Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 141

net synthesis and accumulation than the H 2 0 controls (Table 4.2 , B). It is also
apparent that the plastids prepared from kinetin-pretreated tissues were more potent
in converting the nascent Proto into Mpe and Pchlide than the water controls as
evidenced by the lower amounts of Proto accumulation (Table 4.2 , Ba vs Bb).
Altogether these results indicated that we may have succeeded with the fore-
discussed treatment in uncoupling the simultaneous prothylakoid membrane
biosynthesis from Pchl(ide) biosynthesis and in preparing etiochloroplasts
containing excess Pchl(ide)-binding prothylakoid proteins.
It was conjectured that If the above hypothesis is correct and if the Pchlide
biosynthesis-enhancing effect of the kinetin pretreatment is due to the pigment-
uncoupled accumulation of prothylakoid proteins which are devoid of stochiometric
amounts of bound Pchlide, then the addition of kinetin to incubated plastids should
have no enhancing effect on the reactions of the Pchlide biosynthetic pathway
per sec. This was found to be precisely the case (Daniell and Rebeiz 1982a ).

4.4.3 Biosynthesis and Accumulation of Chlorophyll
a at High Rates

Once the biosynthesis and accumulation of Mpe and Pchlide was achieved as
described above in Sect. 4.4.2 we directed our attention to the development of
systems capable of high rates of Chl a biosynthesis and accumulation in organello.
This effort is described below.
The high rates of Chl a net synthesis and accumulation were achieved by first
preincubating 3-day-old etiolated cucumber cotyledons with an aqueous solution of
0.5 mM kinetin and 2 mM gibberellic acid for 20 h in darkness. The etiochloroplasts
were then isolated as described in (Daniell and Rebeiz 1982b ) and were resuspended
in a medium modified from that reported in that publication. The medium and
consisted of 0.5 M sucrose, 0.2 M Tris-HCl, pH 7.7, 20 mM MgCl 2 ,2.5mM
EDTA, 1.25 mM methanol, 20 mM ATP, 40 mM NAD, 8 mM Methionine and 1 %
BSA. Each incubation consisted of 1 ml of plastid suspension (12 mg plastid protein),
one additional ml of the suspension medium, 0.1 ml of 10 mM ALA and 0.9 ml of
H 2 O. The plastids were irradiated with white light (320 w/cm 2 ) for 30 s before

incubation. Incubation was carried out at 28 C for 2 h on a reciprocating water bath
operated at 50 oscillations per min. Chlorophyll (ide) a [Chl(ide) a ] net synthesis and
accumulation was induced by exposing the plastids to an alternating light dark regime,
which consisted of a 2.5 ms pulse of red actinic light, followed by 30 min of dark
incubation. The red light pulse was generated by a Sunpack model Auto 611 photo-
graphic flash unit (Berkey Marketing Co., Woodside, NY) (Duggan and Rebeiz
1982b ) shielded by a long wavelength cut-off red filter, Turner No. 25, that excluded
light below 585 nm. In this manner, the Pchlide which was synthesized from the added
ALA in the dark, was converted into Chl(ide) a by the brief red light treatment. During
the subsequent dark incubation, Chlide a was converted into Chl a by esterification,
and more Pchlide was regenerated during the following dark period.

μ