Synopsis - Chlorophyll Biosynthesis and Technological Applications - page 39

The Reactions between porphobilinogen (PBG) and protoporphyrin IX which
are common to the heme and chlorophyll pathways were well covered, backed by
the pioneering work of David Shemin, Lawrence Bogorad and Sam Granick
(Chapter III). However the reactions between Proto and Chl were very tentative.
It relied mainly on the pioneering work of Sam Granick with X-ray Chlorella
mutants (Granick 1948a , b , 1950 ). Since cell free systems for these reactions
were not available, no precursor-product relationships had been established, and
the pathway proposed by Granick was a paper pathway backed only by incomplete
in vivo data. With these facts in mind I concluded that the field of Chl biosynthesis
was still a virgin field ripe for potential discoveries.

2.6.1 Chlorophyll and Carotenoid Research at Tel-el-Amara

2.6.1.1 In Vivo Experimentation

The picture of excised cucumber cotyledons turning green rapidly under laboratory
light within hours after excision was still very vivid in my mind. Therefore, I thought
that etiolated (dark-grown) excised cucumber cotyledons would be a good system to
study Chl biosynthesis as they rapidly turned green under illumination. A search
of the literature revealed that Withrow et al. ( 1955 ) and Hemming Virgin ( 1960 )
had studied the greening of etiolated bean and wheat seedlings upon illumination.
I therefore decided to repeat some of their work using excised etiolated cucumber
cotyledons. The finished work was published in the Journal of the National Research
Institute entitled Magon (Rebeiz 1967 ). The journal Magon was named after a
Carthaginian scientist who published a treatise about agriculture and veterinary
medicine in 22 volumes, about 140 B.C.
Since I knew little about the pigment composition of etiolated cucumber
cotyledons, I decided to investigate the pigment profile of these cotyledons before
undertaking systematic studies of Chl biosynthesis. The pigment composition of the
etiolated cotyledons consisted mainly of carotenoids and was published in Magon
in two articles (Rebeiz 1968a , b ).

2.6.1.2 In Vitro Experimentation

When the above work was completed I reasoned that the best way of moving
the field of Chl biosynthesis forward was to develop cell-free systems capable of
Chl biosynthesis in vitro. I reckoned that such systems would allow a stepwise
investigation of the Chl biosynthetic pathway and establish in vitro, the missing
precursor-product relationships. The research was started by growing in darkness
cucumber cotyledons, then excising the etiolated cotyledons and exposing them
to white light for 3 h to trigger the greening process. The greening cotyledons
were rapidly homogenized, the homogenate was filtered through cheese cloth and
the filtrate was exposed to white light for a few minutes. It was conjectured that