involves the
presence of functional plastids [98,99]. Barbato et al. [100] proposed that the presence of Chl also regu-
lates the light-harvesting chlorophyll a/bbinding protein (Cab) CP29 maturation during greening. The
idea that a signal originating from the chloroplast activates nuclear gene synthesis emerges from these
studies (reviewed in Ref. 101). However, the nature of the signal remains unknown. Pchlide precursors
can correspond to such a signal. This can be deduced from experiments with Chlamydomonasincubated
with a metal chelator [108]. In this condition, the Chl biosynthetic pathway is impaired and Mg-proto-
porphyrin monomethyl ester is accumulated [102] with the consequence that the light-dependent accu-
mulation of Cabproteins [103,104] and of the small subunits of ribulose-1,5-bisphosphate carboxylase
(Rubisco) are inhibited [105]. The mechanism(s) of action remains uncertain. The presence of Pchlide
precursors could decrease the amount of mRNA [103] or interfere with the light-dependent transcription
[104,106]. Another possibility for regulation is the very different affinity of Mg-chelatase for Proto-IX
[107]. This implies that when Mg-chelatase is active, Proto-IX is preferentially used to synthesize Chl.
Chl hemes are turning over [109]. When heme degradation is higher than heme formation, the -ALA
synthesis is stimulated because heme inhibits -ALA formation stoichiometrically [110–112]. Conse-
quently, the heme concentration will rise again and the -ALA formation will be partially inhibited. Such
a mechanism has been proposed for bacteria by Lascelles and Hatch [113] but also seems to occur in plas-
tids [9] regardless of their developmental stage.
After the initial Pchlide reduction, Chl synthesis in angiosperms shows three phases [114]: the lag
phase, the phase of rapid accumulation, and the stationary phase.
The length of the lag phase is dependent on the developmental stage. Precise measurements of the
length of the lag phase as a function of the bean leaf age [28,115,116] have indicated that there exists a
developmental stage, i.e., 3 days old, for which the lag phase is very short. Leaves below this stage, i.e.,
younger leaves, can accumulate Chl only after a very long lag phase. Above this stage, the older the
leaves, the longer the lag phase. A similar conclusion was reached with wheat leaves [117]. Using etio-
lated material, it was shown that the factor limiting Chl accumulation during greening is the synthesis of
-ALA. In fact incubation of seedlings with this compound abolished the lag phase [118]. However, this
is not the case in young seedlings [41,115]. Therefore, the long lag phase observed in young seedlings is
partially due to another factor(s). It is important to mention that during this period, a minimal but func-
tional photosynthetic apparatus is assembled very rapidly after the onset of the illumination. However, the
F 0 level of induction kinetics remains very high during all this period, suggesting that most of the Chl re-
mains not integrated with the photosynthetic units [27,67]. There are several lines of evidence that dur-
ing greening Chl synthesis is coordinated with those of Car and polypeptides composing the photosyn-
thetic apparatus [119–122]. Therefore, the long lag phase can be a consequence of either a deficiency of
the Chl biosynthesis itself or of other pathways (carotenoids, synthesis of Cabproteins; CO 2 fixation,
chemical energy production, etc). Interestingly, Chl, carotenoids, and leaf dry weight, which reflect the
actual CO 2 fixation, present the same lag phase whatever the leaf developmental stage [122]. This exper-
imental fact can be explained as follows: Chl phytol and carotenoids are both synthesized from geranyl-
geraniol, which itself is synthesized from the simultaneously fixed CO 2 [123,124]. Therefore, if the CO 2
fixation activity is low, the pigment synthesis is low. Interestingly, it has been shown that phytochrome
controls the length of the lag phase [125] at the level of -ALA synthesis (reviewed in Refs. 126 and 127),
CHLOROPHYLL BIOSYNTHESIS DURING PLANT GREENING 273