Selected Research Highlights of Constantin A. Rebeiz

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I. Synopsis of Unique Developments

  • 1. Development of seedless cherry fruits (1960).
  • 2. Discovery of the extra-mitochondrial -oxidation activity of fatty acids in plants (1964).
  • 3. Biosynthesis in vitro of protochlorophyll(ide) the immediate precursors of chlorophyll(ide) (1971).
  • 4. Total biosynthesis of chlorophyll a and b in Vitro (1971).
  • 5. Biosynthesis of a photosynthetic membranes in a test tube (1973).
  • 6. Formulation of the concept of Cell-Free Agriculture (1974).
  • 7. Development of a quantitative tetrapyrrole spectrofluorometric methodology (1975).
  • 8. Demonstration of the insertion of Mg into protoporphyrin IX in vitro (1977).
  • 9. Demonstration of the conversion of protoporphyrin IX to protochlorophyllide a in vitro (1977).
  • 10. Demonstration of the conversion of Mg-protoporphyrin monoester to protochlorophyllide a in vitro (1977).
  • 11. Formulation of a two-branched chlorophyll a biosynthetic pathway (1978).
  • 12. Description of an experimental photosynthetic reactor (1978).
  • 13. Discovery of novel chlorophyll and chlorophyll precursors in green plants (1979).
  • 14. Detection of divinyl protochlorophyllide a in higher plants (1980).
  • 15. Detection of divinyl protochlorophyllide a ester in higher plants (1980).
  • 16. Discovery of divinyl chlorophyllide a and divinyl Chl a (1980).
  • 17. Formulation of a four-branched chlorophyll a biosynthetic pathway (1981).
  • 18 . Discovery of fully esterified Mg-protoporphyrins in higher plants (1981).
  • 19. Demonstration of independent acidic and fully esterified chlorophyll a biosynthetic routes in higher plants (1982)
  • 20. Detection of chlorophyllide b in higher plants (1982).
  • 21. Detection of monovinyl Mg-protoporphyrins in higher plants (1982).
  • 22. Demonstration of a biosynthetic bridge between monovinyl and divinyl chlorophyllide a (1982).
  • 23. Biosynthesis of chlorophyll a in a test tube at rates higher than in a whole plant (1982).
  • 24. Formulation of a six-branched chlorophyll a biosynthetic pathway in nature (1983).
  • 25. Discovery of photodynamic (laser) herbicides (1984).
  • 26. Discovery of a new plant classification system based on greening biochemical patterns (1985).
  • 27. Demonstration of the monovinyl and divinyl monocarboxylic chlorophyll a biosynthetic routes in green plants (1986).
  • 28. Discovery of various photodynamic herbicide modulators (1987).
  • 29. Discovery of porphyric insecticides (1988).
  • 30. Discovery of 4-vinyl protochlorophyllide reductase (1988).
  • 31. Discovery of photodynamic insecticide modulators (1990)
  • 32. Discovery of monovinyl protochlorophyllide b in green plants (1991).
  • 33. Doscovery of 4-vinyl chorophyllide a reductase (1992).
  • 34. Destruction of Solid Tumors (1996).
  • 35. Discovery of 4-vinyl Mg-proporphyrin IX reductase (1997)
  • 36. Discovery of the Dark-divinyl/light divinyl/-dark-light divinyl and the Dark-monoviny/light divinyl/-dark-light monovinyl greening subgroups of plants (1997).
  • 37. Discovery of 4-vinyl Chl a reducase(1998).
  • 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

    1. Horticulture

    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 -oxidation of long chain fatty acids

    In 1964, the occurrence of the -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.

    3. Botany

    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

    7. Biochemistry

    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.

    8. Herbicides

    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.

    9. Insecticides

    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.

