Plastids from dark-grown pine cotyledons are differentiated into grana and thylakoids and also con-
tain a PLB (reviewed in Ref. 3). They are called etiochloroplasts. LPOR has been found in PLB as well
as thylakoids and grana [86]. Forreiter and Apel [87] demonstrated that etiochloroplasts, as the etioplasts,
contain two LPORs: the first one (36 kDa) is associated with the PLB, and the second one (38 kDa) is
found in the thylakoids. Although direct evidence for the analogy of these two LPORs to LPORA and
LPORB is lacking, it can be assumed that LPORA is located in the PLB and LPORB is located in thy-
lakoids. Analyses of fluorescence spectra of dark-grown pine tissues indicate the same spectral and chem-
ical heterogeneity of nonphotoactive and photoactive Pchlides as in dark-grown higher plants (cotyledons
[36,88], primary needles [24]). Spectroscopic investigations of the Pchlide-Chlide cycle in angiosperms
are difficult because of the presence of emission bands corresponding to photosystems I and II (PSI and
PSII) [88]. In order to determine the fate of the Chlide resulting from the photoreduction in primary nee-
dles, pine seeds were cultivated in the dark and in the presence of norflurazon, an inhibitor of -carotene
synthesis (reviewed in Ref. 89). In the absence of carotenoid, neither PSI nor PSII assembled [24]. When
Pchlide photoreduction was triggered in such plants, the first product of the photoreduction, C676–688, was
rapidly transformed to C670–675[24]. Therefore a Pchlide-Chlide cycle similar to the one observed in em-
bryonic angiosperm leaves (Figure 2) seems to operate in these conditions.
IV. REGULATION (AN ASSAY)
A. Amount of Pchlide in Nonilluminated Plastids
The total Pchlide and photoactive Pchlide accumulation curves during the development in the dark are
sigmoidal. They reach a stationary level after approximately 7 to 10 days of growth depending on the
species and on the growth conditions (reviewed in Ref. 13). These levels correspond to the maximum
amount of Pchlide that a definite species is able to accumulate naturally for given growth conditions. The
arrest of Pchlide accumulation cannot be explained by feedback inhibition of -ALA synthesizing en-
zymes by Pchlide because they are not very sensitive to Pchlide [90,91]. In contrast, the preferential ac-
cumulation of photoactive Pchlide during dark growth can be explained by the fact that the import and
processing of LPORA precursor (pLPORA) from the cytoplasm into the plastids are dependent on the
availability of the nonphotoactive Pchlide [80].
According to this model, when Pchlide synthesis stops, the import of pLPORA is blocked and pho-
toactive Pchlide is no longer actively accumulated. In contrast, this model cannot explain why under ap-
plication of exogenous -ALA nonphotoactive Pchlide is accumulated [92]. In fact, the saturation level
observed when plants are developing in the dark does not correspond to the maximum capacity of Pch-
lide accumulation inside the plastids. Upon addition of exogenous -ALA, dark-grown leaves are able to
accumulate much more Pchlide than untreated leaves. Consequently, their yellow color, due to
carotenoids, is masked and the leaves appear green! This Pchlide, however, is nonphotoactive [92]. It is
relevant to add here that when leaves fed with -ALA are illuminated, the accumulated nonphotoactive
Pchlide produced so much activated oxygen species that the leaf can be bleached. This is especially ob-
vious with tigrinamutants of barley, which “naturally” overproduce nonphotoactive Pchlide (for pictures,
see Ref. 93). Because carotenoids do not protect Pchlide from photo-oxidation (reviewed in Ref. 59), the
simultaneous arrest of Pchlide and LPOR accumulation during dark growth can be understood as a mech-
anism to avoid production of activated oxygen species.
B. Regulation of the Chlorophyll Accumulation During Greening
Usually, plastids from dark-grown leaves do not contain polypeptides belonging to the photosynthetic ap-
paratus but contain their corresponding transcripts [94]. A very elegant study demonstrated that mRNAs
start to be translated by polysomes into the plastids during dark growth but the translation cannot be com-
pleted because some cofactor(s) is (are) missing [95]. Eichacker et al. [12] demonstrated that the missing
cofactor is not light itself but Chlide (plus phytol). In fact, these authors incubated lysed etioplasts in the
dark with exogenous Chlide plus phytol and observed the synthesis of several polypeptides encoded by
the chloroplastic genome in complete darkness!
On the other hand, Franck et al. [96] demonstrated that the appearance of variable fluorescence after
one single millisecond flash is detected only when the extent of Pchlide phototransformation is higher
272 SCHOEFS