Biology Reference
In-Depth Information
It should be noted that the isolated plastids and the cotyledonary tissue
exhibited different requirements for achieving their highest greening rates.
First, the isolated plastids required much lower light intensities than the
cotyledons (Table
4.3
, A, B, E). Second, while the plastids did very well in the
presence of exogenous ALA [indeed no substantial tetrapyrrole biosynthesis
occurs in the absence of added ALA in organello systems (Mattheis and Rebeiz
1977a
,
b
)], the addition of ALA to the greening cotyledons was detrimental to
Chl(ide)
a
accumulation, even under the low light intensities of exp. B. This is not
surprising however as plant tissues are noteworthy for generating their own ALA
during greening and for failing to accumulate substantial amounts of Chl(ide)
a
from exogenous ALA, under even moderate light intensities (Sisler and Klein
1963
). Second, while pretreatment with hormones was required for achieving
high Chl(ide)
a
biosynthetic rates in organello, this was not observed to be the
case in vivo, as if the tissue generated its own hormonal requirements during
greening in the light (Table
4.4
,A,C,D).
The massive amounts of Chlide
a
detectable in vitro, was not generated by the
hydrolysis of endogenous Chl
a
but was synthesized de novo from exogenous ALA
as evidenced by the lack of Chlide
a
accumulation in dark controls, i.e., in
etiochloroplasts incubated in complete darkness, with ALA, for 2 h. It could have
arisen also from the newly formed Chl
a
. Finally the cell-free system described in
exp. A (Table I) was not optimized for Chl(ide)
b
biosynthesis and accumulation.
The in organello system capable of massive Chl(ide)
b
biosynthesis and accumula-
tion will be discussed in Sect.
4.5.2
below.
4.5 Development of an in Organello System Capable
of High Rates of Chlorophyll(ide) b Biosynthesis
and Accumulation
4.5.1 Preparative Techniques
After removing the hypocotyl hooks of light-pretreated cotyledons, 85-90 g batches
of tissue were homogenized in a Waring blender (2 bursts, 5 s each) under subdued
cool white fluorescent laboratory light (4.0 nmol/m
2
/s) in 230 ml of a homogeniza-
tion medium consisting of 0.5 M sucrose, 15 mMHepes, 30 mM Tes, 1 mMMgCl2,
1 mM EDTA, 5 mM cysteine, and 0.2 % bovine serum albumin (w/v) at room
temperature, and pH 7.7 (Daniell and Rebeiz
1982b
; Rebeiz et al.
1984
).
The homogenate was passed through four layers of cheese cloth and one layer of
miracloth. The plastids were pelleted by centrifuging the homogenate at 200
g for
3 min followed by centrifuging the resulting supernatant for10 min at 1,500
g.
The plastid pellet was gently resuspended in 10 ml of homogenization medium.
The resuspended plastids were further purified by layering 6 ml of the suspension
over 25 cm
3
of homogenization medium containing 35 % Percoll, in a 50 ml
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