    II. References

  • 1. Rebeiz, C. A. and Crane, J. C. (1961) Growth regulator-induced parthenoncarpy in the Bing cherry. Proc. Amer. Soc. Hort. Sci. 78: 69-75.
  • 2. Rebeiz, C. A. and Castelfranco, P. (1964) An extra-mitochondrial enzyme system from eanuts catalyzing the -oxidation of fatty acids. Plant Physiol. 39: 932 938
  • 3. Rebeiz, C. A. and Castelfranco, P. (1971) Protochlorophyll biosynthesis in a cell-free system from higher plants. Plant Physiol. 47: 24-32
  • 4. Rebeiz, C. A. and Castelfranco, P. (1971) Chlorophyll biosynthesis in a cell-free system from higher plants. Plant Physiol. 47: 33-37
  • 5. Rebeiz, C. A., Larson, S., Weier, T. E., and Castelfranco, P. A. (1973) Chloroplast maintenance and partial differentiation in vitro. Plant Physiol. 51: 651-659
  • 6. Rebeiz, C. A. (1974) Cell-free agriculture:fiction or reality. Ill. Res. 16: 3-4
  • 7. Rebeiz, C. A., Smith, B. B. Mattheis, J. R., Rebeiz, C. C. and Dayton, D. F. (1975) Chloroplast biogenesis. Biosynthesis and accumulation of protochlorophyll by isolated etioplasts and developing chloroplasts. Arch. Biochem. Biophys. 171: 549-567
  • 8. Smith, B. B. and Rebeiz, C. A. (1977) Chloroplasts Biogenesis: Detection of Mg-protoporphyrin chelatase in vitro. Arch. Biochem. Biophys. 180: 50-60
  • 9. Mattheis, J. R. and Rebeiz, C. A. (1977) Chloroplast biogenesis: Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts. J. Biol. Chem. 252: 8347-8349
  • 10. Mattheis, James R. and Rebeiz, C. A. (1977) Chloroplast biogenesis: Net synthesis of protochlorophyllide from magnesium protoporphyrin monoester by developing chloroplasts. J. Biol. Chem. 252: 4022-4024
  • 11. Rebeiz, C. A., B. B. Smith, B. B. Mattheis, J. R., Cohen, C. E. and McCarthy, S. (1978) Chlorophyll Biosynthesis: The reactions between protoporphyrin IX and phototransformable protochlorophyll from higher plants. In Chloroplast Development, pp. 59 76. (G. Akoyunoglou and J. H. Argyroundi-Akoyunoglou, Eds). Amsterdam, Elsevier
  • 12. Rebeiz, C. A. and Bazzaz, M. B. (1978) Cell-free agriculture: The concept and its initial implementation. In: Biotechnology in Energy Production and Conservation; (C. D. Scott, Ed.) 8: 453 471. John Wiley & Sons
  • 13. Belanger, F. C. and Rebeiz, C. A. (1979) Chloroplast Biogenesis XXVII. Detection of novel chlorophyll and chlorophyll precursors in higher plants. Biochem. Biophys. Res. Comm. 88: 365-372
  • 14. Belanger, F. C. and Rebeiz, C. A. (1980) Chloroplast Biogenesis. Detection of divinyl protochlorophyllide in higher plants. J. Biol. Chem. 255: 1266-1272
  • 15. Belanger, F. C. and Rebeiz, C. A. (1980) Chloroplast Biogenesis: Detection of divinyl-protochlorophyllide ester in higher plants. Biochemistry. 19: 4875-4883
  • 16. Belanger, F. C. and Rebeiz, C. A. (1980) Chloroplast Biogenesis 30. Chlorophyll(ide) (E459 F675). The first detectable products of divinyl and monovinyl protochlorophyll photoreduction. Plant Sci. Lett. 18: 343-350.
  • 17. Rebeiz, C. A., Belanger, F. C., McCarthy, S. A., Freyssinet, G., Duggan, J. X., Wu, S, M., and Mattheis, R. R. (1981) Biosynthesis and accumulation of novel chlorophyll a and b chromophoric species in green plants. Proc. 5th Int. Cong. Photosynth. (G. Akoyunoglou, Ed.) V 5 pp. 197 212. Int. Sci. Services, Jerusalem, Israel
