38 Formulation of a unified multibranched chlorophyll a/b biosynthetic pathway (1999).
39. Demonstration of resonance excitation energy transfer between anabolic tetrapyrroles and chl-protein complexes (2003).
40. Discovery of the light-independent reduction of protochlorophyllide a ester (2003).
41. Formulation of Chl-protein biosynthetic models (2003).
42. Development of analytical tools for measuring tetrapyrrole-Chl distances in chloroplasts (2004).
43. Formulation of a blue print for reducing the size of the photosynthetic unit (2004).
44. Detection of Delta-aminolevulinic esterases in higher plant and insect tissues (2005).
The above discoveries fall into eleven different research domains namely: (1) Horticulture, (2) Plant Physiology, (3) Botany, (4) Preparative Methodologies, (5) Analytical Methodologies, (6) Organic Chemistry of Natural Products, (7) Biochemistry, (8) Herbicides, (9) Insecticides, (10) Chloroplast Bioengineering, and (11) Animal Biology .
A. Details of Unique Developments
In 1960, it was shown that following appropriate multiple hormonal treatment, flowers of stone fruits such as cherries and peaches developed into mature seedless fruits. This in turn proved for the first time that the development of a flower into a fruit did not depend on a single hormone, but required the cooperation of several hormones.
2. Plant Physiology
a. Discovery of the extra-mitochondrial ß-oxidation of long chain fatty acids
In 1964, the occurrence of the ß-oxidation of long chain fatty acids outside the mitochondrion was described. This discovery paved the way later on for the discovery by others of a new subcellular organelle, the glyoxysome, which is the site of the glyoxylate pathway. The latter is responsible for the conversion of lipids to carbohydrates in plant cells. Animal cells lack such a metabolic activity.
b. Discovery of the divinyl and monovinyl greening groups of plants
In 1985, it was discovered that all green plants belonged to one of three greening groups, depending on which chlorophpyll biosynthetic route they use to form chlorophyll at night and in daylight. The three greening groups have been designated: dark divinyl-light divinyl, dark monovinyl-light monovinyl, and dark monovinyl-light divinyl. Since photosynthetic efficiency appears to be related to the greening group affiliation of plants, induced alterations of the greening process may be used in attempts to improve plant productivity under field conditions.
In 1987, it was demonstrated that the dark divinyl-light-divinyl greening group of plants was the most primitive of the four greening groups and was predominant about 500 million years ago. On the other hand, the dark monovinyl-light monovinyl greening group was shown to be the most advanced and probably evolved about 60 to 65 million years ago. These discoveries have led to the development of an evolutionary taxonomical biological clock, based upon the biochemistry of greening. It is presently being used to probe the phylogenetic origin of flowering plants.
4. Preparative Methodologies
a. Development of cell-free systems capable of chlorophyll biosynthesis in vitro
In 1971, the first total biosynthesis of protochlorophyll and protochlorophyllide ester (immediate precursors of chlorophyll) and of chlorophyll a and b was achieved in vitro, from a simple 5 carbon amino acid, delta-aminolevulinic acid. This replication of the greening process in a test tube made it possible to investigate the biochemistry of the greening process by time-honored biochemical techniques. This in turn has led to the discovery of novel terapyrroles, novel chlorophyll chemical species, and chlorophyll biosynthetic routes in green plants (see below).
b. Development of cell-free systems capable of photosynthetic membrane formation in vitro
This group of discoveries spans a period of 13 years and extends from l973 when grana assembly was first reported in vitro, to l985, when the massive formation of chlorophyll and photosynthetic membranes in vitro was described. These cell-free systems are now being used by molecular biologists for the introduction and expression of extraplastidic genes into the chloroplast. This in turn promises to open the way for the bioengineering of photosynthetically more efficient chloroplasts which can be introduced into protoplasts from which more efficient food-forming plants can be regenerated.
5. Analytical Methodologies
a. Development of spectrofluorometric methodologies
These methodologies span a period of 18 years and extend from 1975 to the present. They fall into three different groups. One group consists of spectrofluorometric techniques which have allowed the detection of various intermediates of the chlorophyll biosynthetic pathway. Another group consists of mathematical equations that have allowed the precise quantitative determination of various intermediates of the chlorophyll biosynthetic pathway from fluorescence spectra. The third group consists of quantitative equations that have allowed the determination of the residual electrical charge on the central metal atom of metallotetrapyrroles. The equations have been used to determine the precise axial coordination state of chlorophyll and other Mg-containing tetrapyrroles. Altogether these techniques have made it possible to demonstrate various chlorophyll biosynthetic routes and eliminated the guesswork from this research domain.
b. Development of analytical precursor-product methodologies
These methodologies consist of mathematical equations that permit the precise determination of precursor-product relationships in complex multibranched biochemical routes. These equations have been successfully used to demonstrate the multibranched nature of the chlorophyll biosynthetic pathway.
