Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 140

Table 4.2 Effect of kinetin-pretreatment of etiolated cotyledons upon the tetrapyrrole biosyn-
thetic capacity of isolated plastids

Δ Change after 2 h

Pchlide MP(E) Proto
in nmoles/100 mg
plastid protein Experiment Treatment

A Cotyledons were harvested in the dark and were pretreated

either with pater a with kinetin; the plastids were
isolatedin the dark, then were incubated with ALA
(a) Water pretreatment

21.58 71.74 91.71

(b) Kinetin pretreatment 56.40 8.86 451.40

2 cm 2 )
of white light and were pretreated either with water or
with kinetin; the plastids were isolated under subdued
laboratory light, and were incubated with ALA
(a) Water pretreatment 18.24 73.86 267.36
(b) Kinetin pretreatment 73.88 85.46 172.02
Cotyledons were harvested with hypocotyl hooks from 3-day old etiolated cucumber seedlings
either in the dark or under subdued laboratory light (6 uw/cm 2 ). They were preincubated either
with distilled water or with a 0.5 mM aqueous solution of kinetin for 20 h in the dark at 28 C. The
plastids were isolated either in the dark or under subdued laboratory light, were given a 30 s
phototransforming light treatment (320 uw/cm 2 of white fluorescent light) then were incubated
with ALA, in the dark. The Δ change refers to the pigment contents of the plastids at the end of the
incubation minus the pigment content before incubation (Adapted from Daniell and Rebeiz 1982a )

B Cotyledons were harvested under 5 ftc (6 μ

biosynthesis in higher plants. Furthermore it was well known that in vivo, Pchl
accumulation rapidly ceases in the dark due to feed back inhibition of ALA
biosynthesis which may be relieved by addition of exogenous ALA (Beale and
Castelfranco 1974 ; Sisler and Klein 1963 ). Altogether these observations raised
the possibility that the forementioned kinetin treatment may have uncoupled the
etioplast prothylakoid biosynthesis from Pchl(ide) biosynthesis which in turn
resulted in the accumulation of excess kinetin-induced prothylakoid membranes
devoid of stochiometric amounts of membrane-bound Pchl(ide).
In order to test the above hypothesis 3-day old etiolated cucumber Cotyledons
were excised, with hypocotyl hooks, then were incubated either with distilled H 2 O
or with a 0.5 mM aqueous kinetin solution, for 20 h in the dark at 28 C.
The plastids were then isolated and their tetrapyrrole biosynthetic capability was
determined by monitoring the conversion of exogenous ALA into Proto, Mpe and
Pchlide (Daniell and Rebeiz 1982a ). As shown in Table 4.2 , A the Pchlide Net
synthesis and accumulation capabilities of the plastids prepared from kinetin
pretreated tissues was about 160 % higher than those of plastids prepared from
the H 2 O-pretreated control.
When the lag-phase of Chl biosynthesis was first eliminated (Rebeiz 1967 )by
exposing the cotyledons to laboratory light (4 h, 320 uw/cm 2 ) prior to the kinetin or
H 2 O dark pretreatment, the biosynthetic capabilities of the plastids that were
isolated from the kinetin pretreated tissues were about 400 %more active in Pchlide