  • 18. McCarthy, S. A., Belanger, F. C. and Rebeiz, C. A. (1982) Chloroplast biogenesis: Detection of a magnesium protoporphyrin diester pool in plants. Biochemistry 20: 5080 - 5087
  • 19. McCarthy, S. A., Mattheis, J. R. and Rebeiz, C. A. (1982) Chloroplast Biogenesis: Biosynthesis of protochlorophyll(ide) via acidic and fully esterified biosynthetic branches in higher plants. Biochemistry 21: 242 - 247
  • 20. Duggan, J. X. and Rebeiz, C. A. (1982) Chloroplast Biogenesis 38. Quantitative detection of a chlorophyllide b pool in higher plants. Biochim. Biophys. Acta. 714: 248 - 260
  • 21. . Belanger, F. C.. and Rebeiz, C. A. (1982) Chloroplast Biogenesis: Detection of monovinyl magnesium protoporphyrin monoester and other monovinyl magnesium porphyrins in higher plants. J. Biol. Chem. 257:1360 - 1371
  • 22. Duggan, J. X. and Rebeiz C. A.. (1982) Chloroplast Biogenesis 42. Conversion of divinyl chlorophyllide a to monovinyl chlorophyllide a in vivo and in vitro. Plant Sci. Lett. 27: 137 - 145
  • 23. Daniell, H. and Rebeiz C. A. (1982) Chloroplast culture IX. Chlorophyll(ide) a biosynthesis in vitro at rates higher than in vivo. Biochem. Biophys. Res. Comm. 106: 466 - 470.
  • 24. Rebeiz, C. A., Wu, S. M., Kuhadja, M., Daniell, H. and Perkins, E. J. (1983) Chlorophyll biosynthetic routes and chlorophyll a chemical heterogeneity in plants. Mol. Cell. Biochem. 58: 97 - 125
  • 25. Rebeiz, C. A., Montazer-Zouhoor, A., Hopen, H. J. and Wu, S. M. (1984) Photodynamic herbicides: Concept and phenomenology. Enzyme and Microbial Technology 6: 390-401
  • 26. Carey, E. E. and Rebeiz, C. A. (1985) Chloroplast biogenesis 49: Differences among angiosperms in the biosynthesis and accumulation of monovinyl and divinyl protochlorophyllide during photoperiodic greening. Plant Physiol. 79: 1-6
  • 27. Tripathy, B. C. and Rebeiz, C. A. (1986) Chloroplast Biogenesis. Demonstration of the monovinyl and divinyl monocarboxylic routes of chlorophyll biosynthesis in higher plants. J. Biol. Chem. 261: 13556-13564
  • 28. Rebeiz, C. A., Montazer-Zouhoor, A., Mayasich, J. M., Tripathy, B. C., Wu, S. M.and Rebeiz, C. C. (1987) Photodynamic herbicides and chlorophyll biosynthesis modulators. In: Ligh-Activated Pesticides, (J. R. Heitz and K. R. Downum, Eds.) ACS Symposium Series 339, pp. 295-328
  • 29. Rebeiz, C. A., Juvik, J. A., and Rebeiz, C. C. (1988) Porphyric insecticides. 1. Concept and phenomenology. Pesticide Biochem. Physiol. 30: 11-27
  • 30. Tripathy, B. C. and Rebeiz, C. A. (1988) Chloroplast Biogenesis 60: Conversion of divinyl protochlorophyllide to monovinyl protochlorophyllide in green(ing) barley, a dark monovinyl light divinyl plant species. Plant Physiol. 87: 89-94
  • 31. Rebeiz, C. A., Juvik, J. A. Rebeiz, C. E. Bouton, C. E. and Gut, L. J. (1990) Porphyric insecticides 2. 1,10-phenonthroline, a potent porphyric insecticide modulator. Pestic. Biochem. Physiol. 36: 201-207
  • 32. Shedbalkar V.P., Ioannides, I.M. and Rebeiz, C.A. (1991) Chloroplast Biogenesis. Detection of monovinyl protochlorophyll(ide) b in plants. J. Biol. Chem. 266: 17151-17157