6. Organic Chemistry of Natural Products
Since 1979 the following novel metabolic tetrapyrrole pools have been discovered in higher plants and have been fully or partially characterized:
a. Divinyl chlorophyll a
b. Divinyl chlorophyll b
c. Monovinyl 10-OH-chlorophyll a lactone
d. Several non-phytol monovinyl and divinyl chlorophyll a species
e. Monovinyl chlorophyllide b
f. Divinyl chlorophyllide a
g. Divinyl protochlorophyllide a ester
h. Divinyl protochlorophyllide a
g. Monovinyl prochlorophyllide b
i. Divinyl Mg-protoporphyrin diester
j. Monovinyl Mg-protoporphyrin diester
k. Monovinyl Mg-protoporphyrin monoester
l. Monovinyl Mg-protoporphyrin IX
a. Demonstration of Mg-protochelatase.
This is the enzyme that inserts Mg into protoporphyrin IX and initiates the Mg-branch of the porphyrin pathway that leads to chlorophyll formation.
b. Discovery and demonstration of the divinyl to monovinyl tetrapyrrole reductases
This group of several enzymes links the divinyl monocarboxylic chlorophyll biosynthetic routes to the monovinyl routes in the dark monovinyl-light divinyl greening group of plants.
c. Demonstration of the paper-chemistry chlorophyll biosynthetic pathway which was proposed by sam granick in 1950.
This involved the first demonstration, in vitro, of the convertibility of protoporphyrin IX and Mg-protoporphyrin IX monoester to protochlorophyllide a. This was achieved with the use of newly developed cell-free systems (see above), twenty-five years after the proposal of these biosynthetic steps by Sam Granick.
d. Proposal and demonstration of the multibranched nature of the chlorophyll biosynthetic pathway
A dual branched pathway based on precursor-product analysis in vivo was proposed in 1970. On the basis of the detection and identification of the novel tetrapyrrole pools described above, a 4-branched pathway was proposed in 1980, a six-branched pathway was proposed in 1983, a 12-branched pathway was proposed in 1988, and a 17-branched pathway was proposed in 2003. The knowledge derived from these discoveries has been used to develop potent and selective photodynamic herbicides (see below). It also had the potential to improve the photosynthetic efficiency of plants under field conditions.
a. Discovery of photodynamic herbicides.
In 1984 a new concept in herbicide methodology and design was described. The main component of the herbicide consisted of a harmless 5-carbon amino acid, delta-aminolevulinic acid, which is the natural precursor of all hemes and tetrapyrroles, including chlorophyll, in nature. Delta-aminolevulinic is highly biodegradable and disappears from the environment within 24 hours. It destroys green plants by forcing them to accumulate excessive amounts of tetrapyrroles which in the light generate destructive singlet oxygen.
b. Discovery of photodynamic herbicide modulators
This discovery extended considerably the scope potency and selectivity of photodynamic herbicides. Essentially thirteen different chemicals and biochemicals were discovered which imparted a considerable degree of photodynamic herbicidal potency and selectivity to delta-aminolevulinic acid, when combined four, three, two, or one at a time with the aminoacid. So far, this has resulted in 3458 possible photodynamic herbicidal formulations.
The discovery of novel insecticides that kill insects by interfering with their heme-cytochrome pathway has been announced in January 1988. These insecticides which are novel in concept design and phenomenology have been named "porphyric insecticides." .
10. Chloroplast Bioengineering
With the formulation of a blue-print for the integration of chlorophyll biosynthesis and the biogenesis of photosynthetic membranes, it has become possible to investigate systematically the bioengineering of more efficient photosynthetic membranes.
11. Animal Biology
The photodynamic porphyric pesticide methodology has been adopted to the destruction of cancer cells. Several thousand laboratories around the world are now involved in the development of delta-aminolevulinic acid-based methodologies to the treatment of many types of cancer.
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