A. Chl Formation in Plants Growing in Conditions Similar to the
Natural OnesUsing radiolabeled precursors, it was shown that the first Pchlide molecules are synthesized 12 hr after
the imbibition of cucumber cotyledons [25]. In situ spectroscopic investigations, especially in vivo ab-
sorbance measurements, at these early developmental stages are very difficult because the leaves are very
small [26,27] and contain only traces of pigments [28]. Using 77 K fluorescence detection, nonphotoac-
tive and photoactive Pchlide were detected in situ during the second photoperiod of greening in bean
leaves, i.e., when the radicle emerges from the seed [14,27]. At this developmental stage, the ratio of pho-
toactive to nonphotoactive Pchlide is in favor of the nonphotoactive form [26,29]. Similar results have
been obtained with other angiosperm tissues (Pisum sativum[30],Triticum aestivum[31]). Photoactive
Pchlide is a ternary complex containing Pchlide, NADPH, and a photoenzyme, the so-called LPOR. In
vivo, individual ternary complexes form aggregates (reviewed in Ref. 32). Reconstitution experiments
suggest that LPOR activity requires at least LPOR dimers [33], which could correspond to the photoac-
tive Pchlide P638–645* observed in vivo. Aggregation of these dimers yields the formation of large aggre-
gates also observed in vivo, i.e., P650–657. Both P638–645and P650–657have been isolated [34]. The behav-
iors of the two photoactive Pchlides are very difficult to analyze separately. Consequently, in this chapter
they have been considered as a single entity that is denoted by P638,650–657. Nonphotoactive Pchlide is de-
noted by P628–633. The biochemical state of nonphotoactive Pchlide is less clear because it is spectrally
and chemically heterogeneous [28,35–38]. Actually, several different nonphotoactive Pchlide forms have
been characterized more: (1) free Pchlide (emission at approximately 625 nm [39], (2) a monomeric Pch-
lide-LPOR complex (emission at approximately 634 nm [39], and (3) an aggregate similar to the pho-
toactive Pchlide but with NADPinstead of NADPH (P642–649[40]).
When a young dark-grown leaf (e.g., 2 days old) is illuminated by a short and saturating flash,
P638,650–657is transformed to the Chlide. The reaction consists of the reduction of ring D of Pchlide (Fig-
ure 3). The Chlide formed has absorbance and fluorescence emission maxima at 676 and 688 nm, re-
spectively (C676–688) [29,41] (Figure 4A). C678–688is an aggregate similar to P638,650–657but containing
Chlide and NADPinstead of Pchlide and NADPH [42]. Then the major part of the Chlide is liberated
from the active site of the enzyme, leaves the aggregate, and a new Chlide spectral form, C670–675, ap-
pears. The liberation of Chlide from the active site of the enzyme is indicated by the simultaneous regen-
268 SCHOEFS*PXYand CXYmean Pchlide and Chlide absorbing at Xnm and emitting fluorescence at Ynm at 77 K, respectively.
Figure 3 Scheme of the photoreduction of Pchlide to Chlide.