  • 33. Parham R. and Rebeiz, C. A. (1992) Chloroplast Biogenesis: [4-Vinyl] chlorophyllide reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme. Biochemistry. 31: 8460-8464
  • 34. Rebeiz, N., Arkins, S., Rebeiz, C. A., Simon, J., Zakary, J. F, and Kelley, K. W. (1996) Induction of Tumor Necrosis by ?-Aminolevulinic Acid and 1,10-Phenanthroline. Cancer Res. 56: 339-344
  • 35. Kim, J. S., and C. A. Rebeiz, (1996) Origin of chlorophyll a biosynthetic heterogeneity in higher plants. J. Biochem. Mol. Biol. 29: 327-334
  • 36. Awad Abd El Mageed, El Sahhar, K. F., Robertson, K. R., Parham, R. and Rebeiz, C. A. (1997). Chloroplast Biogenesis 77: Two novel monovinyl and divinyl light-dark greening groups of plants and their relationship to the chlorophyll a biosynthetic heterogeneity of green plants. Photochem. Photobiol. 66: 89-96
  • 37. Adra, A. N. and Rebeiz, C. A. (1998) Chloroplast Biogenesis 81. Tansient Formation of divinyl chlorophyll a following a 2.5 millisecond light flash treatment of etiolated cucumber cotyledons. Photochem. Photobiol. 68: 852-856
  • 38. Rebeiz, C. A., Ioannides, I. M., Kolossov, V. and Kopetz, K. J. (1999) Chloroplast Biogenesis 80. Proposal of a unified chlorophyll a/b biosynthetic pathway. Photosynthetica 36: 117-128
  • 39. Kolossov, V. and Kopetz, K. and Rebeiz, C. A. (2003). Chloroplast Biogenesis 87: Evidence of resonance excitation energy transfer between intermediates of the chlorophyll biosynthetic pathway and chlorophyll a. Photochem. Photobiol. 78: 184-196
  • 40. Rebeiz, C. A. Kolossov, V. L., Briskin, D., and Gawienoski, M. (2003). Chloroplast Biogenesis 86: Chlorophyll biosynthetic heterogeneity, multiple biosynthetic routes and biotechnological spin-offs. In: Handbook of Photochemistry and Photobiology. Amer. Sci. Publisher, Los Angeles, pp 183-248
  • 41. Rebeiz, C. A. Kolossov, V. L., Briskin, D., and Gawienoski, M. (2003). Chloroplast Biogenesis 86: Chlorophyll biosynthetic heterogeneity, multiple biosynthetic routes and biotechnological spin-offs. In: Handbook of Photochemistry and Photobiology. PP Amer. Sci. Publisher, Los Angeles, pp 183-248
  • 42. Kopetz, K. K., Kolossov, V. L., and Rebeiz, C. A. (2004). Chloroplast Biogenesis 89: Development of analytical tools for probing the biosynthetic topography of photosynthetic membranes by determination of resonance excitation energy transfer distances separating metabolic tetrapyrrole donors from chlorophyll a acceptors. Anal. Biochem. Anal. Biochem. 329:207-219
  • 43. . Rebeiz, C. A., Kolossov, V. L. and Kopetz, K. K. (2004). Chloroplast Bioengineering 3. Photosynthetic efficiency, Modulation of the photosynthetic unit size, and the agriculture of the future. In: Agricultural Applications of Green Chemistry (W. M. Nelson, ed.) ACS Symposium Series 887 pp 81-105
  • 44. Kolossov V. L. and Rebeiz, C. A. (2005). Chloroplast Biogenesis 91: Detection of delta-aminolevulinic acid esterase activity in higher plant and insect tissues. Pest.Biochem. Physiol. 83: 9-20

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